(Advances in Meat Research 9) (Auth.), A. M. Pearson, T. R. Dutson (Eds.) - Quality Attributes and Their Measurement in Meat, Poultry and Fish Products-Springer US (1994) PDF

Advances in Meat Research - Volume 9
Quality Attributes and their Measurement

in Meat, Poultry and Fish Products
The Advances in Meat Research series reviews recent advances in meat
science and technology. Each volume concentrates on one specific topic
and discusses it in depth. The chapter authors are recognized as autho-
rities in their fields and come from various countries.
The following volumes are also available:

Volume 6 Meat and Health
Volume 7 Growth Regulations in Farm Animals
Volume 8 Inedible Meat By-Products
Advances in Meat Research - Volume 9
Quality Attributes and their

Measurement in Meat, Poultry
and Fish Products
Edited by
A.M. PEARSON
Courtesy Professor
Department of Animal Sciences
Oregon State University
and
T.R. DUTSON
Dean, College of Agricultural Sciences
Director of Agricultural Experiment Station
Oregon State University
SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.

First edition 1994
© 1994 Springer Science+Business Media Dordrecht
Originally published by Chapman & Hali in 1994
Softcover reprint of the hardcover 1st edition 1994
Typeset in 10/12 pt Times by Acom Bookwork, Salisbury, Wilts
ISBN 978-1-4613-5906-7 ISBN 978-1-4615-2167-9 (eBook)

DOI 10.1007/978-1-4615-2167-9
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Preface
The theme for this volume was chosen because no previous book has
discussed the quality attributes of meat, poultry and fish and the methods
that can be utilized for their measurement. The topics are not only timely
but of great importance.
Chapter I provides an introduction to the topic and presents a brief
overview of the subject to be discussed. The next two chapters review
information on the importance of color and some color problems in
muscle foods, and explains the basis of color vision and perception of
color before describing the methods that may be used for its measure-
ment. The following chapter discusses water binding and juiciness and
their importance, while Chapter 5 provides the first intensive modern
review on measurement of juiciness that has been published (to the
knowledge of the author and editors).
Chapter 6 reviews the physiology and psychology of flavor and aroma,
which serves as a background for further discussion on the flavor and
aroma of foods. The next chapter discusses the chemistry of flavor and
aroma in muscle foods, while measurement of flavor and aroma are
covered in Chapter 8. Chapter 9 reviews the species-specific meat flavors
and aromas. Chapter 10 reviews some flavor and aroma problems in
muscle foods and their measurement.
The next two chapters deal with the importance of meat texture or
tenderness and methods for its measurement, respectively. The related
topic of panel acceptability and the use of sensory panels in measurement
of palatability of muscle foods is discussed in Chapter 13. The following
two chapters cover microbial growth and factors related to problems in
muscle foods and the usefulness of rapid methods for enumeration of
microbial numbers, respectively. The final chapter discusses the impor-
tance of meat, poultry and fish to the health and well being of man - a
most important topic.
A.M.P.
T.R.D.
Contributors
G.K. Beauchamp Monell Chemical Senses Center, 3500 Market Street,

Philadelphia, Pennsylvania 19104, USA.
K.L. Bett Southern Regional Research Center, Agricultural
Research Service, USDA, PO Box 19687, New
Orleans, Louisiana 70179, USA.
J.G. Brand Monell Chemical Senses Center, 3500 Market Street,
Philadelphia, Pennsylvania 19104, USA.
C.P. Brennand Department of Nutrition and Food Science, Utah
State University, Logan, Utah 84322, USA.
B. ChrystaU Meat Industry Research Institute of New Zealand, PO
Box 617, Hamilton, New Zealand.
D. Cornforth Department of Nutrition and Food Sciences, Utah
State University, Logan, Utah 84322-8700, USA.
H.R. Cross Food Safety and Inspection Service, USDA, Washing-
ton, DC 20250, USA.
E. Dransfield Station De Recherches Sur La Viande, INRA, Theix
63122, St. Genes-Champanelle, France.
T.R. Dutson Agricultural Experiment Station, Oregon State Uni-

versity, Oregon 97331, USA.
R.L. Ellis USDA, Food Safety & Inspection Service, Science &
Technology, Chemistry Division, Washington, DC
20250, USA.
D.Y.C. Fung Department of Animal Sciences & Industry, Kansas
State University, Manhattan, Kansas 66506, USA.
J.I. Gray Department of Food Science and Human Nutrition,
Michigan State University, East Lansing, Michigan
48824, USA.
C.G. Grimm Southern Regional Research Center, Agricultural
Research Service, USDA, PO Box 19687, New
Orleans, Louisiana 70179, USA.
viii CONTRIBUTORS
c.J. Hagyard Meat Industry Research Institute of NZ Inc., Hamil-

ton, New Zealand.
R. Hamm Institute of Chemistry and Physics, Federal Centre for
Meat Research, Kulmbach, Germany.
K.O. Honikel Institute of Chemistry and Physics, Federal Centre for
Meat Research, Kulmbach, Germany.
J. Love Department of Food Science and Human Nutrition,
Iowa State University, Ames, Iowa 50011, USA.
J.R. Lupton Faculty of Nutrition, Department of Animal Science,
Texas A&M University, College Station, Texas 77843,
USA.
D.B. MacDougall Department of Food Science and Technology, Uni-
versity of Reading, White knights, PO Box 226,
Reading RG6 2AP, UK.
F.J. Monahan Department of Food Science and Technology, Uni-
versity of California, Davis, California 95616, USA.
A.M. Pearson Department of Animal Sciences, Oregon State Uni-
versity, Corvallis, Oregon 97331, USA.
G. Reineccius Department of Food Science and Nutrition, University
of Minnesota, St. Pauls, Minnesota 55108, USA.
J.N. Sofos Department of Animal Sciences, Colorado State Uni-
versity, Fort Collins, Colorado 80523, USA.
R.J. Winger Department of Food Technology, Massey University,
Palmerston, New Zealand.
Contents
1 Introduction to quality attributes and their measurement in

meat, poultry and fish products 1
A.M. PEARSON
1.1 Introduction 1
1.2 Color 2
1.2.1 Importance 2
1.2.2 Variability and measurement 2
1.3 Juiciness and/or water-binding 2
1.3.1 Importance 2
1.3.2 Effects of variability and measurement 3
1.4 Flavor 3
1.4.1 Importance 3
1.4.2 Variability 3
1.4.3 Physiology and psychology of flavor/aroma 4
1.4.4 Specific flavors/odors 4
1.4.5 Flavor and aroma problems 4
1.5 Tenderness 5
1.5.1 Importance 5
1.5.2 Some factors influencing tenderness and its measurement 5
1.6 Microbial problems 6
1.6.1 Importance 6
1.6.2 Measurement 6
1.7 Additives and residues 6
1.7.1 Additives 6
1.7.2 Residues 7
1.8 Contributions of meat to human nutrition 7
1.8.1 Proteins and essential amino acids 7
1.8.2 Fats and essential fatty acids 7
1.8.3 Vitamins 8
1.8.4 Minerals 9
1.9 Summary 9
References 10
Appendix 1.1 18
Appendix 1.2 25
2 Color - its basis and importance 34

D.CORNFORTH
2.1 Introduction 34
2.1.1 Retail importance of meat color 34
2.2 Myoglobin and its derivatives 35
2.2.1 Myoglobin concentration in muscle 37
2.3 Factors affecting fresh meat color stability 39
2.3.1 Oxygen tension 39
2.3.2 Bacteria 41
2.3.3 Vacuum-packaging 43
2.3.4 Packaging with oxygen-permeable films 45
x CONTENTS
2.3.5 Modified-atmosphere packaging 46

2.3.6 Effects of pH 48
2.3.7 Temperature 48
2.4 Dark-cutting beef and related dark color problems 49
2.4.1 Characteristics of dark-cutting meat 49
2.4.2 Mechanism by which pH affects color 50
2.4.3 Changes occurring after death 50
2.4.4 Shelf-life of high pH meat 51
2.4.5 Vacuum packaging 51
2.4.6 Minimizing dark-cutters by management 51
2.4.7 Dark, coarse band in beef ribs 52
2.5 Pale, soft, exudative (PSE), porcine stress syndrome (PSS) and dark, firm,
dry (DFD) pork' 52
2.5.1 Importance of PSE, PSS and DFD pork 52
2.5.2 Genetic basis 53
2.5.3 Influence of environmental factors 53
2.6 Enzymatic reduction of metmyoglobin 54
2.6.1 Enzymes involved 55
2.6.2 Variation among muscles 56
2.6.3 Other factors influencing color stability 56
2.7 Non-enzymatic reductants and inhibitors of oxidation 58
2.7.1 Effects of antioxidants and reductants 58
2.8 Irradiation and other antimicrobial treatments 60
2.8.1 Irradiation of fresh and cooked meats 60
2.8.2 Sulfites and meat color 61
2.9 Effects of light, freezing, salt and lipid oxidation on meat color 62
2.1 0 Cooked meat color 62
2.10.1 Pink color in cooked, uncured meat 63
2.11 Cured meat color 64
2.11.1 Role of nitrite 64
2.11.2 Action of cysteine and ascorbate 65
2.11.3 The cured meat pigment 66
2.11.4 Fading of cured meat color 66
2.12 Summary 67
References 68
3 Colour of meat 79
D.B. MACDOUGALL
3.1 Introduction to vision and colour 79
3.2 Colour vision 79
3.2.1 Colour measurement 80
3.2.2 Uniform colour space 81
3.3 Terminology 82
3.4 Instrumentation 83
3.4.1 Trichromatic colorimeters 83
3.4.2 Spectrophotometers 83
3.4.3 Sources of variation among colorimeters and spectrophotometers 84
3.5 Absorption, scatter and pigmentation 84
3.5.1 Reflectance 84
3.5.2 Light scatter 85
3.6 Meat colour 85
3.6.1 Measurement procedure 86
3.6.2 Reflectance spectral changes in meat 90
3.6.3 Colour changes in beef 91
3.6.4 Differences between CIELAB and Hunter scales 91
3.7 Summary 92
References 92
CONTRIBUTORS xi
4 Juiciness - its importance and some contributing factors 94

R.J. WINGER and c.J. HAGYARD
4.1 Introduction 94
4.2 Subjective assessment of juiciness 94
4.3 Relationship to objective measurements 96
4.3.1 Juiciness vs. water-holding 97
4.3.2 The state of water 97
4.3.3 Heating method and end-point temperature vs. juiciness 97
4.3.4 The role of fat 97
4.3.5 Relationship between drip losses and juiciness 98
4.3.6 Relationship between press fluid and juiciness 98
4.4 Factors influencing the juiciness of intact meat 98
4.4.1 Interference from other experimental and textural factors 98
4.4.2 Heating/cooking methods 99
4.4.3 Animal characteristics 102
4.4.4 Factors related to rigor development 105
4.4.5 Restructured meat 108
4.4.6 Processed meats 112
4.4.7 Marinaded meat 114
4.5 Conclusions 115
References 116
5 Measurement of water-holding capacity and juiciness 125

K.O. HONIKEL and R. HAMM
5.1 Introduction 125
5.2 Composition and structure of meat 126
5.3 State of water in meat 127
5.4 Definition of water-holding capacity 129
5.5 General methodology 130
5.5.1 Applying no force 130
5.5.2 Applying external mechanical force 130
5.5.3 Applying thermal force 130
5.5.4 Choosing a method for measuring WHC 131
5.6 Influence of different factors on WHC 131
5.6.1 Evaporation losses 132
5.6.2 Drip losses 133
5.6.3 Filter paper press method (FPPM) 133
5.6.4 Centrifugation methods for uncooked meat 134
5.6.5 Capillary volumeter method 134
5.6.6 Imbibing method 134
5.6.7 Cooking losses 134
5.7 Description of methods and evaluation 136
5.7.1 Drip loss determination 136
5.7.2 Determination of water-holding capacity using the filter paper press
method (FPPM) 139
5.7.3 Determination of water-holding capacity by centrifugation 143
5.7.4 Capillary volumeter measurements 145
5.7.5 Imbibing method 148
5.7.6 Cooking loss measurement 148
5.8 Meat products 152
5.8.1 Manufacturing of cooked sausage 153
5.8.2 Water-holding capacity of unheated batters 154
5.8.3 Water-holding capacity of heated batters 156
5.8.4 Measurement of water-holding capacity in sausage batters 157
5.8.5 Evaluation of methods 157
5.9 Conclusions about water-holding capacity measurements 157
Xli CONTRIBUTORS
5.10 Measurement of juiciness 158

5.10.1 Problems in evaluating juiciness 158
5.10.2 Evaluation of methods 158
5.11 Summary 159
References 159
6 The chemical senses 162

G.K. BEAUCHAMP and J.G. BRAND
6.2 Anatomy and physiology of the chemical senses: taste 162
6.2.1 Overview, 162
6.2.2 Anatomy 163
6.2.3 Reception and transduction 163
6.3 Anatomy and physiology of the chemical senses: olfaction 167
6.3.1 Overview 167
6.3.2 Anatomy 167
6.3.3 Reception and transduction 168
6.4 Sensory responses to food: taste 169
6.4.1 Development of taste preference 169
6.4.2 Effects of aging 171
6.5 Sensory responses to food: olfaction 172
6.5.1 Development of olfactory preference 172
6.5.2 Effects of aging 174
6.6 Specific appetites 175
6.6.1 Salt 175
6.6.2 Amino acids and proteins 176
6.7 Chemosensory mixtures 177
6.8 Conclusions 178
References 178
Acknowledgements 183
7 Flavor and aroma chemistry 184

G. REINECCIUS
7.2 Flavor precursors 185
7.3 The taste of meat 186
7.4 Meat aroma 187
7.4.1 Maillard reaction 189
7.4.2 Lipid reactions 190
7.4.3 Thiamin degradation 193
7.4.4 Role of nitrite in cured meat flavor 193
7.4.5 Volatiles and flavor 197
7.5 Summary 198
References 199
8 Flavor and aroma - its measurement 202

K.L. BETT and C.C. GRIMM
8.2 Difference tests 202
8.3 Descriptive flavor analysis 203
8.3.1 The flavor profile 204
8.3.2 Quantitative descriptive analysis 204
8.3.3 The spectrum method 205
CONTRIBUTORS Xlll
8.4 Instrumental analysis 205

8.4.1 Extraction and concentration of flavor compounds 205
8.4.2 Selection of a concentration-extraction procedure 207
8.4.3 Gas chromatographic (GC) analysis 207
8.4.4 Sniffer-port analysis 209
8.4.5 Flavor analysis by HPLC 209
8.4.6 Piezo-eIectric crystals 210
8.5 Correlation between sensory analysis and gas chromatography 210
8.5.1 Principal component analysis 211
8.5.2 Factor analysis 211
8.5.3 Cluster analysis 212
8.5.4 Discriminant analysis 212
8.5.5 Regression and correlation 213
8.5.6 Response surface methodology 213
8.5.7 Neural networks 214
8.6 Analysis of an example data-set 214
8.6.1 Description of data 214
8.6.2 Statistical methods 215
8.6.3 Results of statistical analysis 217
8.7 Summary 219
References 220
9 Species-specific flavors and odors 222

A.M. PEARSON, 1.1. GRAY and C.P. BRENNAND
9.2 Fatty acids in meats 222
9.2.1 Influence of cooking on the fatty acid composition of meat 223
9.2.2 Volatile free fatty acids and some other possible flavor compounds 223
9.2.3 Free fatty acids in meat 224
9.2.4 Branched-chain fatty acids (BCFAs) 224
9.3 Subcutaneous and perinephric adipose tissue 227
9.4 Synthesis of branched-chain fatty acids 231
9.4.1 Effects of diet on synthesis of branched-chain fatty acids 231
9.4.2 Body secretions of branched-chain fatty acids by meat-producing
animals 232
9.4.3 Odors and flavors of some selected fatty acids 235
9.4.4 Determination of the concentration of short-chain and branched-
chain fatty acids 236
9.4.5 Measurement of alkylphenols and thiophenols 237
9.5 Other flavor or odor compounds localized in the fatty tissues 237
9.5.1 Sex odor or boar 'taint' 237
9.5.2 Pathways for CI9"~16-steroid production 237
9.5.3 Thresholds and odors of the CI9"~ 16-steroids 238
9.5.4 Identification and quantification of the C19-~16-steroids 238
9.5.5 Possible role of skatole 238
9.5.6 Isolation and quantification of skatole 239
9.6 Species-specific flavors 239
9.6.1 Lamb and/or mutton flavor 239
9.6.2 Goaty flavors and odors 241
9.6.3 Odors and flavors in pork 241
9.6.4 Beef and veal flavors and odors 242
9.6.5 Chicken- and turkey-specific flavors and odors 243
9.6.6 Fish-specific flavors and odors 243
9.6.7 'Gamey' flavors and odors 244
9.7 Summary 244
References 245
xiv CONTRIBUTORS
10 Flavor and aroma problems and their measurement in meat, poultry

and fish products 250
l.I. GRAY, A.M. PEARSON and F.l. MONAHAN
10.2 Oxidative rancidity/warmed over flavors 251
10.2.1 Lipid oxidation and meat quality 251
10.2.2 Inhibiting lipid oxidation 253
10.2.3 Effect of lipid oxidation on meat flavor 254
10.2.4 Catalysis of lipid oxidation in meats 255
10.2.5 Measurement of lipid oxidation in meats 257
10.3 Species-specific flavors 261
10.4 Effects of differertt feeds on flavor and aroma 261
10.4.1 Lamb and mutton 261
10.4.2 Veal and beef 264
10.4.3 Pig meat 265
10.4.4 Fish 265
10.4.5 Poultry 266
10.4.6 Other species 267
10.5 'Gamey' flavors 267
10.6 Off flavors due to sex condition 268
10.6.1 Boar odor or taint 268
10.6.2 Ram odor/flavor 269
10.6.3 Sex flavor/aroma in other species 270
10.7 Off-flavors from the environment 270
10.8 Processing-induced off-flavors 271
10.8.1 Irradiation flavor/odor 271
10.8.2 Retort flavor of canned meat 273
10.9 Off-flavors associated with microbial growth 274
10.9.1 Off-odors in fish caused by microbial growth 274
10.9.2 Off-odors and flavors in poultry associated with microbial growth 276
10.9.3 Off-odors and flavors produced in red meats by microbial growth 277
10.10 Summary 277
References 278
11 Tenderness of meat, poultry and fish 289

E. DRANSFIELD
ILl Introduction 289
11.2 Pre-slaughter factors 290
11.2.1 Breed effects 290
11.2.2 Fatness 292
11.2.3 Sex effects 293
11.2.4 Growth promoters 294
11.2.5 Connective tissue 294
11.3 Slaughtering 296
11.4 Rigor development 297
11.4.1 Compositional and structural changes post-mortem 297
11.4.2 Temperature effects 297
11.4.3 Electrical stimulation 298
11.5 Muscle shortening 299
11.5.1 Relationship between muscle shortening and tenderness 299
11.5.2 Carcass suspension effects 300
11.5.3 Hot-deboning 300
11.5.4 Pre-rigor cooking 301
11.5.5 Pre-rigor cooling 301
11.5.6 Electrical stimulation 302
CONTRIBUTORS xv
11.6 Ultimate pH effects 303

11. 7 Effects of post-rigor storage 303
11. 7.1 Mechanism of tenderisation 304
11. 7.2 Pre-slaughter factors 306
11. 7.3 Influence of muscle-shortening 306
11.7.4 Temperature 307
11.8 Artificial tenderisation 308
11.8.1 Proteolytic enzymes 308
11.8.2 Marinading 309
11.8.3 Pressure treatment 309
11.9 Control of tenderness 309
11.10 Summary and research needs. 310
References 311
12 Meat texture measurement 316

B. CHRYSTALL
12.2 Why measure tenderness and when? 317
12.3 Subjective assessments 318
12.4 Objective assessments 319
12.4.1 Shear and biting systems 320
12.4.2 Compression methods 321
12.4.3 Tensile assessments 322
12.4.4 Penetration methods 324
12.4.5 Grinding methods 325
12.4.6 Fragmentation methods 325
12.5 Structural assessments 325
12.6 Chemical measures 326
12.7 Other methods 326
12.8 Samples 327
12.9 Standard protocols 329
12.10 Relationships between assessment methods 329
12.10.1 Between objective assessments 329
12.10.2 Between objective and subjective assessments 330
12.11 Conclusions 331
References 332
13 Product acceptability evaluation 337

J. LOVE
13.2 Affective testing: testing for acceptance and preference 339
13.2.1 Scaling 339
13.2.2 Direct preference tests 341
13.3 Affective vs. analytical sensory testing 342
13.4 Subjects in acceptance tests: the usefulness of trained panels or experts in
evaluating product acceptability 343
13.5 Relating sensory attributes to product acceptability 346
13.5.1 Hedonic scores and intensity of sensory attributes 346
13.5.2 Fat level as a determinant of acceptability 348
13.5.3 Factors that alter the relative importance of appearance, texture
and flavor in consumer acceptance studies 351
13.6 Factors affecting the outcome of acceptance tests 352
13.6.1 Type of test or method 352
XVI CONTRIBUTORS
13.6.2 Context effects 354

13.6.3 Design and control of experiments 355
13.7 Summary 355
References 355
14 Microbial growth and its control in meat, poultry and fish 359
J.N. SOFOS
14.2 Microbial contamination of muscle foods 359
14.2.1 Sources of contamination 359
14.2.2 Types of contamination 362
14.3 Microbial effects'on muscle foods 364
14.3.1 Spoilage 364
14.3.2 Foodborne illness 368
14.4 Control of microbial growth in muscle foods 379
14.4.1 General 379
14.4.2 Decontamination 382
14.4.3 Modified-atmosphere storage 385
14.4.4 Biopreservation 388
14.5 Summary 391
References 391
15 Rapid methods for measurement and enumeration of microbial

contamination 404
D.Y.C. FUNG
15.2 Improvements in sampling and sample preparation 405
15.2.1 Stomacher 405
15.2.2 Hand roller 406
15.2.3 Gravimetric Diluter or Diluflo 408
15.2.4 Other methods 409
15.3 Alternative methods for viable cell count procedure 409
15.3.1 The spiral plating method 409
15.3.2 The Isogrid system 413
15.3.3 The Petrifilm system 413
15.3.4 The Redigel system 415
15.3.5 The Direct Epifluorescent Filter Technique 416
15.3.6 Double-tube method 417
15.4 New methods for estimation of microbial populations 418
15.4.1 ATP estimation 418
15.4.2 Impedance and conductance measurements 419
15.4.3 Radiometry and calorimetry 422
15.4.4 Reflectance colorimetry 423
15.4.5 Limulus amoebocyte lysate and catalase tests 423
15.5 Miniaturized microbiological techniques 427
15.6 New and novel techniques 429
15.6.1 Vitek system 429
15.6.2 DNA probes 430
15.6.3 Target RNA probes 430
15.6.4 ELISA tests 430
15.6.5 The VIDAS immunoanalysis system 431
15.6.6 The polymerase chain reaction 431
15.6.7 Motility enrichment 432
15.6.8 Oxyrase enzyme system 433
15.7 Conclusions 433
Acknowledgement 435
References 435
CONTRIBUTORS XVll
16 Food analysis and chemical residues in muscle foods 441

R.L. ELLIS
16.1 Food analysis 441
16.1.1 Introduction 441
16.1.2 Protein 442
16.1.3 Fat 443
16.1.4 Moisture 443
16.1.5 Salt 443
16.1.6 Sugars 444
16.1.7 Nitrate 445
16.1.8 Nitrite 445
16.1.9 Nitrosamines 445
16.1.1 0 Cholesterol 446
16.1.11 Rapid test methods 447
16.2 Chemical residues 447
16.2.1 Introduction 447
16.2.2 Pesticide-residue analysis 450
16.2.3 Volatile environmental contaminants 456
16.2.4 Halophenols 456
16.2.5 Veterinary drug residue analysis 457
16.3 Summary 473
References 474
17 The contributions of meat, poultry and fish to the health and well
being of man 479
1.R. LUPTON and H.R. CROSS
17.2 Consumption of meat, poultry and fish 479
17.2.1 Factors affecting consumption of meat, poultry and fish 479
17.2.2 How consumption of meat, poultry and fish contributes to nutrient
intake 481
17.3 The nutritional value of meat, poultry and fish 481
17.3.1 Criteria for what makes a food 'nutritious' 482
17.3.2 Specific nutrients supplied by meat, poultry and fish 482
17.4 The role of meat, poultry and fish in human health 487
17.4.1 Meat, poultry and fish and their relationship to heart disease 488
17.4.2 Meat, poultry and fish and their relationship to cancer 493
17.5 Summary 495
Acknowledgement 496
References 496
Index 500
1 Introduction to quality attributes and their
measurement in meat, poultry and fish products
A.M. PEARSON
1.1 Introduction
Although food is essential in that it supplies the nutrients necessary to

sustain life, it must first be consumed in order to supply the needed nutri-
ents. Thus, food must not only be pleasing in taste, aroma, consistency
and appearance to make it palatable but it must also satisfy the nutri-
tional requirements and aesthetic needs of consumers in order to fulfil its
life-sustaining role. Meat, poultry and fish products must meet the same
requirements as other foods in providing not only nutritionally adequate,
but palatable and acceptable products to the diet of humans.
The purpose of this book is to consider the palatability attributes that
influence the acceptability of various meat, poultry and fishery products,
how they may differ or be altered and the methodology that can be
utilized for their measurement. It is also the aim of this book to briefly
review the nutritional contributions of meat, poultry and fish, which were
extensively discussed in an earlier volume of this series by those knowl-
edgeable in the science of nutrition (Pearson and Dutson, 1990). Suffice it
to say that these animal products make important nutritional contribu-
tions to the diet of mankind. Evidence for this viewpoint is supported by
the increase in stature of succeeding generations of Japanese, both in
Japan and among immigrants to the US from Japan, as the per capita
consumption of meat, poultry and fish products has increased.
This volume will concentrate on the palatability attributes of meat,
poultry and fish products, since these characteristics have not been exten-
sively covered in a single publication. First, consideration will be given to
the factors influencing our current concepts and knowledge of each of the
acceptability and/or palatability attributes. Then discussion will focus on
methods of measurement for each of the factors affecting acceptability.
Consideration will also be given to two other key areas that can influence
the acceptability and safety of meat, poultry and fish products, namely,
contamination by microorganisms and residues in these products. Follow-
ing that discussion the last chapter will focus on the nutritional contribu-
tions of meat, poultry and fish products to the health and well-being of
man, which is a subject of great importance.
2 QUALITY ATTRIBUTES IN MEAT, POULTRY AND FISH PRODUCTS
1.2 Color
1.2.1 Importance
Color is known to play an important role in the acceptability of meat,
poultry and fish products. The changes in color associated with the muscle
and blood pigments (myoglobin and hemoglobin, respectively) to a con-
siderable extent determine the attractiveness of fresh red meat, while the
formation of cured meat pigments and their stability influence the accept-
ability of cured meat products (Appendix 1.1). The muscle pigments and
their reactions - both desirable and undesirable - have been reviewed by
several researchers (Fox, 1966; Greene, 1969; Giddings, 1977; Livingston
and Brown, 1981; MacDougall, 1982; Hunt and Kropf, 1987). Further
discussion of meat pigment chemistry and factors affecting their stability
or lack of stability will be discussed in chapter 2. Emphasis on the color
of both fresh and cured meat products can not be overemphasized in view
of the importance of color to attractiveness and acceptability. Color is
known to influence the consumer concept of freshness, which in turn
influences acceptance of meat, poultry and fish products.
1.2.2 Variability and measurement

In the case of poultry and fish products, emphasis on color may be quite
different than for red meats, with a lack of muscle pigmentation being
desirable in turkey and chicken breast meat (Appendix 1.1), which may
also be true for certain species of fish. Conversely, a darker or reddish
color may be desirable in some species of fish, such as salmon, whereas
thigh meat from chickens and turkeys should have enough myoglobin to
make the muscles appear red in the fresh state and be adequate in
quantity to produce a good cured meat color in production of several
cured poultry products (chapter 2). The fact that the muscles from turkeys
and chickens vary in the amount of indigenous muscle pigments creates
special problems in poultry. Sometimes the problems are compounded by
the fact that the poultry bones contain both hemoglobin and myoglobin,
which may be released during processing and/or freezing. In chapter 2
some of these problems and their impact on the color of poultry and fish
products are discussed, while in chapter 3 measurement of color is
covered.
1.3 Juiciness and/or water-binding
1.3.1 Importance
The importance of mouthfeel, which is associated with juiciness, is
another quality attribute that contributes to consumer acceptability of
INTRODUCTION 3
meat, poultry and fish products (Appendix 1.1). The crucial importance of
mouthfeel and the concept of juiciness while eating these animal products
is difficult to describe and quantify but has a profound effect upon the
other sensory attributes of meat, poultry and fish. Dryness is associated
with a decrease in the other palatability attributes, especially with the lack
of flavor and increased toughness.
1.3.2 Effects of variability and measurement

Water-binding refers to the inherent ability of these products to hold or
bind water. Economically, water-binding is important besides its direct
effects upon the sensation of juiciness. The effect of 'weep' or 'drip' losses
extends far beyond its influence on juiciness as indicated by the unattrac-
tiveness observed in pale, soft and exudative (PSE) pig meat as outlined
by Briskey (1964). The effects of poor water-binding in production of
cured meat, poultry and fish products has been discussed by Hamm (1960,
1977) and more recently expanded and explained by Offer and Trinick
(1983). In chapters 4 and 5 of this volume the importance of juiciness in
these products is discussed and also the measurement of juiciness and
water-binding, respectively. In chapter 4 some factors contributing to jui-
ciness are reviewed, while in chapter 5 various procedures that have been
used to measure juiciness and water-binding along with their probable
usefulness are discussed.
1.4 Flavor
1.4.1 Importance
Although consumer-type panels have indicated that tenderness may be the
most important quality attribute influencing the acceptability of meat, this
would assume that the products have a flavor typical of the product being
consumed. Without question, consumers prize the flavors that are
commonly associated with meat, poultry and fish products, which to a
large extent are responsible for the desirability of these products by con-
sumers (Appendix 1.1). However, there normally tends to be less variation
in flavor perception than is true for some of the other sensory attributes
of these products, i.e. tenderness and juiciness.
1.4.2 Variability
Flavor and aroma of meat, poultry and fish products varies widely, with
each species having their own characteristic flavor and aroma. The meaty
flavor from the various kinds of meat products has been shown to be
water-extractable (Crocker, 1948; Bouthilet, 1951a,b; Kramlich and

Pearson, 1958) and to be identical for muscle, regardless of the species
from which it originated (Hornstein and Crowe, 1960; Hornstein et al.,
1963). Conversely, species differences in flavor have been reported to ori-
ginate from the lipid fraction and have been associated with different lipid
components (Hornstein et al., 1963). Thus, the subtle but recognizable dif-
ferences in the flavor of meat, poultry and fish are believed to be the
result of different lipid components in the respective species. The compo-
nents contributing to meat, poultry and fish flavors per se and the mea-
surement of the constituents contributing to such flavors are discussed in
greater detail in chapters 7 and 8.
1.4.3 Physiology and psychology offlavor/aroma

Aroma and flavor physiology and psychology is an area of great current
interest. Tremendous strides have been made in understanding the role of
the nervous system and brain and how they react to various stimuli via
flavor and aroma perception. Nevertheless, our understanding of this
important area is still growing at a rapid pace. Once the interactions are
fully elucidated, other ways of altering our perception of aromas and
flavors may be possible. The basis of flavor and aroma perception no
doubt lies in understanding the mechanism by which mankind responds to
flavors and aromas. In chapter 6 the current state of knowledge on flavor
and aroma physiology and psychology and their interactions is reviewed.
1.4.4 Species-specific flavors/odors

Species-specific flavors and odors are also of great interest as consumers
have distinct flavor/aroma preferences (Appendix 1.1). Much work has
been carried out in attempts to differentiate between the components that
contribute to species-specific flavors, i.e. beef, pork and lamb flavor.
Developments in this area are presented and discussed in chapter 9 and
summarize the current status of our understanding of how species-specific
flavors/aromas are developed and controlled in different meat, poultry and
fish products.
1.4.5 Flavor and aroma problems

Flavor and aroma problems in meat, poultry and fish arise from a variety
of conditions, which include absorption of undesirable odors and flavors
from atmospheric contaminants. For example, the author has observed
absorption of off-flavors by storing fresh chicken meat in a refrigerator
containing a variety of medicinals used for veterinary purposes, which
caused the chicken to taste like medicine even after cooking (A.M.
INTRODUCTION 5
Pearson, unpublished observations). Spoiled meat aromas and flavors may

also be absorbed by fresh meat, poultry and fish even when stored under
refrigeration (A.M. Pearson, unpublished observations). The flavors of
dietary origin (known as feed flavors) can also influence the acceptability
of meat, poultry and fish products (chapter 10).
Oxidized and warmed-over flavors are also of great interest because of
the increased emphasis on marketing of frozen precooked meals, where
both freezing and precooking have been demonstrated to contribute to
off-flavor development in meat, poultry and fish products (Appendix 1.1).
The lipid fraction of these animal products is responsible for both oxida-
tion and warmed-over flavor, which is covered in some detail in chapter
10, along with some suggestions on how such problems can be avoided in
meat, poultry and fish products. Since meat, poultry and fish products are
major components in the precooked frozen meals that are being widely
used in the food service industry and are becoming increasingly important
to home consumers where convenience and ease of preparation are
becoming important considerations, such discussion is of special interest.
1.5 Tenderness
1.5.1 Importance
Consumer surveys have indicated that tenderness of meat is the palat-
ability attribute that is most frequently perceived to be a problem by con-
sumers. Thus, the importance of tenderness and the factors that impact on
it are emphasized. Although it is commonly accepted that the meat from
older animals is tougher than that from younger animals, it is also known
that, for some unexplained reason, a low but significant proportion of old
cows produce tender meat. The ability to identify the reasons that these
cows produce tender meat may help in unraveling the mystery of varia-
bility in meat tenderness.
1.5.2 Some Jactors influencing tenderness and its measurement

The effects of exercise, sex, marbling, fat covering, conformation, aging,
breed, cold shortening, muscle-to-muscle variation, thaw rigor and other
factors that may influence meat tenderness (Appendix 1.1) are discussed in
chapter II, while measurement of tenderness is reviewed in chapter 12.
Consideration will be focused on the most important factors that influence
meat tenderness and will be discussed in considerable detail. Although
tenderness is probably less important in poultry and fish, under certain
conditions it can also be a problem in some of these products (chapter
II ).
1.6 Microbial problems
1.6.1 Importance
Meat, poultry and fish are all highly perishable in their fresh condition.
Microbial contamination, its control and measurement are important
topics (Appendix 1.1) and are discussed in chapters 14 and 15, respec-
tively. Total microbial counts and identification of specific pathogens and
their control are of major concern to producers, slaughterers and pro-
cessors of meat, poultry and fish (Zottola and Smith, 1990). Problems of
microbial origin are certainly among, if not the most important, chal-
lenges facing these industries today. Thus, emphasis must be focused on
steps that can be taken to resolve the most important microbiological
problems in order to not only protect consumers by providing them with
a safe supply of meat, poultry and fish products but also to prevent costly
governmental recalls of already contaminated products.
1.6.2 Measurement
Rapid methods used for measurement and enumeration of microbial con-
tamination are discussed in chapter 15. Implementation of the Hazard
Analysis Critical Control Point (HACCP) concept by the meat, poultry
and fish industries also offers promise in helping to decrease problems
from microbial contamination and can be utilized in recognition of the
critical points that need emphasis to control both microbial hazards and
spoilage.
1.7 Additives and residues
1.7.1 Additives
Additives are added to meat, poultry and fish products for a variety of
purposes, such as nitrite that is used to stabilize the color, to protect
against oxidation and to prevent the outgrowth of Clostridium botulinum
with production of its potent deadly toxin. Other additives include salt,
which preserves meat, poultry and fish products by lowering the water
activity (aw ), and phosphates, which increase water-binding in these same
products. A host of other additives are used in the meat trade, including
erythorbate, carrageenans, hydrolyzed vegetable protein (HVP), white and
black pepper, soy proteins and various other seasoning ingredients. Addi-
tives are covered in greater detail in chapter 16. Toxic compounds
produced during meat processing have been reviewed by Hotchkiss and
Parker (1990) and are also discussed further in chapter 16.
INTRODUCTION 7
1.7.2 Residues
Residues include those from pesticides, insecticides, feed additives or from
antibiotics and sulfa drugs that are used in veterinary medicine. Residues
are often conceived by consumers as being a serious problem, although
Food Safety and Inspection Service (FSIS) inspection provides a con-
tinuous in-place monitoring program as explained by Pullen (1990).
Although residues probably are less of a hazard to consumers than patho-
genic bacteria, concerns of the public about residues could result in even
greater emphasis upon residue monitoring for meat, poultry and fish
products by FSIS and Food and Drug Administration (FDA) inspectors.
Residues in foods of animal origin are discussed in greater detail in
chapter 16.
1.8 Contributions of meat to human nutrition
Discussion of the quality attributes of meat, poultry and fish would be

incomplete without covering the nutritional contributions of these foods,
which will be reviewed in chapter 17. Meat, poultry and fish products
make important contributions to the diet of man in four major areas: (i)
proteins and/or essential amino acids; (ii) fats and essential fatty acids;
(iii) vitamins; and (iv) minerals.
I.B.1 Proteins and essential amino acids

Meat is an excellent source of protein and essential amino acids, with its
contributions to the human dietary needs being recently reviewed by
Pellett and Young (1990). These authors concluded that the high content
of dietary lysine in meat, poultry and fish make these products particu-
larly important in meeting the needs for this indispensable (essential)
amino acid in cereal-based diets. Even though there is no absolute nutri-
tional requirement for meat proteins per se in these products in the human
diet, meat is an excellent source of protein and indispensable amino acids
that are highly palatable to humans (Pellett and Young, 1990). For more
information on proteins and the indispensable amino acids, readers are
referred to chapter 17.
I.B.2 Fats and essential fatty acids

Reiser and Shorland (1990) have summarized some of the considerations
about the relationship of meat fats and fatty acids to coronary heart
disease (CHD), which has become a major health concern of the public.
These authors carefully point out that not all long-chain saturated fatty
acids are atherogenic but may be metabolized differently than some
shorter-chained fatty acids as shown earlier by Hegsted et al. (1965) and

confirmed more recently by Bonanome and Grundy (1988). McNamara
(1990) has reviewed the relationship between dietary and blood cholesterol
and concluded that although there is a relationship, a reduction of dietary
cholesterol by 150 mg per day would, on average, result in only a 3-4 mg.
dl- 1• per day reduction in blood cholesterol or a little less than a 2%
reduction. This does not mean that one should ignore the fat content of
the diet but that the major monounsaturated and saturated fatty acids
found in meat may not increase blood cholesterol but actually be anti-
atherogenic in contrast to some of the shorter-chain fatty acids
(Bonanome and Grundy, 1988). For more details on the effects of the
fatty acids on CHD and health, readers are referred to chapter 17.
Another area of considerable interest is the n-3 fatty acids present in
fish and fish oils (Reiser and Shorland, 1990). The n-3 fatty acids have
recently been shown to control the deleterious effects of excess linoleic
acid, which have markedly increased as a consequence of the use of unsa-
turated fatty acids in an attempt to reduce blood cholesterol levels in man
(chapter 17). Thus, the n-3 fatty acids that are found in high concentra-
tions in fish and fish oils, and to a lesser extent in the tissues of meat
animals, may play an important role in reducing CHD in man (Reiser and
Shorland, 1990). Although an understanding of exactly how n-3 fatty
acids reduce the incidence of CHD is not entirely clear at this time, there
is little doubt about its overall effect on CHD.
1.8.3 Vitamins
The vitamins are usually divided into two groups on the basis of their
solubility, i.e. the fat-soluble vitamins (A, D, E and K) and the water-
soluble vitamins, which include vitamin C and the group generally classi-
fied as the B-complex vitamins (Appendix 1.2).
1.8.3.1 Fat-soluble vitamins. The fat soluble vitamins as shown in

Appendix 1.2 consist of A, D, E and K. Generally, meat is not a good
source of the fat soluble vitamins, although it may make a small but
important contribution to vitamins A and E (Smith, 1990). The impor-
tance of meat, poultry and fish to the intake of the fat soluble vitamins is
discussed further in chapter 17.
1.8.3.2 Water-soluble vitamins. Meat, poultry and fish make little con-
tribution to vitamin C intake but are major contributors to many B-
complex vitamins, especially to thiamin, riboflavin, niacin and pantothenic
acid intake (Windham et al., 1990). Animal products are also major
dietary sources of vitamin B6 and vitamin B12, with these products pro-
viding over 98% of the intake of vitamin Bl2 (Sauberlich, 1990). More
discussion on the role of meat, poultry and fish in meeting the nutritional
INTRODUCTION 9
needs of man for the water-soluble vitamins can be found in chapter 17

and in Appendix 1.2.
1.8.4 Minerals
The minerals required in the diet of humans are generally classified into
two groups, namely the macro- and micro-minerals. Thus the differentia-
tion is based on the relative amounts needed by man, with the former
group being required in larger amounts than the latter, which are fre-
quently called trace minerals (Appendix 1.2).
1.8.4.1 Macro-minerals. The macro-minerals include calcium, phos-

phorus, potassium, magnesium, sodium and chlorine, all of which are
required by man. Meat, poultry and fish are not good sources of calcium
(except in de boned products), potassium or magnesium (Johnson and
Nielsen, 1990; Karanja et al., 1990). Calcium, potassium and magnesium
are discussed in chapter 17 and Appendix 1.2.
Although meat, poultry and fish are important dietary sources of phos-
phorus, there are also several other good sources (Appendix 1.2). Sodium
and chlorine are also present in appreciable amounts in meat, poultry and
fish products, especially in cured items where salt is used as a preservative.
However, excessive salt intake is of greater importance than deficiencies of
sodium and chlorine. Thus, reduction of sodium intake may be more
important than too little intake (Pearson and Wolzak, 1982; Karanja et
al., 1990). All of the macro-minerals will be discussed in greater detail in
chapter 17.
1.8.4.2 Micro-minerals (trace minerals). The trace or micro-minerals

known to be required in the diet of man include iron, copper, cobalt,
selenium, manganese, zinc and iodine, and perhaps others (Appendix 1.2).
Meat, poultry and fish are relatively poor sources of manganese but these
products may help in some unknown way to improve manganese balance
(Johnson and Nielsen, 1990). These animal products also contribute
appreciable amounts of cobalt (vitamin Bl2), copper and selenium to
human diets (Appendix 1.2).
The major trace elements contributed by meat, poultry and fish
products, however, are iron and zinc, where these products play a major
dietary role. Details about the contributions and advantages of meat,
poultry and fish to iron and zinc nutrition are given in chapter 17.
1.9 Summary
The role of color in meat, poultry and fish products and its measurement
are introduced, since color plays a major role in the acceptability of these
items. Flavor is also briefly outlined from the standpoint of both desirable
and undesirable flavors, with emphasis on its physiology and biochemistry
and its measurement. Other important quality traits and their measure-
ment, such as tenderness, juiciness and/or water-binding are also briefly
discussed in this chapter.
Three other important quality traits are also introduced. These include
the microbiology of meat, poultry and fish products and the importance
of their measurement. Additives and residues (both intentional and unin-
tentional) are also introduced. The importance of methods for the identifi-
cation of these problems are of the utmost importance to the meat,
poultry and fish industries and are discussed further in this volume.
The final quality attribute that is introduced is the nutritional contribu-
tion of meat, poultry and fish to the health and well-being of man.
Although consumers eat meat, poultry and fish products because of their
stimulation of the physical senses, consideration of their contribution to
the health and well-being of man is one of the most important quality
attributes. Without the major contributions of these products to nutrition,
there would be little reason to consume them.
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Appendix 1.1 Major quality traits of meat, poultry and fish products - some factors associated with them and their
effects on acceptability of different products
Quality trait Associated factors Effects on different products References
Color Concentration of meat pigments (Mb High concentrations produce deep red Fox (1966); Giddings (1977);
and Hb) and their derivatives; high color that is characteristic of red meats Livingston and Brown (1981);
concentrations of pigments produce dark (beef, lamb and pork); low MacDougall (1982); Hunt and Kropf
colors and low levels produce pale colors concentrations of Mb and Hb produce (1987)
pale colors such as chicken breast
Species and/or muscle-to-muscle Lamb, beef and pork have deep red color Fox (1966); Greene (1969); Giddings
differences as do some species and/or certain chicken (1977); Livingston and Brown (1981);
and fish muscles; chicken legs and thighs Hunt and Kropf (1987)
are red in color while breast is white or
light in color; salmon and lake trout
have an orange or pink color; tuna have
both dark and white muscles, while other
species of fish (catfish) have white flesh
Age effects Older animals, birds or fishes generally Fox (1966); Giddings (1977);
have higher concentrations of muscle Livingston and Brown (1981); Hunt
pigments, being darker in color, while and Kropf (1987)
younger animals are lighter in color due
to lower levels of muscle pigments
Exercise effects Heavy exercise tends to produce more Fox (1966); Giddings (1977); Hunt
muscle pigments and hence a darker and Kropf (1987); Pearson (1990)
color, whereas confinement decreases the
level of pigments and produces a lighter-
colored muscle
Oxygenation and oxidation of muscle Oxygenation of Mb produces OxyMb - a Fox (1966); Greene (1969); Hunt and
pigments bright red pigment; oxidation of Mb Kropf (1987)
produces MetMb - a brown/black
pigment
Electrical stimulation Electrical stimulation of pre-rigor beef Savell (1979); Dutson and Pearson
muscle hastens rigor mortis and improves (1985); Pearson and Dutson
color in beef and probably in lamb; (1985a,b); Smith (1985)
prevents heat ring in beef; effects on
poultry and fish muscle color have not
been investigated
Post-slaughter treatment Chilling immediately post-mortem tends George and Stratmann (1952); Lanier
to enhance bright red color in beef; et al. (1977); Ockerman and Cahill
holding fresh meat for extended periods (1977); Fox (1987)
produces oxidation of myoglobin to form
metmyoglobin, which is accelerated by
increases in time and temperature;
Microbial growth also increases
oxidation of myoglobin with lower
temperatures near freezing delaying color
degradation
Curing - nitrite is the active ingredient Produces bright red or pink pigment, Fox (1966); Giddings (1977); Hunt
which upon application of heat produces and Kropf (1987)
stable pink pigment
Flavor Age effects Generally, flavor increases with age,
being greater in old than in young
animals and poultry; data lacking for
effects of age on flavor of fishes
Species effects Distinct species effects on flavor; appears Kramlich and Pearson (1958);
to be due to differences in the lipid or Hornstein and Crowe (1960);
fatty fractions Hornstein et al. (1963); Minor et al.
(1965)
Fat effects Fat contributes to aroma and flavor; Wasserman and Talley (1968);
some minimum but unknown minimum Wasserman (1979)
amount of fat is needed for flavor and
aroma
Off flavors: Rancid flavors and/or oxidized flavors; Watts (1954, 1962); Tims and Watts
Oxidized and warmed-over flavors accelerated by cooking to produce (1958); Sato and Hegarty (1971); Sato
warmed-over flavor et al. (1973); Pearson et al. (1977)
Absorbed flavors Fresh, meat, poultry and fish can pick up A.M. Pearson, unpublished
flavors/aromas from atmosphere, paint observations
flavors being an example
Feed flavors (grassy flavors) Animal feeds can influence the flavor as Kemp and Varney (1955); Cramer
shown by fact that wild onions and and Marchello (1964); Cramer et al.
alfalfa flavors can be carried over into (1967); Park et al. (1972 a,b)
the meat; grassy flavors have also been
reported
Sex flavors Boar odor or taint in meat from Craig et al. (1962); Patterson (1968);
uncastrated male pig (boar) is due to Berry et al. (1971); Berry and Sink
several steroid compounds; rams (1971); Thompson and Pearson
(uncastrated male sheep) have also been (1977); Williamson and Patterson
claimed to have a strong flavor but no (1982); Williamson et al. (1985);
causative compounds have been Brooks et al. (1986); Brooks and
identified; no sex flavors have been found Pearson (1986, 1989)
in beef; poultry and fish are not believed
to suffer from sexual flavors
Spoiled flavors Spoiled flavors occur in flesh from all A.M. Pearson, unpublished
species and are associated with microbial observations
and putrefactive spoilage
Mutton flavor Mutton flavor is an undesirable flavor or Ziegler (1944); Kemp et al. (1970,
aroma sometimes associated with meat 1972); Field (1971); Jacobs et al.
from the ovine species; some researchers (1972); Crouse et af. (1981)
have suggested that it is related to the
hardness of mutton fat but it has never
been characterized
Cowy flavor Meat from old cows and cull dairy W.E. Kramlich, unpublished
animals sometimes has an objectionable observations; R.L. Dickson,
flavor that has been described as having unpublished observations
a cowy flavor; it may occasionally occur
in young beef; it has never been
characterized.
Piggy flavor (porky flavor) Pork sometimes has a piggy aroma or A.M. Pearson, unpublished
flavor that is reminiscent of the odor of observations
live pigs; piggy flavors and aromas have
never been studied and characterized.
Fishy flavors Strong fishy flavors are objectionable to Love (1958)
most consumers; however, fish flavor per
se is a delicate flavor that is desirable and
in high demand
Juiciness Water-binding and moisture content Water-binding may be associated with Hamm (1960, 1977); Offer and
juiciness, although small differences are Trinick (1983)
probably unimportant
Fat effects Fat content appears to influence juiciness Wellington and Stouffer (1959);
but there are probably limitations Blumer (1963)
Other effects Other factors may influence juiciness but A.M. Pearson, unpublished
there have been very few studies observations
Tenderness Age effects Older animals and birds are tougher than Lawrie (1966); Romans and Ziegler
young; this is true in meat from cattle, (1974)
sheep and pigs as well as poultry; less is
known about fish.
Exercise Exercise is believed to increase toughness Lawrie (1966); Romans and Ziegler
but data are conflicting; does not appear (1974)
to be due to amount of collagen but
could be associated with cross-linking of
collagen
Sex effects Bulls produce tougher meat than steers Lawrie (1966); Romans and Ziegler
and heifers although the differences do (1974)
not become apparent until about IS
months of age; less data are available for
other species
Marbling fat Marbling fat has been shown to be Doty and Pierce (1961); Blumer
related to tenderness in beef, which is (1963); Romans and Ziegler (1974)
probably the case for sheep meat and
pork to a lesser extent; marbling,
however, accounts for only about 10% of
the variability in tenderness of beef
Fat covering Although amount of finish was once used Ziegler (1944)
as a major criterion for grading beef, it
has been shown to have little or no effect
on tenderness
Conformation It was once considered that conformation Pearson (1966); Romans and Ziegler
in beef cattle, sheep and hogs influenced (1974)
cut-out percentage, yield and tenderness;
however, more recent data shows that
conformation does not affect tenderness
and has only a minimum influence on
wholesale and retail cut yields
Aging Holding freshly slaughtered meat at Doty and Pierce (1961); Romans and
temperatures just above freezing for 3-14 Ziegler (1974)
days results in improvement in
tenderness
Breed effects Meat from the Brahman breed has been Carpenter et af. (1955)
shown to be tougher than that from the
British breeds; there are also strain-
within-a-breed variations in meat
tenderness
Electrical stimulation Electrical stimulation prevents cold- Locker and Hagyard (1963); Marsh et
shortening and its related toughness in af. (1968); Locker et af. (1975); Savell
pre-rigor meat; it hastens the onset of (1979); Cornforth et af. (1980); Smith
rigor mortis so that cold does not cause (1985); Pearson and Dutson (1985a,b)
shortening and toughness in red muscle
tissue; cold-shortening does not occur in
white muscles, such as poultry breast
meat and some species of fish
Conditioning and prevention of cold- Although aging and conditioning are Locker & Hagyard (1963); Marsh et
shortening often used interchangeably in the al. (1968); Locker et al. (1975)
literature, it is also used to refer
specifically to holding freshly slaughtered
meat at temperatures of 10-15°C (50-
60°F) until rigor mortis is complete; this
prevents cold-shortening.
Post-slaughter treatment Holding beef carcasses at high Pearson et al. (1983); Dutson and
temperatures for a short period of time Pearson (1985); Pearson and Dutson
post-mortem (several hours) accelerates (1985a,b)
improvement in tenderness; high
temperatures hasten improvement in
tenderness by activation of the
indigenous enzymes
Muscle-to-muscle differences There are large muscle-to-muscle Ramsbottom et al. (1945); Ginger
differences in tenderness, some of which and Weir (1958)
are probably related to the amount and
characteristics of the connective tissues
Rigor mortis and its effects Freshly slaughtered pre-rigor meat is Bate-Smith (1948); Bate-Smith and
tender but becomes tough at the onset of Bendall (1949); Bendall (1951, 1960);
rigor mortis; as it passes through rigor Paul et al. (1952).
mortis and is aged, it increases in
tenderness (see aging effects)
Tenderness Thaw rigor (thaw-shortening) Meat frozen in the pre-rigor state Sharp and Marsh (1953); Marsh
shortens on thawing and exudes large (1966); Newbold (1966); Newbold et
quantities of drip; this is associated with al. (1973)
a high salt flux
Microbial Total microbial counts Although total microbial counts have Lawrie (1966); Romans and Ziegler
contamination and some value for indicating possible (1974); Zottola and Smith (1990)
growth problems from food spoilage and/or
pathogens, such data alone may be
unreliable
Identification of specific pathogens The number and kinds of pathogenic Lawrie (1966); Romans and Ziegler
microorganisms are very useful (1974); Zottola and Smith (1990)
information and can identify potential
problems
Quality Trait Associated factors Effects on different products References
Additives and Additives (intentional) Nitrite and nitrate are added to stabilize Lawrie (1966); Romans and Ziegler
residues meat color, to develop cured meat flavor (1974); Hotchkiss and Parker (1990);
and to help control Clostridium Pullen (1990)
botulinum; however, too much residual
nitrite can lead to formation of the
carcinogenic nitrosamines; amounts
allowed are established by FSIS
Pesticides Levels allowed are established by FSIS, Lawrie (1966); Romans and Ziegler
FDA and EPA; producers and (1974); Pullen (1990)
processors should be careful to follow
directions of the manufacturer
Appendix 1.2 Contributions of meat, poultry and fish products to the diet of humans in the USA
Nutrient(s) Some major metabolic functions Importance of meat, poultry and Relative contribution to RDA References
fish products
Protein, and amino Supply indispensable amino acids Could be important in meeting Varies greatly in different areas Young et al. (1975, 1984);
acids required in diet; may playa role in protein needs of infants and of the world and among Wayler et al. (1983); Pellett
supplying conditionally children; may even be useful in various consumers and Young (1984, 1990)
indispensable amino acids as well meeting amino acid requirements
as meeting non-specific in adults
requirements for nitrogen.
Fats and fatty acids Supply the essential fatty acids There are enough essential fatty The essential fatty acid needs Hegsted et al. (1965);
(e.g. linoleic, linolenic and acids to meet the needs of humans are easily supplied by normal Bonanome and Grundy (1988);
arachidonic) that are needed in from meat, poultry and fish diets Budowski (1988); Reiser and
small amounts by man; although products. The n-3 polyunsaturated Shorland (1990)
meat, poultry and fish products fatty acids found in fish and fish
supply enough essential fatty acids oils control the level of plasma
to meet the requirements of man, triglycerides and inhibit cyc1o-
they also are supplied by vegetable oxygenase activity, thus reducing
fats and oils so deficiency is not the level of thromboxane A2,
normally a problem which is responsible for blood
platelet adhesiveness and the
resultant thrombosis and coronary
heart disease
Fat-soluble vitamins Vitamin A activity is derived from Fish liver oils and liver are the Meat, fish and poultry DeVaney and Munsell (1935a);
(A,D,E and K) retinoids and carotinoids; richest sources of vitamin A products may contribute about Campos et al. (1987); Olson
deficiency leads to blindness and is activity; some vitamin A activity 12-15% of total vitamin A (1987); Smith (1990)
a serious world-wide problem; occurs in adipose tissue activity of diet with much of
studies indicate that vitamin A this coming from liver; lower
levels in the USA may be marginal income groups tend to eat
in some population groups more liver and hence more
vitamin A
fish products
Vitamin D can be activated in the Best dietary source is salt water Needs can be met by as little as DeVaney and Munsell (1935b);
skin by irradiation with ultraviolet fish liver oils; fat and liver contain 10-15 min exposure to sunlight Pugsley et al. (1945); DeLuca
light; inadequate vitamin D activity small amounts of vitamin D two to three times per week (1978); Smith (1990)
impairs intestinal absorption and
renal reabsorption of calcium and
phosphorus leading to skeletal and
bone deformities
.Vitamin E activity is associated Animal fats are generally poor Contributes only 7-8% of Bieri and Evarts (1974); Chow
with the tocopherols and sources of vitamin E; activity is dietary vitamin E intake at a (1979); Horowitt (1986); Smith
tocotrienols; protects fatty tissues limited to fatty tissues fat content of 15% of calories (1990)
against oxidation
Vitamin K plays a role in blood Vitamin K activity is expressed as Meat, poultry and fish Olson (1980); Suttie et al.
clotting; it functions in formation phylloquinone units; data on the probably contribute about (1988); Smith (1990)
of y-carboxyglutamic acid (GLA), concentration of vitamin K in meat 10% of the daily requirement
which is a structural component of products is scare for vitamin K
four proteins that function in the
blood-clotting cascade
Water-soluble vitamins Vitamin C activity in meat is Meat is a poor source of vitamin Meat, fish and poultry Kunert and Tappel (1983); Lee
largely localized in glands and C, with even the adrenal glands products are generally poor (1983)
organs, especially the adrenal providing minimum requirements sources
glands
Thiamin Thiamin (vitamin B,) in its active Meat, poultry and fish make a About 25% of daily intake of Jackson et al. (1945); Beuk et
form exists as a coenzyme, thiamin substantial contribution to the thiamin is provided by meat, al. (1950); Gubler (1968);
pyrophosphate, which functions in thiamin needs of USA consumers; with pork being a particularly Windham et al. (1990)
active aldehyde transfers and serves cooking of meat results in losses of good source; processing and
a specific independent role in thiamin amounting to 15-40% cooking can reduce levels of
transmission of nerve impulses during boiling, 40-50% on frying, thiamin in meat products
30-60% on roasting and 50-75%
during canning
Riboflavin Riboflavin (vitamin B2) in animal Meat, poultry and fish products Approximately one quarter of Cheldelin and Williams (1943);
tissues is linked with phosphoric are important dietary sources of intake of riboflavin comes from Hills et al. (1951); Windham et
acid to form flavin mononucleotide riboflavin in the human diet; meat; recent data suggests that al. (1990)
(FMN) or to adenosine riboflavin is light-labile but is a slight decline in riboflavin
monophosphate to form flavin generally quite stable during intake has occurred in the
adenine dinucleotide (FAD), which processing of meat; however, it is USA as meat consumption has
are the prosthetic groups of the prone to destruction under alkaline decreased
flavoprotein enzymes that catalyze conditions
in vivo oxidation-reduction
reactions
Niacin Niacin includes both nicotinic acid Meat, poultry and fish are the Some 44% of the pre-formed Elvehjem et al. (1938); Spies et
and nicotinamide; dietary major sources of niacin in the diet, niacin in the diet comes from al. (1938); Elvehjem (1940);
deficiencies result in pellagra in although bacteria in the digestive meat; in addition, niacin is Wertz et al. (1958); Windham
man and black tongue in the dog; tract can synthesize it from produced metabolically in the et al. (1990)
in foods of animal origin exists as tryptophan; niacin is the most liver from dietary tryptophan
nicotinamide adenine dinucleotide stable of the BI vitamins; baking so that meat being a good
[NAD(H)] and nicotinamide or roasting of meat may actually source supplies an even greater
adenine dinucleotide phosphate, increase the amount of available amount of niacin
which function in many metabolic niacin
pathways including anaerobic
glycolysis, the citric acid cycle-
electron transport chain and in
both fatty acid synthesis and
oxidation
Pantothenic acid Pantothenic acid is the coenzyme Pantothenic acid occurs in all Recent evidence suggests that Williams et al. (1933);
of acetylation, commonly called animal tissues, especially in brain, highly processed foods contain Lipmann (1945); Windham et
CoA; pantothenic acid functions in heart, kidney and liver; it is also considerably less pantothenate a/. (1990)
CoA and as phosphopantothiene widely found in other foods; than they do before processing;
of the covalently attached pantothenic acid is fairly stable generally, pantothenic acid
prosthetic group of acylcarrier during ordinary cooking and intake appears to be lower
protein (ACP), which are involved storage but sterilization and than previous data would
in over 70 different metabolic storage can result in losses of up to indicate
reactions 50%
fish products
Vitamin B6 Vitamin B6 (pyridoxine) exists in Meat, poultry and fish are the Over 46% of the vitamin B6 in Spies et al. (1939); Gyorgy
three forms - pyridoxine, most important dietary sources of the average US diet comes (1971); Lofgren and Speckman
pyridoxamine and pyridoxal - al\ vitamin B6 in the USA; levels in from meat increasing from (1979); Sauberlich and Canham
being phosphorylated; aU three the diet appear to be marginal; 26% in 1909-1913; The (1981); Sauberlich (1990)
forms are connected to the meat products are highly increase appears to be due to
.coenzyme, pyridoxal phosphate in bioavailable in contrast to an increase in use of meat,
the body, which is involved in vegetables and fruits; losses during poultry and fish
many enzymatic reactions, mostly canning of meat, poultry and fish
associated with protein and amino may amount to 42-49% and in
acid metabolism processing may be as high as 50-
75%
Vitamin B12 Vitamin B12 was originaUy known Practical\y all of the vitamin B12 in Meat contributes over 69% of Riches et al. (1948); Stokstad
as the 'animal protein factor' since the diet comes from animal the vitamin B I2 in the diet, et al. (1949); Schweigert (1949);
it was associated with animal products; meat, poultry and fish dairy products 20% and eggs Marston and Peterkin (1980);
products; the active coenzyme are al\ excel\ent sources of this about 8.5%, with the Sauberlich (1990)
forms of B12 (5- essential vitamin, which is also remainder coming from
deoxyadenosylcobalamin and present in eggs and milk but is fortified cereal products
methylcobalamin) act in several virtually absent in plant foods; a
enzyme systems including deficiency may occur on strict
conversion of methylmalonate to vegetarian diets; retention of
succinate, formation of methionine vitamin BI2 during cooking is quite
from homocysteine and in high with losses general\y falling in
maintaining the sulfhydryl group in a range of 10-30%
a reduced state; it is essential for
normal development and
functioning of the red bloods cel\s
as weU as other essential metabolic
processes
Folate Folate (folic acid or folacin) is Folate is present in many foods, Animal products contribute Wills (1933); Angier et af.
essential for normal hematopoiesis; especially in green, leafy about 20% of the folate in the (l946); Anderson and Talbot
in its absence megaloblastic anemia vegetables; nevertheless, meat and diet; surveys of military (l981); Sauberlich (l990)
occurs; folic acid aids in synthesis dairy products, and eggs provide a personnel indicated that about
of choline, of serine from glycine, sizeable contribution to folate 40-44% of the RDA came
in formate metabolism, in intake; red meat contributes more from animal products, with
pyrimidine and purine biosynthesis folate to the diet than poultry and dairy products providing about
and in the formation of methionine fish; folate is relatively labile with one-half of this intake; all
from homocysteine; both folate as much as 50-90% of that in meats combined contribute
and B12 are required for synthesis foods being destroyed during about 13% of RDA with liver
of thymidylate, which is necessary cooking and processing; however, being the best source from
for DNA synthesis; folate folate in beef appears to be fairly meat, poultry and fish
deficiency is rather common stable during cooking with
retention being from 72-88%
depending on the cooking method
Macro-minerals Calcium (Ca) is one of the two Although the skeleton contains Meat supplies only token Spencer et af. (l965, 1988);
Calcium major structural elements of bone large amounts of calcium, muscle amounts of calcium to the Allen (1982); Bronner and
and plays a diverse role in and organ meats are low in RDA, since muscle tissue is Coburn (1982); Tso et af.
maintenance of cellular functions, calcium; mechanically deboned low; deboned meat, poultry (l984); Karanja et af. (1990)
serving as a second messenger in meat, poultry and fish products and fish, however, may supply
muscle contraction and in contain from 0.50-0.75% of appreciable amounts of
mobilization of energy, as well as a calcium; excessive meat calcium, if widely utilized
cofactor in several enzymatic consumption may increase mineral
reactions losses, resulting in mineral
imbalances, although such data are
not conclusive
Phosphorus Phosphorus (P) is the other major Phosphorus is widely distributed in Meat, poultry and fish supply a Lee et al. (l981); Hegsted et al.
mineral element in the skeleton. It foods, especially those with a high large portion of phosphorus (1965); Zeller (l987); Karanja
also provides the linkage in the protein content, including meat, needs; large amounts of et af. (l990)
nucleotides, thus playing an poultry and fish; the phosphorus are supplied to
important role in growth and calcium:phosphorus ratio should some segments of the
heredity. It also is a constituent of be from 2: I to 1:2 with adequate populations by soft drinks,
many enzymes and metabolic amounts of vitamin D which may change the calcium;
intermediates such as carbohydrate phosphorus ratio to below 1
metabolism and in oxidation-
reduction reactions
fish products
Potassium Potassium (K) is the major Fresh vegetables and fruits are the Meat is not a good source of Whang (1983); Vander (1985);
intracellular cation with its high richest sources of potassium, potassium but still makes a Jansson (1965); Karanja et at.
concentration in the cell being followed in order by fruit juices, small but important (1990)
maintained by the Na + -K + - milk, poultry, shellfish, red meats contribution to total intake
ATPase, which pumps K into the and cereal grains. Homeostasis is
cells and pumps Na into the achieved by acid-base balance,
extracellular spaces; the pump sodium metabolism and certain
adjusts ionic balance, which aids in humoral factors, such as the action
transmission of nerve impulses and of aldosterone
in contractability of muscle
Sodium Sodium is the major extracellular Sodium concentration is moderate Meat, poultry and fish are not Dahl (1960); Prineas and
cation in mammals. Its main to low in most fresh meat, poultry needed to supply the RDA for Blackburn (1985); Grobbee
function is the control of plasma and fish products but can be high sodium; salt-cured products and Hofman (1986); Morgan
osmolality along with maintenance in processed and cured products may be a problem for about and Nowson (1986); Intersalt
of water balance and osmotic where salt is used for curing; one-third of the population Cooperative Research Group
pressure in the extracelluar fluid. It although NaCI restriction may (1988); Karanja et at. (1990)
comprises about 0.2% of the body; lower the blood pressure of some
blood serum levels are maintained individuals, it is not always
at about 145 mEqr', primarily as effective as only about one-half of
a result of renal reabsorption and the hypertensive subjects respond
excretion. to a reduction in salt intake
Magnesium Magnesium (Mg) is the fourth Meats, especially products high in Meat makes only a small Kobayashi (1957); Neri et at.
most abundant cation in the body fat, are relatively poor sources of contribution to the RDAs (1985); Food and Nutrition
and is second to potassium in its magnesium; bacon, sausage and which are 40-70 mg.day-' for Board (1986); Johnson and
intracellular concentration; it frankfurters contain 50 mg. 1000 infants, increasing to 250 Neilsen (1990)
catalyzes over 300 different body kcal-' while lean meat contains mg.day-' by 10 years of age;
enzymes, including those involved from 50-100 mg. 1000 kcal- l RDAs fall in the range of 300-
in the hydrolysis and transfer of 400 mg.day-' for adolescents,
phosphate groups, fatty acid adult males and non-pregnant
degradation, protein synthesis, and non-lactating females but
DNA synthesis and degradation, are 450 mg.day-' for pregnant
cardiac and smooth muscle or lactating women
contraction, and calcium ion
transport and utilization
Chlorine Chlorine (CI) is present at a high Meat is low in chlorine in its fresh The normal human diet usually Smith and Aines (1959);
concentration both in the state, but can provide large contains adequate amounts of Weinberger et al. (1986);
extracellular and intracellular fluids amounts in cured products chlorine; greater quantities of Karanja et al. (1990)
and plays a role in acid-base chlorine may be needed during
balance lactation or heavy work that
causes profuse sweating
Micro-minerals ~ Trace Iron (Fe) functions in oxygen Meat, poultry and fish are the No other food supplies as high Monsen and Cook (1976);
Elements transport and storage as a major most important nutritional sources a level of bioavailable iron; Cook and Monsen (1976,
Iron component of hemoglobin and of iron, since they contain a large although the RDAs vary with 1977); Monsen et al. (1978);
myoglobin, respectively; it is also a proportion (about 50%) of heme age and sex, iron from meat, Finch and Cook (1984);
component or cofactor in many iron; heme iron enhances the poultry and fish will meet the Monsen (1988a,b);
enzymatic reactions; iron deficiency absorption of non-heme iron; in recommendations; pre- Worthington-Roberts and
is one of the most widespread addition, meat contains a factor menopausal women have the Monsen (1984)
specific nutritional problems in the called the 'meat factor' that also highest requirements for iron
world increases the absorption of iron (l8-mg), and have a difficult
from other food sources; heme iron time meeting the RDAs
is 6~ 7 times more available to man without meat in their diets, as
than inorganic iron do others
Copper Copper (Cu) is involved in Copper is widely distributed in No RDAs have been Klevay et al. (1979); Davis and
erythropoiesis, mineralization of foods but is most concentrated in established for copper, Mertz (1987); Johnson and
the skeleton, synthesis of collagen, liver, legumes, shellfish, whole although it is an essential Lykken (1988); Johnson et al.
myelin formation, catecholamine grains and nuts, about 10% of the nutrient; the Estimated Safe (1988); Johnson and Nielsen
metabolism, oxidative average copper intake in the US and Adequate Intake has been (1990)
phosphorylation, protection of diet comes from meat, poultry and set at 2~3 mg.day-I for adults
antioxidants, lipid metabolism, fish; pork contains more copper and adolescents over II years
immune function, cardiac function than beef, while beef and pork of age; most diets in the USA
and regulation of glucose contain more copper than poultry are believed to contain less
metabolism than 2 mg.day-I
Cobalt Cobalt (Co), unlike most other Cobalt is used by microorganisms Meat, poultry, fish, milk and Johnson and Nielsen (1990);
minerals is not required as an ion; to synthesize vitamin B12; eggs are all good sources of Sauberlich (1990)
it functions as the metal ion at the ruminants are the only animals vitamin B 12; there is no
center of the corrin ring as vitamin that can meet their vitamin BI2 requirement for cobalt in the
B 12; vitamin BI2 functions as the requirements by dietary cobalt, human diet except for vitamin
cofactor in at least three with all other animals requiring B12 BI2 (see vitamin Bn); animal
mammalian enzymes, namely, in their diet products supply 98.4% of the
methylmalonyl CoA mutase, DI-5- BI2 in the diet
methyltetrahydrofolate-
homocysteine methyltransferase
(methionine synthetase) and leucine
2,3-aminomutase
fish products
Selenium Selenium (Se), although originally Selenium is widely distributed in The level of selenium proposed Deagan et al. (1987); Behne et
recognized as being toxic, has since most foods, with fish being an for the human diet has been set al. (1988); Willett and Stampfer
been shown to be an essential excellent source and meat and at 50-200 mg.day-I in the (1988); Yang et al. (1988);
mineral in both animals and man; poultry being good sources; it is USA; except for areas where Burk (1990); Spallholz et al.
it is a component of glutathione also found in nearly all foods, the food all comes from (1990); Lane et al. (1991);
peroxidase, which protects against except for those raised on selenium-deficient soils, there Oldfield (1991)
free radical damage, and is a selenium-deficient soils; the best should be no problem in
component of several documented human deficiency is human diets
selenoproteins the functions of Keshan disease (cardiomyopathy)
which are less well characterized; in in China, where the local diet is
addition to its protection against raised on selenium-deficient soils
oxidative damage, it appears to
playa role in the immune system
and may be involved in prevention
of human cancer
Manganese Manganese (Mn) is involved in a Manganese is found mainly in No RDA has been established Rao and Rao (1980); Hurley
great many metabolic processes, plant foods with meat, poultry and for manganese but the (1984); Keen et al. (1985);
such as a component of enzymes fish being relatively poor sources; Estimated Safe and Adequate Hurley and Keen (1987);
involved in protein and energy whole grains, nuts and legumes are Intake has been set at 2.5-5 Kelsay et al. (1988); Johnson
metabolism and in formation of the best sources of manganese; of mg.day-I for adults. Despite a and Nielsen (1990)
mucoploysaccharides; it also plays all animal products liver contains lower manganese intake,
a role in carbohydrate and lipid the most manganese substitution of meat and fish
metabolism, in cartilage and bone for legumes produced a more
formation as well as in brain positive manganese balance
function
Zinc Zinc (Zn) is required for synthesis, Animal products are excellent Meat, poultry and fish supply Prassad et al. (I 96 I); Halsted
repair and structural integrity of dietary sources of zinc, with beef at least 50% of the RDA for et al. (1974); Sandstead et al.
the nucleic acids, for synthesis of being the richest source followed zinc and are more bioavailable (1982; 1990); Gibson et al.
proteins and as a component of a by pork; meat products supply than zinc from plant sources (1989)
number of zinc metalloenzymes about 50% of the zinc in the US
functioning in these systems; zinc diet, with dairy products supplying
also appears to be involved in lipid 20%, while cereals provide about
metabolism; zinc is a key element 25%; the zinc in animal products is
in appetite stimulation and affects more bioavailable than that in
the taste of foods; it also is plant products
required for sexual development in
humans, for gestation, parturition,
brain development and function,
and plays a role in preventing
peroxidation and in immune
development and function as well
as in skeletal development and in
prevention of parakeratosis and
cancer of the esophagus
Iodine Iodine (I) comprises part of the Although iodine is required by the Red meats and poultry are Ensminger and Olentine
thyroid hormones, which are called body in small amounts for generally poor sources of (1978); Whitney and Hamilton
thyroxine, but consist of tri- synthesis of thyroid hormones, the iodine; seafoods, however, are (1984)
iodothyronine or T 3, tetra- only appreciable amount in the good sources; generally,
iodothyronine or T4 and di- bodies of meat animals and poultry iodized salt is used in areas
iodothyronine; these hormones is found in the thyroid glands; where deficiencies occur, such
regulate body temperature, smaller amounts are found in the as the Great Lakes, the
metabolic rate, reproduction, kidneys, salivary glands, hair, intermountain states and the
growth, production of blood cells, stomach, skin, mammary glands northwestern part of USA;
nerve and muscle function and and ovaries; salt water fish and excessive intake of iodine
other body processes other seafoods contain appreciable results in toxicity
amounts of iodine
2 Color - its basis and importance
D.CORNFORTH
2.1 Introduction
2.1.1 Retail importance of meat color

Meat color is the primary criterion by which consumers evaluate meat
quality and acceptability. Consumers prefer bright-red fresh meats, brown
or gray-colored cooked meats and pink cured meats. Any deviations may
result in reduced sales, consumer complaints and returned products. The
relatively short shelf-life of fresh meats is the single greatest concern to
retail meat markets. When brown metmyoglobin reaches 30-40% of total
pigments on the surface of fresh retail beef, consumers make a no-
purchase decision (Greene et aI., 1971). It is estimated that 4-10% of
retail meats are either discounted, processed into hamburger, or even dis-
carded due to brown-color development. Efforts continue to extend fresh
meat color stability through improved sanitation and antimicrobial treat-
ments, temperature control, packaging, and also through diet modification
and improved breeding and handling of live animals.
Pale, soft, exudative (PSE) pork and dark cutting beef continue to be
major color problems and sources of economic loss to the US meat
industry. When fresh cuts from PSE carcasses are wrapped for retail
display, they exude large volumes of drip, markedly lowering product
acceptance. Also, yield of cooked products is lower from PSE carcasses -
by 2% for smoked butt to 10% for Canadian bacon (Briskey, 1964). The
PSE condition was first described in Denmark in 1954. At that time, the
incidence of PSE in Danish pork was 40-60%, and the US incidence was
about 18%. The Danish pork industry, through mandated selection
programs and improved processing, has eliminated PSE. However, in a
study of seven US suppliers of raw pork to Oscar Mayer Foods, the inci-
dence of PSE ranged from 2-18% (Pietraszek, 1989). The 18% PSE inci-
dence occurred in plants that were part of a traditional supply chain -
farmer, marketer, trucker, slaughterer - that made no effort to minimize
PSE. Currently, it is estimated that the US pork supply is about 16% PSE
and 10% dark, firm, dry (DFD), values that should be of serious concern
to the industry (Kauffman et al., 1992).
Dark cutting beef has shorter shelf-life (Gill, 1983) and because it is not
eligible for 'choice' or 'prime' quality grades, it is estimated that dark
COLOR-BASIS AND IMPORTANCE 35
cutting beef cost the industry $1.36 (Nunes, 1992) to $5 for every steer or
heifer marketed (Grandin, 1992). Currently, severe dark cutting occurs in
about 1% of slaughter cattle, with periodic 'epidemics' of 5-8% in some
plants. Inappropriate use of trenbo10ne acetate (a synthetic male
hormone) and a higher percentage of exotic cattle in US feedlots account
for the recent increase in dark cutters (Grandin, 1992). Although estimates
of dollar losses are not available, cooked meat defects, including pink
color in cooked turkey rolls or pre-cooked bratwurst, continue to be sig-
nificant problems for some processors (Cornforth et aI., 1991).
2.2 Myoglobin and its derivatives
Myoglobin is the primary meat pigment, and exists as bright-red oxymyo-

globin (Mb0 2 , purple-red de oxymyoglobin (Mb), or brown metmyoglobin
(MetMb) (Broumand et al., 1958). A spectral comparison of myoglobin
derivatives with other meat pigments is shown in Table 2.1. Myoglobin is
a complex molecule composed of a protein moiety (globin) and a heme
prosthetic group. The molecular weight of myoglobin is about 17 600
(Wang, 1962). The amino-acid ,sequence has been determined on more
than 50 species (Romero-Herrera et al., 1978). Sperm whale myoglobin,
representative of mammalian myoglobins, is made up of 153 amino-acid
residues (Edmundson, 1965). Myoglobin and hemoglobin were the first
two globular proteins to be structurally analysed by X-ray diffraction, an
achievement for which J.C. Kendrew and M.F. Perutz shared the 1962
Nobel prize in chemistry (Dickerson and Geis, 1969). It was found that
the globin polypeptide consists of eight helical segments, forming a
shallow box around the heme moiety (Figure 2.1). The hydrophobic heme
group (Figure 2.2), is oriented such that the vinyl groups are oriented
towards the hydrophobic interior of the box and the propionic acid
groups towards the outer surface of the molecule (Kendrew et al., 1960).
It is the resonant nature of the conjugated double bonds of heme that is
responsible for the ability of myoglobin to absorb visible light. Ferrous
(Fe 2 +) iron in heme can accept six electrons in its outer orbital and can
thus form six coordination bonds, four with pyrrole groups of the por-
phyrin ring of heme and one with histidine F8, which connects the heme
group to globin. The sixth position is available for binding oxygen or
other small ligands such as CO. Oxidation of heme iron to the ferric
(Fe3+) form results in the formation of metmyoglobin, which is incapable
of binding oxygen and thus is physiologically inactive (Shikama, 1990).
Myoglobin is found in highest concentrations in red muscle cells
(James, 1968; Morita et al., 1969; Cassens, 1970), where it serves as a
storehouse for oxygen, until it is needed by the mitochondrial respiratory
enzymes. There is also evidence that myoglobin functions to facilitate
Table 2.1 Spectral properties of selected meat pigments
Pigment Source Color Absorption maxima (nm)

Soret ~ ex
Myoglobin l - 3 Horse Purple 439 555

Oxymyoglobin 1-3 Horse Bright red 420 544 582
Metmyoglobin 1-3 Horse Brown 409 500 630
CO myoglobin l ,3 Horse Bright red 540 577
Oxyhemoglobin4 Man Bright red 415 541 577
Cytochrome c5 Horse Red 415 521 550
Nitrosyl hemochrome6 Cooked pork Pink 540
Denatured globin-CO Cooked beef Pink 425 542 570
hemochrome7
Globin hemochrome 7 Pig Pink 424 530 558
Nicotinamide Pink 420 529 558
hemochrome7,8
I Bowen (1949). 2 Broumand et al. (1958). 3 EI-Badawi et al. (1964). 4 Antonini and Brunori (1971). 5 Girard et al. (1990). 6 Homsey (1956). 7 Tappel (1957a). 8 Cornforth et
al. (1986).
COLOR~BASIS AND IMPORTANCE 37
Figure 2.1 The myoglobin molecule, consisting of heme attached to globin at imidazole F8.
The heme group is situated in a hydrophobic cleft of the molecule where only small ligands
such as O 2 and CO have ready access. Owing to the hydrophobic environment, even water
has limited access to the heme group. (Adapted from Dickerson and Geis, 1969.)
transport of oxygen within the muscle cell (Wittenberg, 1970; Livingston

et aI., 1983). The globin portion of the molecule serves to confer water
solubility upon the hydrophobic heme group, and more importantly,
protects the heme iron from oxidation. Oxidation of Mb0 2 to MetMb is
much slower than the corresponding process for free heme - by a factor
of 108 (Wang, 1962). The Fe02 center of oxymyoglobin is always subject
to oxidation via nucleophilic attack of water from the surrounding
solvent. Thus, myoglobin and hemoglobin have evolved with a globin
moiety to protect the Fe02 center from easy access to water and its con-
jugate ions, OH- and H+ (Shikama, 1990). The chemistry of the reactions
of heme with oxygen and other ligands has been reviewed previously
(Williams, 1956; Giddings, 1974; Shikama, 1990).
2.2.1 Myoglobin concentration in muscle

Muscle fiber type, and thus myoglobin concentration, is affected by
species (Ashmore and Doerr, 1971) and muscle anatomical location
(Beecher et al., 1965). Muscle myoglobin content is also affected by age
(Morita et al., 1969), and exercise (Barnard et aI., 1970). Treadmill
exercise, however, had no effect on myoglobin content or longissimus
-0
0:":':://
C
\ CH
/
CH
II
CH 2
CH
Figure 2.2 Heme, the 02-binding group of myoglobin. The iron may be ferrous (myoglobin
or oxymyoglobin), or ferric (metmyoglobin). The ferric iron of metmyoglobin is not capable
of binding O 2.
muscle color in swine (Weiss et al., 1975). Although Aalhus et al. (1991)
reported that chronic treadmill exercise increased the tenderness of lamb
muscles, they made no mention of meat color. Krzywicki (1982), when
using a rapid spectrophotometric procedure for heme pigment determina-
tion, reported a mean pigment content (myoglobin + hemoglobin) for
beef of 4.43 mg.g- 1 muscle, with values ranging from 3.02-6.54 mg.g- 1.
Agullo et al. (1990) reported total pigment levels of 3.78-4.56 mg.g- 1 for
beef, 2.55 and 2.79 mg.g- 1 for ovine thigh and loin, respectively, and
1.77 mg.g- 1 for pork rib. Myoglobin values of 4-7 mg.g- 1 for lamb, 2.5-
7.0 mg.g- 1 for pork and 1-2 mg.g- 1 for dark poultry meat have been
reported by Ledward and Shorthose, (1971), Topel et aI., (1966), and by
Nishida (1976), respectively. Oellingrath et al. (1990) developed a high-
performance liquid chromatography (HPLC) method for determination of
myoglobin and hemoglobin in beef, and reported values of 6.9 and
0.65 mg.g- 1 meat, respectively. Rickansrud and Henrickson (1967)
reported myoglobin concentrations ranging from 1.99 mg.g- 1 for semi-

tendinosus muscle to 3.64 mg.g- 1 for biceps femoris muscle. Hemoglobin
values, expressed as a percentage of total pigments ranged from 20%
(longissimus dorsi muscle) to 37.7% (psoas major muscle). Hunt and
Hedrick (1977) also observed variability in pigment content among beef
muscles, from 2.4 mg Mb.g- 1 in outer semitendinosus muscle to 4.6 mg
Mb.g- 1 for gluteus medius muscle. For the same muscles, hemoglobin
amounted to 14.4 and 12.4% of total pigment concentration, respectively.
More blood is retained in muscles of older animals. Warriss and
Rhodes (1977) found that the mean hemoglobin concentration in steer
muscles was 0.65 mg.g- 1, compared with 1.00 mg.g- 1 in cow muscles.
Hemoglobin comprised 14.0% of the total pigments in the meat. Only one
in 60 samples of I. dorsi muscle showed any evidence of poor bleeding.
The average residual blood content of meat was only 0.3%. Rhee and
Ziprin (1987) reported total pigment levels of 0.16 and 0.54 mg.g- 1
for white and dark meat of chicken respectively. Myoglobin was undetect-
able in white meat. Pigments were extracted with cool water, using
the method of Rickansrud and Henrickson (1967). Warriss (1979) pointed
out that meat pigments are incompletely extracted in water, and recom-
mended pigment extraction with ice cold, 0.04 M phosphate buffer, with a
pH of 6.8.
2.3 Factors affecting fresh meat color stability
Metmyoglobin formation and resultant brown discoloration is generally

thought to be indicative of bacterial growth but equally as important is
oxygen tension. Commercially, vacuum-packaging and temperature
control are the primary means for extending fresh meat color stability
during distribution. Modified-atmosphere packaging, using elevated levels
of CO 2 in the headspace, also effectively inhibits aerobic psychrotrophs
and extends fresh meat shelf-life. Other important factors affecting fresh
meat color include pH, metmyoglobin reducing activity, retail lighting
conditions, and the effects of exogenous reductants or antimicrobial
agents.
2.3.1 Oxygen tension

When fresh meat surfaces are exposed to air, oxygenation or 'blooming'
occurs within 30 min, as oxymyoglobin is formed from myoglobin. Bloom
development is accelerated by reducing the temperature (Pirko and Ayres,
1957). Brooks (1929, 1938) and Pirko and Ayres (1957) found that the
depth of oxygen penetration into the muscle was determined by the rate of
oxygen diffusion in muscle and by the rate of tissue consumption of
:!::
.... 0. 8 70
C 60
co c:
coc: 0.6
50 .a
0 ~
u
40 '">-0
! 0.4 E
~ 30 ;;
E
.,
~
20
~ 0.2 -Ji.
0
10
~
LL 0.0 0
0 5 1 0 15 20 25 30
Pa rtial press u re of oxyg en (mm Hg)
Figure 2.3 The effect of oxygen tension on rate of myoglobin oxidation in solution (0) or
on the surface of sterile beef slices (.). Myoglobin oxidation rates of pure solutions were
determined at 30 0 e in 0.6 M phosphate buffer, pH 5.69 (George and Stratmann, 1952). Met-
myoglobin accumulation on the surface of beef slices was measured after storage for 12 days
at ooe (Ledward, 1970). The higher oxygen requirement for maximal pigment oxidation rate
in meat is due to O2 utilization by competing reactions, including mitochondrial O 2 con-
sumption.
oxygen. Increasing the temperature tended to decrease the depth of the

oxymyoglobin layer. Below the red layer of oxygenated tissue lay the dark
purple unoxygenated meat. Brooks (1929) found that when beef slices
were covered with thin glass plates (preventing further tissue oxygenation),
a thin brown band of metmyoglobin developed between the red surface
layer and the purple unoxygenated meat. George and Stratmann (1952)
demonstrated that the maximum rate of myoglobin oxidation in pure
solutions occurs at very low oxygen partial pressures of 1-1.4 mm Hg at
30°C and pH 5.69 (Figure 2.3). In meat, however, the maximal rate of
myoglobin oxidation occurs at oxygen partial pressures of 6- 7 mm Hg, as
shown in Figure 2.3 (Ledward, 1970). The higher O2 requirement for
maximal oxidation of myoglobin in meat is due to O2 consumption by
competing reactions, including mitochondrial cytochrome oxidase.
The brown band of oxidized metmyoglobin first appears at the limit of
oxygen diffusion into meat, where the concentrations of myoglobin and
oxymyoglobin are about equal. This is shown in the following summary
equation:
(2.1)
Myoglobin heme iron donates one electron to oxygen. Various one-
equivalent reductants provide the second electron (Giddings, 1977). Deox-
ygenation of oxymyoglobin is an intermediate step. Oxymyoglobin cannot
participate in electron-transfer reactions, accounting for the slower oxida-
tion rate of myoglobin at higher oxygen tensions. Brown and Mebine

(1969) reported that the half-life for autoxidation of bovine myoglobin at
pH 6.5 and 22°C was 26.5 h. Thus the reaction is fortunately quite slow in
physiological terms but significant from the standpoint of meat color,
since meat storage times are normally significantly longer (Livingston and
Brown, 1981).
Applying these concepts to shelf-life extension of fresh beef, MacDou-
gall and Taylor (1975) demonstrated that by holding steaks I day in
100% oxygen at 2°C, the depth of the surface oxymyoglobin layer was
increased by 3-5 mm (to a total thickness of 10 mm in sirloin steaks),
and the display life was extended by at least 20 h. Muscle with fast rates
of pigment oxidation, such as the tenderloin (psoas muscle), benefited
most from the treatment, while longissimus dorsi (top loin) discolored so
slowly that oxygen treatment was only marginally beneficial. Daun et al.
(1971) found that fresh beef color stability could be extended up to 10
days (vs. 6 days for controls) when the steaks were held at 4°C in an
atmosphere of 90% oxygen. Urbain (1960) used oxygen-enriched atmo-
spheres to restore the bright red color to meat irradiated with high
energy electrons.
2.3.2 Bacteria
Bacterial growth has long been suspected to influence meat color (Jensen,
1945). Urbain and Greenwood (1940) found that in bacteria-free solu-
tions of oxyhemoglobin, methemoglobin was not formed even after 60
days storage at 10°C. However, in solutions in which bacteria were
allowed to grow, there was a 50% decrease in oxygen capacity that was
attributed to methemoglobin formation. A conclusive effect of bacteria
on meat color was shown by Butler et al. (1953). In their study, beef
steaks inoculated with Pseudomonas spp. and wrapped in cellophane
became discolored by day 6 of storage at 1°C, while uninoculated
controls did not discolor until day 13. Discoloration was faster at 4°C
than at 1°C. Butler et al. (1953) attributed the rapid discoloration in
inoculated samples to bacterial consumption of oxygen. As discussed
earlier, Brooks (1938) and George and Stratmann (1952) demonstrated
that myoglobin oxidation to metmyoglobin occurred most rapidly at low
oxygen pressures of 1-2 mm Hg. The partial pressure of oxygen in air at
sea level, for comparison, is 158 mm Hg, which is equivalent to 20% 02.
Butler et al. (1953) observed that in inoculated steaks, the metmyoglobin
level peaked on day 7 at about 56%, and declined to less than 20% by
day 14 of storage (Figure 2.4). Surface color turned from brown to
purple, similar to the color of myoglobin in freshly cut beef. The color
reversion was attributed to reducing conditions produced in the presence
of a high bacterial load.
60
z
iii 50
0
..J
CI
0 40
>
:::;;
I-
w 30
:::;;
I-
z 20
w
U
II:
w 10
11-
0
0 2 4 6 B 10 12 14 16 1 B 20
DAYS STORAGE
Figure 2.4 Color reversion (brown to purple), as indicated by percentage metmyoglobin on

surfaces of inoculated beef steaks packaged and stored at I DC (Butler et al., 1953). Inocu-
lated, 0; control, •.
2.3.2.1 Effects of different bacteria on color. The results of Butler et al.

(1953) were confirmed in several subsequent studies (Rikert et al., 1957;
Robach & Costilow, 1961; Satterlee and Hansmeyer, 1974; Bala et al.,
1977; Lin et al., 1977). Robach and Costilow (1961) found that low-level
inoculation of wrapped beef steaks with several strains of aerobes (Pseu-
domonas spp. or Achromobacter liquefaciens) caused rapid brown dis-
coloration, whereas inoculation with greater numbers of the same bacteria
quickly produced the purple color of myoglobin. No brown discoloration
was seen after inoculation with Lactobacillus plantarum, an organism that
does not consume oxygen to any appreciable extent. Taking into con-
sideration the oxygen permeability of polyethylene, bacterial oxygen
demand and respiratory activity of post-mortem tissue, Bevilacqua and
Zaritzky (1986) calculated that greater than 106 CFU per cm 2 of Pseudo-
monas spp. were required before aerobic bacterial growth could reduce
oxygen partial pressure to lower than 30 mm Hg, sufficient to accelerate
metmyoglobin formation in beef under conventional display conditions.
Rikert et al. (1957) reported that ground ham samples (including fat)
had improved color retention when inoculated with Achromobacter spp.
Reddy et al. (1970) reported that inoculation of ground beef with lactic
cultures and 10% sterile skimmed milk or 1% lactose effectively prevented
aerobic microbial growth. A significant increase in numbers of Gram-
negative bacteria was not observed until 7 days of storage. Flavor and
aroma scores were acceptable for samples inoculated with lactics.
Halleck et al. (1958a) and Satterlee and Hansmeyer (1974) both
reported meat color reversion from brown to purple with prolonged
storage. Faustman et al. (1990) similarly found that ground beef homo-
genates inoculated with fluorescent pseudomonads or Brochothrix thermo-
sphacta turned brown initially, but reverted to a red color, which was
associated with a decline in metmyoglobin content, when bacterial levels
reached about 109 CFU.g-'. Culture filtrates were also capable of facil-
itating the brown-to-red color change, indicating production of reducing
substances by these bacteria, or production of some metabolite capable of
forming a red-colored complex with metmyoglobin. Kalchayanand et al.
(1989) noted the production of bright pink purge in spoiled, vacuum-
packaged beef. Pink discoloration was attributed to spoilage by a Clos-
tridium sp.
2.3.2.2 Role of bacteria in green discoloration. Several different micro-

organisms are capable of inducing green discoloration in fresh meats.
Jensen and Urbain (1936a) classified green discoloration of meat products
into two categories of causes: (i) that caused by oxidizing bacteria; or (ii)
that by hydrogen sulfide-producing bacteria. Green discoloration of fresh
meat has been reported due to H 2S production by Pseudomonas mephitica,
resulting in the conversion of myoglobin to sulfmyoglobin (Nicol et al.,
1970). The organism was capable of causing green discoloration when it
was only a small percentage of the total micro flora but only if the oxygen
tension was low (about 1%) and the meat pH was above 6.0. Alteromonas
putrefaciens is another organism capable of H 2 S production at a pH
above 6.0, with deleterious effects upon meat color (Gill and Newton,
1979). Green choleglobin may also result from the oxidation of myoglobin
by hydrogen peroxide (Fox et al., 1974). The source of hydrogen peroxide
may be bacterial (Jensen and Urbain, 1936a; Jensen, 1945), or result from
the reaction of ascorbic acid with oxymyoglobin (Fox, 1966). Hydrogen
peroxide may also be produced by endogenous muscle reactions (Harel
and Kanner, 1985) but not in a sufficient quantity to cause choleglobin
formation. Choleglobin formation after treatment with hydrogen peroxide
is diagnostic for the presence of heme pigments, as opposed to carotenoids
or pigments of microbial origin (Jensen and Urbain, 1936b).
2.3.3 Vacuum-packaging
2.3.3.1 Role in extending shelf-life. Short of freezing, vacuum-packaging
and refrigerated storage is the most effective method currently used for
extending shelf-life of uncooked meats. Vacuum-packaging is central to
the 'boxed beef distribution system, whereby carcasses are fabricated into
primal or subprimal cuts and vacuum-packaged in processing plants for
distribution to retail outlets, where items may be further fabricated into
retail cuts (Seideman and Durland, 1983). Vacuum-packaging lowers total
plate count and favors lactobacilli, whereas pseudomonads usually
dominate the spoilage micro flora of meat wrapped in oxygen-permeable
films (Pierson et aI., 1970; Roth and Clark, 1972; Gill, 1983). Halleck et
al. (1958b) reported that gas-impermeable packaging films increased the

lag phase and the final bacterial counts were not so high as with more
permeable films. Jaye et al. (1962) found that ground beef that was
packaged in impermeable saran had a lower total plate count at 30°F or
38°F compared with gas-permeable cellophane-wrapped samples. Lactics
predominated in the saran-wrapped samples and pH decreased slightly
during storage. Pseudomonads predominated in the cellophane-wrapped
samples, and the pH increased from 5.7 to 6.5 during storage. Carbon
dioxide accumulates in the headspace of vacuum-packaged meats (Ingram,
1962; Seideman et al., 1979) and this phenomenon seems to be responsible
for the change in microflora seen in vacuum-packaged meats (Gardner et
al., 1967). An atmosphere of about 10% CO 2 retards growth of Pseudo-
monas spp. in meat (Clark and Lentz, 1972; Enfors et al., 1979).
2.3.3.2 Influence of oxygen-transmission rates. The shelf-life of vacuum-

packaged meat is affected by film oxygen transmission rate. Savell et al.
(1986) found that beef knuckles that were vacuum-packaged in bags with
oxygen transmission rates (OTR) of 1 or 12 cm2 .m- 2 .24 h-' at 4°C did not
discolor during 21 days storage, while brown discoloration was apparent
as soon as 1 day in bags with OTR of 30 or 400 cm2 .m-2 .24 h-'. Vacuum-
packaged steaks were acceptable after 28 days storage at 2°C when
packaged in bags with OTR of 10 (Hanna et al., 1983). Egan and Shay
(1982) found that beef steaks packaged in film with an OTR of 25 did not
have aroma or flavor defects until 27 days storage at 4°C. Gill and
Harrison (1989) reported a storage life of 4 weeks for pork loin cuts held
at -I.soC and packaged with an ethylene vinyl acetate copolymer-poly-
vinylidene chloride (PVDC laminate, Cryovac) film with an OTR of 40.
Vacuum-packaged beef is now widely accepted for distribution of
primals and subprimals, in part because the retail cuts produced after in-
store fabrication retain the ability to bloom and maintain acceptable color
for 3-4 days of retail display, when repackaged in an oxygen-permeable
film such as polyvinyl chloride (Roth and Clark, 1972). However,
consumer acceptance of vacuum-packaged retail beef has been low. Excel
Corporation (Wichita, Kansas) recently test-marketed 'Excel Brand Beef,
a line of vacuum-packaged retail beef cuts, in several regional markets in
the USA (Meischen et al., 1987). The products were purple-colored but
would bloom upon opening the package. Excel cited several product
advantages, including longer shelf-life (70 days total, 7 days in the con-
sumer's refrigerator, as indicated by a pull-date on the label), and a leak-
proof and freezer-ready package. Initial sales were reportedly good but
declined in part due to the higher price on vacuum-packaged cuts.
Vacuum-packaged chicken or turkey breast meat products have received
greater consumer acceptance than vacuum-packaged beef, since these
lightly pigmented poultry cuts do not turn purple with vacuum-packaging.
100
90
~
80
z
w 70
:::;:
(!l 60
a: 50
~
z
w 40
U
II: 30
w
~
20
10
2 4 6 8 10 12 14 16 18 20
STORAGE TIME (HOURS)
Figure 2.5 Changes occurring in surface pigments of vacuum-packaged beef during storage
at 3°C (Pierson et at., 1970). Oxymyoglobin, 0; myoglobin, 0; metmyoglobin, !::,..
When beef is vacuum-packaged, there is a characteristic loss of bloom

and an increase in metmyoglobin concentration 2-4 h after packaging
(Pirko and Ayres, 1957), followed by a near total conversion of pigments,
including metmyoglobin, to deoxymyoglobin (Pierson et al., 1970) as illu-
strated in Figure 2.5. The amount of metmyoglobin formed is dependent
upon the exposure time in air before vacuum packaging. Pierson et al.
(1970) found that samples held for 48 h in oxygen-permeable cellophane
required over 20 h after vacuum-packaging for complete reduction of
metmyoglobin to myoglobin.
2.3.4 Packaging with oxygen-permeable films

By far the greatest volume of fresh meat sold in over-the-counter retail
trade is presented in styrofoam trays overwrapped with oxygen-permeable
polyvinyl chloride (PVC) film. Display time is usually only about 3 days
at 4°C before discoloration occurs, regardless of how long the meat was
stored in vacuum before repackaging (Roth and Clark, 1972). Landrock
and Wallace (1955) found that film with an oxygen permeability of greater
than 5000 ml.m- 2 .24 h- 1 at 24°C and 100% relative humidity was required
for development and maintenance of bloom in fresh beef for 48 h. Dis-
advantages of presenting meat in this manner are: (i) the short shelf-life;
(ii) the development of drip in the package; and (iii) the messiness asso-
ciated with films that leak or are punctured. However, PVC overwrapping
remains the preferred method of presenting fresh meats, since the meat
develops bright-red color attractive to consumers, the packaging materials
are inexpensive and packaging equipment is available and relatively inex-
pensive, allowing retailers to package products 'in-house'. Centralized pre-
paration of red meats for retail sales remains a goal for many in the meat-
packing industry, eliminating the costs associated with retail cutting and
wrapping. Modified-atmosphere packaging, vacuum-packaging or irradia-
tion are all viable methods for extending the shelf-life of centrally pro-
cessed retail cuts. Color stability along with cost will be major factors
affecting acceptance of these alternative methods for processing and pre-
sentation of retail red meat cuts.
2.3.5 Modified-atmosphere packaging

Modified-atmosphere packaging (MAP) systems are designed to inhibit
the normal aerobic spoilage flora of fresh meats by altering the composi-
tion of the headspace gases. MAP also affects myoglobin oxygenation or
oxidation and thus color. Purple myoglobin predominates in the absence
of oxygen. Low levels of oxygen (1 %) favor formation of brown metmyo-
globin, while high levels favor formation of red oxymyoglobin (Figure
2.6). Several different gas combinations have been tested. However, pure
carbon dioxide or combinations of CO 2 with oxygen and/or nitrogen are
by far the most important (Finne, 1982). The antimicrobial effect of
carbon dioxide has been known for a long time (Pasteur and Joubert,
1877 - as cited in Enfors et al., 1979). Coyne (1932) reported marked
inhibition of several bacteria, including Achromobacter spp. and Pseudo-
monas spp. by packaging them in 25% CO 2 , Optimal inhibition occurred
at 40-60% CO 2 with no additional advantage at higher concentrations
(Coyne, 1933). Enfors et al. (1979) held pork loins at 4°C in atmospheres
of air, 100% N 2 , or 100% CO 2 and found that total aerobic plate count
Bright ~I-&<er-----G.....
Red
Brown
Purple
o 2 4 6 8 10 12 14 16
TIME - DAYS
Figure 2.6 Color changes observed on beef steak surfaces held in various atmospheres. The
total pressure of each atmosphere was adjusted to 750 mm Hg. Samples were stored at 4°C
(Robach and Costilow, 1961). Color changes after 6 days were attributed to microbial
growth. 750 mm O 2 , 0 ; air, 0 ; 75 mm O2 , £.; 10 mm O2 , 6,; N 2 , • •
reached high levels (5 x 106 organisms.cm- 2 ) after 4, 5 or 32 days, respec-

tively. Initially, Acinetobacter calcoaceticus and pseudomonads were the
predominant microflora but after 35 days in the CO 2 atmosphere the
microflora changed towards one completely dominated by lactic acid
bacteria. Gill and Harrison (1989) similarly reported an increase in storage
life of chilled pork stored under CO 2 .
Ogilvy and Ayres (1951) found that CO 2 inhibited meat spoilage
organisms but that levels higher than 25% caused discoloration of the
flesh. Ledward (1970) also observed development of a surface gray tinge
on some samples of beef held for 14 or 28 days in 60% CO 2 ; he sug-
gested the possibility that this may have been due to meat-surface acid-
ification and protein denaturation. Ledward (1970) further showed that
CO 2 concentration per se had no effect on myoglobin oxidation. In
samples of high CO 2 concentration, brown discoloration was related to
low oxygen concentration, favoring metmyoglobin formation. Ledward
(1970) concluded that 'storage of sterile beef in COrenriched atmo-
spheres leads to no increased metmyoglobin formation, provided the
oxygen partial pressure is maintained above a limiting value of about
5%' (equivalent to 38 mm Hg). Gill and Harrison (1989) cited previous
work where meat discoloration in CO 2 atmospheres was prevented by
initial reduction of oxygen to very low levels and by use of an aluminum
foil laminate package to prevent oxygen ingress during storage. Benedict
et al. (1975) reported no problems with color in a procedure involving a
chemical system for continuous CO 2 generation inside packages. Huffman
et al. (1975) reported that a mixture of 70% N 2 , 25% CO 2 and 5% O 2
effectively reduced aerobic plate counts during storage of beef steaks but
that these samples were among the least desirable in color of all treat-
ments studied, in contrast to steaks stored in 100% O 2 which had the
most desirable color through 23 days storage at 1°C. Savell et al. (1981)
reported that beef steaks had unacceptable color during modified atmo-
sphere storage if the subprimals from which they were derived had been
subjected to extended vacuum storage.
Carbon monoxide (2%) exposure before package sealing has been
found to stabilize the color of fresh beef for up to 15 days (EI-Badawi et
aI., 1964). Atmospheres containing 1% CO and 50% CO 2 were shown to
inhibit microbial growth and double the color shelf-life of ground beef
(Gee and Brown, 1978a,b). Seideman et al. (1979) reported that beef
roasts stored in an atmosphere containing 1% CO had less surface dis-
coloration and lower metmyoglobin values than roasts in Orcontaining
modified atmospheres. After exposure of ground beef to 1% CO, it was
found that the CO was lost during storage (TI//2 = 3 days) and there was
an 85% loss of CO after cooking (Watts et al., 1978). Nevertheless, CO is
not currently permitted in the USA as a component for modified-atmo-
sphere storage of meat.
2.3.6 Effects of pH
Living muscle has a near-neutral pH but, due to post-mortem glycolysis
and resultant accumulation of lactic acid, the pH declines. The rate of
acid production and the ultimate pH attained affect many properties of
meat, including color, water-holding capacity, protein solubility and rate
of microbial spoilage. High-pH meat (> 6.0) is dark, including dark-
cutting beef, dark-firm-dry (DFD) pork and the dark, coarse band (DCB)
of ribbed beef carcasses. A gradual pH decline to an ultimate pH of about
5.6 results in normal red meat capable of bloom development upon
exposure to air. However, very rapid pH declines result in excessive
protein denaturation, drip loss and the pale color characteristic of pale,
soft, exudative (PSE) pork (Briskey, 1964). Specific properties of dark-
cutting beef and PSE pork will be discussed in later sections.
It is well known that hemoglobin affinity for oxygen is affected by pH,
resulting in enhanced release of O 2 from oxyhemoglobin in the more acid
environment of exercising muscle. This is known as the Bohr effect (Leh-
ninger, 1975). However, the oxygenation of myoglobin is not affected by
pH (Govindarajan, 1973). Unlike oxygenation, myoglobin oxidation is
affected substantially by pH. Low pH favors myoglobin oxidation, due in
part to destabilization of the heme-protein linkage (Livingston and
Brown, 1981). At pH values below 5.0, myoglobin denatures (Appel and
Brown, 1971), totally exposing heme to oxidants in the medium. In
addition, low pH increases proto nation of oxymyoglobin, favoring one-
electron transfer from heme iron to the bound oxygen, ultimately forming
metmyoglobin and the superoxide anion, O 2- (Livingston and Brown,
1981). At 35°C, the half-life for conversion of bovine heart oxymyoglobin
to metmyoglobin is 3.3 days at pH 9.0, 11 h at pH 7.0 and less than
30 min at pH 5.0 in 0.1 M buffer (Shikama, 1985).
2.3.7 Temperature
As previously discussed, meat discoloration associated with microbial
growth occurs more rapidly at higher storage temperatures (Butler et at.,
1953; Rikert et at., 1957; Jaye et at., 1962; Calkins et at., 1986; Gill and
Harrison, 1989). The rate of metmyoglobin formation and browning of
exposed lean beef carcass surfaces increases with increasing temperature
(0-6.6°C) and air velocity (Lanier et at., 1977), but low relative humidity
(0.85 vs. 0.95) does not promote myoglobin oxidation. Myoglobin oxida-
tion by oxygen (i.e. autoxidation) is also accelerated at higher tempera-
tures. For oxymyoglobin oxidation, Brown and Mebine (1969) reported a
QIO value of 5, over the temperature range -2°C to 22°e. Gotoh and
Shikama (1974) reported a similar value of 5.3 over the range -3°C to
30°C, where QIO is defined as the factor by which the reaction rate increa-
ses for each 10°C increase in temperature. Faustman and Cassens (1990)
pointed out that a QIO of 5 is indicative of high-temperature sensitivity for
this reaction, since QIO values for most chemical reactions range from 2-4
(Mortimer, 1977). Brown and Dolev (1963) reported that tuna oxymyo-
globin oxidized more slowly than beef at 0 to 10°C, but the reverse was
true at higher temperatures. Livingston and Brown (1981) stated that in
general, fish myoglobins are at least 2.5 times more sensitive to autoxida-
tion than mammalian myoglobins and even more so at higher tempera-
tures.
2.4 Dark cutting beef and related dark color problems
2.4.1 Characteristics of dark-cutting meat

Although dark-cutting beef is an old and well understood meat color phe-
nomenon, it has not been eliminated and economic losses from it still
persist. Dark-cutting beef is characterized by high pH (Winkler, 1939),
sticky texture and high water-binding ability (Fischer, 1981), increased
tenderness (Fredeen et ai., 1974; MacDougall et ai., 1979; Dransfield,
1981) and high oxygen consumption (Hall et ai., 1944; Bendall and
Taylor, 1972; Ashmore et ai., 1971), resulting in poor oxygen penetration,
a dark color and inability to bloom in air. Guilbert (1937) postulated that
unknown hereditary factors caused the 'black cutter' condition. More
recently, Lawrie (1958) and Hedrick et ai. (1959) conclusively linked stress
with the increased incidence of dark-cutting beef. Excitable animals that
develop tremors or shivering before slaughter are especially prone to the
dark-cutting condition, according to Lawrie (1958). In USA and Canada,
a seasonal peak has been observed in dark-cutting incidence in November
and again in late spring (Munns and Burrell, 1966), implicating cold, wet
conditions as contributing stress factors. Fasting of steers did not produce
dark-cutting carcasses (Lawrie, 1958), but a 48 h fast period after trans-
port greatly increased the incidence of dark-cutters among young bulls,
compared with control animals slaughtered immediately upon arrival at
the slaughterhouse (Fischer, 1981).
Time and duration of stress also affects meat color. Dark-cutting may
result if stress-susceptible cattle are continuously stressed in the 24 h
period prior to slaughter (Hedrick, 1981) but short-term stress approxi-
mately 1 h before slaughter may result in the pale, soft, watery syndrome.
A similar relation of stress and meat color has been observed for pork.
Low muscle glycogen levels and a higher incidence of dark-cutters may be
induced by subcutaneous epinephrine (adrenalin) injections (Hedrick et
aI., 1959; Hedrick, 1981). This response may be blocked by prior adminis-
tration of propranolol (Ashmore et ai., 1973). However, propranolol was
ineffective in preventing muscle glycogen depletion in young bulls during

mixing stress (McVeigh and Tarrant, 1981).
2.4.2 Mechanism by which pH affects color

Meat pH is thought to affect color in two ways. At high pH (> 6.0),
mitochondrial oxygen consumption is high, and remains so for a con-
siderably longer period post-rigor than is the case for normal meat with a
pH of about 5.6 (Chyah and Cheah, 1971; Bendall, 1972; Bendall and
Taylor, 1972). Consequently, in meat exposed to air, the surface oxymyo-
globin layer is thin and the purple color of myoglobin predominates
(Lawrie, 1958; Ashmore et aI., 1972). Secondly, high pH meat has a high
water-binding capacity, associated with greater translucence and less
scatter of incident light, allowing greater light penetration and absorption,
which makes meat appear darker (Lawrie, 1958; MacDougall and Jones,
1981; MacDougall, 1982). However, high pH beef (either pre-rigor or
dark-cutting) will turn red when chilled in an oxygen atmosphere or when
homogenized with rotenone, conditions that favor oxymyoglobin forma-
tion by reducing or inhibiting mitochondrial oxygen consumption, respec-
tively (Cornforth et al., 1985; Cornforth and Egbert, 1985; Egbert and
Cornforth, 1986).
2.4.3 Changes occurring after death

After slaughter, blood supply to the tissues ceases, forcing the muscle to
rely upon glycolysis for maintenance of energy-linked functions. In
normal muscle, glycogen conversion to lactic acid and its accumulation
results in a pH decline from about 6.8 down to pH 5.5, over a 4-24 h
period, depending upon species, muscle and temperature (Briskey, 1964).
The inhibition of mitochondrial oxygen consumption at pH 5.5-5.6
(Bendall, 1972) allows oxymyoglobin accumulation and bloom develop-
ment when normal meat is exposed to air. In muscle from stressed
animals, however, available glycogen stores have been depleted. Approxi-
mately 100 j.lmollactate per gram muscle may be produced in meat with
normally low ultimate pH of 5.5, but only 40 j.lmol lactate per gram
would be expected in dark-cutting muscle of pH 6.2 (Davey and Gilbert,
1976; Davey and Graafhuis, 1981). In other words, muscle from stressed
animals has less than half the glycogen capacity of normal muscle to
utilize in lactic acid production. Consequently, in high pH meat, mito-
chondria are not inactivated by acid, mitochondrial oxygen consumption
remains high and at meat surfaces less myoglobin remains in the oxyge-
nated form. In general, dark color may be observed at meat pH values
above 6.0, and especially so at pH values above 6.2 (Hedrick, 1981).
However, muscles vary in ultimate pH, so that not all muscles of stressed
animals exhibit dark color (Tarrant and Sherington, 1980; Davey and
Graafhuis, 1981).
2.4.4 Shelf-life of high pH meat

Aerobic microbial growth and spoilage occurs more rapidly on high pH
DFD pork (Rey et aI., 1976; Newton and Gill, 1981; Greer and Murray,
1989). Aerobic spoilage is related to glucose availability. While glucose
serves as the major energy source for meat spoilage microflora, no off-
odors can be detected. When glucose levels become limiting, however, the
bacteria begin to degrade amino acids, producing ammonia and spoilage
odors (Newton and Gill, 1981). Since dark-cutting beef or its equivalent,
dark-firm-dry (DFD) pork, is devoid of glucose, pseudomonads degrade
amino acids without delay, producing odors faster than normal. This
occurs when the cell density exceeds 106 cm- 2 . Spoilage can be delayed by
adding glucose to a concentration similar to normal meat but lactic acid
addition to pH 5.5 has no effect upon time of spoilage onset (Newton and
Gill, 1978).
2.4.5 Vacuum-packaging
Vacuum-packaging of high pH meat allows growth of three facultative
anaerobes that do not usually contribute to anaerobic spoilage. They are
Yersinia enterocolitica (probably non-pathogenic strains), Enterobacter
liquefaciens and Alteromonas putrefaciens (Newton and Gill, 1981). E.
liquefaciens produces spoilage odors at low cell densities on high-pH meat
but spoilage onset can be delayed by glucose addition (Patterson and
Gibbs, 1977; Gill and Newton, 1979). A. putrefaciens is responsible for
hydrogen sulfide production from cysteine or glutathione in vacuum-
packaged high-pH meat, forming green sulfmyoglobin (Nicol et al., 1970).
E. liquefaciens and A. putrefaciens are both inhibited at pH values below
6.0. Addition of citrate buffer to lower the pH of the meat surface to less
than 6.0 reduces greening of high-pH, vacuum-packaged meat by allowing
A. putrefaciens to utilize citrate in preference to amino acids (Gill and
Newton, 1979; Newton and Gill, 1981).
2.4.6 Mimimizing dark-cutters by management

Dark-cutting is more prevalent in bulls (Tarrant, 1981) and remains one
of the major impediments to increased utilization of young bulls, given
their superior growth rate and leaner carcasses. The incidence of dark-
cutting may be reduced by marketing well-nourished cattle, mimimizing
excitement during loading and handling, reducing shipping times, provid-
ing feed after long periods of transport, and avoidance of mixing of
strange cattle, females in estrus or bulls (Hedrick, 1981). To prevent

mounting behavior of bulls penned together, Kenny and Tarrant (1987)
recommended modification of abattoir pens by installation of an overhead
electrical grid.
2.4.7 Dark, coarse band in beef ribs

Dark, coarse band (DCB) is a slightly dark, coarse, depressed area some-
times observed immediately under the subcutaneous fat of the longissimus
dorsi muscle after ribbing beef carcasses in preparation for grading
(Orcutt et al., 1984). The former term 'heat ring', is a misnomer, since the
phenomenon is actually caused by rapid chilling of thin carcasses and the
slight depression is the result of cold-shortening. The rapid chilling of the
exterior of the longissimus dorsi delays glycolysis, so that this portion of
the muscle is still pre-rigor, with pH values above 6.0 and a dark color,
while the remainder of the muscle has entered rigor and exhibits a bright
red color upon exposure to air (Orcutt et al., 1984). DCB is only apparent
in carcasses that have been ribbed 16-24 h post-mortem. At 48 h post-
mortem, carcasses have fully gone into rigor, eliminating DCB (Savell et
aI., 1978; Calkins et al., 1980). DCB is a problem in that it delays accurate
evaluation of marbling score, a critical factor in determining US beef-
quality grades. Acceleration of rigor development by carcass electrical sti-
mulation reduces or eliminates DCB (Savell et aI., 1978; Calkins et aI.,
1980; McKeith et aI., 1981; Orcutt et al., 1984).
2.5 Pale, soft, exudative (PSE), porcine stress syndrome (PSS) and dark,
firm, dry (DFD) pork
2.5.1 Importance of PSE, PSS and DFD pork

As the name implies, PSE pork is typically colored pale gray to white,
with a soft texture and excessive fluid or drip loss from the muscle. The
condition was first described in Denmark, where it was termed 'muscle
degeneration' (Ludvigsen, 1954). Muscles with a tendency to become PSE
have a high proportion of intermediate fibers, with a high capacity for
anaerobic glycolysis (Cooper et aI., 1969). The longissimus dorsi and biceps
femoris muscles are particularly prone to the condition (Briskey and
Wismer-Pedersen, 1961), while adjacent muscles may appear normal,
giving the meat a two-toned appearance. PSE pork commonly occurs in
animals susceptible to porcine stress syndrome (PSS). PSS animals often
die during normal transport and handling. Stress-susceptible hogs may be
identified by their sensitivity to the inhalation anesthetic, halothane. PSS
hogs usually die under the influence of halothane (Heinze and Mitchell,
1991) due to development of malignant hyperthermia (MH), an inherited

myopathy characterized by muscle contracture, hypermetabolism and ele-
vation of body temperature. PSS and halothane-induced MH are, in
effect, the same syndrome. Both produce PSE meat but are triggered by
different stimuli (Heinze and Mitchell, 1991).
2.5.2 Genetic basis

Recently, malignant hyperthermia (PSS) has been shown to result from a
single-point mutation in the receptor for calcium ion release from the sar-
coplasmic reticulum (Fujii et al., 1991), facilitating release of Ca2 + and
consequent stimulation of muscle contraction and glycolysis. PSS and the
tendency towards PSE meat is associated with heavy muscled hogs and is
likely the result of selection for leanness and muscularity in breeding
stock. Fujii et al. (1991) suggest the possibility of producing hybrid
market animals heterozygous for the defective gene but with high lean
body mass and acceptable meat quality.
2.5.3 Influence of environmental factors

Environmental factors play an important role in determining the incidence
and severity of PSE symptoms in susceptible animals. Ante-mortem
exposure of pigs to a hot (45°C) environment for 1 h significantly lowered
muscle glycogen levels and resulted in extreme PSE musculature.
However, further stress imposed by placing animals in a cold water bath
(1°C) for an additional 30 min prior to slaughter (warm + cold treat-
ment) resulted in high pH muscle with very low glycogen reserves, dark
color and associated characteristics of dark, firm, dry muscle (Kas-
tenschmidt et al., 1964). If muscle pH is above 6.0 at 2 h post-mortem,
meat color will be normal (Briskey and Sayre, 1964) but if glycolysis
proceeds rapidly, causing pH to fall to below 5.7 while the muscle is still
hot (> 35°C), the muscle becomes extremely PSE, with a marked loss in
sarcoplasmic and myofibrillar protein solubility (Sayre and Briskey, 1963;
Briskey, 1964; Sayre et ai., 1964), which is associated with very poor fluid-
retention properties (Briskey et al., 1960).
2.5.3.1 Effect of rapid chilling. PSE symptoms may be reduced by rapid

carcass chilling (James et ai., 1983; Crenwelge et al., 1984), or prevented
entirely by partial freezing of cuts in liquid nitrogen (Borchert and
Briskey, 1964). Complete freezing in liquid nitrogen is not recommended,
due to fracturing of skin, fat and muscle (Borchert and Briskey, 1964).
Electrical stimulation of pork carcasses also is not recommended (Cren-
welge et ai., 1984), since this treatment induces PSE musculature by accel-
erating acid production while the muscle is still warm.
2.5.3.2 Myoglobin content. The myoglobin content of PSE muscle has

been found to be similar to that of normal muscle in some studies
(Briskey et al., 1959; Hunt and Hedrick, 1977) but lower in others
(Lawrie, 1960). Briskey (1964) pointed out that some of the discrepancy
may be due to myoglobin precipitation or loss in the drip of PSE muscle.
PSE muscle has greater light scatter and lightness, apparently independent
of myoglobin concentration, which is associated with decreased protein
solubility at lower pH values (MacDougall, 1982).
2.6 Enzymatic reduction of metmyoglobin
Empirical evidence for the presence of metmyoglobin reducing systems in

meat came from the observation that after vacuum-packaging, meat
surfaces with brown discoloration would return to redness (Dean and
Ball, 1960; Pierson et al., 1970). Pirko and Ayres (1957) previously noted
a sharp increase in metmyoglobin formation in meats packaged in films of
low oxygen permeability, followed by appreciable regeneration of myoglo-
bin and oxymyoglobin, which they attributed to 'reducing activity of
muscle'. Walters and Taylor (1963) showed that the metmyoglobin
reducing activity of fresh pork minces was not due to microbial growth,
since metmyoglobin reduction was observed even in the presence of an
inhibitory concentration of the antibiotic, chloromycetin.
Enzymatic reduction of metmyoglobin in meats was first characterized
by Stewart et al. (1965), who oxidized meat pigments with ferricyanide,
then followed the reduction process by monitoring the increase in sample
reflectance at 573 nm during storage. They found that the metmyoglobin
reducing activity (MRA) in antibiotic-treated samples increased with tem-
perature (3-35°C) and pH (5.1-7.1), and was correlated with total
pigments. The MRA declined rapidly in ground meat, compared with
intact cuts. Activity was inhibited by 5% salt or by cooking, which is con-
sistent with an enzymatic process rather than non-enzymatic reduction.
The mitochondrial inhibitors (amy tal and rotenone) inhibited oxygen con-
sumption and MRA in minced pork, while added NAD increased MRA
according to Watts et al. (1966). Rapid loss of metmyoglobin-reducing
ability in ground meat was ascribed to the more rapid loss of NAD in
macerated vs. intact tissue (Severin et aI., 1963; Watts et al., 1966). Watts
et al. (1966) concluded that reduced NADH was a cofactor in enzymatic
reduction of metmyoglobin. In ground meat in the presence of oxygen,
NADH was preferentially oxidized, inhibiting metmyoglobin reduction.
Mitochondrial respiration favored metmyoglobin reduction primarily by
consuming oxygen in the system, which favors establishment of reducing
conditions. Mitochondrial inhibition by addition of arsenate or rotenone
has been shown to increase the rate of discoloration of ground beef
(Govindarajan et al., 1977). Addition of various glycolytic or citric acid

cycle intermediates has been found to increase the MRA of ground beef,
presumably by serving as substrates for generation of reduced NADH
(Saleh and Watts, 1968).
MRA measured by the procedure of Stewart et al. (1965) does not cor-
relate well with color stability during retail display. However, aerobic
reducing ability (ARA) was highly negatively correlated with rate of met-
myoglobin formation in beef muscles (Ledward, 1972). To determine
ARA, thin (2 mm) beef slices were stored at 1°C in atmospheres contain-
ing low (1 %) O 2 , to promote pigment oxidation. After 24 h, metmyoglo-
bin concentration was 50-70% of total pigments. Slices were then
transferred to air and refrigerated for an additional 24 h, during which
time metmyoglobin concentration decreased. ARA was defined as the per-
centage reduction in metmyoglobin concentration after 24 h in air
(Ledward, 1972). Aerobic reducing ability is a measure of both enzymatic
and non-enzymatic metmyoglobin reduction. The lack of correlation of
enzymatic MRA with color stability suggests that the MRA technique
does not accurately measure the activity of enzymatic reduction, or else
aerobic reduction proceeds via a non-enzymatic mechanism (Ledward,
1972).
2.6.1 Enzymes involved

Nonspecific 'diaphorase' enzyme systems for metmyoglobin reduction
have been described in dolphin (Shimizu and Matsuura, 1971) and tuna
(AI-Shaibani et al., 1977). Diaphorase is a term applied to flavoprotein
intermediates capable of transferring hydrogen from NADH to a redox
dye (Nachlas et aI., 1958). With appropriate substrates, the reduced
NADH may be generated by several dehydrogenases, either cytoplasmic
(Bodwell et al., 1965) or mitochondrial (Brooke and Engel, 1966). Non-
specific metmyoglobin reductase activity has also been reported by
Renerre and Labas (1987), Echevarne et al. (1990) and Lanari and
Cassens (1991). Metmyoglobin reduction of muscle minces or extracts was
assayed with addition of NADH and methylene blue, a non-specific
mediator that upon enzymatic reduction can subsequently transfer elec-
trons to several different electron acceptors, including metmyoglobin. By
differential centrifugation, metmyoglobin reductase activity was shown to
be associated with the mitochondrial fraction of beef homogenates (Eche-
varne et al., 1990). Renerre and Labas (1987) and Echevarne et al. (1990)
both reported high reductase activity in the psoas and diaphragma
medialis, muscles, which are unstable muscles, while the color-stable tensor
faciae latae muscles had lower reductase activity. Lanari and Cassens
(1991) similarly reported that muscles of lower color stability (gluteus
medius) had high myoglobin reductase activity, compared with the more
color-stable longissimus dorsi. Thus, the non-specific, methylene-blue-

mediated assay for metmyoglobin reductase varies with mitochondrial
content, and is inversely related to color stability.
A non-mitochondrial enzyme that is specific for metmyoglobin reduc-
tion has been isolated from bovine heart (Hagler et al., 1979; Faustman et
al., 1988) and skeletal muscle (Arihara et at., 1989a). The enzyme contains
flavin, is NADH-dependent and requires the presence of ferrocyanide or
cytochrome b 5 . Based on its similarity in absorption spectra, pH optimum
(6.5), molecular weight (33 000) and other characteristics, Arihara et al.
(1989a) concluded that the heart and skeletal muscle enzymes were iden-
tical. Furthermore, both were probably identical to erythrocyte NADH-
cytochrome b 5 reductase, the enzyme responsible for methemoglobin
reduction. Arihara et al. (1989b) subsequently demonstrated the presence
of the electron-transfer mediator, cytochrome b 5 , in bovine skeletal
muscle. Metmyoglobin reductase (NADH-cytochrome b 5 reductase)
activity has been reported to vary among beef muscles, with higher
actlvlty in tensor Jaciae latae than in the color-unstable psoas muscles
(Reddy and Carpenter, 1991).
2.6.2 Variation among muscles

Variable color stability among beef muscles is well known. Ledward
(1971) reported that the susceptibility of different muscles to metmyoglo-
bin formation was in order of:
biceps femoris muscle > semimembranosus muscle > longissimus dorsi muscle =
semitendinosus muscle.
Color stability differences are especially apparent in the beef 'T-bone'

steak, which is composed of the longissimus dorsi muscle with good stabi-
lity, and the psoas major muscle with very poor color stability (Ledward et
at., 1977; Hood, 1980). In general, muscles of poor color stability tend to
have high oxygen consumption rates (Hood, 1980; O'Keefe and Hood,
1982; Renerre and Labas, 1987; Echevarne et at., 1990; Lanari and
Cassens, 1991). Variability has been reported among breeds of cattle, with
Holstein muscles having a higher oxygen consumption rate and poorer
color stability than muscles from crossbreds (Lanari and Cassens, 1991).
Lamb muscles tend to have a higher post-mortem oxygen consumption
rate than beef and discolor more rapidly during storage (Atkinson and
Follett, 1973).
2.6.3 Other Jactors influencing color stability

Several factors other than oxygen consumption rate have been shown to
influence color stability, including post-mortem meat pH and temperature,
substrate and cofactor availability, and even the resistance of the myoglo-
bin molecule towards oxidation. Ledward (1985) reported that high-tem-
perature low-pH treatment of latissimus dorsi muscle induced by electrical
stimulation and high temperature incubation of excised muscles, resulted
in more rapid color deterioration. He concluded that in practice, electrical
stimulation may have little effect on color stability of longissimus dorsi,
since this muscle normally chills rapidly. However, electrical stimulation
may impair color stability of slower cooling muscles such as the psoas. He
further suggested that, while the oxygen consumption rate is of sig-
nificance in early post-mortem muscle, when rates are high, color stability
at longer storage times is determined primarily by the activity of the enzy-
matic reducing system. Enzymatic reducing activity gradually declines in
post-mortem muscle, due to the lack of substrates (Ledward et al., 1977).
Atkinson and Follett (1973) reported a decreased level of NAD in muscles
of beef, pork and lamb with storage time but there was no apparent
relationship to differences in color stability among species. However,
Echevarne et al. (1990) reported that NADH catabolism was more rapid
in unstable muscles, such as diaphragma medialis, compared with the
stable longissimus dorsi.
Renerre et al. (1992) recently reported that psoas muscle myoglobin was
more prone to oxidation after 8 days storage than was myoglobin from
longissimus dorsi, even though no differences were apparent after 2 h. This
is in agreement with the previous observation by Ledward (1985) that
variable conditions of temperature and pH within a muscle may affect
color stability. Renerre et al. (1992) suggested that environmental condi-
tions within the muscle during storage may have affected globin tertiary
structure, and hence the susceptibility of the molecule towards oxidation.
Of interest and practical application is the observation that duration of
vacuum-aging (conditioning) before fabrication of primals into retail cuts
affects initial meat color and meat color stability. When primals are fabri-
cated for retail display after 3-7 days of vacuum storage, initial bloom
development is more intense than for fresh cuts (Hood, 1980; O'Keefe and
Hood, 1982). This is apparently due to lower oxygen consumption rate in
aged cuts (Bendall and Taylor, 1972; O'Keefe and Hood, 1982), which
results in a deeper oxymyoglobin band after exposure of the cut surfaces
to air. Unfortunately, retail cuts from vacuum-aged beef primals also tend
to discolor more rapidly (Hood, 1980; Bevilacqua and Zaritzky, 1986). A
similar explanation has been advanced to explain the rapid discoloration
of psoas muscle. The higher oxygen consumption rate of this muscle
results in a thinner oxymyoglobin band for psoas muscle (5 mm) than for
longissimus dorsi (7 mm) after 10 days vacuum-aging, followed by 2 days
air exposure of the retail cuts (O'Keefe and Hood, 1982). Consequently,
metmyoglobin formation occurs closer to the surface in psoas muscle and
becomes visible at shorter retail display times. Metmyoglobin reducing
activity of intact psoas muscle is also lower than for other muscles
(O'Keefe and Hood, 1982; Ledward, 1985). Thus, the recognized instabil-
ity of psoas muscle during retail storage is the result of both a higher
oxygen consumption rate and lower metmyoglobin reducing ability than is
the case for other muscles.
2.7 Non-enzymatic reductants and inhibitors of oxidation
2.7.1 Effects of antioxidants and reductants

Ascorbate (vitamin C) and ex-tocopherol (vitamin E) have received the
most attention as antioxidants in meats but metal chelators and synthetic
inhibitors of fatty acid oxidation have also been shown to protect meat
color. Brown and Snyder (1969) reported non-enzymatic reduction of
metmyoglobin by NADH and NADPH but EDTA was also required.
Metmyoglobin reduction was further increased in the presence of photo-
active flavins. It has since been recognized that EDTA itself may act as
reductant, particularly in systems containing flavins (Fife and Moore,
1979). It is therefore reasonable to conclude that NADH and FADH,
cofactors of enzymatic reduction reactions, have little or no capability for
non-enzymatic reduction of metmyoglobin. For ascorbate in particular,
variable antioxidant effects have been reported, depending upon the con-
centration. Watts and Lehmann (1952) found that for hemoglobin solu-
tions held at room temperature, 0.02% ascorbate inhibited discoloration
indefinitely but, at higher concentrations of ascorbate, induced green dis-
coloration. Similarly, Rikert et al. (1957) reported slightly improved color
of some fresh beef cuts treated with 0.05-0.1 % ascorbate (depending
upon the wrapping material) but noted browning of canned samples
treated with 0.5% ascorbic acid.
Greene et al. (1971) reported that both pigment and lipid oxidation of
ground beef was more effectively inhibited by ascorbate plus butylated
hydroxy anisole (BHA) or propyl gallate (PG) than by ascorbate alone.
Govindarajan et al. (1977) reported improved color maintenance in
ground beef with either added ascorbate, BHA, BHT or PG. These com-
pounds had no effect on the initial slow oxidation of myoglobin but
extended by 2-4 days the time before rapid color deterioration began.
Addition of glucose, a reducing sugar, had no effect on color stability.
Oxalate, a chelator of ferrous iron, when added at the 1% level was found
to inhibit color deterioration very effectively. The color-stabilizing effects
of BHA, BHT, PG and oxalate result from inhibition of lipid oxidation.
Lipid oxidation products or intermediates, including oxygen radicals may
either directly oxidize meat pigments or reduce the effectiveness of the
pigment-reducing systems (Greene et al., 1971).
Ascorbate treatment of fresh meats is not known to inhibit bacterial

growth (Greene et al., 1971). Therefore, Okayama et al. (1987) dipped
beef steaks in solutions of ascorbate (3%), (X-tocopherol (0.08%) and 70%
ethanol, in an attempt to obtain both antimicrobial effects and improved
color stability. Bacterial growth was delayed in all ethanol treatments.
Treatments containing ascorbate had an additional 4 days of color stabi-
lity but the (X-tocopherol treatment had no effect on color. Mitsumoto et
al. (1991a) observed no additional improvement in surface color of beef
steaks treated with vitamins E + C compared with ascorbate alone.
Okayama et al. (1987) observed that steaks dipped in ascorbate had
higher metmyoglobin levels than undipped controls through 3 days'
storage, although the color was acceptable. Harbers et al. (1981) pre-
viously reported that psoas muscles dipped in 5% ascorbate exhibited an
initial discoloration but a longer total shelf-life. How~er, Mitsumoto et
al. (1991b) found that spreading a 10% solution of ascorbate on the
surface improved the color of beef steaks. No mention was made of initial
discoloration problems. Hood (1975) demonstrated that intravenous injec-
tion of cattle with sodium ascorbate immediately prior to slaughter
increased the color stability of the psoas muscle.
Treatment of fresh beef cuts with antioxidants was not permitted in the
USA until recently, the rationale being that preservation of the fresh
appearance without inhibiting bacterial growth could compromise the
safety of these products. However, recent reports indicate that a sub-
stantial increase in meat color stability can be achieved by more natural
means through dietary supplementation of feedlot cattle with vitamin E
70
~
60
I:
..c
~ 50
'">-
0
E 40
"Ii
E
., 30
u
~ 20
"
C/)
10
0 2 4 6 8 10 12 14 16
Days di s pl ay ed
Figure 2.7 Effect of dietary vitamin E supplementation and/or vitamin C dip treatment on
surface metmyoglobin content of beef loin steaks during display at 4°C. CNTRL = control;
UN = undipped; SUPP = dietary supplementation with vitamin E; DIP = dipping steaks
in 1% ascorbic acid (Mitsumoto et al., 1991c). CNTRL-UN, 0; CNTRL-DIP, e; SUPP-
UN, 0; SUPP-DIP, •.
(Faustman et al., 1989a,b). In fact, dietary supplementation with vitamin

E (Mitsumoto et al., 1991c) resulted in less surface metmyoglobin on
steaks after 16 days' display than for control steaks dipped in 1% ascorbic
acid (Figure 2.7). The beneficial effects of vitamin E supplementation were
most pronounced in the meat from Holstein cattle. However, supple-
mentation also showed promise for improving the color stability of meat
from other breeds.
The USDA has recently approved the treatment of fresh pork with
ascorbate- or erythorbate-containing solutions. Pork has a highly variable
pH, due partly to ante':mortem handling and the variable incidence of the
PSE syndrome. Cheng (1987) demonstrated that in pork chops of low pH
( < 5.7), metmyoglobin formation and brown discoloration was rapid even
though bacterial numbers remained low. Dipping or spraying of fresh cuts
with a solution of 0.1 % ascorbate, 0.2% citrate or an equivalent chelating
agent, and 2% phosphate buffer was shown to stabilize pork color and
slightly inhibit rancidity without masking product deterioration due to
microbial growth. Product shelf-life was extended further by placing indi-
vidually wrapped retail cuts in modified atmosphere containers (20-80%
CO 2 and 2-30% O2 ) during distribution (Cheng, 1987). The US patent
was assigned to Wilson Foods Corporation, Oklahoma City. A similar
system has been described for treatment and distribution of retail beef
cuts (Manu-Tawiah et aI., 1991). To date, distribution of retail meat cuts
in modified atmosphere containers is not widely practised, in part due to
the general acceptance of the 'boxed beef concept of delivering vacuum-
packaged primals to retail outlets for fabrication on the premises.
2.8 Irradiation and other antimicrobial treatments
Color stability of fresh meats may potentially be increased by several dif-

ferent antimicrobial treatments, including ionizing (gamma) radiation
(Satterlee et al., 1971), ultraviolet radiation (Reagan et aI., 1973; Hood,
1980; Stermer et al., 1987), washing or dipping carcasses or cuts with
dilute acid solutions (Biemuller et al., 1973; Anderson et aI., 1979;
Mendonca et al., 1989), sorbate (Unda et aI., 1990), and ammonium
hydroxide (Gupta et al., 1988). Shelf-life extension has been reported for
cuts coated with acetylated monoglyceride (Keeton et aI., 1988) or calcium
pectinate (Stubbs and Cornforth, 1980).
2.B.1 Irradiation of fresh and cooked meats

Gamma irradiation causes rapid brown discoloration of raw meats
wrapped in oxygen permeable film (Ginger et al., 1955). However, an
objectionable red pigment develops upon irradiation of chicken or other
meats in an inert atmosphere (Tappe1, 1956; Bernofsky et aI., 1959;

Hansen et al., 1963). Satterlee et al. (1971) reported that low-dose gamma
irradiation of metmyoglobin solutions resulted in formation of a red
pigment with many characteristics of oxymyoglobin. Tappel (1956) pre-
viously proposed oxymyoglobin formation upon reaction of metmyoglo-
bin with hydroxyl radicals in irradiated solutions, but Satterlee et al.
(1971) found that the amount of red pigment formed was unaffected by
the presence of tert-butanol, a hydroxyl radical scavenger. Irradiation of
cooked meats in an anaerobic environment results in pink color develop-
ment due to reduction of the cooked meat pigment, denatured globin
hemichrome to the hemochrome form (Tappel, 1957b). The pink color of
cooked meats radappertized (radiation sterilized) in evacuated containers
is not regarded as significant to consumer acceptance, since the normal
brown or gray color rapidly returns after the container is opened, due to
pink pigment oxidation by O 2 (Urbain, 1986). The brown discoloration
and fluid loss of irradiated fresh meats may be inhibited by a double
packaging system (Urbain, 1973), in which individual cuts are dipped in
0.5% sodium tripolyphosphate solution and wrapped in oxygen-permeable
film. Several individually wrapped cuts are then placed in a bulk con-
tainer, which is vacuum-evacuated. After irradiation, the refrigerated bulk
pack may be transported to the retail store, where individually wrapped
cuts may be displayed for retail sale. A variation of the double packaging
system is the recently USDA-approved MAP system for fresh pork
(Cheng, 1987; see also 2.7), in which individually wrapped cuts are trans-
ported in modified-atmosphere containers. With either variation, retail
cuts may be prepared at a central location. An additional feature of both
systems is use of oxygen-permeable wrapping of individual cuts, so that
meat will bloom (turn bright red) upon removal from bulk containers.
2.8.2 Su/jites and meat color

Sulfites are especially effective color preservatives, since they have both
reducing properties and antimicrobial effects. Sulfites are not permitted for
application to fresh meats in the USA but violations have occurred. For
example, the USDA temporarily withdrew inspection services (effectively
halting operations) from a North Carolina firm that illegally added sulfites
to pork sausages (Mocker, 1991). Sulfite addition is considered a mislead-
ing practice and detection methods are available for sulfite in meat
products (Mallinson et aI., 1985). Adverse reactions to sulfited foods have
been reported (1FT, 1986). However, sulfites are permitted preservatives in
fresh pork sausages in the UK, and are reportedly inhibitory to Salmo-
nella spp. and spoilage organisms (Banks et aI., 1985). A US patent has
been assigned to Stay Fresh Inc. for color preservation of fresh meats with
a solution containing ascorbate, citrate, and sulfite salts (Aversano, 1984).
2.9 Effects of light, freezing, salt and lipid oxidation on meat color
Ramsbottom et al. (1951) and Rikert et al. (1957) both reported that light
had little effect on fresh meat color. However, Marriott et al. (1967)
demonstrated increased color deterioration of fresh meat at -1 DC under
direct illumination, and concluded that illumination caused an increase in
meat surface temperature, enhancing bacterial growth. Satterlee and
Hansmeyer (1974) reported enhanced discoloration of fresh beef surfaces
by illumination with S9ft, white fluorescent lamps (250 foot-candles). Cool
flood lamps reportedly caused an increase in pork chop temperature
resulting in a darker color than other types of illumination (Calkins et al.,
1986). The effects of retail display lighting on meat color have been
reviewed by Kropf (1980).
Rapidly frozen beef has a light color (Hanenian et al., 1989; Lanari et
al., 1989). The color stability of frozen beef is increased by vacuum-
packaging as opposed to polyethylene wrapping (Lanari et al., 1989).
Ultraviolet barrier packaging has been reported to increase color stability
of frozen beef products during display (Andersen et al., 1989).
Salt (NaCI, 1-3% solution) markedly increases pigment oxidation. This
has been demonstrated in pure solutions of hemoglobin by Watts and
Lehmann (1952), in myoglobin by Wallace et al. (1982) and in ground
beef by Govindarajan et al. (1977) and Trout (1990). The oxidation rate is
proportional to the concentration of the chloride ion (Wallace et al., 1982;
Trout, 1990). Possible effects of salt on the tertiary structure of myoglobin
need further investigation.
Several reports indicate co-oxidation of meat lipids and pigments, as
previously discussed by Greene et al. (1971) and Govindarajan et al.
(1977). Comprehensive reviews on this topic are available (Govindarajan,
1973; Faustman and Cassens, 1990).
2.10 Cooked meat color
The brown pigment of cooked meat has been characterized as denatured

globin hemichrome (also called metmyochromogen), consisting of a high-
spin ferric porphyrin (hematin), in which the fifth coordination position of
hematin is occupied by a carboxylate ion of heat-denatured globin. The
sixth binding site may be occupied by water but probably not another
denatured globin moiety because of steric considerations (Giddings, 1977).
Since the cooked meat pigment is not extractable in acetone, it cannot
simply be hematin (Tarladgis, 1962a). Tappel (1957a) had earlier suggested
that the cooked meat pigment was a denatured globin-nicotinamide hemi-
chrome but based on the characteristics of the cooked meat reflectance
spectra, Tarladgis (1962a) concluded that nicotinamide was not a ligand.
2.10.1 Pink color in cooked, uncured meat

Consumers often interpret pink or red color in cooked meats as an indica-
tion of undercooking, based on the well-accepted relationship between
color and internal temperature of beef steaks, where rare is equivalent to
60°C (140°F) internal temperature, medium well done is equivalent to
71 DC (160°F), and well done is equivalent to 82°C (180°F). Processors
experiencing problems with red or pink color in cooked products should
first check to assure that the desired internal temperature has been
reached. If pink color persists in products reaching the desired cooking
temperature, processors often suspect that nitrate, nitrite or nitric oxide
contamination of products or other ingredients led to development of
pink, cured meat color. Pink color in cooked beef has been traced to
leaching of nitrate into the raw meat from wetted package adhesive
(Scriven et ai., 1987). Microbial reduction of nitrate to nitrite must also
have occurred, since meat tissue is unable to catalyse this conversion. As
low as 1 p.p.m. nitrite may cause a significant color change in oven-
roasted turkey breast meat (Ahn and Maurer, 1989a). Some soy proteins
used in formulation of turkey rolls may contain 50 p.p.m. nitrite, and
more than 300 p.p.m. nitrate. However, pink discoloration was not
observed in turkey rolls formulated with nitrate- or nitrite-containing soy
protein (Cornforth, unpublished observations). Carbon monoxide in
smoke or combustion gases may also cause pink discoloration of cooked
meat surfaces (Pool, 1956) due to formation of CO-globin hemochromes
(Tappel, 1957a). After slicing, a pink ring is common and desirable in
'Texas BBQ' beef, where meat has been slowly cooked in a heavy smoke
(Cornforth et al., 1991). Interestingly, Pool (1956) reported that some
turkeys cooked in an electric roaster had surface pinking, when the
heating element was set on high and air in contact with the heating
element was circulated over the bird. Apparently, temperatures near the
heating element were high enough to generate nitrous oxides, subsequently
causing pink color development.
When meat pH is high (> 6.0), myoglobin is more resistant to heat
denaturation, causing mildly heated meat products to remain pink or light
red after cooking (Trout, 1989). One example is 'hard-to-cook' hamburger
patties, traced to use of high levels of bull meat (pH > 6.0) in the pattie
formulation (Mendenhall, 1989). Myoglobin has also been identified as
the pigment responsible for red discoloration of pre-cooked, vacuum-
packaged bratwurst by Ghorpade et al. (1992). The brown-to-red color
change was found to be associated with microbial growth. Development
of red discoloration was delayed by the use of sodium lactate and cooking
the product to a higher internal temperature. Spoiled sow meat, character-
ized by high myoglobin content and a pH greater than 6.4, was found to
remain red after cooking to 7PC by Cornforth et al. (1993).
Under anaerobic conditions such as occur in the center of large roasts

or in vacuum-packaged cooked meats, the brown cooked meat pigment
may be reduced to the corresponding denatured globin hemochrome.
Globin hemochromes were first identified as the pink pigments of canned
tuna by Brown and Tappel (1957). The pink hemochromes are a hetero-
geneous group, consisting of complexes between ferrous heme and nitro-
genous side-chains of various denatured proteins. Mixed complexes also
occur with heme, denatured protein and nicotinamide, histidine or other
nitrogenous compounds (Anson and Mirsky, 1928; Brown and Tappel,
1957; Cornforth et al., 1986; Ahn and Maurer, 1990a, b). Exposure of raw
pork to ammonia results in development of a distinct pink color after
cooking, which is similar to some hemochromes (Shaw et al., 1992).
Various hemochromes have been proposed as substitutes for the nitrosyl
hemochromes of cured meats (Howard et aI., 1973; Dymicky et al., 1975).
In commercial canning of beef, a pink color is regularly observed when
the can is filled with raw meat. The pink color, considered undesirable for
baby foods, may be avoided by pre-cooking the meat before canning
(Urbain, 1986).
The pink color associated with denatured globin hemochromes may not
be apparent after cooking but may develop during refrigerated storage of
turkey rolls (Cornforth et al., 1986) or cooked pork roasts (Ghorpade and
Cornforth, 1993). Pink color development in refrigerated turkey rolls can
be inhibited by inclusion of non-fat dry milk in the formulation (Dobson
and Cornforth, 1992). Cytochrome c, although present at low levels, has
greater heat stability than myoglobin (Ahn and Maurer, 1989b), and may
contribute to the pink color remaining in turkey rolls after other pigments
have faded (Girard et al., 1990).
2.11 Cured meat color
2.11.1 Role of nitrite

Sodium (or potassium) nitrite is the agent commonly added to brines to
produce the pink color typical of cured meats. Additionally, nitrite and its
derivatives have antimicrobial and antioxidant properties. Nitrate salts
may also be used as a curing agents. Historically, nitrates were uninten-
tional contaminants of salt used in the curing process. Later, it was recog-
nized that saltpeter (potassium or sodium nitrate) was the compound
actually responsible for cured meat color (Binkerd and Kolari, 1975).
Haldane (1901) demonstrated that nitrite alone could produce cured meat
color. Hoagland (1908) recognized that bacterial and meat-induced reduc-
tion of nitrate resulted in formation of nitrite and nitric oxide (NO). In
1923, the USDA first gave permission for the use of nitrite in curing brines
(Binkerd and Kolari, 1975). Muscle tissues are incapable of reducing

nitrate to nitrite but bacteria perform the reduction in nitrate-cured meats
(Walters and Taylor, 1964). Walters and Taylor (1964) also conclusively
identified NO as the product of nitrite reduction in pork muscle minces.
Enzymatic reduction of nitrite to NO may proceed via mitochondrial
(Walters et at., 1967), or non-mitochondrial (Koizumi and Brown, 1971)
muscle pathways, if sufficient NADH is present. However, in commercial
meat curing, nitrite reduction occurs primarily by non-enzymatic means.
Meat turns brown in presence of nitrite, as myoglobin is oxidized con-
current with nitrite reduction to NO. A metmyoglobin-NO complex
forms, which under anaerobic conditions may be reduced to red colored
NO-myoglobin. The absorption spectra of NOMb is very similar to that
of oxymyoglobin (Reith and Szakaly, 1967b). The overall rate of NOMb
formation is pH- and temperature-dependent. Either NO formation or
NOMetMb reduction is rate-limiting (Fox and Ackerman, 1968). Myoglo-
bin itself is a reductant, since NOMb is formed upon anaerobic incuba-
tion of solutions containing only nitrite and myoglobin (Koizumi and
Brown, 1971). In the absence of other reduct ants, the initial product,
NOMetMb, is capable of slow autoreduction. Apparently, the globin imi-
dazole residues of NO-metmyoglobin are oxidized (Killday et at., 1988),
allowing slow 'autoreduction with time via internal electronic rearrange-
ment' of the ferric heme-NO (Giddings, 1977).
2.11.2 Action of cysteine and ascorbate

Cysteine and ascorbate also reduce nitrite to NO (Reith and Szakaly,
1967a,b; Fox and Ackerman, 1968) but in model systems NO-myoglobin
formation is appreciably slower with cysteine than for ascorbate (Reith
and Szakaly, 1967a). Green discoloration (nitrite burn) was observed
when nitrite was added in great excess (500 moles per mole myoglobin)
according to Reith and Szakaly (l967b). In solution, nitrite ion (N02~)
reaches equilibrium with nitrous acid (HN0 2; pKa = 3.4). Ascorbate
reacts with nitrous acid to form a nitrosoascorbic acid intermediate (Fox
and Thompson, 1963; Izumi et at., 1989), which releases NO, as follows:
2HN0 2 + ascorbate -+ 2NO + dehydroascorbate + 2H20 (2.2)
Meat has sufficient reducing capacity to reduce added nitrite to NO.
However, ascorbate (or its isomer, erythorbate) is commonly added to
brine or sausage emulsions to obtain faster NO production and thus more
rapid development of cured meat color. Care must be taken in use of
ascorbates in curing brines. If ascorbate-containing brines are held for
long periods, or at elevated temperature and acid pH, nitrite will be lost
from the brine as NO gas, reducing the effectiveness of the brine for meat
curing. Brines with ascorbate should be held no longer than a day, at less
than lOOC (50°F) and at alkaline or very slightly acid conditions (Rust,
1977).
2.11.3 The cured meat pigment

Heating of NOMb forms cured meat pigment, nitrosylhemochrome, which
is acetone soluble and exhibits a broad absorption peak from about 535-
565 nm (Homsey, 1956). NO has an unpaired electron, as would a mono-
nitrosylheme complex. Paramagnetic compounds (those with unpaired
electrons) are detectable by EPR spectroscopy. Tarladgis (1962b) con-
cluded that the cured meat pigment was dinitrosylhemochrome, since EPR
spectra of the acetone extract of cured meat showed no indication of
paramagnetism. In support of this conclusion, Lee and Cassens (1976)
found that myoglobin contained twice as much 15N-labelled nitrogen
when heated with ascorbate and 15N-labelled nitrite, compared with
unheated solutions. However, more recent mass spectroscopic studies
indicate that the pigment is actually a mononitrosylheme complex. When
NOMb is heated, the protein is detached from the heme, with one mole of
NO binding to the heme and a second mole of nitrite is incorporated into
the denatured protein (Kill day et al., 1988).
NO pigments may be produced by direct application of NO gas to
pigment solutions (Urbain and Jensen, 1940; Keilin, 1955; Fox and
Ackerman, 1968; Shahidi et aI., 1985) or to meats (McBrady, 1968;
Braddock and Dugan, 1969; Rankin, 1973; Vahabzadeh et aI., 1983).
Although a patent has been issued for meat curing with NO gas
(McBrady, 1968), the process has not been used commercially. NOMb
formation may also occur as the result of NO transfer from nitrosylated
compounds (S-nitrosocysteine, nitrosylated albumin) to myoglobin
(Kanner and Juven, 1980; Ito et al., 1983). Cured meat color may be
obtained in nitrite-free comminuted meats by addition of pre-formed
nitrosylhemochrome (mononitrosyl ferrohemochrome) to raw meat emul-
sions (Shahidi et al., 1985; O'Boyle et aI., 1990; Shahidi & Pegg, 1991),
lowering the possibility of nitrosamine formation in these products during
heat processing. Microencapsulation of the pre-formed nitrosylhemo-
chrome has been reported to increase pigment stability in nitrite-free ham
(O'Boyle et al., 1992).
2.11.4 Fading of cured meat color

The most significant commercial problem associated with cured meat
color is rapid fading in air and light (Urbain and Jensen, 1940; Kraft and
Ayres, 1952; Draudt and Deatherage, 1956; Walch and Rose, 1956), which
can be avoided by vacuum-packaging systems (Urbain and Ramsbottom,
1948). Photocatalyzed oxidation of nitrosylhemochrome is a two-step
process: light-induced dissociation of nitric oxide from heme is followed

by subsequent oxidation of both the heme iron and nitric oxide by oxygen
(Fox, 1966). Reductants, such as cysteine or ascorbate, greatly stabilize
the pigment from fading (Homsey, 1956; Reith and Szakaly, 1967b).
Application of a 10% solution of ascorbate or erythorbate is permitted
before packaging of cured meat cuts to inhibit fading (deHoll, 1981).
2.12 Summary
Fresh meat color intensity is dependent primarily upon myoglobin con-

centration, which varies among species and muscles. This pigment may
exist in three forms; (i) purple deoxymyoglobin (Mb); (ii) red oxymyoglo-
bin (Mb0 2); or (iii) brown metmyoglobin (MetMb). Oxygenation of Mb
to Mb0 2 normally occurs within 30 min of exposure of meat to air.
Mb0 2 predominates at fresh meat surfaces. In the bulk of the meat, the
pigment is Mb. Mb oxidation proceeds most rapidly at low oxygen con-
centration (P02 = 1 torr = 1 mm' Hg). The MetMb concentration increa-
ses gradually at the limit of oxygen diffusion into the meat (ca. 3-5 mm),
forming a brown subsurface band that gradually widens, causing dis-
coloration even in the absence of bacterial growth. Oxidation (ferrous to
ferric heme iron) of meat Mb to MetMb is, in physiological terms, a slow
process and can be measured in hours or days.
Aerobic bacterial growth consumes O 2 , reducing O 2 penetration into the
meat, and favoring MetMb formation near the surface, where it becomes
visible as brown discoloration. At very high bacterial levels characteristic
of spoilage (> 10 7 cfu.g- 1), the surface becomes anaerobic, and reduct ants
from the bacteria or endogenous to the meat reduce MetMb to Mb
(brown to purple color reversion). In vacuum-packaged meat, lactic
bacteria gradually reach high levels but in the absence of oxygen, brown
discoloration does not occur. Modified-atmosphere (elevated CO 2 + O 2 )
packaging is not widely used for fresh meats but offers the simultaneous
benefits of eye appeal (bloom) in the presence of O 2 , with longer shelf-life
due to inhibitory effects of CO 2 on aerobic bacteria.
The effects of meat mitochondrial oxygen consumption rate (OCR) on
color are entirely analogous to the effects of aerobic bacteria. Mitochon-
drial OCR is, in turn, affected by pH, so for dark-cutting meat at pH
greater than 6.0, OCR remains very high, and Mb predominates. At pH
5.6, mitochondrial OCR is lower, but measurable. Unstable muscles such
as the psoas with relatively higher OCR have less O 2 penetration and
faster discoloration than color-stable muscles such as longissimus dorsi.
Endogenous MetMb reducing activity (enzymatic and non-enzymatic) also
affects meat color stability.
Heat denaturation and consequent oxidation of Mb forms denatured
globin hemichrome, the gray pigment of cooked meat. Under anaerobic

conditions, denatured globin hemichrome may be reduced slowly, allowing
the formation of denatured globin hemochrome or related pink hemo-
chromes. Although uncommon, pink color in cooked meats may occur
due to exposure to CO, NO or their precursors. Red or pink color may
persist in meats with pH greater than 6.0 and cooked to less than 74°C,
since high pH stabilizes Mb to heat denaturation. Mono-nitrosylhemo-
chrome is the pink pigment of cured meats. It is susceptible to fading in
light or air. Fading can be retarded by surface application of ascorbate or
erythorbate and vacuum-packaging.
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e
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3 Colour of meat
D. B. MACDOUGALL
3.1 Introduction to vision and colour
The human senses can be separated into two classes, the so-called lower
and higher. The lower senses function by direct reaction of the stimulus
with the sense organ, such as in smell and taste. Thus it requires molecules
or ions in the airstream to the nose or in the saliva in the mouth to elicit
the response to smell and taste. The sense of touch responds to contact or
pressure on the skin. The higher senses of hearing and vision function by
detection of specific ranges of the electromagnetic spectrum, which for
vision, is between 380 and 770 nm.
The sense of vision provides the ability to recognize much of the world
about us provided that there is sufficient energy for our eyes to respond to
the stimulus. At very low levels, light detection is colourless or achro-
matic, whereas, at high or photoptic levels the response is described as
coloured. The interlinked mechanisms of visual detection afford the ability
to recognize such features as object shape, size, location, distance, direc-
tion, perspective and movement in the perceived scene in which colour
response is a major contribution to information input. The intensity of
colour varies with distance from the observer and the level and spectral
composition of the light.
Perception of colour is a psychological phenomenon dependent on the
observer and is not an intrinsic property of the object or scene, although
our common experience causes us to assign the experience of colour to the
object as though it were a property of the object itself. It requires, there-
fore, three factors for 'object' colours to be perceived and recognized.
These are (i) sufficient light within the visible spectrum; (ii) the object; and
(iii) the human vision apparatus. Such is the complex nature and moder-
ating capacity of the visual process that colour perception, although
essentially controlled by these three factors, does not undergo such drastic
changes as would be expected initially if anyone of the factors were
changed.
3.2 Colour vision
The human eye has two types of detecting cells in the retina, the rods and
cones. The rods are sensitive to low levels of light and the sensation is
achromatic. The sensation of colour is dependent upon detection of higher

levels of light by the cones, so-called because of their shape. They are
located in a small area, the fovea, in the central region of the retina. Light
entering the eye is controlled in intensity by the iris and focused onto the
fovea. The signals generated by the cones, which are sensitive to three
wavelength ranges, short or blue (B), mid or green (G) and long or red (R)
(Estevez, 1982), are amplified through the visual pathway (Rodieck, 1979)
to the brain's cortex for interpretation (Zeki, 1980). The sensation is
perceived as though it were located where the light originated from the
object within the scene out in the external world. This leads to the concep-
tional misunderstanding that colour is a property of the light or the illumi-
nated object, whereas, it is actually a sensation in the mind. As such, it is
subject to such modifying influences as adaptation to the spectral composi-
tion and the intensity of the 'colour' of the background illumination.
Thus, white objects, which act as visual references, appear to remain
white even though the light may change in colour quality, e.g. from the
cold blue light of overcast daylight to the warm yellow of sunshine. This
process of adaptation serves to keep the eye in the balance (Boynton,
1979). Although white objects tend to remain constant, changes occur in
the perceived colour quality of coloured objects (Bartleson, 1979a, b) but
the changes are less than would be expected if the scene were not balanced
to white, i.e. the phenomenon of colour constancy. In addition to adapta-
tion, light intensity directly affects object colour brightness (Hunt, 1977).
Owing to the limitation of human memory, the magnitude of chromatic
adaptation on perceived colour change or the apparent maintenance of
colour constancy is not realized by the observer (Brill and West, 1986).
Any system devised for measuring colour, therefore, must take account of
the effects of both light intensity and spectral composition on observer
adaptation.
3.2.1 Colour measurement

Colour is usually considered the most important sensory characteristic in
the appearance of a food, especially of meat, except when interacting with
translucency, e.g. when meat is either pale, soft and exudative (PSE) as in
pork, or dark, firm and dry (DFD) as in dark cutting beef.
3.2.1.1 The CIE system of colour measurement. The International Com-

mission on Illumination (Commission Internationale de l'Eclairage) (CIE,
1986) system transforms the object's reflectance or transmission spectrum
into three-dimensional space by integrating the object spectrum with the
colour-matching functions of the standard observer and the spectral
power distribution of the illuminant. Details of the mathematical proce-
dure are given in any standard text on colour, for example Wright (1980),
COLOUR OF MEAT 81
Judd and Wyszecki (1975) and Hunt (1987). Instead of using 'real' red,
green and blue primaries R, G and B with the necessity of negative
matching, the system uses transformed or 'imaginary' primaries X, Y and
Z, which are all positive. Primary Y, the luminous reflectance or transmit-
tance, contains the entire lightness stimulus. Hence every colour can be
located uniquely in 1931 CIE colour space by Y, its lightness and its chro-
maticity coordinates, x = Xj (X + Y + Z) and y = Yj (X + Y + Z),
for the defined illuminant and observer conditions.
CIE 'source C,' the original but synthetic light representative of
daylight is now being superseded by 'D 65 ' (6500 0 K) based on a cool phase
of white Planckian daylight typical of cloudy overcast northern light,
which includes ultraviolet light and occluded sunlight. The original 2°
colour-matching functions apply strictly only to small objects. The CIE
have now added a 10° human vision field size, more representative of the
bulk of object colours. The current trend in colour measurement, there-
fore, is to use D65 and the 10° observer, although much food colorimetry
continues to use source C and 2°. These CIE recommended procedures
(CIE, 1986) are included in the latest ASTM (1987) standards and also in
Hunt (1987), together with the weighting factors for several illuminants,
including some fluorescent lamps typical of those found in commercial use
(Rigg, 1987).
3.2.2 Uniform colour space

Since the 1931 CIE Y, x, y system is not visually uniform, chromaticity
values of constant hue and chroma are distorted. Construction of
improved colour spacing into near-equal visually perceptual steps has
been attempted by both linear and nonlinear transformations of Y, x, y
(Billmeyer and Saltzman, 1981). Approximate uniform spaces are the
Hunter L, a, b opponent colour space devised in the 1940s (Hunter,
1958) and the recent 1976 CIELUV and CIELAB spaces (Robertson,
1977) that were formulated by the CIE to reduce the many approaches
to scale uniformity to two, which should be applicable to most demands
of practical colour measurement. CIELUV space is used for the mea-
surement of the colour of lights and large colour differences, and
CIELAB for objects and small colour differences. For example CIELAB
has been the colour space used for the development of colour tolerance
measurement equations for 'near-perceptual' differences (McLaren, 1986).
The lightness coordinate (L *) is the same for both CIELUV and
CIELAB but the chromaticness diagrams are constructed differently.
CIELAB (L*, a*, b*) space has found general application in industry. It
is a nonlinear cube-root transformation of the 1931 tristimulus values to
approximate the Munsell colour order system. Except for very low levels
of Y, the formulae are:
L* = 116 (Yjyn )I/3 - 16 (3.1)

a* 500 [(Xj X n)I/3 - (Yj Yn)I/3] (3.2)
b* 200 [(Yjyn )I/3 - (ZjZn)I/3] (3.3)
X n , Yn , Zn refer to the nominally white object colour stimulus. This can
be compared with the Hunter scale, which uses a square root transforma-
tion:
L = lOyl/2 (3.4)
a [17.5(1.02X- Y)]jyl/2 (3.5)
b [7.0(Y - O.847Z)]jyl/2 (3.6)
3.3 Terminology
Colour can be considered as both subjective and objective (Hunt, 1978).

Subjectiveness refers to what is perceived after light enters the eye and
uses terms such as brightness, lightness, hue, saturation, chroma and col-
ourfulness. Colourfulness, a recently introduced term, is that aspect of
visual sensation that describes an area or object with more or less chro-
matic colour. Hue is that attribute described in colour names, red, green,
purple, etc. However, saturation and chroma are less easily understood.
Saturation is colourfulness in proportion to its brightness, whereas,
chroma is colourfulness relative to the brightness of the surroundings. A
similar difference exists between lightness and brightness where lightness is
relative brightness. Lightness is unaffected by illumination intensity
because it is the proportion of light reflected, whereas, brightness increases
with illumination level.
The objective colour terms refer to the stimulus and are calculated from
the spectral distributions of the object's reflectance, the illuminant and the
standard observer's trichromatic (x,y,z) colour-matching functions. Inte-
gration of these three components is the basis of the psychometric quali-
ties that correspond approximately to those perceived. For CIELAB the
most important terms are:
lightness = L * (3.7)
hue = h* = tan-1(b*ja*) (3.8)
chroma = C* = (a*2 + b*2)1/2 (3.9)
Total colour difference, 1\ E*, is calculated from either the colour space
coordinates or from lightness, chroma and hue. Therefore,
1\ E* = [(1\L *)2 + (1\a*)2 + (1\b*)2]1/2 or (3.10)
COLOUR OF MEAT 83
(3.11 )
where H* is used rather than h* because h* is angular. Hence for small
colour differences away from the L * axis:
Li H* = C*Lih*(n/180). (3.12)
3.4 Instrumentation
Colour-measuring instruments, based on human colour perception contain

correction factors for both lighting and human visual responses. These
corrections are incorporated either as filters, as in trichromatic colori-
meters, or as computer programs in automatic colour spectrophotometers.
3.4.1 Trichromatic colorimeters

The trichromatic colorimeter constructed by Hunter (1958) in the 1940s
was highly significant in the development of automated colorimetry. This
valve amplified instrument comprised a stable light source and three wide-
band red, green and blue filters, which approximated CIE standard illumi-
nant C and the 2° observer. The tristimulus values obtained were trans-
formed into the Hunter L, a, b (or aL bd colour space to approximate
visual spacing. Such instruments were less expensive than the more precise
spectrophotometers but were sufficiently accurate at measuring colour dif-
ferences for industrial process control (Patterson, 1987). Modern
computer-driven tristimulus instruments may contain options of several
colour spaces and a variety of sensor heads with different illuminating and
light collecting geometries. A wide range of product types can be
measured even though their surface structures may vary. Hand-held col-
orimeters with optical geometries based on the larger bench-type instru-
ments owe their compactness to the use of high-energy, pulsed xenon arc
lamps and filtered silicon detectors and microchip circuitry. Their com-
parative inexpensiveness has resulted in increased on-line colour control in
several branches of the manufacturing industry.
3.4.2 Spectrophotometers
Spectrophotometers are the most accurate type of colour-measuring
instrument. They are usually fitted with an integrating sphere and a choice
of reflectance geometries. Inclusion or exclusion of the specular or gloss
component depends on which geometry is appropriate for the particular
product application.
The CIE recommends that colour measurement of opaque materials
should be obtained with one of the following conditions of illumination:
• 45 /0 or 0 /45 specular excluded;

0 0 0 0
,
• diffuse/O° or O /diffuse, specular included or excluded.

O
The former is typical of Hunter-type colorimeters or colour difference

meters and the latter of spectrophotometers.
3.4.3 Sources of variation among colorimeters and spectrophotometers

Unfortunately, colour measuring spectrophotometers do not have iden-
tical geometries. The largest source of differences among them is caused
by the specular component (Patterson, 1987). If measurements are to be
compared, or used for computer-match prediction of pigmented materials,
it is better to include the specular (Hunt, 1987), although visual observa-
tion may be more closely related to measurement when the specular is
excluded (Best, 1987).
Other sources of variation among colorimeters and spectrophotometers
are the viewing aperture area and the illuminating light spot. These affect
both the direction and amount of light returned from translucent materi-
als, hence are important variables in measuring meat colour. An increase
in aperture area from 5 mm to 20 mm can exhibit a lO-fold decrease in
K/S for translucent suspensions (MacDougall, 1987). The wavelength
interval used to calculate the tristimulus values is also a potential source of
error. Although the CIE (1986) specifies the standard observer at 5 nm
intervals from 380 nm to 780 nm, 10 nm accuracy is sufficient for most
purposes. The CIE have not yet officially recommended the use of 20 nm
intervals, although some colour spectrophotometers detect at 20 nm
intervals.
3.5 Absorption, scatter and pigmentation
3.5.1 Reflectance
In addition to the reflectance spectrum providing the basis for colour cal-
culations, meat spectra provide information on the state of pigment oxy-
genation or oxidation. The three forms of the muscle pigment myoglobin
have sufficiently different absorption properties such that ratio techniques
can be used to estimate their relative proportions on meat surfaces. A
variety of techniques have been proposed from simple ratios of reflec-
tance, 630 nm:580 nm, which is indicative of development of metmyoglo-
bin absorption at 630 nm, to procedures for estimating all three forms of
the pigment based on K/S ratios of reflectance at specific wavelengths
relative to the isosbestic wavelengths (where absolute spectra from three
different compounds crossover at the same concentration) at 525 nm
(Stewart et al., 1965; Krzywicki, 1979; Trout, 1991). However, reflectance
measurement of meat surfaces cannot provide accurate information on
COLOUR OF MEAT 85
pigment concentration because of variation in the light-scattering proper-

ties of meat. Pigment concentration is usually measured by the cyanmet
procedure for haemoproteins (Drabkin, 1950; Warriss, 1979) or by the
Homsey method (Homsey, 1956), which uses acetone/hydrochloric acid
for pigment extraction. Both methods have drawbacks, the cyanmet pro-
cedure uses extremely toxic cyanide and the use of the acid haematin pro-
cedure on pork may produce . turbidity. Trout (1991) has recently
proposed the use of sodium nitrite to oxidize the aqueous extract and
measure absorption differences between 490 nm and 730 nm. The method
is reported to compare favourably with both the cyanmet and acid
haematin procedures and also that of Krzywicki (1979).
3.5.2 Light scatter

Light scatter in muscle varies from two causes, which may interact with
each other, the intrinsic physiological state of the animal prior to slaugh-
ter and the killing/chilling regime. Immediately after slaughter muscle is
dark in appearance because of its translucence. The rate of post-mortem
glycolysis controls the rate of fall in muscle pH from production of lactic
acid, which, in turn, together with the rate of temperature decrease in the
carcass as it cools, affects the degree of protein denaturation. Ultrafast
production of lactic acid, as is found in carcasses of stress-susceptible
breeds of pigs, can result in a doubling of the light-scattering power of the
meat, i.e. are the cause of the paleness of pale, soft and exudative (PSE)
meat. Alternatively, depletion of glycogen prior to slaughter from
extended animal stress results in an incomplete fall in pH and the meat
remains translucent, i.e. as is found in dark, firm and dry (DFD) pork or
'dark-cutting' beef.
3.5.2.1 Measuring Light Scatter in Meat. It is possible to measure the

light-scattering power of meat by a modification of the Kubelka Munk
analysis (Kubelka, 1948; Judd and Wyszecki, 1975; MacDougall, 1988).
Slices of 2 mm thick meat are mounted on opaque black and white back-
grounds and their reflectance measured. The reflectance values of the white
background, the sample over white and over black are used to determine
the Kubelka Munk scatter and absorption coefficients, Sand K mm- 1,
which are related to reflectance at theoretical infinite thickness, Roo by:
(3.13)
3.6 Meat Colour
The haem pigment myoglobin is responsible for the colour of meat. On

exposure to air the purple ferrous deoxygenated form rapidly oxygenates
on the surface to the brighter red covalent complex oxymyoglobin. During

display this oxidizes to brownish red, then to reddish brown mixtures of
oxymyoglobin and ferric metmyoglobin, and ultimately to brownish green
metmyoglobin (MacDougall, 1982). Some 20% of surface metmyoglobin
is sufficient to cause rejection of the product at retail outlets because of its
dull brownish colour (Hood and Riordon, 1973). Details of the pigment
reactions in fresh, cooked and cured meat are the subjects of chapter 2 of
this book along with the pale, soft and exudative (PSE) and dark, firm
and dry (DFD) conditions.
3.6.1 Measurement procedure

There is no generally recommended procedure for measuring the colour of
meat, poultry or fish because of instrument variability, as previously
described, and the limiting constraints of product availability, sample size
and the particular demands of where the measurements are taken (e.g. in
the laboratory, abattoir or supermarket).
Since most bench-top colorimeters and spectrophotometers have 20-
22 mm apertures, colour measurements under these conditions should
produce similar but not necessarily identical colour values (Kent, 1987;
MacDougall, 1987). However, portable instruments with smaller aper-
tures, typically of lO mm, will give different values because of the wide
range in translucency in the meat that results from differences in the
extent of protein denaturation during post-mortem glycolysis, as affected
by pH and temperature during chilling.
The author has found the following procedure effective in the labora-
tory for simulated supermarket display experiments. The meat should be
sliced at optical infinity. Optical infinity is that thickness that obliterates
the colour of the background on which the product is laid such that there
are no differences in the reflectance spectra when laid on black and white.
This is usually assured at a thickness of lO-15 mm. Since 'optical infinity'
is seldom greater that 6 mm and incursion of subsurface metmyoglobin
from the underside, which could affect upper surface colour, is usually less
than 6 mm, a lO- 15 mm slice is ideal.
When the objective is the routine measurement of bloomed colour, suf-
ficient time should elapse from cutting to measurement. This is accom-
plished by covering the freshly exposed meat surface with oxygen
permeable film and holding at 0-4°C (32-39°F) for 1 h.
Pillowing of the sample into the instrument aperture should be avoided.
Pillowing is caused by excess pressure, which projects the sample surface
forward in a plane nearer the detector than that used for calibration.
During measurement, the operator should note obvious changes or
development of distinct localized patches of colour. If possible, these
should be measured in addition to the overall colour of the slice. Obser-
COLOUR OF MEAT 87
vation should be made under non-distorting lamps with good colour ren-
dering and at an appropriate colour temperature. Fluorescent lamps are
defined by their colour-rendering index. Colour judgment is usually per-
formed with lamps with an index greater than 90% at a colour tempera-
ture of D65 to approximate daylight. It is essential that the operator be
able to distinguish changes in colour and appearance, both from pigment
change and the variation in translucency. Some agreed code might be
(a) 50
40
c:
II)
0 5h
Q;
c- 30
al
0
c:
ra 20
i:)
.!!!
Q;
a: 10
0
350 450 550 650 750
nm
(b) 50 : ......
:#-. ..'
-
c:
II)
40 ../f
;,
.: :
~
II) :........l 1;
c- 30
. ,I
al # - ... ~~
0
c:
ra 20
i:)
II)
Q;
a: 10
0
350 450 550 650 750
nm
Figure 3.1 Reflectance spectra, specular included, of beef semimembranosus muscle and pork
longissimus dorsi muscle, wrapped in oxygen-permeable film, during storage at 4°C. (a) Beef
freshly cut Oh and beef and pork exposed to air for 5 h. (b) Beef after 2 days exposure and
at stage of reddish brown (RB) and brownish green (BG) colour appearance.
Table 3.1 Differences in colour values of beef and pork from CIELAB L *, a *, b*, D 65 , and 10° with change in
illuminant, observer angle, specular inclusion/exclusion, diameter of aperture, and Hunter L, a, b scales
CIELAB Hunter
Specular included
Specular excluded Specular included
1 2 3 4 5 6 7 8
2cm 2 cm 2cm 2cm 1 cm 2cm 2cm (delta)
D 65 , 10° C,lO° D65,2° C,2° D65'lO° D65,10° C,2° C,2°
Beef- L* 48.7 0.0 -0.1 0.0 L 41.6 (-7.1)

Fresh a* 11.2 -0.7 +3.9 +3.0 a 11.8 ( -2.4)
Cut b* 13.6 +0.4 -1.6 -1.1 b 8.8 ( -3.7)
C* 17.6 -0.1 +1.7 +1.3 C 14.7 (-4.2)
h* 50.5 +2.6 -12.0 -9.1 h 36.7 ( -4.7)
Bloom L* 48.8 -0.1 +0.2 +0.3 L 42.0 (-7.1)

5h a* 18.9 -0.9 +3.6 +2.5 a 18.2 ( -3.2)
b* 19.9 +0.3 -1.1 -0.6 b 12.7 ( -6.6)
C* 27.4 -0.3 +1.9 +1.4 C 22.2 ( -6.6)
h* 46.5 +1.8 -6.7 -4.5 h 34.9 (-7.1)
Exposed L* 49.2 +0.1 +0.2 +0.3 -3.8 -1.0 L 42.4 (-7.1)

1 day a* 19.8 -0.9 +3.2 +2.5 -7.2 +0.1 a 19.0 (-3.3)
b* 20.3 +0.4 -1.1 -0.5 -4.6 +0.3 b 13.1 (-6.7)
C* 28.4 -0.4 +1.9 +1.4 -8.3 +0.2 C 23.1 (-6.7)
h* 45.7 +1.9 -5.3 -4.1 +5.5 +0.3 h 34.6 (-7.0)
, Red-brown L* 47.6 +0.1 +0.2 +0.3 L 40.9 ( -7.0)
a* 14.7 -0.7 +2.7 +1.8 a 13.6 ( -2.9)
b* 15.8 +0.3 -1.1 -0.4 b 10.4 ( -5.0)
C* 21.5 -0.2 +1.4 +1.1 C 17.1 ( -5.5)
h* 47.1 + 1.8 -6.6 -4.1 h 37.4 ( -7.6)
Brown-green L* 48.7 0.0 +0.3 +0.4 L 42.0 (-7.1)

a* 7.5 -0.7 +0.9 +0.1 a 6.1 ( -1.5)
b* 14.5 +0.4 -0.7 -0.2 b 9.9 ( -4.4)
C* 16.3 +0.1 -0.1 -0.1 C 11.6 ( -5.6)
h* 62.7 +3.2 -4.0 -0.7 h 58.4 (-3.6)
Pork L* 56.7 0.0 +0.1 +0.1 -6.4 -2.0 L 49.7 (-7.1)

1 Day a* 7.6 -0.7 +2.7 +1.9 -5.1 0.0 a 8.1 ( -1.4)
b* 16.7 +0.4 -1.5 -1.0 -6.6 -0.8 b 11.5 ( -4.2)
C* 17.9 +0.5 +0.5 +0.4 -7.5 -0.3 C 14.1 ( -4.2)
h* 64.9 +3.1 -9.0 -6.1 +11.2 -0.5 h 54.8 ( -4.0)
aMeasur,ed values, specular included, for CIELAB, 2 cm aperture, D 65 , and 10° observer in column 1 and for Hunter,
2 cm, C and 2° observer in column 7.
bColumns 2, 3 and 4 are differences from column 1 for 2 cm, specular included conditions. Differences between
illuminants, D65 and C are less than between 10° and 2° observers.
cColumn 5: differences between 1 cm and 2 cm apertures are greater than between observers.
dColumn 6: differences between specular included and excluded are low for chromaticness (a*, b*) but lightness (L *)
is lower for specular excluded conditions.
eColumn 8: differences between conditions normal for Hunter and equivalent conditions for CIELAB (column 4) are
of same order as the large differences owing to aperture size.
incorporated into the report that allows visual description of the devel-
opment of discoloured areas, which are too small to be measured
directly. This is particularly important when conducting colour-stability
studies.
The number of replications required for assurance depends on several
factors. If an overall mean of the colour values is required to describe a
surface, replication should be such that much of the available surface is
sampled. Therefore, for beef longissimus dorsi muscle and a 20 mm
aperture, four to six adjacent areas across the surface should suffice. The
number of slices available as replicates is usually determined by the lim-
itations of the experiment. Ideally at least two and preferably four areas
should be measured.
3.6.2 Reflectance spectral changes in meat

Reflectance spectra of fresh beef during pigment oxygenation and oxida-
tion will be used to illustrate some of the difficulties in measuring and
interpreting meat colour spectra. The outer portion of a beef semimem-
branosus muscle aged for more than 7 days, with a normal ultimate pH of
5.6, was cut into four 1-1.5 cm thick slices and wrapped in highly oxygen-
permeable film and stored at 4°C. Spectra were measured on each slice at
four locations over a period of 10 days on a Hunterlab Color Quest pho-
todiode array spectrophotometer fitted with an integrating sphere and
choices of including or excluding the specular component and diameter of
viewing aperture of either 1 cm or 2 cm. Reflectance was sampled at
10 nm intervals from 400-710 nm. Mean spectra at five stages of pigment
reaction are shown in Figure 3.1 a and 3.1 b and the colour values
compared with CIELAB D 65 , 10° in Table 3.1 for several geometric con-
ditions and Hunter colour space. A sample of similarly treated pork held
for 1 day is also compared.
On oxygenation of freshly cut meat, there is a reduction in reflectance
at approximately 480 nm and a distinct increase in the red part of the
visual spectrum between 600 and 700 nm. The change in colour as oxy-
myoglobin oxidizes to metmyoglobin is caused by increased reflectance in
the green region of the spectrum, with an accompanying loss in reflectance
in the red region as the ferric metmyoglobin absorption band intensifies at
630 nm (Figure 3.1a and 3.1b). The sp!!ctra are typical of predominantly
reduced myoglobin after cutting and oxymyoglobin after 5 hand 1 day
exposure to air. They appear dull brownish-red after 4 days and total
brownish-green metmyoglobin after 10 days.
The calculated colour values (Table 3.1) for the various sample condi-
tions and measurement procedures are presented as differences from the
data at CIELAB D65 and 10° angle of view and 2 cm sphere aperture.
This illustrates the confusion that can arise in relating values obtained
QUALITY ATTRIBUTES IN MEAT, POULTRY AND FISH PRODUCTS 91
from one measurement condition to another unless conditions are stated

precisely and the instrument type specified (Kent, 1987; MacDougall,
1987).
3.6.3 Colour changes in beef

Within the CIELAB system, the effect of change with time for beef is seen
clearly in the interpretive parameters chroma, C*, and hue angle, h*
(Figure 3.2). The major colour change from beef freshly cut to bloomed at
5 h is a large increase in C*, which continues until oxygenation is nearly
complete as shown for 1 day. The change towards the visual recognition
of red brown rather than bright red is a reduction in C* with h* begin-
ning to increase. The increase in h* continues with further reduction in C*
until the colour finally becomes distinctly green.
The changes in calculated colour values from D65 to source C are con-
siderably less than those from 10° to 2°. Much more important is the
change from reduction in aperture diameter from 2 cm to I cm, accounted
for by the masking of the halo effect of translucence at the smaller
diameter (MacDougall, 1987).
3.6.4 Differences between CIELAB and Hunter scales

The differences between CIELAB and Hunter scales are as expected
because of the concepts used in constructing the formulae. Nevertheless,
70
BG
60
.c
...0 50 5h , 1d
~
~
40
30
10 20 30
C· or C
Figure 3.2 Relationship of change in chroma (C* or C) to hue angle (h* or h) of beef semi-
membranosus muscle, wrapped in oxygen-permeable film, during storage at 4°C. Data pre-
sented in both CIELAB (C*, h*) (e) and Hunter scales (C, h) (.) for source C and 2°
observation. RB = reddish brown; BG = brownish green colour appearance.
92 COLOUR OF MEAT
scrutiny of the delta source C, 2° differences between the two scales,

shows that the values are essentially parallel (Figure 3.2). Thus, although
both scales show the same directionality in the information portrayed, the
magnitude of the differences between the scales is such that equal if not
greater care must be exercised in their interpretation as for differences
caused by variation in aperture diameter within and among instruments.
3.7 Summary
The sensation of colour varies with the observer's vision and the quality
and intensity of light, as well as with the intrinsic chemical and physical
properties of the objects in the area viewed. The measurement of colour,
therefore, requires that these variables be controlled. The human visual
system is reviewed and the procedures developed by the International
Commission on Illumination (CIE) to measure colour are described. The
two major types of instruments used in colour measurement, spectro-
photometers and tristimulus colorimeters are compared and their limita-
tions discussed. Although reflectance spectra are the source of the colour
sensation, other factors affect object appearance, especially variation in
opacity or translucence. This is particularly important when measuring
meat colour because pigmentation and light scatter interact to affect
colour appearance.
No recognized method for measuring meat colour has been established.
The general principles of sample preparation and the need for operator
experience are discussed. To illustrate some of the problems in measuring
meat colour and interpreting the data, the changes in the reflectance
spectra of beef semimembranous muscle during storage were examined.
The calculated colour value coordinates were translated to the more easily
interpreted psychological values of lightness, hue angle and chroma. These
were related to the visual description as the colour changed during storage
from the initial purple of myoglobin to the bright red of oxymyoglobin to
the reddish and greenish brown upon oxidation to produce metmyoglobin.
The effects of instrument parameters on these values were calculated and
aperture size was shown to be equally as important as a source of poten-
tial error and confusion as the differences in values between the two most
used colour scales, the Hunter L, a, b and the newer CIELAB L*, a*, b*
uniform colour space.
References
ASTM (1987) Standards on Color and Appearance Measurement, American Society for
Testing and Materials, Philadelphia.
COLOUR OF MEAT 93
Bartleson, c.J. (1979a) Changes in color appearance with variations in chromatic adaption.
Color Res. Applic. 4, 119.
Bartleson, C.J. (l979b) Predicting corresponding colors with changes in adaptation. Color
Res. Applic. 4, 143.
Best, R.P. (1987) Computer match prediction - pigments, in Colour Physics for Industry (ed.
R. McDonald), Society of Dyers and Colourists, Bradford, pp. 186-210.
Billmeyer, F.W. and Saltzman, M. (1981) Principles of Color Technology, 2nd edn., J. Wiley
and Sons, New York.
Boynton, R.M. (1979) Human Color Vision., Holt, Rinehart and Winston, New York.
Brill, M.H. and West, G. (1986) Chromatic adaptation and colour constancy; a possible
dichotomy. Color Res. Applic. 11, 196.
CIE (1986) Colorimetry, 2nd edn., CIE Publications No. 15.2 Commission Intemationale de
I'Eciairage, Vienna.
Drabkin, D.L. (1950) The distribution of the chromoproteins, haemoglobin, myoglobin,
cytochrome c, in the tissues of different species, and the relationship of the total content of
each chromoprotein to body mass. J. Bioi. Chem. 182, 317.
Estevez, O. (1982) A better colorimetric standard observer for color-vision studies. The Stiles
and Burch 2° color-matching functions. Color Res. Applic. 7, 131.
Hood, D.E. and Riordan, E.B. (1973) Discolouration in pre-packaged beef: Measurement by
reflectance spectrophotometry and shopper discrimination. J. Food Technol. 8, 333.
Homsey, H.C. (1956) The colour of cooked cured pork. J. Sci. Food Agric. 7, 534.
Hunt, R.W.G. (1977) Specification of colour appearance. Effects of changes in viewing con-
ditions. Color Res. Applic. 2, 109.
Hunt, R.W.G. (1978) Color terminology. Color Res. Applic. 3, 79.
Hunt, R.W.G. (1987) Measuring Colour, Ellis Horwood, Chichester.
Hunter, R.S. (1958) Photoelectric color difference meter. J. Optical Soc. America 48, 985.
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Wiley and Sons, New York.
Kent, M. (1987) Collaborative measurements on the colour of light-scattering foods. In
Physical Properties of Foods-2 (Proc. Final Seminar COST), Elsevier Applied Science Pub-
lishers, London, pp. 277-94.
Krzywicki, K. (1979) Assessment of relative content of myoglobin, oxymyoglobin and met-
myoglobin at the surface of beef. Meat Sci. 3, I.
Kubelka, P. (1948) New contributions to the optics of intensely light scattering materials. J.
Optic. Soc. Amer. 38, 448.
MacDougall, D.B. (1982) Changes in colour and opacity of meat. Food Chem. 9, 75.
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Physical Properties of Foods-2 (Proc. Final Sem. of COST), Elsevier, London, pp. 319-30.
MacDougall, D.B. (1988) Colour vision and appearance measurement, in Sensory Analysis of
Foods (ed. J.R. Piggott), Elsevier Applied Science, London, pp. 103-30.
McLaren, K. (1986) The Colour Science of Dyes and Pigments, 2nd edn., Adam Hilger,
Bristol.
Patterson, D. (1987) Instruments for the measurement of the colour of transparent and
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Colourists, Bradford, pp. 35-62.
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McDonald), Society of Dyers and Colourists, Bradford, pp. 63-96.
Robertson, A.R. (1977) The CIE 1976 color-difference formulae. Color Res. Applic, 2, 7.
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4 Juiciness - its importance and some contributing
factors
R.l. WINGER and C.l. HAGYARD
4.1 Introduction
Meat juiciness is an important contributor to eating quality and also plays

a key role in meat texture (Szczesniak, 1963; Jowitt, 1974; Dransfield et
al., 1984a; Hutchings and Lillford, 1988), contributing between 10% and
40% to its variability. Unlike other key aspects of texture, juiciness
remains a uniquely subjective property. The relationship between 'sub-
jective' juiciness of meat and any objective measurement remains elusive
and poorly understood (Hamm, 1960).
The problems related to the measurement of meat juiciness and the
complexity of interpreting sensory data collected by a myriad of methods
from a wide variety of meat sources are discussed in this chapter. The dis-
cussion leads to a summary of the impact of animal characteristics, post-
mortem development of rigor mortis, meat processing, handling and
storage on meat juiciness. The authors believe this to be the first compre-
hensive review of meat juiciness, and it particularly focuses on red meat,
pork and poultry. The exclusion of fish juiciness in this chapter is not an
oversight but rather a recognition that factors impacting upon fish juici-
ness do not appear to overlap significantly with red meat, pork and
poultry factors. As such, fish should merit a chapter in its own right.
4.2 Subjective assessment of juiciness
The only reliable and consistent measure of juiciness is achieved using

sensory methods. Early researchers suggested that meat juiciness could be
separated into two components. The first is the impression of wetness
during the first few chews, produced by the rapid release of meat fluids,
while the second is one of sustained juiciness, apparently due to the sti-
mulating effect of fat on salivary flow and the coating of fat on the
tongue, teeth and other parts of the mouth (Gaddis et aI., 1950; Weir,
1960; Dikeman, 1987). Therefore, early sensory research tended to assess
juiciness using a two-component assessment (initial wetness and sustained
juiciness). With the advent of sophisticated multicomponent evaluation
and multivariate analysis of sensory attributes, it is now established that
JUICINESS 95
initial wetness and overall juiciness can be combined into one factor
(Harries et aI., 1972; Dransfield et al., 1984a). Modern sensory techniques,
therefore, usually measure juiciness as a single attribute.
Despite modifications in sensory methods and a better understanding of
mastication, meat juiciness is still considered to arise from two sources: (i)
moisture released by meat during chewing, and (ii) moisture from saliva
(Harries and MacFie, 1976; Harris, 1976; Horsfield & Taylor, 1976;
Howard, 1976; Gullett et aI., 1984). Thus, 'juiciness' is influenced not only
by meat-related factors, but also by physiological factors inherent within
individual tasters.
The complex sensory experiences that occur during texture and juiciness
testing, from both a perceptual perspective (i.e. what a panelist perceives
as juiciness) and a physical perspective (i.e. applied forces, deformation
rates, viscosity in the mouth) have been reviewed by Christensen (1984).
The review clearly shows that good correlations between complex sensory
attributes (such as juiciness) and objective measurements using either raw
or cooked meat will be fortuitous unless the masticatory experience is
taken into account. Attempts to model juiciness have found it to be highly
complex. One of the best models is a three-dimensional one, involving the
effect of 'time' in the mouth on the 'degree of structure' and the 'degree of
lubrication'. All these variables are needed to allow the model the greatest
flexibility as shown in Figure 4.1 (Hutchings and Lillford, 1988). This is a
Degree of
stru cture
degree of structure
reauced enough to
s'oIaliow plone
lubricated enou gh
to swo l!O\oI plane
Figure 4.1 The mouth process model with special emphasis on juiciness of foods. (Rep-
rinted from Hutchings and Lillford (1988), with permission of the Food and Nutrition Press
Inc. USA) I, Tender juicy steak; 2, tough dry meat; 3, dry sponge cake; 4, oyster; 5 liquids.
three-dimensional model of the effect of time of chewing, degree of food

structure and degree of lubrication. The axes are dimensionless because
this figure relates to a wide range of food products. The model proposes
the degree of structure must be below a certain level (plane ABCD) before
food can be swallowed. Similarly the food must be lubricated above a
certain level (plane EFGH). Eating a juicy steak is exemplified in curve 1,
while curve 2 represents a tougher, dry steak. On chewing a tender juicy
steak, the degree of lubrication is quickly increased above plane EFGH
but it takes longer to reduce the degree of structure sufficient to swallow
the meat. With tougher, drier meats the degree of structure is rapidly
reduced but the meat is not adequately lubricated for an additional pro-
longed chewing time. In some meats (e.g. overcooked chicken), by the
time the meat is properly lubricated, its degree of structure is too low to
swallow. The consumer needs more time to increase the degree of struc-
ture (e.g. make a bolus) before swallowing.
Differences in subjective measurements of juiciness often occur. These
differences are compounded by the lack of a universal 'standard' that can
be used to calibrate the various research groups. Other key textural attri-
butes of meat, such as tenderness, have objective standards. Various end-
point cooking temperatures have been recommended for varying the juici-
ness of samples during panelist training (Cross et al., 1978). Although this
technique has proven valuable for panel training, it does not allow stan-
dardization among research groups. As observed by Gaddis et al. (1950),
juiciness is an extremely complex parameter and much disagreement in the
literature must be caused by differences in interpretation of juiciness.
Despite over 40 years of progress and sophistication in sensory research,
the statement remains relevant today.
One further consideration is worthy of note. Juiciness is often con-
sidered to be a relatively minor component of the eating quality of meat,
being third in importance behind tenderness and flavour. Most researchers
have devised experiments, which have had a significant impact on the first
two components, but simply attempted to measure juiciness as part of the
sensory techniques. Thus, there are relatively few published articles where
the primary objective was the modification or control of juiciness.
4.3 Relationship to objective measurements
The results of studies comparing subjective measurements of juiciness to

measures of water-holding capacity or to quantitative or qualitative
measures of muscle fluid are contradictory. Some studies suggest close
relationships (Tannor et al., 1943; Hardy and Noble, 1945; Bouton et aI.,
1975; Ackerman et aI., 1981; Jones et al., 1985), whereas others show little
relationship (Sartorius and Child, 1938; Lowe, 1948; Gaddis et aI., 1950;
JUICINESS 97
Hamm, 1960; Cover et al., 1962; Ritchey and Hostetler, 1964; Harries et
al., 1972). For further review on this subject readers should read chapter 5
of this book.
4.3.1 Juiciness vs. water-holding

It is not surprising such contradiction exists. Highly significant differences
have been observed among muscles and between replicate trials when
comparing juiciness with percentage moisture (Lowe, 1948; Gullett et aI.,
1984). Overall results suggest neither a consistent nor a significant rela-
tionship. Gaddis et al. (1950) demonstrated that juiciness and the amount
of press fluid were not the same. Hamm (1960) stated that the relationship
between the water-holding capacity of meat and its juiciness required
critical measurement of water-holding capacity of raw meat, the amount
of water released on cooking, and the water-holding capacity of cooked
meat compared with juiciness scores. Such studies had not been made up
to 1960 and remain inconclusive to date.
4.3.2 The state of water

The use of nuclear magnetic resonance (NMR) techniques to measure the
state of water in meat may help to elucidate our understanding of juici-
ness (Pearson et al., 1974). Renou et al. (1985), Fjelkner-Modig and
Tornberg (1986) and Borisova and Oreshkin (1992) have discussed the
condition of water in pork. The interpretation of these data with regard to
juiciness is in its infancy and further work is required but NMR may
provide valuable insights to the subtle changes that are occurring with the
water in meat, especially with regard to juiciness.
4.3.3 Heating method and end-point temperature vs. juiciness

The impact of heating method and maximum end-point temperature
during cooking on juiciness is well described (Lowe, 1948; Clark et aI.,
1955; Cover et al., 1957; Hamm, 1960; Laakkonen, 1973; Cross et al.,
1978; Penfield et aI., 1989). Differences in cooking procedures among
researchers make comparisons difficult.
4.3.4 The role of fat

There is evidence that fat plays an important role in perceived juiciness.
Fat adds flavour to meat and this stimulates saliva flow (Gaddis et al.,
1950). Yet in bland meat, juiciness is the main attribute of consumer
acceptability (Gullett et al., 1984). Different fat levels have been shown to
play an important role in juiciness and acceptability (Cross and Stanfield,
1976; Cross et aI., 1980; Berry et aI., 1985; Savell et al., 1987; Penfield et
aI., 1989). However, other researchers suggest that this relationship is not
significant (Marriott et al., 1988).
4.3.5 Relationship between drip losses and juiciness

The relationship between drip losses and juiciness is conflicting. Some
authors suggest a close correlation (Bramblett and Vail, 1964; Bouton et
al., 1975; Kemp et aI., 1976; Bowers et al., 1987; Rhee et al., 1990), while
others have found the lack of a relationship (Gaddis et al., 1950; Searcy et
al., 1969; Minks and Stringer, 1972; Bouton et al., 1973; Fox et aI., 1980;
Griffin et al., 1981). Interestingly, Bouton et al. (1973) found no relation-
ship between drip loss and juiciness in beef muscle as affected by pH
ranging from pH 5.3 to almost 7.0. This result was in contrast to a later
report from this group (Bouton et al., 1975) indicating a relationship
between drip losses and juiciness.
4.3.6 Relationship between press fluid and juiciness

Gullett et al. (1984) found the relationship between press fluid and juici-
ness was insignificant for beef longissimus dorsi muscle but was significant
for beef semitendinosus muscle (p < 0.05) and highly correlated (p <
0.001) for pork and lamb muscles. Again, this shows the complexity of
comparisons as affected by muscle type or animal characteristics. The
conflicting information correlates closely to the difficulty in establishing a
consistent link between juiciness and any other objective measure.
4.4 Factors influencing the juiciness of intact meat
4.4.1 Interference from other experimental and textural factors

In the statistical testing of a hypothesis, it is usual for the scientific
method to focus on one, or a few variables while controlling as many of
the others as possible. This is especially important in 'analytical' sensory
evaluation (Lawless, 1991). In order to study juiciness of meat, one ideally
should minimize the variation in other attributes, such as tenderness,
flavour and colour (Cross et al., 1978). In reality, however, this is difficult
to achieve. It has been well-documented that tenderness is often related
positively to juiciness in some instances but negatively in others (Cover et
al., 1962). Furthermore, most studies have not tended to focus on juiciness
per se. The combination of these aspects of the scientific method have
resulted in a complex array of contradictory reports.
In addition, the subjective nature of juiciness demands a thorough
JUICINESS 99
understanding and proper manipulation of the perception of texture by

each human panellist. As stated by Christensen (1984), 'Food texture per-
ception spans several different disciplines including physiology, engineer-
ing, psychology and food science; thus, an appreciation of each of these
fields is a prerequisite to conducting valid research in this area'. There are,
however, sound psychophysical approaches to understanding the range
and effects of physical variables on sensory perception (Lawless, 1991).
Further understanding of the psychology and physiology of flavour and
aroma is presented in chapter 6.
The use of computers and multivariate analysis has heralded an era of
sophistication in data analysis. However, these analyses do not supersede
employing the scientific method. Many papers published do not contain
enough methodological information for the scientific appropriateness of
the trial to be assessed. Consequently, relating the results between differ-
ent reports is often difficult.
Early researchers were unable to manipulate reliably tenderness, flavour
and other meat characteristics because techniques to control these vari-
ables consistently have been available only in the last 20 years. Thus, trials
tended to give results that were confounded by variations in tenderness,
flavour and other attributes. In addition, cooking procedures and taste-
panel techniques have varied among research groups.
There are other factors that need to be considered for studies on juici-
ness. These include sample size, sample-presentation temperature, and
handling from end-of-cooking to sample presentation. These factors are
all examples of experimental conditions that are very difficult to control
but are nevertheless important for consistency of juiciness measurements.
For example, Berry et al. (1980) found significant differences in juiciness if
cubes of meat were tasted, but no differences if thin slices were used. Patty
size can also play an important role in beef patty juiciness (Cross and
Berry, 1980).
4.4.2 Heating/cooking methods

4.4.2.1 Temperature gradients. Cooking methods have a distinct impact
on perceived juiciness, which has been discussed by Paul (1975). In eva-
luation of meat cookery, it is important to recognize that the rate and
extent of temperature rise in the sample being cooked represents a con-
tinuum of temperature from that of the external heating source (e.g. oven)
to the centre of the meat being cooked. Laakkonen (1973) has reviewed
the factors related to cooking on meat quality. This is evident when con-
sidering the cooking of a loin chop compared with the entire loin. Tem-
perature penetration of a loin chop is from its cut edges to the centre. The
temperature profile will be relatively flat, and cooking time would be
short. In an entire loin, however, the temperature profile is at right angles
to that of the chop. The temperature profile will be quite steep and there
may be a difference of 40-50°C between the outer edge and the centre
temperatures as shown by Laakkonen (1973). The cooking time for the
entire loin will be relatively long compared with the chop. Mathematical
modelling of these temperature gradients, with respect to juiciness
changes, has not been performed.
4.4.2.2 End-point temperature. End-point temperature is an important

determinant of juiciness, and has been proposed as a method for obtain-
ing meat of different juiciness levels for taste-panel training (Cross et al.,
1978). 'Rare' steaks (end-point of 60°C) were more juicy than 'well done'
steaks (end-point of 80°C), which were more juicy than steaks cooked to
100°C (Cover et al., 1962; Parrish et al., 1973). Bowers et al. (1987) found
that juiciness decreased with increasing internal temperatures from 55-
85°C. The decrease in juiciness occurred at two temperature increments;
the first one between 60°C and 65°C, and the second between 80°C and
85°C. Results are similar for beef (Ritchey and Hostetler, 1964; Martens
et al., 1982), lamb (Woodhams & Mathews, 1965), and pork (Simmons et
al., 1985; Dikeman, 1987; Heymann et al., 1990).
4.4.2.3 Rate of heating. Whether the rate of heating affects juiciness is

more controversial. Pressure cooking beef to an end-point temperature of
112°C resulted in very dry meat compared with beef that had been either
braised or pressure-cooked to an internal temperature of 80°C as found
by Clark et al. (1955). In contrast, pressure cooking or braising beef to the
same internal end-point (80°C) resulted in meat of equal juiciness. These
results suggest that the rate of heating does not affect juiciness, provided
that a constant internal end-point is achieved. Similar results were found
for pork by Bennett et al. (1973) and Flynn and Bramblett (1975). Pork
chops cooked by oven broiling to an end-point of 77°C (average 48.9 min
to cook) had the same juiciness as chops that were deep-fat-fried to the
same end-point temperature (13.5 min to cook). No differences on meat
juiciness have been found between microwave cooking and roasting of
beef muscles to a constant end-point (Griffin et ai., 1981; Ray et al.,
1985).
4.4.2.4 Effects of heating to an end-point temperature. In a study on dif-

ferent methods of cooking to the same end-point (70°C), Schock et al.
(1970) found significant differences in juiciness among oven roasting
(most juicy, score 5.8), deep fat frying and pressure cooking (least juicy,
scores 5.0 and 4.5, respectively) and oven braising (score 5.3). These dif-
ferences related closely to rates of heating, with pressure cooking and
deep fat frying being the fastest, and oven roasting and braising being the
slowest.
JUICINESS 101
Although most researchers cook to a constant end-point (e.g. 70°C), it

appears this often represents the internal temperature at the end of
cooking. In that case, there would be a continuing increase in cooking
temperature after removal of the meat from the cooking medium and that
increase in temperature would be greatest for the more rapidly heated
samples (Paul, 1975). Thus some results may reflect differences in final
internal temperature reached. However, in a carefully controlled experi-
ment comparing a high rate of heating (0.60°C min-I) to a low rate
(0.12°C min-I), Appel and Lofqvist (1978) cooked hams to an identical
product thermal gradient and end-point internal temperature. The slow
heating method produced hams with significantly improved juiciness.
4.4.2.5 Microwave cooking. Microwave cooking offers a four to five

times faster cooking method for meat compared with a conventional oven.
Moore et al. (1980) found microwave cooking resulted in significantly
drier beef round steaks compared with conventional oven roasting. There
was, however, no difference in juiciness between moist and dry-heat
cooking of meat in either the microwave or conventional ovens. Ray et al.
(1985) found no measureable juiciness differences between microwave-
cooked and roasted lamb.
4.4.2.6 Slow vs. rapid cooking. Bramblett and Vail (1964) studied the
effect of different cooking methods on juiciness of beef adductor, biceps
femoris, gracilis, semimembranosus and semitendinosus muscles. Samples
were wrapped in aluminium foil and roasted to an internal temperature of
65°C in ovens set at either 68°C or 93°C (155°F and 200°F, respectively).
Meat cooked quickly (93°C oven) was more juicy than meat cooked
slowly.
Different types of cooking are also important, although this may relate
to rate of heat penetration. Woodhams and Mathews (1965) found that
lamb neck slices were less juicy when cooked in aluminium foil than by
conventional braising. Dransfield et al. (1984a) in an experiment compar-
ing 13 assessors and 66 meats from different species, muscles and cooking
treatments found that grilled beef was very juicy, whereas waterbath-
heated beef, bacon and chicken were among the driest meats. In a com-
parison between roasting (175°C oven to an internal end-point of 70°C)
and broiling (275°C to an internal end-point of 70°C), Cross et al. (1979a)
found beef longissimus muscles to be significantly more juicy when roasted
(slower heating rates). This was confirmed later by Bowers et al. (1987).
Noble et al. (1990) found juiciness differences in restructured roasts
cooked in different types of institutional oven (e.g. conduction, convec-
tion, low-temperature cook-and-hold with or without humidity control).
The relationship to cooking rates was not clear, although end-point tem-
peratures were constant at 71°C.
4.4.2.7 Configuration during cooking. Even the configuration of cooking

can produce significant differences in juiciness. McCready and Mitchell
(1969b) found significant differences between turkeys roasted with their
breast up compared with those roasted with their breast down. Moisture
contents of pectoralis major and pectoralis minor muscles were about 1%
higher in birds cooked with their breast down. The reasons for these dif-
ferences were not postulated. The impact of cooking configuration on
meat juiciness has not received attention since this report.
4.4.2.8 Effect of removal of fat cover before cooking. Cooking beef strip-
loins, top round roasts (semimembranosus), eye of round roasts and arm
pot roasts with the natural external fat cover, or following excision of the
fat cover prior to cooking had no influence on the cooked meat juiciness.
However, briskets showed a significant reduction in juiciness when braised
without the fat cover (Coleman et al., 1988). Rhee et al. (1990) found a
highly significant reduction in juiciness for broiled lamb loin chops if the
subcutaneous fat layer and epimysium were removed prior to cooking.
4.4.3 Animal characteristics

4.4.3.1 Variation among muscles. Different muscles from the same
animal can produce different juiciness values when they are cooked under
the same conditions. Paul et al. (1950) found highly significant differences
in juiciness among eight different beef cuts using roasting or oven broiling.
Cover et al. (1962) found that the biceps femoris and longissimus dorsi
muscles from beef animals exhibited similar trends in juiciness when
cooked from 61°C to 100°C but that the pattern and relative degree of
juiciness changes were different between the muscles. Bramblett and Vail
(1964) found highly significant juiciness differences among five muscles of
beef animals, when all were roasted to an end-point of 65°C. Miller et al.
(1987a) found a difference in beef longissimus dorsi and semitendinosus
muscles but it is not known whether this difference was significant. Fox et
al. (1980) and Heymann et al. (1990) found significant juiciness differences
among various pork muscles and chops, while Paterson and Parrish (1986)
found significant differences among nine muscles from the beef chuck. Jui-
ciness of beef rib steaks was found to be greater than that of loin steaks
(Parrish et al., 1991).
4.4.3.2 Effects of the age of the animal. Animal age had no significant
impact on lamb juiciness for animals ranging from 17-27 weeks of age
(Woodhams et aI., 1966). There was also no impact of bovine animal age
on meat juiciness, i.e. 12 vs. 24 months (Arthaud et al., 1977), 9 vs. 18
months (Riley et al., 1986), or 18 vs. 54 months (Powell, 1991). Con-
versely, Paul et al. (1964a) found that cuts from lambs of 11-12 months
JUICINESS 103
of age were generally more juicy than cuts from 5 Y2-month-01d animals.
'Senior' rabbits (> 1 year of age) are less juicy than fryer rabbits (8-10
weeks of age) according to Coppings and Godwin (1990).
4.4.3.3 Influence of breeding. Breed and sire has no influence on juiciness

in beef (Koch et al., 1979; Knapp et al., 1989; Ockerman et aI., 1984;
Riley et al., 1986), or in lamb (Wilcox and Galloway, 1952; Woodhams et
al., 1966). These results indicate that meat from animals of different
breeds and sires is of relatively consistent juiciness provided that the
animals are of similar age and are raised under the same management
conditions and feeding regimes. This was observed even though there were
large breed and sire differences in fat cover, growth rates and degrees of
marbling in these studies.
4.4.3.4 Effects of sex. Some research groups have found no difference in

juiciness between intact males and castrates (Woodhams and Trower,
1965; Bailey et aI., 1966; Field et al., 1966; Champagne et aI., 1969; Field,
1971; Reagan et aI., 1971; Arthaud et al., 1977; Seideman et al., 1982c;
Ockerman et al., 1984; Griffin et al., 1985b), beef animals of different
sexes (Paterson and Parrish, 1986) or pheasants of different sexes
(McCready and Mitchell, 1969a). However, in a study involving nine sets
of twin calves, Jones et al. (1964) found significant, but small differences
in juiciness between bulls and steers. The authors noted these differences
were not sufficiently large to be detected by all tasters, or sufficiently con-
sistent to be detected in all the pairs of animals. Unruh et al. (1987) found
meat from zeranol-implanted steers was more juicy than that from either
zeranol-implanted bulls or control bulls that were grown without zeranbl.
4.4.3.5 Influence of growth promoters. Studies on the use of growth pro-

moters in beef animals indicate various effects on juiciness. Calkins et al.
(1986) found that zeranol implants used on bulls resulted in their long-
issimus dorsi muscles having improved juiciness compared with untreated
bulls. Unruh et al. (1987) did not find this effect but noticed a significant
improvement in juiciness for castrated bulls (steers) grown using zeranol
implants compared with untreated bulls. In a similar study with steers
only, Nute and Dransfield (1984) found no difference in juiciness of meat
from animals grown with or without zeranol.
4.4.3.6 Relationship of marbling and juiciness. The relationship of

marbling to juiciness is unclear. Blumer (1963) suggested that about 16%
of the variance of juiciness was attributable to variation in marbling fat.
Barbella et al. (1939) reported that the juiciness of beef ribs increased
rapidly with an increase in fatness of the 'edible portion' up to 22.5% fat,
more slowly to 42.5% fat and thereafter increasing fatness had no effect.
Cover et al. (1956) found that fatness accounted for less than 30% of the
variation in juiciness of broiled or braised beef steaks. These authors, as
well as Wanderstock and Miller (1948) concluded that there may be some
relationship between the fatness of carcasses and cooked meat juiciness,
but that the juiciness differences were small.
Parrish et al. (1973) and Garcia-de-Siles et al. (1977) found no sig-
nificant influence of intramuscular fat on juiciness of beef longissimus dorsi
muscles. Miller et al. (1987b) studied the impact of pre-finishing diets (low
vs. high energy) on young bulls grown to the same live weight. There were
no differences in hot carcass weights, although the degree of marbling was
lower in those animals fed the low-energy pre-finishing diet. Juiciness was
not affected. In similar studies, Crouse et al. (1986) and Miller et at.
(1987a) evaluated pre-finishing diets and four finishing times on meat
quality of steers. All animals were slaughtered at the same age (20
months). Those animals on high-energy diets had the fattest carcasses,
including the highest marbling scores. There were no differences in juici-
ness scores among all carcass grades, marbling scores or dietary regimens.
Goll et at. (1965) found only limited relationships between marbling,
maturity and juiciness.
4.4.3.7 Effects of production systems and carcass grade factors. In an

extensive collaborative study on beef between the University of Nebraska,
Kansas State University and the US Meat Animal Research Center in
Nebraska, Dikeman et al. (1979) and Koch et al. (1979) studied breed
types and different production systems. Carcasses of various grades were
found to have no significant differences in juiciness. In contrast, feed10t-
raised animals tended to be more juicy than those grown on pasture but
these differences were considered to be 'not very marked' (Paul e t al.,
1964b). Schroeder et at. (1980) found grainfed cattle to be markedly more
juicy than pasture-fed animals. Parrish et at. (1991) found small but sig-
nificant (p < 0.01) juiciness differences between prime, choice and select
grades of beef animals as measured by both a trained and consumer
panel.
Tatum et al. (1980) found meat from the highest-grade carcasses (high-
choice and average-choice) were more juicy than lower-grade carcasses
(low-choice to high-standard) (panel scores 4.83-5.23 vs. 4.51-4.72,
respectively). Dolezal et al. (1982) found beef rib steaks from 'modest and
higher' marbling groups were more juicy than steaks from 'slight-minus'
and lower marbling score groups. More recently, Smith et al. (1984a) and
Savell et al. (1987) found a clear and highly significant relationship
between marbling score and juiciness in beef longissimus muscle. In a
study involving over 1000 beef animals of different animal maturity grades
(USDA maturity grades A, B, C and E), Smith et al. (1984a) found that
meat from A-grade maturity animals having the highest levels of marbling
JUICINESS 105
(scored as 'moderate to moderately abundant'), was the most juicy (juici-

ness rating 5.31-5.52). Meat with the next range of marbling scores
('slight to modest') was significantly less juicy (rating 4.80-4.97), while the
meat in the lean nest range, ('practically devoid to traces') was relatively
dry (rating 4.58-4.73). Similar trends were found for all four animal
maturities studied. The semimembranosus muscles exhibited a similar trend
to the longissimus muscles but the differences were not so significant.
These results on the relationship between marbling and juiciness agree
with the findings of Breidenstein et al. (1968), Gilpin et al. (1965), McBee
& Wiles (1967), Jennings et al. (1978), Cross et al. (1979b) and Griffin et
al. (1985b).
In contrast, work reviewed by Dikeman (1987) suggests that beef
carcass fat thickness (from < 0.64 cm to > 2.54 cm) had no significant
effect on juiciness. These results reflect those found for longissimus and
biceps femoris muscles from lambs with large differences in fatness levels
(Batcher et al., 1962; Smith et al., 1976). In earlier studies, Smith et al.
(1970a,b) suggested that there was a significant trend of increased juiciness
in lamb leg roasts, rib, loin and sirloin steaks from animals exhibiting
increased 'flank streaking scores'. However, this relationship was not
closely correlated to animal maturity groups or USDA grades.
Some of the confusion seen in the literature might be accounted for by
differences in the muscles being used. Browning et al. (1990) studied ten
muscles of eight lean and eight 'typical A maturity' beef carcasses. These
researchers found that only one muscle (semitendinosus) showed significant
differences in juiciness between lean and 'typical' carcasses. None of the
other nine muscles exhibited any difference in juiciness.
4.4.3.8 Influence of growth rate. Rate of animal growth has little impact
on juiciness. In a study comparing growth rates of young bulls, Crouse et
al. (1986) found no significant changes in juiciness among animals
assigned to one of three 30-day or 60-day finishing treatments to produce
negative, zero or positive weight gains.
4.4.4 Factors related to rigor development

4.4.4.1 Pre- and post-rigor effects. There are few reports assessing the
juiciness of intact meat cooked from pre-rigor muscle. Berry et al. (1980)
found no differences in juiciness between pre-rigor and post-rigor semi-
membranosus roasts if they were served as thin slices. However, when
served as cubes, the pre-rigor muscle was significantly less juicy than the
post-rigor muscle.
Ray et al. (1985) compared hot-boned (pre-rigor) and post-rigor lamb
using conventional electric oven roasting, microwave cooking and a com-
bination of both cooking methods. They found no detectable differences
in juiciness scores for either the pre-rigorjpost-rigor comparison, or the

different cooking methods. Using longissimus muscles, Hoes et al. (1980)
and Wu et al. (1990) found no significant juiciness differences between
hot-boned and cold-boned pork, although the hot-boned product was not
cooked from the pre-rigor state.
In the same study, Hoes et al. (1980) found a significant increase in jui-
ciness if pork was injected with a 5% solution of sodium hexametapho-
sphate and sodium pyrophosphate (to 110% of green weight). There was
no significant difference between hot- or cold-processed meat. However,
the meat was cooked after the development of rigor mortis. In contrast,
the use of hot-boned pork for ham manufacture had a profound effect on
juiciness (Troeger and Woltersdorf, 1987). Although the hams were eval-
uated post-rigor, they were pumped and cooked while still in the pre-rigor
state. Pre-rigor prepared hams, as well as the individual muscles (i.e.
adductor, semimembranosus, biceps femoris), were significantly more juicy
than the post-rigor products. However, shoulders and loins exhibited no
significant juiciness differences whether prepared pre-rigor or post-rigor.
Hot-boned beef biceps femoris muscles were significantly juicier than
those chilled for 40 h on the carcass prior to boning (Griffin et al., 1981,
1985a). However, there was no significant difference in juiciness between
hot-boned (1 h post-mortem) and conventionally boned (48 h post-
mortem) longissimus dorsi steaks (Cross and Tennent, 1980).
4.4.4.2 Effects of electrical stimulation. Results of studies examining the

effect of electrical stimulation on meat juiciness have been variable. Some
studies suggest no influence of electrical stimulation on juiciness of beef
(Griffin et aI., 1981, 1985a; Schroeder et al., 1982) or of goats (McKeith et
al., 1979). In contrast, Medeiros et al. (1989) found that although elec-
trical stimulation of beef animals had no impact on the juiciness of long-
issimus muscles, it deleteriously affected the juiciness of semimembranosus
muscles from forage-fed animals. Miller et al. (1987b) found a significant
improvement in juiciness of longissimus dorsi muscle from electrically sti-
mulated beef but the difference was small (panel scores 5.4 vs. 5.3 ± 0.4).
Similar results were reported by Powell (1991), who found that electrically
stimulated longissimus dorsi muscles were significantly more juicy than
non-stimulated muscles (p < 0.05). Seideman et al. (1979) found that
electrical stimulation reduced the juiciness of hot-boned longissimus
muscle but did not affect the semimembranosus.
Some differences in results among researchers may relate to the post-sti-
mulation handling of the carcasses and muscles. Reagan & Honikel (1985)
found that pork loin chops from animals that had been electrically stimu-
lated and then held at 7°C for 24 h, were significantly less juicy than those
that had been hot-boned (with or without electrical stimulation), or
controls excised after 24 h at 7°C from animals that had not been elec-
JUICINESS 107
trically stimulated. Unruh et al. (1986) found that low-voltage stimulation

significantly reduced the juiciness of beef longissimus muscles but had no
effect on semimembranosus muscles. Wiley et al. (1988) in a study of the
palatability of ham slices found that electrical stimulation caused a reduc-
tion in juiciness of hot-boned compared with the hot-boned non-stimu-
lated product. Electrical stimulation had no influence on ham juiciness
from conventionally chilled and boned pork carcasses.
4.4.4.3 Influence of altered posture. Tender stretching of carcasses invol-

ving altered posture hanging during rigor development (Bouton and
Harris, 1972; Hostetler et al., 1972), compared with electrical stimulation
caused a significant increase in juiciness of beef longissimus dorsi muscles
but had no impact on semimembranosus steaks (Mitchell et aI., 1991).
4.4.4.4 Effects of blade tenderization. Three studies have found that

blade tenderizing using a commercial machine had no effect on juiciness
(Cordray et al., 1986; Medeiros et aI., 1989; Wheeler et aI., 1990b). When
electrical stimulation and blade tenderizing were used together, however,
the resulting meat was significantly less juicy than for either treatment
alone (Medeiros et al., 1989). Other researchers, however, have reported a
significant reduction in juiciness following blade tenderizing of beef, lamb
and goat meat (Bowling et al., 1976; Glover et al., 1977; Savell et aI.,
1977). Reasons for these differences are not clear.
4.4.4.5 Influence of fabricating subprimals. Miller et al. (1985) evaluated

the impact on juiciness of fabricating subprimals from beef striploins. In
this study, steaks cut from striploins prior to vacuum-packaging and
storage were compared with those cut after storage (immediately prior to
cooking). The fabricated (cut pre-storage) steaks were significantly less
juicy than steaks cut from intact striploins following 14 days storage at
1-3°C. Rapid post-mortem chilling of beef (using air at - 70 C for 5 h)
D
resulted in loin steaks that were more juicy than conventionally chilled
(-7 C for 24 h) meat (Bowling et al., 1987).
D
4.4.4.6 Effects of PSE and DFD conditions. A common occurrence in

pork processing is the high incidence of PSE and DFD meat. Researchers
have consistently found DFD pork to be significantly more juicy than
either 'normal' pork, or PSE pork. Of the three, when differences were
found, PSE pork invariably scored lowest for juiciness (Lewis et al., 1962;
Kauffman et al., 1964; Sayre et al., 1964; Bennett et al., 1973; Cassens et
al., 1975; Flynn and Bramblett, 1975; Kemp et al., 1976; Topel et aI.,
1976; Fox et aI., 1980; Jeremiah, 1986; Jeremiah et al., 1990). However,
some studies suggest no differences in juiciness between PSE and normal
pork meat (e.g. Searcy et al., 1969). Interestingly, however, Bouton et al.
(1973) found no relationship between ultimate pH (ranging from about

pH 5.3-7.0) and juiciness in beef muscles.
4.4.4.7 Influence of post-rigor ageing. Post-rigor ageing has been reported

to have a limited impact on meat juiciness in beef (Minks and Stringer,
1972; Jennings et al., 1978; Lee et al., 1990; Mitchell et al., 1991; Powell,
1991). In contrast, Wheeler et al. (1990b) found only small decreases in
juiciness of top loin steaks aged at 2°C (13 vs. 20 days) but no difference
in sirloin steaks. Stewart et al. (1945) reported significant reduction in jui-
ciness upon ageing chickens, which was also found by Bouton et al. (1958)
using beef. It is quite possible that the ageing temperature may affect meat
juiciness. McCready & Mitchell (1969a) found ageing pheasants at 3°C
produced meat with much improved juiciness compared with ageing at
18°C. On aseptic storage of chicken, van den Berg et al. (1964) concluded
that juiciness changed dramatically upon ageing but that the degree and
direction of change depended on the temperature. For example, at O°C
juiciness improved on ageing for 1 week.
4.4.4.8 Effects of freezing and thawing. Increasing the length of frozen

storage has been found to increase the amount of thaw loss, drip loss on
cooking, total weight loss and to adversely affect the juiciness of ground
pork stored for 39 weeks at -17°C (Brewer and Harbers, 1991). Similar
deleterious effects of frozen storage on beef juiciness were reported by
Howard and Lawrie (1956) and Law et al. (1967). Stewart et al. (1945)
found similar results for chickens stored up to 79 days at -23.3°C.
In a study involving a range of PSE and DFD pork longissimus dorsi
muscles, however, Jeremiah et al. (1990) found frozen storage at -30°C
for 6 months produced a slightly beneficial effect on juiciness. There are
relatively few reports on meat juiciness and its change during frozen
storage.
4.4.5 Restructured meat

Interest in meat restructuring technology first emerged in the 1940s
(Seideman and Durland, 1983). The 1970s heralded considerable research
on the restructuring of meat products and the advent of modern restruc-
tured meat technology (Mandigo, 1988). Carcasses that were not used for
prime eating meat (e.g. bulls, cows, old animals) and less valuable cuts were
comminuted, especially by flaking, and reassembled into a shape resem-
bling meat from intact muscles. There are advantages in restructuring, such
as improvements in palatability characteristics compared with the original
meat, portion control, reduced cooking losses, and control of components
such as gristle, connective tissue and fat. Red meat restructuring has been
reviewed by Seideman and Durland (1983) and Mandigo (1988).
JUICINESS 109
4.4.5.1 Processing factors. Processing factors associated with the restruc-

turing operation may have an influence on the juiciness of the finished
restructured meat. Particle size achieved by comminution has been found
to have little effect on juiciness (Costello et aI., 1981; Durland et at., 1982;
Seideman et at., 1982b; Noble et at., 1985; Marriott et aI., 1986b, 1987;
Cordray et at., 1989; Liu et al., 1990). However, Chesney et at. (1978)
found a significant improvement in juiciness in restructured pork steaks as
flaked particle size was reduced. Some of these differences may relate to
the combination of muscles used, plate sizes and the shape of the cutting
head as was found by Terrell et al. (1985). These researchers found that
beef knuckles and gracilis muscles ground through a three-hole kidney-
shaped plate were more juicy than bottom and top round muscles that
were hand-cut into cubes before restructuring.
Overmixing of comminuted meat during the reassembly of the product
has been shown to have a deleterious effect on juiciness (Durland et aI.,
1982). However, Noble et al. (1985) found no impact of mixing time on
juiciness. Juiciness was not influenced by mixing and pre blending of red
meat in a vacuum, compared with a non-vacuum (Booren et at., 1981b).
Although manufacture of restructured steaks from different muscles was
found to have no influence on juiciness (Booren et al., 198Ia), Terrell et
al. (1985) found that roasts made from the knuckles and gracilis muscles
were juicier than those made from top and bottom rounds.
4.4.5.2 Effects of cooking procedures. As for all meat products, beef

patties cooked to a high internal temperature (77°C) are less juicy than
those cooked to a lower internal temperature (71°C) (Kregel et al., 1986).
In that study, storage of raw or pre-cooked beef patties for 30 days at
-20°C had no significant influence on the perceived cooked juiciness
levels.
4.4.5.3 Effects of adjuncts. The manufacture of restructured meat allows

the use of adjuncts to modify the properties of the final product. Some of
the adjuncts may have a profound effect on juiciness (Seideman and
Durland, 1983). Salt (sodium chloride) is typically added at 0.75-2% to
restructured beef and pork steaks. Primarily needed to extract the muscle
protein and to assist in rebinding of the meat particles, salt also improves
juiciness (Cross and Stanfield, 1976; Schwartz and Mandigo, 1976; Hand
et al., 1981; Marriott et aI., 1983; Seideman and Durland, 1983; Paterson
and Parrish, 1988; Chen and Trout, 1991). The same is true for restruc-
tured lamb roasts (Brewer et at., 1984) where 1-2% salt resulted in roasts
that were more juicy than those made with 0.5% salt.
Some studies have indicated that sodium tripolyphosphate significantly
increases juiciness, but this effect is only slight compared with the impact
of salt (Schwartz and Mandigo, 1976; Huffman et al., 1981; Seideman and
Durland, 1983; Smith et ai., 1984b; Paterson and Parrish, 1988). Con-
versely, some researchers have found tripolyphosphate to have no sig-
nificant impact on meat juiciness (Cross and Stanfield, 1976).
Sodium chloride can be replaced with KCl, CaCh or MgCh without
any effect on juiciness of restructured beef steaks (Miller et al., 1986a).
Marriott et al. (1986a) found that combinations of NaCl, KCl, sodium
tripolyphosphate and lecithin had no impact on the juiciness of restruc-
tured pork chops. In contrast, infusing hot-boned meat with solutions of
NaCl, KCl, glucose, phosphate and pyrophosphate (in various combina-
tions) resulted in a significant improvement in juiciness (WU et al., 1990).
Wheeler et al. (1990a) found NaCI and KCl were more effective at
increasing beef juiciness than MgCh.
Other adjuncts have been used to improve binding, or textural proper-
ties of restructured meats. Some (e.g. dried milk powder) have little
impact on juiciness, whereas others (e.g. soy isolate) may have a sig-
nificant deleterious effect (Brewer et al., 1984). The incorporation of
mechanically separated meat at levels between 10% and 20% does not
affect juiciness in restructured beef (Miller et al., 1986b) but levels around
30% by weight increase juiciness of restructured lamb roasts (Field et al.,
1984). Liu et al. (1990) added various amounts of connective tissue to
restructured beef steaks. Only at levels of 30% added connective tissue
was there a reduction in juiciness. Chen and Trout (1991) found crude
myosin, whey protein, wheat gluten, soy protein isolate and surimi all
reduced juiciness of restructured beef steaks, compared with intact muscle
or restructured steaks made with NaCI/pyrophosphate.
Hand et al. (1981) studied the importance of vital wheat gluten, soy
isolate and 'flavourings' on restructured beef steak palatability. Replacing
13% of the raw meat with gluten or soy isolate had no effect on juiciness.
However, the addition of 'flavourings' (0.44% NaCl, 0.25% sodium tripo-
lyphosphate and 0.31 % hydrolysed plant protein) resulted in a significant
increase in juiciness of all the steaks (meat only, and also meat with
protein replacers). These data were corroborated by Miller et al. (1986c),
who found additions of 10% and 20% of either textured soy protein, or
vital wheat gluten had no significant influence on restructured beef steak
juiciness.
4.4.5.4 Effects of fat content. Fat content has been found to significantly
affect restructured meat juiciness. Cross and Stanfield (1976) found
consumer acceptability of juiciness of restructured steaks containing 30%
fat to be greater than that of steaks containing 20% fat. Berry et al.
(1985) evaluated steaks containing 10-25% fat. The juiciness of steaks
containing 18% and 22% fat was significantly greater than that of steaks
containing 10% or 14% fat. Similar results were obtained by Penfield et
al. (1989) using steaks with fat levels ranging from 15-25%. In contrast to
JUICINESS 111
these studies, Marriott et al. (1988) found that restructured steaks made
from pre-rigor excised (i.e. hot-boned) beef muscles containing lO%, 15%
or 20% fat all had equivalent juiciness scores.
Fat content plays a key role in the acceptability of beef patties.
Huffman and Egbert (1990) found that the overall consumer acceptability
of beef patties increased from unacceptable at 5% fat, to a peak of
acceptability at 20% fat. Acceptability was highly correlated with flavour.
Others have shown a direct correlation between increasing fat content and
increasing juiciness in beef patties (Kendall et aI., 1974; Cross et al., 1980;
Berry and Leddy, 1984; Kregel et al., 1986; Egbert et al., 1991).
4.4.5.5 Use of fat substitutes. The formulation of consumer acceptable,

low-fat beef patties for McDonald's Corporation in the USA arose from a
research study that developed a clever use of food adjuncts to improve the
juiciness of the patties (Egbert et al., 1991). Beef patties are made from
minced meat and differ from restructured products in that the minced
meat is not reformed into an intact, meat-like product. Texture profile
analysis has shown juiciness to be a very important characteristic of patty
acceptability (Dransfield et al., 1984b; Jones et al., 1985). Egbert et al.
(1991) found patties made conventionally with a low fat content (8%) are
significantly less juicy than those made with 20% fat content (panel scores
4.6 and 5.8 respectively). The newly formulated patties, called 'Au Lean',
contain lO% added water, 0.5% iota-carrageenan, 0.4% encapsulated salt
and 0.2% hydrolysed vegetable protein. The resulting patties had a juici-
ness score of 6.7, which was significantly more juicy than patties made
with 20% fat. The improvement in juiciness was a result of the added
water and carrageenan.
Other fat substitutes have been used to improve meat patty quality.
However, their impact has been variable. Troutt et al. (1992) found 20%
fat beef patties were more juicy (score 5.8) than those containing either
5% or lO% fat (score 5.1) and these in turn were more juicy than 5% or
lO% patties containing Polydextrose (RTM Pfizer, score 4.5), sugarbeet
fiber (score 2.5), and combinations of these two products with oat fiber,
potato starch, and pea fiber (score range 4.0-4.4). Giese (1992) provides a
summary of the current developments in low-fat meat products.
4.4.5.6 Influence of pre-cooked meat.The use of pre-cooked meats in the

formulation of restructured products has a significant, deleterious effect
on final product juiciness (Cash and Carlin, 1968; Seideman et al.,
1982a,b).
4.4.5.7 Hot-boned vs. cold-boned meat. Hot-boning has been found to

have no significant effect on restructured beef or pork steak juiciness
(Seideman et al., 1982a; Marriott et al., 1983; Seman et al., 1987). Juici-
ness of restructured meats can be improved by a pre-rigor high pressure

treatment (Macfarlane, 1973; Berry et aI., 1986). In these studies, pre-rigor
meat was subjected to pressures of over 100 MN m- 2 (15000 lb in- 2) for
about 2 min, at 35°C prior to chilling for rigor development.
Beef patties made from hot-boned meat are considered to possess
superior juiciness to patties made from conventionally processed meat
(Cross et aI., 1979b; Jacobs and Sebranek, 1980). However, Contreras et
al. (1981) found the opposite result, i.e. patties from hot-boned beef were
less juicy than those from conventionally processed beef. No differences
were noted between patties made from hot-boned and conventionally pro-
cessed goat meat (Padda et al., 1988).
4.4.6 Processed meats

The terminology used in this chapter differentiates between restructured
meat and processed meats. The former involves the comminution of meat
and its reforming into analogues of whole-tissue meat. Processed meats
involve the comminution of meats, then recombination with a variety of
other ingredients to produce sausages, and similar products, none of
which bears a direct similarity to the original meat.
4.4.6.1 Relationship of processing to juiciness. Juiciness has been shown

to be one of the key textural attributes related to consumer acceptability
of frankfurters and similar sausages (Lee and Patel, 1984; Beilken et al.,
1990, 1991). Intact meat juiciness is a complex, subjective measure that
does not correlate well with any objective test; however, in comminuted
and reformed products, some objective measures have been found to
relate to subjective juiciness. This is true especially for measures of expres-
sible fluid and moisture (Ackerman et al., 1981; Puolanne and Terrell,
1983; Lee and Patel, 1984; Jones et aI., 1985; Lee et al., 1987; Beilken et
aI., 1991). In contrast, Baker et al. (1968) found no correlation between
sensory and objective tests for juiciness when using chicken frankfurters.
Processed meat products often incorporate various amounts of 'waste
recovery' materials, such as scraps and offals, mechanically deboned meat
and non-skeletal meat animal by-products such as offals, hearts, tongues
etc. Mechanically deboned and mechanically desinewed meat has been
found to have relatively little impact on juiciness of frankfurters (Berry et
aI., 1981) and various ground beef products (Miller et aI., 1986b; Wheeler
et aI., 1990a). However, beef by-products (e.g. heart, tripe, tongue,
weasand) have been incorporated into restructured beef products with sig-
nificant effects on juiciness (Ibarra et aI., 1979).
4.4.6.2 Effects of old animals. The use of older animals (e.g. spent laying
hens, mutton and boner cows) is common in meat processing. Bushwayet
JUICINESS 113
al. (1988) evaluated mutton:old fowl frankfurters against pure beef frank-
furters. Significant differences were found among the beef, 50:50 mix
(mutton:fowl) and 67:33 (mutton:fowl) frankfurters. Beef was the most
dry, while those with the highest proportion of mutton were the most
juicy.
4.4.6.3 Effects of extenders. In an effort to reduce the cost of processed

meat products, various extenders are often used, e.g. flour or meals from
various grains and legumes. In a study where chickpea flour was used to
extend mutton, pork and beef sausages (Verma et aI., 1984), flour sub-
stitution of meat to 40% significantly changed the sUbjective juiciness
measurements of mutton sausages. The juiciness of mutton sausages
decreased when 10% and 20% chickpea flour was incorporated, was the
same as the control (no added flour) when 30% flour was used, and was
more juicy than the control with 40% added flour. There were no juiciness
differences with pork and beef sausages at 30% and 40% added flour
(10% and 20% additions were not evaluated).
Sison et al. (1975) and Sison and Almira (1975a,b) evaluated various
extenders, including camote, cassava, wheat, rice and potato flours, on
sausage quality. No significant effects of these flours on JUlcmess were
found in salami sausages using up to 15% incorporation of flour (Sison et
al., 1975). In fresh sausages, however, there was a tendency for reduced
juiciness as the amount of flour was increased to 15%. For wheat flour,
15% added flour resulted in a significant reduction in juiciness (Sison and
Almira, 1975a). Overall, these differences in juiciness were small, or insig-
nificant. The use of cassava flour and cornstarch in meat loaf formulae
resulted in a significantly drier product compared with products made
using non-fat dry milk and wheat flour (Sison and Almira, 1975b).
4.4.6.4 Influence of pre-rigor meat. The use of pre-rigor meat in sausage

formulations has been promoted for its impact on water-holding capacity,
water binding and fat emulsification (Ramm, 1960). The use of pre-rigor
meat does not appear to improve the juiciness of the sausages, however
(Puo1anne and Terrell, 1983; Abu-Bakar et al., 1989).
4.4.6.5 Effects of fat content. The effect of fat on the juiciness of pro-
cessed meat products has received relatively little study. Lee et al. (1987)
found that juiciness was not influenced by fat content in beef and pork
frankfurters over a range of 16-27% but increased significantly between
27% and 31 % of added fat. Interestingly, the panelists disliked the
increase in juiciness. These researchers concluded that the fat level for
optimum juiciness 'desirability' was 20-27%, though their data on desir-
ability using a trained panel are questionable. Reagan et al. (1983) eval-
uated fat levels ranging from 30-45% in pork sausages. Juiciness was
higher in sausages containing 40% and 45% fat, compared with those con-
taining 30% and 35% fat. These studies suggest that fat levels influence
juiciness, but only to a slight extent since it took over a 10% difference in
fat to elicit any significant variation in juiciness. In a study evaluating 12
types of commercially available sausages in Australia, Beilken et al. (1991)
found a significant relationship between juiciness and fat content.
4.4.6.6 Effects of other adjuncts. Adding water to sausages is a routine

procedure that is facilitated by other adjuncts, such as salt and tripoly-
phosphate. In a study comparing various combinations of fat and water
(15%, 25%, 35% fat with 3% and 13% added water), Ahmed et al.
(1990) found increases in juiciness for both added fat and water. However
the impact of added water (13% vs. 3%) was not consistent over all fat
levels used, with an increase in juiciness seen for 15% and 35% fat but
not 25% fat.
The addition of various salts to processed meats has a similar impact on
juiciness to that exhibited in restructured meats. Sodium chloride is the
primary salt that modifies the juiciness of processed meats. Polypho-
sphates, such as sodium acid pyrophosphate and tripolyphosphate, have
no, or relatively little impact on juiciness of frankfurters (Hargett et aI.,
1980; Puolanne and Terrell, 1983).
Barbut et al. (1988) found that 2.5% NaCl provided optimal juiciness in
turkey frankfurters. In this study, the addition of tripolyphosphate to
frankfurters containing 2.5% NaCl resulted in no change in juiciness.
However, the addition of tripolyphosphate, orthophosphate and acid pyr-
ophosphate to frankfurters with reduced NaCl contents (1.5% and 2%)
resulted in slight but significant improvements in juiciness, as samples
were found to be as juicy as, and in some cases more juicy than, the
control (2.5% NaCl) product.
As for restructured meat, different comminution treatments have been
found to have little, if any impact on juiciness of processed meat products.
Reagan et al. (1983) studied three different grinding treatments: 13 mm +
6.5 mm hole plates; 13 mm + 3.3 mm plates; 13 mm + 6.5 mm +
3.3 mm plates. None influenced juiciness.
4.4.7 Marinaded meat

Marinading meat has been practised for centuries. Traditionally mar-
inades have been used as both a preservation method (using vinegar, wine,
spices and salt) and as a means for improving the palatability of meat.
Gault (1991) in his recent comprehensive review of marinaded meat, sug-
gested that the tenderising influence of acid marination was primarily due
to water absorption and its retention during cooking. Wenham and
Locker (1976) studied the effect of marinading on beef sternomandibularis
JUICINESS 115
and longissimus dorsi muscles. The samples were marinaded for 43 h at

2°C in acetic acid and salt (0.15 M), with or without sucrose (12%). Taste
panels could find no difference in juiciness compared with unmarinaded
products. The only real organoleptic benefits of marinading have been
towards tenderness (Gault, 1991).
4.5 Conclusions
It is clear that juiciness is a poorly understood aspect of the eating quality

of meat. Few researchers have studied factors on intact meat that have a
primary impact on juiciness, as distinct from other organoleptic qualities.
Methods for measuring juiciness, including cooking, sampling and all
aspects of sensory techniques, vary from one research group to the next.
There is no carefully defined methodology accepted for this testing proce-
dure. With intact meat, almost every aspect of factors affecting meat juici-
ness appears to be contradicted by other research publications. This review
was complicated by the observation that most researchers have con-
centrated on controlling or manipulating tenderness or flavour, while juici-
ness has been measured merely as part of the sensory profile. Thus, the
apparent contradictions may be caused by subtle differences in the experi-
mental design, which cannot be evaluated easily from scientific reports.
Processed meat products have received more focused research on juici-
ness. Despite this, there is still some confusion over the real factors that
directly affect juiciness rather than other meat attributes.
The relationship between meat juiciness and objective properties of the
raw and cooked meat have been elusive. Modern objective techniques,
such as NMR spectroscopy, may provide a different insight to water and
fat properties of cooked meat; this may bridge the gap. However, such
work requires a careful consideration of the masticatory process: what is
happening in the mouth and how juiciness relates to such factors as saliva
release. There is an opportunity for some ingenious research into the rates
and quality of saliva release, rheological properties of meat in the mouth
and the psychological perception of juiciness.
The importance of juiciness to the consumer is also poorly understood.
There are several interesting observations to suggest that meat juiciness
may be important in some instances. Our modern technology is able to
produce consistently meat of acceptable tenderness, a success that reflects
nearly 30 years research on meat tenderness. If meat is consistently and
acceptably tender and there is no off-flavour, then juiciness becomes the
sensory characteristic that is the primary determinant of meat quality. It
might be argued therefore, that juiciness will become the most important
meat quality factor. If that is the case, then we need to understand more
about its control.
This review has posed more questions than it may have answered.
However, it has focused on the salient points of meat juiciness, the
problems of experimental method and the difficulty of designing trials for
evaluating interactions among animal and meat characteristics and cooked
meat juiciness. The review accentuates the fertile research area still to be
tackled.
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JUICINESS 123
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5 Measurement of water-holding capacity and
juiciness
K.O. HONIKEL and R. HAMM
5.1 Introduction
The measurement of water-holding capacity (WHC) of meat is carried out

in many different ways all over the world. Some of the methods need only
a few minutes to obtain results, others require days. All of them measure
the inherent ability of the cellular and subcellular structures of meat to
retain excess water compared with the amount of the other muscular con-
stituents. Variability occurs for three reasons:
1. The water molecules are not linked in uniform amounts to the various
cell constituents;
2. These constituents have a variable ability to associate with the water
molecules; and
3. The cellular components are changed by processing.
Various amounts of water are released by the methods applied at different
times after slaughter. In the same piece of meat, the drip loss within 24 h
may amount to 3%, the cooking loss to 25-35% and, with the filter paper
press method, about 60% of the water may be released. From these
results, it is evident that the water must have been lost from different
cellular structures. This means the term water-holding has no uniform
meaning because the methods measure various forms of water retention,
which means that the results may not be comparable with each other.
Thus, everyone who measures WHC must be aware of what is required
to be measured and which method is to be applied for that purpose. In
this respect, the chapter will serve as a guideline.
Juiciness can be looked at as another way of measuring WHC, which is
measured by chewing the meat and using the human senses. While the
physical methods for WHC determination can be described and standar-
dized exactly, in juiciness evaluation even standardization or calibration is
difficult, not to mention the evaluation itself. Calibration of juiciness can
be performed only in a group, where all members of a panel try to agree
on a hedonic scale by judging reference samples. Unlike tenderness or
flavour, which can be calibrated by testing reference samples, this is
impossible with juiciness as standardized samples have not been described

or produced.
Furthermore, as already mentioned in chapter 1, juiciness, the trans-
formed sensation of a part of the mouthfeel, is influenced by and influ-
ences the impression of toughness and flavour. Additionally, mouthfeel
has an indistinguishable fat component (chapter 4). Well-marbled beef
appears to be more juicy and is often also more tender than a leaner piece
of meat, even if the water content of the cooked products is equal.
As a consequence, this chapter will not provide sophisticated methods
for the evaluation of juiciness in the same sense as they are presented for
WHC.
5.2 Composition and structure of meat
Meat originates from cross-striated muscular and adhering fatty tissue.

The lean muscular tissue consists of muscle cells arranged in bundles,
which are surrounded by connective tissue. Between the muscle bundles
there is a variable amount of fat (less than 1% and up to 10%), part of
which appears as marbling of the meat. Muscle cells contain 72~ 75%
water, while 21 ~24% of muscle weight consists of different proteins. The
remainder is made up by small amounts of salt, lipids, nitrogen com-
pounds of low molecular weight and traces of carbohydrates. The main
protein groups of meat are the extracellular connective tissue proteins
(mainly collagen) and the cellular sarcoplasmic, myofibrillar, and cytoske-
letal proteins. The connective tissue protein content of muscle accounts
for 0.5~4% of the muscle weight, while the actual muscle cell proteins
account for 15~23% by weight (Linke and Arneth, 1970; Honikel and
Wellhauser, 1993). Even if the connective tissue protein content is fairly
low in comparison with the other muscle cell proteins, a small amount is
quite important for the tenderness and WHC of the meat.
Within the muscle cell, two main groups of proteins are important in
the changes occurring in meat during processing and manufacture. The
first group are the soluble sarcoplasmic proteins, with many of them being
enzymes, which comprise about one third of the total muscle proteins.
The second group of proteins are those that are insoluble at physiological
salt concentration (ca. 0.15 M) and pH (7.0), namely, the insoluble myofi-
brillar proteins. The latter are organized in fibrils and their substructures,
the filaments, and are very important constituents of meat with regard to
various aspects of meat quality.
In cross-striated muscles, from which the meat we eat comes, the fila-
ments and fibrils are subdivided into sarcomeres limited by the Z-discs.
These are the basic contractile units of the muscles. These sarcomeres are
able to contract in the presence of adenosine triphosphate (A TP), which
WATER-HOLDING CAPACITY 127
remains available in the muscles during about the first 10 h post-mortem.

In the pre-rigor state, Ca2 + ions are released into the myofibrillar space
by the membranes surrounding the cell substructures (the sarcoplasmic
reticulum (SR) and mitochondria) causing the muscles to contract (Buege
and Marsh, 1975; Hamm, 1979). Contracted muscles going into rigor
mortis exhibit a change in WHC, which is expressed by an enhanced drip
loss.
5.3 State of water in meat
The structure of the muscle and its substructures, especially the highly
organized insoluble myofibrillar proteins, are responsible for the retention
of (about 75%) water in the muscular tissue. A water:total protein ratio
of 3.3 to 3.6 and a water:myofibrillar protein ratio of about 5 exists in the
lean tissue of the major meat animals, namely, beef and pork.
It is generally accepted that the water in meat is bound in different
ways. A small part of the water within the muscle is the constitutional
water which comprises about 0.5 g H 20 per 100 g protein, i.e. about 0.1 %
of the total tissue water that is located within the protein molecules.
Protein- water binding energies for this water are much greater than those
existing in normal water binding (Fennema, 1977). A further part of tissue
water (5- 10% of total water) shows a relatively restricted mobility
(Hamm, 1972, 1975). According to the classification proposed by
(a) (b) (e)
Figure 5.1 Scheme of the various forms of water within a muscle cell. Depending on the pH
and salt concentration, meat protein structures such as myofilaments and fibrils shrink or
swell. On the left (a), the scheme shows the shrunken state where protein chains are close to
each other and a large amount of water is 'free' (horizontal lines). In the swollen state (b), a
considerable part of the 'free' water becomes immobilized (net-like structure). Infinite
swelling leads to dissolving of the protein molecules (i.e. they move among each other
without major interference), which is shown in (c). Around the protein threads (thick lines),
the interfacial water is shown (parallel lines to the protein threads).
Fennema (1977), this fraction of tissue water might be defined as inter-

facial water (Figure 5.1). This water is bound at the surface of the
proteins, probably in multilayers and in small crevices. The interactions
result in decreased mobility of the water molecules and represent an inter-
mediate stage between constitutional water and the bulk water with a
reduced vapour pressure and melting point. A part of this water remains
liquid even after freezing below -20 e.
D
The question arises as to whether the remaining bulk of cellular water

(around 90-95%) is free in the physical sense of this term, which means
that it can move without becoming attracted by proteins. The interpreta-
tion of nuclear magnetic resonance (NMR) studies about the state of
water in muscle has aroused some controversy. Numerous authors believe
that essentially all of the water in skeletal muscle exists in a physical state
different from that of pure water or diluted electrolyte solutions (Blan-
shard and Derbyshire, 1975; Peschel and Belouschek, 1979). The alter-
native model, again citing NMR evidence, claims that most of the muscle
water behaves like bulk water in a diluted salt solution but that only a
small fraction of the water adjacent to the protein molecules has a
modified molecular structure. This fraction of tissue water would corre-
spond to the constitutional and interfacial water mentioned above. Some
scientists have concluded from their results that in most of intracellular
water no dramatic changes in the characteristics of molecular motion have
been imposed by the constitutents of the muscle cell (Blanshard and Der-
byshire, 1975; Garlid, 1979). The water within muscle, however, is limited
in motion by the sarcolemma or cell wall.
In addition to the intracellular water, there also exists in muscle an
extracellular space filled with water. Hamm (1986) reported that about
lO% of the total water in living muscle may be associated with the extra-
cellular space. The mobility of the extracellular water is supposed to be
somewhere between that of pure water and that of cellular water. The
dimension of the extracellular space depends on the state of swelling.
Swelling of muscle fibres by increasing pH or addition of divalent cations
decreases the extracellular space, whereas shrinkage of fibres as a result of
development of rigor mortis and fall in pH is associated with an increase
in the extracellular space (Rome, 1967; Offer & Trinick, 1983; Hamm,
1986; Offer and Knight, 1988). Therefore, swelling and shrinkage of
muscle fibres in the intact tissue cause movement of water between the
intracellular and extracellular spaces.
In summary according to the literature it is reasonable to suppose that
water is held in muscle: (i) by capillarity, mostly in the interfilamental
spaces within the myofibrils; (ii) a substantial part is situated in the spaces
between the myofibrils; and (iii) water that is located in the extracellular
space. However, methods for an exact and reliable differentiation between
capillary forces and other forces restricting the mobility of water in animal
tissues do not exist and, therefore, it is not clear whether and to what
extent water is ordered in this biosystem.
Water in muscle that is not bound as interfacial water or constitutional
water, is more or less expressible during application of any force but it is
not yet known if the differences in the state of water in muscle have any
meaning in the measurement of WHC by physical methods. Therefore, the
contradictory results of studies on the state of water in muscle mentioned
above are of very limited value for understanding the different changes in
the water-holding properties of meat.
The remarkable changes in WHC occurring during storage and proces-
sing of meat are determined by the extent to which the bulk of the water
is immobilized within the microstructure of the intact or comminuted
tissue (Hamm, 1972). Fennema (1977) called this immobilized bulk water
'entrapped water' because it is physically entrapped in a fashion similar to
that found in gels. The authors prefer the expression immobilized water
(Figure 5.1) because this term has been commonly used since the first
review on the WHC of meat (Hamm, 1960). It should be noted that the
changes or differences in WHC of meat that are of practical importance,
are not related to the fractions of constitutional or interfacial water
(Hamm, 1960, 1972, 1986).
There seems, however, to be a more-or-less continuous transition from
water that is strongly immobilized within the tissue to the 'free water' that
can be squeezed out by very low pressure. It is not possible, therefore, to
present absolute figures for the immobilized part of bulk water because
the amount of immobilized water measured depends on the method used;
nevertheless by using a standardized method one can measure relative dif-
ferences in WHC quite accurately.
5.4 Definition of water-holding capacity

There exists a wide variety of methods in research and practice to measure
WHC (Hamm, 1986). Before we enter into the description and compar-
ison of the methods the authors will try to define the expression WHC
and explain in general the methods used.
WHC is the ability of meat - or more generally of meat systems - to
hold all or part of its own and/or added water. This ability depends on
the method of handling and the state of the system. As the state of meat
and its treatment differ considerably, the meaning of WHC varies widely.
Therefore, the methods applied and the state of meat at the time of mea-
surement must be defined exactly in order to obtain comparable results. In
spite of all the variation in the methods used, there are three main ways of
treatment that can be divided in three different basic procedures for mea-
suring WHC, i.e. (i) applying no force, (ii) applying mechanical force and
(iii) applying thermal force.
5.5 General methodology
5.5.1 Applying no force

To this group of methods belong the measurement of evaporation and
weight loss, free drip, bag drip, weep (Penny, 1977; Honikel et al., 1986),
cube drip and related methods (Howard and Lawrie, 1956), whereby the
meat is left to itself under different environmental conditions. The only
force on the meat is gravity. These methods are very sensitive but time-
consuming, requiring from one to several days and are often hastened by
the other two methods that are discussed below.
5.5.2 Applying external mechanical force

By using positive or negative pressure the WHC of meat can be detected
within a few minutes or up to an hour. In this group are the centrifuga-
tion methods (Wierbicki and Deatherage, 1958; Honikel and Hamm,
1983), the filter paper press method (Grau and Hamm, 1957), the capillary
volumeter method (Fischer et aI., 1976) and imbibing methods (Monin et
al., 1981; Kauffman et al., 1986). With these methods the amount of water
released is higher than with the methods discussed under applying no
force (5.5.1) since the pressure applied forces the release of water from the
intra- and extracellular space of the muscle tissue. In drip loss measure-
ments, only extracellular water exudes from the meat (Offer, 1984). There-
fore, factors must be known to evaluate the actual drip loss of the meat
by these mechanical force methods. The matter becomes even more com-
plicated as the state of meat changes during conditioning and ageing,
which also influences the WHC. Therefore, methods applying mechanical
force reveal, in most cases, only a tendency as to how the meat may
behave during the following days but absolute values that can be recalcu-
lated as drip loss cannot be obtained.
5.5.3 Applying thermal force

Since meat usually is consumed after cooking, the WHC of cooked meat
is of central interest. Cooking losses can be measured by a wide variety of
methods (Bendall and Restall, 1983). During heating, the meat proteins
denature and the cellular structures are disrupted, which have a strong
influence on the WHC of meat. Extra- and intracellular water will be
released by the meat sample on cooking. The influence of the method of
heating and the final temperature have a great effect on WHC. The influ-
encing parameters are, however, not fully recognized. Also the relation-
ships between WHC measurements that apply no force, those applying
mechanical force and those using thermal force are not known.
5.5.4 Choosing a method for measuring WHC

The different methods of measuring WHC in research and practice are
due to the different interest of people who handle meat. For example,
people who slaughter, condition and age meat are mainly interested in
weight and drip losses. They understand WHC in these terms. They apply
methods of no force and external mechanical force for evaluating WHC.
In contrast, those who produce 'ready to eat' meat and also consumers
are interested in cooking losses. They use mainly method (5.5.3) or in
laboratories sometimes in combination with method (5.5.2). Manu-
facturers of meat products are concerned about WHC and the fat emulsi-
fication capacity of salted meat products. They generally use methods
(5.5.2) and (5.5.3)
It becomes evident from the different ways of treatment and interest
that WHC means different things to different people, which explains why
different methods are used to evaluate WHC. Therefore, it is and will be
impossible to find a single method that gives reliable information on all
aspects of WHC that are of practical interest.
5.6 Influence of different factors on WHC

The WHC of all methods used in practice depend on the pH of the meat
(Figures 5.2 and 5.3), which changes after death from around 7.0 to about
70
-
_?fi.
c:
60 -
50 -
-
m 40 -
Cl
30 -
.c x
Cl
Q) 20 0
3: 10
o
4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0
pH of meat
Figure 5.2 Water-holding capacity of beef expressed as an increase in weight of a homo-
genate in dependence of the pH of the meat (Grau and Hamm, 1957). Small cubes of beef
(0.3-0.5 cm diameter) were inserted for several hours in 0.15 M salt solutions at the pH
values indicated. After that time the cubes were gently dried by absorbing tissue and
weighed. The symbols show two sets of experiments with different beef samples.
5.5 under normal circumstances due to the formation of lactic acid. The
final pH of meat, however, ranges between 6.8 and 5.4. Furthermore, the
WHC depends on the muscle type (Figure 5.4) and the species of animal
as a result of their varying composition and structure.
5.6.1 Evaporation losses

Evaporation losses depend on the following factors :
1. The surface cover of the muscle tissue, such as adipose tissue, covering
or wrapping material;
2. The size of the sample (large or small);
3. The length of the measuring period;
4. The temperature of meat and chilling room; and
5. The air speed and air humidity within the chiller.
Evaporation losses may be standardized in laboratories but, in practice,
standardization becomes rather difficult. Useful measurements, however,
15 ..
••••
•
•• PSE
•• •
• • ••
••• x
•
••
x •
•
v. .x. 0
~o
o )( 0 ox 0
~o~x~
OXxxOo 0 0
x
x ~ 0
N
• )( 0
~
OX
<9 x o~ 000
• o 0 0
• 0
.x 0 0
•• • 0
0
OF[)
5.3 5,8 6.3
Figure 5.3 Drip loss of various pork muscles over a wide range of pHI. Three different
muscles were used: (.) longissimus dorsi; (x) semimembranosus muscle; and (0) adductor
muscle. The drip-loss measurements lasted from 24-96 h post-mortem. According to the
released drip within this period of time, limits were set for DFD, normal and PSE pork. If
the time of measurement was shorter, e.g. 24- 48 h post-mortem, the limits of drip loss for
the three classes were reduced to less than I % for DFD, 1-5% for normal and above 5% for
PSE pork.
12 F oSS
10 I
8 1
6~
4~
2L
o --~----~----------------------------------~
o 2 4 6 8 10 12 14 16
days of measurement
Figure 5.4 Drip losses of various bull muscles during 14 days of measurement. Day 0 is
24 h post-mortem. Cubes of meat (25~30 g) as described in the text were used. There was no
sarcomere shortening with all sarcomere lengths being between 1.7 and 1.9 !lm. The pH
values of all four muscles had a mean of 5.55~5.60. The number of samples for each point
was 33. longissimus dorsi, 0; psoas major, +; semitendinosus, A; supraspinatosus, o.
are possible in the same plants under prevailing conditions of the chilling
facilities. Nevertheless, they might not be comparable to those data from
other companies.
5.6.2 Drip losses

Drip losses also depend on several other factors namely:
1. size of sample;
2. shape of sample;
3. treatment during the conditioning period (Figures 5.5-5.7) and pro-
cessing (Figure 5.8);
4. temperature of chilling; and
5. length of measuring period (Figure 5.6).
Drip losses in meat samples can be measured under defined conditions
and an optimum procedure can be recommended. The sum of the eva-
poration and drip losses is the total weight loss, in which the individual is
interested.
5.6.3 Filter paper press method ( FP PM)

The FPPM was developed by Grau and Hamm (1957). This method
(scheme shown in Figure 5.9) is easy to apply and very rapid. Thus it has
been widely used. Besides pH (Figure 5.10), the results depend on the fol-
lowing factors:
1. The amount of pressure applied (Figure 5.11);

2. The length of time that the pressure is applied (Figure 5.11); and
3. The 'viscosity' or plasticity of the meat, i.e. the pre-rigor state or after
grinding and salting.
5.6.4 Centrifugation methods for uncooked meat

Besides the factors influencing weight loss, the centrifugation losses
depend additionally on the following conditions:
1. Speed of centrifugation (xg);
2. Length of time of centrifugation; and
3. The plasticity of the meat, which is influenced by state of meat and
additives.
5.6.5 Capillary volumeter method

This method makes use of an apparatus developed by Hofmann (1975),
which is shown and described in Figure 5.13. The measurement is faster
than the FPPM with a single measurement requiring 30-120 s. The results
depend on the duration of the measurement and the amount of pressure
applied.
5.6.6 Imbibing method

The imbibing method of Kauffman et al. (1986) is the procedure devel-
oped most recently. With a 3 s measuring time, this is the fastest available
method. The results depend on the length of imbibing and the time
between cutting the fresh surface and the application of the method.
5.6.7 Cooking losses

In addition to pH (Figure 5.16, Table 5.1) cooking losses depend on:
1. The shape and size of sample;
2. Temperature profile during cooking (Figure 5.17);
3. Final cooking temperature (Figure 5.16); and
4. The environment during cooking (in water on salt solution as shown
in Table 5.1, air, wrapping).
A standardized procedure for cooking concerning shape, size and environ-

ment is possible and will be given. The profiles of heating and final tem-
perature, however, are dependent upon the consumers' attitudes in
different countries and may differ from the standardized method
proposed.
Table 5.1 WHC (cooking loss") as % of muscle, unsalted and salted muscleb homogenate (50% water added) at different post-mortem pH values and
temperatures
Temperature pH 6.8 pH 6.1 pH 5.9 pH 5.5

CC)
Muscle Homogenate Muscle Homogenate Muscle Homogenate Muscle Homogenate
Unsalted Salted Unsalted Salted Unsalted Salted Unsalted Salted
0.5 34 52 24 42 58 36 44 59 40 55
4 34 50 16 40 53 31 41 55 34 44 60 51
5 36 53 20 41 56 32 42 56 37 45 57 50
7.5 37 51 28 40 56 29 42 57 35 45 60 49
10 27 49 22 38 55 28 40 55 35 45 55 51
14 33 53 22 39 54 22 41 55 22 44 58 53
17 32 49 21 40 57 36 43 57 41 44 58 54
20 37 48 27 42 58 36 43 58 40 44 60 51
20 35 42 16 39 50 24 41 52 27 43 57 48
23 33 55 31 40 57 32 42 59 33 44 59 54
24 37 47 26 44 58 33 46 59 34 53 59 50
27 37 53 28 39 57 32 40 58 34 42 60 47
30 35 52 26 41 56 32 42 57 34 45 60 56
Means 34.4 50.3 23.6 40.4 55.8 31.0 42.1 56.7 34.3 44.8 58.6 51.5
SD c ±2.8 ±3.4 ±4.6 ±1.6 ±2.3 ±4.3 ±1.7 ±2.0 ±5.2 ±2.7 ±1.6 ±2.7
aMeat heated for 30 min to 95°C.

bGround meat (homogenate) includes 50% water, resp. 50%,4% NaCI solution added.
cSD = standard deviation.
Out of this wide variety of possible methods, the authors have selected
methods and evaluated them critically and recommend standardized pro-
cedures.
5.7 Description of methods and evaluation
5.7.1 Drip loss determination

Drip losses can be measured on whole cuts or parts of cuts. For practical
reasons it is advisable to use a piece of meat with a defined shape and
weight. The reason for this is, as already mentioned, that drip loss is
dependent upon the surface area and weight of the sample. In larger
pieces, a smaller percentage of the weight is lost as drip. Furthermore, the
fibre direction in the sample taken for drip loss determination is impor-
tant. Therefore, it is recommended that one generally use a cube of meat
cut parallel to the fibre direction at a weight of about 30-100 g. In special
cases, such as the widely used longissimus dorsi muscle, a slice of a muscle
may be preferred to a cube. Within the European Community (EC), a
recommendation for measuring drip on the longissimus muscle was
proposed 10 years ago by Honikel, as discussed in the following section.
0/0
--. -.
-
--
50 \ •
\ ./'
~ 40 \
I
/
•
•
\
.;
~
§u \
~ 30 \ /
a •\ I
0>
\ • /
.~
c 20 /
\
~
/
0 \ / "$1'
00
L
Ul 10 'r--- u..
u..
«
CD
£
0 4 8 12 16 20 24 28 32 36°C
temperature
Figure 5.5 Influence of the storage temperature between I and 24 h post-mortem on sarco-
mere shortening in beef sternomandibularis muscle; 0% shortening is equal to a sarcomere
length of 1.9 ~m.
(%)
from day 2. oOe
8
7
6
5
Vl
Vl
.S' 4
~3
-0
2
1
0
o 4 8 12 16 20 21.. 28 32
muscle temperature at 0-2 4 hou r s
Figure 5.6 Drip loss of beef muscle cubes (about 30 g) from 1-7 days stored at the first day
post-mortem between O°C and 35°C. Samples were those used in Figure 5.5. The days of
measurement are shown on the right side of the diagram.
5.7.1.1 Method for drip loss. Before carrying out the determination, it is
advisable to record the pH of the sample, the time post-mortem and the
type and age of the animal. Knowledge of these parameters is important
when evaluating the results obtained. The method is described for the
widely used longissimus dorsi muscle.
A slice (2.5 cm thick) of the longissimus dorsi muscle is removed
between the eighth thoracic and first lumbar vertebrae, with only freshly
cut surfaces being used for the measurement. Associated adipose tissue
and parts of spinalis and multifidus dorsi muscles should be removed. The
fascia should remain around the muscle. Room temperature during
cutting should be similar to the temperature of the meat. The slice of
muscle should be weighed and suspended by means of a net or thread
inside a plastic pouch and sealed under atmospheric pressure. The samples
are then held at 0-4 C for at least 24 h. The duration of storage must be
D
reported. The pouches should hang in such a way that the exudate
dripping from the meat does not remain in contact with the meat. At the
end of the measuring period the muscle is taken from the pouch, dried
gently with an absorbing tissue and reweighed. During weighing, care
must be taken that no condensation of water vapour occurs on the cold
surface. Drip loss is expressed as the weight loss in mg.g- 1 of the original
weight of meat or, better, as the percentage of original weight. Finally, the
pH of the muscle should be measured again. If other muscles are used for
drip loss determination, the difference in the way of handling is in regard
10
8
0
......, 0
"-
•
6 • '" 0, •
.' .
(f)
"-
(f)
0
4
"-
,
Q. , 0
beef
u
...... pork
2
~
0
0.7 0.9 1.1 1.3 1.5 1.7 1.9 ( }Jm )

final sarcomere length
Figure 5.7 Relationship between sarcomere length and drip loss in beef and pork muscles.
to the shape of the sample only. As noted above, a cube cut parallel and
perpendicular to the fibre direction is recommended.
Following the drip loss determination, the sample can be used immedi-
ately for cooking loss measurements. If there is a delay before cooking
loss measurement, the sample must be wrapped to avoid drying out of the
surface.
5.7.1.2 Evaluation of the method. The method as described above uses a

minimum time requirement of 24 h. A longer period of 2 days or more
lowers the variability and increases the correlation coefficient (Table 5.2)
with regard to long-range storage time. If beef muscle is to be aged for 2
weeks, then the reliability of a 24 h drip-loss measurement (r = 0.57) is
Table 5.2 Linear correlation coefficients between

drip-loss measurements and time post-mortem a
Drip losses b
Days
post-mortem 1 day 3 days 6 days 8 days
3 0.88
6 0.70 0.92
8 0.63 0.87 0.97
14 0.57 0.82 0.93 0.96
an = 565 beef samples .

bDay 0 is 24 h post-mortem.
vplocity of frPe'Zing (-10 bis -7"C) during

12 frppzing thawing
slow slow O,OJOC/min

10
fast slow
slow O,05°C/min fasl 1"C lmin
8
fast 50C/min fast
unfrozpn
5 6 7 days after thawing
Figure 5.8 Drip loss of thawed beef. The meat was kept unfrozen or frozen post-rigor at the
indicated velocities to about - 18°C. After 2 days of frozen storage, the meat was thawed as
indicated. On reaching one, the cubes were cut and weighed. Measurements started at 2°C.
rather poor. A 3-day measurement increases the correlation coefficient

considerably to a value 0.82.
The samples should not be taken from the carcass or muscles before the
onset of rigor mortis. Small pre-rigor cubes may shorten and results may
be obtained similar to those shown in Figures 5.5-5.7.
Thawed or slightly frozen samples should be measured with care as
their drip loss depends on the method of freezing and thawing (Figure
5.8). Determining drip for 1 day in this case may give false results.
5.7.2 Determination of water-holding capacity using the jilter paper press

method (FPPM)
The procedure is simple and rapid. It is applicable to both intact and
ground tissue, with and without added water. Heat-denatured meat can
also be used and only 0.3 g of tissue is necessary. The technique requires
only a few minutes and the result is fixed permanently on the filter paper.
There are, however, disadvantages of the FPPM; for example the techni-
que is not applicable to samples containing salt and large amounts of fat
and/or water such as sausage emulsions (Grau and Hamm, 1957; Tsai and
Ockerman, 1981) because their plasticity (fluidity) is very high.
In the original FPPM developed by Grau and Hamm (1957), 300 mg of
muscle tissue are placed on a filter paper (Schleicher and Schull, Dassel,
Germany, 2040b resp., Whatman No. 1 or a similar filter paper) with a
defined moisture content and pressed between two plexiglas plates to a
thin film . The water, which is squeezed out, is absorbed by the filter paper
(Figure 5.9). The area of the ring of fluid, which is obtained by subtract-
ing the area of the meat film from the total area, is proportional to the
140 QUALITY ATTRIBUTES IN MEAT, POULTRY AND FISH PRODUC'I:S
(a) (b) (c)
Figure 5.9 Diagram of results of the FPPM obtained with meat of different WHC: (a) pre-
rigor meat with high WHC; (b) post-rigor meat of higher pH and medium WHC; and (c)
post-rigor meat with low pH and low WHC. The area of the outer ring zone represents the
fluid ring, the centre zone is the area of the meat film, which is spread out by the pressure
applied. (From Hofmann, 1982.)
amount of water. The pressure produced by tightening the plates with

screws and by hand is so great (see Figure 5.l1) that individual differences
in pressure do not influence the fluid area. This simple technique can be
carried out in a slaughterhouse. The accuracy of the method is satisfac-
tory provided that the sample is weighed precisely.
2
em
Q)
10
--'
U
lfl
:J
8
E
.....
u
6
.....
0 0
c 8 0
Ol
t.
c
L
-
\J 2
:J
--'
0
6.7 65 6.3 6 .1 5.9 5.7 5.5 pH
post rigor
Figure 5.10 Dependency of fluid ring area of various pork muscles (e.g. PSE, normal, DFD)
on the final pH value.
- I -----,---r - ,----.---- r- - - , - -,----,- ,--

M RZ
(a) (b)
-'" 70 7 -
u
:;
E 60' 6
/' WL
a
~ 50
/
If,
'"E
e
~ 1.0 '"
15 ~
.~:::I 30
fr om R
:;:
'"
OJ
20 /~- ----- --- 1)
OJ /
cr: 10 I 12
/
20 {,O 60 80 100 5 6
Load (kp) Time of pressi ng (m in)
Figure 5.11 Parameters of importance for the filter-paper press method: (a) influence of load
(kp) on the amount of released juice, measured by weight loss (WL) or calculated from the
fluid area (RZ) ( - = meat with lower WHC; - - - = meat with higher WHC); (b) influ-
ence of time of pressing on the area of meat (M) and fluid area (RZ). (See Figure 5.9.)
5.7.2.1 Method. A piece of filter paper (size about 6 x 6 em) is placed

on a plexiglass plate and 0.2-0.4 g of meat (originally exactly 0.3 g) is
placed in its centre. A second plexig1ass plate is put on top and pressed by
a weight of 50 kg or some other device for 5 min. After removal of the
top plate, the meat area is marked with a pencil and, if necessary, with
light meat (e.g. chicken), also the fluid area. The size of the area is
measured by a planimeter or an electronic area measuring device.
5.7.2.2 Evaluation of the method. When using exactly 0.3 g of meat, the
WHC can be expressed as the area of the fluid ring (Figure 5.10) or as the
amount of loose water in it and is related either to the weight of sample,
to the total water content, or to the protein content of the sample. With
regard to measurement of WHC for samples of raw, unground tissue from
the muscles, the coefficients of repeatability reported in the literature vary
from 0.68-0.98 (Fewson and Kirsammer, 1960; Gravert, 1962; Fewson et
al., 1964; Brendl and Klein, 1971). The error of variance found by Fewson
et al. (1964) was below 5%. Several modifications of this technique have
been suggested. In most of them the application of defined pressure is
recommended (Wierbicki and Deatherage, 1958) or the amount of
released water is determined by weighing the meat sample or the filter
paper before and after pressing (Hamm, 1960, 1972).
The assumption that almost all of the water released during pressing of
the meat sample is located in the fluid area outside the meat area (Grau
and Hamm 1957; Wierbicki and Deatherage, 1958) cannot be correct

because the amount of expressible water, determined by weight loss, is
higher than the amount of water in the fluid area, as shown in Figure 5.11
(Hofmann, 1977; Hofmann et al., 1982). Therefore, the fluid that is
squeezed out by pressing must be adsorbed also by the filter paper below
the meat area. With increasing pressure the amount of released water,
determined by the weight loss of the meat sample, increases to about 60%
of the original weight of the sample until a load of about 40 kg is imposed
(Figure 5.11). A further increase in load does not result in any additional
release of juice. It is interesting that the amount of bound water in the
tissue, which cannot be squeezed out, is in the range of 10-15% of the
original tissue water. The amount of non-expressible water corresponds
closely to the amount of constitutional and interfacial water in muscle (see
section on 'States of water in meat'). The total area is much less depen-
dent on WHC than the fluid area (Figure 5.9). From this observation, it
must be concluded that fluid ring is determined primarily by the meat area
of the meat film rather than by the amount of expressible water
(Hofmann et al., 1982).
The magnitude of force depends on the deformability or plasticity of
the meat sample, which is mainly a function of the protein-protein inter-
actions in the myofibrillar system and, therefore, is correlated with WHC.
In meat with a weak protein-protein interaction (e.g. in the pre-rigor state
at a high pH value), a higher proportion of the total water is immobilized
than in post-rigor meat with a strong protein-protein interaction. Conse-
quently, under a certain pressure, meat with high WHC (high pH) is more
easily deformed than meat with a low WHC (low pH). This explains the
highly significant correlations between FPPM and other methods for the
determination of WHC, which are actually pH correlations. Furthermore,
it must be considered that, under certain circumstances, differences in the
deformability of the meat sample might not be related to differences in
WHC but to other factors. This certainly plays a role if large amounts of
water or fat are added to minced meat. With the FPPM, the deformation
of the meat sample to a thin film occurs very quickly; indeed, after a few
seconds the meat film has reached its maximum size (Figure 5.11). The
loosely bound water is squeezed out at the same rate but the adsorption
of the released fluid by the filter paper takes a longer time. It takes several
minutes for the fluid areas to reach their maximum (Figure 5.11).
Originally the FPPM used the fluid area with exactly 300 mg as a
measure for WHC. Following our proposal, George et al. (1980) ex-
pressed WHC in terms of the ratio of meat to fluid. Hofmann et at. (1982)
proposed using the ratio of meat area to total area because within a
certain weight range (200-400 mg) this ratio is independent of the weight
of sample and determined by WHC only.
In studies on the influence of different factors on WHC of meat, Tsai
and Ockerman (1981) found highly significant correlations (r = 0.88-

0.99) between the FPPM and a centrifugation method. According to
Ubertalle and Mazzocco (1979), the FPPM (Grau and Hamm, 1957) IS
quicker and more easily reproduced than the centrifugation methods.
5.7.3 Determination of water-holding capacity by centrifugation

The centrifugation methods are applicable for intact tissue and ground
meat. In the technique of Bouton et al. (1971, 1972) a weighed intact
muscle sample (3-4 g) is centrifuged at 100 000 xg for 1 h in stainless steel
tubes. Fischer et al. (1976) also found that with centrifugation at
60 000 xg for 30 min good results could be obtained. The juice that is
released from the meat is decanted off as quickly as possible in order to
avoid readsorption of water. The meat sample is removed from the tube
with a forceps, dried with tissue paper and then reweighed to determine
liquid loss. If the residue is dried in the tube at 105°C, the total water
content of the sample can be determined, and WHC can be expressed as
released or bound water as a percentage of total water. The coefficient of
variation is less than 5% and, although the conditions are arbitrary, they
are easily reproducible. The need for a high-speed centrifuge makes it
almost impossible to use this type of method outside a laboratory.
Using a relatively low speed and with ground meat, however, the
remaining amount of juice at the end of centrifugation is very small
(Wierbicki and Deatherage, 1958) because the fluid is readsorbed at the
end of centrifugation. In some techniques, readsorption of water is
avoided by separation of the released juice from the sample. The meat can
be put on a fritted glass disk or glass beads at the bottom of the tube,
thus separating the released juice from the tissue by centrifugation (Wier-
bicki et at., 1957). In the method of Penny (1975), the meat is placed on
perforated teflon disks, covered with pieces of fine nylon mesh and placed
at the bottom of the centrifuge tube, which is partially filled with glass
beads. The juice released by centrifugation can also be adsorbed by using
other absorbing materials like gypsum (Hofman et at., 1980) that is placed
on the bottom of the centrifuge tube.
5.7.3.1 High-speed centrifugation method. In each of two centrifuge

tubes, or a multiple of two, with a volume of about 50 ml, 10 g of meat
are weighed as accurately as possible. After balancing the tubes they are
placed in a fixed angle rotor and centrifuged for 20 min at about 5-10 °C
and about 50 000 xg. In a series of the authors' own unpublished experi-
ments, it was shown that the same rotor and speed must be used. Com-
parison with other rotors and speeds is limited. After the centrifugation,
the tubes are at once turned upside down on filter paper for about a
minute, then the samples are taken by a forceps from the tube, dried
Table 5.3 Linear correlation coefficients between high-speed centrifugation losses (30 min
at 50000 xg), capillary volumeter measurements and drip losses and time post-mortema
Drip lossb
Days Centrifugation
post-mortem 1 day 3 days 6 days 8 days 14 day losses at 48h
Centrifugation
loss at 48 h
post-mortem 0.12 0.34 0.46 0.47 0.50
Capillary
volumeter at
48 h post-mortem 0.29 0.37 0.35 0.33 0.30 0.38
an = 480/495 beef samples. bDay 0 is 24 h post-mortem.
gently with absorbent paper and weighed. The centrifugation loss is

expressed as percentage centrifugation loss to the original weight.
5.7.3.2 Low-speed centrifugation method. The procedure is very similar to

the high-speed method discussed above. However, before putting the meat
in the tubes, glass beads or other absorbing materials are accurately
weighed into the tubes, after which the meat is placed above the glass
beads or absorbant. The speed of the fixed angle or swing out rotor
should be from 5000-10 000 xg. After the centrifugation, the tubes should
not be turned upside down but the meat should be removed directly by
forceps and handled as described in the high-speed method.
5.7.3.3 Evaluation of the methods. Different centrifugation methods with

low speeds (1200-4000 r.p.m.) and varying duration (8-20 min) using
meat and tissue homogenates were found to be accurate and reproducible
(Grosse et al., 1979). Highly significant correlations between the results of
different centrifugation methods (about 1000 xg) and the FPPM have
been reported by Brendl and Klein (1971). Other authors, using smaller
samples for the FPPM (0.3-0.5 g) came to the conclusion that the cen-
trifugation method (with about 1000 xg calculated) is more exact and
more reproducible (Gravert, 1962; Steinhauf et ai., 1965). Penny (1975)
found a highly significant correlation. (r = 0.88) between 'spun drip'
(1700 xg) of frozen, thawed meat measured with the centrifugation
method and the amount of 'free drip' (released without centrifugation).
An interesting relationship between high-speed centrifugation losses
(50 000 xg) for 30 min and drip losses is shown in Table 5.3. Drip-loss
measurements for 24-48 h post-mortem samples (day 1) had a rather poor
correlation to the centrifugation losses at 48 h. The correlation coefficients
increased considerably for drip losses for 3-14 days but, in the end, still
% centrifugation loss
young bull steer heifer

sex
_ long.dorsi ::::::J psoasmajor

![§ill semitend. _ supr aspinam
Figure 5.12 Influence of the sex of cattle and muscles on centrifugation loss at 48 h post-
mortem. The method used is the high-speed centrifugation method described in the text.
Each group contains 13-34 muscles.
had only a moderate correlation coefficient of 0.50. The causes of this

relatively low relationship are unknown. One reason is easily recognizable
by comparing Figures 5.4 and 5.12. While the drip losses in the four
muscles with which the experiments were carried out are rather different
for supraspinatus muscle only, centrifugation losses are additionally lower
with semitendinosus and psoas major muscles compared with longissimus
dorsi. Further variations between the sexes occur. The reason may be the
plasticity of different muscles, which is important with centrifugation.
5.7.4 Capillary volumeter measurements

A newer type of method for the evaluation of WHC of meat is based on
the application of capillary forces to the muscle tissue (Hofmann, 1975;
Fischer and Hofmann, 1978). A gypsum plate is placed onto the surface
of the intact tissue for a definite time (30-120 s).
The loosely bound water is sucked up into the porous material by the
effect of capillary forces. The air displaced from the capillaries by the
meat juice goes into an V-shaped, calibrated capillary glass tube contain-
ing a coloured fluid (Figure 5.13). The volume of the displaced air read
from the shift of this fluid is equal to the volume of released water and is
inversely proportional to the WHC of the tissue. It is not necessary to
adjust the gypsum to a certain humidity but, for each measurement, a new
disk of gypsum has to be used. The thickness of meat has no influence on
the results. However, the volume of the water sucked up by the porous
material depends on the pressure used. The volume increases with an
~_-Cylinder guide
Capillary tube
It -I---+---+-__Scale
Rising liquid -+-+--__ Cylinder

Cylinder handle
. ~\ Hose connection
Test Block_---t.-
~_- Stand
__
Meat sample
r==~~~~~==~ Base plate
Stand base
Figure 5.13 Drawing of the capillary volumeter. The test block contains the gypsum plate.
It can be screwed off and exchanged. The cylinder handle lowers the cylinder (weight about
800 g) onto the meat sample.
increase in load up to 300 g, then reaches a plateau, while at loads greater

than 1000 g the volume increases again with the rising load. In the low-
pressure range, the capillary forces of the gypsum are probably dominat-
ing. Between 300 g and 1000 g the capillary forces seem to be equal to the
WHC, while at loads greater than 1000 g the meat juice is squeezed out
by the high pressure (Fischer et al., 1976). It could be that the plateau of
the volume-pressure curve indicates the border between extracellular and
intracellular water. Surprisingly, with the capillary volumeter method, the
same values of WHC in terms of volume of released water are obtained if
the tissue is cut either parallel or perpendicular to the fibre direction
(Hofmann, 1975).
5.7.4.1 Procedure for capillary volumeter method. A freshly and evenly

cut surface of lean meat of at least 4 x 4 cm is placed below the gypsum
plate. The gypsum plate is then combined with the capillary and a weight
is placed on the meat for a constant time of 30-120 s. At the end of mea-
surement, the amount (~.tl) of meat juice that penetrates into the gypsum
plate is read on the capillary.
mikroliter
young bull steer heifer

sex
_ long.dorsi _ psoasmajor
_ semi tend. _ supraspinam
Figure 5.14 Influence of the sex of cattle and different muscles on capillary volumeter values
measured at 48 h post-mortem. The method is described in the text.
5.7.4.2 Evaluation of the method. The methoJ is highly reproducible, the

coefficient of variation being 4%. It has been used successfully for the
evaluation of pork quality. Fischer et al. (1976) have used simple equip-
ment based on the same principle. A significant correlation coefficient
between the results of the capillary volumeter method and those of the
centrifugation method of Bouton et al. (1972) was found. The correlation
between the capillary volumeter method and the FPPM of Grau and
Hamm (1957) was smaller but was still significant (Fischer et al., 1976).
The correlation between capillary volumeter method and drip losses was
astonishingly poor (Table 5.3). One reason again may be the different
behaviour of various muscles. When comparing Figures 5.4 and 5.14 the
differences become obvious. As already observed in comparison of cen-
trifugation losses (Figure 5.12) and drip losses (Figure 5.4), there are also
differences in WHC between muscles, which are detected with drip loss or
capillary volumeter measurements. It should be mentioned, however, that
capillary volumeter measurements with beef muscles are not easily applied
as the enhanced size of the fibre bundles of beef makes it rather difficult to
get the gypsum plate airtight onto the meat surface.
Nevertheless, the method has its advantages. No weighing of the sample
is required, the data obtained are directly related to WHC and also kinetic
measurements can be carried out. It has the disadvantage that the data
cannot be applied to studies on minced meat or homogenates (e.g. sausage
emulsions).
5.7.5 Imbibing method

The FPPM and the capillary volumeter method are fast and can be
carried out without the need of electric power. The equipment is easy to
carry and can be handled by any trained person. However, Monin et al.
(1981) and Kauffman et al. (1986) indicated the procedure was too com-
plicated. Both groups developed methods which use a simple filter paper,
getting results within seconds.
5.7.5.1 Imbibing method of Kauffman et al. (1986). The method of

Kauffman et al. (1986) uses a muscle surface that is cut and left for 10-
15 min. Then a 45 mm diameter filter paper (Kauffman et al., 1986 used
filter paper No. 300 204 of Schleicher and Schull, Dassel, Germany) is
placed on the meat surface for about 1-2 s. The paper is evaluated imme-
diately on a wetness score of 0-5 (0 = 0% of paper wet, 1 = 20%, 2 =
40%, 3 = 60%, 4 = 80%, 5 = 100% of the paper wet) or the weight
gain is measured at once (weight gain of less than 20 mg to 150 mg).
5.7.5.2 Evaluation of the method. The authors (Kauffman et al., 1986)

found correlation coefficients between drip loss at 48 h and their methods
(either by wetness score or by weight gain) to be 0.90 or higher. More
than 86% of the variation in drip loss could be accounted by variation in
filter paper score. However, pH variation in the meat sample might have
been responsible for the high correlation coefficient.
This method is cheap, easy to handle and, according to the data, very
accurate. There are, however, a few precautions that must be observed.
During the 10-15 min waiting time neither condensation nor evaporation
of water must be allowed to occur. This means that the room temperature
must be very similar to that of the meat and the humidity in the room
must be between 90% and 96%. Second, the method does not work on
meat with a large amount of fat on the surface. Usually this is a minor
problem.
The imbibing method is a rapid alternative to drip loss measurements.
However, its use as a fast method with regard to other WHC measure-
ments must be proven.
5.7.6 Cooking loss measurement

There exists a widespread opinion that meat with a high drip loss also has
a high cooking loss. This, however, has not been shown necessarily to be
the case. In Figure 5.15, an experiment with beef, which was obtained at
1 h post-mortem and stored at various temperatures (O°C, 8°C and 25°C)
within the first day after slaughter is shown. From day 2 on, it was stored
at O°e. Drip loss as already discussed depends on the state of contraction
(/)
(/)
6
~
0- 4
....
l:J
0
0-
2
0
50
(/)
J'c __E~~----~R--~~~~~r-~~=-~~--
(/)
0 40
01
C
oX
30 •
0
0
u 20
-ge
10
0 2 3 4 5 6 7 8 days
time of storage
Figure 5.15 Influence of time and temperature of storage on drip loss and cooking loss
of the sternomandibularis muscle of beef. Muscle cubes (about 30 g) were stored at 70°C 0,
SoC 6 and 25°C . within I h post-mortem and held for 24 h at these temperatures. From
day 2 onwards they were stored at O°C . Drip and cooking losses were determined by the
methods described in the text.
(cold shortening) and increases with the time of storage. The cooking
losses of the meat, however, stayed fairly constant within the period of
storage and were independent of the temperature of storage (shortening
on the first day) and the drip released.
Cooking losses depended much more on the end-point temperature
(Figure 5.16) and the speed of heating (Figure 5.17). The higher the final
temperature and the slower the velocity of heating, the higher were the
cooking losses. Between 50°C and 70°C, there was nearly a linear rela-
tionship between the final temperature and cooking losses (Figure 5.16).
These relationships have been investigated in detail by Laroche (1982a,b).
Taking these considerations into account, the authors have worked out a
standardized cooking procedure, which is described for the longissimus
dorsi muscle and has been used for drip loss measurements.
5.7.6.1 Measurement of cooking losses. According to Boccard et al.

(1981) a slice of loin of known weight (70-100 g) and pH is placed in a
thin-walled polyethylene bag (the bag must be waterproof and withstand
48 -
/x
44 x
40 - //
I 0x' ,6
36 - x .
32 -
/i
I
28 -
//' !,/
x ,0 /
, ,
I •
, l
~ 20 -
.2
I
.° , . .
I.
(J1
.~ 16 I ,
""oo I "
X
v 12 ,,:/ .
I .
8 , I
I /
4 / ~ ./
// /
~ .- .
40 50 60 70 80 90 100°C
temperature
Figure 5.16 Influence of end heating temperature and pH of meat on cooking loss. The
meat was heated at a rate of about 2°C.min- 1 to the indicated end-points. x, pH 5.5; 0, pH
5.8; . , pH 6.3.
75°C) and sealed under moderate vacuum (about 150 mm Hg) to remove
the air trapped between the meat and the wall of the bag. This facilitates
complete immersion of the package in water. If air pockets remain in the
pouch, part of the meat may float above the water level. If the bag is not
sealed, care must be taken that the sides of the bag are in contact with the
meat surface in order to allow optimum heat flow. The mouth of the bag
must remain above the water level. Glass beads or stainless steel rods
should be put in the bottom of the bag in order to keep all of the meat
immersed in the bath.
The pouch is placed in water at 75°C for 50 min and then placed in
running tap water (about 15°C) for 40 min, after which the meat is taken
from the bag, mopped dry and weighed. The heat loss is expressed as
heating loss (in g) per initial weight before cooking (in g) or as percentage
heat loss.
There will be occasions when the history of the meat is unknown or
insufficiently known. In such cases, it is advantageous to measure the
0/0
40
39
38 xxx
I,{)
I,{) 37 xx
.9 ><
><
(Jl
c 36 ><
-'"
0
x
0
u 35
34
0 4 8 12 16 20 24 28 32 36
1/ velocity of heating (sec/oC)
Figure 5.17 Influence of reciprocal heating velocity on cooking losses in pork. The end-
point of heating was 95°C.
moisture content of the meat and relate the loss to total moisture content,
or if appropriate, to dry matter content. The method of moisture determi-
nation is described by Boccard et a/. (1981). Results are expressed as
heating loss (in g)/protein (in g). After measuring heating loss, the sample
may be used for other quality evaluations such as tenderness measure-
ments.
5.7.6.2 Evaluation of the method. Cooking-loss measurements are used in

a wide range of conditions. As shown in Figures 5.16 and 5.17, many
factors influence cooking losses. Another factor is rendering of the fat in
meat with a high amount of fat. In the literature, fat rendering is often
included in cooking losses. Some scientists, however, like to separate
aqueous cooking juice from liquid fat, especially if this method is applied
to meat batters.
As shown in Figure 5.18, cooking losses and drip losses are apparently
dependent on different meat characteristics. Cooking losses do not depend
on shortening (Figure 5.15) or on the PSE-condition, which can lead to
high drip losses. This is shown on Figure 5.18 where several PSE muscles
and also cold-shortened muscles are compared with normal and unshor-
tened meat samples. Heating causes denaturation of the proteins and dis-
integration of membranes, which has a tremendous influence on the bulk
phase water so that shortening or the amount of extracellular space
becomes unimportant.
,
I
0/0
16 l- • ••
••
14 l- • • • -
•• •
12 - : I -
• • \ ••
-
\Il
>.
10-
•
0
-0
(T) B • -
•
L..
(l)
13
\Il
6-
•• -
\Il
.2 4- •
•
l
.
0..
L..
-0
• •
2,
:
0 I I
10 20 30 40 50 0/0
Cooking loss after 3 days
Figure 5.18 Comparison of cooking and drip losses of pork longissimus dorsi muscles at 3
days post-mortem. All muscles had an ultimate pH of 5.5-5.75. PSE, cold-shortened and
normal muscles were included in the group. Drip losses were determined between 24 h
and 72 h post-mortem, and cooking losses at 72 h post-mortem according to the method
described in the text but after reaching a final temperature of 95°C.
5.8 Meat products
Meat products are generally prepared by the addition of salt and other
additives. In some products, e.g. cooked or raw ham and bacon, the
muscular structure of the meat remain.s fairly intact. In most of the meat
products, however, the fibrillar structure of the meat is destroyed by
cutting or comminuting. The salt and other additives, water and fatty
tissue are finely comminuted with the meat. In many cases, especially in
cooked sausages, a homogeneous batter is obtained that is stabilized by
heating. During heating, cooking stability (the prevention of water and fat
rendering out) is the most important criterion.
Cooked comminuted meat products of the frankfurter type are the
largest in terms of volume and the most thoroughly studied meat

products. In these types of products, WHC is an important characteristic;
thus, the methodology used to detect changes in WHC and the manu-
facturing of cooked sausages will be described briefly.
5.B.1 Manufacturing of cooked sausage

Raw meat of various species (e.g. beef, pork, chicken and others) is cut
in a bowl chopper with the salt, additives, spices and ice or water.
Cutting destroys the cellular and fibrillar structure and facilitates the
action of salt and water, transforming the meat from a state of limited
swelling (Figure 5.la) to one of unlimited swelling (Figures 5.1 b, c) as
explained by Hamm (1975). Salt ions decrease the attractive forces
between adjacent protein molecules, and the added water allows an
increase in volume. The distance between protein molecules is enlarged
and the meat undergoes swelling (Figure 5.1 b). If the swelling of protein
molecules is unlimited, their attractive forces become small enough that
they are solubilized (Figure 5.lc). These salt soluble proteins, discussed
later, are considered to play an important role in meat batter stability.
The degree of swelling and solubilization depends on the pH and rigor
state of the meat, the salt concentration (Hamm, 1972) and the tem-
perature.
Addition of salt not only causes swelling but also results in a change
in the conformational structure of the myofibrillar proteins that in-
creases their hydrophobicity (Voutsinas et at., 1983). The increase in
surface hydrophobicity facilitates the interaction between proteins and
fat particles, forming a covering layer. This allows the hydrophobic
regions of the proteins to interact with each other and to form a
three-dimensional structure upon heating. Thus, the addition of salt
produces three effects: (i) the swelling and dissolving of myofibrillar
proteins, which increases the amount of immobilized water; (ii) the
increase in surface hydrophobicity of muscle proteins that leads to fat
binding; and (iii) the formation of a heat-stable network by hydro-
phobic forces upon heating.
None or little of the water and fat should render out on heating. It is
the intention of the manufacturer to prepare a stable product, which he or
she knows is stable before heating. Therefore, the objective in producing
cooked sausage is to manufacture a stable, cooked sausage. Direct mea-
surements of WHC and fat-rendering of the batter are applied. Also mea-
surements which are supposed to have a relationship to the cooking
stability are used. These include viscosity or emulsification capacity mea-
surements and detection of WHC of the batter before heating, e.g. by cen-
trifugation. Some of the methods used for evaluating cooking stability of
batters will be discussed briefly.
5.8.2 Water-holding capacity of unheated batters

Before cooking, the colloidal batter system is stable due to the low tem-
peratures in the batter and its high viscosity. Gel formation, however, has
not yet taken place. Gel formation on heating depends on the type and
state of the proteins in the batter and the degree of comminution as well
as the heating process itself, i.e. the velocity and final temperature of
heating. Therefore, results obtained with raw material can give hints only
about the stability of the heated product.
The preferred method for evaluating the WHC in the raw batter is the
centrifugation method. Centrifugal forces on the batter are supposed to
release water by differences in density within the batter, but the viscosity
of the batter is also important. The relationship between viscosity and
centrifugation losses is shown in Figures 5.19 and 5.20. In both figures,
the same batters were measured. On increasing the salt content of the
batters above 1%, the authors observed an increase in viscosity and a
decrease in centrifugation losses. This indicated that a higher viscosity,
which is due to a higher interaction between the particles, increased the
3500
o /'
/
/'
" 0
o
o
8 ",/ '"
,
~
..
"" 0 . / ./
300 '....... 8 . . . . ,- •
.............. -J:J _ _ - - - 0
~ o
___ 0 ___ , _.
,
••
(g 2500
u
Ul
>
2000~~__- L_ _~_ _L-~_ _~_ _~_ _~

o 0.5 1.0 1.5 2.0 2.5 3.0 3.5 f..0
% Noel in meat + ice
Figure 5.19 Changes in the viscosity of unheated meat batters with increasing salt con-
centrations and without (0) or with (e) 0.75% lecithin as an emulsifier. Viscosity was
measured with a rotational viscosimeter (Haake Rotovisko, Berlin, Germany) as described by
Honikel and Hamm (1983).
~
o
(/)
(/)
52
c
o
-+-'
d
01
:::J
~
'--
-+-'
C
<!J
u
% NaCl in mea t + ice
Figure 5_20 Centrifugation losses (45000 xg for 30 min) of unheated meat batters as a
means of determining WHC with regard to salt concentration and lecithin in the batter.
Batters were the same as shown in Figure 5.19. The method is described by Honikel and
Hamm (1983).
WHC, as measured by centrifugation. In addition to the addition of salt,

lecithin was also added to the batters as an emulsifier in a second set of
experiments. Addition of lecithin decreased the viscosity of the batters
over the entire range of salt concentrations studied. It also lowered cen-
trifugation losses. Thus, salt increased the viscosity and WHC, while
lecithin decreased the viscosity but increased the WHC. Both batter ingre-
dients exhibited different relationships to centrifugation losses and viscos-
ity. However, the opposing relationship between centrifugation "' losses and
viscosity have also been reported in other studies. Pre-rigor, salted meat
has a low viscosity but an excellent WHC due to a low interaction
between actin and myosin (Hamm, 1972). The same situation applies on
addition of di- or polyphosphates to salted meat (Hamm, 1972).
Viscosity is an expression of the interaction between molecules and par-
ticles within an unheated batter. As the examples above indicate, the visc-
osity or interaction between batter components and centrifugation losses
of unheated batters as an expression of WHC may give conflicting results.
5.8.3 Water-holding capacity of heated batters

If viscosity measurements are not always in accordance with centrifuga-
tion losses, how does centrifugation relate to cooking losses? Centrifuga-
tion and heating losses are not always in accordance with each other
(Honikel and Hamm, 1983). Batters with or without lecithin (Figure 5.21)
give different results. Centrifugation losses in batters with lecithin are
always lower than those without lecithin, while cooking losses of batters
with lecithin have higher salt concentrations (above 1.2%) and a higher
cooking loss (cookout) than batters without lecithin. Lecithin, which is an
emulsifier, interacts with hydrophobic regions of the meat proteins
(Honikel and Hamm, 1983), decreasing the ability of the batter to form a
stable gel for coating the hydrophobic surface of the proteins. In unheated
batters, however, lecithin lowers the viscosity by reducing the attractive
forces between protein molecules.
In conclusion, using viscosity and centrifugation measurements as
methods to predict the WHC of heated batters may result in erroneous
28
24
.-.i. 20
• • <III
<J
>.
.~ 12
• • 15
~
0
8 10
- 0 ___
- --0
d
5
1.0 2.0 3.0 4.0

% Noel in meat + ice
Figure 5.21 Influence of salt concentration and lecithin on jelly and fat rendering. The
batter was heated to 95°C within 45 min (open symbols without lecithin; closed symbols
batter with 0.5% lecithin).
predictions of cooking stability. The latter should be measured preferably

by cooking batters under similar conditions, as used for sausage manu-
facturing.
5.8.4 Measurement of water-holding capacity in sausage batters

In principal, the same methods are used as described in the section on
cooking loss measurement in this chapter. Since the rendering of fat will
often occur, the quantity of batters used should be larger than that of the
meat, as weighing of two fluids is difficult. Measuring the volume of the
aqueous phase and the fat phase requires more material. Thus, in actual
tests, a sample of 100-200 g of batter is stuffed in impermeable casings or
in cans and heated to the desired temperature. The batter is then cooled,
preferably overnight and rewarmed to about 45-50°C. (The cans should
not be opened at high temperatures.) Some juice will be lost by over-
pressure. The jelly and fat are fluid and can be poured via a funnel into a
measuring cylinder. After a few moments, jelly and fat will separate and
the volume can be easily measured.
5.8.5 Evaluation of methods

Owing to the changes in the meat proteins described earlier, none of the
methods used for WHC measurements of the raw meat will give a hint
about the WHC of the meat in a cooked sausage batter. If there is any
relationship between the WHC of the meat and batters, the pH of the
meat, which also influences the pH of the batter, will be the decisive
factor. It is easier to measure the pH of the meat than its WHC.
Some papers have reported an intermediate method for WHC between
the meat and batter. The meat is minced or comminuted with salt and
water and heated. The results allow some conclusions but the cookout is
usually somewhat higher than that after comminuting with fat. This can
be explained by the positive action of the fat in batters. The fat particles
stabilize the batter, acting as spacers in the protein network.
In conclusion, the WHC of batters can be measured best in pilot
charges of batters. Predictions by other methods always have their short-
commgs.
5.9 Conclusions about water-holding capacity measurements
WHC is due to three factors: (i) the various forms of water in meat; (ii)
compartmentalization within the cellular and subcellular structures of
meat; and (iii) the changes occurring post-mortem that alter the amount
of water in different forms and compartments.
WHC can vary widely. As the methods of measurement apply various

forces, different amounts of water are released from the meat. For
example, in drip-loss measurements, a small percentage drip may exude
from the surface within 24 h, while with the FPPM, 60% of the water is
squeezed out within a few minutes. Therefore one should avoid the claim
of measuring the WHC of meat, but report that drip losses or cooking
losses have been measured. The proper method should also be used.
Cooking-loss measurements in slaughter plant are unnecessary but drip-
loss measurements would be the appropriate method. A plant that
produces ready-to-eat meals must cook its meat under conditions similar
to those used to produce its products. In all publications, the methods
must be described carefully.
5.10 Measurement of juiciness
5.10.1 Problems in evaluating juiciness

Evaluation of juiciness can be regarded as another way to evaluate WHC
as stated earlier. Expression of juiciness in objective terms, as with WHC
by applying physical methods, has not been reported to date. Several
obstacles appear to prevent this, including the following.
1: Juiciness is a part of mouthfeel, which is influenced by tenderness and
aroma, so it may be changed by these impressions.
2. Mouthfeel includes the sensation of fat, which enhances the impression
of juiciness.
3. There are no definite units for expressing juiciness, such as ml or per-
centage in WHC.
4. Since there is not an objective measurement, calibration of taste panels
is difficult or impossible.
5.10.2 Evaluation of methods

These problems can be overcome by a series of reference samples with
which the taste panel can be calibrated. However, calibration must be
repeated from time to time as no one can keep reference meat samples for
long. Owing to the many factors influencing mouthfeel, e.g. fat content,
juiciness cannot be related to the water content, neither in the uncooked
nor the cooked meat. If any relationships exist, they are, in most cases,
due to other attributes, such as pH or toughness from cold-shortening.
Thus, evaluation of juiciness by a good, trained taste panel can be
reproducible and result in reduced variability; however, because of the
problems outlined above it will not have the reliability of well-defined
chemical and physical WHC measurements.
Books have been written about sensory evaluation of foods (e.g.

Lawless and Klein, 1991) and hundreds of papers have measured WHC
and evaluated juiciness simultaneously. From these, it becomes clear that
the results are conflicting. The authors recommend that WHC and juici-
ness evaluations be examined separately, since both have some" common
but many different factors that influence them.
5.11 Summary
Water-holding capacity of meat is the ability of meat to hold all or part of

its own and added water. The chemically bound portion of water in meat
is rather small. Fibrillar protein structures, which are the cellular struc-
tures of muscles, and capillary forces retard or prevent the movement of
water molecules. The main water-retaining meat compounds are the myo-
fibrillar proteins, which are organized in myofibres.
The amount of water immobilization depends on the pH value, which
falls from about 7.0 in live muscle to around 5.5 in meat. The minimum
water-holding capacity of meat exists around pH 5.3 since this is near the
isoelectric point of the myofibrillar proteins.
Measurement of water-holding capacity is performed in many ways,
which can be divided into three chief areas: (i) no force is applied other
than the gravity of earth - these methods (e.g. drip-loss measurement)
require a considerable length of time; (ii) mechanical forces such as
pressure or increased gravity speed up the methods described - centrifuga-
tion-loss measurements or the filter paper press method belong to this
group; and (iii) thermal forces release water from the meat upon heating.
The various forces applied cause release of different amounts of water.
A few percentage up to 60% of the weight of meat are released during
measurement. Due to this wide range, comparision of methods is limited.
Therefore, methods should be stated clearly. Also the proper method for
the specific purpose must be used.
Sensory evaluation of juiciness can be regarded as another way to
evaluate water-holding capacity. Even though the results of determining
the water-holding capacity with physical methods can vary among
methods, it is possible to calibrate the methodology and instruments, not-
withstanding the fact that human senses are difficult to calibrate.
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6 The chemical senses
G.K. BEAUCHAMP and J.G. BRAND
6.1 Introduction
The flavor of a food can be defined as the combination of its taste, smell,
chemical irritation (the common chemical sense), and temperature. In this
chapter, the contribution of taste and smell will be discussed. In the first
sections, basic anatomy and physiology of the chemical senses will be
described. Some indication of the rapid strides in understanding reception
and transduction will also be provided. Much of this information has
recently been reviewed in detail by Brand and Shah (1992). Leaning
heavily on this review, in the beginning of this chapter an overview of this
rapidly advancing area will be provided. In subsequent sections the psy-
chological aspects of the chemical senses, with particular emphasis on the
basis for preference and aversions will be concentrated upon. In the final
section issues related to specific appetites for salt and protein and to
mixtures will be dealt with briefly.
6.2 Anatomy and physiology of the chemical senses: taste
6.2.1 Overview
Although it is still controversial (Erickson and Covey, 1980), most investi-
gators (McBurney and Gent, 1979) believe taste to be composed of a
restricted set of qualities or categories, namely sweet, sour, salty, bitter
and perhaps a few others, such as umami, the flavor associated with
monosodium glutamate (MSG). It is hypothesized that this small number
of qualities evolved because of the fundamental importance of the primary
stimuli (e.g. sugars, acids, sodium chloride, bitter toxins) in nutrient selec-
tion (Beauchamp and Mason, 1991).
One practical consequence of this classification scheme in taste research
has been to limit the range of stimuli examined, thus some potentially
interesting classes of chemical stimuli, such as amino acids, have not been
used so often as might be desirable. A second consequence is that anato-
mical and biophysical studies of taste have concentrated mainly on these
primary stimuli. Although this is reasonable and has led to substantial
advances in our understanding, it has also served to limit our future
CHEMICAL SENSES 163
knowledge. Although considerable work on amino-acid detection and

transduction exists in studies of aquatic organisms (Brand et al., 1989),
more work with amino acids in mammals, including humans, is needed.
6.2.2 Anatomy
The sense of taste is initiated by specialized neuroepithelial receptor cells
embedded in taste papillae located in the oral cavity. In humans, taste
receptors are innervated by three cranial nerves. Papillae of the anterior
two-thirds of the tongue are innervated by the chorda tympani branch of
the VIIth cranial nerve. The posterior one-third, including the receptor
cells of the vallate and foliate papillae, is innervated by cranial nerve IX.
Cranial nerve X (the vagus) innervates receptors on the soft palate, glottis
and epiglottis. There is some overlap among these fields. Evidence
suggests that some portions of the tongue are more sensitive than others
to particular qualities, for example, the front being sensitive to salt and
sweet and the rear more responsive to bitter, although all qualities can be
detected in all regions (Miller and Bartoshuk, 1991).
Taste cells have a life-span of approximately 9-14 days (Beidler and
Smallman, 1965; Farbman, 1980). They tend to respond to more than a
single class of chemicals such as salts, bitters and so on (Ozeki and Sato,
1972; Sugimoto and Teeter, 1991), and single fungiform papillae, contain-
ing even a single taste bud, are also responsive to multiple taste qualities
(Harper et aI., 1966). This suggests that single taste cells contain receptor
and transductive elements that allow them to recognize more than a single
taste quality.
Taste cells serve to change the firing rate of sensory nerves located in
close contact with them. These sensory nerves first synapse in the hind-
brain in the nucleus of the solitary tract. Taste signals are processed here
and elsewhere in the central nervous system (eNS, Scott, 1987). Taste
information is integrated with other visceral sensations in the mid-brain
and plays a critical role in regulating food selection (Friedman et al.,
1991).
6.2.3 Reception and Transduction

Each classical quality makes apparent use of a unique transductive
sequence and has associated with it a set of stimuli with varying, but in
many cases, internally consistent structural attributes. This diversity of
transductive sequences and structural or physical similarities of stimuli
supports the existence of classical taste qualities (Kinnamon, 1988; Teeter
and Cagan, 1990) as do electrophysiological and behavioral studies
(McBurney and Gent, 1979).
6.2.3.1 Salt taste: reception and transduction. Only NaCl (salt) and LiCI
are described as primarily salty at concentrations above 0.1 M (Murphy et

at., 1981). It has been impossible thus far to find an acceptable tasting salt
substitute. This implies that the reception/transduction events for salt are
likely to be highly specific.
It is now generally accepted that salty taste transduction occurs via a
sodium/lithium epithelial ion channel that is sensitive to the diuretic
amiloride (Schiffman et at., 1983; DeSimone et aI., 1984, 1989; Brand et
at., 1985; Avenet and Lindemann, 1989). An influx of sodium (Na) into
the cell depolarizes the cell membrane leading to alterations in voltage-
sensitive ion channels, which ultimately modulate intracellular calcium ion
activity leading to neurotransmitter secretion. Sodium ions are removed
from these cells via a Na-K ATPase pump (DeSimone et at., 1989).
Amiloride does not completely eliminate responses due to NaCl. This
suggests a stimulatory action of the chloride (Cl) anion (Formaker and
Hill, 1988) and/or that anions modulate differentially the diffusion of
sodium to amiloride insensitive or inaccessible sites (Ye et at., 1991).
Amiloride will inhibit the neural response due to NaCl and LiCl in the rat
but not that due to KCl, RbCl or CsCI. The antagonistic activities of
amiloride can be localized to multiple sites or multiple processes. These
recent discoveries concerning the mechanism of salt-taste transduction
may provide avenues to the development of salt enhancers (substances
that heighten the saltiness of low levels of salt) in the future.
6.2.3.2 Sour taste: reception and transduction. Sourness is related to the

solution activity of the proton. However, using pH alone one cannot
predict completely the perceived sour intensity of the solution nor its
overall quality. The anion probably plays a role independent of the
activity of the proton. Perception of sourness has been linked to various
measures of acidity, including titratable acidity (Beatty and Cragg, 1935),
the quality and character of the anion and the undissociated acid (Norris
et at., 1984; Ganzevles and Kroeze, 1987) and, with organic weak acids,
lipophilicity (Gardner, 1980).
The primary sour-taste transduction event in mammals may be an
influx of protons through an amiloride-sensitive channel (Gilbertson et aI.,
1991). The role of the anion in the perception of sourness has not been
studied rigorously at the receptor level. It would appear that it has
multiple roles, since its presence has been linked to effects on taste percep-
tion and salivary flow (Norris et at., 1984; Christensen et at., 1987). The
latter events can alter intraoral pH (Christensen et at., 1987) and may
come about as a result of stimulation of the trigeminal nerve.
6.2.3.3 Sweet taste: reception and transduction. Sweet-tasting compounds

include selected members of such diverse chemical classes as carbohy-
drates, halogenated sugars, amino acids, aminoacyl sugars, peptides and
CHEMICAL SENSES 165
proteins, terpenoids, N-sulfonyl amides, sulfamates, anilines, polyketides,

ureas and heavy metal salts (e.g. lead acetate) as shown by Walters et a/.
(1991). From a structure/activity viewpoint, many sweet-tasting com-
pounds are found to have a proton-donor group at a critical distance
from a proton-accepting group (Shallenberger and Acree, 1967). More
inclusive theories have recently been proposed, with some becoming quite
complex (Walters et at., 1991). The intense search for sweeteners, with its
possible huge market payoff, has provided receptor scientists with several
useful molecular tools that are only just now being exploited.
The transductive events in sweet-taste recognition have only recently
begun to be characterized. The existence of inhibitors for sweet-taste
transduction and the specificity shown by these agents had long argued
for sweet taste transduction being a receptor-mediated event. Yet, no
receptor protein has yet been unequivocally identified.
The sequence of events (transduction) triggered by sweet stimulus
binding to a presumed receptor(s) has been characterized. Sweet-taste
transduction involves activation of a G-protein, which results in produc-
tion of the second messenger cyclic AMP (cAMP). The cAMP produced
intracellularly probably activates a cAMP-dependent protein kinase
(Avenet et at., 1988; Kinnamon, 1988; Tonosaki and Funakoshi, 1988;
Striem et at., 1989; Lancet and Ben-Arie, 1991). Phosphorylation stimu-
lated by the kinase may lead to alterations in the membrane ion channel
activities, which ultimately bring about changes in intracellular calcium
ion activity, which trigger neurotransmitter release.
The question of whether there is more than one sweet-taste receptor is
still open. It is known, for example, that all animals do not share respon-
ses to common chemical stimuli. Some animals, particularly carnivores,
are insensitive to sweet-tasting carbohydrates (Beauchamp et at., 1977).
Omnivores and herbivores, while generally sensitive to carbohydrates that
humans find sweet, may not find them acceptable to the same degree, nor
rank them in the same order of intensity vs. concentration (Kare, 1971).
Among mammals, considerable divergence is found in the ability to taste
artificial sweeteners. Sweet-taste transduction in primates occurs on fibers
relatively specific for this modality, and these fibers carry information
from both natural and artificial sweeteners (Hellekant and Ninomiya,
1991).
6.2.3.4 Bitter taste: reception and transduction. Many compounds with a

great diversity of chemical structure taste bitter. While many of these
compounds can be grouped into major structural classes - the quaternary
amines, acetylated sugars, alkaloids, amino acids (primarily L-isomers)
and certain inorganic salts - no chemical or physical features have been
found that are common to all stimuli. Thus, there must either be more
than a single receptor for bitterness, or no receptors at all but rather these
stimuli achieve their taste effect by altering the lipid/protein environment

of the plasma membrane of the taste cell to such a degree that intracel-
lular ionic changes can take place via a change in membrane voltage
(Kumazawa et al., 1986, 1988). The well-known genetic dimorphism on
the ability to taste the thioureas (PTC, Lawless, 1980) as well as studies
on bitter taste in well-defined strains of mice (Whitney et al., 1991) argue
for a multireceptor/transduction hypothesis but cannot at this time rule
out the non-receptor postulate for at least some bitter compounds.
The receptor and transductive events in bitter taste received little direct
attention until the mid-1980s. At this point, some key observations were
made. The picture that has recently emerged (Akabas et al., 1988;
Spielman et al., 1989, 1991) is one involving a receptor coupled to a G-
protein that activates production of IP 3 (inositol trisphosphate, another
second messenger), which releases calcium ions from internal stores. The
calcium ions and/or the diacylglycerol produced could act to close
outward-going K-channels and activate inward-going Ca2 + channels,
causing membrane depolarization and neurotransmitter release.
It is important to recognize that the initial receptor recognition step is
not absolutely required for transduction of some stimuli so long as they
are capable of causing the release of calcium ions or closure of K-
channels. Thus, quinine, a well-known hydrophobic bitter stimulus and
K-channel blocker, could conceivably act directly on K-channels in the
taste cell. Other hydrophilic bitter-tasting stimuli may presumably act via
a receptor.
6.2.3.5 Other tastes: reception and transduction. Other taste primaries,

such as metallic, soapy, etc. have been postulated from time to time but
their inclusion in a universal taste field has not received wide acceptance.
Many of these 'off-tastes' may be the result of olfactory stimulation or sti-
mulation of the trigeminal nerve and some are considered phantom tastes,
often associated with neurological damage or pathology (Shafar and
Glasg, 1965; EI-Deiry and McCabe, 1990).
One primary taste not associated with the classic four, but perhaps
equivalent to them, is that for the prototypical stimulus - monosodium
glutamate (Kawamura and Kare, 1987). This stimulus imparts a taste
quality often called 'umami', the Japanese word for delicious or savory.
The taste of MSG has the interesting property of being enhanced by
certain 5' -ribonucleotides, thereby forming a true taste synergism (Yama-
guchi, 1987, 1991). That the umami taste is a primary taste quality is sup-
ported by work at the psychophysical level and the receptor level.
Biochemical receptor-binding studies and recent results from reconstituted
taste-receptor membranes suggest the existence of a receptor site for gluta-
mate and a mode of transduction involving an ion channel whose activity
is controlled by stimuli that impart an umami taste, for example, the
CHEMICAL SENSES 167
stimulus, monosodium glutamate (Brand et at., 1991). These types of ion

channels are said to be 'gated' by the stimulus and are often considered to
be receptor/ion channel complexes.
6.3 Anatomy and physiology of the chemical senses: olfaction
6.3.1 Overview
In contrast to the sense of taste, there is no widely accepted classification
scheme for odorous chemicals. In fact, a major unresolved issue in olfac-
tion is whether there exist a few primary odors from which all others are
derived by combinational rules, or whether, as in the immune system,
there are many, perhaps thousands, of more or less independently recog-
nized odors. Although recent evidence from molecular biological studies
(outlined later in this chapter) argue in favor of the latter hypothesis, this
question is still an issue. A practical consequence of this scientific disarray
is the absence of an agreed set of stimuli to use in sensory tests. Thus,
olfactory stimulus selection tends to be based upon availability and
presumed safety or upon presumed ecologically significant odors, such as
food odors.
Also unlike the sense of taste, olfaction serves functions in addition to
those involved with foods. For many organisms it is intimately involved in
inter- and intraspecies communication. Sexual and social activities are
organized around odors and olfactory information transfer (Macdonald et
at., 1991).
6.3.2 Anatomy
Unlike taste receptor cells, which are innervated epithelial cells, the
receptor cells for the sense of smell are bipolar neurons of the olfactory
nerve, cranial nerve I. The receptor elements for olfactory stimuli are
located on the ciliary processes that emanate from a terminal knob on
olfactory receptor neurons. These neuronal receptors have a lifetime of
approximately 30 days and are the only neuronal cell type that is con-
tinuously renewed in the adult. Renewal and replacement of olfactory
receptor cells ensures continued chemosensitivity in spite of environmental
assaults on these receptor areas. The cilia of the olfactory neurons float in
mucus and detect odors that are solubilized in the mucus sheet. The
proximal end of the olfactory neuron makes synaptic contact with mitral/
tufted cells in the glomeruli of the olfactory bulb. Processing takes place
within the bulb before the signals are sent out of the bulb to the CNS for
further processing (Scott and Harrison, 1987). It is presumably within the
CNS that olfactory information is integrated with taste information (as
168 CHEMICAL SENSES
well as other sensory information) to provide an overall sensation of

flavor.
6.3.3 Reception and transduction

It has been estimated that several hundred to thousands of different odors
can be recognized. Yet, these estimates are based on very little true
experimental evidence. In reality, no one knows how many qualitatively
distinguishable odors exist. The reception and transduction events for
odors, which play an important role in food flavors, are now beginning to
be understood for the first time. It is now apparent that the initial recog-
nition of odors occurs on one or more of many receptors. These are
linked to one of at least three transduction mechanisms: (i) increased pro-
duction of cAMP; (ii) increased production of IP 3 ; or (iii) activation of a
stimulus (odor)-gated ion channel.
The existence of receptors that bind olfactory stimuli and are unique to
the olfactory epithelium had been postulated for many years. The recep-
tors have been characterized biochemically from several models, most
notably from aquatic species (Rhein and Cagan, 1983; Bruch and Rulli,
1988; Novoselov et al., 1988). Nevertheless, the molecular nature of these
olfactory receptors remains elusive. Recently, Buck and Axel (1991)
reported the cloning of nearly two dozen members of a large multi-gene
family of putative olfactory receptors from the rat. These clones encode
integral membrane proteins of the seven transmembrane domain types.
Subfamilies of these clones can be arranged that exhibit sequence diver-
gence within the transmembrane domains, which suggests that each sub-
family may bind distinct structural classes of odor stimuli.
From biochemical studies showing odorant-stimulated production of
second messengers, it has been hypothesized that olfactory receptors
interact with GTP-binding regulatory proteins (Pace et aI., 1985; Huque
and Bruch, 1986). At least one G-protein is unique to the olfactory system
of the rat (Jones and Reed, 1989), while another may be unique to the
olfactory system of catfish (Bruch and Kalinoski, 1987). These G-proteins
modulate production of second messengers, at least two of which have
been implicated in olfactory transduction. One of these second messen-
gers, cyclic AMP (cAMP), is produced in response to a wide variety of
odor stimuli (Pace et aI., 1985; Sklar et al., 1986; Breer et al., 1990), while
another, IP 3, is produced primarily, although not exclusively, in response
to odors that impart a putrid or otherwise unpleasant sensation (Boekhoff
et al., 1990; Breer and Boekhoff, 1991).
Metabolic and ion-channel events subsequent to the formation of
cAMP and IP 3 indicate that the olfactory system contains ion channels in
the ciliary membrane, which are stimulated directly by cAMP and IP 3 and
that phosphorylation events and alterations in intracellular calcium-ion
CHEMICAL SENSES 169
activity are important regulatory and perhaps desensitization mechanisms

in olfaction. Nakamura and Gold (1987) discovered a cAMP- and cyclic
GMP (cGMP)-stimulated ion channel in olfactory cilia, while Restrepo et
al. (1990) reported the existence of an IP 3-stimulated ion channel in olfac-
tory cilia. The former is a non-specific cation channel, while the latter is
primarily a calcium-ion channel. Calcium entry into olfactory cells from
the extracellular (mucosal) space is observed upon stimulation by some
odors (Restrepo et al., 1990).
6.4 Sensory responses to food: taste
6.4.1 Development of taste preference

This topic has been reviewed in detail (Beauchamp et al., 1991a); this
section will provide an overview and summary of that review. Taste cells
first appear in the human fetus at 7-8 weeks of gestation and morpholo-
gically mature cells can be found at about 14 weeks (Bradley, 1972;
Bradley and Stern, 1967). Fetal taste receptors may be stimulated by che-
micals present in the amniotic fluid. Reported differential fetal swallowing
following injections of sweet or bitter substances into the amniotic fluid of
pregnant women may suggest that fetuses may show a preference for
sweet and a rejection of bitter (De Snoo, 1937).
Direct studies of taste in the pre-term infant are rare. Tatzer et al.
(1985) studied non-nutritive sucking of pre-term infants exposed to brief,
intraoral presentations of very small amounts of glucose solution and
water. They found that glucose solution stimulated more non-nutritive
sucking than water. Recently, Maone et al. (1990) developed a method for
administering a taste to infants that does not necessitate the delivery of
any fluids and may permit more extensive study of taste perception in pre-
term infants. The taste substance is embedded in a nipple-shaped gelatin
medium that releases small amounts of the substance when it is mouthed
or sucked. Infants born pre-term and tested between 33 and 40 weeks
post-conception produced more frequent, stronger sucking responses when
offered a sucrose-sweetened nipple compared with a latex nipple. It
appears that infants who were born prior to term and have had little or
no extra-uterine taste experience possess the ability to detect sucrose and
glucose and show an avidity for it that might be attributed to a hedonic
process.
At birth, not surprisingly, recognition of many tastes is well developed.
Steiner (1977) has reported that consistent, quality specific facial expres-
sions can be elicited in the first few hours of life by sweet (sucrose,
0.73 M), sour (citric acid, 0.12 M) and bitter (quinine sulfate, 0.003 M)
stimuli (see also Ganchrow et al., 1983). Steiner (1987) has also reported
that distinctive positive facial expressions are elicited by soup to which

MSG has been added compared with the soup diluent alone. MSG alone
does not appear to elicit those facial responses, however, the question of
exactly what it is about the soup-MSG mixture that is of apparent
positive hedonic valence remains.
Rosenstein and Oster (1988) have conducted a recent detailed study of
facial response to taste stimuli. They report considerable overlap in the
distribution of not only discrete facial actions but also full-face configura-
tions elicited by the different taste stimuli. Considerable individual varia-
tion in facial responses was also observed. Moreover, although they found
that hedonically negative facial components were more common in
response to all three non-sweet tastes than in response to sweet tastes,
untrained observers found only the responses to the bitter taste to be
highly negative. Even with their fine-grained analysis and a concentrated
salt stimulus (0.73 M), Rosenstein and Oster (1988) were not able to
identify a distinctive facial response to salt.
Differential intake has been used more frequently than any other
response to study taste preference in infancy. Studies have demonstrated
repeatedly strong acceptance of sweet tastes by infants at birth and shown
that newborns respond to even dilute sweet tastes and can differentiate
varying degrees of sweetness (Desor et al., 1973, 1975b). However, they
have generally failed to demonstrate differential response to relatively low
concentrations of sour or bitter stimuli, as well as to salt. This absence of
differential response to salt is consistent with results from the studies of
facial expression described above. The absence of response to bitter and
sour could be due to some combination of methodological and stimulus
concentration differences (Beauchamp et al., 1991a). Finally, as was the
case for premature infants the study of sucking patterns has been used as
an alternative to the measurement of intake in newborns. Results from the
work are generally consistent with use of other methods (Beauchamp et
al., 1991a).
There are relatively few studies of taste perception in older infants and
children compared with neonates. In the context of studies on effects of
protein-calorie malnutrition on taste, Vazquez et al. (1982) evaluated
sucrose, citric acid, urea and salt acceptability in well-nourished and mal-
nourished Mexican infants 2-24 months of age. They reported that,
relative to water, infants under 1 year of age preferentially ingested 0.1
and 0.2 M NaCl. A preference for sucrose was observed for infants at all
ages. The bitter (urea: 0.12 M and 0.24 M) and sour (citric acid: 0.006 M
and 0.012 M) stimuli were tested with mild sucrose solutions as diluent.
Citric acid was rejected relative to the diluent as were both concentrations
of the bitter urea.
Since newborn infants are indifferent to or reject salt, whereas older
children clearly prefer salty foods, a developmental change in response to
CHEMICAL SENSES 171
salt is likely. This suggestion of a developmental shift in salt acceptability

was supported in studies of Beauchamp et al. (1986) who reported that in
a sample of children from the USA, preferential ingestion of salt water
relative to plain water emerged at approximately 4 months of age. Experi-
ence with salty tastes probably does not play a major role in the shift
from apparent indifference to salt at birth to acceptance in later infancy;
rather, this change in response may reflect post-natal maturation of
central and/or peripheral mechanisms underlying salt-taste perception,
allowing for the expression of a largely unlearned preference for saltiness.
Following infancy and childhood, responses to taste continue to exhibit
changes with age. Children exhibit preferences for higher concentrations
of sweet (Desor et al., 1975a; Desor and Beauchamp, 1987) and salty
(Beauchamp and Cowart, 1990) relative to adults. Peripheral differences
are not known to underlie these differences in preference, which may
instead reflect larger physiological need for calories and minerals in
younger organisms.
Hedonic responses to many taste stimuli are clearly innate as they are
evident in newborn and premature infants. However, sensory experience
may act to modify responses to taste. Very few studies have tested this in
humans, although there is some evidence that experiences mold our taste
preferences.
For infants and children, Harris and Booth (1985) have reported some
evidence that the amount of salt an infant is exposed to may influence its
willingness to consume salty foods. This study remains tentative, however,
due to small sample size. Similarly, Beauchamp and Moran (1982)
reported some effects of dietary experience on acceptability of certain
sweet stimuli but this effect was very specific and not likely to be involved
with overall preference for the sweet taste.
There are several studies with adults indicating that the preferred level
of certain tastes are modulated by experience. The most compelling case is
for salt where evidence indicates that the optimal level of salt in food is a
function of an individual's habitual level of consumption. When experi-
mental subjects are placed on lowered sodium (e.g. Bertino et al., 1982;
Blais et al., 1986) or increased sodium (Bertino et al., 1986) diets, their
preferences are changed such that optimal salt levels decline or increase,
respectively. In a correlational study, Moskowitz et al. (1975) reported
that among Indians who consume a diet that is high in sour and bitter
components, there is an elevated preference for these tastes compared with
Indians on less bitter/sour diets.
6.4.2 Effects of aging

Considerable work has recently been devoted to the study of aging and
taste perception and preference. This has been reviewed (Cowart, 1989;
Bartoshuk, 1989) and will only be discussed briefly here. In general, taste
sensitivity is relatively resistant to the effects of aging. The ability to detect
low concentrations of sweet, sour, salty and bitter stimuli, although
perhaps declining somewhat (and perhaps differentially according to the
quality), remains remarkably intact in the elderly (Cowart, 1989; Bar-
toshuk, 1989). Some work (Cowart, 1989; Stevens et al., 1991) suggests
that older people perceive the intensity of stronger tastes in food as well
as in pure water as less intense, suggesting more loss than had previously
been indicated; however, more work is needed in this area. It is well-
known that older people often complain that food no longer 'tastes' good
but whether this is due to changes in taste per se, olfaction or is due to
non-sensory factors still needs to be determined.
6.S Sensory responses to food: olfaction
6.5.1 Development of olfactory preference

This area has also been reviewed recently (Beauchamp et al., 1991a;
Mennella and Beauchamp, 1992) and will be summarized here. The ability
to detect odors may develop prenatally. The olfactory bulb and receptors
have the adult pattern by the middle of the 11 th week of gestation.
Several lines of evidence (Beauchamp et al., 1991a) suggest that around 28
weeks the olfactory system is capable of detecting chemical stimuli
although definitive studies remain to be conducted.
Several studies have shown that infants can detect and discriminate
among a variety of qualitatively distinct odors (Beauchamp et al., 1991a).
Collectively, these early studies indicate that infants respond with respira-
tory and/or behavioral changes to a wide variety of odorous chemicals.
Some evidence suggests that the neonate may also have remarkable sen-
sitivity to odors such as the homologous alcohols (e.g. Rovee, 1972).
Newborn infants respond with a suppression of motor activity when pre-
sented with the odor of their mother's breast or neck. This suggests excep-
tional olfactory acuity in the newborn since such discriminations are
difficult for adults.
Although newborn infants clearly can detect odors, there is some con-
troversy as to the development of preference for and aversion to odors.
This is particularly puzzling since hedonic valence is the most salient psy-
chological attribute of an odor for adults (Schiffman, 1974) and anatomic
olfactory structures have relatively direct access to brain structures that
govern emotion (Kandel and Schwartz, 1985). Innately determined
hedonic reactions to odors could be hypothesized to playa role in mediat-
ing components of food selection.
Engen (1982) has argued that all odor preferences and aversions are
CHEMICAL SENSES 173
learned through associational learning processes; even the smell of a

skunk would be perceived as pleasant if experienced under appropriate
conditions. This view is primarily based on several studies that failed to
find adult-like odor preference patterns in children less than 5 years of age
(Engen, 1982).
Methodological difficulties could contribute to these apparent age-
related differences. Recent studies employing more age-appropriate
methods provide partial support for this hypothesis. Essentially, adult-like
odor-preference patterns were obtained for a set of nine odorants when 3-
year-old children were asked to give 'good smells' to a likeable television
character (Big Bird) and 'bad smells' to a grouchy character (Oscar the
Grouch) (Schmidt and Beauchamp, 1988).
In fact, some of the neonatal studies implicate affective odor reactions
in early infancy. Although the tendencies of newborn infants to turn
toward maternal odors and away from the smell of ammonia may reflect
a tendency to avoid any novel or strong olfactory stimuli, they may also
be associated with, or mediated by, hedonic reactions. Additionally,
Steiner (1977) has reported that a variety of food-related odors elicited
adult-like facial expressions of 'enjoyment' and 'disgust' in newborns with
no prior feeding experience. However, other scientists have failed to
observe such reliable facial reactions with other odorants of opposite
hedonic valence (Beauchamp et aI., 1991a).
The issue of whether very young infants experience odors as pleasant or
unpleasant, as they seemingly do for certain tastes, must be considered
open. Further research in this area is needed.
Following infancy, there is some evidence that preference and aversions
change throughout adolescence and adulthood. Moncrieff (1966) con-
ducted one of the most extensive studies on the development of olfactory
preferences to date. One approach entailed testing many individuals (n =
559), ranging in age from 4-82 years, with 10 odors. Subjects were asked
to rank the odors on the basis of 'pleasantness'. Moncrieff (1966) reported
that substantial changes in the ranking occurred as a function of the sub-
ject's age, with the greatest changes occurring during the first 20 years of
life. In particular, strawberry was more likely to be judged the most
pleasant odor by subjects under 20 years of age than by those older. In
contrast, lavender odor and neroli odor (orange blossom) increased in
ranking among subjects over 20 years of age. Other developmental gen-
eralizations were that odors commonly disliked by adults (e.g. rape oil,
chlorophyll) were best tolerated by children younger than 8 years and that
there were substantial age-related changes during adolescence and adult-
hood to the odor of musk-lactone, a presumed 'sexual smell'.
In 1986, the National Geographical Society and the Monell Chemical
Senses Center conducted the largest smell survey in history (Gilbert and
Wysocki, 1987; Wysocki and Gilbert, 1989). The survey, completed by
approximately 1.4 million people world-wide, consisted of a 'scratch and

sniff' test with questions regarding the detection, identification, intensity
rating and perceived pleasantness of six odorants. Pleasantness ratings
showed some variation as a function of age and odorant but there was
remarkable consistency in the overall ordering of odorants across age
groups. That is, the same ranking of the most pleasant to the least
pleasant odor was evidenced in virtually all of the age groups. These data
suggest that, while there is heterogeneity in the perception of odor plea-
santness across the life-span as exemplified by Moncrieff's (1966) study,
there is considerable consistency as well.
Finally, in a recent series of developmental studies of one odor, the
steroid androstenone, it was reported that the ability of individuals, parti-
cularly males, to perceive this odor appeared to diminish with age between
3 years and young adulthood (Schmidt and Beauchamp, 1988; Dorries et
ai., 1989). It appears that virtually all young children can detect this odor,
which is characteristic of 'boar taint' but that by post-adolescence
approximately one-half or more of all males can no longer detect it.
6.5.2 Effects of aging

There is now a large body of research demonstrating that people lose their
ability to detect and identify odors as they age (Murphy et aI., 1989). This
loss appears to represent a sensory as well as cognitive decline in function.
There is some controversy as to whether this decline in sensitivity is
uniform across odors (Cain and Stevens, 1989) or is more evident for
some odors compared to others (Wysocki and Gilbert, 1989). It may
require extensive, ideally longitudinal, studies to resolve this issue.
The mechanism(s) underlying the decline with age is still unknown
(Doty, 1989). Since olfactory receptors are localized on the cilia of bipolar
neurons that synapse in the olfactory bulb and exhibit the unusual
property of regeneration, the decline in olfactory function with age could
be the result of a declining ability to produce new receptor or cellular
elements and/or more rapid dying-off of older elements. Alternatively or
additionally, the receptors and/or their transductive elements could
become less efficient with age. Using modern techniques of biophysics, this
hypothesis could be investigated with very small biopsy samples of tissues
from individuals of different ages. Finally, the decline in olfactory
function could represent changes at more central stages of the olfactory
pathway.
The practical effects of olfactory loss with age are most evident in the
loss of an appreciation for the subtle flavors of foods. Since their loss is
usually gradual, many people do not recognize the source of the problem
but a common complaint among the elderly, as mentioned earlier, is that
food does not 'taste like it used to.' This most likely is correct. The devel-
CHEMICAL SENSES 175
opment of strategies to increase flavor intensity for foods consumed by

older persons has been suggested to overcome these problems (Schiffman
and Warwick, 1988) but the efficacy of this reasonable recommendation
has not been tested widely. Since the population of many of the highly
industrialized countries (e.g. USA, much of Europe, Japan) is, on average,
growing older, this issue will only grow in significance.
6.6 Specific Appetites
6.6.1 Salt
Specific appetite can be defined as a desire to consume a nutrient for
which an organism has a need. The classic example of this has been the
appetite for salt in salt-depleted animals. This area has been extensively
reviewed by Denton (1982). Briefly, many studies demonstrate that herbi-
vorous animals, that are often sodium deficient, will go to great length to
obtain salt. Evidence from herbivores as well as from studies with rats
indicates that the ability to recognize the needed nutrient (Na) is innate;
the first time a salt-depleted rat tastes salt solution it responds to it with
avid consumption.
Much study has gone into attempts to understand behavioral and neu-
rophysiological control of appetite for salt in salt-depleted animals
(Denton, 1982; Epstein and Sakai, 1987; Stricker and Verbalis, 1988).
Several generalizations come out of this work. First, there are substantial
species differences in the mechanisms stimulating salt ingestion; changes in
specific hormones (e.g. angiotensin, aldosterone), temporal variations in
exposure to salt (Hill and Prezkop, 1988) as well as sodium levels in body
fluids have been implicated. Surprisingly, even after all the research effort
devoted to this area, controversy still exists attesting to the complexity
involved even in such an apparently simple system as regulation of sodium
balance. In fact, recently Tordoff et al. (1990) have demonstrated that salt
appetite is stimulated as much or more by calcium depletion compared
with sodium depletion. The explanation for this intriguing finding remains
to be elucidated.
For humans, the evidence that a specific appetite for salt is stimulated
by sodium loss is surprisingly weak. A recent review of the clinical litera-
ture (Beauchamp et al., 1991 b) was able to find only a few cases where
documented salt-wasting led to substantial increases in salt consumption.
Interestingly, almost all of these cases had their beginnings in childhood.
This, combined with some animal evidence (e.g. Hill and Prezkop, 1988)
suggests that the immature organism is particularly sensitive to the effect
of sodium depletion.
The few experimental studies with adults support the view that sub-
stantial salt loss is not necessarily followed by an elevated desire for salt.
In one study (Beauchamp et al., 1990), volunteers were depleted of sodium
by feeding them very low sodium diets and treating them with sodium-
depleting diuretics. Sodium loss was monitored and verified by urinary
loss. Following substantial sodium depletion, subjects exhibited a
moderate increased desire for foods high in salt but very few expressed a
specific desire to consume salt itself. Perhaps even greater depletion would
have led to a frank desire to consume pure salt. In summary, sodium
depletion (as well as calcium depletion) in many non-human animals
reliably elicits a drive to consume salty-tasting substances. In humans, a
similar phenomenon has been observed in some clinical cases but this
requires depletion early in life and/or depletion so extreme as to be very
rare under most conditions.
6.6.2 Amino acids and proteins

A substantial body of animal model studies indicates that deprivation of
essential amino acids or their imbalance leads to depression of food
intake. Surprisingly, it has also been found that if animals are offered a
choice between a diet that is protein-deficient and a diet that has an
imbalance of amino acids, they will choose the former even though it
cannot survive on the protein-free diet (Rogers and Lueng, 1977). It
would appear that the major mechanism underlying an animal's ability to
recognize amino-acid imbalances is through learning. Apparently, unba-
lanced diets lead to a feeling of malaise or illness. This stimulates the
animal to investigate and ingest available substances. If anyone of these
contain the missing amino acids, an association is formed between the
flavor of that substance and recovery from illness. Subsequently, the
animal exhibits a preference for that flavor. Fuller discussions of these
issues can be found in Rozin (1976).
Whether there exists a specific appetite for protein is controversial. It
has been argued that protein-depleted rats exhibit an innate recognition
and preference for several protein sources (e.g. Deutsch et al., 1989; Hein-
richs et al., 1990), although learning has also been implicated (Gibson and
Booth, 1985). To our knowledge, very few human studies have been pub-
lished that have investigated sensory changes in individuals depleted of
protein. Vazquez et al. (1982) and Beauchamp et al. (1987) reported that
protein-calorie malnourished infants exhibited an elevated preference for
casein hydrolysate solution in a soup diluent relative to well-nourished
and recovered malnourished control infants. Both malnourished and well-
nourished infants preferred the soup with added MSG compared with the
soup alone. In unpublished studies, the authors attempted to place adults
on very low protein diets (6% calories from protein) for several weeks and
determine whether increased desires for protein-rich foods were observed
CHEMICAL SENSES 177
(R.D. Mattes and G.K. Beauchamp, unpublished data). Results were

mixed; some individuals reported a craving for high-protein foods whereas
others did not. The diet necessary to reduce protein intake to approxi-
mately 6% of calories was highly unpalatable and compliance was difficult
to monitor. The issue remains open.
6.7 Chemosensory mixtures
Most of the research previously discussed has involved sensory responses

to individual chemicals that have tastes or smells. This focus, which is
characteristic of much work in the field, permits the investigator to
control the stimulus and to make specific inferences about mechanisms
and functions. However, the flavor of most foods is the result of combi-
nations of tastes and odors. Much less physiological or perceptual work
has been performed in the area of complex mixtures. However, a good
introduction is provided in Laing et al. (1989).
In general, mixtures of tastes tend to suppress one another although
suppression may not be completely reciprocal (McBride, 1989). For
example, salt suppresses the taste of the bitter substance quinine hydro-
chloride (QHCl), whereas the QHCI has little suppressive effect on salti-
ness (Schifferstein and Frijters, 1992). With the exception of MSG and
certain 5' -ribonucleotides already mentioned, there is little evidence that
one taste substance increases the intensity of another. Thus, taste mixtures
in general tend to be as intense or less intense than the sum of their
unmixed components, and since, as indicated, suppression is not com-
pletely reciprocal, the quality of a mixture cannot easily be predicted from
the qualities of the mixing components.
Odor mixtures, as would be common in the complex aroma of foods,
have been reviewed by Engen (1982), Laffort (1989) and Laing (1989). As
with taste, odors in mixtures tend to suppress each other, some in a non-
reciprocal fashion and others reciprocally. However, it is difficult to
predict how specific odors will interact. Unlike the sense of taste, which
has been described as being analytical (generally the qualities of the taste
stimuli can be discerned in a mixture), in mixtures of odors it is often very
difficult to identify the qualities of the individual components that make
up the mixture (Laing et al., 1991). For example, in mixtures of four or
five volatile substances it may be difficult or impossible to identify any of
the individual components. The reason for this remains unknown. Perhaps
when there is a clearer idea of how the olfactory system operates, as
recent work reviewed above suggests is imminent, researchers will be able
to make sense of and thus develop specific predictive rules for mixtures of
odorous compounds.
Even less is known about the psychological and physiological effects of
mixtures of taste stimuli and odorants (Hornung and Enns, 1989). One
interesting observation is that the sensation of volatile (odor) compounds
of a mixture that is placed in the mouth is generally perceived as originat-
ing completely in the mouth and is labeled a taste, even though it is the
olfactory receptors that are stimulated. Thus, sips of low to moderate
concentrations of citral, which has no taste itself (i.e. cannot be dis-
criminated from water with the nostrils closed), will be identified as
having a distinct taste by individuals with normal olfactory function
(Murphy and Cain, 1980; Rozin, 1982). Similarly, many of the volatile
compounds of a complex food, such as meat, which give it its distinctive
flavor, are localized in the mouth rather than in the nose. This phenom-
enon results in the common misapprehension that people lose their ability
to taste whe'n they have a cold or when the olfactory sense is lost through
disease, accident or aging.
6.8 Conclusions
Under most conditions, humans will not consume foods that 'taste' bad
and will seek out food that 'tastes' good. Thus, the study of flavor - its
chemistry, biology and psychology - is a central task for those interested
in world-wide nutrition.
Great progress has been made in our understanding of the initial
(receptor, transduction) events in taste and smell. This understanding
should provide the knowledge necessary to develop rationally novel tastes,
odors and their enhancers and suppressors. A new era in the creation of
flavors is ahead. Much less well understood is the more central processing
of food and flavor information. Although there is good information on
why some foods are preferred and others rejected, much remains to be
discovered. A further challenge for the future is to understand how the
different sensory characters of a flavor are integrated into the singular
sensation we experience.
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Acknowledgements
Preparation of this review has been supported in part by NIH DC-00882,

DC-00356 and DC-00327; and the Veterans Affairs Department.
7 Flavor and aroma chemistry
G. REINECCIUS
7.1 Introduction
The flavor of meat develops largely through the cooking process. Raw
meat is generally characterized as being salty, metallic and 'bloody' tasting
with a sweet aroma (Wasserman, 1972). During cooking numerous non-
volatile precursors react to form the characteristic taste and aroma one
associates with beef, pork, chicken and so on. These flavor compounds
arise via such major reaction mechanisms as the Maillard reaction and
lipid oxidation. Several other less studied yet key reactions also occur to
contribute additional flavor compounds (e.g. thiamin degradation). The
result is a very complex flavor profile with the total number of volatile
substances being present in cooked meats probably exceeding 1000
(Maarse and Visscher, 1989, 1992).
Different meats have a similar gross composition (e.g. fat, carbohydrate,
protein, etc.) and it is these components that undergo various chemical
reactions to form meat flavor. Since the composition is similar among
meats and they are processed (heated) in a similar manner, meat flavors
from various species have a common basic profile. There is little question
that the various species of meat also have unique sensory notes, which
differentiate the species. These differentiating notes will be discussed in
chapter 9 of this book, while this chapter will focus on the chemistry of
meat flavor, which is common for all species.
The general area of meat flavor has attracted much research over the
years. There are few, if any, foods that have been studied by so many and
for so long as meat. This may be due to any number of reasons but
possibly includes its importance to the human diet, cost, the academic
challenge and the ability to attract research funding. In the last few years
several exceedingly in-depth reviews have been published on meat flavor
(Dwivedi, 1975; MacLeod and Seyyedain-Ardebili, 1981; Danehey, 1986;
Shahidi, et af., 1986; Mottram, 1991). These reviews have discussed analy-
tical approaches for studying meat flavor, precursors and reactions
involved in flavor formation, model reaction systems yielding meat flavor
and volatile compounds found in meats. This chapter will not offer the
depth that is found in the reviews cited so the reader is encouraged to
seek out these reviews if more detail is desired. This chapter will give an
FLAVOR AND AROMA CHEMISTRY 185
overview of meat flavor including a historical perspective and will focus

mainly on the chemistry of basic meat flavor.
7.2 Flavor precursors
Prior to the development of sophisticated gas chromatography/mass spec-

trometry, most research on meat flavor focused on studying meat flavor
precursors. In the late 1950s and early 1960s several investigators noted
that the meaty note of beef was associated with the water-soluble fraction
of beef (Wood and Bender, 1957; Bender et ai., 1958; Kramlich and
Pearson, 1960). Further work in the 1960s focused on determining the
constituents in the water-soluble fraction that were responsible for the
meaty flavor. The dialyzable fraction of meat extracts was found to
produce a meat flavor on heating (Hornstein and Crowe, 1960a; Batzer et
at., 1960, 1962; Wasserman and Gray, 1965). Thus, research focused on
determining the composition of the dialyzable fraction of meat and then
which of the components (or mixtures of components) yielded a meaty
flavor on heating (Macy et ai., 1964a,b, 1970a,b,c,; Mabrouk et at. 1967;
Wasserman, 1979; Wasserman and Spinelli, 1972). Qualitative and quanti-
tative studies on the dialyzable fraction demonstrated that substantial
quantities of free sugars, sugar phosphates, nucleotide-bound sugars, free
amino acids, pep tides, nucleotides, glycopeptides, organic acids, creatine
and creatinine were lost on heating, thus, likely served as precursors of
meat flavor. Of these components, the greatest losses were noted in
cysteine and ribose. This basic research led to one of the classic patents on
meat-like flavors in which ribose and cysteine were heated to generate a
beef flavor (Morton et at., 1960).
Hornstein and Crowe (1960b, 1963) reported that aqueous extracts of
beef, pork and lamb produced similar meaty aromas on heating, while
heating the fats from these same species gave unique species-characteristic
aromas. This observation has led to a generalization (not absolutely true
as will be discussed later) about fats being necessary to give the unique
species notes to meats. This theory was evaluated further by Wasserman
and Talley (1968) who used ground veal as a base for the addition of
adipose tissue from pork, beef and lamb. Each ground mixture was
cooked and then panelists were asked to identify the species of meat based
on the flavor. Panelists were able to identify correctly the species of meat
based on the kind of adipose tissue that had been added.
Hornstein and Wasserman (1987) have summarized the research on
meat flavor precursors as follows.
1. The flavor precursors of lean meat are water-soluble.

2. A non-enzymatic reaction between sugars and amino acids may playa
major role in producing basic meaty flavor.

3. The lean-meat flavor common to meats in general is similar since the
water-soluble fraction of these meats is similar.
4. Intact meat proteins contribute little to meat flavor; and
5. Lipids may contribute to species flavor owing to their unique compo-
sition and by serving as a reservoir for odorous or reactive fat-soluble
components.
7.3 The taste of meat
Raw meat contains several constituents that are non-volatile and con-
tribute to its taste. While these constituents are present in raw meat, they
undergo substantial changes in proportions during the cooking process.
MacLeod (1986) has provided a list of the taste components in raw meat;
this is reproduced in Table 7.1. The sweet notes of raw meat come from
glucose, ribose and fructose. The salty notes come from a variety of inor-
ganic salts as well as sodium glutamate and sodium aspartate. While lactic
acid is the major free acid, several other acids contribute to the acidic taste
attribute. The brothiness is associated with numerous free amino acids as
well as the monosodium glutamate (MSG) and 5'-nucleotides. Both MSG
and the 5'-nucleotides contribute to taste by giving the urn ami character to
meat, as well as serving as general flavor-enhancers for meat flavor.
MacLeod (1986) has provided substantial discussion on the role of these
flavor potentiators in meat flavor, which is summarized below.
Inosine monophosphate (IMP) and MSG are the major flavor enhan-
cers found in raw meat (Figure 7.1). IMP is derived from adenosine-5'-tri-
phosphate (ATP) breakdown during the aging process. IMP and MSG
Table 7.1 Taste compounds present in meat"
Taste Compound
Sweet Glucose, fructose, ribose, glycine, alanine, serine, threonine, lysine, proline,
hydroxyproline
Salty Inorganic salts, sodium glutamate, sodium aspartate
Sour Aspartic acid, glutamic acid, histidine, asparagine, succinic acid, lactic acid,
pyrrolidone carboxylic acid, o-phosphoric acid
Bitter Creatine, creatinine, hypoxanthine, anserine, carnosine, other peptides,

histidine, arginine, methionine, valine, leucine, isoleucine, phenylalanine,
tryptophan, tyrosine
'Umami' MSG, 5'-IMP, 5'-GMP, certain peptides
"Taken from MacLeod (1986).

(b)
oII
(a)
HN
/CjC:~
I I
COOH ~ N
O=~~O-:~
I
H 2 N--C""H
I
CH z f 20
I OH
CH z
I _ +
COO,Na HO OH
Figure 7.1 The structure of (a) monosodium glutamate and (b) inosine monophosphate.
have synergistic properties with each other as well as with certain amino
acids (Mabrouk, 1976) such as glycine, and dipeptides (Baines and Mlot-
kiewicz, 1984) such as asparagine-L-aspartate. The flavor-potentiating
effects of MSG and IMP have been shown to increase the sensory notes
described as meaty, brothy, mouth-filling, dry and astringent. Notes such
as sulfurous and hydrolyzed vegetable protein are suppressed, while sour,
sweet, oily, fatty, starchy and burnt are not influenced (Kuninaka, 1981).
While MSG will undergo some losses during the cooking of meat (e.g.
through the Maillard reaction and conversion to pyrrolidone carboxylic
acid), sensorially significant quantities remain in cooked meats. IMP will
also suffer some loss during cooking of meat but the losses are generally
considered to be minor. Thus, both IMP and MSG are present in cooked
meats and are credited with contributing to the desirable attributes of
meat flavor via their inherent umami character and flavor-enhancing
properties (MacLeod, 1986).
7.4 Meat aroma
As noted earlier, raw meat lacks the aroma properties that generally char-
acterize cooked meat. Cooked meat aroma is formed virtually entirely
during the heating process from non-volatile precursor compounds. The
most important mechanisms responsible for these flavor compounds are
Maillard browning, lipid reactions and thiamin degradation. Of these
reactions, about 90% of the volume of flavor compounds in cooked meat
arise due to lipid reactions. This leaves only about 10% of the volatiles
coming from Maillard browning (Bailey, 1983) and thiamin degradation.
Of these two reactions, the Maillard reaction produces a much greater
number and quantity of volatiles than thiamin degradation. The fact that
both thiamin degradation and the Maillard reaction produce a small
portion of the volatiles in cooked meat does not diminish the contribution
of either of these pathways to meat flavor. It is well-documented in the
literature that the compounds that are present in the least amounts in an
aroma profile may make the greatest contribution to aroma. Sensory sig-
nificance does not depend upon absolute quantity of a compound but its
quantity relative to its sensory threshold and the corresponding relation-
ship between concentration and perceived intensity. Thus, one cannot
underestimate the role of either thiamin degradation or the Maillard
reaction in contributing to meat aroma.
Mottram (1991) noted that there have been more than 120 publications
presenting qualitative data on meat volatiles. He has summarized the
data, which are presented in Table 7.2, and commented on trends. Beef
has the largest proportion of sulfur compounds (20% of the total vola-
Table 7.2 Numbers of volatile compounds of different chemical classes reported in cooked
meats. a.b
Pork
Compound Beef Cured Uncured Lamb/mutton Chicken
Hydrocarbons 193(105) 39(30) 37(14) 43(15) 84(26)

Alcohols and phenols 82(28) 64(38) 25(7) 20(6) 53(24)
Aldehydes 65(9) 38(5) 41(3) 39(4) 83(27)
Ketones 76(21) 32(19) 31(6) 20(1) 53(30)
Carboxylic acids 24(1) 29(3) 30- 51(6) 22(20)
Esters 59(30) 21(12) 33- 11(6) 16(12)
Lactones 38(6) 8(8) 12- 14(2) 24(23)
Furans and pyrans 47(5) 16(9) 28(1) 5- 16(9)
Pyrroles and 39(11) 12(11) 16(12) 19(2) 24(23)
pyridines
Pyrazines 51(3) 22(21) 44(8) 16(3) 22(21)
Other nitrogen 28(1) 22(14) 9- 2- 7(3)
compounds
Oxazoles and 13(6) 3(3) 1- 4(4) 5(5)
oxoazolines
Non-heterocyclic 72- 20(11) 17- 7- 17(1)
sulfur compounds
Thiophenes 35(1) 4- 15(4) 2- 7(3)
Thiazoles and 29(14) 6(4) 17(14) 13(9) 18(18)
thiazolines
Other heterocyclic 13- 4(1) 1- 4- 6(5)
sulfur compounds
Miscellaneous 16(5) 7(6) 4(1) 1(1) 11(6)
compounds
Total 880(235) 347(195) 361(70) 271(59) 468(256)
aTaken from Mottram (1991).

bFigures in parentheses are numbers of volatiles reported for the first time in the particular
meat species since 1983.
tiles) of all the meats. Sulfur compounds appear to playa major role in
determining beef flavor. Lamb is noted to contain more carboxylic acids
than other meats and chicken more lipid-derived volatiles. Aldehydes and
ketones are major classes of volatiles in chicken meat. Cured pork
contains more alcohols and phenolic substances than other meats, pre-
sumably since most cured meats are smoked.
An abbreviated discussion of the mechanisms responsible for meat
aroma will follow. This will be an overview only since, as noted earlier,
numerous current detailed reviews are available in the literature.
7.4.1 Maillard reaction

It is well-documented in the literature that meaty, brothy notes are
produced by heating the aqueous soluble fraction of meats. During
heating, numerous amino acids and most free sugars are virtually
consumed. These precursors are consumed via the Maillard reaction - a
reaction between amino acids and reducing sugars - to yield several
aroma compounds. A significant proportion of these aroma constituents
are heterocyclic compounds, which generally have characteristic aromas
and low sensory thresholds (Bailey, 1983). The Maillard reaction is con-
sidered to be catalyzed by amines and readily progresses at the normal
cooking temperatures of meats.
While there is an abundance of literature on factors that influence the
Maillard reaction (e.g. pH, water activity, time and temperature of
heating, system composition), most studies have been related to either
color formation or the loss of an essential amino acid (e.g. lysine). There
have been few detailed mechanistic or kinetic studies whose goal has been
to understand better the Maillard reaction and flavor formation. Most of
the studies performed in Maillard reaction chemistry and flavor have
involved model systems heated under various conditions and then the
volatiles formed have been identified. While significant understanding of
the Maillard reaction and flavor has been obtained in this manner, such
information is still lacking since nearly all of the pathways for the forma-
tion of flavor compounds are simply hypothetical. Few if any researchers
have been sufficiently rigorous to perform labeling studies and confirm
mechanisms of formation. Additionally, few studies have been designed to
elucidate the kinetics of the Maillard reaction. This would require a
rigorous experimental design and accurate quantification of the flavor
compounds formed.
Another shortcoming of the research in this area is that few investiga-
tors have chosen to determine the sensory contribution of the volatile
compounds identified in their work. Thus, there are long lists of com-
pounds identified in various meat products, but with little understanding
of which compounds make the greatest contribution to meat flavor or the
concentration/balance of volatile compounds needed to produce meat

flavor. One of the few exceptions is the study by Gasser and Grosch
(1990), which will be discussed later. Thus, it is unfortunate to read page
upon page of reaction mechanisms (proposed and not proven) and long
lists of volatile compounds identified in meats or meat model systems and
yet have such a poor understanding of meat flavor.
Despite the shortcoming of the available data, one can make some
comments on the role of the Maillard reaction in meat flavor. It is clear
that heterocyclic compounds formed during the heating make significant
contribution to meat aroma. Heterocyclic compounds identified in meats
include furans, furanones, pyrans, pyrazines, thiophenes, thiazoles, thiazo-
lines, oxazolines and heterocyclic polysulfides. Some combination of these
volatiles will give the 'basic' meaty flavor common to all meat species. As
noted earlier, there is to date only a poor appreciation of the compounds
or their concentrations that are most important.
7.4.2 Lipid reactions

Lipid-based reactions that occur during the cooking of meat are generally
considered to be contributors to the species flavors of meats. While beef
and pork will develop species-specific flavor without the presence of fat,
all other species require the presence of fat to develop their characteristic
flavor. The role of lipid degradation and meat flavor will be discussed
briefly in this chapter since the subject is discussed in detail later in
chapter 10 and reviewed thoroughly by Grosch (1982).
Lipids make their contribution to flavor via thermally induced oxida-
tion reactions. The mechanisms involved are very similar to those
observed during room-temperature lipid autoxidation except the high
levels of energy available during heating favor certain reactions that are
otherwise energetically unfavorable at room temperature. Thus, one does
not obtain a rancid oxidized flavor during thermally-induced lipid oxida-
tion, which is characteristic of autoxidation; instead very desirable notes
are obtained. Some of the unique properties of thermally-induced lipid
oxidation have been summarized by Nawar (1989).
Contrary to low storage temperature oxidation, high-temperature oxi-
dations are not characterized by the build-up of hydroperoxides. The rate
of hydroperoxide decomposition parallels that of hydro peroxide formation
at high temperatures. This is illustrated in a study on the oxidation of
ethyl lin oleate at 70°C vs. 250°C. Peroxide content reached a maximum of
1777 mEq.kg- 1 after 6 h of heating at 70°C, while the same system at
250°C reached a maximum of only 198 mEq.kg- 1 after 10 min of heating.
Low-temperature oxidations also are characterized by the formation of
the C9 and C l3 hydroperoxides (from linoleic acid) while thermally
induced oxidation results in significant proportions of the five other posi-
tional isomers (C g-C 14). The formation and subsequent degradation of

these additional hydroperoxides contributes to a much greater number of
volatile Compounds being present in thermally oxidized lipids. This is
most evident in the aldehydes coming from oxidative processes. Hexanal
characterizes low-temperature oxidations, while 2,4-decadienals pre-
dominate in high-temperature oxidations (Swoboda and Lea, 1965;
Kimoto and Gaddis, 1969; Henderson et ai., 1980). The 2,4-decadienals
are associated with a 'french fry' aroma, while hexanal is characterized as
being 'green or grassy'.
While saturated fatty acids are very stable at low storage temperatures,
temperatures in excess of 150°C will result in their decomposition (Nawar,
1989). Alkanes, alkenes, propene and propanediol esters, oxopropyl esters,
ketones and fatty acids will readily be formed via non-oxidative degrada-
tion of these saturated triglycerides. The saturated triglycerides will also
undergo oxidative changes at high temperatures. Oxygen is believed to
attack preferentially the methylene groups near the ester linkage of the tri-
glyceride. Oxidative degradation of these saturated fatty acids contributes
additional methyl ketones, aldehydes, lactones and hydrocarbons
(Crossley et ai., 1962; Crnjar et ai., 1981;). Thus, high-temperature reac-
tions of lipids introduce a large group of volatiles that are minimally
formed during low-temperature oxidation, which further contributes to
the different sensory profiles.
When considering the role of lipids in contributing desirable notes to
meat flavors, one also has to consider the many secondary reactions that
will occur during heating. The vast variety of volatiles formed via ther-
mally-induced oxidations will undergo further reactions (degradations)
themselves or react with other food constituents to expand further the
variety of volatiles found in heated fats. Various aspects of this source of
flavor compounds have been studied by several researchers (Mottram and
Whitfield, 1987; Huang et ai., 1987; Ho et aI., 1989).
An important source of flavor compounds involving lipid-degradation
products is their participation in the Maillard reaction. Since the Maillard
reaction requires only a carbonyl group and an amine, the variety of alde-
hydes produced via thermally-induced lipid oxidation will contribute
readily to Maillard browning. There have been several heterocyclic flavor
compounds found in foods that have long side-chains (four to seven
carbons). These long side-chains probably originate from the reaction of
amino acids with carbonyls formed from lipids. This mechanism was
demonstrated by Ho et al. (1989) when they heated 2,4-decadienal with
cysteine. As shown in Table 7.3, numerous long-chain alkyl-substituted
heterocyclic compounds were found in this system. The participation of
beef fat in Maillard reactions was demonstrated by Ohnishi and Shiba-
moto (1984), who heated beef fat with glycine and found butyl- and pen-
tylpyridines and numerous other volatile products.
Table 7.3 Some heterocyclic compounds identified from thermal interaction of

2,4-decadienal and cysteine a
Amount produced
Compounds (mg/mol)
Furans
2-Butylfuran 12.8
2-Pentylfuran 6.4
2-Hexylfuran trace
Thiophenes
Thiophene 3.5
Tetrahydrothiophene-3-one 10.5
2-Butylthiophene 57.2
2-Formyl-3-methylthiophene 29.8
2-Pentylthiophene 13.1
2-Hexylthiophene 42.0
2-Heptylthiophene 1.8
2-Formyl-5( or 3)-pentylthiophene 15.6
Thiazoles
Thiazole 25.6
3-Methylisothiazole 2.0
2-Acetylthiazole 2.2
Cyclic polysulfides
3,5-Dimethylc 1 ,2,4-trithiolane (isomer) 122.8
3,5-Dimethyl-1,2,4-trithiolane (isomer) 18.2
3-Methyl-5-pentyl-1 ,2,4-trithiolane 14.3
2,4,6-Trimethylperhydro-1 ,3,5-thiadiazine 828.5
2,4,6-Trimethylperhydro-1 ,3,5-dithiazine 284.2
2,4-Dimethyl-6-pentylperhydro-1,3 ,5-dithiazine 18.9
2-Pentyl-4 ,6-dimethylperhydro-1,3 ,5-dithiazine 28.7
Pyridine
2-pentylpyridine 501.2
aTaken from Ho et al. (1989).
Other researchers have suggested that lipids may influence indirectly the
formation of meat flavors. Whitfield et a/. (1988) heated some amino acid/
ribose mixtures in the presence and absence of phospholipids. They noted
that the inclusion of phospholipids in the model system reduced the
amounts of heterocyclic compounds formed during heating. They postu-
lated that this occurred as a result of the phospholipid degradation
products (i.e. carbonyls) capturing free hydrogen sulfide (H 2S), which was
necessary to form many of the sulfur-containing heterocyclic compounds.
Later work by Farmer and Mottram (1990) on this same reaction system
confirmed this effect but also noted that the inclusion of phospholipids in
the reaction system increased meaty notes upon heating. They suggested
that the phospholipids increased the intensity of meaty notes by forming
1-(2-methyl-3-furylthio)propan-2-one and 2-methyl-3-furyl tetra-hydro-
furyl sulfide as unique meaty flavor components.
Gasser and Grosch (1990) postulated that lipids might influence flavor
formation in meats in a different manner. In their studies on cooked meat

flavor, they noted that chicken contained about the same quantity of 2-
methy1-3-furanthiol as beef yet had substantially less of the oxidized form
(bis-2-methy1furyl) disulfide. They suggested that the high levels of oxidiz-
able lipid in chicken may result in the consumption of the available
oxygen, thereby protecting the 2-methyl-3-furanthiol from oxidation.
Since there is a substantial difference in the sensory properties of these
two compounds, the relative proportions of them would be expected to
have a significant impact on the overall flavor character.
7.4.3 Thiamin degradation

Of the vast number of precursor compounds in meat that are degraded
during cooking, no one compound is considered more important to beef
flavor than thiamin. Research on thiamin degradation and its importance
to meat flavor began in the late 1960s and has continued to be a very
active area of study (Guntert et aI., 1990; Werkhoff et aI., 1990). In
addition to the number of publications on the volatile compounds formed
as a result of thiamin degradation, its importance to meat flavor is
demonstrated further by the frequency of its use in synthetic meat flavors
(MacLeod and Seyyedain-Ardebili, 1981; MacLeod, 1986).
Initial work on the contribution of thiamin to meat flavor focused on
the liberation of H 2 S as a major degradation product (Dwivedi and
Arnold, 1971, 1972). Hydrogen sulfide has been shown to be an important
flavor compound in itself and also will react with furanones to give an
intense meaty flavor. Later work has studied the minor compounds
formed upon the heating of thiamin in model systems (van Dort et aI.,
1984; Buttery et al., 1984; Guntert et aI., 1990; Grosch and Zeiler-Hilgart,
1992). As a result of this work, many volatile compounds have been iden-
tified and mechanisms for their formation proposed (Figure 7.2). While
many of these thiamin degradation products probably make a significant
contribution to meaty flavors, research has focused on the intense meaty-
flavored compound 2-methyl-3-furanthiol. This compound is considered to
be the most important volatile compound in beef and ox broth and
undoubtedly plays an important role in other meaty flavors (Gasser and
Grosch 1990).
7.4.4 Role of nitrite in cured meat flavor

The curing of meat involves the addition of mtnte (with or without
nitrate), salt and, optionally, sugar, ascorbates, phosphates and season-
ings. The most important ingredient in terms of flavor development is the
nitrite, since it imparts indirectly the characteristic cured meat flavor.
While. there is not total agreement on what gives cured meat products
[ 'T'H,]
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Figure 7.2 Volatile compounds resulting from thiamin degradation and pathways proposed
for their formation. Reprinted with permission from Guntert et al. (1990). Copyright 1990
American Chemical Society.
their characteristic flavor, there is agreement that mtnte is an effective

antioxidant and this property strongly influences flavor development
during cooking (Gray et al., 1981). Several studies have been performed
on cured meats, which demonstrate that the volatile profile of cured
meats is much simpler than that of the corresponding uncured meats
(Cross and Ziegler, 1965; Ramarathnam et ai., 1991) (Figure 7.3). The
components, which are absent or greatly reduced in concentration, are
products of lipid oxidation. Cross and Ziegler (1965) proposed that the
flavor of cured meats is simply the 'basic' meat flavor i.e. it is the
common flavor of meats minus the contributions of fat that has been
proposed to give the species notes to cooked meats. The theory of Cross
and Ziegler (1965) was supported by Ramarathnam et at. (1991) but has
been questioned by Dumont et al. (1990). The latter group noted that the
nitrite ion is a very reactive species that probably would undergo
numerous reactions some of which might make a significant contribution
to cured-meat flavor. They also noted that there were several compounds
that inhibited warmed-over flavor (WOF) in meats, which arises from
lipid oxidation. Of the inhibitors of WOF, only nitrite yields a cured-
meat flavor. If cured-meat flavor were simply a lack of lipid oxidation
products, cured-meat flavor should be produced by several antioxidants
and it is not. Thus, while Dumont et al. (1990) have not identified what
is responsible for cured-meat flavor, they have a reasonable argument
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Figure 7.3 Gas chromatographic profiles of the volatiles found in cured (bottom) and uncured pork (top). Reprinted with permission from Ramar-
athnam et al. (1991). Copyright 1991, American Chemical Society.
that it is the product of reduced oxidation and some unique flavor com-
pounds associated with the nitrite reactions.
7.4.5 Volatiles and flavor

Despite the amount of research that has been performed on meat flavor,
no one can define what key volatile compounds and the ranges in con-
centration of these key compounds that will yield each of the meat flavors.
Long lists of compounds have been identified, but there is a poor under-
standing of what compounds are important to meat flavor. In the last 10-
15 years many researchers have chosen to smell the gas chromatographic
effluent from various meats and assigned sensory attributes to most or all
components in the gas chromatographic profile. However, the question as
to which of the several hundred aroma compounds give meat flavor is yet
to be answered. Gasser and Grosch (1990) have carried out the best work
in this respect, by using aroma-extract dilution analysis (AEDA) on
chicken, beef and ox broths. The key results of their work are presented in
Table 7.4. Interpretation of the data in this table suggests that the com-
pounds present at the greatest flavor-dilution (FD) factor are most impor-
tant to flavor. Their work shows that all three broths contain substantial
Table 7.4 Comparison of flavor-dilution (FD) factors of odor-

ants appearing in broths from chicken, cow and ox meats a.b
Compound FD factor
Chicken Cow Ox
2-Methyl-3-furanthiol 1024 512 512

Bis (2-methyl-3-furyl) disulphide <16 2048 1024
2-Furrurylthiol 512 512 256
2, 5-Dimethyl-3-furanthiol 256 <16 <16
3-Mercapto-2-pentanone 128 32 32
Methional 128 512 1024
2, 4, 5-Trimethyiazole 128 <16 <16
2-Formyl-5-methylthiophene 64 <16 <16
Phenylacetaldehyde 16 64 32
2 (E), 4 (E)-Decadienal 2048 64 32
2, 4-Decadienal 128 <16 <16
2-Undecenal 256 <16 <16
y- Dodecalactone 512 <16 <16
y- Decalactone 64 <16 <16
Nonanal 128 <16 <16
2 (E)-Nonenal 64 32 64
2 (E), 4(E)-Nonadienal 64 <16 <16
B-Ione 64 64 64
P-Cresol 64 <16 <16
aTaken from Gasser and Grosch (1990).

bThe compounds mat appeared in one of the meat species with a
FD factor of at least 64 are compared.
quantities of 2-methyl-3-furanthiol, 2-furfurylthiol, 3-mercapto-2-penta-

none and methional. These compounds could be considered as the key to
the 'basic' meaty flavor. Chicken meat, which depends upon lipid oxida-
tion for its unique notes, was expectedly high in lipid-oxidation products
2(E),4(E)-decadienal (most important), 2-undecanal, 2,4-decadienal, and
deca- and dodecalactones, as well as some other unsaturated aldehydes.
One would have to interpret these data as implying that beef flavor is a
'basic' meat flavor and chicken flavor deviates by adding lipid-oxidation
notes.
As mentioned earlier, many researchers have identified other meaty
notes by smelling the gas chromatographic effluent of various meat flavor
isolates but no group other than Gasser and Grosch (1990) has attempted
to assign significance to their identifications. One can have a meaty note
in a product at or below sensory threshold and it still will not make any
practical contribution to product flavor. More studies similar to that by
Gasser and Grosch (1990) are needed; these need to be continued further
by obtaining the desired chemicals and blending them to evaluate the
validity of the results.
7.5 Summary
While a host of meat constituents either degrade or undergo reaction with

other components, only a limited number are considered important for
flavor formation. The key precursor reactions are:
1. sugar and amino acid (particularly cysteine) reactions via the Maillard
reaction;
2. lipids via thermal oxidation;
3. thiamin by way of thermal degradation; and
4. nitrite-based reactions and associated anti oxidative effects (in cured
meats).
The long-held theory that the Maillard reaction is responsible for the
'basic' meat flavor and lipid-based reactions for the species flavor has been
proven incorrect in some meats. Very good beef and pork flavors arise
from lean tissues and can also be generated in model systems without
lipids. Nevertheless, the flavor of lamb, mutton and chicken depends upon
the lipid to develop each of the characteristic cooked-meat flavors of these
meats.
The scientific community is divided on the role of nitrite in producing
cured-meat flavor. Some believe that the cured-meat flavor is the 'basic'
meat flavor as a result of the nitrite blocking any significant flavor con-
tribution from the lipids. Others have pointed to weaknesses in this theory
and feel that nitrite must be involved in the reactions that produce unique
flavor constituents, which, together with the anti oxidative effects, yield a
cured-meat flavor. There is no definitive answer to this question at this
time.
There have been many studies identifying the volatile constituents in
meats, with the number of volatiles identified approaching 1000; however,
there has been little work to determine which of these constituents are
truly responsible for each species flavor. The limited work performed on
significance has confirmed the theory that lipid oxidation is a key con-
tributor to chicken flavor. Beef (or ox) flavor is not characterized by lipid
oxidation products but by degradation products of thiamin. Thiamin may
well prove to be the key constituent of beef, yielding the characteristic
flavor. Recent work suggests that beef flavor may be common to all
species if unmodified by lipid contributions.
Additional work needs to be performed to identify the flavor con-
stituents responsible for meat flavor - not just those present in the flavor
profile. Compounds must be identified, quantified, purchased and added
back to deodorized meats to determine what compounds and what pro-
portions are required to simulate the true flavor character of cooked
meats. Despite hundreds of research publications on meats, there is still
some distance to go to understand meat flavor.
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phospholipid on the formation of volatile heterocyclic compounds in heated aqueous solu-
tions of amino acids and ribose. J. Sci. Food Agric. 42, 261.
Wood, T. and Bender, A.E. (1957) Analysis of tissue constituents. Commercial ox-muscle
extract. Biochem. J. 67, 366.
8 Flavor and aroma - its measurement
K.L. BETT and C.c. GRIMM
8.1 Introduction
The acceptance of a food in the marketplace is largely dependent upon the

flavor quality of that food. There are several sensory methods for mon-
itoring or measuring flavor quality. Carefully designed experiments using
difference type sensory methods, such as paired-comparison or ratio-
scaling, can be used to measure flavor changes with an untrained or inex-
perienced panel. Descriptive flavor analysis, which requires extensive
training of panelists, is used to measure flavor intensity and monitor
flavor changes.
Flavor is 'the impressions perceived via the chemical senses from a
product in the mouth' according to Caul (1957). Flavor impressions per se
cannot be measured by instrumental means, although the presence of
compounds that contribute to flavor can be monitored by instrumental
techniques, such as gas chromatography, high-performance liquid chroma-
tography (HPLC) or piezo electric crystals-mass balance measurement.
The objectives of this chapter are to present methods of measuring flavor
and flavor compounds, and methods of correlating instrumental and
sensory data, and to present an example of measuring meat flavor.
8.2 Difference tests
Flavor changes can be measured by difference tests (Larmond, 1977).

These tests can be accomplished with untrained panelists. A brief orienta-
tion on the test methods is all that is required. These methods include
triangle tests, paired comparison tests, duo-trio tests, multiple comparison
tests, ranking tests, scoring and ratio-scaling tests, and are described in
detail by Larmond (1977). Meilgaard et al. (1991) divided these methods
into two groups, overall difference tests, and attribute difference tests.
Overall difference tests, such as duo-trio or triangle tests, determine
whether panelists can detect overall sensory differences, but are not good
means for measuring flavor differences and do not isolate the flavor attri-
butes from other sensory attributes.
These simple difference tests allow the investigator to determine if an
ingredient change or process change causes a noticeable difference in the
FLAVOR AND AROMA - ITS MEASUREMENT 203
sensory perception of the product. More appropriate evaluation of flavor

involves the use of attribute difference tests, such as paired comparison,
multiple comparison, scoring or ratio-scaling tests (Meilgaard et aI., 1991).
The investigator may be interested in simple differences, directional differ-
ences of the defined criterion or preference differences (Amerine et aI.,
1965).
These test methods have the advantage that panelists do not require any
previous training, although orientation about the test method is necessary.
Experience in sensory evaluation and/or with the product being tested
may improve the panelist's chances of finding differences. The number of
panelists needed for these tests varies, with 20-40 persons being the
normal number. If differences are large, as few as 12 persons may be
needed in determining differences; however, if minute differences exist then
the test may require 50-100 persons to achieve confidence in the results
(Meilgaard et at., 1991). Paired comparison tests only indicate if a differ-
ence exists or not but do not give information about the direction and
magnitude of the difference. Ranking tests indicate the direction of differ-
ence but not the magnitude. Ranking tests also have a limitation in that
no more than 3-7 samples can be evaluated at one session. Also, ranking
tests may require some training in identifying the particular attribute
being evaluated in order to assure the investigator that panelists are eval-
uating the desired attribute.
The ratio-scaling method indicates the direction of the difference and is
also useful in determining its magnitude. New panelists are not so percep-
tive as experienced panelists, which means that training and experience are
necessary for reliable results. This increases the cost and time involved to
achieve results. If more details on the sensory changes are desired,
descriptive sensory analysis is recommended. This method can be used for
flavor, texture, appearance or noise properties. This chapter exclusively
deals with flavor properties.
8.3 Descriptive flavor analysis
Descriptive flavor analysis had its beginnings at Arthur D. Little, Inc. in

the late 1940s. This company began to supplement existing sensory
methods with descriptive words and then to assign intensities to the words
to develop what is now called the flavor profile (Caul, 1957). Many
descriptive methods have been developed since, but only a few have
remained as standard methods (Meilgaard et at., 1991).
The basic idea behind descriptive analysis is that total flavor is broken
down into flavor terms, notes or descriptors. These descriptors are words
that specifically characterize the perceived flavor, such as 'painty', which is
used to describe one of the flavors associated with lipid oxidation. The
descriptors are precisely worded definitions that specifically portray the

flavor. A properly functioning descriptive flavor panel is exposed to refer-
ences or examples that represent the individual flavor descriptors. A
descriptive panel evaluates the intensity of the descriptors using a specified
intensity scale. The scale depends on the method in use.
Descriptive panels typically consist of five to 20 members. They are
selected based on their normal abilities to taste and smell, availability,
interest and ability to function in a group setting. They need to be able to
detect and describe differences in sensory perceptions. After selection, the
panelists are trained in the basic principles of sensory perception, food-
flavor analysis, descriptor development and flavor,.,intensity measurement.
The training period requires many hours of practising to perfect evalua-
tion skills. A few of the common descriptive flavor methods will be pre-
sented briefly; these have been described by Caul (1957), Stone et al.
(1974) and Meilgaard et al. (1991).
8.3.1 The flavor profile

The flavor profile method analyses the intensity of the product's aroma,
flavor and aftertaste using a simple universal scale common to all products.
The scale consists of: (i) not present (below threshold); (ii) threshold; (iii)
slight; (iv) moderate; or (v) strong (Caul, 1957). The four to six trained
panelists form a consensus for each character note (descriptor). This
method places importance on the order of appearance of the character
notes because of the importance that order plays in flavor. For example, if
an undesirable· flavor occurs as the first or last note, then the flavor
impression is usually undesirable. The character notes are developed by the
panel, which then comes to a consensus on the term and its definition.
The flavor profile includes the evaluation of amplitude, which is the
overall impression of everything included in aroma and flavor (Caul,
1957). Meilgaard et al. (1991) describe amplitude as 'the blend or balance
of the individual flavors'. Amplitude rating is half-learned and half-intui-
tive and must be approached with caution (Meilgaard et al., 1991). It is
not a single number that represents the overall acceptance of the sample,
but represents the degree to which the individual flavors blend together. In
some products, a high degree of blend is desirable, while in other products
a dominant flavor characteristic is desired.
8.3.2 Quantitative descriptive analysis

Another commonly used descriptive flavor analysis method is Quantitative
Descriptive Analysis (QDA). Trained panelists identify and quantify, in
order of occurrence, the sensory properties of the product. The intensity
scale consists of a 6" (15.2 cm) line with anchor points 0.5" (1.3 cm) from
each end and usually, but not necessarily, a third anchor at the mid-point.
Each anchor is labeled with a word that expresses intensity. The panelist
places a vertical line at the point that reflects their impression of the
intensity of the flavor descriptor. The development of the terms is a group
process but principal component analysis is used to determine the primary
sensory variables; in contrast to the flavor profile method, it is not a
group consensus. Multiple evaluations on each sample are required to
improve the accuracy of results. This method was developed for using sta-
tistical analysis (Stone et aI., 1974; Meilgaard et al., 1991).
8.3.3 The spectrum method

The third method to be discussed is the spectrum method. It is similar to
the flavor profile and QDA methods in that it separates the overall flavor
impression into descriptors. The descriptors are derived by a trained panel
and must describe characteristics for which concrete references are
supplied. This minimizes confusion between descriptors. Intensity is eval-
uated with a universal scale based on extensive reference points. The
reference-point intensities are standardized by specific flavors in commer-
cially available foods. Numerous reference points reduce panel variability
and allow comparison of data across time and between products by mini-
mizing panelist drift (Meilgaard et al., 1991).
8.4 Instrumental analysis
Much has been written about methodology for measuring flavor com-
pounds by gas chromatographic analysis. A practical reference for mea-
suring flavor volatiles by gas chromatographic methodology is Burgard
and Kuznicki (1990). The review herein will be an overview of some
established instrumental methods and a discussion on the sniffer port for
qualitative evaluation of flavor compounds.
8.4.1 Extraction and concentration of.f/avor compounds

The first step in gas chromatographic analysis is collection of volatile
compounds from the food sample, which is commonly referred to as
sample preparation. In general, food samples consist of rather complex
matrices of proteins, carbohydrates and lipids, with varying amounts of
water present. The compounds that constitute the flavor components are
often locked into these matrices by both chemical and physical bonds.
Heat, water or an organic solvent are used routinely to denature the
matrix and release the flavor components. During this process new flavor
components may be formed.
Several methods are employed to extract the volatile flavor components

from the food sample; however, each method has its own shortcomings.
The method used will depend upon the objectives of the experiment.
Methods for collecting the flavor volatiles include steam distillation,
solvent extraction, removal of headspace gases and supercritical fluid
extraction.
8.4.1.1 Steam distillation. In steam distillation the volatile components

are steam-distilled from an aqueous suspension of the food sample. Simple
steam distillation has evolved to include a capture solvent, such as
employed in the Likens-Nickerson continuous extraction apparatus
(Likens and Nickerson, 1964). The steam distillate is brought into contact
with a non-polar solvent and the flavor components partition between the
two phases. The extracted aqueous layer is then returned to the sample
flask and the process is repeated. The repeated cycle of distillation and
extraction results in an enrichment of the non-polar solvent with the
volatile components. The solvent can be methylene chloride, freon, an
alkane or an alkane-ether mixture. Large quantities of sample may be
used with relatively small amounts of solvent, which results in concentra-
tion by one to two orders of magnitude. In some cases, the extract may be
injected directly onto the gas chromatograph (GC) but normally the
solution must be concentrated further.
Steam distillation is rather indiscriminate and relatively large quantities
of undesirable compounds may be carried over. Upon injection into the
GC, these compounds are not volatilized, remain in the injection port or
at the head of the column as will be described later and, after successive
injections, hamper chromatographic performance. Another problem with
steam distillation is artifact formation, which can result from holding the
sample at elevated temperatures.
8.4.1.2 Solvent extraction. Solvent extraction can be used with liquid or

finely ground solid samples. Common solvents are pentane, hexane,
methylene chloride, diethylether, ethanol, methanol, or mixtures thereof.
The solvent is mixed with the sample and allowed to separate from the
sample by gravity in a separatory funnel or other extractor. A given solute
partitions between the two phases at a constant ratio dependent upon its
physical interaction with the two solvents. Repeated washings of the
aqueous phase with fresh solvent are used to extract a high percentage of
the components. The washings are then combined and the sample is con-
centrated by evaporation of the solvent. Some of the more volatile com-
ponents may be lost during the evaporation/concentration step but solvent
extraction is relatively free of artifact formation. Methylene chloride pos-
sesses a specific density greater than that of water, which lends itself to
convenient use in a standard separation funnel.
8.4.1.3 Headspace gas analysis. Headspace analysis determines the com-

position of the volatiles present in the gaseous phase above a sample in a
sealed container. Headspace analysis is ideal for lighter more volatile
compounds. Dynamic headspace analysis continually removes the head-
space gases by sparging a carrier gas through the sample and collecting on
a trap or cryofocusing at the head of a capillary column. Solid adsorbent
traps such as charcoal, Tenax®, Porapak® or Chromosorb® materials are
used extensively to trap the volatile compounds (Zlatkis et al., 1973). The
adsorbents also tend to adsorb volatiles at room temperature. The com-
pounds can be desorbed either by heating the trap or washing it with
solvent. Thermal desorption is usually followed by cryofocusing the vola-
tiles at the head of the column. The presence of moisture introduces
problems in reproducibility. The retention of polar compounds can be
affected adversely by the presence of water in the column (Takeoka et al.,
1988). Direct cryofocusing of the headspace gas captures moisture as well
as the desired volatiles and can only be used with packed, or mega-bore
capillary columns. Trapping with a solid adsorbent permits dry purging of
the trap at ambient temperature while venting to remove the water.
8.4.1.4 Supercritical fluid extraction. Supercritical fluid extraction as a

sample preparation method by gas chromatographic analysis is a special
form of solvent extraction. Liquid carbon dioxide is the most commonly
used solvent. The pressure and temperature conditions are such that the
solvent (C0 2) remains neither in a liquid or gaseous phase but in a
'supercritical' state. Supercritical fluid is passed through the sample to
extract the flavor components. The CO2 can be vented directly onto the
column, or back-extracted into a solvent such as methylene chloride
(Hawthorne et al., 1990).
8.4.2 Selection of a concentration-extraction procedure

Anyone of the above methods is appropriate and selection should be
based on the experimental objectives. If low-boiling compounds are of
interest, th~n solvent extraction and evaporation would not be appropriate
because a high percentage of the recovered compounds would be lost
during the evaporation and concentration steps. In this case, purge and
trap followed by thermal desorption would be more appropriate. A high
moisture content present in the sample would warrant the use of a carbo-
pack trap over that of Tenax®. The sample matrix and the volatiles to be
analyzed determine the method employed.
8.4.3 Gas chromatographic (GC) analysis

The parts of a gas chromatograph include the injection port, the column
oven, the column and the detector. A brief overview of the parts and their
function will be presented.
8.4.3.1 Injection of sample. The sample is introduced into the instrument

through the injection port. A small portion (1-5 III of liquid or gas) of
the sample extract or headspace gas is injected onto the column in one of
two modes. Splitless mode is where the entire volume goes into the
column and split mode is where a precise fraction of effluent is injected
onto the column, while the remainder is vented out of the GC. The injec-
tion port may be held at high temperatures to avoid condensation of high-
boiling compounds. However, high temperatures may result in thermal
degradation of some compounds.
Alternative methods of injection are direct on-column injection and
cryofocusing. Coolon-column injection is where the sample extract is
placed at the head of the column held at near ambient temperature. It
requires that the column be cooled below the boiling point of the solvent.
An ultra-thin needle of less than 0.2 Ilm outer diameter (OD) is inserted
into the capillary column and the sample is placed directly on the column.
Cryofocusing is used in conjunction with either headspace analysis or
thermal desorption. In either case, the head of the column is held at sub-
ambient temperatures and the volatiles are condensed at that point. At the
completion of the headspace sweep, or the thermal desorption process, the
head of the column is flash-heated to volatilize the compounds. The char-
acteristics of the sample and the equipment available will determine the
injection technique.
8.4.3.2 Column oven. The column oven is a temperature-programmable

oven that contains the column. Isothermal analysis is appropriate in a few
cases when a sample contains only a few compounds or when only one
compound is being analyzed. Temperature-programming allows the
column to be exposed to a range of temperatures so that it passes through
the boiling points of several compounds, resulting in enhanced chromato-
graphic resolution. The range of teinperatures is determined by the char-
acteristics of the sample, the injection technique and the thermal limits of
the column. To analyze complex samples, high-efficiency capillary columns
are needed. Columns are coated or bonded with a variety of stationary
phases and columns are selected based on the analytes to be analyzed. The
important parameters are polarity of the stationary phase, bonded or
coated stationary phase, capacities and sometimes thermal ranges.
8.4.3.3 Detectors. The detector responds to the presence of a chemical

compound and converts the response to an electrical signal that is trans-
mitted to a computer monitoring system, integrator, or chart recorder.
The flame-ionization detector is the most universally used detector. It
responds to any compound that is combustible in a hydrogen flame and is
suitable for most general applications. Element-selective detectors are
available for organic compounds containing halogens, nitrogen, sulfur and
FLAVOR AND AROMA ~ ITS MEASUREMENT 209
phosphorus. The flame-photometric detector is selective for organic sulfur

and phosphorus compounds. Their sensitivity is usually equal to or better
than the flame-ionization detector. Electron-capture detectors provide a
high sensitivity for compounds containing electron withdrawing groups,
such as halogens and nitroxides. All of these detectors may be used for
quantification.
Positive identification of eluted compounds using qualitative detectors
such as Fourier transform infrared spectroscopy and mass spectrometry
are used to confirm the retention times of the volatiles. With the advent of
low-cost high-performance personal computers, operation of these
'hyphenated analytical techniques' has become commonplace. The latest
generation of these instruments is relatively low-cost and low-main-
tenance. They are routinely equipped with database spectral libraries and
automated search routines, which simplify data interpretation. Chemical
derivatization can provide compound identification information but has
been largely substituted with these qualitative detectors.
8.4.4 Sniffer-port analysis

Another method of detection, which is not new but is becoming more
advanced, is the use of a sniffer port. A portion of the column effiuent is
diverted from the end of the column, prior to the detector and routed
through a port to be sniffed by a human subject for qualitative evaluation.
Splitting of the effiuent permits simultaneous human evaluation and
instrumental quantification. This method allows for the important
odorous compounds to be identified in a food sample. While instrumental
detectors can quantify the individual components of a food sample, the
peak areas do not necessarily correspond to flavor intensity. Compounds
with larger peak areas sometimes contribute little to the flavor of the
sample, whereas very strong aromas may be associated with very small
peaks. Many times the human nose at the sniffer port is more sensitive
than the instrumental detector. The aroma intensity can be evaluated by
sensory evaluation methods, such as threshold determination, magnitude
estimation or universal intensity scale.
Software programs have been developed to be used in conjunction with
the. sniffer port to aid the analyst in identifying and logging the smell
(Acree et al., 19~4). Flavor lexicons for various foods can be developed
using these programs. Once a method has been developed, the compounds
producing flavors of interest can be identified by repetitive analysis of the
same sample by their retention times and isolated for identification.
8.4.5 Flavor analysis by HPLC

Volatile flavors are readily amenable to analysis by gas chromatography.
However, polar compounds, such as sugars, amino acids and fatty acids,
must first be derivatized prior to analysis. Liquid chromatography, which

is performed at ambient temperature, permits the analysis of these com-
pounds but with somewhat less separation efficiency than gas chromato-
graphy. A wide variety of detectors is available including UVjvis,
electrochemical and refractive index. Pre- or post-column derivatization
may be used to enhance sensitivity. A promising application of liquid
chromatography (LC) to food flavors is its use to fractionate a mixture of
compounds and to collect the fractions of interest. Selected fractions can
then be injected into a gas chromatograph for further separation and
identification. This allows the detection of compounds present in subparts-
per-million concentrations to be separated from analytes present in parts-
per-thousand levels. Whereas gas chromatographs are coupled routinely to
a mass spectrometer for qualitative analysis, HPLCjmass spectrometry is
not yet routine, although great strides have been made in the interfacing
of these two analytical techniques. Relatively low-cost commercial instru-
mentation is now available, although application is more of an art than a
science for HPLCjMS.
8.4.6 Piezo-electric crystals

Piezo-electric crystals can be used to measure very small amounts of
material. Electrodes are attached to a crystal to monitor the frequency at
which it oscillates. Molecules adsorbing onto the surface of the crystal
alter the frequency. By coating the surface with a polymer specific for a
class of compounds, such a device can crudely mimic the human nose.
Molecules specific to the polymer are adsorbed. The concentration can
be determined by the magnitude of the shift in the frequency of the
crystal. Presently, the specificity of the coating polymers is limited to
classes of compounds, whereas the human nose can distinguish thou-
sands of individual compounds (Muramatsu et al., 1990; Chang et al.
1991).
8.5 Correlation between sensory analysis and gas chromatography
Flavor is very complex and, in most foods, multiple compounds are

responsible for a particular flavor descriptor. It is of great interest to the
food industry to know the chemical compounds that constitute specific
flavors. Also, compounds need to be identified to indicate that flavors
will be present at detectable intensities. Determining the compounds that
contribute to a flavor descriptor is a tedious process. It requires precise
GC methods, analytical sensory methods and advanced statistical
methods or computer-modeling. The investigator must be aware that
small data-sets with many variables may not be robust enough to reflect
authentic trends accurately. Therefore, caution should be used when

applying statistical or computer-modeling to any data-set.
A detailed GC analysis may result in 200 to 1000 peaks (MacLeod and
Ames, 1986; Gasser and Grosch, 1988). Many of these peaks can be
grouped into categories. One way of doing this is by compound type.
Another valid approach is the use of statistical data-grouping or variable
reduction techniques. Cluster analysis, discriminate analysis, principal
component analysis and factor analysis are statistical methods used.
Principal component analysis, factor analysis and cluster analysis are used
to discover the variables in the data-set that form coherent subgroups that
are independent of one another. These methods may reveal hypothetical
structures that are generated by mathematical combinations (equations)
measured by the observed variables (Tabachnick and Fidell, 1983).
8.5.1 Principal component analysis

Principal component analysis can be used to indicate the relationship
among multiple groups of variables within a data-set (Piggott and
Sharman, 1986). It can be used to reduce the dimensionality or collapse
the number of variables in the data to form principal components. This
can group sensory attributes with physical parameters if any relationships
exist. Researchers can choose to do statistical analysis on the principal
components rather than on the original variables. This method should
only be used when all variables are measured in the same or comparable
units or when the data are standardized (Johnson, 1988). The correlation
matrix can be used in the analysis to standardize the data. Principal com-
ponent analysis is, however, not always readily interpretable. It reparti-
tions all of the observed data variability into orthogonal (statistically
independent) components. The analysis does not take into consideration
that there may be correlation among these components due to the under-
lying data structure. Therefore, if a correlation exists, other methods of
analysis are more appropriate. This is not the choice method for grouping
instrumental measurements and sensory attributes.
8.5.2 Factor analysis

Factor analysis reduces a large set of variables to a smaller set of unob-
servable or latent variables called factors (i.e. groups of variability in
common among the original variables) that, together with any remaining
unique variability, explain the totality of variability observed in the
original variables. It overcomes the problem of having multiple scale units
where principal component analysis falters (Johnson, 1988). One problem
with factor analysis is that there is an infinite number of possible rota-
tions, all accounting for the same amount of variance from the original
data but each rotation defines factors differently. The rotation choice
depends on the researcher's assessment of scientific utility and interpret-
ability (Tabachnick and Fidell, 1983). In the example data-set presented
later, this method was most effective for finding instrumental/sensory rela-
tionships.
8.5.3 Cluster analysis

Cluster analysis is a general procedure that groups variables. It identifies
and classifies variables so that each variable responds to treatments
similar to other variables in its cluster. The simplest form of cluster
analysis is to plot the objects on a two-dimensional graph and to observe
natural groupings. Data within a cluster will be close together when
plotted geometrically, while data in other clusters will be further apart.
Computers are used to determine mathematically distances between
objects in order to find natural groupings. Computerized cluster analysis
is necessary when more than two or three dimensions exist in the data
(Hair et al., 1987; Jacobsen and Gunderson, 1986). Clustering methods
can detect clusters that do not exist and/or various clustering methods
can produce different clusters (Johnson, 1988). Some of these data-
grouping techniques can be used to group two sets of data, such as
instrumental data and sensory data. This allows the researcher to deter-
mine which sensory characteristics respond to the independent variables
(experimental treatments) in a manner similar to the instrumental respon-
ses. This is a good exploratory tool for determining the best variables to
be used in regression models that will be described below. The scales used
for measuring variables can vary in a data-set, and cluster analysis can be
run on the data without normalizing the data. Caution should be used in
interpreting the analysis when using multiple scales but care should
always be taken when interpreting the results of multivariate analysis
(Jacobsen and Gunderson, 1986). In the following example, data-set
cluster analysis gave results somewhat similar to those found with factor
analysis.
8.5.4 Discriminant analysis

Discriminant analysis is used to classify measured data (metric indepen-
dent variables) into groups based on non-metric response variables (cate-
gorical dependent variables) (Hair et al., 1987). The main difference
between discriminant analysis and the other methods already described is
that it is only good for separating data into nominal categories (Powers
and Ware, 1986; Resurreccion, 1988). This method is not the appropriate
method for grouping physical parameters with analytical sensory para-
meters.
The researcher must make judgements on the validity of the results of

these grouping methods and be cautious of the interpretations. It is advi-
sable to subject the data to several methods and/or rotations to see if
there are similar trends across methods. If so, one can be confident of
natural groupings in the data. In addition, the design of the experiment
must be planned before starting the data collection. A poorly designed
experiment leaves little chance of answering the desired questions. These
methods are not appropriate for salvaging poorly designed experiments
(Tabachnick and Fidell, 1983).
8.5.5 Regression and correlation

Another statistical approach to relating sensory measures with flavor-
emitting compounds is to compare the chemical response curve of a set of
samples with the flavor intensity responses from the same samples. Corre-
lation coefficients indicate the relationship of two variables as they are
affected by outside influences, while regression analysis deals with how
one variable changes because of the influence of the other (Steel and
Torrie, 1980). Linear correlation relates an independent variable with a
dependent variable. When using multiple regression, one analyzes the rela-
tionship of a single dependent variable and several independent variables.
Canonical correlation relates multiple independent variables to multiple
dependent variables. Linear and multiple regressions require metric or
quantitative data, while canonical correlations can be used with metric
(quantitative) or non-metric (qualitative) data (Hair et al., 1987). Some
researchers view canonical correlation as a 'last chance' effort because of
its lack of restrictions on the data (metric vs. non-metric) (Hair et al.,
1987). Crippen et al. (1992) have used regression analysis to relate
chemical compounds to peanut-flavor attributes.
8.5.6 Response surface methodology

Response surface methodology (RSM) is a method of optlmlzmg the
levels of multiple factors. It is ideal for use in product optimization. The
factors can be various ingredients and the levels can be the various con-
centrations of those ingredients. RSM is a statistical method that uses
quantitative data to maximize simultaneously the concentrations of two or
more ingredients, which results in optimum quality attributes (Pearson et
al., 1962; Giovanni, 1983). In flavor measurement, it can be used to
optimize flavor acceptance. Bodrero et al. (1981) and Fooladi et al. (1986)
used RSM to determine optimal combinations of meat flavor compounds
that resemble beef aroma. It is not suitable for exploring relationships
between physical data and sensory data.
Table 8.1 Means of data used in multivariate analyses
Chuck steak T-bone steak

o days 2 days 4 days 0 days 2 days 4 days
Beefy/meatya 3.7 3.5 3.0 3.7 3.4 3.3

Brothya 2.3 2.2 1.8 2.1 2.0 1.9
Paintya 0.4 0.6 0.9 0.4 0.8 0.8
Browned caramel a 2.8 2.3 2.1 2.6 2.5 2.3
Cardboard a 0.5 0.7 0.9 0.5 0.8 0.8
Bittera 0.5 0.6 0.7 0.7 0.7 0.7
Sweeta 1.3 1.1 0.9 1.2 1.0 1.0
Sour a 0.7 0.8 0.8 0.7 0.8 0.7
Hexanal b 0.4 1.4 0.8 0.6 1.4 1.6
Benzaldehyde b 0.5 0.6 0.6 0.6 0.6 0.4
Limonene b 0.2 0.3 0.2 0.1 0.1 0.2
2,3-dichloropyrazine b 0.4 0.6 0.4 0.3 0.4 0.4
Nonanal b 0.9 2.1 3.1 0.6 1.7 4.5
aIn Spectrum Universal intensity flavor scale units.

bIntegrated peak areas normalized to benzothiophene.
8.5.7 Neural networks

The above methods are based on 'if-then-else' decisions. Neural networks
are non-algorithmic and based on a set of highly interconnected simple
units with local memories. They can identify patterns from noisy, incom-
plete data (Smith and Walter, 1991). The number of applications pub-
lished in the literature is limited. Holley and Karplus (1989) used neural
networks to determine a protein's secondary structure. Much of what has
been done is proprietary or still in the developmental stages. As soon as a
neural networking chip is developed this methodology should experience
rapid growth (Smith and Walter, 1991).
8.6 Analysis of an example data-set
8.6.1 Description of data

To illustrate some of the multivariate techniques, a data-set that consists
of descriptive flavor attributes and gas chromatographic response data will
be used. Descriptive flavor data and gas chromatographic data were col-
lected on ground beef samples. The treatments were:
1. two different cuts of beef, T-bone (longissimus and psoas muscles) and
chuck (longissimus, infraspinatus and supraspinatus muscles);
2. time at 4°C storage (O-day, 2-day and 4-day).
The descriptive flavor analysis was accomplished according to Love (1988)
and St. Angelo et al. (1993). Beefy/meaty, brothy, browned caramel and
sweet are desirable characteristics. Paint and cardboard are undesirable
characteristics and these flavors increase with storage of pre-cooked meat.
Bitter and sour are undesirable if their intensities are great enough. The
means for each treatment combination are presented in Table 8.1. The gas
chromatographic data were collected from ground beef patties, which were
minced and placed in a 250 ml round-bottomed flask equipped with a
nitrogen sparger. An internal standard consisting of 10 III of a 10 Ilg.llr1
solution of benzothiophene and 2,3-dichloropyrazine in hexane was
added. The flask was placed in a 65°C water bath and volatiles were col-
lected on a 200 mg Tenax® trap. Nitrogen flow was set at 40 ml.min- 1
and a vacuum was applied at the top of the trap. After 2 h the trap was
removed and washed twice with redistilled diethyl ether. The ether was
concentrated down to 10 Ill, and 1 III was then injected on to an HP5890
gas chromatograph equipped with a flame-ionization detector and a flame-
photometric detector. A 30 m, 0.53 mm capillary column with a DB-5
coating of 1 11m film thickness was used. The effluent from the column
was split and simultaneous chromatograms were collected for each
detector. Only the FID data were used in these analyses. The oven tem-
perature was held at 35°C for 5 min then ramped to 250°C at 3°C.min-l
and held at 250°C for a total run time of 90 min. Compounds were iden-
tified by injecting on an HP5988A GCjMS. Integrated peak areas from
the chromatograms were then collected and normalized to benzothio-
phene. Three complete replications were used, which included different
beef samples. The compounds included in this data-set were selected based
on their odor significance during sniffer-port analysis. These compounds
were identified using mass spectra and retention times of standards con-
sisting of these compounds.
8.6.2 Statistical methods

Principal component analysis was performed on the correlation matrix
using SAS (SAS, 1988). The correlation matrix rather than covariance was
used because the gas chromatographic data and the sensory data were
combined. The number of principal components having associated eigen-
values greater than one is a good indicator of the number of latent
(common) factors in the data. A factor analysis specifying the appropriate
number of latent factors to extract from the data can then be conducted.
The two general types of rotation applicable to factor analysis are
orthogonal and oblique. Orthogonal rotation required that axes represent-
ing each factor remain at 90° angles to one another. Oblique rotation
lacks this requirement to allow correlation among factors (Hair et al.,
1987). An indicator of which rotation to use is if any correlations exceed
0.3 in the factor correlation matrix when an oblique rotation is requested
Table 8.2 Eigenvectors from the principal component analysis on the

correlation matrix
Prin. 1" Prin. 2 Prin.3 Prin.4
Beefy/meaty -0.37 b -0.12 0.23 0.09

Brothy -0.37 0.07 0.27 -0.12
Painty 0.33 -0.21 -0.19 0.15
Browned/caramel -0.35 -0.10 -0.05 0.17
Cardboard 0.33 -0.22 0.17 0.16
Bitter 0.28 -0.20 0.34 0.18
Sweet -0.40 -0.01 -0.16 -0.03
Sour 0.18 -0.03 0.63 -0.29
Hexanal 0.17 0.35 -0.37 0.12
Benzaldehyde 0.09 0.30 0.16 0.66
Limonene 0.11 0.50 0.13 -0.38
2,3-dichloropyrazine 0.12 0.55 0.03 -0.05
Nonanal 0.22 -0.27 -0.30 -0.43
aprincipal component No. 1.

bBold numbers indicate that absolute values are greater than 0.30
Table 8.3 Factor analysis, oblique rotation, interfactor

correlations
Factor 1 Factor 2 Factor 3 Factor 4
Factor 1 1.0
Factor 2 -0.15 1.0
Factor 3 -0.18 -0.05 1.0
Factor 4 -0.04 0.16 0.05 1.0
with the number of factors specified by the number of eigenvalues greater

than one. If the correlations are less than 0.3, use of an orthogonal
rotation method is acceptable (Tabachnick and Fidell, 1983). Factor
analysis was performed using the orthogonal rotation, Varimax rotation
in SAS (1988). The oblique rotation (Promax in SAS, 1988) was not
necessary for this data because it did not meet the criteria described
above. Scree plots and eigenvalues greater than one were used to deter-
mine the number of factor solutions. Cluster analysis was performed using
oblique principal component clustering (Varclus in SAS, 1988). The Tree
procedure was used to determine the number of clusters. The result
(number of clusters) was 2.l (three clusters).
Discriminant analysis was not performed because this was metric data
and not categorical data. Simple and multiple correlations were not per-
formed because the authors feel that many people are familiar with these
methods. In addition the data were inappropriate for response surface
analysis because optimization was not the intent of the analysis. Neural
Table 8.4 Rotated factor pattern using orthogonal (Varimax) rotation
Fl F2 F3 F4 Commonality
Beefy/meaty 0.81 0.88

Brothy 0.93 0.87
Painty -0.88 0.81
Browned/caramel 0.65 0.73
Cardboard -0.75 0.80
Bitter -0.58 0.59 0.75
Sweet 0.79 0.89
Sour 0.93 0.91
Hexanal 0.58 0.72
Benzaldehyde 0.89 0.87
Limonene 0.96 0.96
2,3-dichloropyrazine 0.91 0.92
Nonanal -0.59 -0.69 0.83
Variance 4.79 2.76 1.93 1.49 10.97
aIndicates absolute value less than 0.5.
networking software is unavailable to the authors, so it was not demon-

strated.
8.6.3 Results of statistical analysis
8.6.3.1 Principal component analysis. Principal component analysis was

run on the full set of data that included the descriptive flavor data and the
GC normalized quantity data. There were four eigenvalues greater than
1.0 so the analysis was run, requesting four principal components. Table
8.2 displays the eigenvectors. On the first principal component, the flavors
beefy/meaty, brothy, browned caramel and sweet had negative values with
similar magnitude, which means that they had equal loadings on this prin-
cipal component. These are the desirable flavor characteristics. The unde-
sirable flavors, painty, cardboard and bitter loaded positively on this
component with similar values. This could be called the desirable/undesir-
able principal component. In the second principal component, several
other compounds that were monitored loaded positively at an arbitrary
level of 0.3 or higher. This component seems to measure the chemical
compounds. The third and fourth components do not have obvious inter-
pretations. Principal component analysis failed to identify any relationship
between flavor descriptors (not basic tastes) and the chemical compounds
measured, although hex anal and nonanal were inversely related to sour
and bitter.
8.6.3.2 Factor analysis. The data was analyzed with an oblique rotation
(Promax rotation in SAS, 1988). The absolute value of the inter-factor
Table 8.5 Coefficient of determination values from clus-

ter analysis
Variable Within Next 1_R2

cluster closest ratio
R2 cluster
Cluster 1
Beefy/meaty 0.81 0.21 0.24
Brothy 0.82 0.21 0.23
Painty 0.67 0.27 0.45
Browned caramel 0.68 0.21 0.41
Sweet 0.79 0.49 0.42
Nonanal 0.36 0.11 0.72
Cluster 2
Hexanal 0.55 0.11 0.50
Benzaldehyde 0.31 0.01 0.70
Limonene 0.71 0.03 0.30
2,3-Dichloropyrazine 0.93 0.03 0.07
Cluster 3
Cardboardy 0.76 0.44 0.42
Bitter 0.81 0.27 0.26
Sour 0.59 0.07 0.44
correlations was less than 0.3 (Table 8.3). Therefore an orthogonal

rotation (Varimax in SAS, 1988) was used for the factor analysis and all
loadings could be interpreted as the correlation of a variable with the
factor. Scree plots and eigenvalues indicated that a four-factor solution
may give the best results. The results are found in Table 8.4. Nonanal
loaded negatively on factor 1 along with cardboard and painty. Beefy/
meaty, brothy, browned/caramel and sweet loaded positively on factor 1.
This indicates an inverse relationship between desirable and undesirable
flavors, and a relationship between nonanal and lipid oxidation related
flavors. Hexanal, limonene and the internal standard, 2,3-dichloropyrazine
loaded on factor 2, which is independent of flavor attributes. Factor 3
loaded the basic tastes bitter and sour. Factor 4 loaded benzaldehyde and
nonanal. Nonanal loaded on two factors, factor 1 and factor 4. This indi-
cated that the variable is not clearly associated with either factor. The use
of other orthogonal rotation methods (Equamax and Quartimax in SAS,
1988) did not clarify it. The relation between flavor evaluation and
chemical analysis resulted in a somewhat different pattern in factor
analysis than in principal component analysis.
8.6.3.3 Cluster analysis. The results of the cluster analysis are found in
Table 8.5. Cluster 1 included beefy/meaty, brothy, painty, browned/
caramel, sweet and nonanal. Cluster 2 included hexanal, benzaldehyde,
limonene and the internal standard, 2,3-dichloropyrazine. Cluster 3
included cardboard, bitter and sour. If the 'within cluster R2', is large it is
better, and should be higher than between cluster R2. If the 'next closest'
value is low, it means the clusters are well separated. If the 'I_R2 ratio'
value is low, it indicates good clustering. Using these criteria, non anal in
Cluster 1 was not strongly related to other variables in Cluster 1. It had a
low R2 within the cluster and the I_R2 ratio did not indicate good cluster-
ing.
8.6.3.4 Conclusions from example data-set. This example illustrates some

of the points brought out in the discussion above on statistical methods.
The small number of samples limited the number of variables that could
be analyzed in order to keep the analysis robust. Flavor descriptors
usually consist of multiple compounds and not just one compound, there-
fore the limited number of compounds analyzed lowered the chances of
finding relationships.
All three multivariate statistical methods produced similar results, indi-
cating a relationship among flavor descriptors and flavor compounds.
Beefy/meaty, brothy, browned/caramel and sweet are desirable flavors in
beef, and tend to be related. Painty, and most of the time, cardboard
flavors negatively correlate with the desirable flavors. Nonanal tends to
relate to painty and maybe cardboard but it is not a solid relationship.
The other flavor compounds did not relate to any descriptive flavors in
these samples. The fact that differences occurred between the multivariate
methods indicates that the relationships are not solid. A data-set with
more samples that have extreme variation would result in more sub-
stantial results. These results indicate more research would be needed to
understand the relationship between beef-flavor attributes and the
chemical compounds responsible for their perception.
8.7 Summary
The concept of analytical descriptive flavor and aroma analysis was

reviewed. Instrumental methods for measuring flavor-producing com-
pounds were presented. Included is the concept of gas chromatograph/
sniffer-port analysis. Sensory methods adequately measure flavor and
aroma but can be very expensive to perform. Instrumental methods are
more economical to perform on a routine basis but do not always reveal
the complete flavor story. Both types of flavor-measurement methods are
important and have their place in flavor investigation. To be effective the
instrumental measurements have to be related to the sensory evaluations.
Multivariate statistical methods are presently the routine methods for
relating the sensory attributes with instrumental measurements. Several
types of statistical procedures were presented. The concept of neural net-
working was presented as a futuristic method of relating sensory and

instrumental data. A small data-set was used to illustrate some of the sta-
tistical methods, and associated limitations.
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Acknowledgements
The authors wish to acknowledge Dr Arthur Spanier for the gas chroma-
tographic data, and Dr Bryan Vinyard for the thought-provoking discus-
sion on the statistical analysis. A special thanks goes to both of these
scientists for reviewing the manuscript. Appreciation goes to Gioconda
Lau and Larry Boihem for preparing the data-set for statistical analysis,
and to Carolyn Vinnett for her descriptive panel leadership.
9 Species-specific flavors and odors
A.M. PEARSON, 1.1. GRAY and C.P.'BRENNAND
9.1 Introduction
Although meat flavor per se was shown to reside in the water-soluble

fraction by Crocker (1948), Bouthilet (1951a,b) and Kramlich and
Pearson (1958), it was demonstrated later that the lipid fraction was the
main contributor to species-specific flavors and odors (Hornstein and
Crowe, 1960; Hornstein et aI., 1963; Minor et at., 1965). The latter obser-
vation was confirmed by the taste-panel studies of Wasserman and Talley
(1968) who showed that the fatty tissues contained the species-specific
flavor and odor compounds. Addition of fat from any species significantly
improved the number of correctly identified lean meat samples. This
provided conclusive proof that the fatty tissues were responsible for the
species-specific flavors and odors in various kinds of meat.
In spite of the importance of the studies mentioned above, generally
there has been a paucity of information on the compounds that are
responsible for the species-specific odors and flavors in meat, poultry and
fish products. Since the fatty tissues are definite contributors to the
species-specific odors and flavors, the fatty acid composition of the
adipose tissues from some of the common meat-producing species will be
discussed briefly. Following this the lipid-soluble components that are
known to playa role in the genesis of species-specific flavors and odors in
meats will be outlined. Finally, discussion will center on the species-
specific flavors and odors and the responsible compounds.
9.2 Fatty acids in meats
Fatty acids in meats have been of interest for a variety of reasons. Com-
parisons have typically been concerned with those fatty acids containing
10 carbons or more and their degree of unsaturation. From a flavor
standpoint, however, the short- to medium-chain fatty acids are the most
important contributors (Brennand, 1989). They occur in very small quan-
tities in meats and are frequently ignored in research studies on the fatty
acids because methods for their accurate measurement were not available
until recently.
SPECIES-SPECIFIC FLAVORS AND ODORS 223
The fatty acids in adipose tissue of beef, pork and lamb, in descending
order of concentration are: oleic, palmitic, stearic, linoleic, myristic and
palmitoleic (Anderson et al., 1975, 1977). These six fatty acids account for
over 90% of the fatty acids in the depot fat of ruminants. A greater pro-
portion of stearic acid in lamb fat than in beef or pork fat results in a
higher melting point and the undesirable mouth-coating properties of
lamb and mutton fat (Cramer and Marchello, 1964).
9.2.1 Influence of cooking on the fatty acid composition of meat

There are mixed reports on whether cooking of meat changes the ratio of
total fatty acids in meat. Janicki and Appledorf (1974) found a significant
loss of Cl6 during cooking (e.g. boiling, broiling, frying or microwaving)
of raw ground beef. There was an increase in C18:1 and C18:2 fatty acids
of the cooked meat compared with the raw samples. Cooking of the meat
resulted in a decrease of about 50% of the total fat content, mainly due to
losses of C9 to C18:1 fatty acids in the drippings.
Terrell et al. (1968) reported deceases in C18:3 of the neutral triacylgly-
cerol fraction and in C14 and C15 of the phospholipid fraction on
broiling of beef steaks. They also found an increase in the percentage of
C8 in the phospholipid fraction. The sex of the animal made a greater dif-
ference than broiling. Steers had significantly higher percentages of C16
and C18 in the neutral fraction than heifers but less C18:1. Values for
octanoic acid were only 0.44% and 0.53% in raw and broiled meat
compared with 0.63% and 1.20% for the sex differences, respectively.
Campbell and Turkki (1967) did not find any difference in the fatty acid
contents of raw and cooked beef but noted the concentration of linoleate
in the phospholipid fraction was higher for cooked than for raw pork.
Siedler et al. (1964) reported that the fatty acid distribution in beef variety
meats was not greatly changed during braising. Differences in the fatty
acid composition between raw and cooked meats were suggested by
Chang and Watts (1952) to be due to a nonuniform distribution of tri-
acylglycerols in the raw meat rather than a large difference in the extent of
destruction of the unsaturated fatty acids during cooking.
9.2.2 Volatile free fatty acids and some other possible flavor compounds
Brennand and Lindsay (1992a,b) determined the volatile free fatty acids
(VFFAs) in various fractions from mutton during cooking by roasting,
frying or boiling. They found that among the various VFF As from C4 to
C11, 4-methyloctanoic, 4-ethyloctanoic and 4-methylnonanoic were always
in the condensates of the cooking vapor and in the meat samples. These
VFFAs were present in sufficient concentrations to provide the character-
istic 'mutton-like' and 'goat-like' aromas and flavors.
9.2.2.1 Thiophenols and alkylphenols. In addition, to the VFFAs,

Brennand and Lindsay (1992b) found thiophenol, 0-, m- and p-cresol,
phenol, 2-isopropylphenol, 3- and/or 4-isopropylphenol, thymol and car-
vacrol in the cooked mutton fractions in sufficient concentrations to
provide distinctive flavor notes that are associated with mutton. Thus, the
results indicate that the species-related flavors in mutton are, at least in
part, due to the presence of some potent BCFAs and alkylphenols. The
latter group of compounds was shown to increase in concentration during
cooking.
9.2.3 Free fatty acids in meat

As a group, free fatty acids (FFAs) have greater implications for the
flavor of meat than bound fatty acids but data on the content of FFAs in
meat are limited. This is especially true for changes in FFAs as related to
heating. Hornstein and Crowe (1960) and Hornstein et al. (1963) reported
that 99% of the FFAs in lamb are accounted for by oleic, stearic and
palmitic acids compared with 85% of the FFAs in beef and pork. In
addition to the relative concentrations of the fatty acids, the total amount
of the FFAs was lower in lamb than in beef or pork (0.4%, 3.7% and
5.5%, respectively). Variations caused by heating were not obvious from
the data.
9.2.4 Branched-chain fatty acids (BeFAs)

Certain patterns in the occurrence of BCFAs in adipose tissue have been
observed, especially for the longer-chained members. Species, diet and
tissue source all play a role in the concentration and the type of BCFAs
found. Iso (H3C-CH(CH3)-R-COOH) and anteiso (H3C-CH2-CH(CH3)-R-
COOH) acids are the most common long-chain BCFAs in the fat of cattle
and pasture-fed sheep. Other positions for branching exist. According to
Wong et al. (1975a-c), those BCFAs with methyl groups at the fourth
carbon from the carboxyl group are especially important to the undesir-
able or so-called 'soo' flavor of lamb. These workers reported that steam
distillates of fatty acids from lamb and mutton contained a homologous
series that included n, 2-methyl, 4-methyl, anteiso, iso, 4,6-dimethyl, Cl,~
unsaturated, unsaturated (designated a and b), oxo and aromatic acids.
The even-numbered n-chain acids were the most abundant group quanti-
tatively. The same acids were present in cooked lean meat plus adipose
tissue and in cooked ground adipose tissue. 'Sheep-like' odors were found
to be associated predominantly with the trace acids lying between n-C8
and n-ClO upon gas chromatographic separations. Thus, BCFAs having
8-10 carbon atoms contribute to the 'soo' flavor of cooked mutton, espe-
cially 4-methyloctanoic and 4-methylnonanoic acids (Wong et al., 1975b).
Table 9.1 Concentrations of volatile fatty acid constituents in the fat of mutton, beef and
goat meat"
Acid concentration (p.p.m.)

Animal n6 n7 n8 n9 nl 4MeClOb 4MeC8 c
Goat 86 24 157 45 1180 122 48

Muttond
B 28 28 148 58 2030 36 38
M 57 7.3 111 15 924 4.6 10.5
S 136 24 165 17 1860 9.6 6.9
G 49 5.7 190 12 1395 2.4 4.8
L 124 6.7 164 22 784 2.6 4.7
F 41 3.1 121 8.3 990 1.0 2.0
N 145 4.9 122 13 850 1.6 2.0
Beef 58 8.4 115 14 596 0.7
"From Wong et al. (1975a).

b4MeClO = 4-methylnonanoic acid.
c4MeC8 = 4-methyloctanoic acid.
dLetter designations were used to indicate the diet or sample source as follows: B = barley;
F = clover pasture; G = grass pasture; S = sunflower oil-protected supplement; L = lamb;
M = mutton-control; N = lamb.
Differences in goat, mutton and beef fat were manifested primarily in the
relative proportions of odd-numbered and BCFAs present, as demon-
strated by the data shown in Table 9.l.
Fat from a goat and a barley-fed sheep (B, Table 9.1) contained the
highest amounts of 4-methyloctanoic and 4-methylnonanoic fatty acids.
Neither of these fatty acids was found at appreciable levels in the distillate
from beef adipose tissue samples. Addition of 4-methyloctanoic and 4-
methylnonanoic acids to ground mutton resulted in a trained sensory
panel concluding that these samples had a more intense 'mutton flavor'
than the control. However, Wong et al. (1975c) noted a relatively high
threshold value (45 p.p.m.) for 4-methylnonanoic acid compared with the
level they found in meat (1-36 p.p.m.). Thus, these workers concluded
that this fatty acid was likely not to contribute directly to 'mutton-like'
flavor but that it could exert an effect through a synergistic action on 4-
methyl octanoic acid, which has a lower threshold (0.5 p.p.m.). Concentra-
tions of 4-methyloctanoic acid varied from 2-38 p.p.m. in mutton,
depending on the animal's diet (Wong et al., 1975c).
Ha and Lindsay (1990a) identified and provided quantitative data on
additional fatty acids from butanoic through to 2-ethyldecanoic in a
variety of meat fats. They found 4-ethyloctanoic acid in male goat, ram,
ewe and lamb depot fats but not in veal, beef, pork or horse depot fats.
Previously 4-ethyloctanoic acid had been identified in the sebaceous gland
secretion of the male goat (Sugiyama et al., 1981, 1986). This compound
has an extremely low threshold (Boelens et al., 1983) and a pronounced

'goat-like' aroma (Brennand, 1989; Brennand et aI., 1989; Brennand and
Lindsay, 1992a,b).
9.2.4.1 Quantities and sources of BCFAs. Long branched-chain fatty

acids in ruminant fats are typically present in relatively low concentrations
when compared with long n-chain fatty acids. Subcutaneous adipose tissue
from pasture-fed lambs contained from 2-4% BCFAs according to
Duncan et al. (1972) and Hansen and Czochanska (1976). In studies with
light and heavy animals, Busboom et al. (1981) found that fat from rams
had 15.6% and 6.9% BCFAs, while fat from wethers only had 3.4% and
3.5% BCFAs, respectively. Low concentrations of certain longer BCFAs
have also been found in ox fat (Duncan and Garton, 1978). Trace
amounts of branched C12:0 and C16:0 fatty acids were reported in bovine
adipose tissue by Terrell et al. (1967). They also found from 0.8-2% of
odd-numbered fatty acids. Booren et al. (1973) compared beef and
antelope fat and noted that the latter had a significantly higher quantity
of branched-Cl6 fatty acid than beef fat. Fallow deer fat also contained
an unusually high level of long-chain BCFAs according to Smith and
Duncan (1979). These workers found that the perinephric adipose tissue
of fallow deer contained 15.5% BCFAs, with iso- and anteiso-acids being
present in the largest quantities (8.96% and 2.85%, respectively) followed
by other monomethyl fatty acids (1.73%). Di- and poly-methyl branched
fatty acids also occurred at low levels.
Goats' milk and cows' milk have 2.02% and 3.07% of primarily long-
chain BCFAs, respectively (Massart-Leen et al., 1981). The addition of 4-
ethyl-2-octenoic acid to milk resulted in a 'goaty flavor' (Smith and Parks,
1982) but this compound has not been isolated from milk fat or animal
fats. Long-chain iso- and anteiso-acids predominate in both goats' and
cows' milk but the former also has other monomethyl branched fatty
acids. In cows' milk, the monomethyl branched-fatty acids are virtually
absent with the exception of trace amounts of 6-methylhexadecanoate
(Massart-Leen et aI., 1981).
However, in several types of cheese made from cows', sheeps' and
goats' milk, Ha and Lindsay (1990b, 1991 b) found a wide variety of
volatile BCFAs. In cows' milk cheese 4-methyloctanoic acid was present
in low concentrations, whereas, cheese made from sheeps' and goats' milk
contained significant amounts of both 4-methyloctanoic and 4-ethylocta-
noic acids. These fatty acids appear to contribute 'mutton-like' and 'goat-
like' characteristics to both sheep and goat cheeses (Brennand et al.,
1989).
BCFAs also have been reported in whales by Morii (1980). The short-
chain fatty acids in whale adipose tissue are predominantly branched
acids, with isovaleric accounting for more than 90% of the fatty acids
with chain lengths under C6. Among the long-chain fatty acids, the most
common BCFAs are iso-tetradecanoic and iso-hexadecanoic acids. The
long-chain fatty acids also include odd-numbered chain length acids in
both the iso- and anteiso-configurations.
9.2.4.2 Structures of branched-chain fatty acids. Most long-chain BCFAs

identified in ruminant tissues and milk are saturated with methyl substitu-
tions on even-numbered carbon atoms (Smith et al., 1979; Hansen and
Czochanska, 1976; Massart-Leen et al., 1981). Acids with one methyl
group are most abundant but di-and tri-methyl fatty acids have also been
reported (Smith et al., 1979). Iso- and anteiso- fatty acids are found in a
wide variety of species but other branching patterns are more common in
ovine fatty tissues according to Smith et al. (1979). Among the BCF As, 4-
methyl branching occurs in lamb triacylglycerols most frequently, followed
by 6- and 8-methyl and 4,6-dimethyl branching. The latter three branching
positions occur at about one-half the concentration of those with 4-methyl
branching.
9.2.4.3 Effect of cooking on branched-chain fatty acids. Harsh methods

of cooking, including high temperatures, extended cooking times and
large surface areas, resulted in lower concentrations of BCFAs in the
meat and larger amounts in the drippings and condensed volatile frac-
tions in studies reported by Brennand and Lindsay (1992b). Boiling
reduced the amount of BCFAs in the meat because they were partitioned
into the broth.
9.3 Subcutaneous and perinephric adipose tissue
Variations in fatty acid composition between the subcutaneous and peri-

nephric adipose tissues have been noted. Garton et al. (1972a,b) found
that sheep fed a diet high in rolled barley or a propionate-supplemented
diet contained BCFAs and odd-numbered fatty acids, which were present
for the most part in the subcutaneous triacylglycerols. The perinephric
triacylglycerols also had high levels of BCFAs in barley-fed sheep,
although a composite of the BCFAs (excluding iso- and anteiso-acids)
was lower than in the subcutaneous fat. The smaller proportion of
BCFAs in perinephric triacyglycerols than in subcutaneous tissue was
confirmed by Duncan et al. (1974a-c). Hansen and Czochanska (1976)
compared the fat composition in pasture-fed lambs and reported that
respective differences between subcutaneous and perinephric fats existed
in the quantities of palmitic (20.4% and 16.6%, respectively) and stearic
(15.7% and 23.9%, respectively) acids. Subcutaneous fats included 3.6%
of odd-numbered fatty acids while perinephric fats had 3.1 % of odd-
(a) (b)
Isoleucine leucine
o
II
H,C--H,C .......... / C ..........
C S-CoA
/H
H,C
o
II
H,C--H,C .......... / C ..........
C OH
/H
H,C
2-Methy lbutanoic acid 3-Methylbutanoic acid
Figure 9.1 Scheme for synthesis of (a) 2-methylbutanoic and (b) 3-methylbutanoic acids
through transamination and decarboxylation of isoleucine and leucine, respectively (Ha and
Lindsay, 1990a).
numbered fatty acids. Subcutaneous adipose tissue also has a higher pro-
portion of fatty acids synthesized in the tissue than the perinephric fats
(Christie, 1981).
The softer outer region of ram subcutaneous tissue has a lower melting
point than the inner area and also contains a greater concentration of
o o 0
II
~SCoA + ENZ. - Biotin - CO, ..
Propionyl- CoA
carboxy lase
.. g
7",Y )\ "SCoA
Propionyl - CoA CH]
MethylmaIonyl- CoA
+
ENZ. - Biotin
o 0 o
~ II II
/c'" A
eO Y "S-ACP AAS-ACP ~
CH3
\...,... .. ~S-ACP
Methylmalonyl-S-ACP ~ CH3
co,
2-Methylhexanoyl-S-ACP
0
/
II
--- AS-ACP
o
II
~OH
CH 3
4-Methyloctanoic acid
Figure 9.2 Proposed biosynthetic pathway for production of 4-methyloctanoic acid (Ha and
Lindsay, 1990a).
BCFAs (Johnson et al., 1988). However, the proportions of medium-

chain-length fatty acids other than C9 did not differ statistically between
the layers.
Brennand and Lindsay (1992a) have quantitatively determined the dis-
tribution of the volatile BCFAs in various lamb tissues. They found that
the BCFAs were more concentrated in the subcutaneous adipose tissue
than in either the perinephric adipose tissue or in the intramuscular fat.
Perinephric adipose tissue contained relatively high concentrations of n-
chain even-numbered fatty acids and low levels of BCFAs. In all the fat
o 0
;C"Y A
Butyryl- CoA
carboxylase
.. ~ "SCoA
C,H,
Ethylmalonyl - CoA
+
ENZ. - Biotin
o 0 o
~ II II
A
~
/C"
eO Y "S-ACP AAS-ACP
C,H,
Ethylmalonyl-S-ACP
~ .. ~S-ACP
C,H,
CO,
2-Ethylhexanoyl-S-ACP
0
/
II
--- AS-ACP
o
II
~OH
C H, 2
4-Ethyloctanoic acid
Figure 9.3 Proposed biosynthetic pathway for production of 4-ethyloctanoic acid (Ha and
Lindsay, 1990a).
samples tested, 4-methyl- and 4-ethyl-octanoic acids were present at con-

centrations considerably above threshold values and thus, seem to be con-
tributors to the flavor of lamb. Conversely, 4-methylnonanoic
concentrations from lamb fat ranged from non-detectable levels to greater
than the threshold level. Therefore, this compound does not always
appear to be a contributor to the flavor and/or odor of lamb or mutton.
Lean meat contained only low levels of 4-methyl- and 4-ethyloctanoic
acids, especially when compared with adipose tissues (Brennand and
Lindsay, 1992a). This adds further proof to the earlier work of Wasser-
man and Talley (1968) that the species-specific flavors and odors are loca-
lized mainly in the adipose tissues. Trace amounts of BCFAs in lean meat
are no doubt derived from the relatively small amounts of intramuscular

fat in the lean tissue.
9.4 Synthesis of branched-chain fatty acids
Several mechanisms for the formation of methyl-branched chain fatty

acids have been suggested by Christie (1981). These include the incorpora-
tion of carbon skeletons from branched amino acids (such as leucine and
isoleucine), the incorporation of propionic acid and the addition of a
carbon from methionine.
The incorporation of leucine or valine, and isoleucine can result in iso-
and anteiso-acids, respectively (Gurr and James, 1971). In Figure 9.l.,
taken from Ha and Lindsay (1990a), the pathways for the formation of
each type are illustrated. Iso-acids are considered to be formed biosytheti-
cally through methylmalonyl Co-A (Smith et ai., 1979). Of the branched
fatty acids, the iso-acids occur in the highest concentrations in the fat
from cattle or pasture-fed lambs and in milk (Hansen and Czochanska,
1976; Massart-Leen et ai., 1981).
Propionic acid, produced by microflora in the rumen, can also lead to
the formation of BCFAs. When propionic acid is substituted for acetic
acid in fatty acid synthesis, the reactions yield methylmalonyl-CoA. Fatty
acids with methyl substitutions on the even number carbons then arise
from the incorporation of methylmalonyl-CoA into the fatty acyl chain.
Suggested pathways for the formation of the 4-methyl- and 4-ethyl-branch
positions have been proposed by Ha and Lindsay (1990a) and are shown
in Figures 9.2 and 9.3, respectively.
Methylmalonate can accumulate when the propionate presented to the
liver exceeds the capacity to metabolize it. Methylmalonate then competes
with malonate for inclusion into fatty acid synthesis (Duncan and Garton,
1978). The availability of propionyl-CoA as a primer unit would account
for the presence of odd-numbered chain as well as even-numbered chain
compositions of BCFAs (Smith et ai., 1979.
9.4.1 Effects of diet on synthesis of branched-chain fatty acids.

Fat from barley-fed lambs has a higher proportion (6-10%) of BCFAs
and also of fatty acids with an odd number of carbons than that from
pasture-fed lambs (Duncan et ai., 1972, 1974a; Garton et aI., 1972b;
Pearce and Chestnutt, 1974; Smith et ai., 1979). When sheep, goats and
cattle were fed a similar barley-rich ration, there was a high proportion of
BCFAs with methyl groups in positions different from iso- and anteiso-
positions in sheep and goats but not in cattle. The barley-rich ration did
not affect radically the levels of iso- and anteiso-BCF As but both the
number and proportions of BCFAs were greater in barley-fed than m

grass-fed sheep.
Lambs fed a ration containing 21 % of their metabolizable energy as
propionate salts had higher amounts of BCFAs than lambs consuming a
control, acetate-, or butyrate-enriched diet (Garton et al., 1972b). Barley-
fed lambs produced a high amount of propionic acid in the rumen
(Duncan et al., 1972), whereas perennial rye grass and white clover diets
resulted in a high concentration of acetic acid but a low amount of pro-
pionic acid (Hansen and Czochanska, 1976). Corn diets also result in an
increase in BCFAs in lamb tissues (Miller et aI., 1980). Lambs raised on
high-energy diets with increased BCFAs and odd-numbered fatty acids
tend to have soft, oily carcass fat.
Interspecies differences exist in the metabolism of propionic acid
between cattle, sheep and goats. The differences appear to lie in the effec-
tiveness with which each species of animal metabolizes propionate and its
carboxylation product, methylmalonyl-CoA (Smith et al., 1979; Massart-
Leen et aI., 1981). Thus, this biochemical process may ultimately provide
the basis for much of the observed differences in flavor between meats for
the various species of ruminants.
9.4.2 Body secretions of branched-chain fatty acids by meat-producing

animals
Compounds that are found in one biological system also frequently occur
in other locations in the same or other species of animals. Sugiyama et al.
(1981) have reported 4-ethyloctanoic, 4-ethyldecanoic, 4-ethyldodecanoic
and 4-ethyltetradecanoic acids to be the odoriferous substances secreted
by the sebaceous gland of the mature male goat. These investigators also
noted that 4-ethyloctanoic acid had an intense and characteristic 'goaty
odor'. However, fatty acids containing a 4-methyl branch were not found
in the sebaceous gland secretion. Smith and Parks (1982) notecl that over
50% of the sebaceous gland lipids in male goats were branched-chain
fatty acids.
Unprocessed sheep wool or fleeces have a definite 'sheep-like' odor that
may be partially derived from volatile BCFAs. Wool grease and wax are
products of the sebaceous gland. Wool fat consists of 11 % free acids,
44% combined acids and 46% of unsaponifiable matter according to
Truter (1956). Wool wax acids consist of alkanoic, (X-hydroxy and 0)-
hydroxy fatty acids, with each of these three groups containing normal,
iso- and anteiso-series of various chain lengths. Practically all of the acids
are saturated (Motiuk, 1979). In addition to BCFAs, wool wax also
contains branched alcohols and branched hydrocarbons according to
Motiuk (1979, 1980). Motiuk (1980) noted a structural similarity between
the hydrocarbons, acids and aliphatic alcohols found in wool wax, with
Table 9.2 Qualitative aroma properties of selected fatty acids'
Fatty acid Odor description Odor

concentration thresholds
(p.p.m.) (p.p.m.)
2-Methylpropionic 5.3
10 Sweaty fatty acid-like
50 Sweet, rotten apple, sweaty
Neat Sweet, apple-like, fatty acid-like
2-Methylbutanoic 3.2
5 Rotten fruit, estery, sweet
10 Slightly sweet, fruity, waxy, sweaty-fatty acid
Neat Slightly sweet, fruity, waxy, sweaty-fatty acid
3-Methylbutanoic 0.Q7
5 Sweaty socks, fatty acid-like
10 Sweaty socks, fatty acid-like, slightly sweet
Neat Sweaty, slightly green apple note
2-Ethylbutanoic 5.7
1 Very mild, slightly sweet
5 Very distinct, fruity, pleasant
Neat Slightly sweaty fatty acid, very slightly fruity
Pentanoic 6.5
5 Cheese-like, nutty, butyric-like
10 Nutty, butyric-like, waxy
Neat Cheese-like, butyric-like, waxy
2-Methylpentanoic 11.8
5 Swiss cheese-like, smooth fatty acid
25 Sweet, sourish, fatty acid, early cooking fried
hamburger
Neat Rotten fruit, raw sheep wool, sheep pen, slight apple
note
0.5 Wet pinewood, sheep wool, fatty acid-like
1 Phenolic, sheep-like
Neat Sweet, fruit-like, slightly woody, fatty acid-like
5 Slight cheese-like, waxy
25 Cheese-like, sweet, dirty sweaty socks
Neat Cheese-like, hexanoic-like
2-Methylhexanoic 55.3
5 Fruity, fatty acid-like
50 Slight sweaty, waxy
Neat Musty-sweaty, apple-like
4-Methylhexanoic 7.3
10 Woody, sweet-sweaty
20 Sweaty, sour, woody-waxy
Neat Sweaty, sheepy, waxy
Table 9.2 (continued)

(p.p.m.) (p.p.m.)
2-Ethylhexanoic 82.4
5 Phenolic, waxy
10 Phenolic, waxy, woody
Neat Heavy, sweet, waxy, cheese-like
Heptanoic 0.28
1 Soapy, fatty acid-like
25 Sweet, soapy fatty acid
Neat Woody, sweat, waxy, fatty
6-Methylheptanoic 0.84
0.5 Woody, cheese-like
2.5 Cheese-like, sheep wool, sweaty
Neat Cedar, fruity, waxy
Octanoic 2.2a
5 Waxy
50 Goaty, waxy, soapy
Neat Goaty, waxy, soapy
4-Methyloctanoic 0.02
1 Waxy, goaty
10 Goaty-muttony
Neat Waxy, sweaty, perfumy
6-Methyloctanoic 4.1
0.5 Sheep wool-like, fatty acid-like
2.5 Phenolic, wet woody, slightly sweet
Neat Sweet, wet wood, fatty
4-Ethyloctanoic 0.006
5 p.p.b. Goaty
1 Goaty
Neat Very goaty
Nonanoic 2.4
1 Soapy, fatty acid-like
10 Waxy, fatty, soapy
Neat Soapy, waxy
4-Methylnonanoic 0.65
1 Waxy-sweet, soapy, fatty acid-like
25 Muttony, wet wood, fatty
Neat Muttony, wet wood, fatty
8-Methylnonanoic 0.66
2.5 Soapy, decanoic-like
10 Sheepy, waxy fatty acid
Neat Sharp woody, fatty, slightly sweet

(p.p.m.) (p.p.m.)
2-Ethyldecanoic 0.02
1 Musty, musky, sweet, woody
10 Musky, perfumy, woody
Neat Soapy-waxy, slight citrus, slight woody
9-Decenoic 4.3
5 Sweet, fatty
10 Sweet, soapy
Neat Heavy, sweet soapy
Undecanoic 0.1
5 Soapy
10 Soapy, waxy
Neat Harsh, fatty, sweet, soapy, waxy
10-Undecenoic 2.3
5 Soapy, sweet
50 Medicinal (athletes' foot ointment)
Neat Medicinal (athletes' foot ointment)
"Data from Brennand et al. (1989). bOdor thresholds are for samples for octanoic acid, which
is for flavor and was run at pH 3.2 (Baldwin et al., 1973).
all three groups of compounds containing a normal, iso- and anteiso-

series.
Humans also produce unique fatty acids in the secretion from their
sebaceous glands. Nicolaides (1974) noted that most of the 200 different
fatty acids found in human skin were not normally encountered in the
internal tissues. About 16% of these fatty acids were branched. Excluding
the iso- and anteiso-branched-chain fatty acids, the maximum amount of
methyl-branching occurs at the fourth carbon for all chain lengths. Under
the testing conditions used, however, only long-chain fatty acids were
detected.
9.4.3 Odors and flavors of some selected fatty acids

The descriptive odors of the major BCFAs and some straight-chain fatty
acids found in lamb and/or mutton fat (Brennand et ai., 1989) are pre-
sented in Table 9.2. The values given in the table were all, except for one,
determined at pH 2.0, at which Brennand et al. (1989) found the panelists
to be generally more sensitive than at higher pH values. It should be
pointed out, however, that cooked meat has a pH of about 6.0 and raw
meat seldom gets below pH 5.3.
It is also interesting to note that the sensory descriptors used by the

panel may differ greatly, depending on the concentration tested (Table
9.2). For example, octanoic acid at low levels (5 p.p.m.) was described
as having a waxy aroma but at 50 p.p.m. as being 'goaty, waxy and
soapy'. On the other hand, 4-ethyloctanoic acid was described as 'goaty'
at low concentrations (5 p.p.m.) but increased to 'very goaty' at high con-
centrations (Table 9.2). Of special interest are octanoic, 4-methyloctanoic
and 4-ethyloctanoic acids, which were described as being 'goaty' or
'muttony' at some or all concentrations tested (Table 9.2). Other fatty
acids that may be related to 'mutton' or 'lamb' flavors and odors include
2-methylpentanoic, which was described as having a 'sheep wool' or
'sheep pen' odor, 4-methylhexanoic, which was characterized as being
'sheepy' and 6-methylheptanoic that was described as having a 'sheep
wool' aroma.
Table 9.2 also gives threshold values for each of the fatty acids, which
varied from 0.02-82.4 p.p.m. The lowest threshold values were for 4-
methyloctanoic, and 2-ethyldecanoic, while the high value was for 2-ethyl-
hexanoic. These values were determined by Brennand and Lindsay (1989)
at pH 2.0 and differ from the threshold values reported by other workers
determined under different conditions, which were collected and summar-
ized by the above researchers.
Although those fatty acids having odors described as being 'goaty' or
'muttony' are more likely to be contributors to 'mutton' or 'lamb' flavors
and odors, one cannot rule out the possible interactions between mixtures
of compounds to produce the characteristic species-specific flavor notes. In
general, the short-chain (C4 to Cll) BCFAs are more likely contributors
than the longer-chained fatty acids (CIO to C22). Persistent information
would lead one to believe that 4-methyl- and 4-ethyloctanoic acids may
make major contributions to the species-specific flavor of lamb and
mutton (Brennand and Lindsay, 1992a,b). Other compounds, however,
may also playa role in lamb and mutton flavors and odors.
9.4.4 Determination of the concentration of short-chain and branched-

chain fatty acids
Adipose tissue and meat samples were first ground and the lipids from the
meat samples were then extracted by a modified Bligh and Dyer (1959)
method, and those in the adipose tissues by using a modification of the
Folch et al. (1957) procedure. Samples were then subjected to concentra-
tion, extraction and saponification according to the procedures outlined
by Brennand and Lindsay (1992a). Volatile n-chain and branch-chain fatty
acids were concentrated using a simultaneous distillation and extraction
procedure that was described earlier by Ha and Lindsay (1990b). After
esterification, the fatty acids were identified and quantified by gas
chromatography (GC) and mass spectrometry (MS) according to the

methods outlined by Ha and Lindsay (1990b).
9.4.5 Measurement of alkylphenols and thiophenols

Extraction, concentration and measurement of the alkylphenols and the
thiophenols were accomplished by selected ion monitoring using the GC-
MS methods described by Ha and Lindsay (1991) and Heil and Lindsay
(1988). These methods have been described in detail by Brennand and
Lindsay (1992a) and used for identifying and quantifying the alkylphenols
and thiophenols in mutton.
9.5 Other flavor or odor compounds localized in the fatty tissues
9.5.1 Sex odor or boar 'taint'

Craig and Pearson (1959) and Craig et al. (1962) first demonstrated that
the objectionable 'urine-like' or 'perspiration-like' odor associated with the
meat of the sexually mature uncastrated male pig or boar was not only
confined to the fatty tissues but was actually localized in the unsaponifi-
able fraction of the fat. The objectionable odor is commonly referred to as
'boar odor', 'sex odor' or 'boar taint' in the literature and was identified
as being associated with the steroid compound, 5Cl-androst-16-en-3-one or
androstenone by Patterson (1968) and shown by Desmoulin et at. (1982)
to be localized in the fat. Later, it was shown that four other steroids
having slightly different structures and all belonging to the C 19-d 16-steroid
group possessed similar odors and could be isolated from the unsaponifi-
able fraction of mature boar fat (Berry and Sink, 1971; Berry et al., 1971;
Thompson et at., 1972).
9.5.2 Pathways for C 19-d 16-steroid production

Based on an exhaustive series of studies by Gower and his associates
(Ahmad and Gower, 1968; Katkov and Gower, 1970; Loke and Gower,
1971, 1972; Gower, 1972; Brophy and Gower, 1972, 1973, 1974; Katkov
et at., 1972; Saat et al., 1972, 1974; Gower and Fotherby, 1975) many of
the pathways for the biosynthesis of the Cwd 16-steroids have been
worked out. The metabolism of these compounds (Bonneau and Terqui,
1982) has been summarized by Brooks and Pearson (1986). The pathway
for production of the Cwd 16-steroids beginning with pregnenolone
through the Cwd 16 -steroids is presented in a review, which shows the
entire scheme for all steroid hormones (Brooks and Pearson, 1986). The
Cwd 16-steroids are not in the same part of the pathway as the sex
hormones, testosterone and 17J3-estradio1, although all originate from the

same precursor compound, pregnenolone. Although the sex hormones are
directly involved in the reproductive process, the C w Ll l6-steroids play an
indirect role in the pig by functioning as pheromones (Melrose, et al.,
1971; Reed et al., 1974; Perry et aI., 1980).
9.5.3 Thresholds and odors of the Cw Ll l6 -steroids

Brooks and Pearson (1989) have determined the odor thresholds, the simi-
larities and differences for the C W Lll6-steroids that have been identified as
contributors to 'boar odor' (Thompson et al., 1972). Thresholds for 5,16-
androstadien-3J3-01 and 4,16-androstadien-3-one were about 10 !!g.g-l,
whereas those for 5cx-androst-16-en-3-one, 5cx-androst-16-en-3cx-ol and 5cx-
androst-16-en-3J3-01 were about 1 !!g.g-l The differences in the threshold
values were statistically significant (P < 0.05). Results from triangle tests
demonstrated that panelists could differentiate between the odors of the
compounds possessing ketone groups and those containing alcohol
groups. Panelists, however, were unable to distinguish between the differ-
ent ketone compounds or between the various alcohol-containing com-
pounds.
9.5.4 Identification and quantification of the Cw Ll l6 -steroids

Although Patterson (1968) isolated and identified androstenone (5cx-
androst-16-en-3-one) as the compound responsible for 'boar taint', the
other C W Lll6-steroids were not identified by the GC procedure used.
Using steam distillation and GC methods, the other C w Ll l6-steroids were
isolated and identified (Berry and Sink, 1971; Berry et al., 1971). Quantifi-
cation by the early procedures was difficult, if not impossible. Thompson
and Pearson (1977) used GCjmass spectrometry in order to quantify
androstenone more accurately based on the use of deuterium-labeling.
Thompson and Pearson (1982) described the synthesis of deuterium-
labeled C W Ll l6-steroids, which was essential for quantification. Brooks
and Pearson (1986) have compared the various methods for identifying
and measuring the C w Ll I6-steroids, including the use of radio-
immunoassays for androstenone (Claus, 1974; Andresen, 1975, 1979) and
an enzyme-linked-immunoabsorbant-assay (ELISA). Once deuterium-
labeling is achieved, this assay, as described by Brooks et al. (1986),
appears to be the most sensitive and accurate and can detect all of the
C w Ll l6-steroids simultaneously.
9.5.5 Possible role of skatole

Lundstrom et al. (1980) and Hansen et al. (1980) reported that skatole
contributes to the objectionable odor of boar meat. Results from these
studies led them to conclude that skatole is a major contributor to 'boar

taint' and could be used as a screening test for the objectionable odor
associated with boar meat. On the basis of these investigations, Danish
slaughtering cooperatives have adopted on-line testing for skatole as a
means of identifying those carcasses with high levels of 'boar taint'. This
permits removal of the carcasses having objectionable levels of skatole
from the line and allows their use in other products.
9.5.6 Isolation and quantification of skatole

Hansson et al. (1980) improved the extraction of skatole from boar fat by
using simultaneous steam distillation and extraction. In this procedure,
both skatole and indole are distilled and extracted from the fat samples
and then concentrated. Skatole quantification is then achieved by subject-
ing the concentrated sample to Gc. Lin et al. (1991) compared GC and
high-performance liquid chromatography (HPLC) for measurement of
skatole and demonstrated that results from the two methods were highly
correlated and about equally sensitive.
9.6 Species-specific flavors
Discussion in this section will be divided into species-specific flavors for

lamb and/or mutton, goat, pork, beef, chicken and/or turkey, fish and
game animals.
9.6.1 Lamb and/or mutton flavor

Although Cramer et al. (1967) working in New Zealand were the first to
demonstrate that the diet of lambs can alter the flavor of the meat, more
extensive research was carried out in Australia by Park et al. (1972a, b).
This work is reviewed in greater detail in chapter 10 of this volume. It was
also shown by Cramer and Marchello (1964) that seasonal effects can alter
the fat composition of lamb, which may thereby influence the acceptability
of the meat. However, in common with the early studies, Hofstrand and
Jacobson, (1960), Pearson et al. (1973) and Caporaso et al. (1977) were
unable to identify any compounds that contributed directly to the species-
specific flavor and odor of lamb/mutton.
Although several researchers have studied ram meat with the objective
of identifying any sex-related flavors and/or odors (Kemp et al., 1970,
1972; Field, 1971; Jacobs et al., 1972; Busboom et al., 1981; Crouse et al.,
1981), they have not identified any compound(s) that contribute to off-
odors in the uncastrated sexually mature sheep. Nevertheless, it is
commonly accepted that ram meat may have a strong flavor and/or odor
(Ziegler, 1944). This has been discussed in some detail by Pearson (1990),
who suggested that ram flavors and odors may be associated with changes
in sex-hormone levels during the breeding season.
Wong et al. (1975a, b) were the first to report on the identification of
compounds contributing to the species-specific flavor in lamb meat. They
demonstrated that the undesirable flavor of lamb was associated with the
presence of the BCFAs, particularly with 4-methyloctanoic and 4-methyl-
nonanoic acids. More recently, Brennand and Lindsay (1982) were able to
isolate certain BCFAs from ovine fat after steam distillation and to
identify 4-methyloctanoic and 4-methylnonanoic acids and to show that
they contribute to 'lamb-like' flavors. They reported flavor thresholds for
the above two BFCAs of 0.6 and 2.4 p.p.m., respectively, in water at rc.
Although 4-methylnonanoic acid was described by the sensory panels as
being more 'lamb-like' than 'beef-like' or 'pork-like', ovine fat-containing
broths were rated as being even more 'lamb-like' than the pure
compound. This indicates that other compounds and/or different con-
centrations of compounds may playa role in the full 'lamb-like' flavor or
odor.
Upon examination of a whole series (n = 23) of branched-chain, odd-
numbered or unsaturated fatty acids, Brennand et al. (1989) found that
fatty acids having branch-chains at the 4-position had 'goaty/mutton/
sheep' aroma notes, as was also the case for some other fatty acids com-
prising 8-carbon structures. Shorter branched-chain fatty acids were char-
acterized frequently by panelists as having 'cheese-like' odors.
Brennand and Lindsay (1992a, b) concluded that 4-methyloctanoic and
4-ethyloctanoic acids were present in lamb fat in sufficient concentrations
to make a major contribution to lamb or mutton flavors. Certain alkyl-
and thiophenols have also been shown to contribute to lamb and mutton
odors by Brennand et al. (1989) and by Ha and Lindsay (1991). These
studies together with those of Wong et al. (1975a-c) leave little doubt that
the BCFAs contribute to the undesirable 'mutton flavor' but are less than
definitive as to their contributions to the desirable flavor attributes of
lamb. It may be that the alkyl- and thiophenols are responsible for the
desirable flavor attributes of lamb meat or that the BCFAs at lower levels
are the species-specific flavor compounds or even that a combination of
the two different groups of compounds may interact to produce the desir-
able flavor notes of lamb. Further studies will be needed to resolve these
unanswered questions.
Although much research has concentrated on identifying the undesir-
able flavor components that contribute to the objectionable flavors and
odors of lamb and mutton, experiments on the desirable flavors and odors
must still be carried out. This difficult but important area of research
remains virtually unexplored.
9.6.2 Goaty flavors and odors

The chemical 4-ethyloctanoic acid was identified in the secretion of the
sebaceous glands from male goats (Sugiyama et aI., 1981) and later in the
fat from male goats, rams, ewes and lambs (Ha and Lindsay, 1990a).
Table 9.2 contains the aroma descriptors of some selected fatty acids that
were characterized by a sensory panel as having 'goaty' odors. These
include octanoic, 4-methyloctanoic and 4-ethyloctanoic acids, which had
odor thresholds of 2.2, 0.02 and 4.1 p.p.m., respectively, at pH 2.0
(Brennand et al., 1989). These same compounds may also be involved in
mutton and/or lamb flavor (Brennand and Lindsay, 1982, 1992a,b;
Brennand et aI., 1989). Thus, the same BCFAs seem to be involved in
mutton and goaty flavors.
Although the same BCFAs may playa major role in 'goaty flavors' as
in the 'soo flavor' of lamb and mutton, no uniquely different odor or
flavor compounds have been associated with goat or chevron meat.
Whether the same low level of the BCFAs is responsible for the desirable
flavor or odor of chevron is not known at present. It is possible that
uniquely different compounds may be responsible for the desirable and
undesirable flavor and odor compounds in goat meat but further research
will be needed to establish whether this is the case.
9.6.3 Odors and flavors in pork

Brennand and Lindsay (1982) found that it was difficult to distinguish the
flavors from porcine, bovine or ovine fatty tissue preparations. Steam dis-
tillates from the fatty samples of pork tissues were rated as being sig-
nificantly more 'pork-like' than 'beef-like' or 'lamb-like'. This indicated
that porcine adipose tissue is the source of the pork-specific flavor com-
pounds and adds credence to the earlier research of Hornstein and Crowe
(1960) and Wassermann and Talley (1968), who had suggested this to be
the case. Wasserman and Talley (1968) found that water washing of the
chloroform/methanol extract from pork adipose tissue resulted in a loss of
the characteristic 'pork-like' flavor. Nevertheless, compounds responsible
for the 'porky' flavor have not been identified if one ignores the informa-
tion available about 'boar-odor' or 'sex-odor' from pork.
Mottram et al. (1984) have demonstrated that the flavor compounds
present in fresh and cured pork are different. They found that nitrite plays
an important role in developing the cured flavor of pork as disclosed in
chapter 10.
9.6.3.1 Boar odor or taint. Boar odor or taint and the contributing com-
pounds were discussed in chapter 10 and earlier in this chapter. Readers
are referred to the studies of Craig et al. (1962), Williams et al. (1963),
Berry and Sink (1971), Berry et af. (1971), Thompson et af. (1972) and
Brooks and Pearson (1986, 1989) for further information.
There have been several attempts to prevent 'boar-odor' by immuniza-
tion against some or all of the C 19 -d 16-steroids, with variable degrees of
success (Williamson and Patterson, 1982; Williamson et al., 1985). Brooks
et al. (1986) were able, by immunization, to decrease the intensity and fre-
quency of the occurrence of boar odor by about 80% but, as is common
with other immunization procedures, complete elimination of the Cwd 16_
steroids was not achieved.
There are other objectionable and also desirable odors generated on
heating porcine adipose tissue (A.M. Pearson, unpublished observations)
but their roles in the flavor and odor of pork are not clear. It was sug-
gested by the late 1. Wismer-Pedersen (personal communication to the
senior author) that pork flavor may be due to low concentrations of the
compounds responsible for 'boar odor'. There may be some basis for this
viewpoint since Brooks et al. (1986) found very low concentrations of 5cx-
androst-16-en-3-one in the fatty tissues from castrates (barrows). Thus,
very low levels of Cwd 16-steroids may be responsible for the desirable
pork flavor and odor, even though the strong penetrating odors of these
compounds would suggest such is not the case.
Thus, research is needed to identify not only the compounds that con-
tribute to the desirable flavor and odor notes in pork but also to unravel
the conflicting opinions concerning 'boar odor'. Isolation and identifica-
tion of the flavor compounds contributing to the species-specific compo-
nents that produce the desirable flavor of pork are needed to elucidate the
true nature of 'pork flavor and odor'.
9.6.4 Beef and veal flavors and odors

Although the meat from bovines appears to have uniquely different
flavors and odors than that from sheep, goats and pork (Wasserman and
Talley, 1968; Brennand and Lindsay, 1982), no species-specific flavors and
odors have been isolated or identified from beef or veal. There is no
evidence that sexually mature cattle, either cows or bulls, have any sex-
related flavors and odors (Field, 1971). Although grassy flavors and odors
sometimes occur in beef (Schroeder et al., 1980; Melton et al., 1982;
Larrick et ai., 1987), these problems are discussed in greater detail in
chapter 10. Some meat scientists, however, have observed what they call a
'cowy flavor or odor' in meat from cows but no specific compounds have
been identified (W.E. Kramlich and R.L. Dickson, personal communica-
tions).
Although veal is known to have a milder flavor than beef (Ziegler,
1944), no specific flavor or odor compounds responsible for the differences
have been found. Nevertheless, beef flavor is highly desirable and gen-
erally liked by consumers. This suggests that there are probably unique
flavor and odor compounds in beef and veal. Thus, the species-specific
flavor and/or odor compounds in beef and veal should be a fruitful field
of research.
9.6.5 Chicken- and turkey-specific flavors and odors

Although Minor et al. (1965) identified several compounds in the volatiles
from cooked chicken, none of them have been demonstrated to be species-
specific. Most if not all of these compounds have also been identified as
contributors to 'meaty flavors' and are not unique to chicken. Never-
theless, the lipids in chicken and turkey tend to be shorter in chain length
and their fats are softer than those of the red meats. It does not appear to
be unreasonable to expect differences in the flavor and/or odor com-
pounds from poultry meat, which suggests that this could be a useful area
of research.
9.6.6 Fish-specific flavors and odors

Fish flesh deteriorates rapidly, even at refrigeration temperatures, and
produces off-odors and flavors, which have been described by a number of
researchers (Davies and Gill, 1936; Obata and Yamanishi, 1952; Shewan
et al., 1960; Adams et al., 1964; Herbert and Shewan, 1975, 1976;
Crawford and Kretsch, 1976). These spoiled odors and flavors develop as
spoilage proceeds and lead to rejection of seafoods by consumers. Hence
only fresh products are acceptable.
Trimethylamine increases in concentration in the flesh of fish as spoilage
ensues and can be used as an index of spoilage for fish (Davies and Gill,
1936; Dyer & Mounsey, 1945; Stansby, 1962; Wong et al., 1969). It is a
breakdown product of trimethylamine oxide (Watson, 1939). Although
trimethylamine also can occur in meat from other species, it does not
accumulate in sufficient quantities to be useful for detection of spoilage.
However, there is no evidence that trimethylamine contributes to the
desirable odors and flavors of fish and seafoods (Castell and Greenough,
1957, 1959).
Flavors and odors in fish vary widely depending on the kind of fish
and the nature of their diet, which is reviewed in greater detail in chapter
10. Some species, however, have a stronger, more fishy flavor than
others. On the other hand, most fish and seafoods generally have a mild
and delicate flavor and odor. To date, no single compound has been
identified that is responsible for the desirable flavors and odors of fish. It
is likely that the pleasant odors and flavors of fish could be due to more
than one compound. Even low quantities of trimethylamine may have a

function in the desirable flavors and contribute by blending with other
compounds. It is clear that much work remains to be done in identifying
the compounds that contribute to the desirable flavors and odors of fish
and seafoods.
9.6.7 'Gamey' flavors and odors

As already mentioned in chapter 10, the 'gamey' odors and flavors may,
at least in part, be associated with partial decomposition. Nevertheless,
there may be compounds that are specific to the meat of game animals
and game birds, some of which may be derived from the diet. At any rate,
the nature of 'gamey flavors and odors' is virtually unexplored and needs
further research.
9.7 Summary
Species-specific flavors have been found to be associated with the lipid

fraction of meat. Although progress has been made in identification of the
undesirable flavors and odors, such as 'mutton flavor' or boar 'taint', the
possibility of specific compounds contributing to desirable flavors and
odors is virtually unexplored.
'Mutton' and 'goaty' flavors and odors have been related to the
presence of the branched-chain fatty acids, especially to 4-ethyl- and 4-
methyloctanoic acids. Other short-chain fatty acids may also have
'muttony' or 'goaty' odors. Some evidence has also indicated that other
short-chain and branched-chain fatty acids may contribute to the undesir-
able odors and flavors. Certain alkyl- and thiophenols may also be
involved in the flavor and odor of lamb but their importance has not been
fully elucidated.
'Boar taint' is due to the presence of the C w '\ 16-steroids. There is less
persuasive evidence for the involvement of skatole, which is a breakdown
product of tryptophan. Whether these compounds contribute to the desir-
able flavors and aromas of pork are not clear at this time. Further work
will be needed to resolve these related problems.
The entire area of beef- and veal-specific flavors and odors, chicken-
and turkey-specific flavors and odors, fish- and seafood-specific flavors
and odors, as well as 'gamey' flavors and odors is less than clear. Exten-
sive research will be needed before species-specific odors in different kinds
of meats are fully understood. The entire area of species-specific flavors
and/or odors provides a real challenge but could be a rewarding research
area.
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10 Flavor and aroma problems and their
measurement in meat, poultry and fish products
1.1. GRAY, A.M. PEARSON and F.1. MONAHAN
10.1 Introduction
Meat, poultry and fish products are normally highly desired for their dis-
tinctive and highly prized flavors, which have been discussed in chapters 7
and 9. Departures from normal flavors, however, are not uncommon in
these products and result in poor acceptability or even rejection by con-
sumers.
Off-flavors, however, may be serious to some consumers, whereas others
may find such flavors to be preferable or highly desirable. An example of
this is the special market for game animals and birds that exists in
Europe, especially in Germany, where consumers will pay a premium for
meat from wild animals and birds with a 'gamey' flavor. Still another
example of such preference is the demand for sheep meat, particularly for
mature or older animals that exists in some areas of the world (e.g. Aus-
tralia, New Zealand and UK), while US consumers eat only token
amounts of lamb and mutton. In other countries (e.g. Mexico, Spain and
Portugal) goat meat is highly prized. All of these preferences indicate that
what some consumers consider to be undesirable may be desirable to
other individuals and groups.
In this chapter, although the existence of personal preferences is recog-
nized, it is also realized that any objectionable aroma or flavor can influ-
ence consumption and affect total consumption of meat, poultry and fish
products. Thus, objectionable off flavors that occur will be discussed and,
for purposes of this review, be classified into several unrelated but impor-
tant groups, namely those related to:
• oxidative rancidity and/or warmed-over-flavor,
• species-specific flavors,
• feed-derived flavors due to different feeding regimens,
• 'gamey' flavors that are commonly associated with the meat from
species of wild animals and game birds,
• off-flavors associated with the sex condition of the animal or bird,
• undesirable flavors that are taken up from a contaminated environment,
• processing-induced off-flavors, and
• spoiled flavors that occur as a result of microbial spoilage.
FLAVOR AND AROMA PROBLEMS 251
Discussion in this chapter will focus upon each of these causes of undesir-
able flavors in meat, poultry and fish products. Causes of the various off-
flavors and how they can be measured will be considered herein, as will
some problems in attempting their measurement. It is only through con-
sideration of all such factors that the causes can be avoided and the
problems and complaints about off-flavors and off-odors by consumers
can be avoided. Even under ideal conditions, some off-flavors and odors
will develop and can be barriers to the acceptability of meat, poultry and
fishery products.
10.2 Oxidative rancidity/warmed over flavors
10.2.1 Lipid oxidation and meat quality

The development of oxidative rancidity has long been recognized as a
problem occurring during the storage of meats. The propensity of meats
and meat products to undergo oxidation depends on several factors
including the fatty acid composition and the presence of pro-oxidants in
the muscle. The susceptibility of muscle lipids to oxidation depends on
their degree of unsaturation. The polyunsaturated fatty acid content of
muscle varies between species and decreases in the order: fish > poultry
> pork > beef> lamb (Allen and Foegeding, 1981). Susceptibility to
lipid oxidation in the presence of Fe2 +, Cu 2 + and C0 2 + ions was shown
to be in the same order (Tichivangana and Morrissey, 1985). Conversely,
the resistance of meat lipids to oxidation results from the presence of anti-
oxidants, such as vitamin E, which function as free radical scavengers to
effectively break free radical chain mechanisms (Monahan et al., 1992).
10.2.1.1 Factors affecting lipid oxidation during meat processing. In pro-

cessed meats, the occurrence of lipid oxidation is influenced not only by
fatty acid, pro-oxidant and antioxidant content, but also by processing
conditions. These include: (i) reduction in meat particle size; (ii) cooking,
and (iii) various additives used in the formulation, e.g. salt, nitrite, phos-
phates, extenders and fillers (Gray and Pearson, 1987; Gray and Crackel,
1992). Reduction in particle size by grinding, chopping, flaking or emulsi-
fication results in disruption of cell membranes and incorporation of air
into the tissues. Both of these actions increase tissue susceptibility to oxi-
dation and hasten the development of oxidative rancidity (Sato and
Hegarty, 1971; Gray and Pearson, 1987).
10.2.1.2 Action of cooking on lipid oxidation. Lipid oxidation has been

observed to increase rapidly upon cooking of meats (Younathan and
Watts, 1959, 1960; Keller and Kinsella, 1973; Igene et al., 1979). The
latter researchers showed that heating facilitated the release of inorganic

iron from muscle pigments and proposed that this free iron was respon-
sible for the rapid rate of oxidation in cooked meats. Similar views were
expressed by Tichivangana and Morrissey (1985).
10.2.1.3 Effects of salt on lipid oxidation. Of the ingredients frequently

used in the preparation of meat products and which affect the rate of lipid
oxidation, salt has a significant pro-oxidant effect while others possess
anti-oxidant activity. Salt (sodium chloride) is added to processed meats
for its sensory, functional and preservation properties (Pearson et al.,
1977). In restructured meats, for example, salt is critical for the extraction
of the salt-soluble proteins that provide the cohesiveness of meat particles
in the finished product. However, it is known to initiate adverse flavor
and color changes in meat but its mechanism of action is poorly under-
stood. Literature references allude to possible salt-catalyzed oxidation by
lip oxygenase (Lea, 1937) and catalysis of oxidation by metal impurities in
the salt (Olson and Rust, 1973). Two recent studies are worth noting with
regard to the possible mechanism of salt as a catalyst of oxidation.
Kanner et al. (1991) reported that the catalytic effect of salt is derived
from the enhancement of the pro-oxidant activity of chela table iron ions.
They concluded that the effects of salt in both model systems and ground
turkey muscle seemed to be derived from the capability of salt to displace
iron ions from binding macromolecules. Osinchak et al. (1992) studied the
effect of salt on catalysis of lipid oxidation by the soluble fraction of fish
muscle and determined that chloride was the active component of salt in
this system. They also determined that redox iron was involved in the
chloride stimulation of lipid oxidation by the soluble extract. More studies
of this nature are required to elucidate fully the mechanism of salt-cata-
lyzed lipid oxidation in muscle foods.
The pro-oxidant activity of salt in processed meats can be minimized by
the judicious selection of ingredients. In cured meat products, nitrite,
phosphate and ascorbate each play a role in inhibiting oxidation (Gray
and Pearson, 1987). The anti-oxidant effect of nitrite is well-documented
and several mechanisms have been suggested. Igene et al. (1985) proposed
that nitrite may form a complex with heme compounds thereby preventing
the release of iron during cooking, stabilize unsaturated lipids in the
membranes, or interact with metal ions to prevent their catalysis by oxi-
dation. Freybler et al. (1989) confirmed the nitrite stabilization of heme
pigments and membrane lipids in cured meats and, in addition, demon-
strated the anti-oxidative properties of nitric oxide myoglobin.
10.2.1.4 Influence of phosphates. Phosphates act as anti-oxidants, pre-

sumably by chelating heavy metals such as iron and copper (Tims and
Watts, 1958). Citrate is also an effective chelator and has been shown to
inhibit lipid oxidation in ground beef (Sato and Hegarty, 1971; Benedict
et al., 1975). Ascorbate, on the other hand, can function both as a pro-
oxidant and an anti-oxidant. At low levels « 100 mg.kg- 1), it has been
shown to catalyze lipid oxidation in meat products (Tims and Watts,
1958; Sato and Hegarty, 1971). However, at levels in excess of
1000 mg.kg- 1, ascorbic acid is an effective inhibitor of oxidation. Sato and
Hegarty (1971) suggested that at low concentrations, ascorbic acid acts to
maintain a portion of the iron in the ferrous state, while at higher con-
centrations it shifts the balance between ferrous and ferric iron or acts as
an oxygen scavenger. Kanner et al. (1986) demonstrated that iron in the
presence of ascorbic acid stimulated muscle membranal lipid peroxidation.
10.2.2 Inhibiting lipid oxidation

In food systems, the most effective anti-oxidants function by interrupting
the lipid oxidation free radical chain mechanism (Dugan, 1976). The use
of synthetic anti-oxidants in meats has been studied widely. Butylated
hydroxyanisole, butylated hydroxy toluene, propyl gallate and tertiary
butylhydroquinone have all been shown to be effective in retarding lipid
oxidation (Greene, 1969; Chastain et al., 1982; Crackel et al., 1988a). A
major problem in using such anti-oxidants is that they are approved for
use in only a limited number of meat products. In addition, public
concern about the presence of chemical additives in food has led to
Table 10.1 Odor properties and thresholds of some compounds that may
contribute to oxidized off-flavors a
Compound Odor character Threshold in oil p.p.b.
Hexanal Green 80
Heptanal Oily, putty 55
Octanal Fatty 40
Nonanal Tallowy 200
trans- 2-Hexenal Green 600
trans-2-Heptenal Putty, fatty 500
trans- 2-0ctenaL Fatty 150
trans- 2-N onenal Tallowy, cucumbers 40
cis-2- Heptenal Creamy, putty 0.5
trans-2,trans-4- Hexadienal Fatty, green 40
trans- 2 ,trans-4- Heptadienal Fatty, oily 100
trans-2,trans-4-Decadienal Deep-fried 20
trans- 2 ,cis-6-Nonadienal Fresh cucumbers 1.5
I-Penten-3-ol Sharp, irritating 4200
1-0cten-3-ol Mushroom 7.5
1-Penten-3-one Fishy, oily 3
1-0cten-3-one Metallic 0.1
3,5-0ctadien-2-one Fruity, fatty 300
aTaken from Mottram (1987).

renewed interest in the use of natural anti-oxidants in foods (Gordon,

1987). Naturally occurring substances with anti-oxidant activity include
edible products from vegetables, fruits, oil seeds and grains, as well as
spices, herbs and protein hydrolyzates (Houlihan and Ho, 1985; Rhee,
1987; Gray and Crackel, 1992). For example, rosemary oleoresin is an
effective inhibitor of warmed-over flavor in cooked beef patties stored at
4°C (St. Angelo et al., 1990). Vitamin E is also a powerful anti-oxidant
and functions as a lipid-soluble anti-oxidant in cell membranes. Dietary
supplementation of vitamin E for the subsequent benefit of increased lipid
stability in muscle foods has been reported extensively for poultry
(Marusich et al., 1975; Lin et al., 1989), pork (Buckley et al., 1989;
Monahan et aI., 1990, 1992), veal (Shorland et al., 1981; Engeseth et al.,
1993), and beef (Faustman et al., 1989). Miles et al. (1986) and Whang et
al. (1986) showed that (X-tocopherol was effective in retarding lipid oxida-
tion in restructured pork products when added as a processing ingredient.
Oxidative deterioration of lipids directly affects several quality char-
acteristics in meat and meat products. These include the flavor, color,
texture, nutritive value and safety (Pearson et al., 1983). Traditionally,
researchers have been concerned with the organoleptic attributes of foods,
particularly odor and flavor characteristics. More recently, consumer
demands for healthier foods have led researchers in the lipid oxidation
area to examine factors that may contribute to these foods having a less
'healthy' image.
10.2.3 Effect of lipid oxidation on meat flavor

Lipid oxidation occurring in fresh, frozen or cooked meat is generally
associated with the development of rancid flavors and odors and a con-
comitant reduction in the acceptability of meat. The term 'warmed-over
flavor' (WOF) was first introduced by Tims and Watts (1958) to describe
the characteristic off-flavor that develops in cooked meat after cooking
and storage. WOF differs from the common rancidity encountered in raw
meats, fatty tissues, rendered fat or lard, which is normally not apparent
until they have been stored for weeks or months (Pearson et aI., 1977).
Nevertheless, WOF also develops in raw meat that is ground and exposed
to air (Sato and Hegarty, 1971) and in unheated products such as
mechanically separated and restructured meats in which the muscle struc-
ture is disrupted and air is incorporated (Gray and Pearson, 1987).
Lipid hydroperoxides, the primary products of lipid oxidation, are
odorless (Paquette et al., 1985). However, on decomposition they yield a
complex mixture of low molecular weight compounds with distinctive
odor and flavor characteristics (Table 10.1), including alkanes, alkenes,
aldehydes, ketones, alcohols, esters and acids (Mottram, 1987). The con-
tribution that a particular compound makes to the flavor or aroma of a
meat product depends on the concentration at which it is present, its odor

threshold and the relationship between concentration and sensory inten-
sity. Aldehydes, unsaturated alcohols and vinyl ketones have relatively
low odor thresholds, some have distinct aromas at concentrations below
1 p.p.b. and are likely, therefore, to contribute flavor if present in food
systems (Mottram, 1987).
Lipid oxidation during cooking may also be a source of intermediates,
which then react with other components to give important constituents of
desirable meat flavor and aroma (Enser, 1987). Mottram (1987) has stated
that, while oxidation of lipids during storage and during thermal proces-
sing follows the same basic pathways, subtle differences in the precise
mechanisms of oxidation may determine whether the particular profile of
lipid oxidation products formed has an overall desirable or undesirable
effect. If oxidized off-flavors have already developed before cooking,
however, they are not removed by the cooking process (Mottram, 1987).
Furthermore, Drumm and Spanier (1991) concluded that the decrease in
desirable flavor observed during refrigerated storage of cooked meat may
be attributed to the masking of desirable flavor notes by the increased
content of undesirable flavor compounds rather than to the degradation
of desirable flavor compounds.
Of apparent importance too, are the interactions of lipid and lipid
degradation products with other meat components during cooking.
Mottram and Edwards (1983) and Whitfield et al. (1987) demonstrated
that lipid interactions with Maillard reaction products contribute to the
desirable meaty flavor in cooked meat. The most likely pathways by which
these interactions take place were summarized by Farmer and Mottram
(1990) as follows: (i) the reaction of carbonyl compounds from lipids with
the amino groups of cysteine and ammonia produced by Strecker degra-
dation; (ii) the reaction of the amino group in phosphatidylethanolamine
with sugar-derived carbonyl compounds; (iii) the interaction of free
radicals from oxidized lipids in the Maillard reaction; and (iv) the reaction
of hydroxy and carbonyl lipid oxidation products with free hydrogen
sulfide.
Efforts have been made to correlate concentrations of carbonyl com-
pounds derived from the decomposition of lipid hydro peroxides with
sensory flavor scores. Some specific examples of these studies will be dis-
cussed later.
10.2.4 Catalysis of lipid oxidation in meats

In muscle, the biochemical changes that accompany post-mortem aging in
the conversion of muscle to meat lead to a situation in which the process
of lipid oxidation is no longer tightly controlled and the balance of pro-
oxidative factors/anti-oxidative capacity favors oxidation (Sanders, 1987;
Willson, 1987). In meat systems, therefore, proteolytic events, pH changes

and post-slaughter destruction of cell compartmentalization require that
pro-oxidant factors, which would normally be controlled in vivo, must be
considered. It is generally believed that lipid oxidation in meats is initiated
in the phospholipid fraction of cells which is associated primarily with cell
membranes (Love and Pearson, 1971; Igene and Pearson, 1979; Gray and
Pearson, 1987). This view is supported by the fact that membranal lipids
are high in polyunsaturated fatty acids that are particularly susceptible to
oxidative attack (Gray and Pearson, 1987). The phospholipid fraction has
been shown to contribute approximately 90% of the 2-thiobarbituric acid-
reactive substances in fat from chicken (Pikul et al., 1984 a,b). In further
support of this hypothesis, processes that disrupt membrane structure,
such as cooking and grinding, promote oxidation, presumably by exposing
the phospholipids to a pro-oxidative environment containing oxygen,
enzymes, heme pigments and metal ions (Sato and Hegarty, 1971; Asghar
et aI., 1988).
10.2.4.1 Effect of iron on oxidation. Attention has focused in the past on

the pro-oxidant potential of the iron-containing fractions of muscle (Sato
and Hegarty, 1971; Apte and Morrissey, 1987a,b), which are believed to
be significant contributors to lipid oxidation during storage. The relative
catalytic effects of heme and catalytic 'free' iron on lipid oxidation in
meats have not been clearly defined despite many studies with muscle
model systems (Love and Pearson, 1974; Tichivangana and Morrissey,
1985; Kanner et al., 1986, 1988; Johns et al., 1989). Until the early 1970s,
myoglobin and other heme compounds that are present at high con-
centrations in muscle, were considered to be major catalysts of lipid oxi-
dation (Robinson, 1924; Younathan and Watts, 1959; Tappel, 1962). In
contrast to earlier views, Sato and Hegarty (1971) and Love and Pearson
(1974) reported that in muscle model systems non-heme iron rather than
heme iron was the major catalyst of lipid oxidation. Tichivangana and
Morrissey (1985) also found that non-heme iron had a greater pro-
oxidant effect than metmyoglobin in raw and heated muscle model
systems. However, several more recent studies have cast some doubt on
the contention that non-heme iron as opposed to heme iron has greater
pro-oxidant activity in muscle (Verma et al., 1985; Johns et aI., 1989).
Johns et al. (1989) found that, at levels approaching those present in
muscle, heme iron was a powerful catalyst of lipid oxidation while non-
heme iron appeared to have little pro-oxidative activity. It is clear that
inconsistencies exist in the results reported to date. For example, Igene et
al. (1979) proposed that iron released from heme proteins during cooking
was responsible for the increased rate of oxidation observed in cooked
meats, yet Love and Pearson (1974) showed no increase in oxidation in a
heated metmyoglobin-containing muscle model system compared with an
unheated system. Furthermore, the levels of inorganic iron incorporated

into muscle model systems often far exceeded the levels likely to be
present in meats. Hazell (1982) reported that beef, pork, lamb and
chicken had mean low molecular weight iron levels of 0.5, 0.3, 0.1 and
0.1 I!g.g-l, respectively.
Apte and Morrissey (1987a,b) demonstrated that ferritin did not con-
tribute to lipid oxidation in raw muscle model systems but that it was
highly catalytic in cooked systems. They speculated that heating denatured
the ferritin molecule and released free iron, which had the capability of
catalyzing lipid oxidation. The release of free iron from ferritin in raw
muscle is currently being investigated as a potential cause of initiation of
lipid oxidation (Decker and Welch, 1990; Kanner and Doll, 1991).
10.2.4.2 Influence of membranal systems. In addition, the subcellular

membranes contain enzymic systems and other co-factors, for example
components of the electron transport chain, which could conceivably
generate reactive species capable of initiating oxidation of the neighbor-
ing phospholipids. NADPH-dependent lipid oxidation has been reported
in muscle microsomes from several species (Slabyj and Hultin, 1984).
The potential for microsomal enzymes, peroxidases, lipoxygenases and
cyclo-oxygenases to initiate lipid oxidation in vivo in animal tissues and
ex vivo in foods is reviewed by Hsieh and Kinsella (1989). Lipoxygenase
and cyclo-oxygenase catalyze the controlled oxidation of unsaturated
fatty acid substrates to yield hydroperoxides and endoperoxides with
important biological functions, namely the leukotrienes and pros-
taglandins. Both of these enzymatic conversions, if perturbed, can cause
uncontrolled lipid peroxidation especially post-mortem. Peroxidase in
animal tissues is generally associated with the antimicrobial activity
involving phagocytosis and protein halogenation but has been shown to
catalyze lipid oxidation and ~-carotene oxidation (Kanner and Kinsella,
1983a,b).
10.2.5 Measurement of lipid oxidation in meats

Many techniques ~ ranging from sensory evaluation to chemical and
physical methods ~ are available for assessing the extent of oxidation in
lipid-containing foods. The latter methods have traditionally been divided
into those that measure primary changes and those that measure second-
ary changes (Coxon, 1987).
1. Primary changes
(a) Oxygen uptake
(b) Loss of polyunsaturated fatty acids
(c) Formation of hydroperoxides (peroxide value)
2. Secondary changes
(a) Formation of carbonyls (as dinitrophenylhydrazones or by GC)
(b) Formation of malonaldehyde (TBA test)
(c) Formation of hydrocarbons (e.g. pentane)
The former group may be classified as those that quantify loss of reac-
tants (e.g. unsaturated fatty acids, oxygen) or formation of primary lipid
oxidation products (e.g. hydroperoxides). In situations where oxidation
occurs rapidly, as in cooked meats, and where primary products decom-
pose to stable secondary products, measurement of secondary lipid oxida-
tion products as an index of lipid oxidation is then more appropriate.
Melton (1983) has reviewed comprehensively the methodology for follow-
ing lipid oxidation in muscle foods, therefore, in this section, only the
thiobarbituric acid (TBA) test and hex anal quantitation will be discussed.
10.2.5.1 The TBA test. The TBA test is the most frequently used method
for assessing lipid oxidation in meat and meat products. The extent of
oxidative rancidity is normally reported as TBA numbers or values and
expressed as milligrams of malonaldehyde equivalents per kilogram of
sample. Malonaldehyde is a relatively minor lipid oxidation product origi-
nating from oxidizing polyunsaturated fatty acids and reacts with TBA
reagent to produce a colored complex with an absorption maximum at
530-532 nm. The red pigment results from the condensation of 2 moles of
TBA with 1 mole of ma10na1dehyde (Sinnhuber and Yu, 1958; Yu et a!.,
1986). The intensity of the color formed was originally believed to be a
measure of malonaldehyde concentration (Tarladgis et aI., 1960, 1964) and
has been reported to correlate well with sensory scores of oxidized and
warmed-over flavors in meat (Zipser et al., 1964; Igene and Pearson, 1979;
Greene and Cumuze, 1981).
The TBA procedure, however, should only be used to assess the extent
of lipid oxidation in general rather than to quantify malonaldehyde. While
some of the malonaldehyde detected in the TBA test is formed during the
oxidation process, most is generated by decomposition of lipid hydroper-
oxides during the acid-heating stage of the test, a process that is acceler-
ated by transition metal ions (Gutteridge and Quinlan, 1983; Gutteridge
and Halliwell, 1990). Furthermore, other products of lipid oxidation, such
as alka-2,4-dienals, also react with TBA reagent to form a red complex
having the same absorption maximum as the malonaldehyde-TBA
complex (Marcuse and Johansson, 1973). Thus, the term TBA-reactive
substances (TBARS) is a much better term and is now commonly used in
place of TBA number or value.
Many forms of the TBA test have evolved over the years and these
have been reviewed adequately by Hoyland and Taylor (1991). It can be
performed:
• directly on the food product, followed by extraction of the colored

complex (Pokorny et al., 1985);
• on an aqueous or acid extract of the food sample (Witte et al., 1970;
Pikul et al., 1983, 1989; Salih et al., 1987);
• on a portion of the steam distillate of the food (Tarladgis et aI., 1960;
Hoyland and Taylor, 1989); and
• on extracted lipid from the food (Y ounathan and Watts, 1960; Pikul et
al., 1983, 1989).
The distillation procedure is the most popular method for measuring

TBARS in muscle foods and has been used extensively without modifica-
tion to assess the oxidative deterioration of lipids in beef, pork and
poultry products (Melton, 1983). In recent years, it has been suggested
that anti-oxidants be added at the blending or distillation stages of the
procedure to minimize further lipid oxidation during the test (Rhee, 1978;
Pikul et al., 1983; Crackel et al., 1988b). The distillation method of Tar-
ladgis et al. (1960) has also been modified for nitrite-cured meats (Zipser
and Watts, 1962). Residual nitrite in cured meat can react with mal-
on aldehyde during the distillation step, leading to underestimation of
TBARS. Sulfanilamide, when added to cured meats before distillation,
reacts with residual nitrite to produce a diazonium salt and eliminates
interference.
The popularity of a particular version of an analytical procedure does
not necessarily mean that it is the best. For example, Witte et al. (1970)
claimed that the solvent extraction method is easier to use than the dis-
tillation method, uses less equipment and heating is not essential. Salih et
al. (1987) and Pikul et al. (1989) concurred that solvent extraction
methods are faster and easier to perform than distillation. In addition,
while the latter investigators reported high correlation between solvent
extraction and distillation methods, it has been noted by several research-
ers that the solvent extraction method gives lower TBARS than the dis-
tillation method for duplicate samples (Witte et al., 1970; Salih et aI.,
1987). It has been suggested that the lower value is due to reduced sample
autoxidation during the extraction method (Pikul et al., 1983).
The TBA assay procedure has been critically reviewed by Ward
(1985) who concluded that the assay is operator-dependent, method-
dependent and suffers from interference. Ward (1985) further suggested
that the test can only be of limited value, either in assessing the degree
of chemical oxidation or in relation to organoleptic response without
knowledge of: (i) the exact nature of the TBA active substrate(s), (ii)
what TBA-adduct(s) is (are) formed, (iii) the compositional profile of the
lipid system in question, (iv) the oxidative pathways taken by compo-
nents of the lipid system leading to the formation of TBA active sub-
strate(s); and (v) the relationship of the TBA active substrate(s) to
flavor-producing molecules, if the assay is to be used to indicate flavor

degradation,
Despite its many limitations, the TBA test, if used wisely, can provide
useful data on the state of lipid oxidation in meats, If all TBARS are
determined by a single method, the change in TBARS for that particular
situation can show the relative amount of lipid oxidation during the pro-
cessing and storage of a single meat. It is, however, preferable to measure
lipid oxidation by a complementary procedure, such as hexanal determi-
nation, and relate both sets of analytical data to sensory evaluation data,
Carbonyls, produced by the degradation of lipid hydroperoxides, have
been quantified by reaction with 2,4-dinitrophenylhydrazine followed by
colorimetric determination of the hydrazones formed (Schwartz et aI.,
1963; Keller and Kinsella, 1973), Melton (1983) reviewed the limitations
of total carbonyl and monocarbonyl determination as a measure of lipid
oxidation in muscle foods, Like the TBA test, production of carbonyls
during the preparation of samples may lead to an overestimation of the
carbonyls present in the food (Pradel and Adda, 1980), Melton (1983) also
concluded, in agreement with Mai and Kinsella (1979), that direct quanti-
fication of peroxide decomposition products by gas chromatography may
be a more accurate method than either the total carbonyl assay or the
TBA test for determining oxidative changes in food,
10.2.5.2 Rexanal concentration. Rexanal, one of the major secondary

products formed during the oxidation of linoleic acid (Frankel, 1991), has
been used successfully to follow lipid oxidation in meat products. Shahidi
et al. (1987) reported a linear relationship between hex anal content,
sensory scores and TBARS of cooked ground pork. The higher the
hex anal content, the lower the acceptability of the meat. In addition,
hexanal determination was shown to be a more sensitive measure of the
oxidative state of cooked meats in the early stages of storage. While
TBARS after 2 days of storage for a variety of meat samples were similar,
their hex anal contents were different. St. Angelo et al. (1987) also indi-
cated that instrumental analyses by direct capillary gas chromatography
can be used to assay beef flavor quality. They demonstrated that volatile
secondary products of lipid oxidation, including hexanal, pentanal and
2,3-octanedione could be used as markers to follow the development of
WOF in beef. The increase in many of these compounds was parallel to
the increase in TBARS and correlated with sensory evaluations. These
results have been supported by the recent findings of Stoick et al. (1991)
and Drumm and Spanier (1991).
It is apparent that, while the TBA test with all of its problems still
remains the most convenient method for following lipid oxidation in
meats, other methods are now available that specifically identify indivi-
dual oxidation products that contribute to off-flavor development in
meats. Such methodology should be employed, by itself or in combination

with the TBA test, to effectively monitor and evaluate lipid oxidation in
meat and other biological tissues.
10.3 Species-specific flavors
Meat flavor resides in the water-soluble fraction as shown by the early

studies on meat flavor (Crocker, 1948; Bouthilet, 1951a,b; Kramlich and
Pearson, 1958). Later it was shown that the characteristic meaty flavor is
essentially the same for all species (Hornstein and Crowe, 1960; Hornstein
et al., 1963; Hornstein and Wasserman, 1987), whereas the species-specific
flavors are localized in the lipid fraction (Hornstein et aI., 1963; Minor et
al., 1965). Wasserman and Talley (1968) unequivocally demonstrated that
the fatty tissues contribute the components that are responsible for the
species-specific flavors of beef, lamb, veal and pork.
Although the flavor of raw meat from all species is 'serumy' or 'blood-
like' in taste (Crocker, 1948; Bouthilet, 1951a,b), both the characteristic
meaty flavor and the species-specific flavors require heat for their forma-
tion (Kramlich & Pearson, 1958). Several research groups (Wong et al.,
1975; Kunsman & Riley, 1975; Cramer, 1983; Field et al., 1983; Ha and
Lindsay, 1991) have attempted to unravel the mystery surrounding lamb
and mutton flavor, with variable success. Since chapter 9 discusses the
details of species-specific flavors and aromas, including the responsible
chemical compounds, the topic will not be covered further in this chapter.
10.4 Effects of different feeds on flavor and aroma
There is a considerable amount of information available on undesirable

flavors originating from different feeds or feed ingredients, which has been
extensively reviewed by Reineccius (1979). Since the effects are sometimes
quite different for various species, this review will discuss the effects of
feed-induced off-flavors separately by species wherever such information is
available.
10.4.1 Lamb and mutton

10.4.1.1 Effects of different pasture crops. More is known about the
effects of diet on the feed-derived flavors in lamb and/or mutton than for
any of the other red meat-producing species. Cramer et al. (1967) first
showed that the type of forage fed to lambs had a marked influence on
the acceptability of their meat, with white clover (Trifolium repens) pro-
ducing a more intense flavor and aroma compared with that from lambs
grazed on perennial ryegrass (Lotium perenne). The meat from animals

grazing the white clover pasture was found to be less desirable in flavor
and aroma than that produced by lambs grazing the ryegrass pasture.
Results were based solely on panel studies and did not identify the
responsible components. A few years later, Park et al. (1972a,b) confirmed
the fact that various forage crops produced different flavors and/or
aromas in the meat from lambs. The authors reported that the meat from
animals grazed on oats was described by sensory panels as being
'pungent', 'rotten egg-like' or 'mercaptan-like'. Nevertheless, the panel did
not feel these off-flavors were sufficiently intense to affect consumer
acceptability adversely. The sheep fed on vetch were described as having a
'sweetish aroma' and a 'stronger meaty flavor', but were not deemed suffi-
ciently strong in flavor to be objectionable. Pasturing of lambs on rape
(Brassica napus en Rangi) produced a 'sickly' or 'nauseating' odor that
was reminiscent of 'boiled cabbage', which Park et al. (1972b) postulated
was derived from the glycosinolates in the rape that could break down to
form aliphatic sulfides, disulfides or mercaptans and thus produce objec-
tionable flavor/aroma.
The removal of sheep from the rape pasture and putting them on per-
ennial ryegrass for 10 days prior to slaughter resulted in significant
improvement in flavor in studies carried out by Wheeler et al. (1974). Park
et al. (l972a) had previously reported that grazing lambs on 'grass pasture'
for 1-2 weeks prior to slaughter significantly reduced the complaints of
off-flavors in the meat from animals previously grazing lucerne (alfalfa)
pastures. However, Nicol and Jagusch (1971) had obtained inconsistent
results from a panel comparing the meat from animals grazed on ryegrass
and lucerne. They suggested that the meat from lambs grazing lucerne
pastures was less acceptable when the plants were young, leafy and
growing rapidly than when they were more mature and growing at a
slower rate. Later Park et al. (1975) found that the intensity of the off-
flavor in meat from lambs grazing lucerne increased with time on pasture
over a 6-month period. However, the intensity of the objectionable flavor
was greatest during the cooler season of the year, confirming the seasonal
effects of pasture growth on the intensity of undesirable flavors in the meat
from lambs grazing alfalfa pasture (Nicol and Jagusch, 1971).
Although most of the studies on off-flavors in meat from lamb and
mutton seem to implicate legumes (Cramer et al., 1967; Nicol and
Jagusch, 1971; Park et al., 1972a,b, 1975; Wheeler et aI., 1974), Park and
Minson (1972) demonstrated that not all legumes produce off-flavors in
the meat of grazing lambs, notable exceptions being sirato (Phaseolus
atropurfureus) and silverleaf desmodium (Desmodium unicinatium), which
showed no off-flavors in meat after grazing for 15 and 6 weeks, respec-
tively. Although meat from lambs grazed on Dolichos axillaris for up to 4
months occasionally exhibited a characteristic and objectionable off-flavor,
it was not deemed to be less acceptable than meat from similar lambs
grazed on grass. Interestingly, lambs grazed on Glycine wishtii for up to 6
weeks produced meat that had an objectionable off-flavor soon after
putting the lambs on pasture but which decreased and/or disappeared as
the grazing time was extended.
Thus, alfalfa and clover (white) pastures appear to be the particular
legumes that produce off-flavors in the meat. Although alfalfa and clover
hays, especially the former, are commonly used in feedlot rations for fat-
tening lambs in the USA, the hays do not appear to produce off-flavors
and aromas as there are no reports of flavor problems in the meat from
lambs fed these legumes in the feedlot.
10A.1.2 Influence of protected lipid supplements. The development of a

protected lipid supplement to bypass the rumen, which decreases the
normally occurring microbial hydrogenation process as shown by Cook
et al. (1970) and Scott et al. (1971), has the potential to change the lipid
composition of the animal and influence the flavor of the meat. Ford and
Park (1975) have demonstrated that this is indeed the case and have
shown that the flavor of meat from lambs fed a protected lipid supple-
ment is quite different from the meat of animals grazed on grass pastures
or fed in feedlots. The meat from pasture-fed and feedlot-fed lambs was
significantly preferred by taste panels over the meat from lambs fed the
protected lipid supplements. The meat from the pasture-fed lambs was
rated as having a 'more intense meat flavor' and a less 'unusual' aroma,
while that from the group given the protected lipid supplement was
observed to have an 'oily' aroma 'similar to chicken and pork' together
with a 'sweet' or 'fruity' aroma and flavor. The 'sweet' character was
believed to be due to the presence of cis-tX-dodeca-6-enolactone in the fat
of the animals fed the protected lipid supplement (Park et al., 1974),
while the 'oily' odor was due to the presence of trans,trans-deca-2,4-
dienal. The panel responses to 'sweet' and 'oily' were closely related to
the concentrations of the unsaturated lactone and 2,4-decadienal, respec-
tively.
Although the 'sweet' aroma was evident in the raw meat from lambs fed
the protected lipid supplement, the 'oily' character was thought to be
developed by cooking (Park and Ford, 1975). It was suggested by Park
and Ford (1975) that the 'oily' characteristic was due to formation of 2,4-
decadienal upon heating of the meat, which contained a relatively high
linoleic acid content in the presence of water. The off-flavors developed in
the meat from lambs fed the lipid protected diet were shown to develop as
a function of time on feed by Park and Ford (1975). The descriptions of
'grassy' or 'cooked vegetable' for the meat from the lambs on the control
diet were changed to 'oily', 'sweet', 'chemical-like' or perfume-like' for the
meat from animals fed the protected lipid supplemented diet after 3-4
weeks, The 'oily' designation became evident after only 2 weeks of

feeding, Park et al. (1976) reported that the panel responses to 'sweet' and
'oily' paralleled increases in the concentrations of the unsaturated lactone
and 2,4-decadienals, respectively,
Park et aI, (1976) found that the type of protein used in the protected
lipid supplements influenced the flavor of the meat from lambs, Meat
from lambs using a protected lipid supplement composed of sunflower
seed-casein was less frequently criticized for the 'sweet' aroma than that
from lambs fed a safflower oil-casein supplement. The latter treatment
resulted in more frequent criticisms of having an 'oil' or 'paint-like' off-
flavor, with the panel scores for these off-flavors being related to the con-
centrations of unsaturated dodecalactone and 2,4-decadienal.
As already indicated, lamb has a milder and less pronounced flavor
than mutton but there is little or no information on the differences in the
amount or kinds of chemical compounds generated on cooking of lamb
and mutton. Nevertheless, mutton is recognized as having a stronger
flavor and aroma, although concrete information is lacking on both the
responsible compounds and their concentrations.
10.4.2 Veal and beef

Few studies have compared the flavors of veal and beef, which is thought
to be a matter of degree of maturity. A panel study by Wasserman and
Talley (1968), however, indicated that beef and veal had some character-
istic differences in flavor, which may be associated with differences in
their diets, with veal calves being reared on milk while beef comes from
cattle fed roughages and grain. This supports the concept that diet
instead of the age of the animal may be a contributor to the differences
in the flavor of veal and beef.
Although there are less data available on the effects of feeds on the
flavor of beef than is the case for lamb and mutton, the carry-over Of
the undesirable flavor of wild onions to the meat of beef cattle was
reported by Kemp and Varney (1955). More recently most of the
research has concentrated on the differences between meat from beef
cattle finished on low-energy (forage) vs. high-energy (grain) diets, with
the flavor of beef from the animals fed the high-energy diets being rated
by sensory panels as being more desirable (Reagan et al., 1977; Bowling
et al., 1978; Brown et al., 1979; We sterling and Hedrick, 1979; Schroeder
et al., 1980; Tatum et al., 1980; Melton et al., 1982a,b; Hedrick et al.,
1983; Larick et al., 1987). Although the beef from animals fed on high-
energy (concentrate) diets was rated as being more desirable, these
studies all came from the USA where consumers have become accus-
tomed to beef from cattle finished on high-grain rations. It seems
possible that consumers accustomed to meat from cattle fed only grass
may prefer that meat. For example, most beef in Argentina is fattened
on alfalfa pastures, yet it has an excellent reputation for its quality and
flavor.
Most of the earlier studies comparing grass-fed and grain-fed beef have
used taste panels to measure differences. However, Larick et al. (1987) not
only subjected the meat to taste panels for evaluation but also identified
the volatiles using gas liquid chromatography/mass spectrometry (GLCj
MS) to help in identifying the chemical components that the panels found
to be objectionable. They identified some 20 compounds that were nega-
tively correlated with panel scores and that appeared to decrease with the
time that concentrates were fed. Two compounds, namely, delta-tetradeca-
lactone and delta-hexadecalactone were negatively correlated with 'grassy'
flavor and may be indicative of grain-fed beef. These findings may
account for the higher flavor scores reported for higher USDA beef
quality grades (Smith et aI., 1983; Cross, 1987).
10.4.3 Pig meat

Much of the early work on the quality of pig meat dealt with the problem
relating to 'soft pork', which Ellis and Isbell (1926a,b) have described. The
'soft pork' problem is due to feeding pigs a diet high in unsaturated fatty
acids, which was common when hogs were finished by 'hogging-off
peanuts, soybeans or chufas. These feeds were all high in unsaturated
fatty acids, which the pig tends to deposit in its body fat. The resulting
bellies and rendered lard produced were soft and undesirable, not so much
because of their flavor but because of their oily nature. The 'soft pork'
problem has largely disappeared with the majority of hogs in the USA
now being raised in confinement, where mixed feeds avoid too much oil in
the diet.
It is possible that in the southern part of the USA a few small farmers
may still finish their pigs by 'hogging-off peanuts or soybeans. The hams
from peanut-fed pigs have been said to produce a desirable flavor that
was in special demand for producing 'country-cured hams.' However, the
major producers of 'country-cured' hams now use meat from confinement-
reared hogs (N.G. Marriott, personal communication), although develop-
ment of the characteristic flavor still relies on oxidative breakdown of the
fatty tissues during the aging process (Ockerman et al., 1964; Skelley et
al., 1964).
10.4.4 Fish
The flavor of fish can be altered by the type of feed as reviewed by Rein-
eccius (1979). Maligalig et al. (1973) demonstrated that flavor of the meat
from pond-reared catfish could be altered by feeding turkey livers in com-
parison to a floating cereal diet, with a noticeable 'liver flavor' becoming

apparent after 19 days on the liver diet and a 'cereal' flavor after 33 days
on the cereal diet. Motohiro (1962) observed a 'petroleum-like' flavor in
chum salmon that was traced to their eating Limacina helicina that con-
tained dimethyl-~-propiothetin, which is thermally degraded to dimethyl
sulfide to produce the undesirable 'petroleum-like' flavor. 'Blackberry' off-
flavor in cod from the Labrador coast has been attributed to the presence
of dimethyl sulfide by Sipos and Ackman (1964) and Ackman et al. (1966,
1967).
Another off-flavor in fish that has been traced back to the diet is the
'earthy' taint found in salmon. It has been shown to be due to the salmon
eating a species of Actinomyces that was present in their watery environ-
ment (Thaysen, 1936; Thaysen and Pentelow, 1936). Since some of the
aquatic food sources are contaminated by their environment, there are a
great many other possible dietary effects that can alter the flavor of fish.
Contamination from the environment and its influence on flavor will be
discussed in greater detail later in this chapter.
10.4.5 Poultry
The flavor and/or aroma of poultry meat (i.e. chicken and turkey) is
easily altered by diet, with relatively unsaturated lipids having a marked
influence on both the composition and flavor/aroma of the carcasses
(Reineccius, 1979). Feeding turkeys highly unsaturated fats in their diets
results in meat with a 'fishy' flavor according to Crawford et al. (1975),
which is not a characteristic of the long chain (C 16 -C 1S ) fatty acids
themselves (Reineccius, 1979). Crawford and Kretsch (1976b) concluded
that the 'fishy' flavor is due to an oxidative process that occurs during
the cooking process. In an effort to determine the chemical compounds
that were responsible for the 'fishy' flavor Crawford and Kretsch
(1976a) identified the volatiles from turkeys that had been fed a tuna
oil (2%) diet. They found 71 compounds were present in the meat from
the tuna oil-supplemented birds that were not present in the meat from
birds fed a standard control diet. They narrowed the list to 21 com-
pounds, which they felt may contribute to the 'fishy' flavor. However,
they did not determine the most important contributors to the undesir-
able flavor.
The 'fishy' flavor in turkey meat could be reduced by feeding the birds
an (X-tocopherol-supplemented diet in studies reported by Crawford et al.
(1975). Feeding (X-tocopherol acetate at 200 mg.kg- 1 of diet completely
eliminated the 'fishy' flavor, which supports the concept that the 'fishy'
flavor results from an oxidative reaction. Removal of the tuna oil from
the unsupplemented diet for 2 weeks prior to slaughter reduced the inten-
sity of the 'fishy' flavor. Injection of (X-tocopherol into the live turkeys 1-2
days before slaughter also reduced the off-flavor as effectively as feeding

the ct-tocopherol.
Rape seed has been implicated as causing a 'fishy' flavor in eggs
(Hawrysh et ai., 1975) but has not been reported to cause flavor problems
in chicken or turkey meat. This suggests that eggs are more likely to be
oxidized during cooking than the meat. Beside the flavor problems in
poultry that are associated with diet, however, it should be pointed out
that diets containing relatively large amounts of unsaturated fatty acids
result in more unsaturated body fat, which is more susceptible to oxida-
tion and the undesirable flavor changes related to rancidity (Reineccius,
1979).
10.4.6 Other species

Little is known about the contribution of feeds to the flavors of other
species, i.e. game animals and birds, although it is logical to assume that
diet affects their flavor. It has been claimed that mule deer differ in flavor
depending on the diet, with those having abundant succulent grazing
(alfalfa fields) being more desirable in flavor/aroma than those having
poor grazing and eating large amounts of sagebrush and evergreens (R.L.
Dickson, personal communication). However, New Zealand researchers
were unable to discern any flavor differences between wild and farmed red
deer (A.H. Kirton, personal communication).
The entire area of desirable feed flavors is interesting but is virtually
unexplored. Indeed, can desirable flavor be induced in flesh by giving
various diets?
10.5 'Gamey' flavors
Meat from wild animals of different species is known to have quite differ-
ent flavors. Available information on this topic is covered in chapter 9.
Quite aside from the species-specific flavors, which may be related to dif-
ferences in diet, the matter of 'gamey' flavors should be mentioned briefly.
Before discussing this topic further, however, it is re-emphasized that the
'gamey' flavor associated with meat from different species of animals can
be desirable or objectionable depending on the likes and dislikes of indivi-
dual consumers.
'Gamey' flavor was said by Ziegler (1944) to be due to higher con-
centrations of nitrogenous extractives, such as creatine, creatinine and the
purines, which were believed to increase with exercise and the age of the
animal. If this were the case, pasture-fed animals would be expected to
have more 'gamey' flavor than drylot or stall fed animals, yet there is no
evidence to support this viewpoint.
Since game animals are usually killed by shooting and are often not
dressed and handled properly, the so-called 'gamey' flavor could be due to
over-ripening of the meat with a rapid increase in the microbial popula-
tion. It is well-known that some consumers, i.e. especially those in
Germany and some other local areas of Europe, prefer the strong flavors
associated with aged game animals. This flavor preference has resulted in
a special market for game animals and birds; however, consumers of these
specialty products would not deem them to be spoiled, although they may
have above normal microbial counts. Thus, the 'gamey' flavor that many
consumers find objectionable may be due to 'over-aging' and the dis-
tinctive species flavor of the game animals and/or birds. For more details
on the species-specific flavors, readers are referred to chapter 9.
10.6 Off flavors due to sex condition
Although there are no major reported differences in the flavor of the meat
due to sex condition of cattle, or most birds and fish, the male pig (boar)
and perhaps the male sheep (ram) and goat (billy) may develop distinctive
objectionable odors/flavors. It is also possible that meat from some male
game animals, i.e. the deer, antelope, elk, etc., may develop sexually
related flavors; however, concrete evidence is lacking. Thus, discussion
here will concentrate on 'boar odor' or 'boar taint' and possible off-odors
in the meat from rams and billy goats.
10.6.1 Boar odor or taint

Lerche (1936) described an objectionable odor emanating from the cooked
meat of uncastrated male pigs as being 'perspiration-like', 'onion-like' or
'urine-like'. Although Craig et a/. (1962) demonstrated that the objection-
able odor was present in the fatty tissues and was localized in the unsapo-
nifiables, their attempts to isolate and identify the compounds responsible
were unsuccessful. Sink (1967) proposed that the CwA 16- steroids may be
responsible for boar odor. Soon after that, Patterson (1968) was able to
identify 5cr-androst-16-ene-3-one as a contributor to the undesirable odor/
flavor. Later it was shown by other workers (Berry and Sink, 1971; Berry
et al., 1971; Thompson et aI., 1972) that several related C w A16 -steroids
are also contributors to boar odor. Brooks and Pearson (1986) have
reviewed the information concerning sex odor in the pig and have
proposed some pathways for production of these steroid hormones in the
live pig. Much of the data on metabolic pathways for their formation
comes from the work of Gower (1979) and his associates (Gower et aI.,
1970, 1972; Katkov and Gower, 1970; Brophy and Gower, 1972; Saat et
al., 1974).
The boar odor (sex odor) problem has been confused by information
provided by Self (1957) indicating that sex odor occurs as frequently in
gilts and sows as in boars. This has not been shown to be the case in
other studies (Williams et al., 1963; USDA, 1968) for reasons explained by
Reineccius (1979). Further complications have arisen from studies suggest-
ing that boar odor may be caused by skatole as claimed by Swedish
workers (Hansson et aI., 1980; Lundstrom et al., 1980). The latter
problem has not been resolved, although both skatole and the Cwd 16_
steroids may be involved. Some confusion has also arisen from the fact
that male and female panelists differ in their ability to detect and their
sensitivity to the objectionable sex odor (Griffiths and Patterson, 1970).
10.6.1.1 Measurement and prevention of boar odor. Although the C w

d 16-steroids that are responsible for boar odor were first detected by gas
liquid chromatography, quantification is difficult. Trained panels have
most often been used as by Brooks and Pearson (1989) but the number of
responsible compounds and their variable odor thresholds makes determi-
nation of their importance extremely difficult. Thompson and Pearson
(1982) have labeled the responsible compounds with deuterium and been
able to quantify their levels in boar tissue by GCjMS techniques
(Thompson and Pearson, 1977).
Although castration will ultimately result in removal of the objection-
able odor/flavor from the meat, clearance is slow, requiring as long as 68
days (Lerche, 1936). The difficulty of the operation, risks from infection
and the low value of the animals result in most farmers marketing them
directly as boars. Boar meat can be used in some meat products without
any problem from the odor/flavor (Pearson et al., 1969).
Probably the most useful procedure for preventing boar odor in pork is
an immunological procedure (Brooks et al., 1986), which prevents the
problem by immunizing pigs against the Cwd 16 -steroids. This procedure
is patented by Pearson et al. (1986) and makes it possible to take advan-
tage of the greater efficiency of young boars in conversion of feed to lean
meat, while blocking production of the Cwd 16-steroids. Although several
other investigators have attempted to use essentially the same procedure,
their efforts have been less successful (Shenoy et al., 1982; Williamson et
al., 1985).
10.6.2 Ram odorlflavor

Although ram odor/flavor has not been investigated as thoroughly as the
sex-associated odor/flavor of the boar pig, sheep producers frequently
mention the strong flavor of the meat from sexually mature rams.
Attempts to identify differences between rams and wethers and/or ewes,
however, have not been successful to date (Kemp et al., 1970, 1972; Field,
1971; Campion et al., 1976; Crouse et al., 1981; Field et al., 1983). Never-
theless, the objection to the 'bucky' flavor/aromas seems to be justified by
the opinion of sheep breeders and meat scientists. It has been suggested
that the undesirable aroma/flavor is seasonal, peaking during the breeding
season and then declining. This viewpoint is supported by data on lutei-
nizing hormone (LH) in the blood serum of Finnish Landrace and Suffolk
rams (Schanbacher and Lunstra, 1976). It is also known that seasonal
fluctuations in the blood serum concentrations of testosterone in fallow
deer result in growth of the splenius muscle just prior to the rutting season
(Field et al., 1985). Thus, controlled studies are needed to ascertain if ram
flavor is indeed a problem.
10.6.3 Sex flavor/aroma in other species

The situation concerning sex aroma/flavor in other species is understood
even less than in the ram. However, if off-flavors are associated with the
meat from rams, it is likely they also exist in the male (billy) goat. Billys
have a highly objectionable odor that may carryover to the meat,
although this has not been proven. Sexually mature billy goats develop
the objectionable odor as a result of urinating on themselves, which is
apparently a sexual attractant or pheromone to the female (nanny) goat.
It is not known if the odor carries over to the meat, although in some
cases the meat may be contaminated during dressing. Definitive studies
are needed to determine if billy goat flavor/aroma is indeed a problem in
the meat of goats or if the objectionable odor is due to contamination.
There is no indicator that sexual condition has any effect on meat from
cattle, fowls (chickens and turkeys) or fish. Nevertheless, there is a dearth
of knowledge on the subject.
10.7 Off-flavors from the environment
Environmental contaminants, such as creosote and various other chemi-

cals used on facilities and/or on the animals themselves can also cause off-
flavors in meat, poultry and fish. However, such off-flavors are usually
serious enough to result in rejection of the contaminated products.
Examples of such contaminants causing off-flavors are the 'petroleum-like'
flavor observed in canned chum salmon by Motohiro (1962), which was
found to be due to thermal degradation of dimethyl-~-propiothetin to
form dimethyl sulfide as described earlier in this chapter. The 'earthy'
taint found in salmon is also derived from eating Actinomyces from a
contaminated environment (Thaysen, 1936; Thaysen and Pentelow, 1936).
Oil spills have also resulted in off-flavors in aquatic species with Shipton
et al. (1970) and Vale et al. (1970) describing a 'kerosene' taint in mullet
taken from the Moreton Bay area of the Brisbane River in Australia. The
off-flavors were associated with fish caught around docks or industrial
sewage outlets. The authors cited above suggested the 'kerosene' taint was
derived from hydrocarbons in the environment.
Reineccius (1979) in his review states, 'It is generally true that if a fish is
reared in waters containing organic volatiles, it will pick up the flavor of
these volatiles'. Although the red-meat species and chickens are not reared
in an aquatic environment, their meat will pick up off-flavors from fence
posts, feedlot facilities and housing treated with organic preservatives.
Lovell and Sackey (1973) reported that catfish developed an 'earthy-
musty' taint from odor-producing algae. In catfish, the rapid absorption
of off-odors from the environment can occur directly through the gills
(Thaysen, 1936; Lovell and Sackey, 1973; Shumway & Palensk, 1973;
Maligalig et at., 1975a,b), while in poultry or meat animals absorption is
believed to be through either the digestive tract or skin.
Maligalig et at. (1975b) found that refrigerated storage was ineffective in
removal of either dimethyl sulfide or 2-pentanone from catfish flesh.
However, purging with fresh dechlorinated tap water, along with fasting
of the live fish considerably reduced the content of these two con-
taminants within 24 h. As pointed out by Reineccius (1979) one would
expect the time required to purge off-flavors and odors from the tissues
would depend on the kind of contaminant, its concentration, the flow rate
of the purging water and the density of the fish population. Nevertheless,
the contaminants can generally be removed from the meat by a combina-
tion of a clean environment and a time period sufficiently long for the
metabolic processes to clear the tissues.
10.8 Processing-induced off-flavors
Although processing may result in development of both desirable and

undesirable flavors, the following discussion will focus on the undesirable
flavors. Two main groups of undesirable flavors will be discussed, namely
irradiation and canned meat flavors.
10.8.1 Irradiation flavor/odor

One of the major drawbacks to the preservation of meat, poultry and fish
products by irradiation has been the development of an off-odor/flavor
that has been described as being 'metallic', 'sulfide', 'wet dog', 'wet
grain', 'goaty' or 'burnt' (Huber et at., 1953; Mehrlich, 1966; Batzer et
aI., 1959). The undesirable odor/flavor becomes more intense as the irra-
diation dosage is increased (Batzer et at., 1959). The entire subject of
irradiation odor/flavor has been reviewed extensively by Reineccius (1979)
so the discussion will summarize some of the pertinent facts in that

review,
After fractionation of meat into the aqueous and lipid fractions
followed by treatment with radiation, Batzer and Doty (1955) concluded
that the sulfur compounds derived from the proteins are responsible for
irradiation odor. Batzer et ai. (1959) studied the effects of pre- and post-
irradiation storage time and temperature, irradiation dosage and grade of
beef on odor and color changes. They concluded that neither pre- nor
post-irradiation storage influenced panel acceptance. In contrast, Pearson
et al. (1959) found post-irradiation storage of cooked meat resulted in an
improvement in flavor, which is probably related to the differences
between fresh raw and cooked meat, with heating being necessary to
prevent further flavor deterioration caused by the indigenous enzymes
during storage of raw irradiated meat.
Merritt et al. (1959) were able to identify 10 compounds in the vola-
tiles isolated from irradiated beef using a low-temperature vacuum dis-
tillation procedure. They showed that of these 10 compounds (i.e.
methyl mercaptan, acetaldehyde, dimethyl sulfide, acetone, methanol,
ethanol, methyl ethyl ketone, dimethyl disulfide, ethyl mercaptan and
isobutyl mercaptan), only dimethyl disulfide and isobutyl mercaptan did
not increase with irradiation dosage. Wick et al. (1961, 1967) used a
distillation technique to isolate the volatiles from irradiated meats and
identified them using GLC. They identified 12 compounds but by
sniffing the GLC effluent concluded that methional made a major con-
tribution to the characteristic unpleasant odor of irradiated beef. Other
researchers (Merritt et ai., 1959, 1975, 1978; Dubravcic and Nawar,
1968; Champagne and Nawar, 1969; Angelini et al., 1975; Nawar, 1978)
have identified many compounds in the volatiles from irradiated meats,
including carbonyls, sulfur compounds and hydrocarbons. In spite of
the large number of compounds identified, it has not been possible to
identify any single compound or mixture of compounds that produces
the characteristic odor/flavor of irradiated meat, poultry or fish
products. Interestingly, the early findings of Huber et al. (1953) still
seem to be the best for producing irradiated meat, poultry and fish
products with a minimum of off-flavors. They found that irradiation
odors/flavors could be reduced by using low temperatures (below
freezing and the lower the better) during irradiation, minimizing the
oxygen content in the packages (either by cellular metabolism, vacuum-
packaging or using an inert gas for purging the air from the package)
and by using anti-oxidants. Furthermore, a single high dosage of irra-
diation for a short time was found to result in less irradiation odor/
flavor than several lower doses over a longer period of time. These
results have been confirmed by using heat-inactivated products followed
by removal of oxygen by vacuum-packaging and flushing with nitrogen
Table 10.2 Compounds found in retorted beef, their concentrations

and threshold levels a
Compounds Absolute Threshold level

concentrations (p.p.b.)
Hydrogen sulfide 1l00b 0.47

Methyl mercaptan 1800b 2.1
Ethyl mercaptan 190b 1.0
Dimethyl sulfide 600 b 1.0
2,3-Butanedione l.4 b 0.82
Ethanal 850b 210
Butanal 14c 9.0
Hexanal 55 c 4.5
Heptanal 25 c 3.0
2-Methyl prop anal 140C 0.9
3-Methyl butanal 130c 0.15
d
Ethylene sulfide 51 b
d
Methyl ethyl sulfide 6.4 b
d
3,5 Dimethyl-1,2,4-trithiolane 3.0
d
2,3-Pentandione 2.4
d
Furan 750
d
2-Pentyl furan 4.7
aTaken from Reineccius (1979).

bBeef heated at 121°C for 30 min.
cBeef heated at 121°C for 60 min.
dUnavailable.
gas before irradiating at a temperature of around -150°C as explained

by Urbain (1986).
10.8.2 Retort flavor of canned meat

Although retorting or canning of many foods is a well-accepted process,
meat, poultry and fish products develop a 'canned meat' flavor, which is
generally considered a defect. However, attempts to quantify the serious-
ness of retort flavor revealed that some consumers prefer the flavor of
retorted beef (A.M. Pearson, unpublished data). Nevertheless, it is
commonly accepted that retorting alters the flavor of meat. The problem
has been discussed in a review by Reineccius (1979), which will be sum-
marized.
Brennan and Bernhard (1964) have identified hydrogen sulfide, metha-
nethiol, ethanethiol, propanethiol and butanethiol in the headspace of
retorted beef and attributed the off-flavor to the latter two compounds.
Although Luh et al. (1964) monitored hydrogen sulfide (H 2S) and metha-
nethiol in retorted and high-temperature short-time sterilized beef, they
did not find any methanethiol, even though three times the threshold con-
centration of H 2S was present. Later Perrson and von Sydow (1973, 1974)
and Perrson et al. (1973) identified 95 different constituents in the head-
space volatiles of canned beef; with the exception of ethylene sulfide, pro-
pylene sulfide, 2-methylfuran and some thiophenes, all of the other com-
pounds have been identified in the volatiles from cooked beef. Table 10.2
presents the concentration of several compounds found in the headspace
of canned beef and their threshold values, which are· thought by Perrson
and von Sydow (1973) to contribute to 'retorted' flavor. These workers
acknowledged that the data in Table 10.2 gives threshold values in pure
systems, which may be quite different for meat systems, and that the
synergism of subthreshold compounds also may contribute to odor/flavors
in meat systems. Perrson and von Sydow (1973) then concluded that
sulfur compounds probably contribute to 'retort' flavor since their con-
centrations exceed threshold levels by a factor of 200 to 2000 but they did
not attempt to determine the contribution of each compound to canned
meat flavor.
Perrson and von Sydow (1974) demonstrated that thin slices of beef
developed less 'retort' flavor than thicker slices. This showed that the
intensity of 'retort' flavor was related to the time of holding at the proces-
sing temperatures required to achieve sterilization during canning.
In summary, Reineccius (1979) concluded that the odor/flavor of
retorted beef is believed to be the result of an increase in the concentra-
tion of certain volatile aldehydes and sulfur compounds above that found
in cooked beef. The objections to 'retort' flavor can be reduced by heating
thin slices of meat at higher temperatures for shorter periods of time,
although the flavor may be atypical (Luh et al., 1964). Addition of L-
arginine, disodium fumarate, disodium malin ate and potassium sorbate
before heat processing resulted in higher sensory scores and reduced
'retort' flavor according to Luh et al. (1964). The improvement in flavor
appeared to caused by the reaction of the volatile aldehydes with the
amino acids and by the binding of the sulfur compounds to the organic
acids.
10.9 Off-flavors associated with microbial growth
Reineccius (1979) has reviewed the information available on off-flavors

due to bacterial growth. Most of the material covered in this section is a
summary of that review. For purposes of this discussion, the topic is dis-
cussed under fish, poultry and the red meats (i.e. beef, pork and lamb).
10.9.1 Off-odors in fish caused by microbial growth

Reineccius (1979) stated that fresh fish has but little odor, but during
storage at temperatures above ooe, the 'fishy' odor develops. The off-
odor is often described as being 'intense', 'putrid' and 'foul'. These odors
are probably the result of microbial enzymes and can be followed by

increases in the volatile acids, amines, volatile reducing substances, carbo-
nyls and so on. The flavor changes that occur during spoilage are not due
to indigenous tissue enzymatic activity (Partmann, 1966) but the spoilage
odors are primarily due to bacterial action.
The major organisms that cause spoilage of fish are Gram-negative
bacteria of the genera Pseudomonas, Achromobacter and Vibrio (Herbert
and Shewan, 1976). Less than 10% of the initial bacterial population on
fish produce the objectionable spoiled odors according to Adams et al.
(1964), although their proportion of the total flora increases during the
early stages of storage and declines later.
The volatile odor/flavor compounds that increase during spoilage
include amines, acids, carbonyls, sulfur compounds, aromatics and total
volatiles, which are all indicative of spoilage. Volatile amines have been
followed by Beatty (1938), Dyer and Mounsey (1945), Dyer and Fraser
(1959), Jones and Murray (1961), Stansby (1962) and Wong et al. (1967)
and are useful indices of spoilage. Volatile acids have been measured by
Hillig et at. (1958) and Hughes (1960) but are not useful for measuring
spoilage. The same is also true for the carbonyl compounds (Ota, 1958;
Diemair & Schams, 1962) and aromatics (Chen et al., 1974). Although
there is an increase in the sulfur compounds (Herbert and Shewan, 1975;
Herbert et al., 1975) and total volatiles (Wong et al., 1967), other factors
can influence their concentrations.
The significance of individual compounds to 'fishy' odors has been
determined by Obata and Yamanishi (1952) who ascertained the role of
pyridine, pyrrolidine, piperidine, delta-aminovaleraldehyde and delta-ami-
novaleric acid in several types of off-odors in fish. Trimethylamine has
been investigated as an index of 'fishy' odor (Davies and Gill, 1936;
Stansby, 1962) and its usefulness summarized in a review by Reay and
Shew an (1949). Trimethylamine oxide is the natural source of trimethy-
lamine in fish muscle, with the latter compound being formed by reduc-
tion through the action of bacterial enzymes involving the coupled
oxidation of lactic acid to acetic acid and carbon dioxide (Watson,
1939).
The odors and volatile compounds produced by pure cultures of
bacteria have also been studied as an approach to understanding the
species of bacteria responsible for causing off-odors. Achromobacter and
Pseudomonas strains have been shown to produce the 'sulfidy' off-odors
(Castell et al., 1959; Shewan et al., 1960) in stored cod. Chai et al. (1968)
found P. fluorescens and P. putrefaciens to be the major spoilage organ-
isms on haddock. P. putrefaciens produced propanal, methyl mercaptan,
dimethyl sulfide, dimethyl trisulfide, 3-methyl-l-butanol and trimethyla-
mine during growth on fish muscle according to Miller et al. (1973c).
Achromobacter produced the same compounds but it did not produce
dimethyl trisulfide. The above results indicate that these organisms

produce the 'sulfidy' odor in fish.
Herbert and Shewan (1975, 1976) demonstrated that not only Pseudo-
monas and Achromobacter organisms produce the 'sulfidy' odor but they
also found that the Vibrio genera also could contribute to the 'sulfidy' off-
odor. These authors attributed the off-odor to hydrogen sulfide, methyl
mercaptan and dimethyl sulfide. By the use of radio labeled precursors,
they found that hydrogen sulfide was derived from cysteine, while methio-
nine was the precursor for methyl mercaptan. They then postulated that
dimethyl sulfide was formed by condensation of two molecules of methyl
mercaptan.
Fish muscle can develop a 'musty, potato-like' odor during the growth
of P. perolens (Castell and Greenough, 1959; Castell et al., 1959).
Although Miller et al. (1973a) isolated and identified several compounds,
they determined that 2-methoxy-3-isopropylpyrazine was responsible for
the 'potato-like' odor.
Castell and Greenough (1959) first described a 'fruity' or 'ester-like' off-
odor in fish muscle, which was shown to be caused by P. fragi (Castell
and Greenough, 1959; Castell et al., 1959). Miller et al. (1973b) identified
several chemical compounds in the headspace of fish muscle inoculated
with P. fragi and concluded that ethyl esters of acetate, butyrate and
hexanoate are responsible for the 'fruity' off-odor.
In summary, although numerous volatile compounds have been identi-
fied in spoiled fish, only a few of these compounds are actually respon-
sible for the off-odors. The 'sulfidy' odor has been attributed to
trimethylamine, while the 'potato-like' off-odor has been shown to be
due to 2-methoxy-3-isopropylpyrazine. The 'fruity' off-odor appears to
be due to ethyl acetate, ethyl butyrate and ethyl hexanoate. Most of the
off-odors are the result of growth of Pseudomonas, Achromobacter and
Vibrio organisms, which comprise less than 10% of the total microbial
flora.
10.9.2 Off-odors and flavors in poultry associated with microbial growth

Poultry meat undergoes essentially the same breakdown process due to
microbial growth as with fish, except for the fact that the quantity of tri-
methylamine formed is much less. Thus, trimethylamine is not a good
index of off-odors in poultry meat (McMeekin, 1975, 1977; McMeekin
and Patterson, 1975), although sulfur breakdown products comprise a
major proportion of the volatiles (McMeekin and Patterson, 1975). The
mechanisms involved in spoilage in poultry meat have been of more
interest than the off-odors (McMeekin, 1975, 1977). This may be because
the off-odors occurring during meat and poultry spoilage, although
equally objectionable, were less unique and better understood as the

importance of prevention of spoilage by low-temperature preservation is
well recognized (Ziegler, 1944; Lawrie, 1966).
10.9.3 Off-odors and flavors produced in red meats by microbial growth

A considerable amount of research has been carried out on the nature of
microbial spoilage in red meat but most of it has concentrated on the
mechanisms involved in the spoilage process (Brown and Weidman, 1958;
Ayres, 1960; Jay, 1967; Jay and Kontou, 1967; Hasegawa et aI., 1970a, b).
Although the mechanisms involved in production of off-odors and flavors
in red meats are believed to be similar to those involved in poultry and
fish, little trimethylamine is evolved. Thus, trimethylamine concentration
is not useful as an index of spoilage in red meat, as is the case for fish
(Reay and Shewan, 1949). As with fish, a 'putrid' note is probably indica-
tive of the presence of large quantities of sulfur breakdown products
(Herbert and Shewan, 1975, 1976).
Similar to fish, it seems probable that the 'sulfury' odor emanating from
spoiled red meats is due to the presence of hydrogen sulfide, methyl mer-
captan and dimethyl sulfide. In fish, those compounds have been shown to
be derived from breakdown of cysteine and methionine and from con-
densation of two molecules of methyl mercaptan, respectively (Herbert
and Shewan, 1975, 1976), which is probably true also for the red meats.
Thus, readers interested in the cause of spoilage-induced off-odors in meat
should review studies referred to under that topic.
10.10 Summary
Meat, poultry and fish products suffer from a variety of off-odors/flavors,

as discussed. These undesirable odors and/or flavors represent serious
impediments to consumption and can be eliminated by taking certain pre-
cautions and some of these are also discussed.
The off-odors covered in this chapter include those due to oxidative
rancidity and/or warmed-over flavor, which are related to breakdown of
the lipids during storage. Species-specific flavors include those that are
associated with a particular species, such as 'mutton' flavor or 'goaty'
odors/flavors. Flavors/odors due to the diet are discussed in considerable
detail and vary from the soft-pork problem to wild onion flavors. They
can be circumvented by altering the diet before slaughtering, although the
length of time required will depend upon the nature of particular feed
involved. Even though objectionable odor/flavor problems have been
found in the meat derived from animals grazing particular crops, legumes
and other forages, these off-odors/flavors can be circumvented by removal
from the feeds involved for a relatively short period of time. Feed flavors
can also be altered to be desirable as has been shown for meat from
animals fed protected lipids, where species-specific flavors can be altered
to desirable ones. Fish and poultry flavors are also affected by diet, which
is discussed in some detail.
'Gamey' flavors are complicated by over-aging and other factors, but
are probably a result of diet. Sex-related flavors are known to exist as is
the case for 'boar odor' in the pig. Although not well-documented, it is
also possible that rams and billy goats may develop off-odors or flavors.
Other species do not appear to have any sex-related flavors or odors.
Environmentally-caused off-flavors can lead to rejection by consumers,
such as has been found to occur with the flesh from fish and birds after
oil spills.
Processing-induced off-odors/flavors are discussed, with two of the best
known ones being those associated with the irradiation and canning of
meat products. Finally, the off-odors/flavors that originate from microbial
growth have been discussed; these are usually derived from microbial
autolysis and ultimately spoilage. Suffice it to say that 'spoiled' off-odors/
flavors are serious problems that can usually be avoided by proper pre-
cautions in handling.
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11 Tenderness of meat, poultry and fish
E. DRANSFIELD
11.1 Introduction
Variability in the quality of meat has long been a concern of the consumer
and recent surveys have shown that consumers have difficulty in selecting
beef because they are unsure of the quality, particularly its texture
(Dransfield, 1985), which is of primary concern in the beef industry
(Morgan et at., 1991).
Mechanical (tenderness) and juiciness (succulence) components con-
tribute to meat texture (a subjective attribute) and form the basis for the
marketing of different beef cuts. Texture also is a major criterion in
poultry meat quality and is an important determinant of preference of
fish, especially those of mild flavour (Wesson et at., 1979). Many factors
affect meat tenderness (Ashgar and Pearson, 1980), thus, a multi-
disciplinary approach is essential to understand the fundamental mechan-
isms controlling tenderness and rationalisation of animal production and
meat processing.
Muscle contains about 75% moisture of which about 10-15% is bound
to the proteins. The dry matter is made up of about 70% protein, 10%
fat, 3% carbohydrate and 5% salts. Muscle fibre bundles, which are sur-
rounded by the perimysium, are composed of finer muscle fibres, which
are surrounded by the endomysium. These finer fibres are composed of
myofibrils of about 1 J.lm in diameter. They function as contractile units
and comprise the sarcomeres, each about 2 J.lm in length. Collagen in the
endomysium and perimysium is often thought to form 'background
toughness' but this implies an inert role, which is misleading, since its
contribution cannot be separated from the myofibrillar component in
most sensory and instrumental shear tests.
Tenderness is affected by variations in the degree of cooking, which
differs considerably between countries and according to individual pre-
ferences. However, despite the end-point temperatures varying from 60-
75°C across European countries, the ranking of tenderness was similar at
different research centres (Dransfield et at., 1982) and allows comparisons
of results across laboratories. Standardized cooking methods for meat
research have been developed in the European Community (EC, Boccard
et at., 1981) and the USA (Cross et at., 1978).
This chapter concentrates on the development of tenderness in carcass
Table 11.1 Sensory tenderness in different beef

breedsa
Breed Mean Variance
Pure breeds
Aberdeen Angus 3.7 1.2
Devon 2.9 3.9
Friesian 2.9 2.2
Friesian cross
Aberdeen Angus 1.7 1.2
Charolais 2.7 3.1
Devon 3.3 2.7
German Simmental 1.9 0.7
Hereford 0.2,2.8 6.3,0.9
Limousin 2.2 1.6
Swiss Simmental 1.8 1.8
Ayrshire cross
Friesian 2.5,4.3 4.0,0.8
Simmental 4.8 0.6
aScored from - 7 to + 7 .
meats, which forms the basis for development of acceptable further pro-
cessed products.
11.2 Pre-slaughter factors
The meat industry has changed, over the past decade, from being produc-
tion led to becoming demand driven by consumers who want good quality
meat with the minimum amount of fat at a reasonable price. While the
industry continues to respond, it has done so cautiously because of the
fear that radical changes in production would lead to a reduction in ten-
derness and a loss of consumer confidence.
11.2.1 Breed effects

11.2.1.1 Breeds of cattle. To meet the needs of modern farming prac-
tices, traditional British beef cattle breeds, such as Hereford and Aberdeen
Angus, are being replaced by dual-purpose cattle such as Friesian and by
other breeds that were introduced to increase lean meat yields. The
inherent tenderness of meat from the Aberdeen Angus was thought to be
derived from its finer grained muscles, with early trials showing that the
Angus had a higher number of cattle in the more tender group than any
other breed. However, other trials have found little difference between
meat from Angus and that from Devon and Hereford breeds. Meat from
TENDERNESS 291
the dairy breeds can be as tender or more tender than that from most of
the conventional beef breeds. Using 108 steers over a 3-year trial in the
UK, only small differences in the tenderness of loin roast were attribu-
table to differences in the breeds: Aberdeen Angus, Charolais, Devon,
Friesian, Hereford, Simmental, South Devon and Sussex (Chadwick et aI.,
1979). Data collected over several years at Langford (Table 11.1) showed
that pure breeds and crosses had similar average tenderness ratings, which
were often quite variable within breeds. The variabilities in tenderness
within breeds and between replicate trials (with Hereford cross Friesians
and Friesian cross Ayrshire) were often larger than the variability between
breeds.
Bos indicus breeds are particularly advantageous in semi-tropical and
tropical climates for their heat and disease resistance but their meat is
usually less tender than that from Bos taurus (Koch et al., 1988). Brahman
bulls, slaughtered at ages between 8 and 87 months, had tougher meat
than Shorthorns but tenderness did not differ between Hereford, Angus
and Brahman x Angus (Burns et aI., 1958). Brahman steers were less
tender than the average of the Angus, Charolais and Hereford steers
(Luckett et al., 1975). Meat from Sahiwal crosses was less tender than that
from Hereford x Friesian crosses (Whipple et al., 1990). The cause of the
toughness in Bos indicus is probably a reduced tenderisation during ageing
because of the higher stability of the proteinase inhibitor (Wheeler et al.,
1990; Whipple et al., 1990).
11.2.1.2 Breeds of sheep. The sire breeds, Texel (introduced for its leaner
meat), Dorset Down, Suffolk, Oxford, Cotswold and Southdown were
without any effect on the eating quality of lamb (Dransfield et al., 1979).
It is unlikely, therefore, that other breeds would significantly influence
texture which enables lamb to be marketed without reference to its breed
origin.
11.2.1.3 Breeds of pigs. There is some evidence for pigmeat that the rela-
tionship between texture and carcass quality is different for the Hamp-
shire, Duroc (Hiner et aI., 1965), Swedish Landrace and Yorkshire breeds
(Malmfors and Nilsson, 1977), which suggests that breed should be taken
into account when evaluating the influence of rearing and the influence of
carcass quality on texture. However, differences in tenderness of pork
from different breeds also depend on the type of cooking and can be
eliminated by higher temperatures of cooking (Fjelkner-Modig, 1985).
11.2.1.4 Breeds of poultry. In poultry, breed, sex and diet were shown
generally to affect meat tenderness to a lesser extent than stress and elec-
trical stunning of the birds, or the method of scalding, plucking, chilling
or freezing of the carcass (Jones and Grey, 1989).
11.2.1.5 Species of fish. Seventeen species of fish were compared for

texture and arranged using multidimensional techniques based on
sensory data (Cardello et al., 1982). The general consensus was that loss
of acceptability in fresh fish is initially attributable to a loss of some
valued fish flavours, and, subsequently, to the development of disliked
flavours by bacterial action. Acceptability of fresh white fish could be
explained entirely on the basis of flavour, while texture was found to be
neutral and appearance either neutral or negative (Bennett and Hamil-
ton, 1986).
11.2.2 Fatness
11.2.2.1 Beef Some of the fattiest meats, which contain up to 20% fat,
are produced in the USA. They are higher in fat than those generally
produced in UK and Ireland, while most other EC countries produce even
leaner meat. The presence of fat was traditionally thought to be essential
for tender meat.
Considerable work has been performed in the USA relating quality to
USDA grading according to thickness of subcutaneous fat and marbling
(visible intramuscular fat). Of consumers who rated rib-eye from Angus,
Hereford x Angus and Holstein and Shorthorn steers, 60-70% found
no or a slight preference related to marbling; however, the effect was so
small that the order of tasting had more influence than marbling on pre-
ference. With steers from the Angus, Brahman, Hereford and Charolais
breeds, less tender steaks were found in lower grading (leaner) carcasses.
However, they also cooled more rapidly than the fatter carcasses
(Luckett et al., 1975), which may have been the origin of the reduced
tenderness. Canadian work has also shown that maturity and marbling
did not affect the texture or overall acceptance of silverside or loin
roasts. Finishing cattle on high-forage (low-energy) rations produced
leaner meat but tenderness was little affected and the meat was good
overall. The conclusion, confirmed in many trials, is that marbling or
intramuscular fat content accounts for about 10% of the variation in
tenderness or texture. Using a range of breeds and crosses on 500 steers
raised in a 3-year study, it was concluded that the USDA quality grade
factors were of minimal value in predicting tenderness and that it would
be more practicable to set a guideline of low fat content rather than to
attempt to reflect quality by the degree of marbling (Campion et al.,
1975).
11.2.2.2 Pig meat. Pig carcasses have become consistently leaner as a

result of improvement in breeding and management. Using Large White,
Gloucester Old Spot and crossbred pigs, only 9% of the vanatlOn in
tenderness was accounted for by variation in percentage lean or fat
TENDERNESS 293
(Wood et aI., 1979), and variation in the intramuscular fat content from
1-4% did not affect the tenderness of roast pork (Rhodes, 1970). Studies
in Denmark have shown that, with increases in intramuscular fat content
up to 2.5%, quality also increased but, at higher levels of fat, there was
no relationship between intramuscular fat content and quality (Kirkgaard
et al., 1979). This conclusion is, therefore, very similar to that found for
beef in USA.
11.2.3 Sex effects

11.2.3.1 Bull and steer meat . . Eating quality of beef from young bulls
has been studied in several countries and evidence can be cited for and
against the existence of differences in tenderness of meat from bulls and
steers. Researchers in the USA have suggested that bull beef cannot be
marketed at the same USDA grade with equal confidence regarding
palatability. Similar conclusions have been reached in Ireland and in the
UK. In some trials, in which shear tests showed that beef from steers
was more tender than that from short-scrotum or normal bulls, experi-
enced assessors and consumers found no significant difference between
steers and bulls. In home tests, 37% of consumers thought bull beef
better, 48% thought it the same and 15% thought it worse than that
from steers. Although most people thought bull beef poorer than steer
beef, nearly 85% thought it was better or equal to their usual purchase
(Dransfield, 1985). Comparing meat from twin steers and young bulls in
commercial trials and consumer tests in the UK, it was found that
roast meat was slightly tougher in bull beef but that the tenderness of
grilled and casserolled cuts was similar to those from steer beef
(Dransfield et at., 1984). In Denmark, meat from bulls from the early-
maturing breeds, which had less intramuscular fat and more heat-labile
collagen, tended to be less tender than that from late-maturing bulls
(Liboriussen et aI., 1977). In EC countries, wide variation in the texture
of beef steaks was not attributable to the sex of the animal (Dransfield
et al., 1982).
11.2.3.2 Ram and wether meat. Entire ram lambs, despite their meat
having a greater proportion of collagen, had similar tenderness to wethers
(Dransfield et al., 1979; Edwards et al., 1982), cryptorchids or partially
castrated males (Alvi, 1980). Toughening in lean ram lamb carcasses,
which can arise readily under normal commercial chilling rates, can be
alleviated by electrical stimulation to produce tender meat (Solomon et
al., 1986).
Lack of castration in beef, sheep and also in pigs, therefore, has neither
more nor less effect on texture than breed (Lloyd et al., 1980) or feed
(Crouse et al., 1985).
11.2.4 Growth promoters

Steroids were among the first growth promoters to be studied and early
reports in the 1950s, using oestrogen implants, were at variance, with
some workers finding tougher meat while others found it similar in ten-
derness to that from non-implanted controls. In combination with hex-
oestrol or oestradiol, trenbolone acetate gave meat of similar quality to
controls (Verbeke et aI., 1976). Increased growth rates over a range of
breeds of cattle upon using zeranol have been shown to have little effect
on tenderness (Ntunde et ai., 1977).
With the Ee ban on the use of some growth promoters, there is an
urgent need to find acceptable alternatives that increase growth rate and
reduce carcass fatness without compromising product quality. The intro-
duction of ~-adrenergic agonists represents the latest use of pharmaco-
logically active compounds, which have opened up new prospects for
improving the efficiency of meat production. The ~-agonists, clenbuterol,
fenoterol and cimaterol, were shown to exhibit marked effects on muscle
and adipose tissue growth in animals. At a concentration of 2 p.p.m. of
~-agonist, growth rates in cattle and sheep increased by about 20% over
a 2-4 month period. Fat was decreased and lean content was raised by
about 10%. The response in pigs is generally less than in ruminants and
in broilers there is only about a 2% increase in growth rate. ~-adrenergic
agonists fed to meat animals increased the toughness of meat in sheep
(Hamby et ai., 1986), cattle (Tarrant, 1987) and, to a lesser extent, in pigs
(Warriss et al., 1991). Although not all muscles are toughened, the effect
can be dramatic and reduce significantly the quality of the meat. Our
understanding of the action of ~-agonists is incomplete, but they appear
to increase nutrient supplies and the levels of mRNA involved in myosin
synthesis. Their use also reduces the levels of the proteases, cathepsins B
and D, and calpain I, and increases the level of calpastatin, a specific
proteinase inhibitor (Kretchmar et al., 1990). Since the activities of cathe-
psins and calpains regulate protein turnover (Dayton et al., 1981) and
post-rigor tenderisation, it is interesting to speculate that their reduced
activity following ~-agonist feeding may be responsible both for the
induced myofibrillar hypertrophy (due to reduced myofibrillar protein
degradation) and for the toughening (due to a reduction of post-mortem
ageing). This mechanism could account for the reversal of the toughening
effect of ~-agonists following infusion of the meat with calcium salts,
which would stimulate proteolysis by activation of the cal pains (Kooh-
maraie and Shakelford, 1991).
11.2.5 Connective tissue

Variations in the connective tissue content in beef muscles form the basis
of differentiation between cuts for grilling, roasting and stewing in many
TENDERNESS 295
14
12 a
10
III
III
a
Q) a rlil
c: 8
.r::
C>
:l
a a
0
I- 6 a a aa a
a III
4 a a
-. •• • • •
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a
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2 • • ••
0
4 6 8 10 12 14
Total Collagen
Figure 11.1 Relationship between the total collagen and toughness. Toughness (kg force)
from a shear test is related to the total collagen content (mg.g- 1 wet tissue) for beef muscles
cooked at 60°C for 20 min (simulated grilling; open symbols) and 90°C for 3 h (stewing;
closed symbols). The muscles were (left to right): psoas major, longissimus dorsi, gluteus
medius, rectus femoris, gastrocnemius, infraspinatus, triceps brachii, rectus abdominis, semi-
membranosus, serratus ventralis, biceps brachii, pectoralis profundus, supraspinatus, semi-
tendinosus, latissimus dorsi, biceps femoris, extensor carpi radialis, and complexus.
countries and, in France, removal of visible collagenous tissue provides a

means of upgrading meat. Early studies by Ramsbottom and Strandine
(1948) found a linear relationship between the amount of visible con-
nective tissue in raw meat and toughness in cooked meat. Tender meat is
also associated with a low collagen content in meat from double muscled
cattle (Bailey et aI., 1980). The contribution from connective tissue
depends on the degree of cooking. From 40- 45°C, the myofibrils
denature, moisture is lost and toughness increases about two to three-fold.
Between 50- 65°C, the collagen shrinks and causes a further moisture loss,
which relates to a further two-fold increase in toughness. The temperature
at the start of the second rise in toughness increases at 50°C at 2-3
months of age to above 60°C in 12-year-old beef cattle and was shown to
be related to the connective tissue strength of the muscles (Shorthose and
Harris, 1990). At temperatures above 80°C, collagen will solubilise and the
meat becomes more tender (Davey and Gilbert, 1974).
With increasing animal maturity, the total collagen content of muscles
does not increase, and may decrease slightly, but the amount of heat-labile
collagen decreases. During cooking, denatured collagen exudes from veal
and forms a gel on cooling but remains within the meat from older cattle.
In 9-week-old calves about 22% of the collagen is soluble in hot water, in
1O-month-old steers about 12% is soluble and in old cows only about 4%
is soluble (Tuma et al., 1962). The decrease in solubility is undoubtedly a
reflection of the type of cross-links present in the collagen but attempts to
quantify the relationship between soluble collagen and tenderness have met
with only limited success. Soluble collagen was poorly related to tenderness
in beef muscles from a range of breeds in Canada (Jeremiah and Martin,
1981) and, in USA studies, was found to account for less than 10% of the
variation in tenderness of meat from young and old cattle (Cross et al.,
1973). In none of the studies has more than 50% of the variation in ten-
derness been accounted for by the content of heat-solubility of the
collagen. These weak relationships may have arisen because other factors
that affect tenderness, such as myofibrillar shortening and pH, cannot be
controlled. Thus, with turkeys, younger or smaller birds may chill faster
and give rise to tougher meat than that from older or larger birds. A mul-
tivariate approach, using measurements of total collagen, heat-soluble
collagen, fat, moisture and pH, in 18 hot, deboned beef muscles (which
had similar sarcomere lengths), showed that total collagen was the best
predictor of tenderness in grilled or roasted meat but that there was little
variation when the muscles were stewed (Dransfield, 1977). In Figure 11.1
a reasonable relationship between total collagen and meat tenderness
under mild cooking conditions, which was removed by stewing, is shown.
Heat-soluble collagen content would, therefore, appear to contribute to
variation in tenderness among animals of different ages but total collagen
content seemed to be the best predictor of tenderness among muscles.
11.3 Slaughtering
Stunning fish either by electrical shock, CO 2 narcotisation, hypothermia, a
blow on the head or by gill and tail bleeding have been studied in relation
to fish quality. There appears to be little consistent effect on tenderness
except that CO2 generally results in soft flesh. The softening effect has
been attributed to degradation of myofibrillar proteins, occurring more
rapidly at pH 6 or less, with no effect on the texture for rainbow trout of
slaughter methods with pH above 6.4 (Azam et al., 1989). Texture of
catfish, however, was not related to the method of killing using electrical
stunning, CO 2 immobilisation, bleeding or ice immobilisation (Boggess et
al., 1973).
In poultry, pre-slaughter electrical stunning can improve tenderness
(Lee et al., 1979) but poorer quality meat can result from the use of high
voltages or a long stunning period. Pre-slaughter heat-stress also de-
creased tenderness. Decreased struggling occurring at or during slaughter
increases the tenderness of turkey breast muscle and broiler chickens.
TENDERNESS 297
Higher temperatures or longer scalding times cause toughening, as does

machine plucking (Jones and Grey, 1989). Most of these effects appear to
cause changes in the rate of post-mortem rigor development, which is
likely to be the origin of the changes in tenderness.
11.4 Rigor development
11.4.1 Compositional and structural changes post-mortem

Although the slaughtering operation causes the death of the animal within
minutes, the muscles continue to metabolise while ATP is present. The
energy to maintain the internal environment is derived from dephos-
phorylation of ATP, which is replenished initially from creatine phosphate
and later through glycogen catabolism. When the ATP content falls below
about 20% of its initial value, cross-bridging of the thick and thin fila-
ments occurs and the muscle becomes progressively stiffer (Bendall, 1979).
Rigor development can take up to 30 h in beef. In chicken and in turkey
breast muscle rigor develops between 0.5 and 6 h, but in extreme cases
rigor can occur within 5 min post-mortem (Ma and Addis, 1973). The fall
in pH causes protein denaturation, membrane breakdown and deregulated
proteolysis. Myosin denaturation, particularly when the fall in pH is rapid,
causes pale, soft and exudative (PSE) muscle and a slight toughening of the
meat. At death, the free calcium ion concentration in the sarcoplasm is
only about 10- 7 M but failure of the calcium pump in the sarcoplasmic
reticulum causes the level to increase to about 10- 4 M. Partial disintegra-
tion of the lysosomal membranes causes release of at least some of the
lysosomal enzymes into the sarcoplasm. Proteolysis also continues out of
control as the activities of the enzymes change with the falling pH and
changes in the levels of cofactors. During the fall in pH, the level of cal-
pastatin, a specific inhibitor of calpains, decreases to about 70% of its
initial value (Vidalenc et aI., 1983). The inhibitory activity of calpastatin is,
therefore, gradually removed and the level of neutral proteinases (calpains)
decreases, particularly below pH 6.2. As acid accumulates, phospho-
fructokinase, which is essential for glycolysis, is inactivated and glycolysis
ceases, preventing a further decline in pH. The normal ultimate pH is
about 5.5 for red meats and slightly higher (5.7-6.2) in poultry muscle.
11.4.2 Temperature effects

Temperature has little effect on the ultimate pH but affects the rate of pH
decline depending on the muscle type. The rate of rigor development and
associated muscle shortening are at a minimum when the muscles are
maintained at about 17°C. Shortening occurs to a greater extent as the
temperature is raised or lowered. Excised muscles typically shorten by
140
en
en
Q)
c::
-g,100
o
I-
60
20 L---~----~--~----~----~--~----~--~----~
0 .2 0 .6 1.0 1 .4 1.8
Muscle Lenglh (relative to slack length)
Figure 11.2 Relationship between toughness and muscle length in unaged and aged beef.
about 30-40% of their relaxed length when held throughout rigor at 0° or

40°C compared with only 10% shortening at about 17°e. Shortening can
be produced either by pre-rigor cooling (cold-shortening, as explained by
Locker and Hagyard, 1963), thawing of pre-rigor meat (thaw-shortening)
or heating (heat- or rigor-shortening) during rigor development. Most
attention has been paid to cold-shortening of meat. During cooling, if the
temperature of meat reaches 11°C before the pH has fallen below 6.2, the
muscles will contract. In beef, this pH occurs at about 10 h, in pigs at 3 h
and in chicken at about 20 min. The extent of shortening in carcasses
depends on the rate of glycolysis, the rate of chilling and the mechanical
restraint on the muscles, which vary considerably between species and
individual carcasses.
There are clearly differences in the processing of different species. In
beef, rigor mortis normally takes place during cooling, which is con-
trollable, while in poultry rigor may develop during the scalding and
plucking operations - two stages that have been implicated in toughening.
Extensive rigor shortening may occur in beef carcasses held at 35°C for
3 h (Lee and Ashmore, 1985) or during very slow chilling following elec-
trical stimulation, particularly in the centre of the round. Contraction in
poultry muscle can be induced by electrical stimulation, beating, freeze-
thawing and heating (Klose et aI., 1970).
11.4.3 Electrical stimulation

Electrical stimulation of carcasses soon after slaughter was developed to
hasten rigor development and reduces the time required to reach pH 6 to
TENDERNESS 299
a few minutes. This reduces the risk of cold-shortening in beef and lamb
carcasses but increases the risk of rigor-shortening in pig and poultry
meats. Various types of application have been used with a range of
voltages, frequencies and times of application, and are practised through-
out the world for beef and sheep meats. The types of electrical stimulation
and their application have been reviewed extensively in a book edited by
Pearson and Dutson (1985).
Electrical stimulation also increases the associated structural and pro-
teolytic changes, resulting in a greater release of lysosomal enzymes into
the sarcoplasm and greater degradation by ~-glucuronidase (Dutson et al.,
1980) and neutral proteinases (Dransfield et al., 1992b).
11.5 Muscle shortening
11.5.1 Relationship between muscle shortening and tenderness

The classical demonstration by Marsh and Leet (1966) that toughness of
cooked meat was dependent on the degree of shortening, irrespective of
how the shortening was derived, has had a lasting impact on under-
standing variation in meat tenderness. Despite its importance and its long
history, however, an explanation for the shape of the toughness-short-
ening curve remains obscure (Figure 11.2). Initially, explanations were
sought in relation to the degree of overlap of the thick and thin filaments,
but maximum tension and crosslinking in living muscle is generated with
less overlap of filaments than is present at maximum toughness. At 40%
shortening, and maximum toughness, the sarcomere is only about as long
as the thick filaments and, on cooking, they denature to form a tough
continuum with filaments of adjacent sarcomeres. The gradual increase in
toughness with shortening may then be due to the frequency of the occur-
rence of the tough (short) regions within the muscle.
Connective tissue has also been implicated in the development of tough-
ness in shortened muscles. Rowe (1974) suggested that crimp length of the
collagen fibres of the perimysial connective tissue in cold-shortened meat
was pulled-out or eliminated, and concluded that the overall change in the
strength of collagen was dependent on the change in collagen fibre
numbers and the angle that the collagen fibres make with the muscle
fibres. Tension developed in the connective tissue network may also con-
tribute to the toughness. The tension in unheated meat is likely to be a
minimum at the relaxed length. On heating stretched meat, collagen
aligned in the direction of the muscle fibres shrinks along the direction of
the muscle fibres, reducing the tension. In shortened muscle, the tension
developed in the raw state would be enhanced by heat shrinkage and will
then bind tightly around the heat denatured myofibrillar and sarcoplasmic
proteins producing a structure that has greater resistance to deformation.

This can be demonstrated by allowing shortened meat to age and then
stretching it. In this case the sarcomeres remain short but the collagen is
pulled out and the meat is more tender than non-stretched meat (Drans-
field and Rhodes, 1976).
11.5.2 Carcass suspension effects

The degree of shortening and susceptibility to shortening depend on the
restrictions imposed on the muscle by its attachments to the skeleton and,
therefore, on the posture of the carcass prior to full rigor. When hung in
the conventional manner from the Achilles tendon, the fibre direction in
the psoas muscles is almost vertical. The muscle fibres are highly stretched,
which, in part, accounts for the tenderness. Adjacent to the psoas is the
longissimus muscle, but its fibres lie almost horizontally, making it very
susceptible to shortening. Pelvic suspension (hanging from the Aitch bone
or 'Tenderstretch'; Hostetler et al., 1972) for beef and the standing posture
for lamb, applied soon after stunning, stretch the muscles in the hind-
quarter and back. When maintained in this position through rigor, the
muscles are more tender than those from conventional hanging. However,
the technique has been rarely used commercially, probably because of the
unconventional handling and shapes of the hindquarter joints. It is
gaining renewed interest for tenderisation of pig meat since it avoids the
potential problems with electrical stimulation of pig carcasses (M011er and
Vestergaard, 1987; Dransfield et al., 1991).
11.5.3 Hot-deboning
Hot-deboning has been practised for many years in developing countries
and has attracted research in developed countries wishing to improve their
slaughtering industry by increasing economic efficiency, adding value to
meat and reducing the energy requirement for chilling. Hot-deboning of
individual muscles will allow them to shorten to their relaxed length and,
after cooking, they will be tougher than cold deboned muscles. When hot-
deboned muscles are stored at about 15°C to minimise shortening, the
muscles are, on average, toughened by about 10%. This toughening is not
likely to be commercially significant, particularly as less shortening occurs
in cuts of meat that cool slowly and are restricted from shortening. Short-
ening and toughening will occur if hot-deboned meat is chilled immedi-
ately but tenderness can be maintained if it is held for about 8 h prior to
chilling. Electrical stimulation can also be used prior to hot-de boning to
allow more rapid chilling.
The growth of fast food operations and the ease with which poultry
meat may be converted into a value-added food have produced a move
TENDERNESS 301
towards portion-cut or boneless cuts. Hot-deboning soon after slaughter

toughens broiler meat but the effect is reduced as the interval between
stunning and deboning is increased, so that, after 4 h, there is no effect on
tenderness (Lyon et aI., 1985). This time can be shortened by increasing
rigor development using higher temperatures or electrical stimulation,
provided that excessive rigor shortening is avoided. In normal commercial
operations, at least 4-6 h should elapse before portioning.
11.5.4 Pre-rigor cooking

Pre-rigor muscle shortening can be extreme if poultry meat is heated soon
after stunning and can lead to tough meat (Abugroun et aI., 1985).
However, tenderness can be maintained if the rate and the time of heating
are controlled. With high rates of heating, deboned muscles will go into
rigor very rapidly and shorten by up to 40%; however, glycolysis is vir-
tually prevented by the rapid denaturation so that the ultimate pH
remains high. The toughening, which would be expected from the short-
ening, is nullified by the tenderness due to the high pH and tender meat is
obtained. With slow cooking, shortening still occurs but the pH can fall to
the normal level before it is stopped by denaturation above 50°C. The
resulting shortened meat of normal pH is very tough. The effect of rate of
cooking diminishes as the time of cooking after stunning is increased and
it has no effect post-rigor (Dransfield and Rhodes, 1975).
11.5.5 Pre-rigor cooling

Temperature legislation has been introduced in many countries to control
bacterial growth and, coupled with the economic requirement to maximise
throughput and minimise weight loss, rapid chilling is being increasingly
applied to carcasses. The EC requires that meat should be cooled immedi-
ately and reach a maximum internal temperature of 7°C for intracommu-
nity trade. The relatively low rate of glycolysis and the small size of the
carcass makes lamb especially susceptible to cold-shortening under con-
ventional commercial chilling practices. In beef sides, the heat capacity
and thickness of the sides make it more difficult to reduce the centre tem-
perature to 7°C within 24 h, nevertheless this can be achieved by using
sub-zero temperatures for a short period followed by equalisation. In such
carcasses, a surface layer of the carcass, about 5 cm deep, can be tough-
ened (Dransfield and Jones, 1978). If the meat is frozen pre-rigor, the
amount of shortening will depend on the rate of thawing. When thawed
quickly, 50% shortening was obtained in neck muscles but this could be
reduced to 20% when held at -3°C for 24 h and then thawed (Behnke and
Fennema, 1973).
Variation in time of the start of chilling, the position of carcass within
the chiller and fat cover will affect the rate of cooling. Fatter carcasses
will cool more slowly than leaner carcasses but selection for leaner car-
casses should be compensated for by regard to reduced chilling times.
Cold-shortening can be avoided by delayed chilling. A delay of about 6 h
for beef and lamb, 3 h for pig meat and about 20 min for chicken car-
casses under average chilling conditions will generally avoid toughening.
11.5.6 Electrical stimulation

When meat is conventionally or rapidly chilled, electrical stimulation will
improve its tenderness by preventing or reducing cold-shortening tough-
ness (Carse, 1973). The mechanism of tenderisation has been the topic of
research by several investigators. The powerful contractions produced
during stimulation may induce fibre rupture to produce weak, stretched or
fractured zones in the muscle, which may be accompanied by super-
stretching, tearing and the loss of the Z-lines (Savell et al., 1978;
Sorinmade et al., 1982). The disruption, however, appears not to occur
directly as a result of the contraction but by earlier and enhanced proteo-
lysis (George et al., 1980; Sorinmade et al., 1982; Fabiansson and Libelius,
1985; Dransfield et al., 1992a). Rapid reduction in the pH to 5.4 in 4 h
and holding at elevated temperatures (above about 30°C) can induce
toughening in beef (Marsh et aI., 1987), probably because the muscles are
exposed to conditions that induce rigor-shortening. Clearly the beneficial
effects of prevention of cold-toughening by electrical stimulation must be
weighed against the possibility of heat (rigor)-shortening.
The use of electrical stimulation in poultry has produced conflicting
effects on tenderness. Stimulation before scalding produced tenderisation
of turkey meat (Maki and Froning, 1987) but toughening of chicken meat
(de Fremery and Pool, 1960). Both toughening and tenderisation can be
obtained in poultry by using electrical stimulation, depending on the rate
of rigor development and the rate of cooling (Wakefield et al., 1989).
Cold-shortening can be induced when chilling is done, while the pH is
above 6.4, but can be reduced by electrical stimulation. Rigor-shortening
can be induced if the pH of the muscle is less than 6.0 and the tempera-
ture is high during scalding. Such toughening may be enhanced by elec-
trical stimulation. Under a given set of slaughtering and chilling
conditions, the amount of tenderisation or toughening, therefore, depends
on the variation in rigor development among carcasses. This suggests that
optimal use of electrical stimulation can be obtained by using it only in
those carcasses in which the glycolysis develops slowly. This necessitates
selection of carcasses prior to stimulation.
Electrical stimulation can also be used in conjunction with hot deboning
of poultry meat. Applying 94 V stimulation to broilers after bleeding and
holding the carcasses at chill temperatures for 2.5 h will improve tender-
TENDERNESS 303
ness, reduce the variability in tenderness between birds and allow

deboning to take place earlier than for non-stimulated birds.
Electrical stimulation can produce slight gains in tenderness when
combined with pelvic suspension to counteract the toughening effect of
blast-chilling of pig meat. However the precise effect will depend upon the
rate of rigor development and the rate of cooling but, similar to poultry
meat, could have no tenderising or a toughening effect with slow cooling
(Dransfield et al., 1991).
11.6 Ultimate pH effects
When glycogen levels in the live animal are low, little lactate is produced
by glycolysis and the ultimate pH is above 5.5. In the extreme conditions
the pH may be above 6.2, in which case the meat appears dark, firm and
dry (DFD). The incidence of dark-cutting beef (DFD) beef is about 1-5%
for steers and heifers, 6-10% for cows and 11-15% for young bulls
(Tarrant, 1981). With such a low incidence, the effect of pH on tenderness
has usually been studied experimentally following pre-slaughter injection
of epinephrine and/or iodoacetate, which increases the ultimate pH of the
meat. Tenderness is greater at high pH in beef, venison, rabbit and
mutton.
The relationship between pH and tenderness of beef (Bouton et al.,
1973) and pig meat (Dransfield et aI., 1985) is usually found to be quad-
ratic, with maximum toughness at a pH between 5.7 and 6.0. Tenderness
clearly follows the water losses during cooking but in raw meat the
strength is largely unaffected by pH (Dransfield, 1981). The relationship
between pH and tenderness appears to differ slightly between species and
muscles. In mutton, minimum tenderness was found at pH 5.6 for biceps
femoris, 5.9 for semitendinosus and 6.1 for longissimus muscles. In beef
(e.g. topside roast, sirloin roast and grilled rump) minimum tenderness
occurred in the pH range of 5.8-6.1. Cooking mutton at 65°C produced
minimum tenderness at pH 5.9 but a linear relationship existed when
cooked at 90°C, suggesting involvement of connective tissue in the devel-
opment of toughness during cooking.
11.7 Effects of post-rigor storage
At a constant temperature above freezing, toughness of meat decreases

approximately exponentially with post-rigor storage time. Thus, most of
the tenderisation occurs early and the amount of tenderisation decreases
with time. Tenderisation is slowest in beef, which requires about 3 weeks
storage at chill temperatures to achieve maximum tenderness.
Table 11.2 Optimal pH for lysosomal

and non-lysosmal proteinases
Proteinase pH activity range
Calpains 6.0-8.5
Cathepsins Band L 3.0-6.5
Cathepsin D 2.5-4.5
11. 7.1 Mechanism of tenderisation

Tenderisation is usually believed to start at or near rigor but proof of this
is lacking because measurement of the mechanical properties during rigor
is confounded with changes in stiffness and pH due to rigor itself. One
approach has been to determine the changes post-rigor and to use a model
to predict the pre-rigor changes in muscle. Using this technique, weaken-
ing due to ageing in raw meat could not be detected prior to the time of
maximum stiffness (Dransfield et al., 1986). In cooked meat, Dransfield et
al. (1992a) used back-extrapolation of tenderisation modelled at different
temperatures and found maximum tenderness to occur at about pH 6.1,
which is in broad agreement with the finding in raw meat. So far, model-
ling has only been applied to beef pectoralis muscle held at 1.2 times its
resting length. It remains to be seen if these findings are universal.
During ageing of beef, the tensile strength along the direction of the
muscle fibres decreases from about 20 N.cm- 2 to 2 N.cm- 2 , which is said
to be the strength at which the I bands fail in aged meat (Davey and
Dickson, 1970). However, breakdown of much weaker (0.06 N.cm- 2)
structures may be involved in tenderisation according to Dransfield et al.
(1986).
Photomicrographs of conditioned muscle suggest that fracture of the
myofibrils occurs near the Z-discs located between adjacent sarcomeres
but that this type of fracture usually occurs after initial tenderisation
(Davey and Dickson, 1970). It is not known how the fractures lead to
tenderisation. Regular cracking at 17 ~m periodicity has also been
observed in meat, similar to that which occurs during extension of man-
made fibrous composites (Dransfield et al., 1986). This analogy suggests
that weakening could occur around the muscle fibre, directing attention to
the sarcolemma and associated structures. Changes in the mucopoly-
saccharides and ground substance have been suggested since the late
I 940s. Indeed, the recent finding of Eggen and Buer (1991) that a reduc-
tion in the size of the high density glyconjugate occurs after 14 days
ageing could be the cause of such weakening is worthy of further study.
There is general agreement that tenderisation during the storage of meat
occurs by enzyme action; thus the enzymes and their substrates most
TENDERNESS 305
Table 11.3 Times taken for tenderisation of pector-

alis muscle at 1°C
Time (days)
50% tenderisation 80% tenderisation
Beef 4.3 10.0

Veal 4.1 9.5
Rabbit 4.1 9.5
Lamb 3.3 7.7
Pork 1.8 4.2
Chicken 0.1 0.3
likely to be involved have been reviewed by Ashgar and Bhatti (1987).

Two proteolytic systems have been implicated. Calcium-activated neutral
proteinases (cal pains I and II) degrade myofibrillar and cytoskeletal
proteins (Dayton et al., 1981) and mimic post-mortem histological
changes observed in myofibrils. Lysosomal acidic proteinases (cathepsins
B, D and L) also hydrolyse myofibrils and isolated proteins. Rapid ten-
derisation is caused by the addition of calcium ions (Koohmaraie et al.,
1988a,b; Alarcon-Rojo and Dransfield, 1989) or calpains (Penny et al.,
1974). Conversely, myofibrillar degradation is inhibited by EDTA (Davey
and Gilbert, 1969; Koohmaraie, 1988a) and tenderisation is prevented by
the addition of Zn ions (Koohmaraie, 1990). All of these phenomena
suggest the primary involvement of calpains. In vitro, however, their
optimum activity near neutral pH has suggested that they are unlikely to
be involved at the later stages of conditioning when the pH is less than
6.0, although considerable tenderisation takes place at the limit value of
pH (Table 11.2). Activity of cathepsins may be more important at these
later times at the limit pH (Calkins and Seideman, 1988). Therefore, a
cooperative mechanism involving both groups of enzymes has been sug-
gested (Ouali and Valin, 1981). Proteinase inhibitors also play an impor-
tant role because the levels of calpastatin (an inhibitor of calpains) and
cystatin (an inhibitor of cathepsins) relate to the tenderness of aged meats
(Shakelford et aI., 1991).
Another interesting enzymatic feature is that the level of cathepsins
remains constant throughout storage but the level of calpains decreases.
The level of calpain I shows a lag until the pH reaches 6.2 and then an
exponential decay similar to that occurring in tenderisation (Dransfield
et aI., 1992a,b). Electrical stimulation advances the decrease in calpain I
in beef, rabbit and chicken in a way similar to the enhancement of ten-
derisation. These results suggest that the tenderisation process may be
related, in some way, to the loss in activity of cal pain I and that tender-
isation will cease when the enzyme is exhausted. However, these
mechanisms are likely to remain circumstantial until ways are discovered

to determine the activities, rather than the levels, of the enzymes in
meat.
11.7.2 Pre-slaughter factors

The rate of tenderisation differs widely between the species (Table 11.3),
the extremes being beef, which takes about 10 days at l°e to achieve 80%
of the total tenderisation, and chicken in which the same tenderisation
occurs in less than 1 day. The level of tenderness of these meats may vary
according to other factors. For example, veal has more heat-labile
collagen and, therefore, is initially more tender than beef and remains
more tender throughout ageing. To achieve a desired level of tenderness,
therefore, veal may be aged for a shorter time than beef, with acceptable
tenderness being obtained in 5 days at l°e. There is no adequate explana-
tion at the structural level or in terms of the enzyme activities to account
for the differences among species. The influence of breed on tenderisation
is unknown but many studies have shown that the rate of conditioning is
not dependent on the level of finish or marbling in steers, bulls and heifers
nor on the weight of steers.
Differences in the rate of tenderisation among muscles are less clear.
Although differences have been reported for beef muscles, the influence
of other factors, such as shortening, connective tissue and temperature,
has also varied. Few data are available for other species. There is no
evidence that variation in the ultimate pH of meat affects the rate of
tenderisation.
11.7.3 Influence of muscle shortening

New Zealand workers were the first to demonstrate the influence of
muscle shortening on the extent of post-rigor tenderisation during storage
(Figure 11.2). Using beef neck muscle, they showed that the extent of ten-
derisation decreased with increased muscle shortening. When a muscle
shortened to 40% of its rest length, no tenderisation occurred. Therefore,
tenderisation does not occur following severe cold-shortening and tough
meat results no matter how long the meat is stored. In practice a wide
range of degrees of shortening are likely in carcass meats and some ten-
derisation will take place on storage but to a lesser degree than in non-
shortened muscles. Variations in sarcomere length between muscles on the
carcass could account for some of the observed differences in the extent of
tenderisation observed in different muscles.
Electrical stimulation will hasten rigor so tenderisation will start earlier
and occur faster at the prevailing higher temperature. Soon after rigor,
electrically stimulated meat will be more tender but the amount of
TENDERNESS 307
improvement will decrease with storage time so that ultimate tenderness

will be the same as in non-stimulated meat. When chilled similarly relative
to the development of rigor, electrically stimulated meat was the same in
tenderness as non-stimulated meat (Dransfield et aI., 1992a). The reduc-
tion in storage time that can be achieved depends on the rapidity in the
reduction of pH and the temperature, particularly during the early stages
of tenderisation. Thus, in meat from carcasses given high- or low-voltage
electrical stimulation and slow cooling, adequate ageing in beef can be
obtained in about 5 days, thus reducing the requirement and cost of
storage.
11.7.4 Temperature
Temperature has a large effect on the rate of tenderisation. From 0-
40°C, the rate of tenderisation increases nearly 2.5-fold for every lOoC
rise in temperature. Above 60°C the rate reduces rapidly owing to
enzyme denaturation. Therefore, tenderisation is stopped by the cooking
process. Initial cooling of the carcass is particularly important when the
temperature of the muscle is reduced from 37°C to about 4°C. During
the first 24 h after slaughter, holding at a high temperature post-rigor
can produce as much as 86% of maximum tenderisation, while at chill
temperatures as little as 8% of tenderisation occurs (Dransfield et at.,
1992a). With normal commercial chilling of beef carcasses, most tender-
isation occurs between 1 and 4 days after slaughter, with 80% of the
tenderisation occurring in 10 days at laC. The same degree of tenderisa-
tion will occur in 4 days at lOoC and in only 1.5 days at 20°e.
The tenderisation process is halted by freezing. Maintaining the meat
frozen prevents tenderisation but after thawing tenderisation will recom-
mence. Practically, beef should be conditioned prior to freezing since a
long time is required after thawing; in contrast, poultry meat can be
aged after thawing in less than 1 day. Such conditions can be achieved
conveniently by thawing poultry in a domestic refrigerator overnight.
The rate of freezing affects the subsequent rate of tenderisation after
thawing. Fast freezing (freezing in about 1 h) more than doubles the
rate of tenderisation after thawing, while very fast freezing (in less than
1 min) increases the rate 3-fold. Freezing is known to cause structural
damage during the formation and growth of ice crystals. Storage at
-70°C also causes loss of the inhibitory activity of calpastatin. The result-
ing release of enzymes and increase in proteolysis could account for the
increased rate of tenderisation after thawing. However, it is unlikely that
the rates of freezing obtained in commerce are high enough to have any
significant effect on the rate of tenderisation after thawing. Even repeated
freeze-thaw cycles at commercial rates of freezing and thawing are
unlikely to affect the subsequent rate of tenderisation.
11.8 Artificial tenderisation
11.8.1 Proteolytic enzymes

The Mexican Indians have long practised wrapping meat in paw-paw
leaves to tenderise it. The active component, papain, is a cysteinyl-protei-
nase and has the unusual properties of a very high temperature optimum
and considerable thermal stability. It will degrade myofibrillar proteins
over a range of temperatures but collagen must be denatured above SO°C
before it is solubilised by papain, with the maximum amount of break-
down occurring at about 6S°C. The amount of tenderisation depends on
the concentration of papain, the muscle and the temperature, with
maximum tenderisation occurring at 70°C. Less tenderisation is obtained
at SO°C due to incomplete denaturation of the proteins and at 80°C as a
result of greater enzyme denaturation (Rhodes and Dransfield, 1973). The
papain is not completely destroyed until about 90°C.
Proteolytic enzymes (e.g. ficin, bromelin or papain) were first used as
dips but, as expected, penetration into the meat was poor and limited
their use to thin slices or to the surface of larger cuts of meat. Penetration
can be improved by rehydrating meat in an enzyme solution or by
pumping it into holes or by needle injection. Injection of a marinade con-
taining papain (O.S g papain.kg- 1) into chilled meat improved the tender-
ness of drumsticks, breast and thigh meat from yearling turkeys and 16-
month baking hens (Cunningham and Tiede, 1981). In the ProTen
process, developed by Swift and Co., a concentrated solution of papain is
injected into the jugular vein of cattle about 10 min prior to slaughter.
This permits distribution of the enzyme by the vascular system but results
in high concentrations of enzyme in the vascular glands, such as the liver
and kidney, so that their commercial value may be reduced because of the
risk of over-tenderisation. The amount of enzyme administered is calcu-
lated to give about 2-S p.p.m. in the muscle, which is sufficient to tender-
ise the average of the different skeletal muscles. Tender muscles (fillet) are
at risk of over-tenderising, while tough muscles (shin) may receive too
little tenderisation. The active enzyme cannot be injected directly into the
live animal because of a severe shock reaction. Thus it is injected in an
inactive, stabilised form which is accomplished by oxidation to the dis-
ulphide form. In the reducing environment of the carcass the oxidised
enzyme is reactivated. Used in this way, papain can overcome the cold-
shortening toughness caused by rapid chilling in lamb carcasses (Rhodes
and Dransfield, 1973). The major drawback with papain is its activity
during the cooking process, which removes control of tenderisation from
the industry.
Rapid tenderisation, while avoiding over-tenderisation, can be obtained
by enhancing the activity of endogenous enzymes. Injecting calcium
TENDERNESS 309
chloride solutions into lamb muscles soon after rigor enhances the activity
of the calpains causing tenderisation (Koohmaraie, 1988b) and opening-up
prospects for its commercialisation.
11.8.2 Marinading
Marinading meat in acid solutions of wine or vinegar is a traditional
culinary technique used to enhance flavour and tenderness, but conflicting
results on its effectiveness in tenderisation have been reported. Recent
studies using discs of meat stored in acid marinades, ranging from pH
3.0-5.0, resulted in tenderisation over the pH range of 4.1-4.6 (Rao and
Gault, 1991). Although tenderisation was seen mainly in the tensile rather
than the adhesive characteristics, the contributions of coagulation and
shrinkage of myofibrillar proteins, connective tissue swelling and possible
gelatinisation have not been clarified.
11.8.3 Pressure treatment

By subjecting muscle to high pressure (1 hat 1500 atmospheres), the myo-
fibrillar structure is disrupted, and at 45-60 a C, the myofibrillar proteins
denature and substantial tenderisation occurs, even in cold-shortened meat
(MacFarlane, 1985). The mechanism was thought to be caused by
membrane damage, release of calcium ions and activation of proteolysis
by the calpains. Slightly milder treatment (1 min at lOOO atmospheres)
applied to pre-rigor meat causes a rise in pH of about 0.6 units at 15-
30 a C. Although this treatment induced some muscle shortening, the meat
was tender, probably because of enhanced proteolysis following the
release of lysosomal enzymes into the sarcoplasm. A commercial develop-
ment, applying 4 atmospheres for 3 weeks at 2a C is claimed to cause ten-
derisation (Aemig, 1990). Such storage, however, in the absence of severe
shortening should produce tender meat even without any pressure treat-
ment.
11.10 Control of tenderness
The rate of rigor development and the temperature profile during the early
stages after slaughter largely determine the tenderness of meat. Since the
temperature can be controlled, regulation of rigor should provide the best
method for process control and optimization of tenderness. Since it is
inconceivable that rigor development could be controlled by selective
breeding and control of animal transportation and slaughter, several
groups of researchers are investigating ways of predicting or monitoring
rigor development.
Measurement of the body temperature of pigs prior to slaughter using

infrared detection can differentiate successfully between rapid glycolysis
(PSE) and slow glycolysis (DFD) from normal meat (Gariepy et aI.,
1987). Its implementation in a commercial lairage warrants further inves-
tigation. The dielectric loss factor, determined at 1 h after slaughter,
increases 10-fold in PSE meat (Kleibel et ai., 1983) but would appear to
have little advantage over direct measurement of pH. A direct measure-
ment of the mechanical properties can be obtained from the pressure
required to inflate a rubber bulb inserted deep into the ham by extension
of a leg muscle using' a portable rigorometer (Swatland, 1986), or by
manually vibrating the carcass with the foreleg and watching the
response. An automatic method of measuring mechanical properties,
using a standardized impact on the carcass, relates muscle stiffness and
pH in pig, poultry and rabbit carcasses and is undergoing further
abattoir development (Dransfield, 1991).
11.10 Summary and research needs
Variations in animal production methods generally have relatively little

effect on tenderness, while the major effects are caused by muscle short-
ening and ageing. Research into tenderness should, therefore, continue
to concentrate on these areas to optimise the storage process and to
achieve better control of tenderness. Better prediction of tenderness is
needed to optimise on-line processing and reduce expensive diagnostic
testing. The rate of rigor development is clearly of paramount impor-
tance and methods should be sought for its prediction or monitoring.
Such results should be combined with the temperature profiles for
cooling and scalding and be used to determine the best operating condi-
tions.
Over the past decade, major advances have been made in understanding
the ageing of meat but much work is needed to identify the structural
components responsible for muscle fibre weakening and the related ten-
derisation. Knowledge of regulation of the levels and activities of calpas-
tatin and cal pains in vivo would aid in optimisation and control of
tenderisation. An explanation for the variation in enzyme and inhibitor
levels between tissues and within the same tissue depending on the age,
sex or nutritional status would be invaluable in unravelling the incon-
sistencies of meat tenderness (Ashgar and Bhatti, 1987). In addition, most
of the work on tenderness is done on cooked meat. Therefore there is also
a need for a better mechanical model of meat texture, incorporating the
structural components and the changes that occur on cooking and their
relationship to sensory evaluation.
TENDERNESS 311
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Wood, J.D., Dransfield, E. and Rhodes, D.N. (1979) The influence of breed on the carcass
and eating quality of pork. J. Sci. Food Agric. 30, 493.
12 Meat texture measurement
B. CHRYSTALL
12.1 Introduction
Many consumers rate tenderness as the most important factor determining

the quality of meat. The author of this chapter supports this view but
only so far as quality is perceived after the product has been cooked and
is being consumed. Tenderness is of little concern if the product is not
attractive enough to entice someone to buy it and prepare it for consump-
tion. The attractiveness can be considered in terms of both appearance
and smell. There are very few people, at least, in affluent nations, who will
be prepared to cook meat that smells putrid or has the appearance of
being spoiled.
It is important at the outset to make sure that the terms being used are
defined so that there is no confusion. The two terms that are often used
interchangeably are texture and tenderness. Texture is a sensory property
of food embodying all the mouth feel characteristics, i.e. kinesthetics
(Kramer, 1973). Tenderness is one attribute of texture, being the resis-
tance to shear or the hardness of the meat (Eadie et ai., 1990). Although it
too is a sensory characteristic, mechanical means are commonly used to
provide a measure of tenderness. The texture of meat may also be influ-
enced by the degree of marbling and there have been numerous studies
attempting, without much success, to relate the US quality grades to
eating characteristics. These studies will not be considered in this review.
Texture and tenderness of meat are recognized not only in consumer
expectations but also in the price that people are prepared to or are
expected to pay. For example, most Westerners accept that beef tenderloin
steak (fillet steak) is the most tender meat available and will pay a
premium for the product. It is generally considered to be tender no matter
from which animal it is derived. Other muscles or groups of muscles are
relegated to lower values and often to different cooking procedures. For
example, forequarter cuts of beef are commonly used for stews and not
for grilling. Not only do the muscles from different parts of an animal
vary in their connective tissue content and characteristics but the same
muscle from animals of different ages may vary greatly. In general, older
animals will have more highly crosslinked connective tissues that are less
soluble than those from younger animals (Shimokomaki et ai., 1972;
Horgan et ai., 1991). The crosslinks in older animals are more numerous
TEXTURE 317
and more resistant to heat, resulting in a residue that persists even after
hydrolysis. The changes are also likely to vary between muscles. In beef,
the loin and rump muscles are less affected by the age of the animal than
are leg muscles (Shorthose and Harris, 1990). Before the early 1960s,
when cold-shortening was discovered and the relationship between short-
ening and toughness defined, the connective tissue components were
regarded as the main cause of toughness. One of the early methods of
physically measuring tenderness was developed to measure connective
tissue in meat (Lehmann 1907).
In some areas, consumers rely on their local butcher to advise them on
the tenderness of product they purchase. In a recent study, Broekhuijsen
and van Willigen (1990) compared the performance of skilled butchers
and a consumer panel in terms of assessment of tenderness. Although the
butchers were less imprecise than the consumers, the relationship of the
butchers' assessments to the ultimate sensory characteristics was poor.
12.2 Why measure tenderness and when?
The philosophical arguments on why and when to assess tenderness could

fill a chapter on their own. There are a variety of reasons for tenderness
measurement that influence the choice of method:
• to compare the tenderness of products available at retail,
• to determine the effectiveness of processing treatments,
• to determine the differences between muscles or between groups of
animals, and
• to assess the processing requirements for a given product.
The timing of any assessment must be considered carefully. Although eva-
luation could take place at almost any time, the subsequent changes, such
as aging of the meat or mechanical treatments, must be considered as they
can considerably alter the interpretation of results. Evaluation of a
product at the retail level is made after completion of all processing influ-
ences except those that still occur during holding and cooking. If the
assessment is made after cooking, then only the changes that can occur
during a variable holding period and perhaps reheating are missed. The
cooking procedure used can have an influence on the results and for any
comparative trial must be standardized.
Assessment of product to compare the effects of different processing
treatments will generally be carried out either soon after the procedures or
at a time that mimics the normal handling of the product. Delays in
assessment may mean that the changes of interest are lost or masked by
subsequent events. Assessments to determine the differences between
muscles or between different groups of animals must ensure that proces-
sing differences do not confound the results. Many differences previously

attributed to production treatments have been confounded by processing
treatments. Processing treatments can have marked influences on the
ultimate level of tenderness, but, unless appropriate assessments are used,
the results can be confusing and can present incomplete answers.
Obviously the ultimate assessment of quality in terms of tenderness is
the sensory assessment by consumers of the cooked product. This fact
should be recognized when a physical method is used. Physical methods
may measure specific mechanical characteristics of raw or cooked product
with tremendous precision but the results may not reflect the actual per-
ceptions of the consumers. Consumers will generally not be specific in
their comments on tenderness or acceptability. They might say 'Yes, that
was a beautiful piece of steak', or they might say 'No, I didn't like that
piece of steak; it was dry'. Mechanically the latter steak could have given
the same results as the former sample that was juicy and scored highly.
Cooking methodology and degree of 'doneness' will influence the
ultimate perception of tenderness. The ultimate test, therefore, is that
applied to meat prepared in the manner normally used for the particular
cut by the population eating it. It is pointless to evaluate a product
cooked to the well-done stage if it is almost always eaten rare. Care must
be taken in extrapolating results obtained from one evaluation method to
another. Texture and tenderness measurement can be applied to almost
any product. In the case of meat it might refer to a roast, a steak, a
ground product (mince) or a processed product. The assessment methods
used can vary considerably but the broad approach will be the same. In
this chapter, although the emphasis will be on whole-tissue meats, some of
the assessment methods can be applied to processed products and will be
mentioned.
12.3 Subjective assessments
The simplest subjective test is to use consumers (or panelists) to test the
product and ask them to score the meat in terms of their impression of
tenderness. The problem is that different people may score the same
sample quite differently. Consumers will determine their scores in relation
to their normal consumption patterns. A person who normally eats beef
tenderloin steak will score a rib steak as tough, whereas a person who
normally eats tough meat might consider the rib steak to be very tender.
In attempts to remove some of this bias, some approaches have used chew
count, and introduced descriptions of other sensations related to the
physical characteristics of the product and how easily the product is
broken down. Harries et al. (1972) used a multivariate analysis procedure
based on eight sensory variables in an attempt to reduce the number of
TEXTURE 319
assessments used; they showed that a juiciness and texture factor could
replace the series of measures normally employed. Much subsequent
research has continued to use a multiplicity of assessments, for example,
Jeremiah et al. (1990) used five surface properties, five first-bite character-
istics, 14 masticatory properties and six after-feel properties to describe
texture profiles in pork loins. The sensory assessment of tenderness will be
covered more fully in chapter 13.
Meat tenderness and texture are the most relevant to the ultimate
consumer, indeed any assessment method purporting to examine tender-
ness of a product destined for the market must, therefore, have some rela-
tionship to that assessment. However, this does not preclude the use of
other measurements, which may have only a remote relationship with the
ultimate consumers' views but may provide important information for
product process control or development of a new process.
Unless the product is very homogeneous, such as a meat loaf or a finely
comminuted sausage, it is unrealistic to expect a uniformity of textural
responses. Most consumers expect a textural experience when they are
eating whole-tissue meat. If they want something totally uniform, they will
eat emulsion-type products. Measurements within a single muscle often
show a variability of nearly 20%. When a consumer is asked to assess this
type of product for tenderness, is it the tender portions or the tough
portions that have most impact on the sensory assessment? It is unlikely
to be an average. Obviously this could have a profound influence on the
relationship between objective and subjective assessments.
In this chapter, the focus is on the objective measures that have been
and are being used to assess meat texture. It is important to consider
methods that are used on raw meat and cooked meat, which are also used
to assess properties of processed products.
12.4 Objective assessments
There have been many reviews of texture assessment methodology. There-

fore there is no claim that the list in this chapter is exhaustive, although
many of the methods are mentioned. Some of the major reviews on meat
texture measurement are Pearson (1963), Szczesniak (1963, 1973), Szczes-
niak & Torgeson (1965), Finney (1969), Voisey (197Ia) and Tsuji (1984).
The list of mechanical devices that have been used to assess the
mechanical properties of meat is long (Table 12.1), although incomplete.
There are obviously many variations in the ways that a product is tested.
In this chapter, individual instruments will not be reviewed; instead the
focus will be on the different approaches and relationships.
Mechanical and chemical assessments are used either to measure the
tenderness of meat or to predict what the ultimate tenderness will be after
processing, distribution, sale and preparation. It seems apparent that

because there is little control over when a product is consumed, the
assessments must either provide a value from which the ultimate tender-
ness range can be deduced or at least provide comparative tenderness
values of the meat at the time of testing. Changes beyond that time might
still result in further change. The methods that are used will depend on
the information that is required. Measurement of some parameter on the
carcass soon after slaughter, then using that measurement to predict the
ultimate tenderness, is the dream of many processors and has been the
vision of many researchers. There have been some very well-publicized
systems. The Armour penetrometer is one of the most well known. This
device, consisting of a multi-needled probe connected to a load cell
(Hansen, 1972), was used to segregate 'tough' and 'tender' sides in the
chiller.
There is still considerable interest in being able to determine the tender-
ness of product soon after slaughter. Attempts are being made to measure
characteristics that are highly correlated with tenderness. Mineral content,
iron/zinc ratios, connective-tissue shrink tension, and 3 h pH values have,
among other factors, been measured as possible indicators of tenderness in
the final product.
12.4.1 Shear and biting systems

When people eat meat they tend to bite through pieces with their front
teeth then grind them with their molars (Boyar and Kilcast, 1986). The
biting action is used as the basis of many devices designed to provide a
measure that will closely relate to human assessment. It must be recog-
nized that deformation rates used in mechanical systems often do not cor-
respond to the variable rates used in human masticatory motions (Bourne
1977; Peleg and Normand, 1982). These differences may influence the rela-
tionships between subjective and objective assessment results.
12.4.1.1 Warner-Bratzler shear. The most common biting or shearing

type system is the Warner-Bratzler shear. Although this has been claimed
to measure shearing forces, there is a considerable tensile component,
especially in less well-cooked samples. There are variations in the basic
instruments. The traditional Warner-Bratzler shear, for example, uses a
triangular hole in a shear plate that is drawn between two bars. Cylind-
rical cores of meat, often cut with a motorized corer (Kastner and Hen-
rickson, 1970), are used and are compressed as they are drawn into the
apex of the triangle. A more recent modification (Harris and Shorthose,
1988) uses a square hole and square cross-sectioned cores. This approach
ensures that the shear-blade sample contact area is constant. Although the
original device used a spring balance and a needle to record the peak
TEXTURE 321
force, many of the more recent reports of the device's use show that mea-
surements are often made with the shearing forces recorded on a load cell
of a universal testing device. The time-deformation curves can, therefore,
be extensively analyzed to gain more information than is provided by the
peak force alone (Voisey, 1971b; Bourne, 1976; King and Jones, 1983;
M0ller, 1980-1981; Suess and Honikel, 1990; Purchas and Aungsupakorn,
1993).
The Warner-Bratzler device is relatively cheap and, provided that the
methodology is standardized, results should be reasonably comparable
between laboratories. Voisey and Larmond (1974) and Purslow (1987)
have listed factors that have been reported to affect the results of Warner-
Bratzler tests and have reinforced the calls for standardization of test
methodology and equipment. The multiplicity of sample configurations
and cooking methods, as well as variations in analysis, mean that, often,
results are not readily comparable.
12.4.1.2 Kramer shear. The Kramer shear (Kramer et al., 1951) differs in
that it is a multi-bladed device originally used to measure the texture of
particulate items, such as peas. The sample to be sheared is often of
variable configuration so that a given weight of product is placed into the
holder so that the alignment is random. The device has been used for
slices of meat or a given weight of mince, etc. The result is an average of
the forces required to cut through the sample of variable geometry.
Analysis of factors affecting the Kramer shear performance and the results
from the device by Timbers et al. (1985) have confirmed the dependence
of the results on the product and on the number and thickness of the
blades. Presumably the thicker blades include more of a compressive com-
ponent, whereas the thinner blades behave more like knives.
12.4.1.3 Nip tenderometer. One of the more interesting devices is the

handheld Nip tenderometer device of Purchas (1973), which it was hoped
could be used on raw meat. The correlation coefficient between results on
raw meat and sensory assessments on the cooked product was poor. This
is perhaps not surprising in that it attempted to measure a shear on the
raw meat. The cooking process, by gelling the proteins, changes the
physical characteristics in a manner that is non-linear.
12.4.2 Compression methods

There are many devices, the MIRINZ (Meat Industry Research Institute
of New Zealand, Inc.) mechanical device (Macfarlane and Marer, 1966),
MIRINZ pneumatic device (Frazerhurst and Macfarlane, 1979), the
Volodkevitch instrument (V olodkevitch, 1938), Winkler device (Winkler,
1939) and a variety of universal testing instrument-based devices (e.g.
Rhodes et al., 1972) that use a wedge-shaped tooth to compress a

standard-sized sample. These instruments have been used to provide
'shear force', work to shear and other measures, which correlate by differ-
ent degrees with the consumer evaluation of tenderness. There is a con-
siderable tensile component to the forces measured as the sample is
compressed below a wedge (Davey and Gilbert, 1967, 1977). Pur slow
(1987) claims rightly that these devices suffer from many of the same
faults as the shear devices, in that they provide measures that are the
result of complex patterns of stresses and strains set up in the meat during
the tests. The results, although of reduced value in interpreting changes in
the mechanical properties of meat, are still useful assessments of the ten-
derness of meat. Recently Vincent et al. (1991) analyzed the wedge-
fracture test as a means of obtaining fracture parameters of brittle and
semi brittle foods. Some meat samples might fall into this category.
Compressive methods are extremely variable and can be expected to
suffer from many of the same drawbacks as shear measurements. Lepetit
et al. (1986) used sinusoidal compression testing of a meat sample with a
SATA device (Sale et al., 1984) to assess the rheological properties of raw
and cooked meat. There are indications that some of the limitations of
shear type devices may be overcome by the complex analysis of the results
from this instrument. The device has also been applied to analysis of raw
meat (Lepetit, 1989) to assess the influence of strain directions in relation
to the connective tissue network. The application of the device, however,
appears to be restricted to France.
Compressive methods are common in processed products. For example
the punch-and-die system has been used for beefburgers (Jones et al.,
1985) and a gel extrusion system similar to that of Bourne and Moyer
(1968) and Bourne (1982) was used by Camou and Sebranek (1991) to
examine gelation of proteins from PSE pigs. The results of multiple com-
pressions are the basis of texture profiling used by Singh et al. (1985) to
assess frankfurters made with different formulations and cooked in differ-
ent ways. A novel Instron attachment has been used by Prusa et al. (1982)
to evaluate the textural characteristics of poultry meat frankfurters.
Although the device contains simulated molars, it relies on compression
rather than the grinding action of the human teeth.
12.4.3 Tensile assessments

Devices measuring tensile characteristics are considered from a purely
mechanical analysis viewpoint as 'purer' assessments, which are likely to
provide more readily interpretable information for understanding textural
changes. This does not mean that they are better assessments of the
ultimate textural acceptance by the consumer. As shown in Table 12.1,
tensile strength and/or fiber extensibility have been used in many studies
TEXTURE 323
Table 12.1 List of some approaches to tenderness/texture measurement in meat.
Methods References
Physical methods
Shear devices
Shear jaw Shockey et al. (1944)
Lee Kramer shear press Szczesniak and Torgeson (1965)
Warner-Bratzler shear Warner (1928)
Rotating knife Bjorksten et al. (1967)
Biting devices
Lehmann Lehmann (1907)
Volodkevitch Volodkevitch (1938)
MIRINZ - mechanical Macfarlane and Marer (1966)
MIRINZ - pneumatic Frazerhurst and Macfarlane (1979)
MIT denture tenderometer Proctor et al. (1956)
KT Biting device Kelly et al. (1960)
General Foods Texturometer Friedman et al. (1963)
Instron RV1 Shorthose et al. (1988)
Nip tenderometer Purchas (1973)
Compressive methods
Swift's tenderness tester Palmer (1962)
Orifice device Howe and Bull (1927)
Sinusoidal compression system Sale et al. (1984)
Tensile methods
Wang fibre extensibility Wang et al. (1956)
Smith tensiometer Bramblett et at. (1959)
Land W Yield meter Locker and Wild (1982)
Penetration devices
Christel texturemeter Miyada and Tappel (1956)
Armour penetrometer Hansen (1972)
Slice tenderness evaluation Kulwich et al. (1963)
Instron compression Bouton and Harris (1972)
Grinding methods
Mincer Miyada and Tappel (1956)
Fragmentation methods
MFI Davey and Gilbert (1969a)
Structural measures
Fiber diameter Hiner et at. (1953)
Fiber size Herring et al. (1965)
Shortening Locker (1960)
Chemical methods
Trace elements Vavak et at. (1976)
Collagen characteristics Seideman (1986)
30000 Dalton component MacBride and Parrish (1977)
and, as with other approaches, show considerable variation. Currie and

Wolfe (1980) measured the extensibility of dumbbell-shaped muscle strips
parallel to and at right angles to the fiber axis with an Instron and special
clamps. Locker and Wild (1982) used a crude device to assess extensibility
parallel to the muscle fiber axis of large samples during aging changes.
The method was more sensitive to many of the aging changes than was
the MIRINZ tenderometer. Other work (Locker and Carse, 1976; Locker
and Wild, 1984) showed that the responses were not uniform between
muscles, reinforcing the need for systems of measurement to be custo-
mized to the task in hand. Davey and Gilbert (1977) illustrate some of the
specific tensile responses of beef sternomandibularis muscles from bulls and
steers.
The approaches to measurement of tensile characteristics range from
the crude large samples of Locker and Wild (1982) to the assessments on
small samples of perimysial connective tissues isolated from cooked
samples (Lewis and Purslow, 1989).
Gripping of samples can be critical with tensile measurements. Locker
and Wild (1982) used nylon tubile and a piece of pressure rubber tubing
applied with an expanding tool. These were then clamped in PVC jaws.
No attempt was made to produce dumbbell shapes. Pneumatic jaws have
been used by, among others, Lewis and Purslow (1990). Tensile assess-
ments transverse to fiber direction are claimed to give a measure of fiber
adhesion that is considered to be a valid measure of connective tissue
(Shorthose and Harris, 1990). Tests parallel to fiber direction give a
measure of tensile strength (Currie and Wolfe, 1980). A new approach to
assessing tensile strengths in raw or cooked meat is being explored by
Phillips (1992) using a system of rotating pins that are inserted into the
meat. The device is being evaluated against sensory panel assessments.
12.4.4 Penetration methods

Traditional butchers often gain their assessment of tenderness by pushing
a finger into raw meat to judge firmness and thereby tenderness. This
approach has been converted to penetrometer methods. Tressler and
Murray (1932) used a needle-type penetrometer but correlations with ten-
derness scores were poor. The Slice Tenderness Evaluator (Kulwich et aI.,
1963) was a combination penetrometer-shear device, which gave encour-
aging results.
Penetrometer methods are common among the instruments to measure
some textural characteristics on the processing line. The Armour tender-
ometer (Hansen, 1972) is a multi-needled penetrometer that can be
pushed into the ribbed carcass. Although results from the Armour pene-
trometer were correlated with muscle firmness (Parrish et al., 1973), the
correlation with taste-panel assessments of tenderness was low. The
TEXTURE 325
method appears to be able to differentiate between ages of animals but

was unable to differentiate between products that were cold-shortened to
increase toughness or aged to increase tenderness. Unpublished work
(Winger and Chrystall, personal communication) showed that similar
Armour penetrometer results were obtained for the two sides of a carcass,
even if the sides were subjected to chilling and aging treatments that
resulted in tenderness differences that were obvious to an untrained
consumer panel.
A recently patented penetrometer (Johnston, 1986) is a single-needled
device to be inserted transversely into the loin to give a measure of ten-
derness. No results from this device are available but since it does not
employ new principles, it is unlikely that it will be much more successful
than other penetrometer methods.
12.4.5 Grinding methods

Since eating involves grinding motions with variable muscle fiber orienta-
tions and variable amounts of connective tissue and fat, consideration has
been given to assessing the work required to mince a standard-sized
sample of meat (Miyada and Tappel, 1956). Although some use was made
of the approach in the early 1960s, no recent applications have been
found. The grinder method would be very dependent on the condition of
the auger and blades and the amount and rate of supply of the material
being fed into the grinder. These variables may be part of the reason for
the lack of use of the method.
12.4.6 Fragmentation methods

One sensory characteristic often scored is the ease of fiber fragmentation.
Physical measurement of this same characteristic has been used as a
measure of tenderness. Davey and Gilbert (l969a) used it as a measure of
the changes during aging; since then it has been used extensively by many
different groups with many different variations of the method. The proce-
dure has been applied to both raw meat (Calkins et at., 1980) and cooked
meat (Davis et aI., 1980). The results depend on the physical operations
and the equipment used. Although fragmentation results obtained by a
given technician or within a given laboratory may be reproducible, it is
doubtful whether there would be much relationship between results from
different laboratories on comparable samples.
12.5 Structural assessments
Since meat is a fibrous material, it is not unexpected that description of

the fibres has been used to assess tenderness. Muscle fiber diameter and
its degree of shortening and breakdown have all been examined as

possible predictors or indicators of tenderness. Development of rapid
methods of sarcomere length measurement (Voyle, 1971) has been driven
by the desire to have a quick assessment that could be used to predict
tenderness.
12.6 Chemical measures
The measurement of chemical changes that could provide a means of ten-

derness assessment is desirable. Changes in myofibrillar or connective
tissue components can sometimes be measured, yet may not be reflected in
changes in the sensory properties. The connective tissue description in
terms of solubility, crosslinking and general extractability has been a
common measure (Seideman, 1986). The aging changes in the myofibrillar
component are shown by assessing changes in electrophoretic patterns.
Changes in the 30000 Dalton component (MacBride and Parrish, 1977)
are considered to be an index of the aging changes. Most of these
chemical measures are slower and require more skill and equipment than
most of the physical assessments and, therefore, are unlikely to be used as
tools in quality assurance schemes. Although the work of Marsh et al.
(1981) led to the proposal that the 3 h pH could be used as a predictor of
the tenderness in beef, there has been no common acceptance because of
research showing that tenderness differences cannot be attributed to the
measured value of pH.
12.7 Other methods
There are many approaches that could perhaps be explored and lead to
new devices or approaches to provide measures of tenderness and texture.
Provided that the results are highly correlated with the ultimate consumer
satisfaction for tenderness/texture, the measure itself does not necessarily
have to be a physical measure. As a meat scientist, one is sometimes con-
strained too closely by one's own knowledge and experience. It is often
the uninformed questioning mind that provides insight that will lead to a
new and innovative approach.
Ultrasonics have been suggested as a means of determining texture
(Bradbury, 1991). Fluorescent probes can be used to measure connective
tissue (Swatland, 1991) and mineral ratios in heart muscle (Vavak et al.,
1976) have been suggested as an indicator of the tenderness of other
muscles in the animal. Although tests may show ultimately that none of
these are of value in assessment of tenderness/texture, they should not be
dismissed as useless without a valid trial.
TEXTURE 327
Texture measurements may be applied to whole tissue products, e.g.

steaks, roasts, etc., or may be used on manufactured products of all types.
The texture of sausages may be measured by any of the multitude of
shearing and compression devices (Voisey et at., 1975). An axial compres-
sion test has been used by Thomsen and Zeuthen (1988) to assess yield
strength and elasticity characteristics of model sausages prepared with and
without mechanically deboned meat.
The texture of sausages or uncooked batters may be measured by gel-
strength tests. A compression approach, back-extrusion method, was
used by Camou and Sebranek (1991) to assess the gelation characteristics
of PSE muscle proteins. The thermal scanning rigidity monitor (TSRM)
of Barbut and Mittal (1991) provides a measure of the gelation of
muscle batters as the gel is heated and the results can be correlated with
the ultimate texture of the sausage. Comparison of tensile adhesive
strength (T AS) and punch-and-die tests suggests that T AS measurements
provide a valuable objective measure of sensory attributes (Savage et at.,
1990). Both tensile and shear adhesive strengths have been used as tests
of the meat-myosin junction in processed meats (Donnelly and Purslow,
1987). The torsional rigidity of samples assessed in a Brookfield Visc-
ometer can be used to assess gels (Wu et at., 1985) and could also be
used as a tenderness assessment. The torsional rigidity of whole tissue
could be assessed but the need to 'turn' or cut the dumbbell shape
results in operational difficulties. A simple lathe-like grinding device has
been developed (Hamann, 1983; Wu et at., 1985) for use with protein
gels and sausage-type products but has not been used for whole-tissue
meat.
The wide variety of methods used to test product characteristics
probably stems from the availability of equipment rather than a percep-
tion that certain tests relate more closely to the actual sensory perfor-
mance. As with whole-tissue testing, the variety of methods makes
comparison difficult. There are some attempts to develop test methods,
which can be used universally to assess the bind characteristics of meats
and the texture of the sausages into which they are made.
12.8 Samples
As any mechanical engineer knows, sample dimensions affect physical

properties. This is definitely the case with meat. Although in many cases
the exact configuration of muscle fibers and so on is not specified, their
alignment can influence the results. Meat is a fiber-reinforced matrix,
therefore the direction of shear or compression will influence the results.
In general, shear results are obtained perpendicular to the fiber direction
but different information can be obtained by shearing parallel to the fiber
direction. Similarly, tensile assessments are often made parallel to the

fibers but, in some cases, assessments transverse to the fibers will provide
useful information (Shorthose and Harris, 1990).
The size of samples will also influence results. Although results are
often given per unit cross-section, there are some indications that by
manipulating sample size the chance of measuring certain of the con-
nective tissue components is reduced (Lewis and Purslow, 1989). The
manipulation of sample size can, therefore, increase the information that
can be obtained.
Depending on the type of test being carried out, sample size can influ-
ence the results in a manner that is not dependent on the different amount
of material being sheared and so on. This is especially so in compressive
tests where restriction of the lateral spread can change the actual results.
Davey and Gilbert (l969b) studied the sample size in terms of the
MIRINZ device and developed a method to adjust for the influence of
sample size. Each type of device will respond differently to sample size,
and it is important that the effects of sample size and shape for the parti-
cular test system be understood.
The configuration of the sample in terms of fiber direction is also
important. Not only will it influence the magnitude of measurements but
the relative magnitude of assessments will change as the animal ages. The
differences will depend on whether the test method attempts to shear
through all the components, or whether the material can be left intact at
completion of the test. Compressive devices often leave an unbroken
residue at completion of the test, whereas shearing devices usually cut
through everything. In some tests, e.g. the Kramer shear test, the direction
of shear or compression is random, greatly simplifying the measurement
and sample preparation (Bentley et al., 1988).
Most meat is eaten cooked, therefore the majority of assessments are
made on the cooked product. The properties being measured can be
modified greatly by the cooking process. Ideally, testing should be after
the same cooking treatment that the meat will receive prior to serving to
the consumer. However, the variety of cooking treatments used by con-
sumers means that, in general, only a few cooking methods are employed
for tenderness testing. Standard methods have been recommended for
cooking and sensory evaluation of meat (AM SA, 1978) and standard
procedures should be used for mechanical testing. This does not mean
that no further research and experimentation should take place in the
cookery and assessment area, rather that, where comparisons are likely,
the tests should be able to be related to each other. It would be ideal if
the tests could be performed on the raw meat and then the cooked results
predicted from those values. Unfortunately systems measuring the tender-
ness of raw meat have not been shown to provide good indications of the
tenderness of cooked product.
TEXTURE 329
12.9 Standard protocols
All of the measurement systems mentioned are of little value if the sample
preparation and processing introduce artifacts. Although not all groups
using tenderness assessments will want to use the same procedure, it is
important that, where interlaboratory comparison is desirable or where
the tenderness assessment is the basis of a quality assurance program, then
the methods used must be strictly defined and monitored.
In New Zealand, the New Zealand Meat Producers Board operates a
quality assurance scheme for lamb tenderness. As part of that program,
processing plants are expected either to adhere strictly to well-specified
processing regimes designed to eliminate processing toughness, or to use
any scheme provided that their product passes the tenderness tests. The
majority of the testing is carried out using a standard portion of the loin
so that the variation that occurs because of position is minimized. The
actual test procedure may not be totally acceptable for all tenderness
testing situations but is reliable, reproducible and simple. It has been
based on water-bath cookery procedures and then testing on a MIRINZ
tenderometer. There is no doubt that it is an imperfect system, but that it
is a positive move which provides a numerical value for total quality man-
agement of the operation. Improved performance in total management of
the processes can be measured.
12.10 Relationships between assessment methods
12.10.1 Between objective assessments

Although different research groups may use different methods to assess
tenderness, it is possible, in some cases, to convert results obtained with
one type of instrument to those obtained with another. Conversion must
take into account the sample size, sample configuration, cooking condi-
tions and any other differences in the samples. Graafhuis et al. (1991)
provided some information comparing the MIRINZ tenderometer results
with those obtained on the same samples (rectangular cross-section
samples) assessed with a Warner-Bratzler type shear assembly in an
Instron instrument. Under these conditions, it has been possible to calcu-
late a regression equation to convert data from one muscle on a parti-
cular instrument to give the result expected from the other machine.
When the muscle also differs and the cooking treatment (e.g. end-point
temperatures, rates of heating, wet or dry) varies, the relationships do
not always hold. This is particularly noticeable with muscles, such as the
sternomandibularis, that are high in connective tissues. Changes in sample
shape will also have a major influence on the values obtained by any test
methodology. Often tests using the Warner-Bratzler apparatus use a

cylindrical core, whereas some other devices use rectangular samples.
Both samples could be of similar cross-section but give differ,ent values in
the tests. The relative directions of fiber axis and force will also affect the
results.
12.10.2 Between objective and subjective assessments

In many cases, attempts to relate subjective and objective assessments
have relied on linear-correlation analysis with the assumption that the
consumers and mechanical devices will give a linear response to increas-
ing levels of toughness (Bratzler and Smith, 1963; Sharrah et al., 1965;
Szczesniak and Torgeson, 1965). Although the correlation coefficients
between the subjective assessments and the objective assessments can be
quoted (Szczesniak and Torgeson, 1965), they are of little value unless
accompanied by descriptions of the methodologies and the range of
values covered. Testing over a narrow range of tenderness values is likely
to give a high correlation, whereas over a broader range there could be
significant deviations from linearity. The notion that a large number of
samples gives credence to the accuracy of the correlation coefficient must
be tempered with the view that it only affects the accuracy over the
range covered in the data. Provided that the limits of accuracy are
known, it is appropriate to use linear correlation coefficients generated
over that range.
Since the human has limits to the force that can be applied during
chewing and has not developed cranial ridges characteristic of a gorilla
chewing small branches, it is not surprising that the relationship
between sensory assessments and mechanical assessments is non-linear.
There is an upper hardness limit of material that can be bitten (Mioche
et aI., 1991). Attempts to bite something that requires a greater force
to penetrate or cleave will cause breakage of teeth, or the material will
not be bitten intentionally but may perhaps be sucked or swallowed
whole. The curvilinear nature of the relationship between mechanical
and sensory assessments also means that there is probably a threshold
of acceptability for products. Recently, several groups seem to have
recognized this (Gilbert et al., 1990; Devine et al., 1990; Shackelford et
al., 1991). The work of Gilbert et al. (1990) considers assessments of
acceptability of lamb, in terms of tenderness, conducted in the UK.
The conclusion of the study was that meat with a shear force greater
than 10 kgF determined on cooked meat using a MIRINZ tender-
ometer, is considered to be at least moderately tough and unacceptable.
Product with a mean force less than 10 kgF is considered acceptable
and below 8 kgF desirable. Devine et al. (1990) found a similar thresh-
old for New Zealand consumers and the 10 kgF figure has become the
TEXTURE 331
critical limit for lamb and table cuts of beef exported from New
Zealand.
Shackelford et al. (1991) concluded that for the 1.27 cm diameter cores
a 4.6 kg value was the margin between tough and tender. Extrapolating
from the data of Gilbert et al. (1990) and Devine et al. (1990), it would be
reasonable to suggest that this figure is approximately equivalent to the
10 kgF limit for the 1.0 x 1.0 cm cross-section samples used in New
Zealand. It is, therefore, possible to get an approximate relationship
between data from different studies.
12.11 Conclusions
This review does not claim to be exhaustive. There are some comparative
trials being conducted, at the time of writing, of devices and methods
claimed to be able to assess the mechanical characteristics of raw meat
and then predict cooked meat tenderness. The results of these trials may
indicate that different procedures can stratify meat into potential tender-
ness groups even though subsequent chilling regimes and aging conditions
may modify the tenderness of those groups. The benefit will be to be able
to sort carcasses early and then modify the processing of those groups to
produce a uniform product. Attempts to define accurately the mechanical
characteristics of raw and cooked meats so that the measured properties
can be compared between laboratories are commendable but new
methods need to be based on sound principles and well researched.
Developments that eliminate the need for exact sample preparations
would speed up the processing of samples and could eliminate some
sources of error and bias.
Assessment methods applied to protein gels and batters early in the
processing operation and to finished processed products must relate ulti-
mately to the perceived qualities and textures of the final products. If the
assessments provide no guide as to the final sensory characteristics, the
value of the results to the consumer is zero.
It is important to remember that the ultimate judge of tenderness is the
final consumer. Regardless of what mechanical device or objective assess-
ment is made during processing, if consumers say they do not like a
product because it is tough, or too tender, they are the final arbiters. It is
rare for a whole-tissue product to be judged too tender but with processed
products the texture can be too soft. Measurement purely for scientific
assessment of the changes, rather than as a measurement related to
sensory assessment, is appropriate in some instances but in general there is
justification for common methods that .predict accurately the end-point
texture as judged by the consumers.
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13 Product acceptability evaluation
1. LOVE
13.1 Introduction
The sensory attributes of foods are widely considered to be an important

determinant, perhaps the most important determinant, of acceptability.
Other factors that determine whether a food will be acceptable to an indi-
vidual include the physiological state of the individual and the social,
institutional and motivational context of eating, serving, cooking or
purchase (Booth, 1981). Amerine et al. (1965) defined acceptance as either:
(i) an experience or feature of an experience characterized by a positive
(e.g. approach in a pleasant) attitude; or (ii) actual utilization (e.g.
purchase, eating). They cautioned that the two definitions are often highly
correlated but not necessarily the same. The factors that influence food
choice and intake are shown in Figure 13.1.
The terms 'food acceptance' and 'acceptability' are used in association
with several types of research methodologies. For example, attitudinal
studies may be conducted using questionnaires with the names of foods as
stimuli in order to determine the overall acceptability of foods or the
relative importance of different factors or sensory attributes to overall
acceptability. Other studies involve sensory evaluation of actual foods.
The foods presented are chosen or manipulated by the experimenter to
obtain the reaction of consumers to variations of particular interest. Often
these samples vary only slightly in sensory attributes and the expectations
of consumers about the samples would be similar until they had actually
tasted the samples.
When reaction to the sensory attributes is desired, samples are usually
presented with blind codes so the sample identity or brand is unknown. A
study by Gacula et al. (1986) demonstrates the bias that brand identifica-
tion can have on sensory acceptance tests. Subjects discriminated sig-
nificantly among frankfurters labeled with brand names even though they
were tasting the same product falsely labeled. When the different samples
(unbranded) wer.! evaluated, no significant differences in overall liking
were found.
The interplay between sensory response and other factors that affect
food purchase and choice is an area that deserves closer attention. The
reactions of consumers to certain kinds of marketing information believed
to be important to purchase decisions may differ depending on whether
Food Person Economic
-
and social
Perception of
I
Physical/chemical sensory attributes Price
properties e.g. Availability
Nutrient content Brand
\
appearance, aroma, Social/cultural
taste, texture
Psychological factors~
e.g.
personality,
experience, mood
Physiological effects beliefs - - Attitudes
1
e.g. e.g. to:
satiety, hunger, sensory properties,
thirst, appetite health/nutrition,
~ ~ price/value
Food choice
+
Food intake
Figure 13.1 Some factors affecting food choice and intake (Shepherd, 1988). Source: Nutri-
tion Society, 1988. Reprinted with the permission of Cambridge University Press.
the food items of interest are actually presented. For example, Cheng et
al. (1990) demonstrated that consumers reacted differently to some types
of marketing information (e.g. brand, price, nutritional information, etc.)
in the presence and absence of restructured meat products. The avail-
ability of restructured beef steaks seemed to cause consumers to ignore
nutritional information, which was ranked as more important than brand
or price when no actual product was presented.
Several qualitative techniques can be used to explore information about
consumer understanding of and desire for products (Cohen, 1990). Focus
groups or panels and in-depth interviews can be used to generate ideas
about new or improved products or explore consumer reactions to product
concepts. Considerable probing may be required to determine true belief
and practice (Chambers and Smith, 1991). An example of the incongruity
of expressed preferences in the absence of a product and preferences when
actual product was evaluated is provided by Fishken (1988). Qualitative
research (a focus group) indicated that consumers wanted more cheese and
more meat on a brand of pizza. However, when actual samples were pre-
sented, with formulations systematically varied, preferences were somewhat
different, more cheese but not more meat being desired.
Presenting blind-coded samples in sensory acceptance tests does not
address many of the variables that affect real-life food choices. In spite of
this limitation, sensory acceptance testing is vital to ensure that products
in the marketplace consistently have sensory characteristics that are accep-
table to consumers. In this chapter sensory methods used to determine the
ACCEPTABILITY EVALUATION 339
acceptability of foods and current knowledge about the way that sensory
characteristics determine acceptability of meat and meat products are
dealt with. It will begin with a general overview of tests that can be used
to determine acceptance and preference and of the differences between
trained and consumer panels. Current knowledge about the relationship
between sensory attributes and consumer acceptability of meat will be dis-
cussed and specific tests and rating scales will be compared for their utility
in consumer tests.
13.2 Affective testing: testing for acceptance and preference
Acceptability is the quality of being satisfactory, agreeable or pleasing.

When someone is asked to judge the acceptability of a food, one is asking
for their personal opinion. Since a judgement is asked for based on
feelings about a product, sensory tests to determine whether products are
acceptable are classified as affective tests (Table 13.1). Participants in
sensory acceptance tests are usually asked to use some kind of rating scale
to indicate how acceptable they find a product to be or to show how
much they like or dislike the product or the specific sensory attributes of
the product. A scale used to measure the degree of liking or disliking is
usually called a hedonic scale, since hedonics is the branch of psychology
dealing with pleasurable or unpleasurable states of consciousness. Much
use in food research has been made of the hedonic scale developed by
Peryam and Pilgrim (1957). The general form of this scale is shown in
Figure 13.2a.
13.2.1 Scaling
Scales with other types of descriptive phrases are also used to measure
affective response to foods. Subjects can, for example, be asked to rate
attributes of a sample on a 'just right' scale (Figure 13.2b), where the
rating is relative to their own ideal about the attribute. Scales can also use
phrases that imply action, for example the intent to purchase (Figure
13.2c). The food action rating (FACT) scale (Schutz, 1965) uses nine cate-
gories labeled with action phrases, for example, 'I would eat (use) this at
every opportunity 1 had' at one extreme and 'I would eat (use) this only if
1 were forced to' at the other extreme. Schutz (1965) found that the corre-
lation between FACT and hedonic scores was 0.97 when subjects respon-
ded to names of foods listed on a questionnaire. He then suggested that
responding to an action statement may require a more realistic attitude
than giving an affective response, thus accounting for the greater sensitiv-
ity of the FACT scale to food differences. Shepherd et al. (1989) found
that maximally preferred concentrations were similar whether measured
Table 13.1 Classification of sensory evaluation methods and panels·
Classification of methods by Appropriate methods Type and numbers of panelists

function
Analytical: Evaluates differences or Screened for interest, ability to discriminate

similarity, quality and/or quantity of differences and reproduce results trained to
sensory characteristics of a product function as a human analytical instrument
Discriminative Paired-comparison Normal sensory acuity
Difference: Measures simply Duo-trio Periodic requalification
whether samples are different Triangle Panel size depends on product variability and
Ranking judgement reproducibility
Rating difference/scalar difference No recommended 'magic number' - a number often
from control used is 10; a recommended minimum number is
generally 5, since any fewer could represent too
much dependence upon one individual's
responses
Sensitivity: Measures ability of Threshold
individuals to detect sensory Dilution
characteristic( s)
Descriptive: Measures qualitative and/ Attribute rating
or quantitative characteristic(s) Category scaling
Ratio scaling (magnitude
estimation)
Descriptive analysis
Flavor profile analysis
Texture profile analysis
Quantitative descriptive analysis
Affective: Evaluates preference and/or Pair-preference Randomly selected

acceptance and/or opinions of Ranking Untrained
product Rating Representative of target population
Hedonic (verbal or facial) Consumers of test product
scale No recommended 'magic number' - minimum is
Food action scale generally 24 panelists, which is sometimes
considered rough product screening; 50-100
panelists usually considered adequate
'Source: Institute of Food Technologists, 1981.

(a)
Like extremely
Like very much
Like moderately
Like slightly
Neither like nor dislike
Dislike slightly
Dislike moderately
Dislike very much
Dislike extremely
(b)
Not nearly Just right Much too salty

salty enough
(c)
r Definitely would buy

Probably would buy
Might buy/might not buy
Probably would not buy
Definitely would not buy
Figure 13.2 Examples of scales to measure acceptance of or intent to purchase products: (a)
nine-point hedonic scale; (b) 'just right' scale; and (c) purchase intent scale.
using relative-to-ideal or hedonic ratings but that variability in responses

was greater for the hedonic ratings. Vie et al. (1991) suggested alternatives
to hedonic and just right scales. They used signal-detection analysis to
give probability values indicating preferences from simple hedonic ranking
and rating procedures.
13.2.2 Direct preference tests

Direct tests of preference are another kind of affective test. People partici-
pating in a paired-preference test are asked to choose one sample over
another based on pleasantness or liking. For three or more samples, a
ranking test can be used to establish preferences. It is important to
remember that indicating a preference for one sample over another does
not necessarily mean that the preferred sample is acceptable or well liked.
Preference tests do not indicate where products fit on the affective con-
tinuum. They are usually used when a researcher knows how well liked
one of the products is, and is 'testing against' that product. Of course,
significant differences in acceptability ratings for products also indicate
preferences.
13.3 Affective vs. analytical sensory testing
Testing to determine the affective response of current or potential users to

a product or products is one of two general uses of sensory evaluation,
the other being analytical testing (Table 13.1). In analytical sensory eva-
luation, the goal is to determine the sensory discriminability of similar
samples or to describe (identify and quantify) the sensory attributes of
food products. Sometimes the same general type of test or scale may be
used in either an affective test or in analytical sensory evaluation. For
example, a category scale can be used either to collect information about
acceptability of a product or a sensory attribute (affective) or about inten-
sity of a sensory attribute (analytical). A paired comparison test may
require a subject to compare two samples to determine which is more
intense in some specified attribute (analytical) or which is preferred
(affective). Presumably because the nine-point hedonic scale is familiar to
many food scientists, nine-point scales for measuring the intensity of
specific attributes are sometimes mistakenly called hedonic scales. Also,
scales that measure degree of liking for specific sensory attributes are
sometimes referred to as 'descriptive analysis scales', even though descrip-
tive analysis by definition refers to methods that require highly trained
panelists to detect and describe the intensity of the perceived sensory attri-
butes of a product.
In affective tests, the goal is to determine the personal feelings or
opinion of the subjects to or about the food, while in difference or
descriptive testing a panel is used as a 'laboratory instrument' to find out
something about the sensory characteristics of the food. This difference
means that the criteria for choosing participants for these two kinds of
tests should differ. When the goal of sensory evaluation is to find out
something about the product, the authors often use subjects who have
been screened and trained or who are experts in identifying defects in
order to optimize the chances of finding differences or consistently char-
acterizing the attributes of the products. This is to make these panelists
as close to 'instruments' as is possible. Although trained judges or
experts are also consumers, their opinions and preferences about food
products are not likely to be representative of people in the general
population. When our goal is to predict consumer response, input is
needed from untrained people outside the industry or research environ-
ment who are representative of people who might buy or use the
product.
13.4 Subjects in acceptance tests: the usefulness of trained panels or

experts to evaluating product acceptability
In spite of the general agreement among sensory scientists that affective

data should be obtained from subjects selected to represent the population
of consumers (Table 13.1), the practice of having small trained or experi-
enced panels make affective judgements (e.g. liking, acceptability, palat-
ability, desirability, etc.) in addition to rating intensity of attributes is
fairly common. This section examines some of the literature on perfor-
mance differences between trained and untrained or consumer panels.
McBride and Finlay (1989) compared perception of taste mixtures by
experienced sensory panelists and subjects who had never participated in
experiments with taste. There was good correspondence between the two
groups especially for judgements of total intensity. The suppression of
sourness by sweetness was, however, less pronounced for the experienced
panel than the novice panel. Chambers et al. (1981) evaluated the perfor-
mance of 'semi-trained' and 'trained/experienced' panelists in evaluating
the flavor and texture of frankfurters and suggested that highly trained
and experienced panels are needed for evaluation of foods with complex
flavors. Sawyer et at. (1988) found that consumer judgements of fish flavor
were in good agreement with trained panel judgements for 'heavy' notes
but that they were in poor agreement for subtle flavors.
Cardello et at. (1982) found that trained texture-profile panelists gen-
erally had a broader perceptual range than untrained panelists, as shown
by the slopes of regression equations relating trained to consumer panel
scores for attributes of fish (Table 13.2). This difference was also noted for
bread texture and seemed to alter the affective response. Liking for bread
decreased much more quickly as a function of increasing modulus of elas-
ticity and density for trained than for untrained panelists (Figure 13.3).
Moskowitz et al. (1979) found agreements between types of panels (e.g.
objective, expert and consumer) on some texture attributes but not on
others. Predictive equations can be developed to take into account percep-
tual differences between trained panels and consumers.
Research to examine the degree of agreement on acceptability between
trained and consumer panels has not given consistent results, but often
shows that preferences differ between trained and untrained panelists
(Shepherd et at., 1988). Wesson et al. (1979) reported that overall pre-
ference scores from a small panel trained for descriptive analysis indicated
that this group was more critical of fish samples than a consumer panel
but that consumers readily discriminated flavor and texture quality
Table 13.2 Regression equations, Pearson product-moment correlation coeffi-

cients and coefficients of determination for the relationship between trained
and consumer panel judgements of texture and appearance a
Regression Correlation Coefficient

equation b coefficient of determination
Flakiness T = 2.42 C - 5.33 0.77'* 0.59

Hardness T = 1.66 C - 3.44 0.75'* 0.57
Chewiness T = 1.58 C - 2.14 0.84" 0.71
Fibrousness T = 1.52 C - 0.06 0.72*' 0.52
Moisture T = 0.79 C - 0.62 0.53 0.28
Oily mouthcoating T = 1.59 C - 0.87 0.73*' 0.57
Darkness T = 1.04 C + 0.76 0.91*' 0.82
aS ource : Cardello et al., 1982.

~ = trained panel ratings; C = consumer panel ratings .
•• p < 0.01
•
I
16 20
Modulus of elasticity
Figure 13.3 Consumers. and trained • panel ratings of liking/disliking as a function of

the modulus of elasticity (Cardello et aI., 1982).
extremes in fish prepared by a procedure yielding a relatively bland

product.
Gacula (1987) reported that laboratory panels predicted consumer
dislikes in frankfurters but that these types of panels were less capable of
predicting consumer likes. Even if panels are untrained, their ability to
predict consumer response should be questioned if they consist of employ-
ees or people who have an atypical degree of product knowledge. Lawless
(1990) suggested that the ability of an untrained internal or employee
panel to predict acceptance by the broader consumer population can be
verified only if some of the products that score poorly with the internal
panel are consumer tested, as well as the highest scoring samples.
Two studies with meat products illustrate further that trained and con-
sumer panels do not always agree on product acceptability. Shackelford et
al. (1990b) trained an eight-member panel to evaluate sensory attributes of
bacon. The panel also judged overall palatability. An untrained consumer
panel tested the samples in their homes. The trained panel scored the
control sample as the most palatable, samples from pigs fed safflower, sun-
flower and animal fats intermediate, while the canola-oil sample was
deemed the least palatable. Consumers rated pigs fed on safflower oil and
control samples highest for eating quality, while a diet of animal fat rated
intermediate and sunflower and canola diets rated the lowest.
Griffin et at. (1985) presented cooked loin steaks from bulls and steers
to an eight-member experienced panel and compared results with those
obtained from a consumer in-home survey. The sex of the beef animal sig-
nificantly affected the experienced panel scores for juiciness, muscle fiber
tenderness and overall tenderness, and did not affect scores for connective
tissue amount, off-flavor or palatability. For consumers, however, loin
steaks from bulls and steers were judged to be different in flavor, tender-
ness and desirability but not in juiciness.
These are examples of studies in which the trained/experienced panel
only partly predicted the acceptability of products to consumers partici-
pating in in-home tests. Tests conducted in different locations (laboratory,
central location or in-home) can have different outcomes because the
choice of location affects several other factors: (i) the products may have
been prepared differently; (ii) the temperature at the time products were
evaluated may have been different; (iii) the quantity of product consumed
may not have been the same; (iv) different score sheets or descriptive
terms may have been used, and so on. Thus tests in different locations
with the same samples may yield different results, even when consumers
are used for all tests. However, it is likely that the trained judges give
responses reflecting their unique background and experience. It is not wise
to assume that a trained panel or an untrained panel internal to the
company or the academic department can predict consumer likes/dislikes,
unless evidence to the contrary has been collected in a carefully designed
study. McDermott (1990) suggested that sensory researchers should follow

the same criteria used by market researchers to select consumer panelists.
Some individuals are 'experts' in identifying defects that detract from
product 'quality'. Is the judgement of these experts any more likely to
predict consumer acceptance than that of trained panels? Sawyer et al.
(1988) concluded that care should be used when generalizing the opinions
of industry experts to consumer perceptions. Data from surveys on the
characteristics of several species of fish (no samples were actually pre-
sented) of industry experts did not correlate with either consumer or
trained panel data except for flavor intensity and color. Moreover, the use
of small groups of industry experts to evaluate quality of products or
identify defects in products may raise other problems or limitations. For
example, when experts arrive at rating by consensus rather than statistical
averaging of scores, dominant individuals may have undue influence on
the outcome. McBride and Hall (1979) criticized defect-oriented dairy
judging methods for failing to predict consumer acceptance. In general,
the judgements of experts about product quality should not be expected to
predict consumer acceptance.
Terms such as 'tenderness' and 'juiciness' are frequently used both with
trained panels and in consumer studies. It seems reasonable to consider
whether meat texture could be described more adequately by trained
panels if the terminology and training protocols used in the texture profile
method or an adaptation thereof were used in place of the traditional ter-
minology. Further studies would be needed to determine the relationship
between the classic texture profile terms and consumer understanding of
meat 'tenderness' and 'juiciness'.
13.5 Relating sensory attributes to product acceptability
13.5.1 Hedonic scores and intensity of sensory attributes

In order to produce food products that are consistently liked by con-
sumers, one needs to know how acceptance or liking relates to specific
sensory characteristics. In meat research, it is sometimes assumed that
there is a direct relationship between intensity of attributes such as tender-
ness, flavor and juiciness and the degree of liking. Trant et al. (1981)
pointed out some fallacies in the use and interpretation of hedonic scores
and a survey of recently published meat research reveals that some of
these practices persist. For example, researchers sometimes use hedonic
scales (or scales for palatability, desirability, degree of appeal, etc.) to
measure panelist response, then discuss outcomes in terms of increases or
decreases in intensity of characteristics. Occasionally some attributes
(usually flavor) are rated using hedonic scales, while others (usually ten-
derness and juiciness) are rated using intensity scales; outcomes often are
treated erroneously as if the two scales were identical. Hedonic scales
differ from other category scales in that responses are not always expected
to increase with increasing magnitude of a physical stimulus.
In addition the methods sections of research papers sometimes report
that panels were 'trained' even when the scales used were hedonic scales.
Presumably panelists might be trained to make intensity judgements even
though scale descriptors relate to degree of liking/disliking, although this
would seem to be unnecessarily confusing to panelists. It is even more dif-
ficult to suggest why investigators sometimes conclude that products are
not different because hedonic ratings for the products did not differ sig-
nificantly. Certainly products can be quite different and liked (or disliked)
equally.
In general, care in making interpretations based on a single type of
sensory test is warranted. Some examples from the literature are illus-
trative. For example, with restructured or reformed products Chesney et
al. (1978) and Popenhagen and Mandigo (1978) suggested that greater
acceptability implies greater strength of particle adhesion. In another
instance, Savage et al. (1990) found that added myosin had no effect on
mean consumer scores for liking of product texture or overall liking for
fabricated meat products, although a trained panel could detect differ-
ences in attributes relating to adhesion. Examination of the consumer data
revealed that some of the 55 people tested consistently liked weakly bound
products, while others preferred strongly bound products. Beilken et al.
(1991) found that meat patties most liked by consumers had properties
akin either to 'meatiness' or 'mushiness'. These studies illustrate that the
preferences of consumers for textural attributes of patties and fabricated
meat products may not correspond to the expectations of researchers.
Caution in making assumptions about the relationships between sensory
attributes and hedonic response to meat products is warranted.
Papadopolus et al. (1991) found that consumers rated control cooked
beef top round and products treated with 3% sodium lactate the same for
desirability of saltiness, even though scores for saltiness intensity were sig-
nificantly different (Table 13.3). The saltiness of the treated sample was
seen as a desirable feature of the product by some people and as undesir-
able by others. Clearly, different levels of intensity can be liked equally
well.
Civille (1991) presents a general approach that can be used to study
how attribute variation affects consumer acceptance and to set specifica-
tions for quality control. In this approach, products that represent a range
of variation for key attributes are evaluated by a descriptive analysis panel
and submitted to a large-scale consumer study for overall acceptance and
acceptance of appearance, flavor or texture and to establish key consumer
terms. From this data, the impact of altered characteristics on accept-
Table 13.3 Mean sensory scores from consumer panelists (n =

110) for cooked beef roasts containing either 0% or 3% sodium
lactate a
Sodium lactate content (%)

0 3
Hedonic scoresb
Overall liking/disliking 5.01 ± 0.12d 5.71 ± 0.12e
Overall flavor 4.84 O.12d 5.64 0.12e
Beef flavor 5.08 0.13 d 5.73 O.13 e
Saltiness 4.89 0.12d 5.08 0.13 d
Sourness 4.86 0.12d 5.18 0.12d
Texture 5.16 0.14d 5.93 O.13 e
Intensity Scores c
Overall flavor intensity 4.56 O.13 d 5.63 0.12e
Beef flavor 4.58 0.13 d 5.43 0.12e
Saltiness 3.83 O.l1d 5.57 0.12e
Sourness 3.60 0.13 d 4.19 0.13 e
aSource: Papadopoulos et aI., 1991.

bSamples evaluated on end-anchored, 9-point hedonic scale (1
= dislike extremely, 9 = like extremely).
cSamples evaluated on 9-point intensity scale (1 = none, 9 =
strong).
d.eMeans within same row with different superscripts, differ
significantly (P < 0.05).
ability can be established. The type of relationship may be expected to

vary for different attributes. For example, product defects such as off-
flavor may exert a strong negative effect on liking responses across a rela-
tively narrow range of intensities. Some flavors are generally pleasant but
can become unpleasant at a sufficiently high level. Other attributes might
vary over a wide range without having much impact on acceptability.
Mathematical modeling of food acceptance based on its relationship to
sensory properties is discussed by Lawless (1990, 1991a).
McBride (1990) questions the necessity of using descriptive analysis to
link product development to consumer preferences and stresses the direct
use of consumers in product research. Booth (1990) and Conner and
Booth (1992) discuss approaches that emphasize the importance of deter-
mination of individual preferences and propose data collection and
analysis at the individual level as a way of linking sensory attributes and
hedonic response.
13.5.2 Fat level as a determinant of acceptability

In general, additional systematic and careful study is needed before the
relationship between specific sensory attributes and acceptability can be
specified for meat and meat products. Considerable meat research has
focused on establishing the relationship of marbling to acceptability, with
emphasis on determining the minimum level of marbling fat consistent
with acceptability (Dikeman, 1987). Savell and Cross (1988) also reviewed
the role of fat in determining the palatability of beef, pork and lamb.
They document the role of fat in influencing sensory characteristics, as
determined by trained panels, but point out the lack of any studies of the
impact on consumer perceptions. The National Consumer Retail Beef
Study (Savell et aI., 1987) was designed to determine whether consumers
could detect differences in steaks that differed in marbling and to examine
preferences of consumers in different regions of the USA. Some of the
results from this study are shown in Figure 13.4, which indicates that con-
sumers in different regions responded differently to marbling. Consumers
in Philadelphia were more critical of the lower grades of beef than con-
sumers in San Francisco, perhaps because the Philadelphia consumers
tended to cook meat to a higher degree of 'doneness'. Other portions of
this study showed that 'taste' was identified by consumers most often as
an important factor in the purchase of beef steaks and roasts, while
'fatness' was a negative influence. In this study, retail cuts from both US
Choice and US Good (Select) carcasses were highly acceptable to con-
7.40
0>
c
~
....CU 7.20
>-
.-:::
:0
CU
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'w
Q)
-0 6.80 l
CU
"-
Q)
> 660
0
6.40 1 I ')
1: E
~
Q)
iii (ij
c T§ Q)
"0 E .Q> .Ql
(1j Q)
0 C/)
U5 U5
"0
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0 :;::
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.0 ~
(1j
>-
E Marbling
.Q>
U5
Figure 13.4 Regression lines, by city, for overall palatability ratings as influenced by
marbling levels (Savell et aI., 1987).
sumers but for different reasons. The cuts from choice animals rated
higher for sensory characteristics, while consumer objections dealt with
fatness. The cuts from US Select carcasses were liked for their leanness,
while consumers' objections dealt with juiciness and tenderness. Mederios
et al. (1987) reported that a household panel perceived that broiled steaks
from concentrate-fed steers were more juicy, tender and flavorful and
more desirable overall than steaks from range-grazed steers. However, in a
test-market study, the same samples were neady equally acceptable
because consumers were concerned about perceived leanness and health-
fulness as well as desirable sensory qualities.
Dikeman (1987) concluded that consumers are segmented when it
comes to the degree of fatness desired in red meat cuts, i.e. no single
fatness level will satisfy all consumers. Consumer tastes are also dynamic
across time. It is likely that, at least for segments of the market concerned
with health and nutrition, the fatness levels preferred in consumer studies
discussed by Dikeman (1987) will no longer be preferred.
The impact of fat reduction on consumer acceptance of ground and
processed meat is also an important topic. Pearson et al. (1987) sug-
gested that acceptable low-fat meat products can be produced by sub-
stituting water for fat; indeed, recent research supports this contention,
at least for some products. Park et al. (1990) reported that frankfurters
with 14% or 16% fat (added high-oleic sunflower oil) and a high
moisture content (about 70%) were considered, by a consumer panel, as
undesirable as control products with 29% fat (animal fat). Other work
with fermented summer sausage from high-oleic pork showed that reduc-
tion of fat from 25% to 15% resulted in sensory characteristics more like
the control (Shackelford et al., 1990a). Additional studies are needed to
determine consumer acceptability and to verify the reports of off-flavor
development during storage of meat products with altered fatty acid
composition.
Reduction in fat alters the sensory properties of ground beef patties.
Egbert et al. (1991) reported that overall acceptability of ground beef
patties peaks at about 20% fat. They reported that 'Au Lean', a low-fat
« 10%) ground beef product formulated with 10% added water, 0.5%
iota carrageenan, 0.4% encapsulated salt and 0.2% hydrolyzed vegetable
protein, had sensory properties somewhat different from either a tradi-
tional ground beef patty or a low-fat product (Table 13.4). Taki (1991)
describes other low-fat ground beef formulations and presents data on the
sensory attributes of the products but does not give data from consumer
studies.
Ahmed et al. (1990) found that a group of 78 untrained subjects
(faculty, staff and students in an Animal Science Department) rated the
visual appearance of raw pork sausage with 35% fat lower on a hedonic
scale than samples with 15% or 25% fat. Scores for eating quality were
Table 13.4 Sensory physical properties of cooked beef paties
Property 20% - fat 8% - fat Au lean Standard error

patties patties of the mean
Juiciness 5.8c 4.6 d 6.7 b 0.12

Tenderness 5.6c 5.2c 6.6b 0.22
Connective tissue 6.8c 6.8 c 7.3 b 0.06
Mealiness 6.5 c 6.1d 6.9 b 0.05
Beef flavor intensity 5.2c 4.6 c 6.4 b 0.16
Shear force (kg/g of sample) 3.5 d 4.4 b 4.0c 0.06
aSource : Egbert et al. ,1991.

b_dJuiciness, tenderness, connective tissue, mealiness and beef flavor intensity were rated on
an 8-point scale where 1 = extremely dry, extremely tough, abundant connective tissue,
abundant mealiness and extremely bland flavor, and 8 = extremely juicy, extremely tender,
no connective tissue, no mealiness, and extremely intense flavor. Means within a row
followed by different super are significantly different (P < 0.05).
not affected significantly by fat level. However, sausage with 13% added
water was liked less than sausage with 3% added water.
13.5.3 Factors that alter the relative importance of appearance, texture

and flavor in consumer acceptance studies
For beef steaks and roasts cooked by dry heat methods, tenderness is
usually considered the most important determinant of consumer accept-
ability. Appearance (i.e. fatness and color) is obviously an important
factor in the decision to purchase. Lynch et al. (1986) showed that for
74% of ground beef consumers color was important to the purchase
decision. Information about vacuum-packaging increased the purchase
intent for purple-red vacuum-packaged ground beef. The previous section
addressed consumer willingness to trade desirable sensory characteristics
for leanness. More research is needed to understand the relative impor-
tance of flavor, texture and appearance when determining preferences for
processed meat products.
Off-flavors may cause consumers to reject products, even when the
appearance and texture are desirable. Wesson et al. (1979) examined the
importance of flavor and texture to overall acceptability for a variety of
fish products and concluded that texture was an extremely influential
determinant of preference when samples had moderate-to-low intensities
of fishy and/or oxidized flavors. Flavor is often said to be a more impor-
tant determinant of fish acceptability than texture (Hamilton and Bennett,
1983; Sawyer et ai., 1988) with off-flavors being most important (Laslett
and Bremner, 1979). Sawyer et al. (1988) showed that texture was of
greater relative importance for those who dislike fish.
Using in-home tests, Rounds et al. (1992) reported that odor may be an
important factor in determining fish acceptability. Appearance may
become important when fish flavors are not pronounced (Wesson et al.,
1979). Rounds et al. (1992) suggested that color was a more important
determinant of acceptability in salmonids than in fish with white flesh.
13.6 Factors affecting the outcome of acceptance tests
13.6.1 Type of test or method

A person conducting a consumer test must choose specific tests or scales
to be used. This section will address the question of which specific tests
have applicability in consumer testing and whether some methods are
superior to others.
The first group of methods to be considered are the sensory difference
tests, in other words, those tests that are used to determine: (i) whether
people can detect any sensory difference at all between samples; and (ii)
whether a specified attribute differs in intensity between samples or
whether one sample is preferred over another. The most straightforward
procedure for a difference test would seem to be simply presenting a
subject with two samples, allowing the subject to evaluate the samples,
then asking whether the samples are the same or different. However, the
tests that are typically used do not rely on the subject's willingness to
judge two or more products as the same or different, rather they force a
choice between two samples based on criteria for comparison described by
the experimenter.
The most frequently discussed of the forced-choice procedures are the
directional paired comparison, duo-trio and triangle tests. All of these
tests are used to determine whether a sensory difference exists between
two products. Judges base their decisions on the characteristics of the
products that they are asked to examine and the instructions given in the
test. The triangle and duo-trio tests allow the investigator to present
samples for comparison without defining the basis on which the decision
is to be made. In the triangle test, the participant knows that two samples
are being presented and that one is presented twice. The participant is
asked to choose the odd (or the most different) sample. In the duo-trio
test, the task is to indicate which of two coded samples matches (or is
most similar to) a reference sample. The triangle or duo-trio tests can be
used with consumers when the specific intent is to determine whether
untrained subjects can detect an overall difference in two samples. Some
researchers use these tests, then ask judges to indicate which sample is
preferred (this practice would generally be less useful in guiding research
or product development than measuring the size of attribute differences
and their importance to consumers). In the paired comparison test, the

task is to choose the sample that has the most or least of some specified
attribute, or that is most liked.
Comparisons generally indicate that the directional pair test is more
powerful or efficient than the duo-trio or triangle tests (Fran90is and
Sauvageot, 1988; Ennis, 1990). Thieme and O'Mahoney (1990) suggest
alternatives to the traditional duo-trio and triangle tests when differences
can not be spelled out in advance.
Several varieties of rating scales are in common use to assess the inten-
sity of sensory attributes and the acceptability of products. Category
scales, line scales and ratio scaling by use of magnitude estimation all
seem to be in common use by food scientists. In category scaling, the
subject is asked to rate the intensity of a particular stimulus or degree of
liking or acceptability by assigning it a category on a limited scale.
Category scales can be constructed with words, numbers or both. Gen-
erally, words are converted to numbers by the experimenter. With
unstructured line scales (sometimes called graphic scales), the subject rates
the sample by placing a vertical mark on a horizontal line. The ends of
the line are labeled to indicate the direction of increasing intensity or
affective response. The mark corresponds to the perceived intensity of the
stimulus or, in the case of a hedonic scale, degree of liking or disliking or
acceptability.
Magnitude estimation methods are the most commonly used methods
of ratio scaling. The application of magnitude estimation to food testing
has been discussed by Moskowitz (1978). The two basic approaches to
magnitude estimation differ in whether a standard is used. In one
approach, the standard is introduced and assigned a number against
which all other samples are assessed as ratios. Alternatively, each assessor
is allowed to assign any number to the first sample, and to assess the rest
of the samples as ratios of this number. Numbers assigned to samples
have no absolute meaning and are normalized as a part of the analysis of
results. There are no restrictions on the numbers that can be used except
that negative numbers and zeros are not allowed. It is essential that asses-
sors understand that they should use numbers as ratios.
Tests have been conducted to compare various combinations of
category scaling, line scaling and ratio scaling techniques for intensity and
hedonic ratings. For examples of these studies, readers are referred to
Pearce et al. (1986) and Lawless (1989). So long as experiments are con-
ducted carefully and conditions are optimized for each type of test, there
seems to be little to indicate the general superiority of anyone method,
either for intensity or hedonic scaling.
In general, much care should be exercised in interpreting results from
anyone type of sensory test since different methods may lead to different
conclusions. For example, Lawless and Schlegel (1984) presented
mixtures of sucrose and citral to 30 subjects in a triangle test and to 10

subjects for intensity scaling for sweetness and lemon odor. Results with
the two types of tasks did not always seem to be consistent. When pairs
of samples with equal citral but low and high sucrose were presented in
the triangle test, the correct choice was made only 41 % of the time.
However, with direct scaling, the same samples were given significantly
different sweetness ratings. Focusing on specific attributes may reveal dif-
ferences not apparent in overall difference tests, while discrimination tests
may reveal interactions not apparent when individual characteristics are
rated.
When attention is directed to specific attributes (and thus away from
others) panelists may miss significant attributes, perhaps ones that might
be noted by consumers and thus affect consumer acceptance. Browning
et al. (1990) found no significant differences in the scores that trained
panels gave to juiciness, tenderness, flavor or the amount of connective
tissue in cooked semimembranosus muscles from lean and typical car-
casses. Nevertheless, a 30-member untrained panel found differences
when samples were presented in triangle tests. The untrained subjects
presumably found differences in some attribute that the panel was not
evaluating. This finding illustrates the need for care in interpretation of
results from tests in which panelists evaluate the intensity of a few attri-
butes presumed to be of key importance in the product. Without a well-
understood mechanism for scaling or at least reporting other attributes,
their existence might be undocumented by a trained panel, even though
easily detected by untrained consumers.
13.6.2 Context effects

Humans do not generate numbers in the same way that other laboratory
instruments do. Indeed, humans constantly recalibrate themselves and
push judgements around on rating scales, relative to recent experience.
Ratings given to samples will depend on the context in which they are
presented (Riskey, 1986). A particular scale value will not always be asso-
ciated with a particular stimulus but will depend on factors such as
stimulus range and stimulus frequency. The range effect is due to subjects
tending to adjust the center of the rating scale in the direction of the
center of the stimulus range. The stimulus frequency and spacing also
affect ratings. McBride (1985) and Riskey (1986) discuss context effects
when using hedonic ratings. These effects mean that the hedonic ratings
for a sample are not absolute but must be considered a function of the
test situation. The extent to which the tendency to make relative intensity
judgements can be overcome by repeated training and use of standards is
not known.
13.6.3 Design and control of experiments

Cross et al. (1986) discusses the selection of cooking methods for sensory
studies, as well as presentation of meat samples to panelists. Gacula et at.
(1986) give examples of studies that have shown how presentation order
can bias response in hedonic tests. For additional information on the
design and control of experiments, readers can consult recent publications
by Lawless (1990) and Meilgaard et al. (1991).
13.7 Summary
The success of new meat products and new technologies in livestock pro-
duction and meat processing ultimately depends on the consistent main-
tenance of desirable sensory characteristics in meat. To ensure that
products are acceptable to consumers, research with representative groups
of current or potential users is needed. Palatability ratings by trained
panels and experts do not necessarily predict consumer response. Addi-
tional research is needed to define adequately the impact of sensory attri-
butes on meat acceptability and to understand the way that preferences
for sensory attributes interact with factors such as nutrient content, con-
venience, price, packaging, etc. An improved theoretical framework for
the understanding of acceptance would be beneficial to sensory scientists,
the meat industry and the consumer.
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14 Microbial growth and its control in meat, poultry
and fish
J.N. SOFOS
14.1 Introduction
Foods of muscle ongm are sensItIve to contamination and support

growth of microorganisms involved in spoilage and foodborne illness. In
fresh, unprocessed products, microorganisms multiply rapidly, especially
at non-refrigeration temperatures, resulting in loss of quality and/or
public health problems. Thus, various methods of processing and pre-
servation are being applied to inactivate or inhibit microbial growth in
order to extend product shelf-life, while maintaining palatability and
safety. In recent years, however, consumers prefer meat products or meat
entrees subjected to minimal processing and preservation treatments,
which at the same time offer convenience, long shelf-life and safety. The
initial high palatability of these products can be compromised by
increased potential for growth of pathogenic bacteria, especially the psy-
chrotrophs of recent concern. Since the vast subject of muscle food
microbiology has been discussed extensively in various books (Brown,
1982; Pearson and Dutson, 1986; Cunningham and Cox, 1987), chapters
and review papers, this chapter will concentrate on certain aspects of
recent research interest and matters of current concern. The topics
include sources, types and removal of microorganisms from muscle foods,
spoilage and foodborne illness, especially by Listeria monocytogenes, and
the control of microbial growth, especially by modified-atmosphere
storage and biopreservation.
14.2 Microbial contamination of muscle foods
14.2.1 Sources of contamination

The types of microorganisms present on muscle food products and their
numbers depend on: (i) the sanitary conditions in the environment from
which the food came: (ii) the properties and microbiological quality of any
added ingredients; (iii) the extent to which the product was processed and
handled; and (iv) the conditions involved in subsequent storage, handling
and distribution.
The origin of microbial contamination in muscle foods has been dis-

cussed extensively in several publications (Nottingham, 1982; Gill, 1983;
Grau, 1986; Niven, 1989). Therefore, only a few highlights of the subject
are presented here. The first microorganisms present in muscle foods may
be introduced through the vascular system during the sticking, bleeding
and scalding operations. At subsequent stages of slaughter, processing and
overall handling, muscle tissues are subject to additional contamination
from various sources associated with the animal and its environment. In
general, microorganisms found on fresh muscle foods originate from air,
water, soil, manure, feed, hides, intestines, organs, lymph nodes, proces-
sing equipment and humans (Ayres, 1955, 1960; Anon., 1980). Thus, the
type and extent of contamination varies with individual animals, herds or
flocks and seasons of the year.
14.2.1.1 Red meats. The cleanliness of the animals prior to slaughter is

dependent on factors such as climate, farm or feedyard location, holding
conditions and method of transportation to the slaughter facility. This
affects the extent and type of contamination during slaughter (Notting.
ham, 1982). Cattle and sheep originating from pastures will carry more
microorganisms of soil origin compared with those from feedlots, which
may contain more bacteria of fecal origin. Higher fecal contamination is
usually associated with more pathogenic bacteria. An evaluation of the
cleanliness of the animals in holding pens prior to slaughter may be bene-
ficial when determining speeds of slaughter, dehiding and evisceration
lines. In cases of higher external animal contamination, it may indicate the
need for more careful trimming and washing of the carcasses prior to
chilling. Incision of lymph nodes and bile ducts during veterinary post-
mortem inspection may increase cross-contamination among carcasses
(Cortesi and Catellani, 1982). Carcass contamination before cutting is
more severe in areas close to skin incisions and where the hands of
workers touch the carcass (Stolle, 1981). During cutting the surface area
exposed to contamination is increased and cross-contamination becomes
greater. In general, the extent of contamination increases with degree of
handling.
Final carcass contamination before chilling depends not only on the
extent of contamination during the slaughtering process but also on
decreasing microbial numbers on carcass surfaces through washing or
through decontaminating spray treatments. Additional contamination can
occur, however, during chilling and carcass fabrication, as indicated
above, through contact with floors, walls, water, air, equipment and per-
sonnel. Final contamination on pig carcasses after slaughter is determined
by the extent of bacterial removal and destruction taking place during
scalding and singeing, as well as by the effects of dehairing, scraping, evis-
ceration and washing (Grau, 1986; Gobat and Jemmi, 1991).
MICROBIAL GROWTH 361
14.2.1.2 Poultry. Similar to red meat animals, healthy birds carry exten-
sive microbial contamination on their feathers, skin and intestinal tract
(Anon., 1980; Cunningham, 1982; Cunningham and Cox, 1987). Wing
flapping during the hanging and bleeding operations generates aerosols,
which distribute contamination on the defeathered carcasses. In addition,
the moist and warm equipment spreads contamination among carcasses
(Grau, 1986). In general, contamination occurs during all processing steps
including stunning, bleeding, scalding, defeathering, washing, evisceration,
and washing and chilling in ice-water or in cold air (Mead, 1982). Cross-
contamination, which is most important during defeathering and eviscera-
tion, becomes a major problem due to the high on line operational speeds.
The scalding water can become a point of major cross-contamination,
unless its temperature is held at 58-60°C, which helps keep its level of
contamination low. Carcass washing decreases contamination, while
immersion chilling can reduce, increase or spread poultry contamination,
depending on the temperature and time of exposure. Chilling in clean cold
air or in water containing chlorine or acids can reduce poultry carcass
contamination (Cunningham, 1982).
14.2.1.3 Seafoods. Similar to land animals, the muscles and organ tissues
of healthy fish should be free of microbial contamination, but micro-
organisms are present on skin, gills and intestines (Liston, 1990). The
extent of bacterial contamination of seafood depends on its quality and
the sanitation prevailing during fishing, processing and storage. Sources of
contamination include water, contact surfaces and humans (Anon., 1980).
Contamination is influenced by the method of fishing, fishing vessel sani-
tation, handling, processing and storage conditions (Ward and Baj, 1988).
Contamination is usually higher on fish from warmer waters, and from
those originating in areas of untreated human waste disposal. Molluscs,
which depend on filter feeding, concentrate bacteria and viruses from
polluted waters and may carry various pathogens. These products should
not be eaten uncooked or undercooked, and should not be allowed to
cross-contaminate other foods (Anon., 1980).
Fish contamination can increase during unloading where it may involve
use of pumps, conveyors and water and result in introducing new micro-
organisms and redistribution of existing ones. With more handling,
bruising of fish muscle tissues increases and this can allow bacteria to
enter and invade the tissues. Contamination is also introduced from nets,
ropes, ship decks, boxes, equipment, utensils, human hands and clothing.
Ice and water for chilling may introduce more contamination but can also
inhibit growth and result in selection for psychrotrophs, which eventually
become the predominating micro flora of refrigerated fish and other muscle
foods (Anon., 1980). Contamination may also increase during open-
market display, also fish cutting, which exposes internal surfaces to
external contamination. Application of good hygiene and sanitary proce-

dures, however, should keep contamination of all muscle foods to a
minimum.
14.2.2 Types of contamination

In addition to the amount of contamination, the types of microorganisms
present in a muscle food depend on sanitation, hygienic practices and
handling during harvesting, processing, storage and distribution. Initial
contamination is generally the result of poor hygiene, while in the finished
product amount and type of contamination is also affected by processing
and storage conditions (Anon., 1980; Kotula et aI., 1987). Processes such
as hot-boning, electrical stimulation and mechanical de boning that can be
involved in the processing of fresh meats are thought not to have sig-
nificant effects on the microbial flora if employed under sanitary condi-
tions (Field, 1976; Froning, 1981; Kotula, 1981; Jay, 1992). The effect of
electric current on microbial survival, however, may need further experi-
mentation (Dickson and Crouse, 1989; Slavic et al., 1991).
14.2.2.1 Red meats. Carcass contamination of beef, pork and lamb after
slaughter and chilling is usually variable and may consist of 10 1_10 5
aerobic mesophiles per cm2 , depending on plant, carcass and site on the
carcass (Nortje and Naude, 1981; Smulders and Woolthuis, 1983). The
rate of chilling affects the proportion of psychrotrophs relative to
mesophiles, which in turn depends on temperature, time, and air velocity
and humidity. Initially, surface contamination with psychrotrophs is less
than 102 and contamination with Enterobacteriaceae less than 10 1_10 2 per
cm 2
Common contaminants of carcass meat are Gram-negative rods and
micrococci, including Pseudomonas spp., Moraxella spp., Acinetobacter
spp., Alcaligenes spp., Flavobacterium spp., Aeromonas spp., Staphylo-
coccus spp., Micrococcus spp., coryneforms, Enterobacteriaceae and fecal
streptococci (Anon. 1980; Nortje et al., 1990). In addition, the lactic acid
producing bacteria, Brochothrix thermosphacta, Shewanella (previo~sly
Alteromonas) putrefaciens, Bacillus and Clostridium spores, yeasts, molds
and enteric viruses may be present in lower numbers (Anon. 1980).
Although contamination is variable, pathogens may include Salmonella
spp., Staphylococcus aureus, Yersinia enterocolitica/pseudotuberculosis,
Campylobacter jejuni/coli, Listeria monocytogenes, enteropathogenic
Escherichia coli, Bacillus cereus, Clostridium perfringens and Clostridium
botulinum. The source of these pathogens is either the intestinal micro-
flora or the environment (Stolle, 1981; Nottingham, 1982). Some of these
pathogens are associated with meat from some species more than others,
such as Y. enterocolitica in pork (Fukushima et al., 1991).
Carcass processing and subsequent meat handling determines the fate of

microorganisms originally present on meat. In general, psychrotrophs
such as Pseudomonas, Moraxella-Acinetobacter, Flavobacterium, Lactoba-
cillus and Brochothrix will predominate in refrigerated meat, with numbers
depending on initial pH and gaseous atmosphere. Degree and variety of
contamination, however, increase with product handling and comminution
(Jay, 1992). Contamination of comminuted meats increases because they
usually consist of extensively handled trimmings, which provide a greater
surface area for increased microbial growth, especially of aerobic spoilage
psychrotrophs. Comminution also increases cross-contamination with
grinders and utensils, especially when used extensively without proper
sanitation, or when heavily contaminated portions are mixed with meat of
better sanitary quality.
Yeasts and molds are not a major concern for fresh muscle foods,
because they develop slowly and predominate only on carcasses after
prolonged storage and aging, which reduces growth of bacteria due to
surface drying. They include Torulopsis, Candida, Rhodotorula, Crypto-
coccus, Trichosporon, Sporotrichum, Cladosporium, Thamnidium, Mucor,
Chaeotostylum, Penicillium, Rhizopus, Aspergillus, Monilia, and Alternaria
(Nottingham, 1982; Kotula et aI., 1987; Dillon and Board, 1991).
14.2.2.2 Poulty meats. Similar to red meat, the microbial contamination

of chilled poultry carcasses reflects that of the live birds, and that added
or modified during slaughter and dressing. Although variable, contamina-
tion includes mesophilic aerobes (102-10 5 .cm- 2, psychrotrophs (10 1-
103.cm- 2, Enterobacteriaceae (103- 104. cm-2), E. coli (101-10 5.cm- 2), S.
aureus (103.cm-2), C. perfringens « 102.cm- 2), Salmonella « 30.g- I ), C.
jejuni/coli and L. monocytogenes (Mead et al., 1982; Campbell et al., 1983;
Grau, 1986; Miller et al., 1990). As is the case for red meat, the extent of
contamination increases with subsequent cutting of poultry carcasses.
14.2.2.3 Seafoods. The initial type of contamination present on fresh

seafood depends on its place of origin. Cold water fish are generally con-
taminated with Gram-negative psychrotrophs, while fish from the tropics
are mostly contaminated with Gram-positive mesophiles. Gram-negative
contaminants include Pseudomonas, Moraxella, Shewanella, Acinetobacter,
Flavobacterium, Aeromonas, Cytophaga and Vibrio spp., while Gram-
positive contaminants are Micrococcus and Bacillus spp. (Hobbs, 1983).
Bacterial numbers present on the skin surface of fish range from 102_
10 7 .cm- 2 and on the gills and the intestine from 103_10 9 .g- l . Frozen
products, however, should have lower microbial loads. Pathogens found
on seafood include Vibrio spp., Klebsiella spp., Staphylococcus spp., Aero-
monas spp., L. monocytogenes and viruses (Liston, 1982; Hobbs, 1983;
Kotula et aI., 1987; Venugopal, 1990).
14.2.2.4 Processed products. Contamination of processed (i.e. cooked,

uncooked, cured, uncured, heated, smoked, fermented, etc.) muscle
products will reflect contamination introduced with raw meat and non-
meat ingredients, additional contamination added during processing and
after processing and microbial modification caused by processing treat-
ments, such as heating, drying and freezing. Non-meat ingredients not
only may introduce additional and diverse microbial contamination but
they can also influence the level of contamination prevailing in the
finished product because they may provide additional nutrients for micro-
bial growth or enhance microbial inhibition as direct antimicrobials or by
altering the properties of the product (e.g. water activity and pH). Overall,
however, the shelf-life of processed muscle foods should be longer than
that of fresh, raw products, especially when stored anaerobically.
14.3 Microbial effects on muscle foods
14.3.1 Spoilage
14.3.1.1 General. Initial contamination, type, compoSItIon and proces-
sing of a muscle food, and storage conditions determine the predominat-
ing spoilage microflora and its effects on product quality. Several
publications have reviewed the subject of microbial spoilage and loss of
quality of muscle foods (Ayres, 1960; Cunningham, 1982; Gill, 1983;
Dainty et al., 1983; Egan, 1984; Kraft, 1986; Egan et al., 1988; Venugopal,
1990). A muscle food is classified subjectively as spoiled when certain
products of enzymatic (natural but mostly microbial) metabolism make it
unacceptable, offensive and unpalatable to the human senses. The types of
bacteria commonly predominating in spoilage of muscle foods include
Pseudomonas spp., Enterobacteriaceae, Brochothrix thermosphacta, lactic
acid bacteria of the genera Lactobacillus, Carnobacterium, Pediococcus,
Streptococcus, Lactococcus and Leuconostoc, as well as Aeromonas spp.
and Shewanella putrefaciens (Egan et al., 1988; Dainty et al., 1989b; Stutz
et al., 1991; Jay, 1992). In some products, spore-forming bacteria such as
Clostridium and Bacillus, as well as microaerophilic yeasts and molds may
be involved in spoilage.
Initial bacterial metabolism is based on use of low-molecular-weight
soluble constituents, such as glucose, glucose-6-phosphate and amino
acids. Subsequently, and in the presence of oxygen, proteins are metabo-
lized into peptides and amino acids, while under vacuum they are
degraded into odoriferous sulfur-containing compounds. Lipase enzymes
hydrolyze triglyceride lipids and phospholipids to form glycerol and free
fatty acids, or nitrogenous bases and phosphorus, respectively. This results
in undesirable flavors and odors from free fatty acids and lipid oxidation
products. Metabolism of natural traces of carbohydrates in fresh meats or

added sugars in processed products results in formation of various end
products including organic acids and alcohols. Thus, .major changes
occurring through microbial enzymatic action in muscle foods include
undesirable odors and flavors, discolorations, softening of the texture, and
formation of slime.
14.3.1.2 Aerobic. Fresh muscle foods are especially perishable under

aerobic conditions. Oxidation of meat pigments on the surface of meat by
bacteria results in the first sign of unacceptable change. Specific defects
caused by aerobic bacteria include green, brown, gray or other discolora-
tions, surface slime and odors, off-flavors and taints. The predominant
microorganisms in refrigerated, aerobically stored fresh meat belong to the
genus Pseudomonas, which cause putrefactive odors and slime when the
number of cells exceeds 107. cm-2 (Ayres, 1960; Gill, 1983; Egan, 1984;
Egan et aI., 1988). These organisms initially metabolize glucose, which is
followed by degradation of amino acids and proteins. Degradation of
these compounds results in formation of ammonia, amines (e.g. cadaver-
ine, putrescine and isobutylamine) and sulfides (e.g. hydrogen sulfide,
methyl sulfide), which result in off-odors and off-flavors caused not only
by proteolytic but also lipolytic problems.
In muscle foods of higher ultimate pH (> 5.8) or at higher storage tem-
peratures, Moraxella and Acinetobacter spp. may become significant in
spoilage. These organisms do not metabolize hexoses but attack amino
acids directly. In addition, facultative anaerobic bacteria of the family
Enterobacteriaceae may be involved in aerobically stored meat. These
organisms initially metabolize glucose-6-phosphate followed by amino
acids. Their growth, however, is slow at cold temperatures (Gill, 1983).
Brochothrix thermosphacta, a Gram-positive facultative anaerobe, is
another species that uses mostly glucose and may be present in spoiled
meat, such as in heavily contaminated lamb, especially when the gas
atmosphere and pH favor its growth (Dainty and Hibbard, 1980; Grau,
1988). Meat spoilage is more rapid in the absence of glucose, such as in
dark-firm-dry meat and on fat. The storage time needed for aerobically
stored fresh meat to spoil depends on the initial extent of contamination
with psychrotrophic bacteria and on the temperature of storage. The
average storage life at O°C is 2 weeks and it is reduced by half for every
5°C increase in storage temperature (Egan, 1984; Egan et aI., 1988).
Since pseudomonads require oxygen for their growth and are inhibited
by 20% carbon dioxide, they are not involved in spoilage of meat stored
under vacuum or a carbon dioxide-enriched atmosphere. Enterobacter-
iaceae, however, may cause putrefactive spoilage problems under both
aerobic and anaerobic conditions since they are inhibited only at carbon
dioxide levels above 40%, especially when the pH is 6.0 or higher (Egan et
aI., 1988). Brochothrix thermosphacta may produce chemical or dairy

odors in presence of oxygen, or souring in vacuum packages, where it
grows better at pH values above 6.0. Lactic acid bacteria, however, can
cause souring, discoloration and gas production under any conditions of
storage. Aeromonas spp. and S. putrefaciens grow better at pH values
above 6.0 and also produce hydrogen sulfide, which results in putrefactive
odors and greening, even in vacuum-packaged meat (Egan et aI., 1988).
Hydrogen sulfide can react with myoglobin to form a green pigment.
Hydrogen sulfide may also be produced by other Gram-negative bacteria,
such as Serratia liquefaciens and Hafnia alvei but their significance in
greening of commercially vacuum-packaged meat is unknown (Dainty et
aI., 1989b).
14.3.1.3 Anaerobic. Bacterial spoilage under anaerobic conditions, which

occur in vacuum-packaged products or in meat interiors where oxygen
availability is limited, is described as souring, taint or putrefaction. After
cooking and during consumption, spoiled vacuum-packaged meat is first
described as cheesy, sour and acid but later in storage it may be described
as bitter or liver-like (Egan, 1984). The changes in sensory quality are
generally caused by accumulation of the end products of metabolic
activity by lactic acid bacteria, which usually dominate the flora and reach
populations of 107.cm~2 (Seideman et al., 1976; Hanna et al., 1983). As
discussed by Egan (1984), however, the total count is not a useful indi-
cator of spoilage in vacuum-packaged meat of normal ultimate pH,
because significant spoilage is not usually detected by sensory evaluation
until several weeks after the count of lactic acid bacteria exceeds 107.cm~2.
In general, the shelf-life of vacuum-packaged beef of pH less than 5.8-6.0
stored at ooe can be as long as 10-12 weeks, provided that it is produced
under good manufacturing practices, good temperature control and that
package permeability is low.
Some specific souring defects are round sour, ham sour and bone sour,
which describe sour or putrid odors that may be associated with tissues
around bones. These defects are caused by anaerobic or facultative
bacteria originating from infected bone joints or lymph nodes or which
may have entered the meat during processing and storage. Souring
becomes a problem when chilling rates of carcasses or primal cuts are
slow. In general it is caused by accumulation of organic acids formed
through bacterial metabolism of complex molecules but it may also be
associated with formation of gases (Niven, 1989).
In addition to traditional types of bacteria involved in meat spoilage
(e.g. heterofermentative lactics in vacuum packages), lesser known micro-
organisms may predominate and spoil muscle foods under certain condi-
tions. Such spoilage may be favored by supersanitation procedures and
cold temperatures, which may eliminate the traditional spoilage flora to
such an extent that proliferation of unusual spoilage bacteria, such as

Carnobacterium spp., Leuconostoc carnosum, Leuconostoc gelidium and
Lactobacillus sake (Jay, 1992) is allowed. A motile, Gram-positive, spore-
forming, anaerobic psychrotroph was isolated from spoiled vacuum-
packaged refrigerated beef of normal pH within 1 week of storage at 2a C
by Kalchayanand et al. (1989) and Ray et al. (1989). The organism has
been designated as Clostridium laramie and causes spoilage characterized
by accumulation of excessive amounts of gas with the odor of hydrogen
sulfide. Spoilage is also characterized by extensive proteolysis and color
changes from normal purple to pinkish-red and finally green. These
authors also have unpublished data indicating isolation of the same
organisms from vacuum-packaged refrigerated roast beef, and growth at
temperatures as low as ~3ac. Dainty et al. (l989a) also reported similar
spoilage of vacuum-packaged uncooked beef under refrigeration (2aC) by
an unusual Clostridium spp. In addition, two different homo fermentative
lactobacilli and a Leuconostoc strain were isolated from vacuum-packaged
cooked meat products showing ropy-slime spoilage (Korkeala et al.,
1988). A yellow discoloration of cooked cured meat products was identi-
fied as being carotenoid in nature and was attributed to a highly heat-
resistant Streptococcus spp. (Whiteley and D'Suza, 1989).
14.3.1.4 Yeasts and molds. Yeasts may develop, especially under aerobic
or microaerophilic conditions, and cause spoilage problems similar to
bacteria, such as sliminess, discoloration, lipolysis and off-odors. Common
mold defects during long-term storage of meat at near freezing tempera-
tures include: whiskers (i.e. white, fuzzy appearance); odors, off-flavors
and tastes (i.e. musty); color defects (e.g. white, green and black spots by
pigmented mold mycelia); surface stickiness (i.e. surface mold growth);
and fat breakdown (e.g. off-odors and flavors).
14.3.1.5 Processed products. Several meat processing treatments reduce

the numbers of bacteria but only canning-sterilization completely elim-
inates microorganisms. Processing may also introduce additional micro-
organisms and select the types that can proliferate and cause spoilage
during storage. Processing also may involve use of non-meat ingredients
that may serve as nutrients (e.g. sugars) or inhibitors (e.g. salt, nitrite) of
microbial growth.
Fresh sausages (e.g. pork sausage or fresh bratwurst) undergo spoilage
similar to that for fresh meat, even though the salts and spices may inhibit
certain microorganisms. At the other extreme, commercially sterile canned
meat should spoil only due to underprocessing or failure of the can integ-
rity and leakage of microorganisms into the container after processing.
Luncheon or delicatessen meats undergo spoilage similar to fresh meat,
depending on the type of package, pH and storage temperature. Since
they are usually vacuum-packaged and refrigerated, they mostly spoil

from lactic acid bacteria. Fermented dry sausages generally spoil from
growth of acid-tolerant organisms that can grow at reduced water activity,
such as molds. In general, perishable meats, which should be kept refri-
gerated, are usually cured and pasteurized and may spoil due to Gram-
positive bacteria, yeasts and molds. Since they are normally vacuum-
packaged and refrigerated, they spoil from facultative organisms able to
grow at cold temperatures, such as lactobacilli and leuconostocs. Spoilage
in these products is detected as a sharp acid flavor and presence of a
milky, viscous liquid in the package, or as a ropy slime. Hetero-
fermentative lactics also produce carbon dioxide gas, which causes
swelling of the package. In general, defects caused by lactic acid bacteria,
which usually dominate in cured meats, include souring, greening, slime
formation and gassing (Egan et aI., 1988).
In addition to lactics, other bacteria that may grow in cured meats
include Staphylococcus, Micrococcus, Microbacterium, Bacillus and Clos-
tridium spp., while in salty products, salt-tolerant organisms present may
include Vibrio and Sarcina spp. (Kraft, 1986). The final flora, however,
depends on factors such as composition and non-meat ingredients, tem-
perature of processing, smoking, slicing, packaging and storage condi-
tions.
14.3.1.6 Poultry and seafood. Spoilage principles involved in red meat

products also apply to poultry and seafood products. Spoilage of chilled
raw poultry meat is detected by off-odors followed by formation of slime
and discolorations similar to beef (Cunningham and Cox, 1987). As with
fresh meat, storage of seafood under refrigeration or in ice results in dom-
inance of Gram-negative, rod-shaped bacteria (e.g. Pseudomonas and
Shewanella spp.), which are also favored by their ability to use low-mol-
ecular-weight non-protein nitrogen compounds. Final products of metabo-
lism include ammonia, free fatty acids and amines, such as trimethylamine
(Malle and Poumeyrol, 1989). Other compounds associated with odors of
spoiled fish include sulfur-containing mercaptans and sulfides, as well as
diacetyl, acetaldehyde, propionaldehyde, ethanol, methanol, acetoin,
butanol and methyl butanal.
14.3.2 Foodborne illness

14.3.2.1 General. Of the documented foodborne disease outbreaks in the
USA in the period 1973-1987 (Bean and Griffin, 1990; Bean et aI., 1990),
beef, pork, turkey, chicken, fin-fish and shellfish caused 9%, 7%, 4%, 3%,
15% and 6%, respectively. The respective percentage of cases for each of
the above muscle foods were 10%, 5%, 6%, 3%, 2% and 3%. Thus,
muscle foods accounted for 44% of the total outbreaks and 29% of the
total cases of reported foodborne illness in the USA in the period 1973-
1987 (Bean and Griffin, 1990). Data for foodborne illness in Canada were
presented by Todd (1992), while Hackney and Dicharry (1988) and Liston
(1990) have reviewed the foodborne diseases transmitted by seafood.
The various etiologic agents and their involvement in outbreaks of
illness from consumption of muscle foods in the USA for the period
1973-1987 (Bean and Griffin, 1990) are summarized in Table 14.1.
Common bacteria involved in food borne illness from consumption of
meat products include Salmonella spp., S. aureus, C. perjringens, C. botuli-
num, B. cereus and E. coli. Bacterial pathogens transmitted from con-
sumption of seafoods include C. botulinum, Salmonella spp., Vibrio spp.,
C. perjringens, E. coli, S. aureus and Shigella spp. It should be noted that
many outbreaks are from unknown etiologic agents (Table 14.1). This
would suggest that either some of these pathogens are involved in addi-
tional outbreaks or that other, presently unsuspected, pathogens are
involved in foodborne illness. Details on meat and other food products
involved in foodborne illness have been presented by Genigeorgis (1986),
Bryan (1988), Bean et al. (1990) and Todd (1992).
Major factors contributing to foodborne disease outbreaks include
improper cooling (46 %), time lapse between preparation and serving
(21 %), infected persons touching the food (20%), inadequate processing/
cooking (16%), improper hot storage (16%), inadequate reheating (12%)
and contaminated raw food (11 %) (Zottola and Smith, 1990). It is inter-
esting to note that less than 10% of the documented outbreaks have their
origin in commercial food processing operations compared with homes
and food service establishments (Genigeorgis, 1986). The estimated annual
economic impact of food borne illness is in the range of billions of dollars
(Todd, 1989; Zottola and Smith, 1990).
In addition to the pathogens listed in Table 14.1, other documented or
suspected bacterial pathogens of potential concern in muscle foods include
Erysipelothrix rhusiopathiae, Proteus spp., Coxiella burnetti, Franciscella
tularensis, Mycobacterium spp., Bacillus anthracis, Chlamydia psittachi,
Leptospira spp., Plesiomonas shigelloides and Pseudomonas spp. (Bryan,
1979; Doyle, 1989; Jay, 1992).
14.3.2.2 Common pathogenic bacteria. Foodborne bacterial pathogens

have been discussed in several publications, including books (Doyle,
1989), and can be divided into traditional or common pathogens with a
minimum temperature for growth in the range 5-1O°C as well as patho-
gens of emerging significance, some of which can proliferate even at tem-
peratures below 5°C. The most common of the bacterial pathogens are
only presented briefly, while some of the emerging pathogens, such as
Listeria monocytogenes, considered of importance to the safety of refri-
gerated muscle foods, are presented in more detail.
Table 14.1 Number of foodborne outbreaks from muscle foods by etiologic agents for the period 1973-1987 in the USA"
Muscle Food Total

Etiologic agent Beef Pork Turkey Chicken Fin·fish Shellfish Muscle All
foods foods
Bacterial:
Bacillus cereus 3 0 1 1 1 2 8 58
Brucella spp. 0 0 0 0 0 0 0 4
Campylobacter spp. 0 0 1 2 0 0 3 53
Clostridium botulinum 2 1 1 1 35 0 40 231
Clostridium perfringens 51 8 19 9 3 2 92 190
Escherichia coli 3 0 0 9 3 0 15 20
Salmonella spp. 77 25 36 30 5 3 176 790
Shigella spp. 0 0 1 3 3 4 11 104
Staphylococcus aureus 22 96 20 14 3 2 157 367
Streptococcus spp. 1 0 0 0 1 0 2 19
Vibrio cholerae 0 0 0 0 2 3 5 6
Vibrio cholerae non-Ol 0 0 0 0 0 2 2 2
Vibrio parahaemolyticus 0 0 0 0 1 18 19 23
Yersinia enterocolitica 0 0 0 0 0 0 0 5
Qther bacterial agents 0 0 0 1 0 0 1 7
Total bacterial 159 130 79 70 57 36 531 1879
Viral:
Hepatitis A 1 3 1 0 1 9 15 110
Norwalk 0 0 0 0 0 1 1 15
Other viral 0 0 0 2 0 1 3 10
Total viral 3 2 11 19 135
Parasitic:
Giardia spp. 0 0 0 0 1 0 1 5
Trichinella spira/is 8 55 0 0 0 0 63 128
Other parasitic agents 1 0 0 0 5 0 6 7
Total parasitic 9 55 0 0 6 0 70 140
Chemical:
Ciguatoxin 0 0 0 0 232 0 232 234
Paralytic shellfish 0 0 0 0 0 21 21 21
Histamine (Scombroid) 0 0 0 0 199 0 199 202
Other chemical agents 3 0 0 0 4 1 8 240
Total chemical 3 0 0 0 435 22 460 697
Confirmed total 172 188 80 72 499 69 1080 2851
Unknown 155 64 53 57 44 144 517 4617
Total 327 252 133 129 543 213 1597 7468
aExtracted from Bean and Griffin (1990).

Salmonella spp. are a leading cause of food borne infection and more
than 2000 serotypes are found in most animals (Flowers, 1988). Raw meat
and especially poultry are major sources of this pathogen (Tauxe, 1991).
Staphylococcus aureus is a ubiquitous organism that causes 20-40% of
the reported foodborne illnesses per year (Newsome, 1988). It is often
found on the skin and in nasal passages of animals and people from
where it may contaminate meat and other foods. The illness is caused by
several heat-resistant enterotoxins produced when the bacterial cells pro-
liferate in foods. Products of animal origin, such as meats, are often
involved in outbreaks of staphylococcal intoxication (Genigeorgis, 1986).
Clostridium perfringens is another common cause of food borne illness
(Labbe, 1988). The bacterium is widely distributed in nature and con-
taminates foods, especially those of animal origin. Common vehicles of
this food borne toxicoinfection are undercooked, slow-processed or
reheated meat products.
Clostridium botulinum causes a food borne intoxication that is often
fatal. The disease is caused by neurotoxins formed in the food (traditional
botulism) or in the intestine (infant botulism). Proper refrigeration should
inhibit the proteolytic strains of this anaerobic bacterium, while non-pro-
teolytic strains grow at refrigeration (3.3°C) temperatures (Sugiyama and
Sofos, 1988).
Non-proteolytic strains of C. botulinum include all type E and certain
type Band F strains. These psychrotrophic strains differ from the other
cold-tolerant pathogens in that they form spores, which are more heat-
resistant than bacterial cells but less than the spores of the proteolytic C.
botulinum strains. Although C. botulinum type E spores are mostly asso-
ciated with seafood products, non-proteolytic type B spores have been
isolated from meat and are more heat-resistant than type E spores (Scott
and Bernard, 1982; Simunovic et al., 1985; Baker et al., 1990). Although
infrequent, botulism is considered as a very potent foodborne illness. The
non-proteolytic strains of C. botulinum are thus of interest because they
can cause safety concerns in muscle foods, especially those involving
minimal heat treatments and vacuum-packaging. The time needed for for-
mation of the toxin depends on temperature of storage, size of inoculum,
heat treatment, type of food, other contaminants and the specific strains.
14.3.2.3 Pathogens of emerging concern. Campylobacter jejuni was recog-

nized as a cause of foodborne illness only during the 1980s and it is
believed that it causes more cases of human enteric illness than Salmonella
spp. (Palumbo, 1986; Doyle, 1988a; Franco, 1988). Outbreaks have been
associated with raw or inadequately cooked foods of animal origin, espe-
cially milk, and including chicken, turkey and hamburger. The incidence
of the bacterium has been found to be 5-10% in red meats and up to
30% in poultry (Stern et al., 1984).
Bacillus cereus is an aerobic spore-forming bacterium, which has also

been implicated as a cause of food borne illness manifested either as a
diarrheal or an emetic syndrome (Johnson, 1984; Kramer and Gilbert,
1989). The organism may be a common contaminant of meat products
and is considered as one of the emerging foodborne pathogens (Konuma
et al., 1988).
The most common Vibrio spp. associated with human illness are the
traditional V. cholerae, as well as the newer species of V. parahaemolyticus
and V. vulnificus (Madden et al., 1989; Twedt, 1989; Oliver, 1989). All
three species are associated with seafood, especially when harvested in
contaminated waters and undercooked. The newest in the group, V. vulni-
ficus, is a very invasive and lethal pathogen associated with wound infec-
tions but having the potential to cause fatal foodborne illness.
The group of psychrotrophic pathogenic bacteria, which can grow at
temperatures even below 5°C, also includes most of the organisms recog-
nized as foodborne pathogens in the past 5-15 years. In addition to the
fact that they are able to grow at cold temperatures and to their patho-
genicity, some of these bacteria also share the property of being able to
multiply in low- or no-oxygen environments. In addition to non-proteoly-
tic C. botulinum types, these organisms include Listeria monocytogenes,
Yersinia enterocolitica, hemorrhagic Escherichia coli strains, and Aero-
monas hydrophila. A major concern with these pathogens is whether they
will be able to proliferate in raw or cooked, cured or uncured, refrigerated
or slightly abused meat products before spoilage is detectable by con-
sumers.
Strains of Escherichia coli are generally considered as harmless, but
some are known to cause gastrointestinal diseases in humans (Doyle,
1984). The organism is a Gram-negative, facultative, non-spore-forming
rod of animal origin, which can grow at temperatures as low as I-5°C
(Palumbo, 1987; Frank, 1988). The normal habitat of the organism is the
intestinal tract, and it is commonly used as an indicator of fecal con-
tamination in food products and water. The E. coli type of current
concern in meat products has been designated as 0157:H7, and causes
hemorrhagic colitis, or bloody diarrhea, and hemolytic uremic syndrome
(Riley et al., 1983; Pai et al., 1984). It was first recognized as a human
pathogen in 1982 when it was associated with two outbreaks linked with
consumption of undercooked ground beef sandwiches (Doyle, 1984).
Several other outbreaks from undercooked meat have been confirmed in
the USA and Canada since 1982.
Yersinia enterocolitica is another of the psychrotrophic pathogenic
bacteria of current concern. It is a Gram-negative, facultative, non-spore-
forming rod of the family Enterobacteriaceae and it is able to proliferate
at temperatures of 0-42°C. Interest in this organism was initiated in the
1970s following several foodborne outbreaks involving chocolate milk,
reconstituted powdered milk, turkey chow mein and tofu (Palumbo, 1987;
Doyle, 1988b). It causes a severe foodborne infection and pseudoappendi-
citis. The organism is widely distributed in nature and it has been isolated
from raw or rare cooked meats (including beef, pork, lamb and poultry),
other foods and water. Pigs are the most important animal source but
most strains are considered non-invasive.
Aeromonas hydrophila is a Gram-negative, facultative, non-spore-
forming rod of water and animal origin and can grow at 4-5°C and
possibly at 1°C (Palumbo, 1987; Buchanan and Palumbo, 1985). The
organism has been a recognized pathogen of fish and, more recently, it
has been of concern to the food chain because it is frequently isolated
from cases Of human gastroenteritis (Abeyta et al., 1986). Thus, since the
1970s, it is discussed as one of the possible causes of human diarrheal
disease (Buchanan and Palumbo, 1985). Being an aquatic microorganism,
it is associated not only with seafood but also with meat, poultry and raw
milk. Actually, it has been found as a component of the microbial flora of
retail samples of refrigerated meat and other animal products (Palumbo et
al., 1985; Stern et aI., 1987). One study demonstrated that it can pro-
liferate in vacuum-packaged raw ground pork at 5°C but growth was
reduced in presence of natural meat microflora (Palumbo, 1988). Studies
with fish indicated that cooked mince and low-salt surimi supported its
growth at 5-25°C (Ingham and Potter, 1988a,b).
Listeria monocytogenes, a pathogen of increasing concern for the meat
industry, is a Gram-positive, catalase-positive, non-spore-forming rod,
which can grow best in small amounts of oxygen but which proliferates
well even in the presence or absence of oxygen. It survives refrigerated
storage and it can grow at temperatures as low as O°C (Miller et al.,
1990). Manifestations of the disease (listeriosis) include abortion, perinatal
septicemia, meningitis and encephalitis. In pregnant women it causes
abortion and delivery of stillborn or very ill infants. Listeriosis infections
are usually associated with pregnant women, the newborn and other indi-
viduals with underlying diseases and deficient immune systems. Death is
rare in healthy adults but the death rate may exceed 30% in sensitive
populations.
Evidence of involvement of meat and meat products in foodborne
human listeriosis is limited (Johnson et al., 1990a). Almost 40 years ago,
sheep were infected experimentally with L. monocytogenes, which indi-
cated that meat products could thus be contaminated and infect humans
(Osebold and Inouye, 1954). Also, mice were infected when fed meat con-
taminated with the pathogen (Temper, 1961) and four neighbors in
Sweden were infected probably from consumption of meat from the same
source (Olding and Philipson, 1960). Consumption of meat from a still-
born calf was also presumed to have resulted in listeriosis (Kampe 1-
macher, 1962).
In recent years, the Centers for Disease Control conducted an epide-

miologic case-control study to determine risk factors for occurrence of
sporadic listeriosis in the USA (Schwartz et at., 1988). It was concluded
that case patients were significantly more likely than controls to have
consumed undercooked chicken or hot dogs not reheated after processing,
with odds ratios of 20.5 and 12.3, respectively. This conclusion, however,
has been considered controversial because it is based only on epidemiolo-
gical evidence and is without established cause-and-effect association
(Johnson et aI., 1990a).
There are at least two cases of documented listeriosis from consump-
tion of poultry products. One of these involved consumption of commer-
cial cooked and chilled chicken, which resulted in materno-fetal infection
(Kerr et aI., 1988). The second incidence involved consumption of con-
taminated turkey frankfurters by an immunocompromised cancer patient
(Anon., 1989). This infection occurred in April 1989 in Oklahoma and it
was confirmed by finding a L. monocytogenes serotype 1/2a strain with
identical isoenzyme types in the patient, an opened package of turkey
frankfurters in her refrigerator, and in two out of five unopened packages
of the same brand purchased in local stores. The most probable number
(MPN) of L. monocytogenes cells in the finished product from the
unopened retail packages was 0.3.g- 1, while the MPN in frankfurters
from the opened package found in the refrigerator was greater than
1100.g- 1. Subsequent to the latter incident, Wenger et at. (1990) evaluated
the facility that produced the turkey frankfurters. The pathogen was
isolated from only 2/41 environmental samples from the plant per se, but
from 12/14 (86%) samples from the frankfurter peeler-conveyor, which
implicated this piece of equipment as the major contributor to the
problem.
The organism is found widely in nature, including soil, water and
vegetation, whence it can contaminate animals, humans and the food
supply. Several studies have examined contamination of meat and poultry
plant environments and products, and seafood (Weagant et aI., 1988; Car-
osella, 1990; Johnson et at., 1990a; Baker et at., 1990; Motes, 1991). In
general, studies from throughout the world have demonstrated that 0-
90% of muscle food samples examined were contaminated with L. mono-
cytogenes.
Processed meat products have also been found to be contaminated with
L. monocytogenes, including fermented sausages (5-33%), wieners (21 %),
luncheon meats (13%), sliced meats (14%), pork sausage (32%) and pro-
cessed, ready-to-eat meat products (53%) (Farber et at., 1988; McLain
and Lee, 1988; Tiwari and Aldenrath, 1990a,b; Grau and Vanderlinde,
1992). Wilson (1988) of the American Meat Institute reported that a 1987
survey found that 11.5% of the samples of ready-to-eat meat products
examined were positive for L. monocytogenes, while the incidence was
13% in 1988. In 1988, 38% of the frankfurter, 7% of the luncheon meat

and 7% of the ham samples examined were positive for L. monocytogenes
(Wilson, 1988). Results of the national monitoring program of the
USDA's FSIS indicated the incidence in raw beef at 6.23% (41/658) for L.
monocytogenes, 1.67% (36/2151) for Salmonella spp. and 0% (0/906) for
Escherichia coli 0157:H7. The incidence of L. monocytogenes in canned-
cured meats was 0% (0/140) but was 2.2% (3/136) in Prosciutto ham
(Carosella, 1990). Its incidence in cooked beef through October 1990 was
2.78% (44/1580), while in ready-to-eat meat and poultry products in the
period of October to December, 1990, there were 48 positive samples
(Anon., 1991). It can be concluded that L. monocytogenes is a frequent
contaminant of meat and poultry products and that it is virtually impos-
sible to completely avoid its presence. Thus, it must be assumed that the
pathogen will be present in the raw material and processing environment
and that procedures must be designed to eliminate it or to avoid its pro-
liferation, especially when introduced after product processing.
The organism can grow in the pH range of 5.0-9.5, can survive high
concentrations of salt for long periods of time, and is relatively resistant
to drying. The pathogen is inhibited by various acids and antimicrobials
(Farber and Peterkin, 1991; Ryser and Marth, 1991). Nitrite is expected to
be effective only when combined with reduced pH, cold temperatures and
sodium chloride in cured meats (Junttila et al., 1989). Available scientific
publications have indicated that L. monocytogenes may be of higher heat
resistance than other non-spore-forming pathogenic bacteria (Mackey and
Bratchell, 1989; Boyle et al., 1990; Schoeni et aI., 1991). Heat resistance
increases with increasing temperature of culture propagation (Bhaduri et
al., 1991); the presence of curing ingredients (Yen et aI., 1991); heat
shocking or tempering (e.g. 48°C) before thermal processing (Farber and
Brown, 1990; Linton et al., 1992); or after slow temperature rise (Quinta-
valla and Campanini, 1991). Reported D-values in uncured beef have
ranged from 81 min at 51.7°C to 0.15 min at 70°C (Fain et al., 1991),
while actual destruction in cured products at 71°C has been in the range
of 3-8 log.g-l (Yen et al., 1991; Zaika et al., 1990). It is advisable that
when new formulations, processing or cooking procedures are developed
for muscle foods, their effect on thermal destruction of L. monocytogenes
should be evaluated.
It appears that cell numbers at refrigeration temperatures either remain
constant or increase with storage time of products, such as lamb meat, beef
and liver (Gouet et al., 1978; Johnson et al., 1988; Shelef, 1989; Grau and
Vanderlinde, 1990). In seafood, L. monocytogenes numbers also remained
constant or multiplied at different rates during cold storage depending on
the type of product (Farber, 1991a; Harrison et aI., 1991). Concerns asso-
ciated with studies examining the fate of L. monocytogenes in muscle foods
are that they used only a limited number of strains and that the cultures
used were propagated at temperatures (30-35°C) optimum for growth,

while such cultures may behave differently when stored at colder tempera-
tures.
Reduced numbers of the pathogen can survive fermentation, drying and
refrigerated storage of hard salami and pepperoni (Glass and Doyle,
1989b; Johnson et al., 1990a). Since there was no growth in these
products, the lactic acid cultures used in the fermentation process reduced
cell numbers and controlled growth. The fate of the pathogen in cooked
meat products appeared to be related to product type and pH (Glass and
Doyle, 1989a). Growth was generally more pronounced in products of pH
6.0 or higher and was inhibited in products of pH 5.0 or lower. Storage
of cooked chicken meat at 10°C also allowed increases of L. mono-
cytogenes cell numbers within 3-10 days (Harrison and Carpenter,
1989a,b; Carpenter and Harrison, 1989a,b). The pathogen also pro-
liferated in previously sterilized chicken loaves stored under modified
atmospheres (Ingham et al., 1990). In general, the fate of L. mono-
cytogenes in meat depended on the product pH, temperature of storage,
amount of fat and lean tissue, strain of the pathogen, other contaminants
present and chemical additives.
Most strains of L. monocytogenes isolated from meat and poultry
products have been identified as serogroup Ij2, and to a lesser extent as
serogroups 3 and 4 (Farber et al., 1989; Johnson et aI., 1990a,b). Wilson
(1988) in an American Meat Institute survey found 46% of the meat
isolates to be serotype 3a, 23 % were serotype 1j2a, 31 % were serotype
Ij2b and none were serotype 4b. A French study reported that strains of
L. monocytogenes isolated from meats were different to human isolates
(Anon., 1990). Serotypes from human patients were 66.5% 4b, 17.4%
1/2b and 12.7% 1/2a. In contrast, meat isolates were 63% 1/2c and 34%
1j2a. This indicates a significantly different distribution of L. mono-
cytogenes serotypes among those found in meat products and those that
had infected humans. Another study serotyped 144 human isolates and
found 30% to be Ij2a, 32% Ij2b and 34% 4b (Broome et al., 1990). It is
also significant to indicate that the serotype implicated in the major food-
borne outbreaks of the 1980s was 4b (Schlech et al., 1983; Ho et aI., 1986;
Linnan et al., 1988). It appears that strains involved in human illness are
mostly of serotype 4b and that serotypes isolated from humans are gen-
erally different than those found in meats. Thus, there is the unanswered
speculation as to whether meat is a source or can support survival and
growth of pathogenic serotypes. It should be indicated, however, that
isolates from meat products have been pathogenic to mice, that 40-50%
of isolates found in processed meats in contrast to fresh meats have been
identified with human illness (Wilson, 1988), and that the documented
infection from consumption of turkey frankfurters was caused by a
serotype Ij2a isolate, which is found in meat.
14.3.2.4 Mycotoxins. Fungi are generally unable to grow rapidly in

animal tissues and are only of minor concern to meat products. In dried
and cured products, especially country cured hams and aged salamis,
however, fungi may compete effectively with bacteria to form mycotoxins,
which may also be present in meat products as tissue residues introduced
through consumption of contaminated feeds (Pestka, 1986). In general,
spoilage or toxigenic fungi are outgrown by bacteria in muscle foods.
Under conditions restrictive to bacterial growth, such as reduced water
activity and cold temperatures, psychrotrophic and xerotolerant yeasts
and molds may dominate. Certain products are believed to benefit in
terms of flavor from growth of fungi but mycotoxin production should be
suspected .only in heavily molded products. The risk to human health
from mycotoxin intake through consumption of muscle foods is con-
sidered minor compared with that from other foods.
14.3.2.5 Parasites. Parasites involved in foodborne illness from muscle

products in the period 1973-1987 (Table 14.1) include Trichinella spiralis
and Giardia spp. Other parasites of concern in meat foods include Tox-
oplasma gonda, Sarcocystis spp., Taenia spp. and Echinococcus spp. (Fayer
and Dubey, 1985; Greiner, 1989; Jackson, 1990; Kotula et al., 1990, 1991).
Fish also may harbor various parasites most of which are harmless to
humans and include Clororchis (Opisthorchis) spp., Heterophyes hetero-
phyes, Diphyllobothrium spp., Metagominus yokogawai, Gnathostoma spini-
gerum, Capillaria philippinensis, Anisakis simplex and Plocanema spp.
(Higashi, 1985). Parasites are generally sensitive to destruction by pasteur-
ization or cooking. Other treatments that inactivate parasites include che-
micals, freezing and ionizing radiation (Engel et al., 1988; Thayer et al.,
1986).
14.3.2.6 Viruses. Viruses such as hepatitis A and Norwalk-like agents

have been implicated in several outbreaks of gastroenteritis from con-
sumption of seafoods as well as meat products (Table 14.1). The viruses in
muscle foods may originate from animal infection, which is of relatively
minor concern but especially from secondary contamination (Blackwell et
al., 1985; Richards, 1985; Cliver, 1988). Viruses of human origin may con-
taminate muscle foods through feces and water, when poor hygienic prac-
tices are employed or vectors, such as insects, are involved. Adequate
cooking inactivates viruses but raw or undercooked shellfish from con-
taminated water can cause problems (Gerba, 1988; Liston, 1990). In
general, inadequately cooked muscle foods may serve as a vehicle for
transmission of human viruses under unsanitary conditions.
14.3.2.7 Other toxins. Outbreaks of illness from contamination of

seafood with chemical agents (Table 14.1) include ciguatera (or sigua-
toxin), paralytic shellfish poisoning and histamine (or scombroid fish poi-
soning). These etiologic agents are derived from microorganisms and have
been recently reviewed by Taylor (1988) and Liston (1990). Poisoning
from histamine and other biogenic amines may also be associated with
other heavily contaminated or fermented foods such as cheeses (Smith and
Palumbo, 1981; Stratton et al., 1991). Levels of biogenic amines in fer-
mented meats are variable but generally low, and no cases of confirmed
poisoning have been associated with sausages. Fermented fish products
may contain high levels of histamine and the USA has set a hazard action
level (50 mg. 100 g-l) for histamine in tuna. The defect action level is set at
10-20 mg.g- 1 (Stratton et al., 1991).
14.4 Control of microbial growth in muscle foods
14.4.1 General
14.4.1.1 Principles. Fresh as well as processed muscle foods provide
excellent substrates for growth of various microorganisms. Microbial
growth on muscle foods or other food products is affected by the type and
extent of initial contamination and by factors intrinsic to the product, as
well as by the environmental conditions surrounding the food (Jay, 1992).
The intrinsic parameters influencing the rate and extent of microbial
growth include moisture content and water activity, product pH, oxida-
tion-reduction potential or Eh, nutrient content, biological structures or
barriers, the physical state of the food and antimicrobial agents. The
extrinsic or environmental factors affecting microbial growth include tem-
perature, relative humidity, and composition of the gas atmosphere sur-
rounding the product. Of these, the most important factors, especially for
fresh muscle foods, are temperature of storage and gas atmosphere, while
other factors such as pH, water activity and antimicrobials become more
important in processed items. The influence of these factors on microbial
growth in muscle foods and other items has been discussed extensively in
the scientific literature.
Numerous methods are employed for the preservation of muscle food
palatability and safety through decontamination and control of microbial
growth. Control of microbial growth is based on modification or employ-
ment of factors such as refrigeration, freezing, salting, sugar addition,
curing, pasteurization, canning, drying, freeze-drying, acidification, fer-
mentation, smoking, irradiation and packaging in modified or controlled-
gas atmospheres, which have been extensively discussed in various review
papers and books. The objective of each of these methods is to maintain
product quality and extend shelf-life through elimination or inhibition of
spoilage and pathogenic microorganisms. In addition to extreme modifica-
tion of individual factors affecting microbial growth, preservation is often

achieved with combinations of factors that interact to yield the 'hurdle' or
barrier effect (Leistner, 1987, 1988). One chemical hurdle, which received
increased attention as a meat preservative in the 1980s, is sodium lactate
(Maas et ai., 1989; Debevere, 1989; Harmayani et ai., 1991; Papadopoulos
et ai., 1991).
14.4.1.2 Newer concerns. The methods discussed above have tradition-

ally provided effective means of muscle food preservation, resulting in an
exemplary safety record for commercially processed and marketed
products. In recent years, however, there is increasing commercial and
consumer interest in preparation and marketing of pre-cooked, uncured
meat products, or meals containing meat, which can be consumed without
or with only minimal preparation (e.g. microwave reheating). These
products meet consumer demands relative to changing lifestyles and
health concerns but need to be distributed under refrigeration in order to
maintain their quality and safety (Lechowich, 1988; Scott, 1989). Such
products should have adequate shelf-life to allow for centralized prepara-
tion and to decrease the frequency of deliveries to retail establishments.
Often retail establishments prepare, handle and sell such sensitive meat
items on the premises with the help of inadequately trained personnel.
Since minimally processed, high-moisture products contain very little or
no additives in their formulation, they rely almost exclusively on refrigera-
tion for maintenance of their quality and safety. Partial or regular
cooking of such products also destroys the natural spoilage bacteria,
which in uncooked meat can produce undesirable quality changes warning
the consumers of product spoilage, or if present can compete and often
outgrow food poisoning bacteria. In the case of product recontamination
with pathogens (after cooking and during cutting, slicing and packaging),
or if the pathogens have not been completely inactivated during cooking,
there is a possibility that they may develop to a degree that will cause
foodborne illness without any indication of product spoilage. If low doses
of ionizing radiation were approved and accepted by consumers, they
could be a viable alternative for preservation of muscle food products
(Kampelmacher, 1983; Giddings, 1984; Lambert et ai., 1991a-c). The
products may also be packaged in modified-atmosphere environments,
such as in a vacuum, to extend their shelf-life. Reduced oxygen atmo-
spheres, however, inhibit development of spoilage bacteria and fat oxida-
tion, which increases product shelf-life, but they may enhance the growth
of several pathogens.
Of particular concern in these products are the emerging pathogens
because they are able to grow at temperatures even below 5°C and close
to O°C and they are either anaerobic or facultative. Thus, normal refrig-
eration temperatures and vacuum-packaging may not inhibit their growth,
if they are present in the product. Since cooking has eliminated or mini-
mized the normal spoilage microfiora, the emerging pathogens may grow
even more rapidly than usual and with no warning to the consumer. Thus,
proper refrigeration will not guarantee safety from psychrotrophic food-
poisoning bacteria. It should be mentioned, however, that storage at
colder temperatures not only reduces the rate of growth of spoilage
bacteria and the rate of chemical deterioration reactions but it also slows
down growth of the psychrotrophic pathogenic bacteria. The question,
however, is whether the race will be won by spoilage or by pathogens.
With fresh raw muscle foods, such potential problems should not be
encountered because the natural spoilage microfiora is still intact, which
should outcompete the pathogens by producing either offensive odors in
aerobic packages or acid that will inhibit pathogens in vacuum packages.
In addition, fresh raw meats are handled as being more sensitive to
spoilage and they are cooked before consumption. If, however, the USDA
regulations on cooking and cooling schedules for beef are followed, most
of the non-spore-forming pathogens should be absent from the cooked
products, provided that post-processing contamination is avoided. In
addition, application of the Hazard Analysis Critical Control Points
(HACCP) concept and of Good Manufacturing Practices (GMP), such as
proper sanitation and good hygiene, should minimize potential problems.
The HACCP system (Corlett, 1989; Bauman, 1990; Tompkin, 1990;
Garrett and Hudak-Roos, 1991) consists of an assessment of hazards
associated with operations, a determination of critical control points
necessary to prevent or control the identified hazards and the establish-
ment of procedures to monitor the critical control points. The system is
an effective and natural approach to the assurance of food safety. The
National Academy of Sciences/National Research Council has endorsed
its use and FSIS of USDA has initiated efforts to apply it to meat inspec-
tion (NAS, 1985).
Since the processors cannot be certain of complete absence of undesir-
able bacteria even after decontamination treatments and maintenance of
the product at very cold temperatures, they should take measures to
assure product shelf-life and safety. This means that refrigeration as an
inhibitor of microbial growth should be complemented with additional
barriers or hurdles that will make microbial growth more difficult. Thus,
there is a need for modification of product formulations or the environ-
ment, which will add barriers to microbial growth. Use of traditional
barriers, such as acidification, fermentation, drying and chemical addi-
tives, which are present in cured meat items, are undesirable in these
products because they will change their identity and the purpose of their
existence will be lost. Other barriers, however, that are milder or cause no
major changes in product identity may be beneficial in maintammg
product quality and safety. These may include storage under modified
atmospheres, including vacuums and flushing with carbon dioxide, and

the use of natural lactic acid bacteria or their metabolites. Even then,
however, inhibition of pathogens may sometimes fail, considering the var-
iation in the raw material (meat), the microbial species and strains, and
the factor of human error. For example, meat of high ultimate pH spoils
faster because of an increased rate of microbial growth. Even in vacuum
packages the products may spoil rapidly if the initial pH is above 6.0
because it allows anaerobic growth of species of higher spoilage potential
than normal lactic acid bacteria, which predominate in lower pH vacuum-
packaged meat. Pathogens also grow faster at higher pH values.
14.4.2 Decontamination
14.4.2.1 General. The microbiological quality of carcasses can be
improved by simple washing of the animals prior to slaughter, by carcass
washing with high-pressure cold or hot water and by brushing or
trimming to remove the bacteria physically. In order to improve their
sanitary status even further, carcasses or meat cuts may be subjected to
washing, spraying, immersing or other sanitizing procedures to reduce
microbial contamination. Commonly evaluated sanitizing agents include
hot water, chlorine and organic acid solutions. The effectiveness of these
compounds varies with the concentration used, temperature, contact time,
nature of microbial contamination, temperature of solution, application
pressure and the step in processing at which they are applied.
Although the most widely evaluated meat decontaminants are chlorine,
and acetic and lactic acids, other compounds tested include citric, pro-
pionic, formic, ascorbic and succinic acids, chloramphenicol, stannous
chloride, quaternary ammonium compounds, poly[hexamethylene-bigua-
nide hydrochloride], ozone, potassium sorbate, hydrogen peroxide,
ethanol, sodium chloride, sodium hydroxide, potassium hydroxide and
various combinations (Dickson, 1988; Dickson and Anderson, 1992), as
well as gamma, ultraviolet and infrared radiations (Stermer et al., 1987;
Lebepe et al., 1990; Nerkar and Bandekar, 1990). Acceptable sanitizing
treatments, however, should exert a strong, rapid and non-selective micro-
bicidal action. They should leave no residues that are detrimental to
human health, and should have no adverse effects on product color,
general appearance, odor and taste.
Total reduction in microbial counts on carcass surfaces following
various types of sprays and washes is in the range of 101-103.cm-2,
depending on water pressure, temperature, time, the chemical agent and
its concentration and also the type of carcass. Although higher pressures
are more effective, there is some concern that they may physically drive
bacteria into muscle tissue; thus the maximum recommended washing
pressure for beef carcasses is 2070 kPa (De Zuninga et al., 1991). The
most widely used temperature is in the range 50-55°C, although hot water
at 80°C has also been evaluated (Anderson and Marshall, 1990). Recom-
mended concentrations of chemical agents are 1-2%, because higher levels
may cause problems, such as bleaching of the carcass.
One critical factor in determining the decontaminating effectiveness of
carcass spray washing is the design of the equipment. Automated equip-
ment has been designed and the physical parameters involved in the
process have been evaluated by Anderson et al. (1987) and Crouse et al.
(1988). Several patents have been issued that are based on the use of
automated, multi-nozzle, oscillating sprayers with variations in configura-
tion and nozzle size (Anderson et al., 1981, 1982; Davey and Smith, 1989).
14.4.2.2 Acetic and lactic acids. Although reductions in surface microbial

counts with the traditional decontaminant chlorine have been as high as
103 .cm- 3 , and it is used in poultry slaughter, Stevenson et al. (1978) and
Johnson et al. (1979) found no significant effect with chlorine sprays. The
variation may have been the result of differences in experimental design
and microbial flora. Addition of a slow-releasing chlorine dioxide solution
to turkey rinse and/or chilling water reduced the incidence of salmonellae
on contaminated carcasses from an average of 70% after evisceration to
25% after chilling (Villarreal et al., 1990). In recent years, however, two
common organic acids, acetic and lactic, have received considerable
testing as decontaminants of meats and poultry, especially because of their
favorable antimicrobial properties and their status as generally recognized
as safe (GRAS) substances (Acuff et al., 1987; Lillard et al., 1987; Hamby
et aI., 1987; van der Marel et aI., 1988, 1989; Izat et al., 1989; Anderson
and Marshall, 1990; Dickson and Anderson, 1992).
Most studies have reported reductions in bacterial counts of 1-3 orders
of magnitude immediately after application of the decontaminant. In
practical terms, however, it is more important to know the effect of such
treatment on the final shelf-life of the meat after cutting and packaging
and during shipment and retail storage (Smulders, 1987). Some studies
found that loins and steaks decontaminated with acidic solutions were not
greatly different in total bacterial counts than controls during simulated
retail storage (Acuff et aI., 1987; Dixon et al., 1987, 1991; Prasai et al.,
1991). In contrast, however, other studies have found a residual anti-
microbial effect during storage of acid-treated meat. This effect is believed
to be due to initial sublethal injury caused by the acid treatment, which
becomes irreversible when the stresses of refrigeration and absence of
oxygen are added during storage (Cacciarelli et al., 1983; Mossel and van
Netten, 1984; Snijders et al., 1985; Smulders and Woolthuis, 1985;
Anderson et al., 1988).
Different microorganisms show variable sensitivities to acids. Concerns
that acid decontamination of meat may selectively eliminate antagonistic
spoilage flora, while allowing pathogens to predominate have been refuted

by van Netten et a/' (1984), Woolthuis et al. (1984) and by Gill and
Penney (1985). There is concern, however, that the high degree of sanita-
tion achieved may eliminate common antagonistic bacteria and allow
lesser-known spoilage organisms to cause product defects such as undesir-
able odors, colors and swelling of vacuum packages. Another concern in
using chlorine or acid sprays may be the potential for equipment corro-
sion problems originating from such application.
The time of application of organic acid solutions to decontaminate car-
casses or cuts of meat may be important. Carcass spraying with lactic acid
(1 %) before chilling (i.e. 45 min post-mortem) caused a greater bacterial
reduction than spraying carcasses that were chilled (Snijders et al., 1985).
Cells of bacteria that become attached to tissues may be significantly more
resistant to sanitizing solutions than unattached cells (Lillard et al., 1987;
Dickson, 1991). Overall, an acid treatment soon after slaughter appears to
be very effective in removing bacteria that have not yet become attached
to the muscle or fatty tissues (Snijders et a/', 1985; Dickson and
Anderson, 1992).
Although most studies have employed a single carcass spray treatment,
usually after evisceration and before chilling, some recent patents suggest
use of two washing and sanitizing treatments, one at hide removal and a
second after carcass evisceration and splitting (Clayton and Bowling,
1989a,b). Studies with model systems and carcasses have indicated,
however, that use of one treatment after evisceration may be adequate, as
well as more practical and economical than spraying before evisceration
or at both locations (van der Marel et al., 1988; Dickson and Anderson,
1991; Prasai et a/', 1991). Under the high operational speeds of slaughter
lines, however, double spraying may be more useful.
The effect of acid decontamination treatments on the quality of meat
products is also important. Acid treatment may affect meat color and
flavor depending on the concentration and intensity of application. Meat
discoloration should be only slight and reversible at acid concentrations of
less than 1-2%, while use of higher concentrations (2-4%) may result in
irreversible discoloration (Smulders and Woolthuis, 1985; Smulders et al.,
1986; Prasai et al., 1991). However, some studies have reported that treat-
ment with up to 2-5% lactic acid had no adverse effects on meat flavor
(Gill and Penney, 1985; Woolthuis and Smulders, 1985; Smulders et al.,
1986).
Although acetic and lactic acid are GRAS substances, they are not yet
approved officially for widespread use as meat decontaminants in the
USA and the EC. This may be because of concerns that their allowance
might lead to disregard for good hygiene and sanitary practices during the
slaughtering operation. Good manufacturing practices during slaughter
and dressing are critically important to the subsequent shelf-life of meat.
Although appropriate hygienic practices are effective in reducing initial

levels of contamination, the use of an additional hurdle, such as carcass
decontamination, has been suggested as beneficial in increasing the shelf-
life of fresh meat and meat by-products, especially for meat exports to
overseas markets. In any case, however, carcass decontamination, if intro-
duced, should be part of an overall HACCP program for assurance of
meat product quality and safety ..
The Food Safety and Inspection Service (FSIS) of the US Department
of Agriculture (USDA) has granted approval on a provisional basis for
red meat and poultry slaughtering establishments to use organic acid solu-
tions as decontaminating agents. Such approvals are granted for a limited
time with the objective of collecting data in order to evaluate the applica-
tions. The approval period can be extended when the application is incor-
porated into an approved partial quality control (PQC) program, which
ensures that the finished products are not adulterated. The reason FSIS
grants only provisional approval is that they believe that the information
available is not yet adequate to determine whether continuous use is
justified.
14.4.3 Modified-atmosphere storage

14.4.3.1 General. Modified-atmosphere storage of muscle foods, which
has been examined in several reviews (Restaino and Hill, 1981; Egan, 1984;
Genigeorgis, 1985; Gill, 1986, 1990; Hintlian and Hotchkiss, 1986; Egan
and Shay, 1988; Egan et al., 1988; Baker and Genigeorgis, 1990; Lambert
et al., 1991a), was introduced in long-distance shipment of red meat more
than 50 years ago. Modified-atmosphere storage of poultry meat has not
been as extensively studied, because frozen storage and shipment of these
products is more acceptable by consumers compared with red meats.
Packaging under carbon dioxide, however, has extended the shelf-life of
poultry meat approximately five-fold compared with aerobic packaging
(Gill et al., 1990). Seafood is also marketed frozen; however, in recent
years there has been a tendency to enhance shelf-life of fish through
modified atmospheres for shipment and marketing in the unfrozen state.
14.4.3.2 Vacuum-packaging and carbon dioxide atmospheres. Storage

under vacuum-packaging and other modified atmospheres is used exten-
sively, especially for long-term storage and shipment of raw meats (Egan
et aI., 1988; Gill, 1990). Vacuum-packaging, however, is not widely used
for display packaging of red meat because the anoxic conditions result in
a dull, purple color that is undesirable to consumers (Young et al., 1988;
Gill, 1990). Extension of shelf-life by vacuum-packaging can be as long as
five-fold compared with aerobic storage of primal cuts of normal pH, and
two-fold for smaller cuts (Seman et aI., 1988; Egan et aI., 1988). Extension
of storage life by vacuum-packaging is shorter in pork and lamb (4-8

weeks) compared with beef (10-12 weeks).
In addition to raw meats, vacuum-packaging reduces micrpbial growth
in cured and other pre-cooked meat products (Carr and Marchello, 1986,
1987; Anderson et al., 1989; Ahvenainen et ai., 1989, 1990). Common dis-
advantages of cooked vacuum-packaged meat include increased accumula-
tion of exudate and product deformation. These problems may be
overcome with improvements in packaging techniques involving shrink
packs, surface sealing, skin packs and high-vacuum packs (Ahvenainen et
ai., 1990).
In general, the storage life of chilled meat is extended under vacuum,
and can be lengthened even more if the atmosphere is enriched with
carbon dioxide (Blickstad and Molin, 1983, 1984; Daniels et al., 1985; Gill
and Reichel, 1989; De Pablo et al., 1989; Farber, 1991b). The inhibitory
effect of carbon dioxide is enhanced at colder temperatures, and it may be
diminished above lOoC (Gill and DeLacy, 1991). Also, inhibition of
bacteria by carbon dioxide is more effective on meat with low initial con-
tamination, which emphasizes the need for good sanitation. Bacteria that
have passed their lag phase may be more resistant to inhibition by carbon
dioxide (Holland, 1980). It should be mentioned that application of
modified atmospheres with high amounts of gas may pose some technical
problems (Egan, 1984). For adequate microbial inhibition there may be a
need for the volume of gas to be twice that of the meat. With time of
storage, however, carbon dioxide is dissolved and absorbed into the meat.
Although modified atmospheres extend shelf-life, there is some concern
relative to their effects against pathogens. Toxin formation in raw ground
beef inoculated with C. botulinum type A and B spores and stored under
vacuum at 25°C occurred in 6 days and was always accompanied by sig-
nificant organoleptic changes (Hauschild et al., 1985). For type E spores
of C. botulinum inoculated in fish stored under various atmospheres, time
to toxin detection depended on type of fish, gas atmosphere and storage
temperature (Post et al., 1985). Garcia et ai. (1987) reported that toxin
formation by type B, E and F spores in salmon fillets was affected by
temperature of storage, modified atmosphere, storage time and interac-
tions of these factors. Yersinia enterocolitica inoculated as a pure culture
in vacuum-packaged raw meat of pH 6.0-6.2 proliferated at 5°C. A.
hydrophila was isolated from pork under vacuum (Myers et ai., 1982) and
nitrogen (Enfors et ai., 1979), and from fat of pork loins stored under
100% carbon dioxide (Blickstad and Molin, 1983) when all products were
stored at refrigeration temperatures. A study by Hintlian and Hotchkiss
(1987) found that a combination of 75% carbon dioxide, 15% nitrogen
and 10% oxygen was the most effective for inhibition of P. fragi, S. typhi-
murium, S. aureus and C. perfringens on cooked, sliced roast beef at
12.8°C and 26.7°C. The cold-tolerant pathogens, L. monocytogenes, Y.
enterocolitica and A. hydrophila were able to grow on vacuum-packaged

high pH raw meat as shown by Gill and Reichel (1989). Actually the
latter two organisms grew at temperatures of -2°C to lOoC, and the first
at O-lOOC in vacuum-packaged inoculated slices of high pH beef. In an
atmosphere of carbon dioxide, Y. enterocolitica proliferated at 5-lOoC
and the other pathogens only at lOoC. Chicken loaves inoculated with L.
monocytogenes and Pseudomonas fragi allowed growth of both organisms
at 3-11 °C under 50-80% carbon dioxide (Ingham et aI., 1990). Modified-
atmosphere packaging of raw chicken substantially inhibited the aerobic
spoilage flora but allowed L. monocytogenes to proliferate (Wimpfheimer
et al., 1990). Also, modified-atmosphere storage of pre-cooked chicken
nuggets selectively inhibited spoilage bacteria, such as P. jiuorescens, over
pathogens like L. monocytogenes (Marshall et al., 1991).
A practical concern in using modified atmospheres for storage of raw
and cooked products is whether anaerobic or facultative pathogens can
grow before non-pathogenic, spoilage or fermentative organisms, which
would warn the consumers or lower the pH and prevent growth of the
pathogens, respectively (Lambert et aI., 1991b,c). Several studies have
examined toxin production by non-proteolytic C. botulinum spores and
detectable spoilage in raw fish stored in modified atmospheres. Stier et al.
(1981) indicated that spoilage of fish was usually evident before detection
of toxin, while Post et al. (1985) found that toxin sometimes preceded
spoilage of fish in modified atmospheres at 4-26°C. Garcia et al. (1987)
indicated that toxin formation preceded spoilage detection at 8°C and
12°C but followed spoilage at 4°C. In general, it is believed that below
lOoC microbial spoilage precedes botulinum toxigenesis (Genigeorgis,
1985; Lindroth and Genigeorgis, 1986).
14.4.3.3 Oxygen-enriched or oxygen-free atmospheres. Modified atmo-

sphere packaging systems involving high oxygen levels (e.g. 30% carbon
dioxide, 70% oxygen) have been introduced to extend color stability and
delay microbial spoilage of products on display (Gill, 1988, 1990). This
type of packaging, however, is unsuitable for bulk or prolonged storage
because storage life is only doubled and the volume ratio of pack to meat
is 3: 1. In modified-atmosphere packages of low oxygen (carbon dioxide
has displaced most of the air) the red meat rapidly discolors because of
limited amounts of oxygen. Such packaging is more useful for poultry and
seafood (Gill, 1990). Complete elimination of oxygen from the package,
however, may prevent meat discoloration, and microbial shelf-life may be
greatly extended when the air is completely replaced with carbon dioxide
in a controlled-atmosphere package (Egan et aI., 1988; Gill, 1990).
Complete elimination of oxygen may be commercially difficult and use of
high levels of carbon dioxide may present technical problems and require
use of completely impermeable films. Scientists in New Zealand have
reported development of a system (the Captech system) that overcomes

these problems (Gill, 1988, 1990). The system was developed for lamb and
involves packaging of cuts in permeable bags that are heat-shrunk, several
of which are then put in a larger pack of highly impermeable film that is
flushed with excess carbon dioxide and sealed. The permeability of the
inside packs allows penetration of carbon dioxide during storage and
shipment, as well as oxygen during display for formation of oxymyoglo-
bin. Under this system, a storage life of 16 weeks at -1°C has been
reported. Requirements for the success of this controlled-atmosphere
packaging system include reliable evacuation equipment and totally
impermeable pouches made of an aluminum foil laminate with good
sealing and puncture-resistant properties. The system was tested and
found effective in extending the shelf-life of several other muscle foods
and vegetables. It is, therefore, presented as an alternative to freezing for
storage of products when freezing is undesirable (Gill, 1990).
14.4.4 Biopreservation
14.4.4.1 Lactic acid bacteria. Lactic acid producing bacterial starter
cultures are used in fermented meat products where they shorten the fer-
mentation time, achieve products of desirable, distinct and consistent
quality, ensure product safety and extend shelf-life (Smith and Palumbo,
1981, 1983). In addition to its tangy flavor, lactic acid denatures meat
proteins, which affects product texture. The USDA classifies meat starter
cultures as flavoring agents, protectors and developers and, as such,
allows their use in fermented products. In addition to fermented products,
starter cultures are also permitted in bacon where the reduced pH aids in
the depletion of residual nitrite, which reduces formation of nitrosamines
during frying. In case of temperature abuse, growth of the starter culture,
such as L. plantarum in presence of added sugar, also inhibits growth of
C. botulinum with the reduced pH (Tanaka et al., 1985).
The meat starter cultures used predominantly in the USA are strains of
Pediococcus acidilactici and L. lantarum. Micrococci (Micrococcus varians)
or coagulase-negative staphylococci are also used, especially in Europe,
for their ability to reduce nitrate to nitrite, for their unique flavors and for
production of catalase, which decomposes hydrogen peroxide and
prevents quality defects.
Lactic acid bacteria, as starter cultures or natural contaminants, have
also been reported to extend the shelf-life of fresh ground beef, steaks and
poultry meat (Raccach et al., 1979). Although inhibition is believed to be
mostly due to production of acid and reduction in pH, there is evidence
that other factors, such as hydrogen peroxide, bacteriocins, diacetyl, other
unspecified compounds and secondary reaction products, such as hypo-
thiocyanite generated through the action of lactoperoxidase on hydrogen
peroxide and thiocyanate, may contribute to antimicrobial activity

(Daeschel, 1989; Gombas, 1989; Tiina and Sandholm, 1989).
Lactic acid bacteria, however, can also impart undesirable qualities to
products, which should not be fermented, as well as in fermented items.
Heterofermentative lactobacilli can produce hydrogen sulfide and
hydrogen peroxide, which cause deterioration of flavor and color (Juven
et ai., 1988). Lactic acid accumulated into the product may promote
growth of pseudo monads when the product is exposed to oxygen (Nassos
et ai., 1985). Although pseudomonads preferentially metabolize glucose,
they can also use amino acids and lactic acid when the glucose is
depleted.
Another problem to be avoided is potential formation of histamine,
which has been associated with several outbreaks of foodborne illness
(Taylor, 1986). It is formed by decarboxylation of histidine and may be
found in spoiled as well as in fermented foods (Smith and Palumbo,
1981). Thus, starter cultures should be screened for absence of excessive
amino acid decarboxylase activity, which results in formation of biogenic
amines (Anderson, 1988). It is also important that when mixed cultures
are used they should be screened for symbiotic rather than antagonistic
relationships (Houle et ai., 1989).
14.4.4.2 Bacteriocins. Although fermented foods have been in existence

for thousands of years, and commercial use of starter cultures in meat fer-
mentation was initiated a few decades ago, in recent years there has been
increased interest in research to develop muscle food-preservation systems
based on the metabolic activities of lactics. This interest has focused
mostly on various strains producing bacteriocins. Research on the subject
has dealt with isolation of inhibitors, determination of antimicrobial
activity and conditions supporting such activity, as well as basic studies on
biotechnological or other modifications to enhance production and
activity of bacteriocins. The long-range objective is to develop cultures,
extracts or purified compounds that could be approved for use in pre-
servation of sensitive foods such as muscle products. The term often used
to describe this concept is biopreservation or 'lactic antagonism'.
Bacteriocins, which have been isolated at an increasing rate, are effec-
tive inhibitors or inactivators of microorganisms and they are produced
by a variety of bacteria (Daeschel, 1989). They include nisin (Hurst, 1981),
lactosin 27 (Upreti and Hinsdill, 1975), acidolin (Hamden and Mikolajcik,
1974), diplococcin (Davey and Richardson, 1981), lactasin (Barefoot and
Klaenhammer, 1984), pediocin A (Daeschel and Klaenhammer, 1985),
pediocin PAC 1.0 (Pucci et ai., 1988) and pediocin AcH (Bhunia et ai.,
1988). They have been produced by lactobacilli such as L. fermentans
(DeKlerk and Smit, 1967), L. heiveticus (Upreti and Hindsdill, 1975;
Joerger and Klaenhammer, 1986), L. acidophilus (Muriana and Klaen-
hammer, 1987), L. sake (Schillinger et aI., 1991) and L. plantarum (West

and Warner, 1988) and by pediococci, including p, acidilactici (Bhunia et
al., 1988; Hoover et al., 1988) and P. pentosaceus (Graham alld McKay,
1985; Daeschel and Klaenhammer, 1985).
The bacteriocins are peptide-containing compounds and attach to
specific receptors on sensitive microbial cells. The organisms that produce
them have plasmid-borne determinants for their formation (Daeschel,
1989). They are effective inactivators or inhibitors against various Gram-
positive bacteria including pathogens, such as L. monocytogenes, at least
in culture media (Hoover et ai., 1988). They are less effective against
Gram-negative bacteria. Their antimicrobial activity, however, appears to
be strain- and species-specific. As peptides, bacteriocins are sensitive to
proteolytic enzymes and their sensitivity to heat must also be examined.
Pediocin AcH is resistant to heat and organic solvents and is active in a
wide pH range; however, it is inactivated by enzymes, such as trypsin and
proteases but not by lipase (Bhunia et al., 1988).
Bacteriocins produced by Lactococcus lactis subsp. lactis, La. sake, Ca.
piscicola, Pe. acidilactici and Pe. pentosaceus have inhibited several food-
borne pathogens, including B. cereus, C. perjringens, S. aureus, L. mono-
cytogenes, A. hydrophila and E. coli 0157:H7, and Salmonella spp.
(Spelhaug and Harlander, 1989; Nielsen et al., 1990; Ahn and Stiles, 1990;
Motlagh et al., 1991; Schillinger et al., 1991; Yousef et al., 1991). Bacter-
iocin production may occur independent of carbohydrate fermentation
and may reduce L. monocytogenes numbers during fermentation. The
bacteriocin, however, was not effective in eliminating L. monocytogenes
that had survived the heating process (Berry et al., 1990, 1991).
Also the bacteriocin, nisin, is produced by strains of Streptococcus
( Lactococcus) lac tis and is approved for use in cheeses to inhibit gas pro-
duction by clostridia (Hurst, 1981). The peptide was found to be inhibi-
tory against C. botulinum in culture broth (Scott and Taylor, 1981). Its
antibotulinal activity, however, was decreased in cooked meat medium
and in meat products, where it probably binds to meat particles and is
neutralized (Rayman et al., 1983; Taylor et al., 1985).
In addition to bacteriocins, other (mostly unidentified) compounds
produced by microorganisms have been demonstrated to have anti-
microbial activity. One such compound is reuterin, which is believed to be
a low-molecular-weight, non-protein product of the heterofermentative
organism La. reuteri (Daeschel, 1989). It was found to inhibit many types
of microorganisms, including Salmonella, Shigella, Staphylococcus, Listeria
and Clostridium. Addition of 50 units.g- 1 of ground beef was cidal to coli-
forms in refrigerated storage (Daeschel, 1989). Another such antibiotic
substance is bulgarican produced by La. bulgaricus strain DDS14 (Reddy
et ai., 1983). Still another antimicrobial substance was recently isolated by
Chen and Tseng (1989) from Monascus spp. such as M. pilosus and M.
purpureus 31499. The crude isolate was heat-resistant and was thus con-
sidered as a non-peptide. It inhibited S. aureus at a concentration of
2 mgr 1, and it decreased total counts in pork at 0-4°C. It is hoped that
effective biopreservation systems that may eventually be developed will
undergo relatively easy regulatory approval and consumer acceptance as
preservatives of muscle products and other foods, because of their origin
from natural food-grade microorganisms.
14.5 Summary
The highly nutritious and palatable muscle foods can be contaminated

easily with microorganisms from the environment during the various
stages of harvest, slaughter, processing, storage, distribution and prepara-
tion. Proliferation of contaminating microorganisms results in spoilage,
making the products unpalatable, or in the development of foodborne
illness from pathogenic microorganisms. In addition to the traditional
pathogens, the more recently recognized pathogenic bacteria such as L.
monocytogenes have become of major concern in muscle foods. These
newer pathogens are unique because of their ability to proliferate under
adverse conditions of storage, such as refrigeration and vacuum, and
because they can be fatal when infecting immunocompromized segments
of the population. In addition to recently recognized concerns for certain
bacteria as potential food borne pathogens, modern sanitation, packaging
and storage practices may result in dominance of lesser-known micro-
organisms by causing spoilage of muscle foods, other than that of tradi-
tional putrefaction and souring. These concerns are exemplified by
consumer demands, not only for food safety but also for improved
product quality and palatability achieved with as little processing as
possible. Current responses to these concerns include research develop-
ments in animal carcass and meat decontamination processes, modified-
atmosphere packaging, and biopreservation. The objective is to extend
product shelf-life and safety by maintaining palatability and inhibiting or
inactivating pathogens. These factors could then be used as hurdles or
barriers of microbial growth in a HACCP system for perishable muscle
food products.
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15 Rapid methods for measurement and enumeration
of microbial contamination
D.Y.C. FUNG
15.1 Introduction
Measurement and enumeration of microbial contamination in meat,

poultry, and fish products have been the subject of research since the
beginning of bacteriology. Conventional methods of detection, enumera-
tion, identification and characterization of microbes are described in refer-
ence books such as Compendium of Methods for the Microbiological
Examination of Foods (Vanderzant and Splittstoesser, 1992), Official
Methods of Analysis of the AOAC (AOAC, 1990), Bacteriological Analy-
tical Manual (FDA, 1992), Standard Methods for the Examination of Dairy
Products (APHA, 1985) and Modern Food Microbiology (Jay, 1992).
The advantages of conventional methods are their long history of devel-
opment and usage and recognition by regulatory agencies, nationally and
internationally. The disadvantages of some of the conventional methods
are the time required in performing the tests; the time delay in obtaining
results; labor intensity; cumbersome methodology; and costliness in terms
of usage of a large number of tubes, plates, bottles, incubation spaces and
so on.
Applied microbiologists are always interested in developing procedures
and methods for the early isolation, detection, characterization and enu-
meration of microorganisms and their products in clinical, food, industrial
and environmental samples. The search for rapid methods started almost
immediately with the development of applied microbiology at the tum of
the century.
Interest in the field of rapid methods and automation in microbiology
has been growing steadily on an international scale in recent years. The
First International Symposium on Rapid Methods and Automation in
Microbiology was held in Stockholm, Sweden in 1973. Subsequently
symposia were held in: Cambridge, England (1976); Washington DC
(1981); Berlin (1984); Florence, Italy (1987); and Helsinki, Finland (1990).
The Seventh International Congress on Rapid Methods and Automation in
Microbiology and Immunology was held in September 1993 in London,
UK. The proceedings of the above symposia are published in book form
by Heden and Illeni (1975a,b), Johnson and Newsom (1976), Tilton
RAPID METHODS 405
(1982), Habermehl (1985), Balows et al. (1989), Vaheri et al. (1991) and
Spencer et al. (1994).
Other important publications on the subject of rapid methods for
medical specimens, water, food, industrial and environmental samples are
in a series of papers by Fung and colleagues (Cox et al., 1984, 1987a,b;
Fung, 1980, 1985, 1989, 1991, 1992; Fung & Cox, 1981; Fung et al., 1984,
1987, 1988b, 1989; Goldschmidt & Fung, 1978, 1979), and books such as
Mechanizing Microbiology (Sharpe and Clark, 1978), Foodborne Micro-
organisms and Their Toxins: Developing Methodology (Pierson and Stern,
1986), Rapid Methods in Food Microbiology (Adams and Hope, 1989), and
Instrumental Methods for Quality Assurance in Foods (Fung and
Matthews, 1991).
The purpose of this chapter is to review the basic principles and prac-
tical applications of a variety of instruments and procedures that are
directly and indirectly related to improved methods for microbiology in
quality assurance and research in food science and technology.
The six major areas of concern in food and meat microbiology are: (i)
sample preparation; (ii) total viable cell counts; (iii) differential viable cell
counts; (iv) pathogenic microorganism monitoring; (v) cell mass and com-
ponent monitoring; and (vi) enzyme and toxin monitoring. Researchers
have tackled all these areas in terms of improving methods for food and
meat microbiological analyses.
15.2 Improvements in sampling and sample preparation
In order to carry out a microbiological analysis, the scientists must first

treat the food sample such that meaningful microbiological evaluations
can be made. The subject of food sampling plans and initial sample
handling as well as preparation of food homogenates is described in the
aforementioned reference books.
15.2.1 Stomacher
One of the most useful instruments developed for sample preparation is
the Stomacher (Tekmar, Cincinnati, Ohio). This instrument is designed to
massage food samples in a sterile bag. The food sample (e.g. meat, poultry
or fish) is first placed in the sterile disposable plastic bag and appropriate
sterile diluents are added. The bag with the food is placed in the open
chamber. After the chamber is closed, the bag is then massaged by two
paddles for a suitable time period, usually from 1-5 min. No contact
occurs between the instrument and the sample. During massaging, micro-
organisms are dislodged into the diluent for further microbiological
manipulation. Massaged slurries are then used for microbiological analysis
Figure 15.1 The author with a Stomacher and Stomacher bag with sample.
(Figure 15.1). Sharpe and Jackson (1975) and Emswiler et al. (1977) have
shown that satisfactory results can be obtained by this method compared
with the conventional blending of foods. The advantages of the Stomacher
instrument compared with conventional blending are: (i) use of disposable
sterile bags, thus eliminating the need for large numbers of glass or metal
jars to be cleaned and continuously resterilized; (ii) there is no generation
of aerosols, such as those created by conventional blending (this becomes
crucial when potentially dangerous microorganisms such as Salmonella
and Listeria are in the food sample); (iii) there is no heat generation
during 'stomaching' compared with blending, which may generate con-
siderable amounts of heat during prolonged operation; (iv) the bag with
the homogenized sample can be used as a storage bag for time-course
studies; and (v) the ease of operation. The disadvantages include possible
breakage of the bag by sharp objects in samples such as bones, hard
fibers, nut shells, metal pieces, wood chips, straws, and so on, and the
initial cost.
15.2.2 Hand roller

In the author's laboratory a hand roller (Figure 15.2) procedure was
developed and tested against the stomacher instrument in recovery of
Clostridium perfringens and total count in meats (Ali and Fung, 1991).
The hand roller was rolled across a stomacher bag containing meat at the
RAPID METHODS 407
Figure 15.2 Hand roller.
Figure 15.3 Massaging meat sample with a hand roller.
rate of three strokes per second for 1.5 min (Figure 15.3). The data indi-
cated that both the roller procedure and the stomacher procedure gave
similar counts (P > 0.05) for C. perfringens from meats (2.2 x 104 vs.
2.3 X 104, n = 10) as well as for aerobic plate counts from meat
(1.3 X 106 vs, 1.3 X 106 , n = 10). The roller is much cheaper than the
stomacher instrument and readily available in hardware stores.
15.2.3 Gravimetric Diluter or Diluflo

Another new instrument that holds promise for sample preparation is the
Gravimetric Diluter (Spiral Biotech, Bethesda, Maryland). Food and meat
microbiologists almost always have to dilute a food sample before micro-
biological testing. One of the most time-consuming procedures of routine
microbiological work is to measure aseptically a sample of food (e.g. 5 g
meat) and then aseptically add an exact amount of sterile diluent (e.g.
45 ml) to make a desired dilution (1:10). With the Gravimetric Diluter,
the analyst needs only to measure an amount of food (e.g. 5.3 g), asepti-
cally place it into a prepared Stomacher bag or a sterile blending jar, set a
desired dilution (1: 10 dilution), then set the instrument to deliver the
appropriate amount of sterile diluent (e.g. 47.7 ml). Thus, the dilution
operation can be performed automatically. The dilution factor can be
chosen by the analyst to satisfy the need (1:10, 1:50, 1:100, etc.) simply by
Figure 15.4 Dilullo (used with permission from Spiral Biotech Inc., Bethesda, Maryland).
RAPID METHODS 409
punching the correct numbers into the instrument. Manninen and Fung
(1992a) evaluated this instrument and found that, depending on the
volume tested, the accuracy of delivery for most samples was in the range
90-100%. A new version of this instrument is called Diluflo (Figure 15.4)
and has been in use satisfactorily in the author's laboratory since 1992.
15.2.4 Other methods

Several devices have been designed to remove meat samples for micro-
biological analysis. For example, the stainless steel template of Yokoya
and Zuluka (1975), the surgical dermatome-like assemblage of Davidson
et al. (1978), the coring device of Emswiler et al. (1978), and the sterile
core system of Hone et al. (1975) were all designed for aseptically
removing meat from specimens for microbiological analysis.
A review by Lee and Fung (1986) documented many of the useful ways
to obtain surface samples from meats and other foods. Fung et al. (1980)
developed an adhesive tape method that can effectively 'peel' viable cells
from meat surfaces and later deposit them on agar surfaces to obtain
viable cell counts of meat surfaces.
Truly automated sample-preparation systems belong to the field of
robotics. The excellent book Laboratory Robotics: A Guide to Planning,
Programming, and Applications by Hurst and Mortimer (1987) should be
consulted on this subject.
15.3 Alternative methods for viable cell count procedure
The conventional viable cell count or Standard Plate Count method is

time-consuming both in terms of operation and collection of data. Several
methods have been explored to improve the efficiency of the viable cell
count procedure.
15.3.1 The spiral plating method

The spiral plating method is an automated system for obtaining viable cell
counts (Spiral Biotech, Bethesda, Maryland). The instrument can spread a
liquid sample on the surface of agar contained in a petri dish in a spiral
shape (the Archimedes spiral) with a concentration gradient starting from
the center and decreasing as the spiral progresses outward on the rotating
plate. The volume of liquid deposited at any segment of the agar plate is
known. After the liquid containing the microorganisms is spread, the agar
pla~e is incubated overnight at an appropriate temperature for the
colonies to develop. The colonies appearing along the spiral pathway can
be counted either manually or electronically.
Figure 15.5 Spiral plater. Samples are spread on the plate from the center to the perimeter
as the stylus moves outwards on a rotating plate and deposits liquid at a decreasing volume
(used with permission from Spiral Biotech Inc., Bethesda, Maryland).
Figure 15.6 Growth of colonies from spiral plater. The six plates contain six separate
samples serially diluted with the lowest density sample on the top left and the one with
highest density on the bottom far right (used with permission from Spiral Biotech Inc.,
Bethesda, Maryland).
The basic spiral system is shown in Figure 15.5. The stylus in the center
is first placed in a liquid sample contained in a small cup to the left and a
sample is obtaihed through suction by a vacuum source shown on the
right. The stylus is then placed on the agar surface. At the start of the
operation, the platform holding the petri dish starts to rotate at a
constant rate and the stylus moves outward. Simultaneously the liquid
sample is forced out at a decreasing volume as the stylus moves outward.
RAPID METHODS 411
After the stylus reaches the edge of the petri dish, it is automatically
lifted. The plate is then incubated. The results of growth of colonies on six
agar plates from six serial dilutions (1:1 0 dilution each step) of a dense
bacterial culture are shown in Figure 15.6. It is evident that the spiral
system can handle bacterial cultures of a variety of densities.
15.3.1.1 Counting spiral-plated colonies. The colonies along the spiral can
be counted manually. The agar plate with growth is placed on top of a
template marked with segments. The analyst counts a segment of the
template and multiplies the number with a designated factor for the parti-
cular area, converting the count into organisms per milliliter of sample.
This method is simple but it requires some training and judgement of the
analyst. To make counting even more automated, a laser counter has been
developed along with appropriate computer software for its operation.
The plate is placed on the stage of the counter, the instrument is set, and
the laser counter will automatically count the number of colonies and
convert the number to organisms per milliliter. The spiral system has been
used in the USA for a variety of foods with satisfactory results (Schalk-
owsky, 1986). The laser counter is also designed to count colonies on con-
ventional agar plates, making the instrument very versatile in a food
microbiology laboratory.
Manninen et af. (1991) evaluated the spiral system and laser colony
scanner for enumeration of microorganisms and found excellent correla-
tion between pour plate and spiral plate using both manual and laser
counting procedures for bacteria (Table 15.1). As for yeasts and molds, a
similar observation was made except for Rhizopus o/igosporus (Table 15.2).
Manninen and Fung (1992b) also evaluated the spiral plater and laser
colony scanner for enumeration of microorganisms in meat and devised a
convenient protocol combining the conventional aerobic plate-count
Table 15.1 Comparison of pour plate and spiral plate counted manually
and by laser for bacterial cultures
Pour plate Spiral plate

Test cultures Manual Laser Manual Laser
LoglOcfu. ml- 1
Escherichia coli 8.86 8.85 8.73 8.85

Salmonella enteritidis 8.76 8.66 8.78 8.92
Pseudomonas aeruginosa 8.00 8.00 8.00 8.00
Staphylococcus aureus 8.04 7.78 8.18 8.18
Lactobacillus plantarum 9.48 9.40 9.60 9.69
Streptococcus spp. 7.73 7.66 8.00 8.08
Bacillus cereus 7.26 7.15 7.15 7.26
Micrococcus luteus 7.40 7.32 7.51 7.57
Table 15.2 Comparison of pour plate and spiral plate counted manually
and by laser for yeast and mold cultures
Pour plate Spiral plate

Test cultures Manual Laser Manual Laser
LoglOcfu.ml~l
Yeasts
Candida macedoniensis 7.11 6.97 7.30 7.38
Hansenula subpelliculosa 5.00 4.99 5.11 5.15
Hansenula wingei 7.04 7.04 7.15 7.00
Saccharomyces cerevisiae 6.38 6.43 6.28 6.46
Saccharomyces tragi/is 7.34 7.36 7.23 7.34
Molds
Aspergillus fiavus 7.04 7.41 7.23 7.56
Penicillium camemberti 6.89 6.86 7.04 7.48
Rhizopus oligosporus 6.51 7.54 6.59 7.49
method with the spiral system to evaluate surface microbial loads on pork
loins. In this procedure, a surface (25 cm2 , 50 cm2 , or 125 cm2) is swabbed
and then placed in 9 ml of sterile buffer. For each sample both the con-
ventional and the spiral plating method were used. For conventional
plating 1 ml was used. For spiral plating the liquid was plated without
further dilution. In this protocol when the number of cells was too
numerous to count by conventional plating (more than 300 per plate), the
spiral-plated sample will provide an accurate count. Conversely, when the
number of cells is very low (1-250 cells per plate), the conventional pour
plate method will provide an accurate count, while the spiral plated
sample will have virtually no colonies. Using four plates (two plates are
really needed), a large range of microbial loads on meat surfaces can be
counted effectively.
15.3.1.2 The Autoplater. A new version of the spiral plater was recently
introduced named 'Auto plater' (Figure 15.7). In the instrument, an
analyst needs only to present the liquid sample and the instrument com-
pletely and automatically processes the sample, including resterilizing the
unit for the next sample.
One of the problems of the spiral system is the clogging of the dispens-
ing stylus by food particles. Konuma and Kurata (1982) modified a Sto-
macher bag by placing a filter in the bag so the homogenized liquid
poured from the bag will be free of particles. The liquid presented to the
spiral system will not clog the stylus. The spiral system is listed as an
alternative method for the examination of foods in the Standard Methods
for the Examination of Dairy Products (APHA, 1985).
RAPID METHODS 413
Figure 15.7 The AutoPlater (used with pennission from Spiral Biotech Inc., Bethesda,
Maryland).
15.3.2 The Isogrid system

Another method for viable cell count is the Isogrid system (QA Labora-
tories Ltd., San Diego, California). This system consists of a square filter
with hydrophobic grids printed on the filter to form 1600 squares for each
filter. Food samples are weighed, blended and enzyme-treated before
passage through the membrane filter containing the hydrophobic grids.
The filter is then placed on agar containing a suitable nutrient for growth
of the bacterium, yeast or mold. The hydrophobic grids prevent colonies
from growing further than the square grids; thus all colonies have a
square shape. This facilitates counting of the colonies both manually and
electronically. This method has been used successfully to make viable cell
counts for a variety of foods, including milk, meat, black pepper, flour,
peanut butter, mushrooms, rice, shrimp, and oysters (Entis, 1983, 1984,
1986; Entis et al., 1982; Sharpe and Peterkin, 1988). Lin et al. (1984)
developed a trypan blue agar for use in conjunction with the Isogrid
system for the effective enumeration of yeasts and molds from foods.
15.3.3 The Petrifilm system

In the Petrifilm system (3M Co., St. Paul, Minnesota), rehydratable nutri-
ents are embedded into a series of films. When needed, the first layer of the
protective cover is lifted, I ml of liquid sample is introduced to the center
of the unit, and the cover is replaced. A plastic template is placed on the
cover to make a round mold. The rehydrated medium will support the
growth of microorganisms after a suitable incubation time at a suitable

temperature. The colonies can be counted directly in the unit. The unit is
about the size and thickness of a plastic credit card, thus providing great
savings of space in storage and incubation. Petrifilm units have been devel-
oped for total bacterial counts, coliform counts, fecal coliform counts, and
yeast and mold counts. Petrifilm has been used successfully to count
organisms from milk and meat (Ginn et aI., 1986; Smith et al., 1986). Fung
et al. (1987) obtained a 0.99 correlation coefficient comparing Petrifilm
system with the conventional viable cell-count method for seafood analysis
for mesophiles (Table 15.3). Bishop and Juan (1988) found that the Petri-
film technique was not statistically different from the agar pour plate
method for enumeration of psychrotrophs in raw milk samples.
The advantages of the Petrifilm system include its ease of operation,
Table 15.3 Comparison of the standard plate count method

and the Petrifilm method for viable cell counts of shrimp,
perch, cod and whiting"
Colony-forming units.g- 1
Standard plate
Seafood Sample b count Petrifiim SM
Shrimp 1 2.1 x 104 0.9 X 104

2 9.3 X 103 6.8 X 103
3 1.5 X 104 1.2 X 104
4 4.4 X 104 2.0 x lif
5 2.0 x 104 0.9 X 104
Perch 1 1.4 x 103 1.2 X 103

2 5.4 X 102 3.7 X 102
3 8.0 X 102 4.8 X 102
4 5.0 X 102 3.2 x lQ2
5 1.0 x 103 1.2 X 103
Cod 1 1.2 x 102 1.1 X 102

2 2.2 X 102 2.4 x lQ2
3 1.0 x 104 1.0 x lif
4 2.1 x 104 2.3 X 104
5 1.2 X 104 1.2 x 1if
Whiting 1 3.0 x 102 3.1 x 1Q2

2 5.8 x 102 5.6 X 102
3 1.2 X 103 1.1 X 103
4 5.4 X 102 5.2 x 1Q2
5 8.8 x 102 8.4 X 102
"Source: Fung et al. (1987).

bSamples were massaged in a stomacher for 1 min in sterile
diluent. Viable cell counts were made according to standard
method. Incubation time was 48 h at 32°C. All samples were
done in duplicate. Correlation coefficient between the two
methods is r = 0.99.
RAPID METHODS 415
savings of storage and incubation space, its long shelf-life because of the
use of dehydrated medium in the film, and independence from heat treat-
ment compared with the conventional agar pour plate method.
15.3.4 The Redigel system

Another convenient system is the Redigel system (RCR Scientific Inc.,
Goshen, Indiana). This system consists of sterile nutrients with a pectin
gel in a tube. A 1 ml sample is first pipetted into the tube. After mixing,
the sample is poured into a special petri dish previously coated with
calcium. When the liquid comes in contact with the calcium, a Ca-pectate
gel is formed and swells to resemble conventional agar. After an appro-
priate incubation time at an appropriate temperature, the colonies can be
counted. Fung and Chain (1991) compared 18 different foods (i.e. pas-
teurized milk, raw milk, Cheddar cheese, chocolate chips, rice, wheat
germ, corn meal, whole wheat, flour, peanuts, pecans, ground beef,
chicken, ground black pepper, thyme, broccoli, mushrooms and turkey
pot pie, 20 samples each) and obtained a correlation of 0.964 (Figure
15.8). Roth (1988) reported a comparative analysis of nine different non-
dairy and dairy food products and found that the temperature-indepen-
dent pectin gel method (Redigel) provided statistically significant results
compared with the conventional method. Also Roth and Bontrager (1989)
evaluated the coliform count of cream, Cheddar cheese, cottage cheese,
homogenized milk, raw milk, sour cream and yogurt by the Redigel
method and the conventional pour plate method and again found statisti-
cally significant results.
FOOD MEANS
(N- 17)
R
G
•
R=.964
8M
Figure 15.8 Comparison of Redigel method and conventional method.

The four methods already mentioned have potential as alternatives to

the conventional agar pour plate method. Chain and Fung (1991) made a
comprehensive analysis of all four methods against the conventional
method on seven different foods (i.e. skinless chicken breast, fresh ground
beef, fresh ground pork, packaged whole shelled pecans, raw milk, thyme
and whole wheat flour, 20 samples each) and showed that the new systems
and the conventional method were highly comparable and exhibited a
high degree of accuracy and agreement (r = 0.95+).
15.3.5 The Direct Epifluorescent Filter Technique

Another alternative viable cell count method is the Direct Epifluorescent
Filter Technique (DEFT). In this method, the sample is first passed
through a filter that retains the microorganisms, the filter is then stained
with acridine orange, and the slide observed under ultraviolet microscopy.
'Live' cells usually stain orange-red, orange-yellow, or orange-brown,
whereas 'dead' cells fluoresce green. The slides can be read by the eye, but
it is a tiring and time-consuming process. Alternatively, the slides can be
read by a semi-automated counting system marketed by Bio-Foss. A
'viable cell count' can be made in less than an hour. With the use of an
image analyzer, an operator can count 50 DEFT slides per hour (Petti-
pher, 1986). This method has been used satisfactorily for counting viable
cells in milk and other food samples such as fish (Pettipher, 1989; Petti-
pher and Rodrigues, 1980, 1982; Pettipher et aI., 1980).
Although viable cell counts have been satisfactorily made by the DEFT
test for milk samples, some investigators noted that cells treated by heat,
irradiation and so on, can also fluoresce orange even if they are dead. The
exact mechanism for the phenomenon is not known. Sjoberg et al. (1990)
actually used this phenomenon to ascertain the irradiation status of spices.
In their study, they found that non-irradiated samples had high DEFT
counts as well as high aerobic plate counts, whereas irradiated samples
had high DEFT counts but very low aerobic plate counts. By observing
the difference between the DEFT orange cell count and aerobic viable cell
count, one can ascertain whether spices have been treated by irradiation.
A somewhat similar method to estimate the viability of cells treated
with chemicals was described by Czechowski and Banner (1989), who
treated Yersinia enterocolitica and Salmonella typhimurium cells with
hypochlorite for a few minutes, after which the cells were exposed to
0.004% nalidixic acid and incubated for 18 h at 25°C. Since nalidixic acid
allows live cells to grow but not to divide, one can estimate the popula-
tion of live cells vs. dead cells by staining these cells with acridine orange
and observing them under fluorescence microscopy. Elongated orange
cells in this system are those that were not killed by the hypochlorite
solution.
RAPID METHODS 417
Anaerobic systems are very important in applied microbiology and

clinical microbiology, yet in food microbiology most food scientists avoid
working in this area because of the equipment and cumbersome proce-
dures involved. In the author's laboratory a simple double tube method
was developed, which has been shown to be surprisingly easy to use and
yet effectively recovers anaerobic organisms that are important in food
microbiology.
15.3.6 Double-tube method

The 'Double tube' is a simple, rapid and convenient method developed for
the cultivation, recovery and enumeration of anaerobic bacteria such as
Clostridium spp. (Fung and Lee, 1981). A thin layer of agar that is pro-
tected from air is created by inserting a smaller glass tube (diameter,
16 mm; length, 150 mm) into a larger tube (diameter, 25 mm; length,
150 mm) containing warm (48°C) agar ('TSC agar') and the test sample.
Clostridium perfringens will form large, black colonies in this system after
~ lO h of incubation at 42°C (Figure 15.9). The system can also be used
for the recovery of Clostridium sporogenes. Ali et al. (1991), when com-
paring several rapid methods for the isolation and enumeration of strains
of C. perfringens in meat, concluded that the recovery of three strains was
significantly higher (P < 0.05) using the double-tube method than with
the other three methods tested. Subsequently, Ali and Fung (199lb, 1992)
showed that the system is very useful for the enumeration of C. perfrin-
gens in ground beef and ground turkey.
TSC Agar
- LargerGI...
Tub. with
Scraw Cap
Smaller
Black Colonia. Glas. Tuba
of Clostridium
perfrlngens
Figure 15.9 Double tube.

A modified tube method for the cultivation and enumeration of anaero-

bic bacteria was reported by Ogg et al., (1979) and is commercially avail-
able as the Lee Tube system. The system consists of an inverted tube
within a test tube. Samples and selective agar are introduced into the
cavity between the double wall. A variety of strict anaerobes can grow in
this simple system.
These methods for viable cell counts of microorganisms in foods
provide an intermediate step toward replacing the cumbersome conven-
tional methods. These rapid methods need to be tested in more labora-
tories with more classes of foods before they can completely replace the
time-honored standard plate count method.
15.4 New methods for estimation of microbial populations
In the previous section a variety of methods that improve upon the viable
cell count procedure were described. Many of those methods, however,
still rely on the growth of the microorganisms to form visible colonies for
counting, which takes valuable time. Many methods have been developed
to estimate the total number of microorganisms by parameters other than
the viable colony count. In order for a new method to be acceptable, it
must be correlated directly with the total viable cell count. Thus, new
methods need standard curves correlating parameters, such as the adeno-
sine triphosphate (ATP) level, detection time of electrical impedance or
conductance, generation of heat, radioactive CO 2 , and so on, with viable
cell counts in a series of samples. In general, the larger the number of
viable cells in the sample, the shorter the detection time of these systems.
A scattergram is then plotted and used for further comparison of
unknown samples. The basic assumption is that as the number of micro-
organisms increases in the sample, these physical, biophysical and bio-
chemical events will increase accordingly. This assumption mayor may
not be exactly correct but it serves as a functional way to estimate indir-
ectly the number of microorganisms.
Theoretically, these methods can detect as low as one viable cell in the
sample if the incubation period is long enough (days or weeks). On the
practical side, usually the limit is 104 cells.ml- l . When a sample has 106 _
10 7 organisms.mr l detection can be achieved in about 4-6 h.
15.4.1 ATP estimation

All living things use A TP. In the presence of a firefly enzyme system
(luciferase and luciferin system), oxygen and magnesium ions, ATP will
facilitate the reaction to generate light. The amount of light generated by
this reaction is proportional to the amount of A TP in the sample, thus the
RAPID METHODS 419
light units can be used to estimate the biomass of cells in a sample. The
light emitted by this process can be monitored by a variety of fluorimeters
such as LKB-Wallac (Turku, Finland), Turner Designs (Mountain View,
California), Lumac (Landgraaf, the Netherlands), Luminometer
(Dynatech Laboratories, Chantilly, Virginia), BioTrace (San Diego, Cali-
fornia), and Enliten-Promega (Madison, Wisconsin). Furthermore, these
procedures can be automated for maximum handling of large numbers of
samples. Some of the instruments can detect as little as 10 2 -10 3 fg ATP.
The amount of A TP in one colony-forming unit has been reported as
0.47 fg with a range of 0.22-1.03 fg. Yeast cells have about 100 times
more ATP than bacterial cells. Using this principle, many researchers have
tested the efficacy of using ATP to estimate microbial cells in foods and
beverages. Littel et al. (1986) indicated that the A TP procedure was able
to predict bacterial levels within 0.5 loglo of the actual count for beef and
chicken samples. Minimum sensitivity is 5 x 104 colony-forming units.g- I
of meat sample. Ward et al. (1986) also found a positive correlation
between the ATP method and the conventional method in evaluating fish
samples. The ATP method has been used to evaluate microbial loads in
meat (Baumgart et al., 1980; Stannard and Wood, 1983a; Kennedy and
Oblinger, 1985), milk (Bossuyt, 1982; Waes and Bossuyt, 1982), water
(Daly, 1974; Levin et al., 1975), fruit juice (Stannard and Wood, 1983b;
Graumlich, 1985), samples in a winery and a brewery (Hysert et al., 1976;
Pavelka, 1987) and sweeteners and syrups (Moritz, 1990).
Lumac (Landgraaf, the Netherlands) markets several models of ATP
instruments and provides customers with test kits with all necessary
reagents, such as a fruit juice kit, hygiene monitoring kit, etc. The
reagents are injected into the instrument automatically and readout is
reported as relative light units (RLUs). By knowing the number of micro-
organisms responsible for generating known RLUs, one can estimate the
number of microorganisms in the food sample. In some food systems,
such as wine, the occurrence of any living matter is undesirable, thus
monitoring of A TP can be a useful tool for quality assurance in the
winery. Recently, much interest has been expressed in using ATP estima-
tion not only for total viable numbers but as a sanitation check. Reviews
on the principles and application of ATP have been made by LaRocco et
al. (1986) and Stannard (1989).
15.4.2 Impedance and conductance measurements

As microorganisms grow and metabolize nutrients, large molecules change
to smaller molecules in a liquid system and cause a change in electrical
conductivity and resistance in the liquid. By measuring the changes in
electrical impedance, capacitance and conductance, scientists can estimate
the number of microorganisms because the larger the number of micro-
organisms in the fluid, the faster the change in these parameters. A

detailed analysis on the subject of impedance, capacitance and con-
ductance in relation to food microbiology has been made by Eden and
Eden (1984).
15.4.2.1 The Bactometer. The Bactometer (Figure 15.10) is an instru-

ment designed to measure impedance changes in foods. Samples are
placed in the wells of a l6-well module. After the module is completely or
partially filled, it is plugged into the incubator unit to start the monitoring
sequence. At first, there is a stabilization period for the instrument to
adjust to the module, then a baseline is established. As the microorgan-
isms metabolize the substrates, changes in impedance increase sharply,
and the monitor screen shows a slope similar to the log phase of a growth
curve. The point at which the change in impedance begins is the 'detection
time', and this is measured in hours from the start of the experiment. The
detection time is inversely proportional to the number of microorganisms
in the sample. By knowing the number of microorganisms per milliliter in
a series of liquid samples and the detection time of each sample, one can
establish cut-off points to monitor certain specifications of the food
products. For example, if one finds that meat with 106 organisms.g- 1 will
result in a detection time of 5 h, then one can use 5 h as a cut-off point
for an indicator that meat samples have fewer than 106 organisms.g- 1. In
newer models of the Bactometer, the screen displays bars with one of
three colors instead of an impedance curve. A red bar signals that the
food being analyzed is 'out of spec', a green bar signals that the food is
'in spec', and a yellow bar signals an 'intermediate caution spec'. These
new developments are designed to be 'user friendly'. Hardware and
software are provided for users to monitor conveniently their food
products so far as impedance microbiology is concerned.
Impedance methods have been used to estimate bacteria in milk, dairy
products, meats, and other foods (Eden and Eden, 1984; Waes and
Bossuyt, 1984; Zindulis, 1984; Bishop and White, 1985). Of particular
Figure 15.10 The Bactometer. On the left side is a temperature-controlled incubator. In the
center is the computer unit with screen display. At the right-hand side is the printer for hard
copies (used with permission from bioMerieux Vitek Inc., St Louis, Missouri).
RAPID METHODS 421
interest is the application of this method for determining the shelf-life

potential of pasteurized whole milk by Bishop et a/. (1984).
15.4.2.2 The Malthus system. An instrument quite similar in principle

and operation is the Malthus system (Malthus Instrument, Crawley,
England) (Figure 15.11). The Malthus system works by measuring the
conductance of the fluid as the organisms grow in the system. It also gen-
erates a conductance curve similar to the impedance curve of the Bact-
ometer; it also uses detection time in monitoring the density of the
microorganisms in the food. Furthermore, the screen also displays red,
green and yellow bars to signify 'fail', 'pass' and 'caution' levels of micro-
organisms in particular food products. The major difference between the
two systems, besides the scientific principle (impedance v. conductance), is
the incubation units. In the Bactometer system, the size of the well (about
2 ml in capacity) in the l6-well module is fixed. No modification is
possible because the module is designed to fit into the incubator chamber.
The Malthus system, however, allows analysts to choose three sizes
ranging from 2-100 ml samples, depending on the sample involved.
Another important difference is that the modules of the Bactometer are
disposable, whereas the jars, tubes and electrodes of the Malthus system
are autoclavable and reusable. In terms of performance, the systems are
equivalent in sensitivity and detection time.
Figure 15.11 The Malthus 2000 system. Samples are placed in the incubator on the left.
Conductance is measured by the instrument on the right. The unit is linked to a computer for
processing of data (used with permission from Malthus Instrument, Crawley, England).
The Malthus system has been used for microbial monitoring of brewing
liquids (Day, 1983; Kilgour and Day, 1983; Evans, 1985), milk (Visser and
de Groote, 1984a,b) and fish and seafoods (Ogden, 1986; Gibson and
Hobbs, 1987; Gibson and Ogden, 1980) and hygiene monitoring
(McMurdo and Whyward, 1984).
Besides estimating viable cells in foods, both the Bactometer and the
Malthus systems can detect specific organisms by the use of selective and
differential liquid media. New developments of these two systems are
constantly being made. For example, the Malthus system has developed
a tube system to detect CO 2 production by yeast using indirect con-
ductance measurements. They also introduced disposable units in the
system.
15.4.3 Radiometry and calorimetry
15.4.3.1 The radiometric method. The radiometric method was developed

to monitor the production of radioactive CO 2 by microorganisms in a
sample containing a radioactive substrate, such as glucose. The theory
behind this method is that, as microorganisms metabolize the sugar, they
release radioactive CO 2 and the amount of radioactive CO 2 generated is
in direct proportion to the biomass in the liquid. An instrument such as
the Bactec can be used to monitor radioactive CO 2 , It has been used to
detect bacteria in urine specimens in medical microbiology. The use of
this instrument in foods was reported by Limpi et al. (1974) and Rowley
et al. (1970). Owing to consumer sensitivity about using radioactive
materials, a new generation of Bactec uses infrared to monitor the gen-
eration of CO 2 ,
15.4.3.2 Microcalorimetry. When microorganisms grow, they produce

heat. The heat generated is proportional to the number of microorganisms
in the substrate. By the use of very sensitive instruments, one can measure
minute heat changes and thus estimate the number of microbes in the
sample. This is the principle of microcalorimetry. Studies on microcalori-
metric measurements in ground meat were made by Gram and Sogaard
(1986), who showed that by using an instrument called the BioActivity
Monitor they could estimate bacterial levels in the range of 105_10 8
colony-forming units.g- 1 in less than 24 h.
Limpi et al. (1974) evaluated both radiometry and microcalorimetry.
They used the systems to detect pathogens such as Staphylococcus aureus,
Salmonella typhimurium, Clostridium botulinum spores, and so on, as well
as to monitor the indigenous flora of meat loaf. Their conclusions indi-
cated that both methods have good promise for applied food micro-
biology.
RAPID METHODS 423
15.4.4 Reflectance colorimetry

A new instrument called the 'Omnispec bioactivity monitor system'
(Wescor Inc., Logan, Utah) is a tri-stimulus reflectance colorimeter that
monitors dye-pigmentation changes mediated by microbial activity. Dyes
can be used to produce color changes as a result of pH changes, changes
in the redox potential of the medium, or the presence of compounds with
free amino groups. Samples are placed in micro titer wells and are scanned
by an automated light source with computer interface during the growth
stages (0-24 h). Manninen and Fung (1992c) evaluated this system in a
study of pure cultures of L. monocytogenes in food samples and found
high correlation coefficients of 0.90-0.99 for pure bacterial cultures and
0.82 for minced beef between the colony counts predicted by the colori-
metric technique and the results of the traditional plate count method.
They also showed that detection times for bacterial cultures such as
Enterobacter aerogenes, E. coli, Haffnia alvei and several strains of L.
monocytogenes were substantially (2-24 h) shorter using the instrument
than using the traditional method and concluded that the colorimetric
detection technique employed by the 'Omnispec' system simplifies the
analysis, saves labor and materials, and provides a high sampling capacity.
15.4.5 Limulus amoebocyte lysate and catalase tests

15.4.5.1 The limulus amoebocyte lysate method. The limulus amoebocyte
lysate (LAL) method is a sensitive method for determining the endotoxin
level of lipopolysaccharides of Gram-negative cell walls. Levin and Bang
(1964) first mentioned the use of amoebocyte lysate of horseshoe crab
(Limulus) to detect the presence of endotoxins. At first, the test was aimed
at medical uses to detect pyrogens in medical supplies and for diagnosis of
Gram-negative infections. Gradually, this method has been applied to
detect Gram-negative bacteria in foods. Jay (1989) made a detailed
account of all aspects of the LAL test, including its history, the mechan-
ism of the LAL reaction and the various methods by which the LAL test
can be performed. He reported the use of the LAL test in milk, dairy
products, sugar and meat samples. One advantage of this test is that it
can be completed within an hour and requires very little skill. This test is
quite useful in estimating Gram-negative bacterial populations in foods,
such as meat, fish and poultry, that are cold-stored under aerobic condi-
tions. However, this test is not suitable for foods that contain mainly
Gram-positive organisms, such as fermented foods.
15.4.5.2 The catalase test. The catalase test is another rapid method for
estimation of microbial populations in certain foods. Microorganisms can
be divided into catalase positive and catalase negative. Both groups are
SEALED END
GAS COLUMN
LIQUID COLUMN
PASTEUR PIPETTE
Figure 15.12 Catalase detection tube.
important in food microbiology; however, under certain food-stonge con-

ditions, a certain group predominates. Most perishable foods (commercial
as well as domestic) are cold-stored under aerobic conditions. The organ-
isms causing spoilage of these foods are psychrotrophs. The predominant
psychrotrophic bacteria are Pseudomonas spp., which are strongly catalase
positive. Other important psychrotrophs such as Micrococcus, Staphylo-
coccus and a variety of enterics are also catalase positive. Thus, one can
make use of the presence of catalase to estimate the bacterial population.
Fung (1985) described a simple catalase detection tube method for rapid
estimation of bacterial catalase (Figure 15.12). A Pasteur pipette is first
sealed at the narrow end by heat. From the wider end, 0.05 ml of a liquid
(with or without catalase) is introduced, after which 0.05 ml of 3% H 20 2
is added. The liquids are allowed to mix by rapidly moving the pipette in
three circular motions. The entire liquid column is then 'shaken' into the
RAPID METHODS 425
narrow portion of the pipette by a quick jerk of the wrist similar to

shaking a mercury column in a thermometer. After 5 s, the pipette is
inverted. Surface tension holds the liquid column in the narrow part of
the Pasteur pipette. Gas bubbles, when generated, will accumulate at the
narrow tip of the unit. To minimize the effect of various diameters of
Pasteur pipettes, the gas column is expressed as percentage of the total
column (i.e. gas column/total column x 100 = percentage gas column). In
general, catalase-positive bacteria will form gas bubbles when there are
105 colony-forming units.ml- I . As the number of bacteria increases, the
gas column also increases.
Another method to detect catalase activity is by use of an instrument
called the Catalasemeter. The principle is based on the flotation time of a
paper disk containing catalase in a tube containing H 20 2 . The reaction
between catalase and H 2 0 2 generates molecular oxygen, which causes the
paper disk to float. In the presence of a high level of catalase, indicating a
high level of catalase-positive microorganisms, the flotation time will be
short (in seconds). Conversely, the flotation time will be long (100-1000 s)
in the presence of a low catalase concentration. When there is no catalase,
the disk will not float. Wang and Fung (1986) made an extensive study of
Logarithm of Percentage Gas Co lum n

2.0 1.5 1.0 0.5 0
MiQro!'.tQ~CUS ~
~<Il 8
'c
2-
Ol
c: 7
E
0
"-
>- 6
c:
0
0
0
E 5
~
;;;
Ol
0
..J 4
0 2 3 4
~ g~~.ri!.hlTl Flg!~ ! igfl Ti.lTl~ (~ ~~ l
Figure 15.13 Measurement of catalase activity of Micrococcus luteus by the catalasemeter

and gas column method. (From Wang and Fung, 1986, used with permission). Log % gas
column, . ; log flotation time, •
E
~
:::J
u..
~
u :3
"-
E u
:= E
.~
0>
:=
.~
0
..J 0>
0
..J Log CFU = 8 .8680 - 0.3435(2.2067l L09 FT
9.0 9
8.0 8
7
7 .0
6.0 6
5.0 5
4
4 .0
3.0 3
2.0 2
1.0 2.0 3.0 4.0
Loga ri thm Fl otat ion Tim e (sec )
Figure 15.14 Catalase activity of psychrotroph population from cold-stored (70C) chicken
samples in pH 3.3 phosphate buffer using catalasemeter with membrane-filter method. r =
0.93 (p < 0.0001); n = 59. (From Wang and Fung, 1986, with permission.)
the use of catalase to monitor bacterial cultures as well as bacterial popu-

lations on the surface of chicken. The measurement of catalase activity of
Micrococcus luteus by the catalasemeter and gas column method is shown
in Figure 15.13, indicating a direct relationship between the log of the
percentage of gas column vs. the log of the flotation time (i.e. when cells
contain high catalase activity, the flotation time is very short and the per-
centage gas column is very high). Figure 15.14 shows the relation between
cell numbers and flotation time using chicken samples. The major problem
of the catalase system is interference of non-bacterial catalase, such as that
in blood. In the study mentioned, the problem was minimized by acid-
ification of the sample before analysis. Although more research and devel-
opment are needed for the catalasemeter method as well as the catalase
tube method to be successful, the speed of the test (in terms of seconds)
warrants continued investigations.
Monitoring cleanliness of work areas with the catalase test. Yet another
way to use the catalase test is as an index of cleanliness of meat-proces-
sing areas. Preliminary data from the author indicate that a simple swab-
catalase test can determine the degree of cleanliness after clean-up of a
RAPID METHODS 427
meat-processing plant. A moist swab was applied to a 2 x 2 inch area and

then the swab was placed in 2 ml of 3% H 2 0 2 in a test tube. The amount
of bubbles generated in the tube can be scaled as 0, 1 +, 2 +, 3 +, 4 + and
5 +, depending on the activity of the sample. A bacterial count of the
immediate adjacent area is also taken. Results from the author's study
showed that, prior to slaughter, the room had very low catalase activity,
which indicated that a low number of bacteria were present, as well as
blood or other particles that may carry catalase activity. During the
slaughter operation, the catalase activity was high because of meat parti-
cles, blood and bacteria in the environment. After proper clean-up, the
catalase activity in the area fell to the original level. Bacterial counts did
not correlate directly with the catalase activity. This is to be expected
because the swab picks up material, other than bacteria, that can generate
bubbles in the presence of H 2 0 2 . Further work is in progress to ascertain
the value of this simple test for monitoring environmental sanitation.
Differentiation between bovine and bacterial catalase. Furthermore,

catalase-like-activities can be used to differentiate between 'bacteria
catalase' and 'bovine catalase'. In the author's laboratory, it was dis-
covered that 'bovine catalase' is inactivated at 65°C for 2 min, whereas
'bacterial catalase' is not. Thus, by heating a piece of ground beef (2 g) at
65°C for 2 min and then measuring the catalase activity researchers can
estimate the bacterial load of the meat. This method is sensitive at about
log 5 bacteria per gram of ground meat.
Determination of end-point temperature of cooked chicken meat by the

catalase test. The catalase test can be used to determine the end-point
temperature (EPT) of meats. A study of chicken meat (Ang et at., 1993)
indicated that chicken samples with EPT of less than or equal to 68°C
always showed positive reactions and those with EPT of greater than or
equal to noe always showed no reaction. At 71°C (the USDA regulation
for cooked poultry meat), 97-99% of the time the catalase test is
negative. Thus, the catalase test offers a simple means for determination
of EPT between 69°C and 71°C for pre-cooked chicken breast and leg
meat.
15.5 Miniaturized microbiological techniques
Identification of microorganisms is an important part of quality assurance

and control programs in the food industry. The author has developed
many methods to reduce the volume of reagents and media (from 5-10 ml
to about 0.2 ml) for microbiological testing in micro titer plates. The basic
components of the miniaturized system are the micro titer plates for test
cultures, a multiple inoculation device, and containers to house solid

media (large petri dishes) and liquid media (another series of microtiter
plates). The procedure involves placing liquid cultures to be studied into
sterile wells of a microtiter plate to form a master plate. Each microtiter
plate can hold up to 96 different cultures, 48 duplicate cultures, or various
combinations as desired. The cultures are then transferred by a sterile
multi-point inoculator (96 needles protruding from a template) to solid or
liquid media. Each transfer represents 96 separate inoculations in the con-
ventional method. After incubation at an appropriate temperature, the
growth of cultures on solid media or liquid media can be observed and
recorded and the data can be analyzed. These miniaturized procedures
save a considerable amount of time in operation, effort in manipulation,
materials, labor and space. These methods are ideal for studying large
numbers of isolates or for research involving challenging large numbers of
microbes against a host of test compounds.
The miniaturized methods have been used to study large numbers of
isolates from foods (Fung and Miller, 1970, 1971; Fung and Hartman,
1975; Lee et al., 1982, 1985) and to develop bacteriological media and
procedures (Fung and Miller, 1973; Fung and Petrisko, 1973; Fung and
Neimeic, 1977; Chein and Fung, 1991). Miniaturized methods for studying
food yeast were also developed in the author's laboratory (Lin et al.,
1984; Lin and Fung, 1985, 1987; Fung and Liang, 1989). Currently, these
miniaturized methods are being used to study food mycology (Hart and
Fung, 1990).
Fung and Kraft (1968, 1969) and Fung and LaGrange (1969) used min-
iaturized concepts to develop viable cell procedures and most probable
number (MPN) tests. This involves using micro titer loops (0.025 ml) to
dilute liquid samples in microtiter wells (each containing a known volume
of sterile buffer) and then aseptically transferring a small volume
(0.025 ml) of diluted samples onto agar surfaces to form spots. The agar
plates (each holding four or six spots) are then incubated and colonies are
counted after bacterial growth. This method was successfully tested
against the conventional method in a collaborative study involving four
laboratories (Fung et al., 1976).
Another concept is to dilute a sample of liquid food (e.g. raw milk) in a
three-well series with nutrient broth in the microtiter plate to form a min-
iaturized three-tube MPN. These tests were developed for efficient opera-
tion of the viable cell count as an alternative to the standard plate count
method and the standard MPN count procedure.
On the commercial side, many diagnostic kits to identify microorgan-
isms have been developed and marketed since the 1970s. Currently, API,
Enterotube, RIB, Minitek, Spectrum 10, MicroID and IDS are available.
Most of these systems were first developed for the identification of enterics
(Escherichia coli, Salmonella, Shigella, Proteus, Enterobacter spp., etc.).
RAPID METHODS 429
Later, many of the companies expanded the capacity to identify non-fer-

mentors, anaerobes, Gram-positive organisms and even yeasts and molds.
Most of the early comparative analyses centered around evaluation of
these kits for clinical specimens. Cox et al. (1977) and Fung and Cox
(1981) studied these systems from the standpoint of food microbiology
and concluded that they generally provide 90-95% accuracy when
compared with conventional methods. Comparative analysis of diagnostic
kits and selection criteria for miniaturized systems were made by Fung et
al. (1984) and Cox et al. (1984). The general conclusions are that these
miniaturized systems are accurate, efficient, labor-saving, space-saving,
and cheaper than the conventional procedures for diagnostic microbiology
in clinical, industrial, environmental and food samples. Their usefulness
in clinical and food microbiological laboratories will continue to be
important.
15.6 New and novel techniques
15.6.1 Vitek system

Many sophisticated instruments have been developed to identify isolates
from clinical specimens. One of the most automated systems for the iden-
tification of isolates (clinical and foods) is the Vitek system (Figure 15.15).
The system depends on the growth of target organisms in specially
designed media housed in tiny chambers in a plastic 'card'. The card is
then inserted into the incubation chamber. The instrument periodically
scans the wells of the cards and sends information to the computer, which
then matches the database and identifies the unknown cultures in the
cards. The system is entirely automated and computerized and provides
Figure 15.15 The Vitek system. The far left is the injection port where the sample is pneu-
matically forced into the 'cards'. Next is the incubator that can hold up to 240 samples. The
next is the computer printer. The far right is the computer screen and keyboard. (Used with
permission of bioMerieux Vitek Inc., St. Louis, Missouri.)
hard copies for record-keeping. Most evaluations of the usefulness of the

Vitek system had previously used clinical specimens. Bailey et al. (1985)
were successful in using the Vitek system to identify Enterobacteriaceae
from foods. The system is capable of identifying enterics, yeast, Bacillus,
selected Gram-positive pathogens and other organisms.
15.6.2 DNA probes

Recently, several miniaturized kits and procedures have been developed to
detect the presence or absence of target food pathogens. These new
methods have cut the detection time for negative screens from 7 days to
48 h. However, if the test is positive, the conventional method must be
used to confirm the presence of target food pathogens. Concepts and
applications of miniaturized kits, immunoassays and DNA hybridization
for recognition and identification of food borne bacteria are the subjects of
a paper by Cox et al. (l987b). The DNA probe (Genetrak) is a sensitive
method to detect pathogens such as Salmonella spp., Listeria spp. and
Escherichia coli. At first, the system used radioactive compounds for
assay. The second generation of probes uses enzymatic reactions to detect
the presence of pathogens. Fitts (1985) and Flowers (1985) showed that
the DNA probe technique is as sensitive or more sensitive than the con-
ventional method for Salmonella detection.
15.6.3 Target RNA probes

Another major change in this area is the development of probes to detect
target RNA. In a cell there is only one copy of DNA, however, there may
be 1000 to 10 000 copies of ribosomal RNA. Thus, the new generation of
probes is designed to probe target RNA. Currently, kits are available for
Salmonella, Listeria, Campylobacter, Yersinia, etc. As the need arises more
organisms will be added to the list.
15.6.4 ELISA tests

Competing with DNA/RNA probe systems are enzyme-linked immuno-
sorbent assay systems (ELISA). The immunoassay screening procedure
(ELISA) method commercialized by Organon Teknika (Durham, North
Carolina) uses two monoclonal antibodies specific for Salmonella detec-
tion. In a comparative study involving 1289 samples, Eckner et al. (1987)
found that there was no significant difference between the conventional
method and the ELISA method for foods sampled, except for cake mixes
and raw shrimp. The Tecra system (International BioProducts, Redmond,
WA) was developed in Australia using a principle similar to that of
ELISA but using polyclonal antibodies to detect Salmonella. These
RAPID METHODS 431
methods have been used to detect Listeria and E. coli also. Many compa-
nies are providing a host of monoclonal and polyclonal antibodies for a
variety of diagnostic tests, some including food pathogens (Fung et al.,
1988a).
15.6.5 The VIDAS immunoanalysis system

A new development in automation occurred recently in immunology. The
VIDAS™ (Vitek ImmunoDiagnostic Assay System) is a multiparametric
immunoanalysis system developed and marketed by bioMerieux Vitek Inc.
(Figure 15.16). The system uses the enzyme-linked fluorescent immu-
noassay (ELF A) method, which is a version of the well-known ELISA
system. According to the manufacturer, 'The end result of the test
protocol is a fluorescent product and the VIDAS reader uses a special
optical scanner that measures the degree of fluorescence. From the
moment the solid-phase receptacles and the reagent strips are placed in
the instrument, the VIDAS is fully automated' . This is a revolutionary
development because one of the drawbacks of the ELISA test is the many
steps necessary for adding reagents and washing test samples. In the
VIDAS system, all intermediate steps are automated.
Figure 15.16 VIDAS system for automated enzyme-linked fluorescent immunoassay (ELFA)
(Used with permission from bioMerieux Vitek Inc. , St Louis, Missouri.)
15.6.6 The polymerase chain reaction

One of the newest developments in rapid methods is polymerase chain
reaction (PCR) technology (McNamara, 1992). With this technology,
theoretically one can amplify one piece of target DNA to many millions
of copies in several hours, thus enabling a scientist to detect target
microorganisms rapidly. To be successful with this technique, one must
know the sequence of two known regions of the target DNA and use
two oligonucleotides to serve as primers for a series of polymerization
reactions that will result in millions of copies of the DNA. Typical PCR
protocol involves denaturation of DNA at 96°C (30 s to several

minutes), primer annealing at 55°C (30 s) and primer extension at noc
(1.5 min). The reaction components include target DNA, bases, DNA
polymerase (TAQ), Mg2+ or Mn2+, primers, buffer (pH 8.0-8.3) and
mineral oil. The PCR products can be detected by gel electrophoresis,
Southern blot, dot blot, or ELISA tests. Automation and improvements
of the entire system are constantly being made, especially by Perkin
Elmer Cetus in Norwalk, Connecticut. This highly sophisticated proce-
dure started in research laboratories for molecular biology and is now
slowly finding its way into applied microbiology. Hill et a/. (1991)
reported identification of Vibrio vulnificus in artificially contaminated
oysters by PCR. In the 1992 annual meeting of the American Society for
Microbiology in New Orleans, a seminar entitled Nuclear Acid Amplifi-
cation and Other Innovative Detection Systems was held, and the use of
PCR in food microbiology was discussed in detail. Certainly, PCR will
be used in all areas of applied microbiology, including meat micro-
biology, in the near future.
15.6.7 Motility enrichment

Motility enrichment is a very interesting concept in rapid isolation and
identification of food pathogens. Twenty years ago, Fung and Kraft
(1970) described a motility flask system for rapid detection and isolation
of Salmonella spp. from mixed cultures and poultry products. The system
involves a flask with a side-arm that contains several agar layers. Lactose
broth is placed in the flask and then a sample (with or without salmo-
nellae) is inoculated into the lactose broth. When salmonellae are present,
they will swim through the first level of agar, which contains selenite
cysteine and sodium lauryl sulfite that inhibits other organisms but allows
salmonellae to pass through. Once salmonellae pass the first layer, they
can grow and metabolize compounds in the second and third layers. By
looking at the color changes in the second and third agar layers, one can
make an assumption that salmonellae were in the sample that was put
into the lactose broth.
Not much happened in motility enrichment until about 7 years ago,
when a commercial system called Salmonella 1-2 test (BioControl,
Bothell, Washington) was developed, which uses motility as a form of
selection. The food sample is first pre-enriched for 24 h in lactose broth,
and then 0.1 ml is inoculated into one of the chambers in an L-shaped
system. The chamber contains selective enrichment liquid medium. There
is also a small hole connecting the liquid chamber with the rest of the
system, which has a soft agar through which salmonellae can migrate. An
opening on the top of the second chamber allows the analyst to deposit a
drop of polyvalent anti-H antibody. If the sample contains salmonellae
RAPID METHODS 433
from the lower side of the L-unit, salmonellae will migrate through the
hole and up the agar column. Simultaneously, the antibody against
fiagellae of salmonellae will move downward by gravity. When the
antibody meets the salmonellae they will form a visible 'immunoband'.
The presence of an immunoband in this system is a positive test for Sal-
monella spp. The system is easy to use and has gained popularity because
of its simplicity.
15.6.8 Oxyrase enzyme system

Recently, it was shown that an enzyme, patented under the name
'Oxyrase' (Oxyrase Inc., Ohio), can stimulate the growth of many
important facultative anaerobic food pathogens. In the presence of a
hydrogen donor, 'Oxyrase' can convert O2 to H 20, thereby reducing the
oxygen tension of the medium and creating anaerobic conditions that
favor the growth of facultative anaerobic organisms. In a medium con-
taining 0.1 units.mr i , of the enzyme, the growth of Listeria mono-
cytogenes, Escherichia coli 0157:H7, Salmonella typhimurium, Strepto-
coccus faecalis and Proteus vulgaris was greatly enhanced. Colony counts
were greater by 1-2 log units, after incubation in the presence of the
enzyme for 5-8 h at 35-42°C, depending on the initial count and the
strain studied.
By combining the Oxyrase enzyme and a unique 'U'-shaped tube, Yu
and Fung (199Ia,b, 1992) developed an effective method to detect Listeria
monocytogenes and Listeria spp. from laboratory cultures and meat
systems.
There are many other systems that involve modern biochemistry, chem-
istry and immunology. For example, one can use protein profiles for
microbial 'fingerprinting' (AMBIS system, San Diego, California), or cell
composition as a way to identify bacterial cultures (Hewlett-Packard, Palo
Alto, California).
15.7 Conclusions
It is not possible to include all types of tests and instruments in this

chapter. A reader can scan through publications such as American Clinical
Laboratory, American Laboratory, Laboratory Equipment, American Bio-
technology Laboratory, and Life Science Lab Products and find literally
hundreds and thousands of instruments for analytical work related to
microbiology. The emphasis of this chapter is on those systems, instru-
ments and kits that are related directly to food microbiology in terms of
quality assurance and research.
Fung et al. (1989) conducted a survey of 55 professional micro-

biologists and obtained data concerning the number of plate counts and
coliform counts performed per year (ranging from a few tests to
840 000 tests conducted by one laboratory), the kinds of pathogen
detection tests routinely performed (e.g. up to 250 000 E. coli tests per
year), and the types of instruments and diagnostic kits routinely used.
The data indicated that indeed food microbiology laboratories are cur-
rently using automated and rapid methods and that food micro-
biologists will continue to adopt these methods in their routine and
research work.
Faced with so many systems and possible kits to use, what should an
analyst look for in a system? The important points one should consider in
either buying or trying to develop an automated microbiology assay
system are listed as follows:
1. Accuracy for the intended purpose:
(a) sensitivity should be within minimal detectable limits;
(b) specificity of the test system;
(c) versatility and potential applications;
(d) comparison to referenced methods.
2. Speed-productivity in obtaining results and in the number of samples
processed per run or per day.
3. Cost of initial outlay, per test, reagents and other things should be
borne in mind.
4. Acceptability by scientific community and by regulatory agencies.
5. Simplicity of operation in sample preparation, operation of test
equipment and computer versatility.
6. Training, i.e. whether on-site, for how long and what the quality of
training personnel is.
7. Reagents in terms of reagent preparation, stability, availability and
consistency.
8. Company reputation.
9. Technical service in terms of speed and availability and cost scope of
technical background.
10. Utility and space requirements.
Every analyst should try some of these systems and compare them with
existing methods before making a decision to adopt or purchase a parti-
cular system or procedure.
The field of rapid methods and automation in microbiology is exciting.
It will continue to flourish in years to come because applied micro-
biologists will always want to find more sensitive, efficient and cheaper
methods to enumerate, detect, isolate and identify microbes from food
and the environment to ensure the safety of our food supplies, and there-
fore, the health of consumers.
RAPID METHODS 435
Acknowledgement
Contribution No. 93-115-B, Agricultural Experiment Station, Kansas

State University, Manhattan, Kansas 66506. This material is based on
work supported by the Cooperative State Research Service, US Depart-
ment of Agriculture, under agreement No. 889034187 4511.
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16 Food analysis and chemical residues in muscle
foods
R.L. ELLIS
16.1 Food analysis
16.1.1 Introduction
Meat is inherently a simple combination of muscle, fat, moisture and very
small amounts of non-combustible material (ash). However, when defining
the term, it becomes somewhat more complicated. For regulatory
purposes, the Code of Federal Regulations (9 CFR Sec 301.2) defines
meat as:
'The part of the muscle of any cattle, sheep, swine, or goats, which is skeletal or
which is found in the tongue, in the diaphragm, in the heart, or in the esopha-
gus, with or without the accompanying and overlying fat, and the portions of
bone, skin, sinew, nerve, and blood vessels which normally accompany the
muscle tissue and which are not separated from it in the process of dressing. It
does not include the muscle found in the lips, snout, or ears. This term, as
applied to products of equines, shall have a meaning comparable to that
provided in this paragraph with respect to cattle, sheep, swine, and goats.'
This clearly identifies meat as a rather complex matrix and provides an
explanation of why analytical methods are somewhat more difficult to
develop for regulatory programs than might be otherwise expected.
The growing interest and focus on the nutritional value of foods in
general and the body of evidence on the relationship of diet and health,
has led to the promulgation of new laws (the Nutrition Labeling and
Education Act) and regulations on the nutritional value of foods. For
meat products, these new label requirements include mandatory analysis
for labeling: calories, calories from fat, total fat, saturated fat, cholesterol,
sodium, total carbohydrate, dietary fiber, sugars, protein, vitamins A and
C, calcium and iron. Other analytes are voluntary. They include calories
from saturated fat, polyunsaturated and monounsaturated fats, and other
vitamins and minerals, such as thiamin, riboflavin and niacin.
This recent focus will require a critical review of our existing, validated
methods to meet today's needs. For the present, however, one must build
on traditional, and in some instances, rather historical methods that trans-
cend two centuries of use. New technologies and processes for preparing
meat products challenge the limits of our methods, while at the same time
making us focus on applications of new technology to meet tomorrow's

needs. This review of current regulatory methods is designed to serve as a
foundation upon which to build better analytical methods.
16.1.2 Protein
For more than 100 years, protein determinations have been performed on
meat food products by procedures that measure the nitrogen content of
the sample. Percentage nitrogen is converted to the equivalent protein
content by an appropriate numerical factor (i.e. since meat protein is 16%
nitrogen, a factor of 6.25 is used).
The most common analytical procedure for the determination of the
nitrogen content of a food sample is the traditional Kjeldahl method
(McMillin, 1928). The nitrogen in the meat food sample is converted to
ammonium bisulfate during the digestion of the sample using con-
centrated sulfuric acid and a catalyst. Ammonia is liberated from the
digested sample by the addition of excess sodium hydroxide and is col-
lected in an excess of standard acid solution. The excess acid is titrated
with a standard alkali solution. The percentage nitrogen found is con-
verted to percentage protein by multiplying by 6.25.
The Block Digester method (Suhre et al., 1982) offers an option for
determining protein in a meat food sample and is a variation of the tradi-
tional Kjeldahl procedure. The Block Digester is an aluminum alloy
heating block used to digest samples. In addition to the catalyst and
sulfuric acid normally used in the Kjeldahl procedure, a small amount of
30% hydrogen peroxide is used to increase the digestion efficiency. After
digestion is complete the cooled sample is processed in the same manner
as the standard Kjeldahl procedure.
An automated Kjeldahl procedure has been used in repetitious food
protein determinations (Noel, 1976). Once initiated, the automated
procedure carries out the complete Kjeldahl process without further atten-
tion.
Another automated procedure for determining nitrogen in meat food
samples uses a continuous digester and the formation of a blue dissociated
form of indophenol as a result of ammonia reacting with hypochlorite and
phenate in alkaline solution to produce quinonechloramine, which reacts
with additional phenate ion (Ganrenbein, 1973). The blue dissociated
form of indophenol has a maximum absorbance at 630 nm in alkaline
solution. The sample absorbance is compared with a standard curve of
absorbance vs. mg N.mrl to determine the percentage nitrogen, which is
converted to percentage protein.
The AOAC International (1990) recently adopted as a first-action status
a combustion method for the determination of nitrogen in meat samples.
The nitrogen in the sample is liberated by high-temperature combustion in
MUSCLE FOOD ANALYSIS 443
pure oxygen. The nitrogen is measured by thermal conductivity and con-

verted to the equivalent percentage protein in the meat sample.
16.1.3 Fat
The percentage fat in a meat sample is usually determined by solvent
extraction of the fat from the meat sample (AOAC, 1960). An extraction
thimble composed of pressed and formed filter paper material is used to
hold the sample. Sand is mixed with the sample to facilitate ether extrac-
tion of the fat. The thimble containing the sample and sand is placed in a
forced-air drying oven to remove moisture. After the fat has been extrac-
ted, the ether is evaporated and the fat residue is dried to constant weight.
A rapid specific gravity method described by Pettinati and Swift (1977)
uses tetra-chloroethylene to extract the fat. The extraction agent, meat
sample and a drying compound are mixed in a closed-steel vessel, which is
placed in a motor-driven orbital shaker. After mixing, the extract is
filtered and the specific gravity is determined and converted to percentage
fat using a pre-calibrated chart.
A rapid microwave solvent-extraction method developed by Bostian
(1985) uses methylene chloride to extract the meat fat from the microwave
dried samples in an automatic extraction system. The extracted sample
residue is dried by microwave heating to remove the residual solvent. The
weight loss from extraction is converted to percentage fat.
Owing to the fat definition used in the new nutrition labeling rules, total
nutritional fat (the sum of the fatty acids liberated by hydrolysis from a
lipid extract) will be required. With current techniques this analysis would
consist of a Folch (methanol-chloroform) extraction, followed by hydro-
lysis of the fatty acids and quantification by gas chromatography, being
reported as triglyceride equivalents. No validated standard method is
available for meat products at this time.
16.1.4 Moisture
The percentage moisture in meat and meat food products is usually deter-
mined gravimetrically by heating a meat sample to constant weight. The
heating of the meat sample may be accomplished by the use of a moisture
oven (Windham, 1953), a vacuum oven (although it is unsuitable for high-
fat products such as pork sausages) (AOAC International, 1990) or by a
microwave procedure (Bostian et al., 1985).
16.1.5 Salt
The Volhard method (AOAC International, 1990) is the only officially
approved method for the determination of sodium chloride in meat and
meat food products. An excess of silver nitrate solution is added to the

meat sample before the addition of concentrated nitric acid. This order of
addition is critical to ensure complete precipitation of the chlorides. If
nitric acid is added first, loss of chlorine by volatilization as hydrogen
chloride could occur because hydrogen chloride has a higher vapor
pressure than nitric acid. The addition of concentrated potassium perman-
ganate assists in the oxidation of any organic matter not digested by the
nitric acid. Following boiling, cooling and dilution, a coating agent, such
as diethyl ether, is added and the excess silver nitrate solution is back-
titrated with potassium thiocyanate solution, employing ferric ammonium
sulfate solution as an indicator. The indicator reacts with an excess of
thiocyanate, forming the salmon-colored complex, indicating the end-
point. The addition of a coating agent protects the precipitated silver
chloride from reaction with the thiocyanate solution.
16.1.6 Sugars
Lactose is almost always the sugar of interest in meats. The analysis of
other sugars such as dextrose, sucrose or maltose is rarely called for. A
meat sample is analyzed for its lactose content to determine the amount of
non-fat dry milk (NFDM) or calcium-reduced dry skim milk (CRDSM).
These two analytes are regulated for use as binders in certain cooked-
sausage products. The NFDM and CRDSM each contain approximately
50% lactose. Methods currently in use for lactose determination are the
Benedict solution method (Cook, 1958), the gas-liquid chromatography
method and the high-performance liquid chromatography method
(USDA, 1986). The liquid chromatography method also provides for the
determination of dextrose, maltose and sucrose. Maltose may also be
determined by the fermentation procedure outlined by USDA (1986).
In the Benedict solution method (USDA, 1986), lactose is extracted
from the meat sample with an aqueous solution and then treated with
phosphotungstic acid to remove any soluble proteins. If corn syrup solids
are absent in the meat sample, the aqueous solution is neutralized just to
the acid side of bromothymol blue indicator. If corn syrup solids are
present, the sample is neutralized just to the acid side of chlorophenol red
indicator and a buffer solution (pH 4.8) is added. A specially prepared
yeast suspension is used to ferment any sugars other than lactose. The
lactose in the sample is reacted with Benedict's solution to form cuprous
oxide. The cuprous oxide is dissolved by a volume of standard iodine
solution. The excess volume of iodine is titrated with standard sodium
thiosulfate solution using starch solution as an indicator. The iodine/thio-
sulfate ratio and the lactose/iodine ratio must be determined.
The percentage lactose in a meat food sample determined by the gas-
chromatographic method (USDA, 1986) is a much simpler and more
straightforward procedure than the Benedict solution method. The extrac-

ted meat sample is dried using freeze-drying procedures, silylated and
quantified by gas chromatography using a flame-ionization detector.
A procedure using liquid chromatography described by USDA (1986),
can be used for the determination of dextrose, sucrose, maltose and
lactose. Following an aqueous initial extraction, the extract is con-
centrated and purified with a silica-gel cartridge and two ion-exchange
columns. The purified extracts are concentrated and analyzed by liquid
chromatography using a normal-phase amino column. The analytical
range of this procedure is from 0.22-0.55% for any of the sugars already
mentioned.
16.1.7 Nitrate
The procedures for determination of the nitrate content (USDA, 1986;
AOAC International, 1990) are based on the fact that nitrates yield nitric
acid upon treatment with sulfuric acid. The nitric acid liberated from the
sample then nitrates meta-xylenol, yielding ortho-nitroxylenol, which is
steam distillable. The distillate is collected in aqueous sodium hydroxide.
The colored sodium salt thus formed (which follows Beer's law) is deter-
mined spectrophotometrically. Since nitrites, chlorides and proteins inter-
fere, they must be removed prior to the nitrate determination. Nitrites are
oxidized to nitrates using potassium permanganate. Chlorides are pre-
cipitated using silver ammonium hydroxide and proteins are precipitated
using phosphotungstic acid.
16.1.8 Nitrite
The amount of free nitrite remaining in a sample of cured processed meat
that has not combined with myoglobin to form nitrosomyoglobin is an
important food-safety concern and is limited to 200 mg.kg- 1 (p.p.m.) by
regulation (9 CFR Sec 318.6). The spectrophotometric analytical proce-
dure (Fiddler, 1977; USDA, 1986) is based on reaction of an aromatic
primary amine with an acidified solution of a nitrite to produce a diazo-
nium salt. The diazonium salt is then coupled with another chromogenic
primary aromatic amine, forming an aminoazo compound, which obeys
Beer's law.
16.1.9 Nitrosamines
The one-trap mineral oil-vacuum distillation procedure (USDA, 1991), for
the determination of nitrosamines in fried-bacon samples requires the use
of a Thermal Energy Analyzer (TEA). This detector measures photo-
decomposition of an excited state nitrogen oxide to detect nitrosamine
compounds. A weighed, fried bacon sample is placed in a flask, covered

with mineral oil and vacuum-distilled with gradual heating to 120°C and
the volatile materials are trapped cryogenically. The residues in the cold
trap are dissolved with methylene chloride, rinsed with sodium chloride
solution, dried and concentrated. An aliquot is then subjected to gas chro-
matograph analysis using a TEA detector. The method is suitable for
volatile nitrosamines, with a limit of determination in 1-10 Ilg.kg-1
(p.p.b.) concentration.
Another method of determining nitrosamines is low-temperature
vacuum-distillation (USDA, 1991). A pre-fried sample is treated with a
strong basic solution. The sample flask is maintained at 45-46°C in a
water bath and is vacuum-distilled. The aqueous distillate is acidified and
extracted with dichloromethane. The extract is purified by washing with
acid and base. Volatile nitrosamines are determined and reported as
above.
A solid-phase extraction method for volatile nitrosamines in meat
samples is described by Pensabene et al. (1992). This procedure requires
the sample to be ground in a mortar and pestle with propyl gallate, anhy-
drous sodium sulfate and celite. The ground, mixed sample is transferred
to a glass chromatography column and the nitrosamines are eluted with
dichloromethane. The eluate is concentrated and quantitatively transferred
to a silica gel column. The column is rinsed and the nitrosamines eluted
with 30% ether in dichloromethane. Nitrosamines are determined and
reported as already described before.
16.1.10 Cholesterol
Cholesterol occurs in foods of animal origin with it being concentrated in
the fat of meat and dairy products. Cholesterol is initially extracted from
the fat using a mixture of methanol, water and chloroform. The crude
extract is saponified with potassium hydroxide, and the unsaponifiable
fraction is extracted using a liquid-liquid procedure. Two alternatives are
available for determining cholesterol as described by Shepperd et al.
(1977) and Newkirk and Shepperd (1981).
For gas chromatographic determination, the purified extract is deriva-
tized with hexamethyldisilizane and analyzed using a flame-ionization
detector. The alternative method involves an isocratic, reversed-phase
high-performance liquid chromatographic procedure. Using this detection
procedure, cx-cholestane is used as a reference standard and the analyte
is detected using a variable wavelength ultraviolet detector. The detec-
tion limit is 10 ng cholesterol. Owing to the large volumes of organic
solvents used in this procedure, an alternative method from the FDA
Lipid Manual is being evaluated for use in nutritional analysis (FDA,
1992a).
16.1.11 Rapid test methods

A summary of rapid food chemistry methods studied by the United States
Department of Agriculture, Food Safety and Inspection Service is
provided in Table 16.1. This table is not intended to imply use of any of
these methods for regulatory analysis. Rather, it is a summary of our
efforts to evaluate them for this purpose. Comments noted reflect our
assessment of these procedures and instrumental methods and so on.
16.2 Chemical residues
16.2.1 Introduction
A key aspect of food safety in todays' environment is the control of
residues in food that may result from the use of animal drugs and pesti-
cides, or from incidents involving environmental contaminants. In the
USA, three agencies have major roles in protecting consumers from
unsafe residues. The Environmental Protection Agency regulates pesticide
use in food production as well as other industrial substances that have the
potential to contaminate food. This includes setting tolerances (residue
limits) for pesticides and other chemicals. The Food and Drug Adminis-
tration regulates and inspects foods other than meat and poultry and also
regulates animal feeds. Using a risk-management approach, the FDA
determines if animal drugs can be introduced safely into veterinary
practice. This includes establishing tolerances for residues of veterinary
drugs in edible tissues. The US Department of Agriculture has responsi-
bility for determining residues of pesticides, environmental contaminants
and veterinary drugs in meat and poultry products. The USDA's Food
Safety and Inspection Service is responsible for ensuring that meat and
poultry sold in interstate commerce is safe, wholesome and accurately
labeled. This food-safety responsibility for chemical residues is accom-
plished through its National Residue Program.
Residue analysis of meat products for agricultural pesticides, environ-
mental contaminants or veterinary drugs, whether for quality assurance or
regulatory control purposes, can be an onerous task. Whereas food com-
ponents are measured in parts per hundred or thousand, xenobiotics are
often measured in parts per million or parts per billion. Analysis for
analytes at these concentrations generally requires more complex and
sophisticated equipment, technology and procedures to achieve the desired
performance.
To accomplish the objectives of a residue-control program that may
examine 10 or more categories of xenobiotics and more than 75 individual
compounds, requires a wide variety of analytical and microbiological
Table 16.1 List of rapid methods available for analysis of animal tissues
Analysis Instrument/technique Comment
Protein P-lOO Protein Analyzerllow-resolution Too expensive, fast readout but long
pulsed NMR sample-preparation time
Block digestion automated distillation AOAC official method
and titration
706 Nitrogen-Protein Analyzer/ Sample size too small
chemiluminescence
UDY Protein Analyzer/dye binding May be suitable for quality control
but too variable for regulatory
analysis
Carlovera Nitrogen Analyzer Sample size too small unless freeze-
dried first
Protein Assay Kit/dye binding Results too variable
Protimeter/IR Reflectance Not suitable for meat analysis
Moisture KF-4B Aquameter/Karl Fischer Low bias

PR-I03/pulsed N Sample preparation time too long,
very expensive
Compu-Trac/IR moisture oven Too variable
Moisture Meter Model 919A1IR For powders and grains only
reflectance
Metrohm Model 633/Karl Fischer Low bias
Accu-DryllR oven-drying Suitable for plant quality control
Moistu-TraC/PM-80/IR reflectance Suitable for plant quality control
Models CA-50, V A-O Moisture Low bias
Measurement System/Karl Fischer
Automatic 4586 Moisture Tester/IR Low moisture range (0-40%)
reflectance
Infra Dry/IR oven Suitable for plant quality control
Model G8R and G9/Radiofrequency For powders and granular samples
power loss only
Fat Rapid Dry Column/dichloromethane- High bias (0.6%), measures total

methanol extract of celite - sample lipids, not crude fat
column
SFC-900/solid fat analysis Not suitable for meat; acceptable for
lard, butter, etc.
Anal-Ray Fat Analyzer/X-ray Suitable for plant quality control
absorption (production line and
lab-table top models)
DjME-IOO Ground Meat AnalyzerllR Suitable for plant quality control,
reflectance ground beef only
MIRAN-80/IR reflectance For dairy products and snack foods
only
Analysis Instrument/technique Comment
Soxtec HT/ether extraction (Randall) AOAC official method

Foss-Let/specific gravity AOAC official method
Salt Chlor-o-Stat TitrationiCoulometric Suitable for plant quality control

titration
Dicromat/conductivity Suitable for plant quality control
NOVAlspecific ion electrode Too variable unless carefully
controlled
Fat & CEM/microwave oven (generic AOAC official method

moisture microwave method)
Protein, Noetec Model102INIR reflectance Too variable for regulatory work;

moisture acceptable for plant quality control
& fat
Automatic meat analyzer Model Acceptable for plant quality control,
FMP-llmicrowave oven not for regulatory work
Infra Analyzer 400/IR reflectance Acceptable for plant quality control,
not for regulatory work
methods. Examples include screening tests for use in meat inspection facil-
ities or laboratories using either animal tissue or biological fluid. Com-
plementing these screening tests, and comprising the majority of methods
useq in a regulatory testing program, are the various quantitative labora-
tory methods using different extraction, isolation, purification, concentra-
tion and detection procedures. Rigorous confirmation of an analyte is
performed using methods that identify or verify the structure of the xeno-
biotic.
Owing to this extensive combination of analytical detection methods
and procedures, this review will focus on validated regulatory methods
rather than on non-validated published research methods. Validated
methods are those that have usually been subjected to rigorous assessment
by multiple laboratory and/or analyst performance-based protocols.
An excellent, encompassing review, focused on newer technologies and
including some methods that have been used for veterinary drug-residue
analysis in food animal tissue regulatory programs, was recently published
by Shephard (1991). The review describes many applications of newer
physicochemical and immunochemical technologies used for residue detec-
tion and quantification. The Commission of the European Communities
(1992) has also published an excellent manual of reference methods and
materials.
16.2.2 Pesticide-residue analysis

There are several hundred pesticides registered for use in the USA for
agricultural purposes (NAS, 1987). However, the number registered for
use in food animal products is small compared with the more than 8000
food tolerances listed in the Food, Drug and Cosmetic Act, Section 408
and 409. When pesticides are not used according to label instuctions or
good agricultural practices, unacceptable concentrations of residues may
occur. Pesticide residues may also occur in food animal products as the
result of environmental contamination. As a result of public health
concerns about pesticides in foods by consumers and legislators alike, the
US Congress requested the Office of Technology Assessment (OTA, 1988)
to review the status of pesticide residues in food. This report reviews
extensively federal capabilities, research initiatives and technologies for
residue detection and is recommended reading.
16.2.2.1 Halocarbon pesticides. Chlorinated hydrocarbon pesticides and

many chlorinated organophosphate pesticides are lipophilic substances.
Residues are extracted from rendered animal fat or by direct extraction
from low-fat meat products using an organic solvent to separate the pesti-
cide residues of interest. The isolation procedures generally rely on either
adsorption or gel-permeation chromatography (GPC). Adsorption chro-
matography is based on the interaction between a chemical dissolved in a
solvent and an absorptive surface. Gel-permeation chromatography is a
technique that separates compounds on the principle of molecular size.
The advantage of GPC over adsorption chromatography is that there is
no loss of pesticide on the analytical column by irreversible adsorption or
by chemical reaction. The disadvantage of GPC is that it requires more
expensive equipment. The extraction time for either technique is usually
less than an hour. The sample clean-up step is most often the limiting
factor in pesticide-residue analysis.
Most methods used for pesticide residues in animal foods are based on
the fatty food methods originally developed by Mills (1959). The extrac-
tion and clean-up procedures described earlier for oils, fats and animal
products are basically the same as those currently used for these products.
The extensive application of the basic procedure has been reviewed by
Burke (1971). The review examines the 21 studies on variables of the
method, 19 method-extension reports, and 9 AOAC collaborative studies.
Pesticide analysis for meat products is based on the extracted or
rendered fat. For the Mills (l959)-based adsorption chromatog-
raphy methods, the sample is dissolved in an organic solvent and
partitioned with water. The residues are purified by florisil column
chromatography. Pesticide residues are measured quantitatively by gas-
liquid chromatography and confirmed by mass spectrometry (GCjMS).
Examples of some typical compounds detected by this methodology are as

follows:
• Aldrin
• BHC
• Chlordane
• Dieldrin
• DDT
• DDE
• TDE (DDD)
• Endrin
• Heptachlor
• Heptachlor epoxide
• Lindane
• Methoxychlor
• Toxaphene
• PCBs (Aroclor 1016, 1242, 1248, 1254, 1260)
• HCB
• Mirex
• Nonachlor
• Strobane
A rnodification of this procedure employs deactivated alumina for column
chromatography (USDA, 1991).
A more versatile approach for pesticide-residue analysis in meat
products is the gel-permeation chromatography (GPC) procedure devel-
oped and validated by Ault and Spurgeon (1984). In this procedure the
liquefied animal fat is dissolved in a 1: 1 mixture of methylene chloride:
cyclohexane. The residues are purified by using an automated GPC
system and quantified by gas-liquid chromatography. The analytes in the
validation study are shown in Table 16.2, along with other compounds
that the method has been extended to include for quantification (USDA,
1991). Note that some of the compounds are chlorinated organophos-
phates.
The confirmatory procedure used by the Food Safety and Inspection
Service for structure determination is a non-validated gas chromato-
graphy/mass-spectrometry method using select-ion monitoring. The
method is capable of confirming chlorinated hydrocarbon pesticides at
concentrations of 10-80% of the action level (recommended by EPA)
using the extract from the GPC- or Mills-based methods. The absolute
sensitivity varies for each compound. For confirmation, six ions are mon-
itored for each component. Compounds for which the method is applic-
able include BHC, HCB, lindane, heptachlor (and its epoxide),
oxychlordane, DDE, dieldrin, endrin, TDE, DDT, methoxychlor and
mirex.
Table 16.2 Compounds applicable to gel-permeation chroma-

tography procedure
A. Compounds for which the method has been validated

Aldrin Dieldrin Heptachlor
a-BHC o,p'-DDT Heptachlor epoxide
Lindane p,p'-DDT Hexachlorobenzene
cis-Chlordane p,p'-DDE Methoxychlor
trans-Chlordane p,p'-TDE Mirex
Oxychlordane Endrin
B. Compounds to which method applies for quantification

Toxaphene Halowaxes Nonachlor
Coumaphos-O Coumaphos-S Ronnel
Phosalone Kepone Captan
Linuron Endosulfan I Endosulfan II
Chlorpyrifos PCBs Stirophos
Carbophenothion Chlorfenvinphos
C. Compounds for identification only

Dichlofenthion Chlordene o,p'-TDE
f3-BHC Leptophos o,p'-DDE
y-BHC
16.2.2.2 Organophosphates. Non-chlorinated organophosphate pesticides

are more commonly found in liver tissue than are the chlorinated hydro-
carbon and chlorinated organophosphate compounds. As a consequence,
however, residue-analysis procedures usually result in a more con-
taminated extract and require a more extensive purification procedure. The
available regulatory methods are based on the procedure of Storrer (1971),
even though the method was originally designed for use in non-fatty foods.
The procedure requires organic solvent extraction of homogenized tissue,
partitioning and a charcoal column clean-up procedure (Watts et al.,
1969). The eluant is concentrated and analyzed by gas chromatography
using a flame-photometric detector. Examples of organophosphate com-
pounds analyzed quantitatively by this procedure include dioxathion,
diazinon, methyl parathion, fenitrothion, malathion, ethyl parathion,
ruelene, gardona, ethion, trithion and coumaphos. Other organopho-
sphates such as chlorpyrifos, disulfuton and trichlorfon can be quantita-
tively and qualitatively recovered by this procedure but GLC retention
times may overlap (USDA, 1991; EPA, 1992). An internal standard is used
for quantitative analysis. Gas chromatographic retention times are reported
in EPA and FDA manuals. These manuals are published annually.
Organophosphates act by inhibiting cholinesterase. Advantage of this
mechanism of action has led to the development of laboratory bioassay
and field screening tests. The procedure of Clear et at. (1977) consists of
thin-layer chromatography for separation of 12 organophosphates using
cholinesterase enzyme extracted from flies. In current practice, commercial
cholinesterase extracts are used. The procedure has been adapted to a

simple screening test for a wide variety of food commodities but requires
a water-based extract for colorimetric analysis. By employing an oxidation
step with bromine water, the bioassay 'ticket test' can differentiate
between the oxygen and sulfur analogs (Enzytec product bulletin). Its
application to meat products has not been verified.
In as much as organophosphate pesticides tend to rapidly metabolize in
biological systems, validated confirmatory methods for incurred residues
are not readily available. Confirmatory procedures are usually designed by
using either a gas or a high-performance liquid chromatographic separa-
tion procedure coupled with mass spectrometry for compound identifica-
tion. Whenever possible, six unique mass fragments are used for
verification of structure for regulatory purposes, including pesticides
(Spohn, 1978). With rapid metabolism of organophosphates, it is extre-
mely difficult to satisfy this criterion for mass spectrometry confirmation
because of the usually low-molecular-weight fragments. At least three ion
ratios, relative to the most intense ion, must be within 20% of those
found in the recovery sample spiked at the level detected in the suspect
sample for confirmation.
Exceptions to confirmation are generally limited to those halogenated
organophosphates that tend to be more lipid soluble. For these analytes
there is a non-validated method using capillary column separation and
electron impact ionization for detection, using either gel permeation,
micro alumina or florisil column clean-up. The method is used to confirm
the presence of ronnel, chlorpyrifos, chlofenvinphos, stirophos, phosalone,
coumaphos, linuron, endosulfan and carbophenothion (USDA, 1991).
16.2.2.3 Carbamates. Carbamates are primarily systemic insecticides and

acaricides that act as cholinesterase inhibitors and are usually neurotoxic.
Almost all carbamates are rapidly metabolized at the carbamate side-
chain. Most procedures are based on this pattern. Methods do not detect
carbamates as parent molecules, rather they are detected indirectly as the
hydrolyzed amine side-chain. Carbamates are extracted from liver tissue,
followed by gel-permeation chromatography. Eluates are further purified
then quantified using high-performance liquid chromatography using
hydrolysis and post-column derivatization with ortho phthaldehyde and
fluorescence detection (Ali, 1989). Compounds demonstrated to be
detected and quantified by this method are as follows:
• Aldicarb
• Aldicarb sulfoxide
• Bufencarb
• Carbofuran
• 3-Hydroxy carbofuran
• Methiocarb
• Methomyl
• Promecarb
• Aldicarb sulfone
• Bendiocarb
• Carbaryl
• Dioxacarb
• Isoprocarb
• Methiocarb Sulfoxide
• Ox amyl
• Propoxor
Unvalidated immunoassay methods have been reported for carbamate
pesticides (Newsome and Shields, 1981; Newsome and Collins, 1987;
Brady et aI., 1988).
Using the same extraction and clean-up procedure from the above
quantitative method, the carbamate eluate may be purified for confirma-
tion using a reversed-phase solid-phase extraction chromatographic clean-
up followed by derivatization with heptafluorobutyric anhydride. The
derivatives are analyzed by gas chromatography/mass spectrometry using
electron impact ionization and select-ion monitoring. Analytes confirmed
by this procedure include bendiocarb, bufencarb, carbaryl, carbofuran, 3-
hydroxycarbofuran, dioxacarb, isoprocarb, methiocarb, promecarb and
propoxor (Ali, 1989).
16.2.2.4 Pyrethroids. The naturally occurring pyrethrins are too short-

lived to be of commercial use as insecticides. The only compounds of this
class that have been registered for use are the synthetic pyrethroids, all
containing a 3-phenoxyphenyl moiety. All are stereochemical mixtures
that complicate gas chromatographic separation and may result in co-
eluting peaks. Identification for analytes of this class is by gas chromato-
graphy/mass spectrometry as described by USDA (1991). In the determi-
native method, rendered fat samples are extracted and partitioned with
petroleum ether and acetonitrile using a modified version of the chlori-
nated hydrocarbon method. Florisil column chromatography is used to
purify the sample extracts. Residues are measured quantitatively using
capillary gas chromatography and may be confirmed using capillary gas
chromatography coupled to a mass selective detector in the select-ion
monitoring mode. The method is suitable for permethrin (cis and trans
isomers), cypermethrin (isomers A, B and C), flucythrinate (isomers A and
B), fenvalerate (isomers A and B) and deltamethrin.
For multi-residue screening and determinative procedures, enzyme-
linked immunosorbent assay (c-ELISA) procedures have been employed
for pesticide-residue analysis (Mumma and Hunter, 1988; OTA, 1988;
Stanker et aI., 1991). Stanker et al. (1989) have reported a monoclonal

anti-pyrethroid antibody with acceptable analytical sensitivity suitable for
detecting permethrin, cypermethrin, deltamethrin, phenothrin and fenpro-
pathrin. Flucythrinate and fen valerate are weakly detected, while tetra-
methrin is not recognized by the antibody. Identification and
quantification of the individual pyrethroids detected are not possible
because the ELISA response is integrated over the spectrum of all detect-
able compounds. Results are reported as permethrin equivalents. In this
procedure, the rendered fat is extracted with a mixture of acetonitrile and
water and subsequently partitioned into hexane. Following alumina chro-
matography, the eluate is concentrated, adjusted to 6% acetonitrile in
phosphate-buffered saline and analyzed using the competitive ELISA
(USDA, 1991).
Although the ELISA procedure is an effective screening procedure, it
also highlights the need to conduct an extensive clean-up procedure prior
to using an immunoassay procedure. This is a common occurrence with
residue analysis from animal tissue matrices (such as fat) that has limited
application for this type of screening procedure. As progress is made in
developing aqueous-based extraction procedures and immunoassays are
developed that are more tolerant of aqueous organic solvent mixtures,
these immunoassay procedures are expected to have more extensive appli-
cation in tissue-residue analysis.
16.2.2.5 Chlorinated triazines. A regulatory method for the detection,

quantification and confirmation of chlorinated triazines has been reported
by the USDA (1991). Chlorinated triazine herbicides are widely used in
the production of grain and cereal crops. Since these grains may be used
in animal feed, there is the potential for indirect contamination of food-
producing animals resulting from their use. The method has been used to
quantity and confirm propazine, terbuthylazine, atrazine and simazine in
rendered fat of bovine, porcine and avian species.
The chlorinated triazines are extracted with acetonitrile, partitioned
with solvents and purified by florisil column chromatography. Detection
of the isolated triazine compounds is accomplished by capillary gas chro-
matography using a nitrogen-phosphorus detector. Quantitation limits are
10-40 ~g.kg-l. The method is unsuitable for the expected metabolic
residues of these chlorinated triazines. This is important in as much as
exposure to animals most commonly would be through indirect exposure
from contaminated grains.
The confirmatory method invokes the same sample-preparation proce-
dure. The extract is reconstituted into toluene for gas chromatography!
mass spectrometry analysis using select-ion monitoring. The confirmatory
procedure is applicable to cyprazine in addition to the four previously
mentioned analytes.
16.2.3 Volatile environmental contaminants

Particularly in the western world, meat has constituted an important part
of the diet because it is a good source of protein and for its sensory flavor
properties. An extensive review of the volatile and semi-volatile flavor
components has been published (MacLeod and Seyyedain-Ardebili, 1981).
Occasionally, however, meat products may be exposed to volatile com-
pounds that are used in a variety of industrial applications. The ability to
distinguish between those compounds occurring naturally and those
occurring from inadvertant exposure becomes a challenging endeavor. A
typical example is airborne styrene residues from use of certain adhesives
in floor applications. Based on limited controlled studies, up to 99% of
the fortified styrene is absorbed in fat (USDA, unpublished study). This
high absorption capability causes some difficulty in determining residues
because the vapor headspace procedure may require temperatures that
result in some thermal polymerization of the analyte.
Styrene can be detected at concentrations of 1-10 mg.kg- 1 by capillary
gas chromatography/mass spectrometry analysis of headspace vapors from
tissues exposed to styrene. For GCjMS confirmation, the select-ion mon-
itoring mode is used (USDA, 1991). A similar procedure has been devel-
oped for gasoline vapors in animal fat. In this headspace procedure, a
flame-ionization detector is employed. Typical analytes detected with this
procedure include the petroleum ethers, isomeric xylenes and ethyl
benzene. Quantification is not possible because the instrument response is
non-linear. Repeatability, however, with the same tissue is good. The limit
of sensitivity is 0.1 mg.kg- 1.
16.2.4 Halophenols
Halophenols such as pentachlorophenol are widely used as wood pre-
servatives in food animal production facilities. This analyte is highly lipid-
soluble and stable, particularly in fatty tissues. Chemical stability is used
in the analytical procedure. Pentachlorophenol is extracted into cyclohex-
ane following removal of the interfering compounds by a strong sulfuric
acid digestion treatment. The use of an internal standard reference
compound, pentabromoethylbenzene, eliminates the need for quantitative
transfers in the analytical procedure. The analyte and internal reference
standard are quantified directly using a gas-liquid chromatograph
equipped with an electron-capture detector (Gillard et al., 1988). The
collaboratively studied method reports a detection limit of 20 f..lg.kg-1 and
a quantification limit of 50 f..lg.kg-1. Confirmation is by gas chromato-
graphy/mass spectrometry. An extraction method suitable for fat and liver
tissue has been reported by Ryan et al. (1985) as part of a survey for
chlorinated dioxins in Canadian poultry and pork. This procedure
requires treatment of the purified extract with diazo methane prior to gas-
chromatographic analysis. No reference was made to this being a vali-
dated method.
16.2.5 Veterinary drug residue analysis

The Code of Federal Regulations (21 CFR Sections 556 and 558) lists 86
specific chemical entities having a residue tolerance in food-producing
animals and 62 specific chemical entities and veterinary drug combinations
approved for use in animal feed. Of these, 42 compounds are listed in
both sections of the Code of Federal Regulations. This list does not
include, however, those substances that have been approved for use in
food-producing animals that have a zero withdrawal period because the
total residues have been determined to be below a safe concentration
when used according to their approved conditions of use.
One of the general provisions for approval of a veterinary drug requir-
ing a tolerance, in addition to animal and human toxicological considera-
tions, is the availability of a practical analytical method to determine the
quantity of residue in edible tissue. These methods must be sensitive and
reliable at the established tolerance. In certain instances, the residue-detec-
tion methods may require adequate performance characteristics at con-
centrations where it is deemed safe in light of the low toxicity of the drug
residue and the unlikelihood of residues exceeding the tolerance.
The condition of acceptance of a method for drug approval, is that it
must pass a method trial in Federal regulatory laboratories. Although
there has been substantial improvement in the methods submitted for
residue analysis, there are some methods that are more research-study-
oriented than regulatory-analysis-oriented. That is, they may not satisfy
the desired characteristics of a regulatory method. For example, they may
use reagents or equipment that are not commercially available, use exces-
sive volumes of solvents or be too time-consuming to allow high-volume
analysis at the concentrations of interest. As a consequence, there is
usually only a limited number of validated methods for tissue residue
analysis for the compounds of interest. This is not a national issue, it is an
international one. For example, of the veterinary drugs considered by the
Codex Committee on Residues of Veterinary Drugs in Food throughout
1992, of the 14 compounds with recommended maximum residue limits,
the Committee has only accepted eight methods that meet established
method performance characteristics and adopted a provisional status on
14 others (Codex Alimentarius Commission, 1992). Some compounds do
not have any methods approved for determining compliance with Codex
food safety (maximum residue limit) standards.
The discussions on analytical methods for specific classes of veterinary
drugs, for reasons elaborated above, is limited primarily to methods con-
sidered to meet validated regulatory method requirements. An excellent

review on method-development advances for veterinary drug-residue
analysis in tissue has been published by Shepherd (1991), apd is recom-
mended complementary reading.
16.2.5.1 Antibiotics. The historical procedures for determination of anti-

biotics involve microbial inhibition assays and, in several instances,
remain the primary methods for residue-analysis screening. The Food and
Drug Administration is encouraging development of chemical methods for
supplementary quantification and confirmation for older and new anti-
biotics. The basic procedure for determining antibiotic residues in animal
tissues is dependent upon having suitable test organisms that can be
incorporated into an agar layer for the microbial inhibition bioassay.
Current laboratory procedures use a variety of test organisms for these
multi-residue assays. Table 16.3 is a summary of the analyte class and test
organisms (USDA, 1974) used for identification purposes. Through use of
standards, fingerprint profiles for the classes of antimicrobials have been
developed.
The generalized response of microbial inhibition assays is a strength
because it detects almost all members of several antibiotic classes simulta-
neously at relatively low cost. However, drawbacks may occur where some
analytes have reduced detection sensitivity to the microorganism or inhibi-
tion by possible co-extractives (e.g. lipids from animal tissues or
lysozyme). Sheppard (1991) describes some schemes used to minimize the
influence of co-extractive materials.
Chemical methods, particularly for the older antibiotics, are rather
limited because the methods often used for regulation purposes were
microbial-inhibition assays and chemical methods were only used for
structure identification. For example, thin-layer chromatographic methods
were developed for this purpose. Applications of analytical methods, such
as high-performance liquid chromatography, for antimicrobials have been
described by Moats (1984, 1985, 1990). There are no published reports
Table 16.3 Analyte class and test organisms for antimicrobials
Analyte class Test organism
Penicillin (susceptible) Sarcina lutea ATCC 9341a

Tetracycline (susceptible) Bacillus cereus var. mycoides ATCC 11778
Streptomycin (susceptible) Bacillus subtilis ATCC 6633
Tetracycline (resistant) Staphylococcus epidermidis ATCC 12228
Streptomycin (resistant) Sarcina lutea ATCC 9341a
Neomycin (susceptible) Staphylococcus epidermidis ATCC 12228
Erythromycin (susceptible) Sarcina lutea A TCC 9341a
Neomycin (resistant) Sarcina lutea A TCC 9341
Erythromycin (resistant) Sarcina lutea A TCC 15957
that any of these methods have been subjected successfully to multi-

laboratory validation studies.
In some instances, a combination of thin-layer chromatography and
microbial inhibition has been employed. The former for separation, and
the latter for identification. An example of this technique is apramycin.
The analyte is extracted from tissues following treatment with aqueous
potassium hydroxide. Potential interfering substances are removed by pre-
cipitation and centrifugation following treatment with trichloroacetic acid.
The supernate is then passed through an ion-exchange column and the
analyte is eluted with ammonium hydroxide. An aliquot of the eluate is
spotted on a silica gel plate, developed and covered with an agar contain-
ing Bacillus subtilis as the test organism. The zone of inhibition is used to
estimate the analyte concentration (USDA, 1991).
Typical of the lack of analytical chemistry methods for antibiotics are
the aminoglycosides - for example, neomycin, gentamicin and streptomy-
cin. These analytes are very polar compounds with poor to non-existent
chromophores that could be used for analytical determination methods.
Analysis for these compounds in animal tissue is difficult and no validated
methods are available. The limited number of published methods for
tissue-residue analysis (kidney is the target tissue) are high-performance
liquid chromatography methods using fluorescence detection following
post-column derivatization with ortho-phthaldehyde or its derivatives.
Most published but non-validated methods for tissue analysis have detec-
tion limits of approximately 100 Ilg.kg-1 or above (Shepherd, 1991).
The most sensitive published method for aminoglycosides appears to be
a fluorescence polarization immunoassay system for the determination of
gentamicin in milk and bovine and ovine kidney and muscle (Brown et aI.,
1990). Reported detection limits are approximately 20 Ilg.kg-1. A specific
and reproducible liquid chromatographic procedure has been developed
for the quantitative determination of gentamicin in porcine tissue (USDA,
1991). Concentrations of 0.4 mg.kg- 1 can be determined without inter-
ference from other chemical substances. Tissues are extracted with dilute
sulfuric acid and purified using column chromatography, liquid-liquid
extraction and solid-phase extraction chromatography. Again, detection is
carried out by post-column derivatization using netimicin as a retention-
time standard.
Macrolide antibiotics may be complex mixtures of closely related com-
pounds whose composition can depend on the source. There are, however,
some members of this class that are generally pure substances. Effective
chemical methods for their detection are limited and usually confined to
confirmation of identity following tentative identification by microbial
inhibition assays. In addition, sensitivity is commonly lacking since many
of the members of this class have marginal functional groups or chromo-
phores upon which to develop detection systems. Perhaps fortunately,
members of this class such as erythromycin, novobiocin and tylosin, are

not so broadly used as penicillins. They are generally less potent than
penicillins and drugs, such as oxacillin, which are penicillinase-resistant
and have been developed.
Although there are no collaboratively studied chemical methods, some
determinative methods have been reported (Shepherd, 1991). A regulatory
determinative method has been reported for novobiocin and virginiamycin
(USDA, 1991). Additionally, the method provides quantitative supporting
data for the official biological method. Residues are extracted with
methanol, filtered, and an aliquot submitted to liquid chromatography
directly for novobiocin. A second aliquot of the original extract is parti-
tioned with dichloromethane for virginiamycin. Both compounds are
determined in a single-gradient liquid-chromatographic system. The
method is suitable for bovine, porcine and avian species using liver,
muscle or kidney tissue. A unique feature of the method is that it has been
automated for high-sample throughput using a commercial robotic
system.
Penicillins in animal tissue may be detected using biochemical and phy-
sicochemical methods. Biochemical assays are based on using penicillins as
substrates for the enzyme penicillinase. These methods are more specific
than microbial-inhibition assays. Physicochemical methods usually employ
high-performance liquid chromatography. These physicochemical methods
tend to be more specific, precise and have lower limits of sensitivity;
however, none have been subjected to a successful interlaboratory study.
Screening methods commonly employed for penicillins use either direct
contact of a freshly cut tissue surface or exudate obtained from, or soaked
onto, absorbent discs from fresh or frozen meat. In some instances,
however, non-specific inhibitors may give false-positive readings. Proce-
dures, such as use of semipermeable membranes between the meat sample
and the absorbent disc tend to reduce false-positive results from this type
of screening test. Some recent analytical method approaches use solvent
extraction and are usually more sensitive because they involve a con-
centration step but they are more labor-intensive.
Promising physicochemical methods for penicillins involve liquid chro-
matography using ultraviolet detection (Moats, 1984; Teyzkowska et al.,
1989; Boison et al. 1991). Analytical detection limits usually range from
50-100 J.1g.kg-'. Boison et al. (1991) coupled the liquid chromatography
separation procedure to a mass spectrometer for confirming the presence
of the analyte in milk but its use for tissue analysis has not been reported.
A sensitive, determinative method for amoxicillin from bovine and swine
tissues has been reported using aqueous extraction, removal of potential
interfering substances with trichloroacetic acid and liquid chromatography
following heat treatment to generate a fluorescent compound (USDA,
1991). Detection limits are reported to be 10 J.1g.kg-'. Very little develop-
mental work has been performed on the cephalosporins. One reason for
this is their relatively high safe concentration residue limits.
Tetracyclines have been used extensively as veterinary drugs. Although
tetracyclines have been analyzed by chemical and microbiological
methods, good analytical methods are rare. The classical method for
residues in milk and animal tissues is a microbiological procedure
(Kramer et ai., 1968). The assay is an agar-diffusion method using Bacillus
cereus (A TCC 11778) as the test organism. The assay has a sensitivity
limit of 2-200 l!g.kg- 1 depending on the tissue matrix being analyzed.
Tetracyclines are very stable as crystalline solids, yet solutions of these
materials are susceptible to decomposition. These analytes are also rela-
tively labile to mild heating in acid solutions, which destroys the tetra-
cycline ring structure. This property was explored as an approach to a
reliable method, but without success, by Ashworth (1986).
Most of the recent efforts to develop reliable methods have focused on
liquid chromatography and, to a lesser extent, on thin-layer chromato-
graphy methods because of the polar characteristics of the tetracyclines.
Mild acidic extraction methods are almost universal for residue analysis.
Use of acidic extraction media to obtain suitable recoveries is usually
attributed to the tenacity of tetracyclines to bind to tissue. This is most
evident on extraction of incurred tetracycline residues. The second attri-
bute affecting method development and analyte recovery is their ability to
form chelates, which leads to poor recoveries.
A creative approach to an acceptable method has been developed which
turns the chelating capability into an asset by using a chelated sepharose
adsorbant in a copper(II) form as part of the chromatographic clean-up
procedure (Farrington et ai., 1991). Using this approach, acceptable re-
coveries have been reported with detection limits approaching 10 l!g.kg- 1 .
This analytical procedure has not been studied collaboratively. Another
promising approach has been reported that uses a solid-phase extraction
procedure (Oka et ai., 1985). Recoveries have been reported for seven tet-
racyclines in honey at 20-50 l!g.kg- 1 and 10 l!g.kg- 1 in bovine and swine
tissue. An interlaboratory method validation study is pending.
Few veterinary drugs have been targeted for method development activ-
ities like chloramphenicol. Owing to the toxicological properties of chlor-
amphenicol, method development has focused at residue concentrations at
or below 10 l!g.kg- 1. Somewhat uncommon, is that muscle tissue is the
matrix of choice for determining chloramphenicol residues in red-meat
animals. One particular benefit of this is that such methods are applicable
to domestic and import residue-testing programs. Approaches include
various immunochemical and virtually every mature chromatographic and
instrumental procedure and, in some instances, combinations thereof
(Sheppard, 1991). Several methods have been subjected successfully to
interlaboratory validation (Aerts et ai., 1989; Codex Alimentarius Com-
mission, 1992), although many others have not. Results from many pub-
lished studies may, therefore, be inconclusive and only provide general
information on chloramphenicol residues.
Methods that have been evaluated successfully in multi-laboratory
studies include high-performance liquid chromatography (Aerts et al.,
1989), gas-liquid chromatography following derivatization (Epstein et al.,
1986) and negative-ion chemical-ionization mass spectrometry. Data from
the high-performance liquid chromatography procedure indicate that it
has a throughput of about 20 samples per day with recoveries of approxi-
mately 55% at 10 Ilg.kg-1 and a limit of detection of 1.5 Ilg.kg-1. The gas
chromatographic procedure (Epstein et aI., 1986) suitable for urine and
muscle tissue includes the use of a glucuronidase digestion step to convert
any glucuronide metabolites to the parent drug. This permits determina-
tion of free and at least one metabolite of chloramphenicol. This is rela-
tively important as the presence of this enzyme in the human digestive
system would readily hydrolyze this metabolite to the parent drug. Mod-
ifications of this method (USDA, 1991) include use of meta-
chloramphenicol as an internal standard and recovery index, and the use
of a solid-phase extraction step to provide a cleaner extract. As a result of
these changes, the procedure can be used to readily determine chlor-
amphenicol residues at concentrations of 1 Ilg.kg-1 in tissue and urine.
The only other published method with suitable analytical data in this
range is a radioimmunoassay procedure with a limit of detection of
0.2 Ilg.kg-1 (Balizs and Arnold, 1989).
16.2.5.2 Anabolic hormones Few veterinary drugs have had as much

impact on public health, consumer interest or international trade as
anabolic hormones - in some instances, with reason. The synthetic
anabolic hormone diethylstilbestrol (DES) has been banned worldwide as
a veterinary drug because of its carcinogenic and other adverse human
health effects. The naturally occurring anabolic hormones, however, in
some instances have been claimed to yield meat products with unsafe
residues, although scientific assessments affirm their safety (WHO, 1988).
The EC has prohibited the use of natural and synthetic hormones for
growth promotion but the natural anabolics are permitted for certain
therapeutic purposes. When used according to good veterinary practices,
residue levels are usually in the low parts per billion region. To determine
reliably these analytes, detection limits of less than 1 Ilg.kg-1 are required.
This requirement usually dictates extensive isolation and clean-up before
employing mass spectrometry procedures. In some instances, immu-
noassays with extensive clean-up have been used with mass spectrometry
confirmation for a wide variety of anabolic hormones (Dixon and Russell,
1986).
Methods of analysis for the naturally occurring anabolic hormones (e.g.
estradiol, progesterone and testosterone) and their metabolites, require

highly sensitive methods to detect sub-parts per billion or parts per trillion
concentrations (US Code of Federal Regulations, 1992). Using these
methods, increases in concentrations compared with controls have been
demonstrated, however, the increases are small (ng.kg- I differences). In
addition, the variability of these methods at these low concentrations is
insufficient to measure a specific analyte with a high degree of accuracy. A
second critical factor in the analysis of these natural hormones is that the
small increases in residue concentrations determined in treated steers,
heifers and calves are smaller than the variations seen in cycling heifers
and much smaller than the large increases in estrogen residue concentra-
tions seen in pregnant heifers (WHO, 1988).
Several methods of analysis have been reported and they have been
reviewed by Metzler (1989). Some of these methods have been used for
regulatory analysis (Covey et at., 1988). Sample-preparation procedures
for these compounds usually include enzyme or acid hydrolysis because
the anabolic drugs exhibit tissue-dependent conjugation to sulfates and
glucuronides. A second important feature of these analytes affecting
method development and performance is the phenolic hydroxyl group that
influences pH-dependent partitioning and extraction during sample clean-
up. As a result, ratios of the cis:trans isomers detected may vary and
influence recovery estimates. Regulatory methods reflect appreciation of
these two chemical properties.
A regulatory method for DES and zeranol (USDA, 1991) uses a novel
three-phase solvent extraction system of aqueous buffer, acetonitrile,
dichloromethane and hexane to remove effectively the majority of the tri-
glycerides and highly non-polar materials prior to solid-phase extraction
clean-up procedures. A strongly basic anion exchange resin is used to
extract the analytes of interest. Following additional solvent washes, the
analytes are derivatized and subjected to gas chromatography/mass spec-
trometry for quantification and confirmation using zeralane and deuter-
ated DES as reference standards. The procedure may be automated for
high sample throughput. The method will detect DES, zeranol and taler-
anol at or below 1 Ilg.kg-I.
The progestational agent, melengestrol acetate, has been analyzed using
a gas chromatography/chemical-ionization mass spectrometry procedure
following thin-layer chromatography clean-up of sample extracts,
although some difficulty has been experienced with achieving highly repro-
ducible chromatographic results (Neidert et at., 1990). A method using
similar technology to the DES"zeranol method already mentioned has
been developed for melengestrol acetate but it employs normal-phase on-
line liquid chromatography column switching (Chichila et at., 1989). The
detection limit for liver is reported to be 5 Ilg.kg-I. The method is suitable
for bovine fatty tissue.
The glucocorticosteroid, dexamethasone, has been quantified and con-

firmed in bovine muscle and liver using a similar approach (McLaughlin
and Henion, 1990; USDA, 1991). One notable difference in this procedure
is the use of a cyano-packed column in place of the silica analytical
column because of the physicochemical differences with the other steroidal
materials. The detection system involves coupled-column normal-phase
liquid chromatography and further clean-up on-line with the analytical
determination. Time-programmable switching valves control the chroma-
tography. The limit of detection is approximately 5 I-lg.kg- 1, whereas the
method provides acceptable method performance for accuracy and preci-
sion at 10 I-lg.kg- 1•
Methods used for analysis of anabolic hormones at concentrations less
than 1 I-lg.kg- 1 rely on gas chromatography/mass spectrometry procedures.
A multi-residue method for this purpose has been developed that permits
limits of detection of 0.02-0.05 I-lg.kg- 1 for estrogens and androgens and
is reported to have been collaboratively tested successfully (Sheppard,
1991). The method has a very extensive clean-up procedure. Other mass-
spectrometry procedures are not so sensitive and interfering substances
tend to demand very careful clean-up procedures.
16.2.5.3 Anthelmintics. Benzimidazoles are the principal class of com-

pounds having anthelmintic efficacy. A typical structural feature of these
compounds is a 5-substituted-2-carbamoylbenzimidazole moiety. Few
methods for the analysis of benzimidazole residues are available, although
some of them have been used for regulatory analysis. Typical of the
methods used and available are those submitted by the drug sponsor.
These methods are, however, usually unsuitable for multi-residue analysis
without modification because they were intended to be compound-specific.
Contributing to the complexity of residue analysis of this class of com-
pounds is the metabolism of the 5-substituted side-chain and hydrolysis of
the carbamate moiety. In the case of the 5-thio-substituted compounds,
oxidation occurs readily to give the corresponding sulfoxide and, subse-
quently, the corresponding sulfone as well as products of aromatic ring
hydroxylation (Sheppard, 1991). The result of these metabolic paths is
that suitable methods must be applicable to the parent drugs as well as
their oxidative metabolites. Compounds fitting this pattern include
febantel (which undergoes in vivo cyclization to fenbendazole), fenbenda-
zole and oxfendazole. Exceptions to these benzimidazole metabolic
pathways are cambendazole, albendazole and thiabendazole, although
they exhibit some of the same metabolic pathways, depending on their
specific structural features.
Methods for the benzimidazoles typically employ organic extraction,
solid-phase extraction and subsequent high-performance liquid chromato-
graphy for quantification. Confirmation typically requires mass spectro-
metry methods (Tai et al., 1990; USDA, 1991). A novel sample clean-up
procedure involves a technique described as matrix solid-phase dispersion
(Long et al., 1990a). This procedure tends to shorten sample preparation
and purification but has not been collaboratively studied for residues in
meat products.
A typical example of the multi-residue method used for regulatory
analysis involves use of reversed-phase high-performance liquid chromato-
graphy with ultraviolet absorbance detection. Residues determined using
this procedure include thiabendazole and its metabolite, albendazole 2-
aminosulfone, carbendazim, oxfendazole, mebendazole, cambendazole and
fenbendazole (USDA, 1991). The sensitivity of the method is approxi-
mately 25 Ilg.kg-1 based on a 10 g sample. The method takes advantage of
the weakly basic properties of the benzimidazoles for effective extraction
and clean-up. A modification has been developed for samples where
matrix effects prevent satisfactory liquid chromatography analysis by using
a mildly acidic ethanol solution with a C2 solid-phase extraction column
and subsequent elution with ethyl acetate. The eluate is determined by
liquid chromatography. Confirmation requires derivatization for gas
chromatography/mass spectrometry analysis using select-ion monitoring.
A second but limited class of anthelmintics are the substituted tetra-
hydro pyrimidines - morantel and pyrantel tartrate. The residue assay is
designed to detect and quantify the parent drug-related metabolites that
are hydrolyzable to N-methyl-l,3-propanediamine in swine liver and
muscle tissue (Lynch and Bartolucci, 1982). Tissues are hydrolyzed in
aqueous potassium hydroxide, yielding the propanediamine common to
both compounds. Following hydrolysis, both diamines are converted to a
bis nitrotrifluorobenzene derivative, purified with thin-layer chromatog-
raphy and quantified by gas chromatography using an electron-capture
detector. An internal standard is used to aid quantification. Confirmation
focuses on the aromatic component generated by the initial hydrolysis
used for quantification, the specific anthelmintic thienyl molecules, using
select-ion monitoring mass spectrometry. This procedure provides specific
identity and quantification of either parent drug.
A less commonly used anthelmintic compound, dibutyltin dilaurate, has
been used for treating hexametiasis and parasitic worms in poultry. Owing
to its potential toxicological properties and the presence of residues, its
use has been largely curtailed. Early methods proved to be non-specific,
simply identifying elemental tin. A specific procedure has subsequently
been developed (Epstein et al., 1990). Dibutyltin dilaurate is extracted
from tissue, followed by a silica 'sep-pac' clean-up and quantified using
high-performance liquid chromatography with post-column derivatization
and a fluorescence detector. The method is applicable to concentrations of
0.1 mg.kg- 1 in liver and muscle tissue. Studies using this method demon-
strated that substantial residues can occur in turkeys (Epstein et al., 1990).
16.2.5.4 Coccidiostats. The most common class of compounds having

antiprotozoal activity are the nitroimidazoles - dimetridazole, ipronida-
zole and ronidazole. Metronidazole is less commonly used. Dimetridazole
is used for prevention and control of histomoniasis in turkeys and dysen-
tery in swine. Ipronidazole and ronidazole are used for prevention and
control of histomoniasis in turkeys. The drugs are administered in water
or feed.
The use of these compounds at permitted concentrations in feed and
drinking water in poultry and swine produce residues that deplete below
detectable concentrations 2-3 days post-dosing (to less than 1-2 f.lg.kg-1
in tissue), while total residues are orders of magnitude higher based on
radio tracer studies. Since residue concentrations for the parent compound
and their primary metabolite (oxidation of the alpha carbon at the C-2
position) are below the limit of detection at times beyond 2 days, it has
not been possible to establish any relationship between the total residue
and a compound to be used as a marker residue for determining com-
pliance with a regulatory tolerance. Coupled with the carcinogenic proper-
ties of these nitroimidazoles, residue analysis has been limited by the
sensitivity of the methods.
Metabolism work that has been performed identifies two metabolic
pathways occurring as a result of fission of the nitroimidazole ring. In one
pathway, fission occurs prior to oxidation of the side-chain. The second
pathway involves oxidation prior to ring fission. Subsequent metabolism
suggests that at least some of these small fragments become incorporated
into natural components and become unidentifiable. It is possible that
acetamide, a known carcinogen, can result from the fragmentation of
dimetridazole (WHO, 1974). A further concern relating to these com-
pounds is the possibility of the formation of 'bound residues', which result
from the reaction of reactive metabolic intermediates with an endogenous
tissue component. The result is that the total residue picture for these
compounds has not been fully characterized.
Method sensitivity for the nitroimidazole class of compounds is noted
above. Initial methods were gas-chromatographic procedures presented by
drug sponsors (Kane, 1961; MacDonald et al., 1969). The early methods
of analysis relied on polarography with a sensitivity of 0.05 mg.kg- 1, to
detect parent and intact C-2 oxidized side-chain compounds. The sensitiv-
ity was improved by using differential pulse-polarography techniques (to
1-2 f.lg.kg-1) but the lack of specificity of these methods likely resulted in
detection of all nitroimidazole residues present in the sample (Craine et
al., 1974).
More recent methods have relied on high-performance liquid or gas-
liquid chromatography with ultraviolet detectors. A liquid-chromato-
graphic method for dimetridazole residues in poultry tissue has been
developed and successfully collaborated (Hobson-Frohock and Reader,
1983). It has a detection limit of 10 Ilg.kg-1, although the extracts are suf-
ficiently clean to suggest improvements in sensitivity are possible. More
rapid methods have been reported (Carignan et aI., 1988) as well as
methods with greater sensitivity (Newkirk et al., 1990) but they have not
been validated for regulatory use. Other methods used include derivatiza-
tion of sample extracts following solvent partitioning clean-up and gas-
liquid chromatographic analysis (USDA, 1991).
Owing to the residue and toxicity considerations of the nitroimidazoles,
several other drugs have been introduced. Included in this list are the
ionophore polyethers, halofuginone, nicarbazin and decoquinate. Among
the ionophores are monensin, narasin, salinomycin and lasalocid. These
ionophore polyethers are challenging analytes because, with the exception
of lasalocid, they have no useful chromophore to facilitate design of
suitable detection systems. In addition, they tend to metabolize rapidly
and unchanged drug represents only a minor fraction of the total residues.
Most validated methods for monensin, narasin and salinomycin couple
column chromatography with bioal,ltography detection (Analytical
Methods Committee, 1986). These methods were originally developed by
drug sponsors for registration purposes. Typically, the analytes are extrac-
ted with methanol, solvent-partitioned and subjected to silica gel or
alumina-column chromatography. The semipurified analytes are subjected
to silica gel thin-layer chromatography and the analytes detected by
bioautography using Bacillus subtilis as the assay organism. Residue con-
centrations are estimated by comparing zones of inhibition with external
standards. The method is usually applicable to muscle, liver and kidney if
tetrachloromethane is used for solvent partitioning, and to fat samples if
acetonitrile is used (USDA, 1991).
Lasalocid residue analysis has been developed based on its fluorescence
in the ionized form and the marked increase in fluorescence when the pH
is raised from 3.2 to 8.3 (MacDonald, 1978). Tissue is extracted with acet-
onitrile, partitioned with solvent, evaporated and the residue redissolved
in water that is saturated with mobile-phase solvent and analyzed by high-
performance liquid chromatography with fluorescence detection. Quantifi-
cation is calculated from an external standard curve made up from for-
tified, enzyme-deactivated liver tissue (USDA, 1991).
Clopidol is an effective co~cidiostat used in poultry feed. A regulatory
gas-chromatographic method has been developed (Mtema et al., 1984).
Poultry tissues are extracted with methanol followed by alumina and
anion-exchange column chromatography clean-up. The eluate is deriva-
tized with diazomethane, extracted and subjected to gas chromatography
using an electron-capture detector.
Halofuginone is used primarily in young chickens and turkeys. It is,
however, prohibited from use during the last 4 days before slaughter. The
regulatory method is based on the new animal drug application developed
by the sponsor (Hazelton Biotechnologies Corporation, 1982, 1984;

USDA, 1991). The drug residue is extracted from enzyme-digested liver as
a free base, partitioned into an ammonium acetate buffer solution, con-
centrated with a silica solid-phase extraction system, eluted, concentrated
and dissolved in the liquid chromatography mobile phase. Detection is
with a variable wavelength ultraviolet detector. The limit of quantification
is 0.05 mg.kg- I . The confirmatory method is a negative-ion chemical-ioni-
zation mass spectrometry/mass spectrometry procedure. The method is
applicable to chicken and turkey liver.
Nicarbazin is a 1:1 molar mixture of 4,6-dimethyl-2-pyrimidinol (HDP)
and 4,4-dinitrocarbanilide (DNC). The residue is extracted from tissue,
filtered and evaporated. The residue is purifed by liquid-liquid partitioning
and alumina column chromatography. Analytical separation and determi-
nation are accomplished by high-performance liquid chromatography with
ultraviolet detection of the DNC portion of the nicarbazin complex
(USDA, 1991). The method is applicable to chicken muscle and liver.
16.2.5.5 Sulfonamides. Sulfonamides have been used broadly in human

and veterinary medicine for decades as antibacterial agents. They are
usually employed as feed additives or boluses in food-producing animals
as prophylactic agents. Their broadest application is in porcine and
bovine species and they have been particularly noteworthy for their persis-
tence as a veterinary drug residue in pigs. This broad use and potential for
drug residues has resulted in extensive method development, however, as
with many other classes of analytes, few methods have been collaborated
successfully. The target tissue for sulfonamide residue analysis is the liver,
although kidney and muscle tissue are also frequently analyzed.
The most frequently used sulfonamide and the one to which persistence
of residues in swine production is attributed is sulfamethazine (also known
as sulfadimidine). In efforts to understand better the persistence of sulfa-
methazine residues, several unpublished USDA studies have been con-
ducted. Studies indicate that its metabolic half-life and its electrostatic
properties as a powdered feed additive contribute significantly to drug
residues.
To better understand the metabolic pattern of sulfamethazine, con-
trolled feeding studies have been conducted to determine if tissue:fluid
relationships exist that would enable producers to improve quality control
during animal production (Randecker et al., 1987). Results from these
studies indicated that predictive relationships exist, such that tissue con-
centration is a function of fluid concentration. For serum, the mean
tissue:fluid ratios in muscle, liver and kidney were 0.24, 0.90 and 0.53,
respectively. For urine, the respective ratios were 0.08, 0.27 and 0.16.
Variability attributed to biological, analytical and other factors is
acknowledged. These data have been used by the Food Safety and Inspec-
tion Service to develop a thin-layer chromatographic semi-quantitative

screening procedure that may be used for in-plant testing of pig urine to
identify suspect animals and carcasses for sulfonamide residues.
Sulfonamide metabolism occurs through conjugation of the terminal
aromatic amine via acetylation, conjugation with reducing sugars, such as
the glucopyranosyl derivative and glucosamine. These metabolites are
converted reversibly to the parent drug by use of mild acid hydrolysis,
which is often employed in analytical schemes. Metabolism also proceeds
by loss of the terminal aromatic amine but this metabolite is not deter-
mined by most residue methods. To account for these metabolites and
others, methods focus on analysis of the parent drug (the residue marker)
as a function of total sulfonamide residues. Most sulfonamide residue tol-
erances have been established at 0.1 mg.kg- 1 in tissue.
Residue methods for sulfonamides usually involve thin-layer, gas-liquid
or high-performance liquid chromatography for quantitative determina-
tion. Confirmatory methods tend to be gas chromatography/mass spec-
trometry procedures following derivatization of the sulfonamide. The
sulfonamide nitrogen is usually alkylated with diazomethane, followed by
acylation of the terminal aromatic amine (USDA, 1991). A gas chromato-
graphy/mass spectrometry procedure has been developed and subjected to
collaborative study for quantification and confirmation of sulfonamides in
milk (Weber and Smedley, 1989). Analytical sensitivities of 5-20 J..Lg.kg- 1
for 10 sulfonamides are reported, although some have variable recoveries.
Although most methods use tissue homogenization and solvent extraction
as the initial isolation procedure, a matrix solid-phase extraction proce-
dure has been developed that permits digestion of the sample, typically
with a C-8 or C-18 silica-extraction cartridge followed by column chroma-
tography of the dried mixture (Long et at., 1990b). This procedure may
have promise for reducing solvent use in sulfonamide residue analysis.
Sulfonamides tend to be well-suited for residue analysis using reversed-
phase liquid chromatography with ultraviolet detection. Many recent pro-
cedures have used this approach and coupled it with solid-phase extrac-
tion procedures. Using this technique, sulfonamides are extracted from
homogenized tissue with an organic solvent, isolated on a silica solid-
phase extraction cartridge, eluted with a small volume of solvent and
analyzed by liquid chromatography (Haagsma et at., 1987). This proce-
dure has been used in regulatory laboratories and has been proven reliable
although some solvent difficulties have been noted (Sheppard, 1991).
Detection limits are reported to be 10 J..Lg.kg- l . A multi-residue method for
sulfonamides using thin-layer chromatography has been developed and
studied collaboratively using fortified and incurred tissue samples in red
meat and poultry (Thomas et at., 1983). The procedure couples thin-layer
chromatography with fluorometric scanning densitometry for quantifica-
tion. After addition of an internal standard, the homogenized tissue is
extracted, partitioned with a glycine buffer and re-extracted from the pH-
adjusted aqueous solution. Separation of the analytes from any co-extrac-
ted material is carried out on a silica-gel plate containing a pre-adsorbent
spotting area. Although visualization is quite satisfactory, the fluorescent
derivative fades with time. The limit of detection is 0.02 mg.kg- 1 with the
limit of quantification being 0.05 mg.kg- 1.
The method has been extended to 13 sulfonamides with confirmation
using gas chromatography/mass spectrometry (USDA, 1991). For con-
firmation, the sulfonamides are extracted from tissue as described above,
methylated with diazomethane and acylated with a perfluoroacid anhy-
dride before mass-spectrometry detection using electron-impact select-ion
monitoring.
Some procedures have been reported using immunochemical assays,
usually for sulfamethazine. One commercial test developed for use with
diluted pig plasma has performance characteristics published from a vali-
dation study (Singh et ai., 1989). The limit of detection was 10 J.lg.kg- 1
and the inter-well coefficient of variation at 100 J.lg.kg- 1 was less than 7%.
Intra- and interassay results showed coefficients of variation of less than
18% and 9%, respectively. Some cross-reactivity (12%) was reported with
sulfamerazine and less than 1% with other sulfonamides studied.
16.2.5.6 Tranquilizers. Veterinary tranquilizers consist of a broad group

of organic bases. Within this category of compounds are ~-blockers and
neuroleptic sedatives. Representatives of these two categories are carazolol
and azaperone, respectively. The rather widespread use of these com-
pounds is to control stress and anxiety in food-producing animals, typi-
cally in animal transport, but other uses are to control behavior in farm-
production practices. Use in the first case contributes to residues in food
because there is usually insufficient time to permit the drugs to clear the
animals. These compounds tend to concentrate in the kidneys and, for this
reason, kidney is the target tissue for residue analysis. Multi-residue
methods have been developed for residue control programs based on thin-
layer chromatography (Haagsma et ai., 1988) and high-performance liquid
chromatography (Keukens and Aerts, 1989).
The thin-layer chromatography procedure involves digesting the tissue
with hot sodium hydroxide solution, extraction with ether, passing the
extract onto a pre-treated silica-gel column and eluting with a dilute
methanol/hydrochloric acid solution. Following concentration of the
eluate, the supernatant is applied to pre-coated high-performance thin-
layer chromatographic plates. Development is two-dimensional, using
two solvent systems. Analytes are detected with ultraviolet light. The
procedure will detect azaperone, carazolol, acepromazine, propioproma-
zine, chlorpromazine, haloperidol, azaperol and xylazine. The detection
limit for azaperone and azaperol is 50 J.lg.kg- 1 . This procedure seems
appropriate as a screening method but it has not been studied collabora-

tively.
A method that has been used in a regulatory program for detection and
quantification is the liquid chromatography procedure by Keukens and
Aerts (1989). Initial sample extraction involves tissue homogenization and
extraction with acetonitrile. The extract is washed and passed onto a
silica-gel cartridge. The analytes are eluted, washed and chromatographed
using a reversed-phase liquid chromatography system. Detection is with a
fluorescence and ultraviolet-visible dual-detector system. Recoveries for all
analytes noted above were greater than 90%, except for xylazine (about
50%), at concentrations of 1O-40l1g.kg-1 in kidney. Limits of detection
were 0.3-6 I1g.kg-l.
A multi-residue approach has been developed for regulatory purposes
that combines liquid and thin-layer chromatography (van Ginkel et at.,
1989). This procedure uses basic hydrolysis, solvent extraction, purifica-
tion with a solid-phase extraction cartridge and analyte separation with a
reversed-phase liquid chromatography system and ultraviolet detector.
Three sample eluates are collected. Following another solvent wash of the
three eluates, the analytes are determined by thin-layer chromatography.
Detection limits are 1-10 I1g.kg-1. Results are comparable with those of
the liquid chromatography method.
16.2.5.7 Macrocyclic tactones. One of the most widely used drugs for
veterinary purposes is ivermectin. It is a potent endo- and ectoparasitic
agent with a broad spectrum of activity in several animal species and
humans. It is a mixture of two closely related macrocyclic molecules that
are chemically modified fermentation products isolated from Streptomyces
avermitilis. The mixture of the two components (often referred to as aver-
mectins) contains at least 80% H2Bla and no more than 20% H2Blb.
Using radiometabolism studies, the H2Bla component is metabolized
more slowly than others and occurs in tissue as the major unmodified
residue. The drug has two target tissues - liver and fat. These tissues are
used for the determination of the residues in food-producing animals. The
two ivermectin components have little functionality for residue detection
and must be converted chemically to a fluorescent compound for quantifi-
cation and confirmation. The fluorescent residue marker compound is
then quantified using an ultraviolet detection system.
The analytical procedure used for residue analysis in animal tissue was
developed by the drug sponsor (Tway et at., 1981). The analyte is extrac-
ted from tissue with iso-octane, initially purified by precipitation of extra-
neous matrix components, followed by extensive liquid-liquid partitioning,
dehydrated to yield the fluorescent derivative and analyzed using high-
performance liquid chromatography. Recoveries average about 75% over
a concentration range of 5-60 I1g.kg-I, with a limit of detection of about
3 I-lg.kg- 1• The method is rather labor intensive and recent efforts have
focused on developing a modified procedure that can be automated for
greater laboratory sample throughput efficiency (FSIS Eastern Technical
Support Laboratory, personal communication).
Attempts have been made to develop a mass-spectrometry confirmation
procedure but, despite extensive sample purification efforts, a satisfactory
procedure has not been forthcoming. The method of choice is a modifica-
tion of the quantitative procedure (USDA, 1991). Part of the purified
extract from the quantitative procedure is converted to a monosaccharide
and an aglycone using 1% sulfuric acid in isopropanol and methanol,
respectively. After solvent extraction and partitioning, the solvent is
removed and the extracts dehydrated to yield the fluorescent derivatives.
After organic solvent extraction, the mixture is purified with a silica-gel
extraction cartridge and subjected to liquid chromatography. All three
peaks must be semi-quantified. These methods are suitable for muscle as
well as liver, fat and plasma in red-meat species.
16.2.5.8 Beta-agonists. Beta-agonists have limited approvals for respira-

tory purposes in animals but they are now being used to increase the rate
of growth and the lean:fat ratio in food-producing animals. The high
level of pharmacologic activity of clenbuterol has led to its use for this
purpose. Residues of clenbuterol in cattle liver have been identified as the
responsible agent of acute toxicity-related illnesses in Spain and France.
Acute toxicity in humans occurring as a result of residues from a veter-
inary drug in food of animal origin is a limited phenomenon. Public
health considerations are usually with chronic toxicity (e.g. mutagenicity
or carcinogenicity). Analytical schemes have been developed for animal
urine and animal tissue for residue control programs (Schilt et al., 1990;
van Ginkel et aI., 1992). Liver is typically the tissue of choice. For residue
analysis of the beta-agonists, the concentration of interest is 1 I-lg.kg- 1 or
lower.
The general structure of these compounds is similar to the epinephrines.
However, within this class of compounds, there are two major categories -
the substituted phenols and substituted anilines. These two categories have
dissimilar chemistry, which limits development of a multi-residue method.
The phenols and amines have weakly acidic and basic properties, respec-
tively. The substituted anilines require pH adjustments to partition them
between organic solvents and aqueous media. The substituted phenols
tend to be charged species under the pH conditions suitable for purifying
the aniline derivatives. Another consideration in method development is
that these compounds are protein bound and enzymatic digestion with a
protease is necessary to get acceptable recovery.
The structural similarity of the aliphatic side-chain provides a suitable
substrate for using an immunochemical approach to a multi-residue
method. An application of this approach involves use of an immunochem-

ical (polyclonal antibody) reagent as an isolation/purification procedure
for residue analysis. An immunoaffinity chromatography/gas chromato-
graphy method (Schilt et al., 1990) has demonstrated a multi-residue limit
of determination of 1 Ilg.kg-1 in liver for clenbuterol, cimaterol, terbuta-
line and salbutamol. The antibodies are bound to an activated sepharose
support, resulting in an immunoaffinity column suitable for selective pre-
concentration of the analytes of interest. Tissue is homogenized, extracted
into a buffer solution, centrifuged and the aqueous layer pH-adjusted and
purified with a solid-phase extraction cartridge, prior to application to the
immunoaffinity column. The eluates may be quantified by use of a deuter-
ated clenbuterol internal standard. For structure identification, the
analytes are derivatized and subjected to gas chromatography/mass spec-
trometry using a combination of electron-impact and positive chemical-
ionization detection modes. The method is applicable to feed, urine and
liver. In cross-reactivity studies, the polyclonal clenbuterol antibody had
less than 5% cross-reactivity with the analytes noted above.
A similar analytical strategy has been developed for the detection of
beta-agonists containing an n-tertiary-butyl and an n-isopropyl phenetha-
nolamine structure (van Ginkel et aI., 1992). Extraction of the analytes is
based on immunoaffinity chromatography, with structure identification by
gas chromatography/mass spectrometry. The polyvalent antibodies were
prepared against clenbuterol and cimaterol and coupled to an activated
sepharose matrix. The immunoaffinity columns gave quantitative recov-
eries of 200 ng of clenbuterol, salbutamol, terbutaline, cimaterol and
mabuterol. Recoveries from liver tissue ranged from 40-70%, with limits
of detection by mass spectrometry being from 0.05-0.2 Ilg.kg-1 and limits
of identification 1-2Ilg.kg-1. Repeatability was less than 15%. The
method is applicable to animal feed, urine and tissue.
16.3 Summary
Although on the surface, meat is a simple matrix primarily consisting of

protein, fat and moisture, analytical strategies for meat composition,
meat-processing additives, pesticides, environmental contaminants or
veterinary drugs are, more often than not, rather specialized and complex.
Certainly, determination of analytes at parts per hundred or thousand
(percentage and fractions thereof) is generally not so complex as residue
analysis at parts per million (mg.kg- 1) or parts per billion (llg.kg-1). Some
may argue that one of the most difficult analytical determinations is
protein analysis. This is supported, for example, by the inherent difficulty
for distinguishing between meat protein and non-meat protein in a reg-
ulatory program.
Public health and safety considerations have been the primary focus of
recent years and this is reflected in the applications of technologies includ-
ing materials science, electronics and computers, analytical detectors and
instrumentation and maturation of scientific disciplines, such as micro-
biology, immunochemistry, organic chemistry and analytical chemistry.
These new technologies have been largely beneficial to regulatory
programs, while at the same time presenting new challenges for existing
food safety laws. The Delaney clause of the Food, Drug and Cosmetic
Act is such an example. Things that were incapable of being detected a
decade ago, have, in several instances, become rather common, resulting
in difficult decisions on how to interpret and apply this new science in a
regulatory environment.
Development of new technologies and their application to food compo-
sition and safety is expected to continue. In one sense, this attempt to
review most major regulatory analyses will have a built-in obsolescence.
Ten years from now, new technologies and strategies will provide new and
better analytical systems. Growing emphasis on reducing use of organic
solvents and environmental waste will dictate new procedures. Everyone
should prepare themselves for this eventuality.
Acknowledgements
The author acknowledges the support of Mr. H.J. Barth for portions of
the food chemistry text and assistance in typing portions of this manu-
script by Jessica Grantling and Valerie Sewell.
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17 The contributions of meat, poultry and fish to the
health and well being of man
J.R. LUPTON and H.R. CROSS
17.1 Introduction
Meat, poultry and fish (MPF) make important nutritional contributions

to the diet of mankind which have been extensively discussed in an earlier
volume of this series (Pearson and Dutson, 1990). The aim of this chapter
is to review briefly the contribution of MPF to the diet and to the health
and well-being of man. Special consideration will be given to the role of
MPF in the etiology of heart disease and certain cancers. The word 'meat'
is used to refer to red meats (e.g. beef, pork, lamb/mutton and veal).
'Poultry' is limited to chicken, turkey and duck, while 'fish' will include
fresh and saltwater fish and seafood. All nutrient data presented are from
the latest issues of the USDA Agriculture Handbook 8 series (1963, 1979,
1983, 1987, 1988, 1989, 1990a).
17.2 Consumption of meat, poultry and fish
For a food to contribute to the nutritional status of man, it must first be

consumed. In 1990, preliminary data showed that total per capita con-
sumption of MPF was 191.5 Ib (USDA, 1991). These data are retail
weight, and consumption represents total supply minus exports and non-
food use. Of that 191.5 Ib, red meat comprised 112.3 Ib, poultry 63.8 Ib
and fish 15.4 lb. The 1990 data showed a 113% increase in consumption
of chicken compared with 1965 and a 146% increase in turkey consump-
tion (Bunch, 1987). Fish consumption increased 37% between 1965 and
1990, while red-meat consumption decreased by 10% (USDA, 1991).
17.2.1 Factors affecting consumption of meat, poultry and fish

Many factors affect intake of MPF, including age, sex, income level, eth-
nicity, education, and health beliefs. In one study comparing 254 low-
income elderly Mexican Americans and non-Hispanic whites, ethnicity
was the major variable influencing food intake (Bartholomew et al., 1990).
Mexican Americans consumed more poultry and organ meats and less
beef than non-Hispanic whites (Bartholomew et al., 1990). In contrast, sex
was the major variable influencing food intake in a recent study examining
MPF consumption in the healthy elderly (Koehler et al., 1992). Women's
mean daily intake of meat/poultry/fish was 87.7 g (52.1 g meat, 23.6 g
poultry, and 12.0 g fish). Men's intake of meat/poultry/fish was 121.2 g
(78.4 g meat, 21.2 g poultry and 21.6 g fish). Of the total red meat, more
than 50% was beef, 20% was pork or ham, and more than 10% was pro-
cessed meat. For women, meat/poultry/fish provided 14% of total dietary
energy, 37% of protein, 13-32% of selected B vitamins and iron, 20% of
fat and 34% of cholesterol. Percentages were similar for men. In one
study in which men and women were selected randomly from the
Auckland, New Zealand general electoral rolls (Scragg et al., 1991), men
were found to consume significantly more fat and cholesterol than
women, consistent with their increased intake of red meat (median
servings per month = 28 for men, 23 for women) and their greater
tendency to eat fried meat (80.3% vs. 71.7%). In contrast, women
consumed more carbohydrate and fiber than men, consistent with their
increased intake of vegetables and fruit (Scragg et aI., 1991).
Of particular importance to this review is the influence of diet/health
messages on intake of MPF. Recent changes in the consumption patterns
of MPF are thought to be due, in part, to consumer response to health
messages. Changes are evident in the American diet, particularly in
response to the message to decrease consumption of fat. In clinical trials,
when participants are instructed to decrease fat intake, the diet modifica-
tions most frequently made are reductions of butter, margarine, eggs and
red-meat consumption. In addition, what one family member eats affects
the food intake of other family members. For example, in the Women's
Health Trial, the intervention women consumed 21 % of total calories
from fat vs. 38% for control women at 6 months post-randomization
(White et al., 1991). When husbands of the women in the trial were
surveyed, intervention husbands consumed 34% of total calories from fat
vs. 36% for controls (White et aI., 1991).
Consumers have also opted for lower-fat versions of the same product.
Intakes of high-fat beef and pork decreased between the two most recent
Nationwide Food Consumption Surveys (1977-78 and 1987-88), while
low-fat beef, pork, poultry and fish intake increased (Popkin et al., 1992).
This would seem to be in response to diet/health messages to decrease fat.
However, consumption of many non-MPF sources of calories and fat (e.g.
high-fat desserts, butter and margarine) changed little between 1977 and
1987 (Popkin et al., 1992). This report, and others, suggests that con-
sumers may be responding to diet/health messages, but that their response
may not always be appropriate.
Of interest is the finding that, while Americans are attempting to
decrease consumption of MPF and increase consumption of complex car-
bohydrates, the opposite is true for developing countries. At present, the
HEALTH CONTRIBUTIONS 481
Chinese eat primarily a plant-based diet as shown by the finding that

plant-derived foods supply 93% of energy, 87% of protein and 55% of fat
(Kantha, 1990). Among animal foods, pork is the most common and least
expensive form of meat, contributing more than 90% of China's total
meat production, excluding poultry and fish (Kantha, 1990). However,
MPF consumption is increasing in China, while carbohydrate consump-
tion is decreasing (Kantha, 1990).
17.2.2 How consumption of meat, poultry and fish contributes to nutrient

intake
One of the most important dietary guidelines is to eat a variety of foods
(USDAjDHHS, 1990). It is one thing for a food to be a good source of
particular nutrients but if the food is not consumed its nutritional value is
zero. The extent to which people follow the guideline to eat a variety of
foods was recently evaluated by scoring consumption of items from the
food groups and numbers of servings from those groups using data from
the second National Health and Nutrition Examination Survey (Kant et
al., 1991a). MPF scored the highest in consumption with the proportion
of the population consuming at least the desired number of servings from
each food group - dairy (51 %), meat (71 %), fruit (29%), vegetables
(29%) and grains (61 %). In a second study, food-group intake patterns
were evaluated using dietary recall data from the second National Health
and Nutrition Examination Survey (Kant et aI., 1991b). Each 24 h dietary
intake recall was evaluated for the presence or omission of five broad food
groups - dairy, meat, grain, fruit and vegetable. The five most prevalent
patterns and the proportion of the population reporting them were as
follows: (i) all food groups, 33.6%; (ii) no fruit, 23.9%; (iii) no dairy and
fruit, 9.0%; (iv) no dairy, 8.0%; and (v) no fruit and vegetable, 5.6%. The
pattern of eating all food groups was the only one that provided mean
amounts of all of the key vitamins and minerals at levels greater than or
equal to the Recommended Dietary Allowances (RDAs) (Kant et aI.,
1991b).
17.3 The nutritiomd value of meat, poultry and fish
When talking about MPF as food one is concerned primarily with the
skeletal muscle of the animal. Different muscles contain different ratios of
protein:fat. In general, the more a muscle is used, the less fat it contains.
The type and amount of fatty tissue may vary with the species, genetics,
the age of the animal and its diet and treatment during growth. This fact
is of particular importance with meat, since intramuscular fat (marbling)
contributes both to tenderness and to fat intake. Water is the most
abundant constituent of MPF. The fatter the animal, the lower the per-
centage of water. Beef muscle from a mature, moderately fat animal may
contain 45% water in contrast to 72% water in veal muscle from a young
lean animal (National Livestock and Meat Board, 1991). Other important
nutrients in MPF are vitamins and minerals, which will be discussed in
detail in this chapter.
17.3.1 Criteria for what makes a food 'nutritious'

Although there is no set rule as to what makes a food nutntIous, in
general terms, if a serving supplies at least 10% of an individual's Recom-
mended Dietary Allowance (RDA), it is considered a good source of that
nutrient. The RDAs are tables of amounts of essential nutrients con-
sidered adequate to meet the needs of healthy individuals. The tables are
divided by sex and age and the values contain a margin of error to
account for nearly all healthy people in the USA. The RDAs are used by
nutritionists and dietitians and as the basis for most public health
programs. The RDAs are shown in Table 17.l.
Since most people are also calorie-conscious, the fewer calories required
to obtain the nutrient, the better. For example, the RDA for protein for
women 25 years of age and over is 50 g per day, while the recommended
energy intake is 2200 calories. One serving of lean beef (3 oz) contains
approximately 25 g of protein at a cost of 183 calories. Thus, a 3 ounce
serving of lean beef would provide the reference woman with half of her
RDA for protein at a cost of only 8.3% of her recommended energy
intake. This is a lot of nutrition for the energy dollar. The concept of a
food being high in a particular nutrient relative to the calories provided is
called 'nutrient density'. Any food that provides a greater portion of the
individual's RDA for a particular nutrient than it does for that indivi-
dual's RDA for calories would be nutrient-dense with respect to that par-
ticular nutrient. In Figures 17.1 and 17.2 are shown the major nutrients
contributed by MPF as a percentage of the RDAs for women aged 25-50
years (Figure 17.1) and men aged 25-50 years (Figure 17.2). Information
on the specific nutrients shown is provided below.
17.3.2 Specific nutrients supplied by meat, poultry and fish

Specific nutrients contributed by MPF include protein, lipids, vitamins
(particularly thiamin, riboflavin, niacin, vitamin B6 and vitamin B12) and
minerals, including iron and zinc. In Chapter 1 is included a summary
table of the contributions of meat, poultry and fish products to the diet of
humans in the USA (Table 1.2). This section in this chapter will discuss
the positive contributions of MPF to human nutrition.
Table 17.1 Recommended Daily Dietary Allowancesa,b
Characteristics of population Protein Fat-soluble vitamins Water-soluble vitamins Minerals
"C
~
1§'
tl 00
~ ~ Oil Oil Oil Oil
OIl
"
Oil OIl OIl Oil Oil ~ S S
~
Oil 5 -3 ~ Oil
~ -3 -3 -3 z ~ N Oil
~
'00 5 5 5 OIl OIl S S Oil -3
<: Q ~ ~ U 5 ,:; rtf ~ ~
~ ~ ~ e..2.- g ,:; '0: ~
S ,:;
-3 e
0 .~ Oil Oil -3 S
,:; ,:; ,:; <l)
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,:; S S <l)
~
b'" 1: 1:
OIl
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.§ ~ ,:; 5
<.)
,:;
OIl 0 's
1:OIl § 'u OIl
.e!' (5
'sos 'u 0 ,:; ,:;
.0 " '6
'il 'il
'.::is '§os .§ 's
OIl 'il <l) '" '0'" OJ ..c: '"il
'"
'""
<:'" ~ ~ ::t: ::t: ~
... ~ ~ ;; ~ ~ ~'" ~ z'" ~'" ~ ~'" U ~ '"
~ .§ N ..s '"
Infants 0.0-0.5 6 13 60 24 13 375 7.5 3 5 30 0.3 0.4 5 0.3 25 0.3 400 300 40 6 5 40 10
0.5-1.0 9 20 71 28 14 375 10 4 10 35 0.4 0.5 6 0.6 35 0.5 600 500 60 10 5 50 15
Children 1-3 13 29 90 35 16 400 10 6 15 40 0.7 0.8 9 1.0 50 0.7 800 800 80 10 10 70 20

4-6 20 44 112 44 24 500 10 7 20 45 0.9 1.1 12 1.1 75 1.0 800 800 120 10 10 90 20
7-10 28 62 132 52 28 700 10 7 30 45 1.0 1.2 13 1.4 100 1.4 800 800 170 10 10 120 30
Males 11-14 45 99 157 62 45 1000 10 10 45 50 1.3 1.5 17 1.7 150 2.0 1200 1200 270 12 15 150 40
15-18 66 145 176 69 59 1000 10 10 65 60 1.5 1.8 20 2.0 200 2.0 1200 1200 400 12 15 150 50
19-24 72 160 177 70 58 1000 10 10 70 60 1.5 1.7 19 2.0 200 2.0 1200 1200 350 10 15 150 70
25-50 79 174 176 70 63 1000 5 10 80 60 1.5 1.7 19 2.0 200 2.0 800 800 350 10 15 150 70
51+ 77 170 173 68 63 1000 5 10 80 60 1.2 1.4 15 2.0 200 2.0 800 800 350 10 15 150 70
Females 11-14 46 101 157 62 46 800 10 8 45 50 1.1 1.3 15 1.4 150 2.0 1200 1200 280 15 12 150 45
15-18 55 120 163 64 44 800 10 8 55 60 1.1 1.3 15 1.5 180 2.0 1200 1200 300 15 12 150 50
19-24 58 128 164 65 46 800 10 8 60 60 1.1 1.3 15 1.6 180 2.0 1200 1200 280 15 12 150 55
25-50 63 138 163 64 50 800 5 8 65 60 1.1 1.3 15 1.6 180 2.0 800 800 280 15 12 150 55
51+ 65 143 160 63 50 800 5 8 65 60 1.0 1.2 13 1.6 180 2.0 800 800 280 10 12 150 55
Pregnant
women 60 800 10 10 65 70 1.5 1.6 17 2.2 400 2.2 1200 1200 320 30 15 175 65
Lactating 1st 6 months 65 1300 10 12 65 95 1.6 1.8 20 2.1 280 2.6 1200 1200 355 15 19 200 75
women 2nd 6 months 62 1200 10 11 65 90 1.6 1.7 20 2.1 260 2.6 1200 1200 340 15 16 200 75
'Source: Recommended Dietary Allowances. Revised (1989). bThe allowances, expressed as average daily intakes over time, are intended to provide for individual variations
among most normal persons as they live in the US under usual environmental stresses. Diets should be based on a variety of common foods in order to provide other
nutrients for which human requirements have been less well defined. CWeights and heights of Reference Adults are actual medians for the US population of the designated
age, as reported by NHANES II. The median weights and heights of those under 19 years of age were taken from Hamill et al. (1979). The use of these figures does not
imply that the height-to-weight ratios are ideal. dRetinol equivalents: 1 retinol equivalent = 1 I'g retinol or 6 I'g tJ-carotene. eAs cholecalciferol: 10 I'g cholecalciferol
= 400 IU of vitamin D. 'Tocopherol equivalents: 1 mg d-a-tocopherol = 1 aTE. gl NE (niacin equivalent) is equal to 1 mg of niacin or 60 mg of dietary tryptophan.
Nutrient
Calories "
I
Protein
Thiamin
...
~
Riboflavin
~
Niacin
Vitamin B6
Vitamin B' 2
Phosphorus
~
Iron
~P
Zinc
~
o ~ ~ ~
o o o o
6 6L() o 6
N
• Beef ~ Veal 0 Pork []J Lamb 6!l Chicken 0 Turkey • Fish
Figure 17.1 The contribution that 3 oz of cooked lean meat, poultry or fish (MPF) makes
to the Recommended Dietary Allowances (RDAs) for women aged 25-50 years. These calcu-
lations are based on a daily caloric intake of 2200 calories and the 1989 Recommended
Dietary Allowances. Each horizontal bar represents the percentage of the RDA for that par-
ticular nutrient supplied by 3 oz of beef, veal, pork, lamb, chicken, turkey or fish (codes for
these foods are shown at the bottom of the figure). If a serving of MPF supplies a greater
percentage of the RDA for a particular nutrient than it does for calories (shown at the top of
the figure) then it is a particularly good source of that nutrient. For example, a 3 oz serving
of MPF supplies less than 10% of the RDA for calories and 40-50% of the RDA for
protein. One 3 oz serving of beef, lamb or fish supplies over 100% of vitamin B12. Sources:
Data for beef, veal, lamb and pork were published in National Live Stock and Meat Board,
1991; data for chicken are for broiler breast, flesh only, roasted (USDA, 1979); turkey data
are for light meat without skin, roasted (USDA, 1979); and fish data are based on flatfish,
flounder, dry heat (USDA, 1987).
Nutrient
,
..
Calories·
Protein
Th iamin
~
Riboflavin
Niacin
-&
Vitamin B6
Vitamin B'2
Phosphorus
~
Iron
~
Z inc
~
<ft <ft <ft <ft <ft <ft ::!!
0 <ft <ft <ft <ft <ft ::!!
0
0
0 0 0 0
0
a
0
a
0
aN
0
0
0
a
0
ci
0
0
0
ci
.....
a aen a0 0 aN
" '"
(") <D (X)
111 Beef 0 Veal [J Pork (]I Lamb ~ Chicken 0 Turkey • Fish
Figure 17.2 The contribution that 3 oz of cooked lean meat, poultry or fish (MPF) makes
to the Recommended Dietary Allowances (RDAs) for men aged 25- 50 years. These calcula-
tions are based on a daily caloric intake of 2900 calories and the 1989 Recommended Dietary
Allowances. Each horizontal bar represents the percentage of the RDA for that particular
nutrient supplied by 3 oz of beef, veal, pork, lamb, chicken, turkey or fish (codes for these
foods are shown at the bottom of the figure). If a serving of MPF supplies a greater percen-
tage of the RDA for a particular nutrient than it does for calories (shown at the top of the
figure) then it is a particularly good source of that nutrient. For example, a 3 oz serving of
MPF supplies less than 10% of the RDA for calories and 30- 40% of the RDA for protein.
One 3 oz, serving of beef, lamb or fish supplies over 100% of vitamin B12. Sources: Data for
beef, veal, lamb and pork were published in National Live Stock and Meat Board, 1991; data
for chicken are for broiler breast, flesh only, roasted (USDA, 1979); turkey data are for light
meat without skin, roasted (USDA, 1979); and fish data are based on flatfish, flounder, dry
heat (USDA, 1987).
17.3.2.1 Amount and type of protein contributed to the diet by meat, poultry
and fish. MPF contribute both quantity and quality of protein to the
diet. As shown in Figures 17.1 and 17.2, just 3 ounces of lean cooked
MPF contribute from 38% to over 50% of the protein RDAs for adult
men and women, respectively. In addition, the protein is of high quality.
A high-quality protein is defined as one that supplies all of the essential
amino acids in amounts relative to the body's need for protein synthesis.
Although vegetable proteins are not of so high a quality as animal
proteins, their deficiencies can be compensated for by eating a combina-
tion of vegetable and/or animal proteins. The single most limiting amino
acid in plant proteins is lysine, which is high in MPF, making the combi-
nation of plant foods and MPF a complementary one (Pellet and Young,
1990).
17.3.2.2 Vitamins contributed by meat, poultry and fish. A summary table

of the contributions of MPF to the diet of humans in the USA is shown
in chapter 1 (Table 1.2), which provides an overview of the contribution
of both the fat- and water-soluble vitamins. MPF in general, are not par-
ticularly good sources of the fat-soluble vitamins (D,E,A,K), although
liver is the largest single food source of vitamin A and also is a good
source of D and K. MPF are, however, the major sources of five of the
water-soluble vitamins: thiamin, riboflavin, niacin, vitamin B6 and vitamin
B12 (Figures 17.1 and 17.2). Thiamin, riboflavin and niacin are all
required as coenzymes for the pathways of the complete metabolism of all
energy-providing nutrients, and MPF are excellent sources of all of these
water-soluble vitamins. Pork, for example, provides more thiamin than
any other food commonly eaten (National Livestock and Meat Board,
1991). Although niacin is considered an essential vitamin, it can also be
synthesized in the body from the amino acid tryptophan. Thus, MPF are
doubly good sources of niacin, i.e. as niacin itself and as its precursor,
tryptophan. Vitamin B6 is needed for the transfer of amino groups. Thus,
the more protein the body needs to handle, the higher is its need for B6.
Fortunately, MPF are excellent sources of both protein and vitamin B6.
Vitamin B12 is necessary for DNA synthesis, which is required every time
a cell divides. Rapidly proliferating tissues, such as fetal tissue, have high
B12 requirements. Vitamin B12 is only found in animal products.
17.3.2.3 Minerals contributed by meat, poultry and fish. Meat is a major

dietary source of iron, zinc and phosphorus as shown in Figures 17.1 and
17.2. Although phosphorus is not usually limiting in most US diets, high
amounts of this mineral are found in MPF. Iron deficiency, however, is
considered a public health problem in the US today, and national surveys
designed to monitor nutrient status have shown low iron intakes in certain
populations (Kant et al., 1991a). Not only are MPF excellent quantitative
sources of iron but the bioavailability of this essential mineral is higher

from MPF than from non-heme sources. Dietary iron is found as heme or
non-heme iron, with heme iron coming from the myoglobin and hemoglo-
bin of MPF. Heme iron accounts for approximately 50-60% of the iron
in beef, lamb and chicken and 30-40% of that in pork, liver and fish
(National Livestock and Meat Board, 1991). Humans absorb heme iron at
a rate about 5-10 times greater than non-heme iron (Cook and Monsen,
1976). In addition, the mixing of heme iron with sources of non-heme iron
(e.g. small amounts of MPF in a stir-fry vegetable mixture) improves the
absorption of the non-heme iron.
Like iron, zinc is differentially absorbed from vegetable products vs.
MPF. Absorption of zinc may be as high as 40% from MPF and as low
as 10% from cereals. Zinc deficiency occurs in individuals and populations
whose diets are low in sources of readily bioavailable zinc, such as red
meat, and high in unrefined cereals that are rich in phytate and dietary
fibers (Sand stead, 1991). Nearly half of an adult woman's RDA for zinc
can be met by only 3 ounces of lean beef (Figure 17.1). When the health
and nutritional status of 44 Dutch vegetarians, aged 65-97 years, was
investigated for long-term consequences of a vegetarian diet, the results
indicated that the elderly vegetarians were at a higher risk for a marginal
iron, zinc, and vitamin B12 deficiency than non-vegetarians (Lowik et aI.,
1990).
17.4 The role of meat, poultry and fish in human health
There is increasing emphasis today on the relationship of diet to human

health. Not only are foods expected to supply nutrients but they are
selected to impart health benefits or avoided if considered 'unhealthy'.
Based on epidemiological data, certain populations are considered to have
'healthy' diets, while others are not. In general, the western-style diet is
considered to be less healthy than diets from developing countries because
of its relatively high fat and low complex-carbohydrate content.
- Certain populations stand out as being exceptions to the hypothesis that
high fat/low carbohydrate diets are major contributors to chronic disease.
For example, Alaskan natives are often cited in diet/health studies because
rates of heart disease, cancer and diabetes are lower in Alaskan natives
than in US whites (Nobmann et aI., 1992). However, Alaskan natives
consume fewer fruits and vegetables than the general US adult population
and they also consume significantly more energy, protein and fat
(Nobmann et al., 1992). This seeming incongruity is 'explained' by the
finding that Alaska natives also consume six times more fish than the
general US adult population (Nobmann et aI., 1992). Fish is considered to
exert a protective effect, which overcomes the excess of calories and fat
and lack of fruits and vegetables (Nobmann et al., 1992). It should also be
pointed out, however, that the rate of iron-deficiency anemia for Alaskan
natives continues to be higher than that in the general population.
Several investigators have noted that humans subsisted for many thou-
sands of years on diets high in vegetables and low in animal products.
Certain of these investigators suggest that meat may not be essential for a
balanced diet. A recent review assesses the evidence for and against the
consumption of meat and its relationship to disease (Anon., 1991). In
contrast, others see the increased emphasis on limitation of red-meat con-
sumption as a return to the 'clean-living' movement of the late 19th
century and a limitation of individual choice regarding personal health
behavior (Engs, 1991).
17.4.1 Meat, poultry and fish and their relationship to heart disease
There are strong epidemiological data to suggest that blood serum choles-
terol is correlated positively with an increased risk for CHD (Ernest and
Cleeman, 1988; Keys, 1970; McNamara, 1990). However, the mechanisms
by which blood cholesterol can be most effectively lowered, and the degree
to which specific dietary factors influence serum cholesterol are less clear.
The primary nutrients in MPF that are investigated for their effect on
CHD are cholesterol and fat (both amount and type). These are discussed
in detail in the following.
17.4.1.1 Cholesterol content of meat, poultry and fish. Even though

dietary cholesterol has less effect on serum cholesterol than saturated fat
in the diet (Gotto, 1991), consumers are still concerned with their choles-
terol intakes. Animal products contribute 100% of the dietary cholesterol,
which translates into a concern over excess consumption of MPF. Meat,
poultry and fish have similar cholesterol levels (around 72-76 mg per 3 oz
(84 g) serving). The National Cholesterol Education Program recommen-
dation that intake of cholesterol be limited to 300 mg per day (Ernest and
Cleeman, 1988) means that one serving of MPF would supply about one-
quarter of the day's suggested intake.
17.4.1.2 Relationship of dietary cholesterol to serum cholesterol. One of

the most comprehensive diet/CHD epidemiological studies showed no
relationship between dietary cholesterol and CHD, nor did it show a cor-
relation between dietary cholesterol and plasma cholesterol levels, despite
providing strong evidence for the positive relationship between plasma
cholesterol levels and risk of CHD (Keys, 1970). Similarly, the Framing-
ham study (Gordon, 1970) and others (Gertler et al., 1950; Moore et al.,
1976; Morris et aI., 1976; Garcia-Palmieri et al., 1977) have found no
relationship between dietary cholesterol intake and plasma cholesterol
levels. In contrast, several other studies, including the Western Electric

study (Shekelle et al., 1981), the Zutphen study (Kromhout, 1983), the
Ireland-Boston Diet Heart study (Kushi et aI., 1985) and the Honolulu
Heart Program (Kagan et aI., 1981) have shown a positive relationship
between dietary cholesterol, plasma cholesterol and risk for CHD. Results
of clinical trials are equally equivocal, with some showing a positive asso-
ciation between dietary cholesterol and serum cholesterol (Keys et at.,
1965; Edington et aI., 1989), while others show no such relationship (Keys
et at., 1956; Kestin et at., 1989). The issue of the magnitude of the effect
(if any) of dietary cholesterol on blood cholesterol values remains unre-
solved. Obviously, this issue is of importance to the consumption of MPF
because animal products are the sole sources of dietary cholesterol. When
estimating the impact of dietary cholesterol intake from MPF on serum
cholesterol values, McNamara (1990) estimated that the cholesterol in
MPF contributes only 3-4 mg.dl- 1 of our plasma cholesterol levels or
approximately 1.5-2% of the total.
17.4.1.3 Amount and type of fat contributed to the diet by meat, poultry
and fish. Although there is no RDA for lipid in the diet, the recommen-
dation by the National Cholesterol Education Panel and by most major
health organizations is that calories from fat be limited to 30% of total
calories (Ernest and Cleeman, 1988; USDAjDHHS, 1990). Since excess fat
in the diet has been identified as a dietary problem, efforts have been
made to decrease fat in meat products and to switch from high- to lower-
fat food choices. Americans have decreased the proportion of calories
from fat from 40-42% in the 1950s and 1960s to the current value of
around 36% (Stephen and Wald, 1990). Industry has responded to the
demand for a leaner product. The meat animal industry has implemented
strategies to depress fat deposition and increase lean tissue gain (Bergen
and Merkel, 1991). Strategies used include the use of late-maturing
animals and exogenous agents, such as anabolic steroids (Bergen and
Merkel, 1991). Modification of production and breeding processes for
poultry to produce a leaner bird have also occurred at a rapid rate.
Actual lipid composition of meat depends on a variety of factors
including cut, grade, cooking method, and whether the fat is trimmed
prior to or after cooking. For a recent review of amount and type of fat
in beef and chicken meat, readers are referred to Byers et at. (1992). For
example, the percentage of fat in different cuts of beef (cooked) can range
from 8.8 in top round to 42.0 in short ribs (USDA, 1990). Choice of
grade can make an equally significant difference. In braised beef chuck
blade, the amount of fat in the separable lean ranges from 20.5% fat for
Prime to 13.7% for Select grade (NASjNRC, 1988). Most important is
whether meat is trimmed of all visible fat before eating because, on
average, beef is approximately 27% separable fat (USDA, 1990).
Pork, like beef, has become leaner over the past 30 years (Byers et al.,
1992). For example, the separable lean for whole pork loin contained
11.4% fat in 1963 (USDA, 1963) compared with 7.5% in 1983 (USDA,
1983). There are dramatic differences in percentage fat for different cuts of
pork, with fried bacon containing 49% fat compared with 5.5% fat in
extra lean roasted ham (USDA, 1983). Pork grades are not usually
provided on retail products. Lamb and veal contribute only 1-1.5 Ib per
capita annually in the USA, so neither is a significant source of fat in the
diet. Lamb is one of the few meats in which the fat content has actually
increased during the last 20 years (Cross et aI., 1991). However, USDA
has recently revised the official US standards for grades of lamb. This
action is expected to result in a leaner product to meet consumers' pre-
ferences for lean meat. Fat composition data on lamb are provided by
Ono et al. (1984). Veal is generally a lean meat, ranging from 2.98% fat in
rib roast from animals slaughtered at less than 4 weeks of age to 9.74% in
a braised loin chop from special fed veal animals slaughtered at about 16
weeks of age (Ono et al., 1986).
The fat content of poultry depends upon the species, cut, cooking
method and whether the skin is removed. For example, light turkey meat
is about 3.2% fat, while duck is over 28% fat (Byers et al., 1992). Differ-
ences due to cooking technique are equally dramatic, stewed chicken
breast without skin containing 3% fat and a flour-coated and fried wing
containing 22% fat (USDA, 1979).
The fat content of fish, like that of poultry, varies considerably with the
cooking technique. Steamed shrimp, for example, has less than 1% fat,
whereas, the same shrimp breaded and fried contains 12% fat (Byers et
aI., 1992). In general, however, steamed or broiled fish are low in fat,
ranging from less than 1% to 3% (Byers et al., 1992).
The fatty acid composition of the product will depend on species, cut
and cooking technique. In beef, for example, approximately 51 % of the
total fatty acids are saturated (Byers et aI., 1992). However, about one-
third of the saturated fat in beef is stearic acid, which is not considered to
have the same cholesterol raising property as shorter-chain saturated fatty
acids (Bonanome and Grundy, 1988; Reiser and Shorland, 1990). The
lipids in chicken are approximately 31 % saturated (Byers et al., 1992).
The principal contribution of fish that differs from poultry and meat is the
high amount of omega-3 fatty acids. Several sources report that the ratio
of omega-6 to omega-3 fatty acids in the diet at one time was in a 1: 1
ratio, while today this ratio is approximately 10: 1 to 20-25: 1 (Simopoulos,
1991), suggesting a need for increased consumption of fish. An excellent
review on the role of omega-3 fatty acids in health and disease and in
growth and development was published by Simopoulos (1991).
Although the fatty acid composition of MPF can be determined using
the appropriate tables from USDA Handbook 8, it is important to keep
in mind that often the majority of fat in the final product is added fat
(such as in batter frying). The resultant fatty acid composition will be
more reflective of added fat than of the MPF product itself.
17.4.1.4 The role of saturated fat. Fat, and particularly saturated fat,
appears to be the major dietary factor associated with high serum choles-
terol. Most of the saturated fat in the US diet (35% of total fat
consumed) is obtained from meat, poultry, fish and dairy products
(approximately 60%) (Dupont et al., 1991). The contribution of saturated
fatty acids to the US diet, their metabolism and effects upon plasma cho-
lesterol have been recently reviewed by Dupont et al. (1991). In addition,
the Food and Drug Administration published extensive summaries of
studies relating to dietary lipids and cardiovascular disease, and dietary
lipids and cancer in the Federal Register of November 27, 1991. One of
the first experimental studies to show a differential effect of saturated and
unsaturated fatty acids on serum cholesterol was performed by Ahrens
(1957), who provided metabolic ward subjects with a variety of fats at a
constant level of 40% of total calories. When polyunsaturated vegetable
oils were substituted for saturated fat, plasma cholesterol levels dropped.
Results of most clinical trials and epidemiological studies are consistent
with these findings. Intake of saturated fatty acids was significantly corre-
lated with risk for CHD and with plasma cholesterol levels in the Seven
Countries study (Keys, 1970). It now appears, however, that not all satu-
rated fatty acids are equally cholesterolemic.
17.4.1.5 The special case of stearic acid. Stearic acid appears to be an

exception to the rule that saturated fatty acids raise serum cholesterol.
Since beef fat is approximately 20% stearate, this is an important issue for
consumers. The original hypothesis that stearate might not be hypercho-
lesterolemic was based on observations from studies performed with cocoa
butter, which is high in this fatty acid but does not raise cholesterol values
(Grande et aI., 1970). Reiser et al. (1985) confirmed that stearate from
beef fat was not hypercholesterolemic. Using liquid-formula diets,
Bonanome and Grundy (1988) compared the effects of palmitate, stearate
and oleate on serum lipids. Both stearate and oleate resulted in lowered
low-density lipoprotein cholesterol (LDL-C) compared with palmitate. As
a result of these and other studies, some individuals remove stearic acid
from the saturated fatty acid category when expressing the poly-
unsaturated to saturated fatty acid content of MPF products.
17.4.1.6 The role of monounsaturated fat. Interest in the contribution of

monounsaturated fatty acids to serum cholesterol levels was sparked by
data from Mediterranean populations, which show a lower total mortality
rate from coronary heart disease than their Northern European counter-
parts (Keys, 1970). The Mediterranean diet is characterized by a relatively

high consumption of fish, olive oil (high in monounsaturates), vegetables
and fruit, and by a lower consumption of meat and animal fat (Buzina
et al., 1991). When comparing the Mediterranean diet to others (e.g.
Northern vs. Southern Italy), total fat is similar but the composition of
the fat is different. Recent evidence suggests that the Mediterranean diet is
changing, particularly in regard to an increase in meat and dairy product
consumption (Buzina et al., 1991). There also appears to be a concomitant
increase in serum cholesterol values in this population. In a study of 387
Cretan bank employees, the mean cholesterol concentration has risen by
36% over a 26-year period. Dietary intake has changed, with the con-
sumption of meat, fish and cheese increasing, and consumption of bread,
fruit and potatoes decreasing (Kafatos et al., 1991).
The factor in the Mediterranean diet responsible for decreased incidence
of CHD is hypothesized to be the high amounts of monounsaturated fatty
acids. Ferro-Luzzi et al. (1984) compared the effects of a western diet to a
Mediterranean diet and concluded that the Mediterranean diet was prefer-
able because it did not increase low-density lipoprotein (LDL) levels of
cholesterol. Most recent evidence suggests that monounsaturated fatty
acids (primarily oleic acid) appear to have a preferential effect on serum
lipids in that they selectively lower LDL-C and not HDL cholesterol
(Matson and Grundy, 1985; Grundy, 1986; Grundy et ai., 1988; Mensink
and Katan, 1989).
17.4.1.7 The role of omega-3 fatty acids. The fatty acid composition of
fish, particularly the presence of omega-3 fatty acids and their relationship
to diabetes (Malasanos and Stacpoole, 1991), heart disease (Wallingford
and Yetley, 1991) and cancer, has been the subject of considerable investi-
gation. Fish oils have been reported to lower blood pressure (Cobiac et
ai., 1992), decrease triglycerides and cholesterol (Simopoulos, 1991) and
decrease platelet aggregation (Simopoulos, 1991), although not all dietary
intervention studies produced consistent results. It appears that the effects
of omega-3 fatty acids on serum lipids depend on both the previous status
of the patient and whether the amount of saturated fatty acids in the diet
is held constant. For example, in hyperlipidemic patients, omega-3 fatty
acids lower LDL-C if the saturated fatty acid content is decreased, other-
wise, there is a slight increase (Simopoulos, 1991). At high doses, they
lower LDL-C. The two consistent effects of omega-3 fatty acids are a
lowering of triglycerides and an antithrombic effect.
17.4.1.8 Problems in interpretation of diet/CHD studies. When interpret-

ing studies that correlate MPF consumption to increased incidence of
CHD, it should be kept in mind that MPF consumption is also associated
with other dietary- and health-status indicators. For example, using cross-
sectional data from the longitudinal Coronary Artery Risk Development

in Young Adults (CARDIA) study, Slattery et al. (1991) found that indi-
viduals who ate red meat and poultry less than once per week were less
likely to drink alcohol. They reported more physical activity, consumed
diets higher in carbohydrates, starch, fiber, vitamins A and C, and calcium
and lower in energy, fat and protein, and had smaller body sizes. The
finding that these individuals also had lower concentrations of total serum
cholesterol, low-density-lipoprotein cholesterol, and triglycerides
compared with individuals who consumed meat more frequently could
thus be related to any of these factors in addition to actual meat con-
sumption (Slattery et al., 1991).
Further complicating the issue is the finding that different subpopula-
tions may have different dietary and behavior characteristics. For
example, a recent epidemiological study showed that specific consumer
characteristics and dietary behaviors significantly differentiated male and
female groups with low, moderate and high cholesterol levels (Sharlin et
al., 1992). Among the discriminators for high serum cholesterol in men
were obesity, lack of exercise and beef intake. For women, predictors of
high serum cholesterol were beef intake, fat and saturated fat intake and
lack of carbohydrates and dietary fiber (Sharlin et al., 1992). In addition,
men and women differ in their high-risk and low-risk behaviors. For
example, Randall et al. (1991) reported that women are more likely to
trim fat from meat, consume less alcohol and smoke less than men.
An additional but very real problem in diet studies is the reproducibility
of the food-intake data. In one study designed to estimate the reproduci-
bility of food frequency measurements, the authors found that informa-
tion on smoking and use of alcohol and coffee was quite reproducible,
while frequency of meat, vegetable and fruit intake had low reproduci-
bility (Morabia et aI., 1990).
17.4.2 Meat, poultry and fish and their relationship to cancer

Surveys of the epidemiological data have attempted to delineate high-risk
and low-risk behavior patterns for cancer. What has emerged from these
studies is that assessing risk associated with dietary patterns sheds more
light on disease relationships than studies of single nutrients (Randall et
al., 1991). The specific relationship of meat and cancer has been reviewed
previously in detail by Kritchevsky (1990).
17.4.2.1 Colorectal cancer. Of all cancer sites, colon and breast cancer
appear to be the most highly correlated with diet. Several studies show
that the rates of colorectal cancer in various countries are strongly corre-
lated with per-capita consumption of red meat and animal fat and inver-
sely associated with fiber consumption. Whether meat itself, fat or meat
protein is the risk factor for colon cancer is not clear. Other contributing
factors may be the method by which meat is prepared. A very recent
study by Giovannucci et al. (1992) examined the relationship between col-
orectal adenomas (considered precursors to colon cancer) and dietary
factors. Data were obtained from 7284 male health professionals, 170 of
whom had documented cases of adenomas of the left colon or rectum.
After adjustment for total energy intake, saturated fat was positively asso-
ciated with risk of colorectal adenoma, while dietary fiber was inversely
associated with risk of adenoma. Subjects on a high-saturated fat, low-
fiber diet had a relative risk for adenoma of 3.7 compared with those on a
low-saturated fat, high-fiber diet. The ratio of the intake of red meat to
the intake of chicken and fish was positively associated with risk of
adenoma (p < 0.02). The authors concluded that Americans should sub-
stitute chicken and fish for red meat in the diet and increase their intake
of vegetables, fruits and grains to reduce the risk of colorectal cancer.
Indeed, one population that basically abstains from meat, Seventh Day
Adventists (Sabate et at., 1991) also has a lower incidence of all cancers
unrelated to drinking or smoking than those in the general US population
(Phillips, 1975). However, as Kritchevsky (1990) points out: Mormons,
who eat large amounts of meat, also have significantly less colon cancer
than the US average. (Lyon et aI., 1976). Other problems with the inter-
pretation of correlation data include confounding variables. As noted
above, individuals who eat red meat and poultry less than once per week
are less likely to drink alcohol, are more physically active and consume
diets higher in carbohydrates, starch, fiber, vitamins A and C, and
calcium, which are also lower in energy, fat and protein, and they have
smaller body sizes. Thus, correlation of MPF intake with cancer incidence
may be confounded by these other variables (Slattery et aI., 1991). When
MPF consumption data are expressed in quintiles, the trends are incon-
sistent. The pattern that appears to emerge is that high levels of MPF
intake result in higher cancer incidences at several sites. Again, modera-
tion appears to be the issue.
17.4.2.2 Breast cancer and cancers at other sites. Breast cancer also
seems to be related to fat and fiber intake. In one study, dietary habits in
the USA and Northern and Southern Italy were compared and related to
mortality rates for breast cancer (Taioli et aI., 1991). Death rates were
lowest in Southern Italy, followed by Northern Italy and the USA. Meat
intake was twice as high in the USA as in Southern Italy. In contrast,
bread, pasta and fruit intakes were almost twice as high in Italy as in the
USA (Taioli et aI., 1991).
Other cancers thought to be related to food intake include pancreatic
and gastric cancer and cancers of the prostate, ovary, endometrium and
kidney. In a 29-country epidemiological survey, a direct and significant
correlation between mortality rates from cancer of the pancreas and per-
capita consumption of eggs, milk and meat was found. In addition, con-
sumption of vegetable calories correlated with decreased rates of mortality
from pancreatic cancer (Ghadirian et aI., 1991). Results of this survey are
compatible with earlier data from 33 countries linking animal protein and
the development of pancreatic cancer (Lea, 1967). In a case-control study
of risk factors for renal adenocarcinoma, incident cases consumed more
meats and fewer vegetables than controls, with those eating 3 oz of beef
per day having a 3.4 times greater risk than those eating less (Madure and
Willett, 1990). Data from 34 198 non-Hispanic white Seventh-day Adven-
tists in California revealed a significantly increased risk of bladder cancer
with high consumption of meat, poultry and fish (Relative Risk = 2.57)
(Mills et al., 1991).
Although gastric cancer death rates are decreasing worldwide, this
cancer is still a major public health problem in many parts of the world.
Within Europe, Italy has one of the highest gastric cancer rates. A
recently reported study showed that consumption of traditional soups,
meat, salted and dried fish, cold cuts and seasoned cheeses, as well as the
intake of animal proteins and nitrites, were related to an increased
gastric cancer risk (Cipriani et al., 1991). In contrast, consumption of
fresh fruit, raw vegetables, spices, garlic and olive oil, and vitamins C
and E and beta-carotene intake were found to be protective factors
(Cipriani et al., 1991). The only consistent finding in all of these diet/
cancer studies appears to be that the overall dietary pattern is more
important than the consumption of specific foods or nutrients. The
pattern that consistently emerges as the healthiest diet is one containing
moderate amounts of MPF and high amounts of fresh fruits, vegetables
and whole grains.
17.5 Summary
Meat, poultry and fish make an important contribution to human health

throughout the world. While individuals in developed countries are con-
suming less of these products, those in the developing nations are con-
suming more. In a recent survey in the USA the only population to meet
its RDAs was the one consuming servings from all food groups, including
MPF (Kant et al., 1991b). MPF are excellent sources of high-quality
protein and supply significant amounts of B-vitamins (particularly
thiamin, riboflavin, niacin, vitamin B6 and vitamin B12) and important
minerals (particularly iron and zinc). The relationship of MPF to human
health, independent of its contribution to nutritional status, is an ongoing
subject for discussion. All major health organizations support the con-
sumption of moderate amounts of lean MPF as a healthful practice
(American Heart Association, 1988; USDA, 1990; US Department of

Health and Human Services, 1988).
Excess amounts of MPF, like excess amounts of any food, may put
individuals at increased risk for heart disease and certain cancers. Mod-
eration is the key. There is little doubt that lower-fat diets are to be pre-
ferred to those high in fat. However, the way to achieve a lower-fat diet
should be in judicious reduction of foods from all categories rather than
the avoidance of particular classes of foods (Dupont et aI., 1991). With an
increased awareness of the importance of nutrition in long-term health
care, there has been a concomitant increase in misconceptions about what
foods are 'good' or 'bad' for you. This is illustrated most clearly in the
finding that consumption of red meat has declined while consumption of
fats and oils has actually increased (Popkin et aI., 1992). It is time for sci-
entists and health professionals to work together to educate the public
about the many nutrient-dense, low-fat food choices available in a well-
balanced diet (MacDonald, 1991), which may include moderate amounts
of MPF.
Acknowledgement
The authors wish to acknowledge Dr. Dana R. Smith, for her contribu-
tion to the section on MPF and heart disease.
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intervention. Am. J. Prevo Med. 7, 319.
Index
absorption 84-85 canned meat, retort flavor of 273-274

acceptance testing see product capillary volumeter 134, 145-147
acceptability evaluation carcass suspension effects 300
additives 6 catalase tests 423-427
adipose tissue 227-231 centrifugation 134,143-145
affective testing see product acceptability chemical
evaluation measures 326
aging residues in muscle foods 447-474
effects on olfaction 174-175 senses 162-178
effects on taste 171-172 chemistry of flavor and aroma 184-199
alkylphenols 237 chemosensory mixtures 177-178
amino acids, essential 7 chicken-specific flavor and odors 243
and specific appetites 176~ 177 cholesterol 446
analytical sensory testing see product CIELAB 91-92
acceptability evaluation CIE System 80-81
anatomy and physiology of the chemical cluster analysis 212
senses color
olfaction 167-169 absorption 84-85
taste 162-167 bacteria 41-43
animal characteristics 102-105 changes, in beef 91
antimicrobial treatments 60-61 CIELAB 91-92
antioxidants 58-60 CIE System 80-81
appetites, specific cooked meat 62-64
and amino acids 176-177 cured meat 64-67
and proteins 176-177 dark color problems 49-52
and salt 175-176 fading in cured meat 66-67
aroma see flavor and aroma freezing 62
artificial tenderisation 308-309 green discoloration and bacteria 43
ascorbate 65-66 Hunter scales 91-92
ATP estimation of microbial population importance 2
418-419 instrumentation for measurement of
83-84
bacteria 41-43 light 62
batters 154-157 light scatter 84-85
BCFAs see branched-chain fatty acids lipid oxidation 62
beef flavors and odors 242-243 measurement procedure 80,86-90
effects of feeds on 264-265 modified-atmosphere packaging 46-47
biopreservation 388-391 oxygen-permeable films 45-46
boar odor or taint 237,268-269 oxygen tension 39-41
body secretions of BCFAs 232-235 pH, effects of 48
branched-chain fatty acids (BCFAs) pigment, cured meat 66
224-227 pigmentation 84-85
synthesis of 231-237 pink in cooked, uncured meat 63-64
breed effects 290-292 reflectance 84-85
spectral changes 90-91
calorimetry 422 retail importance of 34-35
cancer 493-496 salt 62
502 INDEX
color 'contd' DNA probes 430

spectrophotometers 83-84 double tube method 417-418
stability, factors affecting 39-49 drip losses
sulfites 61 and juiciness 98
temperature 48-49 in measuring WHC 133, 136-139
terminology 82-83
trichromatic colorimeters 83 electrical stimulation 298-299,302-303
uncured meat 63-64 ELISA tests 430-431
uniform colour space 81-82 end-point temperature vs. juiciness 97
vacuum-packaging 43-45 environment, off-flavors 270-271
variability and measurement 2 enzyme-linked immunosorbent assay
vision 79-82 tests see ELISA tests
colour see color essential fatty acids 7-8
compounds, flavor see flavor, compounds evaporation losses 132-133
compression methods 321-322 external mechanical force 130
concentration-extraction procedure 207
conductance 419-422 factor analysis 211-212
connective tissue 294-296 fading of cured meat color 66-67
consumption, factors affecting human fat 443
479-481 and juiciness 97-98
contamination of muscle foods, microbial level, product acceptability 348-351
sources 359-364 fatness and tenderness 292-293
types 362-364 fats, essential 7-8
cooked meat fatty acids in meats 222-227
color 62-64 feeds see flavor and aroma, feeds
irradiation of 60-61 filter paper press method (FPPM) 133-
cooked sausage 153 134,139-143
cooking fish
influence on fatty acids 223 effects of feeds on flavor and aroma
methods and juiciness 99-102 265-266
pre-rigor 301 off-odors and microbial growth
waterIoss 134-136,148-152 274-276
cooling, pre-rigor 301-302 fish-specific flavors and odors 243-244
correlation 213 flavor
cured meat 64-67 compounds 205-207
cysteine 65-66 fish-specific 243-244
precursors 185-186
dark color problems 49-52 profile 204
dark, coarse band (DCB] 52 flavor and aroma
dark cutting beef boar taint 237
and pH 50-51 branched-chain fatty acids, synthesis of
characteristics 49-50 231-237
dark, coarse band (DCB) 52 chemistry 184-199
dark cutters, minimizing by fatty acids in meats 222-227
management 51-52 feeds 261-267
death, changes occurring after 50-51 importance 3
vacuum-packaging 51 measurement 202-220
dark, firm dry (DFD) pork 52-54 data-set, example analysis 214-220
DCB see dark, coarse band (DCB) meat aroma 187-199
decontamination 382-385 meat taste 186-187
descriptive flavor analysis 203-205 physiology and psychology 4
DFD pork see dark, firm dry (DFD) pork problems 4-5,251-278
diet 231-232 feeds, effects on flavor and aroma
Diluflo 408-409 261-267
Direct Epifluorescent Filter Technique gamey flavors 267-268
416-417 off-flavors 268-78
direct preference tests 341-342 from the environment 270-271
discriminate analysis 212-213 microbial growth 274-277
INDEX 503
processing-induced 271-274 International Commission on Illumination

oxidative rancidity 251-261 (CIE) see CIE System
warmed over flavors 251-261 irradiation
sex odor 237 and off-flavours 271-273
species-specific 4,222-244,261 and other antimicrobial treatments
steroids 237-238 60-61
subcutaneous and perinephric adipose Isogrid system 413
tissue 227-231
variability 3-4 juiciness
flavour see flavor importance 2-3,94-116
food analysis in muscle foods 441-447 influencing factors 98-115
foodborne illness 368-379 measurement 3,158-159
force 130 objective meaurements 96-98
FPPM see filter paper press method subjective assessment of 94-96
(FPPM)
fragmentation, meat texture
measurement 325 lamb
free fatty acids 224 effects of feeds on flavor and aroma
freezing, effects on meat color 62 261-264
fresh meats, irradiation 60-61 flavor 239-240
light, effects on meat color 62
light scatter 84-85
gamey flavors and odors 244,267-268 Limulus Amoebocyte Lysate tests
gas chromatographic (GC) analysis 423-427
207-209 lipid oxidation
gas chromatography 210-214 catalysis 255-257
GC analysis see gas chromatographic effect on meat flavor 254-255
(GC) analysis effects on meat color 62
genetic basis of PSE, PSS and DFD pork inhibiting 253-254
53 measurement in meats 257-261
goaty flavors and odors 241 meat quality 251-253
Gravimetric Diluter see Diluflo lipid reactions 190-193
green discoloration, bacteria 43
grinding, meat texture measurement 325
growth promoters 294 macro-minerals 9
Maillard reaction 189-190
marinading 309
halophenols 456-457 marinated meat 114-115
hand roller 406-408 measurement of color 80,86-90
health of man see human health meat color and sulfites 61
heart disease, relationship of meat, poultry meat texture measurement 316-331
and fish to 488-493 assessment methods
heated batters 156-157 objective 329-331
heating methods and juiciness 97, 99-102 and subjective 330-331
hedonic scores 346-348 assessments
hot-deboning 300-301 objective 319-325
HPLC analysis 209-210 structural 325-326
human color vision 79-82 subjective 318-319
human health and meat, poultry and fish chemical measures 326
cancer 493-496 samples 327-329
consumption 479-481 tenderness 317-318
heart disease 488-493 metmyoglobin, enzymatic reduction of
nutritional value 7-9,481-487 54-58
role 487-495 microbial contamination
Hunter scales 91-92 growth control 359-391
off-flavors 274-277
imbibing method 134,148 problems
impedance 419-422 importance 6
instrumental analysis 205-210 measurement 6
504 INDEX
microbial contamination 'contd' pigment, cured meat 66

rapid methods for measurement and pigmentation 84-85
enumeration 404-435 pink in cooked, uncured meat 63-64
cell count procedure, alternative polymerase chain reaction 431-432
methods 409-418 porcine stress syndrome (PSS) 52-54
estimation of population 418-427 pork flavors and odors 241-242
miniaturized techniques 427-429 post-mortem compositional and structural
new and novel techniques 429-434 changes 297
sampling and sample preparation post-rigor storage 303-304
405-409 poultry
micro-minerals 9 effects of feeds on flavour and aroma
minerals 9 266-267
modified-atmosphere off-odors and flavors 276-277
packaging 46-47 pre-rigor
storage 385-388 cooking 301
moisture 443 cooling 301-302
motalityenrichment 432-433 pre-slaughter factors 306
muscle shortening 306-307 press fluid 98
mutton see lamb pressure treatment 309
myoglobin 35-39 principal component analysis 211
content of PSE muscle 54 processed meats 112-114
product acceptability evaluation
neural networks 214 acceptance tests 343-346,352-355
nitrate 64-65, 445 affective testing 339-342
nitrite 445 affective vs. analytical sensory testing
in cured meat flavor 193-197 342-343
nitrosamines 445-446 sensory attributes 346-352
nutritional value of meat, poultry and fish proteins 7, 442-443
7-9,481-487 and specific appetites 176-177
odor see flavor and aroma proteolytic enzymes 308'-309
odors and flavors, selected fatty acids PSE pork see pale, soft exudative (PSE)
235-236 pork
off-flavours 268-278,271-277 PSE, PSS and DFD pork 52-54
olfaction 167-169,172-175 PSS see porcine stress syndrome (PSS)
olfactory preference 172-174 psychology see flavor and aroma,
oxidation, non-enzymatic reductants and physiology and psychology
inhibitors of 58-60
oxidative rancidity 251-261 quantitative descriptive analysis 204-205
oxygen-permeable films 45-46
oxygen tension 39-41 radiometry 422
oxyrase enzyme system 433 ram odorlflavor 269-270
Rapid Chilling 53
pale, soft exudative (PSE) pork 52-54 rapid methods see microbial
penetrations, meat texture measurement contamination, rapid methods for
324-325 measurement and enumeration
perinephric tissue 227-231 rapid test methods 447
pesticide-residue analysis 450-455 reception and transduction
Petrifilm system 413-415 of olfaction 168-169
pH of taste 163-167
and color 48 red meats, off-odors and flavors 277
and dark cutting beef 50-51 Redigel system 415-416
and tenderness 303 reductants 58-60
physiology reflectance
chemical senses see anatomy and and color 84-85,90-91
physiology of the chemical senses colorimetry 423
flavor 4 regression 213
piezo-electric crystals 210 residues 7
pig meat, effects of feeds on flavor and response surface methodology (RSM)
aroma 265 213-214
INDEX 505
restructured meat 108-112 pre-slaughter factors 290-296

retail importance of color 34-35 rigor development 297-299
rigor development 105-108,297-299 slaughtering 296-297
RSM see response surface methodology tensile assessments 322-324
texture measurements, meats 316-331
thermal force 130
salt 443-444
thiamin degradation 193
and specific appetites 175-176
thiophenols 237
effects on meat color 62
transduction see .eception and
sausage batters 157
transduction
scaling 339-341
trichromatic colorimeters 83
sensory analysis 210-214
turkey-specific flavors and odors 243
sensory attributes 346-352
sensory responses to food 169-175
uncured meat 63-64
sex
unheated batters 154-156
and tenderness 293
uniform color space 81-82
condition leading to off-flavors 268
flavor/aroma 270
odor or boar taint 237 vacuum-packaging
and dark-cutting beef 51
shear and biting systems 320-321
oxygen transmission rates 44-45
short-chain fatty acids 236-237
role in extending shelf-life 43-44
slaughtering 296-297
variability of flavor 3-4
sniffer-port analysis 209
species-specific flavors and odors 222-244 veal
spectrophotometers 83-84 effects of feeds on flavor and aroma
264-265
spectrum method 205
flavor 242-243
spiral plating 409-413
veterinary drug residue analysis 457-473
spoilage 364-368
VIDAS immunoanalysis system 431
stability of color 39-49
vision, color 79-82
steroids 237-238
vitamins
stomacher 405-406
subcutaneous tissue 227-231 fat-soluble 8
water-soluble 8
sugars 444-445
Vitek system 429-430
sulites 61
volatile
environmental contaminants 456
taint see boar odor or taint free fatty acids 223
target RNA probes 430 volatiles and flavor 197-198
taste 162-167
preference, development of 169-171 warmed over flavors 251-261
temperature water-binding
and color 48-49 importance 2-3
andtendernes 297-298,307 variability and measurement 3
tenderisation water-holding capacity
artificial 308-309 definition 129
mechanism of 304-306 measurement 125-159
tenderness influence of different factors 131-136
artificial tenderisation 308-309 meat, composition and structure
control of 309-310 126-127
importance 5 meat, state of water in 127-129
measurement 5 methodology 130-131
meat texture 317-318 methods and evaluation 136-152
muscle shortening 299-303 water-holding vs. juiciness 97
post-rigor storage 303-307 WHC see water-holding capacity

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Advances in Meat Research - Volume 9

Quality Attributes and their Measurement

The following volumes are also available:

Quality Attributes and their

SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.

ISBN 978-1-4613-5906-7 ISBN 978-1-4615-2167-9 (eBook)

~Printed on acid-free text paper, manufactured in accordance with ANSI/

G.K. Beauchamp Monell Chemical Senses Center, 3500 Market Street,

T.R. Dutson Agricultural Experiment Station, Oregon State Uni-

c.J. Hagyard Meat Industry Research Institute of NZ Inc., Hamil-

1 Introduction to quality attributes and their measurement in

2 Color - its basis and importance 34

2.3.5 Modified-atmosphere packaging 46

4 Juiciness - its importance and some contributing factors 94

5 Measurement of water-holding capacity and juiciness 125

5.10 Measurement of juiciness 158

6 The chemical senses 162

7 Flavor and aroma chemistry 184

8 Flavor and aroma - its measurement 202

8.4 Instrumental analysis 205

9 Species-specific flavors and odors 222

10 Flavor and aroma problems and their measurement in meat, poultry

11 Tenderness of meat, poultry and fish 289

11.6 Ultimate pH effects 303

12 Meat texture measurement 316

13 Product acceptability evaluation 337

13.6.2 Context effects 354

15 Rapid methods for measurement and enumeration of microbial

16 Food analysis and chemical residues in muscle foods 441

Although food is essential in that it supplies the nutrients necessary to

1.2.2 Variability and measurement

1.3 Juiciness and/or water-binding

1.3.2 Effects of variability and measurement

water-extractable (Crocker, 1948; Bouthilet, 1951a,b; Kramlich and

1.4.3 Physiology and psychology offlavor/aroma

1.4.4 Species-specific flavors/odors

1.4.5 Flavor and aroma problems

Pearson, unpublished observations). Spoiled meat aromas and flavors may

1.5.2 Some Jactors influencing tenderness and its measurement

1.6 Microbial problems

1.7 Additives and residues

1.8 Contributions of meat to human nutrition

Discussion of the quality attributes of meat, poultry and fish would be

I.B.1 Proteins and essential amino acids

I.B.2 Fats and essential fatty acids

shorter-chained fatty acids as shown earlier by Hegsted et al. (1965) and

1.8.3.1 Fat-soluble vitamins. The fat soluble vitamins as shown in

needs of man for the water-soluble vitamins can be found in chapter 17

1.8.4.1 Macro-minerals. The macro-minerals include calcium, phos-

1.8.4.2 Micro-minerals (trace minerals). The trace or micro-minerals

2.1.1 Retail importance of meat color

2.2 Myoglobin and its derivatives

Myoglobin is the primary meat pigment, and exists as bright-red oxymyo-

Pigment Source Color Absorption maxima (nm)

Myoglobin l - 3 Horse Purple 439 555

transport of oxygen within the muscle cell (Wittenberg, 1970; Livingston

2.2.1 Myoglobin concentration in muscle

reported myoglobin concentrations ranging from 1.99 mg.g- 1 for semi-

2.3 Factors affecting fresh meat color stability

Metmyoglobin formation and resultant brown discoloration is generally

2.3.1 Oxygen tension

oxygen. Increasing the temperature tended to decrease the depth of the

tion rate of myoglobin at higher oxygen tensions. Brown and Mebine