FOOD PROTEIN ANALYSIS

Quantitative Effects on Processing

R. K. Owusu-Apenten
The Pennsylvania State University
University Park, Pennsylvania

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 FOOD SCIENCE AND TECHNOLOGY

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Owen R. Fennema University of WisconsinMadison
Y.H. Hui Science Technology System
Marcus Karel Rutgers University (emeritus)
Pieter Walstra Wageningen University
John R. Whitaker University of CaliforniaDavis

Additives P. Michael Davidson University of TennesseeKnoxville

Dairy science James L. Steele University of WisconsinMadison
Flavor chemistry and sensory analysis John H. Thorngate III University of
CaliforniaDavis
Food engineering Daryl B. Lund University of WisconsinMadison
Food proteins/food chemistry Rickey Y. Yada University of Guelph
Health and disease Seppo Salminen University of Turku, Finland
Nutrition and nutraceuticals Mark Dreher Mead Johnson Nutritionals
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For Mum and Dad, Elizabeth, James, Richard, Candida, and Aa
Preface

There is no book dealing with food protein analysis exclusively, that is, with
the analysis of proteins in the food system. This books attempts to ll this
niche. Protein analysis comes in two forms: 1) Quantitative analysis, and 2)
fractionation and characterization. The rst activity is described here. This
publication provides a reference for planning, performing and interpreting
assays for food proteins. Many approved methods derive from the late-19th
century, but they have undergone rigorous testing and modernization. This
book does not focus on reviewing the latest research methods for protein
analysis. With the exceptions of Chapters 6 and 7, each of the 14 self-
contained chapters describes one protein assayprinciples, practices, and
expected results.
This book describes the effect of food processing on protein assay
results with the emphasis on how to analyze proteins in real foods. A
number of ``Methods'' sections provide instructions for specic tests.
Sample pretreatment and clean-up procedures are described. General
pretreatment strategies help in the avoidance of interference. More specic
clean-up methods apply to particular protein assays and are described along
with these. Example results, performance characteristics, case reports, and
practical problems and solutions related to a wide range of foods are
detailed in numerous gures, tables, and references.

v
vi Preface

Food protein analysis is a hugely important activity performed by

thousands worldwide. The book should appeal to professionals interested in
food proteins and anyone working in the food system formerly called the
food chain. This includes researchers and workers in agricultural produc-
tion, food processing, and wholesale and/or retail marketing. It provides
information for the grain or dairy farmer, extension worker, agricultural
scientist, food scientists and technologists, or college professor. Some
techniques described in this book were rst used by clinicians, nutritionists,
and veterinary scientists. The book may also be of interest to those in small
businesses, private or government laboratories, research institutes, colleges,
and universities. It will be useful to undergraduate, postgraduate, or
postdoctoral students. Sections dealing with mechanisms assume graduate
level chemistry and/or analytical biochemistry.
Any shortcomings of this project are wholly my responsibility. I thank
all those colleagues worldwide whose research is reported here. My thanks
to Anna Dolezal, Mr. DeSouza and Professor Arthur Finch for teaching me
to think for myself. I am grateful to my past students: Drs. Yetunde
Folawiyo, Despina Galani, Michael Anaydiegwu, Kiattisak Duangmal,
Pitaya Adulyatham, Kwanele Mdluli, Halima Omar and Sripaarna Banerjee
for raising my awareness of protein assay issues and for reading parts of the
manuscript. Thanks to Dr. Bob Roberts (The Pennsylvania State
University) for his advice on combustion methods. I am grateful to Dr. S.
Khokhar and Marcel Dekker, Inc., for their commitment. I am also grateful
to my family for their support.

R. K. Owusu-Apenten
Contents

Preface v

Part 1. Fundamental Techniques

Chapter 1. Kjeldahl Method, Quantitative Amino Acid Analysis and
Combustion Analysis 1
1. Introduction to Food Protein Analyses 1
2. Kjeldahl Analysis 7
3. Colorimetric Analysis of Kjeldahl Nitrogen 18
4. Quantitative Amino Acid Analysis 25
5. Combustion Nitrogen Analyzers 29
References 38

Part 2. Copper Binding Methods

Chapter 2. The Alkaline Copper Reagent: Biuret Assay 47
1. Introduction 47
2. The Alkaline Copper Reagent Protein Assay 48
3. Chemistry of the Alkaline Copper Reagent Protein
Assay 50
4. Interference Compounds 53

vii
viii Contents

5. Sample Pretreatment and Avoiding Interferences 55

6. The Micro-Biuret or Ultraviolet Biuret Protein Analysis 56
7. Applications of the ACR Solution for Food Protein
Analysis 57
References 64
Chapter 3. The Lowry Method 69
1. Introduction 69
2. The Lowry Protein Assay 70
3. Chemistry of the Lowry Assay 73
4. Calibration Features 77
5. Interference Compounds 80
6. Sample Pretreatment, Avoiding Interferences, and
Ensuring Accuracy 86
7. Applications of Lowry Assays to Food Protein Analysis 87
References 93
Chapter 4. The Bicinchoninic Acid Protein Assay 99
1. Introduction 99
2. The BCA Protein Assay 103
3. Chemistry of the BCA Protein Assay 105
4. Calibration Features 109
5. Interference Compounds 110
6. Sample Pretreatment, Avoiding Interference, Ensuring
Accuracy 112
7. Automated BCA Protein Assays 113
8. Applications of the BCA Assay to Food Protein
Analysis 116
References 121

Part 3. Dye Binding Methods

Chapter 5. The Udy Method 125
1. Introduction 125
2. The Udy Method 127
3. Solid-Phase Dye-Binding Assays 131
4. The Chemistry of Dye-Binding Protein Assays 133
5. Interference Compounds and Their Avoidance 147
6. Applications of Dye-Binding Assays for Food Protein
Analysis 147
References 160
Chapter 6. The Bradford MethodPrinciples 169
1. Introduction 169
Contents ix

2. Theory of the Bradford Assay 171

3. Effect of Protein-Dye Binding Parameters on the
Bradford Assay 183
4. Linearization Plots for the Bradford Assays 184
5. Assay Sensitivity and the Maximum Number of Dye
Binding Sites 184
6. Solid-Phase Dye-Binding Assays 185
7. Interference Compounds and Sample Pretreatment 186
References 191
Chapter 7. Bradford AssayApplications 195
1. Introduction 195
2. Coomassie Brilliant Blue Dye-Binding Assays 195
3. Performance Characteristics of CBBG Dye-Binding
Assays 201
4. Applications to Food Protein Analysis 204
References 218

Part 4. Immunological Methods for Protein Speciation

Chapter 8. Immunological Assay: General Principles and the Agar
Diffusion Assay 221
1. Introduction 221
2. Immunological Methods 225
3. Speciation of Proteins by Agar Gel Double
Immunodiffusion Assay 230
References 241
Chapter 9. Speciation of Meat Proteins by Enzyme-Linked
Immunosorbent Assay 247
1. Introduction 247
2. Raw Meat Speciation by Indirect ELISA 252
3. Raw Meat Speciation by Sandwich ELISA 255
4. Muscle Protein Antigens for ELISA 257
5. Cooked Meat Analysis by ELISA 260
6. Monoclonal Antibodies for Meat Speciation 265
7. Fish and Seafood Identication by ELISA 268
8. Performance Characteristics for Different ELISA
Formats 270
9. Meat Testing for Transmissible Spongiform
Encephalopathy Agents 271
References 274
x Contents

Chapter 10. Speciation of Soya Protein by Enzyme-Linked

Immunoassay 281
1. Introduction 281
2. Sample Pretreatment and Analysis of Soy Protein 281
3. Structure, Denaturation, and Renaturation of Soybean
Proteins 285
4. Solvent-Extractable Soybean Protein 289
5. Thermostable Antigens for Soybean Protein Analysis 289
6. Other Nonmeat Proteins 292
References 292
Chapter 11. Determination of Trace Protein Allergens in Foods 297
1. Introduction 297
2. Soya Bean Protein Allergens 301
3. Peanuts 305
4. Wheat and Related Cereals 312
References 329

Part 5. Protein Nutrient Value

Chapter 12. Biological and Chemical Tests for Protein Nutrient Value 341
1. Introduction 341
2. Human and Other In Vivo Assays for Protein Nutrient
Value 346
3. Small Animal Bioassays for Protein Nutrient Value 348
4. In Vitro Methods for Assessing Protein Nutrient Value 354
5. Protein Digestibility 366
References 374
Chapter 13. Effect of Processing on Protein Nutrient Value 381
1. Introduction 381
2. Milk and Milk Powders 381
3. Infant Formulas 384
4. Feedstuffs and Concentrates for Livestock 386
5. Legumes and Oilseeds 393
6. Cereal and Cereal Products 398
7. Improving Cereal Protein Quality by Screening 401
References 402
Chapter 14. Protein DigestibilityCorrected Amino Acid Scores 411
1. Introduction 411
2. Protein Digestibility 411
3. Protein Denaturation 414
4. Chemical Deterioration of Protein Ingredients 416
Contents xi

5. Matrix Effects on the Rate of Deterioration of Protein

Ingredients 420
6. Protein DigestibilityCorrected Amino Acid Scores
(PDCAAS) 427
References 440

Index 447
1
Kjeldahl Method, Quantitative Amino
Acid Analysis and Combustion
Analysis

1. INTRODUCTION TO FOOD PROTEIN ANALYSES

Protein analysis is a subject of enormous economic and social interest. The

market value of the major agricultural commodities (cereal grains, legumes,
our, oilseeds, milk, livestock feeds) is determined partly by their protein
content. Protein quantitative analysis is necessary for quality control and is
a prerequisite for accurate food labeling. Proteins from different sources
have varying aesthetic appeal to the consumer. Compliance with religious
dietary restrictions means excluding certain protein (sources) from the diet.
The variety of protein consumed is also extremely important in relation to
food allergy. Detecting undeclared protein additives and substitutions is a
growing problem. Proteins show differing nutritional quality or ability to
support dietary needs. In summary, protein analysis has legal, nutritional,
health, safety, and economic implications for the food industry (1).
The estimated global food production total for 1988 was 4 billion
metric tons. Allowing an average of 10% protein in foodstuffs yields 400
million metric tons of protein annually (2). Nonetheless, sensitivity is a
major consideration for protein analysts. Some immunological methods can
detect nanomole (10 9 mole) amounts of protein. Other important
considerations when choosing a method for food protein analysis include

1
2 Chapter 1

TABLE 1 Approximate Chronology for Methods for Food Protein Analysis

Date Technique

1831 Dumasa
1843 Nessler's reagenta
1849 Biuret method
1859 Alkali-phenol reagent or Bethelot's methoda
1883 Kjeldahla
1927 Folin-Ciocalteau
1944 Dye bindinga
1951 Lowry
1960 Direct alkaline distillation
1960 Near-infrared reectance (NIR)a
1971 Modied Berthelot reaction
1975 Modied Lowry method (Peterson)
1976 Bradford method (Coomassie Blue binding method)
1985 Bicinchoninic acid (BCA) method
a
Techniques for which semiautomated or fully automated apparatus has been manufactured.

high sample throughput, simplicity, and low capital costs. Some of the most
signicant methods (Dumas, Kjeldahl, and biuret assays) date from the
late 1800s (Table 1). Techniques for food protein analysis are described
in this book. I will focus on the techniques that feature most often in
the food science literature. Infrared analysis of food proteins is not discussed
here.

1.1. Characteristics of Food Protein Assays

Techniques for food protein analysis need to be robust. This means one of
several things. Foremost is compatibility with fresh produce (cereals, fruits,
vegetables, meat, milk) and processed foods. Samples in various physical
states (powders, slurries, dilute liquids, emulsions, gels, pastes) should be
analyzable. A robust assay will also deal effectively with foods from either
animal or plant sources. Such techniques are unaffected by the presence of
dyes or pigments that absorb infrared, visible, or ultraviolet light. A robust
protein assay needs mimimal sample pretreatment, which increases error
and decrease analytical precision. Sample cleanup also increases the time per
analysis (reduces sample throughput) and adds to costs. In the worst-case
scenario, pretreatment can be too invasive, thereby invalidating results. In
summary, a robust protein assay is simple, quick, sensitive, and reliable. It is
also compatible with a diverse range of foods. The economic imperative
Kjeldahl Method 3

leads to a preference for techniques requiring low capital expenditure and

minimum training. Laboratories handling more than 8000 analyses per year
tend to select techniques on the basis of their speed and ease of operation. A
high sample throughput is usually achieved by automation or continuous
ow analysis (CFA). A rough ``time line'' for some food protein assays is
given in Table 1. Common descriptive terms for protein analysis are dened
in Table 2.
Kjeldahl analysis gives accurate protein readings no matter what the
physical state of the sample. This technique has approved status and is the
reference method adopted by many national and international organiza-
tions. However, the use of hazardous and potentially toxic chemicals in
Kjeldahl analysis is creating concern. The Dumas combustion method is
comparatively quicker, cheaper, easier to perform, safer, and more
environment friendly; it is now considered on equal terms with Kjeldahl
analysis in the United States, Canada, and Western Europe. Dye binding is
another robust test for proteins (3,4). The biuret method is widely used,

TABLE 2 Some Important Calibration Indices and a Brief Explanation of Their

Meaning

Calibration feature Explanation

Linear dynamic range Range over which signal is proportional to

analyte concentration
Sensitivity Slope of the calibration graph; analytical
response per unit change in protein
concentration. cf. parameters a, a0 in Eqs.
(1)(4)
Accuracy Degree of agreement of results with a true
value
Precision, repeatability, or Agreement between repeated measurements
reproducibility taken with a single sample or with
different paired samples
Specicity Ability to discriminate between protein and
interfering substance. Ratio of sensitivity
for the analyte and interference
Reliability A composite parameter combining
specicity, accuracy, precision, and
sensitivity
Lower limit of detection (LLD) Minimal protein concentration detectable
above background noise
Sample throughput (time per Numbers of samples analyzed per unit time,
analysis) speed of analysis
4 Chapter 1

especially for cereal proteins (5). Procedures involving copper-based

reagents (Lowry and bicinchoninic acid assays) continue to be important.
Finally, a range of empirical (viscosity, refractive index, specic gravity)
measurements are used for protein quantitation within industry.

1.2. Calibration and Statistical Principles

The two common forms of calibration are (a) method calibration and (b)
sample calibrations. With method calibration a set of food samples are
analyzed using a new test method and a reference method that has been
validated by a committee of the Association of Ofcial Analytical Chemists
(AOAC). A calibration graph is then drawn by plotting results from the
reference method (% Kjeldahl protein) on the Y-axis and the test results on
the X-axis. The Xi and Yi observations are usually related by an equation for
a straight line:

Yi aXi b 1

where a is the gradient and b is the intercept for the calibration graph. For
each Xi result we can determine the calculated % Kjeldahl protein value
(Ycalc) via Eq. (2).

Ycalc aXi b 2

Values for Yi and Ycalc can be compared in order to evaluate the test method
(see later). Some investigators choose to plot the Kjeldahl results on the X-
axis. Therefore, rather than Eq. (1) we get

Yi* a0 X * b0 3

where Xi* is % Kjeldahl protein and Yi* is the test result. To compare Eq.
(1) and Eq. (3), notice that a0 1/a and b0 Yi (Xi / a).
For sample calibration, the assay technique is assumed to be valid. We
analyze a set of (standard) samples containing known amounts of protein.
In Eq. (1), Xi now represents a range of known protein concentrations and
Yi are the corresponding instrument responses. Calibration factors (a, b,
etc.) can be determined from simple algebra or statistical analysis of paired
(Xi, Yi) results. From the principles of least-squares analysis,
P
Xi Xm = Yi Ym
a P 4
X i X m 2
Kjeldahl Method 5

and
b Ym aXm 5

where Xm and Ym are the mean values for all Xi and Yi observations.
Agreement between the reference and test results is measured by the
correlation coefcient (R); R&1 shows excellent agreement. When Yi and
Ycalc observations are poorly correlated, R & 0. The squared correlation
coefcient (R2) can be calculated from Eq. (6). Most handheld calculators
can perform this operation automatically.
" #
2 Yi Ycalc 2
R 1 P 6
Yi Ym 2

Precision is another measure of the (dis)agreement between Yi and

Ycalc values. This can be expressed as the standard deviation (SD) or
coefcient of variation (CV). High-precision methods produce low values
for the SD and CV.
P
2 Yi Ycalc 2
SD 7
n 2

CV SD=Ym 6100 8

We can also measure precision (commonly called error) from n-replicate (Yi)
measurements on a single test sample. Thereafter, the numerator in Eq. (7)
becomes (Yi Ym)2, which is the square of the differences between
individual observations and the mean for all observations. A low CV
implies good agreement between successive test results.

1.3. Assay Performance

Calibration parameters can provide a great deal of other information about
assay performance (Table 2). The linear dynamic range is the concentration
range over which a linear relationship exists between the instrumental
response and protein concentration. Sensitivity is the slope of the calibration
graph, and the lower limit of detection (LLD) is smallest quantity of sample
that triggers an instrumental response above the background noise. The
LLD is dependent on the instrument baseline quality and assay sensitivity.
It is common to refer to ``sensitivity'' when we mean the LLD.We
differentiate between sensitivity and LLD via the following exercise.
Measure the instrument baseline noise by recording the output (Yo) and
6 Chapter 1

the standard deviation (SDo) using a sample blank. The smallest

instrumental response that can be distinguished from ``random noise'' in
95% of all cases is Yo +2:326SDo . Now substitute for Yi (Yo 2.326 SDo)
and Xi ( LLD) in Eq. (1), leading to the following expression:
Yo 2:326SDo b
LLD 9
a
Usually Yo and b are both set to zero when the analyst sets the instrument
baseline response to zero. Consequently, Eq. (9) becomes
LLD 2:326SDo =a 10

This relation shows that LLD decreases with increasing assay sensitivity and
with increasing baseline quality (see decrease in the value for SDo). In order
to ensure high sensitivity, it is important to obtain a stable instrumental
baseline.

1.4. Calibrating Protein Assays

The Kjeldahl method is used for calibrating other protein assays. Duda and
Szot (6) evaluated six methods for analyzing porcine plasma protein during
its manufacture. The techniques are simple and therefore of wider interest
(Table 3). The protein content of porcine plasma was 5.58% (w/v). All
techniques showed a good correlation with Kjeldahl results (R 0.905
0.952). The precision for density and Kjeldahl assays was the same
(CV 10.8%). The sensitivity of the former method was better. With
appropriate calibration, density or viscosity measurements could be suitable
for the routine analysis during the manufacture of plasma proteins.

TABLE 3 Some Simple Methods for Evaluating Porcine Plasma Protein

Method Instrument

Densitometry Standard picnometer

Refractometry
Modied refractometry
UV absorbance (215/225 nm)
UV absorbance (241 nm)
UV absorbance (280 nm)
Laboratory refractometer
Laboratory refractometer
UV spectrophotometer
UV spectrophotometer
UV spectrophotometer
Kjeldahl Method 7

Williams et al. (7) calibrated beer protein analyses using quantitative

sodium dodecyl sulfate polyacrylamide gel electrophoresis (QSDS-PAGE).
A range of test methods were investigated including biuret, bicinchoninic
acid (BCA), Bradford, Kjeldahl, Lowry, and pyrogallol-red molybdate
(PRM) assays. QSDS-PAGE revealed that beer has between 0.5 and
1 mg mL 1 protein. Only the Bradford and PRM assays gave accurate
results (Fig. 1). The main sources of error were low-molecular-weight
interferences. Beer contains plant pigments, starch, sugars, alcohol, and
natural dyes of barley origin. Both Kjeldahl and combustion analyses were
subject to interferences by nonprotein nitrogenous (NPN) compounds.
Dialysis did not improve accuracy for BCA, Lowry, and biuret assays,
which were affected by high-molecular-weight Cu- reducing agents such as
pectin and starch.
Calibration issues are discussed in two articles by Pomeranz and co-
workers (8,9). They considered the reliability of several test methods (biuret,
dye binding, infrared reectance, alkaline distillation method) for analyzing
proteins in hard red winter wheat varieties from the American Great Plains.
The test methods were highly correlated with the Kjeldahl assay (R 0.976
0.992). The order of precision was Kjeldahl > biuret > dye binding >
infrared analysis. Pomeranz and More (9) also considered the reliability
of four ``rapid'' methods for barley or malt protein analysis.* A summary of
assay performance statistics is given in Table 4. For barley samples, the
precision and sensitivity of analysis were highest for the Kjeldahl and
infrared analyses. The use of Kjeldhal analysis to calibrate protein assays for
dairy products was discussed by Luithi-Pent and Puhan (10) and also Lynch
and Barbano (11).

2. KJELDAHL ANALYSIS

Johan Kjeldahl was born on August 16, 1849 in the town of Jaegerpris in
Denmark. In 1876 he was employed by the Carlsberg brewery to develop an
improved assay for grain protein. The Kjeldahl method was published in
1883. The original technique has been extensively modied. Key steps for
the assay are (a) sample digestion, (b) neutralization, (c) distillation and
trapping of ammonia, and (d) titration with standard acid. An exhaustive

* For the purposes of calibration, 44 samples of barley and 49 samples of malt were analyzed
with biuret, dye binding, infrared, alkaline distillation, and Kjeldahl tests. Such results were the
basis for deriving calibration relations between Kjeldahl and each test method. Then a further
76 samples of barley and 72 samples of malt were analyzed using only the rapid test methods.
Each Xi result gave rise to a corresponding Kjeldahl protein (Ycalc) value.
8 Chapter 1

FIGURE 1 Apparent protein concentrations in stout beer as determined by seven

methods. (Data from Ref. 7.)

TABLE 4 Barley Protein Analysis Using a Range of Techniques

Regression line
Analysis time and correlation Standard error of
Test method (min) coefcienta analysisb

Biuret 10 Y 0.857 cP 0.336 and 0.2336

1.942
R 0.972
Dye bindingc 15
Infrared 0.51.0 Y 1.060 cP 0.8381.980
1.03 R 0.96
Alkaline 90 Y 1.070 cP 2.383 and 1.540
distillation 0.670
a
cP, crude protein determined by Kjeldhal method (N 6 6.25); Y, response from the test
method. P
b
Assay standard error calculated from Ycalc Yi 2 =n.
c
No information given.
Source: Ref. 8.
Kjeldahl Method 9

account of the Kjeldahl method can be found in the monograph by

Bradstreet (12). The book is divided into ve chapters: 1, introduction to the
Kjeldahl method; 2, the Kjeldahl digestion; 3, digestion procedure (for
fertilizers, leather, cereals, foods and proteins, coal and fuels); 4, the
distillation and detection of ammonia. Chapter 5 is an extensive
bibliography. A standardized Kjeldahl procedure appears in the Interna-
tional Standard ISO-1871 (13). Further descriptions are given by Gaspar
(14) and Osborne (15).
Initially, only sulfuric acid was used for sample digestion. Then solid
potassium permanganate was added to facilitate sample oxidation. Mercuric
oxide was introduced as a catalyst in 1885. During the acid digestion phase,
the food sample is heated with concentrated sulfuric acid, which causes
dehydration and charring. Above a sample decomposition temperature,
carbon, sulfur, hydrogen, and nitrogen are converted to carbon dioxide,
sulfur dioxide, water, and ammonium sulfate [Eq. (11)].

NH2 CH2 p COOH q 1H2 SO4 ?p 1CO2 qSO2

4pH2 O NH4 HSO4 11

Digestion is complete when the mixture turns clear (light green color),
usually after 2030 minutes of heating. A further (after-boiling) period of
heating is necessary to ensure quantitative recovery of nitrogen. Data from
McKenzie and Wallace (cited in Ref. 14) show that adding X (mg) of
potassium sulfate per mL of sulfuric acid increases its boiling point
according to the relation Y (8C) 55.8X 331.2. A maximum boiling point
elevation of 1308C is achivable by adding 2 mg (potassium sulfate) per mL
acid. A high boiling point reduces the sample digestion time. Sample
digestion can also be facilitated by using a catalyst; the order of effectiveness
for metal oxide catalysts is Hg > Se > Te > Ti > Mo > Fe > Cu > V >W > Ag
(16). A proprietary brand of Kjeldahl catalyst (Kjeltabs from Foss
Electric Ltd.) comes as tablets. Each tablet contains 0.25 g of mercuric
oxide and 5 g of potassium sulfate. A working selenium catalyst can be
formulated with potassium sulfate (32 g), mercuric sulfate (5 g), and
selenium powder (1 g). Chemical oxidants (hydrogen peroxide, perchloric
acid, or chromic acid) can be added to the sulfuric acid to speed up sample
digestion.
Ammonium sulfate is rst neutralized with alkali to form ammonia.
This is then distilled and trapped using 4% boric acid. Ammonium borate is
then titrated with standard acid in the presence of a suitable indicator. Low-
cost Quick-t glassware is readily available for distillation and titration.
Sophisticated semiautomatic distillation systems are also available. The
10 Chapter 1

processes of neutralization, distillation, and titrimetric analyses are

summarized as follows.
distill
NH4 HSO4 2OH ? NH3 2H2 O SO24 12

NH3 H3 BO3 excess?NH4 H2 BO3 ?NH

4 H2 BO3 13

H2 BO3 HCl?titration?H3 BO3 14

Suitable titration indicators include methyl orange, methyl red, Congo red,
and Tashiro indicator (a 1:1 mixture of 0.2% methyl red solution and 0.1%
methylene blue).

2.1. Nitrogen-to-Protein Conversion Factors

The Kjeldahl technique measures sample nitrogen (SN) as ammonia. The
value for SN is later converted to crude protein (cP) by multiplying by a
Kjeldahl factor, FK.
cP% S N F K 15

The units for SN are g-N 100 g 1 (g-nitrogen released per 100 g of sample).
The Fk (g-protein g 1 N) is the amount of protein that produces a gram of
nitrogen. Fk is also called the nitrogen-to-protein conversion factor. AOAC-
recommended values for FK for meat and other food are summarized by
Benedict (17). Frequently, FK is given a default value of 6.25 or 5.7 for
animal and plant proteins, which are assumed to have an average N content
of 16% and 17.5%, respectively. In fact, most proteins deviate signicantly
from these averages (18). FK is also affected by the presence of NPN (e.g.,
adenine, ammonia, choline, betaine, guanidine, nucleic acid, urea, free
amino acids). Soya beans have 310% NPN, which increases to about 30%
for immature seeds. The amount of NPN also changes with growth
conditions as well as with geographic factors. There is generally no
correlation between NPN and protein content (19). No single FK value
applies to all food types. Ideally, FK should be determined for each
individual food type (Table 5).
FK can be calculated from amino acid data (18,2024). Table 5 lists
the 20 naturally occurring amino acids along with their formula weight,
number of nitrogen atoms, percent nitrogen, and the value for FK. For
arginine, FK is 3.11 100=32:15. An idealized protein having all 20 amino
acids in equal numbers has a nitrogen content of 14.73%. The FK value is
therefore 6.79 (100 g/14.73). Evaluating FK from amino acid data (for
TABLE 5 Determination of the Kjeldahl Factor for Milk Protein Using Amino Acid Composition Dataa
1 2 3 4 5 6 7 8 9 10
Amino Formula %N Amino AA composition AA-N AA Mole fraction
Acid weight (bi) N atoms Acid FK (mg/g N) (mg/g N) (moles/g N) (Xi) biX

Arginine 174.2 4 32.15 3.11 234.0 75.2 0.001343 0.027082 4.71761

Kjeldahl Method

Histidine 155.2 3 27.06 3.70 188.0 50.9 0.001211 0.024422 3.79022

Asparagine 132.1 2 21.20 4.72 292 61.9 0.00221 0.044564 5.88694
Glutamine 146.1 2 19.16 5.22 783 150.1 0.005359 0.108048 15.7859
Lysine 146.2 2 19.15 5.22 487.0 93.3 0.003331 0.067157 9.81828
Glycine 75.1 1 18.64 5.36 128.0 23.9 0.001704 0.034362 2.58058
Alanine 89.1 1 15.71 6.36 219.0 34.4 0.002458 0.049553 4.4152
Tryptophan 204.2 2 13.71 7.29 90.0 12.3 0.000441 0.008886 1.81447
Serine 105.1 1 13.32 7.51 338.0 45.0 0.003216 0.064837 6.81433
Proline 115.1 1 12.16 8.22 571.0 69.5 0.004961 0.100016 11.5118
Valine 117.1 1 11.96 8.36 428.0 51.2 0.003655 0.073687 8.6288
Threonine 119.1 1 11.75 8.51 278.0 32.7 0.002334 0.047059 5.60469
Cystine 121.1 1 11.56 8.65 47.0 5.4 0.000388 0.007825 0.94756
Isoleucine 131.2 1 10.67 9.37 290.0 30.9 0.00221 0.044563 5.84662
Leucine 131.2 1 10.67 9.37 600.0 64.0 0.004573 0.092199 12.0964
Aspartic acid 133.1 1 10.52 9.51 214.0 22.5 0.001608 0.032415 4.3144
Glutamic acid 147.1 1 9.52 10.51 574.0 54.6 0.003902 0.078669 11.5723
Methionine 149.2 1 9.38 10.66 148.0 13.9 0.000992 0.019999 2.98379
Phenylalanine 165.2 1 8.47 11.80 341.0 28.9 0.002064 0.041615 6.87481
Tyrosine 181.2 1 7.73 12.94 297.0 22.9 0.001639 0.033045 5.98774
Sum 5669.7 943.5 0.04960 1.0 132.0
Average 14.73
FK(1) 6.79 FK 6.01
a
FK(1) is determined from the average nitrogen content for all amino acids, i.e., 14.73%. FK(2) value of whole protein or sum of amino acid nitrogen
divided by sum of weight of nitrogen. FK 5669.7/943.5. Columns 810 contain data for calculating total protein content from quantitative amino
acid analysis (Sec. 4).
11
12 Chapter 1

skimmed milk) can be achieved in the following steps: (a) express each
amino acid as mg per gram of total nitrogen (see column 6 of Table 5) and
(b) calculate the mass of nitrogen derived from each amino acid (column 7 in
Table 5). FK is the weight of amino acids divided by the weight of amino
acid nitrogen (AA-N).

total weight of AA 5696:7

FK 6:01 16
total weight of AA N 943:5

Some typical values for FK are listed in Table 6 for a range of foods. The use
of FK values for quantitative amino acid analysis is discussed in Sec. 4.

TABLE 6 Nitrogen-Protein Conversion (Fk) Factors for Selected Food Protein

Sources

Food product Fk Food product Fk

Dairy products and egg Roots and tuber

Casein 6.15 Carrot 5.80
Milk 6.02 Beet 5.27
Cheese 6.13 Potato 5.18
Egg 5.73 Potato protein 5.94
Egg white solids 5.96
Meat and sh products Fruit
Beef 5.72 Tomato 6.26
Chicken 5.82 Banana 5.32
Fish 5.82 Apple 5.72
Leafy vegetables Microbial and fungal
Lettuce 5.14 Yeast 5.78
Cabbage 5.30 Mushrooms 5.61
Cereals and legumes
Wheat 5.715.75 Buckwheat 5.53
Rice 5.615.64 Oats 5.50
Corn 5.72 Millet 5.68
Sorghum 5.93 Mustard seed 5.40
Field pea 5.40 Rapeseed meal 5.53
Dry bean 5.44 Sunower meal 5.36
Soya bean 5.69 Flax meal 5.41
Source: Data from Refs. 18 and 24.
Kjeldahl Method 13

2.2. Macro- and Micro-Kjeldahl Analysis

Kjeldahl digestion methods are discussed in this section. Illustrative
examples are given to establish a pattern of work. Individual results are
described in later parts of the chapter.

A. Grain and Cereals

Kaul and Sharma (25) analyzed a range of legume and cereal grains by
micro-Kjeldahl analysis. About 200 mg of each sample was weighed into
several 75-mL Kjeldahl digestion tubes. Concentrated sulfuric acid (3 mL),
hydrogen peroxide (1.5 mL), and one Kjeltab tablet were added. The tubes
were heated using a Tectator digestion block at 3748C for exactly 25 minutes
and then allowed to cool. The contents of each tube were diluted to 75 mL
and any ammonia produced quantied by colorimetric analysis (Sec. 3).

B. Potatoes
Mohyuddin and Mazza (26) analyzed proteins from 14 potato cultivars.
Potato tubers were peeled, sliced, diced, and dried in a vacuum oven at 708C
and 48.8 mm Hg pressure. Each sample was milled and sieved through a 40-
mesh sieve. Potato our (100 mg) was added to each 100-mL Kjeldahl ask,
followed by concentrated sulfuric acid (3 mL), hydrogen peroxide (30%
solution; 1.5 mL), and commercial catalyst (500 mg; 10:0.7 w/w ratio of
potassium sulfate and mercuric oxide). Heating for 45 minutes digested the
samples. After cooling to room temperature, the contents of Kjeldahl asks
were diluted to 75 mL and then subjected to colorimetric analysis to
determine ammonia.

C. Dried Milk Powder

Venter et al. (27) described a semimicro-Kjeldahl analysis for low-fat,
medium-fat, and high-fat dried milk. About 200250 mg of sample was
mixed with 2.1 g of selenium catalyst and digested by heating with 10 mL of
concentrated sulfuric acid. The digest was cooled and diluted to 100 mL with
distilled water. Then 60 mL of 45% (w/v) NaOH solution was added and the
liberated ammonia was distilled into 20 mL of 4% (w/v) boric acid solution.
Titration was with 0.02 M HCl to an end point of pH 4.8. The results agreed
well with the macro-Kjeldahl method [International IDF Standard (1962)
No. 20].
14 Chapter 1

D. Beer Protein
Concentrated sulfuric acid (2 mL) was added to 50 mL of beer (bitter, lager,
or stout) and the mixture was heated until nearly dry (7). Kjeldahl catalyst
(10 g) and more sulfuric acid (20 mL) were added, followed by further
heating for 25 minutes. After cooling for 2 hours, water (250 mL) was added
and the Kjeldahl ask was connected to a condenser with one end immersed
in a 2% boric acid solution (200 mL). Bromocresol green was used as
indicator. Sodium hydroxide (10 M, 70 mL) was added, followed by heating
until the distillate tested neutral. The borate solution was later titrated with
0.1 N HCl. The nitrogen content in beer was calculated from the relation

%N 14Va =Wd 17

where Va (mL) is the volume of HCl required to neutralize ammonia and Wd

(g) is the dry weight of beer. Table 7 summarizes characteristics of the
Kjeldahl method used in the brewing and allied industries (28).

TABLE 7 Macro-Kjeldahl Procedures Currently in Use in the Brewing

and Allied Industries

Parameter Commenta

Instrumentationb Kjel-Tec 1030 (Auto), Kjel-Tec 1007/ 1002*;

Kjel-Foss 16120y
Sample weight 0:92+0:23 g (barley or malt) range 0.51.5 g, 14+8:4
mL (beer) range 525 mL
Catalyst (weight range)c K2SO4 CuSO4 TiO2; 3.515.8 g
Volume of conc. H2SO4 16+4:3 mL, range 1025 mL
Digestion temperatured 411148C, range 3804308C
Digestion timee 75 min or 9.6 min
End-point detection Mainly colorimetric
a
Average values unless otherwise stated.
b
Local suppliers *Tectator Ltd. and Perstop Ltd., both of Bristol, UK, { Foss Electric (UK)
Ltd., The Chantry, Bishopthorpe, York, UK.
c
A large amount of a single catalyst or a smaller quantity of a combination catalysts was used.
HgO was used in 2 of the 25 laboratories.
d
Digestion temperatures were not reported for seven laboratories using a manual Kjeldahl
technique.
e
Digestion time plus after-boiling time. The digestion time is 9.6 min when H2O2 is used as a
prooxidant.
Source: Summarized from Ref. 28.
Kjeldahl Method 15

2.3. Automated Kjeldahl Analysis

Kjeldahl analysis has undergone three forms of automation. The Kjel-
Foss1 instrument mechanizes the entire micro-Kjeldahl procedure (diges-
tion, neutralization, distillation, and titration). The Kjel-Tec1 technique
uses a digestion block in conjunction with apparatus for automated
distillation and titrimetric analysis. The nal form of automation is the
Technicon AutoAnalyzer Instrument1, which uses continuous ow analysis
(CFA).

A. The Kjel-Foss Instrument

The Kjel-Foss instrument (N. Foss Electric Ltd., Hillerd, Denmark)
performs the entire Kjeldahl procedure automatically (2931). Automation
reduces the analysis time from 3 hours to 6 minutes. The rst analysis is
completed in 12 minutes and succeeding analyses every 3 minutes. The
sample throughput is 120160 analyses per day. The Kjel-Foss instrument
requires a reliable supply of electricity and tap water for installation and
adequate drains and ventilation. A fume cupboard is not essential.
Accuracy, precision, and economics of the Kjel-Foss method were
compared with those of the manual Kjeldahl method, neutron activation
analysis, proton activation analysis, combustion analysis, and the Kjel-Tech
method (32). Results of the Kjel-Foss and manual Kjeldahl methods were
highly correlated.
Fish meal was analyzed using the Kjel-Foss instrument by Bjarno (33).
Seven collaborators compared the efciency of antimony versus mercuric
oxide as catalyst. After modifying the Kjel-Foss procedure slightly with
higher acid settings, the differences in recovery and repeatability of the two
procedures were < +1%. Using mercuric oxide catalyst poses environ-
mental concerns if the efuent from the Kjel-Foss instrument is to be
disposed of through the sewers. McGill (34) compared the Kjel-Foss
method with an improved AOAC Kjeldahl method for meat and meat
products. Over 80 analyses were performed with low (25%) fat, high (40%)
fat, and dry sausage (50% fat). As shown in Fig. 2 and Table 8, the two
techniques were highly correlated (Y 0.9904X 0.1797; R2 0.9896).
There was no systematic error in the Kjel-Foss technique over the range
of protein concentrations examined by McGill. This work validated the
Kjel-Foss instrument for meat product analysis.
Suhre et al. (35) also evaluated the Kjel-Foss instrument for meat
analysis using the AOAC Kjeldahl method as reference. Twenty-three
different laboratories analyzed six meat products having 1030% crude
protein. Eight laboratories used the automated Kjel-Foss instrument, ve
used the ofcial AOAC method, and eleven used a block digester with steam
16 Chapter 1

FIGURE 2 Calibration graph for the Kjel-Foss automated method for protein
determinations. (Data from Table 1 of Ref. 34.)

distillation. Recommendations from this work led to the block digester

steam distillation method being awarded ``rst action'' status.

B. Automated Kjeldahl Continuous Flow Analysis

The Technicon AutoAnalyzer has two reaction modules (36). The rst
module digests water-dispersible samples. The digest is then pumped to a
second module (AutoAnalyzer Sampler II). Colorigenic reagents are added
in quick succession before the ow stream passes to a delay coil to allow
color formation. Ammonia is detected using Berthelot's reaction or the
ninhydrin assay (Sec. 3). The AutoAnalyzer was applied for protein
determinations in plant material (37), feedstuffs (38), grain our (39),
instant breakfasts, meat analogues (40), meat products (41), and over 40
assorted canned and processed foods (42,43). In general AutoAnalyzer
results agreed with micro-Kjeldahl analysis.
The AutoAnalyzer digestion unit is heated in two stages at 3804008C
and 3003208C. To achieve efcient digestion, the ratio of acid to sample is
Kjeldahl Method 17

TABLE 8 A Comparison of the Automated Kjel-Foss and Approved Kjeldahl

Method for Protein Analysis in Sausage Samples

Sausage type Kjel-Foss (% protein) Kjeldahl (% protein)

Low-fat sausages
Bologna 14.64 14.54
Polish 14.42 14.43
Krakow 16.76 16.60
Liver 16.64 16.74
Hungarian 16.01 16.02
Medium-fat sausages
Breakfast 14.40 14.52
Italian 14.77 14.80
Pizza 17.42 17.33
Pork 14.09 14.21
Dry sausages
Pepperoni 14.33 14.41
Summer 18.87 18.86
Source: Ref. 34.

higher than normal for batch digestion. A superheated layer of acid forms,
which facilitates sample digestion (44). Later tests showed that the recovery
of nitrogen from refractory materials (arginine, creatine, or nicotinic acid)
was only 70%. Davidson, et al. (45) concluded that the AutoAnalyzer
digestion module was not reliable if an accuracy of 1% was desired for
Kjeldahl analysis. Over 70 different animal feeds (corn grain, wheat, barley,
rice, alfalfa, mixed feeds, feed concentrates) were analyzed using the
AutoAnalyzer digestion module. The recovery of nitrogen was 8890% (46).
In contrast, using a Technicon block digestor followed by AutoAnalyzer
Sampler II led to 100% recovery of nitrogen from cattle supplement, swine
ration, pig starter, and poultry ration (47). Suitable catalysts include copper
sulfate and oxides of mercury, selenium, or titanium. Ammonia was
detected using alkaline phenol reagent (Sec. 3.1). Quantitative recovery of
nitrogen was also demonstrated by Kaul and Sharma (25), who used a
Tectator1 heating block to digest 23 assorted strains of rice and 15 other
cereal-legume mixtures. The electrically heated block digests 40 samples per
hour under controlled temperature conditions. Samples were then trans-
ferred to the AutoAnalyzer Sampler II for ammonia detection using the
alkaline phenol reagent.
18 Chapter 1

3. COLORIMETRIC ANALYSIS OF KJELDAHL NITROGEN

Colorimetric analysis simplies Kjeldahl analysis and increases the sample

throughput. Other benets include increased sensitivity and a greater
potential for automation. Reagents for colorimetric Kjeldahl-N analyses
include (a) alkali-phenol reagent (APR), also called indophenol reagent; (b)
ninhydrin (indanetrione hydrate) reagent; (c) Nessler's reagent; and (d)
acetylacetone formaldehyde reagent. These colorimetric techniques are
reviewed next.

3.1. Alkali-Phenol Reagent (Indophenol) Method

The alkali-phenol reagent is frequently used for the Technicon AutoAna-
lyzer. Under alkaline conditions, ammonia, sodium hypochlorite, and
phenol react to form a blue product. Berthelot rst reported this reaction in
1859. The principles of the APR assay have been reviewed (4851) although
the underlying reactions remain uncertain. Ammonia probably reacts with
hypochlorite to form chloramine (NH2Cl). This reacts with phenol to form
N-chloro-p-hydroxybenzoquinone monoimine or quinochloroamine (I).
NH3 OCl ?NH2 Cl

PhO NH2 Cl?I

PhO I?II
(I) Quinochloroamine and (II) Indophenol

Alternatively, ammonia may rst react with phenol to form p-

aminophenol, which is then oxidized by hypochlorite to form (I).
Irrespective of how (I) forms, it reacts with 1 mole of phenol to form
indophenol (II). The yellow compound ionizes under strongly alkaline
conditions (pKa 13.4) giving a blue anion De 1 26104 L mol 1 cm 1 .
Substituted phenols (o-chlorophenol, m-cresol, and guaiacol) undergo the
indophenol reaction. However, para-substituted phenols and some meta-
derivatives do not react. Color intensity and the rate of color formation
increase in the presence of manganese (2) ions or acetone. Sodium
nitroprusside (1040 mg L 1) is another catalyst.
A simple APR assay suitable for detecting 3 ppm ammonia is
described in Ref. 48 (Fig. 3). Indophenol formation is pH and temperature
dependent. The linear dynamic range for ammonia was 0.33 ppm with a
sensitivity of 0.3284 (absorbance units/1 ppm NH3). The assay precision (for
1 ppm NH3) was +3%. Although performed with boric acid as the
background medium, the simple APR assay is probably not suitable for
Kjeldahl Method 19

FIGURE 3 Calibration graph for ammonia determination using the alkali-phenol

reagent assay. (Drawn from results in Ref. 48.)

Kjeldahl-N determination. Copper, zinc, and iron salts were found to act as
interferences.
Tetlow and Wilson (49) added ethylenediaminetetraacetic acid
(EDTA) to APR to reduce metal ion interference. Temperature control
was also improved using a thermostated water bath. An outline protocol is
described below.

Method 1
Analysis of ammonia using the APR assay (48,49).

Reagents
1. Phenol (crystalline,  85% pure)
2. Sodium hydroxide solution (5 N)
3. Sodium hypochlorite solution (or commercial bleach)
4. Ammonium chloride solid
5. EDTA (6% w/v)
6. Acetone
20 Chapter 1

Procedure
Preparation of alkali-phenol reagent. Place 62.5 g of solid phenol in a
500-mL beaker and add 135 mL of sodium hydroxide (5 N) slowly
with stirring. Caution: Use an ice bath to avoid excessive heat
buildup. Add 12 mL of acetone and make up the volume to 500 mL
with deionized water.
Sodium hypochlorite (1% w/v available chloride). Prepare by diluting
commercially available bleach.
Ammonium chloride standards (1000 ppm NH3 and 100 ppm NH3).
Dissolve 314.1 mg of solid NH4Cl in 100 mL of water and then dilute
10-fold. Prepare a working standard solution (0.5 ppm NH3) daily.
The APR assay sequence. Place 1 mL of sample (or standard) in a test
tube. Add EDTA solution (100 mL) with gentle shaking. Next, add
APR (1 mL) and hypochlorite (0.5 mL) in quick succession, mixing
after each addition. Finally, add 2.5 mL of water and incubate at
258C for 60 minutes. Take A625 readings for samples. Prepare a
reagent blank as described next.
Reagent blank (reverse addition method). First, mix hypochlorite
(0.5 mL) and APR (1 mL) solutions and allow to react for 510
minutes. Next add EDTA (100 mL) followed by 3.4 mL of water (or
the designated blank solution).

When reverse addition is used, hypochlorite reacts with phenol rst.

Traces of NH3 present in the blank are not detected (48,49). Reverse addition
is useful where ammonia-free water is not available for sample preparation.
After optimization, the linear dynamic range for ammonia analysis was 50
500 ppb. Assay sensitivity was 200% greater than the results shown in Fig. 2.
Color formation with 50800 ppb NH3 was virtually complete after 15
minutes at 14308C. Temperature variations had little effect on the reaction.
Thermostating at 258C for 60 minutes improved the precision.
Addition of acetone to APR increased the response to ammonia by 10-
fold. The color yield with 500 ppb ammonia declined by 2.65%, 4.8%, and
6.8% for 4.5-hour-, 1-day- or 5-day-old APR. Addition of EDTA prevented
interference from 100 ppb copper. The intervals between addition of various
reagents must not exceed 1 minute to ensure optimum precision. Comparing
results for normal and reverse addition provides a means for detecting very
small amounts of NH3 in samplessuch as water. Otherwise, ammonia-free
water is needed for preparing reagents and blanks. The calibration curve for
the APR assay is described by the equation A625 0.7120X, where X is the
concentration of nitrogen (ppm). The analytical sensitivity was 0.7120
(absorbance units) for 1 ppm ammonia. The SD for the reagent blank was
+0:0005 ppm. These values lead to an expected LLD for ammonia of 1.6
Kjeldahl Method 21

TABLE 9 The Comparative Costs of Manual APR Assay and Other Techniquesa

Capital Running cost Analysis Cost per

Technique costb per yearc per year analysisd

Manual APR 6000 (1) 5200 (1) 8000 0.72 (1)

method
Micro-Kjeldahl 12000 (2) 7000 (1.3) 2000 4.1 (5.4)
AutoAnalyzer 81,000 (13.5) 17,000 (3.3) 32,000 0.78 (1)
APR test
a
Costs are given in deutsche marks. At current exchange rate 2.8 DM $1.4 1. All methods
employ a digestion unit costing DM6000.
b
Capital costs for the micro-Kjeldahl method include the cost of a distillation unit and an
autotitrator.
c
The running cost includes DM5000 for miscellaneous chemicals.
d
Calculated for a 10-year period as capital cost/10 running cost)/no. of samples. Ratios of
costs are given in parentheses for each column.

ppb. Assuming a default FK of 6.25, the LLD for protein is 10 ppb. The
APR assay is widely used in conjunction with the AutoAnalyzer.The
composition of the APR used in CFA is pretty much the same as described
earlier (45,52).
Kaul and Sharma (25) describe a rare attempt to deploy a manual
Kjeldahl-APR assay for protein analysis. They used a Tectator heating
block for micro-Kjeldahl digestion of grain. Sample nitrogen was then
analyzed by the APR assay. The analytical performance was similar to
results obtained with the AutoAnalyzer-APR assay or the conventional
micro-Kjeldahl analysis. From Table 9, the capital cost for the manual
Kjeldahl-APR assay was 2 times lower than for the micro-Kjeldahl and 13.5
times lower than for the AutoAnalyzer method. Running costs were also
lowest for the manual APR assay.
For laboratories handling 40 or more analyses per day, it may be
worth investing in an automated technique. The manual Kjeldahl-APR
analysis was advantageous for small laboratories lacking the wherewithal to
purchase an AutoAnalyzer. Mohyuddin and Mazza (53) used the manual
Kjeldahl-APR assay to analyze potatoes (see Sec. II.B.2). The mean protein
content for 14 potato cultivars was 10.65 (+1.23)% by the manual APR
assay and 10.53 (+1.13)% using the AutoAnalyzer.

3.2. Nessler's Reagent

Ammonia reacts with alkaline potassium iodomercurate II (Nessler's
reagent) to form a colloidal complex (lmax 430460). The linear range
22 Chapter 1

for analysis extends to 75 mg (ammonia) ml 1. A possible reaction scheme

for Nesslerization is

2K2 HgI4 3KOH NH3 ?OHg2 NH2 I 2H2 O 7KI 18

Hach et al. (54) developed a commercial Nesslerization reagent for use in

Kjeldahl analysis. A sulfuric aciddigested sample (0.4 mL) is diluted with
24.6 mL of 0.01% (w/w) polyvinyl alcohol (PVA) solution. One ml of
Nessler's reagent is added and the sample is agitated mechanically before
absorbance measurements are recorded at 430 nm. As the product of
Nesslerization is colloidal in nature, spectrophotometric analysis is sensitive
to the degree of sample agitation. PVA acts as a colloidal stabilizer and
improves the precision of the Nessler method.

3.3. Acetylacetone-Formaldehyde Reagent

The acetylacetone-formaldehyde assay is based on the Hantzsch reaction for
the synthesis of pyridine (55). Prediluted digest is reacted with a mixture of
acetyltacetone and formaldehyde in the presence of sodium acetate. The
yellow product (3,5-diacetyl-1,4-dihydrolutidine) is measured at 410 nm
De 1:46103 Lmol 1 cm 1 . The color-forming reaction is shown in Eq.
(19).

19

Acetylacetone-formaldehyde reagent was used for the analysis of

medicinal agents such as paracetamol, sulfanilamide, and chloropramide.
The potential for colorimetric Kjeldahl analysis is obvious.
Kjeldahl Method 23

Method 2
Determination of ammonia using acetylacetone-formaldehyde reagent (55).

Reagents
Acetylacetone-formaldehyde reagent. Place 15 mL of formaldehyde
(37% w/v) and acetylacetone (7.8 mL) into a 100-mL ask. Make up
to 100 mL with distilled water.
Sodium acetate (2M). Dissolve sodium acetate (82 g) in 1 L of distilled
water.

Procedure
Add prediluted Kjeldahl digest (< 2 mL; 25100 mg N) to a 25-mL
conical ask followed by sodium acetate solution (3 mL) and
acetylacetone-formaldehyde reagent (3 mL). Incubate the mixture at
97.88C for 15 minutes and cool to room temperature.
Bring the total volume to 25 mL and record A412 values using a 1-cm
cuvette.
The linear dynamic range for the preceding assay was 0.56.0 mg N
(per nal reaction mixture). The calibration graph was described by
A412 9:8610 2 X 4:2610 3 R2 0:9999, where X is the amount of
nitrogen present in the nal (25-mL) reaction mixture. The new method
shows levels of accuracy and precision equal to those of the micro-Kjeldahl
method.

3.4. Ninhydrin (Indanetrione Hydrate) Assay

Ninhydrin* reacts with amino acids (Fig. 4) in two stages: (a) the amino acid
is oxidized to aldehyde and ammonia while ninhydrin is converted to
hydrindantin and (b) hydrindantin and 1 mole of ninhydrin react with
ammonia to form Ruhemann's purple (56,57). For ammonia determination
an added reducing agent is necessary to convert ninhydrin to hydrindantin.
Ninhydrin solution is available commercially. Results from the ninhydrin-
Kjeldahl assay agree closely with those from Kjeldahl analysis (5658). The
linear dynamic range for colorimetric Kjeldahl assay depends on the extent
of sample dilution just before ninhydrin analysis. A 2-mL standard
ammonium sulfate solution containing 5.6 mg ( or 2.8 ppm) reacted with
2 mL of ninhydrin solution yields an A570 reading of 0.805. Interference was
noted for concentrations of selenium above 86 mg mL 1 (prediluted digest).
No interferences were observed with Fe, Zn, Pb, Cu, Ca, Ba, Al, Mg, Co, or

* Ninhydrin is often encountered in detective novels as a reagent for ngerprint analysis.

24 Chapter 1

(a)

(b)

FIGURE 4 Reaction scheme between amino acids and ninhydrin reagent.

Kjeldahl Method 25

FIGURE 5 Colorimetric ninhydrin analysis of Kjeldahl nitrogen calibrated against

the conventional macro-Kjedahl analysis. (Drawn from data in Ref. 58.)

Ni at concentrations of 50 mg mL 1 or from Hg at 30 mg mL 1. The overall

impression is that the ninhydrin assay is resistant to metal ions.
Quinn et al. (58) analyzed rapeseed our, rapeseed concentrate,
soybean concentrate, and bovine serum albumin using the ninhydrin assay
in conjunction with an AutoAnalyzer (Fig. 5). The precision of analysis was
1.401.76%. A manual Kjeldahl-ninhydrin assay has not been reported
recently. This seems a pity. Compared with other colorimetric methods, the
ninhydrin assay is more resistant to interferences from metal catalysts. The
color is also formed at a more easily buffered pH between 4.9 and 5.4.

4. QUANTITATIVE AMINO ACID ANALYSIS

The following steps are involved in quantitative amino acid analysis: (a)
hydrolyze a sample of food using concentrated hydrochloric acid, (b)
determine the amino acid prole, (c) calculate the concentration of each
amino acid in the sample, and (d) calculate the weight of each amino acid.
Quantitative amino acid analysis is reportedly one of the most reliable
methods for protein quantitation (5961).
26 Chapter 1

4.1. Principles of Quantitative Amino Acid Analysis

Crude protein (cP) is expressed by Eq. (20), where Ci (mole) is the amount of
each amino acid in the sample and bi (g mole 1), is the formula weight for
each amino acid.
X
20
cP Ci bi 20
i1

However, amino acid proles are reported in terms of mole fraction of each
amino acid (Xi):
Xi Ci =Cnet 21

where Cnet is the net concentration of amino acids found in the sample.
Substituting Ci Cnet Xi in Eq. (20),
X
20
cP Cnet bi Xi 22
i1
P
The term (biXi) is called the mean residue weight, W ( g mole 1).
This is the average formula weight for all amino acids in the sample adjusted
for their frequency.*
X
W g mole 1 bi Xi 23

and
cP WCnet or cP FCnet 24

In Eq. (24), F is the mean residue weight. Usually, W (g mole 1) is adjusted

to take into account two routine errors in amino acid analysis: (a) many
colorimetric reagents for amino acids do not react with proline and (b)
tryptophan is destroyed during acid hydrolysis of proteins. Proline and
tryptophan are usually determined by separate experiments. After correcting
for such errors, one gets the conversion factor, F (g mole 1):
P
18 P
18
bi Ci bi Xi
i1 i1
F or F 25
Cnet CPro CTrp 1 XPro XTrp

where XPro and XTrp are the mole fractions of proline and tryptophan. For a

* Horstmann called this parameter the weight equivalent (WE).

Kjeldahl Method 27

range of meat products, F (g mole 1) was 1020% larger than W [Eq. (23)].
Calculations of F (g mole 1) appear in last three columns of Table 5. Typical
values for F (g mole 1) are given in Table 10. Values for F range from 100 to
125 g mole 1 for most proteins.Most protein sources are now routinely
analyzed for amino acid scores during nutritional evaluations (Chapter 14).
This yields all the information necessary for total protein estimation.
Zarkadas and co-workers in Canada are strong exponents of quantitative
amino acid analysis (Table 11).

4.2. Quantitation of Specic Proteins

Meat collagen was determined by measuring 5-hydroxylysine (5OH-Lys).
This amino acid is found in collagen and no other meat protein. The
concentration of collagen (Pj) was calculated from a modied Eq. (24)
(64,65);
1000Wj
Pj Ci 26
nj M i

TABLE 10 Corrected Mean Residue Weights (F) for

Selected Food Protein Sources

Protein source F (g mole 1)

Barley our 129.84

Soya bean our 119.82
Pea our 118.85
Fish meal 112.70
Beef sausage 107.06109.01
Pig skin (rind) 94.02
Bone meal 104.21
Soya bean protein
Flour 114.43
Isolate 114.48
Concentrate 115.69
Wheat
Flour 113.13116.00
Gluten 108.4108.52
Egg white solids 118.43
Potato protein 108.52
Milk (nonfat) powder 112.98
Source: Data calculated or taken from references in Table 11.
28 Chapter 1

TABLE 11 Protein Determination by Quantitative Amino Acid Analysis

Sample/comments Reference

NASASkylab meals Heidelbaugh et al. (59)

Mushroom total protein Weaver et al. (62), Braaksma and
Schaap (63)
Porcine muscle and connective tissue Zarkadas et al. (64), Zarkadas et al. (65)
proteins (myosin, actin, elastin, and
collagen
Actin, myosin, and collagen in Karatzas and Zarkadas (66)
composite meat products; mixed meat
sausages, bologna, frankfurters,
sausages, hamburgers
Additives and ingredients for meat Zarkadas et al. (67)
products including soybean, wheat
products, potato protein
Porcine skin (rind) Nguyen et al. (68)
Chicken meat and connective tissue Karatzas and Zarkadas (69)
Apple ower buds Khanizadeh et al. (70)
Bone isolates Zarkadas et al. (71)
New soybean cultivars Zarkadas et al. (72)

where Ci is the concentration of 5OH-Lys in the meat hydrolysate, Wj is

mean residue weight from the amino acid prole for collagen (averaged for
the different collagen types), nj is the number of 5OH-Lys residues per 1000
residues in collagen, and Mi is the formula weight for 5OH-Lys. Typically,
Wj 91:01 g mole 1 , Ni 10 residues, Mj 145:18, and consequently

Amount of collagen 62:755OH-Lys 27

Similarly, the concentration of Nt-methylhistidine and 4-hydroxypro-

line was the basis for assessing the amount of myobrillar protein and
connective tissue (collagen and elastin) in meat. These methods are
satisfactory for meat and meat products. They may have questionable
validity for composite foods. Plant foods may contain 4-hydroxyproline
rich glycoproteins (extensins, lectins, salt-extractable glycoproteins). For
example, alfalfa protein and potato protein contain signicant levels of
4OH-Pro.
Kjeldahl Method 29

4.3. Examples and Relation to Kjeldahl Method

Quantitative amino acid analysis is arguably one of the most accurate
methods for food protein quantitation. One source of error is the high
concentration of free amino acids in some foods. The protein content in nine
strains of Agaricus was 28% (+3.4)% by quantitative amino acid analysis
(62). The results were poorly correlated R 0:4 with Kjeldahl results
(22.4% protein per dry weight basis). A more recent analysis of freeze-dried
mushroom powder led to estimates of 7.0% protein by dry weight (63). In
the later study, mushroom powder was extracted with 0.5 M NaOH and
precipitating with trichloroacetic acid (TCA) before analysis. This removed
large amounts of TCA-soluble NPN associated with mushrooms. Of the
total NaOH-soluble nitrogen extracted from mushrooms, 20% was protein,
60% was urea or ammonia, and 20% was free amino acids. Food samples are
now routinely extracted with organic solvents to remove NPN before
quantitative amino acid analysis (Table 11). Advocates for quantitative
amino acid analysis point to its compatibility with plant proteins. There is
no interference from phenols, tannins, and lignin. By contrast, the Kjeldahl
method is unsuitable for plant tissues regardless of the conversion factor
used (73).

5. COMBUSTION NITROGEN ANALYZERS

The Dumas assay predates Kjeldahl analysis by 50 years (Table 1). The
former technique was invented by Jean Baptiste Dumas. Early applications
include the analysis of plant materials (74,75), meat (76), casein, whole
powdered milk, soybeans, and maize our (77). The rst-generation
instruments for the Dumas method were not user friendly. The volume of
nitrogen gas produced by combustion was determined with a manometer.
The advent of easy-to-use and highly accurate combustion nitrogen
analyzers (CNAs) rekindled interest in the Dumas method.
CNAs from various manufacturers work on the same principle. The
sample is dropped into a 95010508C furnace, purged free of atmospheric
gas, and lled with pure (99%) oxygen. Complete sample combustion leads
to CO2, water, SO2, NO2, and N2. The product gases are cooled and a
portion is passed through tubing packed with hot lead chromate, copper,
sodium hydroxide (solid), or phosphorus pentoxide to remove SO2, O2,
CO2, and water, respectively. The NO2 is then reduced to N2 and measured
with a thermal conductivity detector (TCD). Sample protein content is
calculated by taking into account the mass of sample injected, the
30 Chapter 1

proportion of the combustion gases analyzed, and the nitrogen-protein

conversion factor (FK). The calculations are now automated.

5.1. Collaborative Studies and Approved Status for CNAs

CNAs were calibrated with the Kjeldahl method. Interlaboratory studies
appearing after 1987 are listed in Table 12. Such trials led to CNAs receiving
approved status from the AOAC (Association of Ofcial Analytical
Chemists), AOCS (American Oil Chemists' Society), ASBC (American
Society of Brewing Chemists), AFI (American Feed Industry), BRF-
International (Brewing Research Foundation-International), IOB (Institute
of Brewing), and EBC (European Brewing Convention).
The Canadian Grain Commission and U.S. Department of Agricul-
ture (USDA) Federal Grain Inspection Services (FGIS) approved CNAs in

TABLE 12 Food Protein Analysis Using the Dumas Combustion Method

Samplea Reference

Animal feedstuffs, fertilizers Sweeney and Rexroad (78), Schmitter

and Rhihs (79), Sweeney (80), Sachen
and Thiex (81), Tate (82)
Beer ASBC (83), Johnson and Johansson
(84,85)
Brewing grainsbarley, malt, rice ASBC (86), Buckee (28,87), Krotz et al.
(88), Johansson (89), Angelino et al.
(90)
Cereal grainswheat, barley, corn, Bicsak (91), Bicsak (92), Williams et al.
sorghum (93)
Dairy productsskimmed, powdered Wiles and Gray (94), Wiles et al. (95),
milk etc. chocolate milkshake, Simonne et al. (96,97)
cheeses, etc.
Fruitguava, peaches, plum Simonne et al. (96,97), Huang et al. (98)
Infant food Bellemonte et al. (99)
Meat and meat products, sh (raw, sh King-Brink and Sebranek (100),
in oil, tuna) Simonne et al. (96,97), Buschmann
and Westphal (101)
Oilseeds (soybean, canola, sunower, Bicsak (91), Duan and DeClercq (102),
corn) Berner and Brown (103)
Potatoes Young et al. (104)
Vegetablescabbages, broccoli, Simonne et al. (96,97)
ketchup, tomato
a
Approximate sample classication; classes contain the other foodstuffs.
Kjeldahl Method 31

TABLE 13 Advantages of the Dumas Method

1. Greater ease of operation

2. Higher operator safety owing to the nonrequirement for harzadous chemicals
3. The absence of wetchemistry
4. Reduced time of analysis
5. Higher performance characteristics (greatar accuracy, repeatablility)
6. Absence of waste disposal concerns (Table 14)
7. Simple instrument installation without a requirement for specialized ventilation
8. Low cost per analysis

1994 and 1996, respectively (9193). Trials for the combustion method
usually follow guidelines described by Youden and Sleiner (105):
1. The number of laboratories ranges from 7 to 12. Studies involving
as few as three laboratories have been reported.
2. All studies compare CNAs with Kjeldahl analysis.
3. Interlaboratory studies focus on a single food group. Therefore,
CNAs tend to receive approval for one food group at a time
(Table 12).
4. Trials usually test a ``generic combustion method'' and are
independent of the choice of instruments.
Minimum performance guidelines for CNA instruments include (a) a
furnace temperature of 9508C, (b) a separation system for trapping CO2 and
water, (c) a thermal conductivity detector for nitrogen, (d) sufcient
accuracy to produce results within 0.15% of the mean (% nitrogen) results
for 10 successive measurements using a standard compound, and (e)
sufcient precision to produce a relative standard deviation of 0.01%.The
LECO FP-428 analyzer was used by about 80% of the laboratories involved
in collaborative trials. The Foss-Heraue Macro-N analyzer, Carlo Erba
NA-5000, and Perkin Elmer PE2410 also feature. The LECO FP-2000
combustion analyzer appears in the latest trials.

5.2. Advantages of the Combustion Method

The modern CNA has advantages over the Kjeldahl method (Table 13).
There is greater speed of analysis and greater operator safety stemming from
the nonuse of aggressive chemicals. The estimated cost for analysis is $0.37
$0.50 per sample with the LECO FP-2000 protein analyzer (LECO
Corporation, Saint Joseph, MI) compared with $1.0 per test for the
Kjeldahl method (106109).
32 Chapter 1

TABLE 14 A Comparison of Materials Reqirement for the Kjeldhal and Dumas

Methods (74,84)

Requirement Kjeldahl Dumas

Chemical Conc. H2SO4, 40% NaOH, Air, oxygen, helium,

requirements K2SO4, TiO2/CuSO4 (or copper, turnings, EDTA,
HgO), H3BO3 KH, nitrogen catalyst, Mg
phthalate, methyl red, perchlorate, sodium
phenolphthalein, pumice, hydroxide, alumina oxide
water pellets
Other suppliesa Kjeldahl and Erlenmeyer Tinfoil squares, brushes, tin
asks, burettes, acid, alkali capsules, combustion,
and water dispensers, reduction and absorption
stirring equipment, tubes, cotton wool, steel
large containers for wool, particle lters, tubing
acid, etc.
Ancillary equipment Ductwork for corrosive Ductwork for warm airb
fumes, acid-resistant fans,
fume washer, fans, etc.
Disposal of chemical Collected, professionally Nontoxic, wastebin or sink
disposed disposal
Time per analysis 120 min (24 samples) 3 min
Degree of hazardc 6 2
Accuracy 7098 100
Precision (CV %) 1.2 0.7
a
Does not include main equipment (Kjeldahl digester and distillation apparatus, or CNA
instrument
b
Optional, but advisable for large-scale testing.
c
Arbitrary scale of 110, with 10 being extremely hazardous and 1 completely safe. There may
be a risk of burns when maintaining the combustion instrument.

A more detailed discussion of the relative costs of protein analysis by

Kjeldahl or combustion analysis has to consider factors such as number of
analyses per year, capital costs for instrumentation, depreciation, main-
tenance costs, and savings of labor, chemicals, and other consumables costs
(106). It has been suggested that the combustion method provides cost
savings of about 30% with a payback period within 2 years. For research
institutes, universities, and small-scale laboratories, the safety of modern
CNAs probably outweighs cost considerations. Further comparisons of the
CNAs and Kjeldahl analysis are summarized in Table 14.
Kjeldahl Method 33

5.3. Combustion Analysis of Feeds, Cereal Grains, and

Oilseeds
Combustion analysis rst received AOAC approval for feeds in 1968. The
classical instruments (Coleman model 29A nitrogen analyzer) used a
manometer for the volumetric assay of nitrogen (110). Comprehensive
testing using modern TCD-based CNAs appeared in 1987 (78). A nine-
laboratory collaborative trial to determine nitrogen in feeds was successfully
completed in 1989 (80). The AOAC approved CNAs for animal feed testing
in 1990.
The small sample sizes (150500 mg) used with modern CNAs raised
concerns about sampling. Extensive grinding and mixing are essential to
ensure sample homogeneity and representative sampling. Sweeney and
Rexroad (78) analyzed 14 different animal feeds using the LECO FP-228
instrument with a prescribed sample size of <150 mg. Estimates of feed
nitrogen agreed closely with results from Kjeldahl analysis. The precision of
analysis was signicantly lower (0.0130.052%) for the combustion method
as compared with Kjeldahl analysis (0.0060.035%). Schmitter and Rihs (79)
increased the sample size for the LECO-F228 analyzer from 150 mg to 1 g by
palletizing before loading into the instrument port. Adding a few drops of
polyethylene (2% w/w in ethyl acetate solvent) prevented aking of the
pellets. Table 15 shows nitrogen and protein data for feeds determined using
the CNA (78,79). Results have been averaged for samples of sizes 0.151.0 g.
Protein values are calculated as %N 6 6.25. The results agree favorably with
Kjeldahl analysis (Fig. 6). There appeared to be signicant positive bias for
feedstuffs having < 2% nitrogen (Fig. 7). The bias was ascribed to plant-
derived materials containing high levels of nitrate. The Kjeldahl method
achieves low recoveries of nitrogen from refractory compounds (75) with
N22O or N22N bonds (nitrite; nitrate; oximes; azo-, nitro-, nitroso-
compounds).
Sachen and Thiex (81) found that CNAs showed a  1.38% (protein)
bias for hay samples. They attributed such results to ``atmospheric error''
arising from air being trapped in the interstices of the (uffy) hay samples.
Compressing samples to remove trapped air led to agreement between the
Kjeldahl and CNA results (81). The LECO FP-2000 nitrogen analyzer was
not subject to an atmospheric blank because of improvements in instrument
design and efcient purging of atmospheric gases before sample combustion.
Sachen and Thiex examined a range of pelleting equipment and procedures
for eliminating the atmospheric blank for the LECO FP-428 instrument.
They proposed that powdered cellulose could be analyzed to check for an
atmospheric error (81). Not all investigators agree about the nature of the
atmospheric error.
34 Chapter 1

TABLE 15 Nitrogen and Protein in Feedstuffs Determined by Combustion Method

Sample N (g kg 1) Protein (%)

Straw 0.620 3.88

Corn silage 1.175 7.34
Porc soup 1.185 7.41
Hay 1.280 8.00
Corn grain 1.375 8.59
Barley 1.515 9.47
Grass silage 1.835 11.47
Oats 1.760 11.00
Triticale 1.835 11.47
Wheat 1.985 12.41
Dried grass 2.240 14.00
Cow premix 2.755 17.22
Alfafa pellets 2.770 17.31
Hog feed 3.390 21.19
Broiler nisher 3.420 21.38
Milk powder 4.365 27.28
Bone meal 4.325 27.03
Rapeseed 5.710 35.69
Protein conc. 6.045 37.78
Soybean meal 6.485 40.53
Cattle conc. 6.690 20.91
Yeast 6.895 43.09
Peanut meal 8.450 52.81
Meat meal 9.300 58.13
Fish meal 9.960 62.25
Gluten 11.730 73.31
Feather meal 13.610 85.06
Soy protein conc. 14.020 87.63

Bicsak (91) described a collaborative study to extend AOAC-approved

status to cereal grains and oilseeds. Seven laboratories analyzed 15 matched
pairs of samples (soybean, canola, sunower, wheat, barley, corn, sorghum)
having protein levels of 813%, 1723%, or 3540%. Six of the seven
collaborators used the LECO FP-428 instrument. With 210 samples the
average protein reading was 28.26% by combustion analysis and 28.01% by
Kjeldahl analysis. Repeatability and reproducibility statistics were compar-
able. A recommendation to extend the AOAC combustion method to cereal
grains and oilseeds was approved. The following non-English publications
Kjeldahl Method 35

FIGURE 6 Comparison of protein results for feeds determined using the combustion
method and the Kjeldahl method. Micro-CNA and macro-CNA refer to
the use of 150-mg and 1-g sample sizes with the combustion nitrogen
analyzer. List of feedstuffs is given in Table 15. (Data derived from Ref.
78.)

FIGURE 7 Residuals from Fig. 6 showing no systematic differences in results.

36 Chapter 1

describe collaborative tests leading to approved status for combustion

analysis of cereal and cereal products including wheat, wheat bran, pasta,
sorghum, and maize (111,112).

5.4. Barley, Malt, and Beer

The combustion method was subjected to an 11-member interlaboratory
trial for brewing grain (rice, barley, malt, spent grain) analysis. Agreement
was reached in 1992 to include the Dumas method in the methods of
analysis for brewing grains (86). The trial results showed that most
commercial CNAs had a linear range of 09.5% nitrogen and an LLD of
0.0321%. The repeatability CV ranged from 0.013 to 0.055% compared with
a reproducibility of 0.0420.067%.
Further collaborative studies to evaluate CNAs for barley, malt, and
beer analysis were reported by the UK Institute of Brewing in 1996 (28).
Fifteen of the 25 participating laboratories employed the LECO FP-428
instrument. Another ve laboratories used the Foss Heraeus Macro-N
apparatus. The range of Kjeldahl techniques used is shown in Table 7. All
laboratories examined eight samples each of barley, malt, and beer. CNAs
gave slightly higher values for total nitrogen as compared with the Kjeldahl
method. Essentially identical results were obtained when results from the
Kjel-Foss instrument were omitted. The reproducibility was 0.030.07% for
barley and 0.0360.065% for malt analysis. These values were independent
of total nitrogen over the range 1.171.71% (w/w) (barley), 1.452.03% (w/
w) (malt), and 2681020 mg L 1 (beer). Based on such results, the IOB
Analysis Committee (UK) approved combustion analysis for use alongside
Kjeldahl analysis. However, the precision for beer analysis was low, perhaps
because the trial participants lacked expertise with liquid samples. CNAs
were judged unsuitable for beer protein determination.
CNAs were approved for beer analysis in Europe in 1999 (84,85). In
the collaborative trial organized by the IOB and EBC, ve samples of beer
and malt were analyzed in duplicate by 18 laboratories from the brewing
and allied industries. The collaborators used the following CNAs: LECO
FP-428 instrument (11 laboratories), LECO FP-2000 (3 laboratories), and
Macro-N analyzer (3 laboratories). Glycine, Tris, or EDTA was used as the
calibrant. Samples of beer were found to contain 3621159 mg (nitrogen)
L 1 and malt samples had 0.5340.706% nitrogen. Repeatability and
reproducibility statistics for beer analysis were deemed satisfactory, leading
to method approval.
Kjeldahl Method 37

5.5. Milk and Related Dairy Products

Despite the recent widespread use of CNAs for food protein analysis, few
applications in the dairy eld have been published. Wiles et al. (95)
described an 11-member interlaboratory study from New Zealand. They
compared milk protein analysis by CNAs and the Kjeldahl method. Samples
included ultra-heat-treated (UHT) whole milk, infant formulas, whole milk
powder, skimmed milk powder, whey protein concentrate, casein, and
sodium caseinate. Nine of the eleven laboratories employed the LECO FP-
428 instrument. Results for CNAs agreed closely with Kjeldahl ndings.
There was no systematic bias associated with the former results. Indeed, no
evidence was found for a systematic difference between CNA and Kjeldahl
results reported between 1968 and 1997 (95).

5.6. Baby Foods and Infant Formulas

Bellemonte et al. (99) analyzed ve categories of baby foods using the Carlo
Erba model 1500 nitrogen analyzer. This instrument uses a high furnace
temperature (18008C) combined with an oxygen-rich atmosphere to achieve
complete sample combustion. Nitrogen is quantied using a TCD. The
analysis time for this instrument is reportedly 3 minutes. All sample types
were analyzed successfully. Results obtained by the Kjeldahl method were
14% lower than those obtained with CNAs (Table 16). Results from both
techniques compared favorably with protein values declared by food
manufacturers. Compared with the Kjeldahl method, CNAs are convenient
for baby food analysis. The sample throughput and safety considerations
favor the Dumas method as described in Sec. 5.2.

5.7. Meat
King-Brink and Sebranek (100) described a 12-laboratory trial to evaluate
CNAs for meat product analysis. Participants in the trial used the LECO
FP-428 instrument (9 laboratories), the Foss Heraeus Macro-N analyzer (2
laboratories), or the Perkin Elmer PE2410 analyzer. In all, 15 pairs of meat
products, purchased from 30 different manufacturers, were analyzed. All
participants used CNAs satisfactorily judging from (a) the low number of
data outliers and (b) the high precision of results for standard compounds.
The CNA results were slightly higher than Kjeldahl gures: 15.75% versus
15.59% (w/w). Estimates for repeatability and reproducibility were
comparable. A recommendation that the Dumas method should be adopted
as a reference test for meat proteins was approved by the AOAC. A 14-
laboratory trial for analysis of meat and meat products was reported in
38 Chapter 1

TABLE 16 Analysis of Protein in Five Categories of Baby Foods (97)

Declared
Samplea protein Kjeldahlb Dumasb

1. Formula milk (7) 15.55 14.94 15.36

2. Cereal-based products (6) 10.28 10.12 10.35
Cream of rice,
semolina honey, wheat
our milk oats, milk
soup cereal fruit, milk
soup cereal apples
3. Biscuits (2) 9.50 9.40 14.70
4. Lyophilized products (2) 54.20 52.15 53.20
Veal, ham, and eggs
5. Homogenized products (6) 9.52 9.62 9.72
Beef, beef ham, chicken,
veal brain, turkey,
chicken carrot potatoes
a
Numbers in parentheses represent number of different foods in the category analyzed. Protein
values are averaged for each food category
b
Protein values were calculated with a conversion factor (Fk) 6.38 for milk formula or 6.25 for
all other categories of baby food.

Germany. This trial, too, concluded that the performance of the Dumas
method was comparable to that of the Kjeldahl assay but that the former
method was quicker and more environment friendly (101).

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2
The Alkaline Copper Reagent:
Biuret Assay

1. INTRODUCTION

Copper (Cu2) ions react with proteins to form a blue complex at high pH.
The intensity of the color is proportional to the amount of protein present.
The oldest copper binding technique for protein analysis is called the biuret
method. The name, although quite deeply entrenched in the literature, is
unfortunate. The assay has little to do with biuret. A more accurate name is
the alkaline copper reagent (ACR) test. Older literature features almost
exclusively the ``biuret reagent.'' Depending on the context, both these
names will be used. First we will discuss the history and developments of the
ACR assay. Modern and (where relevant) old protocols involving ACR are
described. Applications in food protein analysis are then considered.
Berthelot reported that biuret (NH2CONHCONH2) forms a blue
complex with copper ions under alkaline conditions. Proteins were reported
to undergo a similar reaction in 1873. Early developments of the biuret
method are described by Hiller (1) and also by Robinson and Hodgen (2).
The two publications are some of the earliest accounts of the ACR assay.
Blood plasma was diluted with 25 volumes of 0.9% saline and adjusted to
10% (w/v) TCA. The protein precipitate formed was recovered by ltration
and redissolved in 3% sodium hydroxide solution (5 mL). Then copper

47
48 Chapter 2

sulfate solution (20% w/w; 2.5 mL) was added. After 12 hours the brown
precipitate (Cu2 hydroxide) produced was removed by centrifugation. A
linear relationship was found between DA560 readings and protein
concentration (00.2 mg mL 1). The assay sensitivity was 6 DA560 units
per (mg mL 1) protein. The formation of copper (hydro)oxide precipitate
was a nuisance. Samples had to be centrifuged before DA560 readings could
be taken.

2. THE ALKALINE COPPER REAGENT PROTEIN ASSAY

Mehl (3) added ethylene glycol to ACR to arrest the formation of Cu2
hydroxide. Ethylene glycol (100 mL), 60% (w/v) sodium hydroxide (40 mL),
and 4% (w/v) copper sulfate were mixed and the solution was diluted to
400 mL with water. The mixture was heated to precipitate copper hydroxide
and then ltered. Extra sodium hydroxide was added to bring the nal
concentration to 1011% (w/w). The resulting ACR was stable for several
months. Dissolved egg albumin ( 3 mg mL 1) was analyzed by mixing with
an equal volume of ACR and taking DA550 readings after 90 minutes. The
blue color, once formed, was stable for about 20 hours. Sols (4) used
glycerin as a stabilizer for ACR. The amount of glycerin required was 100
times lower than ethylene glycol. ACR solution was prepared by mixing
glycerin (2 mL), copper sulfate (5% w/v; 80 mL), and sodium hydroxide
(20% w/v; 400 mL) and making up to 1 L with water. To perform analysis,
1 mL of test solution (< 12% w/v protein) was mixed with an equal volume
of ACR solution. Colorimetric readings were recorded at two wavelengths,
accounting for the unpopularity of the procedure.
Weichselbaum (5) produced a modied ACR by adding potassium
sodium tartrate as the stabilizer for Cu2. Gornall et al. (6) evaluated several
modied ACR solutions, nding Mehl's copper reagent difcult to
reproduce. Weichelsbaum also considered the effect of changing copper
sulfate and sodium hydroxide concentrations and the reaction temperature
on color formation. This work revealed some tolerance for variations in
regent composition and experimental conditions.
The ACR developed by Weichselbaum was employed by Gornall et al.
for protein analysis in clinical samples. This variant ACR became more
popular after Layne's account of it appeared in Methods in Enzymology in
1957 (7). ACR is now widely used for food protein analysis. A summary of
this popular method is given next.
The Alkaline Copper Reagent: Biuret Assay 49

Method 1
Protein analysis using alkaline copper reagent (5,6)
Reagents
1. Copper sulfate CuSO4
5H2 O
2. Rochelle salt (potassium sodium tartrate)
3. Sodium hydroxide (10% w/w solution)
Preparation of ACR. Dissolve copper sulfate (1.5 g) and potassium
sodium tartrate (6 g) in 500 mL of water. Add 300 mL of sodium
hydroxide (10% w/w) solution with constant mixing. Adjust the
total volume to 1 L with distilled water. This solution should keep
indenitely. Discard if it shows signs of forming a black or reddish
precipitate. The nal concentrations of copper sulfate and sodium
hydroxide are 0.015% and 3% w/v, respectively.
Procedure
Add 8 mL of ACR solution to 2 mL of protein solution. Mix and allow
to react for 30 minutes. Record DA540 using a 1-cm cuvette.
Dilute plasma (protein) in 0.9% saline before analysis. To separate the
albumin and globulin, dilute 0.5 mL of plasma with 9.5 mL of
sodium sulfate solution (22.6% w/v). Remove 2 mL of the solution
for biuret analysis of total plasma protein. Add 3 mL of ether to the
remaining solution, mix for 30 seconds, and centrifuge. Remove
2 mL of the supernatant and analyze to nd the plasma albumin
concentration. The difference (total protein plasma albumin) is
the plasma globulin concentration.
The linear range for protein analysis with Method 1 was 110% (w/v)
protein. The results were strongly correlated with Kjeldahl nitrogen.

TABLE 1 Effect of Different Proteins on

the Color Yield from the ACR Assay

Protein Relative color

intensity (%)

Serum albumin 100

Serum globulin 98.8
Egg albumin 98.6
Casein 88.1
Gelatin 74.2
Zein 92.1
Source: Ref. 6.
50 Chapter 2

Different proteins gave slightly different color yields (Table 1). Casein, zein,
and gelatin produce low amounts of color compared with serum albumin.
The biuret method cannot be used in the presence of ammonium salts. The
ACR formulation is one of the most popular modications of the biuret
reagent and is the standard method for clinical samples (8). The ACR
formulation is also widely used for the analysis of animal proteins.

3. CHEMISTRY OF THE ALKALINE COPPER REAGENT

PROTEIN ASSAY

Strickland et al. (9) titrated proteins with increasing amounts of ACR. After
each addition, the absorption spectrum was recorded between 425 and
850 nm. Sparingly soluble proteins were dissolved in 8 M urea, which had no
deleterious effects on color formation. By plotting the maximum absorbance
change versus copper concentration, the maximum amount of protein
required to bind all Cu2 was found.
Table 2 shows that lmax for ACR-protein mixtures is 535590 nm. The
extinction coefcient for the Cu2-protein complex (De, M 1 cm 1 with
respect to protein concentration) increased with the molecular weight and
the number of peptide groups per protein. The amount of Cu2 bound to a
xed weight of protein is shown in Table 2. Note that gliadin and gelatin
exhibit low color yields per unit weight of protein. This characteristic is
partly related to the high proline content in these proteins. Other amino acid
side chains also alter the specic color yield from protein-Cu2 binding.
Cysteine binds copper strongly, forming Cu-mercaptide. Therefore cysteine-
rich proteins show a reduced color yield during ACR analysis. High
concentrations of glutamate and aspartate compete with the peptide
backbone for Cu2 binding.
Cu2 binding with biuret, glycinamide, and glycine oligopeptides was
reviewed by Sigel and Martin (10). Cu2 binding reects the acid-base
properties of the peptide bond. Delocalization of the peptide nitrogen lone-
pair electron leads to a planar geometry with partial double-bond character.
The resulting canonical structures have a high electron density at the peptide
oxygen. The amide hydrogen acts as a weak acid, becoming ionized only
under very alkaline conditions (Fig. 1). Binding of Cu2 to the peptide
group occurs via oxygen at neutral pH. In a highly alkaline medium
(pH  13), complexation involves the nitrogen group. Under alkaline
conditions, 2 moles of biuret bind with each Cu2 atom, forming a six-
member ring (Fig. 2). Cu2 forms a tetradentate complex. The amide
nitrogen becomes deprotonated in the process. Peptide-metal ion binding at
high pH values occurs in competition with metal ion hydrolysis. Cu2
TABLE 2 Selected Parameters for the ACR Analysis

Cu atoms
Test Molecular
"a Peptides "c (mole "d
b
sample weight max per M [P] (g/Cu) per Cu per M ligand) 1 per kg

Biuret 103 515 27.8 206 4.0 13.9 0.5 134.95

Triglycine 189 590 90.3 189 3.0 90.3 1.0 477.78
Gliadin 27,000 555 143.4 700 6.0 5,531.1 38.6 204.86
b-Lacto- 36,800 544 151.4 620 6.0 8,986.3 59.4 244.19
globulin
Zein 45,000 535 150.0 638 6.0 10,579.9 70.5 235.11
Gelatin 75,000 554 144.2 670 6.3 16,141.8 111.9 215.22
The Alkaline Copper Reagent: Biuret Assay

Serum 180,000 545 154.0 586 5.0 47,303.8 307.2 262.80

globulin
Edestin 310,000 550 163.6 635 6.0 79,867.7 488.2 257.64
a
Extinction coefcient with respect to Cu2 (M 1 cm 1).
b
Grams of protein bound to 1 mole of Cu2.
c
Extinction coefcient with respect to protein (M 1 cm 1).
d
Extinction coefcient with respect to protein (kg 1 cm 1).
Source: Adapted from Ref. 9.
51
52 Chapter 2

FIGURE 1 Resonance hybrid forms of the peptide bond. Structures (1) and (2)
predominate under physiological conditions at a ratio of 60:40. At neutral
pH the electron density is highest at the oxygen atom. At the pH of the
biuret reaction (pH 14), deprotonation of the amide nitrogen leads to
signicant amounts of (3) with high electron density at the nitrogen atom.

binding with the OH ion leads to an insoluble precipitate. A model for the
2:1 biuret- Cu2 complex at high pH is shown in Fig. 2.
Three kinds of Cu2 complexes are formed with polypeptides. At low
pH Cu2 interacts exclusively with side-chain residues forming ``type S''
complexes. At intermediate pH values there is binding to both side chains

FIGURE 2 Schematic diagram of the Cu2-(biuret)2 complex.

The Alkaline Copper Reagent: Biuret Assay 53

and deprotonated amide nitrogen, forming type SP complexes. Lastly, when

pH >1012, the Cu2 interacts mainly with four peptide nitrogens to form a
biuret-like (type B) complex. Cu2 binding does not involve consecutive
amino acid residues. Interactions leading to type B complexes are
incompatible with regular (a-helical or b-sheet) forms of polypeptide
secondary structure. Interestingly, some (type B) polypeptide complexes
with Cu 2 have rudimentary enzymatic (peroxidase and catalase) activity
(10). The interactions just described can be usefully contrasted with
Cu2 binding to native proteins, where binding occurs at the N-terminal
region.

4. INTERFERENCE COMPOUNDS

ACR dissolves extraneous plant colors and dyes with a high absorbance
at 530550 nm (11).* To overcome this problem during ACR analysis,
Jennings (11) diluted samples with high backgroud readings 100-fold using
standard Lowry reagent A (see Chapter 4). The samples were then
analyzed by treating with Lowry reagent B (11). Pinckney (12) reduced the
interference from phopholipid by adding carbon tetrachloride to ground
meals before analysis. Johnson and Craney (13) noted that isopropanol
reduced the interfering effects from plant dyes. Some of the substances
capable of interfering with the ACR method are listed in Table 3.
Possible interferences during wheat protein analysis using ACR were
investigated by Mitsuda and Mitsunaga (14). They extracted whole wheat
our or bran with 0.5% (w/v) sodium hydroxide. The alkali-soluble extracts
were identied as starch, glucose, pigment, and lipids (Table 4). The effects
of each component were evaluated by adding known amounts to model
solutions consisting of 0.5 or 5 mg mL 1 ovalbumin and then assaying the
mixture by the ACR method. Glucose (<5 mg mL 1) reduced the color yield
for the ACR assay, probably by competing with the peptide group for Cu2
binding. The interference was less pronounced when colorimetric readings
were taken after 30 minutes rather than 2.5 hours. The Cu2 reaction with
glucose takes place more slowly than the Cu2 binding to protein. The
different rates of reaction offer an opportunity for improving analytical
performance (see later). Lipid and starch suspensions cause light scattering
and an apparent increase in absorbance readings. Starch is also hydrolyzed

* Examples of strongly colored grain include barely and oats, which have blue or black chaff
(pericarp, aleurone layer, and outer endosperm).
54 Chapter 2

TABLE 3 Some Interfering and Noninterfering Compounds for ACR Protein Assay

Reactive compounds

Colors Nonpeptide compounds

Anthocyanins and other avonol DNA
Lycopenes (carotenoids and related) Glucose
Pigmentschlorophyll and related Glucoseamine
Starch
Amino acids (histidine and cysteine)
Biopolymers Buffer components
Lignin and low molecular-weight Ethanolamine, ethylenediamine, Tris
derivatives

Unreactive compounds

Amino acids Amides

Gly, Tyr, Arg, Met, Phe, Asp, Glu, Acetamide, sulfanilic acid,
Iso, Lys dimethylformamide, urea
Organic acids N-methylacetamide
Formic, acetic, lactic
Amines
Ammonia, ethylamine
Others
Creatine, betaine, EDTA
Source: Compiled from various sources.

to glucose during high-temperature ACR assays. For small amounts of

sample, protein determinations can be performed after defatting by Soxhlet
extraction or by stirring with cold acetone or hexane.

TABLE 4 Quantities of Alkali-Soluble Components Extracted from Wheat (mg g 1)a

Component Whole wheat Bran

Glucose 9.0 8.2

Starch 670.0 120.0
Protein 130.0 140.0
Pigment 0.0 7.4
Lipid 121.0 32.5
a
A 1-g sample was extracted with 0.5% NaOH (100 mL) (14).
The Alkaline Copper Reagent: Biuret Assay 55

TABLE 5 Effect of Hydrogen Peroxide Addition on ACR Assay of Colored Grain

Regression
Sample/method equationa R SE (%)

Barleyb Y 13:3x 7:73 0.54 1.24

Wheat bran Y 40:8x 3:60 0.79 1.18
hydrogen
peroxideb
Barleyc Y 49:3x 0:48 0.99 0.21
Wheat branc Y 58:5x 0:14 0.98 0.28
a
Y % Kjeldahl protein, x A550.
b
Analysis by the method of Johnson and Craney (13).
c
Analysis after pretreatment with CCl4 and mixture made 1% with repeat to H2O2 for 30
minutes before A550 readings.

5. SAMPLE PRETREATMENT AND AVOIDING

INTERFERENCES

Pinckney (12) successfully analyzed full-fat our after precipitating lipid

with carbon tetrachloride. The presence of 10% (v/v) tetrachloromethane or
dichloroethane also reduced the solubilization of wheat starch in 0.5% (w/v)
sodium hydroxide by 90%. These and other organic solvents (CF3CCl3,
cyclohexane) probably exert their effect by complexing with starch.
Chloroform, 1,2-dibromomethane, n-hexane, n-octane, and n-decane had
no effect.
Strategies for dealing with extraneous plant colors were also
investigated by Mitsuda and Mitsunaga (15). Barley meal and wheat bran
protein were analyzed by the method of Johnson and Craney (13).
Colorimetric measurements were calibrated with the Kjeldahl method. All
analytical parameters (sensitivity, linearity, standard error of analysis) were
altered by the strong background color associated with barley grain and
wheat bran. Adding hydrogen peroxide (1% v/v nal concentration) about
30 minutes before A550 readings improved assay sensitivity and LLD
(Table 5).
Jennings (11) and Williams (16) suggested that strongly colored grains
cannot be analyzed using the ACR assay. They found ACR results were in
poor agreement with Kjeldahl results for barley and wheat bran. Hydrogen
peroxide provides a key ingredient needed for the Fenton reaction.

H2 O2 Cu2 ? HO OH Cu1 1
56 Chapter 2

TABLE 6 Sensitivity of the UV Biuret

Method for a Range of Proteins

Protein
A263a

Insulin 5.2
Bovine serum globulin 6.7
Bovine serum albumin 5.3
Histone 6.3
Ribonuclease 5.9
Ovalbumin 5.0

Protein
A310a

Gelatin 1.56
Histone 1.71
Casein (Hammersten) 1.74
Ovalbumin 1.98
Trypsin 2.17
Bovine serum albumin 2.21
Lysozyme 2.20
a 1
Value for 1 mg mL protein solution.

The highly reactive hydroxyl radical  HO catalyzes the oxidative

destruction of extraneous plant colors.

6. THE MICRO-BIURET OR ULTRAVIOLET BIURET

PROTEIN ANALYSIS

The UV ACR assay is performed at 255320 nm. The technique is 10- to 15-
fold more sensitive than the conventional ACR protein analysis. The use of
UV absorption measurements in conjunction with the ACR method dates
back to 1957. The method became popular owing to its rediscovery by
Ellman (17). Itzhaki and Gill (18) used a similar approach with minor
modications. More recently, Kanaya and Hiromi (19) used a stopped-ow
version of the UV biuret assay.
The sensitivity of the UV biuret assay to gelatin was lower than
observed with other proteins (17) (Table 6). Similar sensitivity differences
are observed at visible wavelengths (see Table 1). The dipeptide L-ProGly
had a high molar extinction coefcient e 176 M 1 cm 1 when analyzed
by the UV biuret method. In contrast, L-GlyPro had zero extinction. The
biuret-positive dipeptide has a ``normal'' peptide nitrogen atom, whereas the
peptide bond in L-GlyPro is formed from a secondary amine nitrogen
The Alkaline Copper Reagent: Biuret Assay 57

without an ionizable hydrogen. In consequence, the complexation of Cu2 is

severely weekened (18).

7. APPLICATIONS OF THE ACR SOLUTION FOR FOOD

PROTEIN ANALYSIS
7.1. Cereal Proteins
High-protein wheat our is suitable for bread making. Low-protein soft
wheat our is used for manufacture of biscuits and cookies or for animal
feed. Barley for brewing should have a low protein content in order to
minimize haze formation. Understandably, the commercial value of grain
depends on its protein content. Grain suppliers and purchasers are
interested in rapid and accurate methods for protein determination. Protein
analysis is also important in connection with plant-breeding programs.
Pinckney (12) was the rst to analyze wheat proteins using ACR.
Flour protein was extracted (peptization) before analysis. Jennings (11) and
also Williams (16) later developed a one-step assay by adding ACR directly
to cereal our. Johnson and Craney (13) and Craney (20) eliminated the
need to prepare ACR by adding solid copper carbonate directly to our
suspended in alcoholic alkaline solvent. Noll et al. (21) produced an ACR
solution containing 50% (w/w) isopropanol to reduce the interference from
plant dyes. Furthermore, the time taken for analysis was reduced from
about 60 minutes to 5 minutes (22,23). Most of the early applications of the
ACR centered on wheat grain and our analysis (1223). A smaller number
of reports deal with the analysis of proteins from rice (24), barley and malt
(25,26), corn (27), and sorghum and peal millet (28).
Developments of the ACR analysis should be seen against a
background of a number of perceived limitations of the manual Kjeldahl
analysis. The method is considered slow and costly. There is a relatively high
capital cost and a need for highly skilled staff (Chapter 1). By comparison,
the ACR method is simple, fast, and affordable. As it is a colorimetric
technique, there are nominal instrumentation costs. The requirement for
personnel training is also minimal. Such considerations provided some of
the impetus for the rst applications of the ACR for cereal protein analysis.

A. Wheat Proteins
Pinckney (12) described a method for extracting wheat protein from our
using a 1:10 volume mixture of carbon tetrachloride and dilute (0.05 N)
potassium hydroxide. The mixture was centrifuged and the aqueous phase
analyzed using ACR stabilized with 0.32% glycerol. About 100 samples of
58 Chapter 2

hard red winter wheat, 36 samples of hard spring wheat, and 28 samples of
hard white wheat were analyzed by Pinckney (12). The A550 readings were
highly correlated with Kjeldahl protein (R 0.9250.976). The standard
error for the ACR assay was 0.300.32 with a reproducibility of 0.1%. Sources
of error include suboptimal peptization by dilute potassium hydroxide. The
rate of color formation was also undesirably slow, requiring 2040 hours.
Jennings (11) developed a one-step assay by adding ACR directly to
our. Protein extraction and color development occurred simultaneously as
the our was agitated with ACR. This innovation, by eliminating a separate
peptization stage, led to a signicant increase in the speed of ACR analysis.
Jennings also found that K-Na tartrate was a better stabilizer for Cu2 than
glycerol. Samples of the former seemed to contain fewer impurities and the
likelihood of copper oxide formation was reduced. The Cu2-tartrate
complex (lmax 675 nm) also interfered less with A550 readings compared
with the Cu2 glycerol chelate (lmax 630 nm). During the one-step assay
the protein extraction efciency was 8497%. With barley varieties having a
black or blue aleurone layer, there was interference from extraneous plant
colors. Unidentied chromogenic species with lmax 500800 nm were
dissolved at high pH. The interference was especially acute for the analysis
of whole-meal wheat our or unhulled oats. The extraneous color was
associated with oat bran (29).
A one-step ACR method was employed to assess protein levels in 45
varieties of brown and milled rice (24). There was a high correlation between
A550 readings and crude protein levels (N 6 5.95).* The calibration equation
for milled rice was cP 15:48A550 0:063, where cP is the % Kjeldahl
protein. The linear range extended to A550  1.0 with a regression coefcient
of 0.964 and a standard error of 0.46. A further ACR analysis of 42 brown
rice samples led to the regression equation cP 16:04A550 0:233
(R 0.981) with a standard error of analysis of 0.30. Other results for
ground rice samples are summarized in Table 7.
Further improvements of the ACR assay were introduced by Johnson
and Craney (13). First, they modied the ACR assay for use with strongly
colored cereals such as barley, oats, and grain sorghum. Next, solid copper
carbonate was added directly to our suspended in alkaline-isopropanol
solution, thereby doing away with a need for a prepared ACR solution.
These changes led to a method that was signicantly faster than any
previous ACR assay (Method 2).

* Ground rice (1 g) and 2 mL of carbon tetrachloride were added to several (25  150 mm) test
tubes along with ACR (40 mL). Samples were shaken with a mechanical shaker for 90 minutes.
Aliquots (15 mL) from each sample were centrifuged and A550 readings were recoreded.
The Alkaline Copper Reagent: Biuret Assay 59

TABLE 7 Protein Content of Ground Rice Determined Using the Biuret Assay

Milled rice Brown rice

Method Mean (%) Range (%) Mean (%) Range (%)

Micro- 7.50 5.7411.69 8.25 6.1511.74

Kjeldahl
ACR or 8.18 5.9512.50 8.85 6.3012.45
biuret
Source: Data from Ref. 24.

Method 2
Rapid analysis of wheat proteins (13).

Reagents
1. Copper carbonate (solid).
2. Potassium hydroxide pellets
3. Isopropanol
Alkaline isopropanol solvent. Add potassium hydroxide (5.61 g) to
isopropanol (600 mL). Make up to 1 L with water.

Procedure
Place 1.00 (+ 0.001) g of our in a 250-mL Erlenmeyer (conical) ask
and add solid copper carbonate (1.0 + 0.1 g). Suspend the mixture
in alkaline isopropanol solution (50 mL) and shake vigorously using
a mechanical shaker for 15 minutes. Allow the sample to stand for a
further 15 minutes and lter through a glass ber lter with vacuum
suction. Take A550 readings against a reagent blank.
About 391 cereal samples were analyzed, including grain sorghum (47
samples), corn (48 samples), oats (40 samples), barley (44 samples), wheat
(165 samples), and hard and soft wheat our (47 samples). The ACR assay
results are highly correlated with crude protein levels determined by
Kjeldahl analysis (Table 8). The former technique could be applied to a wide
range of cereal grains. Using multiple sample shakers, a throughput of
0.2 min 1 could be achieved. Compared with the preprepared ACR
solution, solid copper carbonate is more stable for prolonged storage. The
isopropanol reduced interference by extraneous plant dyes and also reduced
the solubility of starch in the alkaline reaction medium. By eliminating a
peptization stage, the time for the ACR analysis was reduced from 3540
60 Chapter 2

TABLE 8 Analysis of Cereal Proteins Using the Method of Johnson and Craney

Regression Sensitivityb
Number of equationa A550 per Analytical
Cereal samples (Yi ) R (%) error (%)

Grain 47 13:36Xi 0.98 0.0748 0.19

sorghum 2:64
Corn 48 11:486Xi 0.95 0.0871 0.14
4:0
Oats 40 15:93Xi 0.99 0.0627 0.17
1:84
Barley 44 20:36Xi 0.97 0.0491 0.23
1:33
Wheat 165 16:07Xi 0.99 0.0622 0.18
(whole 1:47
meal)
Wheat four 47 16:68Xi 0.99 0.0599 0.14
(rened) 0:26
a
Regression equation with Yi % Kjeldahl protein and Xi absorbance at 550 nm.
b
Specic color change for 1% increase in sample protein content.
Source: Summarized from Ref. 13.

minutes to about 10 minutes. An example of the one-step biuret procedure

for cereal protein analysis is described by Strong and Duate (30). On the
downside, repeated measurement of solid copper carbonate for ACR
analysis is unappealing. Availability of preweighed copper carbonate tablets
could remove some of the objections to ``solid'' reagents. The pharmaceu-
tical industry has been dispensing medicinal tablets for many years. Kjeldahl
catalysts are also available in tablet form.

B. Sorghum and Millet

Deosthale and Visweswara-Rao (28) at the National Institute of Nutrition,
Hyderabad (India) employed the Johnson and Craney method for sorghum
and peal millet protein analysis. The results were compared with the micro-
Kjeldahl method. For sorghum protein A550 0.04131 cP 3.854
(R 0.943), where cP represents Kjeldahl protein (%N 6 6.25). The
calibration graph for pearl millet was expressed by A550 0.07135
cP 1.488 (R 0.9413). Comparing with data in Table 8, note that the
current graphs were drawn with % Kjeldahl protein on the ordinate axis,
and sensitivity for sorghum protein is lower here. Analysis of sorghum
The Alkaline Copper Reagent: Biuret Assay 61

protein using the biuret and Kjeldahl methods is also described by Belavady
et al. (31).

7.2. Meat Proteins

The procedure of Torten and Whitaker (32) is a good example of the
application of the ACR method for meat sample analysis. The method is
summarized next.
Method 3
Analysis of meat proteins using the ACR (32).
Reagents
1. Prepare the ACR solution as described in Method 1.
Procedure
Meat comminution and alkaline digestion. Grind raw meat samples by
passing through a Horbart meat grinder to produce a ne paste.
Place comminuted meat samples (0.91.2 g) in a 50 mL Erlenmeyer
ask. Add 20 mL of sodium hydroxide (0.5 N) and heat the
suspension for 10 minutes over a boiling-water bath. Allow to cool
and lter using Whatman No. 3 paper to remove fat. Shake 15 mL
of ltrate with an equal volume of petroleum ether to remove
remaining fat. Centrifuge using a solvent-resistant centrifuge tube at
* 4000 rpm and collect the claried extract for protein analysis.
Add 4 mL of ACR solution to a xed volume (0.41 mL) of meat
extract. Bring the nal volume to 5 mL with distilled water, mix, and
allow to stand for 30 minutes. Using a colorimeter take, A550
readings against an appropriate reagent blank.
Torten and Whitaker (32) added varying amounts of fat to
comminuted lean beef, chicken, cod, or pork to provide a range of protein
values. For a range of 9.924.2% protein the A550 readings were linearly
related to Kjeldahl results (R > 0.98). The optimal time for meat protein
dissolution using alkali was 10 minutes. Longer heating times led to peptide
bond hydrolysis. Defatting with petroleum ether was efcient. Other
defatting solvents include carbon tetrachloride and diethyl ether. Table 9
shows the range of protein values for raw meat samples as determined by the
ACR analysis.
A very high correlation exists between the Kjeldahl and ACR
methods. Therefore, a comparative calibration exercise may be adopted
by analyzing standard protein samples, e.g., serum albumin. Further
examples of meat protein analysis using the ACR assay are reported by
Lasztity et al. (33) and Reichardt and co-workers (34). Brooks and others
62 Chapter 2

TABLE 9 Meat Protein Analysis Using the Biuret Assay

% Protein
Sample (n)a ACRb / biuret Kjeldahl % Moisture % Fatc

Beef (10) 24.2  0.3 24.3  0.3 60.3  0.11 20.3

Pork (11) 23.1  0.3 22.4  0.3 55.2  0.18 26.7
Chicken (9) 25.2  0.3 24.6  0.2 58.8  0.13 21.2
Cod (8) 18.8  0.2 19.2  0.2 64.5  0.07 20.3
a
Meat sample and number replicate analysis.bACR alkaline copper reagent or biuret method.
c
Approximate fat content estimated as difference between protein and moisture.
Source: Ref. 32.

(35) showed that the ACR method is probably the most accurate of
colorimetric method for assaying whole-body protein concentration in test-
animal carcasses during feeding trials. Silgjnic and Samardzija (36)
considered the best ways to dissolve meat samples for biuret analysis.
Beef, pork, chicken meat, frankfurters, sausage, and sh did not dissolve
fully at high pH, leading to losses during the subsequent analysis. Dissolving
samples at lower pH using concentrated urea solutions provided a better
alternative.
Protease action affects the results of the ACR assay for meat protein.
Errors may arise for meat samples with high amounts of endogenous
proteases. Horse muscle is thought to contain unusually high levels of
catheptic activity. Autolysis leading to loss of some peptide bonds may
affect results obtained using ACR analysis. Turgut (37) has shown that
differently treated sh muscle gives different results with ACR analysis,
probably as a result of sh muscle autolysis. Prusa and Bowers (38) used the
biuret assay to determine the solubility of turkey muscle protein under the
inuence of nonmeat ingredients (sodium nitrite, sodium chloride, and
phosphate salts).

7.3. Meat Process End-point Temperatures

For safety reasons, processed meat should be heated to certain minimum
end-point temperatures (EPTs). With adequate heating, agents responsible
for viral diseases such as foot-and-mouth disease, foul pest, Newcastle
disease, and African swine fever are inactivated. Potential bacteriological
hazards associated with underheated meat include Escherichia coli and
Salmonella. Different classes of meat have different prescribed EPTs. A
protein coagulation test has been suggested as the means for establishing
The Alkaline Copper Reagent: Biuret Assay 63

whether meat has undergone adequate heat treatment. Heating leads to a

reduction in the muscle proteins extracted with 0.9% saline. A reduction in
the ratio of extractable biuret-positive compounds (EBPRs) can be used to
monitor heat treatment. The extractable biuret-positive compound ratio is
dened by the empirical relation
test sample protein solubility
EBPR 2
reference sample protein solubility
Heating meat to the USDA-prescribed EPT produced an EBPR value of 1.1
+ 0.012 (3941).

7.4. Dairy Proteins

A collaborative trial of the biuret method for assessing protein solubility
was undertaken by Morr et al. (42). The tests of sodium chloride solubility
at pH 3 or 7 were compared with Kjeldahl analysis. At pH 7 the range of
solubility values determined by Kjeldhal analysis was 86.994.2% (whey
protein), 7099.9% (sodium caseinate), 17.319.7% (soy protein isolate), or
94.299.2% (egg white protein). Biuret results were correlated with Kjeldahl
values but showed signicantly lower precision. The biuret assay was used
for the measurement of insolubilized protein. Casein from milk was
precipitated using acetate buffer. The precipitate was recovered by
centrifugation and biuret reagent was added directly to the precipitate.
After 30 minutes, A540 readings were recorded. For 10 milk samples the
mean casein content was 2.62.7% (w/v) (43). The method probably works
because casein dissolves in the high-pH biuret reagent. Analyses of other
dairy proteins including cheese have also been described (44).

7.5. Yeast Proteins and Fermentation Monitoring

Analysis of yeast protein raises difculties owing to high levels of NPN,
mainly nucleic acid. Yeast cells are also surrounded by a tough cell wall
composed of glucans, mannan, and chitin as well as protein. The ACR
method was successfully applied for yeast protein analysis by Ihl and Tagle
from the University of Chile, Santiago (45). The method described in detail
next should be applicable to a wider range of single-cell proteins.
64 Chapter 2

Method 4
Analysis of yeast protein by the ACR method.
Reagents
1. ACR (Method 1)
2. Aqueous toluene solution (water containing 10 ppm toluene)
3. Sodium hydroxide solid
4. Torula yeast
Procedure
Yeast protein solubilization. Lyse dried yeast (3 g) by suspending in
100 mL of aqueous toluene solution. Shake or stir the suspension of
cells in a water bath at 508C for 6 hours. Add sodium hydroxide to
bring to 0.5 M and heat at 75808C for 30 minutes. Freeze the
sample at 208C overnight, thaw, and reheat for a further 30
minutes.
Protein analysis. To 1 mL of yeast protein extract add 4 mL of ACR
solution and allow 30 minutes for color formation. Record A540
readings.
Yeast protein reacted with the ACR solution forming a purple-violet color
with lmax 540 nm. The ACR assay results were strongly correlated with
Kjeldahl results (%N 6 6.25). About 12% of nitrogen in dried yeast was
NPN. Protein monitoring during fermenter operation was addressed by
Nielsen et al. (46). They set up a laboratory scale fermenter and examined
various ow analyses for monitoring sugars, lactic acid, biomass, and
protein in the feed stream. Growth media components (yeast extract and
peptone) contain high levels of NPN and glucose. Despite such difculties,
the ACR assay was successfully used to monitor the utilization of feed
protein during the fermentation by lactic acid bacteria.

REFERENCES

1. A Hiller. Determination of albumin and globulin in urine. Proc Soc Exp Biol
Med 24:385386, 1927.
2. HW Robinson, CH Hodgen. The biuret reaction in the determination of
serum proteins. 1. A study of the conditions necessary for the production of a
stable color which bears a quantitative relationship to the protein concentra-
tion. J Biol Chem 135:707724, 1940.
3. JWC Mehl. The biuret reaction of proteins in the presence of ethylene glycol. J
Biol Chem 157:173180, 1945.
4. A Sols. An improved biuret reaction of proteins and the two-standard
colorimetry. Nature 160:89, 1947.
The Alkaline Copper Reagent: Biuret Assay 65

5. TE Weichselbaum. An accurate and rapid method for the determination of

proteins in small amounts of blood serum and albumin. Am J Clin Pathol
10(Tech Suppl):4049, 1946.
6. AG Gornall, CJ Bardawill, MM David. Determination of serum proteins by
means of the biuret reaction. J Biol Chem 177:751766, 1949.
7. E Layne. Spectrophotometric and turbidimetric methods for measuring
proteins. Methods Enzymol 3:447454, 1957.
8. GH Grant, JF Kachmar. The proteins of the body uids. In NW Tiettz,
ed. Fundamentals of Clinical Chemistry. London:WB Saunders, pp 298
376.
9. RD Strickland, ML Freeman, FT Gurule. Copper binding by proteins in
alkaline solution. Anal Chem 961:545552, 1961.
10. H Sigel, RB Martin. Coodinating properties of the amide bond. Stability and
structure of metal ion complexes of peptide related ligands. Chem Rev 82:385
426, 1982.
11. AC Jennings. Determination of the nitrogen content of cereal grain by
colorimetric methods. Cereal Chem 38:467479, 1961.
12. AJ Pinckney. Wheat protein and the biuret reaction. Cereal Chem 26:423443,
1949.
13. RM Johnson, CE Craney. Rapid biuret method for protein content in grains.
Cereal Chem 48:276282, 1971.
14. H Mitsuda, T Mitsunaga. Evaluation and elimination of the interference by
starch in the biuret determination of wheat protein. Agric Biol Chem 38:1649
1655, 1974.
15. H Mitsuda, T Mitsunaga. A convenient method for rapid determination of
cereal proteins: a device to eliminate the effect of color substances on the
biuret procedure. Agric Biol Chem 38:22652266, 1974.
16. PC Williams. The determination of proteins in whole wheatmeal and our by
the biuret method. J Sci Food Agric 12:5861, 1961.
17. GL Ellman. The biuret reaction: changes in the ultraviolet absorption spectra
and its application to the determination of peptide bonds. Anal Biochem 3:40
48, 1962.
18. RF Itzhaki, DM Gill. A micro-biuret method for estimating proteins. Anal
Biochem 9:401410, 1964.
19. K-I Kanaya, K Hiromi. Determination of low concentrations of protein by
the biuret method using the ``stopped-ow time difference analysis'' technique.
Agric Biol Chem 51:18851892, 1987.
20. CE Craney. A quick biuret method for protein in wheat. Cereal Chem 49:496
497, 1972.
21. JS Noll, DH Simmonds, W Bushuk. A modied biuret reagent for
determination of protein. Cereal Chem 51:600616, 1974.
22. WT Greenway, RM Johnson. Five-minute biuret method for protein content
of wheat. Baker's Dig 48(2):3839, 72, 1974.
23. DH Simmonds, JA Ronalds. Rapid protein determination in cereal grains
using the biuret reaction. Baker's Digest 49(4):3640, 51, 1975.
66 Chapter 2

24. LC Parial, LW Rooney, BD Webb. Use of dye-binding and biuret tech-

niques for estimating protein in brown and milled rice. Cereal Chem 47:3843,
1970.
25. M Gullord. Studies on the biuret method for determination of protein in
cereals. Meld Nor Landbrukshoegsk 51(13):6 pp 1972.
26. Y Pomeranz, RB Moore, FS Lai. Reliability of ve methods for protein
determination in barley and malt. J Am Soc Brew Chem 35(2):8693, 1977.
27. PS Misra, R Barba-Ho, ET Mertz, DV Glover. Studies on corn proteins. V.
Reduced color response of opakue-2 corn protein to the biuret reagent, and its
use for the rapid identication of opaque-2 corn. Cereal Chem 50:184190,
1973.
28. YG Doesthale, K Visveswara-Rao. Application of rapid biuret technique for
protein estimation in sorghum and pearl millet. Indian J Nutr Diet 14(3):65
69, 1977.
29. PC Williams. The determination of proteins in whole wheatmeal and our by
the biuret method. J Sci Food Agric 12:5861, 1961.
30. FC Strong, AMA Duate. A room temperature, rapid method for the
determination of protein in wheat and other grains by the biuret reaction.
Cereal Chem 69:659664,1992.
31. B Belavady, MM Subramanya, KNRK Murthy, S Ramachandra, AN Bagali,
SA Hosamani. Appropriate sample for determination of protein in sorghum
(Sorghum bicolor (L) Moench) raised in agricultural trials. J Sci Food Agric
37:207210, 1986.
32. J Torten, JR Whitaker. Evaluation of the biuret and dye-binding methods for
protein determination in meats. J Food Sci 29:1681174, 1964.
33. R Lasztity, D Torley, F Orsi. Contribution to the protein determination in
meat products. Proceedings of the European Meeting of Meat Research
Workers 24:L1:1L1:6, 1978.
34. W Reichardt, J Mueller, S Mueller, B Eckhert. Beef and pork. Direct
determination of connective-tissue-protein-free pure protein content [Rind
und Schweineeisch. Zur direkten Bestimmung des bindegewebseiweissfreien
Reineiweissgehaltes (Rein-BEFFE)]. Fleischwirtschaft 74:13271329, 1994.
35. SPJ Brooks, BJ Lampi, G Sarwar, HG Botting. A comparison of methods for
determining total body protein. Anal Biochem 226:2630, 1995.
36. D Smiljanic, S Samardzija. Biuret method for determination of proteins in
food. I. Conditions of dissolving the sample. Technol Mesa 38:153157, 1997.
37. H Turgut. Drawbacks in the use of the biuret method for determination of
the same protein in differently treated sh samples. Food Chem 4:161165,
1979.
38. KJ Prusa, JA Bowers. Protein extraction from frozen, thawed turkey muscle
with sodium nitrite, sodium chloride, and selected phosphate salts. J Food Sci
49:709713, 720, 1984.
39. WE Townend, JE Thomson, JR Hutchins. ``Coagulation test'' for cooked
meat temperature: effect of sample preparation methods. J Food Sci 50:1179
1180, 1186, 1985.
The Alkaline Copper Reagent: Biuret Assay 67

40. CE Davis, AJ Bracewell, JB Andersen, JO Reagan. Time temperature heating

effects on biuret-positive water-extractable porcine and bovine muscle
proteins. J Food Prot 48:215220, 1985.
41. CE Davis, BG Lyon, JO Reagan, WE Townsend. Effect of heating on water
soluble biuret-positive compounds of canned cured pork picnic shoulder. J
Food Prot 50:681684, 1987.
42. CV Morr, B German, JE Kinsella, JM Regenstein, JP van Burent, A Kilara,
BA Lewis, ME Mangino. A collaborative study to develop a standardized
food protein solubility procedure. J Food Sci 50:17151718, 1985.
43. T Cheng, YB Sun, J Li. Determination of casein content in milk by biuret
method. China Dairy Ind 28:3335, 2000.
44. W Reichardt, B Eckert, Determination of protein in milk, cheese and meat by
means of the biuret reaction [Zur Bestimmung des Proteingehaltes von Milch,
Kaese und Fleisch mit Hilfe der Biuret-Reaktion]. Nahrung 35:731738, 1991.
45. M Ihl, MA Tagle. Estimation of protein in yeast. J Sci Food Agric 25:461
464, 1974.
46. J Nielsen, K Nikolajsen, S Benthin, J Villadsen. Application of ow-injection
analysis in the on-line monitoring of sugars, lactic acid, protein and biomas
during lactic acid fermentation. Anal Chim Acta 237:165175, 1990.
3
The Lowry Method

1. INTRODUCTION

The protein assay of Lowry et al. (1) is simply called the Lowry assay. The
prototype assay has a number of drawbacks. The standard curve is
nonlinear. The technique uses unstable reagents, which are generally
prepared daily. The classical method is also subject to a variety of
interference compounds. Nevertheless, the Lowry assay remains highly
important. Peterson's modication of the Lowry assay (2) is robust,
sensitive, and impervious to most interferences. This chapter contains
descriptions of the Lowry assay, the underlying principles (Sec. 3),
calibration features (Sec. 4), interference compounds, and common sample
pretreatment strategies for ensuring accurate results (Sec. 5 and 6).
Applications of the Lowry method to food protein analysis are reviewed
in Sec. 7.
The design of the Lowry protein assay can be traced to the
investigations of Wu (3,4). These were concerned with the use of tungstate-
molybdate reagent for protein analysis. Folin and Ciocalteu (5) also
determined tyrosine and tryptophan in protein hydrolysates using tung-
state-molybdate. Herriot (6) achieved a 3- to 15-fold increase in sensitivity of
the tungstate-molybdate assay in the presence of copper (Cu2) ions.

69
70 Chapter 3

2. THE LOWRY PROTEIN ASSAY

A mixture of Cu2 sulfate and sodium-potassium tartrate reacts with

proteins. Then Folin-Ciocalteu reagent is added. A blue-purplish color
forms that is measured at 750 nm (A750). The Lowry method is reviewed by
Lane (7) and Peterson (8,9). The following procedure is based on
descriptions from Refs. 1 and 7. Preparation of the Folin-Ciocalteu reagent
(4) is described in Method 2.

Method 1
Analysis of soluble proteins using the Lowry method.

Reagents
1. Sodium carbonate (Na2CO3)
2. Sodium hydroxide (0.1 M)
3. Copper sulfate CuSO4
5H2 O
4. Sodium potassium tartrate
5. Folin-Ciocalteu reagent
6. Bovine serum albumin

Procedure
Prepare stock solutions for reagents A, B, and E as described below.
Mix reagents A and B in a volume ratio of 50:1 to produce reagent
C, which is a copper sulfate solution stabilized with tartrate.
Reagent A (2% sodium carbonate in 0.1 N sodium hydroxide). Dissolve
sodium carbonate (2 g) in 100 mL of 0.1 M sodium hydroxide.
Reagent B (0.5% copper sulfate in 1.0% sodium-potassium tartrate).
Add 0.5 g of copper sulfate and 1.0 g of tartrate to 100 mL of
distilled water.*
Reagent C (alkaline copper tartrate). Mix 50 mL of reagent A and
1 mL of reagent B. Prepare daily.
Reagent E (Folin-Ciocalteu reagent). Dilute the commercial (2 N)
reagent 1:1 with distilled water.
Standard protein assay. Mix 0.2 mL of protein sample (5100 mg){ with
1 mL of reagent C. Incubate at room temperature for 10 minutes.
Now add 0.1 mL of reagent E and mix immediately. After 30

* Copper sulfate and tartrate salts have a low solubility in alkali. Dissolve these separately in a
small volume of water before adding to reagent A.
{ The prescribed reagent volumes lead to a 6.5-fold dilution of protein standards. The
concentration of protein should be 20500 mg mL 1, leading to a ``within cuvette'' concentration
range of 15.477.0 mg mL 1.
The Lowry Method 71

minutes take A750 readings or else A500 readings for strongly

concentrated solutions.
The Folin-Ciocalteu reagent is now widely available. The commercial
reagent is stable for months at room temperature. There seems little
justication for preparing reagent E in house. In the absence of a
commercial supplier, the Folin-Ciocalteu regent may be prepared as follows.
Method 2
Preparation of the Folin-Ciocalteu reagent
Reagents
1. Sodium tungstate Na2 Wo4 ? 2H2 0 100 g
2. Sodium molybdate Na2 MO4 ? 2H2 O 25 g
3. Water 700 mL
4. Phosphoric acid (85% w/w) 50 mL
5. Hydrochloric acid (conc.) 100 mL
Procedure
Add the preceding compounds and some antibumping granules to a
1.5-L ask. Fit a condenser and reux gently for 10 hours in a fume
cupboard. Switch off the gas or electric heater.
Remove the condenser and add (a) 150 g of lithium sulfate and (b)
50 mL of water followed by (c) a few drops of liquid bromine. Bring
the mixture to a boil for 15 minutes to remove excess bromine.
Allow to cool, make up the volume to 1L with distilled water, and
lter. The nished reagent, which should have no green tint, should
be stored in an amber bottle.
Reference 1 is one of the most often cited papers in analytical
biochemistry.* It is one of the scientic papers for which the description
classic is probably deserved. Much of what is known about the Lowry
method was anticipated in the original publication 50 years ago. A number
of investigators have modied the Lowry assay to improve its performance
characteristics. Bensadoun and Weinstein (10) reacted protein samples with
sodium deoxycholate and trichloroacetic acid (TCA) before performing the
Lowry assay. This pretreatment effectively frees protein samples from TCA-
soluble compounds, thereby improving accuracy. When 5 and 50 mg of
protein were precipitated with TCA, the recovery ranged from 7 to 91%.
Pretreatment with DOC led to the quantitative recovery of protein no
matter its initial concentration. With samples containing high concentra-
tions of the interferences, two cycles of precipitation were used. The initial

* As of June 2001 Ref. 1 had 65,535 citations in the Science Citations index.
72 Chapter 3

protein-DOC precipitate was resuspended in distilled water and reprecipi-

tated for a second time. Peterson (2) used DOC-TCA precipitation to
develop an assay, now available in kit form (Sigma-Aldrich Ltd.), that
rendered the classical Lowry method obsolete.*
Method 3
Peterson's modication of the Lowry protein assay (2,8,9)
Reagents
1. Copper sulfate
2. Sodium potassium tartrate
3. Sodium carbonate
4. Sodium dodecyl sulfate (SDS) stock solution (5% w/v)
5. Sodium deoxycholate solution (0.15% w/v)
6. Sodium hydroxide stock solution (0.8 M)
7. Trichloroacetic acid (72% w/v)
8. Folin-Ciocalteu reagent (2 N)
9. Bovine serum albumin (BSA) (0.5 mg mL 1) as protein standard.
Add 1 mg mL 1 sodium azide as preservative and store frozen as
small aliquots.
Procedurey
Copper-tartrate-carbonate (CTC) stock solution. Dissolve copper
sulfate (0.1 g) as well as sodium potassium tartrate (0.2 g) each in
about 10 mL of distilled water. Add both solutions to sodium
carbonate (10 g in 50 mL of distilled water) and make up to a nal
volume of 100 mL.
Preparation of reagent A. Mix CTC, SDS, and sodium hydroxide stock
solutions in a volume ration of 1:2:1. Reagent A is stable at room
temperature for 23 weeks. Refrigeration at 48C will double the
useful life. SDS precipitates in the refrigerator. Redissolve by
holding the reagent bottle under a running hot-water tap.
Preparation of reagent B. Dilute commercial 2 N Folin-Ciocalteu
reagent 1:1 with distilled water. This is stable for many months at
room temperature.
Protein analysis. Add sufcient water to bring the protein sample (5
100 mg) volume to 1 mL. Now, add 0.1 mL of DOC solution,
followed 10 minutes later with 0.1 mL of TCA solution. Centrifuge
the mixture at 10,000 g (mark 12 on a microcentrifuge) for 10
minutes. Decant the supernatant and place the upturned Eppendorf

* Peterson's modication of the Lowry assay had received 6130 citations as of June 2001.
{ It is convenient to employ (1.5-mL) microcentrifuge (Eppendorf) tubes for this procedure.
The Lowry Method 73

tubes over tissue paper to drain. The precipitate of DOC-protein

may just be seen as a gray plaque on the wall of the microcentrifuge
tube.
Add reagent A (1 mL) and mix gently to redissolve the protein
precipitate. After 10 minutes add reagent B and allow to stand for
30 minutes. Take A750 readings using a 1-cm (1.7 mL capacity)
disposable plastic cuvette.
The distinctive features of Peterson's method can be summarized as follows:
1. The increased concentration of copper tartrate increases the
stability of the Lowry reagents from 24 hours to 2 weeks.
2. SDS (1% w/v) reduces interferences from nonionic detergents and
dissolves membrane proteins.
3. Protein precipitation with DOC-TCA eliminates nonprotein
interferences.
4. The precipitation step concentrates samples such that &1m g of
protein can be detected accurately.

3. CHEMISTRY OF THE LOWRY ASSAY

3.1. Reactions of the Lowry Protein Assay
The following account is based on Refs. 1114. The Lowry assay involves
two reactions. First, a protein-Cu2 complex forms. Six peptide bonds
surround a central Cu2 atom. The high-pH solvent (OH &0:1M; pH 13)
induces protein denaturation. Loss of native structure precedes binding with
Cu2 to form a type B protein-Cu2 complex (Chapter 2). Protein
denaturation at high pH also exposes tyrosine and tryptophan residues,
which then ionize.
The second stage of the Lowry assay is a redox reaction with Folin-
Ciocalteu reagent via two pathways. First, Mo6/W6 reacts directly with
amino acid side chains (histidine, cysteine, asparagine, tyrosine, trypto-
phan). A high pH is not required for these reactions. Second, Cu2 mediates
the dehydrogenation of the polypeptide via metal ioncatalyzed oxidation.
The electrons are transferred to Mo6/W6, leading to a color change.
Features of the Mo6/W6 reaction with Cu2 and protein are summarized
in Table 1.

3.2. Metal IonCatalyzed Oxidation of Proteins

The Cu2 catalyzes the oxidative degradation of polypeptides. The process
involves the formation of Cu3 and Cu4 ions. It is possible to produce
74 Chapter 3

TABLE 1 Some Important Reactions for the Lowry Assay

1. Mo6/W6 reacts with reducing agents via a one-electron [e.g., FeCN46 , Fe2,
Sn2] or two-electron transfer (ascorbic acid and peptides). Reactions with
tryptophan and tyrosine may involve four-electrons per residue.
2. Reduction of Mo6/W6 proceeds rapidly under acidic conditions. Adjusting the
pH from 1 to 10 leads to deprotonation followed by a slow structural
rearrangement and 1.7-fold increase in color.
3. Cu2 is not required for the reaction of Mo6/W6 with nonpeptide reductants.
4. The color yield is 3200 (+100) M 1 cm 1 per electron transferred to Mo6/W6.
5. Peptides without oxidizable side chains react with Mo6/W6 only if they can form
a tetradentate peptide-Cu2 complex.
6. Each tetradentate Cu2 complex transfers approximately two reducing equivalents
to Mo6/W6. With well-dened peptides there is a correlation between the color
yield from the biuret and Lowry methods.
7. Color yield increases for polypeptides with oxidizable side chains.
8. Color yield decreases with the number of side chains with Cu2 complexing ability
(glutamate, aspartate).
9. Cu1 is not involved in color formation.
Source: Summarized from Ref. 14.

Cu3 by chemical or electrochemical oxidation.

Cu2 ?Cu3 e; E 0:63V vs: NHE* 1

Metal ioncatalyzed oxidation (MCO) of tetraglycine-Cu2 has been

investigated. One proposal is that in the presence of a strong oxidant
(Qx), e.g., sodium chloroiridate NaIrCl26 , tetraglycine-Cu2 (designated
as RH2 2Cu2) is degraded in three stages: (a) RH22Cu2 is oxidized to
RH22Cu3 [Eq. (2)], (b) RH22Cu3 rearranges to (IIa) or (IIb), which is a
dehydropeptide-Cu1 species [Eq. (3)], (c) compound IIb is hydrolyzed to
diglycinamide, glycoxyglycine, and Cu1 [Eq. (4)]. It has been suggested that
the structure for IIb is probably an iminopeptide (Fig. 1).

Qxn RH22Cu2 I k?
1
Qxn 1
RH22Cu3 II 2

k2
RH22Cu3 II / ? ? R22Cu2 IIa H
3
/ ?R22Cu1 IIb H

* NHE, normal hydrogen electrode.

The Lowry Method 75

FIGURE 1 Suggested mechanism of Cu2-mediated dehydrogenation of peptides

leading to an iminopeptide or a dehydropeptide. (Adapted from Ref.
14.)

R22Cu1 IIb H2 O ?Cu1

4
diglycinamide glycoxyglycine

With molecular oxygen as oxidant, reaction (2) would lead to the superoxide
radical [Eq. (5)], which probably remains protein bound as a ternary
complex (III).

RH22Cu2 I O2 k?
1
2Cu3 II  O2
RH2
5
/ ?O222R22Cu1 III H

Intramolecular oxidation of (III) then generates Cu2-hydroperoxide [Eq.

(6)].

O222R22Cu1 III ?RO2 22Cu2 IV 6

76 Chapter 3

Formation of (IV) occurs via two-electron reduction of oxygen to produce a

protein-bound hydroperoxide anion. Breakdown of the hydroperoxide (Fig.
1) accounts for the carbonyl compounds detectable using 2,4-dinitrophe-
nylhydrazine (DNPH).
A superoxide radical may be involved in the initial oxidation of
RH22Cu2 to RH22Cu3 [Eq. (2)] provided that O2 is released from the
protein in Eq. (5). In summary, RH22Cu2 can be oxidized by strong
oxidants (NaIrCl26 , hydrogen peroxide, and presumably Mo6/W6). The
formation of RH22Cu3 can also be initiated by atmospheric oxygen and
superoxide species O2 and RO222Cu2 . The RH22Cu3 may also form via
the disproportionation of 2 moles of RH2 2Cu2 to produce RH22Cu3 and
1
RH22Cu .
Fragmentation of tetraalanine-Cu2 by IrCl26 produces alanylalanine
amide (HAla2NH2) and pyruvyl alanine (PyrAlaOH). These were identied
by reacting with ninhydrin or DNHP and by analysis with high-voltage
paper electrophoresis. Peptide fragments are also formed during the analysis
of polyalanine by the Lowry method. In one study, the blue molybdate-
tungstate complex was removed by adsorption with cross-linked poly-
acrylamide (Bio-gel P). The eluted (colorless) product reacted with DNPH.
Hydrazone derivatives formed were analyzed by proton nuclear magnetic
resonance (NMR) and by gel ltration on Sephadex G-25. In this manner,
the products of polyalanine MCO were identied as Ala6 or Ala8 peptide
fragments. Tests showed that DNHP reacts with a-iminoisobutyric acid but
not with dehydropeptide analogues such as N-acyldehydroalanine methyl
ester. Therefore, MCO leads to an imino peptide and the dehydropeptide
(Fig. 1). The initial oxidation involves electron abstraction from the lone-
pair electrons on the peptide nitrogen rather than from the methylene group.
According to Livitski et al. (12), the compound II is further oxidized as
shown in Eq. (7). Hydrolysis of the dehydrogenated Cu222R leads to Cu2
[Eq. (8)] and not to Cu1 as shown in Eq. (4).


R22Cu2 II Qxn k?
1
Qxn 1
 R22Cu3 ?R22Cu2 7

R22Cu2 H2 O ?Cu2 diglycinamide glycoxyglycine 8

Evidence for Eq. (8) comes from the deployment of 2.20 -biquinoline
(2,2 DQ), which should form a purple complex with Cu1. Addition of
0

2,20 DQ failed to detect the presence of Cu1. Therefore, the copper ion
probably cycles between oxidation states 2 , 3 , and 4 under strongly
oxidizing conditions. Failure to detect free Cu1 shows that the sequence of
reactions during the Lowry assay is probably Eq. (2)?, Eq. (3)?, Eq. (7)?
The Lowry Method 77

Eq. (8). The net reaction is shown in Eq. (9).

Polypeptide Cu2 Qxn H2 O

?Cu2 Qxn 2
hexapeptide fragments 9

MCO reactions involving proteins are discussed further in Chapter 5.

3.3. Kinetics of the Lowry Protein Assay

The duration of the Lowry assay is 40 minutes. Proteins react with Cu2
(reagent C) in 10 minutes. A further 30 minutes is needed for the reaction
with reagent E before A750 readings are recorded. The speed of the Lowry
assay depends on one or more of the reactions described in Sec. 3.2.
1. The time course for the alkali denaturation of proteins can be
prolonged for stable proteins (e.g., lysozyme).
2. The oxidation of RH22Cu2 by atmospheric oxygen [Eq. (1)]
occurs with the rate constant (k1) of 5.5 6 10 4 (s 1). The time for
99.9% completion (5/k1) is 151 minutes.
3. Oxidation of RH22Cu2 by IrCl6 (and presumably by Mo6/
W6) occurs with a rate close to the diffusion-controlled limit.
Reduction of Mo6/W6 by simple reductants is nearly instanta-
neous, being delayed only by the reagent mixing time.
4. Reduced tungstate-molybdate undergoes slow deprotonation and
structural changes at high pH. At 258C this process takes about 30
minutes.
5. Rearrangement of RH22Cu3 to form a radical species [Eq. (2)] is
a slow process. With IrCl26 as oxidant, k2 is 1.66 6 10 3 (s 1) with
a 99.9% completion time of 36 minutes. Clearly, it is not possible
to identify a single rate-limiting reaction for the Lowry assay of
proteins.
A high reaction rate is essential for automated protein analysis. Anderson
and Marshall (15) and also Huang et al. (16) adapted the Lowry method for
continuous ow analysis. The sample throughput was 30 per hour.

4. CALIBRATION FEATURES

Calibration graphs for the Lowry assay are usually curved. A linear
response is obtained for simple compounds (Sn2, tyrosine, etc.) (14). One
explanation for the nonlinear calibration graphs for proteins is that Mo6/
78 Chapter 3

W6 is degraded at high pH with a half-life of about 8 seconds (1).

Assuming this explanation is correct, the color yield during protein analysis
reects a balance between Mo6/W6 decomposition by alkali and its
reduction to form a blue complex. Inorganic reductants react rapidly with
Mo6/W6 before its degradation. Nonlinearity was also ascribed to the
declining copper/protein ratio as the concentration of protein is increased.
Attempts to improve the linearity by increasing the concentration of copper
led to higher readings for the sample blank.
Readings of A750 may be converted to protein concentration [P] using
a nonlinear graph. This practice emphasizes data points adjacent to the
unknown value. By contrast, a linear graph gives equal weighting to all the
experimental points. In chemical analysis, ``linearity is next to cleanliness.''
Most analysts feel a sense of relief where calibration data conform to a
linear function;
DA750 FP 10

where F is the slope of the calibration graph. The line described by Eq. (10)
passes through the origin and hence
P DA750 =F 11

Stauffer (17) tted Eq. (12) or its logarithmic form [Eq. (13)] to data from
the Lowry assay.

A700 oPF 12

log A700 F logP w 13

where F and o are constants and w log o. For a protein concentration

range of 4400 mg mL 1 a plot of log A700 versus logP was linear. The
unknown protein concentration can be determined from Eq. (14).

P 10Y w=F
14

Notice that Y log A750 is the value for the unknown sample. Coakley
and Jones (18) used reagent volumes three times higher than in the standard
method.* Their calibration results were described by a hyperbolic curve.

* A 0.6-mL portion of protein standard solutions was reacted with 3.0 mL of Lowry reagent C.
After standing for 10 minutes, 0.3 mL of Folin-Ciocalteu regent was added. The A750 readings
were taken 30 minutes later.
The Lowry Method 79

P
A750 15
wP F

Equation (15) applies for within-cuvette BSA concentrations of 0.01

0.77 mg mL 1 and for A750 values of 0.182.2.* The constants F and w were
determined by calibration. The protein concentration can be found using
Eq. (16).

FA750
P 16
1 wA750

For routine use, Eq. (16) can be linearized using a double-reciprocal

transformation.

1 F
w 17
A750 P

Equation (17) allows a two-point calibration: (a) analyze the unknown

sample with two protein standards and (b) determine the constants F and w
from Eqs. (18) and (19).
y1 y2
F 18
x1 x2

where y1 1/A750 (1) and y2 1/A750 (2). Likewise, x1 1/ [P]1 and x2 [P]2.
The concentrations for the protein standards should be [P]1 0.1 mg mL 1
and [P]2 510 mg mL 1 BSA. The intercept of Eq. (17) (w) is found from
averages for y1, y2 and x1, x2.

w y F
x

where

y1 y2 x1 x2
y and x 19
2 2

The previous treatment applies for a protein concentration between 0.1 and
10 mg mL 1. With protein concentrations below 0.2 mg mL 1, a straightfor-
ward calibration graph can be used with little loss of accuracy.

* Allowing for the 6.5 times dilution during the Lowry assay, a protein stock solution of 0.065
5 mg mL 1 leads to a ``within-cuvette'' concentration of 0.010.77 mg mL 1. Investigators are
liable to cite either of these protein concentrations in their work.
80 Chapter 3

TABLE 2 Some Potential Interfering Compounds for the Lowry Assay

Biochemical classication Food additives

1. Amine derivatives Acids and acidulants

2. Amino acids Amino acids
3. Buffers Colors
4. Chelating agents Dyes
5. Detergents (e.g., Triton X-100, Sweeteners
Tween)
6. Drugs Articial antioxidants (BHA, BHT, etc.)
7. Hexosamines Starch
8. Lipids and fatty acids Polysaccharides
9. Miscellaneous compounds Sulfur dioxide, sultes
10. Cryoprotectants Uric acid
11. Polyvinylpyrrolidone Fe2
12. Nucleic acids
13. Organic solvents
14. Phenols and polyphenols
15. Polysaccharides
16. Reducing agents
17. Salts
18. Sugars

5. INTERFERENCE COMPOUNDS

Interference compounds for the Lowry assay are either chelators or reducing
agents. Chelators diminish color formation by sequestering Cu2. Reducing
agents react with the Folin reagent to create extra color. Some interfering
compounds act as both chelators and reducing agents. Dyes having an
absorbance maximum near 600750 nm are also interfering compounds.
Peterson classied interfering compounds into more than 13 classes
comprising over 180 chemicals (Table 2).
Many of these compounds are encountered in raw and processed
foods.* Ascorbic acid and other reducing compounds are found in foods of
plant origin (fruits, juices, pastes, concentrates). Wines and certain
beverages have high concentrations of phenols and related compounds.
Carbohydrates (simple sugarssucrose, glucose, and fructose) and poly-
saccharides (pectin or starch) occur in foods including jams, preserves, and

* However, the analyst may not be aware of the presence of interfering compounds in a given
sample.
The Lowry Method 81

wheat products. Synthetic reductants, acidulants, and dispersants are just

some of the other additives associated with foods. Low-molecular-weight
interfering substances can be removed by dialysis. Another approach is to
recover the protein from the surrounding solvent medium by TCA
precipitation (Method 3). Where possible, sample pretreatment should be
avoided for routine and high-throughput analysis. The effects of selected
interferences on the Lowry assay are discussed in the following.

5.1. Buffers, Chelating Agents, and Detergents

Peters and Fouts (19) showed that BICINE [N,N-bis- (2-hydroxethyl)
glycine] and HEPES [N-(2-hydroxyethylpiperazine-N-2-ethanesulfonic
acid)] produce color in proportion to their concentration. Ethylene diamine
tetraacetic acid (EDTA), tris-hydroxythethylaminomethane (Tris), TRI-
CINE, citrate, and Triton X-100 also produced similar effects (20). These
interfering compounds increase the absorbance value for the reagent blank.
However, they reduce the color yield from proteins. These effects are
attributed to complex formation with copper. Interference by buffer
components can easily be corrected using the appropriate buffer solution
as the reagent blank. Sodium phosphate buffer had no effect on the Lowry
assay.

5.2. Carbohydrates
Reducing sugars (mainly hexoses) react with Cu2 and the Folin reagent.
Nonreducing sugars (sucrose, oligosaccharides, polysaccharides) form
complexes with Cu2. Tagatose, sucrose, and inulin interfered with the
Lowry assay after exposure to hot alkali or acid (21). These solvents are
routinely used to dissolve proteins. As well as reducing Cu2 to Cu1, sugars
reduce tungstate-molybdate. The effects of simple sugars are signicant at
concentrations above 1 mM. The order of effectiveness is fructose >
sorbose > xylose > rhamnose > mannose > glucose (22).
Toldra (23) considered protein analysis in connection with the
production of high-fructose syrup. Effective control of this process requires
the determination of a-amylase and glucoamylase specic activity (hence
protein concentration) in samples containing up to 3040% sugar. A
solution of 1% (w/v) glucose or maltose produced an A750 response
equivalent to that of 18.8 or 16.6 mg mL 1 BSA. Protein analysis is also
necessary to determine the activity of fungal pectinases produced in
submerged culture using high concentrations of pectin as inducer (24).
Concentration of 0.11.0% (w/v) pectin interfered with both the classical
and modied Lowry assays. Calibration graphs showed a decrease in the
82 Chapter 3

slope and increased intercept. Assay sensitivity decreased while the LLD
increased with increasing pectin concentration (Chapter 1). The color with
protein-free samples increased linearly with the amount of added pectin.
These results imply complex formation between pectin and Cu2. The
Bradford method (Chapters 6 and 7) coped better with samples containing
< 0.5% (w/v) pectin. In our experience, pectin forms a gelatinous mass
during the Lowry assay. To avoid this problem, samples are exposed 0.1 M
CaCl2 and then centrifuged before protein analysis. Berg (25) warned that
``anyone attempting to determine protein contamination in polysaccharide
preparations by the Lowry procedure could be misled by positive results
which may be attributed to the carbohydrate.''

5.3. Chlorophyll
Leaves, stems, and green fruit contain chlorophyll. Addition of chlorophyll
(100 mg mL 1) to BSA (300 mg mL 1) increased A750 values by 400% (26).
For accurate analysis, leaf protein must be separated from chlorophyll.*

5.4. Cryoprotectants, Sucrose, Glycerol, and

Polyhydroxyalcohols
Ethylene glycol, polyvinylpyrrolidone (PVP), and dimethyl sulfoxide
(DMSO) are used to protect proteins against freeze damage. Glycerol,
probably acting in a manner similar to sucrose, inhibits color formation. In
the absence of copper, PVP reacts with Mo6/W6, leading to a blue
coloration (27). Glycerol (25%) interferes with the Lowry procedure (28)
but DMSO has no effect.
Many foods and condiments contain very high concentrations of
sucrose. Sucrose is also used for gradient ultracentrifugation. Sucrose by
itself gives a positive response with the Lowry assay (29). However, protein
analysis in the presence of sucrose leads to a reduced color yield. The
interference becomes progressively worse with 065% sucrose. Effects can be
reduced to tolerable levels (< 5% analytical error) by (a) diluting samples to
< 10% sucrose or (b) doubling the concentration of copper in Lowry reagent
B. Quadrupling the copper concentration led to an unstable reagent with a
tendency to form colloidal copper (30).

* Leaf protein can be extracted with 0.1 N alkali. Next, precipitate the protein with 10% (w/v)
TCA. A second approach is to treat freeze-dried leaf powder with acetone. This decolorized
powder can be stored before analysis (this work is discussed in detail later).
The Lowry Method 83

Sucrose (and other polyhydroxy compounds) appear to complex with

Cu2 when monitored by ultraviolet absorbance spectrophotometry. In the
absence of copper, sucrose or glycerol does not interfere with the tyrosine
reaction with Folin-Ciocalteu reagent. Hydrolysis of sucrose by heat, alkali,
or invertase generates fructose and glucose, which affect the Lowry assay as
reducing sugars (31). Ficoll (synthetic polymer of sucrose) yields an intense
blue color when analyzed by the Lowry procedure (32) (Fig. 2).
Concentrations of 236% (w/v) coll are used for density gradient
centrifugation of cell fractions. Because of its high average molecular
mass (*400 kDa), it is not removed from samples by dialysis. Addition of
the Folin-Ciocalteu reagent to coll leads to formation of color with a time
course of over 18 hours at room temperature. Ficoll had no effect on the
yield of color when added to protein samples after the Folin-Ciocalteu
reagent. In the presence of xed concentrations (0.211.88%) of coll, the
calibration graph of standard BSA solutions had progressively lower slopes
while the intercept increased. Ficoll forms a UV-detectable complex with
copper.

FIGURE 2 Effect of increasing coll concentrations on the Lowry assay. Boxed

legend shows Ficoll concentrations. (Drawn from data from Ref. 32.)
84 Chapter 3

5.5. Lipids
Lipids increase sample turbidity and A750 values. The products of lipid
autoxidation also react directly with the Folin reagent. Heating lipids with
alkali (a common method of protein solubilization) leads to Lowry-positive
products. The problem has been examined with arachidonic acid,
phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol
(33). Samples may be delipidated by shaking with chloroform or petroleum
ether and then redissolved with 0.5 M NaOH by storing at 378C overnight
or by heating at 1008C for 30 minutes. Solvent extraction will not be wholly
successful if the protein and proteolipid complexes dissolve within an
organic solvent phase. Emulsion formation can also lead to apparent loss of
protein from the sample.
Lees and Paxman (34) modied the Lowry assay for proteolipids and
lipoproteins. Their method is fast and does not require heating.* Markwell
et al. (35) added 1% SDS directly to Lowry reagent C to dissolve membranes
and other lipid. The concentration of copper sulfate was also increased from
0.5 to 4% (w/w) to reduce interferences from sucrose and EDTA. The
modied method gave results identical to those obtained with lipoprotein
samples delipidated using petroleum ether. However, the new method
allowed higher rates of analysis and greater convenience. There was no
interference from 40200 mM sucrose. SDS also features in Method 3,
which is therefore able to deal with membrane lipids routinely. According to
Kirazov et al. (36), treating membrane-containing fractions with 1 M NaOH
or 0.5% SDS reduced the stability of A750 readings and did not reduce the
interference from lipids.

5.6. Sulfhydryl Agents and Other Reducing Compounds

The effects of various SH compounds are also related to their standard
electrode potential (E8) measured against a normal hydrogen electrode
(NHE) or a calomel electrode. At concentrations below 1 M, the redox
potential is described by the Nernst equation:
RT
E E ln C 20
nF
where n is the number of electrons transferred, E the observed redox
potential corresponding to an activity of C (mol L 1), F the Faraday

* Treat protein samples (2070 mg) with 0.5 mL of alkaline SDS solution (5% SDS, 0.5 M NaOH),
vortex, and let stand at room temperature for 3 hours. Add 2.5 mL of Lowry reagent C followed
10 minutes later with 250 mL of Folin (1 N) reagent. Take A695 reading after 45 minutes.
The Lowry Method 85

constant, and R the gas constant. For dilute solutions, C becomes equal to
concentration. Therefore, the effect of reducing compounds on the Lowry
assay is related to the ``intrinsic'' redox properties (E8 and n) and the
concentration of reducing agent.
Dithiothreitol (DTT), 2-mercaptoethanol (2ME), reduced glutathione
(GSH), and oxidized gluthathione (GSSG) produce color with the Lowry
assay (37). Reducing compounds react directly with the Folin-Ciocalteu
reagent. Table 3 shows the degree of color formation with some SH
compounds. As the underlying reaction is a redox process, calibration
graphs for SH compounds will be nonlinear. For each SH compound De
(M 1 cm 1) was determined from the steepest slope in Fig. 3. Values for De
determined in this manner (Table 3, top half) agree with results from Ref.
14. DTT is one of the most effective interfering SH compounds. Samples
with > 0.2 mM DTT have high A750 high background absorbances.

TABLE 3 The Color Yield from the Lowry Assay of Some Reducing Compounds
1
Reducing agent
" (M cm 1)a nb E1/2(mV)c

Dithiothreitol 7625 (176) 2

2-Mecaptoethanol 3900 1
Glutathione 3333 1
(reduced)
Cysteine 3400 1 0.398
Glutathione 1870 (760) 0.5
(oxidized)
Cystine 1702 (822) 0.5

Cysteine 3150 1
Fe2 3150 1
Sn2 6100 2
Ascorbic acid 6700 2
Phenol 12400 4
Tyrosine 12800 2 (4) 0.398
Indole 13000 2 (4) 0.253
Tryptophan 13200 2 (2) 0.205
a
The De (M 1 cm 1) values in the top half of Table 3 are calculated from the maximum slope in
Fig. 3. Data in the bottom half of Table 3 from Ref. 14.
b
n electrons transferred to Mo6/W6 from one molecule of reducing compound. Values for n
were determined by colorimetry,c Half-wave electrode potentials were determined by cyclic
voltammetry, using standard calomel electrode (i.e., 0.242 V vs. NHE), from Ref. 78. Note
that for dilute solutions E1/2 E8 Eref.
86 Chapter 3

FIGURE 3 The Lowry assay of selected SH compounds. Concentrations of

interference compounds were analyzed in the absence of protein.
(Numerical data from Ref. 37.)

Reduction of the Mo6/W6 produces an absorbance change of 3200 units

(M 1 cm 1) per mole of electrons transferred (see Table 1).

6. SAMPLE PRETREATMENT, AVOIDING

INTERFERENCES, AND ENSURING ACCURACY

Dialysis provides a simple test for the presence of low-molecular-weight

interferences. A sample is judged free of interferences when all attempts to
remove such compounds meet with no success.

6.1. General Strategies

A number of general strategies are available for dealing with interferences:
1. Use an appropriate reagent blank. Where the identity and
concentration of interfering compound are known, a reagent
blank can be prepared. Absorbance readings for the blank are
then subtracted from the results for the sample.
The Lowry Method 87

2. Prepare a reagent blank by analyzing a deproteinized sample.

3. Mask the interfering component using chemical additives. Excess
copper sulfate will compensate for the effect of chelators.
4. Remove the interference. Lipids can be removed by defatting with
an organic solvent. Pectin is removed by treating the sample with
calcium chloride followed by centrifugation. Dialyze to remove
low-molecular-weight interferences.
5. Precipitate the protein from the sample matrix before analysis
(Method 3).

6.2. Destruction of SH Compounds

The effect of SH compounds on the Lowry assay can be annulled by (a)
oxidation with hydrogen peroxide (38), (b) carboxymethylation with
iodoacetate (39), or (c) treatment with chloramine-T or CAT (40). The
oxidation of SH groups by hydrogen peroxide in the presence of Cu2
involves the hydroxyl radical  OHsee Chapter 2. Carboxymethylation
recties analytical error due to  150 mM SH compounds. However,
another popular SH-blocking agent, N-ethylmaleimide (NEM), was not an
effective substitute for iodoacetate. NEM produced coloration with the
Lowry assay.
Interferences due to SH compounds were greatly reduced by oxidation
with CAT. For instance, samples containing 20 mM Cys, 2ME, GSH, or
DTT gave A750 readings above 2. When treated with CAT, the same samples
gave absorbances of 0.113, 0.032, 0.530, and 0.308. The unusually high
reading from GSH was attributed to the peptide nature of this SH
compound. Pretreatment with CAT and TCA also reduced inferences by
KCN, ascorbic acid, NaHSO3, Na2SO3, FeSO4, GSSG, and Cys-Cys.
However, the sensitivity of the Lowry assay was reduced by about 50%. This
effect is probably due to the oxidation of amino acid side chains (tyrosine,
tryptophan, cysteine) by CAT, rendering them unavailable to react with
molybdate-tungstate. Another consequence of the CAT procedure is that
the resulting calibration curves are linear. Higuchi and Yoshida (41) found
that using CAT under neutral conditions maintains analytical sensitivity at
the level obtained with the classical Lowry procedure.

7. APPLICATIONS OF LOWRY ASSAYS TO FOOD PROTEIN

ANALYSIS

Table 4 lists examples of food protein analysis using Lowry procedure.

Selected cases are described in detail next.
88 Chapter 3

TABLE 4 Some Food Commodities and Processes Analyzed by the Lowry

Procedure

Commodity Reference

Beer/brewing Hii and Herwig (42), Williams et al. (43)

Cereal or legume our Padhye and Salunke (44), Sathe and Salunkhe
(45), Sebecic et al. (46), Paredes-Lopez et al.
(47), Seguchi (48,49)
Leaf proteins Eze and Dumbroff (25)
Milk proteins Huang et al. (50), Kroening et al. (51)
Various condiments Ogunbunmi and Bassir (52)
Potato protein Hoff (53), Vigue and Li (54)
Tomato seed proteins Latlief and Knorr (55)
Yeast, microbes, fermentation Vananuvat and Kinsella (56), Gierhart and Potter
(57), Abramov et al. (58), Kovar et al. (59),
Oliveira et al. (60)
Emulsions Galluzzo and Regenstein (61), Tran and Einerson
(62)
Proteolysis Juffs (63), Ory and Sekul (64), Kwan et al. (65),
Siddiqui et al. (66), Ramana-Murthy et al. (67)

7.1. Cereal, Legume, and Flours

Sathe and Salunkhe (45) extracted proteins from the great northern bean
(Phaseolus vulgaris L) before assaying by the Lowry method.* Results were
standardized against the Kjeldahl method (%N 6 6.25). Whole bean our
had 26.1% (w/w) protein (per dry weight) or 22.17% (w/w) crude protein.
About 21.8% of the total protein was water soluble (albumin). Another
73.4% could be extracted with dilute salt solutions (globulin). Table 5 shows
virtually quantitative recovery of bean protein using solutions of sodium
carbonate (0.5%), potassium sulfate (5%), SDS (5%), or sodium hydroxide
(0.02 N or 1 N). Sodium phosphate buffer was not an efcient extractant for
bean our protein. Urea and SDS, also commonly used as protein-
solubilizing agents, did not perform so well.
The extent of protein recovery by extraction is usually uncertain.
Depending on the solvent, different protein fractions (albumin, globulins,
prolamins, etc.) are extracted to different extents. The direct addition of
Lowry reagent C to our samples should be investigated. This approach

* Suspend whole bean our (1 g) in 25 mL of solvent with thorough mixing. Let stand for 12
hours and then centrifuge (5000g) for 45 minutes. Analyze for protein content using Method 1.
The Lowry Method 89

TABLE 5 Analysis of Bean Flour Protein Extracts Using the Lowry Assay

Solventa pH Total protein (%)

Sodium chloride 6.2 61.9

Sodium sulfate 6.3 65.3
Sodium acetate 6.5 52.9
sodium carbonate 10.0 93.4
Sodium dihydrogen phosphate 6.75 55.2
Sodium phosphate 5.0 38.3
Potassium chloride 6.7 57.4
Potassium sulfate 5.9 75.9 (100)
Urea 7.2 69.8 (100)
SDS 7.1 60.2 (100)
HCl 1.5 75.6
Dimethylformamide 6.0 41.6
a
All salts were used at concentrations of 0.5% (w/v) aqueous solution. Values in parentheses are
% extraction using 5% (w/v) solutions.
Source: Adapted from Ref. 45.

worked well for biuret analysis of our (Chapter 2). After centrifugation,
Lowry analysis could then be completed by adding Folin-Ciocalteu reagent.
Sebecic (46) considered the Lowry procedure as a ``new'' method for
wheat protein determination. Forty-ve varieties of Yugoslav wheat were
analyzed.* The Lowry and Kjeldahl methods were highly correlated. The
sensitivity of these methods was 100-fold higher than that of the biuret
assay. Sebecic concluded that the Lowry procedure was a good substitute
for the Kjeldahl or Kjel-Foss method. It was certainly faster, simpler, less
expensive, and easier to perform than the Kjeldahl method.
Seguchi (49) analyzed starch granule surface proteins after extraction
with 1% SDS overnight. Although present in very small amounts
(0.06% w/w), the specically bound protein may have important effects on
the functional properties of wheat our. Treating wheat our at high
temperatures (e.g., 1008C for 40 hours or 608C for 24 days) or storing at
room temperature for long periods (130 days) caused greater than a 300%
increase in wheat starch granule surface proteins.

* Wheat our (0.4 g) was suspended in 50 mL of distilled water and heated at 958C for 2 minutes
to gelatinize starch. The mixture was cooled and brought to a total volume of 100 mL. To the
prediluted samples (with 417 mg mL 1 protein) were added 10 mL of Lowry reagent C. Folin-
Ciocalteu reagent (1 mL) was added after 30 minutes and A540 readings recorded 30 minutes
later.
90 Chapter 3

7.2. Leaf Proteins and Protein from Other Chlorophyllous

Tissue
Adding 100 mg mL 1 chlorophyll to model BSA solutions (300 mg mL 1)
increased the Lowry assay response by 400%. The interference from
chlorophyll can be avoided by (a) precipitating the solubilized plant
proteins with TCA, (b) decoloring the homogenized leaf extract by
acetone treatment, or (c) extracting the freeze-dried leaf powder with
acetone (25). The last strategy is most attractive.* Attempts to decolorize
the leaf homogenate led to signicant losses of protein. The preceding
method has general applicability. Most chlorophyllous tissue could be
treated in this fashion: green fruits, stems, shoots, and green aquatic
plants. Algae are another potentially rich source (3070% dry weight) of
crude protein (68).

7.3. Emulsions and Nondairy Creamers

Protein stabilized emulsions are centrifuged to separate the cream (oil-rich)
and aqueous phases. The aqueous layer is then analyzed for protein content.
Tran and Einerson (62) used the Lowry assay to assess the stability of
nondairy creamers. Emulsion samples were diluted (0.2% w/w) with distilled
water to which salts had been added to simulate hardened water.{ The
aqueous phase protein concentration, following emulsication, gives an
indication of protein emulsifying capacity. Galluzzo and Regenstein (61)
dened emulsifying capacity as the amount of oil emulsied per mg of
protein. The order of emulsifying capacity for muscle proteins was
myosin > actomyosin ( ATP added) > actomyosin > actin.

7.4. Monitoring Proteolysis and Digestion

Anson's (69) method for monitoring proteolysis is straightforward. Adjust
the sample to 10% nal concentration of TCA and centrifuge or lter. Take
A280 measurements for the TCA-soluble supernatant and a quartz cuvette.
The assay sensitivity is increased 10-fold by reacting with Folin-Ciocalteu

* Shake freeze-dried or ground leaf tissue (50 mg) with 15 mL of acetone (62). Filter and store
the decolorized powder for analysis. Extract protein from the decolorized leaf powder and
analyze as usual.
{ Emulsions were centrifuged and to the aqueous phase (0.5 mL) was added 0.5 mL of SDS
(10% w/w) as a dispersant. Lowry reagent C (5 mL) was added followed, 10 minutes later, with
Folin-Ciocalteu reagent (1 mL). Absorbance readings were taken some 30 minutes later. Turbid
samples were claried by one or more freeze-thaw cycles before spectrometric measurements.
The Lowry Method 91

reagent.* Hull (70) used this approach to monitor proteolysis in milk. The
results were expressed as tyrosine equivalents by referring to a calibration
graph for tyrosine. Juffs (63) found that tyrosine equivalents were
signicantly correlated with milk bacterial count, age of the cow, period
of lactation, and milk yield. There were variations in tyrosine equivalence
for normal milk, and it was not possible to set a value for proteolyzed milk
samples.
The concentration of phosphotyrosine residues can also affect milk
tyrosine values. Phosphotyrosine does not react with the Folin-Ciocalteu
reagent (71). The total tyrosine value was determined after treating samples
with added alkaline phosphates. It is therefore suggested that the Folic-
Ciocalteu phenol reagent be used as basis for assaying phosphotyrosine
phosphatase. Finally, milk proteolysis was monitored using the Lowry,
uorescamine, or trinitrobenzene sulfonate method or by A280 readings (65).
The Lowry and Anson methods were highly correlated (R 0.99). Although
10-times more sensitive, the Lowry assay showed less agreement with
techniques that monitor free amino groups. Ory and Sekul (64) recom-
mended that milk proteolysis should be routinely measured with quantita-
tive SDSpolyacrylamide gel electropho (SDS-PAGE) as well as
spectrophotometry.
Gut uid is a challenging milieu for protein analysis. Crossman et al.
(72) compared the modied Lowry assay and other techniques (quantitative
amino acid analysis,{ BCA, and Bradford method) for monitoring protein
levels in the gut uid from the marine herbivorous sh Kyphosus sydneyanus.
The Lowry and BCA assays gave similar results. All spectrophotometric
methods showed a low correlation with quantitative amino acid analysis.
The results were also dependent on the mode of sample collection and
pretreatment. In was important to freeze sh digesta immediately after
collection. The sample could be thawed and gut uid removed closer to the
time for protein analysis. The Lowry assay was also applied in the study of
dietary protein effects on sulde production by bacteria in the human large
intestines (73).

7.5. Yeast and Other Single-Cell Proteins

Single-cell protein (SCP) is produced by growing microorganisms on a range
of substrates including hydrocarbons and sweet whey (68,74,75). Growth

* Lowry reagent C is not required.

{ Quantitative amino acid analysis is described in Chapter 1. Descriptions of the BCA and
Bradford methods appear in Chapters 4 and 5.
92 Chapter 3

media contain a range of interferences for protein assay. Two examples will
illustrate the protein analysis issues. Vananuvat and Kinsella (56) grew
Saccharomyces fragilis on spray-dried crude lactose (2% w/w) supplemented
with ammonium sulfate (0.5%), peptone (0.5%), yeast extract (0.5%), and
urea (0.03%) as nitrogen source. Microbial cell numbers and protein and
nucleic acid concentrations were monitored during the fermentation.
Protein concentrations from the Lowry method were always lower than
Kjeldahl protein (56). Sample pretreatment to remove low-molecular-weight
substances is recommended. Given the complexity of growth media,
quantitative SDS-PAGE is probably a good idea for protein quantitation.
Orban et al. (76) examined the production of yeast autolysates from
Kluyveromyces fragilis grown on pasteurized whey (2%) supplemented with
ammonium sulfate (0.4% w/v), potassium dihydrogen phosphate (0.2% w/v),
and yeast extract. The cells were grown for about 8 hours in a batch
fermenter and harvested by centrifugation. Autolysis was initiated by
suspending freeze-dried cells (10% w/v) in dilute saline (5% w/v NaCl)
solution. Sample results are given in Table 6. Determinations of SCP are
usually performed after washing the wet cells free from growth medium. By
washing the cells thoroughly, interferences from culture medium compo-
nents (sugars, peptides, and proteins) can be avoided. However, Lowry and
Kjeldahl results were consistently different because of interferences by the
high levels of ribonucleic acid found in yeast cells.

TABLE 6 Determination of Protein and Other Components in Kluyveromyces

fragilis cells before and after Autolysis

Dried yeast Autolysatea

Parameter (% dw) (% dw)

Kjeldhal protein (N 6 6.25) 49.7 37.3

Lowry protein 40.4 19.7
TCA-soluble nitrogen 2.0b 5.8b
Amino nitrogen 2.0 3.9
RNA 7.7 9.0
Carbohydrate 41.0 17.0
Moisture 2.2 2.92
a
Optimal conditions of autolysis10% yeast cells are suspended in 5% sodium chloride
and incubated at 548C for 8 hours.
b
Determined from Kjeldhal nitrogen analysis of material soluble in 20% TCA.
Source: Data from Ref. 76.
The Lowry Method 93

TABLE 7 Improved Lowry Methods for Collagen Analysis

Sensitivity
(
A562/ Linear Color Sample
Method g 1) LLDa (g) range (g) stabilityb volume (L)

Modied 0.0033 4 4100 0.85 200

Enhanced 0.0085 3 380 0.85 200
a
LLD, lower limit of detection.
b
Color stability % nal absorbance change in 10 minutes.

7.6. Collagen
The sensitivity of the Lowry assay to collagen is low (0.1 A750 mL mg 1)
because of the low content of aromatic (oxidizable) amino acids (*1%).
Copper binding is also inhibited by the high (hydroxyl) proline content
(> 25%) and the triple-helix structure of collagen, which is apparently stable
in alkaline media. Komsa-Penkova et al. (77) described two procedures for
improving the Lowry assay sensitivity for collagen. In the ``standard
modication'' protocol, samples of collagen (200 mL) are heated with 180 mL
of Lowry reagent A (see Method 1) at 508C for 20 minutes. Reagent B is
added and the mixture is allowed to react at room temperature for 10
minutes. Finally, Folin-Ciocalteu reagent (600 mL) is added and the mixture
heated at 508C for another 10 minutes before taking absorbance readings at
562 nm. With the ``enhanced protocol,'' collagen is reacted with Lowry
reagent C at 508C for 20 minutes, cooled to room temperature, and then
Folin-Ciocalteu reagent is added. Absorbance readings are taken 10 minutes
later. Table 7 shows the performance characteristics of the proposed Lowry
methods for collagen.
The relative response to type I, type II, and type IV collagen was the
same and 50% lower than the response obtained with type V collagen. The
modied methods were 10- to 20-fold more sensitivity than Method 1 for
collagen analysis. The sensitivity increase is due to the denaturation of
collagen by thermal treatment. Gelatin can be analyzed with the normal
Lowry method at room temperature.

REFERENCES

1. OH Lowry, NJ Rosebrough, AL Farr, RJ Randall. Protein measurement with

the Folin phenol reagent. J Biol Chem 193:265273, 1951.
94 Chapter 3

2. GL Peterson. A simplication of the protein assay method of Lowry et al.

which is more generally applicable. Anal Biochem 83:346356, 1977.
3. H Wu. Contribution to the chemistry of phosphomolybdic acid, phospho-
tungstic acids and allied substances. J Biol Chem 43:189220, 1920.
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The Lowry Method 95

22. J O'Sullivan, GE Mathieson. Interference by monosaccharides with the

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26. JMO Eze, ED Dumbroff. A comparison of the Bradford and Lowry methods
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38. PJ Geiger, SP Bessman. Protein determination by the Lowry method in the
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39. E Ross, G Schatz. Assay of protein in the presence of high concentrations of
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40. M Higuchi, F Yoshida. Lowry determination of protein in the presence of
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98 Chapter 3

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4
The Bicinchoninic Acid Protein Assay

1. INTRODUCTION

The bicinchoninic acid (BCA) protein assay was developed by Smith et al.
(1). The plan was to substitute BCA for the Folin-Ciocalteu reagent in the
Lowry assay. The advantages of the BCA method include decreased
sensitivity to interferences, a need for one working reagent, and color
stability. The BCA protein assay has the same sensitivity as the Lowry
method. Reagents for the BCA assay are available commercially. As yet,
the BCA assay does not feature greatly in the food science literature. As
of June 2001, there were 16 references to bicinchoninic acid or
bicinchoninate in the Food Science and Technology abstracts. General
citations in the Science Citation Index number over 8500. The BCA assay
is without a doubt more popular than indicated by the low number of
formal citations.
The characteristics of the BCA protein assay are reviewed in this
chapter. Section 1 is an account of the history of BCA reagent and its use for
chemical analysis. In Sec. 2, the basic BCA procedure is presented, followed,
in Sec. 3, by a discussion of the chemistry underlying color formation. In
Secs. 46 are descriptions of calibration features, interfering compounds,
and sample pretreatment strategies for avoiding error. After a discussion of

99
100 Chapter 4

automated formats (Sec. 7), we turn to application of the BCA assay to food
protein analysis in Sec. 8.

1.1. Determination of Copper Using BCA

BCA is the trivial name for 2,20 -diquinolyl-4,40 -dicarboxylic acid. Early
applications include the analysis of Cu2 in metal alloys and blood sugars.
Hoste (2) evaluated 2,20 -diquinolyl (2,20 DQ) and nine related heterocyclic
compounds for Cu2 analysis after reducing Cu2 to Cu1 with
hydroxylamine hydrochloride. He found that 2,20 DQ forms a 2:1 complex
with Cu1 (Fig. 1) having maximum absorbance (lmax) at 540 nm and a
molar extinction coefcient (De540) of 5490 M 1 cm 1. To determine Cu1,
the working solution of 2,20 DQ (0.02% w/v in ethanol, 10 mL) was added to
3 mL of sample. The mixture was diluted with 25 mL of ethanol and A540
measurements were recorded. The Cu1 was quantitatively determined in
the presence of Cu2, with which there is no reaction.
Kertesz (3) employed 2,20 DQ (0.05% w/v in glacial acetic acid) for the
analysis of copper at the active site of the enzyme tyrosinase. To calibrate
the assay, 2 mL of 2,20 DQ was added to enzyme-free samples containing

FIGURE 1 Diagram of the 2:1 complex formed between 2,20 -diquinolyl (R H) or

2,20 -bicinchoninic acid (R COOH) and Cu1
The Bicinchoninic Acid Protein Assay 101

< 11.5 mg of Cu1 in 2 mL of phosphate buffer (0.05 M, pH 6.8). First, Cu2

was reacted with hydroxylamine hydrochloride to produce Cu1. Upon the
addition of 2,20 DQ, a purple complex formed in 510 minutes at room
temperature. There was no reaction with Cu2, although preincubating
Cu2 with BSA or conalbumin led to reactivity with 2,20 -DQ. Initial studies
using 2,20 DQ suggested that tyrosinase had a Cu1 atom at the active site.
Fesenfeld (4) also investigated the reaction of tyrosinase with 2,20 DQ. He
proposed that in the absence of substrate mushroom tyrosinase had an
active site Cu2 species and that this might easily be reduced to Cu1 by a
protein SH group.
Colorimetric analysis of Cu1using BCA was studied by Gershuns et
al. (5). They showed that Cu1 forms a complex with BCA and that this was
insoluble at pH < 4. At high pH the red-violet complex had a lmax value of
560 nm. The linear range for Cu1 analysis was 1100 mg mL 1. The
analytical precision was 1%. There were no interferences from common
metal ions when present at 612 6 103-fold mole excess. The Cu1 (BCA)2
complex was reportedly stable for a few hours at pH 412, although the
optimal pH for Cu1 analysis was pH 6. As with previous investigations,
Cu2 was analyzed after reduction using hydroxylamine hydrochloride.
Nakano (6) also employed BCA for the analysis of Cu2. In a long-
running study, the characteristics of several 4,40 -substituted DQ derivatives
were investigated. As shown in Table 1, BCA was the second most
colorigenic derivative. Values in Table 1 are with water or isoamyl alcohol
as solvent. The ratio of BCA to Cu1 found in the complex was conrmed as
2:1. The Cu1 (BCA)2 complex had a lmax value of 565 nm and De565 equal

TABLE 1 Properties of Cu1 Complexes with 2,20 DQ derivativesa

1
4,40 Substituent (R) max (nm)
" (M cm 1)

2COOHb
2 560565 79208000
2
2CONHEt 565 3100
2
2CONHCHMe2 566 7100
2
2CONMe2 560 6130
2
2CONEt2 561 6200
2CON (CHMe2)2
2 556 28100
2
2CONBt2 561 5150
2
2COOEt 576 6600
2
2COOBt 576 6020
a
Values are with water or isoamyl alcohol as solvent.
b
R COOH for bicinchoninic acid (BCA).
Source: Compiled from Ref. 6.
102 Chapter 4

to 8000 M 1 cm 1. The color produced from BCA and Cu1 is reportedly

stable for 48 hours at temperatures < 658C and at pH 3 to 13. The linear
range for Cu1 analysis was 0.0220 mg mL 1.
Reports of the analysis of Cu2 using BCA were produced by
Mustan et al. (7) and Tikhonov (8). Noskova (9) used BCA for the
photometric determination of copper in blood serum. The dissociation
constant (Kd) for the Cu1 (BCA)2 complex was estimated as 1 6 10 11 M at
208C by Buhl et al. (10). Capitan et al. (11) developed a solid-phase analysis
of copper in natural water. The microdetermination of copper involved the
reduction of Cu2 to Cu1, reaction with BCA, and adsorption of the
resulting complex with a dextran cation exchange resin. Absorbance
measurements were recorded directly using the resin phase. Copper was
determined at concentrations of 120 6 10 9 g L 1. The relative standard
deviation for analysis was 1.7%. The typical sample size was 2 L.
Brenner and Harris (12) determined serum copper levels using BCA.
Blood plasma (0.75 mL) was deproteinized by treating with 0.25 mL of TCA
(30% w/w). After microcentrifugation, 0.5 mL of deproteinized serum was
added to 0.1 mL of ascorbic acid (352 mg% w/w) to reduce Cu2 to Cu1.
Then 0.4 mL of buffered BCA reagent was added and absorbance readings
were measured. For calibration, standard amounts of copper were analyzed
using 0.1 M sodium phosphate buffer as solvent.
A second lmax value was reported for the Cu1 (BCA)2 complex at
354.5 nm (13). At this wavelength the sensitivity toward copper is six to
seven times greater (De354.5 4.6 6 104 M 1 cm 1) than the sensitivity
562 nm (De562 7.7 6 103 M 1 cm 1). For a system containing 50 mM
BCA, the linear dynamic range for copper detection was 225 mM. The
upper limit of detection is consistent with the 2:1 stoicheometry for BCA
binding to Cu1. The pH stability characteristics of the Cu1 (BCA)2
complex can be seen from Table 2. These results show that Cu1 (BCA)2 is

TABLE 2 The pH Stability of the Cu1 (BCA)2 Complex

Solution pH
"354.5 /104 (M 1

cm 1)

4.5 3.95 (0.1)

7.0 4.58 (0.1)
9.5 4.43 (0.2)
10.5 4.73 (0.2)
12.5 3.88 (0.2)
Source: Adapted from Ref. 12.
The Bicinchoninic Acid Protein Assay 103

stable at pH 313; a similar conclusion is supported by absorbance

measurements at 560565 nm. At the time of writing, no protein assays have
been conducted at 354.5 nm.

1.2. Analysis of Sugars Using BCA

Between 1971 and 1973, researchers afliated with Pierce-Warner Chemical
Company used BCA for sugar analysis. An early iteration of the BCA
reagent developed by Grindler (14) and Mopper and Grindler (15) was
prepared from two stock solutions. Reagent A comprised BCA (170 g) and
sodium carbonate (27 g) dissolved in 500 mL of distilled water. Reagent B
contained copper sulfate (1 g) and aspartic acid (2.55 g) dissolved in 500 mL
of distilled water. A working BCA solution was prepared by mixing equal
volumes of reagents A and B. The analysis of sugars was performed in a ow
system using a reaction coil at 808C. The reaction time was 25 minutes and
absorbance readings were recorded at 562 nm.
McFeeters (13) described a manual BCA assay for sugars. Reagent A
comprised 0.085% (w/v) BCA in 1.0 M phosphate buffer (pH 8.5). Reagent
B contained 25 g of aspartic acid and 33.4 g of sodium carbonate
predissolved in 500 mL of water to which copper sulfate (1.34%, 500 mL)
was then added. A working BCA reagent was prepared from 23 parts of
reagent A and 1 part of reagent B. In a typical analysis, 3 mL of working
BCA reagent was added to a 1-mL solution of sugar. The mixture was
heated in a boiling-water bath for 10 minutes. After allowing the samples to
cool, absorbance readings were taken at 560 nm. The following sugars were
assayed: galacturonic acid, glucuronic acid, glucose, galactose, fructose,
mannose, xylose, maltose, melibiose, and cellobiose. The average color yield
was 1.9 (+1.0) DA560 per mmole. The utmost color yield was obtained with
fructose (10.76 DA560 per mmole). For a within-cuvette fructose concentra-
tion of 120 6 10 9 mole, DA560 was 1.29.

2. THE BCA PROTEIN ASSAY

As described earlier, the BCA protein assay was developed by a 10-member

team (1) from the Biochemical Research Division, Pierce Chemical
Company, Rockford, Illinois.* Their objective was to nd an alternative
to the Folin-Ciocalteu reagent for detecting Cu1, which is formed when
Cu2 is reduced by proteins. Actually, the BCA assay may be a totally new

* In Europe, Pierce and its sister company HyClone trade under the name Perbio Science.
104 Chapter 4

concept in protein analysis. The underlying reactions are signicantly

different from those for the Lowry assay. The distinctiveness of the BCA-
protein reaction probably accounts for the greater resistance to certain
interferences. BCA reagent is prepared from two stock reagents. Reagent A
and B are then mixed in a ratio of 50:1 to produce the working BCA
solution.

Method 1
The bicinchoninic acid protein assay (1).

Reagents
1. Reagent A (alkaline BCA reagent). Add BCA (10 g), sodium
carbonate (20 g), sodium tartrate (1.6 g), sodium hydroxide (4 g),
and sodium hydrogen carbonate (9.5 g) to 500 mL of distilled
water. Adjust to pH 11.25 using sodium hydroxide or solid sodium
carbonate. Make up to a volume of 1 L. Reagent A is apparently
stable indenitely at room temperature.
2. Reagent B (4% copper sulfate). Dissolve 4 g of copper sulfate
CuSO4
5H2 O in 100 mL of deionized water. Reagent B is,
reportedly, stable indenitely at room temperature.
3. Working BCA solution. Mix 50 volumes of reagent A with 1
volume of reagent B. Prepare the working BCA solution daily.*

Procedure
Add 100 mL of sample (20120 mg protein) to exactly 2 mL of BCA
working reagent. As long as the 1:20 volume ratio is maintained,
other sample and BCA reagent volumes can be used. Prepare a
reagent blank by replacing 100 mL of sample with the same volume
of distilled water.
Incubate the mixture at 378C (30 minutes) or at room temperature (2
hours). The assay time may be reduced to 1015 minutes by
performing the reaction at 608C. Record A562 against a reagent
blank.
The BCA assay was characterized with respect to the linear dynamic
range, assay temperature, reaction pH, effect of interferences, and reagent
stability. Performance characteristics were also compared with those from
the Lowry assay. At room temperature, DA562 readings increased slowly
over 21 hours. Heating the reaction mixture at 378C for 60 minutes

* This is a precautionary measure. The working BCA reagent was stable for over 7 days. A
calibration graph produced using the 7-day-old BCA working reagent was virtually identical to
a graph produced with a freshly prepared reagent.
The Bicinchoninic Acid Protein Assay 105

produced a similar amount of color. Performing the assay at 608C increased

the assay sensitivity four- and vefold (1). The effect of pH on the BCA
assay was evaluated by altering the pH of reagent A with sodium hydroxide
or solid sodium carbonate. The DA562 readings showed a bell-shaped
response over the pH range 10.212.0. The optimal color yield occurred at
pH 11.25. The working BCA reagent had sufcient buffer capacity to
provide accurate results for protein samples containing 0.1 N sodium
hydroxide or 0.1 N hydrochloric acid. Because Cu1(BCA)2 is stable at pH
313 (Table 2), the pH range of Smith's assay is possibly dictated by the
effect of pH on the protein-copper reaction.
Protein-protein variations in the color yield follow a pattern similar to
those for the Lowry assay (Chapter 3). The order of increasing color
formation is gelatin < avidin < BSA < immunoglobin G < chymotrypsin <
insulin < ribonuclease. The color yield from gelatin was only 50% of the
value for avidin. A high concentration of (hydroxyl) proline residues
probably reduces Cu2 binding to collagen. There is a further loss of color
yield assay owing to the low concentrations of tyrosine and tryptophan in
the gelatin chain (see later). Protein-protein variations in assay results are
lower at 608C. For samples incubated at room temperature or 378C, DA562
readings increased slightly (0.25% per minute) after the prescribed assay
time. For increased precision, the absorbance changes for all samples should
be taken within a short time of each other.

3. CHEMISTRY OF THE BCA PROTEIN ASSAY

Cu2 reacts with the protein to produce to Cu1, which then binds to BCA.
The reaction is different from those taking place for the Lowry assay
(Chapter 3). In the latter case, the concentration of Cu1 remains very low
due to the action of Mo6/W6. This strong oxidant converts Cu1 to Cu2
and Cu3.

3.1. Reactions of the BCA Protein Assay

Two reactions occur between proteins and Cu2 during the BCA assay. The
rst is a temperature-insensitive reaction between Cu2 and oxidizable
protein side chains (tyrosine, tryptophan, cysteine). The second reaction is a
temperature-sensitive process involving the binding of Cu2 to the peptide
backbone. Once bound, Cu2 undergoes reduction to Cu1. Assays
performed at high temperatures assist the peptide pathway. Because the
peptide backbone is the same for different proteins, a high-temperature
BCA assay lessens protein-protein variations in results. Wiechelman et al.
106 Chapter 4

TABLE 3 Some Features of the BCA Protein Interactions

1. Four amino acids (cysteine, tryptophan, tyrosine, and phenylalanine) react with
BCA working reagent.
2. Biuret and dipeptides (unable to form a tetradentate Cu2 complex) do not react
with the BCA reagent.
3. Di- or tripeptides containing tyrosine or tryptophan react with the BCA reagent.
4. There is no correlation between the redox potential and the color yield from the
BCA assay.
5. The colors from amino acids and peptide backbone are not additive.
6. Oxidizable side chains do not react completely in the BCA assay at 378C.
7. The BCA assay is susceptible to interfering compounds that reduce Cu2 to Cu1.

(16) assessed the reactivity of various model compounds with BCA and
Cu2. They also determined the redox potential for some of these
compounds. Their ndings are summarized in Table 3. Clearly, the two-
tier reaction scheme proposed by Smith et al. (1) is correct. However,
oxidizable side chains do not react easily at either 37 or 608C. The following
half-reactions probably lead to the reduction of 2 moles of Cu2.

2Cys?Cys Cys 2e 2H 1

Tyr?semiquinone radical?L dopa 2e H 2

TABLE 4 Determination of Reducing Compounds Using BCA

1
Compound
" (M cm 1)a Moles per Cu1b

Fructose 43,000 0.2

Glucose 6,120 0.8
Indole 3,621 2.2
Tryptophan 1,589 5.0
Tyrosine 1,172 6.8
Cysteine 1,357 5.8
Acetol 10,000 0.8
Ascorbic acid 3,030 0.26
2,4-Dinitrophenyl 16,400 0.5
hydrazine
Hydroxylamine 3,230 2.5
a
Values for the apparent extinction coefcient are from Ref. 16 and 22.
b
Moles of each compound necessary to reduce 1 mole of Cu2 to Cu1. Value is calculated
as De (M 1 cm 1) divided by 8000 (M 1 cm 1).
The Bicinchoninic Acid Protein Assay 107

The majority of the half-reactions leading to the reduction of Cu2 are

unknown. The
" values for reactions with BCA are given in Table 4.
Dividing
" by 8000 (M 1 cm 1) yields the probable number of moles of
each compound needed to form 1 mole of Cu1(BCA)2 complex. Clearly,
acetol undergoes a one-electron reaction with Cu2. One mole of ascorbic
acid apparently yields four electrons. 2,4-Dinitrophenylhydrazine contri-
butes two electrons per mole. The reactions with protein side chains are
difcult to describe in quantitative terms. Between 5 and 6.8 moles of
tyrosine, cysteine, or tryptophan appear to be involved in the reduction of
one Cu2.

3.2. Metal IonCatalyzed Oxidation of Proteins and

Polypeptides
Metal ioncatalyzed oxidation (MCO) reactions require a transition metal
ion (Cu2, Zn2, Fe3, or Ni2), hydrogen peroxide, and a reducing agent
(cysteine, glutathione, or 2-mecaptoethanol, ascorbic acid). The reducing
agent transform Cu2 to Cu1, which then reduces hydrogen peroxide to
form the highly reactive hydroxyl radical  OH.
The literature concerning MCO of protein was reviewed by Levine et
al. (17) and also Pacici and Davies (18). Possible initiation reactions for
MCO involving ascorbic acid are shown next.

2Cu2 ascorbic acid?2Cu1 DHA* 3

H2 O2 Cu1 ? OH OH Cu2 4

Hydrogen peroxide can also be produced from a two-electron reduction of

oxygen.
O2 ? O2 ?H2 O2 5

Attack by .O2 or .OH can initiate a free radical reaction leading to covalent
modications of amino acid side chains. Depending on the reaction
conditions, MCO can result in subtle changes in protein tertiary and
quaternary structure, aggregation, or fragmentation into small polypep-
tides. Madurawe et al. (19) and also Bush and Lumpkin (20) showed that
lactate dehydrogenase (LDH) is inactivated by MCO in the presence of
added ascorbic acid, Cu2, and hydrogen peroxide. Inactivation of LDH
by MCO also occurred without added ascorbic acid. Under such

* DHA=dehydro ascorbic acid.

108 Chapter 4

circumstances, the reducing power is probably derived from oxidizable

protein side chains (tyrosine, tryptophan, cysteine). The MCO process is site
specic. Protein structural modications seem to occur close to the site for
Cu2/Cu1 binding. The degradation of hydrogen peroxide via Fenton-type
reactions leads to a protein-bound .OH that attacks adjacent groups.
BCA inhibits MCO by forming a complex with Cu1. Other MCO
inhibitors include catalase, thiourea, and ethylenediaminetetraacetate. These
inhibitors remove hydrogen peroxide, quench free radicals, or chelate Cu2,
respectively. Inhibitors did not wholly eliminate MCO when LDH was
exposed to Cu2 alone in the absence of reducing compound. Superoxide
dismutase, mannitol, isopropanol, and sodium formate were also ineffective
MCO inhibitors under such circumstances. The failure of radical scavenges
to eliminate MCO may be partly explained by the localized nature of these
reactions. Protein-bound free radicals may be inaccessible to dissolved
inhibitors.
Lactate dehydrogenase modied by MCO showed increased suscept-
ibility to proteolysis due to changes in the enzyme quaternary and tertiary
structure. The structural changes induced by MCO are subtle. Protease
susceptibility was increased even when the residual LDH activity after MCO
was > 95%. There was no fragmentation and cross-linking after LDH
activity was reduced to < 50% by MCO. One view is that MCO alters the
charge on histidine residues. The consequent changes in enzyme charge
generate conformational changes including subunit dissociation. Due to the
inhibitory effect of BCA on the progress of MCO reactions, we expect that
proteins will not be degraded extensively during the BCA protein assay.

3.3. A Note on Protein-BCA Interactions with Copper

Cu -protein binding (Kd *10 6 M) is signicantly weaker than Cu1
1

binding to BCA (Kd 10 11 M). Consequently, Cu1 will be sequestered as

the Cu1 (BCA)2 complex. On the other hand, Cu2 will mainly complex
with tartrate and protein. As BCA does not bind Cu2, the formation of a
Cu1 (BCA)2 complex will alter the Cu2/Cu1 redox equilibrium in favor
of Cu1.

Cu2 e?Cu1 ; E 0:153 6

In the presence of BCA, the apparent redox potential (E8*) for the
Cu2/Cu1 redox couple can be estimated from Eq. (7) (21).

E * E  0:059 log1=Kd 7
The Bicinchoninic Acid Protein Assay 109

From the Kd value given for Cu1 (BCA)2 the E8* calculated from Eq. (7) is
approximately 0.802. The BCA increases the oxidizing power of the Cu2/
Cu1 system. By comparison, Cu2 is a relatively mild oxidizing agent
(E8 0.153). This suggests that BCA is not a passive ligand for Cu2 but
also plays an integral role in the protein detection mechanism.

4. CALIBRATION FEATURES

The linear dynamic range for the BCA protein assay extends to  40 mg of
protein. The standard graph for 20120 mg of BSA is curvilinear. The linear
range is approximately the same for gelatin, avidin, immunoglobulin G,
chymotrypsinogen, insulin, ribonuclease, and soybean trypsin inhibitor (1).
The limit of linearity is probably an intrinsic feature of the BCA assay.
Depending on the protein, the maximum DA560 12 units 120 mg 1 of
protein.
The calibration graph for the BCA protein assay is nonlinear just like
the standard curve for the Lowry assay (Chapter 3). The two assays have
roughly equal sensitivities. Protein-protein variations in color yield are
also similar. The Lowry assay for inorganic reductants leads to straight-
line graphs. Nonlinearity was ascribed to a slow reaction with proteins
that allows time for the degradation of Mo6/W6 at high pH. With the
BCA assay, the instability explanation for nonlinearty is less tenable. In
contrast to Mo6/W6, BCA is stable in alkaline media. What is clear is
that the reduction of Cu2 to Cu1 becomes less quantitative at high
protein concentrations. Some possible causes for nonlinearity are listed in
Table 5.
A hyperbolic standard curve can be readily explained in terms of
saturation phenomenon. Protein binding to a xed concentration of Cu2 is
according to Eq. (8)

P Cu2 ?PCu2 8

Therefore, Kd PCu2 =PCu2 and total number of sites

Cu2 0 Cu2 PCu2 . Therefore, the fraction of Cu2 bound is
 
b PCu2
 2    9
Cu2 0 Cu PCu2

where b is the concentration of the PCu2 complex. Substitution for [PCu2]

( [P][Cu2]/Kd) and a brief rearrangement of Eq. (9) give the hyperbolic
110 Chapter 4

TABLE 5 Plausible Explanations for Nonlinear BCA Protein Assay and Their
Rebuttal

Explanations
1. Incomplete reduction of Cu2 by proteins.
2. Cu1 is bound to the protein and unavailable to BCA,
3. Cu1 is lost as the insoluble hydroxide,
4. Cu2 concentration is limiting in the BCA assay.

Rebuttals for points 13

1. High temperature increases sensitivity or extent of reaction. There is no effect on
linear range for analysis.
2. The binding afnity of BCA for Cu1 is 10,000 times greater than the afnity of
proteins for Cu1.
3. Formation of insoluble copper precipitates is curtailed by tartrate or BCA.

function.

Cu2 0 P
b 10
P Kd

The absorbance change from the BCA reaction is proportional to the

amount of bound copper ions; therefore,
A562 nm eb1 11

As with the Lowry assay (19), the calibration graph for the BSA-protein
assay can be linearized using a range of transformations (Chapter 3).

5. INTERFERENCE COMPOUNDS

Smith et al. (1) evaluated 41 potential interferences for the BCA assay.
These are either chelators or reducing agents. A further group of
miscellaneous compounds disrupt the assay by virtue of their strong color.
Glucose (100 mM) caused a large increase in the reagent blank. Sucrose
(10%) had little effect on the BCA assay. EDTA, guanidine hydrochloride,
sorbitol, and ammonium sulfate produced signicant color losses compared
with control samples. A list of additional interfering compounds includes
biogenic amines, pharmaceutical agents, and Benedict-positive compounds
(Table 6). The Benedict test is a method for determining easily oxidizable
compounds in biological samples. Upon heating with the test sample, Cu2
The Bicinchoninic Acid Protein Assay 111

TABLE 6 List of Potential Interferences for the BCA Protein Assaya

Interference Reference

Biogenic amines. Slocum and Duepree (23)

Dopamine, norepinephrine, epinephrine,
tyrosine, serotonin (5-HT),
tryptophan
Buffers ( ) Kaushal and Barnes (24), Lleu and
Ada, Ampso, Bes, Bicine, Bistris, Caps, Rebel (25)
Epps, Hepes, Hepps, Mes, Mops,
Pipes, Tes, Tricine
Benedict-positive compounds
Acetol, aminophenol, ascorbic acid, 2,3- Chen et al. (22,26)
butanedione, glucose, glyoxal, 2,4-
dinitrophenylhydrazine, pyruvic
aldehyde
Drugs
Chlorpromazine, caffeine ( ), carbachol Marshall and Williams (27)
( ), chloramphenicol ( ), codeine
phosphate ( ), lidocaine ( ),
penicillin G, paracetamol
Lipids
Phosphatidylcholine, phosphatidyletha- Kesller and Fenestil (28)
nolamine, phosphatidylserine,
phosphatidylinositol, cardiolipin,
sphingomyelin
Phenolsb
Gallic acid, tannic acid, pyrogallic acid, Kamath and Pattabiraman (29)
pyrocatechol
a
( ) With the exception of Tricine, buffers showed no response with BCA reagent.
b
Effects have been carefully documented in food systems (see Sec. 5.2).

is reduced, forming a brown precipitate of Cu1 (hydr)oxide. Chen et al.

(22) used BCA to detect Cu1 formed in the Benedict procedure.
Biogenic amines interfered with the BCA assay of proteins from brain
or adrenal medulla (23). Catecholamines were highly reactive with the BCA
reagent. In protein-free samples, there was a linear response for 1
100 6 10 9 mole of biogenic amine. Very small quantities (1 6 10 9 mole)
of biogenic amine affected protein results. Indeed BCA was suggested as a
highly sensitive reagent for assaying biogenic amines.
112 Chapter 4

Zhang and Halling (30) showed that samples containing high

concentrations of NaOH (1 M) gave unexpectedly low readings with the
BCA assay. The interference was higher for the microassay format, where
sample volume constitutes up to 50% of the total assay volume. The normal
BCA response was restored by neutralizing samples. To analyze highly
alkaline samples, the BCA reagent was adjusted to pH 10.27 in order to
improve its buffer capacity.

6. SAMPLE PRETREATMENT, AVOIDING INTERFERENCE,

ENSURING ACCURACY

The TCA-DOC precipitation method (31) was adapted for the BCA assay
by Brown et al. (32). Protein pellets were prepared as described in Method 3
of Chapter 3 and resuspended in 50 mL of alkaline SDS solution (5% w/v
SDS dissolved in 0.1 N NaOH). Then 1 mL of a standard BCA working
solution was added, followed by sample incubation at 378C for 30 minutes.
Interferences by glucose, DTT, 2ME, and ammonium sulfate were
eliminated after the TCA-DOC procedure. Protein was recovered quantita-
tively from media containing various proprietary ampholytes or polybuffer.
Shahabi and Dyers (33) proposed a two-point kinetic assay for dealing
with interferences. Their approach exploits differences in the kinetics of the
BCA reaction with proteins and interfering compounds. Many interferences
(ascorbic acid, cysteine, uric acid ) react with BCA according to rst-order
kinetics. By contrast, the reaction with proteins is usually zero order. This
means that interfering compounds produce a xed amount of background
color rapidly. In contrast, the absorbance change in the presence of proteins
increases linearly with time. Using the rate of color formation as the index of
protein concentration eliminated the effect of interferences.
Solid-phase assays are another means for avoiding interferences. Gates
(34) adsorbed protein samples (50 mL) on 1 6 2-cm strips of cationized
nylon membrane (Zeta-Probe, Biorad Ltd.). The bound protein was rinsed
free of interferences and then air dried. Each membrane was placed in a
1.2 6 10-cm test tube and 2 mL of BCA working solution was added. The
reaction was incubated at 608C for 30 minutes and the concentration of
nylon-immobilized protein determined from DA562 measurements. The
sensitivity of the solid-phase protein assay was 78 (+5)93 (+3.4)% of the
sensitivity obtained with the conventional solution assay. The limits of
detection (*10 mg protein) and the precision of analysis were the same as for
the conventional BCA assay. Accurate results were obtained in the presence
of glucose (20 mM), Tris-HCl buffer (200 mM), or DTT (0.1% w/v). Drying
the protein solution facilitated binding to the nylon support.
The Bicinchoninic Acid Protein Assay 113

7. AUTOMATED BCA PROTEIN ASSAYS

Automated BCA protein assays were developed using microwell plate

readers or ow injection analysis. Microwell plates are widely used for
enzyme-linked immunoassay (EIA) and other colorimetric assays. The
advantages of microwell plates assays include (a) a requirement for a small
sample size (510 mL), (b) reduced reagent requirements (50200 mL), (c)
compatibility with multichannel pipettes and dispensers, (d) automated
measurement using microwell plate spectrophotometers, and (e) the ability
to download results to a computer for enhanced data processing. Flow
injection analysis and continuous ow analyzers allow rapid and more
precise sample analysis compared with conventional batch analysis.

7.1. Microwell Plate Assays

A microwell plate BCA assay was applied for the rapid analysis of protein
fractions from sucrose gradient centrifugation by Redinbaugh and Turley
(35). The assay involved a commercial BCA reagent kit from Pierce
Chemical Company with BSA as the standard protein. Protein samples
(10 mL) were placed in the 96-well microwell plate, to which BCA working
reagent (200 mL) was added. The samples were incubated at 228C for about
14 hours (i.e., overnight) or at 608C for 2 hours. Absorbance readings were
recorded at 570 nm. Lane et al. (36) also described a BCA microwell plate
protein assay, as have Sorensen and Brodbeck (37). Typically, color
formation took place at 378C for 30 minutes. Absorbance readings for 96
samples could be completed in 5 minutes.
The performance of microwell plate-based BCA protein assays
depends on a range of factors including sample size, volume of protein to
BCA working reagent, incubation temperature, and time. With the standard
assay (1:20 ratio of protein to BCA reagent), the linear dynamic range for
analysis was between 1 and 12 mg of protein (compare with a linear range of
10120 mg for the conventional assay). The calibration graph remains
curvilinear (1). The proposal that microwell plate-based BCA assays are 10-
fold more sensitive than the conventional assay is not supported by the
available evidence when results are normalized for differences in the assay
temperature or for proteinprotein variations in the response.
The maximum absorbance expected for a given amount of protein can
be estimated for the BCA assay. Consider 10 mg of BSA in a 210-mL assay
volume. First, six peptide bonds bind each Cu2 ion. Taking the molecular
weights of BSA and amino acids as 66,000 and 110, then 100 moles of Cu2
ions will bind to 1 mole of BSA (see Chapter 2, Table 2). Consequently,
10 mg of protein (1.51 6 10 10 mole BSA) will bind with 15.1 6 10 9 mole of
114 Chapter 4

Cu2, which is equivalent to 72 6 10 6 M of Cu2 in the 210-mL reaction

volume. Assuming that the bound Cu2 is fully reduced to Cu1, the
maximum absorbance change expected (A ecl) is 0.58. In practice, the
maximum absorbance change obtained for 10 mg of BSA during a microwell
plate assay was between 0.29 and 0.93 (Table 7). To account for the higher
than expected absorbance change, note that the preceding calculation did
not consider the role of oxidizable protein side chains. Groups such as
tyrosine, tryptophan, and cysteine react directly with Cu2. It was also
assumed that each sextet of peptide groups undergoes a one-electron redox
reaction with bound Cu2. Table 4 shows that this assumption is probably
unwarranted.

7.2. Flow Injection Analysis and Continuous Flow Analysis

Using BCA Reagent
The principles of ow injection analysis (FIA) were described in the
monograph by Ruzicka and Hansen (38) and also by Valcarcel and Luque
de Castro (39). In FIA, a pump delivers reagents and analyte to a mixer
(chamber) and then to a reaction coil. The products of the reaction then ow
to a detector and from there to a waste receptacle. Electrochemical and
colorimetric detectors are popular. Davis and Radke (40) illustrated the use
of BCA in a simple FIA system for proteins. They employed a peristaltic
pump to impel BCA reagent (containing 0.52% BCA) through a owline of
Teon or polyethylene tubing [inside diameter (ID) 0.5 mm]. Protein
samples were injected into the ow stream via a septum using a (25- or
50-mL) Hamilton syringe. The mixture then passed through a 3-m length of
Teon tubing incubated in a constant-temperature water bath. The color
formed was monitored with a spectrophotometer tted with a ow-through
cuvette. Absorbance (peak height) measurements were recorded using a strip
chart recorder. The BCA-FIA system's performance depends on variables
such as sample volume (1050 mL), length of the reaction coil, ow rate (0.5
or 1 mL min 1), and reaction temperature (721008C).
Note that the BCA assay is a kinetic method. The color-forming
reactions do not go to completion. This is especially true for FIA when the
sample residence time in the reaction coil is short. Precision can be ensured
only by keeping the sample ow rate (and hence residence time) constant.
To improve the color yield (peak height) during FIA, sample ow rate may
be reduced, thereby increasing the sample residence time in the reaction coil.
Another strategy is to use a longer reaction coil. Assay sensitivity can also
be increased by using higher reaction temperatures and/or concentrations of
BCA.
TABLE 7 Microwell Plate-Based BCA Protein Assaysa

Assay Linear range BSA/BCA volume Sensitivity
A/g %

format (g) (L) (BSA) R Error

Redingbaugh and
Turley (35)
The Bicinchoninic Acid Protein Assay

Microassay 0.22 10:200A 0.113 (0.003)

Standard assay 110 10:200A 0.093 (0.002)
Lane et al. (36)
Microassay 0.52.5 10:50B 0.0547 0.9940 3.3
Standard assay 212 10:200C 0.0245 0.9967 3.5
a
Results with bovine serum albumin as standard. Samples were incubated at (A) 228C overnight, (B) 608C for 30 minutes, or (C) 378C for 30
minutes.
115
116 Chapter 4

The FIA devised by Davis and Radke (40) employed a sample ow

rate of 0.5 mL per minute, a sample size of 10 mL and a reaction temperature
of 808C. The reaction coil was 3 m in length. With such design features, the
sample residence time was 4.7 minutes. The throughput achieved with this
system was 60 samples per hour. The linear range for analysis was 110 mg of
protein. The precision of analysis was surprisingly good (1%) in view of the
crude system for sample injection. With BSA, ovalbumin, hemoglobin, beta-
lactoglobulin, conalbumin, and myoglobin as standard proteins, the average
sensitivity for analysis (DA560/mg) was 0.024 (+0.0013). Therefore, the
sensitivity for FIA is comparable to that obtained for batch analysis (Table
7). At reaction temperatures above 808C, the ow tubing became blocked by
an unidentied precipitate.
A further example of protein FIA using BCA reagent was described by
Wolfe and co-workers (41,42). Their setup was essentially as described by
Davis and Radke (40) with the following modications: (a) sample injection
was via a Rheodyne2 sample valve or an autosampler device with a 20-mL
sample loop, (b) samples were thoroughly degassed before use, (c) the
carrier stream was phosphate buffer (0.1 M, pH 7.4) with 1% Triton X-100,
and (d) protein analysis was performed at 808C with a ow rate of 0.9 mL
min 1. The linear range for analysis was 0260 mg mL 1 (05.6 mg).
The Technicon AutoAnalyzer was adapted for the BCA assay by
Hawkes and Craig (43). The reagent, air, and sample ow lines were
assembled from 2.4-mm (ID) glass tubing. The sample stream was
segmented with air bubbles before mixing with BCA reagent. It then passed
to a reaction coil incubated at 558C at a ow rate designed to produce a
residence time of 4.2 minutes. Color formation (peak heights) was
monitored at 570 nm. Calibration graphs for standard proteins were
curvilinear and tted a polynomial equation. To save on the reagent cost,
commercial BCA reagent was diluted 1:4 before use. The sensitivity of
analysis decreased with decreasing concentrations of BCA reagent. Protein-
protein variations in assay results were similar to those observed with the
batch assay: gelatin < BSA < chymotrypsinogen < RNA < insulin. The
within-day precision of analysis was 1.042.99% and day-to-day precision
was 1.6214%, depending on the nature and concentration of the protein
analyzed.

8. APPLICATIONS OF THE BCA ASSAY TO FOOD PROTEIN

ANALYSIS

There are only a small number of reports describing the use of the BCA
assay for food protein analysis. No doubt, the numbers of applications will
The Bicinchoninic Acid Protein Assay 117

increase in the near future.* Applications reported in the published

literature are discussed next.

8.1. Solid-Phase Analysis of Cereal Proteins

Chan and Wasserman (44) determined protein in corn meal our.
Commercial corn meal samples and/or zein (27 mg) were placed in
microcentrifuge tubes. The BCA working reagent (1 mL) was added and
the mixture was incubated with intermittent shaking at 378C for 30 minutes.
Samples were cooled over an ice bath for 5 minutes and particulate material
removed by centrifuging (13,000g; 10 minutes). Thereafter, 0.2 mL of
supernatant was diluted in 1 mL of BCA reagent A (see Method 1 for the
reagent composition) and A562 readings were recorded. The BSA (50
400 mg) was used as the standard protein while method calibration involved
Kjeldahl analysis.
Fig. 2 shows generally good agreement between Kjeldahl and BCA
results. However, the former technique gave higher values for protein than
the BCA assay. The discrepancy between Kjeldahl and BCA assays was

FIGURE 2 Correlation between Kjeldahl protein and BCA assay of corn meal
protein and samples of zein. (Drawn from Ref. 44.)

* From personal experience, use of the commercially available reagent is underreported.

118 Chapter 4

ascribed to the presence of NPN. It is also likely that BSA is not a

satisfactory standard for corn protein analysis.

8.2. Analysis of Forage Plant Leaf Proteins

Some 23 forage plant (leaf) samples were analyzed using the BCA assay by
Messman and Weiss (45). These included alfalfa (fresh, wilted, hay, silage,
leaves), crown vetch (fresh, wilted, silage), spinach (fresh), perennial
ryegrass (fresh), orchard grass (fresh, wilted), corn plant (silage), and peal
millet (fresh). For sample pretreatment, leaves were lyophilized and ground
using a 1-mm Wiley mill. The resulting powders were extracted with borate-
phosphate buffer (0.1 M ionic strength) containing SDS (1% w/w). Sonicat-
ing the suspension of leaf powder for up to 2 minutes facilitated protein
extraction. In most cases, protein recovery was 85%. The protein extracts
were analyzed with the BCA and Kjeldahl methods (N 6 6.25).
The BCA method gave unreliable estimates of leaf proteins. There was
poor agreement between BCA and Kjeldhal results. Leaf samples contain
numerous interfering compounds (plant pigments, peptides, sulfhydryl
compounds, and phenol derivatives) that can interfere with both the BCA
and Kjeldahl methods. Attempts to circumvent interferences using the
DOC-TCA procedure were not successful. The yield of leaf protein
recovered by precipitation with DOC-TCA ranged from 40 to 80%. Protein
recovery by cold-acetone precipitation was not signicantly higher.

8.3. Analysis of Seed Proteins in the Presence of Phenolic

Compounds
Salt-soluble proteins from soybean, tamarind, ragi, jack fruit, mango kernel,
and sorghum were analyzed by Kamath and Pattabiraman (29). Whole
meals were extracted with 0.3 M NaCl (buffered with 20 mM phosphate
buffer, pH 6.9). Protein extracts were then analyzed using the BCA,
Bradford, and Lowry assays. A range of pure proteins (BSA, casein,
chymotrypsinogen, lysozyme, myoglobin, trypsin, zein) were also analyzed.
The BCA, Bradford, and Lowry assays showed differences in protein-
protein variations in color yield. Apparently, endogenous seed compounds
affected the results. High responses were obtained with sorghum, mango
kernel, and other samples known to have high concentrations of total
phenol. The BCA reagent was more sensitive to phenolic substances than to
protein. On a scale of 1.0 for BSA, the color yields from a range of phenolic
compounds were pyrogallic acid (86), gallic acid (2.1), pyrocatechol (106.0),
tannic acid (9.3), and phenol (0.8). The BCA response to protein and
phenolic substances was additive. There was a linear response between A567
The Bicinchoninic Acid Protein Assay 119

values and the concentration of phenol. Most seed contained comparable

amounts of protein and phenolic compounds. Therefore, the BCA response
to these systems is likely to be due to the phenol. The BCA analysis of
soybean protein was error free owing to the low concentrations of phenolic
compounds in this seed.

8.4. Identication of High-Lysine Cereals Using the BCA

Assay
The A562 readings for ribonuclease, chymotrypsinogen, insulin, and BSA
apparently showed a high degree of correlation with the number of lysine
residues in each protein (R 0.99; P <.01). Therefore, the BCA assay
response was determined for four high-lysine mutants of barley (Hordeum
vulgare) and four normal-lysine variants. Whole meals were extracted with
4% SDS solution in 0.15 M Tris-HCl buffer (pH 7) at 608C for 20 minutes.
The extracts were diluted three fold with distilled water before analysis using
the BCA assay at 258C for 2 hours.
There was a signicant correlation between BCA protein assay results
and lysine content expressed per weight of barley meal (R 0.974, P <.001)
(46). Available lysine was also determined using the TNBS (trinitrobenze-
nesulfonic acid) method of Kakade and Liener (47) (see Chapter 12). A
signicant correlation was also observed between BCA results and sample
protein content as determined by the Kjeldhal method or by UV
absorbance. As a corollary, the sensitivity of the BCA assay for a range
of synthetic polyamino acids increased in the order Asp & His < Glu < Arg
 Lys.

8.5. Determination of Plant Protein in the Presence of

Reagents for Electrophoresis
Extraction buffers for SDS-PAGE, two-dimensional (2D) electrophoresis,
and isoelectric focusing of plant proteins are incompatible with many
protein assays. The 2D electrophoresis samples may contain 9 M urea,
2,2ME, SDS, and ampholyte. Orr et al. (48) assayed such samples accurately
using the BCA assay.

8.6. Analysis of Animal Carcass Total Protein

Analysis of protein nutrient quality sometimes requires a quantitative
determination of total carcass nitrogen (see Chapter 12). Brooks et al. (49)
assayed whole-body nitrogen for male Sprague-Dawley rats using serveral
methods including the BCA assay. Their approach should be applicable for
120 Chapter 4

TABLE 8 Effect of Extraction Solvent on the Apparent Carcass Protein

Concentrationa

Assay method Extraction solvent [Protein] (g/100 g sample)

BCA Water 2.05

SDS-NaOH 12.03
GnHCl 10.43
Biuret Water 3.41
SDS-NaOH 14.70
GnHCl 12.11
Bradford Water 7.30
SDS-NaOH 102.00
GnHCl 6.10
a
Whole-body protein concentration was 16.3% by quantitative amino acid analysis.

other animal carcasses with roughly similar fat content. This work might be
usefully compared with that of Toten and Whitaker (50) described in
Chapter 2. Carcasses were passed through a Horbart meat grinder (model
4812) once. The skin (including hair) was cut into pieces with scissors and
added to the meat. Homogenizing in a Waring blender and a Brinkman
polytron homogenizer further reduced the meat particle size. Finally, 1 g of
protein was solubilized by homogenizing with (a) water, (b) 5% SDS
dissolved in 0.5 M sodium hydroxide, or (c) 6 M guanidine hydrochloride
solution.
Samples were analyzed by the BCA method, biuret assay, or Bradford
assay. Protein extracts were also analyzed by three so-called absolute
methods, i.e., quantitative amino acid analysis, protein hydrolysis followed
by ninhydrin analysis, and Kjeldahl analysis. Quantitative amino acid
analysis yields a value of 16.3 (+0.5) g protein/100 g sample (n 15
replicates). However, Kjeldahl results (N 6 6.25) were 34% higher than
expected. Accurate results were obtained by using Fk 5.51. The ninhydrin
assay results depended on the choice of amino acid standard. The BCA and
biuret methods were not adversely affected by the presence of SDS or
guanidine hydrochloride. The Bradford procedure was unusable in the
presence of SDS (Chapters 6 and 7). These results are summarized in the
following.
Apparently, SDS-NaOH was the most efcient protein extraction
solvent tested. Thereafter, the accuracy of results varied in the order biuret
> BCA >> Bradford. The performance of the BCA assay may be better
than indicated from results in Table 8. First, the BCA assay was not
adversely affected by SDS-NaOH or GnHCl when serum albumin was
The Bicinchoninic Acid Protein Assay 121

employed as the standard protein. Inadequate explanation was given for the
ability of the BCA assay to detect only 75% of the carcass protein.
Calibration graphs for the BCA are nonlinear. However, a hyperbolic
function was not applied by the authors although such an equation was
tted to results from the Bradford assay. There are good prospects that the
BCA method can be adapted for animal protein analysis, perhaps with SDS-
NaOH as the extraction solvent.

8.7. Analysis of Proteins from Freshwater Algae

There were several sources of error during attempts to analyze proteins from
freshwater algae. Meijer and Wijffels (51) noted that the efciency of protein
extraction from cells was variable. Attempts to facilitate extraction using
chemical means led to interferences with the Bradford and BCA protein
assays. Proteins could also undergo severe damage under harsh extraction
conditions such as boiling with alkali. Such harsh treatments could lead to
standard proteins being less representative of the sample.
Boiling Chlorella cells with 1 M NaOH for 30 minutes led to a recovery
of between 3% (Bradford assay) and 14% (BCA assay) of the crude protein.
By comparison, extracting yeast cells under similar conditions produced
protein recoveries between 76% (Bradford method) and 85% (BCA method).
When BSA standard protein was exposed to boiling 1 M NaOH for 30
minutes, there was 32% (Bradford method) or 85% (BCA assay) of the
response recorded for the untreated proteins. Apparently, chemical damage
due to heating at high pH is only partly responsible for the poor assay
results. Chlorella protein was efciently extracted by sonicating 50-mL
samples of fresh algae suspended in sodium phosphate buffer (25 mM)
containing 1% SDS. After sonication for 0.5, 1, and 3 minutes, there was 36,
80, and 104% recovery of crude protein as determined by the BCA assay. To
avoid foaming and a rise in sample temperature, the period of sonication
was divided into six 30-second intervals. In the presence of SDS, the
Bradford assay could not be used.

REFERENCES

1. PK Smith, RI Krohn, GT Hermanson, AK Mallia, FH Gartner, MD

Provenzano, EK Fujimoto, NM Goeke, BJ Olson, DC Klenk. Measurement
of protein using bicinchoninic acid. Anal Biochem 150:7685, 1985.
2. J Hoste. On a new copper specic group. Anal Chim Acta 4:2337, 1950.
3. D Kertesz. State of copper in phenoloxidase (tyrosinase). Nature 180:506507,
1957.
122 Chapter 4

4. G Felsenfeld. The determination of cuprous ion in copper proteins. Arch

Biochem Biophys 87:247251, 1960.
5. AL Gershuns, AA Verezubova, ZhA Tolstykh. Photocolorimetric determina-
tion of copper with 2,20 -bicinchoninic acid. Izv Vyssh Uchebn Zaved Khim
Khim Technol 4 (1):2527, 1961.
6. S Nakano. 2,20 -Biquinoline derivatives VI. Copper (I) chelates of the 4,40 -
substituted 2,20 -biquinoline derivatives and the determination of copper by 2,20 -
bicinchoninic acid. Yakugaku Zasshi 82:486491, 1962.
7. IS Mustan, NS Frumina, VS Kovashova. Determination of copper in various
samples with 2,20 -bicinchoninic acid. Zavod Lab 29:782785, 1963.
8. VN Tikhonov. Highly selective titration method for determining copper with
2,20 -bicinchoninic acid. Zh Anal Khim 27:673677, 1972.
9. NN Noskova. Photometric determination of copper in blood using 2,20 -
bicinchoninic acid. Mikroelem Sib 9:159161, 1974.
10. F Buhl, Z Gregorowicz, B Piwowarska. Complex compound of 2,20 -
bicinchoninic acid with copper (I) ions. Pr Nauk Uniw Slask Katowicach
91:2733, 1975.
11. F Capitan, JMR Navarro, LF Capitanvallvey, Solid-phase spectrophotometric
microdetermination of copper. Anal Lett 24:2011217, 1991.
12. AJ Brenner, ED Harris. A quantitative test for copper using bicinchoninic acid.
Anal Biochem 226:8084, 1995 [see Anal Biochem 230:360, 1995 for erratum].
13. RF McFeeters. A manual method for reducing sugar determination with 2,20 -
bicinchoninic acid reagent. Anal Biochem 103:302306, 1980.
14. EM Grindler. Automated determination of glucose via reductive formation of
lavender Cu(I)-2,20 -bicinchoninate chelate. Clin Chem 16:519, 536, 1970.
15. K Mopper, EM Grindler. A new noncorrosive dye reagent for automatic
chromatography. Anal Biochem 56:440442, 1973.
16. KJ Wiechelman, RD Braun, JD Fitzpatrick. Investigation of the bicinchoninic
acid protein assay: identication of the groups responsible for color formation.
Anal Biochem 175:231237, 1988.
17. RL Levine, D Garland, CN Oliver, A Amici, I Climent, A-G Lenz, B-W Ahn, S
Shaltiel, ER Stadtman. Determination of carbonyl content in oxidatively
modied proteins. Methods Enzymol 186:464478, 1990.
18. RE Pacici, KJA Davies. Protein degradation as an index of oxidative stress.
Methods Enzymol 186:485502, 1990.
19. RD Madurawe, Z Lin, PK Dryden, JA Lumpkin. Stability of lactate
dehydrogenase in metal-catalyzed oxidation solutions containing chelated
metals. Biotechnol Prog 13:179184, 1997.
20. KD Bush, JA Lumpkin. Structural damage to lactate dehydrogenase during
copper iminodiacetic acid metal afnity chromatography. Biotechnol Prog
14:943950, 1998.
21. DA Skoog, DM West. Fundamentals of Analytical Chemistry. 3rd ed. New
York: Holt, Rinehart and Winston, 1976, p 305.
22. Q Chen, N Klemm, I Jeng. Quantitative Benedict test using bicinchoninic acid.
Anal Biochem 182:5457, 1989.
The Bicinchoninic Acid Protein Assay 123

23. TL Slocum, JD Duepree. Interference of biogenic amines with the measurement

of proteins using bicinchoninic acid. Anal Biochem 195:1417, 1991.
24. V Kaushal, LD Barnes. Effect of zwitterionic buffers on measurement of small
masses of protein with bicinchoninic acid. Anal Biochem 157:291294, 1986.
25. PL Lleu, G Rebel. Interference of Good's buffers and other biological buffers
with protein determination. Anal Biochem 192:215218, 1991.
26. Q Chen, N Klemm, G Duncan, K Jeng. Sensitive Benedict test. Analyst
115:109110, 1990.
27. T Marshall, KM Williams. Drug interference in the Bradford and 2,20 -
bicinchoninic acid protein assay. Anal Biochem 198:352354, 1991.
28. RJ Kesller, DD Fanestil. Interference by lipids in the determination of protein
using bicinchoninic acid. Anal Biochem 159:138142, 1986.
29. P Kamath, N Pattabiraman. Phenols interfere in protein estimation by the
bicinchoninic acid assay method. Biochem Arch 4:1723, 1988.
30. J-X Zhang, PJ Halling. pH and buffering in the bicinchoninic acid (4,40 -
dicarboxy-2.20 -biquinoline) protein assay. Anal Biochem 188:910, 1990.
31. A Bensadoun, D Weinstein. Assay of proteins in the presence of interfering
materials. Anal Biochem 70:241250, 1976.
32. RE Brown, KL Jarvis, KJ Hyland. Protein measurement using bicinchoninic
acid: elimination of interfering substances. Anal Biochem 180:136139, 1989.
33. ZK Shahabi, MS Dyers. Protein analysis with bicinchoninic acid. Ann Clin Lab
Sci 18:235239, 1988.
34. RE Gates. Elimination of interfering substances in the presence of detergent in
the bicinchoninic acid protein assay. Anal Biochem 196:290295, 1991.
35. MG Redinbaugh, RB Turley. Adaptation of the bicinchoninic acid assay for
use with microtiter plates and sucrose gradient fractions. Anal Biochem
153:267271, 1986.
36. RD Lane, D Federman, JL Flora, BL Beck. Computer-assisted determination
of protein concentrations from dye-binding and bicinchoninic acid protein
assays performed in microtiter plates. J Immunol Methods 92:261270, 1986.
37. K Sorensen, U Brodbeck. A sensitive protein assay method using microtiter
plates. Experientia 42:161162, 1986.
38. J Ruzicka, EH Hansen. Flow Injection Analysis. 2nd ed. New York: Wiley,
1988.
39. M Valcarcel, MD Luque de Castro. Flow Injection Analysis: Principles and
Applications. Chichester, England: Ellis Horwood, 1987.
40. LC Davis, GA Radke. Measurement of protein using ow injection analysis
with bicinchoninic acid. Anal Biochem 161:152156, 1987.
41. AC Wolfe, DS Hage Automated determination of antibody oxidation using
ow injection analysis. Anal Biochem 219:2631, 1994.
42. CAC Wolfe, MR Oates, DS Hage. Automated protein assay using ow
injection analysis. J Chem Educ 75:10251028, 1998.
43. WC Hawkes, KA Craig. Adaptation of the bicinchoninic acid protein assay to
a continuous ow analyzer. Lab Robot Autom 3:1317, 1990.
124 Chapter 4

44. K-Y Chan, BP Wasserman. Rapid solid-phase determination of cereal proteins

using bicinchoninic acid. Cereal Chem 70:2728, 1993.
45. MA Messman, BP Weiss. Extraction of protein from forages and comparison
of two methods to determine its concentration. J Agric Food Chem 41:1085
1089, 1993.
46. H Ahokas, L Naskali. Bicinchoninic acid protein assay reveals high-lysine
contents in barley seed protein extracts. J Cereal Sci 7:4348, 1988.
47. ML Kakade, E Liener. Determination of available lysine in proteins. Anal
Biochem 27:273280, 1969.
48. A Orr, BA Wagnaer, CT Howard, OA Schwartz. Assay of plant proteins with
bicinchoninic acid for high-resolution two-dimensional polyacrylamide gel
electrophoresis. Plant Cell Rep 7:598601, 1988.
49. SPJ Brooks, JJ Lampi, G Sarwar, HG Botting. A comparison of methods for
determining total body protein. Anal Biochem 226:2630, 1995.
50. J Torten, JR Whitaker. Evaluation of the biuret and dye-binding methods for
protein determination in meats. J Food Sci 29:1681174, 1964.
51. EA Meijer, RH Wijffels. Development of a fast, reproducible and effective
method for the extraction and quantication of protein of micro-algae.
Biotechnol Tech 12:353358, 1998.
5
The Udy Method

1. INTRODUCTION

Proteins react with certain organic dyes to produce insoluble complexes. The
quantity of dye bound is proportional to (a) the dye-binding capacity
(DBC)* and (b) the protein concentration. Farm-gate prices for milk (in
Australia, Denmark, France, Netherlands, New Zealand, United States) is
partly determined by its protein content. Dye-binding assays are widely used
for milk protein determination. Amido Black 10B (C.I. 20470), Acid Orange
12 (C.I. 15970), and Orange-G (C.I. 16230) are the three main dyes used.
Dye-binding assays are presented in Sections 13 of this chapter. Section 4
covers the chemistry of protein binding with azo dyes. Section 5 is a review
of interference compounds and other sources of error. Section 6 covers
applications of dye-binding assays in food protein analysis.
Protein-dye interactions are of interest in relation to (a) dyeing of
natural bers and textiles, (b) dyeing of tissue sections for microscopic
observation, (c) the use of dyes to evaluate renal function, (d) application of
dyes as pH indicators in protein-rich media, and (e) uptake of dyes by
photographic materials. In 1925 Grollman (1) described phenol red

* DBC is the amount of dye bound by a gram of protein.

125
126 Chapter 5

(phenolsulfonphthalein) binding using the Freundlich adsorption isotherm:

1=n
Db =cP KDf 1

where Db (moles) is the dye bound by cP (grams) of crude protein and Df is

the equilibrium concentration of free dye. The empirical constants K and n
were identical for serum albumin samples from healthy and diseased persons
(2). Eq. (1) applied to solid adsorbents (charcoal, casein, granular gelatin) or
soluble proteins (serum albumin). The Df was inversely related to serum
albumin concentration. Robinson and Hogden (3) also showed that Df
(measured as a decreases in A565) was inversely related to protein
concentration (03 mg mL 1).
Schmidt and co-workers (46) found that several proteins (gelatin,
casein, brin, edestin) bound with acidic acid dyes (Biebrich Scarlet,
Naphthylamine Brown,* Metanil Yellow, Tropaeolin O) at pH < 4.0 with
maximum binding at pH 1.562.5. Contrary to earlier ndings, binding did
not conform to the Fruendlich isotherm.{ The DBC was proportional to the
concentration of basic amino acids in different proteins. Binding with basic
dyes (Methylene Blue, Safranine-Y, and Induline Scarlet) depended on the
number of acidic groups in the protein. The DBC was the same for soluble
or granular gelatin. Fraenkel-Conrat and Cooper (7) conrmed that
Orange-G binds to basic amino acids. The basic dye Safranine-O bound
with acidic amino acid side chains (carboxylic, phenolic, sulfhydryl groups).
This study is recognizably the rst protein dye-binding assay.{ The number
of basic (guanidyl, imidazole, amino) groups in several proteins is the same
whether determined by titration or by Orange-G binding (Fig. 1).}
The application of dye-binding assays to food proteins was developed
and later commercialized by Udy (8,9). This subject has been reviewed by
McGann (10), Lakin (11,12), Hartley (13), McGann (14), Lowe (15), and

* Naphthylamine Brown is also called Amido Black 10B (C.I. 20470), Amidoschwarz 10B,
Naphthylamine Black 10BR, Aniline Blue Black, Naphthol Blue Black, Acid Black 1, or
Pontacyl Blue Black SX.
{ From the Freundlich equation, log(Db/cP) log K (1/n) log Df. Thus, a plot of log(Db/cP)
versus log Df should give a straight line graph with a slope (1/n) and an intercept log K.
{ About 5 mg of protein (egg albumin, beta-lactoglobulin, casein, brin, and zein) was placed in
a 15-mL test tube, wetted with 1 mL of buffer followed by the addition 15 mL of dye solution
(e.g. 0.1% w/v Orange-G in 0.2 M citrate0.1 M phosphate buffer, pH 2.2). After shaking for
2024 hours, the suspension was centrifuged to remove the insoluble protein-dye complex. The
supernatant was diluted 100-fold with buffer and absorbance readings were taken. The free dye
concentration was determined from a standard curve of absorbance readings plotted against
known dye concentration.
} Both collagen and casein appear to have a typical dye-binding characteristics.
The Udy Method 127

McGann (16). The literature through to 1967 was discussed by Cole (17).
Sherbon (18) reviewed dye-binding assays for milk proteins.

2. THE UDY METHOD

The Udy assay using Acid Orange 12 is described here (Method 1).
Information in the public domain deals with milk and dairy products. Other
applications developed by Udy Corporation during the 1960s were probably
commercially sensitive and not published (Table 1). The Udy method is the
basis of Udy Protein Systems2.

2.1. Protein Determination Using Acid Orange 12

Method 1 is equivalent to AOAC Method 967.12 for milk protein analysis
(19). Instrument requirements include a spectrophotometer, short-path-
length ow cuvette, and automatic pipettes. For small-scale analysis, a
degree of improvisation is possible. For large numbers of samples the
required accessories include the Udy calorimeter, a 40-mL dye regent

FIGURE 1 Basic amino acid concentration in a range of proteins as measured by

Orange G binding and by titration. Units of the Y-axis are 6 10 4 moles
per gram protein. (Drawn using data from Ref. 7.)
128 Chapter 5

TABLE 1 A Range of Commodities Analyzed by the Udy Method

Alfafa Gaines burger Oats Soybean

Barley Gram Oat groats Soybean hulls
Beans Grass peas Peanut meal Soybean meals
Bermuda grass Lentils Peas Sunower meal
Caseinate Linseed meal Pigeon peas Triticale
Cheese (hard) Malted barley Rapeseed meal Urd beans
Chickpeas Meat Rice Wheat
Corn silage Milkuid Rye Wheat germ
Cottonseed meal Milkpowders Safower meal Wheatgluten
Cowpeas Mung beans Sesame meal Wheydelactose
Fish meal Mustard meal Sorghum (milo) Wheyfresh
Wheypowdered
Source: Adapted from Udy Corporation advertising literature.

dispenser, and a React-R-Shaker* for highly efcient mixing of powdered

samples with dye solution.

Method 1
Acid Orange 12 dye binding (2022).

Reagents{
1. Acetic acid (glacial)
2. Acid Orange 12
3. Oxalic acid
4. Potassium dihydrogen phosphate

Procedure
Purication of Acid Orange 12. Dissolve 400 g of dye in 400 mL of
boiling water. Add 400 mL of reagent-grade ethyl alcohol. Cool to
room temperature and refrigerate at 058C overnight. Vacuum lter
dye solids using a Buchner ask-ltration unit tted with a
polypropylene lter. Wash with cold ethyl alcohol and dry the
resulting solid in an oven at 1258C.

* Udy and React-R-Shaker are trademark terms for the Udy Corporation, 201 Rome Court,
Fort Collins, CO 80524. Fax: 1-970-482-2067. Telephone: 1-970-482-2060. Internet address:
http://www.udycorp.thomasregister.com
{ Other requirements include 2-oz polyethylene bottles or 125-mL conical asks, automatic
pipettes for dispersion of 40 mL of reagent, syringe pipettes (25 mL), sample mill, and ltration
equipment or a low-speed centrifuge.
The Udy Method 129

Phosphate buffer (0.05 M, pH 1.81.9). Dissolve potassium dihydro-

gen phosphate (3.4 g) and oxalic acid (2 g) in 100 mL of warm water.
Add to 800 mL of water containing phosphoric acid (3.4 mL), acetic
acid (60 mL), and propionic acid (1 mL) and dilute the mixture to
1 L.
Working Acid Orange 12 dye reagent (0.13% w/v). Dissolve 1.3 g of
Acid Orange 12 in 100 mL of warm phosphate buffer. Allow to cool
and dilute to 1 L with phosphate buffer.
Reference dye solution (0.06% w/v). Dilute the working dye reagent
with phosphate buffer. Prepare further dilutions and produce a
calibration curve of free dye concentration versus A480 readings.
Performing a dye-binding assay. Place 1.52.4 mL of liquid sample (or
0.250.5 g of solid) in a 2-oz plastic polyethylene bottle. Add 40.44 g
(40 mL) of working dye solution and shake vigorously for 30
seconds. Solid samples may be shaken for 5 minutes.
Centrifuge at 3500 rpm for 30 minutes or lter to remove insoluble
dye-protein complex. Dilute* the supernatant 100-fold with
phosphate buffer and record A480 readings versus an appropriate
buffer blank. Determine the amount of free dye from the calibration
graph.
Calibration for protein determination. Determine dye binding for
samples with known amounts of crude protein (%N 6 6.25).
Establish the regression equation relating Db versus crude protein.

Analyses of 73 whole milk or 34 spray-dried milk samples by Orange

G binding led to a highly signicant correlation between crude protein
(%N 6 6.38) and the amount of free dye (23);

V1 D V2 Df
cP 100 2
kEm

where k (226 g mole 1) is the dye equivalent weight,{ E (moles g 1) is the

DBC expressed as equivalents of dye bound, and m is the weight of sample
in grams. For a solid sample V1 & V2 (i.e., volume of sample & volume of

* Colorimetric measurements are possible without dilution when using a very short (0.3 mm)
path-length ow-through cuvette.
{ The equivalent weight (k) for an ionic species (g mole 1) is the molecular weight divided by the
number of charges per molecule; k 226 (g mole 1) assuming that this dye has two positive
charges. Note that the number of equivalents of dye bound is DBC/k.
130 Chapter 5

sample dye) and hence

V1 D Df
cP 100 3
kEm
Rearranging Equation (2) leads to Eq. (4) or Eq. (5) for liquid or solid
samples, respectively.
V1 cPEkm
Df D 4
V2 V2

cPEkm
Df D 5
V1
Therefore a graph of Df versus cP yields a straight line with a slope
of mkE. The parameter E was 0.792 (mEq g 1) for spray-dried milk and
0.805 (mEq g 1) for fresh milk. See Section 6 for further discussions of milk
protein analysis.

2.2. Protein Determination Using Orange-G

Fraenkel-Conrat and Cooper (7) employed Orange-G in their seminal study
of 1944. Udy (8) also used Orange-G for protein determination and later
changed to Acid Orange 12 in 1963 (22). The color change for Acid Orange
12 was apparently 100% greater than that obtained with Orange-G.* The
former dye is also less hygroscopic and more easily puried. These days,
high-grade samples of Orange-G are readily available. The use of Orange-G
is described further in Refs. 8,9,24,25, and 26.

2.3. Protein Determination Using Amido Black 10B

Milk protein analysis using Amido Black 10B is important in both North
America and Europe (2730). Commercial instruments such as the ProMilk
Mark II or ProMilk PMA (manufactured by Foss Food Technology Corp.)
use Amido Black 10B. Several investigators reported difculties with Amido
Black 10B staining of plant proteins; some Amido Black 10B samples may
contain impurities with different afnities for plant proteins (31). [Method 2
is adapted from Sherbon (32,33).]

* This view is incorrect (Section 3.2). Proteins bind equal amounts of Orange-G and Acid
Orange 12. The molar extinction coefcients for Orange-G and Acid Orange 12 are also similar.
The Udy Method 131

Method 2
Protein analysis using Amido Black 10B dye binding* (32,33).
Reagents
1. Amido Black 10B
2. Citric acid
3. Disodium hydrogen phosphate
4. Thymol blue (optional preservative)
Procedure
0.05M Citrate0.01 M phosphate buffer. Dissolve citric acid (52.6 g),
sodium dihydrogen phosphate (3.3 g), and thymol blue (1 g) in
660 mL of water.
Working dye solution (0.075% w/v). Dissolve Amido Black 10B (3 g) in
1 L of water by heating to 708C. Mix with citrate-phosphate buffer
and add 3.33 kg of water.
Reference dye solution. Dilute the working dye solution (1 volume)
with 2.5 volumes of distilled water.
Add 1 mL of sample to 20 mL of dye solution. Mix for 0.53 minutes.
Filter to remove insoluble dye-protein complex. Dilute the super-
natant 100-fold with phosphate buffer. Record A620 readings.
Determine the amount of free dye from a calibration graph of A620
plotted against reference dye solutions.{
For calibration analyze at least 10 samples of known protein
concentration in duplicate.
Methods 1 and 2 were readily scaled down by mixing 50100 mL of
sample with 1 mL of dye reagent in a polyethylene microcentrifuge tube. The
protein-dye precipitate was then removed by microcentrifugation (13,000
rpm; 5 minutes). Absorbance readings were recorded after diluting the dye-
supernatant solution 100-fold. For strongly colored samples, the dry weight
for the protein-dye complex was recorded. These micro-dye-binding assays
led to signicant savings of reagent and improved convenience (34).

3. SOLID-PHASE DYE-BINDING ASSAYS

Protein is adsorbed on a lter support such as nitrocellulose, lter paper, or

a glass ber lter. Sometimes, the adsorbed protein is ``xed'' by treating

* Amido Black 10B dye binding is also called the ProMilk method.
{ Absorbance measurements can be taken without dilution if using the purpose-made 0.3 mm
path length cuvette. Specic instructions are given for use with commercial instruments.
132 Chapter 5

with dilute TCA. Exposure to dye solution is followed by a destaining

solution to remove excess dye. The dyed protein spot is then excised and
placed in a test tube with elution solvent, which dissolves the protein-dye
complex. Absorbance measurements are then recorded as before. The
advantages of the solid-phase assay include (a) increased sensitivity and (b)
improved resistance to interfering compounds. In the best cases, an LLD of
0.25 mg is attainable with a linear range extending to 200 mg of protein.

3.1. Nitrocellulose and Cellulose Acetate Membranes

Kuno and Kihara (35) employed nitrocellulose membranes for solid-phase
dye-binding assays.* The linear range for analysis was 1050 mg protein. The
efciency of protein binding was >97%. Compared with the Lowry assay,
there was increased resistance to interference from tyrosine or Tris. Heil and
Zillig (36) analyzed protein (0.52.5 mg) using cellulose acetate membranes.
Staining was with Amido Black 10B (0.24% w/w) dissolved in (10:45:45)
acetic acidmethanolwater solvent. The same solvent was used for
destaining. Protein spots were air dried, excised, and eluted with 0.5 mL
of solvent (glacial acetic acid, formic acid, water, TCA).
Schaffner and Weissmann (37) treated protein samples with 10% (w/w)
TCA and recovered the resulting precipitate with a Millipore (HAWP
025CO) membrane lter. The rest of their methodology is essentially as
already described. The linear dynamic range was 530 mg with a sensitivity
of 0.027 DA630 mg 1 BSA. The LLD was 1.5 mg + 5% for a sample volume
of 2 mL. The following compounds did not interfere: dextran
(100 mg mL 1), polyethylene glycol (0.5 mg mL 1), glycogen (0.5 mg mL 1),
RNA or DNA (30 mg mL 1), NaCl (>1 M), ammonium sulfate (2.5 M),
magnesium chloride (>0.1 M), EDTA (>100 mM), 2-mecaptoethanol
(>100 mM), SDS (1%), and sucrose (20%).
The Schaffner-Weissmann method was modied for protein analysis
in the presence of 1000-fold excess lipid (38).BSA (20 mg) was accurately
determined in the presence of 20 mg phospholipid (43% phosphatidylcho-
line, 30% phosphatidylethanolamine, 27% unidentied lipids). The linear
dynamic range (224 mg BSA) was unaffected by added phospholipid. Assay
sensitivity was also unaffected by the presence of lipid. The A630 was 0.744
(+ 0.054) and 0.775 (+ 0.048) for 20 mg of BSA without and with 20 mg of

* Protein (550 mg) dissolved in 0.2 M magnesium chloride was ltered with a nitrocellulose
membrane under suction. Each membrane was stained with 2 mL of Amido Black 10B
(4 mg mL 1 in acetic acidmethanolwater (1:5:4) solvent). After destaining with 5 mL of acetic
acid (1% w/w), membranes were eluted with 3.5 mL of 10 mM NaOH. The released dye was
measured (A620).
The Udy Method 133

lipid, respectively. There was compatibility with a great many buffer salts
(200 mM): NaCl, KCl, Na phosphate, K phosphate, HEPES, Tris-HCl,
MES, MOPS. However, protein-protein variations in the dye response were
evident. The proceding technique was apparently more accurate than the
modied Lowry assay or biuret method.
The speed for solid-phase analysis was increased by Nakamura and
co-workers (39). They used microltration apparatus* to apply multiple
samples to nitrocellulose membranes. Stained protein spots were measured
directly via a densitometer. The linear dynamic range for analysis was 110
mg. Sensitivity using Amido Black 10B staining was twofold higher than
with Ponceau Red. For both dyes the order of increasing sensitivity was
trypsin < lysozyme < cytochrome c < bovine serum albumin < human serum
albumin < concanavalin A < histone II < human g-globulin. Assay results
were unaffected at pH 3.69. There was no interference from a range of
salts, sugars, amino acids, nucleotides, polyols or EDTA. Detergents (SDS,
Triton X-100, Tweens) or high concentrations of denaturants reduced
protein binding to the nitrocellulose membrane.

3.2. Whatman Paper and Glass Membrane Filters

Protein samples (5200 mg) were dried on to Whatman No. 42 paper
followed by treatment with 7.5% (w/v) TCA (40). McKnight (41) spotted
100 mL of protein (0.55 mg) on glass ber lters (Whatman GF/C), dried
the liquid using hot air, and then xed the protein with 20% (w/v) TCA.
Esen (42) and Almand and Saleemuddin (43) dried protein solutions (5 mL;
14 mg mL 1) on Whatman No. 1 with no TCA xation step. The lter
paperbound protein was stained with Amido Black 10B or Coomassie
Brilliant Blue G250 (see Chapter 7).

4. THE CHEMISTRY OF DYE-BINDING PROTEIN ASSAYS

4.1. Characteristics of Azo Dyes
Azo dyes represent 60% of all known dye structures (44). The rst azo
compounds were synthesized by Peter Gries in 1858 using building units
designated A, D, E, M, and Z (Fig. 2). Mono-azo dyes were formed via
electrophilic attack of a diazotized species (A) on a sulfonated amino-

* BIO-DOT apparatus; Bio-Rad Laboratories, Richmond, CA. Membrane-bound protein was

stained using Amido Black 10B or Ponceau Red (0.1% w/v) dissolved in 7% acetic acid and
destained with 7% (w/v) acetic acid.
134 Chapter 5

FIGURE 2 Building blocks for synthesis of azo dyes with a brief explanation of A,
D, E, M, and Z notation. (Top) Diazotized (group A) compounds (left)
or (right) a tetrazotized (group D) compound. (Bottom) Coupling agents
with a capacity to react with one equivalent or two equivalents of a
group A compound. M is an aromatic amine that can react with A. The
product may be diazotized for a second round of coupling.

naphthol nucleus (E or Z). By altering the reaction pH, temperature, and

concentration of reagents, diazo, triazo, or tetrakisazo dyes may be formed.
The structure of azo dyes can written in shorthand: A?Z refers to a mono-
azo dye produced by reacting a diazotized aromatic compound
(A benzenediazonium chloride) with Z. Examples of A?Z dyes are
Acid Orange 12, T-azo-R, and Acid Orange 1. A typical diazo dye is Amido
Black 10B (1-amino-2p-nitrophenylazo-7-phenylazo-8-naphthol-3,6-disulfo-
nic acid) with the formula A1?Z/A2. The A1 and A2 units are attached to
a central (Z) unit, 1-amino-2-naphthol-3,6-disulfonic acid. These structures
are shown in Figs 35.
All azo dyes possess one or more azo (22N55N22) groups. The
nitrogen-nitrogen double bond allows cis-trans isomerism. The naphthalene
2-hydroxyl group hydrogen bonds to the azo-group nitrogen, thereby
stabilizing the trans isomer. The characteristics of some azo dyes are listed in
Table 2. The absorptivity for Acid Orange 12 (w/v) is 26% higher than for
Orange-G. The molar extinction coefcients for the two dyes differ by only
about 6%. The sulfonic acid group of azo dyes remains ionized at most
The Udy Method 135

FIGURE 3 The structure of T-azo-R and Acid Red 1.

accessible pH values. However, the exact acidity of benzenesulfonate or

naphthylenesulfonate groups is uncertain (pKa * 0.71.5).

4.2. Protein Dye Binding

Azo dyes bind with the guanidino, imidazole, and the e-NH2 side chain of
arginine, histidine, and lysine, respectively (4,5,7,4548). Interactions with
wool (composed of the protein keratin) occur via ionic bonding. Further
136 Chapter 5

FIGURE 4 The structure of Acid Orange 12 and Orange G.

The Udy Method 137

FIGURE 5 The structure of Amido Black 10B.

bonding is by van der Waals and hydrophobic interactions. These increase

with the area of contact between the dye and protein (44). Nonionic
interactions become more important at high dye/protein ratios.
Formerly, the order of sensitivity for dye-binding assays was given as
Amido Black 10B > Acid Orange 12 > Orange-G (21,22,26). Protein assays
using Orange G were thought to be 100% less sensitive than assays using
Acid Orange 12 because the two dyes bound 2 or 1 mole of arginine per
mole of dye, respectively. However, more recent data (47) show that both
Orange G and Acid Orange 12 form 1:1 mole complexes with protein basic
amino acid residues. For Amido Black 10B the ratio of dye bound to basic
amino acids is 1:0.5 (Table 3) (49,50). The small distance of separation
between the sulfonate groups of Orange G may exclude binding to two sites.

TABLE 2 Characteristics of Some Acid Azo Dyes Used for Protein Assay

Dye AB 10B AO 12 OG

C.I. No. 20470 15790 16230

Molecular weight 616.50 350 452.38
Net charge 1 1 2
lmax 620 482 480
e (Abs mL/mg)a 81.5 (43,684) 59.0 (22,066) 46.9 (20,683)
AB10, Amido Black 10B; AO12, Acid Orange 12; OG, Orange G.
a
Extinction coefcient, absorbance per unit concentration of dye bound (mg mL 1). Value in
parentheses is molar extinction coefcient.
Source: Refs. 21 and 26.
138 Chapter 5

TABLE 3 Dye-Binding Capacity for a Range of Samples for Orange G (OG), Acid
Orange 12 (AO 12), and Amido Black 10B (AB 10B)
1
DBC (mmoles g cP)a

Sample OG AO12 AB 10B

BSA 1365.0 1775.7 743.5

HSA 1466.8 1780.0 834.4
HGG 1028.8 1354.3 582.8
k-Casein 692.5 857.1 274.4
Meat meal 630.5 905.7 339.3
Fish meal 708.0 920.0 336.0
Milk protein 800.9 1094.3 490.3
Soybean 984.5 1345.7 555.2
Average 959.6 1254.1 519.5
Ratiob 0.8 1.0 0.54
a
Dye-binding capacity (mmoles of dye bound per gram protein) from Ref. 50.
b
Average dye: BAA ratio. BSA, bovine serum albumin; HSA, human serum albimin; HGG,
human gamma globulin.

In summary, careful perusal of information in Table 3 shows that the

sensitivities of dye-binding assays using Orange-G, Acid Orange 12, and
Amido Black 10B are the same.

4.3. Soluble Protein Dye Complexes

Protein-dye complexes can be studied by spectrophotometry or equilibrium
dialysis (51). To avoid precipitate formation, the concentration of dye used
(110 mM) is 3501000 times below those used for the Udy assay. BSA
binding with azosulfathiozole, Orange I, Orange II, methyl orange, and
tetrazine yellow was investigated by Klotz et al. (52), Klotz (53), and
Sheppard et al. (54). Pesavento and Profumo (55) examined T-azo-R
binding to BSA. Other interesting reports describe protein binding to phenol
red (3), bromophenol blue (56,57), thymol blue (58), and the reactive dye
cibracron blue (5961).
Protein dye binding shifts the equilibrium between nonionized and
ionized dye forms. The extinction coefcient for the bound dye (eb) increases
while the wavelength for maximum absorption (lmax) shifts to lower values.
The hyperchromic effect is explained by reference to the conjugation theory.
The lmax for dye molecules is determined by the energy required for p?p*
electron transition. Protein binding alters the degree of conjugation
The Udy Method 139

involving the p orbital and lowers the energy of the p* state. Lysine,
arginine, or histidyl (auxochromic) groups donate electrons to the dye
molecule, thereby increasing its conjugation extent. A further explanation
centers on the transfer of free dye molecules from a polar low-viscosity
solvent phase to a relatively nonpolar or restricted protein phase. Dye
transfer to more nonpolar solvents and micelles leads to spectral changes
resembling those observed during protein binding (6264).
Usually, a xed concentration of dye is exposed to increasing amounts
of protein. Absorbance readings are recorded with a reference cuvette
containing a dye solution of the same concentration as the sample cuvette
(Table 4). Measuring the ``difference absorption'' (DA) is useful where a dye
solution has a high background. The absorbance change for dye reagent
depends on the total dye concentration (D), extinction coefcient (ef), and
the cuvette path length (1 cm) as described in Equation (6).

A1 ef D 6

Df, protein, and the protein-dye complex are in equilibrium. Db has its own
extinction coefcient (eb). The net absorption change is described by

TABLE 4 A Summary of Symbols Used in Describing Protein-Dye Binding

Symbol Denition

A1 and A2 Absorbance for dye and dye protein

DA ( A2 A1) Difference absorbance
a Fraction of dye bound
D Total concentration of dye
Db Concentration of bound dye
Df Concentration of free dye
ef Extinction coefcient for free dye
eb Extinction coefcient for bound dye
De ( ef eb) Extinction coefcient difference for the free and
bound dye
eapp Apparent molar extinction change when a fraction of
dye is bound
P, Pf Added, free concentration of protein
Kd Conditional dissociation constant
limax Wavelength for maximum absorbance
liso Isobestic wavelength where De 0
n, ns Number of dye molecules bound per molecule
protein; ns strong sites
140 Chapter 5

Equation (7).

A2 ef D Db eb Db 7

From Eqs (6) and (7) it can be seen that A2A1 DA Db(eb ef) and also

A2 A1 Db eb ef
or eapp aeb ef ef 8
D D

and therefore

eapp ef
a 9
eb ef

where a is the fraction of dye bound and eapp ( A2/D) is the apparent
extinction coefcient change when dye is bound.
The isobestic point (liso) is the wavelength at which bound and free
dye molecules have equal absorptivity (ef eb). By running absorbance
spectra with increasing dye or protein concentration, liso can be identied as
the wavelength at which there is no absorbance change (DA 0). The
existence of an isobestic point is indication that the dye exists as two
interconvertible forms (e.g., bound and free). No isobestic point will appear
if ef = eb over the wavelength range examined. The corollary is that protein-
dye binding will not generate an absorbance change if De 0.

4.4. Analysis of Protein Dye-Binding Reactions

The protein dye-binding reaction is summarized by the following equation

Df nPf Db 10

Replacing Db with DA/De, we can dene the dissociation constant (Kd) as

D DA=DenP DA=De
Kd 11
DA=De

The concentration of dye species changes with pH and ionic strength.

Therefore, Kd is a conditional constant with a value that depends on the pH
and ionic strength (52). Depending on the protein/dye ratio Eq. (11) takes
on the two forms described in Cases 1 and 2.
The Udy Method 141

Case 1, Low dye/protein ratio. With excess protein we have

nP (DA/De) & nP in Eq. (11). This approximation is also justied if the
number of binding sites is large; hence,

D DA=DenP
Kd
DA=De

and

nDePD
DA 12
Kd nP

For high protein concentrations (nP >10Kd) DA reaches a maximum

(DAmax) where

DAmax
De 13
D

Equation (13) is the chief means by which De and also eb may be determined
(65). First, invert all terms in Eq. (12). The resulting double-reciprocal
relation [Eq. (14)]* allows the determination of DAmax by graphical means
(see the following).

1 Kd 1
14
DA DeDnP DeD

Multiplying the former relation by DADeD gives Eq. (15). Other linearized
forms result from multiplying Eq. (15) by 1/(DeKd) or 1/(DeKdD).

DAKd
DA DeD 15
nP

DA D DA
16
nPDe Kd DeKd

DA n nDA
17
PDDe Kd DeDKd

* The transformation is analogous to linearization of the Michaelis-Menten equation to give the

Lineweaver-Burke double reciprocal plot, Eadie plot, Hanes plot, etc.
142 Chapter 5

Finally, Equation (17) may be restated as

nD nGP
G 18
Kd Kd
where G Db/P.
To evaluate DAmax, De, and Kd/n, proceed as follows:
1. Add varying concentrations of protein to a xed concentration of
dye (D).
2. For each sample measure DA.
3. Using Equation (14), plot a graph of 1/DA (Y-axis) versus 1/P
(X-axis). From the X 0 intercept nd 1/DAmax ( 1/DeD).
4. Use the estimate for DAmax and nd De from Eq. (13).
5. From the slope and known values for De and D calculate Kd/n. It
is not possible to determine Kd independently using Eqs (14)(17).
An alternative stratagem is to translate DA values to Db (e.g.,
Db DA/De) and Df ( D DA/De). Thereafter, use Eq. (18) to
evaluate all binding parameters.
Note that Eqs (12)(18) are valid only at high protein/dye ratios.
Under these circumstances, only high-afnity protein sites (strong sites) are
occupied. Binding parameters therefore relate to strong sites. The number of
strong binding sites (ns) is distinct from the total number of sites (n).

Case 2. High dye/protein ratio. With excess dye D (DA/De) & D in

Eq. (11); therefore,
DenPD
DA 19
Kd D
Eq. (19) describes protein ligand binding when a small xed concentration
of protein is exposed to varying concentrations of dye. As the concentration
of dye increases (e.g., D > 10Kd), DA increases to a maximum value (DAmax)
and Eq. (19) becomes*
DAmax nDeP 20

Using the De value determined before (see Case 1), nd the total number of
binding sites (n) as follows:
1. Add varying concentrations of dye to a xed concentration of
protein.

* A high dye concentration is dened in relation to Kd and not protein concentration.

The Udy Method 143

2. For each mixture measure DA.

3. Plot a graph of 1/DA (Y-axis) versus 1/D (X-axis). The X 0
intercept yields 1/(nDeP) and the slope is Kd/nDeP.
4. Calculate the number of binding sites from Eq. (21).
n DAmax =DeP 21

Dividing the graph slope by the intercept gives Kd under

conditions such that both strong and weak binding sites are lled.
In summary, two different experimental designs and analyses for
protein dye binding are possible. Case 1 employs a xed (low) concentration
of dye and varying amounts of protein. The estimates of De, Kd, and n
obtained are related to high-afnity sites. Case 2 employs a xed (low)
concentration of protein and varying (high) concentrations of dye. This
study is useful mainly for determining the total number of binding sites.
Values of Kd are average parameters for both weak and strong dye-binding
sites (57,58,65).
The application of these relations to a study of BSA binding with
T-azo-R is described next. A xed concentration of dye (D 10.8 mM in
5 6 10 3 M HCl solvent, pH 2.3) was titrated with increasing concentrations
of BSA (see Case 1). Fig. 6 shows the pattern of binding of T-azo-R to BSA
(55). At concentrations of dye above 100 mM, an insoluble protein-dye
complex formed. Apparently T-azo-R binding to BSA could not be
analyzed using the Scatchard plot (55). I have reanalyzed such data and
others from Refs. 57, 58, and 65 using Eqs (14), (15), and (18) (Figs 6 and 7).
Parameters for BSA binding with T-azo-R, bromophenol blue, and thymol
blue are shown in Table 5.
The original studies were not designed to measure the total number of
(low- and high-afnity) dye-binding sites. The proportion of basic amino
acids (*110 per mole BSA) functioning as high-afnity binding sites for
T-azo-R did not exceed 50%. With bromophenol blue there was dye binding
to a very small proportion of basic amino acids.

4.5. Solubility Relations for Protein Dye Complexes*

The reaction between a charged dye molecule (D ) and protein (P )
produces a soluble, complex [PD]AQ that later forms an insoluble complex

* There is no strict adherence to the use of squared brackets to indicate concentration. Brackets
are included only where their presence renders equations more readable.
144 Chapter 5

FIGURE 6 Analysis of protein-dye binding. A xed concentration T-azo-R dye

(D 10.8 mM) was titrated with increasing concentrations of bovine
serum albumin (P 06 mM). (Top graph) Difference absorbance
changes monitored at 510 nm (DA510) plotted versus total added protein
concentration (P). (Lower graph) Determination of binding parameters
using a double reciprocal plot of 1/P versus 1/DA. The Y-intercept
is 1/DAmax. Furthermore, DAmax/D De [see Eq. (13)]. The slope
Kd/(nDeD).
The Udy Method 145

FIGURE 7 Analysis of T-azo-R binding with bovine serum albumin. Same data as
shown in Fig. 6. (Top graph) Determination of binding parameters in
accordance with Eq. (15). DA is plotted versus DA/P. The slope is Kd and
intercept is DAmax. (Bottom graph) Determination of binding parameters
according to Eq. (18). As P ? 0, then Db ? nP and Kd * D. Under such
conditions it follows that G n (see Refs. 58 and 59).
146 Chapter 5

TABLE 5 Binding Parameters for Soluble Serum AlbuminDye Complexes

Kd 1
Dye, equation n mM 1 De (M cm 1) n

T-azo-R
Eq. (14) 0.162 4709
Eq. (15) 0.177 5024
Eq. (18) 0.181 62.a
Bromophenol blue
Eq. (14) 3.11 87787
Eq. (15) 2.03 69250
Eq. (18) 2.23 6.0
Thymol blue
Eq. (18) 69.0 1430 26
a
The intercept from Equation (18) n. See Refs. 57 and 58.

[PQ]S.

P D PDAQ ; Kd P D =PDAQ 22

PDAQ PDS ; K2 PDAQ =PDS 23

The net reaction is

P D PSS 24

The overall process is comparable to isoelectric precipitation (4). Precipita-

tion occurs when sufcient numbers of dye molecules bind to neutralize all
protein charges. In contrast, excess protein produces a ``colloidal protective
effect'' that maintains the solubility of protein-dye complexes. From the
denition of solubility product (KS) we have

KS Kd K2 P D 25

The activity for [PQ]S is given a value of 1. To describe the effect of pH on

protein-dye interactions, consider the ionization of dye-binding sites (pKa
*12.5);

P H P ; Ka Pf H =P 26

The total protein concentration (P) [P ) [Pf] and after substituting for
Pf,

Ka P P H =P 27
The Udy Method 147

and
P
P 28
Ka =H 1

The concentration of P changes with the concentration of H in

accordance with Equation (28) and consequently KS is given by Equation (29).
PD
KS 29
Ka =H 1

To attain very low concentrations of soluble protein (in equilibrium with the
protein-dye precipitate) requires a high dye concentrations at a low pH.
Strong protein-dye binding (small Kd) will also facilitate quantitative
precipitation of protein from solution. At pH 4.84 the gelatin complex with
Amido Black 10B yields KS & 4 6 10 12. Under higher acidity conditions
the value KS was too low to determine (4). Refer to Skoog and West (66) for
more information on KS-solubility relations.

5. INTERFERENCE COMPOUNDS AND THEIR AVOIDANCE

There was no interference from low-molecular-weight compounds, includ-

ing amino acids and peptides. Lipids do not affect protein-dye binding.
Ionic surfactants and benzoic acid derivatives (e.g., p-aminobenzoic acid)
might interfere if present in high concentrations. Chaotropic agents such as
urea are also likely to affect dye-binding results. Dye binding with
nonprotein food components is possible. Calibration graphs for wheat
our protein had a nonzero intercept owing to dye binding with wheat b-
glucan (67). Mass transfer or diffusion limitations may be important for
solid or powdered food materials. Physical effects can be overcome by
achieving high sample agitation, increasing the mixing time, and reducing
sample particle size.

6. APPLICATIONS OF DYE-BINDING ASSAYS FOR FOOD

PROTEIN ANALYSIS
6.1. Milk, Ice Cream, and Dairy Products
Udy (9) analyzed whole milk and spray-dried milk samples by Orange-G
binding. The milk samples and Kjeldahl protein values were supplied by
Ashworth and co-workers at the Department of Dairy Science, Washington
State University (Pullman, WA). Dye-binding studies at Ashworth's
148 Chapter 5

laboratory led to one of the rst Ph.D. dissertations in this area (68).
Literature covering milk or dairy protein analysis using Orange-G and Acid
Orange 12 is summarized in Table 6.
Ashworth et al. (24) analyzed 354 milk samples from six breeds of
cows. Milk powders were also analyzed. The average protein content for
milks was 3.49 (+ 0.273)%. Some 95% of protein determinations were
within + 0.67% of the crude protein content. NPN, proteose peptone, milk
fat, and lactose caused little or no interference. Sample preservatives
(hydrogen peroxide, formaldehyde, or mercuric hydrochloride) also did not
affect the results. Adding mercuric chloride (1.35 mg %) to milk samples
allowed room temperature storage before analysis. Antibiotics were not
effective preservatives.

TABLE 6 Dye-Binding Assay of Milk and Dairy Protein

Dye, application Reference

Orange-G
Milk (fresh, powder) Udy (9), Ashworth et al. (24), Dolby
(25), Ashworth and Chaudry (26), and
Conetta et al. (69), Park and King (70)
Milk (fresh, evaporated, powdered), Ashworth (20)
buttermilk, cheese, sherbet, cream, ice
cream
Ice cream, frozen desert Kroger et al. (71)
Acid Orange 12
Milk (fresh, evaporated, powdered), Ashworth (20)
buttermilk, cheese, sherbet, cream, ice
cream
Milk Sherbon (21)
Chocolate milk drink, buttermilk Sherbon and Luke (72)
Nonfat dry milk powder, ice cream, Sherbon and Luke (73)
half-and-half
Various dairy products Sherbon (74)
Milk Conetta et al. (69), Lakin (75,76),
Wilkinson and Richardson (77),
Cheese Kristoffersen (78)
Various dairy products, NFDM Sherbon and Fleming (79),
Ice cream, ice milk, diet ice cream, Bruhn et al. (80)
dietetic ice cream
Other dyes
Milk powder (delactosed)Ramazol Rawson and Mahoney (81,82)
Blue R
The Udy Method 149

The DBC for milk protein fractions was assessed by Ashworth and
Chaudry (26). Milk protein fractions should have similar DBC values;
otherwise, assays may be affected by variations in milk composition.
Compared with whey proteins, caseins had a lower DBC (Table 7).
Presumably the proportions of casein and whey protein remain fairly
constant in different milk samples. The quantity of protein in several brands
of milk drink (chocolate milk, two-ten, half-and-half, vitamin D milk, etc.)
were determined by Ashworth (20).
The Orange-G binding capacity for milk proteins was remarkably
constant, notwithstanding processing into products such as ice cream and
evaporated milk (Table 8). Fresh milk had a protein content of 3.5%,
whereas evaporated milk contained 7% protein. Cheese manufacture had a
signicant lowering effect on DBC, probably because of proteolysis. Notice
that the values for the DBC are 50% lower than those reported in Table 3
for reasons discussed earlier.

6.2. Ofcial Approval of Dye-Binding Assays

Collaborative studies led to dye-binding assays being granted AOAC
approval for milk protein determination (21,72,83). The studies involved
laboratories attached to the ofces of the U.S. Federal Milk Market

TABLE 7 Apparent DBC for Different Milk Protein Fractionsa

1 1
Milk protein (fraction) AB 10B (mmoles g cP) Orange G (mmoles g cP)

Whole milk 566.6 389.4

Skim 561.7 396.0
NFDP 595.8 407.1
Whole casein 589.3 446.9
Paracasein 556.8 415.9
a-Casein 577.9 398.2
k-casein 592.5 420.4
b-casein 504.9 376.1
Whey protein 730.5 539.8
b-Lactoglobulin 763.0 557.5
Proteose peptone 584.4 278.8
Average 602.1 420.6
SD 76.1 76.2
a
DBC was calculated from Ref. 26 using dye molecular weights from Table 2. Values are
between 50 and 100% lower than expected from the basic amino acid content in milk proteins
(see Table 3).
150 Chapter 5

TABLE 8 Dye-Binding Capacity and Protein Content for Different Milk Productsa

Product OG (mmoles g 1) AO12 (mmoles g 1) Protein (%)

Milk
Fresh 393.5 1000.0 3.5
Evaporated 386.8 932.8 7.0
Powdered, nonfat 394.6 1000.0 36.0
Powdered, whole 366.9 963.6 26.0
Buttermilk 381.1 902.0 4.0
Cottage cheeses
Creamed 394.8 966.4 15.0
Dry curd 394.8 966.4 18.0
Cheddar cheese
Mild 306.8 801.1 24.0
Sharp 245.4 700.3 24.0
Cream
2050% fat 394.8 1000.06 2.5
Ice cream
Vanilla 394.8 1000.0 4.0
Chocolate 362.3 963.6 4.0
a
A comparison with data from Table 3 shows that the values for OG are lower than expected.
Source: DBC values calucalated from Ref. 21.

Administrators.* The extent of interlaboratory differences was considered

practically insignicant. The agreement between dye-binding results and
Kjeldahl results was excellent. Dye binding was granted ofcial rst action
status for protein quantitation in fresh milk, dairy chocolate milk drink,
cultured buttermilk, and half-and-half cream.
A collaborative study to examine dye-binding assays for ice cream mix
and nonfat dry milk powder is described by Sherbon and Luke (73). Five
commercial samples of vanilla ice cream mix and 10 samples of nonfat dry
milk were analyzed in powder form or after reconstitution. Seven
laboratories performed dye-binding assays while three did Kjeldahl analysis.
Tests using nonfat dry milk powder gave the same results as for the
reconstituted material. The protein content for nonfat dry milk was 34.946%
as determined by Kjeldahl analysis and 35.141% by dye binding. The
average difference between Kjeldahl and dye-binding results was 0.195%.

* One lot of sterile, canned milk was analyzed by ve laboratories. Three laboratories carried
out crude protein determination by the Kjeldahl method. A further 25 fresh milk samples were
analyzed via 140 replicate determinations.
The Udy Method 151

Ice cream mix had 3.852% protein or 3.968% protein by the Kjeldhal and
dye-binding analysis, respectively. Consequently, dye binding received
AOAC approval as a method for protein determination in ice cream and
nonfat dry milk Analysis of ice cream mix and ice cream proteins using dye
binding is further described by Kleyn (84) and by Bruhn (85). Further
examples of these studies are listed in Table 9.
Sherbon (32) compared the Pro-Milk and Udy methods for the
analysis of milk proteins. The two techniques gave comparable results.
Greater care was needed in calibrating the Pro-Milk instrument. A
recommendation to grant ofcial status to the Pro-Milk method was
deferred pending further work. A year later, a six-laboratory study of the
Pro-Milk method led to AOAC approval (33). In addition to reports cited in
Table 9 the Pro-Milk Analyzer is discussed in Refs. 8691.

6.3. Wheat and Other Cereal Proteins

Udy (8) found that wheat albumin, gluten, albumin gluten, and residue
proteins bound constant amounts of Orange-G regardless of the variety of
seed. These observations paved the way for a quantitative analysis of wheat

TABLE 9 Application of Amido Black 10B for Milk and Dairy Protein Analysisa

Amido Black 10B Reference

Cheese Kroger and Weaver (92)

Condensed milk Lueck (93)
Ice cream, frozen dessert Kroger et al. (71)
Milk Dolby (25), Ashworth and Chaudry
(26), Radcliffe (94), O'Connell (86),
Conetta et al. (69), Sherbon (95),
Kroger (96), Uzonyi (97), Patel et al.
(98), Ng-Kwai-Hang and Hayes (99),
van Reusel and Klijn (100)
Whey protein, casein Roper and Dolby (101), McGann et al.
(102), Renner and Ando (103),
Reimerdes and Flegel (104)
Milk (goat) Grappin et al. (105), Mabon and
Brechany (106)
Milk (mastitis) Waite and Smith (107)
Skimmed milk powder Sanderson (108), O'Connell and
McGann (109)
a
The majority of investigators used the Pro-Milk Analyzer
152 Chapter 5

protein using dye binding (9). In all, 128 samples of whole wheat our and
218 samples of rened wheat our (from 58 known and 34 unknown wheat
varieties) were examined for Orange-G binding capacity at pH 2.2. The
samples were also analyzed for crude protein content by the Kjeldahl
method. The correlation between amounts of dye bound and crude protein
(%N 6 6.25) is described by Equation (30) (rened wheat our) or Equation
(31) (whole wheat our).
cP 1:092Db 4:62 30

cP 1:000Db 5:53 31

The nonzero intercept indicates dye binding to nonprotein components,

probably starch and/or wheat bran. Greenaway (110) at the U.S.
Department of Agriculture (USDA; Beltsville, MD) reported a positive
correlation between dye binding and Kjeldahl results for soft wheat (< 10%
protein), hard winter wheat, hard spring wheat, and durum wheat;
cP 0:8842X 1:7938 R 0:988 32

where X (% protein) is wheat protein content determined from dye binding

and cP Kjeldahl protein (%N 6 6.25). Over 220 protein assays were
performed. Methods were compared with respect to average protein
content, correlation coefcients, and standard error of estimates. The dye-
binding assay gave reliable estimates for protein content for hard red spring
wheat. For other classes of wheat, protein results were slightly lower than
expected from Kjeldahl analysis. The mean difference between Kjeldahl and
dye-binding tests was  0.5%. For wheat samples having less than 10%
protein, dye-binding and Kjeldahl results differed by *1%.
The reliability of the Udy method was compared with that of ve other
techniques (Kjeldahl, alkaline distillation, biuret, Dumas, and infrared
reectance) for wheat protein determination by Pomeranz and More (111).
Reliability encompasses specicity, accuracy, precision, sensitivity, and the
LLD (Chapter 1). Dye binding was the least precise method tested.
Interestingly, no strong case is made for preferring any one method. Forty-
ve varieties of rice produced in the 1966 season in the Philippines by the
International Rice Research Institute (IRRI) were analyzed for protein
(112). A typical set of results highlighting the performance of the dye-
binding assay for rice protein analysis is given in Table 10.
Barley and malt proteins were analyzed by Pomeranz et al. (113) using
ve methods including the Udy method. About 120 samples each of barley
and malt from all over the United States were analyzed. No details of the
dye-binding assay were given other than a reference to Udy's 1971 paper
The Udy Method 153

TABLE 10 Determination of Rice Protein Using Acid Orange 12 Dye-Binding

Assay

Parameter Milled rice Brown rice

Regression linea Y 14.67 13.60A485 Y 14.78 14.12A485

Protein (%) 5.5511.65 6.0011.95
SY.X (%) + 0.48 + 0.26
R 0.961 0.984
a
Y crude protein content (%). Protein (%) is range for 45 samples. SY.X (%) standard
deviation from the regression line. Data are from experiments using a laboratory shaker (112).

(22). The correlation coefcient for dye-binding and Kjeldhal results was
0.974 (barley) or 0.984 (malt). The average mean squared error for analysis
(with the Kjeldahl method as reference) was 0.897%. Using commercial
apparatus, 200 protein determinations were completed daily.
Baker and Hunt (114) evaluated the Pro-meter instrument (Foss
America Inc) for dye-binding analysis of wheat protein. About 107 wheat
samples were ground to pass 20-mesh screen and then analyzed according
the instrument manufacturer's instructions. The graph of instrument
response versus protein content was curvilinear for 50 samples of red wheat
(hard red spring, hard red winter, durum wheat). By contrast, a linear
calibration graph was obtained for 57 white wheat samples. The Pro-meter
instrument was judged satisfactory despite some mechanical difculties. The
lter system was periodically clogged, necessitating dismantling and
cleaning of the measuring unit.

6.4. Legumes and Other Seed Proteins

The Udy method is applicable to range of legumes including, chickpeas,
cowpeas, gram, mungbeans, peas, and soybean (Table 1). Pomeranz (115)
analyzed 24 soy our samples using Orange-G and commercial apparatus
from the Udy Corporation. The results were compared with the biuret and
Kjeldahl methods. A highly signicant correlation was found between
protein content assessed by dye binding (X, %) and crude protein
(N 6 6.25%). For samples containing up to 80% protein, the regression
equation was
cP 1:003X 4:559 R 0:989 33

The standard error of analysis was 1.8213%. Flour particle size had
negligible effects on protein results. Mild heat did not affect dye-binding
154 Chapter 5

results although other studies show that severe heating reduces the DBC of
soy proteins (116120).
Romo et al. (121) assessed seed protein extractability using the Udy
method. The following DA482 changes were noted for the different seed
protein solutions (10 mg mL 1): 1.363 (eld bean), 1.197 (cowpea), 2.976
(rapeseed), 2.2454 (sesame seed), and 1.203 (cotton seed). Clearly, the assay
sensitivity is different for different seed proteins. Medina et al. (122)
proposed that a single calibration graph might be used for cereal, legume,
and oilseed protein analysis. A composite graph would save time. Sesame
our, rapeseed meal, and rapeseed our were analyzed by the standard Udy
(shaker mixing) method. Fig. 8 shows a composite calibration graph for
Acid Orange 12 binding to cereal, legume, and oilseed proteins. The least-
squares equation for the composite graph is*

cP 0:2152Db 4:7333 R 0:981 34

FIGURE 8 A composite calibration graph relating dye binding (X-axis) and crude
protein content for *28 samples of legumes, cereals, and oilseeds.
(Drawn from Table IV in Ref. 123.)

* Actually, the regression equation reported in the literature was Y 0.245X 2.532
(R 0.995). In contrast, Fig. 1 was drawn using only 50% of the experimental data.
The Udy Method 155

Apparently the average DBC for Acid Orange 12 is 465 (+ 17.11) mg g 1

(cP) or 1328.6 mmole g 1.
The Udy method was further optimized for seed protein determination
(122). Vacuum drying (558C) or atmospheric drying (1008C) had no effect
on dye binding. Improving the degree of mixing, extending the shaking time
from 30 to 150 minutes, and/or reducing the particle size to 40 mesh
increased the perceived sample protein content. Table 11 summarizes results
for sesame our, rapeseed our, and rapeseed meal. For all cases, a good
correlation was obtained between dye binding and Kjeldahl results.
Rapeseed protein was also determined by Goh and Clandinin (123).
Twelve commercial meals and two laboratory samples were analyzed using
the Udy method with Orange G dye reagent. The investigators also
examined Acid Orange 12 as a dye reagent. Rapeseed meal had 30.144.8%
(w/w) protein. For Orange-G the least-squares line relating dye binding and
Kjeldahl results was

cP 0:49Db 3:91 R 0:93 35

For Acid Orange 12 dyes the corresponding equation is

cP 0:28Db 0:36 R 0:98 36

From such data it may be shown that the apparent DBC for Orange-G is
204 mg (dye) g 1 (cP) or 490 mmoles (dye) g 1 (cP). With Acid Orange 12 the
DBC is 357 mg g 1 (cP) or 580 mmoles (dye) g 1 (cP). The calibration data
were not affected by our particle size (40 or 60 mesh). However, DBC was
higher for a protein/dye ratio of 2:1 as compared with a ratio of 4:1. Values
for the DBC were proportional to the net concentration of arginine,
histidine, and lysine. The standard deviation for analysis was 1.3% (Orange
G) or 0.80% (Acid Orange). From the higher DBC (per weight), precision,

TABLE 11 Protein Content in Sesame and Rapeseed Products

Determined from Acid Orange 12 Dye Binding and Kjeldahl
Analysis

Sample Protein (% w/w)a

Dye binding Kjeldahl

Sesame our 59.4 (+ 0.524) 58.9 (+ 1.093)

Rapeseed our 59.4 (+ 1.743) 60 (+ 2.91)
Rapeseed meal 36.1 (+ 0.595) 36 (+ 0.338)
a
Values are mean (+ SD).
Source: Summarized from Ref. 122.
156 Chapter 5

and sensitivity of analysis, Goh and Clandinin (123) concluded that Acid
Orange 12 was a more suitable dye reagent for rapeseed protein
determination.

6.5. Fish, Meat, and Egg Products

A. Animal Feedstuffs
Bunyan (124) determined the protein content of feedstuffs containing
animal protein. The procedure using Orange-G was essentially as given in
Method 2. The dye-binding response was dependent on sample particle size.
The extent of dye binding also increased with mixing time. With care, values
of Db could be correlated with the protein content (Table 12). However,
animal feeds were found to be highly heterogeneous owing to their different
manufacturing and thermal histories. A number of meat meal samples had
an unusually high content of gelatin. In one case, meat meal was positively
identied as feather meal (techniques for establishing protein authenticity
are described in Chapters 911). It was concluded that dye-binding assays
were not suited for animal feedstuffs. Differences in processing history,
protein quality, and possible adulteration led to large variations in results.

TABLE 12 Analysis of Protein Content of Animal Feeds Using Orange G Dye

Binding

DBC
Sample (na) Regression line (mmole g 1 cP) % Error (CV)

Meat meal cP 0.278Db 30 796925 6.4

(21)
Whale meat cP 0.216Db 30 842770 2.0
meal (12)
Fish meal (8) cP 0.325Db 24 675 2.3
Soy bean (8) cP 0.217Db 28 1020 2.0
or Groundnut
meals (6)
Miscellaneous cP 0.414Db 12
foodsb
a
n number of different feed samples.
b
Including casein, dried blood protein, egg, brewer's yeast, roller dried milk, and grass meal.
Source: Summarized from Ref. 124.
The Udy Method 157

TABLE 13 Protein Determination in Raw Meat Using Orange G Dye Binding

Sample Regression linea R DBC (mg g 1

cP)

Beef cP 0.301Db 8.18 0.90 DBC 209.2 1.135cP

Chicken breast cP 0.602Db 2.50 0.94 DBC 265.5 3.721cP
Pork loin cP 0.367Db 5.45 0.80 DBC 271.2 4.040cP
Cod llet cP 0.632Db 3.00 0.95 DBC 246.9 3.397cP
a
Symbols cP and Db are as dened previously.
Source: Summarized from Ref. 125.

B. Meat Proteins
Raw beef, chicken, pork (loin), and cod llets were analyzed using Orange-
G or Amido Black 10B* dye binding by Torten and Whitaker (125). Their
procedure was described in Chapter 4. A signicant correlation was
observed between crude protein values (Kjeldhal-N 6 6.25) and Db (Table
13).
The DBC for raw meat proteins decreased linearly with increasing
sample protein (see last column of Table 13). The amount of dye bound
depended on the dye/protein ratio. In general, inadequate amounts of
Orange-G were used in many early studies. Dye limitations and inadvertent
changes in protein/dye ratio for different assays reduced the reliability of
dye-binding assays. The regression equation (cP 0.301Db 8.18) for beef
applies over a restricted range of protein content. The effect of a changing
DBC is shown in the simulations reported in Fig. 9. One set of results are
computed on the basis that the DBC is xed. Where DBC varies the dye/
protein ratio the simulated calibration graphs were nonlinear (Fig. 9). The
curves are remarkably like actual calibration curves reported for ground
pork and chicken (125). These samples showed a high dependence of DBC
on protein content and large deviations from linearity. A linear equation did
t the data but only over a highly restricted range of protein content.
Ground chicken, pork loin, or cod llet having greater than 50% crude
protein content should probably not be analyzed by Orange-G dye binding.
It was on account of the dependence of DBC on protein content that Amido
Black 10B was judged unsuitable for meat protein analysis.

* As Amido Black 10B was found to be unsuitable for raw meat analysis, the following
discussion focuses on results obtained with Orange-G.
158 Chapter 5

FIGURE 9 The effect of a changing dye-binding capacity on calibration graphs for

protein analysis in raw meat samples using Orange G binding. Shaded
squares show normal response according to regression equations in
Table 13. The open circles show simulated graphs for the assay response
when DBC changes with sample protein content.
The Udy Method 159

TABLE 14 Acid Orange 12 DBC of Egg and Meat Products

1
Meat product DBC (mg g cP)a

Egg (whole) 410440

Egg albumin (egg white) 390410
Chicken meat 460480
Chicken liver 360390
Beef or pork (ground) 430440
Beef liver 420440
Proteose peptone 90145
Gelatin 310350
a
Ranges of values are given for analysis performed in the presence of
excess of dye concentration of 0.40.6 mg mL 1.
Source: Ref. 126.

C. Egg, Chicken, and Meat Protein

Egg, chicken, and meat products were analyzed by Ashworth (126) (Table 14).
As he was one of the rst investigators to apply dye-binding assays to foods,
his approach merits attention. Reliable results were obtained provided that
the free Acid Orange 12 concentration (after shaking with the protein sample)
was kept within the range of 0.40.6 mg mL 1. To achieve this, the initial the
dye/protein ratio was kept within a range of 0.640.92. Pork had the same
DBC as beef, which was lower than the value of chicken. The DBC for
mixtures of meat could be deduced from values for individual components.
Dye binding was not affected by the presence of fat or by normal cooking
(1608C, 40 minutes). It was concluded that dye binding is useful method for
composition control in ground meats, eggs, and prepared mixes.

D. Sausage Protein
Seperich and Price (127) determined protein in model sausage emulsions and
muscle components (myobrillar protein, sarcoplasmic protein, and stroma)
from which they were produced. The approach was modied from Ref.
128.* These studies conrmed that protein dye binding was not affected by
sausage emulsion fat content from 20 to 40%. The DBC was a function of

* Sausage emulsion samples (3.5 g) were homogenized with 51 mL of citrate (0.2 M)phosphate
(0.1 M) buffer (pH 5.5). Ten milliliters of the resulting homogenate was retained for Kjeldahl
analysis. The remainder was shaken with 80 mL of Acid Orange 12 (0.563.64 mM; 0.2
1.27 mg mL 1) in a 250-mL centrifuge tube for 30 minutes and then centrifuged (5.680g; 5
minutes).
160 Chapter 5

dye/protein ratio. At the highest dye concentration examined the DBC was
of the order of 400 mg g 1 (cP), in line with values reported by other
investigators. However, DBC decreased to about 3334 mg g 1 (cP) at a dye
concentration of 0.2 mg mL 1.

6.6. Mushrooms
Nine strains of Agaricus bisporus (Lange) Imbach were analyzed by Weaver
et al. (128) using dye binding, Kjeldahl, and quantitative amino
acid analysis.*. The average protein content was 29.4 (+ 6.2)% by Kjeldahl
analysis, 22.4 (+ 2.4)% by dye binding, and 28% (+ 3.4)% by amino acid
analysis. Per wet weight basis, Agaricus had 2.62.8% protein. Quantitative
amino acid analysis was more correlated with dye-binding analysis
(R 0.74) than Kjeldahl analysis (R 0.4). Mushrooms are thought to
contain high amounts of NPN, which could lead to errors in Kjeldahl
analysis. Braaksma and Schaap (129) reported the protein content for
Agaricus as 0.5% fresh weight or 7% per dry weight basis (Chapter 7).

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milk powder by dye binding. N Z J Dairy Sci Technol 5:4648, 1970.
109. JA O'Connell, TCA McGann. Rapid estimation of protein in skim milk
powders. Ir Agric Cream Rev 25(110):1719, 1972.
110. WT Greenaway. Comparisons of the Kjeldahl, dye binding, and biuret
methods for wheat protein content. Cereal Chem 49:609615, 1972.
111. Y Pomeranz, RB More. Reliability of several methods for protein determina-
tion in wheat. Bakers Dig 49:4448, 58, 1975.
112. LC Parial, LW Rooney, BD Webb. Use of dye-binding and biuret techniques
for estimating protein in brown and milled rice. Cereal Chem 47:3843, 1970.
113. Y Pomeranz, RB Moore, FS Lai. Reliability of ve methods for protein
determination in barley and malt. J Am Soc Brew Chem 35:8693, 1977.
114. D Baker, WH Hunt. Pro-Meter evaluation. Cereal Foods World 20:246247,
1975.
115. Y Pomeranz. Evaluation of factors affecting the determination of nitrogen in
soya products by the biuret and Orange-G dye-binding methods. J Food Sci
30:307311, 1965.
116. T Hymowitz, FI Collins, SJ Gibbons. A modied dye-binding method for
estimating soybean protein. Agron J 61:601603, 1969.
117. L Sandler, FL Warren. Effect of ethyl chloroformate on the DBC of protein.
Anal Chem 46:18701872, 1974.
118. IM Perl, MP Szakacs, A Koevago, J Petroczy. Stoichiometric dye-binding
procedure for the determination of the reactive lysine content of soya bean
protein. Food Chem 16:163174, 1985.
119. I Molnar-Peal, M Pinter-Szakacs, D Medzihradszky. Dye-binding ``stoichio-
metry'' and selectivity of cresol red with various proteins. Food Chem 35:69
80, 1990.
120. S Lin, AL Lakin. Thermal denaturation of soy proteins as related to their dye-
binding characteristics and functionality. J Am Oil Chem Assoc 67:872878,
1990.
121. CR Romo, AL Lakin, EF Rolfe. Properties of protein isolates prepared from
ground seeds. I. Development and evaluation of a dye binding procedure for
the measurement of protein solubility. J Food Technol 10:541546, 1975.
The Udy Method 167

122. MB Medina, DH Kleyn, WH Swallow. Protein estimation in sesame seed and

rapeseed ours and meals by a modied Udy dye binding method. J Am Oil
Chem Assoc 53:555558, 1976.
123. YK Goh, DR Clandinin. The estimation of protein in rapeseed meal by a dye-
binding method. Can J Anim Sci 58:97103, 1978.
124. G Bunyan. Orange-G binding as a measure of protein content. J Sci Food
Agric 10:425430, 1959.
125. J Torten, JR Whitaker. Evaluation of the biuret and dye-binding methods for
protein determination in meats. J Food Sci 25:168174, 1964.
126. US Ashworth. Proteins in meat and egg products determined by dye binding. J
Food Sci 36:509510, 1971.
127. GJ Seperich, JF Price. Dye binding procedure for the estimation of protein
content of meat components and sausage emulsions. J Food Sci 44:643645,
1979.
128. JC Weaver, M Kroger, LR Kneebone. Comparative protein studies (Kjeldahl,
dye binding, amino acid analysis) of nine strains of Agaricus bisporus (Lange)
Imbach mushrooms. J Food Sci 42:364366, 1977.
129. A Braaksma, DJ Schaap. Protein analysis of the common mushroom Agaricus
bisporus. Postharvest Biol Technol 7:119127, 1996.
6
The Bradford MethodPrinciples

1. INTRODUCTION

Proteins bind with Coomassie Brilliant Blue G250 (CBBG,* C.I. 42655) to
produce a sparingly soluble complex (1). Protein-dye binding alters the
absorption spectrum for CBBG. This is the basis of the assay developed by
Bradford in 1976 (2). The Bradford assay has several advantages compared
with the Lowry test: (a) four- to tenfold greater sensitivity, (b) tenfold
greater speed, (c) decreased susceptibility to interferences, (d) requirement
for a single reagent, and (e) lower cost. The Bradford assay is quicker than
dye-protein precipitation (Udy assay) as no ltration step is required.
Ready-to-use CBBG dye reagent is available from Bio-Rad Laboratory
Ltd., Pierce Warriner Ltd., and the Sigma-Aldrich Chemical Company.
Principles of the Bradford assay are described in this chapter. Applications
for food protein analysis are discussed in Chapter 7.
Coomassie Blue is the trade name for a group of dyes rst produced by
Imperial Chemical Industries (ICI) Ltd. (UK). Weiler discovered CBBG in
1913. The 1971 edition of the Colour Index (3) lists 40 Coomassie dyes
including Coomassie Blue FF (C.I. 42645), Coomassie Blue R (C.I. 42660),
Coomassie Blue BL (C.I. 50315), Coomassie Brilliant Blue G (C.I. 42655),
Coomassie Blue GL (C.I. 50320), and Coomassie Blue RL (C.I. 13390). The

* Abbreviations: CBBG, Coomassie Brilliant Blue G250; CBBR, Coomassie Brilliant Blue
R250.

169
170 Chapter 6

FIGURE 1 The structure of Coomassie Brilliant Blue G250 (CBBG). Coomassie

Brilliant Blue R250 (CBBR) lacks two methyl groups.

G and R labels refer to dyes having a greenish blue or reddish blue hue.
Samples of dye with 2.5 times greater purity than the standard grade (19
22% purity) are labeled ``250.'' CBBG is also known as C.I. Acid Blue 90,
Xylene Brilliant Cyanine G, or Brilliant Blue G (4).
CBBR* was rst used as a protein stain in 1963 by Fazekas de St.
Groth et al. (5). Cellulose acetate electrophoresis support was soaked in
sulfosalicylic acid to x protein bands and then transferred to the CBBR
solution (0.25% w/v in water). Blue protein bands form against a clear
background. Nonspecic staining increased if CBBR dye was prepared with
methanol rather than water. From a densitometric analysis of polyacryla-
minde gels, the blue color was proportional to protein amount (020 mg). In
later developments, polyacrylamide gels were stained with CBBR dissolved
with a 5:1:5 mixture of methanol, acetic acid, and water or 12.5% (w/v) TCA
(6).
Diezel et al. (1) introduced CBBG as a protein stain for electrophor-
esis. CBBG has two methyl groups more than CBBR (Fig. 1) and is
therefore less soluble in 12% TCA. This lowers dye penetration into
polyacrylamide gels and reduces background staining. The sensitivity of

* CBBR (CI 42660) is also known as Acid Blue 83, Coomassie Blue R, Xylene Brilliant Cyanine
6b, or Supranolcyanin 6B.
Bradford MethodPrinciples 171

CBBG for protein is also signicantly greater than CBBR.* Reisner et al. (7)
used perchloric acid (3.5%) as solvent for CBBG. The free dye exists as a
colorless (leuco) molecule in perchloric acid solvent. Binding to protein
leads to a blue protein-dye complex. There was virtually no background
staining for polyacrylamide gels. Assay sensitivity was comparable to that
obtained with Naphthylamine Black 10 (0.5% w/v) stain.{

2. THEORY OF THE BRADFORD ASSAY

Protein-protein variations in the sensitivity of the Bradford assay (8,9) led to

interest in CBB-protein interactions. Coomassie Brilliant Blue binds with
proteins by electrostatic interactions. The complex is also held together by
van der Waals forces (5). The rst quantitative investigation of CBBR
binding with proteins was reported by Tal et al. (10). Dye binding occurs
only with polypeptides larger than about 3000 daltons. The number of dye
molecules bound per molecule of protein (n) was strongly correlated with
the total number of arginine, histidine, and lysine (Arg His Lys)
residues. However, the average DBC was 100% greater than combined
numbers of basic amino acid residues. Hydrophobic interactions may
account for dye binding when charged protein sites were saturated.
Rosenthal and Koussale (11) determined the critical micelle concentration
(cmc) of nonionic surfactants with the Bradford reagent. Hydrophobic
solubilization of CBBG within micelles led to marked increases in
absorption at 620 nm.

2.1. Overview of CBBG Protein Binding

Compton and Jones (12) assessed the effect of pH, protein, and surfactants
on the absorption spectrum of CBBG. They concluded that CBBG exists in
three ionized forms rather than two. The absorptivity of protein-CBBG
complexes increased with protein molecular weight (13). Signcantly higher
color yields were obtained for CBBG binding with polyarginine, polylysine,
or polyhistidine. There was no dye binding with polyaspartic acid,
polyglutamic acid, and polyproline. Lea et al. (14) recorded anomalous
results with the Bradford assay for highly basic proteins. Chemical

* A polyacrylamide slab gel (10 6 15 cm) is soaked in 40 mL of 12.5% TCA for 5 minutes to x
protein. Then 2.5 mL of CBBG solution (0.25% w/v dissolved in water) is added and the gel is
incubated for 1530 minutes. Transferring the gel to a 5% (w/v) acetic acid solution for 12 hours
increases protein band intensity and reduces background staining.
{ This dye is the same as Amido Black 10B (Chapter 5).
172 Chapter 6

modication to lower the numbers of positive charges increased (rather than

decreased) the assay response for polylysine and histone. The expected
reductions in assay sensitivity occurred when a more concentrated dye
reagent solution was used (15).
Protein-CGGB binding parameters were reported by Chial and
Splittgerber (16), who also characterized the Bradford assay at pH 1 and
pH 7. Congdon et al. (17) reported the dissociation constant for CBBG-
protein binding. Although not involving CBBG, the study of Cibracron blue
binding to lysozyme (18) is a ne example of contemporary methods for
studying protein-dye binding. Atherton et al. (19) apply these principles to
the CBBG system.

2.2. The Compton-Jones Scheme for CBBG Ionization

CBBG dye shows two lmax values at 470 and 650 nm at pH 0.8* (12).
Adjusting the dye reagent to pH 1.2 produced the following changes: (a)
decreased absorbance at 650 and 470 nm and (b) a new absorbance peak at
595 nm. There was no isobestic point, meaning that more than two
interconverting dye species occurred over the pH range examined. In a
different experiment, addition of 140 mg of BSA to CBBG dye reagent
produced a difference spectra (dye protein versus dye) with lmax at 595
nm. Exposure of CBBG to excess SDS diminished the 470 peak and
produced a new peak at 650 nm.
Shareef and Shetty (20) identied a fourth CBBG charged species. The
purple bi-ionic form (CBBG2 , lmax 515 nm) appears at pH 11.5. With
acidic conditions (pH 1.25) Coomassie Blue dye is a positively charged/
cationic/red (CBBGH2)1 species (lmax 475 nm). This is in equilibrium
with other CBBG forms. (CBBGH2)1 is converted to the zero-charge/
neutral/green (CBBGH)0 species (lmax 650 nm) at about pH 1.6. There-
after (CBBG)1 , which is the anionic blue form (lmax 595), is produced
at pH 1.8pH 7. The Compton-Jones scheme for CBBG ionization is
summarized in Table 1. Crystal violet shows a similar three-state ionization
as it changes color from violet to green to yellow at pH 8 to pH 2.4 and
pH 0.6 (21);

R1 violet?RH2 green?RH2 3 yellow 1

Similar transformations occur with many other triphenylamine dyes

(rosaniline, para-rosaniline, malachite green, aniline blue).

* Compton and Jones reported that the Bradford reagent had a pH of 0.8. Our own
measurements found pH 1.25.
Bradford MethodPrinciples 173

TABLE 1 The Compton-Jones Scheme Showing the Three-State Ionization of

CBBG

Dye form Anion Neutral Cation

Structure (CBBG)1 (CBBGH)0 (CBBGH2) 1

Net charge 1 0 1
Color Blue Green Red/leuco
lmax(nm) 595 650 470475
ve charges 1 2 3
ve charges 2 2 2
pH* 1.87 1.6 1.25

2.3. Spectrophotometric Analysis of CBBGProtein

Binding
It is not easy to decide whether protein-bound CBBG is charged or not.
Compton and Jones (12) stated, ``based on the identical lmax for the dye-
protein complex and the blue dye anion, . . . the bound dye species is in fact
the dye anion. The (dye ionization) equilibrium shown above are forced to
the left as the anion is bound by protein.'' CBBG binding to serum albumin
leads to lmax 620 nm when the protein is present in excess (22). By
contrast, lmax 595 nm with excess dye. A lmax value of 620 nm does not
match the absorption maximum for any of the dye forms in solution (470,
595, or 650 nm; Table 2). Two possible models may be proposed for protein-
CBBG binding.

A. Binding Scenario 1Excess Protein

Just prior to binding there is the anionic form (CBBG)1 in solution. Dye
binding is with the positively charged protein site (RNH3)1. Protein-bound
CBBG has a net charge of zero.

CBBG1 RNH3 1 ?CBBG:RNH3 0 2

Electrostatic intereactions will neutralize charges on the dye and protein

molecule, forming (CBBG.RNH3)0. The analogous reaction occurs between
(CBBG)1 and the hydrogen ion:

CBBG1 H1 ?CBBGH0 3

Table 1 shows that lmax is 650 nm for the neutral dye species (CBBGH)0 in
174 Chapter 6

solution. For the protein-bound nuetral dye form (CBBG.RNH3)0 we nd

lmax is 620 nm. To explain the hypsochromic shift from 650 to 620 nm,
consider the different dye environments for (CBBG.RNH3)0 and
(CBBGH)0. The lmax shifts from 650 to 600620 nm when (CBBGH)0 is
internalized within the nonpolar environment of micelles formed by
nonionic detergents. One implication is that charged protein sites for dye
binding are (within) nonpolar environments. With scenario 1, the Bradford
assay can be performed at 620 nm. Eq. (2) partly explains the correlation
between DBC and total numbers of basic amino acids per molecule of
protein. However, excess protein leads to the occupancy of strong binding
sites (Chapter 5).

B. Binding Scenario 2Excess Dye

Just prior to binding, the free dye form is the blue (CBBG)1 species. Dye
binding involves hydrophobic interactions with one of two types of neutral
protein sites, Ro or (CBBGRNH3)0. Protein-bound CBBG has a net charge
of 1.

CBBG1 R0 ?CBBGR1

or

CBBG1 CBBGRNH3 0 ?CBBG2 RNH3 1 4

Dye binding shifts the dye ionization equilibrium toward (CBBG)1 and
produces an absorbance increase at 595 nm. Equation (4) accounts for the
use of A595 readings for the Bradford assay. Dye binding with R0 does not
account for the correlation between DBC and the number of positively
charged basic amino acid residues. In contrast, the number of
(CBBGRNH3)0 sites is determined by the number of basic amino acids.
The Bradford assay can be monitored at either 620 or 595 nm. The
alternative dye-binding scenarios are not mutually exclusive. As described in
Chapter 5, dye binding involves both nonpolar and ionic sites. CBBR binds
to proteins with a dye basic amino acid ratio ranging from 1:1.5 to 1:2 (10).
Compton and Jones (12) suggest that 60% of the Gibbs free energy change
for protein-CBBG binding (*40 kJ mol 1) is due to the nonpolar structure
(benzene and methylene groups) of the dye. The remaining binding free
energy (*30 kJ mol 1) arises from protein interactions with the sulfonate
groups of CBBG.
The metachromatic properties of crystal violet provide relevant
insights (23). In contemporary terms, metachromasia is a change in the
dye absorption spectra due to changes in the dye environment. The lmax
Bradford MethodPrinciples 175

may shift to shorter or longer wavelengths. Sometimes, the absorbance peak

increases without lmax shifting. Crystal violet showed metachromatic
behavior due to (a) increasing dye concentration, (b) addition of low
concentrations of ammonium sulfate, or (c) binding to sites followed by
optically signicant interactions between dye molecules. Low concentrations
of ammonium sulfate induce the dimerization of crystal violet. The singly
charged R (violet) form [see Eq. (1)] binds to phosphate groups from
nucleic acids with a ratio of 1:1. For this reaction, lmax remains unchanged
but the absorption peak increases. Crystal violet binding to agar sulfonate
groups apparently shifts lmax from 585 to 510 nm as the peaks at these
wavelengths decrease and increase, respectively. Each agar sulfonate group
binds several dye molecules. The shift in lmax was ascribed to ``stacking
interactions'' as excess dye molecules adsorb to dye molecules initially
bound by electrostic interactions. Dye stacking is essentially a nonpolar
process like those leading to dimerization. Examples of metachromatic
phenomena were reported for dyes with nonionizing quaternary nitrogen
groups; the process does not require dye ionization.
Evidence from spectral measurements also supports two modes of
CBBG-protein binding. With excess protein lmax 620 and (CBBG)1
binds to positively charged sites. The formation of a neutral 1:1 protein-dye
complex is accompanied by a metachromatic shift of lmax by *30 nm. By
contrast, excess dye favors nonpolar interactions probably involving
stacking. Eq. (4) shows two different hydrophobic binding sites; R0 sites
include the side chains of tyrosine, tryptophan, phenylalanine, leucine, and
isoleucine. (CBBGRNH3)0 is produced when (CBBG)1 binds to a charged
protein group. The (CBBG)1 binding with nonpolar sites accounts for the
lmax value of 595 nm. Perhaps lmax is the same for (CBBGR)1 and
CBBG1 because R0 sites are highly hydrated. Given the amphipathic
nature of the CBBG molecule (Fig. 1), it is likely that the principal binding
sites (R0 and RNH4) are also amphipathic.

2.4. Quantitative Analysis of CBBG Binding to Proteins*

Protein-dye binding parameters (Kd, n, and De) were reported by Klotz
(24,25), Klotz et al. (26), and also Aizawa (27). From the law of mass action,
D DA=DenP DA=De
Kd 5
DA=De

The concentration of bound dye (Db) is DA/De while Df D Db and

* All symbols are dened in Chapter 5, Sections 4.3 and 4.4.

176 Chapter 6

therefore

Df nP Db
Kd 6
Db

Db Kd Df nPDf 7

and
nPDf
Db 8
Kd Df

Eq. (8) describes protein-ligand binding with n independent sites.

Linearization of this relation leads to Eqs (9)(11) for extracting binding
parameters (Kd and n).
1 Kd 1
9
Db Df nP nP

Multiplying this double-reciprocal equation with nPDb and rearrangement

lead to
Db n Db
10
PDf Kd PKd

Substituting G Db/P, we obtain the equation for the Scatchard plot.

G n G
11
Df Kd Kd

The more familiar version of Eq. (11) (Db/Df nP/Kd Db/Kd) is usable
where the protein concentration is kept constant. Most dye-binding studies
employ a constant concentration of dye while the protein concentration is
varied.
Two studies of CBBG-protein binding have been published. Congdon
et al. (17) segregated protein binding sites for CBBG into ``strong'' and
``weak'' sites. To characterize strong binding sites, different amounts of BSA
(041 mM) were added to a xed concentration (20 mM)* of CBBG. From a
graph of 1/DA versus 1/P the x 0 yields a reciprocal for the maximum

* The concentration of CBBG was 0.0166% (w/v) or 200 mM. To a xed volume (100 mL) of dye
reagent solution was added 02.66 mg of BSA. The nal volume of mixture was brought to
1 mL where needed with distilled water. Then DA620 was recorded for each protein
concentration.
Bradford MethodPrinciples 177

absorbance change (1/DAmax) at an innite protein concentration. From

this, De DAmax/D. The absorbance measurements were also used to
estimate the number of strong sites. First, values for Db ( DA/De) and Df
( D Db) were determined for each protein concentration. Then results
were analyzed using Scatchard and related equations.
To determine the total number of binding sites, a xed concentration
of CBBG (133 mM) was exposed to 0.5100 mg of BSA (0.0250.512 mM).
The conditions (excess dye) are similar to those used for the normal
Bradford assay. The number of binding sites was determined from the
relation (16)

dDA
nDe 12
dP

The left-hand side of Eq. (12) is supposedly the maximum gradient from the
Bradford assay standard curve.* The results for CBBG binding to seven
proteins are reported (Table 2).
For BSA binding with T-azo-R, Eq. (11) gives a poor t to the results.
The presence of two classes of binding sites (e.g., strong and weak sites)
should lead to curvature in the Scatchard plot (28,29) although this has not
been demonstrated. Because few graphs for CBBG-protein binding have
been published, I have reexamined the data for BSA binding with T-azo-R

TABLE 2 Parameters for Coomassie Brilliant Blue G250 Binding to Selected

Proteinsa
1
Protein Kd (mM) n(ns) eb(M cm 1)

BSA 18.6 105 (2.7) 48,000

Alcohol dehydrogenase 40.0 30 (7) 57,700
a-Lactalbumin 110.0 14 (2.4) 55,400
Glutamate dehydrogenase 8.9 ? (2.2) 57,900
Chymotrypsin 80.0 13 (2.3) 55,700
Ovalbumin 23.0 33 (1.9) 41,900
Carbonic anhydrase 35.0 28 (2.8) 51,900
a
nS number of strong binding sites (in parentheses) and Kd are average values from the
modied Scatchard plot [Eq. (11)] and Hill plot [Eq. (15)].
Source: Based on results from Ref. 18.

* Eq. (12) is not an appropriate expression for the assay sensitivity. The right-hand expression
should nDeD/Kd (Section 3 of this chapter). The consequence of using Equation (12) to
estimate n is described in Section 5.
178 Chapter 6

(see Chapter 5, Figs 5 and 6 and associated text). The data from Ref. 30
were replotted using the Scatchard equation [Eq. (11)] or modied
Scatchard relations [Eqs (13)(15)].
1 n 1
13
Df GKd Kd

1 Kd 1
14
G nDf n

G 1 n 1 Kd
15
G n nDf

Equations for the straight lines and binding parameters are reported in
Table 3.
The results show a poor t to the Scatchard plot; the regression
coefcient (R) for the graph shown in Fig. 2 was 0.7310. From the equation
of the straight line Kd 11.6 mM and nS 106. Other equations led to more
gratifying transformations of the data. Using Eqs (13)(15), R 0.9941. In
Table 3, two data entries are shown for each graph. In the rst case, values
for Kd and nS were assessed assuming that all data conform to a straight line
for a single class of binding sites.
Figs 25 show deviations from linearity at the extremes. The second
data entry in Table 3 is derived from results tted to the main linear phase in
each graph. Eqs (13) and (15) emphasize data collected at high protein

TABLE 3 Parameters for T-Azo-R Binding to Bovine Serum Albumin Analyzed

Using Scatchard and Modied Scatchard Plots

Linearization
equation Equation Kd (mM) n
6 4
Eq. (11) Y 9.21 6 10 8.62 6 10 X 11.6 106
Eq. (13) Y 1.06 6 107 X 164 6 105 6.1 64
(strong sites)a 3.8 43
8 2
Eq. (14) Y 9.26 6 10 X 164 6 10 5.6 61
(weak strong sites)a 19.61 76
8
Eq. (15) Y 0.984 9.26 6 10 X 5.8 63
(strong sites)a 4.2 48
a
Different equations emphasize data collected at a high protein/dye ratio (strong sites) or a low
protein/dye ratio (weak strong binding sites). Second data entries are calculated using the
major linear phase of each graph.
Bradford MethodPrinciples 179

FIGURE 2 Scatchard plot for T-Azo-R binding with bovine serum albumin. Dye (10
mM) was titrated with 06 mM BSA. Study was performed at pH 2.3.
Data from Ref. 31 plotted in accordance with Eq. (8).

concentrations and dominated by strong binding sites. Eq. (14) emphasizes

both weak and strong dye binding sites. It appears there are 4348 strong
binding sites on the BSA molecule for T-azo-R (Table 3). By comparison,
ns 26 for thymol blue and 6 for bromophenol blue (see Table 5, Chapter
5). The number of strong binding sites depends on the type of dye and also
the reaction conditions. Counting the numbers of weak as well as strong
binding sites using a single experiment may require the approach described
by Rosenthal (28).
Chial and Splittgerber (16) assessed protein-CBBG binding at pH 1
and pH 7. The total number of binding sites (n) was determined using Eq.
(12). Detailed procedures are the same as reported by Congdon et al. (17). A
summary of results is given in Table 4. The assay at pH 7 had decreased
sensitivity. In a number of cases (BSA, a-lactalbumin, carbonic anhydrase)
the sensitivity change from pH 1 to pH 7 could apparently be explained
using Eq. (12). A 40-fold decrease in assay sensitivity for BSA at pH 7
compared with pH 1 could arise from 2-fold and 20-fold decreases in the
values n and De, respectively. Eq. (12) predicts a sensitivity change of 2 6 20
( 40-fold). The second footnote in this section suggests that this agreement
is fortuitous.
180 Chapter 6

FIGURE 3 Modied Scatchard plot for T-Azo-R binding with bovine serum
albumin. Dye (10 mM) was titrated with 06 mM BSA. Study was
performed at pH 2.3. Data from Ref. 31 plotted in accordance with
Eq. (10).

FIGURE 4 Modied Scatchard plot for T-Azo-R binding with bovine serum
albumin. Dye (10 mM) was titrated with 06 mM BSA. Data from Ref.
31 plotted in accordance with Eq. (14).
Bradford MethodPrinciples 181

FIGURE 5 Modied Scatchard plot for T-Azo-R binding with bovine serum
albumin. Dye (10 mM) was titrated with 06 mM BSA. Data from Ref.
31 plotted according to Eq. (15).

In Table 4 the molar absorptivity for protein-bound CBBG at 620 nm

(eb,620) was not affected by solvent pH. Hence eb,620 was 49 (+8.3) 6 103
M 1 cm 1 at pH 1 or 53 (+6.0) 6 103 M 1 cm 1 at pH 7.0. In contrast, the
free dye had an extinction coefcient (ef,620) of 43,700 M 1 cm 1 at pH 7.0
and 8800 M 1 cm 1 at pH 1.* Consequently, we have De 39.2 6 103
M 1 cm 1 at pH 1 and De 9.3 6 103 M 1 cm 1 at pH 7. The reaction time
for protein-dye binding was about 60 minutes at pH 7 compared with about
5 minutes at the pH for the normal assay.

2.5. Identifying CBBG Binding Sites on Proteins

The relative color yield for CBBG binding to different poly-L-amino acids
was poly-L-lysine (1), poly-L-histidine (4.2), poly-L-tryptophan (4.4), poly-L-
tyrosine (4.7), and poly-L-arginine (36) (12). With increasing dye concentra-
tion there was a more similar CBBG response to different basic amino acids

* It is not certain whether these calculations were corrected for changes in the dye ionization
with pH. The low ef,20nm value at pH 1 could be due to a sixfold lower concentration of blue
(neutral) dye species at pH 1 compared with pH 7.
 182

TABLE 4 Effect of pH on Dye Binding Parameters and Sensitivity of the Bradford Assaya
Sensitivityb
1
Protein (DA620 mg 1)6104 De620(M cm 1) n Lys Arg
(pH 1.0)
Alcohol dehydrogenase 127.0 49,200 28 32
Bovine serumalbumin 193.0 39,200 100 86
Carbonic anhydrase 125.0 43,200 25 27
Chymotrypsin 101.0 47,200 16 17
a-Lactalbumin 143.0 44,200 14 13
b-Lactoglobulin 84.0 25,200 18 18
Ovalbumin 87.0 34,200 33 36
(pH 7.0)
Alcohol dehydrogenase 2.6 11,300 2
Bovine serumalbumin 4.8 18,300 5
Carbonic anhydrase 0.9 7,300 1
Chymotrypsin 2.1 1,300 2
a-Lactalbumin 15.0 9,300 6
b-Lactoglobulin 3.0 12,300 12
Ovalbumin 0.9 8,300 2
a 1 1 1 1
ef (620) 43,700 M cm (pH 7) or 8800 M cm (pH 1).b Sensitivity as determined from the maximum slope of the calibration graph.
Source: Ref. 17.
Chapter 6
Bradford MethodPrinciples 183

(10); DA595 readings were 0.92, 1.2, and 1.5 for poly-L-lysine, poly-L-
histidine, and poly-L-arginine, respectively. These studies agree on the
importance of poly-L-arginine as a CBBG binding site. Moreno et al. (13)
reported a relative color yield from different polyamino acids as poly-L-
lysine (1), poly-L-tyrosine (1.9), poly-L-arginine (3), poly-L-histidine (>5.5).
CBBG binding increased with the polypeptide molecular weight, but
differences in color yield are less marked when absorbance changes are
expressed per unit mass (microgram) of material analyzed.
In summary, studies involving CBBG/R binding to poly-L-amino
acids indicate that these dyes bind to basic groups. Nonpolar amino acid
residues, notably tyrosine and tryptophan, are also important binding sites.
Additional nonpolar sites for CBBG binding are created as the anionic dye
molecule binds to positively charged protein sites. However, poly-L-amino
acid results should be treated with caution. Proteins rarely feature the high
density of sites associated with homopolymers. The correlation between
CBBG binding and the number of basic amino acids occurs when ionic
bonding predominates and the dye species is limiting (Chapter 5). The
relations between protein-binding parameters (n, Kd, De) and characteristics
of the Bradford assay are discussed in the next section.

3. EFFECT OF PROTEIN-DYE BINDING PARAMETERS ON

THE BRADFORD ASSAY

The plot of DA versus protein concentration leads to a hyperbolic

calibration graph.*
nDePD
DA 16
Kd nP
Eq. (16) is a result of the equilibrium between free dye, protein, and the
bound dye. At low protein concentrations nP << Kd and
DA nDeDP=Kd 17

Therefore, a graph of DA versus P gives a straight line with a gradient equal

to the sensitivity F, where
SensitivityF nDeD=Kd 18

A linear calibration graph will be obtained when nP << Kd. For a linear
relationship between DA and [P] it is necceary that Kd exceeds protein

* This relation is the same as Equation (12) of Chapter 5.

184 Chapter 6

concentration multiplied by the number of binding sites per molecule of

protein. Eq. (18) also predicts that assay sensitivity will increase with (a)
number of dye binding sites, (b) molar absorptivity change due to dye
binding, (c) protein-dye binding afnity (proportional to 1/Kd), and (d)
increasing dye reagent concentration. Assay sensitivity will also be
independent of the concentration of protein (provided nP << Kd). Another
prediction from Eq. (18) is that protein-protein variations in assay results
are not inevitable. Assume that De is the same for most proteins. Using BSA
as standard, the relative assay sensitivity for different proteins can be stated
as F/FBSA, where
F n=Kd
% 19
FBSA n=Kd BSA

Protein-protein variations in assay sensitivity are related to values for n

and Kd. These parameters are not immutable. Encouraging nonspecic
protein-dye binding may reduce protein-protein variations in sensitivity
(see later).

4. LINEARIZATION PLOTS FOR THE BRADFORD ASSAYS

A double reciprocal plot (1/DA versus 1/[P]) or log(DA) versus log[P] will
extend the linear range of a hyperbolic Bradford calibration graph
(Chapters 3, Sections 3 and 4). Other linearization schemes have been
proposed for the Bradford assay. Sedmak and Grossberg (31) increased the
upper limit of linearity from 10 mg BSA to 50 mg by plotting A620/A456 versus
protein amount. Bearden (32) obtained an upper limit of linearity of 40 mg
BSA by plotting the difference between A595 and A465 readings against the
amount of BSA. Zor and Selinger (33) plotted A595/A450 versus protein
concentration. These linearization schemes allow for (a) the hyperbolic
protein-dye binding prole and (b) the overlap of the absorbance spectra for
the free dye and bound dye.

5. ASSAY SENSITIVITY AND THE MAXIMUM NUMBER OF

DYE BINDING SITES

Eq. (18), which properly describes the sensitivity of the Bradford assay,
reduces to Eq. (12) only when [D] & Kd. Otherwise, the total number of
binding sites calculated from Eq. (12) will be in error by the factor D/Kd.
Bradford MethodPrinciples 185

This error appears in estimates for the total number of sites reported in
Table 2 and Table 4. The CBBG concentration used in the studies was *113
mM and hence values for n need revising downward by 113/Kd. A further
error appears with respect to pH 1 data in Tables 2 and 4. Allowing for a
shift from (CBBGH)0 to the (CBBGH2) species at low pH, ef,620 and hence
De probably remain unchanged with pH (De620 is approximately 18,300). Eq.
(12) then gives n 214 for BSA at pH 1. The corrected number of binding
sites is 35 (i.e., 214 6 Kd/D). A further independent estimate comes from
Fig. 1 of Chapter 7. A plot of DA/P versus D gives the regression line
Y 5.94 6 1010D. The gradient of this graph is n De/Kd [see Eq. (18)].
Assuming that Kd 18.6 6 10 6 M and De 18,300 M 1 cm 1 for BSA (see
Tables 4 and 6), we obtain n 56.
The preceding corrections lead to new estimates for the maximum
number of CBBG binding sites for BSA (n 3556). As BSA has
approximately 117 Arg His Lys sites, only 3048% of available cationic
sites bind CBBG. In Table 4 the reported agreement between numbers of
CBBG binding sites and (Arg Lys) residues is fortuitous.
In any case, the n estimate from Equation (12) (Table 4) exceeds the
total of Arg Lys residues. Hydrophobic interactions were invoked to
explain excess dye binding. CBBR-protein complexes precipitate within the
interstices of polyacrylamide gels. The number of dye binding sites exceeds
the number of protein cationic sites (Lys Arg His) up to 150% (10).
However, Kd values for protein-CBBR binding are similar to those in Table
6.4: lysozyme (35.7 mM), cytochrome c (83.3 mM), RNAse (125 mM), trypsin
(83.3 mM), pepsinogen (37 mM), pepsin (77 mM), and gramicidin S (111 mM).
Between 1.3 and 3.0 CBBR molecules bind for each basic amino acid
residue. Initially CBBR molecules bind to protein via 1:1 ionic interactions.
There then follows the uptake of a second CBBR molecule via nonpolar
interactions. This binding scheme accounts for (a) the correlation between
DBC and numbers of basic residues and (b) the involvement of nonpolar
interactions in protein-dye binding. Wilson (4) suggested the same idea.

6. SOLID-PHASE DYE-BINDING ASSAYS

To perform a solid-phase assay, the protein is rst bound to lter discs and
stained with CBBG dye reagent. Destaining is carried out to remove
nonspecically bound dye and the stained protein spot is excised using a
cork borer and immersed in a solubilization solvent. The amount of dye
solubilized is measured from DA readings at 600630 nm. A calibration
graph can be produced as usual by analyzing standard concentrations of
protein. Solid-phase dye-binding assays are described in Chapter 5.
186 Chapter 6

7. INTERFERENCE COMPOUNDS AND SAMPLE PRETREATMENT

7.1. Interferences and Compatible Solutes
Compounds that interfere with the Bradford protein assay are listed in
Table 5. These include food constituents such as chlorophyll, pectin, and
ethanol and low-molecular-weight surface-active agents. Reducing com-
pounds such as ascorbic acid have a fading effect on triphenylmethane
colors (34). The action is slow and requires about 7 days. Compounds
thought to be compatible with the Bradford method are listed in Table 6.
Many buffer salts, EDTA, hydroxycinnamic acid derivatives, and low
concentrations of avanols apparently do not interfere with protein-CBBG
interactions (Section 7.3).
The preceding information is intended only as a rough guide. Food
components not listed should be compared with their nearest listed relative.
For example, pectin and gum arabic are interfering compounds. Expect
interference from other structural polysaccharides (alginate, carrageenans)
and plant gums (acacia, Tara gum, etc.). Tables 5 and Table 6 are not
denitive guides: (a) potential interferences are usually tested in the absence
of protein, (b) many interferences were tested using the prototype Bradford
method (involving low dye concentrations), and (c) some interferences have

TABLE 5 A List of Interferences for the Bradford Assaya

Interfering substances

Ampholyte (pH 310.0; 1%) Potassium chloride (1M)

Acetone Quercitin
Apigenin rRNA
Chlorophyll Rutin
Chrysin SDS (1%)
Detergents (various) Sodium acetate (2 M)
Ethanol (95%) Sodium chloride (5 M)
Fisetin Sodium hydroxide (2 M)
Glycerol (99%) Sucrose (2 M)
Gum arabic Tannic acid
Hemosol (1%) Tris (2 M)
Kaempferol Triton X-100 (0.1%)
Magnesium chloride (1M) Tween 80 (>0.0001%)
Myricetin Urea (>0.2 M)
Pectin Vanadate
Phenol (5%)
a
Summarized from Refs. 2, 13, 21, 32.
Bradford MethodPrinciples 187

no effect or contrary effects at concentrations below those shown in Table

6-7. Interferences that compete with CBBG for protein binding will not be
detected in the absence of protein. Some interferences have a different effect
if the dye reagent/sample volume ratio is lower than used with the standard
Bradford assay.

7.2. Detergents and Low-Molecular-Weight Surfactants

Nonionic detergent concentrations above the cmc produce a shift in the lmax
value for CBBG from 650 nm to 600620 nm. Thereafter A620 increases at
the expense of A470 and A650 values. CBBG binding to nonionic detergents
leads to an increase in A595 compared with A650. The reverse is observed for
CBBG binding to anionic detergents (SDS). At concentrations below
the cmc, SDS reduces the assay sensitivity by competing with dye molecules

TABLE 6 A List of Compounds Considered Compatible with the Bradford Method

Noninterfering substances

Adenosine (1 mM) Potassium chloride (1 M)

Amino acids Potassium phosphate (pH 7; 1 M)
Ammonium sulfate (1 M) rRNA (0.25 mg/mL)
Asparagine Rutin
ATP (1 mM) Sinapic acid
BES (2.5 M) Sodium acetate (0.6 M)
Cacodylate-Tris (0.1 M) Sodium citrate (0.05 M)
Caffeic acid Streptomycin sulfate (20%)
CDGA (0.05 M) Thymidine (1 mM)
Dethiothreitol (1 M) tRNA (0.4 mg/mL)
DNA (1 mg/mL) Tyrosine (1 mM)
EDTA (0.05 M) Valine
Ethanol (95%) Glucuronic acid
Ferulic acid Sitosterol
Formic acid (1 M) Stigmasterol
Glycine (0.1 M) Maltol
HEPES (0.1 M) Chlorogenic acid
Hexyl-b-D-glucopyranose (<10% w/w) Apigenin
Maleic acid Phloretin
MES (0.7 M) Chrystin
MOPS (0.2 M) Fisetin
PIPES (0.5 M)
Source: Summarized from Refs. 2, 13, 21, 32.
188 Chapter 6

for charged and noncharged protein sites. The spectral changes are due to
the solubilization of neutral (CBBG)0 species inside detergent micelles (11).
The positively charged dye species (CBBG)1 binds to SDS, forming a
neutral complex, which is then solubilized within detergent micelles (35).
To avoid interference, Zaman and Verwilghen (36) reduced the
concentration of SDS in protein samples by precipitating with potassium
salts. The SDS-depleted sample is then assayed using the Sedmak-Grossberg
assay (Chapter 7, Section 2.5). Kapp and Vinogradov (37) removed SDS
from protein samples using a column lled with the ion-exchange resin
AG11A8 (Bio-Rad Laboratories). The SDS binding capacity was 1.7 mg/
mL (wet resin). Binding of the detergent was extremely rm and no simple
techniques were found to regenerate the support. The recovery of protein
from this support ranged from 52 to 90%. SDS may be removed from
samples using charcoal cartridges made in house (38). Preextracting SDS
from PAGE gels (with charcoal placed within a dialysis bag) allows gel
staining using the conventional Bradford reagent. This can therefore serve
as a dual-purpose reagent for protein analysis in solution as well as for
PAGE.
Alkyl-b-D-glucopyranoside detergents are compatible with the Brad-
ford assay. Fanger (39) screened a number of commercially available
detergents for solubilizing membrane proteins.* Low interferences were
obtained with octyl-b-D-glucopyranose and related alkyl-b-D-glucopyrano-
sides. There was tolerance for up to 10% (w/w) detergent in protein samples.
Detergents with low cmc have more adverse effects on the Bradford
assay (Fig. 6). Nonionic detergents showed a bell-shaped concentration
response. Friedenauer and Berlet (40) found the optimum color yield for a
Triton X-100 concentration of 0.008% in the nal assay mixture; the cmc for
Triton X-100 is about 0.01% (w/w). Assay sensitivity increased by 11 to
128% with an average increase of 33% for 15 different proteins examined.
Apparently, Triton X-100 facilitates protein-dye interactions via noncova-
lent bonding. The detergent had no effect on protein conformation. To
enhance sensitivity the detergent should be added to the protein sample
before dye.

7.3. Plant Secondary Metabolites

Aromatic amino acids (tyrosine, phenylalanine, and tryptophan) from the
shikimic acid pathway are not interferences. Hydroxycinnamic, caffeic acid,

* Detergent solutions (10% w/w; 10 mL) were added to 4.9 mL of Bradford dye reagent and A595
and A650 measurements recorded.
Bradford MethodPrinciples 189

FIGURE 6 The effect of detergents on the standard Bradford assay. A 100-mL

portion of detergent solution (10% w/v) was added to 4.9 mL of
Bradford dye reagent and absorbance measurements recorded 595 or
650 nm. (Drawn using data from Ref. 40.)

ferulic acid, and sinapic acid are also compatible solutes. Flavanoids and
related substances possessing a chalcone skeleton (anthocyanins, anthox-
anthins, proanthocyanadins, and tannin) can interfere (41). Other potential
interferences from plants include products of the mevalonate pathway
(carotenoids, lycopenes, terpenes, etc.) and the molanic acid pathway (lipids,
phospholipids, and lipid hydroperoxides). Some phenol-CBBG complexes
have an absorption spectrum that overlaps that for the protein-CBBG
complex. There is no evidence that protein precipitation with tannins is a
source of error. Techniques for preparing plant proteins, to minimize
interactions with phenolic compounds, are reviewed by Loomis and Battaile
(42) and Loomis (43).
Phenolic metabolites bind to proteins via (a) hydrogen bonding to the
peptide oxygen, (b) oxidation to quinones followed by covalent reactions
with the protein e-NH2 or SH group, (c) ionic interactions with phenolate or
carboxylate groups, and (d) hydrophobic interactions between the phenolic
benzene ring and aromatic amino acid residues. Some techniques for
controlling protein-phenol interactions include (a) maintaining sample pH
at just below neutral pH; (b) addition of phenolic adsorbents, e.g.,
polyvinylpryrrolidone (PVP), Polyclar AT, Amberlite XAD-2, XAD-4, or
XAD-7; (c) use of antioxidants and phenol oxidase inhibitors including
190 Chapter 6

chelators, sulte, and ascorbic acid; (d) addition of protective proteins such
as BSA; and (e) use of inert atmospheres such as argon, carbon dioxide, or
nitrogen (42,43).
The Bradford assay is more resistant to interference by phenolics than
the biuret, Lowry, and BCA assays. Indeed, Folin Ciocalteu and BCA
reagents are used for the quantitative analysis of phenolics and other food
antioxidants. These assays are therefore not suited to plant-derived samples
including leafy vegetables, fruits, stem, and tubers. Processed foods such as
beverages (tea, coffee, beer, wine, chocolate) are also not readily analyzed by
the Lowry or BCA assays. The Bradford assay is widely applied to beer and
wine protein analysis (Chapter 7, Sec. 4). Sample pretreatment involves size
exclusion chromatography to remove low-molecular-weight interferences
from beer or wine. Dialysis or treatment with phenolic adsorbents is also
feasible.

7.4. Sugars, Glycation, and Glycosylation Products*

Simple monosaccharides and disaccharides have no direct effect on the
Bradford assay. At concentrations of 1 mM to 1 mM the following sugars
produced no color with CBBG dye reagent: galactose, glucose, lactose,
maltose, mannose, N-acetylglucosamine, and sucrose (44). However, protein
glycation or glycosylation reduces the sensitivity of the Bradford assay. The
formation of advanced glycation products (AGPs) is a feature of the
pathology of diabetes. Incubating human serum albumin (HSA) with 1 M
glucose for 12 weeks also leads to the glycation of lysine and arginine
groups. So does heating dry mixtures of protein and sugar at oven
temperatures overnight. The modied proteins show improved foaming and
emulsication properties. The chemistry of protein-sugar reactions is similar
to the early steps of the Maillard reaction (see Chapter 9).
Brimer et al. (45) observed that nonenzymatic glycation interferes with
the Bradford assay. HSA was underestimated by about 20% after glycation
and exhaustive dialysis. Amino acid analysis showed a reduction of lysine
and arginine residues of 4554% and 2430% due to glycation. There was no
major error in the Bradford assay after HSA was incubated with 25 mM
glucose. Relatively high levels of glycation were needed to cause
interference. The Bradford assay gave 50% lower estimates for native
glycosylated proteins (ovalbumin, brinogen, horseradish peroxidase, IgM,

* Glycation usually describes carbonyl-amine reactions between sugars and proteins in vitro as
well as in vivo. Glycosylation is the attachment of sugar residues to proteins immediately after
synthesis (posttranslational modication) during protein secretion from cells. Many glycosy-
lated proteins contain oligosaccharide units linked to asparagine, serine, and threonine.
Bradford MethodPrinciples 191

and soybean trypsin inhibitor) (44). In some cases, the relative sensitivity of
the Bradford assay was related to differences in protein hydrophobicity. The
rate of color formation was also marginally reduced for some glycosylated
versus nonglycosylated proteins.
Rocher et al. (46) identied six distinct g- and o-secalins by
fractionation of rye storage proteins some of which were glycosylated. A
40-kDa rye protein was exceptional in having signicant absorbance in the
visible region, low CBBG binding, and immunoreactivity. Other naturally
glycosylated proteins [peanut conarachin, soybean conglycinin (47), lupin
conglutin (48)] might show abnormally low reactivity with CBBG. The total
protein content of rapeseed our determined by the Bradford method did
not agree with results of Kjeldahl method (49).
The mechanism by which sugars interfere with the Bradford assay is
uncertain. Sugars have no direct interactions with CBBG. Glycosylated
proteins contain oligosaccharide units linked to asparagine (Asn-X-Ser or
Asn-X-Thr), serine, or threonine residues that are not obviously involved in
protein-CBBG binding (50). Perhaps steric hindrance and the generally
lower hydrophobicity of glycosylated proteins may account for the
reduction in CBBG binding.

7.5. Polysaccharides, Lipids and Nucleic Acids

Interferences by nucleic acids, lipids, and polysaccharides have not been
assessed in detail. Nondialyzable polysaccharides from cane sugar may bind
CBBG (51). Alkorta et al. (52) reported results consistent with pectin
binding with CBBG. Compared with either the Lowry or modied Lowry
assays, the Bradford method was resistant to interference from pectin at
concentrations below 5 g L 1. Other acidic polysaccharides from terrestrial
plants, marine plants, or bacteria are widely used in foods. The possible
effects of gums and stabilizers on the Bradford assay have yet to be
investigated. Interference by lipids is discussed in Chapter 7.

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staining in polyacrylamide gels with a new type of Coomassie Brilliant Blue.
Anal Biochem 48:617620, 1972.
2. MM Bradford. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilising the principle of protein-dye binding.
Anal Biochem 72:248254, 1976.
192 Chapter 6

3. Colour Index. Vol 5. Bradford, W. Yorkshire, England: The Society of Dyers

and Colourants, 1971, p 5400.
4. CM Wilson. Studies and critique of Amido Black 10B, Coomassie Blue R, and
Fast Green FCF as stains for proteins after polyacrylamide gel electrophor-
esis. Anal Biochem 96:263278, 1979.
5. S Fazekas de St. Groth, RG Webster, A Daytner. Two new staining
procedures for quantitative estimation of proteins on electrophoretic strips.
Biochim Biophys Acta 71:377391, 1963.
6. A Chramback, RA Reisfeld, M Wyckhoff, J Zaccari. A procedure for rapi and
sensititive staining of protein fractionated by polyacrylamide gel electrophor-
esis. Anal Biochem 20:150154, 1967.
7. AH Reisner, P Nemes, C Pcholtz. The use of Coomassie Brilliant Blue G250
perchloric acid solution for staining in electrophoresis and isoelectric focusing
on polyacrylamide gels. Anal Biochem 64:509516, 1975.
8. J Pierce, CH Suelter. An evaluation of the Coomassie Brilliant Blue G250 dye
binding method for quantitative protein determination. Anal Biochem 81:478
480, 1977.
9. H Van Kley, SM Hale. Assay for protein by dye binding. Anal Biochem
81:485487, 1977.
10. M Tal, A Silbersteine, E Nusser. Why does Coomassie Brilliant Blue R
interact differently with different proteins: a partial answer. J Biol Chem
260:99769980, 1980.
11. KS Rosenthal, F Koussale. Critical micelle concentration determination of
non-ionic detergents with Coomassie Brilliant Blue G-250. Anal Biochem
55:11151117, 1983.
12. SJ Compton, CG Jones. Mechanism of dye response and interference in the
Bradford protein assay. Anal Biochem 151:369374, 1985.
13. MR De Moreno, JF Smith, RV Smith. Mechanism studies of Coomassie Blue
and silver staining. J Pharm Sci 75:907911, 1986.
14. MA Lea, A Luke, C Martinson, O Velazquez. Inuence of carbomoylation on
some physical properties of basic polypeptides. Int J Peptide Prot Res 27:251
260, 1986.
15. MA Lea, A Luke. Effect of carbomoylation with alkyl isocyanates on the
assay of protein by dye binding. Int J Peptide Prot 29:561567, 1987.
16. HJ Chial, AG Splittgerber. A comparison of the binding of Coomassie
Brilliant Blue to proteins at low and neutral pH. Anal Biochem 213:362369,
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17. RW Congdon, WG Muth, AG Splittgerber. The binding interactions of
Coomassie Blue with proteins. Anal Biochem 213:407413, 1993.
18. AG Mayes, R Eisenthal, J Hubble. Binding isotherms for soluble immobilised
afnity ligands from spectral titration. Biotechnol Bioeng 40:126370, 1992.
19. BA Atherton, EL Cunningham, AG Splittgerber. A mathematical model for
the description of the Coomassie Brilliant Blue protein assay. Anal Biochem
233:160168, 1996.
Bradford MethodPrinciples 193

20. MM Shareef, T Shetty. Effect of vanadate on different forms of Coomassie

Brilliant Blue and protein assay. Anal Biochem 258:143146, 1998.
21. EQ Adams, L Rosenstein. The colour and ionisation of crystal violet. J Am
Chem Soc 36:14521473, 1914.
22. AG Splittgerber, J Sohl. Nonlinearity in protein assays by the Coomassie Blue
dye-binding assay. Anal Biochem 179:198201, 1989.
23. L Michaelis, S Granick. Metachromasia of basic dyes. J Am Chem Soc
67:12121219, 1945.
24. IM Klotz. Spectrophotometric investigations of the interactions of proteins
with organic anions. J Am Chem Soc 68:22992304, 1946.
25. IM Klotz. The application of the law of mass action to binding by proteins.
Interactions with calcium. Arch Biochem 9:109117, 1946.
26. IM Klotz, FM Walker, RB Pivan. The binding of organic ions by proteins. J
Am Chem Soc 68:14861490, 1946.
27. H Aizawa. Interactions of food colours with proteins. Part XII. Studies on the
bindings of the food xanthene colours with trypsin. J Food Hyg Soc Jpn
12:8185, 1971.
28. HE Rosenthal. A graphical method for the determination and presentation of
binding parameters in a complex system. Anal Biochem 20:525532, 1967.
29. JG Norby, P Ottolenghi, J Jensen. Scatchard plot: common misinterpretation
of binding experiments. Anal Biochem 102:318320, 1980.
30. M Pesavento, A Profumo. Interaction of serum albumin with a sulphonated
azo dye in acidic solution. Talanta 38:10991106, 1991.
31. JJ Sedmak, SE Grossberg. A rapid, sensitive and versatile assay for protein
using Coomassie Brilliant Blue G250. Anal Biochem 79:544552, 1977.
32. JC Bearden Jr. Quantitation of submicrogram quantities of protein by an
improved protein dye binding assay. Biochim Biophys Acta 533:525529,
1978.
33. Z Zor, Z Selinger. Linearisation of the Bradford protein assay increases its
sensitivity: theoretical and experimental studies. Anal Biochem 236:302308,
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34. DM Marmion. Handbook of US Colorants: Foods, Drugs, Cosmetics, and
Medical Devices. New York: John Wiley & Sons, 1991.
35. GL Boccaccio, LA Quesada-Allue. Interference of sodium dodecyl sulfate in
the Bradford assay for protein quantitation. An Assoc Quim Argent 77:7988,
1989.
36. Z Zaman, RL Verwilghen. Quantitation of protein solubilized in sodium
dodecyl sulfate mechaptoethanol-tris electrophoresis buffer. Anal Biochem
100:6469, 1979.
37. OH Kapp, SN Vinogradov. Removal of sodium dodecyl sulfate from proteins.
Anal Biochem 91:230235, 1978.
38. RC Duhamel, E Meezan, K Brendel. A charcoal cartridge for the removal of
anionic detergent and electrophoresis stains. J Biochem Biophys Methods
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194 Chapter 6

39. BO Fanger. Adaptation of the Bradford assay to membrane-bound proteins

by solubilizing in glucopyranoside detergents. Anal Biochem 162:1117, 1987.
40. S Friedenauer, HH Berlet. Sensitivity and variability of the Bradford protein
assay in the presence of detergents. Anal Biochem 178:263268, 1989.
41. JH Von Elbe, SJ Schwartz. Colorants. In O Fennema, ed. Food Chemistry.
New York: Marcel Dekker, 1996, pp 651722.
42. WD Loomis, J Battaile. Plant phenolic compounds and the isolation of plant
enzymes. Phytochemistry 5:423438, 1966.
43. WD Loomis. Overcoming problems of phenolics and quinones in the isolation
of plant enzymes and organelles. Methods Enzymol 31:528544, 1974.
44. M Fountoulakis, J-F Juranville, M Manneberg. Comparison of the Coomassie
Brilliant Blue, bicinchoninic acid, and Lowry quantitation assays using non-
glycosylated and glycosylated proteins. J Biochem Biophys Methods 24:265
274, 1992.
45. CM Brimer, RP Murray-McIntosh, TJ Neale, PF Davis. Nonenzymatic
glycation interferes with protein concentration determinations. Anal Biochem
224:461463, 1995.
46. A Rocher, M Calero, F Soriano, E Mendez. Identication of major rye
secalins as celiac immunoreactive proteins. Biochim Biophys Acta 1295:1322,
1996.
47. JR Books, CV More. Current aspects of soy protein fractionation and
nomenclature. J Am Oil Chem Soc 62:13471354, 1985.
48. M Durante, E Cucchetti, P Cerletti. Changes in composition and subunits in
the storage proteins of germinating lupin seeds. J Agric Food Chem 32:490
493, 1984.
49. YL Folawiyo. PhD thesis, University of Leeds, 1996.
50. L Stryer. Biochemistry. 3rd ed. New York: WH Freeman, 1988, p 773.
51. MA Godshall. Interference of plant polysaccharides and tannin in the
Coomassie Blue G250 test for protein. J Food Sci 48:13461347, 1983.
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7
Bradford AssayApplications

1. INTRODUCTION

The principles of the Bradford assay (1) are discussed in Chapter 6.

Applications of the Bradford assay for food protein analysis are discussed in
this chapter. Section 2.2 introduces several modications of the Bradford
assay designed to increase its compatibility with buffer salts, reduce protein-
protein variations in results, and enable analysis of detergent-containing
samples. Protein precipitation using the TCA-DOC method is not suitable
for the Bradford assay. Section 2.3 describes several protein precipitation
methods not using detergents. The performance characteristics of the
different assays are summarized in Section 3. Section 4 of this chapter is a
review of applications of the Bradford assay for food protein analysis.

2. COOMASSIE BRILLIANT BLUE DYE-BINDING ASSAYS

2.1. The Bradford Assay
The design of the Bradford assay owes much to classical dye-binding assays.
There are similarities in the choice of buffer and the relative volumes for
protein and dye reagent:

Method 1
CBBG dye binding assay for proteins (1).

195
196 Chapter 7

Reagents
1. Coomassie Brilliant Blue G250 (Sigma)
2. Ethanol (95% w/v)
3. Phosphoric acid (85% w/v)
4. Bovine serum albumin

Procedure
Preparation of CBBG dye reagent. Dissolve CBBG (100 mg) in 50 mL
of ethanol. Add 100 mL of phosphoric acid (85% w/v) and dilute to
1 L with distilled water. Filter through Whatman No. 1 paper and
store in a stoppered bottle.*
Standard protein assay. Pipette 100 mL of protein (1001000 mg mL 1)
into several 1.2 6 10 cm test tubes. Add 5 mL of dye reagent to each.
Mix and record A595 readings after about 5 minutes. Prepare a
reagent blank with 100 mL of solvent and 5 mL of dye reagent.
Microassay. Pipette 100 mL of protein standard (10100 mg mL 1) into
small test tubes.{ Add 1 mL of dye solution to each, mix, and record
A595 readings against an appropriate reagent blank.
Calibration graph. Plot DA595 (i.e., absorbance corrected for the blank
reading) versus amount of protein (10100 mg).
Determine the concentration of protein in 0.1mL of the unknown
sample by referring to the calibration graph.
A blue color develops within 2 minutes of mixing protein and dye
solutions. The color remains virtually constant for up to 20 minutes and
decreases slowly between 20 and 50 minutes. Assay reproducibility can be
improved by measuring A595 readings within 5 and 60 minutes after mixing
dye and protein. The calibration features of the Bradford assay are
described in Chapter 6.

2.2. Modications and Variants of the Bradford Assay

The Sedmak-Grossberg (2) assay employs perchloric acid (3.5% w/w) as the
solvent for CBBG. The sensitivity is higher than with Method 1 but the new
method is subject to interferences by strongly buffered samples. The use of
perchloric acid solvent for CBBG was rst suggested by Reisner et al. (3).
CBBG is red (lmax 465) when dissolved in 3.5% (w/v) perchloric acid
solution. Addition to protein generates a blue protein-dye complex with

* The nal reagent composition is nominally 0.01% CBBG in 8.5% phosphoric acid and 4.5%
ethanol.
{ Plastic microcentrifuge tubes are convenient vessels for microassay.
Bradford AssayApplications 197

lmax 595 nm. Using a low concentration of mineral acid (nitric or sulfuric
acid) as solvent leads to reagent instability with regard to small changes in
sample ionic strength. If CBBG is dissolved with high concentrations of
mineral acid, there is poor color change when the dye is added to protein.
Weak organic acids (e.g., acetic, formic, and isobutyric acid) are not suitable
solvents for CBBG because very high concentrations are needed to maintain
the dye pH (2).
The Sedmak-Grossberg reagent gives an immediate color reaction
with proteins. The absorbance change is stable for 6090 minutes. With
0.6 M HCl as solvent, the color was stable for 34 hours. Thereafter the
protein-dye complex formed a precipitate. The main disadvantage of this
assay is its susceptibility to pH variations. Samples containing 0.1 M
phosphate buffer (pH 7.0) or Tris-HCl buffer (pH 8.0) are not usable. Gel
ltration fractions with low buffer capacity (*10 mM) can be analyzed
accurately.
Bearden (4) modied the Sedmak-Grossberg and Bradford assays to
(a) increase the color stability, (b) increase assay sensitivity, and (c) reduce
interference by buffer salts. The new dye reagent was prepared as described
in Method 1 but without ethanol. Upon mixing equal volumes of protein
and dye solutions (thereby avoiding the 10- to 50-fold dilution of samples by
the dye reagent), a blue complex is formed in 12 minutes. The absorbance
increases slowly over 6090 minutes, then decreases from 90 to 180 minutes.
After 3 hours, A595 readings were within +8% of the maximum. The
increase in color stability was attributed to the omission of ethanol from the
reagent formulation. The protein-dye complex may be less soluble in
perchloric acid plus organic solvent (ethanol or methanol). Plastic cuvettes
are to be avoided as they may catalyze protein-dye precipitation.
The Read and Northcote (5) assay reduced protein-protein variations
in assay results by increasing the CBBG dye reagent concentration.
The order of color intensity for Method 1 with different proteins is
RNAse < ovalbumin < lysozyme < BSA cytochrome c (6); trypsin < chym-
otrypsin < pepsinogen < lysozyme < BSA < cytochrome c (7). More uniform
responses were achieved by increasing the CBBG concentration. Table 1
lists a range of CBBG dyes from various suppliers (5,8). Commercial
samples vary greatly in dye content because different users require different
levels of purity. Only the highest purity CBBG (98% purity) samples
should be used for protein analysis. The technical grades have 2050% (w/w)
dye matter. Variations in dye purity makes it impossible to compare the
performance of Bradford reagents (prepared with CBBG) from different
suppliers. Ideally, dye reagent formulations should be standardized as
suggested by Sedmak and Grossberg (2). Reagents prepared from different
batches of dye should be diluted to provide a CBBG content of 0.01% (w/v)
198 Chapter 7

TABLE 1 Composition of Coomassie Brilliant Blue G250 Samples Used for Protein
Analysis

Dye purity (%), real dye content, and

Dye (designation and supplier) comments

Coomassie Brilliant Blue G250, Sigma 3660% purity (see Method 1). Bradford
(Poole, UK or St. Louis, MO) reagent has 0.00360.006% (w/w) dye.
Serva Coomassie Blue G, Serva *50% purity. Dye solution (0.01%) has
(Heidelberg, Germany) A550 0.6. Saturated solution
0.018% (w/v) dye.
Serva Blue G, Serva (Heidelberg, 98% purity. Dye solution of 0.01% (w/v)
Germany) has A550 1.18. Saturated
solution 0.01% (w/v) dyea
Coomassie Blue G, Eastman (Rochester, 1922% purity
NY)
a
Dye purity is given as percent dry weight (% DW). This information has not been checked
against current dye grades from suppliers. The formula weight of CBBB 854 Da. Saturating
concentrations correspond to &117 mM.
Source: Compiled from Refs. 5 and 8.

with A550 1.18 (Table 1). This is actually twofold greater than the CBBG
concentration in Method 1.
Stoscheck (9,10) reduced protein-protein variations in assay results by
15-fold. She achieved this by adding NaOH to the assay mixture. Alkali also
increased assay sensitivity by 2.7-fold. Either 520 mL of 10 M NaOH or 50
200 mL of 1 M NaOH should be added to 1 mL of Bradford reagent. The
modest (0.020.12) pH change leads to no changes in dye ionization. NaOH
is thought to produce a salting-out effect leading to enhanced protein-dye
binding by hydrophobic interactions. The quantity of NaOH needed
decreases with increasing CBBG dye concentration. At low dye concentra-
tions about 200 mL of 1.0 M NaOH was needed for optimum effect. Dye
reagent prepared from high-purity Serva Blue G dye required 50 mL of
1.0 M NaOH per mL of dye solution. The NaOH can be added to the
protein sample or to the Bradford dye reagent. The effect of adding other
salts (e.g., ammonium sulfate) on the Bradford assay should be examined. In
summary, the Read-Northcote-Stoschek modication can be implemented
by simply using high-purity CBBG grade (such as Serva grade; Table 1) for
routine analysis. The nal dye reagent should be alkalinized with 50 mL (of
1.0 M NaOH) per mL (dye reagent) before use.
SDS, which is commonly added to electrophoresis buffers, lowers the
sensitivity of the Bradford assay (Chapter 6). With the Zaman and
Verwilghen (11) version of the Bradford assay, SDS is removed from
Bradford AssayApplications 199

samples by precipitation with potassium phosphate. Boccaccio and Qesada-

Allue (12) used potassium chloride as the precipitant for SDS. After
centrifugation, the SDS-depleted samples are analyzed using Method 1. The
linear range for analysis was 050 mg BSA with a sensitivity 10-fold lower
than observed with SDS-free samples. The sensitivity for SDS-depleted
samples reached 80% of the value for control samples when Boccaccio and
Quesada-Allue (12) analyzed insect cuticular protein extracted with the aid
of SDS. This approach should work for crustaceacrabs, lobster, and
related commodities.
Proteins dissolved in electrophoresis buffers (normally containing 2%
w/w SDS and 5% w/w 2-mercaptoethanol) were also successfully analyzed
after simple dilution (13). Typically, 10 mL of sample was diluted to 100 mL
with distilled water. Then 5 mL of Bradford dye reagent was added.
Calibrations with BSA dissolved in electrophoresis sample buffer showed a
linear dynamic range up to 100 mg BSA. Once more the assay sensitivity was
10-fold lower in the presence of SDS but comparable to the sensitivity of the
Lowry assay.

2.3. Protein Precipitation for the Bradford Assay

The DOC-TCA precipitation method (14,15) is not suitable for the
Bradford assay. Normally 100 mL of DOC (5% w/w) solution is added to
1 mL of protein solution followed by 100 mL of TCA (72% w/w). Chang (16)
found that several nonionic detergents (cholate, deoxycholate, Triton X-
100, Triton X-114, Tween 20, Nonidet P-40) formed dark precipitates with
TCA. These interfered with the Bradford, Lowry, and BCA assays. Kirazov
et al. (17) reported that >0.1% (w/w) sodium DOC formed a heavy
precipitate with the Bradford dye reagent. Alternative methods for protein
precipitation that are compatible with the Bradford assay are described
here.
Proteins can be precipitated with TCA at low temperature for the
Bradford assay. With cultured animal cell protein the recovery by TCA
precipitation was 98 + 0.17% (18).* To ensure a quantitative recovery of
protein, the nal TCA concentration in the sample should be 1020% (w/w).

* To 100 mL of protein solution add 25 mL of TCA (72% w/w). Incubate the sample at 08C on ice
for 30 minutes and then centrifuge at 10,000g using a microcentrifuge. Discard the supernatant
and remove excess TCA using absorbent paper. Redissolve the protein pellet with 50 mL of 1 M
NaOH. For refractory samples incubate at 378C for 23 hours. Add 1 mL of Bradford reagent
and record absorbance values at 595620 nm.
200 Chapter 7

Protein precipitation should be performed at 08C. Bensadoun and Weinstein

(14) obtained erratic protein recoveries of 791% by precipitating proteins
using 6% (w/w) TCA at room temperature. Protein pellets from TCA
precipitation dissolve readily with 0.11 M NaOH before analysis with the
standard Bradford assay. Protein coprecipitation with calcium phosphate is
also compatible with the Bradford assay (19). This mild sample pretreat-
ment eliminates inferences from high concentrations of detergents and
lipids.* Accurate results were obtained for protein samples containing
2 mg of cardiolipin, egg yolk phospholipid (mainly lecithin), or
phosphatidylcholine. Other interferences can be annuled by this treatment,
including potassium chloride (0.5 M), glycerol (30% v/v), Triton X-100 (3%
w/w), SDS (0.33% w/w), CHAPS (2.5% w/v), and deoxycholate (0.075%
w/v). With protein coprecipitation, interference effects were reduced by
40120 times.

2.4. Purication of Coomassie Brilliant Blue

The purity of CBBG should be high in order to produce sensitive, accurate,
and precise protein analyses. Impurities may affect the dye absorption
characteristics as well as protein-dye binding. CBBG can be puried by
precipitation with sodium chloride (8){ or ammonium sulfate (20). The
former method was rst applied to CBBR (8) and later adapted for CBBG
purcation (21). Starting with Sigma-grade CBBG (Table 1), dye samples of
98% purity were obtained with a yield of 50%. The purication fold was not
reported for the ammonium sulfate method. Dye contaminants produced an
absorption peak at 442 and 652 nm. Pure CBBG dissolved in 7.5% (w/w)
acetic acid shows three major absorbance peaks at 590, 310, and 210 nm.

* Adjust the sample (100 mg protein) volume to 200 mL with distilled water, if necessary. Add
10 mL of potassium phosphate buffer (0.5 M pH 7.4), 10 mL of calcium chloride (0.25 M), and
1 mL of ethanol (80% v/v); mix after each addition. Centrifuge at 7000 g and remove the
supernatant by aspiration. Wash the protein pellet by adding 100 mL of water and 1 mL of
ethanol. Centrifuge and remove the supernatant as before. Repeat the washing step if necessary.
Add CBCG dye concentrate (100 mL) and allow at least 5 minutes for the pellet to dissolve. Next
add 400 mL of distilled water and record absorbance readings at 595 nm.
{ Dissolve CBBG or CBBR in a minimum volume of water-methanol (2:1) solvent. Add an
equal volume of sodium chloride (5 M). Filter and dry CBBG precipitate at 808C. Alternatively,
dissolve the CBBG solid with methanol. Dry the methanol solution to recover solid CBBG.
Bradford AssayApplications 201

3. PERFORMANCE CHARACTERISTICS OF CBBG

DYE-BINDING ASSAYS
3.1. Linearity, Sensitivity, and Precision
A. Bradford Assay
The calibration graph yields a hyperbolic prole over an extended range of
(5125 mg) protein. An approximately linear equation provides an adequate
t for 050 mg BSA. Later investigators reported a narrower linear dynamic
range of 010 mg of BSA (6,7). The assay reproducibility was 1.2% with a
sensitivity of 0.011 (DA595 mg 1). The accuracy was satisfactory. Equal levels
of color were obtained with HSA, BSA, hemoglobin, chymotrypsinogen,
and cytochrome c (1).
The Bradford microassay is reportedly less sensitive than the standard
assay (1). Analysis of 5 mg mL 1 BSA gave an absorbance change of 0.1
(microassay) or 0.27 (standard assay). The results are due to different
degrees of sample dilution. The volume of dye reagent is 5 mL for the
standard assay and 1 mL for the microassay. To examine the effect of
sample dilution, Spector (22) assayed xed volumes of BSA (0.1 mL; 5500
mg mL 1) with varying amounts of dye reagent (0.5, 1, 2, or 5.0 mL). The
assay sensitivity and linear dynamic range can be seen from Table 2.
With increasing volumes of dye reagent the assay sensitivity decreased
while the linear dynamic range increased. These results remove a common
misconception about so-called micro assays, namely that these have (a)
increased sensitivity and (b) a more restricted linear dynamic range
compared with the ``standard'' format. Where results are normalized for
the total assay volume, sensitivity and linear dynamic range are independent
of dye volume. Dilution effects can easily be addressed by ensuring that the
same sample and reagent volumes are adopted for calibration and for

TABLE 2 Effect of Dye Reagent Volume on the Sensitivity of the Bradford Assay

Assay Sensitivityb Sensitivityc Linear range

volume (mL)a DA595 mg 1 DA595 (mL mg 1) (mg)

5 0.011 0.058 550

2 0.028 0.059 220
1 0.057 0.063 110
0.5 0.099 0.059 0.55
a
Volume of dye reagent added to 0.1 mL of BSA ( 0.5 mg mL 1).
b
Sensitivity determined from the slope (F) of the calibration graph.
c
Sensitivity calculated as F multiplied by nal assay volume.
Source: Adapted from Ref. 22.
202 Chapter 7

sample analysis. Spector (22) suggested that 4- to 5-month-old samples of

CBBG gave lower sensitivity. It is good practice to run several calibration
samples alongside every assay as this adds minimally to the overall effort.
Chiappelli et al. (18) compared the sensitivity of the Bradford and
Lowry assays for proteins in crude cell-tissue extracts. The dye reagent was
reportedly stable for 46 months when stored in a brown glass bottle at
room temperature. The Bradford assay gave consistently lower protein
readings compared with the Lowry assay. The ratio of protein values for the
Lowry and Bradford assay was 1.58 (+0.09). It was proposed that results
from the two assays could be interconverted using the factor 1.58. However,
this conversion factor was not constant. Lowry assay results for TCA-
precipitated proteins from the adrenal medulla were virtually identical to
those from the Bradford assay if BSA was selected as standard protein.
Using bovine g-globulin as standard, the ratio of results from the Lowry and
Bradford assays was 2:1 (23).

B. Sedmak-Grossberg Modication
A plot of A620 versus the amount of BSA (060 mg) produced a curvilinear
graph. A linear response was obtained for 010 mg BSA. An extended linear
graph was produced by plotting A620/A465 versus protein. The reproduci-
bility of the standard and microassay formats was 45% for 10 replicate
measurements with 10 mg of BSA. The sensitivity was 0.044 (DA595 mg 1).
There was no color change with polypeptides smaller than 3000 Da. This so-
called molecular weight selectivity is an important feature of CBBG dye-
binding assays (Section 4). Many low-molecular-weight nonprotein con-
stituents do not interfere with the Bradford assay (2).

C. Bearden's Modication
The linear dynamic range was 040 mg protein. For samples containing
0.021 mg of protein the assay sensitivity was 0.045 (DA mg 1), which is four
times greater than that for the conventional Bradford method (4).

3.2. Effect of Coomassie Brilliant Blue Dye Concentration

Increasing the CBBG dye reagent concentration led to the following effects:
(a) a two- to four fold increase in assay sensitivity, (b) increased rate of color
formation, and (c) reduced protein-protein variations in assay sensitivity.
Optimum assay sensitivity was attained using Serva Blue G dye of 98%
purity (Table 1). Addition of 50 mL of 1.0 M NaOH per mL dye reagent
reduced protein-protein variations by 15-fold. Assay sensitivity increased by
a further 2.7-fold (5).
Bradford AssayApplications 203

Fig. 1 shows the linear relationship between assay sensitivity and

CBBG concentrations above a critical value, C*dye. With BSA as a standard
protein C*dye was < 0.003%. With lysozyme a dye concentration (apparently)
exceeding 0.01% was necessary for dye-protein binding. Concentrations of
dye equal to C*dye might be necessary to act as nucleation sites for dye
binding. Small quantities of dye may induce a conformational change in the
protein before widespread binding occurs. Because C*dye differs for a range
of proteins, it is not related to a ``dye-centered'' phenomenon such as
micellation. Clearly, the sensitivity of the Bradford assay was under-
estimated in most early studies using low-purity CBBG. The maximum
solubility for 98% pure CBBG, is 0.01% (w/v) or 117 mM.*
The optimum dye reagent formulation described by Lofer and Kunze
(24) apparently had 0.017% CBBG (Serva grade) in 10.5% (w/w) phosphoric

FIGURE 1 Effect of CBBG concentration on the sensitivity of the Bradford assay.

Bovine serum albumin (5 mg) was analyzed with dye Serva Coomassie
Blue G (50% ``true'' CBBG dye content). Circles sensitivity, crosses
apparent maximum numbers of dye-binding sites calculated from Eq.
(11) of Chapter 6, i.e., n & sensitivity/De. Assume that De 44200
M 1 cm 1 for CBBG binding with BSA. (Based on results from Ref. 5.)

* Notice from Table 1 that Serva Coomassie Blue G has 50% dye purity. With classical studies
using the dye grade the real dye reagent concentrations are one half the values published.
204 Chapter 7

acid and 2.5% (w/w) ethanol.* Making the dye reagent 0.008% (w/v) with
respect to Triton X-100 increased assay sensitivity 25-fold compared with
the standard Bradford assay. The effect of Triton X-100 and other
detergents on the Bradford assay is described further in the next section.

3.3. Effect of Added Salts and Hydrophobic Interactions

Nonspecic dye-binding interactions are expected to reduce protein-protein
variations in assay results. Stoscheck (9) produced a 15-fold reduction in
protein-protein variations by adding 50200 mL of alkali (1 M NaOH) to
1 mL of Bradford reagent. The assay sensitivity for all proteins tested
increased by an average of 2.7-fold. The added salt probably increased
hydrophobic interactions between protein and dye. The sensitivity increase
with Triton X-100 was also explained in terms of increasing hydrophobic
interactions. Read and Northcote (5) reduced protein-protein variations by
increasing the CBBG dye reagent concentration. Excess dye is thought to
promote hydrophobic dye-protein interactions (Chapter 6).

4. APPLICATIONS TO FOOD PROTEIN ANALYSIS

4.1. Beer
Adequate levels of protein ensure beer foam stability. However, too much
protein leads to haze formation in cold beer. Rapid methods for beer protein
analysis are necessary for improved quality assurance and for monitoring
brewing processes. Beer is an aqueous extract from malted barley to which
hops are added for avor. The nished product contains protein,
polypeptides, NPN, polyphenolic dyes, carbohydrates, and nucleic acids.
The Kjeldahl method, although approved for beer protein analysis, is
subject to interferences by NPN. Plant-derived substances interfere with the
biuret, Lowry and BCA methods. Lewis et al. (25) were rst to use the
Bradford assay for beer protein analysis (Table 3).{

* A dye concentration of 0.01% (w/w) appears to be saturating for a solvent comprising 8.5%
phosphoric acid and 4.5% ethanol. The saturating concentration of CBBG appears to have
doubled for a solvent with higher (12.5%) phosphoric acid and lower (2.5%) ethanol.
{ Beer and wort samples were subjected to ultraltration using a 10 kDa molecular size cutoff
membrane to remove interferents. Pretreatment is also possible by size exclusion chromato-
graphy (SEC) with a Sephadex G50 (2.2 6 40 cm) column. Then 0.10.5 mL of pretreated beer
or wort was added to 5 mL of Bradford reagent with mixing. A595 or A620 readings were
recorded after 1040 minutes.
Bradford AssayApplications 205

TABLE 3 Analysis of Beer Proteins Using the Bradford Assay

Application and comments Reference

First reported analysis of beer proteins Lewis et al. (25)

Assay results (A595 or A620) agree with Hii and Herwig (26)
micro-Kjeldahl analysis of alcohol-
insoluble crude protein
Proposal for organized collaborative American Society of Brewing Chemists
study (27)
Bradford assay results vs. beer foam Dale and Young (28)
stability (head retention index)
Collaborative study; ofcial approval American Society of Brewing Chemists
deferred (29)
Investigation of chillproong using silica Kano and Kamimura (30)
gel
Analysis of foam polypeptides Onishi and Proudlove (31)
Bradford and pyrogallol method for Williams et al. (32)
beer protein

Their results were compared with micro-Kjeldahl and Lowry assays.

Some potential interferences (reducing sugar, total carbohydrates, a-amino
acids) were also measured. The Bradford assay detected high-molecular-
weight (>4 kDa) polypeptides eluted within the column void volume during
size exclusion chromatography. Lowry- and Kjeldahl-positive substances
eluted after the void volume and were not detected. Wort was found to
contain 660 mg L 1 or 4670 mg (protein) L 1 by the Bradford and Lowry
methods, respectively. Simulated beer samples containing 20% (w/v) amino
acids, phenols, nucleic acids, or other common beer constituents showed
that these had no effect on the Bradford assay. Indeed, such preliminary
studies (25) suggest that the Bradford assay can accurately monitor protein
levels during brewing operations (chill proong, proteolysis, mashing,
lautering, and wort boiling).
Hii and Herwig (26) compared the Bradford and Kjeldahl assay for
beer protein. Sample pretreatment involved degassing beer by standing
overnight and/or size exclusion chromatography with the Bio-Rad P2
(2562.5 cm) column.* To avoid interference from NPN, Kjeldahl analysis
was performed with beer protein precipitated by 80% alcohol. Bradford

* Samples were eluted with 0.05 M NaCl (ow rate 84 mL hr 1). Fractions eluting from the
column void volume (>2 kDa molecular size) were collected for analysis; 0.4 mL of sample was
added to 5 mL of Bradford reagent and the assay performed as described in Method 1.
206 Chapter 7

assay results were correlated (R 0.95) with crude protein content

(N 6 6.25). For 29 beer samples with a range of 5250 mg (protein) L 1
the regression equation was

A595 0:001428 cP mg L 1 0:02857 1

The CV for analysis was 5.8%. Sensitivity toward beer proteins was
lower than for lysozyme. Beer total nitrogen had 6380% NPN. The protein
standard should be chosen carefully. Compared with other protein assays,
the Bradford technique had advantages of greater speed, higher resistance to
interferences, and high molecular weight specicity. Dale and Young (28)
examined the correlation between Bradford assay results and beer foam
stability (head retention index). Beer was pretreated by SEC using FPLC*
with Sephadex G25, G50, and G57 columns. Fractions eluted in the void
volume were collected for analysis. There was a signicant correlation
(R 0.91) between foam stability and Bradford assay results. Foam stability
was related to the quantity of polypeptides with sizes >5, >30, or >80 kDa.
Beer contained polypeptides ranging from 2 to 100 kDa.
A collaborative test for the Bradford assay organized by the American
Society of Brewing Chemists was reported in 1987 (27,29).{ The major
ndings of the ASBC study were:
1. The Bradford method is rapid, convenient, and highly sensitive for
beer proteins.
2. The precision of analysis for within-laboratory errors is 2.64.5%.
The between-laboratory errors range from 15.5 to 35.5%.
3. Degassing (14 hours vs. 1 hour standing) and the degree of mixing
beer and dye reagent (3 vs. 5 seconds) have a signicant effect on
the test results.
4. Temperature (208C versus 258C), color development time (1055
minutes), or light exposure has no signicant effect on the
Bradford assay.
5. Results with different dye reagents are unacceptably different
(Fig. 2).
The recommendation to grant the Bradford method ``approved'' status
was deferred in 1987. Between-laboratory differences were considered too
great. Variation arose from the use of different commercial samples of
CBBG in different laboratories (Fig. 2). Ready-made dye reagents were

* FPLC=Fast protein liquid chromatography.

{ Two samples each of premium beer, light beer, and malt liquor were analyzed by 13
laboratories.
Bradford AssayApplications 207

FIGURE 2 The effect of different commercial dye reagents on beer protein analysis
by the Bradford assay. Boxed legend refers to different beer samples.
(Drawn from results of Ref. 29.) Some data points were calculated.

from Piece Ltd. or Bio-Rad Ltd. Other CBBG samples were purchased as
solids and the dye reagent solutions were prepared in house. Within the
remit of the test, it was not possible to determine which dye reagent gave
accurate results. However, the ``true'' protein content of the beers was not
determined. I discussed problems likely to arise from batch-to-batch
differences in CBBG dye purity (Table 1). To improve assay repeatability
(between-laboratory reproducibility), strictly dened dye formulations
should be used.
Williams et al. (32) analyzed protein levels for stout beer, bitter, and
lager from British retail outlets. The CBBG reagent from Bio-Rad Ltd. was
used. Beer was analyzed as is or after exhaustive dialysis to remove low-
molecular-weight components. Results from this investigation are illustrated
in Fig. 1 of Chapter 1. Reliable estimates for beer protein were obtained with
the Bradford and the PMR* assays (Table 4). Both techniques had a CV of
1.7%. Kjeldahl, Dumas, biuret, Lowry, and BCA methods were subject to
error due to the presence of dialyzable inteference compounds in beer.

* PMR=Pyrogallol-red molybdate.
208 Chapter 7

TABLE 4 Protein Levels in Some British

Beers Determined Using the Bradford and
PRM Methodsa

Protein (mg L 1)

Sample Bradford PRM

Stout 460 1050

430 800
Lager 200 630
160 500
Bitter 270 570
250 500
a
Lower values show protein values after
dialyzing samples.
Source: Summarized from Ref. 32.

The effect of different protein assays on the study of beer foam

stabilization was highlighted by Onishi and Proudlove (31). Beer foam
proteins were fractionated by hydrophobic interaction chromatography,
yielding ve fractions. In order of descending hydrophobicity, these were
fraction 5 > 4 > 3 > 2 > 1. Each fraction was repeatedly ultraltered to
remove solutes <3 kDa and then assessed for their foaming characteristics.
Protein quantitation was via the BCA or Bradford assay. Estimates of
protein content from the BCA method were 2.45.4 times greater than
results from the Bradford assay (Table 5). The degree of foam stabilization
by proteins from whole lager, foam protein isolate, or foam protein fraction

TABLE 5 Apparent Protein Levels in Lager

Beer Fractions 15 Separated by Hydrophobic
Interaction Chromatographya
1
Foam Protein (mg L )
fractions BCA Bradford

1 356 66
2 84 32
3 181 114
4 56 22
5 356 144
Total 1036 378
a
Fractions 15 are listed in order of increasing
hydrophobicity.
Source: Values estimated from Ref. 35.
Bradford AssayApplications 209

TABLE 6 Analysis of Wine Proteins by the Bradford Assay

Applications and comments Reference

First assay of wine protein Hsu and Heatherbell (33)

Color reaction takes 6090 minutes. Murphy et al. (34)
Grape proteins, soluble solids, and Murphy et al. (35)
maturation
Phenol-CBBG and CBBG-protein Brenna and de Vecchi (36)
spectra overlap. Pretreatment by SEC
Sensitivity to wine protein is *34% of Waters et al. (37)
response to BSA.
Direct protein analysispolyphenols Marchal et al. (38)
and ethanol cause 3090% error.
Bradford assay underestimates wine Boyes et al. (39)
protein by 10-fold. Pretreat samples
with NaOH.

5 were equal when normalized for protein concentration. The BCA assay
results were affected by reducing substances such as melanoidins.

4.2. Wine and Fruit Juice

Wine protein is frequently analyzed using the Bradford assay (Table 6). Like
beer, wine contains endogenous polyphenols and reducing compounds. The
wide range of protein values reported for wine (1 mg L 11 g L 1) probably
reects inherent differences between types of wines, grape cultivars and
maturity, and processing methods. Some variations are due to simple
measurement error.
The Bradford assay is more resistant to interfering compounds from
wine (see Lowry, biuret, BCA, and Kjeldahl methods). However, claims that
high concentrations of polyphenols (<400 mg L 1) do not interfere with dye
binding were challenged (36). The choice of standard proteins for wine
analysis is crucial.
Research on wine proteins is mainly concerned with haze formation
and identifying the protein fraction(s) responsible for wine instability. Hsu
and Heatherbell (33)* in 1987 were probably the rst to analyze grape and

* Grapes were homogenized with liquid nitrogen using a Waring blender and then freeze dried.
The resultant powder (3 g) was extracted for 2 hours with 30 mL of 0.1 M citrate, 0.2 M
phosphate buffer (pH 5) containing Amberlite XAD-4 (6 g), PVP (3 g), and two protease
inhibitors (diethyldithiocarbamate, 5 mM, and phenylmethylsulfonyl uoride, 5 mM). The
resultant mixture was ltered through muslin cloth and then centrifuged. The clear supernatant
was analyzed by the Bradford assay. Then A595 readings were recorded after 15 minutes.
210 Chapter 7

wine proteins using the Bradford method. Treating grape juice or wine with
ion-exchange resin (XAD-4) and PVP reduced total phenols by 44 and 57%
(Table 7). However, protein-quinone interactions were already complete
during the wine manufacturing process. Electrophoresis analysis achieved
poor resolution of wine proteins despite treatment with Amberlite and PVP.
There was also signicant adsorption of wine proteins to phenol adsorbents.
In particular, it is thought that hydrophilic proteins adsorbed strongly on
tannins, polyphenols, and phenol adsorbents.
The rate of A595 increase during wine protein analysis was examined
by Murphy et al. (34). The A595 reached a maximum after 90 minutes for
wine proteins and after 210 minutes with BSA. The incubation time for
wine protein analysis using the Bradford assay has to be extended and
strictly controlled. Dissolving BSA with protein-depleted wine reproduced
the slow reaction with CBBG; addition of polyphenols to BSA in idealized
buffer solutions did not.
Wine protein values depend on the maturity of the grapes used by the
manufacturer. Gewurztraminer and white Riesling grapes were found to
contain 50120 mg (protein) L 1; such results are usually cited as mg L 1
equivalent to BSA (35). Interestingly, grape juice protein levels were highly
correlated with acidity (R > 0.86). A highly signicant relationship also
existed between wine soluble solids content (1025% w/w) and wine proteins
(25105 mg L 1 eq. BSA). For wines having a solids content of 1820% the
protein value was 40 mg L 1 (BSA eq.). Adsorption of wine proteins with

TABLE 7 Wine Protein Analysis Using the Bradford Method:

Apparent Protein and Phenol Levels in Grapes, Grape Juice,
and Gewurztraminer Wine

Protein Phenola
Sample (mg L 1) eq. BSA (mg L 1)

Grapes 102 + 1.8 98 + 4.2

Db 34% 44%
Grape juice 77 + 1.5 298 + 14
D 25% 49%
Wine 29.4 + 0.5 124 + 6.3
D 29% 57%
a
Phenol levels were assayed using Folin Ciocalteu assay.
b
D percent change after treatment with Amberlite XAD-4, PVP, and
enzyme inhibitors.
Source: Data from Ref. 33.
Bradford AssayApplications 211

bentonite (0.48 g L 1) or other ning agents could be measured by the

Bradford assay.
The Bradford technique is not wholly resistant to interference by low-
molecular-weight wine constituents. The absorption spectra for CBBG
tannic acid or CBBG-proanthocyanidin mixtures overlap with the spectra
for the CBBG-protein complex (36). The interfering compounds were
readily removed by Sephadex G25 column chromatography. Fractions from
the column void volume (1 mL) were mixed with an equal volume of
Bradford reagent and A595 values were recorded. The standard protein was
wine protein isolate. Over the range 10100 mg (protein) L 1, the assay
response was described by

DA595 0:002213 cP 0:00941 2

(R 0.998). Maximum color formation occurred in 10 minutes. No further

DA595 increases were observed after 30, 50, or 80 minutes of incubation.
Interference compounds from pinot noir and chardonnay wines were
assessed for their effects on the Bradford protein assay (38).* Some wine
samples were analyzed without sample pretreatment. Low-molecular-weight
nonprotein substances were removed by ultraltration with a 10- or 3-kDa
molecular size cutoff membrane. Substances possessing a molecular size of
<10 kDa accounted for 2868% of the Bradford assay response. In one
instance, NPN accounted for 93% of the apparent protein. The A595 values
also increased linearly with ethanol levels. This effect led to an overestimate
of proteins by 3050% at normal alcohol levels in wine. Apparently, ethanol
induced changes in the ionization equilibrium for CBBG dye. The impact of
individual interference substances was further analyzed using simulated
wine samples containing BSA.{ Endogenous phenols produced a level of
error equivalent to about 100% of protein. Tannins (10 mg L 1) and pectin
(100 mg L 1) had comparatively little effect on protein analysis in simulated
wines.
Wine protein may be severely underestimated using the standard
Bradford assay. Boyes et al. (39) examined protein levels in gewurztraminer

* With so-called direct wine protein analysis, 0.2 mL of commercial CBBG dye reagent
concentrate (Bio-Rad Ltd.) was added to 0.4 mL of wine and 0.4 mL of distilled water. The A595
readings were taken after 60 minutes. Wine samples were diluted as necessary to keep A595
readings below one. BSA was used for calibration.
{ Model wine solutions contain potassium hydrogen tartrate (0.3% w/v), alcohol (12% v/v),
lysine (0.1% w/w), phenylalanine (100 mg L 1), sulfur dioxide (200 mg L 1) and an unspecied
amount of wine vitamins. Proteins and tannins were added as necessary.
Add 50 mL of NaOH (1 M) to 10100 mL of sample to adjust to pH > 11. After 5 minutes of
incubation add 3 mL of Bradford dye reagent. For the control study omit NaOH.
212 Chapter 7

wine and sauvignon blanc wine. Kiwi fruit juice and corn protein extract
were also analyzed using a new ``alkali'' Bradford assay.* Fruit juice and
gewurztraminer wine protein estimates were *10 times higher with the new
method (Table 8). The alkali Bradford assay is similar to the procedure of
Stoschek (9,10). In both instances 50 mL of NaOH (1.0 M) is added to the
sample before the Bradford reagent. The effect of pH adjustment may be to
reduce protein H-bonding interactions with phenolic compounds.

4.3. Cereal Products

Protein from wheat our, dough, bread crumb, or gluten was estimated with
the Bradford assay by Eynard et al. (40). Samples were preextracted with a
range of solvents before analysis. The calibration graph (with 50350 mg
gluten as standard protein) was curved and described by a second-order
equation, A595 ax2 bx c, where x is the amount of protein. As an
alternative, plotting A595/A460 versus protein gave a linear graph. Protein-
protein variations in sensitivity followed the series serum albumin > water-
soluble protein * acid-soluble protein > gluten * gliadin. Assay sensitivity
matched the total number of CBBG-reactive amino acids
(Arg Lys His Tyr Trp Phe). Water-extractable materials from
our, dough, and bread crumb had protein contents of 19.4%, 22.3%, and
6.9 g%, respectively. By comparison, the acetic acidextractable material
from our, bread dough, and bread crumb has 57.7% protein, 58.2%
protein, and 8.3% protein. Baking led to a large decrease in the solubility of
bread crumb protein.

TABLE 8 Effect of NaOH Addition on Bradford Assay of Fruit Juice and

Wine Protein

Protein (mg L 1)

Bradford assay
Sample ( NaOH) Bradford assay

Pressed kiwi fruit juice 2000

Heat-treated kiwi fruit juice 560830 150410
As above bentonite treated 120 0
Gewurztraminer wine 75250 9.733
( bentonite treatment)
Sauvignon blanc wine <50 <7
Source: Summarized from Ref. 39.
Bradford AssayApplications 213

Changes in the breadmaking quality of wheat due to drying at high

temperature were examined by CBBG dye-binding assay (41). The dried
grain was milled and extracted with phosphoric acid. The amount of soluble
protein assayed by the Bradford assay was correlated with bread baking
quality. Excessive heating during drying resulted in a decrease in the soluble
protein.
Esen (42) analyzed corn protein by solid-phase CBBG dye binding.
The calibration graph was linear for up to 10 mg mL 1 protein. The lower
limit of detection (LLD) for zein was 0.1 mg mL 1 or 1 mg per spot. Results
were highly correlated with micro-Kjeldahl analysis. The quantity of zein
from corn grain was negatively correlated with lysine levels (R 0.896
and 0.946). The Bradford assay was suggested as an indirect method for
screening for high-quality maize. Caution is required so that corn is not
ranked of higher quality by virtue of a low protein content.

4.4. Legumes
Dhillon and Nainawatee (43) found that whole or defatted mungbeen our
had 24% or 24.9% protein (per dry weight, DW) using the Bradford assay
and Kjeldahl (N 6 6.25) analysis.* As a form of sample pretreatment,
legume protein was rst extracted using Tris-SDS-2ME buffer (0.05 M Tris-
HCl buffer, pH 7.0, with 10 mM 2-mecaptoethanol and 2% SDS). The
calibration graph had a linear range of 0100 mg BSA.{ In agreement with
Rubin and Warren (13), assay sensitivity was reduced 10-fold due to the
presence of SDS (<0.05%).
The maturation process for beans (Phaseolus vulgaris L. cv. or de
mayo) was monitored by the Bradford, Kjeldahl, and Lowry protein assays
(44). Results from micro-Kjeldahl analysis of bean our and the dialyzed
our homogenate are shown in Fig. 3. Fig. 4 shows the changes in PBS-
soluble bean protein fractions. Broad beans had an apparent crude protein
content of 40% DW. Dialysis removed approximate 45% of the nitrogen,
which is therefore low-molecular-weight NPN.
Bean protein levels (15% DW) decreased slightly with maturation.
Results in Fig. 4 are for samples freed from NPN by dialysis. There were
inherent differences between results from different assay methods. Using
casein as a standard, the Bradford, micro-Kjeldahl, Lowry, and biuret

* Shake 100 mg of our with 1 mL of Tris-SDS-2ME for 60 minutes. Centrifuge (5000g) and
collect the supernatant. Wash the pellet with 2 6 2 mL of Tris-SDS-2ME buffer and combine
with the supernatant. Dilute (5 mL) pooled extract with water to a nal volume of 40 mL.
Determined protein content by adding 0.1 mL of extract to 5 mL of standard Bradford reagent.
{ Dissolve BSA standards in 0.05 M Tris-HCl buffer containing 0.05% (w/v) SDS.
214 Chapter 7

FIGURE 3 Determination of proteins in bean (Phaseolus vulgaris L., cv. or de

mayo) seeds. Samples were analyzed by the micro-Kjeldahl method. Left
to right: bean our; homogenized and dialyzed bean our extract;
homogenized, centrifuged and dialyzed extract.

methods all gave comparable results. By contrast, protein levels in soy

protein isolate were estimated as 84 (+0.2)% by micro-Kjeldahl analysis,
87.8 (+2.1)% by Lowry assay, 78.3 (+0.7)% by the Bradford method, or
106 (+1.1)% by the biuret method. The differences in assay results are
unexplained. Paredes-Lopez and co-workers (44) suggest that the Lowry,
Bradford, and biuret methods are unsuitable for legume protein determina-
tion. It is essential to remove NPN from our samples (e.g., by washing with
80% ethanol) before protein analysis. Improving bean protein extraction
with SDS may also increase the accuracy (43).

4.5. Potatoes
Potatoes contain high amounts of NPN. The amount and quality of protein
also differ for different potato varieties and states of maturation. Rapid
methods for protein analysis are necessary to assist in attempts to breed
potato varieties having higher protein content. Criteria for selecting
Bradford AssayApplications 215

FIGURE 4 Determination of proteins in bean (Phaseolus vulgaris L., cv. or de

mayo) seeds. Effect of assay method on PBS-soluble protein (dialyzed).

convenient assays for potato breeding include (45) (a) efcient extraction
and solubilization of protein from potato tissue, (b) compatibility with
different potato varieties and tubers at different stages of maturation, (c)
lack of interferences from NPN, and (d) low expense and high throughput
compared with the Kjeldahl method.
Snyder and Desborough (45) determined potato tuber protein using
the Bradford assay.* Bradford test results for potato tubers were strongly
correlated with Kjeldahl results (R 0.93). The six hybrid potato varieties
studied had 315.6% protein per DW in agreement with quantitative amino
acid analysis or micro-Kjeldahl results. The cost per analysis was 100 times
lower and the sample throughput 6 times higher for the Bradford assay

* Dice, freeze, and lyophilize fresh potato. Suspend the ground potato powder (15 g) in 2.5 mL
of water and then add 2.5 mL of 1 M sodium hydroxide. Incubate at room temperature for 2.5
hours. Remove 0.4 mL of extract to another test tube and add 5 mL of Bradford reagent.
Record A595 readings as usual.
216 Chapter 7

compared with Kjeldahl analysis. A more extensive sample pretreatment

is required for the Kjeldahl method. Potato powder was preextracted
with 80% ethanol to remove NPN before Kjeldahl analysis. None of the 20
common amino acids (2 mg mL 1) interfered with the Bradford assay.

4.6. Mushrooms
A wide range of protein values have been reported for mushrooms.
Weaver et al. (46) found 1939% protein (DW) 1. Pecora (47) explained
some of the variations in mushroom protein levels in terms of differences
in developmental stage, growth conditions, harvesting time, varietal
differences, and different amounts of NPN. Also, two Kjeldahl factors
(4.38 and 6.25) are available for mushroom protein analysis. The literature
describing mushroom (Agaricus bisporus) protein analysis is summarized
by Braaksma and Schaap (48). These investigators reexamined protein
levels in mushroom using Bradford, Kjeldahl, and quantitative amino acid
analysis.
Just under 50% of mushroom protein was extractable with phosphate-
buffered saline. The remainder dissolved with 0.5 M NaOH, which is also an
ideal one-step extractant for mushroom protein. Kjeldahl analysis of whole
mushroom indicated a nitrogen content of 63 mg N g 1 DW or 4.66 mg N
g 1 fresh weight (FW). The nominal protein content (%N 6 6.25) was 39%
DW or 2.92% FW. Analysis of TCA-insoluble material using the Bradford
assay or quantitative amino acid analysis showed 7% protein (DW) or a
nitrogen content of 12.8 mg N g 1 DW. Apparently, NPN accounted for
80% of the total nitrogen in whole mushrooms. The water-soluble nitrogen
fraction from mushroom comprised 20% protein, 60% urea-ammonia, and
20% free amino acids. By comparison, the nitrogenous material extracted by
0.5 M NaOH contained 37% protein, 9% urea-ammonia, 15% free amino
acids, and 20% undened constituents, probably glucosamine from the cell
wall. Compared with quantitative amino acid analysis, the Bradford assay
underestimated mushroom protein by 1014%. It is possible that Bradford
assay results are more accurate than those from quantitative amino acid
analysis. Bradford assay results (6.9% protein per DW) were not affected by
NPN.
The pretreatment procedure for mushroom protein determination
starts with powdered mushrooms. Fresh mushrooms were frozen in liquid
nitrogen, freeze dried, and ground into a ne powder. Protein was extracted
with phosphate-buffered saline (50 mM sodium phosphate buffer, pH
7.0 0.5 M NaCl) followed by 0.5 M NaOH. Kjeldahl analysis can be
performed on samples precipitated by 15% TCA (4648).
Bradford AssayApplications 217

4.7. Honey
Bogdanov (49) determined protein levels in honey using the Bradford,*
biuret, and Kjeldahl techniques. The average protein content for 12 different
honey samples was 130 mg 100 g 1 (range 50185 mg 100 g 1). Kjeldahl
results were 30% lower than protein estimates obtained with the Bradford
assay. Biuret analysis gave protein estimates 2.5-fold greater than expected,
probably because of Cu2 binding by honey polypeptides. g-Globulin (120
mg) was the standard protein for the Bradford assay.

4.8. Whey Protein Concentrates

Richard and Paquin (50) compared the Bradford and Kjeldahl assays for
whey protein concentrate (WPC). It was thought desirable to control both
the ionic strength and pH of the solvent. Thus, 1 g of WPC (33.8% protein)
was dissolved in 100 mL of McIlvine buffer adjusted to various pH values.
The solutions were centrifuged (40,000g) and then ltered using Whatman
No. 1 paper. Solubility results from the Bradford and Kjeldahl assays were
3% different (*83% solubility) at pH 69. At pH 3 protein solubility
determined via the Bradford assay was 7% lower than the value from
Kjeldahl analysis (78%). Compared with Kjeldahl analysis, the Bradford
assay was a simpler, faster, and a more affordable technique for monitoring
the solubility of WPC.

4.9. Insoluble Proteins

Gotham et al. (51) assayed BSA, beta-lactoglobulin, egg albumin, or
lysozyme aggregated by heating at 1008C. Protein aggregates were recovered
by microcentrifugation and resolubilized by heating for 24 minutes with
urea-mercaptoethanol solvent (8 M urea 5% w/v 2-ME). Dissolved protein
samples (50 mL) were added to 2.5 mL of the standard Bradford reagent and
A595 values were recorded. Assay sensitivity, precision, and accuracy were
comparable to values obtained with soluble proteins. Guanidine hydro-
chloride was not a suitable substitute for urea as a diluent. Resolubilization
strategies should be checked for particular proteins before use. The
approach has potential for monitoring food protein adsorption and fouling
of process equipment.

* Dissolve 0.5 g of honey with 100 mL of water. To 0.8 mL of diluted honey add 0.2 mL of
commercial CBBG dye regent concentrate (Bio-Rad Ltd.). Use 0.8 mL of water as a reagent
blank. Record A595 readings after 560 minutes.
218 Chapter 7

REFERENCES

1. MM Bradford. A rapid and sensitive method for the quantitation of

microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem 72:248254, 1976.
2. JJ Sedmak, SE Grossberg. A rapid, sensitive and versatile assay for protein
using Coomassie Brilliant Blue G250. Anal Biochem 79:544552, 1977.
3. AH Reisner, P Nemes, C Bucholtz. The use of Coomassie Brilliant Blue G250
perchloric acid solution for staining in electrophoresis and isolectric focussing
on polyacrylamide gels. Anal Biochem 64:509516, 1975.
4. JC Bearden Jr. Quantitation of submicrogram quantities of protein by an
improved protein dye binding assay. Biochim Biophys Acta 533:525529,
1978.
5. SM Read, DH Northcote. Minimization of variation in the response to
different proteins of the Coomassie Blue G dye-binding assay for protein.
Anal Biochem 116:5364, 1981.
6. J Pierce, CH Suelter. An evaluation of the Coomassie Brilliant Blue G250 dye
binding method for quantitative protein determination. Anal Biochem 81:478
480, 1977.
7. H Van Kley, SM Hale. Assay for protein by dye binding. Anal Biochem
81:485487, 1977.
8. CM Wilson. Studies and critique of Amido Black 10B, Coomassie Blue R, and
Fast Green FCF as stains for proteins after polyacrylamide gel electrophor-
esis. Anal Biochem 96:263278, 1979.
9. CM Stoscheck. Increased uniformity in the response of the Coomassie Blue G
protein assay of different proteins. Anal Biochem 184:111116, 1990.
10. CM Stoscheck. Quantitation of protein. Methods Enzymol 182:5068, 1990.
11. Z Zaman, RL Verwilghen. Quantitation of protein solubilized in sodium
dodecyl sulfate mercaptoethanol-tris electrophoresis buffer. Anal Biochem
100:6469, 1979.
12. GL Boccaccio, LA Quesada-Allue. Interference of sodium dodecyl sulfate in
the Bradford assay for protein quantitation. An Assoc Quim Argent 77:7988,
1989.
13. RW Rubin, RW Warren. Quantitation of microgram amounts of protein in
SDS-mercaptoethanol tris electrophoresis sample buffer. Anal Biochem
83:773777, 1977.
14. A Bensadoun, D Weinstein. Assay of proteins in the presence of interfering
materials. Anal Biochem 70:241250, 1976.
15. GL Peterson. A simplication of the protein assay method of Lowry et al.
which is more generally applicable. Anal Biochem 83:346356, 1977.
16. Y-C Chang. Efcient precipitation and accurate quantitation of detergent
solubilized membrane proteins. Anal Biochem 205:2226, 1992.
17. LP Kirazov, LG Venkov, EP Kirazov. Comparison of the Lowry and
Bradford protein assays as applied to protein estimation of membrane-
containing fractions. Anal Biochem 208:4448, 1993.
Bradford AssayApplications 219

18. F Chiappelli, A Vasil, DF Haggerty. The protein concentration of crude cell

and tissue extracts as estimated by the method of dye binding: comparison
with the Lowry method. Anal Biochem 94:160165, 1979.
19. SV Pande, SR Murthy. A modied micro-Bradford procedure for elimination
of interferences from sodium dodecylsulfate, other detergents and lipids. Anal
Biochem 220:424426, 1994.
20. V Neuoff, R Stamm, H Eibl. Clear background and highly sensitive protein
staining with Coomassie Blue dyes in polyacrylamide gels: a systematic
analysis. Electrophoresis 6:427448, 1985.
21. HJ Chial, AG Splittgerber. A comparison of the binding of Coomassie
Brilliant Blue to proteins at low and neutral pH. Anal Biochem 213:362369,
1993.
22. T Spector. Renement of the Coomassie Blue method of protein quantitation.
Anal Biochem. 86:142146, 1978.
23. HG Pollard, R Menard, HA Brandt, CJ Pazoles, CE Cruetz, A Ramu.
Application of Bradford's protein assay to adrenal gland subcellular fractions.
Anal Biochem 86:761763, 1978.
24. B-M Lofer, H Kunze. Renement of the Coomassie Brilliant Blue G assay
for quantitative protein determination. Anal Biochem 177:100102, 1989.
25. MJ Lewis, SC Krumland, DJ Muhleman. Dye-binding method for measure-
ment of protein in wort and beer. J Am Soc Brew Chem 38:3741, 1980.
26. V Hii, WC Herwig. Determination of high molecular weight proteins in beer
using Coomassie Blue. J Am Soc Brew Chem 40:4650, 1982.
27. EJ Knudson. Co-ordination of new and alternate methods of analysis. J Am
Soc Brew Chem 45:99100, 1987.
28. CJ Dale TW Young. Rapid methods for determining the high molecular
weight polypeptide components of beer. J Inst Brew 93:465467, 1987.
29. American Society of Brewing Chemists. High molecular weight protein in beer
(Bradford method). J Am Soc Brew Chem 46:116118, 1988.
30. Y Kano, M Kamimura. Simple methods for determination of the molecular
weight distribution of beer proteins and their application to foam and haze
studies. J Am Soc Brew Chem 51:2128, 1993.
31. A Onishi, MO Proudlove. Isolation of beer foam polypeptides by hydrophobic
interaction chromatography and their partial characterization. J Sci Food
Agric 65:233240, 1994.
32. KM Williams, P Fox, T Marshall. A comparison of protein assays for the
determination of the protein concentration of beer. J Inst Brew 101:365369,
1995.
33. JC Hsu, DA Heatherbell. Isolation and characterization of soluble proteins in
grapes, grape juice and wine. Am J Enol Viticult 38:610, 1987.
34. JM Murphy, JR Powers, SE Spayd. Estimation of soluble protein concentra-
tion of white wines using Coomassie Brilliant Blue G-250. Am J Enol Viticult
40:189193, 1989.
220 Chapter 7

35. JM Murphey, SE Spayd, JR Powers. Effect of grape maturation on soluble

protein characteristics of Gerwurztraminer and White Riesling juice and wine.
Am J Enol Viticult 40:199207, 1989.
36. O Brenna, S de Vecchi. Evaluation of protein and phenolic content in must
and wine. I. Assay of soluble proteins. Ital J Food Sci 2:269273, 1990.
37. EJ Waters, W Wallace, PJ Williams. Heat haze characteristics of fractionated
wine proteins. Am J Enol Viticult 42:123127, 1991.
38. R Marchal, V Seguin, A Maujean. Quantication of interferences in the direct
measurement of proteins in wines from the Champagne region using the
Bradford method. Am J Enol Viticult 48:303309, 1997.
39. S Boyes, P Struebi, H Dawes. Measurement of protein content in fruit juices,
wine and plant extracts in the presence of endogenous organic compounds.
Lebensm Wiss Technol 30:778785, 1997.
40. L Eynard, N Guerrieri, P Cerletti. Determination of wheat proteins in solution
by dye binding in our, dough, and bread crumb. Cereal Chem 71:434438,
1994.
41. EA Tosi, ED Re, L Carbone, M Cuniberti. Breadmaking quality estimation by
fast spectrophotometric method. Cereal Chem 77:699701, 2000.
42. A Esen. Estimation of protein quality and quantity in corn (Zea mays L.) by
assaying protein in two solubility fractions. J Agric Food Chem 28:529532,
1980.
43. S Dhillon, HS Nainawatee. A rapid and sensitive method of protein
estimation in legume grains. Int J Trop Agric 5:231234, 1987.
44. O Paredes-Lopez, F Guevara-Lara, ML Schevenin-Pinedo, R Montes-Rivera.
Comparison of procedures to determine protein content of developing bean
seeds (Phaseolus vulgaris). Plant Foods Hum Nutr 39:137148, 1989.
45. JC Snyder, SL Desborough. Rapid estimation of potato tuber total protein
content with Coomassie Brilliant Blue G-250. Theor Appl Genet 52:135139,
1978.
46. JC Weaver, M Kroger, LR Kneebone. Comparative protein studies (Kjeldahl,
dye binding, amino acid analysis) of nine strains of Agaricus bisporus (Lange)
Imbach mushrooms. J Food Sci 42:364366, 1977.
47. RP Pecora. Determination of protein in edible mushroom (Boletus sp.). Int J
Food Sci Technol 24:207210, 1989.
48. A Braaksma, DJ Schaap. Protein analysis of the common mushroom Agaricus
bisporus. Postharvest Biol Technol 7:119127, 1996.
49. S Bogdanov. Determination of honey protein with Coomassie Brilliant Blue
G. Mitt Geb Lebensm Hyg 72:411417, 1981.
50. JP Richard, P Paquin. Use of a dye binding method for the determination of
the protein content of dairy products. Milchwissenschaft 45:9294, 1990.
51. SM Gotham, PJ Fryer, WR Perterson. The measurement of insoluble proteins
using a modied Bradford assay. Anal Biochem 173:353358, 1988.
8
Immunological Assay: General
Principles and the Agar Diffusion
Assay

1. INTRODUCTION
1.1. Protein Adulteration, Authenticity, and Speciation
Adulteration is any undeclared substitution or addition designed to enhance
the economic value of a food product (1). The more general features of
adulteration include (a) abstraction or omission of valuable constituents, (b)
substitution by undeclared (usually cheaper or less safe) components, and
(c) concealment of intrinsic low quality or damage (Fig. 1). The seriousness
of each infringement is related to the position of a commodity in the food
system, the origin of the foodstuff, the economic impact of the practice,
consumer awareness of the problem, and safety and health considerations.
Certain forms of adulteration may be unintentional but no less negligent.
For instance, cross-contamination arises when poorly cleaned equipment is
used for processing meats from two different species.
Plant proteins can be legitimately added to meat products as
extenders. They introduce new functional properties such as binding meat
pieces, fat, and water. However, undeclared substitution of one protein by
another (meat by nonmeat proteins) is usually undesirable. This and the
following three chapters describe immunochemical tests for food proteins.
The source of a protein affects its aesthetic appeal and economic value.

221
222 Chapter 8

FIGURE 1 Forms of food protein adulteration.

Protein speciation is a subject of legal, religious, and public health interest.

Groups concerned with protein authenticity include consumer organiza-
tions, the food industry (ingredient suppliers, food manufacturers), public
analysts, enforcement agencies, government ofcials, and the legal profes-
sion. Such interest groups work together to identify and advise on areas of
food adulteration and encourage the use of new or existing methods of
authentication. Protein adulteration is also associated with poor labeling.
An accurately labeled food product is always authentic (25).

1.2. Implications of Adulteration

Protein adulteration has health and safety consequences. Adulteration may
expose sensitive individuals to allergens. To destroy meat-borne bacteria
and viruses (foot and mouth, Newcastle disease, African swine fever, etc.),
the United States Department of AgricultureFood Safety Inspection
Services (USDA-FSIS) specicies minimum processing end-point tempera-
tures (EPTs) for a range of imported meats. Processing at the EPT for beef
Immunological Assay: General Principles 223

will lead to health risks if a product contains undeclared chicken or pork.

The USDA-FSIS also periodically lists voluntary recall notices posted by
companies due to accidental inclusion of ``undeclared allergens'' in various
processed food products. In Europe, the use of meat and bone meal as feeds
is subject to legislation.
Religious dietary regimes (kosher, vegetarian, and halal practices)
require accurate labeling information. Certain protein sources are unac-
ceptable to people of the Jewish, Hindu, and Islamic religions. The
geographic origin of meat can also be a signicant issue. Wildlife
conservation requires the differentiation of farmed and free-living species
(wildlife game, antelopes, buffalo, cattle venison). Species substitution may
involve different classes of meat (poultry or beef), closely related meat
(horse or kangaroo for beef or veal, chicken for turkey), or inadvertent
cross-contamination (beef with pork or kangaroo).
Adulteration undermines commerce, which is based on a rational
relationship between pricing and quality. Undeclared substitution leads to
purchasing decisions based on inaccurate product information. The practice
erodes condence. The likelihood of meat adulteration has increased.
According to Patterson and Jones (6), improvements in meat technology
have made it easier to transport and utilize cheap undeclared meats that are
difcult to identify when meat has been deboned and removed from the
carcass. These products present few visual clues to their origin. Large frozen
blocks and aked or comminuted (minced) batches of meat are virtually
impossible to identify by eye.
The economic imperative can lead to the use of cheaper meats for
certain processed foods. Blood (protein) may be added to mince meat to
make it appear leaner. Mechanically recovered meat (MRM) from chicken
may be used in place of beef or pork. High demand for poultry in India can
lead to the adulteration of chicken meat products with beef and other meat
types. Plant proteins may be added to processed meat products to increase
their bulk. Readily available cow's milk can be added to ewe's milk for the
manufacture of some specialty cheeses. The economic impact of adultera-
tion may be estimated from a notional Protein Adulteration Price Index
(PAPI) dened as

PAPI frequency of substitutions6substitution amount %

1
6price dividend

The potential ill gain from adulteration is partly related to (a) the frequency
of species substitution, (b) the percent weight of adulterant added, and (c)
the price dividend from substitution. Multiplying PAPI by the net value of
224 Chapter 8

trade gives the nancial cost to the consumer arising from undeclared
substitutions.
PAPI can be estimated only crudely for several reasons. First,
information about the pattern of protein substitution worldwide is scarce.
Wide variations will occur in the frequency and extent of undeclared
substitutions in different countries and states and regions within any given
country. Next, the incentive for adulteration is probably related to the net
value of trade in that particular commodity. Most protein commodities
could be subject to adulteration but higher value animal proteins are more
likely targets. The estimated rate of undeclared substitutions for meat is
about 16% in the state of Florida (7). For either raw or cooked meat, the
weight of adulterant added was 15% (average 2.5%). The price dividend for
the added meat can be estimated from the formula 1 (Y/X), where Y is the
real price of the adulterant and X is the advertised price. When the added
protein has no intrinsic value, the price dividend is 1.0. Assuming a price
dividend of 0.5, the PAPI estimate for the U.S. meat and poultry market is
then 0.2%. The upper PAPI can be as high as 5%. Thus, the frequency of
substitution is easily 50% in some sectors of the meat industry. A percent
weight substitution of 10% also seems credible. For the worst-case scenario,
we assume a maximum price dividend of 1, leading to PAPI value of 5%.
The value of the U.S. meat and poultry trade is about US$120 billion
according to FSIS gures from 1995. Consumer spending on meat and
poultry products accounts for one third of the annual food budget. Meat
purchases included raw beef, pork, lamb, chicken, turkey, and approxi-
mately 250,000 processed meat products, which are processed foods
containing >2% poultry or >3% meat. Examples include ham, sausages,
sources, soups, stews, pizzas, and frozen dinners. From the preceding PAPI
and net trade values, we may suppose that the U.S. meat consumer is
overcharged by between US$250 million and US$6 billion annually.
The most efcient techniques for detecting protein adulteration are
immunological. Other well-established approaches include quantitative
sodium dodecyl sulfate polyacrylamide gel electrophoresis (QSDS-PAGE),
isoelectric focusing (IEF), and capillary electrophoresis (8). High-pressure
liquid chromatography (9) and fast protein liquid chromatography (10)
have also been employed for the differentiation of milk from different
species. Finally, mention must be made of nonprotein-based methods.
Detection of amino acids, sugars, or fats associated with particular species
can be a clue to adulteration. Species-specic DNA testing using
hybridization probes or polymerase chain reaction (PCR) is also increasing.
The interested reader is referred to papers by Hunt et al. (11), Fairbrother
et al. (12), Lenstra and Buntjer (13), and Wolf et al. (14); the subject is
reviewed by Meyer and Candrian (15).
Immunological Assay: General Principles 225

FIGURE 2 Methods for food protein immunoassay are divided into marker-free and
marker-linked methods.

2. IMMUNOLOGICAL METHODS

Immunoassays are based on the use of antibodies (Fig. 2). The range of
techniques includes marker-free techniques in solution or agar, e.g.,
solution-phase precipitation, agar gel immunodiffusion, immunoelectro-
phoresis, and counterelectrophoresis. The marker-linked techniques involve
enzyme immunoassay (EIA) or radioimmunoassay (RIA). Application of
immunoassays in food analysis and authenticity testing are reviewed by
Samarajeewa et al. (16), Gazzaz et al. (17), Allen and Smith (18), Barai et al.
(19), Lee and Morgan (20), Hernandez et al. (21), Taylor et al. (22), Smith
(23), and Mandokhot and Kotwal (24).

2.1. Methods of Food Protein Immunoassay *

With marker-free immunoassays, antibody-antigen binding is monitored
directly. Excess antibody is added to a sample suspected to contain antigen.

* The term ``antiserum,'' although established in the literature, can lead to confusion.
Descriptions such as rabbit antiserum for horse serum are not helpful. We will use antibody
wherever possible.
226 Chapter 8

Antibody-antigen binding leads to a precipitate whose height and/or volume

is proportional to the antigen concentration. Antibody-antigen binding can
take place within an agar gel matrix (Section 3). Marker-free immunoassays
are easy to perform and give qualitative (yes-no) answers in a relatively
short time. They are, however, relatively insensitive. Marker-linked
immunoassays provide more sensitive, although more expensive, analysis.

2.2. Principles of Immunoassay

Antibody-antigen binding involves a reversible equilibrium reaction,

Ig P B 2

where, Ig is the free antibody concentration and P the free concentration of

antigen. From Eq. (2), the association constant (Ka) for antibody-antigen
binding is
B B
Ka 3
IgP IgT BP

where IgT is the total concentration of antibody and B the concentration of

bound antibody. From this, one can readily write down the Scatchard
equation:
B
B IgT 4
PKa

A graph of [B] plotted versus [B]/[P] yields a straight line with a slope of 1/Ka
and the intercept IgT. Dividing the value of the intercept by the known
molar concentration of antibody gives the number of antigen binding sites
per Ig molecule; ordinarily, the answer should be 2. The Scatchard plot
is usually nonlinear, showing the presence of different populations of
antibodies. Adsorbing Ig to a solid support will also obscure some antigen
binding sites, leading to a range of Ig-antigen binding afnity.
We turn to the effect of antibody-antigen binding parameters on the
immunoassay characteristics. The various terms in Eq. (3) can be expressed
in terms of the fraction of antigen bound, X:
X
Ka 5
IgT PX1 X

where X B/PT and the term PT is total antigen concentration. Rearranging

Immunological Assay: General Principles 227

this relation gives the quadratic relation

aX 2 bX C 0 6

with a 1, b [PT IgT 1/Ka]/PT, and c IgT/PT. Theoretical X vs. PT

graphs showing the fraction of antigen bound versus concentration were
simulated from known values for Ka, IgT, and PT (Figs 3 and 4).
The rst graph shows a binding curve for an immunoassay using two
antibody concentrations (IgT) of 10 8 and 10 9 M. The value of Ka is
constant (1011 M 1). Fig. 3 shows that the analytically useful binding
interactions occur over the antigen concentration where X 1. The
concentration of antigen should not exceed the total concentration of
antibody binding sites, otherwise the fraction bound decreases below 1. The
linear dynamic range depends on the concentration of antibody. Simula-
tions were also performed in which the concentration of antibody was kept
constant while changing Ka from 1010 to 108 and 106 M 1. Fig. 4 shows that
decreasing antibody-antigen binding strength decreases the sensitivity
(slope) of the calibration graph. We expect that the LLD for antigen will
increase with decreasing Ig-antigen binding afnity.

FIGURE 3 Simulated binding curves for food protein immunoassay. A plot of the
fraction of antigen bound versus the total antigen concentration.
228 Chapter 8

FIGURE 4 Simulated binding curves for food protein immunoassay. Effect of

changing antigen binding afnity constant. The Ka is given values of
1010, 108, or 106 M 1.

2.3. Background Immunology

Antibodies are secreted by vertebrates in response to foreign matter or
antigen. An antibody binds strongly to the antigen that triggered its
formation. The antibodies are immunoglobins (Igs), which are proteins that
dissolve in dilute salt solution and function in connection with immunity.
The Igs are produced by blood cells called B lymphocytes. Each organism
has at least 100 million different B lymphocytes. Each B lymphocyte has a
distinct Ig protruding from its surface. One of the 100 million B lymphocytes
is likely to recognize any antigen encountered in the environment. Binding
of antigen to a cell-bound Ig leads to a proliferation of the recognizing B
lymphocyte within the general population (an idea called the clonal selection
theory).
Antigen binding also triggers B-lymphocyte transformation into two
new cell types. One cell type responsible for Ig production is called the
plasma cell. The second cell type, B memory cells, primes the body to
remember its encounter with the antigen. To induce Ig production, an
antigen must possess a molecular weight >5000. Low-molecular-weight
Immunological Assay: General Principles 229

compounds (haptens) act as antigens when covalently attached to carrier

proteins. Large antigens possess multiple sites (epitopes) for Ig binding and
are said to be polyvalent. Each epitope elicits Ig production by a specic B
lymphocyte. The heterogeneous mixture of antibody produced by a
population of B lymphocytes is described as a polyclonal antibody (pAb).
A sample of pAb contains a mixture of Ig specic for the different epitopes
on a single polyvalent antigen. Two proteins sharing a common epitope,
perhaps because of sequence or structural homology, will be recognized by
pAb, leading to cross-reactivity. The diversity of antibodies arises from their
structure and large numbers of encoding genes.
Antibodies are glycoproteins with a molecular mass >150 kDa. Each
antibody comprises two heavy (5575 kDa) polypeptide chains and two
light (25 kDa) chains joined by (three) intermolecular disulde bonds. Each
heavy (H) or light (L) chain has variable (v) amino acid sequences at the C
terminal and constant (c) regions near the N terminal. There are ve
different H chains (g, a, m, d, and e) giving rise to ve classes of antibody;
IgG, IgA, IgM, IgD, and IgE. The different classes of Ig occur at plasma
concentrations of about 12, 3, 1, 0.1, and 0.001 mg mL 1 and differ in their
stability and ease of handling. There are two groups of L chains designated k
and l.
The three-dimensional structures of the H chain comprises ve
domains whereas each L chain is organized into two domains. The Hc
and Lc regions are produced by two gene alleles and Lv and Hv regions are
each encoded by about 300 genes. In all, an organism is able to produce at
least 3002 or 9 6 104 types of antibody. Further diversity comes from a
process of somatic recombination and the translation of messenger RNA
(mRNA) transcripts using different reading frames. As a result of such
mechanisms, vertebrates produce up to 1 6 108 different antibodies and
their corresponding B lymphocytes before exposure to an antigen.
The pAbs for commercial immunoassays are produced through the
agency of live animals. B lymphocytes have not been successfully cultured
in vitro. Such restrictions make the production of pAbs difcult to
standardize. Immunizing a second animal must lead to variations in the
crude reagent, which must then be puried and standardized in some way.
The technology for producing monoclonal antibodies (mAbs) was
developed by Kohler and Milstein (25) of the University of Cambridge,
U.K. Tumor cell lines, which survive indenitely when grown in cell culture,
produce mAbs naturally. To produce mAbs of dened specicity, spleen
cells from immunized mice are fused with tumor cells. The resulting spleen
cellmyeloma hybrid (hybridoma) possesses traits from both parental cell
lines, i.e., immortality and the ability to synthesis specic mAbs derived
from mice spleen cells. The hybridoma can be grown in PVC microwell
230 Chapter 8

plates and the cell supernatant examined for the mAb. It was possible to
isolate a single hybridoma cell line (or clone) that produces the required Ig.
Hybridomas can be grown indenitely and large amounts of mAbs
produced for analytical use. Only a few of the available genes for the H
and L regions are expressed owing to a process called allelic exclusion (25).
Fusion is needed for the production of mAbs. The mere mixing of myeloma
cells and spleen cells (polyethylene glycol as a fusion agent) will not lead to
new Ig.

3. SPECIATION OF PROTEINS BY AGAR GEL DOUBLE

IMMUNODIFFUSION ASSAY

Modern agar gel double immunodiffusion (AGID) assays were developed

by Cutrufelli et al. (26,27). The technique is approved by the AOAC. The
AGID assay is fast, requires minimal equipment, and can be performed by
personnel with minimum training. The tests are based on the two-stage
precipitin reaction. AGID comes in several forms (28): (a) linear diffusion,
(b) double radial diffusion, (c) single radial immunodiffusion (SRID), (d)
immunoelectrophoresis, and (e) counterimmunoelectrophoresis.
With linear diffusion, antibody is added to molten agar and the
mixture is allowed to set in a small tube. The sample antigen is placed on the
column of gel and diffuses downward, forming an opaque precipitin phase.
The distance of migration is a function of antigen concentration. For an
SRID assay the antigen is placed in a circular well cut into the antibody-
agar plate (29). The radius of diffusion, indicated by a precipitin phase, is
related to the concentration of antigen. Double immunodiffusion assays
involve two sets of circular wells cut into an agar gel. Into separate wells are
placed antibody and analyte. They diffuse toward each other, combining to
form an opaque precipitation band. With immunoelectrophoresis, the
protein sample is placed within holes cut into the gel and forced to migrate
under an electrical potential. Then antibody is placed along a rectangular
trough parallel to the direction of electrophoresis. Each protein band reacts
with the diffusing antibody, leading to a series of precipitin arcs (30).

3.1. Procedures for Meat Protein Identication by AGID

Assay
AGID assays are an extension of the test tube precipitation test rst
demonstrated by Uhlenauth in 1901 (cited in Refs. 5 and 31). Further work
between 1902 and 1928 led to the production of less complex antigen
(inoculation) mixtures designed to improve specicity and reduce toxicity to
Immunological Assay: General Principles 231

immunized animals. Gel-phase immunodiffusion assays were described by

Oudin around 1946 and then by Ouchterlony (32). The last study led to a
simple in vitro assay for diphtheria toxin and for toxin-producing bacteria.
Heating diphtheria toxin led to its denaturation and failure to produce a
precipitin reaction. Early serological tests for meat and other food proteins
are described by Oswarld (33).
To produce pAb for analytical applications, a host animal is
immunized with antigen at intervals ranging from several weeks to months.
Mice, rats, rabbits, sheep, goats, or horses are used for antibody production.
Larger animals are preferred by large-scale, commercial, producers. After
about 4 weeks, blood is taken from the immunized animal and centrifuged
to produce antiserum, i.e., blood serum containing antibody. Further
booster injections may be administered and antibody production monitored
until a high plasma concentration (antibody titer) is attained. To avoid
cross-reactivity, pAb may be puried by immunoadsorption. When the titer
of antibodies reaches a high level, larger samples of blood are removed,
allowed to coagulate, and centrifuged to produce crude antibody.
Preservative is normally added before storing at 208C. Important
variables for antibody production include the following:

1. Purity of the antigen. Puried antigen is seldom required.

However, more pure antigens have less toxic effects on the
immunized animal.
2. Choice of host animal. A large animal such as a goat yields more
serum than a mouse. To reduce cross-reactivity, pAb should be
raised in a species similar to the species for which the antibody is
intended to differentiate. Goats or sheep pAb is more able to
differentiate between antigens from cattle, buffalo, and other
bovine species (59).
3. Choice of adjuvant. Most investigators use Freund's complete
adjuvant. This is an oil suspension of inactivated Mycobacterium
tuberculosis. A muramyl peptide from the bacterial wall stimulates
antibody production. Alternatively, antigen is coprecipitated or
adsorbed on insoluble aluminum oxide, synthetic muramyl
peptides, or carbohydrate (34).
4. Mode of immunization. Subcutaneous or intramuscular injection is
common. Several investigators describe injections via the foot
pads of rabbits. Intervals of injections are usually weekly. Larger
animals may be injected at 30-day intervals.
5. Time and method for bleeding. Immunization schedules usually
require trial bleedings at 4- to 7-day intervals after the injection of
232 Chapter 8

booster antigen. Blood may be collected from a vein in the ears or

tail.
6. Purication by immunoadsorption. To remove cross-reactivity with
sheep, goat pAb may be exposed to sheep antigen and then
centrifuged to remove any precipitate formed. The soluble pAb
phase should not now react with sheep. Immunoabsorption can be
performed with soluble sheep antigen or antigen immobilized on
cyanogen bromideactivated support (3539).
AGID tests are performed according to the following steps: (a)
extraction of antigen and immunization of host animal (see Method 1), (b)
production of pAb (Method 1), (c) purication of pAb by immunoadsorp-
tion (see Method 2), (d) preparation of agar gel plates (see Method 3), and
(e) conduction of AGID test. Details of these steps are given next. The
protocols are drawn from reference listed in Table 2 (Sec. 3.4). The
inexperienced worker should seek expert advice regarding humane treat-
ment and immunization of host animals. Commercial pAb should be used
when available. The internet is a good source of addresses of companies
currently supplying immunoassay reagents and ready-to-use kits.
Method 1
Extraction of meat antigens and pAb production (40,59).
Reagents
1. Phosphate-buffered saline (PBS; 0.01 M phosphate buffer, pH
6.87.2 0.1 M KCl)
2. Muslin cloth
3. Freund's adjuvant
Procedure
Fresh meat antigen. Stir ground meat with 1 volume of PBS overnight
at 48C. Filter through muslin cloth and centrifuge at 10,400g for 60
minutes. Filter the supernatant through Whatman No. 3 paper to
remove oating fat droplets. Freeze dry for prolonged storage.
Immunization. Mix the antigen (510 mg mL 1 PBS) with an equal
volume of Freund's complete adjuvant. Inject adult rabbits with
2 mL of antigen suspension at multiple sites. Reinject the animals
with booster doses of antigen dissolved in Fruend's incomplete
adjuvant at weekly intervals. With goats the rst injection can be
4 mL of antigen. The booster dose is injected every other week.
Small amounts of blood should be removed in the intervening weeks
and tested for pAb production.
Treatment of blood and antibody production. Collect whole blood,
allow to coagulate at room temperature for about 3 hours, and then
Immunological Assay: General Principles 233

store at refrigerator temperatures overnight. Centrifuge at 2000g to

remove blood cells. To the supernatant (antibody) add 100 ppm of
methiolate as a preservative and store at 208C until needed.
Immunization schedules are exible, ranging from 34 weeks for
rabbits to several months for goats, sheep, or horses. The pAbs for ORBIT
and PROFIT were produced by injecting goats with 10 mL of antigen
coprecipitated with alum. The same antigen was injected 2130 days later
(5 mL per hind leg), then at 3-weekly intervals. Blood samples were taken
*7 days after each injection to monitor pAb production. The immunization
process is continued until a desired concentration of antibody is attained.
Method 2
Antibody purication by immunoadsorption.
1. A simple immunoadsorption procedure (31,35). To 1 mL of crude
pAb for species A that shows cross-reactivity with species Y, add
20 mg of antigen from species Y. Incubate the mixture at room
temperature for 4 hours. Refrigerate at 48C overnight and
centrifuge (2500g for 20 minutes) to remove pAb-antigen
complexes. The supernatant phase should be pAb with improved
specicity for species A.
2. Immunoadsorption using ethylchrolorformate cross-linked antigen.
The original method was described by Avrameas and Ternynck
(36,37). Adjust a solution of antigen (e.g., 500 mg of whole serum
protein in 10 mL of 0.2 M acetate buffer) to pH 5.0 with dilute
HCl. Add ethylchloroformate (*3 mL) dropwise. Maintain the
acidity at pH 4.55 using 1 M NaOH. Allow to react for about 10
15 minutes, redisperse the resulting gel in a further 10 mL of 0.2 M
acetate buffer, and allow to react for another 60 minutes. Recover
the gel formed by ltration or by centrifuging gently. Wash with
distilled water and keep refrigerated until use. Shake antibody for
species A and with cross-linked antigen from species Y overnight.
Recover the soluble serum phase, which should now be mono-
specic for species A. This method has been demonstrated by
Kang'ethe et al. (59).
Immunosorption can be performed with the dried antigen, cross-linked
antigen, or antigen immobilized on CNBr-activated support.
Method 3
Standard agar gel double immunodiffusion assay.
Reagents
1. Agar (Oxoid Ltd.)
234 Chapter 8

2. PBS buffer
3. 6.5-mm-diameter lter discsoptional

Procedure
Prepare 1% (w/v) molten agar in PBS by heating on a water bath. Pour
about 4 mL of molten agar into a petri dish and allow to set at room
temperature. Refrigerating for a few hours will harden the gel. Cut
out several 6-mm-diameter holes or wells using a cork borer. Place
one well in the center of the petri dish and then surround this with a
quartet of wells each placed 6 mm from the central well. Remove the
circular piece of gel using a Pasteur pipette attached to a vacuum
line. Place a drop of molten agar into each well to act as a seal,
thereby avoiding the migration of samples below the well.
To perform a standard AGID assay, place pAb in the central well. In
the surrounding wells, place one each of the test samples (*20 mL).
Cover the petri dishes to avoid dehydration and allow to stand at
room temperature for 24 hours.

3.2. Agar Gel Double Immunodiffusion Assay for Uncooked

Meat
The rst attempts to identify raw meat using the AGID assays were
described by Warnecke and Safe (38). They found that actomyosin was a
poor antigen, with injections of 20150 mg leading to only moderate pAb
production in rabbits. Rabbit pAB for beef whole serum showed cross-
reactivity for lamb extract. No reactivity was seen for horse meat or pork.
The crude pAb was rendered monospecic for beef by immunoadsorption.
An AGID assay with immunoadsorbed pAb allowed the detection of beef.
Many of the methods described in this report are still in use at the present
time.
Further developments in AGID assays for meat speciation occurred in
the laboratories of the U.S. Department of Agriculture, Beltsville, MD.
Fugate and Penn (39) used AGID assays to identify meat from beef, horse,
pig, and sheep. Of 12 meat samples examined, 11 were correctly identied.
They recommended that the AGID test should be subjected to collaborative
testing. AGID assays using pAb for residual serum albumin from meat were
produced by Hayden (40). Hers was probably the rst dissertation on this
subject. At the Department of Food Science, University of Georgia (Athens,
GA), Helm et al. (41) compared the AGID assay and a simple test tube
precipitin test using (rabbit) pAb for beef, horse, lamb, and pork. The
AGID test detected adulteration at the 2% level.The solution precipitin test
was *three times faster and four times more sensitive. In Australia, Swart
Immunological Assay: General Principles 235

and Wilks (42) differentiated beef, horse, kangaroo, and mutton by AGID
assay. Species identication eld tests (SIFTs) were developed in 1984 by
Mageau et al. (43) at the USDA. Finally, Darwish et al. (44) also described
an AGID assay for detecting beef adulteration with camel and pork. Some
examples of the application of immunodiffusion assays include the detection
of pork in beef mince meat (45), Alaska pollack surimi analysis in meat
products (46), and the detection of various meat types (beef, pork, horse,
poultry) in hamburger (47).

3.3. Species Identication Field Tests (SIFTs) for Uncooked

Meat
SIFTs are designed for eld testing in abattoirs and meat inspection
stations. The rst of these test kits is called ORBIT (43). The acronym
stands for the Overnight Rapid Bovine Identication Test. ORBIT was
followed by PROFIT (Poultry Rapid Overnight Field Identication Test)
(48), SOFT (Serological Ovine Field Test) (49), PRIME (Porcine Rapid
Identication Method) (50), REST (Rapid Equine Serological Test) (51),
DRIFT (Deer Rapid Identication Field Test) (52), and MULTI-SIFT
(Multispecies Identication Field Test) (53). The last kit can simultaneously
test for beef, poultry, pork, sheep, horse, and deer meat. After successful
collaborative trials (26), ORBIT and PROFIT received approval from the
AOAC. These methods can detect meat adulteration levels of about 10% or
greater (27).
SIFTs are not radically different from classical AGID assays.
However, considerable design effort has gone into making SIFTs attractive
and easy to use. Antibody and meat sample extract are adsorbed onto lter
paper discs. Both are then freeze dried, providing stabilized reagent discs
that can be stored for up to 12 months at refrigeration temperature. The
antibody lter discs and all materials needed to perform SIFTs are available
commercially. The tests are used by the USDA and meat inspection services
in the United States.
According to the team responsible for SIFTs, conventional methods
for meat speciation (e.g., electrophoresis, chromatography) share many of
the following disadvantages: (a) tests are usually performed within a formal
laboratory, (b) relatively sophisticated equipment is needed, (c) high levels
of staff expertise and training are necessary, (d) time delays arise due to off-
site testing with an attendant need for transmitting samples and assay results
to and from the analyst, and (e) relatively labile reagents are used. SIFTs
were developed with the aim of avoiding such disadvantages. The assays are
highly reliable, fast, easy to use, accurate, and sensitive. Little expertise or
previous experience is needed for successful testing. Finally, SIFTs use
236 Chapter 8

stabilized reagents (antibody and gel plates) with a shelf life of up to 12

months.
SIFTs use pAb specic for native serum albumin (40). The tests are
therefore intended for raw meat speciation. A key feature is the use of
lyophilized reagents that are more stable for storage. Commercial pAbs as
well as those prepared in house proved usable. Assay results were affected
by the distance of separation between lter discs, with a distance of 45 mm
being optimal. The tests showed considerable temperature tolerance.
Incubating agar gels plates at 25378C had no ill effect. Higher temperatures
led to gel dehydration. Lower temperatures prolonged the time needed for
precipitin formation. Finally, SIFTs were suitable for the analysis of whole
meats, ground meat, meat emulsions, and other formulated meat products,
provided these were not heated. Table 1 summarizes some performance
characteristics of SIFT assays. The MULT-SIFT assay allows simultaneous
testing of an unknown meat sample (antigen) against six different reference
pAbs (Table 1).

3.4. Thermostable Meat Antigens for Cooked Meat Analysis

Ideal antigens for cooked meat analysis survive thermal treatment at 708C
or autoclaving temperatures of 1208C for 15 minutes. Hayden (54)
considered troponin as a heat-stable meat antigen (Table 2). Extensive
research since 1977 has shown that thermostable meat antigens are usually
troponin C, troponin I, or troponin T. Schweiger et al. (55) used puried
turkey troponin T for AGID assay. Thermostable antigens were initially
developed from adrenal gland extracts (56,57) by Milgrom and Witebsky
(58).* Rabbit pAb for boiling-resistant, ethanol-insoluble (BE) antigen from
beef showed cross-reactivity with BE antigen from sheep. There was no
reaction with heated adrenal extracts from pig, rat, guinea pigs, or humans.
The method for BE antigen preparation has remained unchanged for nearly
40 years. Hayden (56,57) used BE antigen for detecting cooked beef sausage
adulteration with horse, sheep, and pig meat.
Radhakrishna et al. (62) showed that (buffalo, goat, oxen) muscle BE
antigen had SDS-PAGE bands corresponding to troponin (C, I, and T) and
tropomyosin. Bhilegaonkar et al. (67) found that the concentration of
(buffalo, sheep, goat, pig) BE antigen was 21130% higher in adrenal tissue

* Boiling-resistant ethanol-insoluble (BE) antigen is produced by boiling an aqueous extract

from bovine adrenal glands or muscle tissue followed by centrifugation. The supernatant is
autoclaved at 1208C for 30 minutes and centrifuged. Then three volumes of 95% ethanol are
added to the soluble fraction. A whitish precipitate forms after *1214 hours at 378C and is
then dissolved in normal saline for immunization.
Immunological Assay: General Principles 237

TABLE 1 Characteristics of Some Species Identication Field Tests (SIFTs)a

Test acronym and Immunized

specicity host Performance

ORBIT, Rabbit, 70 samples. No false

beef (39) goat, sheep positives/negatives, Specicity: bison
( ), bovine ( ), water buffalo ( ),
deer ( ), elk ( ), goat ( ), horse ( ).
PROFIT, Goat 66 samples. No false
poultry (40) / results. Specicity: chicken ( ),
turkey ( ), goose ( ), quail ( ),
partridge ( ), bovine ( ), deer ( ),
horse ( ), pig ( ), sheep ( ).LLD 3%
or 5% in pork or beef
PRIME, Goat 83 samples. No false /
pork (42) results. LLD 5% pork (in beef), 3% pork
in lamb.
SOFT, Calf 90 samples. No false /
sheep (41) results. LLD 3% mutton in beef.
REST, Sheep 101 samples. No false /
horse (43) results. Specicity: deer, ( ), donkey
( ), mule ( ), beef ( ), pork ( ),
sheep ( ), chicken ( ), turkey ( ),
kangaroo ( ). LLD 3% horse
DRIFT, Goat 100 samples. No false /
deer (44) results. Specicity: mule deer ( ), elk
( ), moose ( ), reindeer ( ), beef ( ),
horse ( ), sheep ( ), chicken/turkey
( ).
MULTI-SIFT, Various No false / results.
six different Specicity (as above)
species (45)
a
LLD, Lower limit of detection or minimum % weight of adulterant detectable. All antiserum lter
discs are stable for 45 months at room temperature and 12 months at 48C. ( ) positive test,
( ) negative test.

than in muscle. SDS-PAGE analysis of adrenal BE antigens showed a single

35.5-kDa component (troponin T). Cow and buffalo muscle BE antigen had
three components with molecular masses of 35.5 kDa (troponin T), 19 kDa
(troponin I), and 16 kDa (troponin C). Sheep and goat muscle BE antigen
had the 35.5- and 19-kDa components. Pork thermostable antigen showed
three troponin bands and an extra 66-kDa protein.
238 Chapter 8

TABLE 2 Boiling-resistant Ethanol-Insoluble (BE) Protein as Thermostable Antigen

for Agar Gel Double Diffusions Assay of Cooked Meat

Analysis/comments Reference

Troponin T, as chicken muscle antigen Hayden (54)

BE antigen for sausage analysis
Troponin T antigen for turkey analysis
BE antigen for differentiating domestic
meat sources (beef, goat, and sheep)
from 14 game species
BE antigen for detecting chicken
adulteration by buffalo, sheep, goats,
pig meat
BE antigen identied as troponin T;
isolation from buffalo, cattle, sheep,
goat, and pig adrenals and muscle
tissue
BE antigen for detection of pork in beef,
buffalo, sheep, goat, chicken meat
End-point temperature determination,
myoglobin
Hayden (56,57)
Schweiger et al. (55)
Kang'ethe et al. (59)
Sherikar et al. (60,61)
Radhakrishna et al. (62);
Bhilegaonkar et al. (67);
Sherikar et al. (63)
Reddy and Giridhar-Reddy (64);
Saisekhar and Reddy (65)
Levieux and Levieux (66)

Saisekhar and Reddy (65) isolated troponin T from raw beef and
buffalo meat. An AGID assay based on native troponin T antigen from
buffalo cross-reacted with cattle, goat, and sheep meat. There was no
reaction with chicken or pork. The (rabbit) pAb for buffalo was rendered
monospecic by immunoadsorption with cattle, goat, and sheep antigen.
The AGID assay using monospecic buffalo pAb could detect beef or
mutton adulteration with 1% of buffalo meat. Interestingly, (rabbit) pAb
for bovine troponin T was monospecic for beef without prior immunopur-
ication. No cross-reactivity occurred with buffalo, goat, sheep, or chevon
meat. The LLD was 10% beef addition to samples of buffalo, chevon, or
mutton. These tests based on pAb for native troponin T did not detect cooked
meat. In contrast, pAb for native troponin T from chicken or turkey was
sensitive to (poultry meat ) antigen in fried sausages (54,55). Clearly,
troponin T is not heat resistant. Heating may generate a denatured but
soluble troponin T that functions as a BE antigen.
Much evidence points to troponin T being the major BE antigen.
However, other muscle proteins might also play this role. SDS-PAGE
analysis of muscle proteins extracted by 0.6 M KCl shows bands for myosin
(200 kDa), actinin, actin (42 kDa), and troponin T (37 kDa). In the 3228
kDa region appeared bands for troponin I, tropomyosin, and myosin light
Immunological Assay: General Principles 239

chain (68). After comminution, troponin T was susceptible to proteolysis,

forming a 30-kDa fragment. Proteolysis also occurred for meats up to 7
days old (69). After heating meat to 808C, only bands for troponin T, actin,
and the myosin light chains remained. As actin is highly conserved between
species (70), antibodies for this protein will not differentiate meat from
different mammals (62,67). The pAb for actin could form a general test for
animal tissue.

3.5. Agar Gel Double Immunodiffusion Assays for Cooked

Meat
Myoglobin was not wholly successful as a heat-stable antigen (31). To
produce an AGID test for cooked meat, (rabbit) pAb was prepared using
pure myoglobin preheated to 908C for 15 minutes. Cooking meat samples to
708C led to loss of sensitivity to meat myoglobin. There was cross-reactivity
between (rabbit) pAb for lamb and beef myoglobin. However (goat) pAb
for porcine myoglobin showed no cross-reactivity with beef. Thus 3% pork
could be detected in unheated meat samples. Mild heating raised the LLD
(lower limit of detection) to 10% adulteration of beef with pork. Hayden
(31) proposed that (rabbit) pAb for heated myoglobin might be suited for
developing AGID tests for mildly heated meat from bovine, horse, seal, or
whale. Actually, pAb for heat-denatured pure myoglobin did not recognize
myoglobin heated within a meat matrix. Apparently, the conformation of
denatured myoglobin depends on the heating matrix. The denaturation of
heme proteins involves an in-series mechanism. First, there is a conforma-
tional change and dissociation of the heme group. This binds covalently to
the apo protein and other proteins or else forms heme oligomers (71); it is
possible that some heme degradation products are antigenic. The apo
protein then undergoes incorrect refolding and/or aggregation. The presence
of other meat proteins can easily affect the end point for myoglobin
denaturation.
The AGID assay using (rabbit) pAb for chicken troponin T could
detect the presence of 15% chicken meat in beef sausages cooked to an
internal temperature of 70908C (54). Using (rabbit) pAb for turkey
troponin T, turkey sausages heated to an internal temperature of 708C and
cooked for 1015 minutes were also successfully analyzed. All assays were
also highly sensitive to turkey meat in raw meat balls (mixture of pork and
beef) and extract from poultry fried sausages (55).
Thermostable BE antigen was the basis for the AGID tests for
differentiating meat of domesticated species (cattle, goats, sheep) from 14
240 Chapter 8

wildlife game species.* The tests developed by Kang'ethe et al. (59) from the
University of Nairobi used (goat) pAb. The crude pAb was surprisingly
monospecic in most cases. Cross-reactivity was observed for some closely
related species: buffalo and cattle, bushbuck and cattle, Grant's gazelle and
sheep, and Grant's gazelle and Thomson's gazelle. After immunoadsorb-
tion, each pAb was rendered monospecic for thermostable antigen, cooked
meat extracts, or fresh meat. The domesticated species were clearly
distinguished from the game species. Differentiating between Grant's and
Thomson's gazelle, kongoni and topi, and kongoni and wildebeest remained
problematic. Using goat as the host for pAb production probably accounts
for low cross-reactivity. By comparison, (rabbit) pAb shows lower
specicity and a greater likelihood of cross-reactivity between closely
related species.
Chicken meat is in high demand in parts of Asia owing to religious
restrictions related to the consumption of beef. Sherikar et al. (61,63)
produced an AGID assay for chicken meat adulteration with beef, buffalo,
goat, mutton, or pork. BE antigen was prepared from heart, kidney, liver,
spleen, or lung tissue. Crude (rabbit) pAb for pork was monospecic. The
other (rabbit) pAbs were puried by immunoadsorption. Partially puried
pAbs reacted only with homologous antigen from raw tissue or tissue mildly
heated at 708C. Fully cooked meat could not be detected unless further
processed to BE antigen (by autoclaving, centrifugation, and ethanol
precipitation). Apparently, components in the relatively complex cooked
meat extract interfered with antibody-antigen binding and/or precipitin
formation. AGID assays were performed on samples with BE antigen.
Adulteration of chicken with 10% (w/w) beef, buffalo, goat, or sheep meat
was detectable. The LLD for pork was 5% (w/w).
Reddy and Giridhar-Reddy (64) also produced an AGID assay for
cooked pork. Porcine muscle BE antigen and the corresponding (rabbit)
pAb were prepared as usual. The crude (rabbit) pAb was monospecic for
pork. Samples for analysis were extracted from raw pork or after heating at
1208C for 30 minutes. The AGID assay detected 10% (w/w) pork in
cooked meat from buffalo, cattle, chicken, goat, and sheep. The LLD for
pork in raw meat mixtures was 20% (w/w).
The frequency of undeclared meat substitutions was referred to earlier.
Hsieh et al. (7) examined 806 raw meat and 96 cooked meat samples (mostly

* The species include buffalo (Syncerus caffer), bushbuck (Tragelaphus scriptus), cattle (Bos
indicus), eland (Taurotragus oryx), goat (Capra aegagrus hircus), Grant's gazelle (Gazella
granti), impala (Aepyceros malampus), kongoni (Alcelaphus buselaphus cokii), oryx (Oryx spp.),
sheep (Ovis ammonaires), Thomson's gazelle (Gazella thomsoni), topi (Damaliscis lunatus),
waterbuck (Kobus spp.), and wildebeest (Connochaetes taurinus).
Immunological Assay: General Principles 241

beef and ground veal). The AGID tests involved commercially available
pAb specic for raw sheep, pork, beef, and horse meat. Enzyme-linked
immunosorbent assay (ELISA) kits utilizing pAb for thermostable antigen
were used for the analysis of cooked meats. About 16% of raw meat samples
were adulterated with meat from another species. The frequency of
adulteration increased to 23% for cooked meat. The most frequent
adulterants for ground veal or beef were sheep (47%), pork (42%), or
poultry (31%). There were no substitutions involving horse meat. Cross-
contamination via (improperly cleaned) processing equipment was not
signicant. Ground lamb and pork had adulteration frequencies of 66.7%
and 53%, respectively. So far, immunological methods cannot distinguish
beef from veal or mutton from lamb.

3.6. Agar Gel Double Immunodiffusion Assays for Other

Food Proteins
AGID assays have not proved popular for the analysis of nonmeat proteins.
The analysis of soy protein in meat is reviewed by Llewellyn (5). Hammond
et al. (72) found that cross-reactivity with other legume proteins was
widespread. The reliability of AGID tests for vegetable proteins is poor
because of the effect of heat, extrusion, texturization, and other forms of
processing. More promising are methods based on ELISA (Chapter 10).

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Immunological Assay: General Principles 243

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9
Speciation of Meat Proteins by
Enzyme-Linked Immunosorbent
Assay

1. INTRODUCTION

Enzyme-linked immunosorbent assay (ELISA) was invented by Eva Engvall

during her Ph.D. studies in Stockholm in 1971 (1). A similar assay was
produced by Van Weemen and Shuurs (2). Developments between 1971 to
1981 are reviewed in reference 3. ELISA is an example of an enzyme
immunoassay (EIA). These are immunological tests ending with enzymatic
analysis. Enzyme multiplied immunoassay tests (EMITs) are performed in
solution. ELISA employs immunoglobins attached to a solid surface. EIA is
further categorized as competitive or noncompetitive (Table 1). Secs 25 of
this chapter describe authenticity testing for raw and cooked meat by
ELISA. The meat antigens detected with such tests are mostly residual
serum proteins from the blood or else muscle proteins. Sec. 6 covers ELISA
for meat protein using monoclonal antibodies (mAbs). Seafood speciation is
discussed in Sec. 7, followed by a comparison of the different ELISA
formats in Sec. 8. Attempts to detect meat and bone meal in animal feeds are
described in Sec. 9, as are some new ELISA tests for the bovine spongiform
encephalopathy (BSE) agent.
During EMIT an enzyme-antigen conjugate (Enz-antigen) reacts with
antibody (Ig). Steric hindrance from Ig binding reduces enzyme activity. The

247
248 Chapter 9

TABLE 1 A Range of Enzyme Immunoassays (EIAs)a

Solution-phase EIA Solid-phase EIA

EMIT ELISA
Competitive Competitive
Enz-antigen Enz-antigen, Enz-Ig
Noncompetitive
Direct, indirect, sandwich
a
Enz, enzyme; Ig, antibody. Other acronyms are dened in the
general text.

added sample (with known concentration of antigen) competes with the Enz-
antigen for Ig. This process relieves steric inhibition of the Enz-antigen.
EMITs are suitable for analyzing small molecular weight antigens. The
measured enzyme activity is proportional to the added analyte concentration.
To perform EMIT requires access to pure antigen and Enz-antigen conjugate.
Competitive ELISA uses enzyme-labeled antibody (Enz-Ig or Enz-
Ig0 )* or Enz-Antigen conjugate as reagent:

1. Adsorb pure antigen on microwell plates (Fig. 1)

2. Block excess adsorption sites with milk protein.
3. In a separate step, preincubate Enz-Ig with the sample (containing
an unknown concentration of antigen).
4. Transfer the mixture to the antigen-coated microwell plate.
5. Wash to remove excess reagent.
6. Assay the microwell plate for bound Enz-Ig using enzyme
substrate.

The enzymatic activity is inversely related to the concentration of antigen in

step 3. An alternative one-step competitive ELISA is performed as follows:

1. Coat microwell plates with antibody.

2. Block nonspecic sites (as described above).
3. Add Enz-antigen and sample and allow competition for the
microwell-bound Ig.
4. Wash microwell plates and assay with enzyme substrate.

The enzyme activity is inversely related to sample antigen concentration.

* Enz-Ig enzyme linked to a primary antibody specic for a food protein. Eng-Ig0 enzyme
conjugate with antibody (Ig0 ) specic for the primary antibody, e.g., goat antibody for (rabbit)
Ig.
Speciation of Meat Proteins 249

FIGURE 1 Competitive ELISA using enzyme-labeled antibody or enzyme-labeled

antigen. (Top) Direct competitive ELISA, bound antigen (1) and free
antigen (3) compete for Enz-Ig (2). (Bottom) Enz-Antigen (2) competes
with antigens (3) for bound antibody (1).

Noncompetitive ELISA is performed using a direct, indirect, or

sandwich format. For indirect ELISA the order of reagent addition is
sample, rst antibody, Enz-Ig0 conjugate, and enzyme substrate (Fig. 2).
After each step, wash the microwell plates to remove excess reagents.
The ``indirect'' prex refers to the use of a second antibody (see Enz-
Ig0 ) to visualize the bound antigen. With direct ELISA, the antigen is
visualized (directly) using one antibody (Enz-Ig). Although requiring two
antibodies, indirect ELISA is a more exible and ultimately cheaper. The
second antibody (Ig0 ) is produced by immunizing goat or sheep with the
primary rabbit Ig. Commercially available Enz-Ig0 can be used for
visualization wherever (rabbit) pAb is used for analysis. By contrast, a
distinct Enz-Ig has to be synthesized for every new direct ELISA. The
sandwich ELISA format may be implemented by binding ``capture''
250 Chapter 9

FIGURE 2 Formats for noncompetitive ELISA. Meat antigen (1) adsorbed on the
surface of a microwell plate, (2) species-specic rabbit antibody, (3)
enzyme-antibody directed against the rabbit antibody.

antibody to the microwell plate, followed by antigen. Thereafter one may

adopt either the indirect or direct visualization strategy.
Applications of ELISA for food analysis were rst discussed at a
symposium held at the University of Surrey in September 1983 (4). The same
theme was addressed by Hitchcock (5) at the second symposium on the
application of immunoassays in veterinary and food analysis. Potential
targets for immunoassay are listed in Table 2. Reviews dealing with ELISA
for food analysis and authenticity testing include those by Allen and Smith
(6), Samarajeewa et al. (7), Gazzaz et al. (8), Barai et al. (9), Lea and
Morgan (10), Hernandez et al. (11), Taylor et al. (12), Smith (13), Simpkins
and Harrison (14), Mandokhot and Kotwal (15), and Luethy (16).
Hitchcock and co-workers (17)* were rst to use ELISA for food
protein speciation. Compared with ELISA, other immunological assays
have some of the following disadvantages: (a) a requirement for large

* They were afliated with Unilever Research Laboratories, Colworth House, Bedford (UK).
Speciation of Meat Proteins 251

TABLE 2 Potential Food Analytes (Antigens) for ELISA

Major groups Examples

Trace components/contaminants
Mycotoxins Ochratoxin A1, aatoxins
Bacterial toxins Clostridium, Staphylococcus, E. coli
Hormones and anabolic agents Natural, synthetic reproductive or
growth-affecting hormones
Drugs and Antibiotics
Antinutrients Solanine, trypsin inhibitor
Vitamins
Plant hormones Indoleacetic acid, abscisic acid
Pesticide and residues
Additives
Low molecular weight Colors, avors
High molecular weight Gums, stabilizers, emulsiers
Food protein speciation
Meat and egg Beef, buffalo, camel, poultry
Milk Cow, ewe, goat's milk (speciation),
specic milk proteins
Blood Serum protein speciation
Bacterial and fungal Single-cell proteins
Plant proteins Soya, wheat gluten, pea, potato
Source: Adapted from Refs. 4 and 5.

quantities of antibody; (b) long assay times of between 18 and 24 hours,

although 23 hours have been used for qualitative studies, and (c) high cost
per analysis due to the cost of antibody. The advantages of ELISA
compared with argar gel immunoassays include (18) (a) a 100- and 1000-fold
lower requirement for antibody, (b) small sample size (1 mg of meat) per
assay, (c) increased (10- and 100-fold higher) sensitivity, (d) more easily
interpreted results, (e) screening for multiple adulterations possible, and (f)
increased potential for partial automation.
ELISA is a major technique for meat protein speciation (Table 3).
Implementing ELISA involves the following stages: (a) production of
antibody, (b) purication of antibody by afnity chromatography, (c)
preparation of Enz-Ig conjugate, (d) extraction of meat antigen, and (e)
ELISA. Depending on past experiences, one can perform all operations in
house or use a commercially available ready-to-use kit. Some off-the-shelf
components may be combined with reagents prepared in house. Essential
equipment and accessories include a 96-well microwell plate reader,
microwell plate washer, precision micropipettes, and microwell plates. A
252 Chapter 9

TABLE 3 Speciation of Raw Meat by ELISA with pAb

Analysis/comments Reference

ELISA Engvall and Perlman (1)

Indirect ELISA, horse, beef Kang'ethe et al. (19)
Indirect ELISA, beef, camel, horse, kangaroo, sheep Whittaker et al. (18,20)
Sandwich ELISA, beef, sheep, horse, kangaroo, pig, Patterson et al. (21)
camel, buffalo, and goat
Sandwich ELISA, pork, beef Jones and Patterson (22)
Sandwich ELISA, beef, buffalo Patterson and Spencer (23)
Indirect ELISA, beef, pork, horse Jones and Patterson (24)
Meat identication ELISA kits (UK) Pelly and Tindle (25)
Sandwich ELISA,a beef, pork Martin et al. (26,27)
Sandwich ELISA,a chicken Martin et al. (27)
Indirect/competitive ELISA, direct/competitive Ayob et al. (29)
ELISA, pork, beef
Indirect ELISA, hand or mechanically recovered Stevenson et al. (30)
chickena meat
Seafood (sardine, tuna, and crustacea) Taylor et al. (31)
a
Studies based on muscle protein antigen. Indirect ELISAs are noncompetitive methods unless
stated.

plate reader enables colorimetric readings from microwell plates in situ. The
96 wells can be read within a space of 1.5 minutes. Several precision
micropipettes are necessary for dispensing reagents; most essential are 50-mL
and 100-mL pipettes. A continually adjustable (50200 mL) multiwell pipette
is convenient for rapid dispensing. Polystyrene microwell plates appear to be
the solid phase of choice.

2. RAW MEAT SPECIATION BY INDIRECT ELISA

Noncompetitive indirect ELISA is probably the simplest EIA format. A

reasonably comprehensive description of this technique is provided here to
encourage those wishing to implement this assay for the rst time. This
section also considers the limits and tolerances of this technique.
Method 1
Analysis of uncooked meat by noncompetitive indirect ELISA.
Reagents
1. Detection antibody (e.g., peroxidase-labeled antibody)
Speciation of Meat Proteins 253

2. Antigen standard
3. Coating buffer (0.1 M sodium carbonate buffer, pH 9)
4. PBST (phosphate-buffered saline with 0.05% Tween 20)wash
and diluent buffer
5. Enzyme assay buffer (citrate-phosphate buffer, pH 4.2)
6. Enzyme substrate (varioussee the following)
7. Enzyme stopping solution (varioussee the following)
Procedure
1. Coat microwell plates. Add 100 mL of meat extract (diluted in
PBST) to microwell plates and incubate for 60 minutes at room
temperature. Wash with PBST (100 mL) three times.
2. First antibody. Add 100 mL of rabbit antibody (diluted in PBST).
Incubate for 3060 minutes and wash wells with PBST (100 mL)
three times.
3. Detection antibody. Enzyme conjugate. Add 100 mL of Enz-
antibody conjugate. Incubate for 60 minutes. Wash with PBST
(100 mL) three times.
4. Enzyme assay. Add 100 mL of enzyme substrate. Incubate for 30
minutes. Add stopping solution and record absorbency reading
with the plate reader.
Not counting the PBST washing steps, indirect ELISA involves four steps.
Perform preliminary experiments to establish the optimum sample (antigen)
dilution as well as the required concentrations of antibody and Enz-Ig
conjugate. Incubation times ranging from 30 minutes to 3 hours have been
employed. Horseradish peroxidase (HRP) is the most common enzyme label
for ELISA. Suitable substrates for HRP are ABTS [2,20 -azino-di(3-
ethylbenzthiazoline sulfonate)] and hydrogen peroxide. Other HRP
substrates are listed in Table 4. Alkaline phosphatase, urease, and glucose
oxidase have also been used as labels.
Kang'ethe et al. (19) developed indirect ELISA for horse meat.
Polyclonal antibody (pAb) for horse serum albumin (HrSA) was raised by
immunizing rabbits. Preliminary tests using AGID assay showed that
(rabbit) pAb cross-reacted with BSA and sheep serum albumin (SSA).
Therefore crude (rabbit) pAb for HrSA was puried by column
immunoadsorption. Antibody samples were diluted by about 100 1 and
meat extracts diluted by 200 1 and 3200 1 before assay. Indirect ELISA was
performed essentially as described in Method 1. Substitution of beef with 5
80% horse meat produced the calibration response
 
1 DA492 K 1
%Horse ln 1
C K2
254 Chapter 9

TABLE 4 Enzymes and Substrates for ELISA

Substrate and assay

Enzyme conditions Stopping solution

Horseradish peroxidase ABTS (2 mM) H2O2 30 mL of NaCN (37 mM)

Horseradish peroxidase
Horseradish peroxidase
Horseradish peroxidase
Horseradish peroxidase
Alkaline phosphatase
Source: Compiled from multiple sources in Table 3.
(2 mM), citrate- or 50 mL citric acid
phosphate (0.1 M, pH (0.1 M)
4.2), DA 414 nm
o-Dinisidine (0.08%) 50 mL of H2SO4 (4 M)
H2O2 (0.006%), citrate-
phosphate buffer
(0.1 M, pH 5);
DA 620 nm
o-Diphenylenediamine 50 mL of H2SO4 (12.7 M)
H2O2 in citrate-
phosphate buffer
(0.1 M, pH 5),
DA 492 nm
o-Toluidine (2.5 mM) 50 mL of H2SO4 (12.7 M)
H2O2 (2.5 mM), citrate-
phosphate (0.1 M, pH
4.5), DA 620 nm
3,30 ,5,50 -Tetramethylben- 100 mL of H2SO4 (3 M)
zidine (1 mM) H2O2
(3 mM) in 0.2 M citrate
buffer (pH 3.95),
DA 450 nm
p-Nitrophenyl phosphate 25 mL of NaOH (0.4 N
(1 mg/mL) in NaOH)
diethanolamine buffer,
pH 9.8, DA 405 nm

where C, K1, and K2 are constants and A492 is the absorbance reading. A
simple straight-line equation applied for 060% substitution of beef by horse
meat. The assay precision was 2.38%.
Whittaker et al. (18,20) employed indirect ELISA for the identication
of uncooked meat from cattle, camel, horse, kangaroo, and sheep. To
improve specicity, (rabbit) pAbs for serum proteins were puried via
afnity chromatography. Antigen adsorption to microwell plates was
optimum at pH 56. The working range for analysis was 1080% (w/w)
adulteration.
Speciation of Meat Proteins 255

3. RAW MEAT SPECIATION BY SANDWICH ELISA

The rst step for sandwich ELISA is coating microwell plates with capture
antibody. Then meat extract is added followed by either Enz-Ig conjugate or
the combination of second Ig Enz-Ig0 . Binding meat antigens to a ``bed''
of antibody introduces selectivity. Noncomplementary proteins do not bind
to the rst antibody and can therefore be washed from the microwell plate.
Further specicity derives from the second antibody (Fig. 3).
Patterson et al. (21) were the rst to develop a sandwich ELISA for
meat speciation. Different ELISA tests were produced with specicity for
meat from buffalo (water buffalo),* camel, cattle, goat, horse, kangaroo,
pig, or sheep. Capture pAbs were usually raised by immunizing sheep with
whole serum protein from camel, cattle, goat, etc. Specic pAb for sheep
was produced using cattle as host. The yield of pAb was greater if the host
animal was phylogenetically different from the donor species. Sheep
produced greater quantities of pAb when injected with kangaroo antigen
as compared with beef antigen. Detection pAbs were raised using rabbits.
Samples of crude pAb were puried by afnity chromatography using the
complementary antigens immobilized on CNBr-activated Sepharose.

FIGURE 3 Sandwich ELISA format. (1) Microwell platebound rst (capture)

antibody, (2) meat antigen, (3) second (usually rabbit) antibody, and (4)
enzyme-conjugated goat antibody for rabbit IgG.

* The American plains bison is also buffalo. The buffalo referred to in this chapter is the water
buffalo of Asia and Africa called simply buffalo in the literature.
256 Chapter 9

Tests with 8 pAbs versus 8 meat extracts (64 tests) showed three false
positives. The (cow) pAb for sheep cross-reacted with goat meat. However
(sheep) pAb for goat did not cross-react with sheep. The (sheep) pAb for
beef cross-reacted with buffalo meat, but (sheep) pAb for buffalo did not
react with beef. By choosing the host animal for antibody production
carefully, specic pAb could be produced for sandwich-ELISA. The
relatively high pAb specicity was ascribed to the following factors: (a)
choice of host species (sheep or goats produced more specic antibodies
than rabbit or mice) and (b) choice of antigen. Using whole serum protein
for immunization, rather than a single pure protein, introduces many
antigenic determinants. The pAbs are produced that are more discriminat-
ing between species. Meat samples (1 g) were extracted by 10 mL of solvent
and diluted by a factor of 10 15000 1 before analysis. The LLD was 1%
(w/w) kangaroo meat substitution for beef or 1% (w/w) substitution of goat
meat for sheep. Cross-reactivity between closely related species (beef-
buffalo, goat-sheep, and donkey-horse) was evident.
Patterson and Spencer (23) also produced monospecic pAbs for
buffalo, goat, or donkey using cattle, sheep, or horse as host, respectively.
Each pAb was then puried by immunoafnity chromatography. Thus,
(sheep) pAb for goat was puried with a column of Sepharosegoat serum
protein. Bound pAb was eluted with ammonium thiocyanate (2.5 M, pH
7.0), desalted by gel ltration with Sephadex G25, and concentrated by
ultraltration. The pAb sample containing 8 mg mL 1 protein was divided
into two portions; half was covalently conjugated to HRP for detection
and the other half was used for capture. Enzymatic detection was via
o-toluidinehydrogen peroxide (Table 4). From visual inspection the LLD
was 0.1% (w/w) donkey meat added to horse meat or 0.1% (w/w) goat meat
added to mutton. Beef adulteration with > 1.0% (w/w) buffalo meat was
detectable.
Jones and Patterson (22) showed that it was possible to detect 0.51%
(w/w) adulteration of beef by pork using a sandwich ELISA. The linear
range of analysis was 13%. The capture antibody was (rabbit) pAb for
porcine serum albumin (PSA). The detector pAb was produced with a sheep
host. Both (rabbit) pAb and (sheep) pAb for PSA were puried by a two-
stage afnity procedure. The order of reagent addition was (rabbit) pAb for
PSA, meat extract, (sheep) pAb for PSA, and HRP-pAb conjugate for sheep
Ig. The (sheep) pAb for PSA was unstable when directly adsorbed on
microwell plates.* Presumably, instability prevents the preparation of HRP

* Reagent stability is an important feature of sandwich ELISA. Once coated with capture
antibody, microwell plates may be dried and stored at refrigerator temperatures for 6 months.
Speciation of Meat Proteins 257

conjugate with (sheep) pAb for PSA. Also important for assay design is the
low specicity of (rabbit) pAb for PSA. Using (sheep) pAb for PSA to
complete the ``sandwich'' improved assay sensitivity. For details of the
sample pretreatment see footnote.* The LLD was 1% (w/w) pork in minced
beef, beef sausage mix, or beef burger mix. For 110% (w/w) substitution,
the assay response was described by the relation
A492 0:301 0:153 log%Pork 2

These results should be compared with the analysis of pork in commercial

meat (ham, pork-soy sausages, pate) products (29) by indirect ELISA.
Using afnity-puried (rabbit) pAb for pork, the linear range of analysis
was 140% (w/w) substitution.

4. MUSCLE PROTEIN ANTIGENS FOR ELISA

The ELISAs described so far were specic for residual blood proteins within
meat. These assays are unsatisfactory. Cross-contamination by blood from
another species gives a positive ELISA test (26,30). Furthermore, the
amount of blood lost during the conversion of muscle to meat is variable
(32). Attempts to identify different cuts of meat using ELISA for blood
serum proteins were not successful. There were large variations in the blood
content in different samples (33).
Martin and co-workers (26) developed a sandwich ELISA for
(porcine) muscle protein. Extracts of pork diluted by between 20 1 and
20,480 1 gave absorbance readings of < 1.0. Substitution of beef with
150% (w/w) pork led to the calibration response
A492 0:268 0:114 ln%Pork 3

With experienced personnel, the assay precision was 23%. In another

study, chicken muscle antigen was isolated using an afnity column lled
with Sepharoseprotein A complex with (rabbit) pAb for chicken (28).
Bound antigen was eluted with diethylamine buffer (0.05 M, pH 11.5). An

* The assay was calibrated using samples of 010 g of pork added to 12 kg of lean minced beef.
The mixture was blended with 900 mL of distilled water for 2 minutes, ltered through
Whatman No. 3 paper, and stored at 208C. Other meat products of known formulation were
also analyzed. Ten-kilogram amounts of beef burger and beef sausage mixtures were prepared
according standard recipes. Then 40-g samples were extracted with 360 mL of water and ltered.
The resulting extracts were diluted with PBST for immunoassay. Samples were diluted by 50 1
and 250 1 for assay.
258 Chapter 9

SDS-PAGE analysis followed by Western transfer and immunostaining

showed that the chicken-specic antigen was probably pyruvate kinase. The
antigen was used for sandwich ELISA with both the capture and detector
antibody being (rabbit) pAb. Substitution of beef with 110% (w/w) chicken
meat gave the following response:

A492 0:621 0:161 ln%Chicken 4

The corresponding assay for pork adulteration with 110% (w/w) chicken
meat led to the following performance.

A492 0:590 0:154 ln%Chicken 5

Stevenson et al. (30) developed an assay for mechanically recovered

chicken meat and hand-deboned chicken using indirect ELISA for bone
marrow antigen. Crude (rabbit) pAb was puried by ammonium sulfate
precipitation and afnity chromatography using Sepharose-immobilized
bone marrow antigen extracted with 7 M urea. The ELISA was visualized
using afnity-puried (rabbit) pAb for chicken bone marrow, followed by
commercial HRP-labeled (goat) pAb for rabbit IgG. HRP was assayed with
the substrate 3,30 ,5,50 -tetramethylbenzidine (TMB), which, unlike o-diphe-
nylenediamine, is noncarcinogenic. The nal assay showed only slight
selectivity toward MRM as compared with hand-deboned meat. There was
no cross-reactivity with hand-deboned beef, mutton, or pork. Substitution
of beef with MRM chicken could be detected at levels of 250%.
The identity of muscle protein antigens has not been fully established.
An SDS-PAGE analysis of soluble protein from horse meat revealed *20
proteins. Immunoafnity chromatography using immobilized (rabbit) pAb
for horse muscle reduced the number of SDS-PAGE bands to nine. Three
antigens (37, 70, and 96 kDa) increased in concentration after afnity
chromatography (34). SDS-PAGE also featured in the partial identication
of the bone marrow antigen from chicken (30). Proteins from bone marrow
or muscle were separated by SDS-PAGE followed by Western blot transfer
to a nitrocellulose membrane. The bound protein was visualized with
species-specic (rabbit) pAb and commercially available HRP-labeled (goat)
antibody for rabbit IgG. Immunostaining revealed three antigenic proteins
with molecular sizes of 69, 45, and 96 kDa. The 69-kDa protein was
tentatively identied as chicken serum albumin. The identities of the 45- and
96-kDa proteins are not known.
The case for adopting muscle protein antigens for ELISA is
compelling. However, the extra effort involved in isolating muscle proteins
may require further justication. Fig. 4 shows results for sandwich ELISA
using pAb specic for muscle soluble protein or blood serum albumin. These
Speciation of Meat Proteins 259

FIGURE 4 Blood versus muscle protein antigen for meat speciation using sandwich
ELISA. Adulteration of beef with pork was analyzed using (rabbit) pAb
for porcine muscle protein (open circles) or pAb for porcine serum
albumin (closed circles).
260 Chapter 9

results are comparable. Furthermore, it is not certain that the quantity of

soluble muscle proteins is the same for different meat tissues from any single
species. Myoglobin levels vary with tissue type and levels of exercise.

5. COOKED MEAT ANALYSIS BY ELISA

5.1. Boiling-Resistant Ethanol-Soluble (BE) Antigen
Kang'ethe and Lindqvist (35) found that BE antigen was not wholly suitable
for indirect ELISA. The antigen showed irregular adsorption to microwell
plates because of the presence of extraneous proteins (probably gelatin).
Samples of BE antigen gelled at 48C. Notwithstanding partial purication
by size exclusion chromatography, indirect ELISA using BE antigen yielded
poor sensitivity (36). Tests involved (goat) pAb for partially puried BE
antigen from 4 domesticated species (cattle, camel, pig, and sheep) and 14
games species.* With a total of 324 tests (18 antibody 6 18 meat samples),
no cross-reactivity was observed using pAbs for water buffalo, camel, horse,
topi, and pig. The (goat) pAb for cattle BE antigen cross-reacted with
virtually every species tested. Using the appropriate species-specic (goat)
pAb between 1 and 10% (w/w) adulteration of beef (with buffalo), pork
(with warthog), or goat (with impala) was detectable.
The sensitivity of meat tests using BE antigen was improved 100-fold
by adopting the sandwich ELISA format (37). The higher performance was
attributed to BE antigen binding to capture antibody rather than to a
``bare'' microwell plate. Direct binding to surfaces alters antigen conforma-
tion and can reduce antibody binding (38,39). In all, six sandwich ELISA
tests were developed using (goat) pAb for BE antigen from buffalo, bushpig,
camel, cattle, horse, and pig. Each assay was tested with meat samples from
14 species.* The ELISA test for beef showed cross-reactivity for buffalo,
horse, and bushbuck meat. All other assays were species specic.
Adulteration of beef or pork with 120% (w/w) buffalo or camel meat
was readily detected using the appropriate sandwich ELISA for these
adulterants.

* Buffalo (Syncerus caffer), bushbuck (Tragelaphus scriptus), cattle (Bos indicus), eland
(Taurotragus oryx), goat (Capra aegagrus hircus), Grant's gazelle (Gazella granti), impala
(Aepyceros malampus), kongoni (Alcelaphus buselaphus cokei), oryx (Oryx spp.), sheep (Ovis
ammonaires), Thomson's gazelle (Gazella thomsoni), topi (Damaliscus linatus), waterbuck
(Kobus spp.), and wildebeest (Connochaetes taurinus).
Speciation of Meat Proteins 261

5.2. Thermostable Muscle Protein Antigen

Hsieh and co-workers produced thermostable muscle protein antigens for
analysis of cooked poultry (40), pork (41,42), or red meat (43). Lean meat
paste was blended with three volumes of water or 0.85% saline. The resulting
meat slurry was heated at 1008C for 15 minutes, cooled to refrigeration
temperatures, and shaken gently for 2 hours. The supernatant obtained after
centrifugation (14,000g) was employed as thermostable antigen. SDS-PAGE
and Western blot analyses revealed that thermostable antigens from chicken
skeletal muscle had molecular masses of 2225, 3035, and 120 kDa. The
proteins were not identied but are likely to correspond to troponin C,
troponin T, and myosin fragment. Analysis of the antigen from other animal
sources showed bands with molecular sizes ranging from 14.5 to 26.5 kDa.
Thermal-stable muscle protein antigens were the basis for developing mAb
for ELISA (Sec. 6).
Rencova and co-workers described indirect competitive ELISA tests
for heat-processed meat from horse, kangaroo, poultry, or rat. The tests
involved (rabbit) pAb for heat-stable muscle antigen. This antigen was
prepared by heating meat sample extracts with PBS at 1001208C for 30
minutes. The assay detected chicken or kangaroo meat within commercial
meat products from the retail market in the Czech Republic (44).

5.3. Native Thermostable Antigen

Native thermostable antigens (nTAs) are produced from raw tissue by
ammonium sulfate fractionation and ion-exchange chromatography on
carboxymethylcellulose (45). The isolation process, although technically
straightforward, lasts several days. The yield of nTA was 25 mg per kg of
meat. Sandwich ELISA for using nTA for chicken cross-reacted with turkey
(Fig. 5). No cross-reactivity occurred with red meat (beef, deer, horse,
kangaroo, or sheep). Both cooked and uncooked poultry meat could be
analyzed. This is understandable because a genuinely heat-resistant meat
antigen should be unaffected by thermal treatment. A sandwich ELISA
using nTA for pork was also developed (Fig. 6).
The nTA-based tests can detect chicken or pork in a wide range of
cooked meat products: (a) frankfurters (horse, beef, pork, sheep, deer,
chicken or turkey); (b) bologna (beef, pork, chicken, or turkey); (c) chopped,
pressed, and sliced meats (beef, ham, chicken, turkey); (d) canned baby
foods (beef, pork, lamb, chicken, or turkey); and (e) canned meat spreads
(beef, ham, chicken). In every case, product varieties containing poultry or
pork were correctly identied. The LLDs for chicken and pork were 126 and
250 ppm, respectively. The sensitivity, ascribed to the biotin-streptavidin
262 Chapter 9

FIGURE 5 Specicity of sandwich ELISA for chicken native thermostable antigen.

(Drawn using results from Ref. 45.)

amplication system, was more than adequate to detect meat adulterations

of practical signicance. Assay performance was not affected by the nature
of the meat matrix.
The nTA was also the basis for sandwich ELISA tests for beef, deer,
horse, and mutton (46). The nTAs were routinely puried by immunoad-
sorption (47,48). Even then some cross-reactivity occurred with the
following samples: beef/American bison, goat/sheep, donkey/horse, white-
tail deer/mule deercaribou. With cooked meat adulterants the LLD was
0.16% (w/w). Apparently the assays were less efcient for raw meat. A
commercial ELISA kit utilizing nTA was used by Hsieh et al. (49) to survey
cooked meat adulteration in Florida. Commercial kits, said to be based on
the USDA protocol, are available for cooked beef, pork, poultry, sheep,
horse, and deer meat (ELISA Technologies, Alachua, FL). In the United
Kingdom similar kits are available form Cortecs Diagnostics Ltd (Newtech
Square, Deeside Industrial Park, Flintshire, CH5 2NT, UK). Potential
difculties arising from the analysis of cooked meat samples containing high
amounts of gelatin using commercial ELISA kits were described (50).
Speciation of Meat Proteins 263

FIGURE 6 Specicity of sandwich ELISA for cooked pork based on antibody for
native thermostable antigen. (Drawn using results from Ref. 45.)

The identity of the 50-kDa protein associated with nTA has not been
established. Antibody for nTA did not react with a-acid glycoprotein (also
called a-HS-glycoprotein) (45,46). The list of blood serum proteins includes
immunoglobins (160 kDa), transferrin (85 kDa), albumin (66 kDa), Ig
fragment (45 kDa), a1-antitrypsin (45 kDa), orosomucoid (44 kDa), GC
globulin (51 kDa), a-HS-glycoprotein (49 kDa), g-globulin (25 kDa) and
b2-microglobulin (11.8 kDa). The a-HS-glycoprotein is probably the most
heat-stable serum protein. However, several serum proteins have molecular
sizes around the region 4550 kDa (51).
The thermal stability characteristics of nTAs are unlike those of
other muscle proteins (Table 5, Fig. 7). Levieux et al. (52) heated readily
extractable muscle proteins and analyzed the residual soluble proteins
by QSDS-PAGE. The order of thermal resistance was albumin
> myoglobin lactate dehydrogenase (M4) > IgG > transferrin. All pro-
teins were completely denatured by heating at > 808C for 30 minutes. I
estimate that the half-life (t1/2) of chicken nTA is 213.6 or 52 minutes at 100
or 1258C, respectively. Turkey or beef nTAs were relatively less heat
resistant by comparison with t1/2 of 51.3 or 103 minutes at 1008C. These t1/2
values were reduced to 35 minutes (turkey nTA) or 68 minutes (beef nTA) at
1208C. The heat deactivation mechanism for nTA was also different from
264 Chapter 9

TABLE 5 Thermal Inactivation Parameters for Some Soluble Muscle Proteins

Muscle DH# DS#

protein (kJ mol 1) (J mol 1 K 1) Arrhenius equation

IgG 184.3 280 ln k 58.6 2.23 6 104

(1/T)
LDH (M4) 340.7 740 ln k 113.0 4.05 6 104
(1/T)
Myoglobin 455.7 1040 ln k 136.4 4.90 6 104
(1/T)
Albumin 475.3 1130 ln k 150.6 5.3 6 104
(1/T)
Chkn-nTA 86 49 ln k 17.9 1.03 6 104
(1/T)
Trk-nTA or 22 201 ln k 1.25 2.67 6 103
pork-nTA (1/T)
Source: Based on data from Refs. 45, 46, and 60.

FIGURE 7 Simulated thermal inactivation proles for bovine muscle proteins and
native-thermostable antigen from chicken (Chkn-nTA) or turkey
(Trk-nTa). Samples are held at 20958C for 30 minutes. (Calculated
from data in Table 5.)
Speciation of Meat Proteins 265

that for the other muscle proteins. The activation enthalpy change (DH#)
and entropy change (DS#) for heat deactivation were large and positive for
most of the muscle proteins. This is indicative of large conformational
changes being the rate-limiting step during heat denaturation.
For nTA we nd that DH# < 100 and DS# was negative. Low
transition state parameters are characteristic of conformationally plastic
proteins (53). Such proteins survive heat treatment owing to the ability to
refold once the thermal stress is removed. A low DH# can also arise where
bioactivity (antigenicity) resides in lower order (primary or secondary)
protein structure. Simulated thermal inactivation proles for nTA and some
other muscle proteins are compared in Fig. 7. I have assumed that (a) the
Arrhenius equation (Table 5) applies over a temperature range of 201208C
and (b) thermal deactivation kinetics are rst order (54). Caution is also
warranted because the initial data (45,60) for modeling came from a limited
temperature range of 54668C for muscle proteins and 1001208C for nTA.

5.4. End-Point Temperature Determination

According to USDA guidelines, meat imported into the United States
should be heated to a certain minimum end-point temperature (EPT) to
ensure that it is free from pathogens and viruses. Compliance with the
USDA guidelines on meat EPT can be assessed by ELISA (5559). For
example, Denise Smith and co-workers developed a sandwich ELISA for
chicken or turkey skeletal muscle lactate dehydrogenase (LDH). Heart LDH
was detected with 1000-fold lower afnity. No cross-reactivity occurred
with porcine or bovine skeletal muscle or heart muscle LDH. The limit of
detection was 1 ng mL 1 or 1 ppb. Heating to an EPT of 68.3728C
inactivated muscle LDH. Didier and Annie Levieux (60) also developed
immunological EPT indicators.

6. MONOCLONAL ANTIBODIES FOR MEAT SPECIATION

The production and use of pAbs for protein analysis has several
disadvantages: (a) the process involves live animals and results in batch-
to-batch variation in pAb, (b) there is a requirement for reagent
standardization with respect to pAb concentration and afnity for antigen,
(c) there is limited pAb production from a single animal host, and (d) crude
pAb requires purifying to lessen cross-reactivity. This process is technically
demanding, slow, and expensive. Such disadvantages can be overcome with
hybridoma technology for monoclonal antibody (mAb) production.
266 Chapter 9

Between 1989 and 1999 two research groups explored the use of mAbs
for meat speciation. One group (from Spain) utilized afnity-puried
(unheated) meat antigen for immunization. Hsieh's group at the Depart-
ment of Nutrition and Food Science, Auburn University initially used
(crude) thermostable meat antigens. The comparatively small number of
publications describing ELISA with mAbs are summarized in Table 6.

6.1. Poultry (Chicken and Turkey)

Afnity-puried chicken muscle antigen (28) (Section 4) was used for mAb
production. Three hybridoma cell lines (designated AH4, BC9, and CF2)
were identied that produced mAb for chicken muscle antigen. SDS-PAGE
and immunoblotting revealed that mAb-CF2 was specic for chicken muscle
pyruvate kinase (58 kDa). Both mAb-AH4 and mAb-BC9 bound to a
47-kDa protein tentatively identied as 3-phosphoglycerate kinase. Martin
et al. (62) puried mAb-BC9 by ion-exchange chromatography with a
Mono-Q column for use as capture antibody. The detection antibody for
their sandwich-ELISA was (rabbit) pAb for chicken muscle antigen.
Visualization was by commercial HRP-labeled (goat) antibody for rabbit
IgG.
A sandwich ELISA using mAb-BC9 was selective for raw poultry
(chicken and turkey) meat. No cross-reactivity was observed with beef,
horse, pork, rabbit, or mutton. There was also no reaction with puried
proteins such as casein, gelatin, or soy protein. The sandwich ELISA test

TABLE 6 Monoclonal Antibody for the Speciation of Raw or Heated Meat Proteins
by ELISA

Analysis, commentsa Reference

Chicken Martin et al. (61)

Chicken Martin et al. (62)
Porku,c Morales et al. (63), Chen et al. (41),
Chen and Hsieh (42)
Horse Garcia et al. (64)
Chicken LDHc Abouzied et al. (55)
Turkey LDHc Wang and Smith (57)
Poultryc Sheu and Hsieh (40)
Meat (beef, pork, etc.)c Hsieh et al. (43), Chen et al. (41)
Central nervous system tissue O'Callaghan (65), Schmidt et al. (66)
a
c, Cooked or autoclaved meat, all other samples, and u, unheated samples.
Speciation of Meat Proteins 267

responses were described by

A405 0:455 0:47 ln%Chicken 6

A405 0:404 0:46 ln%Chicken 7

for beef or pork adulteration by chicken, respectively. The linear dynamic

range for analysis was 1100% (w/w). The preceding assays are for raw meat
samples only. Notice that the sandwich ELISAs using mAb are more
sensitive than those with pAb [Eqs (4) and (5)].

6.2. Pork and Horse Meat

A pork-specic mAb-DD9 did not react with beef, chicken, horse, casein,
soy, gelatin, or BSA (63). The mAb-DD9 was prepared from unheated
muscle protein antigen puried by immunoafnity chromatography (26,27).
Indirect ELISA utilizing mAb-DD9 detected beef or chicken adulteration by
1100% (w/w) pork. The calibration response was described by
A405 0:0692 0:7989 ln%Pork 8

A405 0:0745 0:7612 ln%Pork 9

for beef and chicken samples, respectively. The LLD was 0.1% (w/w), which
is below levels probably economically advantageous to the retailer. The
assay sensitivity compares favourably with results expressed in Eq. (3).
Heating meat samples to 658C for 30 minutes had no adverse on the assay
reponse, but autoclaving samples at 1208C for 20 minutes led to loss of assay
sensitivity.
Chen and Hsieh (42) have recently described an mAb-based ELISA
for detecting the presence of pork within cooked or processed meat
products. The assay employs porcine thermostable antigen (Sec. 5.3). The
LLD for pork was 0.5% (w/w) with intrassay and interassay precision of
5.8% and 7.9%. The highly accurate method was able to identify pork in 45
commercial processed meat samples. Sawaya and co-workers (67) also
produced an ELISA test sensitive to cooked pork although they used pAb.
Horse meatspecic mAb-DD3 (64) showed no cross-reactivity for
beef, chicken, pork, soy proteins, casein, gelatin, or BSA. Addition of
050% (w/w) horse meat to beef led to the following ELISA response.
A405 0:4626 0:0314 %Horse 10

The LLD for horse meat was 2% (w/w). The Spanish group suggest that
268 Chapter 9

antigen purication by afnity chromatography may be necessary for mAb

production.

6.3. Cooked Red Meat

The mAb-2F8 had selectivity for beef, sheep, or lamb and reduced
sensitivity for deer meat (43). There was no cross-reactivity with raw
pork, beef, lamb, mutton, or deer. Chicken or turkey was not detected in
either the raw or cooked state. Indirect ELISA using mAb-2F8 was specic
for red meat. Adulteration of poultry (chicken and/or turkey) with 0.515%
(w/w) beef, horse, sheep, or pork was easily detectable. The LLD for deer
meat was * 5% (w/w) addition to poultry. Multiple adulterants produced a
cumulative signal. Red meat is often more expensive than chicken. However,
the preceding test will be useful for detecting the substitution of chicken
meat with less valuable beef or pork trimmings (49). Chicken is also in
higher demand in some parts of Asia where the consumption of beef is
restricted by religious stricture.

7. FISH AND SEAFOOD IDENTIFICATION BY ELISA

Seafood adulteration may be growing in importance for three main reasons.

First, there is a trend away from red meat toward the consumption of sh
and seafood. Fish and seafood are perceived as healthy because of their
higher content of unsaturated fatty acids. Second, world sh stocks continue
to decline, increasing the economic incentive for adulteration. Third,
improvements in processing technology have led to larger markets for
comminuted sh products (68) and sh protein concentrates (69). These
products are difcult to identify visually and offer signicant scope for
adulteration. Speciation by immunological methods is seen as complemen-
tary to traditional methods such as isolectric focusing and SDS-PAGE (70).
Suzuki (71) used an agar gel diffusion assay to identify tuna. Fish protein
authentication using ELISA is discussed in this section.

7.1. Sardine and Tuna

Taylor and Jones (72) and also Taylor et al. (73) described an indirect
ELISA using (rabbit) pAb for soluble antigens from canned sardine, bonito,
yellown tuna, or skipjack tuna. Crude (rabbit) pAb for canned sh antigen
was nonspecic. High responses were obtained for most canned sh samples
(Fig. 8). To improve the specicity, pAb was puried via (a) batch
immunoadsorption, (b) immunoadsorption using antigens immobilized on
Speciation of Meat Proteins 269

FIGURE 8 Noncompetitive indirect ELISA for the identication of canned sh

species. Graph shows the specicity of crude (rabbit) antibody for
different canned sh samples. (Based on data from Ref. 64.)

magnetic beads, and (c) immunoafnity chromatography using the antigen

immobilized on CNBr-activated Sepharose.
Tuna species (albacore, yellown, and skipjack) were difcult to
differentiate from each other or from bonito (a potential tuna substitute).
Attempts to increase the specicity of pAb for tuna species (by
immunoadsorption) led to large reductions in the net concentration of
free pAb. The indirect ELISA for canned sh was able to differentiate
between sh and crustacea (prawn and scampi). The assay is potentially
useful for detecting the adulteration of prawn, scampi, and other crustacean
meat with cheaper sh (74).

7.2. Rock Shrimp

SDS-PAGE analysis of seafood tissue extracts showed that protein M was
unique to rock shrimp (Sicyonia brevirostris). The electrophoresis band
corresponding to protein M was excised, homogenized with buffer, and
centrifuged. The supernatant was dialyzed overnight and utilized as antigen
for mAb production. Indirect ELISA using mAb-4H210D3 successfully
270 Chapter 9

identied rock shrimp from three geographic locations in the United States.
These samples were also correctly differentiated from 23 other seafood
samples including white shrimp (from Colombia, Ecuador, Honduras, and
Peru) and blue shrimp (from Ecuador). The specicity for rock shrimp was
attributed to the use of puried antigen for mAb production. The test was
more sensitive for heated samples probably because protein M was heated
during isolation by SDS-PAGE. The antigen had molecular size of
17.718.5 kDa and made up *20% of the total water-soluble protein (75).

7.3. Red Snapper

Adulteration of red snapper (Lutjanus campechanus) appears widespread.
Approximately 14 out of 15 (91%) sh samples branded as red snapper by
U.S. retailers were inaccurately labeled. Another survey involving 24
samples found 70% mislabeling. The high level of adulteration indicates
genuine error on the part of retailers. Huang et al. (76) developed
mAb-C1C1 and mAb-A1B1 for red snapper. The species-specic antigen
(protein A, pI 5.93 ) was isolated by isoelectric focusing of red snapper
muscle extract. Both mAb-C1C1 and mAb-A1B1 were applied for indirect
ELISA. The detection system was (rabbit) pAb for mouse IgG conjugated
to alkaline phosphatase. In tests involving 24 sh, seafood, and meat
samples, indirect ELISA with mAb-C1C2 gave positive results for red
snapper samples of vermilion snapper, lane snapper, mutton snapper, and
yellowtail snapper. Using both mAb-C1C2 and mAb-A1B1based ELISA
tests simultaneously allowed a clear differentiation of red snapper samples
from most other seafoods.

8. PERFORMANCE CHARACTERISTICS FOR DIFFERENT

ELISA FORMATS

Factors affecting assay performance include (a) the ELISA format, (b) the
type of enzyme label, (c) antibody characteristics, and (d) the nature of the
antigen used. Calibration graphs for sandwich ELISA [Eqs (6) and (7)] or
indirect ELISA [Eqs (8) and (9)] show that the indirect format is more
sensitive. Using pAb for capture can lead to loss of assay response for
sandwich ELISA. Multiple interactions involved in pAb-antigen binding
may leave few epitopes for the detector antibody. Poor assay sensitivity also
arises when a sandwich ELISA utilizes the same mAb for both capture and
detection. Under such circumstances, highly specic epitopes become
occupied by the capture mAb with few left for binding with the (same)
mAb for detection. Comparing the sandwich ELISA tests for muscle antigen
Speciation of Meat Proteins 271

shows that using mAb for capture enhances assay sensitivity by about an
order of magnitude compared with the use of pAb. Finally, whether puried
antigens are necessary for mAb production is contentious. With mAb for
unheated samples it seems necessary to use puried antigen. However,
thermostable antigens appear to induce specic mAb formation (see earlier).

9. MEAT TESTING FOR TRANSMISSIBLE SPONGIFORM

ENCEPHALOPATHY AGENTS
9.1. Meat and Bone Meal
Bovine spongiform encephalopathy (BSE) or ``mad cow disease'' is linked
with feeding contaminated meat and bone meal to cattle. The rst
recognized cases of BSE appeared in the United Kingdom in 19851986.
New-variant Creutzfeldt-Jakob disease (nvCJD), thought to be a human
form of BSE, was detected 10 years after the rst BSE cases (77).
Transmission to other animals was demonstrated in laboratory studies with
cats, pigs, goats, and sheep. The origin of BSE and other transmissible
spongiform encephalopathies (TSEs) remains uncertain. One hypothesis
highlights changes in meat rendering procedures in the late 1970s and early
1980s. The discontinuation of meat rendering processes involving hydro-
carbon solventsteam treatment may have led to a critical (even if slight)
increase in the infectivity of BSE-contaminated feed. The theory, although
compelling, is not necessarily accepted by all (78).
The government of the United Kingdom banned the use of meat and
bone meal in cattle feed in 1988. The incidence of BSE in the United
Kingdom declined as a result of this ban, which was later extended to feed
materials for all farm animals. However, large numbers of cattle remain
affected with BSE. Infected cattle have also been discovered in the European
Community countries including France and Belgium. Public health concerns
about BSE seem likely to continue because the projected incubation period
for nvCJD could extend from 4 to 30 years (79). Methods are being
developed to monitor compliance with the legislation excluding meat and
bone meal from feeds. Adequate heat treatment can also turn out safe meat
and bone for use and/or disposal (80). The inactivation characteristics of the
BSE agent were studied by Taylor and co-workers (79). BSE survives
irradiation or boiling. Heating at 1218C (15 psi) leads to partial inactivation
and thermal treatment at 1348C for 60 minutes produces complete
inactivation. Meat-rendering plants in Germany heat treat samples at
1338C for 20 minutes at 3 bars of pressure.
Klaus Hofmann used the Cortecs ELISA test kit for pork to test for
bone and meat meal. Similar ELISA test kits exist for cooked beef or
272 Chapter 9

mutton. Samples of meat and bone meal heated to a rendering temperature

of 1338C for 20 minutes were fully degraded and undetectable by ELISA
(81,82). In the near future such tests might be used for routine monitoring of
meat-rendering plants in Germany. Collaborative trials involving 21
laboratories from 12 countries (83) showed that these tests had sufcient
reliability as judged by a precision between 11 and 12%. Understerilized
meat and bone meal were clearly distinguishable by a 10-fold greater assay
signal.

9.2. Central Nervous System Tissue

Concentrations of the BSE agent are higher in the central nervous system
(CNS) tissue compared with peripheral nerves. A sandwich ELISA for CNS
tissue was developed using mAb for GFAP (glial brillary acidic protein)
(66). This protein is found only in astrocyte cells in the CNS.* The ELISA
for GFAP had an approximate LLD of 1 ng (GFAP) with a linear range
extending to 40 ng. The within-assay precision was 3.254%. While assay
sensitivity remained constant for different matrices, the LLD increased in
the order ground beef brain tissue > ground beef spinal cord tissue
> puried GFAP standards > brain > spinal cord. The ELISA gave the
concentrations of GFAP in spinal cord tissue (55,000220,000 ng mg 1
tissue), brain tissue (900055,000 ng mg 1 tissue), and cerebral cortex
(17,000 ng mg 1 tissue). Neck muscle and ground beef were free of GFAP.
The antigen is not very stable; therefore CNS tissue could be detected for
only up to 8 days when samples were stored at 48C. CNS tissue was also
analyzed using immunoblot analysis (84). The tests were directed at two
antigens: GFAP and neuron-specic enolase. Immunoblot analysis did
detect CNS tissue if samples were subjected to extreme temperature
processing.

9.3. Direct Immunological Detection of the BSE Agent

As of December 2000, the European Commission accepted ve direct tests
for the BSE agent for further evaluation (85). The tests were produced by

* The details of the sandwich ELISA for GFAP were essentially as described elsewhere. (a) Coat
microwell plates with a commercial pAb for GFAP (supplied by Dako Corporation,
Carpentaria, CA) at 378C for 1 hour or at 48C overnight. (b) Block nonspecic microwell
plate sites with PBSTpowdered milk protein. (c) React pAb-coated microwells with GFAP
standards or samples for 60 minutes. (d) Add diluted mAb for GFAP (supplied by Boehringer
Mannheim, Indianapolis, IN). (e) Add enzyme-labeled antibody, i.e., alkaline phosphatase
labeled (rabbit) pAb for mouse IgG. Assay for enzyme activity.
Speciation of Meat Proteins 273

ID Lalyated, Netherlands
Imperial College of Science and Technology, UK
Institute of Neurodegenerative Diseases, University of California, San
Francisco
Perkin Elmer Life Sciences, UK
Prionics AG, Switzerland
The BSE test produced by Prionics AG, Switzerland (Prionics AG,
University of Zurich, 8057 Zurich, Switzerland) appears to be the favorite.
Prionics-checkTM employs immunoblot analysis. Brain tissue extract is rst
exposed to a protease solution followed by SDS-PAGE and then transferred
to a nitrocellulose membrane by Western transfer. The membrane-bound
proteins are detected immunologically using mAb specic for prion
particles. The test is able to differentiate between the benign prion protein
(PrPC) and the disease-causing PrPSc because the former is susceptible to
protease attack but the latter is not.
According to the advertising literature, Prionics-check is intended for
(a) identication of suspected BSE cases, (b) diagnostic testing in abattoirs
and slaughterhouses, and (c) general monitoring for scrapie and BSE.
Validation of the Prionics-Check tests has been documented (86). Prionics-
check will be used for mandatory BSE testing in the European Union from
2001. Some important characteristics of the Prionic-checks include (a) high
selectivity and specicity, (b) ability to distinguish cattle with BSE from
those with other neurological disease states, (c) detection of subclinical cases
of BSE, (d) ease of use and availability in a kit form, (e) suitability for eld
use, (f) high sample throughput (the time of analysis is reportedly 68 hours
from tissue extraction to nal test results), and (g) current use for Swiss BSE
surveillance for all sick and falling cattle. At this time, Prionics AG
manufacture at least two prion-specic mAbs (6H4 and 34C9) as well as
pAb (RO29). The antibodies are suitable for developing ELISA. The
company also has available the full kit for BSE detection. The life science
diagnostics company Bio-Rad Ltd. currently manufactures an ELISA test
for BSE (PLATELIA2 BSE test). This test is commercially available in the
United Kindom, France, Germany, Belgium, Luxembourg, Norway,
Sweden, Switzerland, Italy, and Spain. At this time the test is not being
sold in the United States (87). The list of companies now entering the
BSE diagnostics market is growing rapidly as shown by the following list
(88).
274 Chapter 9

Abbeymoy Ltd. Altegen Inc. Anonyx Inc.

Bayer
Boehringer-Ingelheim AG
Commissariat a l'Energie Enfer Scientic Ltd.
Atomique
Genesis Biventures
Mary Jo Schmerr
New York State Basic
Research Institute for
Neurological Disorders
Prionics AG
Caprion Pharmaceuti- Bio-Rad Inc.
cals Inc.
Centre Suisse Celcus Inc.
d'Electronique et de
Microtechnique SA
Disease Sciences Ltd.
IGEN International Microsens Biohase Ltd.
Paradigm Genetics Inc. Nen Life Science
Products Inc.
Proteome Sciences Ltd. Prion Developments
Laboratory
Q-One Biotech Ltd. V.I. Technologies Inc.

For further discussions of immunological tests for the BSE agent see
Refs. 89 and 90.

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75. H An, PA Klein, K-J Kao, MR Marshall, MWS Otwell, C We. Development
of monoclonal antibody for rock shrimp identication using enzyme-linked
immunosorbent assay. J Agric Food Chem 38:20942101, 1990.
76. T-S Huang, MR Marshall, K-J Kao, WW Otwell, C Wei. Development of
monoclonal antibodies for red snapper (Lutjanus campechansus) identication
using enzyme-linked immunsorbent assay. J Agric Food Chem 43:23012307,
1995.
77. RG Will, JW Ironside, M Zeidler, SN Coursens, K Estibeiro, A Alperovitch, S
Poser, M Pocchiary, A Hofman, PG Smith. A new variant Creutzfeldt-Jakob
disease in the UK. Lancet 347:921925, 1996.
78. P Brown. On the origins of BSE. Lancet 352:252253, 1998.
79. D Taylor. BSE: our future in the balance. Chem Ind (June):444447, 1998.
80. P Brown. BSE: the nal resting place. Lancet 351:11461147, 1998.
81. K Hofmann. Proof of proper heating at meat-and-bone meals. Fleisch-
wirtschaft 76:10371039, 1996.
82. K Hofmann, K Fischer, E Mueller, V Kemper. Experiments to demonstrate
the effectiveness of heat treatments applied to canned meats and meat-and-
bone meals. Fleischwirtschaft 76:920923, 1996.
83. C von Holst, KO Honickel, W Unglaug, G Kramer, E Anklam. Determina-
tion of an appropriate heat treatment of animal waste using the ELISA
technique: results of a validation study. Meat Sci 54:17, 2000.
84. EH Luecker, E Eigenbrodt, S Wenisch, R Leier, M Buelte. Identication of
central nervous system tissue in retail meat products. J Food Prot 63:258263,
2000.
85. European Commission. DG24. Directorate B, Unit B3. The evaluation of tests
for the diagnosis of transmissible spongiform encephalopathy in bovines (8
July 1999). http:/europe.eu.int./comm/dg24/health/bse.
86. O Schaller, R Fatzer, M Stack, J Clark, W Cooley, K Bifger, S Egli, M
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surveillance method for the diagnosis of bovine spongiform encephalopathy
(BSE). Acta Neuropathol 98:437443, 1999.
87. http://www.fda.gov/oc/opacom/hottopics/bse.html#3anchor.
88. Prion Disease Diagnostics. http://www.mad-cow.org/00/feb01_last.html.
280 Chapter 9

89. LKJ van Keulen, JPM Langeveld, GJ Garssen, JG Jacobs, BEC Schreuder,
MA Smits. Diagnosis of bovine spongiform encephalopathy: a review. Vet Q
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90. D Momocilovic, A Rasooly. Detection and analysis of animal materials in
food and feed. J Food Prot 63:16021609, 2000.
10
Speciation of Soya Protein by
Enzyme-Linked Immunoassay

1. INTRODUCTION

There are usually guidelines for adding plant and other nonmeat protein to
meat products. Food technologists use such ingredients legitimately to
enhance functional properties such as water holding, fat binding, and
gelation. Nevertheless, levels of nonanimal proteins in meat should be
monitored. Much research has appeared in connection with soybean
protein, this being the most important nonmeat protein ingredient. This
chapter describes immunological methods for detecting bulk quantities
(>0.5% w/w) of soybean protein in meat and meat products. The topic is
dominated by methods of sample pretreatment designed to ensure accurate
results no matter the sample processing history.

2. SAMPLE PRETREATMENT AND ANALYSIS OF SOY

PROTEIN

Hitchcock et al. (1) were rst to use ELISA for soy protein analysis. The
assay was designed for a wide range of commercial soy samples including
our, protein isolates, and texturates. To correct for variable (heat)
processing history, samples are pretreated with 8 M urea. Denatured soy

281
282 Chapter 10

protein is then renatured before analysis by competitive indirect ELISA.

Details of this pretreatment regime are given later (Method 1). Develop-
ments leading to the eventual commercialization and ofcial approval for
soybean protein ELISA tests are summarized in Table 1.
With indirect ELISA (1),* the nal absorbance measurement is
inversely related to the concentration of soy protein. The assay was highly
specic with negligible responses toward beef, milk, eld bean, or wheat
proteins. Specicity was for conglycinin, which is the 7S soybean globulin
(Fig. 1). The precision for soy protein determination was 10.5%. Grifths et
al. (3) found that a commercial pAb for native and/or heat-denatured soy
protein was as effective as the laboratory-developed (rabbit) pAb for
renatured soy protein (Fig. 2).
Collaborative testing of a commercial ELISA kit for soya protein
involved 13 laboratories from the United Kingdom (4,7). The kits were
supplied by Biokits Ltd. (Newtech Square, Deeside Industrial Park,
Deeside, Clwyd CH5 2NU, UK), who also organized a 1-day workshop
to familiarize trial members with the test procedures. For the actual trial,

TABLE 1 Determination of Soybean Proteins by ELISA

Analysis and comment Reference

First ELISA for soya protein Hitchcock et al. (1), Grifths et al. (2)
ingredients (our, isolates,
concentrates, and extrudates)
Commercial pAb for soy protein Grifths et al. (3)
ELISA
Commercial pAb for soy protein in Crimes et al. (4)
ELISA, collaborative study
Europe-wide collaborative study Olsman et al. (5)
ELISA of soya protein by Ravestein and Driedonks (6)
immunoblotting
Collaborative study of ELISA kit Hall et al. (7)
AOAC approval for ELISA for soy McNeal (8)
proteins
Detection of soy milk in bovine milk Hewedy and Smith (9)

* The following steps are involved: (a) Pretreat soy sample and standards by denaturation-
renaturation protocol. (b) React soy samples or standards with (rabbit) pAb for soy protein. (c)
Add mixture to a microwell plate precoated with bound (renatured) soy protein. (d) Incubate to
allow antibody-antigen reaction. (e) Wash thoroughly with PBST. (f) Add enzyme-labeled pAb
for rabbit IgG. (g) Wash thoroughly with PBST. (h) Add enzyme substrate. (i) Stop reaction
after a xed incubation time and record absorbance readings.
Speciation of Soya Protein 283

FIGURE 1 Competitive indirect ELISA for soya bean protein using commercially
available (rabbit) polyclonal antibodies (1) or experimental (rabbit)
polyclonal antibodies for renatured soya bean protein. (Drawn from
Refs. 1 and 3.)

FIGURE 2 Effect of cooking temperatures on soy protein analysis in beefburgers

using competitive indirect ELISA. (Drawn from Refs. 1 and 3.)
284 Chapter 10

participants analyzed duplicate samples of raw beefburger mix (three

samples), raw sausages (two samples), cooked pate (three samples), and a
trial sample. The average LLD was 0.7% (w/w) soy protein. The percent
protein detected (accuracy) was 89 (+6.1)% (7).
A soybean ELISA kit was also evaluated by Rittenburg et al. (10). The
accuracy depended on the type of soy additive, type of food matrix, and
subsequent processing. Protein recovery was 93 (+15.5), 75 (+7.5), 81
(+8.5), or 82 (+8.8)% for soy isolate, our grit, concentrate, or texturate,
respectively. For 72 retail meat products (including beef and/or pork
sausages, bacon and ham loaf, and mince), the average recovery for soy
protein was 91 (+12.3)% when added at levels of 1.21.6% (soy protein
isolate), 2.45% (soy our), or 4.8% (soy texturate). Autoclaving (1218C,
15 psi; 20 minutes) produced a signicant decline in accuracy (Fig. 2).
Overall, the performance of the commercial ELISA kit was deemed
acceptable. ELISA results agreed with the soy protein levels declared by
most manufacturers.
Method 1
Denaturation-renaturation sample treatment for soy protein antigens
(1,4,7).
Samples or soy protein standards are dissolved with hot urea-
mercaptoethanol* solution. This unfolds proteins and destroys S22S bonds.
The sample is then transferred to a renaturation buffer. This treatment
encourages protein refolding and restores soy proteins to a baseline
conformation regardless of their previous thermal history.
Reagents
1. Urea
2. Dithiothreitol (DTT)
3. Tris-HCl buffer (0.25 M, pH 8.6)
Procedure
Denaturation-extraction buffer (urea 10.6 M, DTT 18.8 mM in
*25 mM Tris-HCl, pH 8.6). To a 250-mL volumetric ask add
80 g of urea, 20 mL of Tris-HCl buffer (0.25 M, pH 8.6), and warm
to dissolve. Add 30 mg of DTT to the hot solution, dissolve, and
keep solution in a 1008C water bath. Add 60 mL of distilled water.
Renaturation buffer (0.06 M NaCl with 7.5 mM L-cystine). Dissolve
1.8 g of L-cysteine with sodium hydroxide (1.0 M, 20 mL). Add the
solution to 900 mL of 0.06 M NaCl solution. Adjust to pH 9 with
1 M HCl and make up to 1000 mL.

* Mercaptoethanol (2-ME) was later replaced with the less odorous dithiothreitol (DTT).
Speciation of Soya Protein 285

Sample extraction. Homogenize 12 g of nely chopped (meat) sample

with 48 mL of Tris-HCl buffer (0.05 M, pH 8.6) using an Untra-
Turax homogeneizer. Add 2.5 g of meat homogenate or 40 mg of soy
protein standard to a 50-mL ask. Add 7.5 mL of urea-DTT
solution, mix, and heat at 1008C for 60 minutes. Transfer the
mixture to a 508C water bath.
Renaturation. Add renaturation buffer (20 mL, prewarmed to 508C),
mix, and then allow to cool to room temperature. Bring the nal
volume to 100 mL and lter through Whatman No. I paper. Collect
the rst 10 mL of ltrate for ELISA.
Meat samples suspected of containing undeclared soy protein were
extracted with acidic ethanol, followed by acetone, and then air dried to
produce acetone powder. These samples and soy antigen (for immunization
or precoating microwell plates) were dissolved using urea-DTT solvent and
renatured as just described. In theory, the renaturation treatment trans-
forms all soy protein to a baseline renatured state with reconstituted
antigenic determinants. In practice, the recovery of antigenicity is found to
be about 20% for 11S soy globulin and 70% for the 7S soy protein. Reasons
for this are twofold. First, severe heat treatment leads to covalent
modication of protein side chains. The chemical changes (lysinoanaline
formation, cross-link formation, deamidation, etc.) cannot be reversed by
the renaturation procedure. Second, protein refolding is usually less than
100% efcient. Competing side reactions result in the formation of protein
aggregates and misfolded structures with improperly aligned S22S bonds.
The ranaturation procedure reverses protein sulfhydryl/-disulde exchange
and noncovalent interactions produced during food processing.

3. STRUCTURE, DENATURATION, AND RENATURATION

OF SOYBEAN PROTEINS
3.1. Soy Protein Structure
Soya beans contain between 40 and 50% protein by dry weight. The major
protein groups are (a) storage proteins (7080% total), (b) enzymes (notably
lipoxygenase, lactate dehydrogenase), (c) protease inhibitors (notably the
Bowman-Birk and the Kunitz inhibitors), and (d) other storage proteins
(e.g., lectin). The foremost storage protein (*40% total protein) is 11S
glycinin. It is a member of the leguminin family of proteins. The second
storage protein (*30% total protein) is the 7S conglycinin.
Glycinin (molecular mass of 350 kDa) is a hexamer of A-B subunits.
The six acidic (A) and basic (B) subunit pairs are each joined by a disulde
286 Chapter 10

bond. The A-B subunit is synthesized as a single polypeptide chain

containing an intermolecular disulde bond and a linker sequence.
Posttranslational proteolysis removes the linker sequence, leaving the A
and B subunits linked by a disulde bond. Isoelectric focusing reveals ve A-
subunit isoforms (A1, A2, A3, A4, A5). There are at least four B-chain
isoforms (B1, B2, B3, B4). The structural characteristics of the glycinin were
reviewed by Peng et al. (11), Nielsen (12), and Fukushima (13). Conglycinin
(170 kDa) is a trimer. From the structural model proposed by Thanh and
Shibasaki (14), conglycinin has six isomers. They are produced from the
random combination of four subunits; a, a0 , b, and g. Conglycinin contains
about 5% carbohydrate but is virtually devoid of cysteinyl residues or S2 2S
bonds (15).

3.2. Thermal Denaturation

The effects of heating on soy proteins were extensively investigated by
nonimmunological techniques (1621). Turbidimetric measurements showed
that heating glycinin at 1008C (0.5% w/v in potassium phosphate buffer,
I 0.5) led to aggregation. The rate of aggregation increased (while the net
aggregation decreased) in the presence of 2-ME as a source of free
sulfhydryl groups. The kinetics of aggregation conformed to a reaction
order of 1.2. The Arrhenius plot for the reaction was biphasic between 70
and 908C (16). Studies using turbidity and UV difference measurements do
not differentiate between intermolecular and intra-intermolecular effects
such as dissociation, unfolding, and aggregation.
Precise thermal stability data were obtained with differential scanning
calorimetry (DSC). Glycinin (710% w/v) had a denaturation temperature
(TD) of 85948C, whereas the TD for conglycinin was 10128C lower. The
TD increased by a maximum of 308C with increasing salt (02 M NaCl)
concentration for both glycinin and conglycinin (2225). The ratio of
calorimetric and Van't Hoff enthalpies was *1, implying that seed globulins
denature via a one-step (all or nothing) process (24,25). By contrast, the
kinetics of conglycinin and glycinin thermal denaturation is biphasic with
separate dissociation and protein unfolding steps (26,27). Apparently, DSC
studies do not register changes in soybean protein quaternary structure.
The thermal dissociation of 11S glycinin leads to a 7S trimer and then
to individual (3S) A-B subunits. Glycinin trimer has a sedimentation
number equal to that of native 7S conglycinin and is therefore designated 7s .
Further heating produces disulde bond lysis and the separation of the A
and B subunits. The hydrophobic B subunit forms aggregates, leaving the A
units in solution. From the many studies of glycinin denaturation comes the
Speciation of Soya Protein 287

following commonly accepted scheme below.

11S 7s 3S A subunits B subunits

;
B Subunits sol-aggregates 1
;
B Subunits solid-aggregates

Processes in Equation (1) will be modied by soy protein interactions with

muscle proteins, lipid, rusk, and other ingredients. Indeed, TD for soy
protein depends on the moisture level, ionic strength, pH, and the presence
of sulfhydryl compounds including other meat proteins. Within a defatted
our matrix containing about 60% (w/w) moisture, TD for glycinin was
908C. The TD value increased to 1608C for absolutely dry our (28,29).

3.3. Effect of Heat Treatment on Glycinin Structure and

Antigenicity
Preheating soy protein for 60 minutes in a high-ionic-strength medium
(0.035 M phosphate buffer, pH 7.6 with 0.4 M NaCl) at 30808C did not
impair pAb binding with glycinin* at room temperature (30,31). By
contrast, the thermal treatment of glycinin dissolved in low-salt solvent
(0.035 M phosphate buffer, pH 7.6, with 0.15 M NaCl) produced a total loss
of antigenicity (32). Apparently, pAbs for native glycinin bind to the
nondissociated protein [Eq. (1)]. The antibody recognizes discontinuous
epitopes formed by the intact protein.
High salt concentrations stabilize glycinin. Thermal treatment at
1008C (5 minutes) led to a total loss of glycinin antigenicity in the presence
of 00.7 M salt. However, the residual antigenicity increased in the presence
of 0.72.0 M NaCl. Glycinin retained 90% antigenicity at the highest salt
concentrations tested (33). Exposure to extremes of pH (pH < 2 and pH
> 10) also diminished glycinin antigenicity. High acidity or basicity is
known to cause the dissociation of glycinin (34). The order of anitgenicity
for soy protein preparations (with a rabbit host) was 11S 4 A1a*A2 > A3

* The effect of heating on glycinin antigenicity was examined by agar gel double
immunodiffusion (AGID) or single radial immunodiffusion (SRID). The tests involved
crude (rabbit) pAb for native glycinin, puried by immunoadsorption using ethylchloroformate
cross-linked glycinin (see Chapter 8 for a description of this method). AGID assay for soy
proteins showed specicity for glycinin with no cross-reactivity for whey proteins or
conglycinin.
288 Chapter 10

* B1 (35). No antibodies formed against A4, B3, and B4 glycinin subunits

despite multiple injections. Gel immunodiffusion assays conrmed that pAb
for native glycinin did not react with isolated A or B glycinin subunits.

3.4. Effect of Heat Treatment on Conglycinin Structure and

Antigenicity
Immunoanalytical investigations reveal that b-conglycinin is more heat
resistant than glycinin. Heating conglycinin (in 0.035 M K-phosphate buffer
with 00.1 M NaCl) at 1008C for 5 minutes produced 5075% retention of
antigenicity (compared with zero for glycinin). Conglycinin subunits and
soluble aggregates produced by thermal treatment or native subunits (a, a0 ,
and b) isolated by ion-exchange chromatography are able to react with pAb
for the native protein (36). These results are consistent with one or more of
the following conclusions: (a) antigenic sites for conglycinin are continuous,
i.e., involve a contiguous sequence of amino acids formed from lower order
(18 and 28) protein structure; (b) thermal dissociation of conglycinin does
not involve large changes in conformation of subunits; and (c) antigenic sites
for conglycinin (subunits) are surface located and unaffected by the protein
association-dissociation transition (37). Even though it has a lower TD,
b-conglycinin is apparently less vulnerable to thermal processing than
glycinin.

3.5. Renaturing Efciency of Glycinin and Conglycinin

The antigenicity of soy protein is dominated by the 7S protein (Fig. 1). This
is the result of a more efcient renaturation of conglycinin compared with
glycinin. After exposure to urea, extreme pH or high temperature, glycinin
dissociates into its constituent subunits (3739). German et al. (19) showed
that heating then disrupts S22S bonds between A-B subunits. Disulde bond
cleavange is catalyzed by indigenous free sulfhydryl compounds associated
with soy protein. Isolated glycinin B subunits then aggregate via SH/S22S
exchange while the A chains remain soluble (20). On the other hand
conglycinin does not aggregate extensively upon heating. This protein lacks
S22S bonds and also has low levels of SH groups. Extensive heating
eventually produces soluble conglycinin aggregates although SDS-PAGE
analysis shows little irreversible damage.
Conglycinin and glycinin refold with *80% and *70% efciency after
exposure to 8 M urea and dialysis, respectively. However, treatment with
urea2-ME (which disrupts A-B disulde bonds) leads to a glycinin
renaturing efciency of *20%. Breaking the A-B disulde linkage produces
a marked reduction in glycinin renaturation efciency and enhanced
Speciation of Soya Protein 289

aggregation. A small proportion of glycinin molecules also form incorrectly

refolded soluble monomers with altered antigenic properties. Reconnecting
S22S bonds in a correct orientation is the limiting step for glycinin
renaturation (39).
According to Berkowitz and Webert (40), the renaturation procedure
for soybean products, although long and laborious, is inescapable. They
proposed using SDS-PAGE analysis and immunoblotting as a more rapid
ELISA format. During immunoblotting, soybean protein is denatured,
separated by SDS-PAGE, and transferred to a nitrocellulose membrane
before immunoassay. There is no distinct sample renaturation step, which
reduces the assay time.

4. SOLVENT-EXTRACTABLE SOYBEAN PROTEIN

There may be other more convenient methods for preparing soy protein for
analysis. Medina (41) ultrasonicated soy protein standards (12 mg) with
1020 mL of coating buffer (3.2 mM sodium carbonate, pH 9.8 0.1%
thimerosol and 0.05% Tween 20). Samples of cooked sausage (1 g) were
similarly homogenized with 10 mL of carbonate buffer and subjected to
untrasonication and (1000-fold) dilution followed by ltration. Results
using soy protein fractions showed pAb binding to glycinin A subunits.
Pretreating commercial soy protein isolate with reducing agent increased
antibody binding. Assay precision was improved by increasing the time for
pAb coating on microwell plates. The linear dynamic range was 00.2 mg soy
protein per well, or 05% (w/w) soy protein in cooked sausages. For
laboratory-prepared sausages the amount of soy protein found (Y, %) was
described by the relation
Y% 1:22 1:09X 2

where X is the amount of soy protein known to be present in samples. From

the gradient in Equation (2) there was a quantitative recovery of soy protein
antigen from the cooked sausage matrix.

5. THERMOSTABLE ANTIGENS FOR SOYBEAN PROTEIN

ANALYSIS

ELISA for soy products would be improved by using thermostable antigens

that are unaffected by cooking and other forms of processing. Possible
290 Chapter 10

thermostable antigens include protease inhibitors, peptide antigens (natural

or synthetic), and conglycinin.

5.1. Protease Inhibitors

The Bowman-Birk inhibitor is a possible thermostable antigen for ELISA of
soybean additives (42,43). Kunitz soybean trypsin inhibitors (KSTIs), which
rival the Bowman-Birk inhibitor in thermal resistance (44), are another
potential thermostable antigen (45).

5.2. Peptide Antigens

Peptide antigens with continuous epitopes are potentially useful thermo-
stable antigens. Yasumoto, et al. (46) developed a noncompetitive indirect
ELISA for soybean protein predigested with trypsin. The assay was highly
specic with no cross-reactivity for pork, beef, egg, or azuki bean proteins.
Soya protein (10.420.8% w/w) was quantitatively determined for pork
sausages cooked at 808C for 20 minutes. The LLD was 0.4%. The pAbs used
for this assay were raised by immunizing rabbits with intact glycinin.
As a form of pretreatment, glycinin standards were autoclaved at
1208C for 180 minutes and then digested with trypsin for 24 hours.
Microwell plates were coated with the peptide digest and blocked with BSA
to avoid nonspecic binding. Food samples, e.g., cooked sausages, were
pretreated by homogenization with acidic ethanol, followed by acetone
precipitation and drying. The powders were suspended in buffer, autoclaved
for 1208C for 180 minutes, and digested with trypsin for 24 hours at 378C.
Proteolysis was terminated by boiling briey. The supernatant was removed
for indirect ELISA.
Carter et al. (47) used glycinin peptides as antigens for pAb and mAb
production. The absence of discrete protein bands during SDS-PAGE
analysis conrmed a complete hydrolysis of glycinin (1 mg mL 1) by
treatment with 20 mg of subtilisin (in ammonium bicarbonate buffer,
50 mM, pH 8) overnight. The (rabbit) pAbs for glycinin peptides were
nonspecic (Fig. 3). Avidity for intact glycinin was attributed to surface-
located antigenic determinants. There was cross-reactivity with other seed
globulins (b-conglycinin, pea 11S globulin), probably due to partial amino
acid sequence homology. Three mAbs (designated mAbs IFRN024,
IFRN025, and IFRN026) were also produced with interesting specicity
characteristics (Fig. 4). The mAb-IFRN024 was nonspecic and recognized
an epitope that was susceptible to denaturation by SDS treatment. Both
mAb-IFRN025 and mAb-IFRN026 were specic for both intact glycinin
and glycinin peptides. The former mAb showed a 100-fold greater avidity
Speciation of Soya Protein 291

FIGURE 3 Indirect ELISA for soya bean and other seed globulins using polyclonal
antibodies for glycinin peptides. (Drawn from Ref. 47.)

FIGURE 4 Indirect ELISA for soybean and other seed globulins using mAbs for
glycinin peptides. Results for mAb-IFRN0024 and mAb-IFRN0026
were multiplied by 10 before plotting. (Drawn from Ref. 47.)
292 Chapter 10

for these samples. The epitopes recognized by mAb-IFRN025 and mAb-

IFRN026 were resistant to combined SDS and thermal treatment. One
should expect that peptide antigens will be resistant to processing effects
owing to their small size.

5.3. Conglycinin
Structural changes for thermally treated conglycinin were monitored using
mAb-IFRN089 (48). The antigenicity of b-conglycinin increased after
preheating to 658C, which is the TD for this protein. Huang et al. (49) also
monitored heating effects on glycinin using mAb-IFRN025.* Thermal
treatment led to progressive ``denaturation'' at temperatures above 908C
and an increase in protein antigenicity (49). The insoluble protein precipitate
was not analyzed. In summary, the use of mAbs for continuous epitopes
should lead to ELISAs that are suitable for processed foods. Continuous
epitopes comprising consecutive amino acids (protein 18 structure) are more
thermoresistant than conformational or discontinuous epitopes formed
from higher order protein (48, 38, or 28) structure. Beyond these
considerations, sample solubility may still be a limiting factor. Efcient
strategies are needed for resolubilizing proteins that have undergone severe
processing.

6. OTHER NONMEAT PROTEINS

Nonmeat protein additives from cereal sources, milk, and egg have been
analyzed by ELISA. However, there is far greater interest in the detection of
trace amounts of these proteins in relation to their ability to cause allergic
reactions. The analysis of protein allergens is discussed in Chapter 11.

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2. NM Grifths, MJ Billington, W Grifths. A review of three modern
techniques available for the determination of soya protein in meat products.
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* Glycinin (4 mg mL 1 in 0.35 mM K-phosphate buffer 0.1 M NaCl) was heated and then
centrifuged to remove insoluble aggregates. The soluble fraction was analyzed by ELISA.
Speciation of Soya Protein 293

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294 Chapter 10

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22. AM Hermansson. Physico-chemical aspects of soy proteins structure
formation. J Texture Stud 9:3358, 1978.
23. S Damodaran. Refolding of thermally unfolded soy proteins during the
cooling regime of the gelation process: effect on gelation. J Agric Food Chem
36:262269, 1988.
24. AN Danilenko, EK Grozav, TM Bikbov, VY Grinberg, VB Tolstogozov.
Studies of the stability of 11S globulin from soybeans by differential scanning
microcalorimetry. Int J Biol Macromol 7:109112, 1985.
25. VY Grinberg, AN Danilenko, TV Burova, VB Tolstoguzov. Conformational
stability of 11S globulins from seeds. J Sci Food Agric 49:235248, 1989.
26. S Iwabuchi, H Watanabe, F Yamauchi. Thermal denaturation of b-
conglycinin. Kinetic resolution of reaction mechanisms. J Agric Food Chem
39:2733, 1991.
27. K Watanabe. Kinetics of heat insolubilization of soybean 11S protein in
phosphate buffer system. Agric Biol Chem 52:20952096, 1988.
28. DJ Sessa. Hydration effects on the thermal stability of proteins in cracked
soybeans and defated soy our. Lebensm Wiss Technol 25:365370, 1992.
29. DJ Sessa. Thermal denaturation of glycinin as a function of hydration. J Am
Oil Chem Soc 70:12791284, 1993.
30. N Catsimpoolas, EW Meyer. Immunochemical properties of the 11S
component of soybean proteins. Arch Biochem Biophys 125:742750, 1968.
31. N Catsimpoolas, TG Campbell, EW Meyer. Association-dissociation
phenomena in glycinin. Arch Biochem Biophys 131:577569, 1969.
32. N Catsimpoolas, J Kenney, EW Meyer. The effect of thermal denaturation on
the antigenicity of glycinin. Biochim Biophys Acta 229:451458, 1971.
33. S Iwabuchi, K Shibasaki. Immunochemical studies of the effect of ionic
strength on thermal denaturation of soybean 11S globulin. Agric Biol Chem
45:285293, 1981.
34. A Demonte, IZ Carlos, EJ Lourenco, JE Dutra de Oliveira. Effect of pH and
temperature on the immunogenicity of glycinin (Glycine max L.). Plant Foods
Hum Nutr 50:6369, 1997.
35. M A Moreira, WC Mahoney, BA Larkins, NC Nielsen. Comparison of the
antigenic properties of the glycinin polypeptides. Arch Biochem Biophys
210:643646, 1981.
36. S Iwabuchi, K Shibasaki. Immunochemical studies of the effects of ionic
strength on thermal denaturation of soybean 7S globulin. Agric Biol Chem
45:13651371, 1981.
Speciation of Soya Protein 295

37. VH Thanh, K Shibasaki. b-Conglycinin from soybean proteins. Isolation and

immunological and physicochemical properties of the monomeric forms.
Biochim Biophys Acta 490:370384, 1977.
38. RC Roberts, DR Briggs. Isolation and characterization of the 7S component
of soybean globulins. Cereal Chem 42:7185, 1965.
39. K Kitamura, T Takagi, K Shibasaki. Renaturation of soybean 11S globulin.
Agric Biol Chem 41:833840, 1977.
40. DB Berkowitz, DW Webert. Determination of soy in meat. J Assoc Off Anal
Chem 70:8589, 1987.
41. MB Medina. Extraction and quantitation of soy protein in sausages by
ELISA. J Agric Food Chem 36:766771, 1988.
42. D Brandon, AH Bates, M Friedman. Monoclonal antibodybased enzyme
immunoassay of the Bowman-Birk proteinase inhibitor of soybeans. J Agric
Food Chem 37:11921196, 1989.
43. H Frokiaer, L Horlyck, V Barkholt, H Sorensen, S Sorensen. Monoclonal
antibodies against soybean and pea proteinase inhibitors: characterization and
applications for immunoassays in food processing and plant breeding. Food
Agric Immunol 6:6372, 1994.
44. CM DiPietro, IE Liener. Heat inactivation of the Kunitz and Bowman-Birk
soybean protease inhibitors. J Agric Food Chem 37:3944, 1989.
45. RE Oste, DL Brandon, AH Bates, M Friedman. Effect of Maillard browning
reactions in the Kunitz soybean trypsin inhibitor on its interaction with
monoclonal antibodies. J Agric Food Chem 38:258261, 1990.
46. K Yasumoto, M Sudo, T Suzuki. Quantitation of soya protein by enzyme
linked immunosorbent assay of its characteristic peptide. J Sci Food Agric
50:377389, 1990.
47. JM Carter, HA Lee, EN Mills, H Lambert, HW-S Chan, MRA Morgan.
Characterization of polyclonal and monoclonal antibodies against glycinin
(11S storage protein) from soya (Glycine max). J Sci Food Agric 58:7582,
1992.
48. GW Plumb, N Lambert, EN Clare Mills, MJ Tatton, CCM D'Ursel, T
Bogracheva, MRA Morgan. Characterization of monoclonal antibodies
against b-conglycinin from soya bean (Glycine max) and their use as probes
for thermal denaturation. J Sci Food Agric 67:511520, 1995.
49. L Huang, EN Clare Mills, JM Carter, MRA Morgan. Analysis of thermal
stability of soya globulins using monoclonal antibodies. Biochim Biophys
Acta 1388:215226, 1998.
11
Determination of Trace Protein
Allergens in Foods

1. INTRODUCTION

Food allergy is one of a number of adverse reactions to foods (Table 1). The
nontoxic adverse reactions are mediated by the immune system (allergy).
Nonimmune system mediated adverse reaction is termed a food intoler-
ance. Some allergic reactions to food, for example, anaphylactic shock, may
be severe and even fatal. General symptoms of food allergy are reviewed by
Anderson (1), Blades (2), Jones (3), and also Burks and Sampson (4).
Allergenic foods are discussed by Hee et al. (5) and also Taylor et al. (6).
Food allergens are reviewed by Matsuda and Nakamura (7), Bush and Hee
(8), and Hee (9). This chapter describes the analysis of trace amounts of
protein allergens in food. Allergens are associated with eight or nine major
food groups (Sec. 1.1). Soybean, peanut, and gluten allergies are described
in Secs 2, 3, and 4. In each instance we also consider the structure of the
protein allegens and the effect of processing on assay results.

1.1. Food Allergens and Labeling Regulations

Articles by Campbell (10) and Amor (11) provide summaries of current food
labeling regulations related to allergens. It is believed that food may be
rendered safe by providing information related to allergen content.

297
298 Chapter 11

TABLE 1 Adverse Reactions to Foods

Class Compounds Adverse effects

Toxic reactions Natural food Range of effects at high

components, food concentrations
additives
Nontoxic reactions
Immune mediated
IgE mediated Protein allergens Anaphylaxis, oral allergy
syndrome, atopic
dermatitis, gastroin-
testinal effects, etc.
Non-IgE mediated Protein allergens Celiac disease and related
gastroenteropathy
Non-immune mediated Lactase deciency, inborn Various effects
errors of
Food intolerance Metabolism, pharmaco-
logical compounds
Food aversion Psychological avoidance

Therefore, manufactures are not to consider allergens as incidental additives

that are exempt from declaration. On the contrary, voluntary declaration of
allergens is encouraged even where they are obviously parts of proprietary
formulations. Of major concern is the presence of hidden allergens. As of
1996, manufacturers are required to take all necessary steps to prevent,
remove, or otherwise arrange not to have inadvertent allergens in foods.
Steps should be taken to avoid the transfer of allergens from one food
product to another. Firms must make all reasonable efforts to provide
adequate labeling describing the allergenic status of their product.
According to the Food and Drug Administration (FDA), which is
concerned with food regulations in the United States, food allergens are
associated with the following eight commodities: (a) legumes (soybean,
peanut), (b) milk, (c) eggs, (d) sh, (e) crustacean, (f) mollusks, (g) wheat,
and (h) tree nuts. The United Nations Codex Alimentarius Commission also
provide a list of the major sources of food allergens. The Codex list is similar
to the that provided by the FDA although (allergenic food groups are)
dened in a slightly broader manner: (a) peanuts, soybean, and products
made from these; (b) milk and milk products (including lactose); (c) eggs
and egg products; (d) sh and sh products; (e) crustacea and derived
products; (f) tree nuts and nut products; (g) cereals containing gluten
Determination of Trace Protein Allergens 299

(wheat, rye, barley, oats) and derived products; and (h) sulte in
concentrations of 10 mg kg 1 or greater. Future FDA and Codex Commis-
sion policy may require the declaration of foods containing allergenic
proteins produced through genetic engineering. The concern is that genetic
modication may result in the inadvertent transfer of allergenic proteins
from one food source to another via genetic modication (12). Food
regulations and mandatory labeling requirements related to allergens are
also discussed in articles by Nestle (13) and others (14,15).

1.2. Detecting Allergens and Allergies

Some of the major protein allergens in foods have been isolated and
partially characterized. Many are heat resistant and survive common food
processing operations. They also tend to be resistant to digestion. The
presence of antibodies in the circulation suggests that allergens pass through
the digestive tract (16). Allergenic peptides and perhaps whole proteins may
be absorbed intact, especially in infants. Some well-characterized food
allergens are listed in Table 2. The detection of food allergens plays a vital
role in the management of diets for susceptible subjects.
Two groups of analyses are employed for food allergens: (a) in vivo
tests using live patients, in which mild symptoms are induced in the subject
during a skin prick test or the double-blind, placebo-controlled food
challenge (DBPCFC) test, and (b) in vitro detection, which covers three
types of tests involving isolated pAbs. A well-known procedure is the
radioallergoadsorbent test (RAST) for circulating antibody. The food
sample containing antigen is mixed with serum (collected from the allergic
subject). The mixture is then added to a xed amount of antigen
immobilized on CNBr-activated Sepharose. After washing the support,
any bound pAb is determined by reacting with iodine-125-labeled (rabbit)
antibody for human IgE. The amount of radioactivity bound to the solid
phase, measured with a scintillation counter, is inversely related to the
concentration of circulating pAb as well as to the food allergen content.
RAST is essentially a competitive immunosorbent assay using iodine-125-
labeled (rabbit) pAb for ``visualization'' (17,18). The second in vitro assay
for food allergens involves immunoblotting (19,20); examples of these
studies are listed in Table 3. The food sample is analyzed by SDS-PAGE,
transferred to a nitrocellulose membrane, and exposed to serum from the
allergic subject. The bound IgE is visualized using iodine-125-labeled
(rabbit) pAb for human IgE followed by radiography (Table 3). The nal
group of in vitro tests for food allergens involves classical ELISA. Included
in this group are tests described in Chapter 10 for bulk food proteins.
300 Chapter 11

TABLE 2 Some Food Allergens Involved in IgE-Mediated Adverse Reactions

Food group Major allergen, comments

Milk aS1-casein (23 kDa, heat stable)

b-lactoglobulin (18.4 kDa, heat stable)
Egg white Ovalbumin (43 kDa)
Ovamucoid (23-kDa glycoprotein trypsin inhibitor, heat
stable)
Soybean 30-kDa Gly m Bd 30 (oil body protein, papain analogue)
28-kDa allergen, KSTI-16 protein allergen protein in all
Peanut Ara h 1. (65-kDa allergen, conarachin)
Ara h 2. (1721 kDa allergen)
Castor bean 12-kDa allergen (amylase/trypsin inhibitor, heat stable)
Codsh Parvalbumin or allergen M (12-kDa Ca2 binding protein,
heat resistant)
Shrimp and other Tropomyosin (70-kDA muscle protein), heat stable
crustacea
Source: Refs. 7 and 8.

The merits and disadvantages of in vivo or in vitro tests for food

allergens were described by Nordlee and Taylor (49) and also Taylor and
Lehrer (50). One advantage of using human pAb for immunoblotting is that
the results are related to functional allergens (49). The test can be done in

TABLE 3 Analysis of Food Allergens by SDS-PAGE and Immunoblotting with

Human pAb

Allergen source References

Soybean Shibasaki et al. (21), Bucks et al. (22), Bush et al. (23), Herian
et al. (24), Ogawa et al. (25), E-F Ebabiker et al. (26)
Peanut Sachs et al. (27), Barnett et al. (28), Bucks et al. (29), Bucks et
al. (30), Uhlemann et al. (31), Hee et al. (32), de Jong et al.
(33)
Shrimp Hoffmann et al. (34), Nagpal et al. (35), Lehrer et al. (36),
Reese et al. (37)
Brazil nuts Hide (38), Morgan et al. (39), Nordlee et al. (12), Borja et al.
(40)
Almonds Bargman et al. (41), Hlywka et al. (42)
Egg white Hoffman (43), Langeland (44), Leduc et al. (45)
Milk proteins Ball et al. (46), Restani et al. (47), Rosendal and Barkholt (48)
Determination of Trace Protein Allergens 301

the absence of the allergenic individuals. Immunoblotting is the default

method for detecting allergens in food materials. The main disadvantages
are that (a) care is needed in the selection of human donors for pAb and (b)
a requirement for human pAb increases costs. Once a major allergen is
identied and isolated, less expensive sources of pAb or mAb may be
developed.

2. SOYA BEAN PROTEIN ALLERGENS

2.1. Structure and Characteristics of Soya Bean Allergens
Soybean allergy is common in children. The allergic reaction is induced by
soy protein, with soybean oil being comparatively hypoallergenic (51). The
identities of soybean protein allergens were established by SDS-PAGE and
immunoblotting with serum pAbs from allergic patients. The same
approach enabled the identication of food allergens associated with
peanut, shrimp, almonds, Brazil nut, and egg white (Table 3). Research
leading to the identication, characterization, and quantitative assays for
the major soybean allergens is described in this section.
Sixteen soybean allergens were identied SDS-PAGE analysis,
Western transfer to a nitrocellulose membrane, and immunoblotting using
pAb pooled from 361 patients exhibiting atopic dermatitis (eczema). Protein
bands were visualized using iodine-125-labeled (rabbit) pAb for human IgE
and autoradiography (25). Approximately 20% of patients with atopic
dermatitis showed IgE production for soy protein. The major soybean
allergens were associated with the 7S protein fraction (7SF). Of the subset of
69 patients showing sensitivity to soybeans, two thirds produced pAb for a
30-kDa protein thereafter named Gly m Bd 30K. About 23% of patients had
circulating pAb for a 70-kDa protein designated Gly m Bd 70 (Table 4).
Only 1.4% and 2.9% of patients produced pAb for the A subunit of glycinin
and the Kunitz soybean trypsin inhibitor, respectively. It has been suggested
that the pattern of IgE specicity can vary in different populations
depending on (a) the age of the subjects, (b) history of sensitization to the
allergen, (c) route of sensitization (airways vs. digestive tract), and (d)
method of sample pretreatment for immunoblotting. It remains to
determined whether Gly m Bd 30K is the major soy allergen in other
clinical populations.
The foremost soybean allergen (Gly m Bd 30K) seems identical to a
34-kDa oil body protein from soybean seeds. Ogawa et al. (52) found that
native Gly m Bd 30K forms a 350-kDa oligomer that elutes ahead of
glycinin during gel ltration column chromatography. Treatment with SDS
and 2-ME produced a dissociated 30-kDa protein with a pI of 4.5. The
302 Chapter 11

TABLE 4 Major Soybean Allergens Detected by Immunoblotting

with pAb from 69 Patients (average age 6 years)

Protein size Patients with

(kDa) Identity antibody (%)

30 7SF, Gly m Bd 30K 65.0

6870 b-Conglycinin a- 23.3
subunit
28 7SF, Gly m Bd 28K 23.3
6367 7SF 18.8
5255 7SF 14.5
4750 7SF 10.1
4345 7S globulin, b-unit 10.1
3335 7SF 15.9
35 11S globulin, a-unit 1.4
20 2S, KSTI 2.9
Source: Ref. 25.

N-terminal 15-amino-acid sequences for Gly m Bd 30K and 34-kDa oil body
protein were the same. The mAb for either protein cross-reacted with the
other. Following established convention, Gly m Bd 30K, being the rst
soybean allergen identied, was designated Gly m 1.
Kalinski et al. (53) independently characterized the 34-kDa oil body
protein. Within intact cells, the protein was a vacuolar protein designated
P34. Like other storage proteins, protein P34 undergoes post-translational
glycosylation and proteolysis. During processing, P34 appears along the
endoplasmic recticulum, Golgi bodies, and eventually within vacuoles or
protein bodies. Protein P34 had partial sequence homology with cysteine
proteases from the papain superfamily. It is not certain that P34 has
proteolytic activity (54). Its association with soybean oil bodies was an
experimental artifact produced by cell disruption. A survey of a large
number of soybean strains shows that P34 is widespread. The possibility of
eliminating P34 from soybean lines by breeding seems doubtful (55).
The next important soybean allergen (Gly m Bd 70K) is the 70-kDa
b-conglycinin a-subunit. Antibody specic for the a-subunit did not cross-
react with the other two (a0 or b) subunits of b-conclycinin despite some
sequence homology between these polypeptides (56). The third soybean
allergen (Gly m Bd 28K) is a 26-kDa glycosylated protein with a pI of 6.1
(57). It is apparently unstable and present in very small quantities in
defatted soy our (*15 ppm). Perhaps for these reasons, Gly m Bd 28K was
difcult to detect in processed foods containing soy protein. Finally, pAbs
from some soybean-sensitive individuals recognized the A-chain residue
Determination of Trace Protein Allergens 303

from glycinin. The epitope consisted of a 114-amino-acid residue fragment

(58). The epitope for Gly m Bd 30K recognized by mouse mAb was also
identied by Hosoyama et al. (59).

2.2. Quantitative Analysis of Soybean Allergens

Quantitative ELISA for Gly m Bd 30K was developed by Tsuji et al. (60)
using mAb produced by conventional means. Eventually, two cell lines
producing mAb (mAb-F5 and mAb-H6) were isolated. Immunoblot
analysis showed that mAb-F5 and mAb-H6 were both specic for Gly m
Bd 30K. Sandwich ELISA for Gly m Bd 30K employed mAb-H6 as the
capture antibody and an HRP conjugate with mAb-F5 as detector. The
linear range for analysis was 2200, 5500, or 10500 ng protein for reduced/
carboxymethylated-allergen, SDS/2-MEtreated sample, or native soy
protein, respectively. Ten soybean products and ve meat products
containing soy protein isolate (SPI) were analyzed by a sandwich ELISA
for Gly m Bd 30K (61). Results from this study are summarized in Table 5.*
From such data it may be surmised that (a) high concentrations of Gly
m Bd 30K are detectable within a range of processed soybean products, (b)
fermented soy products (miso, soyu, and natto) are free of allergen, (c) Gly
m Bd 30K can be accurately determined in cooked meat products known to
contain soy protein isolate, (d) sandwich ELISA results were corroborated
by immunoblot analysis using pAbs from soybean-sensitive patients, and (e)
Gly m Bd 30K is heat resistant, accounting for its detection in cooked
products. The thermal stability of soybean antigens is discussed in Chapter
10. The allergen was also resistant to digestion by chymotrypsin and trypsin.
Levels of Gly m Bd 30K declined to negligible values in fermented soy
products, probably as a result of digestion by microbial (acid) proteases.
Treating soybean samples with added microbial proteases reduced the level
of allergen, leading to hypoallergenic soybean products (62). Clearly, the
preceding assay has potential uses for monitoring soybean allergen in
processed foods. The ELISA test could also nd use as a quality control tool
for hypoallergenic soybean products.

* As pretreatment each (5-g) sample was homogenized with Na- phosphate buffer (50 mM, pH
8.0 with 1% w/v SDS and 20 mM 2-ME). The extract was centrifuged and SDS was precipitated
with KCl (1 M nal concentration). After centrifugation, the SDS-depleted sample was
analyzed by sandwich ELISA.
304 Chapter 11

TABLE 5 Determination of the Major Allergen (Gly m Bd 30K)

in Soybean-Containing Products using Sandwich ELISA

Sandwich ELISA
results (mg allergen Immunoblot
Product g 1 N) analysisa

Soybean 126 ve
Soymilk 106 ve
Tofu (kinugoshi) 89 ve
Tofu (momen) 65 ve
Kori-dofu 64 ve
Kinako 29 ve
Abura-age 59 ve
Yuba 66 ve
Miso npb
Shoyu np
Natto np
Meatballs 17 ve
Beef croquettes 21 ve
Fried chicken 9 ve
Fish sausages np
Hamburger np
a
SDS-PAGE/immonoblotting with IgE from soy-sensitive patients.
ve positive, ( ) negative results.
b
np, no allergen present.
Source: Adapted from Ref. 61.

2.3. Indirect Monitoring of Soybean Allergens by ELISA

ELISA for bulk soy protein analysis (Chapter 10) may be useful for
monitoring soybean allergens indirectly. Yeung and Collins (63) described a
competitive ELISA using (rabbit) pAb for whole soybean extract. The
assay, which was virtually identical to those used by Hitchcock and co-
workers (6467), was intended to detect bulk soy protein as opposed to trace
allergen. The antigen for immunizing rabbits was prepared from soybean
our defatted with cold acetone.* The competitive ELISA for soy protein
(63) had the following characteristics:

* Soy our was extracted with cold Tris-HCl buffer (10 mM, pH 8, with 1% w/v SDS and
10 mM 2-ME) at 48C for 16 hours.
Determination of Trace Protein Allergens 305

1. Specicity. There was no cross-reactivity with 10 other legumes, 8

varieties of nuts, 11 common food ingredients, and 2 phytoestro-
gens.
2. Acuity. The concentration of soy proteins that produced 50%
inhibition of pAb-antigen binding (IC50) was 35 ng mL 1 and the
linear range was 3117 ng mL 1. For various real foods, the
detection limit was 2 ppm.
3. Recovery. For processed tuna sh having 13.554 ppm of soy
protein, the recovery was 7795%. Similar recoveries (6396%)
were observed with hamburger matrix.
4. Precision. For model solutions and hamburger matrix containing
soy protein, the interassay precision was 3.54.2% and 3.68%,
respectively. The corresponding intra-assay precision 2.45.1% or
1.52.6%. Features such as assay specicity and acuity were not
routinely reported for ELISA designed to quantify soy protein in
meat samples (Chapter 10).

3. PEANUTS
3.1. Peanut Allergy
Peanut (groundnut) allergy occurs in about 1 in 200 of the general
population in the United States. The incidence rate is probably similar in
Western Europe. Emmett et al. (68) found a partial association between
peanut allergy and allergy to tree nut. Peanut allergy also persists for life.
Allergic subjects appear in all age groups and with no gender bias (69).
About 2528% of all cases of food allergy involve peanuts. The gures for
tree nuts are Brazil nut (10.2%), almond (7.8%), and hazelnut (7.1%). The
incidence of peanut allergy also appears related to the frequency of exposure
(70). In a study of 868 children in Singapore, 27% of subjects showed
sensitivity to bird's nest soup. The incidences of other food allergies were
24% (crustacean), 11% (eggs and cow milk), and 7% (traditional Chinese
herbs). There was no recorded adverse reaction to peanuts or tree nuts. The
low incidence of peanut allergy was attributed to the low per-capita
consumption of peanuts and other nuts in Singapore (71).
Symptoms of anaphylactic shock arise in approximately 6% of allergic
reactions to peanuts. The onset of fatal and near-fatal anaphylaxis can be
very rapid. The rst reaction may appear within 530 minutes of contact
with the allergen (72). More frequent clinical effects include atopic
dermatitis or eczema (40%), angioedema (37%), and asthma (14%); digestive
symptoms occur more rarely (1.5%). The quantity of peanut necessary to
produce an adverse reaction ranges from a few micrograms to 1 g (73). The
306 Chapter 11

anaphylactic response can arise from topical contact with peanut residue on
another individual, by inhalation (74), or by exposure to peanuts in
vegetarian ``beef '' burgers (75). Small amounts of peanut protein associated
with peanut oil can also induce an allergic episode. Peanut protein was
found in some infant formula prepared from peanut oil (76,77). Highly
rened protein-free oil apparently poses little threat (51).
Reported fatalities due to peanut allergy are of the order of 125
persons per year in the United States (84). Figures from Pumphrey et al. (78)
suggest that there were 21 deaths from peanut and tree nut allergy in the
United Kingdom during the 5- to 6-year period following 1992. Of 541
patients showing sensitivity to nuts, 90% had serum IgE specic for peanut.
About 67% of patients showed serum IgE for another nut besides peanut,
while 34% of patients had immunoglobins for all nuts tested. The threshold
quantity necessary to induce allergy varies for different subjects. Exposure
to peanut allergens in early infancy (<1 year old) or before birth may
contribute to sensitization (70). It is not yet certain whether pregnant and
lactating women should avoid peanuts and tree nuts in their diet (7880).
Avoidance of peanuts is the main strategy for managing the diet of
sensitive individuals. Hidden allergens from peanut-derived ingredients are a
major issue. Eating outside the home setting can also be problematic.
Restaurateurs cannot provide strict assurances that their ingredients or
recipes are free from peanut allergens. People thought to be highly sensitive
to peanut protein are also advised to have rapid access to epinephrine
(adrenaline) by injection. In summary, peanut and nut allergies present
serious health threats. Reviews by Sampson (81), Hourihane (82), Burks et
al. (83,84), and also Warner (85) deal with the clinical and public health
aspects of peanut allergy. Some distinctive features of peanut allergy are
(a) high incidence in the population, (b) speed and severity of the symptoms,
(c) lack of an effective treatment, (d) widespread use of peanuts and peanut
ingredients in the food chain, and (e) persistence of peanut sensitivity with
age. The availability of tests for trace amounts of peanut will reduce the risk
of exposure to peanut allergens.

3.2. Structure and Characteristics of Peanut Allergens

Proteins from peanuts are predominately globulins (* 90% total).
Approximately 10% of the storage proteins are albumins. Other classes of
storage proteins (glutelins, prolamins, etc.) are not present. Johns and Jones
(86) divided the peanut globulins into two groups, arachin and conarachin.
The foremost peanut allergen (Ara h 1) is thought to be the 6065 kDa
subunit of conarachin. Allergin Ara h 2 corresponds to a 21-kDa arachin
subunit (33). Therefore arachin and conarachin are legitimate targets for
Determination of Trace Protein Allergens 307

ELISA tests designed to monitor peanut allergens. SDS-PAGE analysis of

peanut protein treated with 2-ME revealed 14 bands with molecular masses
of 1070 kDa. Western transfer to nitrocellulose membranes and immuno-
blotting with human pAb led to visualization of 13 of the 14 polypeptides; a
26 kDa protein was not allergenic, probably due to denaturation by heat
and SDS treatment. Six major allergens (i.e., recognized by antibody from
 50% of patients) had molecular masses ranging from 17 to 44 kDa
(Table 6).
Fractionation of arachin and conarachin can be achieved by three
strategies (87,88):

1. Cryoprecipitation. Extract crude peanut protein by stirring

defatted our with 0.2 M phosphate buffer (pH 7.9) for up to
4 hours. Dialyze against several changes of tap water at 48C.
Arachin precipitates while conarachin remains soluble. This can
be recovered by a range of methods, e.g., acid precipitation or
freeze-drying.
2. Ammonium sulfate fractionation. Precipitate arachin and conar-
achin from solution by adjusting to 040% and 6080% saturated
ammonium sulfate, respectively. When this is preceded by
cryoprecipitation, the samples of arachin and conarachin obtained
are homogeneous. Chiou's procedure (89) for isolating conarachin
is simple and effective. Add peanut (acetone) powder directly to
60% saturated ammonium sulfate solution. Centrifuge and then
adjust the supernatant to 85% saturation by adding more
ammonium sulfate. The precipitated protein is conarachin.
3. Ion-exchange chromatography. Fractionate crude peanut protein
using low-pressure chromatography on a DEAE column. With
FPLC, use a Mono-Q support. Three peaks (P1, P2, and P3) are

TABLE 6 Major Peanut Allergens

Molecular size (kDa) Patients with antibody (%)

26.0, 0
29.0, 28.0, 16.0 20
36 2030
71.0, 6064, 17.0 30.040.0
44.0, 40.0, 33.0 5060
21.0, 20.0, 1718 6080
Source: Adapted from Ref. 33.
308 Chapter 11

eluted with a 00.6 M gradient of NaCl. Protein peaks P2

(conarachin) and P3 (arachin) normally elute at 0.23 M and
0.33 M NaCl.

Native arachin (1112S globulin) has a molecular mass of 350 kDa

(90). Treatment with 2-ME followed by SDS-PAGE generates seven
polypeptides with molecular masses of 72.4, 60.3, 39.8, 33.1, 29.0, and
21 kDa.* Native conarachin is a 7S globulin with a molecular mass of
180 kDa. The quaternary structure is a trimer of 60-kDa subunits. SDS-
PAGE analysis of 2-ME-treated conarachin yields eight bands (72.4, 39.8,
33.1, 26.9, 24.0, 21.9, 18.6, and 16 kDa). These results from Monteiro and
Prakash (90) suggest that peanut 7S protein has a different architecture from
soybean 7S protein. The a, a0 , and b subunits of conglycinin appear to be
devoid of S22S bonds. In contrast, peanut 7S protein is affected by 2-ME,
indicating the presence of S22S bonded subunits (91).

3.3. Thermal Denaturation of the 11S Peanut Allergen

(Arachin)
Ara h 2 is either the A or B subunit of arachin. Investigations employing dry
and moist heat treatment show that arachin is very heat resistant. Neucere
(92,93) identied 14 peanut antigenic species by immunoelectrophoresis
using (rabbit) pAb. Moist heat treatment (40% w/w moisture, 1108C) or dry
heat treatment (5% w/w moisture, 1101508C) for 60 minutes reduced the
antigenicity of nonarachin proteins monitored by immunoelectrophoresis,
rocket immunoelectrophoresis, or AGID. To achieve the same effect for
arachin required moist heat treatment at 1508C or dry heat treatment at
1758C. Heating also reduced protein solubility. SDS-PAGE analysis of
peanut proteins heated at 1008C for 15210 minutes showed little changes in
the patterns for arachin (94). Dry and moist heating studies by Chiou (89)
also conrm that arachin is heat resistant. Shokraii and Esen (91) found that
treating peanut protein with SDS and 2-ME had no effect on the
immunological characteristics. This raises the possibility that some epitopes
for peanut allergens are continuous. That is, antibody binding occurs with
consecutive sequences of amino acids. Discontinuous epitopes constructed
from higher order protein structure would be destroyed by denaturants.

* Only six from seven bands were prominent.

Determination of Trace Protein Allergens 309

3.4. Thermal Denaturation of the 7S Peanut Allergen

(Conarachin)
The effect of thermal treatment on the conformation and immunological
characteristics of Ara h 1 (b-subunit of conarachin) was examined by
Koppelman et al. (95). The Ara h 1 was isolated by cation-exchange
chromatography of whole peanut protein. Gel ltration and sedimentation
velocity measurements showed that native Ara h 1 was a 180-kDa trimer.
An SDS-PAGE analysis conrmed the presence of 63-kDa subunits. The
far-UV spectrum for native Ara h 1 was consistent with there being 31%
a-helix, 36% b-sheets, and 33% aperiodic structure. The uorescence
spectrum for Ara h 1 indicated that tryptophan residues were solvent-
exposed in the native protein. Differential scanning calorimetry thermo-
grams showed a denaturation temperature (TD) equal to 878C with a small
transition enthalpy (DHm) of about 120 kJ mol 1 for an undened
irreversible structure change.
Upon heating samples of Ara h 1 for 15 minutes at 20, 50, 80, 90, 110,
or 1408C, protein solubility decreased to 75% and 32% at the last two
temperatures. SDS-PAGE analysis showed the same molecular weights for
heated and unheated protein. Thermal treatment did not produce signicant
changes in IgE binding avidity for the soluble protein fraction. Such
results are consistent with one of the following explanations: (a) Ara
h 1 is heat resistant, (b) the epitopes for IgE binding are in parts of the three-
dimensional structure not affected by denaturation, or (c) the epitopes of
Ara h 1 are of the continuous type (conrmed in the following).
The 3D structure of Ara h 1 was elucidated from computer modeling
studies using the crystal structure of phaseolin as template (96). The
locations of 23 linear/continuous epitopes for human IgE binding to this
allergen were also established. For the native protein, antigenic sites were
clustered in the outer exposed region of the 3D structure. Most epitopes of
IgE binding comprised nine amino acids. There was no other common
structural feature for the epitopes. Single amino acid substitution within
epitopes abolished antibody binding.

3.5. Digestibility of Peanut Allergens

Protein stability and/or resistance to digestion appears to be a feature of
allergenic proteins. Compared with a random group of plant proteins,
peanut allergen (Ara h 2) was more resistant to digestion by simulated gut
uid (97). The same was true for allergens from soybean (b-conglycinin
b-subunit, KSTI, Gly m 1), egg (ovalbumin), or milk (b-lactoglobulin,
casein, and BSA). Analysis by enzyme allergosorbent test (EAST) or
310 Chapter 11

immunoblot analysis showed that simulated gut digestion reduced the

allergenicity of peanuts and hazelnuts by 50 and <10% (98). The digestion
of soy proteins by rats proceeded within 3 days to immunoreactive peptides
(99). Allergic reactions are liable to be invoked by contact with raw, cooked,
or roasted peanuts. Peanut and other food allergens will also have a
tendency to survive common food processing operations.

3.6. Determination of Peanut Allergens

Peanut allergens were initially identied by immunoblotting with human
pAb (Table 3) or using the RAST assay (17,18). Both techniques use iodine-
125 as label. These approaches have limited general utility for several
reasons: (a) there is limited availability of human antibody, (b) human
antibody is difcult to obtain as a standard reagent, (c) there are possible
biohazards associated with handling human body uid as a reagent, and (d)
RAST employs radionuclides for visualization (100). More convenient
ELISAs for peanut allergen are now being developed by research groups in
several countries including the United States (100), Netherlands (101),
Canada (102), United Kingdom (103), and Germany (104). Keck-
Gassenmeier et al. (105) evaluated several ELISA kits for peanut allergens
before adapting one for semiquantitative analysis of confectionery products
(see later).
The rst of the new generation of tests for peanut allergens* was
developed by Hee et al. (100). Results from the sandwich ELISA were
positively correlated with RAST results for a range of samples including
cheesecake mix, chocolate peanut cookies, chocolate-nut granolar bars,
confectionpeanut brittle, and peanut butter. The high correlation
(R 0.85) is to be expected because the mAb used for the ELISA test was
specic for peanut allergens recognized by human IgE. The working range
of the ELISA was 3.23162 mg mL 1 of peanut extract. With ice cream, the
LLD for peanut protein was 40 mg mL 1. This level of acuity is higher than
observed with the traditional skin test for peanut allergens.
A competitive indirect ELISA for trace amounts of peanut protein was
developed by Yeung and Collins (102). The IC50 was adopted as an index of
specicity. The linear range for analysis was 163 ng mL 1 extract (R2
> 0.98) with an LLD equal to *400 ng g 1 (food sample). Negligible
responses were obtained for protein extracts from the following legumes and

* Whole peanut protein was used as antigen. An mAb and biotinylated (rabbit) pAb were used
as capture antibody and for detection, respectively. Visualization was with an HRP-streptavidin
conjugate assayed with OPDhydrogen peroxide substrate.
Determination of Trace Protein Allergens 311

nuts: soybean, green pea, chick pea, lupine, hazelnuts, Brazil nuts, pine nut,
pecan, almonds, walnut, and cashew nut. Also, no interferences occurred
with general foodstuffs such as corn, cocoa, chocolate, milk, sugar, soy
lecithin, peanut lectin, and coconut. Peanut proteins were quantitatively
determined in over 40 processed food items belonging to the following food
groups: potato chips (crisps), cooking oil, pasta sources, plain cookies, ice
cream, and milk chocolate bars. All products labeled as containing peanut
protein were correctly identied with no false-positive results.
A sandwich ELISA using (rabbit) pAb for puried Ara h 1 was
described by Koppleman and co-workers (101). The assay characteristics
can be summarized as follows:
1. Range. The linear dynamic range for peanut protein was
0.0051 mg mL 1. Fried peanut samples gave 4580% of the
response obtained from raw samples.
2. Specicity. No cross-reactivity occurred with 40 food products
including, 9 legumes, 9 nuts, 7 cereals, 4 oil seeds, and 9 animal
proteins. In tests involving 31 processed goods (10 cookie brands,
6 chocolates, 3 cereals, 3 baby foods, and 8 sources and soups) all
products labeled as containing peanuts were accurately identied.
3. Recovery. For samples spiked with 0.00011.0% peanut protein,
recoveries were typically 85105%. Reproducible recoveries were
obtained from high-fat foods.
The competitive indirect ELISA described by Holzhauser and Vieths
(104) employed commercial pAbs for unheated peanut protein. Before use,
pAbs were puried by immunoadsorption with soy protein, white bean, and
marzipan. Precooking peanuts had little effect on the levels of Ara h 1 and
its binding by both human and rabbit pAbs. The nal assay was successfully
applied to over 30 commercial food products.
Three commercial ELISA kits for peanut allergen were evaluated (105)
at the Nestle Research Center (Lausanne, Switzerland). The kits were those
from Cortecs Ltd. (UK), Prolab Ltd. (Canada), and the TNO (Nether-
lands). The last two kits appear to be derivatives of the assays developed by
Yeung and Collins (102) and Koppleman and co-workers (101). Detailed
results concerning the preliminary evaluation were not reported. The
Cortecs kit was further evaluated for a semiquantitative determination of
trace amounts of peanuts in a range of confectionery products. The
particular kit employs a sandwich ELISA format with antibody for
concanavalin A for capture. Biotinylated antibody for concanavalin is
used for detection. Bound antigen is visualized with streptavidin-labeled
HRP assayed using tetramethylbenzidine. Recovery of peanut allergen from
dark chocolate was only 23% when using the manufacturer's extraction
312 Chapter 11

buffer (TBST). Inclusion of 12.5% sh gelatin in the extraction solvent

increased immunogen recovery to 6597%. With the modied sample
extraction procedure, the Cortecs ELISA kit was successfully applied to a
wide range of foods including milk chocolate, chocolate confectionery, ice
cream, sherbet, ready-to-use refrigerated cookie dough, lemon curd, apple
sauce, piccalilli, honey, jelly, and a frozen meal. The test kit originally
intended for a qualitative assay of peanut protein could be adapted for a
semiquantitative analysis (105).
In conclusion, several ELISA assay formats are available for detecting
trace amounts of peanuts and tree nut allergens (106). These highly
sensitivity assays can detect as little as 2 mg (allergens) per kg of food
(2 ppm). Making sure that allergen can be effectively solubilized from highly
processed foods is a limiting factor. Present assays can readily provide a yes/
no indication for hidden peanut allergens. Self-operated diagnostic tests for
peanut allergens would be useful for domestic use.

4. WHEAT AND RELATED CEREALS

4.1. Wheat Allergy
Adverse reactions to cereals and cereal products include dermatitis
herpetiformis, baker's asthma, and celiac disease.* The last condition is
related to the consumption of the alcohol-soluble cereal proteins (prola-
mins) from wheat, barley, rye, or triticale. It is doubtful whether oat is a
source of celiac-active protein (107,108). Rice, millet, sorghum, maize, and
buckwheat are reportedly nontoxic to gluten-sensitive individuals (109,110).
Baker's asthma is ascribed to inhalation of airborne cereal amylase inhibitor
proteins (111). This section focuses on allergy to wheat gluten and related
proteins. Various aspects of celiac disease have been reviewed by Goodwin
and Rawcliffe (112), Baldo and Wrigley (113), Ciclitira and Ellis (114),
Cornell (115), Brozzone and Asp (116), Silano and Vincenzi (117), Feighery
(118) and Hadjivassiliou et al. (119). Marsh (120) wrote a monograph on
this subject.
Celiac disease appears in 1 in 300 of the general population in Western
Europe except in Italy, where the frequency is nearer 1 in 4700. The
incidence of nontropical sprue in the United States is about 1 in 250 (121).
One of the highest occurrences of celiac disease was reported for the
Seharawi tribe from western Algeria and parts of Morocco. Their staple diet

* Celiac disease is also known as gluten-sensitive enteropahathy, gluten-induced enteropathy, or

nontropical sprue.
Determination of Trace Protein Allergens 313

consists of couscous and bread. Catassi and co-workers (122) suggested that
celiac disease has adaptive value in preventing intestinal adhesion and
infection by pathogenic microorganisms such as Vibrio cholerae.
The major lesion accompanying celiac disease is atrophy of microvilli
from the small intestine, duodenum, and jejunum. Microscopic observation
shows a attening of the mucosal lining and reduced surface area. There is
malabsorption of most nutrients. The intestinal absorptive layer, being
compromised, may allow the uptake of a greater range of psychoactive
peptides. Theories explaining the etiology of gluten-sensitive enteropathy
are summarized by Cornell (115), who also describes in vitro methods for
assaying celiac-reactive polypeptides or allergens. Bruzzone and Asp (116)
provide an informative introduction to gluten-sensitive enteropathy starting
from its discovery in 19501953 by Dickie and covering possible links
between diet and the immune system. Progress in the study of gluten-
sensitive enteropathy has suffered owing to the lack of reliable assays for the
condition. Current methods for diagnosis for this condition involve
1. Use of organ culture. Intestinal biopsy spceimens when grown in
cell-tissue culture show normal microvilli within 24 hours.
Addition of gluten (peptides) to culture media reintroduces
abnormalities in microvilli.
2. Detection of circulating antigliadin antibodies. Blood serum from
sufferers from gluten-sensitive enteropathy is used as the basis of
indirect ELISA.
The symptoms of celiac disease in infants and young children include
growth impairment, abnormal stool, abdominal distention, and psycholo-
gical effects, which show up as generally poor temperament. Adults
sometimes exhibit diarrhea, atulence, bulky stools, anemia, fatigue, weight
loss, bone weakness, and neurological disorders including depression and
schizophrenia. Treatment for celiac disease involves the exclusion of gluten
from the diet. United Kingdom regulations state that gluten-free foods
should contain no more than 300 mg (gliadin) per 100 g foodstuff (i.e.,
300 mg % or 0.3% w/w). European Union and WHO limits for gluten-free
foods are between 10 and 1 mg %.
Wheat gluten is an inexpensive protein and therefore widely used as a
``vegetable protein'' ingredient for meat products (123). Wheat our is used
for thickening soups and other processed foods. Gluten appears in
confectionery products and is also used as a binder for pharmaceutical
tablets. Beer contains barley protein. Rye bread is widely consumed in parts
of Eastern Europe. Raw meat products such as sausages may contain wheat
proteins derived from rusk. Hidden allergens need to be considered in
attempts to reduce celiac-active proteins and peptides in the diet. There is
314 Chapter 11

generally inadequate labeling information with respect to products having

wheat or barley protein. Levels of potentially active celiac proteins and
peptides in exotic foods, ethnic cuisine, and restaurant or cafeteria foods are
ill dened. A number of manufacturers specialize in the production of
gluten-free products. Most developed countries have celiac disease societies
that coordinate information useful for sufferers.

4.2. Structure and Characteristics of Dietary Wheat

Allergens
A. Classication of Cereal Proteins
Thomas Burr Osborne divided plant seed proteins into ve classes according
to their solubility in water, 0.1 M sodium chloride, alcohol, or alkali.
Sequentially extracting our using this range of solvents recovers albumins
(in water), globulins (with 0.1 M salt), prolamins (in 4070% ethanol, and
glutelins (with 0.1 M sodium hydroxide). A fth class of protein (called
residue protein) can be extracted only with severe solvents. The glutelins are
extractable in alcohol and 2-ME or DTT. About 7090% of the storage
proteins in cereals consist of nearly equal proportions of prolamin and
glutelins.* An important exception is cereal oat, which contains 80%
globulins. Rice has 15% prolamin and 8090% glutelin. The allergens
responsible for celiac disease are thought to be amino acid repeat sequences
found in cereal storage proteins.
Tatham et al. (124) devised a new classication scheme for cereal
proteins (Table 7). The new scheme acknowledges fundamental structural

TABLE 7 Cereal Prolamins Thought to Be Involved in Celiac Disease

Prolamin Wheat Rye Barley

Sulfur rich
Monomeric g-Gliadin g-Secalin g-Hordein
b-Gliadin
a-Gliadin
Polymeric LMW glutenin B hordein
Sulfur poor o-Gliadin o-Secalin C hordein
HMW HMW glutenin HMW secalin D hordein

* By contrast, legume seeds (soya bean, pea, kidney bean, and peanut) contain globulins as the
major storage proteins. There is relatively little (010%) prolamin or glutelin present.
Determination of Trace Protein Allergens 315

relationships between the different protein fractions. First, all cereal protein
fractions (besides albumins) are soluble in 70% alcohol and are therefore
classed as prolamins. Next, the storage proteins are grouped according to
similarities in their amino acid and cDNA sequences into three classes:
(a) sulfur-rich prolamins (a-, b-, g-gliadin), (b) sulfur-poor prolamins
(o-gliadin), and (c) high-molecular-weight (HMW) prolamins. The sulfur-
poor prolamins are monomers possessing only intramolecular disulde
bonds. This group includes o-gliadin (wheat), o-secalin (rye), and C hordein
(barley). Some sulfur-rich prolamins form HMW polymers. By contrast, a-,
b-, and g-gliadins represent monomeric sulfur-rich prolamins. The three
classes of prolamins have related architectures.

B. Gluten
Gluten is prepared by washing wheat our with water to remove the starch.
This leaves a viscoelastic protein network (gluten) that is responsible for the
gas-retentive properties of wheat dough. Gluten is a network formed by
interacting wheat prolamins (Table 7). Each of the four gliadins can be
prepared on a large scale by ion-exchange chromatography (125).* A single
chromatographic run involving about 54 g crude gliadin yields 1 g of o-
gliadin and approximately 10 g each of g-, b-, and a-gliadin. Electrophoresis
at low pH separates of gliadin into four subtypes. SDS-PAGE analysis
shows the following order of increasing electrophoretic mobility: a-gliadin
> b-gliadin >, g-gliadin > o-gliadin. Glutelin is highly polymerized by S22S
bonds.
The sulfur-rich prolamins possess N-terminal repetitive amino acid
sequences containing high numbers of three amino acids: glutamine (Q),
proline (P), and phenylalanine (F) (Figure 1). The C-terminal areas have
variable sequences bearing a number of half cysteine residues normally
involved in intramolecular S22S bonding. Also noteworthy is the rare
occurrence of SH groups in o-gliadin. Regularities are also evident in the 28
structure of cereal proteins (126,127). Thus, a-gliadin, b-gliadin, and
g-gliadin have 3637% a-helix, 1112% b-sheet, and 5253% aperiodic
structure plus b-turns. The precise quantity of b-turn or hairpin loop
structure is uncertain because this does not produce a distinct circular

* Stir 1 kg of our with water-saturated n-butanol to remove lipids. Next, extract with four
volumes of 70% (v/v) aqueous ethanol. Dry the extract by rotary evaporation and redissolve the
protein in 0.1 M acetic acid solvent. Dialyze against the same solvent and fractionate with a
CMC-cellulose column (10 cm 6 22 cm, containing 1.7 kg support) equilibrated with sodium
acetate buffer (5 mM 1 M dimethylformamide and adjusted to pH 3.5 with acetic acid). Elute
the bound protein using stepwise changes in NaCl concentration gradients.
316 Chapter 11

FIGURE 1 The primary structure of cereal prolamins. (Adapted from Refs. 124 and
126.)

dichroism absorbance peak. However, numerical calculations using amino

acid sequence data suggest that the repetitive sequences are b-turns.
Celiac toxic peptides have been identied at the N-terminal regions of
a-gliadin and b-gliadin (117,128,129). The epitopes recognized by intestinal
T cells appear to be two tetrapeptides, PSQQ and QQQP. The rst occurs in
a-gliadin at amino acid residues 1316, 5053, and 213216. QQQP occurs
at residues 1619, 3336, and 188191. Four out of six potentially toxic
sequences appear within the rst 56 amino acids from the N-terminal. The
epitope bound by mAb-WC2 is probably QQQP, which is apparently
common to all celiac-active proteins (129). Tanabe et al. (130), using
Determination of Trace Protein Allergens 317

antibody from celiac sufferers, identied the sequence QQQPP as being

celiac active. Bromelain digests proline-rich peptides leading to wheat our
that may be useful for producing hypoallergenic bread (131). Ginger
protease has high specicity for proline residues (132).

4.3. Thermal Denaturation of Wheat Allergens

The thermal denaturation of gliadin was examined using 70% (v/v) ethanol
as solvent. Heating g-gliadin led to a conformational change at 20608C as
monitored by far-UV circular dichroism.The spectral changes showed an
isobestic point implying that denaturation was a two-state (helix-coil type)
transition. At elevated temperatures the proportion of a-helix declined by
7% and the structure of gliadin loosened to expose phenylalanyl residues to
the solvent. Puried a-, b-, and o-gliadin behaved similarly. Conformational
changes produced by the relatively short (5-minute) thermal treatments were
reversible (126,127). Heating wheat our matrix revealed that o-gliadin is
more heat resistant than the sulfur-rich gliadins.* The levels of a/b-gliadin
decreased substantially after 20 minutes of heating. The g-gliadin was
resistant for up to 50 minutes of heating at 1008C. There was no change in
the quantity of o-gliadin recovered after 0100 minutes of heating. This
result is due to the virtual absence of half cystine residues in o-gliadin. This
protein can not undergo sulfhydryl-disulde exchange during heating (133).

4.4. Determination of Wheat Allergens

Immunological assays for cereal proteins are generally concerned with the
detection of celiac-active peptides. Some attempts to assess grain varietal
differences by EIA were reported (134). Also of interest is the identication
of (bread making) quality-related polypeptides by immunoassay (135,136);
literature in this area was reviewed by Skerritt et al. (137). Sections 4.4
through to 4.10 consider ELISA tests for trace (nanogram to microgram)
quantities of cereal proteins. ELISAs for wheat allergens and bulk protein
adulterants are not fundamentally different in their design. However, the
former techniques possess greater acuity. SDS-PAGEimmunoblot analysis
is another ELISA format.

* A suspension of wheat our in water was heated at 1008C for 0, 10, 20, 30, 50, and 100
minutes. Flour proteins were then extracted with 1 M urea solution and analyzed by gradient gel
electrophoresis.
318 Chapter 11

4.5. Sandwich ELISA Tests for Gluten Using pAb

McKillop et al. (141) provide a useful introduction to ELISA of wheat
gliadin. Sandwich ELISA and competitive indirect ELISA formats were
implemented using (rabbit) pAb for unfractionated gliadin. The two-
antibody format was selected for full evaluation. The assay characteristics
were as follows:
1. Linear range. 0.056 mg (gliadin) mL 1.
2. Detection limit. A concentration of 23 ng (gliadin) mL 1 produced
absorbances of 2 SD above the reagent blank.
3. Specicity. Gliadin and gliadin-containing foods. Wheat our had
5.7% (w/w) gliadin equivalent protein. The values for oats, rice,
and corn our were 240, 46, and 47 mg %, respectively.
4. Precision. The within-assay reproducibility for analysis ranged
from 5 to 40%. Samples with > 330 ng (gluten) mL 1 were
determined with below + 10% error.
The preceding assay enabled the identication of ``gluten-free''
ingredients. No tests were performed on heated samples. Table 8
summarizes other ELISAs for wheat proteins.
Fritschy et al. (140) used (rabbit) pAb for total gliadin as the basis for
a gluten-sensitive ELISA. Wheat ours (20 different types) were found to
contain 2.96.7% (w/w) gliadin equivalent proteins. The gliadin contents of
many commercial gluten-free foods were insignicant. Rice contained
57.5 mg % (w/w) and sorghum 12.5 mg % (w/w) gliadin on a dry weight
basis. Food samples were extracted with 70% (v/v) ethanol with a recovery
of 77107%. The LLD was 10 ng (gliadin) mL 1 sample extract. The assay
was specic for cereal proteins. It was possible to assay rice, corn, oats, and
barley protein when 12% BSA was included in samples to avoid nonspecic
protein binding to microwell plates.
Troncone et al. (145) puried (rabbit) pAb by afnity chromatography
using Sepharose 4Bimmobilized gliadin. They then implemented a
conventional sandwich ELISA using (rabbit) pAb for capture. The linear
dynamic range for gliadin analysis was 5400 ng mL 1 with an LLD of
0.5 ng (3 ng for the competitive assay). There was cross-reactivity with maize
and oat prolamins, although these were detected with 1000-fold lower
sensitivity. The response for gliadin was 104-fold higher than obtained for
rice prolamin. Rye and barley were not assayed. Interference from maize,
rice, or oat prolamins could be avoided by using high protein dilution.*

* Equal amounts of prolamin from different cereals yield 1000- to 10,000fold differences in
assay sensitivity. The ELISA response for wheat protein (cf. maize, oats, and rice proteins)
measures ``gluten''-like structure and functionality. ELISA results do not necessarily show the
absolute amounts of prolamins present in the different cereals.
Determination of Trace Protein Allergens 319

TABLE 8 Analysis of Gliadin and Other Prolamins by ELISA

ELISA format and

antibody Antigen References

Sandwich, pAb a-Gliadin, gliadin Windemann et al. (138),

Meier et al. (139),
Fritschy et al. (140)
Sandwich, Competitive, Gliadin McKillop et al. (141)
pAb
dot blotting, mAb o-Gliadin, gliadin Skerritt and Smith (133),
Skerritt (142), Skerritt
et al. (143), Freedman
et al. (144)
Sandwich, Competitive, Gliadin Troncone et al. (145)
pAb
Sandwich, mAb Gliadin Freedman et al. (146)
Immunoblotting Gliadin Janssen et al. (147)
Sandwich, pAb Gliadin Ayob et al. (148)
Competitive, pAb Gliadin Friis (149), Chirdo et al.
(150)
Sandwich, mAb a-Gliadin, g-gliadin Mills et al. (151)
Sandwich, mAb AOAC o-Gliadin, glutenin Hill and Skerritt (152),
approved laboratory subunits Skerritt et al. (153),
test Skerritt and Hill
(154,155)
Sandwich, mAb Home kit o-Gliadin, glutenin Skerritt and Hill (156)
subunits
Sandwich-ELISA Gliadin Hekkens and Twist de
Graaf (157)
Sandwich, mAb Gliadin peptides Ellis et al. (158), Denery-
Competitive Papini (159), Ellis et al.
(160), Nicolas et al.
(161)

Gliadin was accurately determined in human milk. Eleven of 18

lactating mothers who had consumed gluten meals 2 hours prior to testing
showed gliadin-reactive material in their breast milk. Gliadin was detectable
in human plasma after heat treatment to denature circulating human
antibodies. Thermal treatment (1218C, 5 minutes) had no adverse effect on
assay sensitivity. There was apparently no immunoreactive material in
blood plasma from celiac patients (145). By contrast, Lane et al. (162) found
320 Chapter 11

signicantly high levels of gluten in the serum of patients suffering from

dermatitis herpetiformis and celiac disease (compared with normal
controls). Different assay conditions used in these studies may explain the
lack of agreement. It is perhaps signicant that Lane and co-workers
calibrated their assay by adding gliadin to human plasma.
The performance of a low-cost sandwich ELISA test for gluten was
compared with two commercial gluten tests produced by Cortecs Ltd. (UK)
and R-Biopharm (Germany) (163). Results for the in-house test were highly
correlated (R 0.0.9967) with both commercial tests. The former was more
sensitive than the Cortecs gluten test (see the following). The in-house test
had the advantage of a 10-fold lower cost. The linear range for the in-house
assay was 50150 ng mL 1 compared to 2080 ng mL 1 for the R-Biopharm
test. The LLD values of the two tests were 0.04 and 0.1% mg %, respectively.

4.6. Competitive ELISA for Gluten Using pAb

A competitive ELISA test for gliadin (149) had a working range of
10250 ng mL 1 with an LLD of 1 ng mL 1. The highly accurate test had an
interassay precision of 33%. There was specicity for a-, b-, g-, and o-
gliadin as well as barley, oats, and rye prolamins. No responses were
obtained for maize, soya, or millet protein. Buckwheat gave a low response.
The competitive ELISA test for gluten was not tested for heat-processed
samples. Chirdo et al. (150) used (rabbit) pAb for competitive ELISA for
gluten in a range of processed foods including cake our, breakfast cereal,
processed meat, soup, and chocolate. The working range for gliadin
standards was 0.6 ng mL 1 to 10 mg mL 1. The LLD of 1 ng mL 1
(0.002 mg %) was the same as reported by Friss (149). The average
within-assay precision was about 9%. It was claimed that this test was useful
for thermally processed foods.

4.7. The AOAC-Approved ELISA Test for Gluten

The AOAC-approved test for gluten employs mAbs for capture and
detection (154). Skerritt and Smith (133) and Skerritt (142) found two mAbs
(mAb-401/21 and mAb-304/13) with specicity for o-gliadin, HMW and
LMW glutenin subunits. Because o-gliadin is heat resistant (Section 4.3),
the preceding tests may be suitable for the analysis of heat-processed foods
(see later). Either mAb-401/21 or mAb-304/13 could be used for capture or
detection, leading to similar results. Preliminary tests (153) showed
specicity for wheat (bread or durum) > rye*barley 4 maize > oats. No
responses were observed for oat, maize, or rice protein at levels comparable
Determination of Trace Protein Allergens 321

to those for wheat gluten. Sorghum was not tested. Assay performance
depended on these factors (152,153,154):
1. Extraction solvent. The optimal solvent for gluten extraction from
a range of food samples was 40 % (v/v) ethanol. Using 70% (v/v)
ethanol, 1 M urea, or 1 mM HCl as solvent led to underestimation
or overestimation of the gluten content.
2. Form of extraction. Homogenization of our samples using an
omnimixer or Ultraturrax mixer for about 30 seconds led to
accurate analysis. Vortexing for 30 or 60 seconds duration (four
times per hour) led to inaccuracy, probably due to shear-induced
precipitation of gluten.
3. Choice of gluten standard. Normal gluten is a suitable standard.
This is relatively soluble, stable in the freeze-dried form, and
usable over a wide concentration range (dilutions of up to
10,000 1 were used for analysis).
4. Choice of solid phase. Polystyrene microwell plates were preferable
to PVC plates, which produced high nonspecic binding. Back-
ground adsorption of gluten was not ameliorated by a range of
blocking solutions including PBST with 0.055% BSA, 0.65 M
NaCl, or 2% Tween.
5. mAb characteristics. The nature and concentration of the capture
and detector antibody and coating conditions (time and tempera-
ture of coating) affected the assay.
Following optimization, the linear dynamic range of the AOAC-
approved ELISA test was 0.00755 mg mL 1 (15 mg % to 10% w/w gluten in
actual foods). The LLD was 0.10.2 mg mL 1, which is equivalent to
0.20.38-mg gliadin per 100 g of our (0.2 0.38 mg %). A partial list of food
types successfully analyzed includes starches (wheat, maize), cake ours
(plain, self-raisin, bread crumb, cookie), gluten-free bread mixes, processed
meat (cooked beef, ham sausages, salami), baby foods (beef or chicken
based with and without our thickener), breakfast cereals, baked goods
(bread crumb mix, sweet cookies, crisp bread), soups, confectionery
(caramel, chocolate), and others (lentils, eggs, milk powder).
322 Chapter 11

4.8. Collaborative Testing of AOAC-Approved ELISA Test

for Gluten
Prior to AOAC approval, the previous ELISA was subjected to a
collaborative trial by 15 laboratories* (155). Eighteen samples including
ve prestudy samples (three wheat starches and two meat-gluten blends)
with known amounts of added gluten were tested. Participating laboratories
familiar with ELISA methodology were supplied with commercial versions
of the Skerritt-Hill test. The linear dynamic range for gluten was 16 mg % to
11% (w/w) basis. Assay reproducibility was 2433% with a repeatability of
1922%. Gluten was accurately determined in a range of foods (164). The
accuracy of the Skerritt-Hill ELISA test is indicated by its high precision.
Gluten levels reported by collaborators were less than +12% different from
expected values. The collaborative study led to AOAC approval for the
Skerritt-Hill ELISA test for gluten.
Potential limitations of the AOAC assay for gluten have been
suggested (165). A minicollaborative trial using a limited number of gluten
test kits was organized by the Celiac Society of Great Britain. Participating
groups were major UK clinical laboratories with longstanding interest in
celiac disease.{ Results from ve laboratories agreed with respect to four
gluten-free foods and nine gluten-containing foods (spaghetti bolognese, egg
and bacon breakfast, crispbread, malted drink, porridge, barley, plain our,
and wheat starches). By contrast, large disparities (ve- to sixfold
differences) were reported for gluten levels in 11 foods including egg and
bacon, beer, low-alcohol lager, stout, gluten-free our, gravy powder,
cornour, and rice pudding. An mAb-based gluten test developed by Mills
et al. (151) showed a linear range comparable to that of the AOAC test.
They used pAbs from chicken{ for capture while mAb-IFRN 033

* The list of participants includes M. Billington (City of Birmighman Public Analysts,

Birmingham, UK), A. Crimes, (Unilever, Bedford, UK), C. Cuncliffer, (Somerset County
Council, Taunton, UK), M. Cutrufelli, (USDA, Betsville, MD), T. McKenny (Cerestar,
Manchester, UK), M. Murlry, (Kraft Research Center, Glenview, IL), N. Paterl, (Campden,
Food and Drink Research Association, Chipping Campden, UK), J. Rhodes, and D. Lord,
(Lancashire County Lab., Preston, UK), B. Ritter, (ABC Research, Gainesville, FL), M.
Scooter, (Food Science Division, MAFF, London, UK), M. Smith, (Avon County Scientic
Services, Bristol, UK), C. Stanley, (Laboratory of Government Chemists, Teddington, UK), P.
Sutton, and S. Cooper, (British Food Manufacturing Industries Research Association,
Letherhead, JK), and B. Taylor and D. Ansell, (Greater Manchester Public Analyst,
Manchester, UK).
{ Participating institutions were St James University Hospital (Leeds), St Bartholomew's
Hospital (London), Western General Hospital (Edinburgh), Radcliff Inrmary (Oxford), and
Bristol Royal Inrmary (Bristol).
{ The pAb is recovered from the eggs of immunized chicken.
Determination of Trace Protein Allergens 323

functioned as the detector antibody. The mAb-IFRN 033 was specic for a-
gliadin and g-gliadin, which together constitute *85% of gliadin. The linear
range for gluten was 0.252.5 mg mL 1 with the LLD being 0.1 mg mL 1.
The preceding tests may be compared with the 1987 sandwich ELISA
test developed by Freedman et al. (146) from Guys and St Thomas's
Hospital, London. They used (rabbit) pAb for capture and mAb for
unfractionated gliadin for detection. The bound mAb was visualized with
alkaline phosphataselabeled (goat) pAb for murine IgG/IgM. The linear
dynamic range for this assay was 10 ng mL 1 to 1 mg mL 1. Strong wheat
our contained 3.7% (w/w) gliadin-equivalent proteins. Several brands of
``gluten-free'' our produced from wheat starch had 1.93.3 mg % of gliadin.
The day-to-day precision of the assay was better than 5%.

4.9. A Cocktail mAb-Based Sandwich ELISA Test for Gluten

Gluten tests should be equally sensitive to all celiac-active proteins from
wheat, barley, or rye. However, proteins from celiac-negative cereals (maize,
rice, millet, etc.) should not interfere. Single mAbs had different sensitivities
for different celiac toxic prolamins (166). Combining different mAbs is one
way to ensure cross-reactivity for a range of celiac-active prolamins. A
cocktail antibody test for gluten was developed by mixing two capture mAbs
(mAb-13B4 and mAb-Rye5) with specicity for barley and rye (167). A
third mAb for rye protein (mAb-Rye3) was chosen for detection. Results
compared favorably with the commercial AOAC-ELISA test for gluten. The
cocktail sandwich ELISA produced comparable calibration graphs for
gliadin, hordein, and secalin with a linear range of 3100 ng mL 1 and
an LLD of 1.5 ng (gliadin) mL 1, 0.05 ng (hordein) mL 1, 0.15 ng
(secalin) mL 1, or 12 ng (avenin) mL 1. The acuity toward barley and rye
prolamin was higher than obtained for wheat. The response toward avenin
was 10100 times lower, which is as expected from the lower toxicity of oats.
Compared with the AOAC test, the cocktail mAb test had an LLD for
hordein or gliadin that was *25-fold and 4- to 10-fold lower. The mAb-
Rye3 used for detection in the cocktail ELISA had low specicity for
promlamins from wheat, barley, and rye. This feature probably explains the
relatively high response for barley and rye.

4.10. A Home Test for Gluten

The Skerritt and Hill (156) home test kit for gluten performed well with
ordinary citizens, who successfully identied gluten-free foods with 82
100% accuracy. The home test agreed closely with results from the AOAC-
approved laboratory test kit. The home test kit can be usefully compared
324 Chapter 11

with the home pregnancy test kits, which are now commonplace. Similar
self-use kits are possible for other food allergens. The home-gluten test
consists of the following components:
1. Polystyrene test tubes (Nunc, Denmark) precoated with mAb-401/
21 and blocked with 1% BSA. The antibody-coated test tubes are
stable at 48C for 12 months or at 208C for 6 months.
2. Graduated tubes for sample extraction.
3. Enzyme-labeled mAb-402/21 for detection.
4. Substrate solution (TMB/hydrogen peroxide).
The kit was eld tested in eastern Australia by 5 dieticians or food
technologists and 47 ordinary citizens registered with the Celiac Society.
Participants were 10- to 77-year-old urban and rural dwellers of varied
educational background. The testers were given six food samples to grade as
having ``low,'' ``borderline,'' or ``high'' gluten. Samples classed in the
borderline (40 mg %) or high (>150 mg %) gluten categories were not
acceptable as ``gluten-free'' foods, whereas those with <10 mg % were
placed in the gluten-free category. For sample pretreatment, 0.5 g of ground
or nely chopped foodstuff was shaken with 5 mL of dilute hydrochloric
acid (2 mM) for 1 minute. There was 100% protein recovery from wheat
starch, 62% recovery from wheat starch having 100 mg % gluten, and 18%
recovery from wheat our with 11% gluten.
Home testing for gluten involved ve simple steps: (a) add 0.8 mL of
reaction buffer (1% BSA solution in PBST) to the antibody-coated test tubes
provided, (b) add one drop (30 mL) of food extract and mix gently for 30
seconds, (c) add three drops of HRP-mAb conjugate, (d) wait about 11=2
minutes and rinse the tubes with running tap water, and (e) add enzyme
substrate solution. A positive test gives a blue coloration within 2 minutes.
Classify tests results as high, low, or borderline by comparing results with
color produced by standard samples.

4.11. Gluten ELISA Tests Using Peptide Antigens

As the benchmark technique, the AOAC-approved ELISA test has been
subject to a range of criticisms, not all of which are justied:
1. Low accuracy. The approved method measures o-gliadin. How-
ever, the amount of o-gliadin fraction may be only weakly
correlated with the total gliadin content (168). Determining total
gluten by extrapolation from the o-gliadin may lead to error.
2. Low selectivity. There is some cross-reactivity with cereal proteins
not proved to be celiac toxic (e.g., starch granule proteins).
Determination of Trace Protein Allergens 325

3. Poor limit of detection. The LLD achieved by the AOAC-

approved gluten test may be inadequate to deal with future,
more stringent legislation regarding gluten content.
4. Unproven range of applicability. Although maize, rice, and oats
were tested by the approved AOAC method, it may be desirable to
test a wider range of materials.
5. Low reliability for certain foods. A commercial ELISA kit based
on the approved method gave discordant results for 11 out of 24
foods measured in ve laboratories (Section 4.8).

Peptides are useful antigens for developing gluten ELISA tests. A

sandwich ELISA for gluten was developed using mAb-WB8 with specicity
for peptide B3144 as the detector (158). B3144 consists of the celiac-toxic N-
terminal residues 356 from a-gliadin (129). The capture antibody was
(rabbit) pAb for unfractionated gliadin. The mAb-WB8 bound a-gliadin, b-
gliadin, g-gliadin, o-gliadin, glutenin subunits, and unfractionated gliadin.
There was also high reactivity toward rye and barley prolamins. The
response for celiac-active proteins was 1254000 times greater than that with
celiac-negative cereals such as oats, maize, millet, or sorghum. Very low
binding occurred with ovalbumin or BSA (Fig. 2).

FIGURE 2 Specicity of two mAbs for celiac-active peptide B3144. Specicity was
tested by noncompetitive indirect ELISA using cereal proteins extracted
with 50% (v/v) ethanol. The Y-axis shows mAb titer from ascite uid.
(Drawn from results in Ref. 129.)
326 Chapter 11

The working range for gliadin detection was 2 ng mL 1 to 1 mg mL 1.

The LLD ( 1.5 SD above background absorbance) was 15 ng mL 1
(extract) or 0.03 mg % of gliadin in wheat our. The corresponding LLD
values for other cereals were 0.03 mg % (rye), 0.25 mg % (barley), and 0.5 mg
% (oats). The LLD for gliadin is four orders of magnitude lower than the
current UK upper limit (300 mg %) for gluten-free foods. The preceding
assay was suitable for uncooked foods only. Prolamins were extracted with
20 volumes of 4050% (w/w) ethanol before analysis. The peptide-based
ELISA test for gluten underwent further renements. A 19-residue peptide
fragment from a-gliadin N-terminal (residues 3149) was used as antigen.
By comparison with mAb-WB8, the new detector antibody (mAb-PN3)
(160) was more selective for unfractionated gliadin and a-gliadin at the
expense of o-gliadin, rye, and barley proteins (Fig. 3). The LLD for gliadin
was 4 ng mL 1 of extract or 0.008 mg % (our). The assay repeatability was
8.79.3% with a reproducibility of 18.822.3%.
Cooking reduced the apparent gluten levels by 70% when measured by
the mAb-PN3-based ELISA. An attempt to increase sample solubility by

FIGURE 3 Sandwich ELISA tests for gluten developed with mAbs for peptide
antigens. The capture antibody is (rabbit) pAb. The dectector antibody
is either mAb-WB8 or mAb-PN3 with specicity for a-gliadin N-
terminal residues 356 or 3149, respectively. (Drawn using results from
Refs. 158 and 160.)
Determination of Trace Protein Allergens 327

extracting with a reducing buffer was unsuccessful. Residual mercaptoetha-

nol affected the stability of antibody during ELISA. The inability to assay
gliadin in heat-processed samples is a serious shortcoming. The test using
mAb-PN3 is also likely to underestimate celiac-toxic potential when a
product contains ingredients from barley or rye. This is because the limit of
detection for wheat protein was 250-fold lower than values for hordein or
secalin. The latter proteins will remain ``hidden'' in foods exhibiting
relatively low amounts of celiacactive proteins.
Denery-Papini and co-workers (159) produced pAbs for a number of
synthetic peptides. Each peptide matched a unique amino acid sequence
found in one of the four classes of gliadins. Each 8- to 12-residue peptide
was covalently coupled to a protein carrier for immunization. The (rabbit)
pAbs for these antigens were evaluated by ELISA as well as SDS-PAGE
and immunoblot analysis. A pAb specic for the N-terminal sequence from
g-gliadin or o-gliadin bound only to the corresponding protein. A particular
pAb specic for the N-terminal sequence of a/b-gliadin was used as the basis
for a competitive ELISA test for gliadin (161). Gliadin levels were found to
be 23335040 mg % using our from 20 wheat cultivars. The results were
highly correlated with gliadin determinations by reverse-phase HPLC
(R 0.82) and Kjeldahl analysis (0.86). The linear range for analysis was
45250 mg mL 1 (extract) with an LLD of *9 mg mL 1. Stirring wheat ours
with 70% (w/w) ethanol for 14 hours led to the solubilization of 48% to the
total protein. The solubilized protein was gliadin with little contamination
from glutenin.
Results from ELISA tests for gluten are summarized in Table 9. This
information was compiled from the limited number of studies involving real
foods (140,141,144,146,149,150,154,160). The agreement between gluten
values is reasonable. Results are routinely reported in milligrams of gliadin
(equivalent protein) per 100 g dry food (mg %). Measuring gluten in this
fashion is convenient because ELISA tests are usually calibrated using
whole gliadin extract. The ideal assay for gluten should show equal
responsivity toward prolamins from all celiac-active cereals. It is necessary
to test samples of alcohol-soluble proteins from each cereal. The results in
Table 9 do not include contributions from glutelin subunits, which
(although celiac active) dissolve poorly in 4070% (v/v) aqueous alcohol.
Glutenin could be more effectively solubilized for immunoassay using
solvents containing SDS or guanidine hydrochloride. Conroy and Esen
(169) found that zein was efciently adsorbed by polystyrene microwell
plates from acetic acid (60% v/v) or 6 M urea (dissolved in carbonate coating
buffer). Sample extraction with these solvents could improve the recovery of
glutenin.
328 Chapter 11

TABLE 9 Apparent Gluten Levels in Range of Foods Determined by ELISA for

Gliadin

Foodstuff or commodity Gliadin (mg %)

Gluten-negative foods
Tropical cereals
Millet, sorghum, rice, jawar Negative
Legumes
Soya our, chickpea our, buckwheat, quinoa
Miscellaneous
Potato our, molasses, skimmed milk powder, sugar
beet
Gluten-free (wheat starches)
Kinderm, Glutan, Wheatex, Nutregen 0.57.75
Wheat starch-based products
Cooked foodsa
Glutan GF ber loaf, Glutan GF white bread with 0.120.24
soya, Lopron white bread, low-protein chocolate chip
cookies
Uncooked products
Glutan GF white mix, ber mix, Rite Diet low- 0.91.4
protein our mix
Wheat-free products
Cooked products
Chocolate digestive biscuits, custard creams, tea biscuits, 0.1441.18
orange avor cream wafers, mince pies
Uncooked products
Glutan pasta spirals, vermicelli, spaghetti, macaroni 0.076.7
Flours
Buckwheatb 4
Sorghumb 13
Maizeb 46
Riceb 4757
Oatsb 100240
Barley 400
Rye 580
Wheat (self-raising, plain) 40008100
a
Levels can be underestimated by 70% after heating.
b
Sometimes nil gliadin is found in these cereals.

A meaningful comparison of ELISA tests results for gluten is difcult

because successive investigators aimed for innovation rather than standar-
dization (170). Slight changes in assay conditions can produce substantial
changes in assay performance. The following general observations seem
Determination of Trace Protein Allergens 329

relevant. The most sensitive tests are ELISA using pAb. The LLD for such
assays is about 0.51 ng mL 1. To achieve high acuity it is necessary to avoid
nonspecic binding of gliadin to microwell plates. Exposing microwell plates
to 3% skimmed milk powder or BSA for 60120 minutes reduces nonspecic
protein binding. Precoating with extraneous protein is better than adding
the coating protein together with the sample. Coating of microwell plates at
48C overnight leads to better assay performance in contrast to coating at
378C for 90 minutes. Using mAb in place of pAb increases the cost of
ELISA tests by an estimated 10-fold. On the other hand, mAbs exhibit
increased specicity and decreased likelihood of detecting celiac-negative
cereals. Immunoafntity-puried pAbs show high selectivity for celiac-
active cereals and are more frequently employed than mAbs.

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12
Biological and Chemical Tests for
Protein Nutrient Value

1. INTRODUCTION

The quality of dietary protein should meet the physiological needs of the
consumer. Protein with a high nutrient value is digestible and provides a full
complement of the essential amino acids. Protein nutrient value (PNV)
raises issues beyond the normal terms of reference for chemical analysis. A
great deal of progress was made in this eld recently and a consensus was
reached about procedures for assessing protein quality.This chapter
considers PNV and its evaluation. Sec. 1 describes some of the most
important indicators of protein quality, factors affecting these indicators,
and an outline of the literature. Human bioassays for protein quality are
discussed in Sec. 2. Investigations using human subjects are slow and
expensive. Alternative tests involve rats, chicks, and other small animals
(Sec. 3). A number of chemical tests for protein quality are also available
(Secs. 4 and 5) that are fast and inexpensive but have yet to undergo
rigorous interlaboratory evaluation. The range of in vitro tests for protein
quality includes measurements of reactive lysine, dye binding, and the rate
of hydrolysis by proteolytic enzymes.

341
342 Chapter 12

1.1. Indicators for Protein Nutrient Value

Indices of protein quality are listed in Table 1. The protein efciency ratio
(PER) and the net protein ratio (NPR) are determined noninvasively by
measuring the body weight and the amount of protein consumed. Another
group of noninvasive indices includes nitrogen balance (B), nitrogen balance
index (NBI), and biological value (BV). They are derived from the amount
of protein ingested (nitrogen intake, I) and excreted in the feces (fecal
nitrogen, F) or urine (urinary nitrogen, U). Finally, the net protein
utilization (NPU) and gross protein value (GPV) require the determination
of total body or carcass nitrogen.
The purpose of dietary protein is to provide amino acids for protein
synthesis. This process occurs at a higher rate in young, actively growing,
animals. Gender, physiological status, and natural differences between
individuals are also important (Table 2). There is some recycling of amino
acids as a consequence of the constant breakdown of old protein and its
transformation into new protein; indeed, the amount of protein resynthe-
sized may exceed the daily intake. Amino acid recycling is not 100%

TABLE 1 Indicators of Protein Nutrient Quality and Their Denition

Quality index Denitiona

weight gained
Protein efciency ratio (PER) PER protein consumed

Net protein ratio (NPR) NPR weight change protein groupnonprotein group
protein consumed

Net protein utilization (NPU) NPU body N proteinNgroup nonprotein group

consumed

Gross protein value (GPV) GPV NPU test protein

NPU casein

Total digestibility coefcient (TDC) TDC I F F0

I

Nitrogen balance (B) B I (F U)

Nitrogen balance index (NBI) NBI (B B0) / I
Biological value (BV) BV 100(B B0) / A
response slope fed group
Relative nutritive value (RNV) RNV response slope basal group

(graphs with common intercept)

response slope fed group
Relative protein value (RPV) RNV response slope basal group

(graphs with no common intercept)

a
Letters with subscript 0 refer to parameters for a protein-free diet. For example, F0 fecal
nitrogen concentration for rats fed a nonprotein diet, also called metabolic nitrogen.
Tests for Protein Nutrient Value 343

TABLE 2 Factors Affecting Protein Quality

Consumer or host related, physiological Species, age, gender, reproductive status

(pregnancy and lactation), general
health and pathological states
(trauma, stress, neoplasia)
Sociological Economic, hygiene, and sanitation
Protein related Amino acid composition, primary,
secondary, tertiary, and quaternary
structure, stability, interactions,
digestibility
Processing history Alkali, chemical, heat, or pressure
treatment etc., storage, contamination
by bacteria, presence of
antinutritional factors
Dietary Total protein, total caloric intake,
dietary ber, frequency of feeding

efcient. Over time there is a gradual net loss of body protein, which must be
recovered from the diet. A maintenance diet provides protein at a level that
is just sufcient to avoid a net loss of body mass. Excess dietary amino acids
are deaminated and then oxidized to supply energy. Such processes lead to a
complicated relationship between protein (nitrogen) intake and utilization.
The large number of variables affecting PNV makes it difcult to measure
accurately.

1.2. Factors Affecting Protein Nutrient Value

High-quality protein meets an organism's requirement for essential amino
acids. These are not synthesized by the body and must be provided in the
diet. The levels of essential amino acids affect how proteins are utilized for
growth and maintenance. Protein quality is related to factors intrinsic to
the protein, e.g., amino acid composition, protein source, presence of
antinutritional factors, processing history, and storage conditions. Extrinsic
factors that can affect protein quality include nonprotein dietary
constituents, total energy intake, and contamination by bacteria or toxins.
Consumer-related variables, socioeconomic factors, and hygiene are also
important considerations. Protein quality also varies with the species of
consumer (Table 2).
344 Chapter 12

High-quality protein is readily assimilated by the organism, leading to

large differences between the nitrogen intake and output. Protein digestion
leads to amino acids and small (310 residues) peptides. These are absorbed
from the gut across the intestinal lining into the blood stream. From here
they pass across cell membranes and into the cytoplasm. Unabsorbed
protein and other products of hydrolysis are lost as fecal nitrogen. Absorbed
but nonmetabolized peptides and amino acids are excreted via the urine.
Some dietary protein is degraded by intestinal microora with no benet to
the consumer. This is especially true for ruminants, although such losses
also occur in the large intestines of monogastric animals.
PNV is especially signicant for certain sections of the population.
These include pregnant women, lactating women, infants and preschool
children, and persons with dietary restrictions (vegetarians, patients on
liquid feeds, weight watchers, and others on low-calorie diets). People may
be on special diets by choice or as a part of medical treatment. Of special
concern are populations with diets comprising mainly plant proteins or
where the average total caloric intake is low. In the United States and
Canada, food manufacturers must provide food labeling information
concerning PNV. The evaluation of PNV is important for monitoring
food product formulation, new sources of protein, and for quality control.
The animal feeds industry is also interested in PNV.
A high protein content (%N 6 6.25) is not a reliable indicator of a
food's ability to support growth. The quality of dried milk decreases during
storage under high-humidity conditions at room temperature. The quality
loss is due to reactions between casein and lactose. In model systems
comprising casein and glucose, losses of amino acid side chains occurred
after 530 days at 378C and 70% relative humidity. Kjeldahl protein levels
remained unchanged. Fig. 1 shows sample results for casein or egg albumin
stored with D-glucose. This diagram was drawn using data from Lea and
Hannan (1) and Tanaka et al. (2). The patterns of amino acid losses are
similar for both casein and egg albumin (mainly ovalbumin). Decreases in
the level of lysine and arginine are most notable. Baldwin et al. (3) obtained
comparable results by heating a mixture of casein, dextrose, and water
(1:1:0.5) at 1218C (15 psi) for up to 260 minutes. Protein quality was
assessed using the rat feeding trial, by dye binding, and by monitoring the
concentration of essential amino acids. Heat treatment caused losses of
essential amino acids within the rst 1580 minutes. The pattern of amino
acid destruction (arginine 94%, lysine 80%, tryptophan 42%, methionine
41%, histidine 32%) was not unlike results in Fig. 1. The declines in arginine
and lysine were closely shadowed by losses in protein quality measured by
bioassay. In conclusion, processing and storage can cause changes in protein
quality, whereas the quantity of protein need not be affected.
Tests for Protein Nutrient Value 345

FIGURE 1 The effect of 5 days of storage on residual amino acid levels for a casein-
glucose mixture or an egg albuminglucose mixture. Samples were stored
dry at 378C, pH 6.3 at an RH of 6870%. (Drawn from data from Refs. 1
and 2.)

The literature covering protein quality is extensive. Since 1973,

procedures for the assessing PNV have been reviewed by Rao and
Shurpalekar (4), Roseneld (5), Friedman (6), Pellet (7), Hopkins (8),
Anderson (9), Friedman (10), Bender (11), Satterlee and Chang (12), Walker
(13), Sarwar (14), and Sarwar and McDonough (15). Reviews have
discussed proteins from various sources, including leaf (16), wheat (17),
potato (18,19), sprouted cereal (20), dried beans (21,22), sh (23), various
plant proteins (24), and single-cell protein (25,26). Friedman (27) surveyed
the PNV of most of the major food protein groups. Conference proceedings
on protein quality were edited by Claassens and Potgieter (28), Porter and
Rolls (29), Friedman (3032), Bodwell et al. (33), and also Phillips and
Finley (34). The following books devote one or more chapters to the subject
of protein quality: Milner (35), Bender et al. (36), White and Fletcher (37),
Olson (38), Whitaker and Tannenbaum (39), Milner et al. (40), Rakosky
(41), Liepa (42), and Damodaran and Paraf (43). The book edited by
Finely and Hopkins (44) is another authoritative source of information on
protein nutrient value. Specic chapters are cited from these sources where
relevant.
346 Chapter 12

2. HUMAN AND OTHER IN VIVO ASSAYS FOR PROTEIN

NUTRIENT VALUE

Protein quality may be measured with human subjects randomly selected

from the population of demographic, ethnic, or geographic interest. To
determine PNV for feedstuffs, farming livestock may be used (45). Precise
and accurate measurements of PNV require that many of the variables listed
in Table 2 are controlled. Careful experimental design and due regard to
statistical methods (sampling, appropriate numbers of repetitions, proper
control experiments) are necessary. The evaluation of PNV using humans is
described by Scrimshaw et al. (4648). The following account is based on
this work. High protein quality is the same as the ability to support growth
in humans.
Indices of human growth such as body weight or height could be used
to monitor PNV. In practice, inducing poor growth in humans is considered
unethical. PNV is usually assessed by the nitrogen balance method (Table 1).
Subjects are fed a diet containing a submaintenance level of protein [0.3 g
kg 1 (body weight) day 1]. The amount of nitrogen excreted is carefully
monitored over the trial period. A nitrogen balance curve is constructed by
plotting nitrogen balance versus the N intake. From such a graph, several
indices of quality are determined. Fig. 2 shows two N balance curves
produced by feeding diets containing varying amounts of egg or whole
wheat protein to human subjects. The equation of the line relating N balance
and N intake is

B 0:5I 44 1

where B N balance and I N intake. The (dimensionless) gradient (0.5) is

a straightforward measure of the efciency of nitrogen utilization. The
``B 0'' intercept [88 mg N kg 1 (body weight) day 1] is the maintenance
protein-nitrogen requirement. This is the nitrogen intake necessary to
maintain the body weight with no net gain or loss.
The ``I 0'' intercept shows the N balance for subjects subsisting on a
protein-free diet. A negative intercept implies that subjects lost 44 mg N
kg 1 (body weight) day 1 on a protein-free diet. This value should be
independent of the identity of the sample protein. In practice, different
``I 0'' intercepts may occur when high N intake values are extrapolated to
zero N intake. In Table 1, some PNV indicators (e.g., the relative nutritive
value, RNV) are calculated assuming that all test proteins have the same
``I 0'' intercept value; i.e., the organism exhibits the same maintenance
nitrogen requirement (for different test samples). Actually, the ``I 0''
intercept from Fig. 2 is simply a mathematical value. The real issue is
Tests for Protein Nutrient Value 347

FIGURE 2 An N balance curve for egg or whole wheat protein. Young men were
rst fed a protein-free diet (I 0) followed by a single diet (e.g., with
40 mg N/kg/day) or at multiple levels of nitrogen. The equation of the
straight line is B 0.5(I) 44 for egg protein and B 0.27(I) 35 for
whole wheat protein. Each data point is averaged from seven test
subjects.*

whether the nitrogen balance curves are linear or not. For completely linear
responses the ``I 0'' intercept is more likely to be independent of the type
of protein sample.
Protein quality can also be determined at a single level of N intake.
PNV is then calculated using two experimental points [e.g., I 0 and
I 40 g kg 1 (body weight) day 1]. This approach is prone to error if the N
balance plot is not a straight line. It is preferable to determine PNV by
feeding several diets with different levels of N intake. According to the slope
method, Eq. (1) parameters and associated PNV indices are determined
from several experimental points. From Table 3, the slope method is more
sensitive and shows clearly that the order of increasing quality is egg
protein > soy isolate > whole wheat protein. The level of protein required
for maintenance is also a sensitive index of PNV. A feeding trial using one
level of nitrogen failed to identify egg protein as possessing superior quality.

* Where B Nitrogen balance and I Nitrogen intake as protein (see Table 1).
348 Chapter 12

TABLE 3 Estimation of Protein Nutrient Value from N Balance Studies with Young
Adult Mena

Protein source, N utilization efciency N required for

levels of N (slope)* maintenance (intercept)**

Single test
Egg 0.63 81
Soy isolate 0.54 94
Whole wheat 0.65 78
Slope method
Egg 0.50 88
Soy isolate 0.43 107
Whole wheat 0.27 131
a 1
Protein quality was determined as the (*) the dimensionless slope or (**) intercept [mg N kg
(body weight) day 1] of the N balance curve.

3. SMALL ANIMAL BIOASSAYS FOR PROTEIN NUTRIENT

VALUE

Bioassays using humans or large mammals are time consuming and

expensive for three reasons: (a) larger animals grow slowly, (b) there is a
requirement for special and more exacting accommodation, and (c) people
(even students) may require remuneration. In contrast, small animals such
as rats exhibit higher growth rates than adult humans and are cheaper to
house. The use of model animals cuts cost and speeds up analysis. Protein
bioassays using weanling rats have received ofcial approval in Canada and
the United States. Many proposed chemical methods for assessing PNV are
routinely calibrated using the rat bioassay. Nevertheless, the use of small
animal models (as opposed to humans, livestock, etc.) represents a
compromise. Rats and chicks have different amino acid requirements than
people and farming livestock.

3.1. The Rat Bioassay

Before 1994 there was a statutory requirement in the United States and
Canada for food labeling information based on the PER. Protein quality is
now measured by a new technique described in Chapter 14. The classic PER
assay remains important. This is usually determined using 4-week-old rats.
The PER technique was thoroughly discussed in the proceedings from the
38th Annual Meeting of the Institute of Food Technologist Dallas, Texas
Tests for Protein Nutrient Value 349

(1978) titled Protein Quality Testing: Industry Problems, Needs, Approaches.

Articles by the following authors provide a comprehensive overview of the
topic: Roseneld (49), Staub (50), Hackler (51), Anderson (52), Burnette
and Russoff (53), and Hsu et al. (54). The proceedings from the symposium
Measuring Protein Quality for Human Nutrition (American Association of
Cereal Chemist, New Orleans, 1976) likewise provides informative
summaries by Steinke (55), Bodwell (56), and also Hackler (57). The
following discussion is based on materials from these sources and a number
of other general reviews (416).
Osborne et al. (58) introduced PER as an index for measuring the
growth-promoting effects of proteins. To determine the PER, cohorts of 10
rats were fed a diet containing 10% protein for 28 days. Their weights were
then measured. PER is the ratio of weight gained (or lost) to the weight of
protein consumed. The procedure was later standardized with respect to test
animals (strain, gender, age) and nonprotein dietary factors by Chapman et
al. (59). They adopted casein as a standard protein. A casein standard diet is
assessed along with the test protein. Results are then adjusted such that the
PER for casein is 2.5. The casein-corrected PER value (cPER) is calculated
from the relation

2:5PERSAMPLE
cPER 2
PERCASEIN

Values for cPER allow more reliable comparisons of assay results from
different laboratories. Bender and Doell (60) incorporated weight loss for
rats fed on a protein-free diet into the PER equation. The resulting index,
called the net protein ratio (NPR), is a measure of the protein requirement
for growth and maintenance. In contrast, PER measures only the protein
requirement for growth. Dividing the NPR by the corresponding value for a
standard protein (casein or lactalbumin) yields the relative NPR value
(RNPR). PER, cPER, NPR, and RNPR represent increasingly robust
indices for protein quality determined using the rat bioassay.
In Europe, the preferred rat bioassay involves the measurement of
carcass nitrogen. To determine net protein utilization (NPU), rats are fed
for 10 days on a protein-free diet. Another group of rats is fed the same
basal diet supplemented with 10% protein. Both groups of rats are sacriced
and their carcasses are dried at 1058C or dissolved in concentrated sulfuric
acid. Whole-body nitrogen is determined by Kjeldahl analysis or else
estimated from the known body moisture content. Rats have a xed
water/nitrogen ratio (61).
The PER, NPR, and NPU assays are single-point assays. These tests
involve a single level of N intake. A slope method or multiple-level tests
350 Chapter 12

involving diets with different levels of protein yield more reliable estimates
of quality. PER measurements based on multiple levels of N intake were
introduced by Hegsted and Chang (62) and Hegsted et al. (63). Feeding
multiple levels of nitrogen led to two slope measurements for protein quality
called the relative nutritive value (RNV) and the relative protein value
(RPV).

3.2. Sources of Error

During PER determination, the test sample should contribute a dietary
protein level of about 10%. Provided that the sample and casein standard
diets have similar proximal composition (moisture, protein, fat, crude ber,
minerals), some leeway is permitted in the amounts of nonprotein dietary
constituents. Values for PER change with the amount of dietary water, ber,
and mineral mix as well as net food intake. The PER test is also more suited
for simple foods or protein concentrates (milk powder, infant formula,
wheat our, soy our). With complex foods (hamburgers, processed meat,
sausages, bread, rice, and ready-to-eat meals), measuring PER requires a
great deal of attention to detail. Samples having one or more of the
following characteristics may pose difculty: (a) high moisture content, (b)
high fat (greater fat content than protein content), (c) high moisture and fat
content, and (d) low (<11%) or very high protein content. To obtain
accurate values for cPER, the protein and casein standard diets should have
a similar proximal composition. PER increases with moisture levels over the
range 030%. Therefore, food samples should be lyophilized and controlled
amounts of moisture reintroduced as necessary.
Food components should also be nely ground to avoid dietary
separation by rats, which can reject certain foods while favoring others. Rats
have a preference for sweetened foods and may consume greater quantities
compared with the unsweetened casein standard. Residual feed should be
routinely tested to conrm that unconsumed food is representative of the
general diet. Other rat-related factors (sex, age, strain) should obviously be
standardized. To ensure greater precision, only healthy weanling rats should
be used. The transfer of rats to the test laboratory should be done carefully
to avoid overly stressful conditions. A 2- to 7-day acclimation period in the
new laboratory environment is recommended with longer acclimation
periods resulting in higher PNV.

3.3. A Critique of Animal Testing

According to Bodwell (56), the chief merits of animal testing are their
convenience and low cost. According to this investigator,
Tests for Protein Nutrient Value 351

1. Animal tests have few other advantages over human tests.

2. Rat bioassays have not been satisfactorily calibrated against the
human tests. An equal number of studies show agreement or
disagreement between human- and rat-based results. The number
of studies comparing PNV results in rats and humans is
inadequate.
3. Protein requirements for growth versus maintenance differ for rats
and humans.
4. The PER assay does not measure maintenance requirements.
5. The PER scale is nonlinear and values are not proportional to
quality. For example, a PER change from 1 to 2 does not mean a
100% improvement in quality.
6. Specic amino acid deciencies lead to different effects in rats and
people.
Finally, Bodwell concludes that the performances of various rat
bioassays are no better than chemical methods.
A number of methodological differences between human and small
animal bioassays were also highlighted by Kies and co-workers (64); they
routinely chose mice (rather than rat) for feeding trials owing to the smaller
size of feeding rations required. Whereas growth measurements are
employed for animal assays, nitrogen balance measurements are used for
human assays. Small animal bioassays usually involve groups of genetically
pure strains of mice. With human subjects, such homogeneity is not possible
or desirable. Therefore, all experimental variables including control diets are
usually applied to all human subjects during successive periods within the
feeding trial.

3.4. AOAC Guidelines for the Rat Bioassay

A collaborative study involving eight laboratories was conducted in order to
establish the best practice for rat bioassays. Standard techniques for
assessing true protein digestibility (TPD) and associated statistical
procedures (65) were developed.* The principles apply to other PNV

* Participants McDonough, Steinke, Sarwar, Eggum, Bressani, Huth, Barbeau, Geraldine

Mitchell, and Philips were afliated with USDA-Beltsville, USDA-Washington, USDA-
Philadelphia, Protein Technologies International (St Louis), Health and Welfare Canada
(Ontario), National Institute of Animal Science (Tjele, Denmark), Institute of Nutrition of
Central America and Panama (Guatemala City, Guatemala), Kraft General Foods Inc.
(Glenview, IL), Virginia Polytechnic Institute and State University (Blacksburg, VA).
352 Chapter 12

indicators (Table 1). In vivo digestibility of the major food protein groups
has been reviewed by Hopkins (66) and Sarwar (67).
Method 1
An outline of AOAC guidelines for conducting a rat bioassay.
True protein digestibility was determined for six samples (casein,
canned macaroni and cheese, tuna sh in water, rolled oats, pinto beans,
and heat-treated milk powder).
Sample Pretreatment
1. Freeze dry food samples and (where necessary) grind to pass
through a 20-mesh screen.
2. Determine the proximate composition (e.g., % nitrogen, fat,
water, ber) for each sample.
3. Assess the nitrogen content for each sample by Kjeldhal analysis.
4. Formulate standard rat diets to provide equal levels of protein-N
(*1.6% or 10% true protein).
5. From the known proximate composition, normalize the level of
fat and ber in each diet.
Table 4 shows the composition of some of the diets used in AOAC
study (65). Notice that diet 1 is a nonprotein diet. Butylated hydroxyanisole
(BHA) was added as antioxidant. Dietary constituents were mixed
thoroughly using a mechanical mixer to avoid selection by rats. Male
Sprague-Dawley rats (5070 g) were acclimated in wire net cages (at 18
268C and 4070% relative humidity) for 2 days on normal rat food. Each
test diet was fed (15 g per day, water available ad libitum) to two groups of
four rats (i.e., eight rats per diet) for a total of 9 days. The rst 4 days was an
adaptation period. The next 5 days constituted the balance period, during
which the weight of food eaten was recorded. Rat fecal output was also
collected, dried, and stored for nitrogen determination. Nitrogen intake was
determined from the weight of feed consumed and the percent nitrogen
content of each diet. TPD was calculated from Eq. (3).

IP FP FO;P
TPD 3
IP

where IP, FP, and FO,P are protein intake, fecal protein, and metabolic
protein. Eq. (3) is a specic form of the digestibility equation (see Table 1)
applied to dietary protein. Digestibility can also be dened in relation to
amino acids (Chapter 14).
Tests for Protein Nutrient Value 353

TABLE 4 Example Diets Used in a Collaborative Rat Assay for Protein

Digestibilitya

Diet
Component
(g/100 g) 1 2 3 4 5

Casein 10.8
Macaroni and 54.8
cheese
Tuna sh 11.0
Rolled oats 57.8
Vitamins 2.0 2.0 2.0 2.0 2.0
Minerals 3.5 3.5 3.5 3.5 3.5
Corn oil 10.0 10.0 1.5 9.7 6.6
Cornstarch 79.5 68.7 34.5 68.8 29.4
Cellulose/ber 5.0 5.0 3.7 5.0 0.7
BHA 0.005 0.005 0.005 0.005 0.005
a
The plan conforms to the AOAC test diets for rat bioassays.
Source: Adapted from Refs. 65 and 121.

3.5. The Chick Bioassay

Choppe and Kratzer (68) used White Plymouth Rock chicks to evaluate
PNV for meat and bone meal. Chicks were fed with normal starter mash
rations until 5 days old. They were then divided into groups of 10 and fed a
basal diet (Table 5) with or without a supplement of test sample. Sufcient
meat and bone meal were added to supply 24% of protein (Kjeldahl,
%N 6 6.25). The chicks were weighed at 5, 10, and 15 days old. Moran et al.
(69) used New Hampshire 6 Delaware chicks to evaluate the effect of

TABLE 5 An Example Basal Diet for Chick Feeding Trials

Basal diet Composition (%)

Cornstarch *75
Mineral mixture 2
Soybean oil (crude, 5
degummed)
Cellulose 4
Choline chloride 12.5
Vitamin supplements 2.16
Source: Adapted from Ref. 68.
354 Chapter 12

heating on the quality of soybean and shmeal. Groups comprising ve

male and female chicks are randomly selected and fed a basal diet with and
without added test sample as a protein supplement. During feeding trials
chicks should be kept under constant temperature and controlled lighting
(usually 15 hours lighting per day). They are also fed on demand. Weight
changes are recorded at weekly intervals for up to 4 weeks. Each
measurement is taken in triplicate or quadruplicate and averaged over 30
40 chicks. Various protein quality indicators (Table 1) are calculated from
the weight changes.

4. IN VITRO METHODS FOR ASSESSING PROTEIN

NUTRIENT VALUE
4.1. Amino Acid Scores
The use of amino acid data to determine PNV was suggested in 1946 by
Mitchell and Block (70). They are also credited with introducing the concept
of protein chemical score. To determine the chemical score, divide the
quantity of each amino acid from the test sample by the amount found in
egg protein or another reference protein [Eq. (4)]. Repeat the calculation for
each of the 910 essential amino acids (EAAs).
amount of EAA sample
Chemical score 4
amount of EAA egg

The chemical score is the smallest ratio found; this also identies the limiting
amino acid. Oser (71) introduced the integrated chemical score, which is the
geometric mean of the ``egg ratio'' for all essential amino acids. By the 1970s
the egg protein standard had fallen into disuse. Protein quality is now
determined in relation to the essential amino acid requirements for humans
(Table 6).
Amino acid scores (AASs) are estimated using the FAO/WHO/UNU
(1985) amino acid requirement pattern for 2- to 5-year-old preschool
children (Table 6). The calculation of AAS follows the same principles as
shown in Eq. (4). First, determine the essential amino acid prole for the test
sample. Next, divide the amount of each essential amino acid by the
corresponding value from the FAO/WHO/UNU (1985) list. The smallest
ratio (a) identies the limiting amino acid and (b) yields a numerical value
for the AAS. It is a relatively simple matter to calculate the AAS value for a
hypothetical mixture of foods. Some investigators have taken the sum of the
chemical scores for the nine essential amino acids (EAA9) as the index for
quality.
Tests for Protein Nutrient Value 355

TABLE 6 Suggested Pattern for Essential Amino Acid Requirements

Suggested pattern of requirement (mg/g protein)

Infanta Infanta 25 1012

Amino acids mean range years years Adult

Histidine 26 1836 19 19 16
Isoleucine 46 4753 28 28 13
Leucine 93 83107 66 44 19
Lysine 66 5376 58 44 16
Methionine cystine 42 2960 25 22 17
Phenylalanine 72 68118 63 22 19
tyrosine
Threonine 43 4045 34 28 9
Tryptophan 17 1617 11 9 5
Valine 55 4477 35 25 13
Total EAA 460 398589 339 241 127
Total nonessential 540 411602 661 759 873
AA
Total 1000 1000 1000 1000 1000
a
Mean and range of values found in human milk.
Source: From FAO/WHO/UNU (1985) [see Refs. 75, 76 of Chapter 14].

4.2. Sources of Error

Errors in amino acid analysis (Chapter 1) will affect values of the AAS.
Ideally, the hydrolysis of proteins using 6 M HCl should produce efcient
release of all amino acids followed by accurate analysis by column
chromatography. In practice, the sulfur amino acids and tryptophan are
partially destroyed during acid hydrolysis. The release of valine and
isoleucine is dependent on the hydrolysis time. In order to obtain data for all
amino acids, multiple protein hydrolysis is necessary. The majority of amino
acids are analyzed after protein hydrolysis in 6 N HCl at 1108C for 24 hours.
The sulfur amino acids are rst oxidized by performic acid treatment to
cysteic acid or methionine sulfone. Tryptophan determination is performed
after alkaline hydrolysis.
Many criticisms of amino acid analysis appear to have been addressed
with the advent of more sophisticated equipment. The precision of amino
acid analysis was thought to be questionable in the past. However, recent
collaborative studies have shown a within-laboratory precision of about 3%.
The interlaboratory precision for assaying labile amino acids was 1617%.
356 Chapter 12

Another disadvantage of AAS is that it takes no account of protein

digestibility. This suggests (incorrectly) that (a) all proteins are optimally
digestible and (b) all amino acid residues are absorbed and are bioavialable
to the same degree. Following recent improvements in amino acid analysis,
protein digestibility corrected amino acid scores (PDCAASs) have become
widely adopted as indicators for protein quality (Chapter 14).

4.3. Available or Reactive Lysine

Lysine is the rst limiting essential amino acid in cereals. The most reactive
essential amino acid also appears to be lysine (Fig. 1). Protein-bound lysine
residues with low chemical reactivity tend to be nutritionally unavailable.
Therefore available lysine is widely monitored as an index of protein quality
(Table 7).*
To measure lysine reactivity, the food sample is exposed to FDNB,
TNBS, MIU, or EVS at high pH for periods ranging from 2 hours (FDNB)
to 4 days (MIU). The chemically modied protein is hydrolyzed by reuxing
with 6 M HCl. The hydrolysate is analyzed by colorimetry to determine the
concentration of reactive lysine. The FDNB (difference) or EVS (difference)
methods involve conventional amino acid analysis. Chemically modied

TABLE 7 Reagents for Measuring Protein-Bound Reactive Lysine

Reagent References

1-Fluoro-2,4-dinitrobenzene (FDNB) Sanger (72), Carpenter (73), Hurrell and

Carpenter (74), Booth (75)
1-Fluoro-2,4-dinitrobenzene (FDNB) Booth (75), Roach et al. (76), Ostrowski
(difference method) et al. (77), Couch (78)
Trinitrobenzenesulfonic acid (TNBS) Kakade and Liener (79)
o-Methylisourea (MIU) Hurrell and Carpenter (74)
o-Phthalaldehyde (OPA) Gondo et al. (80)
Ethyl vinyl sulfone (EVS) (difference Friedman and Finley (81)
method)
Differential dye-binding capacity Sandler and Warren (82)

* Carpenter suggests that ``reactive lysine'' is a more accurate description. Lysine chemical
reactivity is not synonymous with nutritional availabilitysee Section 5.1. However, reactive
lysine is not an effective search keyword in the food science literature. We use ``available lysine''
to imply chemically reactive protein lysine groups.
Tests for Protein Nutrient Value 357

lysine is eluted with a different retention time, allowing unmodied (so-

called bound) lysine to be found.

4.4. The o-Phthalaldehyde Method

The o-phthalaldehyde (OPA) method does not involve protein hydrolysis
(80). The sample (* 25 mg in 0.1 M sodium phosphate buffer, pH 7) is
treated with OPA reagent. Fluorescence intensity is measured after 2
minutes using excitation and emission wavelengths of 340 and 455 nm. The
uorescence emission (calibrated with quinine sulfate) is directly propor-
tional to the concentration of reactive lysine. Corrections are needed for
small peptides because OPA also reacts with the terminal amino groups.
There is usually no interference from common reagents such as urea,
guanidine chloride, imidazole, ethanol, tricine, sodium acetate, or 2-
mecaptoethanol at concentrations of 100 mM. So far, the OPA method
has been applied only to relatively pure protein samples.

4.5. Fluorodinitrobenzene Acid Reactive Lysine by

Spectrophotometry
FDNB introduced by Sanger (72) is the most widely used reagent for
assaying available lysine. Lea and Hannan (1,83) used FDNB to monitor
changes in reactive lysine concentration for a casein-glucose mixture stored
in the dry state. They also followed changes in the reactive lysine content of
dry milk powder. Later, Carpenter and co-workers (73,74) from the School
of Agriculture, University of Cambridge (UK) extended the FDNB
procedure for protein-bound lysine in foods. In Method 2, the sample is
rst modied with FDNB and then hydrolyzed by heating with 6 M HCl.
The hydrolysate is extracted with diethyl ether and the absorbance reading is
recorded for the aqueous phase (Tube A). A regent blank (Tube B) is
prepared by neutralizing the hydrolysate, treating with an acylating agent,
and then extracting with diethyl ether. The reactive lysine concentration is
proportional to the absorbance of Tube A minus the absorbance for Tube
B.
Method 2
Determination of FDNB-reactive lysine (73,75).
Reagent
1. 1-Fluoro-2,4-dinitrobenzene (2.5% w/w in ethanol). Melt solid
FDNB by warming to about 408C and dispense using an
automatic pipette. About 12 mL of FDNB solution is required
per sample.
358 Chapter 12

2. Methoxycarbonyl chloride (MCC; also called methyl chlorofor-

mate).
3. Diethyl ether.
4. Sodium carbonate buffer (8% w/v; pH 8.5). Add NaHCO3 (8%
w/v) to a solution of 8% (w/w) Na2CO3 until this reaches pH 8.5.
5. Concentrated hydrochloric acid (8 M).
Procedure
STAGE 1. Production of FDNB-reactive lysine and acid hydrolyses
of the modied protein.
Place 1.5 g of ground (50 mesh) sample in a at-bottomed ask and
wet using 8 mL of sodium carbonate buffer. Add 12 mL of FDNB
solution and shake at room temperature for 2 hours. Warm the
mixture over a boiling-water bath until it no longer effervesces and
all ethanol has been evaporated.
Add 24 mL of 8 M HCl and reux at near 1008C for 16 hours. Allow
the sample to cool slightly and lter through Whatman No. 541
lter paper. Wash the ltrate thoroughly with water, collect the
washings, and make up to nal volume of 200 mL. Remove 2 mL
(62) samples and label as Tube A and Tube B.
STAGE 2. Prepare sample (Tube A) for absorbency measurements.
Extract the contents of Tube A with 5 mL (64) diethyl ether. Discard
the ether phase and warm the aqueous sample over a boiling-water
bath to remove trace amounts of ether. Make up the contents of
Tube A to 10 mL using 1 M HCl and retain this sample (Tube A-aq)
for absorbency readings.
STAGE 3. Acylate the regent blank (Tube B) for absorbency
measurements.
Extract Tube B with 5 mL of ether once. Discard the ether phase and
warm the aqueous phase to remove trace ether as before. Add one
drop of phenolphthalein indicator and neutralize the sample using
0.1 N alkali until pink. Adjust the sample to pH 8.5 by adding 2 mL
of sodium carbonate buffer.
Acylate the sample with 4550 mL of methoxycarbonyl chloride and
shake vigorously.
Using Tube B, follow guidelines from Stage 2.
STAGE 4. Record absorbance measurements at 435 nm.
Record A435 readings for Tube A (aq) and Tube B (aq) using a 1-cm
path length disposable cuvette. The absorbency difference for Tubes
A and B is proportional to the reactive lysine concentration.
Tests for Protein Nutrient Value 359

Food samples usually contain protein-bound lysine and free amino

acids (lysine, histidine, arginine). One mole of FDNB reacts with protein-
bound lysine via the epsilon amino group (e-NH2), forming e-DNP-Lys.
Free lysine or the N-terminal lysine residue reacts with 2 moles of FDNP to
form Lys(DNP)2. This species (and His-DNP, Arg-DNP, AA-DNP) lacks a
formal charge and therefore partitions into the ether phase (see Stage 2).
The a-NH2 group e-DNP-Lys remains free and ionized (when released by
acid hydrolysis of the DNP-modied protein) and therefore extracts into the
aqueous phase of Tube A. Finally, acylation and extraction by ether remove
all DNP-AA derivatives including e-DNP-Lys (in an acylated form) from the
water phase.
Table 8 shows the distribution of different DNPamino acid
derivatives between the aqueous and ethereal fractions during reactive
lysine analysis. DNPamino acid derivatives (30 mM) were added at stage 1
of the FNDB assay. DNP-Arg remained in the aqueous phases in both
Tubes A and B. The nal absorbency difference between Tube A (aq) and
Tube B (aq) provides an excellent correction for the presence for free
histidine which reacts with FDNP.
The recovery of e-DNP-Lys from animal proteins was 90% during the
measurement of reactive lysine. Therefore, results are normally multiplied
by 1.09 as a correction factor for physical losses (73,74). With plant protein
the recovery of e-DNP-Lys was 6085% owing to one or more of the
following factors: (a) plant proteins have a higher resistance to acid
hydrolysis, (b) the degree of hydrolysis is further reduced by the low
solubility of DNP-protein adducts compared with unmodied proteins, (c)
DNP-Lys adsorbs to plant residues not hydrolyzed by concentrated HCl,
and (d) the NO2 group DNP-Lys may be reduced to 22NH2, leading to a
reduced color yield. The correction factor of 1.09 was inapplicable for plant

TABLE 8 Recovery of DNP Derivatives of Three Basic Amino Acids

Final A435 for tube

Amino acid Tube A (aq)a Tube B (aq) Tube B (eth) A B (aq)

e-DNP-Lys 0.203 0.002 0.201 0.201

DNP-Arg 0.090 0.089 0.008 0.001
a-benzoyl His 0.002 0.007 0.138 0.005
Me ester
a
Tube A (ether phase) discarded.
Source: Based on results from Ref. 73.
360 Chapter 12

proteins. The correction factor should be carefully determined during plant

protein analysis (75).

4.6. Fluorodinitrobenzene Reactive Lysine by

Chromatography
To determine chemically available lysine by chromatography, hydrolyze
FDNB-modied protein with 6 M HCl (see Method 1). Then subject the
hydrolysate to chromatographic analysis. The majority of reports involve
low-pressure chromatography (Table 9). However, these are probably only
of historic interest. They have now been superseded by reverse-phase high-
pressure chromatography (RP-HPLC).
Peterson and Warthesen (92) were rst to describe chromatographic
analysis of e-DNP-Lys using RP-HPLC. Their system comprised a
m-Bondapak C18 column coupled to a Waters model 600A pump, UK6
injector, and a 440 absorbance detector. Samples were separated by isocratic
elution with 20:80 (v/v) acetonitrile0.01 M acetate buffer (pH 4.0) and

TABLE 9 Chromatographic Analysis of Available Lysine

Protein source Column, comments References

Various including oilseeds Amberlite IR-120 Rao et al. (84), Frampton

(cottonseed, sesame, (*1 6 6 cm) wash and Kuck (85), Kuck
peanut, soybean), sh 3 N HCl, eluent 3 N and Frampton (86),
meal, meat meal, HCl Me-Et-ketone Hussein (87)
feather
Various including maize, Nylon powder, Amberlite Blom et al. (88)
gluten, milk replacer, C.G. 12) polarograp-
soybean hic detection
Groundnut meal, arachin, Liquid-liquid chromato- Matheson (89,90)
conarachin graphy with kieselguhr
support
Various including sh Amino acid analyzer, Roach et al. (76), Booth
meal, meat meal, milk subtractive amino acid (75), Ostrowski et al.
powder, wheat, maize, analysisSilcock met- (77), Bailey (91)
soybean, sunower hod
meals
Casein, soybean, gluten, C18 HPLC reverse phase, Peterson and Warthesen
zein, sh meal, e.g., m-Bondapak, Nov- (92), Rabasseda et al.
albumin, lysozyme a-Pak, or Spherisorb (93) Castillo et al. (94)
ODS-2 Hernandez et al. (95)
Tests for Protein Nutrient Value 361

products were detected at 436 nm. Two peaks for 2,4-DNP and e-DNP-Lys
were well separated with retention times of 10 and 12 minutes. Comparing
Carpenter's technique (Method 1) with the HPLC method showed that the
former gave signicantly higher lysine readings for high-carbohydrate
samples.
Rabasseda and co-workers (93) attempted to optimize the RP- HPLC
method using product detection at 274 nm. The LLD was 4.5 6 10 9 g of e-
DNP-Lys or 2 6 10 9 g for unmodied lysine. The precision of analysis was
0.95% for sh meal and 1.66% for soybean meal. Castillo and co-workers
(94) criticized the previous HPLC analysis for the arbitrary choice of assay
conditions (e.g., conditions for sample hydrolysis, choice of mobile phase
pH, choice of detector wavelength). The optimal RP-HPLC assay involved a
C18 column operated at 408C to avoid precipitation of DNP during analysis.
Peak elution was with 22:77 (v/v) acetonitrile0.01 M acetic acid (pH 5)
solvent at a ow rate of 2 mL min 1. DNP-protein was more efciently
hydrolyzed by treating solid samples with an excess of relatively dilute HCl
acid (1 mL 6 M HCl per 1 mg sample) as compared with highly concentrated
acid (1 mL of 10 M HCl per 1 mg sample). Apparently, e-DNP-Lys was
degraded by 10 M HCl. Analytical precision varied with sample type; the
CV ranged from 1.41 to 3.19% for enteral formula, milk, and lentil.

4.7. Trinitrobenzene Sulfonic Acid

The analysis of free amino acids and peptides using TNBS (2,4,6-
trinitrobenzene sulfonic acid) was introduced by Okuyama and co-workers
(96,97). TNBS reacts with primary amines in a manner similar to FDNB.
Habeeb (98) used TNBS to monitor protein-bound lysine under relatively
mild conditions. The BSA reaction with formaldehyde could be followed
using TNBS. Binding of SDS to lysine residues interfered with the TNBS
reaction. Mokrasch (99) used TNBS for the coestimation of amines, amino
acids, and proteins. Kakade and Liener (79) were rst to determine available
lysine using TNBS. The trinitrophenyl (TNP) protein derivatives can be
hydrolyzed by autoclaving with concentrated HCl for 60 minutes as
compared with a minimum hydrolysis time of 4 hours for DNP-proteins.
Analysis of trinitrophenylated products is by spectrophotometry (see
Method 2) or RP-HPLC (100).

Method 3
Determination of reactive lysine using TNBS (79).

Reagents
1. 2,4,6-Trinitrobenzene sulfonic acid (0.1% w/w solution in water).
362 Chapter 12

2. Sodium carbonate buffer (4% w/v; pH 8.5). To a solution of 4%

(w/w) Na2CO3 add NaHCO3 (4% w/v) until the correct pH is
attained.
3. Concentrated HCl.
4. Diethyl ether.
Procedure
Add 1 mL of TNBS solution to 1 mg of sample (dispersed in 1 mL
sodium carbonate buffer) and incubate at 408C for 2 hours.
Add 3 mL of concentrated HCl and autoclave at 1208C (15 psi) for 60
minutes.
Cool to room temperature and adjust to 10 mL with distilled water.
Extract the hydrolysate with 2610 mL of diethyl ether.
Record the A346 reading for the aqueous phase using a 1-cm cuvette.
Calculate the concentration of TNP-Lys; c A/e, where e 1.46 6
104 M 1 cm 1.
The TNBS method gave quantitative readings for lysine in a wide
selection of proteins including BSA (60 mole 1), ovalbumin (20 mole 1),
b-lactoglobulin dimer (29 mole 1), bovine hemoglobin (47 mole 1), and
insulin (1 mole 1). In all cases, the numbers of lysine residues detected
equaled literature values. Examples of the application of the TNBS method
for food samples include the analysis of cotton seed meal (101) and casein
and rapeseed protein (102). High amounts of lactose interfere with the
TNBS method. Overnight dialysis followed by freeze-drying is a simple
strategy for reducing the error from lactose (103,104). Interferences also
occur in the presence of high concentrations of other carbohydrates. Sample
treatment with bleach may ameliorate this problem (105).

4.8. Differential Dye-Binding Capacity or Dye-Binding

Lysine
Sandler and Warren (82) measured DBC before and after acylating protein
samples using ethyl chloroformate. The differential dye binding capacity
(dDBC) is the change in dye-binding capacity produced when lysine residues
are blocked by chemical modication. Hurrell et al. (106) used propionic
anhydride as an acylating agent and called their measurement the dye-
binding lysine procedure. As described in Chapters 57, protein DBC is
determined by the net concentration of basic amino acids. Values for the
dDBC are directly proportional to lysine levels alone and therefore provide
a sensitive indicator of protein quality. The dDBC procedure is summarized
next.
Tests for Protein Nutrient Value 363

Method 4
Determination of differential dye-binding capacity using propionic anhy-
dride (106,107).

Reagents
1. Propionic anhydride
2. As listed for the Udy method (Method 1, Chapter 5)
3. Sodium acetate (0.2 M)

Procedure
Dye-binding capacity. To 20500 mg of ground sample (*2090%
w/w protein) in a 100-mL ask, add three 6-mm glass beads and
2 mL of sodium acetate (0.2 M). Shake for 10 minutes to wet. Add
40 mL of dye solution and shake for a further 60 minutes. Filter the
sample, dilute the supernatant as needed, and read its absorbency.
Determine the DBC (mg g 1 protein) from a dye calibration graph.
DBC for a chemically modied sample. Weigh 20500 mg of sample
and wet with sodium acetate as above. Add 0.4 mL of propionic
anhydride, mix, and allow to react for 1015 minutes. Add 40 mL of
dye solution and treat as before.
Calculate the dDBC as the difference between DBC values for the
modied and unmodied protein.
Protein acylation with propionic anhydride is straightforward and
quantitative. The procedure for determining dDBC is no more protracted
than a conventional dye-binding assay. The lysine content of 24 different
varieties of grain legumes was rapidly determined by the dDBC method.
Hurrell et al. (106) found that the dDBC method had potential for
monitoring plant breeding. The technique was successfully applied for
monitoring processing damage. Values for dDBC (mmoles of dye bound per
16 g N) are numerically equal to the concentration of total lysine (mmoles
per 16 g N) determined by amino acid analysis or using the FDNP technique
(Fig. 3). The dDBC measurements are free from interference by starch, lipid,
or free lysine (up to 30% w/v).
Sandler and Warren (82) found that carbethoxylation of sh meal
required 3060 minutes, with 40 minutes being the least time needed to
ensure complete reaction. The rate of dye binding was slow at 208C
although equilibrium is reached in about 20 minutes at 408C. There was
good agreement between values for DBC and the total basic amino acid
concentration (TBAA) and also between dDBC and lysine values measured
by amino acid analysis (Table 10). Pearl and co-workers (107) determined
the dDBC values for soybean meal using Method 3 with minor
modications. The amount of Orange G dye bound to soybean protein
364 Chapter 12

FIGURE 3 Relationship between the total lysine and the differential dye-binding
capacity (dDBC) for a range of commodities: bovine serum albumin,
skim milk powder, sh meal, pea our, soya our, meat and bone meal,
barley, peanut our, corn gluten, as well as a variety of legumes (winged
bean, pea, green gram, chickpea, lentil, cowpea, dry bean, soybean,
runner bean, and pigeon pea). The equation of the line Y mX C,
where m 1.0328 (+0.013), C 0.071 (+0.3810). R2 0.9967. For
statistical testing, F 6287.38 with 21 degrees of freedom.

TABLE 10 Dye-Binding Capacity and Differential Dye-Binding Capacity in Protein

Mealsa

TBAA DBC Lys* dDBC

Protein source (mmoles) (mmoles) (mmoles) (mmoles)

Fish-a 79 (+8.6) 87 43 (+4.7) 44 (+4.8)

Fish-b 74 (+8.1) 73.6 40 (+4.4) 38 (+3.6)
Soya 57.7 59.4 24.1 36.4
Cashew 47.0 52.1 13.7 17.6
a
TBAA, total basic amino acids, i.e., Lys His Arg concentration (mmole/120 mg of sample);
DBC, dye-binding capacity (mmole dye bound/120 mg sample); Lys, results from amino acid
analysis (mmole/120 mg sample); dDBC, differential dye-binding capacity. Samples were sh
meal (sh-a), defatted sh meal (sh-b), soya meal (soya), or cashew nut meal (cashew).
Tests for Protein Nutrient Value 365

was determined before and after acylation with propionic anhydride. Slight
changes in protein-bound lysine due to thermal treatment were detected.
These results (82,106,107) conrm that the dDBC method is a highly
affordable alternative to amino acid analysis when one wants to measure
lysine levels.

4.9. Comparisons of Methods for Available Lysine

One advantage of the TNBS method (compared with the FDNB technique)
is the shorter time for protein hydrolysis. The solvent extraction steps for
both techniques reduce interference. Hurrell and Carpenter (74) compared
FDNB, TNBS, and other reagents for reactive lysine determination in
model systems comprising ovalbumin, lactalbumin, and glucose (3:2:5
weight ratio).* Samples were stored at 378C for up to 30 days or autoclaved
at 1218C for 15 minutes. The reacted protein-sugar mixtures underwent
large changes in the ability to sustain growth in chicks or rats.
The MIU and TNBS methods underestimate total lysine levels for the
unheated (control) sample (Table 11). The ability to detect mild levels of
damage (378C, 30 days storage) also varies for different techniques. Amino
acid analysis and FDNP and TNBS methods did not accurately measure

TABLE 11 Total Lysine and FDNB-Reactive Lysine in Ovalbumin, Lactalbumin,

and Glucose Mixture by Different Methods

Lysine (mg/g protein)

Methoda Control 378C, 30 d 1218C, 15 min

Total Lys 86.3 50.9 29.3

Total Lys (NaBH4) 81.1 12.2 18.7
FDNB (diff) 84.7 45.7 19.5
FDNB (dir) 81.6 19.6 12.2
TNBS 53.6b 35.9 11.2
MIU 71.9b 15.8 6.5
a
Total lysine, amino acid analysis. Total Lys (NaBH4), amino acid analysis after sample
reduction by sodium borohydride. Other abbreviations are dened in the text.
b
Results are signicantly different from the control.
Source: Summarized from Ref. 74.

* Lactalbumin is spray dried whey protein. It has low solubility owing to heat damage by spray
drying.
366 Chapter 12

mild heat damage. The errors were ascribed to two factors: (a) products of
early Maillard reactions are not acid stable and some glycosylated lysine
may be degraded to free lysine during protein hydrolysis in 6 M HCl, and (b)
early Maillard reaction products may react with TNBS or FDNB. This may
be because the nucleophilic character of the lysine derivatives formed during
the early Maillard reaction is similar to that for unmodied lysine. Both
errors can be ameliorated by the addition of sodium borohydride
(74,101,108). The Schiff base formed between lysine e-NH2 and sugars is
rendered acid stable by treatment with sodium borohydride. Without this
precaution, available lysine assays involving acid hydrolysis at high
temperatures will be subject to error.
By comparison with Method 1, the chromatographic detection of e-
DNP-Lys leads to some of the following improvements:
1. Reduction of interference from other yellow substances formed
during protein chemical modication (e.g., dinitrophenol and o-
DNP tyrosine). Plants also contain brown humin and other dyes
that can act as interferences.
2. Ability to measure free lysine and N-terminal lysine residues in
one experiment.
3. Decreased error in the case of high-carbohydrate samples. The
color yield for Method 1 can be impaired when e-DNP-Lys is
reduced to the diaminophenol derivative. Alternatively, carbohy-
drate may react with FDNB to generate water-soluble derivatives
that interfere with the analysis of e-DNP-Lys.
4. Absence of sample pretreatment. Protein hydrolysate can be
subjected to chromatographic analysis directly without an ether
extraction step.
5. High sample throughput.

5. PROTEIN DIGESTIBILITY

Digestion begins in the mouth. Food is macerated by chewing, mixed with

saliva, and swallowed. Further particle size reduction arises from muscular
contractions of the stomach. Within the gastric environment, protein is
broken down by pepsin (or rennin in young animals). The stomach contents
then pass to the small intestines and are neutralized by pancreatic juice. This
contains the principal enzymes for protein digestiontrypsin, chymotryp-
sin, and elastase as inactive zymogens. Following zymogen activation,
protein is degraded to low-molecular-weight peptides and then attacked by
carboxypeptidase A and B. The products of protein digestion (amino acids,
Tests for Protein Nutrient Value 367

dipeptides, tripeptides) are absorbed in the small intestines. Undigested

protein functions as dietary ber or else undergoes microbiological
degradation in the large intestines. Human nutritionist and veterinary
scientists are concerned with protein digestibility in vivo. Guidelines for
assessing digestibility in the rat were described earlier (see Method). More
articial tests for digestibility are described in this section.

5.1. Denition of Terms

Alternative denitions for digestibility are listed in Table 12. Apparent
protein digestibility (APD) is the dietary protein intake (I) minus fecal
protein (F) expressed as a percentage of the dietary protein intake.
Correcting for the metabolic or fecal nitrogen (F0) yields the true protein
digestibility (TPD). The metabolic nitrogen is that which is excreted by a test
subject fed on a protein-free diet. It is derived from cells sloughed off the
intestinal wall and proteins comprising gastric enzymes and other secretions.
Both APD and TPD are sometimes called nitrogen digestibility because of
being calculated from intake and fecal nitrogen concentrations.* The
formula for TPD is the same as that for (bio)availability (Table 12). Both
indices can be further rened by taking account of urinary nitrogen (U or
U0). In practice, this correction is not often applied for tests using human

TABLE 12 Some Common Expressions for Digestibility

Expressed in terms of
Term Equationa retention

Apparent protein (I F)/I (R U)/I

digestibility (APD)
True protein digestibility (I (F F0))/I (R U F0)/I
(TPD)
Availability/bioavailability (I (F F0))/I or (R F0 U0)/I
(I (F F0) (U U0)/I
True adsorption I (F F0)
Retention (R) I (F U)
Nitrogen balance (B) I (F U)
a
I, intake; F, total fecal excretion; R, retention; U, total urinary excretion. Subscript 0 refers to
metabolic values determined with a protein-free diet.

* These relations also apply to dietary nitrogen, which is assumed to be proportional to crude
protein (N 6 6.25). See Chapter 1 for a more detailed description of Kjeldahl analysis.
368 Chapter 12

subjects or rats. Dietary protein levels probably do not affect the amount of
protein-N secreted via healthy kidneys and hence U U0 is close to zero.
For amino acids and other low-molecular-weight nutrients, ignoring urinary
losses may lead to signicant error (109). The importance of ``digestibility''
can be seen by comparing relations in Tables 1 and 12; notice that
parameters such as NBI and BV (Table 3) also feature digestibility.
Digestibility can be measured for specic amino acids and is called
amino acid availability (AAAv), amino acid digestibility, or simply
availability. The three terms are used interchangeably. The AAAv (unlike
TPD) allows for different adsorption rates for specic amino acids.
IAA FAA FO;AA
AAAv 5
IAA
In Equation (5), the term IAA is the intake concentration of a specied
amino acid, FAA is the fecal concentration, and FO,AA is the fecal
concentration with a protein-free diet.* Protein digestibility is routinely
measured using the rat assay (Method 1). Constance Kies (110) refers to
``protein bioavailability'' owing to the similarity between Eq. (3) and Eq. (5).
TPD is determined by measuring nitrogen (crude protein), and AAAv is
determined from the corresponding amino acid concentrations (111). Most
nutritionists do not distinguish between protein digestibility and protein
(bio)availability.

5.2. In Vitro Protein Digestibility and Protein Quality

In vitro protein digestibility refers to the rate of proteolysis of different
proteins by proteases in vitro. Tests of this type do not take account of the
effect of absorption. The link between PNV and in vitro protein digestibility
is shown by results from Hankes and co-workers (112). Autoclaving casein
for 4 minutes or 20 hours produced no signicant changes in the amino acid
composition compared with the unheated protein. However, in vitro protein
digestibility was impaired during successive treatments with pepsin,
pancreatin, and erepsin (a crude source of peptidase). Similar ndings
were reported by Eldred and Rodney (113). Melnick and Oser (114) found
that heating legume or egg proteins improved their biological value

* Measurement of fecal concentrations does not allow for amino acid degradation by colon
bacteria. With farm animals, ileal digestibility is routinely determined by tting catheters and
collecting digestion products (for nitrogen or amino acid determination) before they reach the
colon.
Tests for Protein Nutrient Value 369

probably through inactivation of protease inhibitors, which improves

proteolysis.

5.3. Determination of in Vitro Protein Digestibility

The food sample is exposed to one or more proteases and the degree of
proteolysis is monitored. Single enzymes or combinations of enzymes can be
used including pepsin, chymotrypsin, trypsin, papain, and peptidase. The
degree of protein hydrolysis is generally measured as TCA-soluble products
at 275280 nm, by following the fall in system pH, or by monitoring the
release of free amino groups using TNBS. Techniques for monitoring
protein digestibility were reviewed by Swaisgood and Catignani (115,116)
and by Boisen and Eggum (117). The AOAC-approved test for in vitro
protein digestibility is based on the three-enzyme method of Hsu et al. (118)
or the four-enzyme approach (119,120). A pH meter or pH-Stat instrument
is used to monitor these multienzyme reactions (see Method 5).

Method 5
Determination of in vitro protein digestibility using three or four enzymes
(118120).

Reagents
1. Porcine pancreatic trypsin (14,190 units per mg)
2. Bovine pancreatic a-chymotrypsin (60 BAEE units per mg)
3. Porcine intestinal peptidase (40 units per g powder)
4. Pronase
5. Dilute (0.1 M) hydrochloric acid and sodium hydroxide
Enzyme solution A. Dissolve trypsin (16 mg), chymotrypsin (31 mg),
and peptidase (13 mg) in distilled water at 378C. Adjust to pH 8
using dilute HCl or NaOH and bring to a nal volume of 10 mL.
Enzyme solution B. Dissolve 65 units of pronase in distilled water at
378C. Adjust to pH 8 using dilute HCl or NaOH and bring to a nal
volume of 10 mL. Store the enzyme solution in ice.

Procedure*
Determination of in vitro protein digestibility via a three-enzyme test.
Add the food sample (62.5 mg or equivalent to 10 mg N) to about 6
8 mL of distilled water and soak for 60 minutes at 378C. Adjust to
pH 8 using dilute HCl or NaOH and bring to a nal volume of

* Pronase (enzyme solution B) is not needed in the three-enzyme method.

370 Chapter 12

10 mL. Add 1 mL of enzyme solution A, incubate for 10 minutes at

378C, and record the pH change at 378C.
Determination of in vitro protein digestibility via a four-enzyme test.
Proceed as above. Exactly 10 minutes after adding enzyme solution
A, add 1 mL of enzyme solution B. Transfer the mixture to 558C for
9 minutes. Return the sample mixture to 378C and record the pH
change. The net reaction time should be 20 minutes.
The decline in pH during proteolysis follows a power law, DpH Btm,
where t is reaction time and B and m are constants. Equation parameters
were determined by regression analysis. Results from in vitro protein
digestibility tests were highly correlated with the apparent protein
digestibility (APD) measured using the rat bioassay:

APD% 210:46 18:10 DpH10 min 6

APD% 234:84 22:56 DpH20 min 7

Fig. 4 shows the relation between in vitro protein digestibility and in vivo
results for over 20 processed and unprocessed food protein sources. The
right-hand axis in Fig. 4 shows the residuals for pH (10 min) results, i.e.,
difference between in vitro and in vivo test results. These is no systematic
trend in the residuals. In vitro protein digestibility estimates using the
multienzyme method were not affected by the buffer capacity of normal
food samples.
In a collaborative test for the three-enzyme method, pH was
monitored with a pH-Stat instrument (121).* For sample pretreatment
fresh or canned foods were freeze-dried and ground into a powder. High-
lipid foods were defatted by extracting with anhydrous cold ether. In vitro
protein digestibility was examined essentially as described in Method 4 with
minor modications. Reaction progress was monitored from the volume of
0.1 N NaOH added to the reaction vessel by the pH-Stat set at pH 8. In
vitro protein digestibility{ was calculated from

IVPD% 79:28 40:74V mL 8

* The test using a pH-Stat is AOAC approved. Participants in the interlaboratory tests were
afliated with the USDA (Beltsville) Health and Welfare Canada (Ottawa), Protein
Technologies International (St. Louis, MO), CIVO-TNO Food Analysis InstituteAJ (Zeist,
The Netherlands), Kraft Inc. (Glenview, IL), National Institute of Animal Science (Tjele,
Denmark).
{ The term in vitro protein digestibility has been substituted for ``percent true digestibility'' in
the original reference to avoid confusion with true protein digestibility (TPD).
Tests for Protein Nutrient Value 371

FIGURE 4 Correlation between in vitro protein digestibility and in vivo apparent

protein digestibility (APD) in the rat. Results for three-enzyme (





circles) or four-enzyme () test. Protein sources are casein, soy isolate,
soy protein concentration, delactosed whey, corn, high-protein wheat
bran our, general wheat our, nonfat dry milk, cottonseed meal, wheat
protein concentrate, yeast protein concentrate, bean protein concentrate,
soy concentrate, and full lactose whey. Triangles show residuals. (Drawn
using results from Refs. 118120.)

where IVPD stands for in vitro protein digestibility and V (mL) is the
volume of alkali added to the enzyme reaction over a 5-minute period. In
vitro protein digestibility values were adjusted according to a scale with
casein assigned an arbitrary value of 100. From 204 independent analyses
(17 samples* 6 2 replicates 6 6 laboratories) the between-laboratory re-
producibility was 0.835.0%. The within-laboratory repeatability ranged
from 0.35 to 1.4%. The multienzyme in vitro protein digestibility method
allows a precise and rapid assessment of digestibility. The correlation
between TPD and in vitro protein digestibility implies that protein

* The 17 protein sources examined were casein, nonfat dried milk, nonfat dried milkheated,
tuna sh, salami, canned sausage, chicken frankfurters, beef stew, macaroni and cheese, peanut
butter, soy isolate, pea concentrate, chick peas, rolled oats, pinto beans, whole wheat cereal, and
rice-wheat gluten cereal.
372 Chapter 12

bioavailability is largely determined by the rate of protein breakdown by

digestive enzymes.

5.4. Sample Pretreatment to Inactivate Trypsin Inhibitors

Suspend the sample (62.5 mg or 10 mg N) with 2.5 mL of water and 2.5 mL
of 0.2 N NaOH. Then incubate at 308C for 30 minutes. The high pH
conditions inactivate trypsin inhibitors. Thereafter, add 5 mL of 0.075 N
HCl and adjust the mixture to pH 8 and a volume of 10 mL (121). Add the
three-enzyme mixture (1 mL) and proceed as described in Method 5.

5.5. Degree of Protein Hydrolysis

Adler-Nissen (122) measured in vitro protein digestibility from the degree of
hydrolysis (DH). This is the number of peptide bonds hydrolyzed (h;
milliequivalents per g)* divided by the total number of peptide bonds (htot).
100h
DH% 9
hTOT
The htot is also equal to the net concentration of amino acids (mmoles) per
gram of protein. The DH was determined using a pH-Stat instrument to
measure the volume of alkali required to maintain a static pH value during
proteolysis (V):
VN b
DH% 10
a MhTOT
where Nb is the concentration of the alkali and M the mass of protein
(Kjeldahl N 6 6.25). The term a (degree of dissociation of the ammonium
groups produced by proteolysis) is dependent on the sample temperature
and pH. During initial rate measurements with DH below 20%, the
following relations apply:

10pH pK
a 11
1 10pH pK

* One milliequivalent millimolar multiplied by the valence of the chemical species.

Tests for Protein Nutrient Value 373

and
298 T
pK 7:8 2400 12
298:T
Under standard conditions used for the approved test (pH 8, T 378C
and pK 6.91) we may suppose that a & 0.91. For a wide range of proteins
the typical value for htot is about 89 meq g 1 protein (122), that is, htot
1/F where F (&112 g mole 1) is the average formula weight for all 20
naturally occurring amino acids (see Chapter 1).

5.6. In Vitro Protein Digestibility with Dialysis

During in vitro protein digestibility tests the concentration of products
increases within an enclosed container. By contrast, digestion products are
continually removed from the digestive tract. To simulate the absorption
process, apparatus for protease digestion coupled with continuous dialysis
was devised by Baldwin et al. (3). Mauron et al. (123) used a rened version
of the dialysis apparatus to examine the PNV for condensed milk and milk
powder. A digestion unit with continuous dialysis was also used by Gauthier
et al. (124) to assess the effect of heat and alkali treatment on casein,
soybean, and rapeseed protein. The device consists of a dialysis bag
(containing enzyme, food sample, and a stirrer) suspended in a cylindrical
vessel containing buffer. The cylinder is tted with a tap so that dialysate
can be periodically withdrawn for analysis and replaced with an equal
volume of buffer. Undigested protein and enzyme are retained within the
dialysis bag. In later studies, dialysate was continuously removed, and
compensatory amounts of buffer were added using a peristaltic pump. Using
the digestion-dialysis method, in vitro protein digestibility is measured in
terms of the ND or the ``nitrogen digestibility index'' (125).
N dialysate
ND 6100 13
N dialysate N dialysis bag

where N(dialysate) and N(dialysis bag) are the amount of nitrogen

remaining in the dialysate and within the dialysis bag after 24 hours. The
main disadvantage of the dialyzedin vitro protein digestibility tests is their
slowness. The 1-kDa cutoff dialysis membrane impedes the release of
products. Kennedy et al. (126) described an in vitro protein digestibility test
employing pepsin followed by dialysis of reaction products. The system was
monitored by Kjeldahl or automated amino acid analysis. The results were
described as the pepsin digest dialysate (PDD) index. This and other
374 Chapter 12

attempts to correct amino acid scores for digestibility are discussed further
in Chapter 14.

REFERENCES

1. CH Lea, RS Hannan. Studies of the reaction between proteins and reducing

sugars in the ``dry state.'' III. Nature of the protein groups reacting. Biochim
Biophys Acta 5:433454, 1950.
2. M Tanaka, TC Lee, C Chichester. Effect of browning on chemical properties
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13
Effect of Processing on Protein
Nutrient Value

1. INTRODUCTION

Chemical and biological techniques for determining protein nutrient value

(PNV) are described in Chapter 12. The effect of food processing on PNV
for specic food commodities is discussed here. Up until 19931994 the
protein effeciency ratio (PER) assay was the reference method for assessing
protein quality (1). It has been superseded by a new measure of protein
quality called protein digestibilitycorrected amino acid score, PDCAAS
(Chapter 14). The PER remains important for comparative purposes and is
still widely used. The present discussion is broadly arranged according to
different food commodity groups.

2. MILK AND MILK POWDERS

Lea, Hannan and co-workers studied the effects of sterilization, drying, and
storage on milk protein quality over 50 years ago (25). Their ndings are
discussed throughout this section. Milk is subjected to various thermal
treatments before drying. A range of temperature-time treatments (728C/
15 sec, 958C/30 sec, 758C/30 min, or 808C/30 min) had no signicant effect
on the available lysine (6). Pasteurization (728C for 15 seconds), domestic

381
382 Chapter 13

boiling (30 seconds), or UHT treatment (1351508C for a few seconds) did
not reduce the PER or digestibility determined using the rat bioassay (7).
From the standpoint of protein quality, there is no nutritional reason for
discouraging heat processing of milk for human consumption. The total
lysine content of milk ranges from 6.2 to 11.1 g 100 g 1 (protein). Roller
drying milk that had been preprocessed by UHT treatment produced 2.5
6.4% reduction in available lysine (8). Scorching during roller drying
produced up to 35% loss of lysine (6), whereas storing dried milk for 6
months produced 15.4% decline in the available lysine (9).

2.1. Spray-Dried Delactose Milk

A high lactose content is undesirable in milk intended for adults. Intestinal
lactase levels are reduced in so-called hypolactasic populations, leading to a
reduced tolerance for dietary lactose. The acceptable oral dose of lactose is
thereby limited to 510 g or 100200 mL of milk. Hydrolysis of milk lactose
using exogenous lactase increases the tolerance of hypolactasic subjects for
milk. Such lactose-hydrolyzed milk has different processing requirements
due to its higher content of reducing sugars. The effect of heat processing on
the protein quality for delactase milk was investigated by Burvall et al. (10).
Normal milk samples were prepackaged in 1-L paper cartons and injected
with lter-sterilized lactase. Enzyme treatment reduced milk lactose levels by
99%. Normal milk and lactose-treated milk were UHT sterilized by steam
injection (1428C, 2 seconds) and evaporated to 25% dry weight and/or spray
dried (nal water content 4%; air inlet and outlet temperatures of 1858C and
858C). The total digestibility coefcient (TDC), biological value (BV), and
net protein utilization (NPU) were then measured.*
Spay drying reduced the BV for delactose milk by 45% compared with
10% for normal spray-dried milk powder (Fig. 1). The NPU was also
signicantly lower for delactosed spray-dried milk. Pre-evaporation allowed
more rapid drying and reduced the loss of quality. There was no visible
browning. Protein quality losses were due to the formation of early Maillard
reaction products. Hence supplementation with lysine improved the nutrient
value of delactosed spray-dried milk.

2.2. Storage Stability and Protein Nutrient Value

The effect of prolonged storage on the PNV of freeze-dried versus spray-
dried lactose-hydrolyzed milk powder was examined by Rawson and

* Measures of protein quality are dened in Chapter 12.

Effect of Processing on Protein Nutrient Value 383

FIGURE 1 Effect of processing on milk protein quality. Y-axis values are compared
with freeze-dried normal milk powder as control. X-axis abbreviations:
N-SD, spray-dried normal milk; LH SD, lactose-hydrolyzed and spray
dried; LH EV SD, lactose hydrolyzedevaporatedspray dried;
LH SD ( Lys), lactose hydrolyzed and spray dried with 1% (w/w)
lysine added. Insert shows TDC, total digestibility; BV, biological value;
NPU, net protein utilization determined with a rat bioassay. (Data from
Ref. 10.)

Mahoney (11,12). Freeze drying is a more expensive method for producing

dry milk. The dried milk powders were stored at 208C or 308C for up to
120 days. As shown in Table 1, spray drying had a more adverse effect on
the initial quality. However, PNV was the same for freeze-dried and spray-
dried milk powder samples after prolonged storage; both lost 4648% of
FDNP-reactive lysine after 120 days storage. The net protein ratio for both
samples also declined by nearly equal amounts. Owing to the lower
processing costs, spray drying may be advantageous for milk powders
intended for prolonged storage. Although Rawson and Mahoney (11,12)
found reactive lysine losses of 18% after spray drying delactose milk, others
have reported higher (50%) losses (10,13).

2.3. Effect of Water Activity, pH, and Temperature

Freeze-dried milk powder was equilibrated with saturated salt solutions of
known water activity (AW 0.110.62). The moisture-equilibrated samples
384 Chapter 13

TABLE 1 Effect of Processing and Storage on Spray-Dried or Freeze-Dried

Delactosed Milk

FDNB-Lys DBCb Total Lys Rat assay

a
Sample (mg g 1) (mg g 1) (mg g 1) NPR

Normal milk (fresh) 28 218 29.1 4.8

Freeze-dried 25 178 28.9
Freeze-dried, 120 d, 208C 25 200 29.1
Freeze-dried, 120 d, 308C 12 120 15.7 3.0
Spray-dried 23 195 23.4
Spray-dried, 120 d, 208C 23 235 23.6 4.0
Spray-dried, 120 d, 308C 17 150 17.8 3.2
a
Processing and storage conditions. bDye-binding capacity determined using Remazo Brilliant
Blue R as described in Ref. 11.

were then stored for 112 months. Protein quality was assessed using the rat
bioassays. Available lysine was assayed using the o-methylisourea method
(13). The loss of available lysine was greater for lactose-hydrolyzed milk
powder compared with normal milk powder. Chemical and biological assays
of available lysine were highly correlated. There was a bell-shaped
relationship between AW and quality loss. Maximum losses of PNV
occurred at AW 0.55. These observations agree with results for the dry
caseinate-glucose system (3). Lea and Hannan (3) investigated the reaction
between dry sodium caseinate and glucose at temperatures of 0708C and
AW < 0.95. The system was adjusted to pH 310 before drying. The decline
in free amino groups after 65 days storage at 378C, followed a bell-shaped
curve with a maximum at AW 0.60.8. At low AW the reaction rate may be
limited by the restricted diffusion of reagents. At high AW reactant dilution
or inhibition by water accounts for the decreased rate of deterioration. The
loss of lysine increased steadily from pH 3 to pH 8 and leveled off at pH 8
10. The effect of temperature (AW 0.75) on the kinetics of lysine loss
conformed with the simple Arrhenius equation ln(k) 14446.7(1/T) 38.0
(r 0.9999), where k is the percent loss of lysine per second and
T temperature in kelvin (Fig. 2). Alternative models involving nonlinear
temperature dependence are described in Chapter 14.

3. INFANT FORMULAS

Mitchell and Grundel (14) compared several chemical methods (total lysine
determination by amino acid analysis, differential dye-binding capacity, in
Effect of Processing on Protein Nutrient Value 385

FIGURE 2 Arrhenius plot showing the effect of temperature on the rate of Lys loss
for casein-glucose mixture at 70% RH. The temperature range studied
was 0708C. Reaction pH (not given) is probably pH 6.3 or near the
natural pH for dried milk. (Drawn from data in Refs. 35.)

vitro protein digestibility) and bioassays (TPD, PER) for evaluating protein
quality for ve infant formulas.* These protein quality indices were
evaluated for their sensitivity to ``humidity-related'' damage. For all ve
infant formulas, in vitro protein digestibility was said to give a poor estimate
of TPD. For example, the in vitro digestibility was was 8889% for soy-
based infant formulas compared with the TPD of 9194%. For milk-based
formulas, the in vitro protein digestibility was 8588% compared with a
preadjusted TPD of 8588%; the latter TPD value was adjusted to 9697%
to allow for lactose intolerance in rats. Apparently there is a digestibility
decit of 11% when rats are fed diets with lactose levels comparable to the
concentration of lactose in infant formulas. The dDBC values agreed with
total lysine results. The rat bioassay showed no protein quality changes for
30-day-old infant formulas; however, there were large drops for in vitro

* The designation and composition of the ve commercial infant formulas were: milk-based I
(skimmed milk, reduced minerals, whey, lactose, oleo, coconut, soy, oleic oils, and lecithin),
milk-based II (skimmed milk, lactose, coconut and corn oils), milk-based III (skimmed milk,
lactose, soy and coconut oils), soy-based I (soy protein isolate, corn syrup solids, coconut and
corn oils), and soy-based II (soy protein isolate, soy oil, lecithin, sucrose, tapioca dextrin).
386 Chapter 13

protein digestibility and dDBC. Mitchell and Grundel (14) concluded that
(a) agreement among the various assays is poor although different in vitro
methods show qualitative similarities in some cases, (b) changes in dDBC
and browning of the stored milk-based and soy-based formulas do not
reect changes in relative PER, (c) in vitro methods are not accurate
predictors of protein quality for the rat, and (d) only certain humidity-
related protein nutritional damage in infant formulas could be predicted by
monitoring dDBC or in vitro protein digestibility (14). An interesting
feature of this study is the assumption that lysine is the limiting amino acid
for soy-based infant formulas (see later).
Sarwar et al. (15) evaluated the impact of amino acid supplementation
on protein quality for four commercial soy-based infant formulas. The
samples were fed to 2-week-old weanling rats as the sole source of protein.
Each diet contained net 8% protein, 20% fat, and adequate amounts of
minerals and vitamins. The RPER and RNPR for casein plus methionine
were assigned a value of 100. By comparison, the RPER and RNPR values
for the infant formulas were 7181 and 7885, respectively. There was no
improvement in RPER or RNPR values when diets were supplemented with
either lysine (0.2%), methionine (0.2%), threonine (0.1%), or tryptophan
(0.05%). Increased levels of amino acids appeared in the serum, indicating
efcient adsorption. Protein quality improved following the supplementa-
tion with four essential amino acids (RPER or RNPR values 100).
Apparently several amino acids are colimiting for soy-based formulas.
Measuring available lysine is a useful way to monitor protein quality where
it has been demonstated that lysine is the (only) limiting amino acid.
Otherwise, focusing on a single amino acid will lead to errors of the type
described in the preceding paragraph. Processing losses and shelf-life studies
for infant formulas have also been reported by other investigators (16,17).
Under optimal conditions, infant formulas can be stored for prolonged
periods (1620 weeks) without losses of available lysine. Poor storage
conditions can lead to a 2030% loss of available lysine.

4. FEEDSTUFFS AND CONCENTRATES FOR LIVESTOCK

Protein concentrates and dietary supplements for livestock are derived from
both animal and plant sources (Table 2). The commercial value for feed
concentrates depends on their total nitrogen content as determined by the
Kjeldahl analysis. Differences in feed protein quality arise due to (a)
intrinsic differences in the quality of raw materials and (b) effects of
processing. Heat processing renders feed material more portable and
palatable. It also improves the chemical and microbiological stability of
Effect of Processing on Protein Nutrient Value 387

TABLE 2 Protein Quality Testing of Feedstuffs and Concentrates (19551960)

Feedstuffs Meat and bone meals

Procedures (all samples)
Procedures (selected samples)
Fish meal
Whale meal
Dried whale solubles
Dried skimmed milk
Soya bean meals
Groundnut meal
Cottonseed meal
Sunower seed meal
Gross protein value (GPV)
Net protein utilization (NPU)
Protein quality index (PQI)
Nitrogen solubility (NS)
Dye-binding capacity (Orange G)
Reactive Lys (FDNB method)
Total Lys (Lueconostoc mesenteriodes assay)
Protein quality test by
Tetrahymena pyriformis
Streptococcus faecalis
Streptococcus zymogens
Total protein (Kjeldahl method)
Biological value (BV)
Net protein ratio (NPR)
Sulfur amino acids
Total free amino groups

feedstuffs. Thermal treatment eliminates antinutritional factors, bacteria,

toxins, and viruses. Excessive heating can lead to a reduction in PNV. An
optimal heat treatment ensures microbiological safety while minimizing the
loss of PNV.
Techniques for testing feed protein quality should provide results that
agree with bioassay data. Proposed methods should also be quick,
inexpensive, and reliable. The Agricultural Research Council (UK)
organized a collaborative study of chemical methods for determining
PNV of animal feeds (18). Participants were companies and research
institutes involved in the production and utilization of protein concen-
388 Chapter 13

trates.* A range of chemical methods were compared with the chick and rat
bioassays for protein quality. Techniques of interest had to be simple
enough to be performed in the average analytical laboratory. They also had
to be rapid, allowing (a) on-line monitoring of quality during feedstuff
production and (b) quality assurance at storage or shipment depots.

4.1. Tests for Feed Protein Quality

Table 2 lists some of the chemical methods for assessing feed protein quality
examined in the ARC study. The tests were compared with a chick bioassay
for gross protein value (GPV) and the rat bioassay for NPU. The test
samples were protein feedstuffs commercially important for feeding
nonruminant livestock. To foster cooperation from participants, the names
of feed manufacturers and sources of feed materials were not recorded.
About 130 samples covering seven classes of feedstuffs were investigated.
Key ndings of this study were that (a) rankings of feed protein quality
based on the GPV and NPU tests did not agree with each other except in the
case of whale meat meal; (b) when all samples taken together there was no
correlation between GPV and the crude protein, nitrogen solubility, or
Orange G binding; and (c) no chemical method was positively correlated
with feeding trials for all protein concentrates.
However, available lysine showed a positive correlation with the GPV
test for meat and bone meals (R 0.82), sh meal (R 0.96), and whale
meat meal (0.94). Orange G binding was a useful index for quality, being
correlated with available lysine. With oilseed protein concentrates, GPV was
correlated with the nitrogen solubility index. No chemical test showed any
correlation with NPU or GP. The presence of antinutritional factors
(trypsin inhibitors in groundnuts and soybean, gossypol in cottonseed) may
be partly to blame, as was bacterial contamination of feedstuffs. In applying
PNV testing to practical situations, the ARC group referred to many issues
summarized in Table 2 of Chapter 12. For example, protein meals are
usually fed as compound rations containing different protein types.
Livestock fed with these materials differed in terms of their variety, strain,

* The participants include J. Bibby & Sons Ltd., Liverpool (UK); Bovril Ltd., London (UK);
Croseld & Calthrop Ltd., Liverpool (UK); Department of the Government Chemist, London
(UK); Glaxo Laboratories Ltd., Greenford (UK); National Institute for Medical Research
(UK); National Institute for Research in Dairying, Shineld (UK); Rowett Research Institute,
Aberdeen (UK); Tory Research Station, Aberdeen (UK); Unilever Ltd., Shanbrook (UK);
University of Cambridge School of Agriculture (UK); U.S. Department of Agriculture
Southern Utilization Research Division, New Orleans; Walton Oaks Experimental Station,
Vitamins Ltd., Tadworth (UK).
Effect of Processing on Protein Nutrient Value 389

and age; older pigs or chickens were less sensitive to low-quality protein.
Chemical methods for assessing the quality of protein concentrates/feeds
were also assessed by Bunyan and Price (19). The meat meal (26 batches),
whale meat meal (16 batches), and sh meal (13 batches) samples were
similar to those described for the ARC collaborative study. However, this
study considered only animal-derived protein concentrates. Results for each
class of protein concentrate were considered separately (Table 3).
Orange G binding capacity was a useful quality indicator for most
protein concentrates. FDNB-reactive lysine was not correlated with
bioassay results for samples other than whale meat meal. Three meat
meal samples with unusually high crude protein content (N 6 6.25) had
somewhat anomalous properties. For a crude protein content > 79% the
true protein value was only 2128%. At least one of the samples appeared to
be adulterated with feather meal judging from the low methionine content
and high amounts of gelatin. The NPR value was 0.42.2 for most meat
meals with a value of 1.6 signifying weight loss during the rat bioassay.

4.2. Orange G Binding

Soybean and sh meal samples were heated in steel pans or autoclaved at
1208C (20). The DBC was positively correlated with PER determined using

TABLE 3 Summary Characteristics of Concentrates and Feedstuffs

Meat meal Whale meat meal Fish meal

Sample and characteristics (n 26) (n 16) (n 13)

Crude protein (%) 4090 2595 6073

NPU (%) 938 1762 2266
TDC (%) 7090 7588 7490
BV (%) 3469 2780
Total Lys (g 16 g 1 N) 5.47.8
Reactive Lys (g 16 g 1 N) 3.04.6 4.07.3 3.46.9
Methionine (g 16 g 1) 0.92.6 1.62.6 1.32.5
Correlations identied a
Dye binding (mg g 1)
1. Crude protein (Y1) Y1 0.278Db 30 Y1 0.261Db 30 0.325
Db 24
2. True protein (Y2) Y2 0.239Db 27 Y2 0.287D 17
3. NPU (Y3) Y3 3.86Db0.43 Y3 6.37Db19.8
Reactive Lys (X1) None BV 7.52X1 4.0 None
a
Y1 crude protein, Y2 true protein, Y3 NPU, Db amount of dye bound (acid equivalents
of dye bound/104 grams of protein), X1 reactive Lys.
390 Chapter 13

a chick bioassay. Signicant decreases in DBC occurred within the rst 15

minutes of heat treatment. The quality of raw soybean meal was lower than
expected from its DBC, probably due to the presence of protease inhibitors
in the sample. The overall impression is that the measurement of DBC is a
useful for monitoring heat process damage. Despite this, the DBC for heat-
treated sh meal was high even though samples showed low levels of
cysteine and low PNV; lysine does not appear to be the limiting essential
amino acid in sh meal. The results from Choppe and Kratzer (21) also
show that Orange G binding is positively correlated with protein quality
results from the chick growth assay.

4.3. Acid Orange 12 Binding

The thermal processing damage suffered by sh meal, groundnut meal,
blood meal, freeze-dried beef steak, and lactalbumin was evaluated using
Acid Orange 12 binding by Hurrell and Carpenter (22). The DBC was also
determined for Remazol Brilliant Blue and Cresol Red. The results were
compared FDNP-reactive lysine and total basic amino acids. With severely
heated samples (1218C,  28 hours), Acid Orange 12 binding capacity
agreed closely with the concentration of histidine arginine reactive lysine
(or the HARL value). Proteins that were subjected to mild heat treatment
(378C for  30 days) and/or experienced early Maillard reactions exhibited
little change in DBC even after a 90% decline in available lysine. The early
Maillard products were dye reactive.
Carpenter and Opstvedt (23) collated results from a collaborative
study of eight commercial sh (capelin) meals. Protein quality was evaluated
from the measurement of Acid Orange 12 binding, chick bioassay and
FDNB-reactive lysine. Interlaboratory variations in results were generally as
large as differences between samples. Outlying results were identied and
those samples reanalyzed. A correlation matrix was established indicating a
to strong relationship between the indices of quality. Much of the variation
between samples was dependent on one sample. Omitting this sample led to
the collapse of all correlation below signicant levels. The numbers of
samples used in this trial should, perhaps, have been larger.
The effect of commercial processing and laboratory heat treatment on
126 rapeseed meal samples was evaluated by Goh et al. (2426). Heat
damage was measured via Acid Orange 12 binding and as FDNB-reactive
lysine. The DBC was 124.4 mg g 1 (sample) or 349356 mg g 1 (cP) for
unheated rapeseed meal. The lysine content for unheated Brassica seed
protein is about 5.45 (+ 0.53) g per 100 gram (27). Autoclaving produced
changes in the DBC at 45120 minutes of heating. The concentration
Effect of Processing on Protein Nutrient Value 391

FIGURE 3 Effect of autoclaving on rapeseed meal protein quality assessed from

Acid Orange 12 binding, FDNB-reactive lysine, and total levels. (Drawn
using data in Refs. 2426.)

of FDNB-reactive lysine declined steadily over the entire heating time

(Fig. 3).
The mismatch between DBC and available lysine values occurs
because DBC is dependent on the total basic amino acid concentration. I
have performed multiple regression analysis of DBC versus the concentra-
tion of lysine, histidine, and arginine yielding the following relations:
DBC 26 +2:3 Lys 203 +11:8 1

DBC 1 +0:067 TBAA 2

DBC 17 +4:7 Lys 74 +37 His 5 +6:4 Arg

30 +80 3

These relations were generated from data in Table 5 of Ref. 26. The ranges
of values used were 2.545.9 g (lysine) 100 g 1 protein, 2.432.74 g (histidine)
100 g 1 protein, and 2.985.65 g (arginine) 100 g 1 protein. The total basic
amino acid range is therefore 7.9514.3 g per 100 g 1 protein. The regression
coefcients for Eq. (1), Eq. (2), and Eq. (3) were 0.9201, 0.9533, and 0.9532,
respectively. The nonzero intercept from Eq. (1) suggests that Acid Orange
12 binds to sites other than lysine. Eq. (2) conrms that the DBC is strongly
392 Chapter 13

correlated with TBAA and that Acid Orange 12 binds no sites other than the
three basic amino acids. Eq. (3) conrms the previous results. The coefcient
for arginine, histidine, and lysine shows their relative contributions to DBC.
Apparently, the order of dye binding to basic amino acids is histidine
> lysine 4 arginine. Nevertheless, heating produced the greatest decline in
lysine with histidine being the least heat susceptible.
The extent of heat damage to rapeseed meal is also a function of the
sample moisture content (Fig. 4). The DBC was reduced to a greater extent
by heating rapeseed meal at 1020% moisture as compared with 2% or 40%
moisture. Reactions producing quality loss are limited by lack of reagent
mobility in low-moisture systems and by dilution effects in high-moisture
systems. Changes in available lysine show a more complex dependence on
moisture levels. With prolonged heating, losses in available lysine were
higher for samples having 2% moisture as compared with 40%.
The assessment protein heat damage from DBC is described further by
Hook (28,29), Peal et al. (30), Randall et al. (31), and also by Kratzer et al.
(32). Protein samples investigated include wheat, soybean, defatted milk,
whole egg, and whole blood proteins. In general, changes in DBC tended to

FIGURE 4 Effect of sample moisture content and autoclaving time on the Acid
Orange 12 binding capacity for rapeseed meal.
Effect of Processing on Protein Nutrient Value 393

lag behind PER. Quality loss was strongly correlated with decreases in
available lysine. Lin and Lakin (33) reported disparities between the DBC
and FDNB-reactive lysine for heated soy meal samples. Steaming soy meal
at atmospheric pressure led to the progressive loss of the nitrogen solubility
index (NSI) due to protein denaturation and insolubilization by covalent
(sulfur-disulde exchange) and noncovalent aggregation. Urease activity
decreased because of enzyme inactivation after 6080 minutes of heating. In
vitro protein digestibility increased, probably due to the inactivation of
soybean trypsin inhibitors. The level of unreactive lysine increased gradually
from 0.14 g (lysine) g 1 protein and leveled off at 0.26 g (lysine) g 1 protein
after heating of soybean meal for 120 minutes. Assuming an initial lysine
content of 6.3 g per 100 g, then 9598% of lysine residues remained available
after heating. Steam treatment led to an increase in DBC, probably due to
heat denaturation of soy protein.

4.4. Hot WaterSoluble Protein and Quality

Meat and bone meal samples had high levels of gelatin measured as the
amount of hot watersoluble protein. There was a considerable difference in
the PNV for individual samples owing to their varied thermal history. For
20 different samples, Choppe and Kratzer (21) found a strong (negative)
correlation between the amount of hot watersoluble bone meal gelatin and
PNV. El (34) suggested a regression equation for predicting PER values for
meat or sh based on the collagen content. Calculated PER values agreed
closely with experimental values for sardine, lamb, bovine liver, chicken
meat, or beef sausages. Collagen content may provide a rapid and
inexpensive assay for estimating PNV for meat.

5. LEGUMES AND OILSEEDS

The structure and characteristics of legume proteins and effect of processing

on their quality were reviewed by Vanderstoep (35), Chang and Satterlee
(36), Sathe et al. (37), Friedman (38), and de Lumen and Uchimiya (39).
Common processing operations for legumes include baking/roasting,
dehulling, cooking, canning, extrusion cooking, fermentation, germination,
and hydrothermal treatment. A list of some legumes for which the protein
quality has been investigated over the last decade is given in Table 4. Most
of these processes improve the quality of legume proteins through the
reduction of protein antinutritional factors (trypsin inhibitors, hemagglu-
tinins/lectins) and chemical antinutrients (oxalates, phytic acid, and
tannins).
394 Chapter 13

TABLE 4 Determination of Protein Nutritional Value for Legumes

Commodity References

Acacia farnesiana, Cercidium Ortega-Nieblas et al. (40)

microphyllium, Cercidium sonorae,
Mimosa grahamii, Olneya tesota,
Parkinsonia aculeata, and Prosopis
juliora.
African locust bean Kapu et al. (41)
Alfalfa protein concentrate Hernandez et al. (42)
Bauhinia purpurea L Karuppanan et al. (43)
Canavalia brasiliensis Oliveira et al. (44)
Egyptian legumes; faba beans, lentils Youssef and Abdel-Gawad (45)
(Lens culinaris), common beans
(Phaseolus vulgaris), cowpea, and
soybeans (Glycine max L.).
Lupin varieties; Lupinus polyphyllus Aniszewski (46), Egana et al. (47)
Lindl var., L. angustifolius, Lupinus
albus cv. Multolupa
Mung beans (Phaseolus aureus), black Khalil and Khan (48)
gram, and wild beans (Vigna
sublobata)
Pea protein Wang et al. (49)
Pigeonpea (Cajanus cajan) Singh et al. (50)
Velvet bean (Mucuna pruriens L.) Permal et al. (51)
Soybean varieties Vibha and Simlot (52)
Sudanese legumes: lupin (Lupinus Ahmed and Nour (53)
terminis), pigeon pea (Cajanus cajan),
two types of cowpea (Vigna sinensis
and Vigna unguiculata), bonavist bean
(Dolichos lablab), faba bean (Vicia
faba), and soybean

5.1. Available Lysine

Reductions in legume available lysine levels in occur during domestic
cooking with little nutritional consequence. Lysine is not normally the
limiting essential amino acid in legumes; methionine is. Heating also
produces benecial reductions in antinutritional factors. Available lysine
levels from dhal (split pulses) of ve legumes (Lens esculenta, Phaseolus
mungo, Lathyrus sativus, Cicer arietinum, and Pisum sativum) was 5.55
9.12 g 100 g 1 (54). Cooking each legume for 30 minutes produced a
maximum of 25% reduction in available lysine (range 4.216.79 g 100 g 1).
Effect of Processing on Protein Nutrient Value 395

Pressure cooking for 10 minutes produced a 35% reduction in available

lysine (3.895.87 g 100 g 1). A similar study involving ve types of beans
(white kidney beans, black eye beans, crab eye beans, butter beans, red
kidney beans) found available lysine levels of 6.34 (+ 0.11) g 16 g N 1 in the
raw beans (55). Pressure cooking (30 minutes at 15 lb in 2) produced a mean
available lysine of 5.42 (+ 0.36) g 16 g N 1, a reduction of about 14.5%
compared with the raw beans. The available lysine values were 5.48
(+ 0.48) g 16 g N 1 and 4.78 (+0.16) g 16 g N 1 after cooking for 2 and 8
hours. These changes did not produce net declines in PNV according to rat
bioassays.

5.2. Steaming and Hydrothermal Treatment

Subjecting whole rapeseed to steam treatment for 10 minutes inactivated the
enzyme myrosinase, decomposed glucosinolates, and improved protein
quality (56). The benets of hydrothermal treatment are also observed with
other legumes. Steam treating beans (Phaseolus vulgaris L) at 102, 119, and
1368C resulted in a loss of lectin and trypsin inhibitor activity. There was
also a decline in the available lysine (57). Inactivation of trypsin inhibitor
involved biphasic rst-order kinetics. Effects of steaming temperature on
rate constants followed Arrhenius-type relations. Both total lysine and
available lysine were reduced to differing extents. Steam treatment at 1198C
for 5 or 10 minutes was proposed as a compromise for inactivating
antinutritional factors while avoiding high degrees of protein damage as
measured by total and available lysine.

5.3. Soaking, Dehulling, and Cooking

Dehulling before cooking leads to greater improvements in protein quality
as compared with cooking alone. The effects of dehulling, sprouting, and/or
steam cooking on rice beans and mung beans were evaluated by Mehta, et
al. (58). As well as the in vitro protein digestibility, they measured proximate
composition, sugars, starch, and trace minerals for the raw and processed
beans. The maximum protein content was 23.5% for rice beans and 26.5%
for mung beans. After a combination of sprouting, dehulling, and cooking,
in vitro protein digestibility increased by 35% for rice beans and 30.8% for
mung beans. Soaking a range of legume [soybean, lupin, bean (Phaseolus
vulgaris)] seeds in 0.5% sodium bicarbonate reduced the level of antinutri-
tional factors (phytic acid, tannin, trypsin inhibitor, and hemagglutinin
activity). Interestingly, protein extractability (in distilled water, NaCl, or
sucrose solutions) was also reduced although in vitro digestibility and
available lysine were improved (59).
396 Chapter 13

Seeds of perilla (Perilla frutescens Linn, Britton) have concentrations

of essential amino acids above FAO/WHO/UNU recommendations for
infants. Lysine was limiting. Longvah and Deosthale (60) found that
dehulling and cooking perilla seeds further increased the net protein ratio
(NPR), NPU, and TPD as determined by a rat bioassay although values
were lower than for a casein-based control diet. Therefore, perilla seed
represents a good source of protein for human and animal nutrition,
particularly after dehulling and cooking. In conclusion, legume protein
quality can be improved by a number of inexpensive processing methods.
These studies show that weaning foods and food supplements may be
produced from locally available foodstuffs.

5.4. Roasting and Irradiation

Roasting and malting led to signicant improvements in the protein quality
for a range of local legumes and cereals examined by Gupta and Sehgal
(61), Dahiya and Kapoor (62), and Gahlawat and Sehgal (63). Plahar et al.
(64) evaluated the effect of roasting (preceded by dehulling) on four
varieties of cowpea (Vigna unguiculata). Dehulling reduced the tannin
content by up to 98%. With cowpea having a highly pigmented coat,
dehulling improved the protein quality. Roasting signicantly improved
digestibility and more than doubled the PER. It was suggested that dehulled
and roasted cowpea may be useful as a protein supplement in cereal-based
weaning foods. The benecial effects of roasting have also been conrmed
with chickpeas (Cicer arietinum) and peanut (Arachis hypogaea), where
cooking led to a decrease in the trypsin inhibitor activity and an increase in
the true digestibility, relative nitrogen utilization, and PER values. Hira and
Chopra (65) found that roasting chickpea or peanuts decreased the trypsin
inhibitory activity, available lysine, and BV (P < 0.05). However, the true
digestibility, relative nitrogen utilization, and PER values were all
improved.
Microwave heating presoaked legume seeds (faba beans, peas,
chickpeas, soybeans, lentils, common beans) reduced levels of protein
antinutritional factors (hemagglutinins and inhibitors) comparable with
conventional cooking. In consequence, PER values were increased.
Microwave processing of dried seeds had less effect. Antinutrients from
common bean were destroyed by microwave heating (66). These effects are
in agreement with more general ones showing that effects of both industrial
and domestic microwave cooking on nutritional characteristics including
proteins are comparable to those of conventional cooking (67,68).
Effect of Processing on Protein Nutrient Value 397

5.5. Multiple ProcessingWeaning Foods

Weaning foods are produced from mixtures of cereal and legumes (and
occasionally milk). The multiple protein sources provide complementary
supplies of essential amino acids cysteine/methionine and lysine. A recent
emphasis is on the production of low-cost weaning foods using materials
locally available in developing countries. Gupta and Sehgal (61), Dahiya
and Kapoor (62), Gahlawat and Sehgal (63) from Haryana University
(Hisar State, India) describe a number of weaning food formulations based
on local cereals and legumes including wheat, pearl millet (bajra), Bengal
gram, green gram (mung beans), groundnuts, peal millet, rice, kangini
(Setaria italica), and sanwak (Echinochloa frumentacea).
Formulations containing two or three components were generally
subjected to a range of processing techniques including sprouting, roasting,
and malting. For commodities such as bajra (peal millet), barley, green
gram, amaranth grain (Amaranthus sp.), and jaggery, malting and/or
roasting led to protein quality indices comparable to those for a commercial
weaning food; PER 2.042.13, BV 79.5680.68, NPU 66.7567.86,
NPR 2.132.76, and PRE (protein retention efciency) 34.1844.18.
Dahiya and Kapoor (62) produced food supplements for preschool children
using malted and/or roasted bajra, Bengal gram, green gram (mung beans),
groundnuts, jaggery, or amaranth leaves. Bajra-based food supplements had
quality indices (PER, food efciency ratio, BV, NPU, NPR, and PER)
signicantly higher than those of wheat-based supplements (P < 0.05). The
authors suggest that the quality of their formulations was equal to that of
Cerelac2, a commercial supplement. However, PNV was found to be lower
(P < 0.05) than the value for casein (standard protein). Rats fed on bajra-
based supplements showed an excellent growth pattern throughout the
feeding trial.
Santos et al. (69) prepared extruded weaning foods using a mixture of
rice, mung bean, and milk (70:25:5). Protein quality was determined by a rat
bioassay. The quality of the weaning food was signicantly improved if rice
and mung bean were extruded rst before milk was added. The optimal PER
was 2.25, which is comparable to the growth-promoting effect of casein.
Extrusion cooking the complete mixture led to a PER value of 1.93.
Supplementation with lysine increased the PER value to 2.10. Obviously,
extrusion cooking destroyed some lysine. Fermentation and supplementa-
tion of a traditional Ghanaian cornmeal weaning food with soybean meal
improved its protein quality (70). Studies also suggest that roasted cowpea,
widely grown in the Sahel, may be suitable as a weaning food supplement
(64,71).
398 Chapter 13

Mahgoub (72) produced ve weaning formulations comprising 8.5%

skim milk powder and/or sorghum, groundnuts (peanuts), sesame seeds,
and chickpeas (various concentrations) also along with 5% sugar and a
vitamin and mineral mixture. The formulations were processed by a twin-
roller drum dryer. Protein digestibility and available lysine PER, NPR, and
NPU were determined for the different formulas. Formulation F3 (60%
sorghum, 20% chickpeas, 5% sesame, 8.5% skim milk powder, 5% sugar,
and 1.5% vitamins minerals) and F2 (55% sorghum, 15% chickpeas, 5%
groundnuts, 10% sesame, 8.5% skim milk powder, 5% sugar, and 1.5%
vitamins minerals) had compositions and properties comparable to those
of Cerelac. In summary, cosupplementation using different protein sources
is a key feature of weaning formulations. The choice and order of processing
applied are also important, with some forms of processing (sprouting,
fermentation, malting) sometimes able to compensate for others (autoclav-
ing) (73).

6. CEREAL AND CEREAL PRODUCTS

Cereal products (bread, biscuits, cooked rice, noodles, pasta, etc.) are part
of the staple diet in much of North and South America, North Africa, and
Asia. Cereals also provide proteins indirectly when used as animal
feedstuffs. We now consider the nutritional quality of cereal proteins.
Bread, biscuit, and pasta making quality etc. are not discussed. The reader
should refer to the following reviews for information on protein functional
quality (7476).
The effect of extrusion cooking on protein quality was reviewed by
Bjoerck and Asp (77), Cheftel (78), and Mercier (79). Salunkhe et al. (80)
reviewed the nutritional quality of cereal proteins in general. Lorenz (81)
considered the effect of sprouting on cereal protein quality. Dixon-Philip
(82) discussed the consequences of milling, baking, extrusion, hydrothermal
processing, and fermentation on the quality of cereal products. Examples of
processing effects on PNV in cereal-based products are described here.

6.1. Amaranth
Protein nutrient value for amaranth grain (Amaranthus cruentus) was
determined using a nitrogen balance study with 12 adult men over three
periods of 9 days each (83). There was no signicant difference in
digestibility of popped and extruded amaranth. The nitrogen balance index
(NBI) was 0.97, 0.98, and 0.96 for cheese, extruded amaranth, and popped
Effect of Processing on Protein Nutrient Value 399

amaranth, respectively. These results indicate that amaranth is a good

source of high-quality protein.

6.2. Maize
Bressani et al. (84) examined the effect of processing maize into tortillas on
their nutritional characteristics. Eleven ordinary maize cultivars and one
variety of quality-protein maize (QPM) called `Nutricta' were processed into
cooked maize or tortillas according to methods used in rural Guatemala.
Protein quality was signicantly higher (P < 0.03) in tortillas than in raw
maize. The QPM cultivar had superior PNV both as raw grain and as
tortilla. Gupta (85) reported increased protein quality for maize after
sprouting provided that radicals and plumules were removed from corn
kernels. The true digestibility was unaffected by sprouting although BV,
NPU, and utilizable protein increased. Gupta and Eggum (86) developed a
process for transforming the by-product from corn oil production into a
food-grade protein meal. Commercial oil cake was extracted with hexane
and 80% ethanol and then sieved to remove undesirable materials. The
defatted maize germ oil cake had 24.7% protein. Albumin, globulin, and
zein decreased while glutelin and residue protein fraction increased. The
meal protein had higher levels of lysine and tryptophan than whole maize
grain. Protein digestibility and BV were improved as compared with the
starting material.

6.3. Rice, Millet-Sorghum

Eggum and Juliano (87) found that rice protein quality was not adversely
affected by simple cooking or parboiling. Extrusion cooking led to adverse
effects on protein quality. Lysine is the limiting essential amino acid in millet
(88). The digestibility of all cultivars was high (Table 5). Autoclaving led to
a 1925% decrease in the digestibility and an overall increase in BV.
Fortication with lysine led to greater increases in BV as compared with the
effect of heating. Geervani (89) reviewed effects of processing on protein
quality in millet sorghum and other cereals important for developing
countries. Changes in amino acid composition and protein quality
characteristics for millet (as well as barley, oats, wheat, rye, and maize)
before and after boiling are discussed. Dry heat processing (e.g., as used for
baking biscuits and bread), frying, fermentation (especially sorghum and
millet products), and germination are also discussed. Pawar et al. (90)
obtained a PER increase from 2.14 to 2.32 for pearl millet by soaking in
0.2 N HCl for 15 hours. Cooking for 20 minutes led to further improvements
400 Chapter 13

TABLE 5 Protein Quality in Millet Cultivars

Index Value

Protein (%) 10.7817.3

Lysine (g 16 g 1 N) 0.9951.39
True digestibility (%) 9599.3
Biological value (%) 48.356.5
Range of samples: Italian millet (Setaria italica), French millet
(Panicum miliaceum), barnyard millet (Echinochloa colona),
Kodo millet (Paspalum scrobiculatum), and Little millet
(P. miliare) were investigated.
Source: Summarized from Ref. 88.

in protein quality. The TPD, BV, NPU, and utilizable protein were also
increased for soaked and/or scaried pearl millet.

6.4. Storage and Insect Infestation

Insect infestation produced a signicant decline in protein quality in wheat
grain. Jood and Kapoor (91) examined the effect on wheat grains of 25, 50,
and 75% infestation by mixed populations of Trogoderma granarium Everts
and Rhizopertha dominica Fabricius. Changes in PNV were evaluated using
the rat PER assay. With a diet containing insect-infested wheat grain (at 50
and 75% infestation) there was a decrease in food intake, body weight gain,
PER, nitrogen absorption, BV, NPU, and dry matter digestibility. Virtually
all protein nutrient quality parameters showed a negative association with
infestation levels. Below 25% grain infestation, protein quality was not
affected signicantly.
The mechanism by which protein quality is reduced by insect
infestation is uncertain. Infestation may elicit physiological changes
associated with plant defense (92). Infested seed samples have increased
levels of polyphenols, protease inhibitors, and other plant defense
metabolites. Infestation of wheat, maize, and sorghum produced signicant
decreases in amino acid scores for all essential amino acids. Levels of
nonessential amino acids were also reduced. Large reductions were found in
methionine, isoleucine, and lysine concentrations for infested wheat, maize,
and sorghum grains, respectively. Insect infestation did not change the order
of rst (lysine) and second (isoleucine) most limiting amino acids.
Effect of Processing on Protein Nutrient Value 401

7. IMPROVING CEREAL PROTEIN QUALITY BY

SCREENING

Much effort is being devoted to breeding high-lysine cereal varieties.

Developments in this eld are discussed by Johnson et al. (93), Whitehouse
(94), Bressani (95), Mertz (96), and also de Lumen and Uchimiya (97). The
breeding programs require methods for the rapid identication of high-
lysine seeds. Suitable methods should enable the evaluation of hundreds of
seed varieties quickly and cheaply. Many investigators used dye-binding
assays to detect high-lysine cereal cultivars. A high correlation is reported
between DBC and lysine concentration determined by amino acid analysis.
The correlation between lysine content and DBC is signicantly improved
by normalizing results for Kjeldahl protein (98100). Example of breeding
programs evaluated using dye binding are listed in Table 6.
There is generally a negative correlation between lysine content and
other grain quality indices. Grain yield, size, and crude protein content

TABLE 6 Identication of High-Lysine Cereal Cultivars Using Dye Binding

Cereal References Country

Barley Doll et al. (98) Denmark

Barley Bhatty and Wu (104), Canada
LaBerge et al. (105),
LaBerge et al. (106)
Barley Lekes and Rozkosna Czechoslovakia
(107)
Barley Saastamoinen (108) Finland
Barley Bansal et al. (101) India
Barley, eld beans, and Gullord (99) Norway
wheat
Oats Young et al. (109) USA
Peal millet Rabson et al. (110) USA
Rice Kaul et al. (111,112) India
Rice Juliano et al. (113) Philippines
Rice and maize Le Thi Xuan et al. (114) Vietnam
Rice Chutima et al. (115) Thailand
Sorghum Jambunathan et al. (116) India
Triticale Knoblauch (117) USA
Wheat Mossberg (118) Sweden
Wheat Sharma and Kaul (119) Germany
Wheat Iqbal-Khan (120) Pakistan
402 Chapter 13

decreased as the lysine content increased (98,101). Chatterjee and Abrol

(102) reported that high-lysine barley varieties were less resistant to damage.
Increased lysine content is probably a consequence of the increased
synthesis of water-soluble protein (albumin and globulins) at the expense
of less soluble proteins (prolamins and glutelins). The soluble protein
fractions have a high total basic amino acid content. This increases the
avarage protein hydrophilicity, increases their DBC, and increases their
electrophoretic mobility. Lawrence et al. (103) examined DBC for seven
wheat protein fractions separated by polyacrylamide gel electrophoresis.
PAGE lanes were stained with Amido Black 10B. Matching protein
bands were excised and analyzed for their amino acid prole. Electro-
phoretic mobility and dye-binding strength are positively correlated with the
arginine and lysine content and negatively correlated with the glutamate/
glutamine levels. Total basic amino acid content varied with electrophoretic
mobility from the top to the bottom of the gel: *2.55% (gliadins), 1819%
(albumins), and 28% (globulins).
The genetic linkage between the high-lysine phenotype, impaired
endosperm development, and decreased yield is not well understood.
Horvatic et al. (121) noted that lysine, methionine, and tryptophan levels in
nine cultivars of wheat were negatively correlated with total crude protein
and with the gluten content. In high-lysine opaque (o2) maize the
endosperm was oury, chalky, and soft. Kernel hardness was also low,
leading to reduced resistance to insect damage and low processability.
Quality maize o2 hybrids had kernels with increased density, vitreosity,
and increased grinding time. Paulis et al. (122) found a positive correlation
between kernel hardness and the content of prolamins. Perhaps prolamins
somehow enhance protein-starch interactions.
Clore and Larkins (123) reported high levels of a protein (designated
EF-1alpha) in high-lysine maize varieties. Grain lysine content was highly
correlated with concentrations of protein EF-1alpha. Using immunocyto-
chemistry and confocal microscopy, they showed that EF-1alpha was
associated with the cytoskeletal network within the developing endosperm.
The network of proteins they suggested might be necessary for the
formation of protein bodies. It is feasible that increasing EF-1alpha and
other lysine-rich proteins may have adverse effects on the cohesiveness of
the starchy endosperm.

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123. AM Clore, BA Larkins. Protein quality and its potential relationship to the
cytoskeleton in maize endosperm. J Plant Physiol 152:630635, 1998.
14
Protein DigestibilityCorrected Amino
Acid Scores

1. INTRODUCTION

The protein digestibilitycorrected amino acid score (PDCAAS) was

adopted as the AOAC-approved index for protein quality in 19931994.
The PDCAAS is the amino acid score (AAS) multiplied by protein
digestibility. Alterations in either parameter changes protein nutrient value
(PNV). PDCAAS is discussed in this chapter. In Sec. 2 considration is given
to digestibility and its relation to protein structure. In Secs 3 and 4 is a
review of protein denaturation and chemical deterioration during food
processing and their effect on the PDCAAS. There is also increasing
realization that moisture-temperature-time relations affect the food matrix
and protein quality. Some possible links between Tg (glass transition
temperature) and PNV are explored in Sec. 5. The determination of
PDCAAS for a range of foods is discussed in Sec. 6.

2. PROTEIN DIGESTIBILITY

True protein digestibility (TPD) measured using a rat bioassay agrees with
in vitro protein digestibility (Chapter 13). This results has two implications:

411
412 Chapter 14

1. Absorption across the intestinal membrane does not limit

(bio)availability. Exceptions to this rule appear in Section 6.7,
where availability-corrected amino acid scores (AvCAAS) are
discussed.
2. Fundamental research on protein structure and susceptibility to
proteolysis can be marshaled to help make sense of the PDCAAS.

2.1. Protein Conformation, Molten Globules, and

Digestibility
Binding interactions between a protease and its substrate follow the
approach of two molecules of comparable sizes. An exposed region on the
substrate protein then enters the protease active site. Linderstrom-Lang et
al. (1) described enymatic digestion via a two-stage reaction. First, the native
protein (N) conformation changes to an unfolded (U) state that exposes
peptide bonds to the external solvent [Eq. (1)].
NU 1

Protein unfolding increases within the stomach. The low-pH, high-chloride

environment transforms globular proteins into a molten globule state. This
is an expanded, more exible structure compared with the N state (2,3). At
low pH, brous proteins (myosin, collagen) exhibit changes in protein-
protein interactions followed by unfolding. Second, the U state is
hydrolyzed by an enzymatic reaction that leads to the irreversibly modied
(I) state [Eq. (2)].
U!I 2

The rate of proteolysis (v) is determined by the fraction of protein substrates

unfolded (FU) and the rate constant for the U ! I reaction (ki):
ki PKeq
v & ki PFU 3
1 Keq

where P is the protein concentration. The preceding relation applies to the

whole protein or to specic segments.*

* Protein unfolding is often described as a two-state (all or nothing) process. In this idealized
model all peptide bonds are either inaccessible or fully accessible to the external solvent. In
contrast to this global (un)folding process, a nontwo-state unfolding event leads to the
localized exposure of bonds.
Corrected Amino Acid Scores 413

2.2. Protein Structure, Stability, and Digestibility

In vitro digestibility is a sensitive probe for protein structure. Under
nondenaturing conditions Keq 5 1 and FU &Keq [see Eq. (3)]. The value of
Keq is nitely large or small but never ``zero.'' Proteases attack the U state
readily and therefore slight changes in the N/U equilibrium [Eq. (1)] alter
protease susceptibility. Proteolytic attack on the N state conformation
occurs at surfaces, exposed peptide loops, beta-turns, and at random
(aperiodic) sequences.* Digestion is more likely between protein domains.
Oligomeric proteins are digested following dissociation into subunits. The
rate of digestion is low within areas of regular secondary structure.
Protein stability is inversely related to digestibility (4). Matthyssens
et al. (5) monitored the thermal denaturation of lysozyme at pH 2 by
measuring the rate of proteolysis by pepsin. With increasing temperatures,
lysozyme unfolds and the rate of proteolysis increases. Imoto et al. (6,7)
monitored the thermal unfolding transition for lysozyme from the kinetics
of proteolysis with pronase. Church et al. (8) used immobilized pronase
activity as a probe for normal or chemically modied lysozyme, beta-
lactoglobulin, and casein. Daniel et al. (9) established an inverse correlation
between protein stability and susceptibility to proteolysis. Ueno and
Harrington (10) monitored structural changes in myosin at 5408C using
papain, chymotrypsin, or trypsin. A relationship between protein digest-
ibility, exibility, surface hydrophobicity, and physical functional properties
(foaming, emulsication) was proposed by Kato et al. (11). Kato et al. (12)
reported a similar relation for heat-denatured lysozyme and ovalbumin. The
sites of autolysis for thermolysin had high segmental mobility as detected by
temperature factors (B values) from X-ray crystallography (13). Unstable
proteins for which the N/U equilibrium lies toward the U state are more
susceptible to proteolytic degradation. Acid-stable proteins (e.g., beta-
lactoglobulin) are resistant to unfolding and hence to protease attack. The
relationship between protein stability and digestibility is altered within the
stomach. The majority of food proteins become destabilized at low pH at
378C. Using the uorescent dye anilino-naphthalene-8-sulfonic acid (ANS).
Folawiyo and Apenten (14) showed that oilseed proteins undergo a
conformational change at pH 12. The degree of ANS binding at low pH
was 10-fold greater than obtained by heating alone. Preheating had little
effect on ANS binding sites at low pH.

* Under nondenaturing conditions, the small proportion of the U state in equilibrium with the
native state is attacked by proteases. It is a moot point whether the N state itself undergoes
proteolytic attack.
414 Chapter 14

2.3. Enzyme Specicity

The rate of proteolysis is also determined by protease specicity. Schechter
and Berger (15) supposed that proteases have up to seven active sites (. . . S3,
S2, S1, S10 , S20 , S30 ) with specicity for six peptidyl sites (P3, P2, P1, P10 , P20 ,
P30 ). The peptide bond subjected to cleavage occurs between P1 and P10 .
Different proteases recognize different P1, P10 residues for bond hydrolysis.
For trypsin, chymotrypsin, and pepsin, P1 lysine or arginine, an aromatic
amino acid, or a hydrophobie residue, respectively. The effect of enzyme
specicity on the rate of proteolysis appears in the ki term of Eq. (3). In
accordance with Michaelis-Menten formalism, ki Vmax =Km (ratio of the
maximum velocity to the Michaelis constant).

3. PROTEIN DENATURATION

Changes in protein structure affect digestibility and PNV. The important

variables are temperature, pressure, pH, ionic strength, presence of surfaces,
and shear rate (Table 1). Changes in protein structure follow a two-stage
process [Eq. (1)Eq. (2)]. Unfolding is either a global process or is restricted
to specic segments of the protein. Exposure to chemical denaturing agents
(urea, guanidine hydrochloride) leads to a U state that is approximately a
random coil. Extremes of pH, extremes of temperature, and/or high
pressure produce the partially unfolded (molten globule) state (Table 1, row
C). The N/U transition [Eq. (1)] is formally reversible. Removing the protein
from stress restores the N state. Irreversible U ! I reactions occur after
prolonged exposure to extreme conditions [Eq. (2)]. Besides Linderstrom-
Lang and co-workers (1), Equations (1) and (2) were also developed by
Eyring and Lumry (16), who applied them to describe protein denaturation.
Proteolysis is an irreversible denaturation process where the U ! I reaction
is peptide bond hydrolysis catalyzed by a protease. Nonenzymatic
proteolysis occurs at low pH and at temperatures of about 1008C.
Structural changes of practical signicance are irreversible. Charles
Tanford's treatment of this subject is still unsurpassed after 30 years (17).
Ahern and Klibanov (18,19) have also discussed denaturation at length.
Kinsella (20) reviewed the chemistry of food protein reactions.
On the relatively long time scale associated with aging and
development, there is a constant leakage from the U state to the I state.
The postharvest state is characterized by increased formation of the I state.
Table 1 (row B) lists factors that are likely to support reversible or
Corrected Amino Acid Scores 415

TABLE 1 Forms of Food Protein Denaturationa

A. Reversible denaturation A*. Irreversible denaturation

NU N!I
B. Conditions for the N U transition B*. Conditions for N ! I transition
Low protein concentration High protein concentration
(<0.1% w/v) (>5% w/v)
pH removed from pI Solvent pH&pI
Presence of sulfhydryl reagents Extremes of pH (pH < 4, pH > 8)
Transient exposure to denaturing Prolonged exposure to denaturant
conditions
Low intensity of denaturant High intensity of denaturant/severe
process conditions
Low ionic strength High ionic strengh
C. Some N U processes C0 . Some N ! I processes
Transient unfolding, expansion Covalent aggregation
Semimolten globule Noncovalent aggregaration
Molten globule Peptide bond lysis
Domain denatured Racemization
Cross-link formation
Carbonylamine reactions
Deamidation
D. Processing variables
High temperatures (sterilization, cooking, drying)
Low temperature (cold storage, freezing)
Moisture control (dehydrated storage, drying)
High pressure (sterilization, low-temperature gelation)
High or low pH (solubilization, texturization)
Procesing chemicals (alcohols, alkali, nitrate, reducing compounds, etc.)
Exposure to interfaces (emulcation, foaming, etc.)
High shear treatment
a
Notes: A, Two forms of protein denaturation; B, conditions that facilitate reversible and
irreversible denaturation; C, types of reversible and irreversible denaturation; D, forms of food
processes leading to denaturation.

irreversible denaturation. Irreversible changes in protein conformation arise

from covalent aggregation via sulfhydryl-disulde exchange or sulfhydryl
oxidation. Physical denaturation processes such as gelation will be reversed
in the low-pH gastric environment.
416 Chapter 14

4. CHEMICAL DETERIORATION OF PROTEIN

INGREDIENTS

Denaturation was initially dened only in operational terms by reference to

the changing physical appearances of proteins. Protein precipitation,
aggregation, gelation, or increases in viscosity were taken as signs of
denaturation. These changes in appearance can be induced (by salting-out,
exposure to organic solvents) without denaturation. After the publication of
the 3D structure for myolglobin in 1963, denaturation was redened in
terms of changes in the 28, 38, or 48 structure. Alterations in protein 18
structure were not included in the denition of denaturation. It seemed
unlikely that changes in 18 structure could occur under normal physiological

conditions (atmospheric pressure, T 037 C). There is now much
sympathy for the idea that proteins from archaebacteria evolved at
temperatures of 801208C. Many organisms persist under ``extreme''
physiological conditions of temperature and pressure where changes in
protein 18 structure are conceivable.
The conventional denition of denaturation is also too restricting in
view of recent advances in protein chemistry and technology. Proteins
exhibit 18 structure changes during food processing. The range of
deteriorative changes includes peptide bond hydrolysis, carbonyl-amine
reactions, racemization to form D-amino acids, and formation of covalent
cross-links. Deamidation of glutamine and asparagine transforms these
residues into glutamate and aspartate, respectively. The SH-disulde
exchange produces cystine cross-links from cysteine. Loss of hydrogen
sulde and dehydration generate dehydroalanine. Heating in the presence of
sugars leads to glycation. Most deteriorative processes are accelerated by
water. Addition of water to intermediate moisture foods encourages
deterioration by lowering the glass transition temperature (Tg). With
dissolved proteins, deterioration follows protein unfolding to expose
reactive amino acid residues to the external solvent.

4.1. Carbonyl-Amine Reactions

Within a polypeptide all except three essential amino acids are protected
from the Maillard reaction. Luise Maillard (21) discovered the reactions
between amino acids and sugars. Nonenzymatic browning leads to the
formation of melanoidins and avor precursors. The reactive protein
residues are lysine (e-NH2), methionine (22S22CH3), and tryptophan (indole
group). With high-carbohydrate (plant) foods, lysine is the most reactive
essential amino acid and the most limiting. Methionine or tryptophan is
usually limiting for animal proteins.
Corrected Amino Acid Scores 417

Carbonyl-amine reactions take place in four stages: (a) addition of the

e-NH2 group of lysine to a carbonyl group of an aldose, (b) elimination of
water to form a cationic Schiff base, (c) proton loss to form an enol, and (d)
enol-keto rearrangement to form 1-amino-1-deoxy-2-ketose. Reactions (c)
and (d) together constitute the Amadori rearrangement. For a keto-sugar
the corresponding reaction is the Heyens reaction leading to 2-amino-2-
deoxyaldose. The effect of carbonyl-amine reactions on protein nutrient
value (PNV) is reviewed by Dworschalk (22), Labuza and Saltmarch (23),
Friedman (24,25), Saltmarch and Labuza (26), Feeney and Whitaker (27),
Hurrell (28), and Feather (29).

4.2. Controlling Factors for Carbonyl-Amine Reactions

The effect of pH, water activity (Aw), temperature, and different sugars on
the rate of the carbonyl-amine reaction was studied by Labuza and
Saltmarch (23,26). Maillard browning shows a bell-shaped pH dependence
with a maximum at about pH 8. Extremes of pH lead to a decline in the rate
of browning by (de)protonating the carbonyl and amino functions. The
dependence on AW is also bell shaped. Browning decreases with decreasing
moisture content due to the high medium viscosity and diffusion
restrictions. With increasing AW the reaction rate increases until sufcient
water is available for monolayer coverage. With still higher moisture
content the rate of reaction decreases due to the dilution of reactants. The
temperature dependence of the Maillard reaction was a described with linear
Arrhenius equation. The activation energy for lysine loss was about 80 kJ
mol 1 (Section 5.3). The relative rates of reaction with different sugars
follow the order lactose > D-ribose > D-fructose > D-glucose; pentose
> hexose > disaccharide.

4.3. Impact on Protein Quality

The products of the carbonyl-amine reactions have two principal effects on
PNV: (a) the bioavailability of lysine is reduced and (b) digestibility is
reduced by the presence of protein-bound sugar residues. The Amadori
compound (1-amino-1-deoxy-2-ketose) is biologically unavailable (30).
After ingestion by rats, a signicant proportion is excreted in the urine.
The nonabsorbed fraction appears in the feces and/or is degraded by
intestinal bacteria. Peptide bonds in the vicinity of glycated lysine residues
are not susceptible to protease attack. Maillard reaction products also
inhibit the absorption of other amino acids. Suggested physiological effects
include increased protein allergenicity, mutagenicity, and effects on the
reproductive capacity of rats. Normal cooking does not affect PNV.
418 Chapter 14

Microwave cooking also had no adverse effects (31,32). Maillard reaction

products form during baking that have a detrimental effect on protein
quality. The effects of the Maillard reaction on available lysine is also
discussed in Chapter 12 and 13.

4.4. Racemization and Cross-Linking

Cysteine, serine, and phosphothreonine residues are converted to dehy-
droalanine at high pH. The process involves the elimination of hydrogen
sulde, water, or the phosphate group, respectively. Dehydroalanine then
reacts with a range of protein groups forming cross-links (Fig. 1).
Racemization also takes place under alkaline conditions (33). The two
reactions proceed via the following common steps: (a) abstraction of a
proton from an a-C atom by a base (B) produces a carbanion ion, (b)
readdition of H to the a-C then generates the corresponding D-amino acid,
(c) b-elimination from the carbanion ion produces dehydroalanine, and (d)
addition of protein side chains (histidine, cysteine, lysine) to dehydroala-
ninie forms cross-links. The subject is reviewed by Hurrell (28), Maga (34),
Otterburn (35), and Swaisgood and Catignani (36).

4.5. Factors Controlling the Rate of Racemization and

Cross-Linking
Friedman and Liardon (37) found that the rate of racemization is sensitive
to ( ) or ( ) inductive effects. For simple amino acids the rate of
racemization was predicted using linear free energy relationships. Free
amino acids are *10-fold less reactive than protein-bound amino acids. The
presence of an adjacent carboxyl group destabilizes the intermediate
carbanion ion. A high pH (>9) increases the rate of racemization by
facilitating the initial proton abstraction. Proline residues (especially when
next to aspartate or aparagine) are more reactive than other amino acid
residues. Polypeptide sequences containing serine and cysteine (e.g., Ser/
Cys22X22Lys or Ser/Cys22X22X) are also highly reactive.
Racemization and cross-linking can be observed by heating model
proteins in alkaline solution (38,39). Similar reactions take place in foods
when proteins are exposed to high pH and temperature. A traditional
method for producing tortilla involves steeping corn in 1% lime solution at
808C for 2045 minutes. The preparation of sh protein concentrates,
texturization of soy protein, recovery of residual protein from bone, alkaline
extraction of plant or yeast proteins, and lye peeling also exposes food
proteins to high pH values. Cross-linking and racemization reduce in vitro
protein digestibility (40,41) as well as PNV (42).
Corrected Amino Acid Scores 419

FIGURE 1 A scheme for amino acid racemization, dehydroalanine formation, and

protein cross-linking. Based on a scheme proposed by Masters and
Friedman (33). B base.
420 Chapter 14

5. MATRIX EFFECTS ON THE RATE OF DETERIORATION

OF PROTEIN INGREDIENTS

The glass transition temperature (Tg) is an important index for molecular

mobility and diffusivity within food matrices (43). Studies of the relation-
ship between Tg and the rate of deterioration of protein ingredients are just
beginning. Matrix-based models are also currently being formulated and
tested in relation to freeze-dried (pharmaceutical) proteins. These studies
and the small number of investigations involving food protein ingredients
are discussed here.

5.1. The Glass Transition Temperature

Changes within highly concentrated, glass-forming, food systems were
described using a polymer chemistry approach by Slade and Levine (44,45).
Key elements of glassy state and its relation to food quality deterioration
can be summarized as follows:
1. Foods and food materials can be treated as classic polymer
systems.
2. The glass transition temperature (Tg) for a glass-rubber transition
is a critical parameter that determines processability, quality,
stability, and safety.
3. At temperatures below Tg the viscosity of a food matrix is
extremely high such that physical and chemical changes are
inhibited.
4. Above the Tg the viscosity of a food matrix decreases and various
relaxation process (or chemical changes) become possible.
5. Food stability may be assured by storing at temperatures below
T g.
6. Water is a ubiquitous plasticizer for both natural and fabricated
food ingredients and products. The plasticizing effect of water
reduces Tg.
7. At temperatures above the Tg materials are in a disordered
(rubbery or amorphous) state.
8. William-Landel-Ferry (WLF) kinetics apply at
Tg < T < Tg 1008C. The kinetics of food change does not
conform to the linear Arrhenius equation.
9. Nonequilibrium glass/rubbery state transitions affect all time-
dependent structural and mechanical properties in real-world
foods; in contrast, thermodynamic concepts such as water activity
are inapplicable.
Corrected Amino Acid Scores 421

5.2. Models for Food Matrices

Food matrices are described using the fringed-micelle model or the
folder-chain lamella model. According to the fringed-micelle model, food
materials are composed of crystalline and amorphous phases. Strong
intermolecular associations between polymer chains account for regions
of high order. Amorphous or disordered regions arise from the lack of
strong polymer-polymer interactions. Heating leads to consecutive order-
disorder transitions involving a glassy/rubber state (at Tg) followed by a
crystalline/amorphous transition at the melting temperature (Tm). At
temperatures below the Tg the system possesses a viscosity of about 1012
Pa s. Most physical and chemical processes (microbial growth,
enzymatic activity, protein deterioration) are severely inhibited. For
pure biopolymers Tg is generally about 1502008C. As Tg 5 Tm a solid
may undergo decomposition before the Tm is reached. According to the
folded-chain lamella model (46), crystalline regions are formed by
intramolecular associations involving a single polymer chain folded back
on itself. Amorphous regions occur at interruption zones or regions of
the polymer possessing defects, kinks, and other irregularities in the
structure.
The plasticizing action of water leads to Tg decreasing with increasing
moisture content. At high moisture levels (>20% w/w) Tg decreases to below
the freezing point for water. Cooling produces a freeze-concentrated system
as pure solvent water solidies into ice. The freeze-concentrated component
then undergoes a liquid/glass phase transition at characteristic temperature
designated Tg0 . Solutes for which the Tg0 value is high exhibit greater
preserving action (see later). The polymer science approach provides
satisfactory explanations for a wide range of food-related phenomena
including microbiological stability, enzymatic activity at low Aw, inhibition
of collapse, and improved freeze drying, cooking, and frying processes. For
low or intermediate moisture foods, the new approach leads to an integrated
discussion of moisture, temperature, and time relations during the
processing or storage. Specic process end points can be achieved using
different time-temperature, moisture-temperature, and moisture-time com-
binations.

5.3. The Glass Transition and Protein Quality

The Tg for gluten was reported by several workers (4752). The Tg has also
been reported for beef proteins (53), caseinate (54), and gliadin (5557).
Noel et al. (58) determined the Tg for fractionated wheat gluten proteins (a-,
g-, and o-gliadins and HMW glutenin) using the Perkin-Elmer DSC2
422 Chapter 14

microcalorimeter tted with a liquid nitrogen cooling accessory (Fig. 2).

Temperature scans from 270 and 370 K showed an exothermic peak at
about 320 K (or 478C) for o-gliadin equilibrated with 12% water. For a
moisture content < 20%, Tg decreased in accordance with the Gordon-
Taylor equation,

w1 Tg 1 w2 Tg 2 k
Tg 4
w1 w2 k

where Tg is the observed glass transition temperature, Tg1 is the glass

transition temperature for the pure protein, Tg2 is the glass-transition
temperature for the pure plasticizer ( 138 K for water), w1 and w2 are
the weight fractions of protein and moisture, respectively, and k is a
constant whose value increases with increasing plasticization. Nonlinear
regression analysis lead to prediction of Tg1 for dry proteins based on the

FIGURE 2 Effect of moisture on the glass transition temperature for fractionated

glutenin proteins. High-molecular-weight glutenin subunits (HMW)
a-gliadin, g-gliadin, and o-gliadin. Proles were generated according the
Gordon-Taylor relation, Eq. (4), using values for Tg1 and k given in Refs.
55 and 56.
Corrected Amino Acid Scores 423

observed Tg values at different moisture levels. Values of Tg1 ranged from

397 K (1248C) to 417 K (1448C) for the four gluten proteins. In the
presence of 20% moisture, Tg decreased to between 270 K ( 38C) and
280 K (78C).
Morales and Kokini (59) measured Tg for 7S and 11S soy globulin
fractions using DSC and mechanical spectrometry. There were two Tg
values due to cross-contaminating amounts of the 7S globulin in the sample
of 11S globulins and vice versa. Single Tg values were obtained for highly
puried soy globulins. With 7S soy protein Tg was 387 K (1148C) to 206 K
(678C) for moisture contents of 0 to 35%, respectively. For the puried 11S
fraction Tg ranged from 433 K (1608C) to 256 K ( 178C) for moisture
contents from 0 to 40%. Soy globulins behave as polymers that are highly
plasticizable by water.
The effect of moisture on the Tg and caking properties of sh protein
hydrolysate was examined by Aguiliera et al. (60). Increasing the relative
vapor pressure from 0 to 0.64 reduced Tg from 352.1 K (79.18C) to 230.2 K
( 42.88C). The plasticizing action of water was described by the Gordon-
Taylor equation [Eq. (4)]. At a xed temperature of 198C (room
temperature) collapse was initiated at a relative vapor pressure of 0.44,
corresponding to the T Tg value of 35.88C. Above a relative vapor pressure
of 0.55 the following quality defects occurred: nonenzymic browning,
collapse, shrinkage, and setting into a sticky, high-viscosity brown liquid.
Using the WLF equation, the viscosity of the matrix at the onset of collapse
was estimated as 105107 (Pa
s).
To preserve protein samples from deterioration it is necessary to
employ storage temperatures below the Tg. However, the glassy-rubbery
state transition takes place over a nite temperature interval. Peleg (6163)
modeled mechanical changes during the glass-rubber transition using
Fermi's equation:

Yg
Y 5
1 expT Tc=A

TC TC;0 exp kw 6

A A0 expk00 w 7

where Y is an apparent stiffness parameter; Yg is the stiffness in the glassy

state; T is temperature; TC is the inexion temperature, which is not
necessarily coincident with the Tg; and A is a parameter that measures the
slope of the Y-temperature graph. Both TC and A are dependent on the %
424 Chapter 14

moisture content (w). We have replicated these simulations (Fig. 3) with the
following two results: (a) the inection temperature decreases with
increasing moisture, and (b) the gradient of each graph increases
with increasing moisture. Therefore, sensitivity to temperature
increases with increasing moisture. To preserve high-moisture foods requires
a low storage temperature and improved temperature control. Drying
allows higher storage temperatures and increased tolerance with respect to
temperature variations.
So far, only a few investigators have applied the polymer science
approach to food protein deterioration. The physical-mechanical basis for
glass-liquid transitions for proteins has not been extensively discussed.
According to Ferry (64), Tg is the temperature below which wriggling
motions and conformational rearrangements within a polymer cease.
Liquids and polymers have a constitutive volume (determined by van der
Waals contacts) and free volume arising from packing irregularities or
defects. During cooling the free volume changes in accordance with the

FIGURE 3 Effect of temperature and moisture on the apparent stiffness of gliadin

near the glass transition temperature. The moisture content (%) is shown
in the boxed legend. The inection temperature for each transition is
138C, 38C, 148C, 288C, and 258C at a moisture content of 11.25%,
13%, 19.25%, 24.4%, and 27.2%. Simulations were performed using
equation parameters from Peleg (56,6163).
Corrected Amino Acid Scores 425

thermal expansion coefcient. The Tg is a narrow temperature region in

which the thermal expansion coefcient, heat capacity, adiabatic compres-
sibility, and specic volume show discontinuity. What can undergo a glass
transition in a protein solid? In answer to this self-posed question, Morozov
and Gevorkian (65) suggested that the glass-liquid transition in globular
proteins involves surface groups and packing defects. These structural
components may or may not be hydrated. There are also aperiodic or
random sequences in most proteins.

5.4. Studies on Freeze-Dried Proteins

The storage stability of pharmacologically active proteins is related to Tg.
Freeze-dried high-value proteins undergo so-called moisture-induced
deteriorative reactions. Most of these reactions have been identied by
accelerated testing (Table 1). Liu et al. (66) found that the storage stability
of freeze-dried proteins was increased by adding excipients, mainly
polyhydroxy alcohols. Costantino et al. (67) reported similar ndings. For
proteins in the dry state additives act as platicizers that reduce Tg in
accordance with the Gordon-Taylor equation. The destabilizing action of
added excipients is inversely related to the Tg of the pure additive. Added
excipients are stabilizing as compared with the equivalent weight of water.
To achieve stabilization the pure additive should have Tg2 higher than water.
Excipients also increase the denaturation temperature (Tm) in direct
proportion to their effect on Tg (6870). Indeed, for some synthetic
polymers the two transition temperatures are strongly correlated:
Tg K 0:66 +0:04 Tm 8

The glass-liquid transition and the crystalline-amorphous transition appear

to be subject to similar constraints (46).

5.5. Protein Conformational Stability, Tm, and Quality

Protein solutions have Tg values below room temperature although Tm
remains high (30908C). Regular 28 structure yields regions of order and
crystallinity within the N state. The net conformational (dis)order
determines the peak temperature (Tm) and enthalpy change (DH) measured
by differential scanning calorimetry (DSC). Equation (8) suggests that a
high Tm (compare Tg) will produce lower rates of protein deterioration in
solution. Protein stability-function relations were discussed by Apenten and
Berthalon (71) and Apenten (72) using the two-stage denaturation scheme.
Reactive amino acid groups are buried in the N state and become accessible
426 Chapter 14

to the solvent after protein unfolding. High conformational stability

therefore attenuates U ! I reactions [see Eqs (1)(3)].
The magnitude of DE # provides useful information about the
mechanism of deterioration. The N/U transition is a highly cooperative
process with a large temperature coefcient (Q10 > 2). Where the rate-
limiting step for deterioration is a conformational change [Eq. (1)] the value
of DE # should be 250700 kJ mol 1. In contrast, when the U ! I reaction
determines the rate of deterioration then DE # is small (2080 kJ mol 1). The
magnitude of DE # is not only determined by the rate-limiting step for
protein deterioration because
1. Deterioration is a multistage reaction [Eqs (1)(3)]. Therefore,
DE # is the sum of values for several reactions.
2. Quality loss might not conform to the N ! U ! I scheme.
Multiple U states (U1, U2, U . . . Ui) and/or U ! I reaction occur.
3. DE # is temperature dependent (71,72).
Labuza and Saltmarch (73) investigated the effect of moisture and
temperature on available lysine and nonenzymatic browning in whey
powder. Labuza et al. (74) examined similar reactions for pasta. The loss of
FDNB-lysine and nonenzymatic browning followed rst- and zero-order
kinetics, respectively. The rates of lysine and PNV losses were two to three
orders of magnitude greater than the rate of browning. The reaction
temperature dependence tted a linear Arrhenius equation at 35558C with
a squared regression (R2) coefcient of *0.601. The DE # for lysine loss and
browning ranged from 52 to 85 kJ mol 1 depending on the prevailing AW.
Adding data from 258C produced curved Arrhenius plots.
I have reexamined these results using a higher order Arrhenius
equation that allows for variations in the value for DE # with temperature.
The simple Arrhenius equation is

DE #
ln k ln k0 9
RT
where k is a rate constant and DE # is the energy required to form an
activated complex. A semilog arithmic plot of ln k versus 1/T leads to a
straight-line graph having a slope equal to DE # =R and an intercept value of
ln k0 . A more comprehensive discussion of reaction rates is based on the
transition-state theory. Here the activated complex from the Arrhenius
model is replaced by a transition state, and ``activation energy'' becomes
DG# (the Gibbs free energy change for producing the transition state).
Comparing the Arrhenius and transition state reaction rate models leads to
the realization that DE # is essentially equal to DH # (enthalpy change for
Corrected Amino Acid Scores 427

producing a transition state) where DH # DE # 2RT. Reactions taking

place ``in water'' can be accompanied by signicant heat capacity change;
DCp# dDH # =dT.*
To allow for a temperature-dependent DH # value, the Arrhenius
equation is expanded to a second-order polynomial.

ln k a bx cx2 10

where a, b and c are constants and x 1=T. From general thermodynamic

principles DE # Rd ln k=dx & DH # and dDH # =dT DCp# and there-
fore
2c
DH # b 11
T 1
and
2Rc
DCp# 12
T 2
Fig. 4 and Table 2 show results for the deterioration of pasta (74). The loss
of FNDB-lysine and browning reactions were adequately described by
Equation (10) (R2 0:98 0:99).
Close to the Tg (1208C) for a dry protein the magnitude of DH # is
large, implying that quality loss may be limited by conformational
transitions. The low DH # value near room temperature is a consequence
of the temperature dependence of this parameter.

6. PROTEIN DIGESTIBILITYCORRECTED AMINO ACID

SCORES (PDCAAS)

There has long been dissatisfaction with the rat PER assay. In 1991 an
FAO/WHO Expert Consultative Group (75) agreed that the PER test
should be replaced by a new method with the following characteristics: (a)
greater accuracy and reproducibility than the PER test, (b) shorter time for
assay, (< 48 hours per test), (c) incorporation of digestibility, (d) wider
applicability to samples without extensive prepretreatment, (e) simplicity

* The heat capacity (Cp; J g 1 K 1) is the amount of heat required to raise the temperature of 1 g
of material by 1 K. The reaction A B ! C D will lead to a heat capacity change (DCp ) if the
reactants and products interact differently with the solvent.
428 Chapter 14

FIGURE 4 Arrhenius plot for protein deterioration in pasta at different

temperatures. Relative vapor pressure is 0.44. (Based on data from
Ref. 74.)

TABLE 2 Activation Parameters for Protein Deterioration in Pasta; Analysis by a

Nonlinear Arrhenius Equation

Parameter Lys loss (RVP 0.44) Browning (RVP 0.44)

a 212.3 192.7
b 1.328 6 105 1.22 6 105
c 1.968 6 107 1.825 6 107
R2 0.991 0.980
DH# (kJ mol 1) 42.2a (272.2) 40.1 (252.6)
DS# (J mol 1K 1) 202.8 (419.1) 229.1 (264.5)
DG# (kJ mol 1) 104.79 (107.6) 110.7 (146.6)
DCp# (J mol 1 K 1) 4395 2359
a
The rst and second values are for temperatures of 378C and 1208C.
Corrected Amino Acid Scores 429

and suitability for routine use, and (f) low cost not exceeding $200 per test at
1991 prices. The panel of eminent nutritionists selected a new test involving
protein digestibility-corrected amino acid score (PDCAAS). They further
agreed that PDCAAS should be based on the FAO/WHO/UNU 1985 list
showing the essential amino acid requirements for 25 yr. preschool children
(76). The values are given in Chapter 12 (p. 355). In 1993 the FDA (USA)
adopted the PDCAAS procedure for the routine evaluation of PNV and for
food labeling purposes. Developments leading to the adoption of the
PDCAAS test are reviewed by Sarwar and McDonough (77), Boutrif (78),
Madi (79), Henley and Kuster (80), and also Kuntz (81). The basic
principles for evaluating PDCAAS and examples of its use are discussed in
Section 6.5. First, however, we review older methods for PNV evaluation
and how the PDCAAS index evolved (Sections 6.16.4).

6.1. Protein Chemical Score

Mendel (82) stated that a protein's quality is related to its minimum quantity
of essential amino acids. In 1946 Mitchell and Block (83) dened the
chemical score for protein quality by calculating the decit of essential
amino acids as compared with essential amino acids from whole egg.
Samples with equal amounts of crude protein (%N 6 6.25) were analyzed
for their essential amino acid content. The results were each divided by the
essential amino acid content for whole egg. The result having the largest
decit compared with egg protein shows the limiting essential amino acid.
EAASAMPLE
Chemical score 13
EAAWHOLE EGG
The chemical score for 28 proteins was positively correlated BV determined
using the rat bioassay (Table 3). Thermal processing produced signicant
changes in BV without affecting the essential amino acid content of proteins
(Fig. 5).
Knorr (84) found that potato protein concentrates prepared in
different ways possessed high chemical scores and may be useful for human
nutrition. Based on their chemical scores, immature durum wheat has a
higher nutrient value than mature wheat (85). Chemical scores for eight
peanut cultivars were highly correlated with RNV (R 0:98) and PER
(R 0:88) (86). Ruales and Nair (87) found that the limiting amino acids in
quinoa our were tyrosine and phenylalanine, yielding a chemical score of
0.86. The chemical score has two shortcomings with regard to protein
quality determination. First, adopting a Kjeldahl factor of 6.25 for two
protein sources with equal nitrogen will lead to error if the samples vary
430 Chapter 14

TABLE 3 Chemical Scores for a Range of Food Proteins Determined Using Whole
Egg Standard

Chemical
Limiting Chemical Digestibility score 6
Protein source EAA score BV (%) (%) Dig/100

Beef muscle Cys Met 0.71 76 100 71.0

Beef liver Ile 0.70 77 97 67.9
Egg albumin Lys 0.69 82 100 69.0
Cow's milk Cys Met 0.68 90 95 64.6
Lactalbumin Met 0.66 84 98 64.7
Beef kidney Cys Met 0.65 77 99 64.4
Beef heart Ile 0.65 74 100 65.0
Casein Cys Met 0.58 73 99 57.4
Sunower seed Lys 0.53 65 94 49.8
Soybean (heated) Met 0.49 75 96 47.0
Rolled oats Lys 0.46 66 93 42.8
Yeast (average) Cys Met 0.45 69 93 41.9
White rice Lys 0.44 66 78 34.3
Corn germ Met 0.39 78 85 33.2
Sesame seed Lys 0.39 71 92 35.9
Wheat germ Ile 0.38 75 95 36.1
Whole wheat Lys 0.37 70 91 33.7
Cottonseed Lys 0.37 61 90 33.3
Whole corn Lys 0.28 60 94 26.3
White our Met 0.28 52 100 28.0
Peanut Met 0.24 58 97 23.3
Pea Met 0.24 48 91 21.8
Gelatin Trp 0.0 25 95 0.0
Human milk Met 0.86
Blood serum Ile 0.44
Hemoglobin Ile 0.10
Flax seed Lys 0.35
Alfalfa Ile 0.45
Source: Adapted from Ref. 78.

greatly in the amount of nonprotein nitrogen (Chapter 1). Second, changes

in protein characteristics unrelated to their essential amino acid content are
not measured by the chemical score. Protease inhibitors will affect PNV
although such effects are not reected by changes in the chemical score.
Corrected Amino Acid Scores 431

FIGURE 5 The chemical score as an index for protein quality. (Top) Chemical score
is strongly correlated with the biological value. (Bottom) Showing the
correlation between biological value and ``chemical score corrected for
digestibility'' (open circles). A low correlation is observed between BV
and digestibility (see closed symbols).
432 Chapter 14

6.2. Pepsin Digest Residue Index

Sheffner et al. (88) introduced the pepsin digest residue (PDR) index in 1956.
The PDR is determined by combining in vitro digestibility with the essential
amino acid pattern. The food sample (containing 1 g of protein) was
incubated with pepsin (25 mg) in 30 mL of sulfuric acid (0.1 N) solution for
24 hours.* Precipitating with sodium tungstate and 0.66 M sulfuric acid
separated undigested protein from the products. The essential amino acid
proles for the substrate and soluble digest (products) are determined by
microbiological assay. Subtraction of these values gave the essential amino
acid pattern for the nonhydrolysed protein residue. For both the digest and
residue, each essential amino acid is expressed as a percentage of the total
content of essential amino acids. The two columns of results were then
divided by the corresponding values for egg protein. Next, the geometric
mean was determined for the ``egg ratios'' and the resulting values were
multiplied by a factor that takes into account the relative amounts of digest
and residue formed by the action of pepsin on the sample and the egg
protein. The PDR index was not easy to calculate. Sheffner et al. noted that
any process that decreases pepsin digestibility will also lower protein
nutritional value.

6.3. Pepsin Pancreatic Digestion Index

Akeson and Stahmann (89) introduced the pepsin pancreatin digestion
(PPD) index in 1964. Samples of proteins were digested with pepsin at low
pH, neutralized, and then treated with pancreatin. The soluble products
were concentrated, freeze dried, and subjected to amino acid analysis by ion-
exchange chromatography. The PPD index was then calculated in the same
way as the PDR index. The PPD index showed an excellent correlation
(R 0.99) with BV determined using the rat bioassay. Kennedy et al. (90)
modied the PPD index by performing the pepsin digestion within a dialysis
cell. The pepsin digest dialysate (PDD) index for a range of protein
ingredients (soy our, gelatin, gluten, casein, whole egg powder, cows' milk
fortied with carbohydrates, proteins, and vitamins) was highly correlated
with BV. Some suggested advantages of the PDD index compared with the
PDR and PPD indices include (a) use of simpler apparatus, (b) requirement

* Some experiments were performed with multiple enzyme digestion using trypsin, pancreatin,
and erepsin. However, the nal method used a single enzyme digestion by pepsin. It was
suggested that the proportion of egg protein digested as chyme leaves the duodenum was about
30%. The in vitro study of Sheffner et al. employed conditions designed to ensure about 30%
digestibility of their samples.
Corrected Amino Acid Scores 433

for only one enzyme, (c) use of modern amino acid analysis instrumentation,
(d) higher reproducibility, and (e) use of computerized calculations.

6.4. Computed-PER Index

Satterlee et al. (91) introduced a computed-PER (c-PER) index in an
attempt to correct AAS for digestibility in 1977. First the essential amino
acid pattern for a food sample was expressed as a percentage of the FAO/
WHO pattern for humans (1973). Then each ``%FAO value'' was multiplied
by protein digestibility determined with a rat bioassay or in vitro. The
digestibility-corrected AASs were assigned a statistical weighting inversely
related to the abundance of each essential amino acid. The sum of the
weighted values was divided by the corresponding value for casein. Finally,
the results were transformed by one of four mathematical functions to
generate c-PER values. A collaborative study involving seven laboratories
found that the c-PER gave the same rankings of protein quality as the PER
assays. The c-PER test could be completed in 72 hours compared with 28
days required for the PER determination.

6.5. Available Amino Acid Score and PDCAAS

The PDCAAS is considered an excellent index for protein quality.
Particularly appealing is the ease of calculation. It combines protein
digestibility and AAS in a simple manner. With 20-20 hindsight the
PDCAAS could have been anticipated earlier. Chemical scores were rst
criticized for not taking digestibility into account in 1946 (92). Satterlee et al.
(91) pretty much calculated the PDCAAS value in 1977 and then proceeded
to transform such data to give the c-PER. In 1984 Sarwar (93) introduced
the term ``available amino acid score'' to describe protein scores* corrected
for protein digestibility. His formula was identical to that used later for
calculating PDCAAS (Table 4).
Published lists for TPD may be used to calculate PDCAAS.
Digestibility data are also available from the FDA. Manufacturers should
note that processing can lead to signicant deviations in the TPD compared
with published values. Where a food source contains several proteins (Pi)

* The protein score is calculated in the same way as the chemical score except that the reference
essential amino acids is the prole for humans.
434 Chapter 14

TABLE 4 Stages for the Calculation of Protein Digestibility

Corrected Amino Acid Score (PDCAAS) for a Food Protein

Procedure

1. Determine sample nitrogen content

2. Calculate protein content (N 6 6.25 or specic conversion factor)
3. Analyze sample for essential amino acids
4. Determine the amino acid score ASS =;
mg EAA per 1 g protein
mg EAA per 1 g FAO=WHO=UNU

5. Determine digestibility
6. Calculate PDCAAS Lowest AAS 6 digestibility

the TPD is replaced by the weighted average (TPDav) value:

P
TPDi 6Pi
TPDav P 14
Pi

All proteins having a PDCAAS in excess of 100% (or > 1) are assigned a
value of 100%. A protein with a PDCAAS value above 100% does not
provide further benet as excess amino acids are utilized for energy.
According to Henley and Kuster (80), the PDCAAS method provides a
measure of protein quality that is directly correlated with human
requirements. The PDCAAS method also has considerable exibility.
Manufacturers and diet planners can provide larger quantities of lower
quality dietary protein in order to meet the recommended daily require-
ments.

6.6. Applications
Sarwar et al. (94) showed that a number of rat bioassays (PER, PER,
RPER, NPR, or RNPR) ranked 20 food products in the same order of
protein quality. The correlations between different rat assays were highly
signicant (r 0.980.99). Chemical scores were also correlated with the
results of rat bioassays. Correcting the chemical score for the digestibility
improved the observed correlation. Carnovale et al. (95) compared PNV for
wild and cultivated species of Vigna. The wild type had a signicantly higher
protein content, trypsin inhibitory activity, and tannin content. Protein
digestibility was lower although PNV assessed in terms of the PDCAAS was
not signicantly different.
Corrected Amino Acid Scores 435

A. Quinoa
Quinoa (Chenopodium quinoa Wild) seed protein quality was evaluated using
amino acid analysis and animal feeding trials. The rst limiting amino acids
were tyrosine and phenylalanine with a chemical score of 0.86. Quinoa
protein (14% w/w of the seed) had levels of lysine, methionine, and cysteine
superior to those found in most other plant proteins. From animal feeding
experiments NPU was 75.7, BV was 82.6, and digestibility was 91.7%. These
results yield an estimated PDCAAS of 79%. This value is higher than the
value for meat (87).

B. Rice
The quality of protein associated with cooked milled rice and a typical rice-
based menu for Filipino preschool children and adults was assessed by
Eggum et al. (96). Digestibility, BV, and NPU were assessed with growing
rats. Digestibility was 88.8% for the preschool child diet, compared with a
BV of 90.0 and NPU of 79.9. For the adult diet digestibility was 87.3%, BV
was 86.6, and NPU was 75.5. On its own, cooked rice had a digestibility of
90.0%, BV equal to 82.5, and an NPU value of 74.3. The availability of
lysine (the limiting essential amino acid) was 95.4% for the preschool child
diet, 95.7 for the adult diet, and 100.0 for rice. For whole diets the chemical
scores were 1.00 for the preschool child diet, 0.92 for the adult diet, and 0.62
for rice. From such gures the PDCAAS may be estimated as 88.8% for the
preschool diet, 80.4% for the adult diet, and 56.0% for cooked rice.

C. Maize
The quality of protein from Canadian maize cultivars adapted to Northern
latitudes (greater than 458 N) was assessed by Zarkadas et al. (97). The
cultivars designated Dent CO251, Flint CO255, and Pioneer 3953 compared
favorably with quality protein maize inbred (QPM-C13). Total protein
levels for maize meal were 7.95% (QPM), 8.2% (Pioneer), 10.5% (Dent), and
11.79% (Flint). Compared with ordinary maize, QPM protein had double
the amount of lysine and arginine, increased levels of tryptophan and
cystein, and lower levels of leucine. QPM protein had a good balance of
essential amino acids limited only in lysine. The PDCAAS was 67% (QPM),
28.5% (Pioneer), 31.0% (Dent), or 33.0% (Flint). PNV for a white oury
maize variety from the Indian Agricultural Program of Ontario (98) was
also evaluated. Maize (IAPO-13) harvested over 1992, 1993, and 1994
seasons had an average protein content of 10.14 (+0.1)%. The limiting
essential amino acid was lysine with an AAS of 0.414. The human
digestibility of maize protein is reportedly 89%, leading to an estimated
436 Chapter 14

PDCAAS for IAPO-13 of 3738%. It was concluded from such studies that
Canadian QPM maize variety had higher quality than common maize and
that breeding maize for high protein quality showed a great deal of promise.

D. Oats
Hull-less or naked oats (Avena sativa var. nuda) from temperate zones in
Asia were studied by a Canadian group for their potential use for both
human and animal nutrition. Zarkadas et al. (99) determined the protein
content for three high-yielding and rust-resistant naked oat cultivars. The
protein levels were 13.67 (+ 0.60)%, 13.93 (+ 0.53)%, and 14.40 (+ 0.55)%
for varieties AC Percy, AC Hill, and AC Lotta. All cultivars had a good
balance of nine essential amino acids. Lysine was limiting, followed by
threonine. Assuming a human protein digestibility of 86%, estimates for
PDCAAS are 54.9% (AC Hill), 56.3% (AC Lotta), and 59.3% (AC Percy).
The PDCAAS values for other protein sources are mechanically dehulled
oats (62%), maize (29%), soybean (86%), and egg (95%).
Zarkadas et al. (100) also evaluated the PNV of two newly released
Canadian oat cultivars (Newman and AC Stewart). Oat wholemeal had a
total protein content of 10.75 (+ 0.23)% and 11.92 (+ 0.06)% for strains
Newman and AC Stewart, respectively. The corresponding total protein
content for the oat groats (dehulled grains) was 13.27 (+ 0.24)% and 12.61
(+ 0.94)%. The limiting amino acid was lysine for oat wholemeal and lysine
plus threonine for groats. Values for PDCAAS based on the FAO/WHO/
UNU pattern (2-year-old preschool children) were reportedly 5862.3%
(strain Newman) or 66.762.3% (strain AC Stewart). Dehulling had no
easily predictable effect on the PNV.

E. Collagen
Connective tissue protein (from skin, rind, tendon, or bone) is potentially
useful for livestock feed supplements. The low PNV of collagen was inferred
from feeding trials conducted in the 1960s. Meat meal protein quality is
also inversely related to the hot watersoluble protein (collagen) content.
Feather meal had a distinctly low PNV during an ARC collaborative study
of animal foodstuffs reported in Chapter 13. A contemporary evaluation of
PNV for collagen-containing meat by-products is reported by Zarkadas et
al. (101). Three commercial batches of demineralized beef bone powder were
subjected to quantitative amino acid analysis to determine total protein
content and PDCAAS. The amino acid proles were consistent with the
type I collagen being the main protein constituent. Glycine, proline, and
hydroxyproline each constituted 20% of the total amino acids. Alanine (10%
total amino acids) and basic amino acids (12.5% total) were also relatively
Corrected Amino Acid Scores 437

abundant. Comparatively low amounts of cysteine (*0.2%), methionine

(*1.241.33%) and threonine (1.172.24%) were found. The protein
content of bone meal ranged from 18 to 18.6%. Taking the average
digestibility of collagen as 90%, the PDCAAS value was reportedly 14.4
16.4%; it is not clear how such gures were determined. Table 5 lists the
essential amino acids prole for the three batches of demineralized bone
powder.
Allowing for rounding-up errors, threonine is the limiting amino acid
for one sample with a chemical score of 0.344. Luecine and isoleucine are the
next limiting amino acids with a score of 0.5100.550. Following the normal
rules, the PDCAAS is between 31.0% and 49% for bone meal products.

F. Beans
Changes in the protein quality of red kidney bean (Phaseolus vulgaris L) due
to autoclaving, domestic cooking, or canning were examined by Wu and co-
workers (102) from Clemson University, South Carolina. A large number of
quality indices were considered: available lysine, c-PER, mean chemical
score for lysine, methionine-plus-cysteine levels, and the essential amino
acids index. In vitro protein digestibility was determined with a multienzyme
method. Cysteine plus methionine was limiting with a chemical score of
0.928. In vitro digestibility was 43.2% for uncooked kidney beans and
7982% for heat-treated beans. The PDCAAS was 40% for raw beans
compared with 7075.4% for cooked beans. Other quality indices showed

TABLE 5 Estimation of the PNV for Bone Meal Products

FAO/WHO/UNUa
Amino acids EAA for collagen (mg/1 g protein)

Histidine 11 19
Isoleucine 16 28
Leucine 35 66
Lysine 335 58
Methionine cystine 15 25
Phenylalanine tyrosine 23 63
Threonine 1222 34
Tryptophan 11 11
Valine 27 35
Total EAA 184195 339
Total nonessential AA 805816 661
a
From FAO/WHO/UNU (1985); [Refs. 7581]; see Table 6 of Chapter 12.
438 Chapter 14

sensitivity to heating when corrected for digestibility. For example, raw and
home-cooked beans had available lysine contents of 6.21 and 6.19 g per
100 g protein, respectively. This yields corresponding in vitro protein
digestibilitycorrected available lysine scores 2.68 and 5.1 g per 100 g
protein. After normalization with the FAO/WHO/UNU (1985) pattern, the
protein digestibilitycorrected available lysine score (PDCALS) was 40% for
raw beans and 74% for cooked beans. Apparently, lysine as well as cysteine
plus methionine may be limiting for red kidney beans.

6.7. Beyond Protein DigestibilityCorrected Amino Acid

Scores
Amino acid availability (AAAv) is a more precise measure of digestibility
than TPD (Chapter 12). Calculations of PDCAAS using TPD assume that
all essential amino acids are released from proteins with equal ease. The rates
of absorption are also assumed to be equal. Batterham (103) has reviewed
the signicance of AAAv. Kuiken and Lyman (104) showed that availability
of essential amino acids from a single protein source can differ. The AAAv
was determined for a range of protein sources (egg, liver extract, roast beef,
cotton seed, peanut, and wheat ours) using a rat feeding trial. Employing a
microbiological assay for amino acids, they determined the dietary and fecal
concentrations of individual essential amino acids. With roast beef, egg, or
liver protein values for AAAv were the same for all essential amino acids.
The amino acids from cottonseed meal protein fell into three groups:
(a) arginine, histidine, and tryptophan with high availability (>90%);
(b) isoleucine, phenylalanine, and threonine with intermediate availability
(7090%); and (c) lysine with low availability (65%).
Sarwar (105) conrmed that AAAv was up to 25% lower than the
TPD for legume protein. The AAAv and TPD were determined for 17
protein sources using male rats. Diets and feces were freeze dried, ground,
and analyzed for amino acid proles and for crude protein. The AAS values
for the different diets were expressed as percentages of human requirements
specied by the FAO/WHO (1985). Finally, these results were corrected for
AAAv. The resultant index for protein quality was termed ``available amino
acid score''; I have renamed this quantity availability-corrected amino acid
score (AvCAAS).* Correcting the chemical scores for TPD led Sarwar to
generate what is probably the rst list for PDCAAS. Both AvCAAS and the

* Sarwar used AAS as an abbreviation for the available amino acid score. I have changed this to
AvCAAS (availability-corrected amino acid scores). AAS is used by many investigators to mean
amino acid score. Some investigators use AASTPB to describe amino acid scores corrected for
true protein digestibility.
Corrected Amino Acid Scores 439

notional PDCAAS were highly correlated with the RNPR for 17 diets
(R 0.92). However, values for PDCAAS were consistently higher than
AvCAAS by between 2% and 8% (Fig. 6).
Wu and co-workers (106,107) found much more signicant differences
in PNV after correcting amino acid scores for TPD and AAAv. With raw
kidney beans, cysteine plus methionine was limiting with a chemical score of
0.944. The TPD value was 15.7% for raw beans, increasing to 7287% for
heat-processed beans. By comparison, AAAv was negative ( 18.6%) for raw
bean protein and positive (39.868.0%) for heat-processed beans. Rats fed
with raw beans had higher concentrations of cysteine plus methionine in their
feces as compared with amounts initially present in their diets. The AAAv
values for several other amino acids (alanine, proline, valine, luecine, and
threonine) were also negative. The AvCAAS for raw kidney bean protein was
therefore 17.6% compared with a PDCAAS of 13.9%. The relative merits
of the PDCAAS and AvCAAS are discussed by Darragh et al. (108).
Estimating TPD using a rat assay is a slow process, requiring about 8
days for completion. In view of the high correlation between TPD and in
vitro protein digestibility, correcting AAS using in vitro protein digestibility
is more cost effective. Rozan and co-workers (109) considered AAS for
soybean, lypine, and rapeseed meal proteins using the FAO/WHO/UNU
(1995) pattern for 2- to 5-year-old preschool children. The AAS values were
then corrected using in vitro protein digestibility measured as the degree of

FIGURE 6 Protein nutrient value estimates based on amino acid scores corrected for
true protein digestibility (PDCAAS) or amino acid availability (AAS).
[Drawn from the data of Sarwar (93).] Y-axis shows PDCAAS minus
AAS.
440 Chapter 14

hydrolysis (DH) or/and nitrogen digestibility index (ND). Degree of

hydrolysiscorrected AAS (DHCAAS) or nitrogen digestibilitycorrected
AAS (NDCAAS) values were highly correlated with values of PDCAAS
(p < 0.001).

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Index
AAAvamino acid availability,
438440
AASamino acid score, 354355, 433
Absorptivity, 171
Accuracy, denition, 3
Acetylacetone, Kjeldahl assay and, 22
Acid Orange 12, 125, 127130, 136
feedstuffs protein and, 390
rice protein and, 153
Acid Red 1, 134135
Activation energy, browning, 427428
Actomysin, antigens and, 234
Acylation, 363
Adjuvant, 231
Adulteration, 221
commerce and, 223
health and, 222
protein prices and, 223
economics and, 222
ELISA tests for, 255257
Halal food and, 223
Kosher food and, 223
[Adulteration]
PCR analysis and, 224
US meat trade and, 224
Advanced glycation products, Bradford
assay and, 190
Adulteration frequency, 240
Adverse reaction, food and, 298
Agar gel double immunodiffusion assay,
230246
Agaricus, Udy assay, 160
Aggregated protein, Bradford assay,
217
AGIDagar gel double
immunodiffusion assay, 230246
Albumin, biuret assay, 4849
Alkali treatment, digestibility and, 373
Alkaline copper reagent, 47
Alkali-phenol reagent, Kjeldahl assay,
18
Allergens, 299300
castor bean, 300
egg-white, 300
447
448 Index

[Allergens] Applications
milk, 300 biuret assay, 5767
peanuts, 305312 Bradford assay, 195
shrimp, 300 Udy assay, 147
soybean, 301305 Ara h 2, peanut allergens and, 308
wheat, 312329 Arachin, 306
Allergy Arginine, dye binding and, 135, 139,
infants and, 306 212
pregnancy and, 306 Arrhenius equation, 384, 426427
Amaranth, 398399 PNV and, 384
Amido Black 10B, 130131 Assay performance, 5
Amino acid analysis (see Quantitative Association of ofcial Analytical
amino acid analysis, QAAA) chemists (see AOAC)
Amino acid availability (AAAv), 368, Atmospheric error, Dumas assay, 33
438 Atopic dermatitis, 305
Amino acid availability, protein quality Authenticity, proteins and, 222
and, 438440 Autoanalyzer, 21, 16
Amino acid score, 354355, 433 Autoclaving, effects on digestibility, 368
errors in, 355 Automated, Kjeldahl assay, 1516
Ammonia determination, 1823 Availability corrected amino acid score,
Anaphylactic shock, 297, 305 438
Anilino-naphthalene-8-sulfonic acid, Available amino acid score (see AAS)
413 Available lysine, 356366
ANS uorescence, 413 chromatographic analysis, 360361
Antigen comparison of assays, 365
actomysin, 234 cottonseed, 360
binding curves, 227, 261 interferences, 359, 362
boiling and ethanol resistant, 236, 239, lactalbumin, 365
240, 260 legumes, 394395
cooked meat and, 236, 238 moisture and, 426
peptides as, 324327 ovalbumin, 365
thermostable proteins as, 236, 261 reagents for, 356
troponin T as, 236, 238, 239 sodium borohydride and, 365366
AOAC, Association of Ofcial temperature and, 426427
Analytical Chemists AvCAAS (see Availability corrected
digestibility, 369 amino acid score)
Dumas assay, 30, 34,36 Azo dyes, 133134,137
dye binding assay, 149, 151
gluten ELISA, 320321, 324325 B lymphocytes, 228229
guidelines, rat bioassay and, Baby foods
351353 Dumas assay, 38
immunodiffusion assay, 230 meat protein detection in, 262
Kjeldahl assay, 15 peanut allergens in, 311
factors and, 10 Baby formula, 3738
Udy assay, 149 Bacteria, feedstuffs quality and, 388
Index 449

Baker's asthma, 312 b-Lactoglobulin, 149, 182,

Barley digestibility, 309
biuret assay, 55 dye binding, 149
Dumas assay, 36 Binding constant, protein-CBBG, 177
dye binding assay, 153 Binding sites, dyes, 140
Kjeldahl analysis, 8 Binding, T-azo-R, 177181
protein analysis, 7, 8 Bioassay, 387, 388
rapid assays for analysis, 7 chick, 354
Udy assay, 152 human, 341
BCA assay, 99122 protein quality and, 346354
algae protein, 121 rat 348353
animal carcass protein, 119121 Biological value (BV), 342
automated, 113 Bio-Rad LTD, 207
calibration features, 109 Biotin-streptavidin detection, 261262
copper analysis, 100 2,20 -Biqinoline, Lowry assay and, 76
ow injection analysis, 114116 Birds nest soup, allergy, 305
interferences, 110111 Bitter, Bradford assay, 207
lysine and, 119 Biuret assay, 47
mechanisms, 105 barley analysis, 55
metal ion catalyzed oxidation, 107 casein determination, 49
method, 104 cereal proteins, 57
microwell plate format, 113115 protein solubility determinations by,
phenolic compounds effects, 118 63
reducing compounds and, 106 Biuret, structure, 47
sample pretreatment for, 112 Boiling and ethanol resistant antigen,
serum copper, 102 236, 239, 240, 260
solid phase assay, 117 Bone meal, 271
sugars and, 103 Bovine spongiform encephalopathy (see
TCA-DOC precipitation and, 112 BSE agent)
BCA, derivatives, 101 Bradford assay
BE antigen (boiling and ethanol resistant advantages, 169
antigen), 236, 239, 240, 260 beer protein, 204205
Bean protein, Bradford assay, 213 beverages and, 190
Beans, PDCAAS value, 437 bread crumb, 212
Beef calibration features, 201
analysis, ORBIT, 237 carotenoids and, 189
croquettes, soy protein detection in, Chardonnay wine, 211
304 compatible solutes, 186, 187
protein, dye binding assay, 157 effect of SDS, 187
Beer interferences, 186
Bradford assay, 190, 204206 legumes, 213
celiac disease and, 312 lipids and, 191
Dumas analysis, 30, 36 mechanisms, 172
Kjeldahl analysis, 8, 14 microassay format, 196
Lowry assay, 88 modied, 196
450 Index

[Bradford assay] [Calibration]

monosaccharides and, 190 Lowry assay, 72, 7780
mungbean our, 213 meat ELISA, 253
mushrooms, 216 pork ELISA, 257, 267
pinot noir, 211 soy ELISA, 284
polypeptide selectivity, 171 Udy assay, 154
polyphenols and, 209210 Camel, ELISA, 252, 254, 255, 260
polysaccharides and, 186, 191, 199 Canned baby foods, ELISA and, 262
reagent pH, 172 Canned sh, ELISA, 268269
sensitivity, 202203 Canned foods
solid phase, 185 digestibility, 370
standard method, 196 Kjeldahl analysis, 16
wavelength of, 173175 Canned milk, 150
Bran protein, 55 Canned tuna, 268
Bread crumb, Bradford assay and, 212 Carbethoxylation, sh meal, 363
Bread making quality, 212213 Carbohydrates
Bread mix, gluten free, 321 available lysine determination and,
Bread wheat, 57 359, 362, 366
Brest milk, 318 Lowry assay and, 81
Brewing grains, Dumas assay, 30, 33, 36 Carbonyl-amine reaction, (see also
British beers, Bradford assay, 208 Maillard Reaction), 416418
BSA binding, CBBG, 185 Caroteinoids, Bradford assay and, 189
BSE agent, 271273 Casein
bone meal and, 271 biuret assay, 49
immunological detection, 272 digestibility, 370
test kits for, 271273 dye binding assay, 126
Buckwheat, celiacs and, 320 PER value, 349
Buffalo, antigen, 238 protein quality, 344
Buffer effects, Lowry assay, 81 Udy assay, 138, 149
Butter milk, dye binding assay, 148 Catalysts, Kjeldahl assay and, 9
BV (see Biological value) CBBC sources, Bradford assay and,
197198
Cake our, gluten free, 321 CBBG, bi-ionic form, 172
Calibration, 37 Celiac disease, 312313
ammonia analysis and, 19, 20, 23 Celiac Society of Great Britain, 322
BCA assay and, 109110, 113 Celiac-negative cereals, 323
biuret assay for rice, 5859 Cereal products
Bradford assay and, 201 Bradford assay, 212
denitions, 3 PNV, 398401
Dumas assay and, 35 Cereal proteins, classication, 314
dye binding assay, 129, 131, 154, 155 Cereals
ELISA format and, 270 allergens, 312329
gliadin ELISA, 323 biuret assay, 57
gluten ELISA, 323 Lowry assay, 88
horse meat ELISA, 267 Udy assay, 151
Index
Cerelac, 397
Chardonnay wine, Bradford assay, 211
Cheddar cheese, dye binding, 150
Cheese, 151
Chemical deterioration, proteins,
416420
Chemical score, 429431
egg protein and, 354
Chemistry
biuret assay, 50
Lowry assay, 77
Udy assay, 133147
Chick bioassay, protein quality, 353354
Chick pea, Udy assay, 153
Chicken
antigen for, 238
determination amino acid analysis, 27
dye binding assay, 157, 159
immunodiffusion assay, 240
Chlorophyll, Lowry assay and, 8182
Chocolate
bars, ELISA, 310
gluten ELISA and, 320, 321
milk, dye binding assay, 148
peanut allergens in, 310311
Chromatographic determination,
available lysine, 360361
Circular dichroism, 317
Citations off, Lowry assay, 71
CNS tissue in feed, 272
Cod llet, dye binding assay, 157
Codex Alimentarius Commission,
allergens, 298
Collaborative testing
Bradford assay, 205206
Dumas assay, 30, 34, 36
gluten ELISA, 322
ice cream analysis dye binding, 150
PNV, 390
soya protein ELISA, 282
three-enzyme assay for digestibility,
370
Collagen
Lowry assay, 93
PDCAAS value, 436437
451
[Collagen]
quantitative amino acid analysis,
2728
Colorimetric assay, ammonia, 1823
Colorimetric Kjeldahl analysis, 1825
Combustion analysis, milk products, 37
Combustion nitrogen analyzer (see
Dumas assay)
Commodities, Udy assay, 128
Compatible solutes, Bradford assay,
186, 187
Compton-Jones scheme, CBBG,
172173
ComputedPER index (see c-PER)
Conarachin, 306
Condensed milk, 151
Confectionary, gluten ELISA, 322
Confectionary products, ELISA, 311
Conformational stability, 425
Conglycinin, 285289, 292
renaturing efciency, 288
Conversion factor, Kjeldahl assay, 10,
12
Cooked beef, ELISA, 260
Cooked meat
antigens for, 236, 238
immunoassay, 239, 247, 260, 268
soy allergens and, 303
Cooked poultry, ELISA, 261, 262
Cooking oil, peanut allergens and,
310
Cooking, gluten analysis and, 326
Coomassie Brilliant Blue G250, 169
Copper analysis, BCA assay and, 101
Copper complex, Biuret assay, 5052
Copper concentration, Lowry assay and,
82
Copper hydroxide, biuret assay, 48
Corn our, gluten detection in, 322
Corn meal, BCA assay, 117
Corn protein, Bradford assay, 212, 213
Correlation coefcient, denition, 5
Cortecs, 262, 271
gluten ELISA kit, 319
peanut ELISA kit, 311
452 Index

[Cortecs] Detergent effect, Bradford assay,

pork ELISA kit, 271 187188
Costs Dialysis assay, digestibility, 373
Dumas assay, 32 Differential dye-binding capacity
PNV tests, 429 (d DBC), 362365
Cottage cheese, dye binding, 150 Differential scanning calorimetry (see
Cottonseed, available lysine, 360 DSC)
Cowpea, Udy assay, 154155 Difculties, Udy assay, 130
c-PER, 433 Digestibility
Creutzfeldt-Jacob disease, 271 denitions of, 367368
Cross reactivity (see Specicity) peanut allergens, 309310
Cross-linking, 418419 three enzyme method for, 369372
Crystal violet, protein binding and, Dimethyl sulfoxide and Lowry assay, 82
172 Diphtheria toxin, 231
Crystalline-amorphous transition, 425 Disulde bonding
Cyoprotectants, Lowry assay and, 82 gluten and, 315
Cysteine, racemization, 418 in soybean protein, 285, 288
Donkey, ELISA, 256, 262
Dairy products Double immunodiffusion, 230
Dumas assay, 30 Dried milk, 344
dye binding, 147 Kjeldahl assay, 13
Dairy proteins, Biuret assay, 63 PNV for, 382384
DBC (see Dye binding capacity) DRIFT, 235
d DBC, differential dye-binding capacity DSC, soy protein, 286
available lysine determination and, DSC2, Tg measurements, 422
362365 Dumas assay, 29
legumes and, 364 Advantages of, 31, 36
soybean meal and, 363 atmospheric error and, 33
total lysine and, 364 beer protein, 30, 36
Deer analysis, DRIFT, 237 feedstuffs, 34
Defatting, meat, 61 instrumentation, 3133
Degree of hydrolysis, 373 ketchup, 30
Dehulling, PNV and 395 malt, 30
Dehydrogenation, Lowry assay, 75 materials, 32
Denaturation, 414419 milk products, 37
meat antigens, 263265 oilseed protein, 33
peanut allergens, 308309 semolina, 38
processing and, 415 Dye binding assay
Denaturation temperature (TD), soy beef protein, 157
proteins, 286287 calibration with Kjeldahl, 150,
Denaturation-renaturation, soy 152155
antigens, 284 chicken meat, 157, 159
Denaturing agents, 414 cod llet, 157
Dent CO251 (see also Maize), 435 Dye binding lysine, 362
Design, Lowry assay, 69 Dye equivalent weight, 129
Index 453

Dye purity, Bradford assay and, [ELISA]

197198, 200 analysis, 256, 257
pyruvate kinase as antigen, 258
Effect of dye volume, Bradford, 201 raw meat, 252, 255
Egg albumin, 344 red snapper, 270
biuret assay, 48, 63 reviews, 250
Bradford assay, 217 rock shrimp, 269270
Egg allergens, 305, 309 sardine, 268
Egg products, dye binding assay, 159 sea food, 268270
Egg protein, 290, 292, 346, 347, 392 sheep, 256
Udy assay, 159 soy protein, 281296
Egg ratio, 354 ELISA test kit
Egg white allergens, 300 BSE, 273
Egg yolk, Bradford assay and, 201 deer meat, 262
Egyptian legumes, PNV, 394 gluten, 323
Electrophoresis (see also SDS-PAGE) meat, 262
CBBG, 170 allergens, 310
samples, Bradford assay, 198, 199 peanut, 311
ELISA pork, 262
aatoxins, 251 soybean, 283284
bacterial toxins, 25 tuna, 268
boiling resistant antigen, 260 working range, 254, 320
BSE agent, 271273 Emulsion proteins, Lowry assay, 90
buffalo, 256 End-point temperature, meat ELISA,
meat, 260 265
cattle, 256 End-point temperatures, Biuret assay,
commercial, 282 62
competitive, 248 Enzyme linked immunosorbent assay
confectionary products, 311 (see ELISA)
cooked beef, 262 Enzyme, protein digestibility and, 366
meat, 260265 Equations, Udy assay, 141142
pork, 262 Equilibrium, Udy assay, 138, 156
poultry, 262 Errors
enzyme substrates, 254 chemical score, 355
format, 248250, 255 PER determination, 350
game meat, 260 Essential amino acids, 355
gliadin, 317329 Ethyl vinyl sulfone (EVS), 356
gluten, 317329 Evaporated milk, protein analysis,
horse meat, 252256, 258, 260263, 149
266268
muscle antigen for, 257260 FAO/WHO/UNU, 433
Ochratoxin, 251 Fatalities, peanut allergy, 306
performance characteristics, 270 FDA, 298
peroxidase assay and, 254 FDNB, 1-Fluoro-2,4-dinitrobenzene
pork, 260 assay, 356
454
[FDNB]
available lysine determination,
356359
correction factor, 359
Fecal nitrogen, 367, 368
Feed supplements, 386
Feedstuffs and concentrates, 386393
Feedstuffs, 387
Dumas assay, 30, 33, 34, 35
dye binding assay, 156, 390
Kjeldahl analysis, 16
Orange G binding, 388390
PNV evaluation, 346, 387393
Udy assay, 156
Fermented soybean products,
allergenicity, 304
Fertilizer, Dumas assay, 30
FIA, BCA assay using, 114
Ficol, Lowry assay, 82, 83
Fish, allergens, 298
Fish and seafood identication, 268271
Fish gelatin, 311
Fish meal, 387, 388, 390
Acid Orange 12 binding, 390
DBC, 389
Kjeldahl analysis and, 15
Udy assay, 138, 156
Fish protein, Tg, 423
Fish protein concentrate, 268
Fish sausages, soy protein detection in,
304
Flint CO255 (see Maize), 435
Flow injection analysis (see FIA)
Flower bud protein, quantitative amino
acid analysis, 27
1-Fluoro-2,4-dinitrobenzene (see
FDNB)
Folin-Ciocalteu reagent, 71
Food allergy, 297
intolerance, 297
matrix, protein quality and, 420425
Foods, Dumas assay, 30
Freeze dried proteins, 425
Fringe-micelle model, 421
Frozen dessert, 151
Index
Fruit juice, Bradford assay, 211
Game meat, ELISA, 260
Gel immunodiffusion assay, cooked
meat, 239241
Gelatin
BCA assay, 105
Biuret assay, 49, 56
complexes with Amido Black 10B, 147
feedstuffs and, 156
Lowry assay, 93
Gelatin granules, 126
Gel-ltration, beer, 206
Glass transition temperature (see also
Tg), 420425
Gliadin, 314317
in human milk, 318
Tg value for, 424
Gluten, 315
available lysine, 360
confectionary products and, 322
ELISA, 317329
home test, 323324
preparation, 315
standards for ELISA, 321
Tg value for, 421
Gluten-free foods, 313, 318, 322, 323
Gly m Bd 30k, as soybean allergen,
301304
Glycation, Bradford assay, 190
Glycinin, 285289
Gordon-Taylor equation, 422425
GPV (Gross protein value), 342
Grain and cereal, Kjeldahl assay, 13
Grains, Dumas assay, 33
Grape juice, Bradford assay, 210
Graphical analysis, Udy assay, 144145
Gross protein value (see GPV)
Ground rice, biuret assay, 59
Half and half milk, protein assay, 149
Hamburger, meat identication and, 235
Heat effects, soy antigens, 287
Heat resistance, conglycinin, 288
Histidine, dye binding and, 135, 139
Index
Home test, gluten, 323324
Honey
Bradford assay, 217
Kjeldahl analysis, 217
Horse
antigen production using, 231
ELISA, 267
immunoassay, 234
immunodiffusion assay, 239
meat, 252256, 258, 260263, 266268
meat protein, immunoassay, 252256,
258, 260263, 266268
muscle antigen, identity, 258, 261
muscle protease, biuret assay and, 62
protein assay, 235
Horseradish peroxidase, 253254
Human milk, gliadin detection in, 318
Hybridoma, 229
Hydrogen peroxide
Biuret assay, 55
Kjeldahl assay and, 9
Hypoallergenic bread, 316
Ice cream dye binding, 148
protein, 150157
Udy assay, 148, 150151
Immunization schedule, 231, 233
Immunoabsorption, 232
Immunoassay, principles, 226
Immunoblotting, 299300, 307
Immunodiffusion assay
chicken meat, 240
meat, 230
Immunological assays, disadvantages,
250
Immunology, 228
Imperial Chemical Industries (ICI), 169
In vitro digestibility, 368374
protein stability and, 413
protein structure and, 413
Incidental additives, 298
Indanetrione assay, Kjeldahl assay, 23
Indophenol reagent, Kjeldahl assay, 18
Infant formula, 384386
amino acid supplementation, 386
455
[Infant formula]
Dumas assay, 3738
peanut protein detection in, 306
PER, 350
PNV bioassay, 385
rat bioassay, 385
Infrared analysis, cereal proteins, 7
Insect protein, Bradford assay, 199
Insoluble protein, Bradford assay, 217
Instant breakfasts, Kjeldahl assay
analysis, 7
Interferences
available lysine, 362, 363, 366
BCA assay list for, 111
biuret assay, 5355
Bradford assay, 186191
d DBC, 363
Lowry assay, 80
ninhydrin assay, 23
plant dyes as, 57, 58, 59
Udy assay, 147
Isobestic point, 140, 172
IVPD (see In vitro digestibility)
Kangaroo meat, immunoassay, 234, 235
Ketchup, Dumas assay, 30
Kinetics, 395
Lowry assay, 77
proteolysis, 412414
Kiwi fruit juice, Bradford assay, 211,
212
Kjeldahl analysis, 1, 725, 344
barley, 8
beer, 8, 14
catalysts, 9
colorimetric, acetylacetone and, 22
comparison with dye binding assay,
150, 152, 153
honey, 217
gliadin, 327
Kjeldahl factor and, 10, 12
nitrogen-protein conversion factor,
10, 12
PER values and, 349
PNV determination, 349
456
[Kjeldahl analysis]
reactions of, 9
reliability, 7
sausages, 17
Kjel-foss, Instrument, 15
Labeling, food allergens, 297
Lactalbumin, PER value, 349
Lactose, protein quality and, 382,
385
Larger, Bradford assay, 208
Leaf protein, Lowry assay, 88, 90
LECO FP-2000, Dumas assay, 31
LECO FP-228, Dumas assay, 33
LECO FP-428, Dumas assay, 3132
Legumes
available lysine, 394395
baking and PNV of, 395
Bradford assay, 213
dehulling and PNV, 395
dye biding, 153156
ELISA and, 312
Lowry assay, 88
PNV, 393398
roasting and PNV, 396
sprouting and PNV, 395
steaming and hydrothermal
treatment, 395
Lesions, celiac disease, 312313
Linear dynamic range, 5
Linear free energy relations, amino
acids, 418
Linearity, Bradford assay, 201
Lipid
Bradford assay and, 191, 200
interferences by, 84
Lower limit of detection (LLD)
denition, 3, 6
ELISA, 265
gluten ELISA, 320, 324
soy bean ELISA, 284, 290
Udy assay, 152
Lowry assay
buffers and, 81
carbohydrates and, 81
Index
[Lowry assay]
cereals, 88
chlorophyll and, 8182
citations of, 71
cryoprotectants and, 82
design, 69
emulsion proteins, 90
leaf protein, 90
legumes, 88
mechanisms of, 77
nondairy creamers, 90
sample pretreatments for, 8687
single cell protein, 91
Lysine
BCA assay and, 119
bioassay and, 344
CBBG binding and, 171
determination, 356366
dye binding and, 135, 138
glycation and Bradford assay, 190
loss, Arrhenius plot 385
Maillard reaction, 382, 416418
available lysine, 366
pH and, 417
Maize
available lysine, 360
cultivars, 435
Dumas analysis, 29
PNV, 399
Malt
Dumas assay, 30, 36
dye biding, 152
Materials, Dumas assay, 32
Matrix effects, protein deterioration
and, 420
Mean residue weight, tables of values, 27
Meat, 247281, 284, 303
analysis for soybean, 285
antigenic components, 232, 251
BCA assay and, 119121
biuret assay, 6163
Dumas analysis, 29
dye binding assay, 157
gel immunodiffusion assay, 230
Index
[Meat]
quantitative amino acid analysis, 27
sample pretreatment for ELISA, 284
soy protein detection in, 303
Udy assay, 156157
Meat analogue, Kjeldahl analysis, 16
Meat and bone meal, PNV, 393
Meat and meat products, Kjeldahl
analysis, 15
Meat antigen, 231
Meat rendering plants, 272
Meat speciation, 247280
Meatballs, soy protein detection in, 304
Mechanism
biuret assay, 50
Lowry assay, 73, 77
Udy assay, 133147
Melanoma, 229
Metachromasia, Crystal violet, 174
Metal ion catalyzed oxidation, Lowry
assay, 73
Michaelis-Menten kinetics, 414
Microassay, Bradford, 196
Microbiuret assay, 56
Micro-Kjeldahl analysis, 21, 13
Microwave heating, 396
Microwell plate, BCA assay using, 113
Milk, 282
allergens, 298, 300
ELISA, 282
powder, 381
powder, PER, 350
prices and protein content, 125
products, Dumas assay, 37
protein, dye binding assay, 147, 148
protein, ELISA plate coating, 248,
272
protein, Udy assay, 147151
soymilk detection in, 281
Millet
PNV, 399400
protein, biuret assay, 60
Mince, soy protein detection in, 284
Model wine solution, Bradford assay,
211
457
Moist heating, allergen stability and,
308
Moisture
food deterioration and, 416
peanut allergens and, 308
PER values and, 350
PNV and, 383384
rapeseed heat damage and, 392
Moisture-temperature-time relations,
411
Molten globule and digestibility,
412415
Monoclonal antibodies
development for ELISA, 265268
gluten ELISA and, 325327
meat analysis and, 265267
pork ELISA and, 267
soy bean ELISA and, 290292
turkey analysis, 266
Monosaccharides, Bradford assay and,
190
Multiple processing, legumes, 397
Mungbean, Bradford assay, 213
PNV, 394
Muscle lactate dehydrogenase, ELISA,
264, 265
Mushrooms
Bradford assay, 216
Kjeldahl analysis, 160
quantitative amino acid analysis, 27
Udy assay, 160
Myoglobin, thermostable antigen and,
238, 239
Naphthylamine brown, 126
NB, nitrogen balance, 342, 346347, 367
NDI, nitrogen digestibility index, 373
Nessler's reagent, Kjeldahl method and,
21
Net protein utilization (NPU), 342
Ninhydrin reagent, Kjeldahl method
and, 2324
Nitrogen balance (NB), 342, 346347,
367
Nitrogen digestibility index (NDI), 373
458 Index

Nondairy creamers, Lowry assay, 90 Peanut allergy, 305312

Nonanimal protein ingredients, 281 average fatalities, 306
Nonfat milk, 149 in infants, 306
NPU, net protein utilization, 342 in pregnancy, 306
Nucleic acid, Bradford assay and, 191 Peanut protein, in confectionary, 311
Nuts Peanuts, chemical score, 429
allergens and, 300301 Pepsin digest dialysate index (PDD
ELISA, 310312 index), 432
digest residue index (PDR index), 432
Oats pancreatin digestion index (PPD
Ac Hill, 436 index), 432
Ac Lotta, 436 Peptide antigens
Ac Percy 436 gluten ELISA and, 324327
biuret assay, 60 soybean ELISA and, 290
PDCAAS value, 436 Peptidyl sites, digestibility and, 414
Oilseeds, Dumas assay, 30, 33 PER (Protein efciency ratio), 342,
OPA (see o-Phthaldehyde) 348351
o-Phthaldehyde, 356 AOAC guidelines, 351
Orange G complex foods and, 350
binding, PNV, feedstuffs, 389390 rat acclimation period, 350
structure of, 136 sweetened foods and, 350
Udy assay using, 130 Perilla, 396
ORBIT, 235 Peterson's Lowry assay, 72
Ovalbumin, 182 Phenol red, 125
Phenol
pAb, for detection of donkey muscle Bradford assay and, 188
protein, 256, 262 BCA assay and, 119
PAGE, 81, 402 Phospholipids, Lowry assay, 84
Pavalbumin, allegen and, 300 Phosphothreonine, racemization,
PDCAAS, 411, 427, 429, 433438 418
applications, 434439 Pierce Warriner LTD, 207
beans, 437 Pinot Noir, Bradford assay, 211
benets, 434 Pioneer 3953 (see Maize), 435
calculation, 434 Plant colors, Biuret assay and, 55
collagen, 436437 Plant metabolites, Bradford assay,
maize, 434 188189
oats, 436 Plant proteins, 221
rice, 434 Amido Black 10B binding, 130
Vigna, 434 Plasticizer, water as, 420
PDR index (see Pepsin, digest residue PNV (protein nutrient value), 341380,
index) 411
Pea globulin, ELISA, 290, 291 amino acid losses and, 344345
Peanut allergens, 300, 306308 animal tests, 348354
cooking oil and, 310 assessment by PDCAAS, 434
preparation, 307 bioassays, 346354
Index
[PNV (protein nutrient value)]
consumer factors and, 343
factors affecting, 343
food labeling and, 344
human assays and, 346348
hygiene and, 343
indicators for, 342
infant formulas and, 384
insect infestation and, 400
Kjeldahl analysis and, 349
legumes, 393398
literature, 345, 348349
meat and bone meal, 393
pregnant and lactating women and,
344
processing and, 381402
quality control and, 344
rat bioassay, 348
soaking and, 395
Polyacrylamide gels, CBBG stain, 170
Polyamino acids, dye binding and, 171,
181, 183
Polyclonal antibody production, 232
Polyethylene glycol, 132
Polymer chemistry, 420
Polymer science approach, 424
Polymerase chain reaction, 224
Polyols, 133
Polypeptide selectivity, Bradford assay
and, 171
Polyphenols, Bradford assay and,
209210
Polysaccharide interference, Bradford
assay, 186, 191, 199
Polyvalent antigen, 229
Porcine rind protein, 27
Pork
analysis, PRIME, 237
dye binding assay, 157
ELISA, 267268
thermostable antigen, 238
Potato protein
Bradford assay, 214216
chemical score, 429
Kjeldahl analysis, 13
459
[Potato protein]
Lowry assay, 88
Poultry analysis, PROFIT, 237
Poultry, ELISA for, 266267, 268
PPPD index, 432
Precision, protein assays and, 5
Preservatives, milk, 148
Prionics AG, 273
Procedure
biuret assay, 49
Bradford assay, 195
Lowry assay, 70
PDCAAS and, 433434
soy protein antigen preparation, 284
Udy assay, 128
Processed foods, 2
digestibility, 370371
gluten detection in, 310, 320
interferences in, 80
Kjeldahl analysis, 16
PROFIT, 235
Pro-meter instrument, Udy assay, 153
Propionic anhydride, acylation, 363
Protein
analysis, signicance, 2
assay, characteristics, 2
binding, CBBG, 173, 175, 177
binding, dyes, 135
concentrates, 386393
digestibility, 366, 411
digestibility corrected amino acid
score, (see PDCAAS)
efciency ratio (see PER)
haze, 209
immunoassay, 225
M, shrimp antigen, 269
molecular weights, 57
nutrient value (see PNV)
precipitation, Bradford assay, 199
carbonyl-amine reaction and,
417418
factors inuencing, 343
milk, 381
segmental mobility, digestibility and,
413
460 Index

[Protein] Reverse phase high pressure

stain, CBBR, 170 chromatography (see RP-HPLC)
Protein-protein-variation, Reviews
BCA assay, 105 celiac disease, 313
Bradford assay, 197, 201, 212 dye binding, 126127
Protein-quinone interactions, 210 peanut allergy, 306
Proteolysis, Lowry assay, 9091 protein quality, 345
Purication, Coomassie Brilliant Blue, Rice, 402, 435
200 beans, protein, 395
protein
QPM (see Maize), 435 biuret assay, 59
QSDS-PAGE, 7 Udy assay, 153
Quantitative amino acid analysis, pork PDCAAS value, 435
rind, 27 PNV and, 399
Quantitative sodium dodecylsulfate RNV, 342
polyacrylamide gel Rock shrimp, 260270
electrophoresis (see QSDS- RP-HPLC
PAGE) available lysine, 361
gliadin analysis, 327
Racemization RPV, 342
amino acids and, 418419
cysteine, 418 Salt effects, Bradford assay, 201
factors controlling, 418419 Sample calibration, 4, 7
rate prediction, 418 Sample pelleting, Dumas assay, 33
Radcliff Inrmary, 322 Sample pretreatment
Radioallergo absorbent test (see RAST) beer, Bradford assay, 205
Rapeseed cereals, 55
heat damage, 392 corn meal, BCA assay 117
meal, PNV, 390392 Lowry assay and, 8687
protein, Udy assay, 154155 meat, 261
RAST, 299 Biuret analysis, 61
Rat bioassay, 411 ELISA, 285
Raw meat, ELISA, 252, 255 Plasma, 49
R-Biopharm, ELISA test kit, gluten, soy bean product, 284
320 Sardine, 268
Reactive lysine (see also Available Sausages
lysine), 356 dye binding, 159
Red snapper, 270 gluten and, 313, 321
Red wheat, Udy assay, 153 Immunodiffusion assay, 239
Relative nutritive value (see RNV) Kjeldahl analysis, 17
Relative protein value (see RPV) soy protein, 284, 289, 290
Reliability Udy assay, 159
Kjeldahl assay, 7 Scenario, dye binding, 141143
Udy assay, 152 Schecter and Berger scheme, 414
Renaturing buffer, 284 Screening, high-lysine cereals, 401402
Index
SDS, Bradford assay, 187
SDS-PAGE, 300, 307, 309
antigen analysis, 237
peanut allergens, 309
wheat allergen, 315317
Seal meat, immunodiffusion, 239
Secondary structure, Gliadin, 317
Seed globulins, ELISA and, 291
Seharawi, celiac disease and, 312
Semolina, Dumas assay, 38
Sensitivity, 1, 5
BCA assay of copper, 102
biuret assay, 55, 56
Bradford assay, 177, 179, 183185,
197, 201204
colorimetric Kjeldahl assay, 18
dye binding, 131,132, 133
gluten ELISA, 329
immunoassay, 227
Lowry assay for wheat protein, 89
meat ELISA, 260
protein analysis, 1, 2, 56
Udy assay, 137
Serine, racemization, 418
Sesame our, Udy assay, 155
Sheep antigen, 231
Sheep, ELISA, 249, 252, 255256, 257,
260, 261, 262, 268
Sheep serum albumin (SSA), 253
Shrimps, 269, 300, 301
Single cell protein, Lowry assay, 91
Skimmed milk
dye binding assay, 151
Kjeldahl factor for 12
Slope assay, protein quality, 347
Small animal bioassay, 348
Soaking, PNV and, 395
Sodium borohydride, available lysine,
365366
Sodium dodecyl sulfate (see SDS)
Solid phase assay
Bradford assay, 185
Udy assay, 131133, 143
Solubility relations, Udy assay, 143
Soluble complexes, Udy assay, 138140
461
Sorghum, 399400
biuret assay, 60
Soup, gluten detection in, 322
Soy
bean allergens, analysis, 303305
ELISA
cooking and, 283
specicity, 283, 291
our, Udy assay, 153
products, ELISA, 289
protein, 285286
in sausages, 289
thermal denaturation, 286287
protein antigen, 284, 285
Soybean
ELISA, 281296
7S protein, 285289
Udy assay, 138
Soybean meal, Orange G binding, 389
Soybean protein, available lysine, 360
Soymilk, 282
Species identication eld test (see
SIFTS), 235
Specicity
denition, 3
gluten, ELISA, 318, 325, 326
meat ELISA, 256, 263
peanut ELISA, 311
soya bean ELISA, 283
Spray dried milk, 382383
dye binding, 129, 130,147
PNV, 382
Udy assay, 129
Sprouted cereals, protein quality, 345
St. Bartholomew's Hospital, 322
St. James University Hospital, 322
Standard assay, Bradford, 196
Starch interferences, 55
Statistical principles, 4
Steaming, PNV and, 395
Stiffness parameter, 423
Stout, Bradford assay 207
Structure
Acid Orange 12 dye, 136
Acid Red 1, 135
462 Index

[Structure] [Thermostable antigen]

Amido Black 10B, 137 soybean and, 289292
bicinchoninic acid, 100 Three enzyme assay, in vitro
biuret, 47 digestibility, 369372
CBBG, 170 TNBS assay, 119, 356, 361362, 365,
CBBR, 170 369
copper-biuret complex, 47 Tomato protein, Dumas assay, 30
dehydropeptide, 75 Tomato seed, Lowry assay, 88
iminopeptide, 75 Tortillas, 399
Orange G, 136 Total digestibility co-efcient (see
T-azo-R, 135 TDC)
Sudanese legumes, PNV, 394 TPD (see True protein digestibility)
Sulfhydryl compounds Trace allergens, 297
BCA assay and, 118 Trinitrobenzene Sulfonic acid, (see
Lowry assay and, 8485 TNBS)
Sulfhydryl group, dye binding, 126 Troponin, meat antigens and, 236,
Sulfhydryl-disulde exchange, 317, 415 238239
Symptoms Troponin T, ELISA antigens and, 261
celiac disease, 312313 True protein digestibility, 367, 433
food allergy, 298 Trypsin inhibitors, protein digestibility,
peanut allergy, 305 372
Tuna, 268
T-azo-R, 134 PER value for, 352
TCA-DOC precipitation soy protein in, 305
Bradford assay and, 118, 195, 199200 Turkey sausages, 239
Lowry assay and, 72 Two-state denaturation, 412, 414415,
TDC (total digestibility coefcient), 342 425
Tectator heating block, 21 Tyrosinase, copper detection in, 99
Test kits, species identication and,
235236 Udy assay, 125126, 138
Tg (glass transition temperature), barley, 152
420425 cereal proteins, 151
sh protein hydrolysate, 423 difculties, 130
gluten, 421 dye-protein solubility and, 143
moisture and, 422 equations for, 141142
protein quality and, 420425 sh meal, 138, 156
proteins and peptides, 421 mechanisms of, 133137
Theory (see Mechanism) milk protein, 147151
Thermal reliability, 152
conductivity detectors, 30 sausages, 159
denaturation, peanut allergen, various commodities, 128
308309 UHT milk, protein quality and, 382
stability, meat antigens, 263265 Ultraltration, 211
Thermostable antigen, 238 Uncooked meat, ELISA, 254257
meat and, 261 immunoassay, 234
Index 463

Water [Wheat]
ammonia determination in, 1920 biuret assay, 53, 5760
PER and, 350, 352 dye binding assay, 151
Water activity, 383384 reliability of analysis, 7
Maillard reaction and, 383384, 417 Udy assay, 151
Water holding, 281 Whey protein, Bradford assay, 217
Water supply, Kjel-Foss instrument and, Whole milk, dye binding, 149
15 William-Landel-Ferry kinetics, 420
Weaning foods, PNV, 397398 Wine, Bradford assay, 190, 209212
Western transfer, 273
Whale meal, 387 Yeast protein, Lowry assay,
Wheat, 345 6364, 91
allergens of, 314317
allergy, 312313 Zein, biuret assay, 49