ISOLATION, IDENTIFICATION AND CHARACTERIZATION OF BIOACTIVE PEPTIDES FROM FERMENTED MILK, DETERMINATION OF ITS GASTROPROTECTIVE, IMMUNOMODULATORY AND ANTI-GENOTOXIC

ACTIVITIES
A THESIS Submitted by

R.BALAJI RAJA
In Partial Fulfillment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY

DEPARTMENT OF BIOTECHNOLOGY SCHOOL OF BIOENGINEERING SRM UNIVERSITY, KATTANKULATHUR- 603 203 MAY 2011

DECLARATION

I hereby declare that the dissertation entitled Isolation, identification and characterization of bioactive peptides from fermented milk, determination of its gastroprotective, immunomodulatory and anti-genotoxic activities to be submitted for the Degree of Doctor of Philosophy is my original work and the dissertation has not formed the basis for the award of any degree, diploma, associateship, fellowship of similar other titles. It has not been submitted to any other University or Institution for the award of any degree or diploma.

Place: Kattankulathur Date: 12.8.11

(R. Balaji Raja)

ii

SRM UNIVERSITY
KATTANKULATHUR 603 203

BONAFIDE CERTIFICATE

Certified that this thesis titled Isolation, identification and characterization of bioactive peptides from fermented milk, determination of its gastroprotective, immunomodulatory and antigenotoxic activities is the bonafide work of Mr. R. Balaji Raja who carried out the research under my supervision. Certified further that to the best of my knowledge the work reported herein does not from part of any other thesis or dissertation on the basis of which a degree or award was conferred on an earlier occasion for this or any other candidate.

(Dr.Kantha D.Arunachalam) SUPERVISOR

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ACKNOWLEDGEMENT
My heartfelt gratitude goes to my guide Dr. Kantha D. Arunachalam who introduced me in to the field of Medical biotechnology, constantly motivated and encouraged. My sincere thanks also goes to the management of SRM University for allowing me to carry out this work in their premises and to use their facilities. Support rendered by Dr.P.T.Kalaichelvan, Professor, CAS in Botany, University of Madras (Guindy campus) is mention worthy. The help and technical assistance provided during animal studies by Dr.Sekar, Veterinary Doctor, Mr. Prabhakaran, Technician, Mr.Patra, Lab technician, Mr.Jayaprakash, Lab assistant was invaluable. Special mention is required for Mr.S.Vijayaraghavan and

Mr.Narayanan, Stores assistants, Dept of Biotechnology (E&T), SRM University who were always kind hearted to help with the required chemicals, reagents and glass wares. My heartfelt thanks also goes to Dr. Maria John, Assistant Professor, Dept of Biotechnology, Shool of Bioengineering, SRM University who helped me with immunofluoresence assay deserves a credit. The support given by various 3rd year B.Tech (Biotech) students of SRM University such as R.Balaji, Abin Biswas, Akanksha Singh, and Chetna Sharma was of paramount importance. The role of Dr. Mary Mohan Kumar, Mr. Anbumani, IGCAR, Kalpakkam, Mr. Jayakrishna Kuruva, CIDR and Mr. D. Ashok Kumar, Research scholar, VIT University, Vellore whom put in their hard work for anti-genotoxic studies was
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critical for the work. A special thanks goes to my sister Ms.R.Archana, who helped me with the graphs. This work would have been impossible without support of my family members who were the pillars of moral support and motivation throughout. I am highly indebted to all my well wishers and friends who chipped in with whatever help was required at that moment of time without whom the work would not have shaped to its present form. Last but not the least I would like to thank god almighty whose presence and guidance has always helped me throughout the work.

(R. Balaji Raja)

ABSTRACT
Milk is a unique food comprising of proteins, carbohydrates, fats, vitamins and minerals. The average pH of the fermented milk by Lactobacillus acidophilus was 5.3 0.091, commercial curd Aavin was 5.3 0.185, commercial curd Dodla was 5.3 0.126 and Lactobacillus bulgaricus 5.16 0.095. The pH of the control sample, i.e. nonfermented milk was found to be 7.05. The mean titratable acidity of the fermented milk by commercial curd Dodla was 91.49 T (Toerners degree), commercial curd Aavin was 91.27 T, Lactobacillus acidophilus was 92.11 T and Lactobacillus bulgaricus was 93.52 T. The titratable acidity of the control sample, i.e. non-fermented milk was 50.78 T. The mean viscosity of the milk fermented by commercial curd Dodla was 7.72 mPas (Milli Pascal), commercial curd Aavin was 8.01 mPas, Lactobacillus acidophilus was 7.12 mPas and Lactobacillus bulgaricus was 7.13 mPas. SEM analysis of fermented milk was carried out to confirm the presence of Lactobacillus species in the fermented milk. After the fermentation process was complete in the milk, Isolation of CPP (Casein Phospho Peptides) was done from fermented milk based on enzymatic hydrolysis method. Antimicrobial activity of CPP was tested against two common clinical pathogens Escherichia coli (MTCC Number 443) and Pseudomonas sp (MTCC Number 1194) using zone of inhibition method. AAVIN CPP produced 14 and 16 mm of zone of inhibition with Escherichia coli and Pseudomonas species respectively whereas DODLA CPP produced 13 and 15 mm, L. acido. CPP produced 16 and 15 mm, L. bulg. CPP produced 12 and 15 mm zone of inhibition with Escherichia coli and Pseudomonas

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species respectively. HPLC and FTIR Analysis of four CPPs isolated from fermented milk by two bacterial cultures and two commercial curd inoculums was performed using milk as the control and they gave characteristic peaks for CPP. Molecular weight of the Casein Phospho Peptide isolated from the fermented milk was determined by SDSPAGE (Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis) and it was found to be 3.5 - 4.0 KD (Kilo Daltons). Animal studies were done to study the positive effect of CPP on weight loss and mortality rate in mice challenged with GUT Pathogens. Three common GUT tract pathogens, Escherichia coli, Salmonella sp. and Shigella sp. were used to challenge the mice. DODLA CPP produced the highest percentage increase in weight of mice as 7.22% fed with it for 10 days. All the four test batches of fermented milk CPPs produced substantial increase in the weight of albino mice in a feeding period of 10 days. As far as the weight increase over the 15 days feeding period, DODLA CPP produced the highest percentage increase in weight of mice as 12.06%. Results indicated that continuous intake of fermented milk products contribute to uniform increase in body weight. The mice mortality rate during post infection state indicated the gastroprotective effect of CPP against the GUT pathogens. Immunomodulatory effect of CPP was evaluated using immunofluorescence assay in which IgA secreting B-Lymphocyte were quantitatively computed. In L. acido. CPP 10 days fed mice infected with E. coli showed the highest number of secretary IgA cells in the intestine and least was observed in L. bulg. CPP 10 days fed mice. For 15 days feeding period of test batches, highest number of secretary IgA cells was produced by L. bulg. CPP and least being produced by L. acido. CPP. In Salmonella and Shigella sp. infected mice the highest number of secretary
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IgA cells was produced by L. bulg. CPP after 10 and 15 days feeding period. CPP was able to bring down the pathogen count in the visceral organs of albino mice compared to the control. Histopathological studies showed the cell protective potential of CPP in intestinal tissues of mice infected with GUT pathogens. The anti-genotoxic role of CPP was tested in albino mice using micronucleus assay where the number of micronuclei, binucleated and multi-nucleated erythrocytes formed was higher in the unfed control mice than the CPP fed test mice cells. CPP was proved of its immunomodulatory activity and anti-genotoxic nature. This potential can be harnessed to produce formulations of CPP based medications which can be used in GUT ailments replacing the conventional antibiotics and to develop a new class of nutraceutical anti-genotoxic which could be of immense help to workers exposed to low background radiation.

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TABLE OF CONTENTS

CHAPTER NO. 1. INTRODUCTION

TITLE

PAGE NO.

1.1 MILK CONSUMPTION AND COMPOSITION 1.2 COMPONENTS IN MILK AND THEIR HEALTH EFFECTS 1.2.1 Protein 1.2.2 Branched chain amino acids and other amino acids 1.2.2.1 Taurine 1.2.2.2 Glutathione (GSH) 1.2.3 Lipids 1.2.3.1 Fatty acids 1.2.3.2 Saturated fatty acids 1.2.3.3 Unsaturated fatty acids 1.2.3.4 Trans vaccenic acid (VA) 1.2.4 Phospholipids and glycosphingolipids 1.2.5 Minerals, vitamins and antioxidants 1.2.5.1 Calcium in milk 1.2.5.2 Selenium in milk
ix

4 4

5 5 5 5 5

6 6

1.2.5.3 Iodine in milk 1.2.5.4 Magnesium in milk 1.2.5.5 Zinc in milk 1.2.5.6 Vitamin E in milk 1.2.5.7 Vitamin A in milk 1.2.5.8 Folate in milk 1.2.5.9 Riboflavin in milk 1.3.5.10 Vitamin B12 in milk 1.4 BACTERIAL FLORA OF MILK 1.5 INTOLERANCE TO MILK COMPONENTS 1.6 INTOLERANCE TO MILK PROTEINS 1.7 PHYSIOLOGICALLY ACTIVE MILK PEPTIDES 1.7.1 Antihypertensive Peptides (ACE Inhibitors) 1.7.2 Antithrombotic Peptides 1.7.3 Caseinophosphopeptides (CPP)

6 7 7 7 7 7 8 8 8 8 9

9 10 11 12 13 16 17

1.7.4 Immunomodulatory Peptides 1.7.5 Opioid Milk Peptides 1.7.6 Miscellaneous Peptides 1.8 Global Probiotic food market in industrialized nations
x

1.9

Indian probiotic market

17

1.10 Current players in Indian Probiotic Market 1.10.1 Yakult danone 1.10.2 Amul 1.10.3 Nestle 1.10.4 Mother Dairy 1.11 Fermented milk 1.12 Motivation and Problem statement 2 REVIEW OF LITERATURE . 2.1 Milk and milk-derived products 2.2 Bioactive peptides 2.2.1 Definition 2.2.2 Mechanisms of production of bioactive peptides 2.2.3 Mechanisms of action of bioactive peptides 2.2.4 Bioactive peptide based commercial dairy products 2.3 Digestion of bioactive peptides 2.4 Bioactive peptide absorption 2.4.1 Physiology of the digestion of proteins and peptides 2.4.2 Physical and chemical characteristics of potentially absorbable bioactive peptides
xi

18 19 19 19 20 21

22

23 24 26 28 30

32 33

2.5 Bioactivities of milk and fermented milk peptides 2.5.1 ACE-inhibition 2.5.2 Immunomodulation 2.5.2.1 Immunomodulatory peptides from milk 2.5.2.2 Microorganisms for the production of fermented milk with immunomodulatory activity 2.5.2.3 Two examples of immunomodulatory Peptides derived from milk proteins 2.3.2.4.1 YGG peptide 2.3.2.4.2 -CN (193-209) peptide 2.6 Prebiotics and Probiotics 2.7 Fermentations and microorganisms 2.8 Probiotics and their role in the human health 2.8.1 GI tract and its Pathogens 2.8.1.1 Salmonella infection in human 2.8.1.2 Salmonella enterica serotype enteritidis 2.8.2 Therapeutic effects of probiotics 2.8.2.1 Acute gastroenteritis
2.8.2.2 Inflammatory bowel disease
xii

35

36 38

40

41 42 42 43

46 47 47 48 49 49

2.9 Allergic diseases 2.10 Lactic acid bacteria and the immune system 2.10.1 Role of cytokines in the immune response 2.11 Interactions between epithelial cells and intestinal 2.12 Casein Phosphopeptide and its uses 2.13 Anti-genotoxic role 2.13.1 Classification of radioprotective agent 2.13.2 Milk and fermented milk as an anti-genotoxic agent 2.13.3 Enzymes and their anti-genotoxic mechanism 3 4 OBJECTIVES MATERIALS AND METHODS 4.1 Selection of milk brands and culture sources 4.1.1 Production of fermented milk using different sources of bacterial cultures 4.1.2 Initial Standardization 4.1.2.1 pH 4.1.2.2 Titratable acidity 4.1.2.3 Viscosity 4.1.3 Microbiological analysis of fermented milk
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50 50 51 microflora 51 52

55 55 57 58

59 59

59 60 60 60 60

4.1.4 Isolation of CPP from fermented milk 4.1.5 Characterisation of four isolated CPPs 4.1.5.1 Antimicrobial activity of CPP 4.1.5.2 HPLC Analysis of CPP 4.1.5.3 FTIR Analysis of CPP 4.1.5.4 Molecular weight determination by SDS PAGE 4.2 Animal studies 4.2.1 Effect of CPP on weight loss and mortality rate in mice challenged with GUT Pathogens 4.2.1.1 Acclimatization of animals 4.2.1.2 Feeding with CPP 4.2.1.3 Challenging with GI Tract Pathogens 4.2.1.4 Pathogen count determination in visceral organs 4.2.1.5 Histopathological studies 4.2.2 Immunomodulatory role 4.3 Anti-genotoxic studies using CPP 4.3.1 Gamma irradiation Experiment with Animals 4.3.1.1 Experimental set up for mice 4.3.1.2 Experimental set up for fish
xiv

60

62 62 62 63

63

63 64 64 65 65 65

66 66 66

4.3.1.3 Irradiation with Co60 source 4.3.2 Micronucleus assay 4.3.3 Enzymatic assays 4.3.3.1 Oxidase test 4.3.3.2 Catalase test 5 RESULTS 5.1 Isolation and characterization 5.1.1 Initial Standardization 5.1.1.1 pH 5.1.1.2 Titratable acidity 5.1.1.3 Viscosity 5.1.2 Microbiological analysis of fermented milk 5.1.3 Yield and production cost of CPP 5.1.4 Antimicrobial activity of CPP 5.1.5 HPLC Analysis of CPP 5.1.6 FTIR Analysis of CPP 5.1.7 Molecular weight determination by SDS PAGE 5.2 Animal Studies 5.2.1 Challenging with GI Tract Pathogens
xv

67 68

69 69

70 70 74 75 76 76 77 79 84 90

91

5.2.2 Post infection studies 5.2.3 Determination of pathogen count in visceral organs 5.2.4 Histopathological studies 5.3 Determination of Immunomodulatory activity 5.4 Anti-genotoxic role of CPP 5.4.1 Micronucleus assay 5.4.2 Enzymatic assays 5.4.2.1 Oxidase enzyme test 5.4.2.2 Catalase enzyme test 6 DISCUSSION 6.1 General consideration 6.2 Initial standardization 6.3 Microbiological and SEM analysis 6.4 Anti-microbial activity 6.5 HPLC and FTIR analysis of CPP 6.6 Molecular weight determination by SDS-PAGE 6.7 Animal studies 6.7.1 Gastroprotective action against E. coli 6.7.2 Gastroprotective action against Salmonella sp.
xvi

108 132 138 146

154

166 166

168 168 169 170 171 171

173 173

6.7.3 Gastroprotective action against Shigella sp. 6.8 Pathogen count determination 6.9 Histopathological studies 6.10 Immunomodulatory activity 6.11 Anti-genotoxic role of CPP 6.12 Application to human model 7 8 CONCLUSION FUTURE PROSPECTS OF OUR WORK 8.1 Current status of probiotics in India 8.2 Factors favoring Indian probiotic market and its players 8.3 Challenges to be considered 9 10 LIST OF REFERENCES LIST OF PUBLICATIONS

174 174 176 176 178 179 180

183 184 186 187 216 217

11 PATENT FILED

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LIST OF TABLES NUMBER 2.1 2.2 TITLE Bioactive peptides from milk proteins Studies which have established the occurrence of peptides in various fermented milk products 2.3 2.4 2.5 Examples of ACE-inhibitory peptides derived from milk Immunomodulatory peptides derived from milk proteins List of microorganisms producing immunomodulatory activity from fermented milk 5.1 pH values of the fermented milk with time after added with Dodla dairy curd 5.2 pH values of the fermented milk with time after added with Aavin dairy curd 5.3 pH values of the fermented milk with time after added with Lactobacillus acidophilus culture 5.4 pH values of the fermented milk with time after added with Lactobacillus bulgaricus culture 5.5 The pH changes for milk fermented using commercial curds Dodla, Aavin, Lactobacillus acidophilus and Lactobacillus bulgaricus. 73 73 72 72 71 35 37 38 PAGE NO. 27 28

xviii

5.6

The titratable acidity values for milk fermented using commercial curds Dodla, Aavin, Lactobacillus acidophilus and Lactobacillus bulgaricus

74

5.7

The viscosity changes for milk fermented using commercial curds Dodla, Aavin, Lactobacillus acidophilus and Lactobacillus bulgaricus

75

5.8

Zone of inhibition formed by CPP against Escherichia coli and Pseudomonas sp.

78

5.9

Effect of AAVIN CPP on body weight of albino mice after feeding for 10 days

92

5.10

Effect of DODLA CPP on body weight of albino mice after feeding for 10 days

93

5.11

Effect of L. acido. CPP on body weight of albino mice after feeding for 10 days

94

5.12

Effect of L. bulg. CPP fed on body weight of albino mice after feeding for 10 days

95

5.13

Body weight of albino mice after feeding normal feed alone without CPP for 10 days

96

5.14

Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice after feeding for 10 days

97

5.15

The effect of AAVIN CPP on body weight in albino mice after feeding for 15 days

100

xix

5.16

The effect of AAVIN CPP on body weight in albino mice after feeding for 15 days

101

5.17

The effect of L. acido. CPP on body weight in albino mice after feeding for 15 days

102

5.18

The effect of L. acido. CPP on body weight in albino mice after feeding for 15 days

103

5.19

Body weight of albino mice after feeding normal feed alone without CPP for 10 days

104

5.20

Comparison of the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice after feeding for 15 days

105

5.21

Comparison between the percentage increase in body weight of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP fed mice for a feeding period of 10 and 15 days

106

5.22

Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice after feeding for 10 days and infected with Escherichia coli.

110

5.23

Percentage of body weight loss in albino mice infected with Escherichia coli fed with AAVIN CPP, DODLA CPP, L. acido.

111

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CPP and L. bulg. CPP for 10 days 5.24 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice after feeding for 15 days and infected with Escherichia coli. 5.25 Percentage of body weight loss in albino mice infected with Escherichia coli fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days 5.26 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice after feeding with them for 10 days and infected with Salmonella sp. 5.27 Percentage body weight loss in albino mice infected with Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days 5.28 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 15 days feeding on the body weight of albino mice infected with Salmonella sp. 5.29 Percentage body weight loss in albino mice infected with Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days 120 119 117 116 114 113

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5.30

The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice after feeding for 10 days and infected with Shigella sp. for 10 days

124

5.31

Percentage of body weight loss in albino mice infected with Shigella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days

125

5.32

The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice after feeding for 15 days and infected with Shigella sp.

127

5.33

Percentage of body weight loss in albino mice infected with Shigella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days

128

5.34

The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 15 days feeding on GUT pathogen count of albino mice visceral organs after infected with Escherichia coli

132

5.35

The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 15 days feeding on GUT pathogen count of albino mice visceral organs after infected with Salmonella sp.

134

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5.36

Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 15 days feeding on GUT pathogen count of albino mice visceral organs after infected with Shigella sp.

136

5.37

Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on IgA secretary cell production in albino mice fed with CPP for 10, 15 days and infected with Escherichia coli

147

5.38

Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 10, 15 days feeding on IgA secretary cell production in albino mice after infected with Salmonella sp.

148

5.39

Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 10, 15 days feeding on IgA secretary cell production in albino mice fed after infected with Shigella sp.

151

5.40

Quantification of micro, bi and multi-nucleated cells of mice fed with CPP for 10 days and subjected to Co60 irradiation at LD50 value (1.9 Gy)

161

5.41

Quantification of micro, bi and multi-nucleated cells of mice fed with CPP for 10 days and subjected to Co60 irradiation at LD50 value (1.9 Gy)

162

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5.42

Quantification of micro, bi and multi-nucleated cells of mice not fed with CPP and subjected to Co60 irradiation at LD50 value (1.9 Gy)

163

5.43

Quantification of micro, bi and multi-nucleated cells of fish fed with CPP for 10 days and subjected to Co60 irradiation at LD50 value (135 Gy)

164

5.44

Quantification of micro, bi and multi-nucleated cells of fish fed with CPP for 15 days and subjected to Co60 irradiation at LD50 value (135 Gy)

165

5.45

Quantification of micro, bi and multi-nucleated cells of fish not fed with CPP and subjected to Co60 irradiation at LD50 value (135 Gy)

166

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LIST OF PHOTOS NUMBER 2.1 TITLE Scheme of the mechanisms by which bioactive peptides can be released from the precursor proteins by microbial fermentation and/or gastrointestinal digestion 2.2 2.3 2.4 2.5 4.1 The anatomic structure of the human stomach Different pathways for intestinal absorption of a compound Invasion of S. enteritidis 857 to Caco-2 cells Gut microflora in inflammation Casein isolated from fermented milk by enzymatic hydrolysis 4.2 Gamma irradiator used in anti-genotoxic studies (External view) 4.3 Gamma irradiator used in anti-genotoxic studies (External view) 5.1 5.2 Fermented milk after the addition of commercial Aavin curd Fermented milk after the addition of the culture Lactobacillus bulgaricus 5.3 Scanning Electron microscopic image of Lactobacillus Species 5.4 Zone of inhibition produced by CPP isolated from commercial curd Aavin 79 77 70 71 68 68 30 32 48 49 61 PAGE NO. 24

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5.5 5.6

Molecular weight determination of CPP Photograph showing the histopathological studies of 15 days CPP fed albino mice liver cells infected with Escherichia coli on seven days post infection staining with Methylene blue

90 138

5.7

Photograph showing the histopathological studies of 15 days CPP fed albino mice liver cells infected with Escherichia coli on seven days post infection staining with Methylene blue

140

5.8

Photograph showing the histopathological studies of 15 days CPP fed albino mice spleen cells infected with Escherichia coli on seven days post infection staining with Methylene blue

142

5.9

Photograph showing the histopathological studies of 15 days CPP fed albino mice small intestine cells infected with Escherichia coli on seven days post infection staining with crystal violet

144

5.10

IgA secretary cell production in albino mice not fed with CPP, fed only with normal feed for 15 days and infected with Escherichia coli

152

5.11

Effect of AAVIN CPP on IgA secretary cell production in albino mice fed with it for 10 days and infected with Escherichia coli

153

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5.12

Effect of AAVIN CPP on IgA secretary cell production in albino mice fed with it for 15 days and infected with Escherichia coli infected with Escherichia coli

153

5.13

Control fish fed with normal feed for 15 days and irradiated with Co60 irradiation for 50 Gy for 940 seconds showing micronucleus formation in the erythrocyte cells

155

5.14

Test fish fed for 15 days and irradiated with Co60 irradiation for 50 Gy for 940 seconds showing absence of micronucleus formation in the erythrocyte cells

155

5.15

Control fish fed with normal feed for 15 days and irradiated with Co60 irradiation for 100 Gy for 1880 seconds showing micronucleus formation in the erythrocyte cells

156

5.16

Test fish fed for 15 days and irradiated with Co60 irradiation for 100 Gy for 1880 seconds showing absence of micronucleus formation in the erythrocyte cells

156

5.17

Control fish fed with normal feed for 15 days and irradiated with Co60 irradiation for 135 Gy for 2538 seconds showing nuclear retraction and karyolysis in the erythrocyte cells

157

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5.18

Test fish fed for 15 days and irradiated with Co60 irradiation for 135 Gy for 2538 seconds showing micronucleus formation in the erythrocyte cells

157

5.19

Control mice fed with normal feed for 15 days and irradiated with Co60 irradiation for 1 Gy for 19 seconds showing micronucleus formation in the erythrocyte cells

158

5.20

Test mice fed for 15 days and irradiated with Co60 irradiation for 1 Gy for 19 seconds showing absence of micronucleus formation in the erythrocyte cells

158

5.21

Control mice fed with normal feed for 15 days and irradiated with Co60 irradiation for 1.9 Gy for 34 seconds showing micronucleus, bi and multi nuclear formation in the erythrocyte cells

159

5.22

Test mice fed for 15 days and irradiated with Co60 irradiation for 1.9 Gy for 34 seconds showing absence of micronucleus formation in the erythrocyte cells

159

5.23

Control mice fed with normal feed for 15 days and irradiated with Co60 irradiation for 5 Gy for 94 seconds showing karyolysis and nuclear retraction in the erythrocyte cells

160

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5.24

Test mice fed for 15 days and irradiated with Co60 irradiation for 5 Gy for 904 seconds showing absence of micronucleus formation in the erythrocyte cells

160

5.25

Oxidase enzyme test for albino mice fed with CPP for 15 days, fish fed with CPP for 15 days and control batch, not fed with CPP, only with standard feed for 15 days

167

5.26

Oxidase enzyme test for albino mice fed with CPP for 15 days and control batch, not fed with CPP, only with standard feed for 15 days

167

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LIST OF FIGURES NUMBER 5.1 TITLE The pH changes for milk fermented using commercial curds Dodla, Aavin, Lactobacillus acidophilus and Lactobacillus bulgaricus 5.2 The titratable acidity values for milk fermented using commercial curds Dodla, Aavin, Lactobacillus acidophilus and Lactobacillus bulgaricus 5.3 The viscosity changes for milk fermented using commercial curds Dodla, Aavin, Lactobacillus acidophilus and Lactobacillus bulgaricus 5.4 Anti-microbial activity of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP against Escherichia coli and Pseudomonas sp. 5.5 5.6 HPLC spectrum of non fermented control milk HPLC spectrum of CPP isolated from milk fermented by commercial curd Dodla 5.7 HPLC spectrum of CPP isolated from milk fermented by commercial curd Aavin 82 80 81 78 76 75 PAGE NO. 74

xxx

5.8

HPLC spectrum of CPP isolated from milk fermented by the culture Lactobacillus acidophilus

83

5.9

HPLC spectrum of CPP isolated from milk fermented by the culture Lactobacillus bulgaricus

84

5.10 5.11

FTIR spectrum of non fermented control milk FTIR spectrum of CPP isolated from milk fermented by commercial curd Aavin

85 86

5.12

FTIR spectrum of CPP isolated from milk fermented by commercial curd Dodla

87

5.13

FTIR spectrum of CPP isolated from milk fermented by the culture Lactobacillus acidophilus

88

5.14

FTIR spectrum of CPP isolated from milk fermented by the culture Lactobacillus bulgaricus

89

5.15

Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the percentage increase in body weight of albino mice after fed for 10 days

98

5.16

Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the percentage increase in body weight of albino mice after feeding for 15 days
xxxi

107

5.17

Percentage of body weight loss in albino mice infected with Escherichia coli fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days

111

5.18

Percentage mortality rate in albino mice infected with Escherichia coli fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days

112

5.19

Body weight loss in albino mice infected with Escherichia coli fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days

112

5.20

Percentage of body weight loss in albino mice infected with Escherichia coli fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days

114

5.21

Percentage mortality rate in albino mice infected with Escherichia coli fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days

115

5.22

Body weight loss in albino mice infected with Escherichia coli fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days

115

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5.23

Percentage of body weight loss in albino mice infected with Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days

117

5.24

Percentage mortality rate in albino mice infected with Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days

118

5.25

Body weight loss in albino mice infected with Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days

118

5.26

Percentage of body weight loss in albino mice infected with Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days

120

5.27

Percentage mortality rate in albino mice infected with Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days

121

5.28

Body weight loss in albino mice infected with Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days

121

xxxiii

5.29

Percentage of body weight loss in albino mice infected with Shigella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days

125

5.30

Percentage mortality rate in albino mice infected with Shigella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days

126

5.31

Body weight loss in albino mice infected with Shigella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days

126

5.32

Percentage of body weight loss in albino mice infected with Shigella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days

128

5.33

Percentage mortality rate in albino mice infected with Shigella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days

129

5.34

Body weight loss in albino mice infected with Shigella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days

129

xxxiv

5.35

The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 15 days feeding on GUT pathogen count of albino mice visceral organs after infected with Escherichia coli

133

5.36

Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 15 days feeding on GUT pathogen count of albino mice visceral organs after infected with Salmonella sp.

135

5.37

Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on GUT pathogen count of albino mice visceral organs fed with CPP for 15 days and infected with Shigella sp.

137

5.38

Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 10 days feeding on IgA secretary cell production in albino mice fed with CPP for 10 days after infected with Escherichia coli.

147

5.39

The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP fed for 15 days on IgA secretary cell production in albino mice after infected with Escherichia coli.

148

5.40

The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 10 days feeding on IgA secretary cell production in albino mice after infected with Salmonella sp.

149

xxxv

5.41

Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 15 days feeding on IgA secretary cell production in albino mice after infected with Salmonella sp.

149

5.42

Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 10 days feeding on IgA secretary cell production in albino mice after infected with Shigella sp.

151

5.43

Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 15 days feeding on IgA secretary cell production in albino mice after infected with Shigella sp.

152

xxxvi

LIST OF SYMBOLS AND ABBREVIATIONS CDC HPLC FTIR

60

Centre for Disease Control (USA) High Performance Liquid Chromatography Fourier Transform Infra Red spectroscopy Cobalt-60 Millimeter Casein Phospho Peptide Tamilnadu Veterinary & Animal Sciences University Institute of Microbial Technology Colony Forming Unit Standard Deviation Structure Activity Relationship Toerners degree Millipascal second Immunoglobulin A Lactic Acid Bacteria Gram

CO

mm CPP TANUVAS IMTECH CFU S.D SAR T mPa.s IgA LAB Gm

xxxvii

CHAPTER 1 INTRODUCTION 1.1 Milk consumption and composition: Bovine milk and dairy products have used traditionally for human nutrition. The significance of milk is reflected in our northern mythology where a primal cow named Audhumla was evolved from the melting ice. She had horn and milk was running as rivers from her teats. This milk was used as the food for Ymer, the first creature ever existing [1]. The consumption of milk and milk products among milk consuming regions vary considerably from about 180 kg/yr per capita in Iceland and Finland to less than 50 kg per capita in Japan and China. In the western societies, the consumption of milk has decreased during the last decades [2]. This trend may partly be explained by the negative claims on health effects that have been attributed to milk and milk products. This criticism has arisen especially because milk fat contains a high fraction of saturated fatty acids assumed to contribute to heart diseases, weight gain and obesity [3]. The association between food and health is well established and recent studies have shown that modifiable risk factors seem to be greater significance for health than previously anticipated [4]. Prevention of disease may be just as important as treatment of diseases in the future. Indeed, many consumers of today are highly aware of health-properties of food, and the market for healthy food and special health benefits is in the increasing trend due to the consumer awareness of health properties of food. Bovine milk contains the nutrients needed for growth and development of the calf, and is a resource of lipids, proteins, amino acids, vitamins and minerals. It contains immunoglobulins, hormones, growth factors, cytokines, nucleotides, peptides,

polyamines, enzymes and other bioactive peptides. The lipids in milk are emulsified in globules coated with membranes. The proteins are in colloidal dispersions as micelles. The casein micelles occur as colloidal complexes of protein, salts of primarily calcium [5]. Lactose and most minerals are in solution. Milk composition has a dynamic nature,
1

and the composition varies with stage of lactation, age, breed, nutrition, energy balance and health status of the udder. Specific milk proteins are involved in the early development of immune response, whereas others take part in the non-immunological defence (e.g. lactoferrin). Milk contains many different types of fatty acids [6]. All these components make milk a nutrient rich food item. 1.2 Components in milk and their health effects: 1.2.1 Protein: Bovine milk contains about 32 g protein/l. The milk protein has a high biological value, and milk is therefore a good source for essential amino acids. In addition, milk contains a wide array of proteins with biological activities ranging from antimicrobial ones to those facilitating absorption of nutrients, as well as acting as growth factors, hormones, enzymes, antibodies and immune stimulants [7]. The nitrogen in milk is distributed among caseins, whey proteins and non-protein nitrogen. The casein content of milk represents about 80% of milk proteins. Caseins biological function is to carry calcium and phosphate and to form a clot in the stomach for efficient digestion. The milk whey proteins are globular proteins that are more water soluble than caseins, and the principle fractions are beta-lactoglobin, alpha-lactalbumin, bovine serum albumin and immunoglobulins. Whey is the liquid remaining after milk has been curdled to produce cheese, and it is used in many products for human consumption, such as ricotta and brown cheese. Concentrated whey is an additive to several products e.g. bread, crackers, pastry and animal feed. The rate at which the amino acids are released during digestion and absorbed into the circulation may differ among the milk proteins, and whey proteins are considered as rapid digested protein that gives high concentrations of amino acids in postprandial plasma [8]. Some of the milk proteins (e.g. secretary immunoglobulin A, lactoferrin, 1antitrypsin, -casein and lactalbumin) may be relatively resistant to digestive enzymes, and the whole protein or peptides derived from it, may exert their function in the small
2

intestine before being fully digested [9]. As several bioactive proteins and peptides derived from milk proteins are potential modulators of various regulatory processes in the body, some of these are produced on an industrial scale, and are considered for application as ingredients in both 'functional foods' and pharmaceutical preparations. Although the physiological significance of several of these substances is not yet fully understood, both the mineral binding and cytomodulatory peptides derived from bovine milk proteins are now claimed to be health enhancing components that can be used to reduce the risk of disease or to enhance a certain physiological functions [10]. Milk protein composition may differ among breeds. For example the concentration of betacasein A1 is low in cow milk in Iceland and in New Zealand. It has been speculated that this proteins may have a role in the development of diabetes and cardiac disease. However, later it was concluded in a review article that there is no convincing evidence that the A1 beta-casein of cow milk has any adverse effect in humans [11]. 1.2.2 Branched chain amino acids and other amino acids: Milk is especially rich in essential amino acids and branched chain amino acids. There is several evidence that these amino acids have unique roles in human metabolism; in addition to provide substrates for protein synthesis, it also suppresses protein catabolism and serve as substrates for gluconeogenesis, they also trigger muscle protein synthesis and promote protein synthesis [12]. The stimulated insulin secretion caused by milk, is suggested to be caused by milk proteins, and as shown by Biller et al., 1995 [13] a mixture of leucine, isoleucine, valine, lysine and threonine resulted in glycemic and insulinemic response resembling the response seen after ingestion of whey. A combination of milk with a meal with high glycaemic load (rapidly digested and absorbed carbohydrates) may stimulate insulin release and reduce the postprandial blood glucose concentration [14]. A reduction in postprandial blood glucose is favourable, and it is epidemiological evidence suggesting that milk may lower risk of diseases related to insulin resistance syndrome.

1.2.2.1 Taurine: The concentration of taurine is high in breast milk (about 18 mg/l) and in colostrum from cow [15]. Goat milk is however very rich in taurine: 4691 mg/l. Taurine is an essential amino acid for preterm neonates, and specific groups of individuals are at risk for taurine deficiency and may benefit from supplementation, e.g. patients requiring long-term parenteral nutrition (including premature and newborn infants); diabetes patients, those with chronic hepatic, heart or renal failure. It is suggested that during parenteral nutrition, supplementation of 50 mg taurine per kg body weight may be required. It is implicated in numerous biological and physiological functions: bile acid conjugation and cholestasis prevention, antiarrhythmic/inotropic/chronotropic effects, central nervous system neuromodulation, retinal development and function,

endocrine/metabolic effects and antioxidant/anti-inflammatory properties [16]. Taurine has been shown to have endothelial protective effects, it may function principally as a negative feedback regulator, helping to dampen immunological reactions before they cause too much damage to host tissues or to the leukocytes themselves, and it is shown to be analgesic. 1.2.2.2 Glutathione (GSH): Fresh milk may be a good source of glutathione, a tripeptide of the sulphur amino acid cysteine, plus glycine and glutamic acid. In the organism glutathione has the role as an antioxidant. Glutathione can be oxidized forming GSSG (oxidized glutathione), and in this reaction it may remove reactive oxygenspecies (ROS), thereby regulating the level of ROS in the cells. Glutathione appears to have different important roles in leukocytes, as a growth factor, as an anti-apoptotic factor in leukocytes and to regulate the pattern of cytokine secretion [17]. GSH, moreover, is also central for antioxidative defence in the lungs, which may be very important in connection with lower respiratory infections including influenza [18].

1.2.3 Lipids: 1.2.3.1 Fatty acids: In average, milk contains about 33 g of total fat /l. 1.2.3.2 Saturated fatty acids: More than half of the milk fatty acids are saturated, accounting to about 19 g/l whole milk. The specific health effects of individual fatty acids have been extensively studied [19]. 1.2.3.3 Unsaturated fatty acids: Oleic acid (18:1c9) is the single unsaturated fatty acid with the highest concentration in milk accounting to about 8 g/litre whole milk. Accordingly milk and milk products contribute substantially to the dietary intake of oleic acid in many countries. 1.2.3.4 Trans vaccenic acid (VA): The main trans 18:1 isomer in milk fat is vaccenic acid, (18:1, 11t, VA), but trans double bounds in position 4 to 16 is also observed in low concentrations in milk fat. 1.2.4 Phospholipids and glycosphingolipids: Phospholipids and glycosphingolipids accounts to about 1% of total milk lipids. These lipids contain relatively larger quantities of polyunsaturated fatty acids than the triacylglycerols. They have functional roles in a number of reactions, such as binding cations, help to stabilize emulsions, affect enzymes on the globule surface, cell-cell interactions, differentiation, proliferation, immune recognition, transmembrane signalling and as receptors for certain hormones and growth factors. Gangliosides are one of these components found in milk. Gangliosides (with more than one sialic acid moiety) are
5

mainly found in nerve tissues, and they have been demonstrated to play important roles in neonatal brain development, receptor functions, allergies, for bacterial toxins etc... 1.2.5 Minerals, vitamins and antioxidants in milk: Milk contains many minerals, vitamins and antioxidants. The antioxidants have a role in prevention of oxidation of the milk, and they may also have protective effects in the milk-producing cell, and for the udder. Most important antioxidants in milk are the mineral selenium and the vitamins E and A. As there are many compounds that may have anti-oxidative function in milk, measurement of total anti-oxidative capacity of milk may be a useful tool. 1.3.5.1 Calcium in milk: The calcium concentration in bovine milk is about 1 g/l. In human nutrition adequate calcium intake is essential. Getting enough calcium in the diet gives healthy bones and teeth. 1.3.5.2 Selenium in milk: The selenium concentration in body fluids and tissues are directly related to selenium intake. It has a role in the immune- and antioxidant system and in DNA synthesis and DNA repair. 1.3.5.3 Iodine in milk: Iodine is an essential component of the thyroid hormones. These hormones control the regulation of body metabolic rate, temperature regulation, reproduction and growth.

1.3.5.4 Magnesium in milk: Magnesium is ubiquitous in foods, and milk is a good source, containing about 100 mg/l milk. Magnesium has many functions in the body, participating in more than 300 reactions. 1.3.5.5 Zinc in milk: Zinc is an essential part of several enzymes and metalloproteins. Zinc has several functions in the body, in DNA repair, cell growth and replication, gene expression, protein and lipid metabolism, immune function, hormone activity, etc... 1.3.5.6 Vitamin E in milk: Vitamin E concentration in milk is about 0.6 mg/l. Recommended intake is 15 mg/day. Observational studies indicate that high dietary intake of vitamin E are associated with decreased risk for cancer and coronary heart disease. 1.3.5.7 Vitamin A in milk: Milk is a good source of retinoids, containing 280 ug/l. The recommended daily intake is 700900 g/day. Vitamin A has a role in vision, proper growth, reproduction, and immunity, cell differentiation, in maintaining healthy bones as well as skin and mucosal membranes. 1.3.5.8 Folate in milk: Bovine milk contains 50 g folate/l. Recommended intake of folate is 400 g/day for adults. Many scientists believe that folate deficiency is the most prevalent of all vitamin deficiencies.

1.3.5.9 Riboflavin in milk: Milk is a good source of riboflavin, 1.83 mg riboflavin/l milk. Daily recommended intake is 1.1 and 1.3 mg for women and men, respectively. 1.3.5.10 Vitamin B12 in milk:

Milk is also a good source of vitamin B12, being 4.4 g/l. The daily recommendation is 2.4 g. Vitamin B12 is found only in animal foods and its deficiency may cause megaloblastic anemia and breakdown of the myelin sheath. 1.4 Bacterial flora of milk in milk: Milk samples from normal healthy mammary glands contain many strains of bacteria. To prevent diseases caused by pathogenic bacteria in milk and to lengthen the shelf life of milk, treatment such as cooling and pasteurization or membrane filtration is needed. To preserve milk, addition of selective, well-documented strains of starter cultures for fermentation is a method that has been used for centuries. 1.5 Intolerance to milk components: The public "belief" that milk causes an inflammatory process and an increase in mucus production has not been confirmed. It has been shown that respiratory symptoms was not associated with milk intake, and concluded that consumption of milk does not seem to exacerbate the symptoms of asthma, but in a few cases people with cow's milk allergy may have asthma-like symptoms after milk consumption. However in cells from another tissue; mucin producing cells of gastric mucosa, alpha-lactalbumin stimulates mucin synthesis and secretion. The intolerance is generally not observed for fermented milk. Researchers found out that patients who had developed intolerance to milk components did not develop the same level of intolerance to fermented milk components.

1.6

Intolerance to milk proteins: There has been speculation if milk proteins may have a role in Attention Deficit

Hyperactivity Disorder (ADHD), autism, depressions and schizophrenia in some cases. There are major supports to the hypothesis that ADHD may be linked to increased levels of neuroactive peptides and increased urinary peptide levels. A diet free of milk, milk products and gluten may in many cases give reduced ADHD symptoms. Further, opioid peptides derived from food proteins (exorphins) have been found in urine of autistic patients [18]. This area of investigation is important and large scale, good quality randomised controlled trials are needed. 1.7 Physiologically active milk peptides: In addition to providing immunodefence systems, milk also contains other major peptide fractions that elicit behavioral, neurological, physiological, and vasoregulatory responses. Often, the peptide displays multifunctional properties. Several articles reviewing this topic have already been published [20, 21, 22, and 23]. Here, we categorize classications of physiologically active peptides based on their primary biofunction. 1.7.1 Antihypertensive Peptides (ACE Inhibitors): Antihypertensive peptides inhibit the angiotensin converting enzyme (ACE) [24, 25]. ACE is a peptidyldipeptidase that cleaves dipeptides from the carboxy terminal end of the substrate. ACE converts angiotensin I to angiotensin II, increasing blood pressure and aldersterone, and inactivating the depressor action of bradykinin. ACE inhibitors derived from casein, or casokinins, have been identied within the sequences of human and -CN [26]. They are also generated by tryptic digestion of bovine s1- and -CN [27]. The C-terminal tripeptide sequence is the primary structural feature governing this inhibitory response [28], and reports indicated that the ACE binding pocket exhibited a preference for hydrophobic amino acids at each of these sites [29]. A second
9

characteristic of ACE inhibitory casokinins is the presence of a positively charged lysine or arginine at the carboxy terminal end [30]. It was shown that removal of this critical amino acid residue from bradykinin, an endogenous ACE inhibitor, resulted in production of an analogue that was essentially inactive [31]. ACE inhibitory peptides are also derived from both s1- and -CN that are generated by the hydrolysis of sour milk with the Lactobacillus helveticus CP790 extracellular protease. These peptides exhibited antihypertensive activity in spontaneously hypertensive rats as monitored by systolic blood pressure [32]. A synthetic seven amino acid peptide, equivalent to a segment found in the -CN hydrolysate, exhibited potent antihypertensive activity in these rats over an 8-h interval after oral administration [33]. A third subclass, -lactorphins, are sequestered within the primary amino acid sequence of bovine -LG and released by trypsin [34]. Lastly, novel angiotensin-I converting enzyme (ACE) inhibition was detected in synthetic peptides that corresponded to sequences within both -LG and -LA. 1.7.2 Antithrombotic Peptides: Antithrombotic peptides are present in milk. Early on, it was learned that the mechanisms involved in milk clotting, dened by the interaction of -Casein (CN) with chymosin and blood clotting processes, dened by the interaction of brinogen wit h thrombin, were comparable. In this regard, the C-terminal dodecapeptide of human brinogen -chain (residues 400 to 411) and the undecapeptide (residues 106 to 116) from bovine -CN are structurally and functionally quite similar. This casein-derived peptide sequence, termed casoplatelin, affected platelet function and inhibited both the aggregation of ADP-activated platelets and the binding of human brinogen -chain to its receptor region on the platelets surface [35]. A smaller -CN fragment (residues 106 to110), casopiastrin, was obtained from trypsin hydrolysates and exhibited antithrombotic activity by inhibiting brinogen binding [36]. A second segment of the trypsin -CN fragment, residues 103 to 111, inhibitedplatelet aggregation but did not affect brinogen binding to the platelet receptor [37]. Later, it was reported that biologically active peptides, isolated from both casein and lactotransferrin, inhibited platelet function [38].
10

Antithrombotic peptides have also been derived from -caseinoglycopeptides that were isolated from several animal species. Bovine -caseinoglycopeptide, the C-terminal end of -CN (residues 106 to 169), inhibited von Willebrand factor-dependent platelet aggregation [39]. Two antithrombotic peptides, derived from human and bovine caseinoglycopeptides, have been identied in the plasma of 5-d-old newborns after breast-feeding and ingestion of cows milk based formula, respectively [40]. The Cterminal residues (106 to 171) of sheep -casein, or -caseinoglycopeptide, decreased thrombin- and collagen-induced platelet aggregation in a dose dependent manner [41]. Lastly, thrombin-induced platelet aggregation was inhibited with pepsin digests of sheep and human lactoferrin. A single peptide peak containing this activity was obtained by reverse-phase chromatography of the hydrolysate [42]. 1.7.3 Casein phosphopeptides: (CPP) Casein phosphopeptides (CPP) have been identied after trypsin release from s1, s2-, and -CN [43]. The phosphate residues, which are present as monoesters of serine, occur mainly in clusters. Most CPP contain three serine phosphate clusters followed by two glutamic acid residues, form soluble organophosphate salts, and probably function as carriers for different minerals, especially calcium [44]. These fractions exhibit different degrees of phosphorylation, and a direct relationship between the degree of phosphorylation and mineral chelating ability has been described [45]. In this event, s2CN > s1-CN > -CN > -CN; however, the distribution of their phosphoserine clusters is not uniform. It was further demonstrated that the specic amino acid composition associated with the phosphorylated binding site also inuences the degree of calcium binding [46]. CPP are mostly resistant to enzymatic hydrolysis in the gut and most often found in a complex with calcium phosphate [47]. This complex formation results in an increased solubility which, in turn, provides enhanced absorption of calcium across the distal small intestines of animals fed casein diets in comparison to control animals fed
11

soy-based diets [48]. This passive transport system is the primary means of calcium absorption under physiological conditions and provides calcium required for bone calcication. Caseinophosphopeptides also inhibit caries lesions through recalcication of the dental enamel. Hence, their application in the treatment of dental diseases has been proposed. 1.7.4 Immunomodulatory Peptides: Immunomodulatory milk peptides affect both the immune system and cell proliferation responses. As discussed previously, -casokinins inhibit ACE enzymes that are responsible for inactivating bradykinin, a hormone with immune enhancing effects. Thus, this chain of events indirectly produces an overall immunostimulatory response. Peptides derived from casein hydrolysates were shown to increase phagocytotic activity of human macrophages against aging red blood cells and augment phagocytosis of sheep red blood cells by murine peritoneal macrophages in vitro [49]. Immunostimulatory activity against Klebsiella pneumoniae was demonstrated in vivo using rats treated intravenously with a hexapeptide obtained by hydrolysis of human -CN. Most recently, lactoferricin B, obtained by hydrolysis of lactoferrin with pepsin, was found to promote phagocytic activity of human neutrophils via dual mechanisms that may involve direct binding to the neutrophil and opsonin-like activity. Small peptides, corresponding to the N-terminal end of bovine -LA (dipeptide) and -CN (tripeptide), signicantly increased proliferation of human peripheral blood lymphocytes [100], while the C-terminal sequence of bovine -CN (amino acid sequences 193 to 209), obtained by hydrolysis with pepsin-chymosin, induced a similar response in rats. Bioactive peptides in yogurt preparations actually decreased cell proliferation with IEC-6 or Caco-2 cells. This report may explain, in part, why consumption of yogurt has been associated with a reduced incidence of colon cancer. In general, the mechanisms by which these milkderived peptides exert either their immunopotentiating effects or inuence proliferative responses are not currently known; however, one example suggests that the opioid milk
12

peptide, -casomorphin, may exert an inhibitory effect on the proliferation of human lamina propria lymphocytes in vitro via the opiate receptor [50]. This antiproliferative response was reversed by the opiate receptor antagonist, naloxone. 1.7.5 Opioid Milk Peptides: The major opioid peptides are fragments of -CN, called -casomorphins, due to their exogenous origin and morphine-like properties; however, they have also been obtained from pepsin hydrolysis of bovine s1-CN fractions (43, 54, 66, and 102). Similar peptides have been reported from human -CN fractions (24, 98), and the Y-P-F sequence, which is common to bovine -casomorphin, was also found to be present in the primary structure of human -CN. Various synthetic derivatives have been made and among these, Y-P-F-V-NH2 (valmuceptin) and Y-P-F(D)-V-NH2 (D-valmuceptin) show high afnity for their receptor [51]. Schuster and co-workers in 1980 reported opioid activity from synthetic tetra- and pentapeptide fragments of human -casein [52]. Opioid peptides have been generated in vitro by enzymatic digestion of -caseins from cows, water buffalo, and sheep. In general, the - and -CN fragments produce agonist responses, while those derived from -CN elicit antagonist effects. Opioid peptides may be further subdivided into classications according to the specic milk protein from which they were derived. It is noteworthy that bioactive peptides are generated from most of the major proteins in both bovine and human milk. a. Structure and function Typical opioid peptides, or endorphins, are derived from proenkephalin, propiomelanocortin, and prodynorphin and exhibit a denite N-terminal sequence Y-GG-F. Milk derived peptides, generated by hydrolysis of various precursor proteins such as - and -CN, -LA, and -LG are called atypical, exomorphic, agonist peptides and exhibit morphine-like activity [53]. Their primary structure (i.e., Y-X1-F or Y-X1-X2-F or Y) differs from the amino terminal sequence of the typical endogenous opioid peptide dened above. With the exception of s1-CN, most share a common sequence
13

feature, dened by a N-terminal tyrosine residue, that is absolutely essential for activity. Typically, a second aromatic amino acid residue, such as phenylalanine or tyrosine, is also present in the third or fourth position. This structural motif ts well into the binding pocket of the opioid receptor. One of the most potent milk-derived opioid peptides, casomorphin-4-amide (or morphiceptin), also contains a proline that is crucial for its function. This residue reportedly maintains the proper orientation of the tyrosine and phenylalanine side chains. Exorphins have been isolated from peptic hydrolysates of casein fractions as well. In general, their structures differ considerably from those of caseinomorphins. Active fractions were shown to be a mixture of two separate peptides derived from -casein fragments #9095 and #9096. The sequences were determined as listed, [R90-Y-L-G-Y-L95-(E96)], in which case the latter peptide proved to be more effective. The N-terminal arginine residue was also reported to be essential for activity. b. Opioid agonists -Casein peptides were among the rst reported opioid peptides. -Casomorphins are fragments corresponding to the 60 to 70th amino acid residues of bovine -CN, considered the strategic zone, and are classied as -type receptor ligands [54]. Three exorphins, derived from bovine s1-CN, were shown to be selective for -receptors. Certain proteolytic bacteria, such as Pseudomonas aeruginosa and Bacillus cereus, also produce high levels of -casomorphins when inoculated and grown in milk. Caseinomorphins are resistant to enzymes of the gastrointestinal tract and have been detected in vivo in the intestinal chyme of mini pigs [55] and human small intestines. Because their absorption in the gut has not been observed in adults, it is generally concluded that the physiological inuences are limited to the gastrointestinal tract with important effects on intestinal transit time, amino acid uptake, and water balance. Once they enter the bloodstream, they are rapidly degraded. In contrast, passive transport of caseinomorphins across intestinal mucosal membranes does occur in neonates, which may experience physiological responses such as an analgesic effect on the nervous system resulting in calmness and sleep in infants. A precursor of -casomorphin was
14

reported in the plasma of newborn calves and infants after ingestion of bovine milk. In pregnant or lactating women, -casomorphins originate in the milk pass through the mammary tissue, and possibly inuence the release of prolactin and oxytocin. More recently, it was shown that many peptides derived from s1-,-, or -CN, and -caseinoglycomacropeptide can be detected in the stomach of adults after consumption of milk or yogurt [54]. Casomorphins, as opioid ligands, modulate social behavior increase analgesic behavior prolong gastrointestinal transient time by inhibiting intestinal peristalsis and motility, exert anti-secretary (anti-diarrheal) action, modulate amino acid transport, and stimulate endocrine responses such as the secretion of insulin and somatostatin. Opioidlike milk peptides also play a regulatory role regarding appetite by modifying endocrine activity of the pancreas, resulting in an increase of insulin output. Presently, data suggest that intracerebro ventricular -casomorphin1-7 stimulates uptake of a high fat diet in rats fasted overnight. Enterostatin inhibited this effect, as did naloxone, a general opioid antagonist. Ligand binding studies indicated that at high dosages, -casomorphin1-7 andenterostatin may interact with the same low afnity receptor to modulate intake of dietary fat [55]. c. Opioid antagonists Opioid antagonists suppress the agonist activity of enkephalin. Mensink et al., 1992 [56] reported that a chloroform and methanol extract from a peptic digest of bovine -CN bound to opioid receptors of rat brain. The peptide was methylated at the Cterminal end and exhibited antagonist effects selective for the - and -type of opioid receptor. The peptide was thus named casoxin. Casoxins A and B have been chemically synthesized and correspond to amino acid sequences within both bovine and human CN. Casoxin C is an opioid antagonist, obtained from tryptic digests of bovine -CN, that also functions as an agonist for C3a receptors. Lastly, casoxin D, puried from human s1-CN fractions, elicits an opioid antagonist response. In general, the chemically
15

modied casoxins are more active that their non-methylated derivatives. Lactoferroxins are antagonists generated from human lactoferrin. Initially, a chloroform and methanol extract from a peptic digest of lactoferrin was assayed for activity, and the results indicated that the opioid properties were similar to those of naloxone, a known antagonist ligand. Peptides derived from pepsin digestion, alone, were minimally effective, while those puried from a methyl-esteried fraction were signicantly more potent. HPLC analyses resulted in purication of three separate active fractions designated lactoferroxin A, B, and C, respectively. It was determined that the -carbonyl group of each was methyl esteried based on comparison of bioactivity measurements and HPLC retention times to those of corresponding synthetic peptides. Like casoxins, the chemically modied peptides may not actually exist in vivo. Lactoferroxin A, residues 318 to 323, showed a preference for -receptors. On the other hand, lactoferroxin B and C, derived from residues 536 to 540 and 673 to 679, respectively, exhibited a higher propensity for -receptors. 1.7.6 Miscellaneous Peptides: Physiologically active peptides that directly affect gastrointestinal functions have also been documented. Casomorphins slow gastric motility and emptying in nonruminants, while caseinomacropeptide, a 64-amino acid glycopeptide released from -CN by gastric proteases, exerts its effects on digestive function by inhibiting gastric acid secretions. Several other milk-derived peptides have been described in the literature. Atrial natriuretic factor, or atriopeptin, is a peptide found naturally occurring in human milk. This peptide functions as a strong diuretic, natriuretic, and vasorelaxant, and plays an important role in circulatory adaptation to extrauterine life. More recently, a peptide, obtained by in vitro proteolysis of bovine -LG, was found to exert its effect on smooth muscle.

16

1.8 Global probiotic food market in the industrialized nations: The most active area within the functional foods market in Europe has been probiotic dairy products, in particular, probiotic yogurts and milks. In 1997 these products accounted for 65% of the European functional foods market, valued at US$889 million, followed by spreads, valued at US$320 million and accounting for 23% of the market. Probiotic dairy products are expected to command the highest market share among all the probiotic foodstuffs, accounting for almost 70% in the year 2009 and reaching a market size of almost $24 billion by the end of 2014. The biggest markets for these products are Europe and Asia; the U.S. market has slowly but surely opened up to these products in the recent past and is expected to grow at a CAGR of 17% from 2009 to 2014, the biggest contributor being probiotic cultured drinks followed by probiotic yogurts. Though the market base of probiotic products is comparatively lesser in the US, the market is expected to grow at an astounding rate of almost 14% in the same period driven by the large scale acceptance of - the probiotic yogurts in spoonable single serve packs, probiotic cultured drinks in single shot packaging form and probiotic dietary supplements. 1.9 Indian probiotic market: Indian probiotic market is valued at $2 million as per 2010 estimates and it is poised to quadruple by 2015 due to the advent of Indian and Multinational companies coming in to the fray. With their advent, the Indian probiotic market turnover is expected to reach $8 million by the year 2015. The existing probiotic market in India majorly comprises of three segments, urban chain, young adults and people with special needs such as pregnancy, lactation, immunodeficiency, geriatric etc India at present accounts for less than 1% of the total world market turnover in the probiotic industry and it is a huge deficit considering the fact that India has the highest cattle population and India being the worlds highest milk producer.

17

Probiotics in India generally comes in two forms, milk and fermented milk products with the former occupying 62% of the market share and later having 38% market share (Indian consumer survey, 2010). Indian probiotic products currently are Dahi (Indian yoghurt), flavoured milk and butter milk. Major pharmaceuticals companies have become active in this space and are devising newer drugs and products, however current drugs are predominant in the area of nutraceuticals. 1.10 Current players in indian probiotic market: 1.10.1 Yakult danone: Yakult Danone India Pvt Ltd (YDIPL), is a 50:50 joint venture between Japans Yakult Honsha and The French- Danone Group, with its offering Yakult, a probiotic drink made from fermented milk, lactobacillus and some sugar. Yakult is a world leader in probiotic drinks and has a rich heritage dating back to 1935. Yakult was launched in India in the late 2007. The brand was initially available only in Delhi. Now Yakult is being launched nationally in a phased manner. Yakult is fermented milk that contains healthy bacteria Lactobacillus casei strain Shirota. According to the brand site, a 65 ml Yakult bottle contains 6.5 bn probiotic bacteria. Yakult has been testing its marketing strategy for around a year and is now ready for the national roll out. The brand is currently available in Delhi, Mumbai, Chandigarh and Jaipur. The entry of Yakult is expected to increase the visibility and growth of probiotic category in India. Yakult is also available in supermarkets. Another interesting fact is about the pricing strategy of Yakult. The 65ml bottle is priced at Rs 10 and the product is available in a pack of 5. The price sounds reasonable for those consumers who are health conscious. The main challenge for this product is to make the consumers believe that the product is delivering benefit to them. Most of the health foods have the problem of giving measurable visible results to the consumers. Yakult primarily targets those consumers who are health conscious and is aware of the importance of functional

18

foods like probiotics. It has adopted the right marketing strategy to educate the consumers and also encourage them to make regular use of this product. 1.10.2 Amul: Amul was the first to foray into the category with its probiotic ice creams Prolife in February 2007. Amul, on the other hand, having tasted success in the probiotics category with its ice cream in February earlier this year, is already in the process of testmarketing pouched lassi (sweetened curd) in Gujarat and some parts of Maharashtra, with plans of introducing it in the other parts of the country soon. Probiotic products contribute to 10 per cent to its ice-cream sales and 25 per cent of its Dahi (Indian yoghurt) sales. 1.10.3 Nestle: Nestle, having recently declared dairy as its key area of growth, is all set to introduce probiotics in its other dairy products as well. The total packaged curd market in India is estimated at 40,000-60,000 tons per annum, of which Nestle has a 30 per cent market share. Internationally, the average contribution of probiotic products to total dairy products is estimated between 10-20 % depending on the country and business. Nestle also has introduced flavoured milk varieties of probiotic nature. 1.10.4 Mother dairy: Mother Dairy Delhi was set up in 1974 under the Operation Flood Programme, a wholly owned subsidy of the National Dairy Development Board (NDDB), whose current chairman is Dr. Amrita Patel. Currently, it is one of the largest milk (liquid/unprocessed) plants in Asia selling more than 25 lakh liters of milk per day, thereby enjoying a market share of 66% of the branded milk sales in New Delhi, capital of India. Other important markets include Mumbai, Saurashtra and Hyderabad. Mother dairy ice- cream was launched in the year 1995 and has shown continuous growth over the years, and today it boasts approximately 62% market share in Delhi and NCR. b-Activ Probiotic Dahi, b19

Activ Probiotic Lassi, b-Activ Curd and Nutrifit (Strawberry & Mango) are the companys probiotic products. Probiotic products are contributing to 15 per cent of the turnover of their fresh dairy products. Over the next 3-4 years, the contribution is expected to go up to 25 per cent and the urban acceptance is making the company to increase their focus on probiotic products. 1.11 Fermented milk: Historically, the seasonal variation in milk production made it necessary to preserve milk. The Nordic countries including Iceland have a long tradition for using fermented milk, and the consumption of fermented milk is about 20 kg per person. During fermentation bacteria and yeasts convert lactose in the milk to various degradation products depending on the species present. Lactobacilli and Streptococci give rise to lactic acid and monosaccarides (especially galactose). Bifidobacteria give rise to lactic acid, acetic acid and monosaccarides, while yeasts, present only in some few fermented milk products, produce CO2 and ethanol. Different bacterias may be used for fermentation, giving products of special flavour and aroma, and with several potential health beneficial metabolites. The bacteria contain cell wall components that bind Tolllike receptors on dendritic cells (and also other leucocytes) found in the mucosa of the small intestine and colon, thus stimulating the Th1 immune response [57]. It has been shown that fermented milk stimulates the Th1 immune response, and down-regulates the Th2 immune response. The immune system may thus be strengthened against cancer, virus infections and allergy. Bacterial DNA has also a similar effect, binding to Toll-like receptor-9. Some bacteria can also improve the intestinal microbial balance, and the fermented milk may have positive health effects both in the digestive channel and in metabolism. During the fermentation of milk, lactic acid and other organic acids are produced and these increase the absorption of iron [57]. If fermented milk is consumed at mealtimes, these acids are likely to have a positive effect on the absorption of iron from other foods. Lactic acid is also a poorer substrate for growth of pathogenic
20

bacteria than glucose and lactose. The low pH in fermented milk may also delay the gastric emptying from the stomach into the small intestine and thereby increase the gastrointestinal transit time. Also, full-fat milk has been shown to increase the mean gastric emptying half-time compared to half-skimmed milk, and accordingly it might be favourable to gastric emptying and thus may have an effect on appetite regulation. 1.12 Motivation and Problem statement: Dairy industry is one of the biggest consumer oriented industries in India. Our country being the largest producer of milk and having the highest cattle population is poised for greater growth in the near future. The potential of peptides found in milk and fermented milk has been little utilized and the distinguishment of milk and fermented milk peptides has not been done entirely. Fermented milk peptides hold a lot of potential in treatment of various gut oriented ailments for which antibiotics are extensively used now. Antibiotics produce a lot of side effects which is totally absent in the case of fermented milk peptides. CPP have been largely used as an anti-hypertensive, but CPP can also be used as an Immunomodulatory agent enhancing the immune system of the humans. CPP can also be used as an anti-genotoxic agent who has anti-genotoxic property. Establishment of these facts about the fermented milk peptides can direct the change over in antibiotics based medicinal system for various diseases and also will serve as an anti-genotoxic agent against low ionizing radiation.

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CHAPTER 2 REVIEW OF LITERATURE

2.1 Milk and milk-derived products: Milk is the secretion of the mammary gland, containing approximately 5% lactose, 3.1 protein, 4% lipid and 0.7% minerals. The components of milk provide critical nutritive elements, immunological protection, and biologically active substances to both neonates and adults. It is not surprising, therefore, that the nutritional value of milk is high. The concept of bovine milk as a biologically active fluid is not new [58], but the identification of factors within bovine milk that may be relevant to improving human health, and the potential development of bovine milk-containing preparations into products with proven health-promoting properties, certainly is. Milk is not only consumed as a raw material but it is transformed in a variety of products to preserve its nutrients. Among all the dairy products, milk fermentation and cheese making are the oldest methods used to extend the shelf-life of milk, and they have been practiced by human beings for thousands of years [59]. Recently, numerous scientific works [60-62] have demonstrated and confirmed that the consumption of fermented milk and cheeses manifests health beneficial effects that go beyond the nutritional value. Indeed, fermented milk consumption has been associated with reduction of serum cholesterol [63], antihypertensive [64] and osteo-protective [65] effects. The mechanisms of action responsible of these properties have been investigated and have been attributed to the numerous bioactive peptides contained in milk and/or released during milk processing. It is not surprising that in recent years intense research interest has been focused on identifying biologically active components within bovine milk and milk-derived products, and characterising the way by which mammalian physiological
22

function is modulated by these components. Not surprisingly, a significant proportion of this research has sought to characterise the potential of bovine milk, milk products or milk components to influence some of the most important body physiological functions, such as blood pressure [66], the immune system [67], and the resistance to the infections [68]. For example, there is now a substantial body of evidence to suggest that the major components of bovine milk, as well as several constituents or even yogurt and cheese, can regulate blood pressure in humans [69, 70]. The most significant advances in this field have been made over the last five to ten years, and this review will focus primarily on the recent advances and current knowledge in this rapidly expanding field. Moreover, particular attention is given to the milk-derived bioactive peptides responsible of some important health properties. 2.2 Bioactive peptides: 2.2.1 Definition: Accordingly to a widely shared definition [71], a bioactive dietary substance is "a food component that can affect biological processes or substrates and, hence, have an impact on body function or condition and ultimately health". In addition, dietary substances should give a measurable biological effect in the range of doses it is usually assumed in the food and this bioactivity should be measured at a physiologically realistic level [72]. Following this definition, milk-derived bioactive peptides are milk components able to influence some physiological functions, finally acting on body health condition. Moreover, among the numerous bioactive substances studied up to now, increasing interest is focused on milk-derived bioactive peptides because at present, bovine milk, cheese and dairy products seem to be extremely important sources of bioactive peptides derived from food.

23

2.2.2 Mechanisms of production of bioactive peptides: Milk-derived bioactive peptides, and more generally food bioactive peptides, are usually composed of 2-20 amino acids and become active only when they are released from the precursor protein where they are encrypted. Different mechanisms can release the encrypted bioactive peptides from the precursor proteins as seen in Photo 2.1 [73]: 1. In vivo, during gastrointestinal digestion through the action of digestive enzymes or of the microbial enzymes of the intestinal flora; 2. During milk processing (e. g. milk fermentation, cheese production) through the action of microbial enzymes expressed by the microorganisms used as starter; 3. During milk processing through the action of a single purified enzyme or a combination of selected enzymes; Photo 2.1 Scheme of the mechanisms by which bioactive peptides can be released from the precursor proteins by microbial fermentation and/or gastrointestinal digestion

24

2.2.2.1 Bioactive peptide release during gastrointestinal digestion through the action of digestive enzymes or microbial enzymes of the intestinal flora Bioactive peptides may be released in vivo during gastrointestinal digestion. These bioactive peptides are mostly the result of the degradation of casein with several proteases such as pepsin, trypsin or chymotrypsin. At present, despite some experimental works on the stimulation of gastrointestinal digestion of eggs and meat proteins [74, 75], the production of milk-derived bioactive peptides in vivo during digestion remain unclear. While the peptide products resulting from milk proteins digestion with site-specific pancreatic proteases, such as trypsin or chymotrypsin are well investigated [76, 77], there are only few papers regarding this primary step of human digestion of milk proteins [78]. Microbial proteolysis can be a potential source of bioactive peptides during milk processing [79]. 2.2.2.2 Bioactive peptide release during milk processing through the action of microbial enzymes Many industrially utilized dairy starter cultures are highly proteolytic. Bioactive peptides can, thus, be generated by the starter and non-starter bacteria used in the manufacture of fermented dairy products. The proteolytic system of lactic acid bacteria (LAB), e.g. Lactococcus lactis, Lactobacillus helveticus and L. delb. bulgaricus, is already well characterized. Rapid progress has been made in recent years to elucidate the biochemical and genetic characterization of these enzymes. Many recent articles and book chapters have reviewed the release of various bioactive peptides from milk proteins through microbial proteolysis [80, 81 and 82]. In addition, a number of studies have demonstrated that hydrolysis of milk proteins by digestive and/or microbial enzymes may produce peptides with immunomodulatory activities [83].

25

2.2.2.3 Bioactive peptide release during milk processing trough the action of a single purified enzyme or a combination of selected enzymes The most common way to produce bioactive peptides is through enzymatic hydrolysis of whole protein molecules. ACE-inhibitory peptides and calcium-binding phosphopeptides, for example, are most commonly produced by trypsin [84-86]. Moreover, ACE-inhibitory peptides have recently been identified in the tryptic hydrolysates of bovine s2-casein [87] and in bovine, ovine and caprine k-casein macropeptides [88]. Other digestive enzymes and different enzyme combinations of proteinases - including alcalase, chymotrypsin, pepsin and thermolysin as well as enzymes from bacterial and fungal sources - have also been utilized to generate bioactive peptides from various proteins [89, 90]. Proteolytic enzymes isolated from LAB have been successfully employed to release bioactive peptides from milk proteins. David and colleagues in 1991 [91] reported that casein hydrolyzed by the cell wall-associated proteinase from L. helveticus CP790 showed antihypertensive activity in spontaneously hypertensive rats. Several ACEinhibitory peptides and one antihypertensive peptide were isolated from the hydrolysate. Kunio et al., 2000 [92] hydrolyzed casein using the same proteinase and identified a casein-derived antihypertensive peptide, the fragment -CN (169-175), whose amino acidic sequence is KVLPVPQ. In a recent study, Julius and colleagues in 2004 [93] measured the ACE-inhibitory activity of casein hydrolysates upon treatment with nine different commercially available proteolytic enzymes. Among these enzymes, a protease isolated from Aspergillus oryzae showed the highest ACE-inhibitory activity in vitro per peptide. 2.2.3 Mechanisms of action of bioactive peptides: It has been already demonstrated that milk-derived peptides show biological effects and are able to influence some specific body function. At present, the bioactivities
26

described for milk-derived peptides includes opiate [94], antithrombotic [95], antihypertensive [96], immunomodulating [97], antioxidative [98], antimicrobial [99], anticancer [100], mineral carrying [101] and growth-promoting properties [102]. In the Table 2.1, a brief summary of bioactive peptides from milk proteins is given. Bioactive milk peptides could express their function in the intestinal tract [103107] or inside the body after being absorbed. This signifies that milk-derived bioactive peptides have to be resistant to gastrointestinal, brush border intracellular and serum peptidases [108]. For this reason, scientific works aiming to evaluate the bioavailability of bioactive peptides in vivo are gaining of importance [109, 110]. Table 2.1 Bioactive peptides from milk proteins

Bioactive peptide Casomorphins -lactorphin -lactorphin Lactoferroxins Casoxins Casokinins Lactokinins Immunopeptides Lactoferricin Casoplatelins

Precursor protein - CN, - CN -LA -LG LF -CN - CN, - CN -LA, -LG - CN, - CN LF

Bioactivity Opioid agonist Opioid agonist Opioid agonist Opioid antagonist Opioid antagonist ACE-inhibitory ACE-inhibitory Immunomodulatory Antimicrobial

27

Phosphopeptides

-CN, Transferrin - CN, - CN

Antitrombotic Mineral binding, Anticariogenic

2.2.4 Bioactive peptide based commercial dairy products: It is now well documented that bioactive peptides can be generated during milk fermentation with the starter cultures traditionally employed by the dairy industry. As a result, peptides with various bioactivities can be found in the end-products, such as various cheese varieties and fermented milks. These traditional dairy products may, under certain conditions, carry specific health effects when ingested as part of the daily diet. Table 2.2 below lists a number of studies which have established the occurrence of peptides in various fermented milk products. An increasing number of ingredients containing specific bioactive peptides based on casein or whey protein hydrolysates have been launched on the market within the past few years or are currently under development by international food companies. Table 2.2 Studies which have established the occurrence of peptides in various fermented milk products Product Cheese type ParmigianoReggiano Example of identified peptide
-CN (8-16), -CN (58-77), s2-CN(83-33)

Bioactivity Phosphopeptides, precursor of -casomorphin Several

Cheddar

s1-CN fragments
28

-CN fragments -CN (58-72)

phosphopeptides ACE-inhibitory

Italian varieties: Mozzarella, Crescenza, Gogonzola, Italico Gouda Festivo

s1-CN (1-9), -CN (60-68) s1-CN (1-9), s1-CN (1-7), s1-CN (1-6)

ACE-inhibitory ACE-inhibitory Immunostimulatory, antimicrobial ACE-inhibitory

Emmental

s1-CN fragments -CN fragments

Manchengo

Ovine s1-CN, s2-CN, -CN fragments

Fermented milks Sour milk

-CN (74-76), -CN (84-86), -CN (108-111)

Antihypertensive

Yogurt

Active peptides not identified

Weak ACEinhibitory ACE-inhibitory

Dahi
SKVYP
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2.3 Digestion of bioactive peptides: Some bioactive peptides can express their activity directly on the gastrointestinal tract but the majority of them have to reach their target site inside the body. They have to remain stable during the digestion process and cross the gastrointestinal barrier maintaining their biological activities. It is thus important to know the physiology of digestion of proteins and peptides in the gastrointestinal tract, more specifically the human GI system, to understand the mechanisms determining the bioavailability of bioactive peptides in vivo [148]. In humans, the most important sites for the digestion of proteins and peptides are the stomach and the small intestine. The stomach is the portion of the GI tract that is located between the cardiac and pylorus valves (Photo 2.2). It can be divided in different regions which differ for the structure and functionality of the glands distributed in the gastric mucosa. Photo 2.2 The anatomic structure of the human stomach

30

Regardless of the mechanisms of absorption, the bioactive peptides that enter the enterocyte undergo the action of the peptidases of the cytosol or the cellular organelles. Indeed, the lysosome contains a massive array of enzymes, estimated over 60 in number, which are capable of degrading any biological macromolecule, including peptides and proteins. The release of ACE-inhibitory peptides upon gastrointestinal digestion of milk proteins or protein fragments, as well as the resistance to digestion of known ACEinhibitory sequences has been tested in several in vitro studies where the gastrointestinal process was mimicked by the sequential hydrolysis with pepsin and pancreatic enzymes (trypsin, chymotrypsin, carboxy and aminopeptidases). These studies showed that gastrointestinal digestion is an essential factor in determining ACE-inhibitory activity [149]. The conditions of the simulated gastrointestinal digestion (enzyme preparation, temperature, pH and incubation time) greatly influence the degree of proteolysis and the resultant ACE-inhibitory activity. The action of brush-border peptidases, the recognition by intestinal peptide transporters and the subsequent susceptibility to plasma peptidases also determine the physiological effect [150]. There is an increasing need to develop in vitro gastrointestinal digestion models that could mimic the human digestion processes. In vitro methods therefore offer an appealing alternative to human and animal studies. They can be simple, rapid, and low in cost and may provide insights not achievable in whole animal studies. In fact, in the last years new in vitro gastrointestinal digestion models incorporating the multi-phase nature of the digestive processes, to mimic the passage the food into the stomach and then into the gut, have been developed or adapted for assessing digestibility of food allergens [151], but a potential application on the study of physiology of the digestion of bioactive peptides could be feasible. Such models have to be sufficiently refined to allow the process of digestion to be followed in some detail and have to be validated against in vivo data. Ideally, an in vitro model should offer the advantages of rapid representative sampling at any time point, testing the whole food matrix (or diet) instead of the isolated protein precursor of the bioactive peptide and be capable of handling solid foods which
31

cannot easily be tested in vivo. Moreover, in vitro digestion models should consider three main stages: (i) processing in the mouth, (ii) processing in the stomach (cumulative to the mouth) and (iii) processing in the duodenum (cumulative of mouth and stomach). 2.4 Bioactive peptide absorption: After digestion, di- and tri-peptides can be easily absorbed in the intestine, but it is not clear if larger bioactive peptides can be absorbed from the intestine and reach the target organs. Some bioactive peptides, in particular C-terminal proline containing peptides, are resistant to proteolysis [152], suggesting that this class of peptides have a better chance to be absorbed in their active form. 2.4.1 Physiology of the absorption of proteins and peptides: Approximately 90% of the absorption in the gastrointestinal tract occurs in the small intestinal region. The specialized epithelial barriers of the gastrointestinal tract separate fluid-filled compartments from each other. They restrict and regulate the flux of substances in both directions. In general, the transfer of all substances, from H+ ions to the largest proteins, across these barriers can occur via paracellular or transcellular routes (Photo 2.3). Photo 2.3 Different pathways for intestinal absorption of a compound.

32

The intestinal absorption of a compound can occur via several pathways: (a) transcellular passive permeability; (b) carrier-mediated transport; (c) paracellular passive permeability, and (d) transcytosis. However, there are also mechanisms that can prevent absorption: (e) intestinal absorption can be limited by P-gp, which is an ATP-dependent efflux transporter; and (f) metabolic enzymes in the cells might metabolize the bioactive peptide The transcellular route requires the transport of the solute across two morphologically and functionally different cell membranes (e.g. the apical and the basolateral membrane), by either active or passive processes. The extent of simple passive diffusion of substances across the membranes depends on their size, charge and lipophilicity and could be facilitated by a carrier system and has been observed for most smaller inorganic and organic solutes [153]. 2.4.2 Physical and chemical characteristics of potentially absorbable bioactive peptides: To exert physiological effects after oral ingestion, it is of crucial importance that milk- derived bioactive peptides remain active during gastrointestinal digestion and absorption and reach the circulation. The bioavailability of peptides depends on a variety of structural and chemical properties, i.e. resistance to proteases, charge, molecular weight, hydrogen bonding potential, hydrophobicity and the presence of specific residues [154]. Indeed, proline- and hydroxyproline-containing peptides are relatively resistant to degradation by digestive enzymes. Furthermore, tripeptides containing the C-terminal proline-proline are reported to be resistant to proline-specific peptidases [155] and have been shown to be stable under simulated gastrointestinal digestion conditions. As already explained before, peptides consisting of two or three amino acids can be absorbed intact from the intestinal lumen into the blood circulation via different mechanisms for intestinal transport. The presence of the milk-derived ACE-inhibitory
33

peptide IPP was recently demonstrated in measurable amounts in the circulation of volunteers that consumed a drink enriched in IPP and VPP [156]. Other characteristics contribute to the resistance to hydrolysis. For example, when isolated, some caseinderived peptides tend to be highly negatively charged and phosphorylated, making them resistant to further proteolysis. Thus, some of the bioactive peptides could be absorbed across the intestinal mucosa to enter the circulation or be retained in the lumen and pass into the colon. The latter is likely based on evidence that ingested casein-derived phosphopeptides can be isolated from rat feces. 2.4.2.1 The absorption of bioactive peptides derived from milk proteins: For some bioactive tripeptides the intestinal absorption has been already demonstrated. For example, VPP was detected in the abdominal aorta of SHR 6 hours after its administration in sour milk, which strongly suggests that it is trans-epithelially transported [157]; more recently the absorption was observed also in humans. Paracellular transport, through the intercellular junctions, was suggested as the main mechanism, since the transport via the short-peptide carrier, PepT1, led to a quick hydrolysis of the internalized peptide. In the case of larger sequences, the susceptibility to brush border peptidases is the primary factor that decides the transport rate. For example, the heptapeptide lactokinins (ALPMHIR) was transported intact, although in concentrations too low to exert an ACE-inhibitory activity, which suggests cleavage by aminopeptidases. 2.5 Bioactivities of milk and fermented milk peptides: Milk-derived bioactive peptides are potential modulators of various regulatory processes in the body, and they can express hormone-like activities. Moreover, the primary sequence of some specific bovine proteins, as caseins, contains overlapping regions, partially protected from proteolytic breakdown, that manifest multifunctional properties and influence different biological functions [111]. In particular, ACE34

inhibitory and immunomodulatory properties seem to be associated, possibly because both are correlated to the presence of short chain peptides such as VPP and IPP formed during milk fermentation with selected bacterial strains [112]. 2.5.1 ACE-inhibition: The inhibition of the Angiotensin-I-Converting Enzyme (ACE) is a key point in the treatment of the hypertension. ACE is carboxypeptidase and catalyzes the cleavage of dipeptides [113]. ACE is responsible for the conversion of angiotensin I, a decapeptide generated by the action of rennin on the substrate angiotensinogen, to the vasoconstrictor octapeptide angiotensin II. Angiotensin II directly acts on blood vessels increasing blood pressure, but it also stimulates the release of aldosterone from the adrenal cortex. Aldosterone increases the reabsorption of sodium and water and the secretion of potassium by the kidney, so the overall effect is an increased blood pressure. Examples of ACE-inhibitory peptides derived from milk is given in Table 2.3 Table 2.3 Some examples of ACE-inhibitory peptides derived from milk Peptide sequence VAP FFVAP FFVAPPFPEVFGK FPEVFGK FGK YKVLPQL Fragment s1-CN (25-27) s1-CN (23-27) s1CN (23-34) s1-CN (28-34) s1-CN (32-24) s1-CN (104-109) s1IC50 (mol/L) 2 6 77 140 160 22

35

LAYFYP DAYPSGAW

CN (142-147) s1-CN (157-164) s1-CN

65 98

2.5.2 Immunomodulation: The immune response can be influenced by various factors. Numerous reports demonstrate that milk bioactive peptides can interact with the immune system at different levels [114]. 2.5.2.1 Immunomodulatory peptides derived from milk: Immunomodulatory milk peptides act on the immune system and cell proliferation responses thus influencing downstream immunological responses and cellular functions. Indeed, in 1981 Cinquina and colleagues in 2003 [115] discovered that a tryptic hydrolysate of human milk possessed in vitro immunostimulatory activity (more specifically, stimulation of phagocytosis of sheep red blood cells and production of hemolytic antibodies against the same cells). In the following years, a number of potentially immunoregulatory peptides were identified encrypted in bovine caseins and whey proteins, which can manifest different effects (Table 2.4). Some casein-derived peptides (residues 54-59 of human -casein and residues 194-199 of s1-casein) can stimulate phagocytosis of sheep red blood cells by murine peritoneal macrophages [116], exert a protective effect against Klebsiella pneumoniae [117] or modulate proliferative responses and immunoglobulin production in mouse spleen cell cultures (fragment 1-28 of bovine -casein, [118]. More recently, lactoferricin B, obtained by hydrolysis of lactoferricin by pepsin, was found to promote phagocytic activity of human neutrophils [119]. Others fragments (fragment 18-20 of -casein, fragment 90-96 of s1-casein) can either stimulate or inhibit
36

lymphocyte proliferation depending upon the concentration used, while some wheyderived peptides can affect cytokine production from leucocytes [120]. Table 2.4 Immunomodulatory peptides derived from milk proteins Protein sequence Bovine s1-CN Fragment s1-CN (1-23) Activity Stimulation of phagocytosis and immune responses against bacterial infections Bovine s1-CN s1-CN (23-34) Stimulation of phagocytosis and immune responses against bacterial infections Bovine s1-CN s1-CN (90-96) Stimulation effect on lymphocytes proliferation, NK activity and neutrophil locomotion

Bovine s1-CN

s1-CN (90-95)

Stimulation effect on lymphocytes proliferation, NK activity and neutrophil locomotion

Bovine s1-CN

s1-CN (194-199)

Stimulation of phagocytosis and immune responses against bacterial infections

37

Bovine s2-CN

s2-CN (1-32)

Stimulatory effect on spleen cells

Bovine -CN

-CN (1-28)

Stimulatory effect on spleen cells

Immunomodulatory milk-derived peptides may contribute to the overall immune response and may ameliorate immune system function. Weinrichtera et al., 2001 [121] suggested that casein derived peptides are involved in the stimulation of the newborn's immune system. It cannot be excluded that the immunostimulating activities may also have a direct effect on the resistance to bacterial and viral infection of adult humans. 2.5.2.2 Microorganisms for the production of fermented milk with

immunomodulatory activity Also in the case of immunomodulatory peptides, milk fermentation contributes to the generation of fermented milk with potential immunological activity (Table 2.5). Amitha and colleagues in 1997 [122] demonstrated that milk fermented with L. helveticus modulates lymphocyte proliferation in vitro. Table 2.5 List of microorganisms producing immunomodulatory activity from fermented milk Microorganism L. helveticus 5089 L. helveticus R389
38

Protein source Caseins Milk

L. paracasei NCC2461 L. casei GG (ATCC 53103) L. casei GG L. acidophilus L. casei rhamnosus GG L. delb. bulgaricus ATCC11842 B. lactis BB12 S. thermophilus DSM4022

Tryptic-chymotryptic hydrolysate of -LG Caseins Milk Milk Caseins Milk Milk Milk

Fermented milks with immunomodulatory properties are not produced exclusively by L. helveticus. Milk fermented by L. paracasei [123] shown to produce peptides from -lactoglobulin that stimulate IL10 production and depress lymphocyte proliferation. Additionally, L. casei was used to produce a casein hydrolysate that suppresses human T cell activation, modulating IL2 expression [124, 125 and 126]. The immunomodulatory activity is independent from the presence of living microorganisms, as evidenced by Perdigon [127] and by Vinderola [128] who reported that the supernatant of fermented milk cultured with L. casei, L. acidophilus and L. helveticus strains increased the immune response independently from the presence of lactobacilli. This result was obtained also by De Simone [129] that tested the INF- production of human peripheral blood lymphocytes in response to filtered yoghurt devoid of microorganisms. More recently LeBlanc examined the antibody production following E. coli O157:H7 infection following the administration of a cell- free supernatant from L. helveticus fermented milk and found that the increased antibody production is not related to viable microorganism
39

[130]. Microorganisms other that bacteria, as a cell-free extract obtained from the yeast S. cerevisiae can be used for milk fermentation, producing a milk hydrolysate with potential apoptosis-inducing effect in human leukemia HL-60 cells, as observed by Rudolf et al., 1990 [131]. In addition, as already demonstrated for milk proteins [132, 133], the bioactive peptides present in yoghurt actually decreased cell proliferation with IEC-6 or Caco-2 cells, which may explain, at least partially, why consumption of yoghurt has been associated with a reduced incidence of colon cancer [134]. The molecular mechanism by which the previous mentioned microorganisms enhance the immune system is not yet clear but the previously peptides discussed released reports in strongly support milk the fact to that the

immunomodulatory

fermented

contribute

immunoenhancing and antitumor properties of dairy products. It should be stressed that the extreme difficulty to establish how immunomodulatory peptides and fermented milks influence the immune function is strictly linked to the immune system complexity. This system comprises a complex interplay between different cell populations and molecules. Thus, when the immunomodulatory activity of a bioactive peptide is assessed in vitro, the single experimental result could demonstrate the specific involvement of a particular milk-derived peptide in an immune mechanism but this result is not conclusive in determining if this peptide its effects would be significant for the whole immune system. 2.5.2.3 Examples of immunomodulatory peptides derived from milk proteins: At present, most attention on immunomodulatory peptides has been focused on lactoferricin, a pepsin-derived peptide from lactoferrin and on glycol-macropeptide, a kcasein-derived peptide (-CN (amino acid sequences 106-169)) present in appreciable amounts in some whey protein concentrates and whey protein isolates. Particular attention has been given to the fragment -LA (amino acid sequences 18-20) (a tripeptide named YGG) and to the long fragment -CN (amino acid sequences 193-209)
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because they have been chosen as model peptides to study the immunomodulatory activity and the absorption mechanism of bioactive peptides derived from milk proteins [135]. 2.5.2.3.1 YGG peptide with immunomodulatory activity: The peptide YGG (Tyr-Gly-Gly) represents an interesting example of cryptic peptide with putative immunomodulating effects, as it can originate from at least two different sources. First, it is the product of the hydrolysis of Leu-enkephalin and Metenkephalin and thus it is an endogenous peptide. In addition, it can be considered as a potential nutraceutical, because it is also encrypted in milk proteins and can be released during the digestion of bovine milk, in particular from - lactalbumin (fragment amino acid sequences 18-20) [136]. It is known that Met-enkephalins, the YGG endogenous progenitor, can enhance human T cell proliferation and IL2 production in vitro in the absence of mitogens, possibly through the activation of opioid receptors present on the cell surface [137]. The enhancement of human peripheral blood lymphocytes proliferation and protein synthesis in vitro was obtained also with YGG administration in presence of ConA [138, 139]. In addition, it was observed that YGG can affect INF- and IL2 secretion in murine splenocytes stimulated with suboptimal concentration of ConA in serum- free medium [140]. Stimulatory effects on cell proliferation were observed also in leukocytes obtained from mice administrated in vivo with either Met-enkephalin or YGG, suggesting that Met-enkephalin effects on the immune cells are mediated by YGG [141]. More recently, the immunomodulatory effect of YGG was confirmed in vivo by the observation that the peptide administration modulated the delayed-type hypersensitivity responses to

tuberculin derivatives in hairless guinea pigs [142]. It is noteworthy to observe that YGG seems to have a biphasic effect on the parameters studied so far, as it showed an enhancing effect at low doses and an inhibitory effect at higher doses [143, 144]. It
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should be noted that YGG is contained several times in the primary structure of bovine casein and -lactalbumin and it could be released during milk fermentation or gastrointestinal digestion from the precursor proteins. In addition, it is a tripeptide and, as already demonstrated for other milk-derived bioactive peptides [145], it can be assumed that it can pass across the intestine by a carrier-mediated peptide transport system in quantitatively significant amounts and, hence, may reach peripheral target sites. 2.5.2.4.2 -CN (193-209) peptide with immunomodulatory activity: The -CN (193-209) peptide is released from the C-terminal end of -casein by hydrolysis with pepsin-chymosin. It is a 17 residues long peptide with the amino acid sequence Tyr-Gln-Glu-Pro-Val-Leu-Gly-Pro-Val-Arg-Gly-Pro-Phe-Pro-Ile-Ile-Val. This peptide was isolated and identified from yoghurt and fermented milks as well as several types of cheese including Feta and Camembert [146]. This peptide displays immunomodulatory properties and shows mitogenic activity on primed lymph node cells and unprimed rat spleen cells, it manifests chemotactive activity on L14 lymphoblastoid cell line and enhances phagocytosis in rat macrophages. In addition, a smaller fragment of -CN (193-209), corresponding to the amino acid sequence Gly-Pro-Val-Arg-Gly-ProPhe-Pro-Ile-Ile, displayed ACE-inhibitory activity, further supporting the concept that ACE-inhibitors may also act as immunomodulatory peptides by acting as bradykininpotentiating peptides [147]. Interestingly, the presence of 4 proline residues within the sequence can protect the long peptide -CN (193-209) from the action of peptidases. So it could be possible that this peptide can cross the intestinal barrier in an intact bioactive form. 2.6 Prebiotics and Probiotics: According to Gibson and Roberfroid, prebiotics are defined as a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and /or activity of one or a limited number of bacteria in the colon [158]. Recently, some
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researches have been conducted to manipulate beneficial bacteria in gastrointestinal tract. Folkenberg et al., 2006 [159] suggested that the use of prebiotics is a promising approach for enhancing the role of endogenous beneficial organisms in the gut. They can be used as potential alternatives to growth promoting antibiotics. Several reports have shown that supplementing a diet with oligofructose (OF) improved growth in nursery pigs and in weaned pigs while other reports by Vinderola et al., 2005 [160] did not find growth effect in young pigs. The reasons for the different results are not clear yet. It may be due to the different chemical structure (degree of polymerization, DP) and compositions of the OF used. Ganji et al., 2004 [161] reported that the site in the gut of pigs where the fermentation of OF occurs depend on the molecular structure of the nondigestible carbohydrates. Bifidobacteria may preferentially utilize non-digestible oligosaccharides with a lower DP, whereas bacteroides degrade preferentially oligosaccharides with a higher DP. Thus, it was hypothesized that OF with a low DP is (DP= less than 10), may be more beneficial than FOS with a high DP (DP=10~60) for the development of bifidobacteria in the large intestine of young pigs. It appears that the places of fermentation in the small intestine and large intestine will determine the conditions in these parts of the GIT. Probiotics are live microbial feed supplements which beneficially affect the host animal by improving its microbial balance. Probiotics have been reported to increase feed intake, growth, immune responses, the numbers of lactobacilli and decrease the numbers of E. coli [162]. 2.7 Fermentations and microorganisms: Fermenting fruits and vegetables can bring many benefits to people. They play an important role in providing food safety, enhancing health and improving the nutrition and social well-being of millions of people around the world. Lactic acid bacteria are the most important bacteria in desirable food fermentations, being responsible for the
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fermentation of sour dough bread, sorghum beer, all fermented milks, and most "pickled" (fermented) vegetables. Lactobacillus acidophilus, Lb. bulgaricus, Lb. plantarum, Lb. pentoaceticus, Lb. brevis and Lb. thermophilus are examples of lactic acid-producing bacteria involved in food fermentations. Some of the species are homo-fermentative, because they produce lactic acid only, while others are hetero-fermentative and produce lactic acid plus other volatile compounds and small amounts of alcohol. Leuconostoc mesenteroides is a bacterium associated with the sauerkraut and pickle fermentations. This organism initiates the desirable lactic acid fermentation in these products. Leuconostoc mesenteroides produces carbon dioxide and acids which rapidly lower the pH and inhibits the development of undesirable microorganisms. The carbon dioxide produced replaces the oxygen, making the environment anaerobic and suitable for the growth of subsequent species of lactobacillus. Several other bacteria, for instance Leuconostoc citrovorum, Streptococcus lactis and Brevibacterium species are important in the fermentation of dairy products. Most lactic acid bacteria work best at temperatures of 18 to 22C and tolerate high salt concentrations. The salt tolerance gives them an advantage over other less tolerant species and allows the lactic acid fermenters to begin metabolism, which produces acid that further inhibits the growth of non-desirable organisms. In general, bacteria require a fairly high water activity (0.9 or higher) to survive. The advantages of the use of starter cultures against spontaneous fermentation are well known and widely spread especially for dairy and meat products, but are not often used in the vegetable fermentations. The spontaneous fermentation of sauerkraut can result in the formation of biogenic amines. The utilisation of starter cultures enables producers to make food products with a standard quality in a shorter time. Selection of starter culture, however, should not be only done considering the lactic acid production of the strains but also their activity for biogenic amine synthesis. Several authors have investigated histamine concentration in commercial sauerkraut samples. Canzi et al.,
44

2000 [163] analysed 50 samples and detected histamine in the range of 9-130 mg/kg. Furthermore the histamine levels increased after fermentation for 10 weeks. Red beet is a well-known vegetable which is a considerable source of vitamins C and B, and minerals such as K, Fe, P and Mg. In addition, red beet contains natural pigments (betalains) which have several biological activities, for example modification of blood pressure and antitumor effect as per Apostolids et al., 2006 [164]. Although numerous studies have been carried out on cabbage, olive and pickle fermentation, little is known on the lactic fermentation of other vegetables. Usually, lacto fermented vegetables are pasteurized and there is no information on the behaviour of lactobacilli during storage of unpasteurized fermented vegetables. With fermentation of beetroot by appropriately selected lactobacilli a juice could be produced which combines benefits of betalains and lactobacilli. 2.8 Probiotics and their role in the human health: The gastrointestinal (GI) microflora plays an important role in the health status of people and animals. The GI tract represents a much larger contact area with the environment, compared to the 2 m2 skin surface of our body [165]. The mucosal surface of the small intestine is increased by forming circular folds, intestinal villi and the formation of microvilli in the enterocyte resorptive luminal membrane. The resulting surface of the GI system is calculated to be 150-200 m2 [166], therefore it provides enough space for the interactions related to the digestion and for adhesion to the mucosal wall. It is estimated, that about 300-400 different cultivable species belonging to more than 190 genera are present in the colon of healthy adults. Among the known colonic microbial flora only a few major groups like 'main flora' dominate at levels around 10 10 /g, all of which are strict anaerobes such as Bacteroides, Eubacterium, Bifidobacterium and Peptostreptococcus [167]. Facultative aerobes are considered to belong to the subdominant flora, constituting Enterobacteriaceae, lactobacilli and streptococci. Minor groups of pathogenic and opportunistic organisms, the so-called 'residual flora' are
45
11 10

always present in low numbers. Bacteria present in the 'normal' intestinal flora may exert beneficial effect and are able to degrade certain food components, produce certain B vitamins, stimulate the immune system and produce digestive and protective enzymes. The normal flora also takes part in the metabolism of some potentially carcinogenic substances and may play a role in drug efficacy. In the last few decades there is an increasing interest for influencing the composition of the gut microflora by foods or food ingredients. The goal of these attempts is to induce the number and the activities of those microorganisms which possess health promoting properties, such as Lactobacillus and Bifidobacterium species [168]. The health promoting effect of lactobacilli was first hypothesized at the beginning of last century. In the last four decades there have been growing attempts to improve the health status of the indigenous intestinal flora by live microbial adjuncts, "probiotics." Although a number of definitions for probiotics have been proposed, an appropriate one was suggested by Makino et al., 2006 [169], according to which probiotics are defined as "mono- or mixed cultures of live microorganisms which, when applied to animal or people, beneficially affect the host by improving the properties of the indigenous microflora". This definition does not restrict 'probiotic' activities to the intestinal microflora, but also to the other sites of the body and it might consist of more than one bacterial species. A new definition for probiotics may better characterize both the specific strains and components used for probiotic purposes. Perdigon and coworkers in 2003 [170] proposed that "probiotics are microbial cell preparations or components of microbial cells that have beneficial effect on the health and well-being of the host". The probiotics do not have to be viable as non-viable forms of probiotics have also been shown to exert health promoting effect. 2.8.1 Pathogens and the intestine: Interactions between the host cells and the pathogenic bacteria initiate infectious diseases. Several enterovirulent bacteria by different physiopathological mechanisms are able to increase the volume of water in stools, resulting from the imbalance between the
46

processes of intestinal absorption and secretion of water [172]. Important feature of Salmonella and other genera is the flagella which confer motility to the bacterium and so contribute to the colonization of pathogen. The filament of members of the genus Salmonella is a multimer of a single protein, the flagellin. Comparison of the amino acid sequences of Salmonella flagellins led to the definition of 8 regions of different variability. The flagellins play an important role in holding the flagellum together. 2.8.1.1 Salmonella infection in human: Attachment of pathogen bacteria to intestinal epithelial cells is the first step of bacterial pathogenicity. It requires specialized factors encoded by the bacteria which directly bind to host cell receptors [173]. Attachment of pathogenic bacteria to intestinal epithelial cell surfaces can lead to colonization, cell damages, internalization, intracellular proliferation and disturbances of regulatory cell mechanisms. The invasive bacteria cross the epithelial membrane, proliferate and promote cell death and exfoliation. Due to these effects the mucosal surface is reduced and is characterized by a large number of immature enterocytes. It was shown that some Salmonella spp. is sensitive against the organic acids produced by the Lactobacilli and it produces other metabolites with anti-Salmonella properties, for example Lb. acidophilus LB secretes a compound into the growth medium with a broad inhibitory spectrum. Wellman and coworkers in 2003 [174] performed one of the most convincing animal studies. Ninety percent of conventional mice fed with Lb. rhamnosus HN001 survived the single dose Salmonella challenge while only 7% of control mice survived. It was shown that leucocyte phagocytosis responses significantly increased and Salmonella translocation decreased in the visceral tissue after administering of probiotics. Similar mechanism was observed for E. coli and Shigella sp. 2.8.1.2 Salmonella enterica serotype enteritidis: Salmonella enterica serotype enteritidis (Salmonella enteritidis, S. enteritidis) is a
47

facultative anaerobe Gram negative rode-shaped bacterium and it belongs to the family Enterobacteriaceae, trivially known as "enteric bacteria". Some species are ubiquitous. Other species are specifically adapted to a particular host. In humans, Salmonella are the cause of two diseases called salmonellosis: (i) enteric fever (typhoid), resulting from bacterial invasion of the bloodstream, and (ii) acute gastroenteritis, resulting from foodborne infection. Similar mechanism was observed for E. coli and Shigella sp.
2. 8. 2 Therapeutic effects of probiotics:

Several clinical studies [177,178] have investigated the application of probiotics, especially lactobacilli and bifidobacteria, as dietary supplements for the prevention and treatments of several gastrointestinal diseases. Photo 2.4 Invasion of S. enteritidis 857 to Caco-2 cells (A: Intact microvilli of Caco-2 cells; B and C: Rearrangemets of cytoskeleton with the formation of membrane ruffles; D: S. enteritidis 857 are present in the vacuoles)

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Photo 2.5 Gut microflora in inflammation

2.8.2.1 Acute gastroenteritis: Rotavirus is one of the most common causes of acute childhood diarrhoea worldwide. After invasion in the small intestinal epithelium the rotavirus replicates and causes the partial disruption of the intestinal mucosa with the loss of microvilli and decrease in the villus /crypt ratio. The most often studied gastrointestinal condition treated by probiotics is acute infantile diarrhoea.
2.8.2.2 Inflammatory bowel disease:

Several lines of observational and experimental evidence implicate the normal flora in the pathogenesis of Crohn's disease and ulcerative colitis [179]. Crohn's disease is a chronic and idiopathic inflammation of the gastrointestinal tract with characteristic patchy transmural lesions containing granulomas. The outbreak of Crohn's disease is thought to require genetic predisposition, immunological disturbance and the influence of intraluminal triggering agent(s), e.g. bacteria or viruses.
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2.9 Allergic diseases: Atopic dermatitis is a common, complex, chronically relapsing skin disorder of infancy and childhood. The prevalence of atopic diseases has been progressively increasing in Western societies. The regulatory role of probiotics in human allergic disease was first emphasised in the demonstration of a suppressive effect on lymphocyte proliferation and IL-4 generation in vitro [180]. The preventive potential of probiotics in atopic disease has been shown in a double blind, placebo controlled study by Schaer et al., 2003 [181]. Administration of probiotics to pregnant women and postnatally to infants for six months at high risk of atopic diseases succeeded in reducing the prevalence of atopic eczema to half compared with that in infants receiving placebo.

2.10 Lactic acid bacteria and the immune system:

The intestinal epithelium with optimal intestinal flora serves as the first line of defence against the invading pathogenic microorganisms, antigens and harmful components from the gut lumen. In addition the mucosal surface of the intestine is essential for the assimilation of antigens. Proteases of the intestinal bacteria degrade the antigenic structure, an important step in the introduction of unresponsiveness to dietary antigens. Specialised antigen transport mechanisms take place in different intestinal lymphoid compartments: mesenteric lymph nodes, Peyer's patches, isolated lymph follicles, isolated T lymphocytes in the epithelium and the lamina propria, as well as at secretary sites [175]. The secretary IgA antibodies in the gut are part of the common mucosal immune system, which includes the respiratory tract, salivary and mammary glands. The hallmark of an inflammatory response is the generation of proinflammatory cytokines including interleukin-1, interleukin-2, tumor necrosis factor- (TNF-) and interferon-. There are several reports indicating that proinflammatory cytokines may be the primary mediators of inflammation in clinical conditions characterized by impaired gut
50

barrier functions. It has in fact been demonstrated by Zisu et al., 2005 [176] that probiotics participate in the exclusion of pathogens. They can help to stabilize the gut microbial environment by producing antimicrobial substances and binding pathogens thereby preventing the generation of inflammatory mediators produced by intraluminal bacteria (Photo 2.6). Attachment of probiotic lactobacilli to cell surface receptors of enterocytes also initiates signalling events that result in the synthesis of cytokines. 2.10.1 Role of cytokines in the immune response: Inflammation, the response of tissue to injury, is characterized in the acute phase by increased blood flow and vascular permeability along with the accumulation of fluid, leukocytes and inflammatory mediators, such as cytokines. IL-4, IL-5, IL-6, IL-7, IL-13 are the cytokines mediating humoral responses and IL-1, IL-2, IL-3, IL-4, IL-7, IL-9, IL10, IL-12, interferons, transforming growth factor-, TNF- and- mediate cellular responses. 2.11 Interactions between epithelial cells and intestinal microflora: The interface between a mammalian host and microflora in the lumen is the mucous gel layer and the underlying cell coat (glycocalix) which consists of glycoconjugates on the apical surface of the epithelium. The intestinal microflora can influence the expression of epithelial glycoconjugates which serve as receptor for attachments of pathogenic microorganisms. There are several studies related to the adhesion mechanisms of pathogenic bacteria by fimbriae (pilus) or flagella, but little is known about the adhesion mechanisms of non-pathogenic bacteria such as lactic acid bacteria. Cesena et al., 2001 [182] suggested that lectin-like components in surfacelayered proteins of lactobacilli play an important role in the adhesion to receptors such as glycoproteins on the surface of intestinal epithelial cells.

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2.12 Casein Phosphopeptide and its uses: CPP are a large group of peptides that have a phosphoseryl residue in common. Phosphopeptides are formed either from casein by proteolytic enzymes during fermentation or in the gastrointestinal tract. CPP increase calcium absorption by forming a hydrophobic complex with calcium, thus preventing the formation of insoluble calcium phosphates. In vitro studies have shown the effects of CPP on calcium absorption by inhibiting the precipitation of calcium in the intestine. Casein phosphopeptides (CPP) are a tryptic hydrolysate of bovine casein, which enhances calcium absorption by increasing calcium solubility in vitro according to Drago et al., 1997 [183]. However, reports on the effect of dietary CPP on calcium absorption in vivo are controversial. Most of the reports have failed to show any enhancing effect of CPP on calcium absorption and retention in vivo. In contrast, Chung et al., 2002 [184] reported that CPP administration was associated with better absorption of co-ingested calcium by postmenopausal women with low basal absorptive performance. It was demonstrated that calcium bound to phosphopeptides could be absorbed from the digestive tract and promote bone calcification in rachide children. The purpose of this study was to evaluate the calcium and phosphorus availability from Cabound casein phosphopeptides (CaCPP) by testing the effect of long-term feeding on the bone loss in aged ovary ectomized rats. In vitro studies demonstrated that CPP can prevent the precipitation of calcium ions as insoluble salts such as calcium phosphate. This suggested the possibility that CPP enhance the amount of soluble calcium in the intestinal lumen, thereby increasing the mineral availability for absorption in the small intestine. Experiments performed on intestinal preparations (everted sacs, loops) provided evidence supporting this possibility [185]. However, in vivo investigations performed on whole animals designed to ascertain a role of CPP in both absorption and bioavailability of calcium, generated some controversial results. In fact, studies on growing pigs, as well as on weaning and adult (female) rats, showed that diets supplemented with CPP influenced neither calcium
52

absorption nor bone mineralization. On the contrary, rats fed a CPP-supplemented soybean protein diet had significantly greater calcium absorption than controls fed soybean alone. Moreover, the bioavailability of calcium appeared to be increased by CPP-enriched infant formula in rat pups, and the presence of CPP in the diet prevented mineral density decline in old ovary ectomized female rats. Finally, CPP were shown to enhance calcium absorption in both rachitic and normal chicks [186]. Interestingly, CPP also induced Ca2+ uptake by boar spermatozoa, facilitating sperm penetration into pig oocytes; the effect was reduced by dephosphorylation of CPP. Tamime et al., 1985 [187] stated that CPP were generally viewed as agents capable of maintaining intestinal calcium in its "soluble" form, thus facilitating the mineral flux through the membranes. However, the presence or absence of substances in the diet such as phosphate or phytate, that are capable of forming insoluble calcium salts or complexes, was not accurately assessed. This may be the basis for the conflicting results in in vivo studies. No determination was made of the direct interactions of CPP with the plasma membrane (particularly that of intestinal cells), which might affect calcium flux through the same membrane, regardless of any calcium-solubilizing action. The present work was designed to explore the possibility of a direct CPP influence on calcium uptake, using as a study model the human intestinal tumor cell line, HT-29, which tends to undergo an enterocytically oriented differentiation in culture. Calcium uptake was monitored as a rise in free cytosolic calcium concentration due to calcium ion movement through the plasma membrane. CPP has always been a widely studied peptide group in dentistry [188]. CPP also has been researched in the areas of sports medicine, anti-hypertensive medicine, remineralisation, and immune-enhancement and immune modulation [189]. The concept of food based nutrition has been practiced and advocated in India from the time immemorial and this has again gained momentum in the recent past due to social trends such as globalization, booming economy, growing purchase power of Indian middle
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class. CPP has the potential of becoming a food based nutritional item and boosting the immune system of humans. Virtual problem associated with any food based item is that it has less shelf life, chances of infection by micro organism become quite high and dissipation rate also increases considerably. If CPP is isolated from the fermented milk, then it reduces the dissipation rate, increases shelf life and the risk involved with the micro organisms decreases considerably [190]. Various parameters like viscosity, titratable acidity and pH are important to be studied and standardized before commencing the work. Viscosity of fermented milk that is prepared in a domestic environment will be lower than the fermented milk which is produced in a commercial firm. Milk fermented by lactic acid bacteria (LAB) have previously been shown to enhance both specific and nonspecific immune responses. Though most related studies focus on the administration of live bacteria, there is a lack of recognition of the possible Immunomodulatory role of the bioactive peptides or other compounds released in the culture medium during fermentation with LAB. Indeed, many beneficial effects have been attributed to bioactive peptides derived from milk, including opiate activity, antimicrobial activity, antihypertension, antithrombotic activity, and immunomodulation. Cell-free supernatants have been used to study the possible role of bioactive compounds released during milk fermentation. Hugenholtz et al., 1999 [191] reported that cell-free supernatants of Lactobacillus helveticus - fermented casein-enriched medium modulated lymphocyte proliferation in vitro. In parallel, De Vin in the year 2005 [192] used cultured macrophages to demonstrate that cell-free supernatants of L. helveticus-fermented milks exhibit higher interleukin-6 (IL-6) production than with lipopolysaccharide alone. More recently, peptide fractions of cell-free supernatants of L. helveticus-fermented milks have been shown to significantly reduce fibro sarcoma in vivo. However, cell-free supernatants of L. helveticus-fermented milks have not yet been implicated in the prevention or attenuation of bacterial infections in vivo.

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2.13 Anti-genotoxicity of CPP: 2.13.1 Classification of radioprotective agent: Radioprotective agents can be classified as: (i) (ii) (iii) chemical radioprotectors, adaptogens, and absorbents

The first group constitutes mainly sulf-hydryl compounds and other antioxidants. Adaptogens act as stimulators of radioresistance. These are natural protectors that offer chemical protection under low levels of ionizing radiations. They are generally extracted from the cells of plants and animals and have least toxicity. They can influence the regulatory system of exposed organisms, mobilize the endogenous background of radioresistance immunity, and intensify the overall nonspecific resistance of an organism. Absorbents protect organisms from internal radiation and chemicals. These include drugs which prevent the incorporation of radioiodine by the thyroid gland and the absorption of radionuclides like 137Cs, 90Sr and 239Pu. Post-irradiation radioprotectors are important when an accidental exposure occurs during operation of equipments with radiation source or intentional exposures during war and such unnatural calamities. This area of radiation biology is a very slowly developing area since it is rather difficult to get such effective protectors. 2.13.2 Milk and fermented milk as an anti-genotoxic agent: In the beginning of the 20th century, the Russian Nobel prizewinner lie Metchnikoff observed high life expectancy in Bulgarian persons who ate large amounts
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of fermented-milk products. One hundred years later, the consumption of fermentedmilk products is still associated with several types of human health benefits [193]. In addition to the favorable effects against diseases caused by an imbalance of the gut microflora, several experimental observations have indicated a potential protective effect of lactic acid bacteria (LAB) against the development of colon tumors. Colon cancer is the second to third most frequent type of cancer in Western industrialized countries. Within the complex gut microflora, which consists of above 1011 CFU living bacteria/g colon content, LAB belong to those bacteria with such beneficial effects. LAB plays an important role in retarding colon carcinogenesis by possibly influencing metabolic, immunologic, and protective functions in the colon. Concentrations of LAB may increase in the colon after the consumption of foods containing probiotics; however, probiotic ingestion also increases the number and metabolic activity of LAB in the colon of humans and animals [194]. In animals, LAB ingestion was shown to prevent carcinogeninduced preneoplastic lesions and tumors. A reduced activity of pro-carcinogenic enzymes in humans also was shown as a consequence of probiotic intake [195]. However, in humans, there is no evidence available on whether probiotics and prebiotics can prevent the initiation of colon cancer. Epidemiologic studies are contradictory; some studies could not find an association between the consumption of fermented-milk products and the risk of colon cancer whereas other studies showed a lower incidence of colon cancer in persons consuming fermented- milk products or yogurt [196]. In one case-control study, yogurt was the only milk product inversely related to the formation of large adenomas [197]. Therefore, the hypothesis that LAB may reduce the risk of developing colon tumors in humans is based mainly on experimental data. Within this context, it is postulated that the protective effects of probiotics and prebiotics can be effectively used in the near future.

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2.13.3 Enzymes and their anti-genotoxic mechanism: Oxidase and catalase enzymes have the potential to be useful as anti-genotoxic agents. Catalase enzyme prevents the nuclear degeneration and thus indirectly preventing the formation of micro nucleus. Alander et al., 1999 [166] have established the antigenotoxic role of catalase enzyme through a series of test and have also stated that the possible mechanism by which the catalase enzyme carries out its role is by preventing nuclear degeneration of the cell which ha been exposed to a genotoxic agent. Work carried out by Perdigon et al., 2003 [170] confirmed the anti-genotoxic role played by oxidase and they state that the possible mechanism of action by which oxidase enzyme is able to bring about the anti-genotoxic role is by stimulating a cellular level resistance which eventually leads to the anti-genotoxicity.

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CHAPTER 3 OBJECTIVES

1. Isolation of Casein Phospho Peptides (CPP) from fermented milk (FM) 2. Characterisation of the CPP isolated from FM by various standard procedures like HPLC, FTIR and SEM 3. Determination of the molecular weight of CPP by SDS PAGE 4. Assessment of the anti-microbial activity against selected GI tract pathogens 5. To prove the Immunomodulatory potential of bioactive peptides isolated from the fermented milk 6. To examine the anti-genotoxic effect of bioactive peptides isolated from the fermented milk

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CHAPTER 4 MATERIALS AND METHODS 4.1 Selection of milk brands and culture sources: Arokya brand milk with 4% fat content was purchased periodically from Tambaram, Chennai and used throughout the studies. Lactobacillus acidophilus, (MTCC number 721) Culture A and Lactobacillus bulgaricus, (MTCC number 738) Culture B were procured from IMTECH, Chandigarh. Arokya and Dodla commercial brand yogurt were used for commercial culture sources.

4.1.1 Production of fermented milk using different sources of bacterial cultures:

Two liters of Arokya brand milk with 4% fat was boiled for 15 minutes and divided in to 4 equal proportions containing 500ml each. To this 5ml of 108 CFU/ml cultures of Lactobacillus acidophilus, (MTCC number 721) designated as Culture A was added to all the 4 portions in triplicate for statistical purpose and incubated for overnight at room temperature. Another batch of 2 liters Arokya milk was divided in to 4 portions containing 500ml. To this 5ml of 108 CFU/ml culture of Lactobacillus bulgaricus, (MTCC number 738) designated as Culture B was added to all the 4 portions in triplicate for statistical purpose and incubated for overnight at room temperature. Another 2 batches were repeated using the same procedure with commercially available curds, Aavin Culture as C and Dodla culture as D. 5 ml of 108 CFU/ml of cultures C and D were added in triplicate and incubated at room

temperature for overnight [29,30]. 4.1.2 Initial Standardization: The effective fermentation parameters were taken in to consideration were pH, titratable acidity and viscosity which have direct impact on the production of CPP.
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4.1.2.1 pH: The change of pH for the all the fermented milk samples were recorded using a pH meter in triplicate (obtained from Mettler Toledo, 2006 model) in a time interval of 1 hour and the standard deviation was also calculated [198]. 4.1.2.2 Titratable Acidity: The titratable acidity of all the fermented milk samples in triplicate was determined titrimetrically by a 0.1 M NaOH with phenolphthalein as an indicator. The volume of the NaOH used up in milliliters to neutralize the 0.1M of the acid in 10 ml of the product expressed in Toerners degree (T) which could also be expressed as T x 0.1 M [32]. 4.1.2.3 Viscosity: Viscosity was measured for all the four fermented milk samples in triplicate using a viscometer (SVM 3000, Stabinger, manufactured by Antony paar) at 25C and was expressed as millipascal (mPas) [33]. 4.1.3 Microbiological analysis of fermented milk: The presence of the lactic acid bacteria in all the four fermented cultures were tested for the confirmation of Lactobacillus sp. using LB agar plates incubated at 37 C for 48 hours [199]. The organisms were also identified by grams staining (Medox suppliers, India). The presence of bacillus bacteria as fermenting agent was confirmed by Scanning Electron Microscopy (SEM) (Quanta 200 FEG) analysis. 4.1.4 Isolation of CPP from fermented milk: Milk was fermented for 24 h using either commercial curd or Lactobacillus species and the pH was adjusted to 7 using 0.5 M NaOH. Enzyme trypsin (Trypsin-T
60

from Medox suppliers, India) was added at enzyme: substrate ratio of 1:100. Then hydrolysis was carried out by mixing the suspension in a water bath using magnetic stirrer at 37C for 30 minutes. The pH of the solution was kept constant at pH 7.0 by addition of 0.1M NaOH solution. After complete hydrolysis the mixture was removed from water bath. The pH of casein hydrolysate was readjusted to 4.6 using 2M HCl. Centrifugation was done at 3000 rpm for 10 min to remove the non-phosphorylated peptides. The supernatant was removed and pH was adjusted to 7.0 using 2 M NaOH. Calcium chloride at 1% level was added to the supernatant and allowed to stand for 1 hour at room temperature. 50 % (V/V) ethanol was added and the precipitate was collected by centrifugation at 6000 rpm for 10 min (Photo 4.1). The CPPs thus obtained was lyophilized (120). The above given procedure was done to isolate the CPP from all the four different fermented milk. 4.1.5 Characterisation of four isolated CPPs: CPPs isolated from all the four sources were characterized using HPLC (High Performance Liquid Chromatography), FTIR (Fourier Transform Infra Red)

spectroscopy, SDS-PAGE and antimicrobial activity was also established. Photo 4.1 Casein isolated from fermented milk by enzymatic hydrolysis

61

4.1.5.1 Antimicrobial activity of CPP: The antibacterial activity of the isolated CPP was determined using Escherichia coli (MTCC Number 443) and Pseudomonas sp. (MTCC Number 1194). The bacteria were grown as 106 bacteria CFU/ml in 1.5% LB agar plates. Wells were created in the LB (Lacto-Bacillus) media and 100l of all the four CPPs were added along with same volume of streptomycin as the control. The plates were incubated at 37C for 48 hours and then the zone of inhibition was measured. The zone of inhibition for control and test were measured in terms of mm with the standard disc diffusion being followed. Antibacterial activity was assayed by the suppression of bacterial growth dependent on application of fractions to the top agar surface [165]. 4.1.5.2 High Performance Liquid Chromatography (HPLC) Analysis of CPP: HPLC (High Performance Liquid Chromatography), (Shimadzu LC 10AT VP model) was performed for the 4 different CPPs obtained from different sources, such as Aavin fermented milk, Dodla fermented milk, Milk fermented by Lactobacillus acidophilus and Lactobacillus bulgaricus. Non-fermented milk was used as the control. The column used was C-17, sample volume of 100l, flow rate of 10l/min, full volume injection without loss of sample, carryover of 0.01% and injection volume accuracy of 1%. 4.1.5.3 Fourier Transform Infra Red (FTIR) spectroscopy Analysis of CPP: FTIR analysis (GE FT09, SR Model) of the milk fermented by commercially available curd and milk fermented by bacterial cultures were performed. The transformation of the interferogram into spectrum was carried out mathematically with a dedicated on-line computer. The Bruker IFS66v FT-IR instrument (VERTEX model, Bruker optics, Germany) consists of globar and mercury vapor lamp as sources, an interferometer chamber comprising of KBr (Potassium Bromide) and Mylar beam
62

splitters followed by a sample chamber and detector. Entire region of 10-10000 cm-1 is covered by this instrument. The spectrometer works under vacuum conditions. Solid samples are dispersed in KBr or polyethylene pellets depending on the region of interest. This instrument has a resolution of 0.1 cm-1. Signal averaging, signal enhancement, base line correction and other spectral manipulations are possible with multitasking OPUS software on the dedicated PC/AT 486. Spectra are plotted on a HP plotter (19 inch plotter, HP, USA) and data was printed. 4.1.5.4 Molecular weight determination by Sodium Dodecyl Sulphate

Polyacrylamide Gel Electrophoresis (SDS PAGE): Polyacrylamide gels containing 2.5% of acrylamide was prepared following the procedures described by Lahov et al., 1996 [135]. The concentration of bisacrylamide was 5%. The buffer contained 36 mM-tris, 30 mM-NaH2 PO4 and 1 mm-EDTA (disodium salt) of pH 7.7 at room temperature was used. The running buffer in the buffer compartments also contained SDS (0-2%). This buffer has a greater buffering capacity and a lower UV absorption than the tris-acetate buffer. Mg buffer was the same as the low-salt buffer but with magnesium acetate (2 mM). The sample was prepared by mixing the protein with the sample buffer in the ratio 4:1. The sample was prepared by boiling for 10 minutes. It was then run at 250 V constant for about 30 minutes total run time. The bands were identified and tabulated after the staining process with staining dye (coomassie dye). Comparison of test samples with the standard marker was enacted. 4.2 Animal studies: 4.2.1 Effect of CPP on weight loss and mortality rate in mice challenged with GUT Pathogens: 4.2.1.1 Acclimatization of animals: About 18 male albino mice of 6 weeks old, weighed 25 - 30 grams were obtained from TANUVAS (Tamil Nadu University for Veterinary and Animal Sciences), Madhavaram, Chennai were placed as 3 mice per cage in 6 cages labeled as A1, A2, B1,
63

B2, Control (C) and Normal (N). They were acclimatized for 15 days period in their respective cages. The mice were fed with ad libitum standard rodent chow and provided with distilled water. All experiments were performed under controlled conditions (temperature [21 2C], humidity and a 12-h light-dark cycle). The individual weights of all the 18 mice were recorded and tabulated. All the animal works were cleared by Institutional ethical committee (The Ethical committee clearance number is SRM-2010027). 4.2.1.2 Injection with CPP: The injection period was divided into 10 and 15 days for 2 sets of test mice. Batches A1 and B1 were fed for 10 days whereas batches A2 and B2 were fed only for the last 15 days. 0.5 ml of CPP was injected through intramuscular route using standard 1ml syringes (Dispo Van, USA). The oral route was not preferred for injection since the CPP will be affected by intestinal enzymes. The dosage was administered in standard time intervals of 24 hours. It was ensured that the mice were healthy and did not pose any health problems during the injection time. The regular mice feed and the distilled water was available ad libitum. The mice were housed under controlled temperature and standard dark light cycle. 4.2.1.3 Challenging with Gastro-Intestinal Tract Pathogens: E. coli (MTCC Number-078) was procured from IMTECH, Chandigarh and was sub cultured in a specific medium (LB Agar medium). All the 18 mice including the control and the test were given 0.1 ml of 104 E. coli through oral route using gastric tube (Merck Instruments, USA) on the 16th day (The day after challenging the injection with CPP was stopped). Prior to infection the weights of all the 18 mice were recorded and tabulated. It was ensured that during challenging all the mice were healthy and did not have any physical wounds. The same procedures were repeated for Salmonella and Shigella species.
64

4.2.1.4 Determination of pathogen count in visceral organs: All mice were taken from all the groups (Normal, test and Control), anaesthetized, dissected and the visceral organs were harvested after 7 days of post infection (Liver, spleen and kidney). The CFU of the E. coli in the visceral organs were determined after 7 days by culturing techniques. A thin horizontal streaking of the harvested visceral organs was done in separate petri plates containing sterilized MS agar media (Medox suppliers, Chennai). The number of CFU present on the surface of the media was counted using a digital colony counter after an incubation period of 48 hours at 37C. The CFU readings were tabulated and recorded. The same procedures were repeated with Salmonella and Shigella species. 4.2.1.5 Histopathological studies: The tissues of liver, kidney, spleen and small intestine were fixed in 4% formaldehyde in PBS (pH 7.4), prior to dehydration in alcohol and embedding in wax. Tissue sections were cut using a microtome at 1.5 mm thickness. The tissues were mounted on a clean pre-sterilized glass slide and allowed to remain in the buffer for 24 hours. Then they were allowed to air dry for 6 hours. Finally the tissues were treated with a staining dye (Methylene blue), washed with washing buffer and air dried again. The slides were kept at room temperature for 48 hours and finally mounted again on the slide with the necessary preservative. 4.2.2 Immunomodulatory role: [169] The number of cells secreting IgA was determined by Direct Immunofluorescence assay method. For each intestinal tissue, 10 slides were prepared with 4mm serial paraffin section. Slides were incubated with 50l of 1/50 dilution of -chain monospecific antibody (Sigma Aldrich, Texas, USA) conjugated with fluorescein isothiocyanate (Sigma Aldrich, Bangalore, India) and left at room temperature for 30 minutes. The slides
65

were washed for 3 minutes with PBS in a coplin jar placed upon the shaker. A small drop of VectaShield medium was added next to each spot so that a thin layer results when the coverslip is put on. It was then observed under Hund H6000 fluorescence light microscope. Results were expressed as the number of fluorescent cells counted in 10 fields of vision at 100X magnification. 4.3 Anti-genotoxic studies using CPP: 4.3.1 Gamma irradiation Experiment with Animals: 4.3.1.1 Experimental set up for mice: The Anti-genotoxic effect of CPP was examined using micronucleus assay in mice. About 18 male albino mice of 6 weeks old, weighed 25-30 grams were obtained per experiment from TANUVAS (Tamil Nadu University for Veterinary and Animal Sciences), Madhavaram, Chennai. They were placed in standard size cages of dimensions 12 x 3 x 6. The mice were divided in to 5 groups with each group having 12 mice. The test batches were subdivided in to 2 groups based on the number of CPP injection days as Test 1 batch T1 Injection period for 10 days and Test 2 - T2 injection period for 15 days. One batch was kept as control without CPP injection. Four groups were designated as test batches to be fed with Aavin CPP, Dodla CPP, L. acido. CPP and L. bulg. CPP. All the mice were acclimatized for 15 days period in their respective cages. The mice were fed once a day with standard rodent chow and provided with distilled water. All experiments were performed under controlled conditions (temperature 21 2C and a 12hours light-dark cycle). 4.3.1.2 Experimental set up for fish: About 50 6 weeks old, 6-7 cm length Pangasius pangasius fishes were procured from A.M. Aqua Farm, Madurai, India and acclimatized in tanks of dimension 15 x 4 x 12 with a capacity of 200 liters. The fishes were divided in to 5 groups with each group having 10 fishes. One batch was kept as control without CPP feed and 4 groups as test
66

batches with Aavin CPP, Dodla CPP, L. acido. CPP and L. bulg. CPP. The fishes were fed with standard fish feed along with CPP for 15 days and experiments were performed under controlled conditions of temperature 21 2C and a 12-hours light-dark cycle). 4.3.1.3 Irradiation with Co60 source: Both the groups of animals were subjected to Cobalt-60 irradiation using gamma irradiator equipment at Radiological safety division, IGCAR (Indira Gandhi Centre for Atomic Energy), Kalpakkam, Tamilnadu (Photo 4.2, Photo 4.3). Mice batches were subjected to irradiation at 0.5, 1 and 5 Gy for duration of 10, 19 and 94 seconds respectively. Fish batches were irradiated at 50, 100 and 150 for duration of 940, 1880 and 2820 seconds respectively. The LD50 values in case of mice and fish were determined using the formula, LD50 = Dose - (a*b) / c Where Dose Dose at which complete mortality was observed (LD100) a Dose difference b Mean mortality c- Mortality observed at LD100 LD50 was found to be 1.9 Gy units and 135 Gy units for mice and fish respectively. A positive control batch was also maintained which were fed with CPP but were not irradiated. Negative control was the batch which was irradiated but not fed with CPP. The blood samples were taken and assayed for micronucleus as an indication of DNA damage.

67

Photo 4.2 Gamma irradiator used in anti-genotoxic studies (External view)

Photo 4.3 Gamma irradiator used in anti-genotoxic studies (inside view)

4.3.2

Micronucleus assay: About 1ml of blood was collected using heparinized syringe from irradiated,

control mice and fish. Thin smear of the blood was prepared on a clean glass slide. The slides were treated in methanol for 10 min and washed with distilled water, dried and
68

stained with 8% giemsa stain allowed to be air dried for 10 minutes and washed with double distilled water. The glass slides were observed under the microscope at 100x magnification in oil immersion. The cells with micronucleus and changes in the nucleus like binucleus, multi-nucleus or any deformation in the 10 field of magnification were observed at 100X. 4.3.3 Enzymatic assays: 4.3.3.1 Oxidase test: Oxidase test was carried out using impregnated oxidase test strip method. Separate solutions of intestinal tissue belonging to test batches of mice and fish were prepared using PBS buffer and designated as test solutions. Control batch mice and fish intestinal tissues were prepared using the same PBS buffer and designated as control solutions. Few drops of the solutions were added slowly on the strip containing the reagent and colour change was observed for the next few seconds. Production of blue colour was considered as the presence of oxidase enzyme. 4.3.3.2 Catalase test: Catalase is the enzyme that breaks hydrogen peroxide (H2O2) into H2O and O2. The bubbling that is seen is due to the evolution of O2 gas. Catalase test was carried out using air bubble formation method. Separate smears of intestinal tissue belonging to test batches of mice and fish were prepared using PBS buffer and designated as test smears after the irradiation. Control batch mice and fish intestinal tissue smears were prepared using the same PBS buffer and designated as control smears. Few drops of the catalase reagent were slowly added on the test and control smears and production of air bubble was observed for the next few seconds. Production of air bubble was considered as the presence of oxidase enzyme.

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CHAPTER 5 RESULTS 5.1 Isolation and characterization: 5.1.1 Initial Standardization: 5.1.1.1 pH: The pH of all the four fermented milk samples showed steady decrease with time due to the fermentation process. Photo 5.1 depicts the milk fermented by Aavin and Photo 5.2 depicts the milk fermented by Lactobacillus bulgaricus as representative samples. The milk samples fermented by L. acidophilus, Aavin brand and Dodla brand showed the pH as 5.3 0.09 and L. bulgaricus showed 5.16 0.13 when compared with the milk samples fermented by commercial cultures (Table 5.1 to 5.4). Figure 5.1 represents the pH value change in the various fermented milk. The titratable acidity was found to be the highest in milk fermented by L. bulgaricus and the lowest in milk fermented by commercial Aavin (Table 5.6). The control had the value at around 50 Toerners degrees (T). Figure 5.2 represents the titratable acidity value change in the various fermented milk. The highest viscosity value was recorded for milk fermented by commercial curd Dodla and the lowest value was in milk fermented by the culture L. acidophilus (Table 5.7). Figure 5.3 represents the viscosity value change for various fermented milk. Photo 5.1 Fermented milk after the addition of commercial Aavin curd. The samples were prepared in triplicate (A, B, C) with 5 ml of Aavin curd added to 500 ml of 4% fat containing arokya milk and allowed to ferment overnight at standard conditions

70

Photo 5.2 Fermented milk after the addition of the culture Lactobacillus bulgaricus. The samples were prepared in triplicate with 5 ml of 108 CFU/ml culture added to 500 ml of 4% fat containing arokya milk and allowed to ferment overnight at standard conditions

Table 5.1 pH values of the fermented milk with time after added with Dodla dairy curd. The values given are mean Standard Deviation of triplicate samples. The values were measured for every hour using a pH meter under standard conditions with number of hours n = 7.

Time (In hours) 0 1 2 3 4 5 6 7

71

pH (Mean S.D) 7.050 0.010 6.175 0.154 6.038 0.182 5.927 0.196 5.790 0.218 5.657 0.183 5.463 0.166 5.255 0.126

Table 5.2 pH values of the fermented milk with time after added with Aavin dairy curd. The values given are mean Standard Deviation of triplicate samples. The values were measured every hour using a pH meter under standard conditions, number of hours n = 7. Time (in hours) 0 1 2 3 4 5 6 7 Mean S.D 7.050 0.010 6.380 0.092 6.447 0.151 6.237 0.108 6.117 0.106 5.792 0.155 5.460 0.107 5.282 0.185

Table 5.3 pH values of the fermented milk with time after added with Lactobacillus acidophilus culture. The values given are mean Standard Deviation of triplicate samples. The values were measured every hour using a pH meter under standard conditions, number of hours n = 7. Time (in hours) 0 1 2 3 4 5 6 7
72

Mean S.D 7.050 0.010 6.028 0.108 6.053 0.112 6.008 0.135 5.910 0.133 5.733 0.191 5.420 0.212 5.297 0.091

Table 5.4 pH values of the fermented milk with time after added with Lactobacillus bulgaricus culture. The values given are mean Standard Deviation of triplicate samples. The values were measured for every hour using a pH meter under standard conditions. Time (in hours) 0 1 2 3 4 5 6 7 Mean S.D 7.050 0.010 6.213 0.107 5.982 0.068 5.705 0.083 5.705 0.083 5.357 0.095 5.238 0.082 5.155 0.095

Table 5.5 The pH changes for milk fermented using commercial curds Dodla, Aavin, Lactobacillus acidophilus and Lactobacillus bulgaricus. The values given are mean Standard Deviation of triplicate samples. The values were measured every hour using a pH meter under standard conditions, number of hours n = 7. Time (in hours) 0 1 2 3 4 5 6 7 Control 7.050 0.010 7.050 0.010 7.050 0.010 7.050 0.010 7.050 0.010 7.050 0.010 7.050 0.010 7.050 0.010 Aavin Curd 7.050 0.010 6.380 0.092 6.447 0.151 6.237 0.108 6.117 0.106 5.792 0.155 5.460 0.107 5.282 0.185
73

Dodla Curd 7.050 0.010 6.175 0.154 6.038 0.182 5.927 0.196 5.790 0.218 5.657 0.183 5.463 0.166 5.255 0.126

L. acidophilus 7.050 0.010 6.028 0.108 6.053 0.112 6.008 0.135 5.910 0.133 5.733 0.191 5.420 0.212 5.297 0.091

L. bulgaricus 7.050 0.010 6.213 0.107 5.982 0.068 5.705 0.083 5.705 0.083 5.357 0.095 5.238 0.082 5.155 0.095

Figure 5.1 The pH changes for milk fermented using commercial curds Dodla, Aavin, Lactobacillus acidophilus and Lactobacillus bulgaricus. The values given are mean Standard Deviation of triplicate samples. The values were measured every hour using a pH meter under standard conditions, number of hours n = 7.

5.1.1.2 Titratable acidity: Table 5.6 The titratable acidity values for milk fermented using commercial curds Dodla, Aavin, Lactobacillus acidophilus and Lactobacillus bulgaricus. The values given are mean Standard Deviation of triplicate samples. The values were measured using acid base neutralization reaction under standard conditions and given with SD.

S.No 1 2 3 4 5

Sample Control Milk Aavin curd Dodla Curd Lactobacillus acidophilus Lactobacillus bulgaricus
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Titratable acidity 50.78 2.91 91.27 3.89 91.49 4.03 92.11 4.42 93.52 4.67

Figure 5.2 The titratable acidity values for milk fermented using commercial curds Dodla, Aavin, Lactobacillus acidophilus and Lactobacillus bulgaricus. The values given are mean Standard Deviation of triplicate samples. The values were measured using acid base neutralization reaction under standard conditions and given with SD.

L. acido. CPP L. bulg. CPP

Control milk

AAVIN CPP

DODLA CPP

L. acido. CPP

L. bulg. CPP

Type of fermented milk

5.1.1.3 Viscosity: Table 5.7 The viscosity changes for milk fermented using commercial curds Dodla, Aavin, Lactobacillus acidophilus and Lactobacillus bulgaricus. The values given are mean Standard Deviation of triplicate samples. The values were measured using a viscometer under standard conditions and given with SD S.No 1. 2. 3. 4. Sample Aavin curd Dodla Curd Lactobacillus acidophilus Lactobacillus bulgaricus Viscosity 8.01 1.93 7.72 1.16 7.12 1.02 7.13 1.04

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Figure 5.3 The viscosity changes for milk fermented using commercial curds Dodla, Aavin, Lactobacillus acidophilus and Lactobacillus bulgaricus. The values given are mean Standard Deviation of triplicate samples. The values were measured using a viscometer under standard conditions and given with SD.

L. acidophilus L.bulgaricus

Dodla curd

Aavin curd

L. acidophilus

L. bulgaricus

Type of fermented milk

5.1.2 Microbiological analysis of fermented milk: The bacterial staining by gram stain showed the presence of gram positive bacteria that is L. acidophilus and L. bulgaricus. The presence of L. acidiophilus was confirmed by Scanning Electron Microscopy (Photo 5.3). 5.1.3 Yield and production cost of CPP: The amount of CPP produced from 500 ml of fermented milk was found to be 6.5 grams. Hence the yield proportion of CPP is calculated as follows, Yield proportion = 6.5/500 x 100 = 1.5 grams/100 ml of fermented milk
76

The cost of 500 ml of fermented milk is Rs.12, hence the production cost of CPP per gram including the reagents such as enzyme, pH adjusting buffers etc would come around Rs.21/Photo 5.3 Scanning Electron microscopic image of Lactobacillus species present in milk fermented by Lactobacillus bulgaricus at a magnification of 1000 X. The bacterium was identified by rod shape, filamented structure visible in the microscopic background

5.1.4 Anti-microbial activity of CPP: Anti-microbial activity of the CPP was proven against the gastrointestinal pathogens like E. coli and Pseudomonas sp. The mean zone of inhibition produced by Aavin CPP against E. coli was 14 mm and 16 mm with Pseudomonas sp. compared with the standard streptomycin (12 and 13mm) when performed in triplicate (Photo 5.4). Dodla CPP produced 13 mm zone of inhibition against E. coli and 15 mm with Pseudomonas sp. L. acido. CPP produced 16 mm zone of inhibition against E. coli and 15 mm with Pseudomonas sp. L. bulg. CPP produced 12 mm zone of inhibition against E. coli and 15 mm with Pseudomonas sp. (Table 5.8).

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Table 5.8 Zone of inhibition formed by CPP isolated from commercial curd Aavin, commercial curd Dodla, Lactobacillus acidophilus and Lactobacillus bulgaricus cultures against Escherichia coli and Pseudomonas sp. The mean of triplicate values were taken with n=3 and the difference in the values were < 0.001 (P < 0.001) S.No Material Zone of inhibition against Escherichia coli (mm) 12 14 13 16 12 Zone of inhibition against Pseudomonas sp. (mm) 13 16 15 15 15

1 2 3 4 5

Streptomycin (control) Aavin CPP Dodla CPP L. acido. CPP L. bulg. CPP

Figure 5.4 Anti-microbial activity of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP against Escherichia coli and Pseudomonas sp. The zone of inhibition above 12 mm was considered to be positive effect against the growth of bacterium

L. acido. CPP

L. bulg. CPP

Type of Fermented Milk 78

Photo 5.4 Zone of inhibition produced by CPP isolated from commercial curd Aavin against 105 CFU/ml of Escherichia coli (a) and 105 CFU/ml of Pseudomonas sp. (b)

(a)

(b)

5.1.5 High Performance Liquid Chromatography (HPLC) Analysis of CPP: All the four CPP (Dodla, Aavin, L. acidophilus, L. bulgaricus) isolated from fermented milk were subjected to High Performance Liquid Chromatography (HPLC) analysis. The peaks observed showed the characteristic features of fermented milk peptides. The peaks from the control, non-fermented milk were quite different from the fermented milk. The CPP isolated from four different sources had minor, salient intra-differences among them. The results are shown in the figures 5.5-5.9.

79

Figure 5.5 HPLC spectrum of non fermented control milk. The area under peaks had significance less than 0.005 (P < 0.005) with number of peaks, n=8. Difference in values significance was calculated to be < 0.005 using ANOVA.

80

Figure 5.6 HPLC spectrum of CPP isolated from milk fermented by commercial curd Dodla. Peaks at 4.03 Rt, 4.30 Rt, and 14.93 Rt showed the characteristic features of fermented milk peptides. The area under peak had significance less than 0.005 (P < 0.005) with number of peaks, n=8.

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Figure 5.7 HPLC spectrum of CPP isolated from milk fermented by commercial curd Aavin. Peaks at 4.01 Rt, 4.26 Rt and 15.03 Rt showed the characteristic features of fermented milk peptides. The area under peak had significance less than 0.005 (P < 0.005) with number of peaks, n=8.

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Figure 5.8 HPLC spectrum of CPP isolated from milk fermented by the culture Lactobacillus acidophilus. Peaks at 4.29 Rt, 5.28 Rt and 15.02 Rt showed the characteristic features of fermented milk peptides. The area under peak had significance less than 0.005 (P < 0.005) with number of peaks, n=8.

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Figure 5.9 HPLC spectrum of CPP isolated from milk fermented by the culture Lactobacillus bulgaricus. Peaks at 3.51 Rt and 14.95 Rt showed the characteristic features of fermented milk peptides. The area under peak had significance less than 0.005 (P < 0.005) with number of peaks, n=8.

5.1.6 Fourier Transform Infra Red spectroscopy analysis of CPP: The FTIR analysis of all the four CPP (Dodla, Aavin, L. acidophilus, L. bulgaricus) given in the figures 5.10-5.13 showed the characteristic peaks at 2808-1 cm, 1654-1 cm, 1130-1 cm, 975-1 cm and 909-1 cm and some of those peaks were absent in the FTIR figure 5.14 of control, non-fermented milk.

84

Figure 5.10 FTIR spectrum of non fermented control milk. The area under peaks had significance less than 0.005 (P < 0.005) with number of peaks, n=8. Difference in values significance was calculated to be < 0.005 using ANOVA.
1.80 1.7 1.6 1.5 1.4 1.3 1.2 1.1
1543

C1

1653

Absorbance

1.0
1404

1098 3286 2929

0.9 0.8 0.7

1243

0.6 0.5 0.4 0.3 0.2 0.1 0.00 4000.0 3600 3200 2800 2400 2000 1800 cm-1 1600 1400 1200 1000 800 600 450.0
544

cm

85

Figure 5.11 FTIR spectrum of CPP isolated from milk fermented by commercial curd Aavin. Peaks at 2878 cm, 1654 cm, 1256 cm, and 977 cm showed the characteristic features of fermented milk peptides. The area under peak had significance less than 0.002 (P < 0.002) with number of peaks, n=24.
1.80 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0
1534

C2
1654

Absorbance

0.9 0.8 0.7

1799

1403 1256 1098

0.6 0.5 0.4

3298 2971 2761 2878 977

688

555

0.3
3726

0.2 0.1 0.00 4000.0 3600 3200 2800 2400 2000 1800 cm-1 1600 1400 1200 1000 800 600 450.0

cm
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Figure 5.12 FTIR spectrum of CPP isolated from milk fermented by commercial curd Dodla. Peaks at 2808 cm, 1659 cm, 1131 cm, 976 cm and 834 cm1 showed the characteristic features of fermented milk peptides. The area under peak had significance less than 0.002 (P < 0.002) with number of peaks, n=24.
1.80 1.7 1.6 1.5 1.4 1.3 1.2 1.1
3048

B
1659 1403

1337

1.0

2808 2881

1534 1131 976 834 2060 1824 1182 688

Absorbance

0.9 A 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.00 4000.0 3600 3200 2800 2400 2000 1800 cm-1 1600 1400 1200

526 3700 3575 3667

1000

800

600

450.0

cm

87

Figure 5.13 FTIR spectrum of CPP isolated from milk fermented by the culture Lactobacillus acidophilus. Peaks at 2808 cm, 1654 cm, 1130 cm, 975 cm and 909 cm1 showed the characteristic features of fermented milk peptides. The area under peak had significance less than 0.002 (P < 0.002) with number of peaks, n=24.
1.80 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0
2051 1308 1357 2318 2352 1827 1183 975 909 1130 1099 2808 2872 2897 2969

A
1654 1543 1405

Absorbance

0.9 A 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.00 4000.0 3600 3200 2800
3697 3776 3574 3916

528

701

2400

2000

1800 cm-1

1600

1400

1200

1000

800

600

450.0

88

cm

Figure 5.14 FTIR spectrum of CPP isolated from milk fermented by the culture Lactobacillus bulgaricus. Peaks at 2808 cm, 1651 cm, 1127 cm, 976 cm and 834 cm1 showed the characteristic features of fermented milk peptides. The area under peak had significance less than 0.002 (P < 0.002) with number of peaks, n=24.
1.80 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0
3032 2879 1542 2808 1098 1127

C
1651 1403

Absorbance

0.9 0.8 0.7 0.6

3715

2317 2351

1831

976 834 928

0.5 0.4 0.3 0.2 0.1 0.00 4000.0 3600 3200 2800 2400 2000 1800 cm-1 1600 1400 1200 1000 800

700

531

600

450.0

89

cm

5.1.7 Determination of Molecular weight of CPP by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE): The CPP isolated from the fermented milk by the cultures of L. acidophilus and L. bulgaricus were run along with CPP from the fermented milk by two commercial curds in SDS PAGE along with the standard markers. The molecular weight of the four isolated CPPs was found to be in the range of 1.5 3.5 kD (Photo 5.5). Photo 5.5 Molecular weight determination of CPP isolated from milk fermented by commercial curd Aavin, commercial curd Dodla, Lactobacillus acidophilus and Lactobacillus bulgaricus using Sodium Dodecyl Sulphate - Polyacrylamide Gel Electrophoresis (SDS-PAGE) having 2.5% of acrylamide and run at 250 V constant for about 30 minutes

Bands coinciding with the test samples

C1 Culture A (Lactobacillus acidophilus), C2 Culture B (Lactobacillus bulgaricus)

90

5.2 Animal Studies: 5.2.1 Challenging with GUT Tract Pathogens: The initial mean body weight of mice fed with AAVIN CPP isolated from milk fermented by commercial Aavin was 31.23 3.27 grams. The final mean body weight after a injection period of 10 days was 33.23 3.08 grams. There was a 2 0.57 grams body weight increase in the mice fed with AAVIN CPP for 10 days (Table 5.9). The initial mean body weight of DODLA CPP fed mice was 31.5 2.62 grams, increased to 33.9 3.69 grams after 10 days of CPP injection showing an increase of 2.59 0.15 grams body weight (Table 5.10). The initial mean body weight of L. acido. CPP fed mice was 32.11 3.36 grams, increased to 34.33 3.03 grams after 10 days of CPP injection showing an increase of 2.22 0.22 grams body weight (Table 5.11).

The initial mean body weight of L. bulg. CPP fed mice was 31.89 2.83 grams, increased to 34.12 2.87 grams after 10 days of CPP injection, showing an increase of 2.28 0.71 grams body weight (Table 5.12). The body weight of control CPP unfed mice after 10 days had a body weight increase of 1.28 0.46 grams (Table 5.13). Table 5.14 and Figure 5.15 give the summary of the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice after fed for 10 days.

91

Table 5.9 Effect of AAVIN CPP on body weight in albino mice after injection for 10 days. The number of animals fed, n = 18 and the difference of significance was not more than 0.001 (P 0.001) calculated using ANOVA and given with SD

S.No

Body weight of mice (gm) Before injection with CPP After injection with CPP Increase +3 +4 +1 +4 +1 +5 +1 +1 0 +2 +3 +1 +3 +2 +4 0 0 +1 2 0.57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mean

25 34 36 27 30 31 32 30 35 29 27 33 34 29 31 33 30 37 31.23 3.27

28 38 37 31 31 36 33 31 35 31 30 34 37 31 35 33 30 38 33.23 3.08

92

Table 5.10 Effect of DODLA CPP on body weight in albino mice after injection for 10 days. The number of animals fed, n = 18 and the difference of significance was not more than 0.001 (P 0.001) calculated using ANOVA and given with SD S.No Before injection with CPP 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mean 31 33 30 34 32 27 31 33 30 34 32 27 33 37 34 30 28 31 31.5 2.62 Body weight of mice (gm) After injection with CPP Increase

32 37 31 36 34 28 32 33 31 33 32 28 39 41 40 34 33 36 33.9 3.69

+1 +4 +1 +3 +2 +1 +1 0 +1 -1 0 +1 +6 +4 +6 +4 +5 +5 2.59 0.15

93

Table 5.11 Effect of L. acido. CPP on body weight in albino mice after injection for 10 days. The number of animals fed, n = 18 and the difference of significance was not more than 0.001 (P 0.001) calculated using ANOVA and given with SD S.No Before injection with CPP 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mean 29 27 31 30 33 37 33 35 32 36 28 29 37 27 35 32 31 36 32.11 3.36 Body weight of mice (gm) After injection with CPP Increase

32 28 33 33 37 39 35 38 33 37 30 32 37 32 36 35 33 38 34.33 3.03

+3 +1 +2 +3 +4 +2 +2 +3 +1 +1 +2 +3 0 +5 +1 +3 +2 +2 2.22 0.22

94

Table 5.12 Effect of L. bulg. CPP on body weight in albino mice after injection for 10 days. The number of animals fed, n = 18 and the difference of significance was not more than 0.001 (P 0.001) calculated using ANOVA and given with SD

S.No

Body weight of mice (gm) Before injection with CPP After injection with CPP 30 38 38 33 34 33 33 33 36 30 34 39 30 34 35 33 39 33 34.12 2.87 Increase +2 +1 +3 0 +4 +1 +2 +3 +2 +1 +2 +3 +2 +3 +2 +3 +3 +4 2.28 0.71

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mean

28 37 35 33 30 32 31 30 34 29 32 36 28 31 33 30 36 29 31.89 2.83

95

Table 5.13 Body weight of albino mice after injection normal feed alone without CPP for 10 days. The number of animals fed, n = 18 and the difference of significance was not more than 0.001 (P 0.001) calculated using ANOVA and given with SD S.No Body weight of mice (gm) Before injection with CPP 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mean 30 28 31 34 32 35 33 31 33 28 32 34 33 27 35 32 29 31 31.56 2.41 After injection with CPP 30 29 31 34 32 34 33 30 35 28 32 34 33 27 35 33 29 32 31.72 2.42 Increase 0 +1 0 0 0 -1 0 -1 +2 0 0 0 0 0 0 -1 0 +1 0.16 0.03

96

Table 5.14 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice after injection for 10 days. The number of animals fed, n = 18 and the difference of significance was not less than 0.002 (P 0.001) calculated using ANOVA and given with SD

S.No

Source of CPP

Initial body weight (gm)

Body weight after injection for 10 days (gm)

Increase in body weight (%)

Control

31.56 2.41

31.72 2.42

0.50

AAVIN CPP

31.23 3.27

33.23 3.08

6.02

DODLA CPP

31.5 2.62

33.9 3.69

7.22

L. acido. CPP

32.11 3.36

34.33 3.03

6.47

L. bulg. CPP

31.89 2.83

34.12 2.87

6.54

97

Figure 5.15 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the percentage increase in body weight of albino mice after fed for 10 days

L. acido. CPP Source of CPP

L. bulg. CPP

AAVIN CPP - CPP isolated from milk fermented by commercial Aavin DODLA CPP - CPP isolated from milk fermented by commercial Dodla L. acido. CPP - CPP isolated from milk fermented by Lactobacillus acidophilus L. bulg. CPP - CPP isolated from milk fermented by Lactobacillus bulgaricus The initial mean body weight of AAVIN CPP fed mice was 31.39 2.59 grams, increased to 34.94 3.28 grams after 15 days of injection, showing an increase
98

of 3.56 0.54 grams body weight (Table 5.15). The initial mean body weight of DODLA CPP was 31.51 2.62 grams, increased to 35.83 3.05 grams after 15 days of injection, showing an increase of 4.33 1.08 grams increase in body weight (Table 5.16). The initial mean body weight of L. acido. CPP fed mice was 32.56 2.99 grams, increased to 36.50 3.19 grams after 15 days of injection, showing an increase of 3.94 1.30 grams in body weight (Table 5.17). The initial mean body weight of L. bulg. CPP fed mice was 30.11 3.25 grams, increased to 33.28 4.03 grams after 15 days of injection, showing an increase of 3.17 1.38 grams in body weight (Table 5.18). The body weight of control CPP unfed mice after 15 days had a body weight increase of 1.56 0.51 grams. The mean body weight of mice at day 0 was 31.12 2.20 and the body weight after 15 days was 32.72 2.42 grams (Table 5.19).

Table 5.20 and Figure 5.16 shows the summary of the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice after injection for 15 days. Table 5.21 gave the comparison between the percentage increase in body weight of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP fed mice for a injection period of 10 and 15 days.

99

Table 5.15 The effect of AAVIN CPP on body weight in albino mice after injection for 15 days. The number of animals fed, n = 18 and the difference of significance was not more than 0.001 (P 0.001) calculated using ANOVA and given with SD

S.No

Body weight of mice (gm) Before injection with CPP After injection with CPP 38 32 40 34 31 34 32 33 34 33 38 31 38 34 36 43 35 33 34.94 3.28 Increase

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mean

32 29 34 31 28 30 28 31 33 30 36 29 34 29 31 37 32 31 31.39 2.59

+6 +3 +6 +3 +3 +4 +4 +2 +1 +3 +2 +2 +4 +5 +5 +6 +3 +2 3.56 0.54

100

Table 5.16 Effect of DODLA CPP on body weight in albino mice after injection for 15 days. The number of animals fed, n = 18 and the difference of significance was not more than 0.001 (P 0.001) calculated using ANOVA and given with SD

S.No

Body weight of mice (gm) Before injection with CPP After injection with CPP 34 38 35 32 37 39 32 34 40 32 36 33 39 41 40 34 33 36 35.83 3.05 Increase +3 +5 +5 +4 +5 +5 +4 +2 +5 +3 +3 +4 +6 +4 +6 +4 +5 +5 4.33 1.08

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mean

31 33 30 28 32 34 28 32 35 29 33 29 33 37 34 30 28 31 31.51 2.62

101

Table 5.18 Effect of L. acido. CPP on body weight in albino mice after injection for 15 days. The number of animals fed, n = 18 and the difference of significance was not more than 0.001 (P 0.001) calculated using ANOVA and given with SD S.No Body weight of mice (gm) Before injection with CPP 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mean 33 35 38 32 27 31 31 36 34 35 30 29 31 36 34 35 30 29 32.56 2.99 After injection with CPP 36 39 40 37 30 36 34 38 38 38 32 33 35 42 40 40 35 34 36.50 3.19 Increase +3 +4 +2 +5 +3 +5 +3 +2 +4 +3 +2 +4 +4 +6 +6 +5 +5 +4 3.94 1.30

102

Table 5.18 Effect of L. bulg. CPP on body weight in albino mice after injection for 15 days. The number of animals fed, n = 18 and the difference of significance was not more than 0.001 (P 0.001) calculated using ANOVA and given with SD S.No Body weight of mice (gm) Before injection with CPP 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mean 34 30 31 29 27 32 27 29 26 24 28 32 33 35 32 36 28 29 30.11 3.25 After injection with CPP 37 32 33 34 30 34 28 32 30 25 30 36 37 38 38 41 32 32 33.28 4.03 Increase +3 +2 +2 +5 +3 +2 +1 +3 +4 +1 +2 +4 +4 +3 +6 +5 +4 +3 3.17 1.38

103

Table 5.19 Body weight of albino mice after injection with normal feed alone without CPP for 15 days. The number of animals fed, n = 18 and the difference of significance was not more than 0.001 (P 0.001) calculated using ANOVA and given with SD

S.No

Body weight of mice (gm) Before injection with CPP After injection with CPP Increase +2 +1 +1 +2 +1 +2 +2 +2 +2 +2 +1 +1 +1 +2 +1 +2 +1 +2 1.56 0.51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mean

29 27 32 30 31 31 29 35 32 28 31 34 33 29 32 31 34 33 31.12 2.20

31 28 33 32 32 33 31 37 34 30 32 35 34 31 33 33 35 35 32.72 2.42
104

Table 5.20 Comparison of the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice after injection for 15 days. The number of animals fed, n = 18 and the difference of significance was not less than 0.002 (P 0.001) calculated using ANOVA and given with SD

S.No Source of CPP

Initial body weight (gm)

Body weight after injection for 15 days (gm)

Increase in body weight (%)

Control

31.12 2.41

32.72 2.42

4.89

AAVIN CPP

31.39 2.59

34.94 3.28

10.16

DODLA CPP

31.51 2.62

35.83 3.05

12.06

L. acido. CPP

32.56 2.99

36.50 3.19

10.79

L. bulg. CPP

30.11 3.25

33.28 4.03

9.53

105

Table 5.21 Comparison between the percentage increase in body weight of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP fed mice for a injection period of 10 and 15 days.

Increase in body weight S.No Source of CPP after 10 days of injection (%)

Increase in body weight after 15 days of injection (%)

Control

0.50

4.89

AAVIN CPP

6.02

10.16

DODLA CPP

7.22

12.06

L. acido. CPP

6.47

10.79

L. bulg. CPP

6.54

9.53

106

Figure 5.16 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the percentage increase in body weight of albino mice after injection for 15 days

L. acido. CPP Source of CPP

L. bulg. CPP

107

5.2.2. Post infection studies:

As given in the table 5.22 in case of E.coli infected control mice, there was a steep decline of body weight from first day till seventh day with a mortality rate of 100 %. But in AAVIN CPP, 10 days fed, E.Coli infected mice there was a steady increase in the body weigh from the day one of post infection till the third day. Starting from the fourth day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 33%. In DODLA CPP, 10 days fed, E.Coli infected mice there was a steady increase in the body weigh from the day one of post infection till the second day. Starting from the third day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 17%. In L. acido. CPP, 10 days fed, E.Coli infected mice there was a steady increase in the body weigh from the day one of post infection till the second day. Starting from the third day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 33%. In L. bulg. CPP, 10 days fed, E.Coli infected mice there was a steady increase in the body weigh from the day one of post infection till the third day. Starting from the fourth day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 17%.

Table 5.22 summarised the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight loss percentage of albino mice fed for 10 days with CPP and infected with Escherichia coli. Table 5.23 and Figure 5.17 gave the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight loss percentage of albino mice fed for 10 days with CPP and infected with Escherichia coli. Figure 5.18 depicted the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the mortality percentage of albino mice fed for 10 days with CPP and infected with Escherichia coli. Figure 5.19 summarised the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice fed for 10 days with CPP and infected with Escherichia coli.

108

As given in the table 5.24 in case of E.coli infected control mice, there was a steep decline of body weight from first day till seventh day with a mortality rate of 83%. In AAVIN CPP, 15 days fed, E.Coli infected mice there was a steady increase in the body weigh from the day one of post infection till the fourth day. Starting from the fifth day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 33%. In DODLA CPP, 15 days fed, E.Coli infected mice there was a steady increase in the body weigh from the day one of post infection till the fourth day. Starting from the fifth day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 33%. In L. acido. CPP, 15 days fed, E.Coli infected mice there was a steady increase in the body weigh from the day one of post infection till the fifth day. Starting from the sixth day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 17%. In L. bulg. CPP, 15 days fed, E.Coli infected mice there was a steady increase in the body weigh from the day one of post infection till the fourth day. Starting from the fifth day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 17%.

Table 5.24 summarised the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight loss percentage of albino mice fed for 15 days with CPP and infected with Escherichia coli. Table 5.25 and Figure 5.20 gave the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight loss percentage of albino mice fed for 10 days with CPP and infected with Escherichia coli. Figure 5.21 depicted the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the mortality percentage of albino mice fed for 15 days with CPP and infected with Escherichia coli. Figure 5.22 summarised the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice fed for 15 days with CPP and infected with Escherichia coli.

109

Table 5.22 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice after injection for 10 days and infected with Escherichia coli. Number of mice n = 6 and the level of significance was not less than 0.005 (P 0.005) and given with SD

Decrease in body weight (in grams) Source of CPP

Control AAVIN CPP DODLA CPP L. acido. CPP L. bulg. CPP 6 32.5 3.24

No. of mice
6

Day 1
29.8 3.07

Day 2
27.5 2.56 35.5 2.71

Day 3
21.2 2.05 36.7 2.83

Day 4
19.7 2.02 32.3 2.21

Day 5
17.3 1.43 33.7 3.54

Day 6
16.7 1.59 32.2 3.85

Day 7
14.7 1.72 30.8 2.31

Mice mortality
6 2

Mortality Rate
100% 33%

34.7 2.50

6 6

30.8 3.12 30.8 3.10

31.0 2.03

28.0 2.45

27.8 3.71

27.2 2.93

25.0 2.55

23.7 2.33

17%

31.5 3.59 33.2 3.68

31.2 2.36 33.7 3.25

27.0 2.71 33.0 3.18

26.5 2.56 32.3 3.16

25.2 2.32 30.5 2.92

23.3 2.80 25.0 2.06

2 1

33% 17%

110

Table 5.23 Percentage of body weight loss in albino mice infected with Escherichia coli fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days

Source of CPP Control AAVIN CPP DODLA CPP L. acido. CPP L. bulg. CPP

Percentage of body weight loss 50.67% 11.24% 23.05% 24.35% 23.08%

Figure 5.17 Percentage of body weight loss in albino mice infected with Escherichia coli fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days

L. acido. CPP

L. bulg. CPP

Source of CPP

111

Figure 5.18 Percentage mortality rate in albino mice infected with Escherichia coli fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days

L. acido. CPP

L. bulg. CPP

Source of CPP

Figure 5.19 Body weight loss in albino mice infected with Escherichia coli fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days

L. acido. CPP L. bulg. CPP

112

Table 5.24 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice after injection for 15 days and infected with Escherichia coli. Number of mice n = 6 and the level of significance was not less than 0.005 (P 0.005) and given with SD Decrease in body weight (in grams) Source of No. of CPP mice
6

Day 1
31.5 1.96

Day 2
25.8 2.14

Day 3
24.3 2.18

Day 4
19.7 1.67

Day 5`
19.0 1.55

Day 6
18.2 1.48

Day 7
17.5 1.36

Mice mortality

Mortality Rate

Control AAVIN CPP DODLA CPP L. acido. CPP L. bulg. CPP

83%

34.0 3.61

36.2 3.65

37.5 3.81

39.0 3.92

37.3 3.74

34.3 3.43

32.2 2.55

33%

31.5 2.87

32.8 2.71

33.7 2.79

34.8 2.64

33.8 2.53

32.3 2.40

31.1 2.23

33%

32.7 2.51

34.0 2.59

34.3 2.60

35.2 2.65

35.5 2.68

33.2 2.33

30.0 2.20

17%

30.7 2.22

32.0 2.34

33.0 2.85

33.7 2.62

32.2 2.71

30.5 2.38

28.5 2.78

17%

113

Table 5.25 Percentage of body weight loss in albino mice infected with Escherichia coli fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days Source of CPP Control AAVIN CPP DODLA CPP L. acido. CPP L. bulg. CPP Percentage of body weight loss 18.60% 5.29% 1.27% 8.26% 7.17%

Figure 5.20 Percentage of body weight loss in albino mice infected with Escherichia coli fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days.

L. acido. CPP
Treatment Source of CPP

L. bulg. CPP

114

Figure 5.21 Percentage mortality rate in albino mice infected with Escherichia coli fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days

L. acido. CPP

L. bulg. CPP

Figure 5.22 Body weight loss in albino mice infected with Escherichia coli fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days

L. acido. CPP L. bulg. CPP

Source of CPP

115

Table 5.26 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice after injection with them for 10 days and infected with Salmonella sp. Number of mice n = 6 and the level of significance was not less than 0.005 (P 0.005) and given with SD

Source of CPP
Control AAVIN CPP DODLA CPP L. acido. CPP L. bulg. CPP

No. of mice
6

Decrease in body weight ( in grams)

Day 1 31.7 2.67 Day 2 30.8 2.49 Day 3 29.7 2.32 Day 4 28.3 2.60 Day 5` 27.3 2.33 Day 6 22.5 1.78 Day 7 17.3 1.45

Mice mortality
4

Mortality Rate
100%

33.0 2.75

33.5 2.73

34.2 2.62

34.5 2.86

33.0 2.65

26.3 2.74

24.3 2.51

17%

31.7 2.64

31.8 2.65

32.7 2.82

28.3 2.39

28.2 2.41

27.2 2.32

25.3 2.17

0%

32.0 2.80

33.3 2.70

34.7 2.69

34.3 3.01

32.5 3.12

30.7 2.92

29.0 2.41

33%

32.0 2.43

33.3 3.14

34.7 3.44

36.0 3.05

35.2 3.61

33.0 2.96

30.8 2.12

17%

116

Table 5.27 Percentage body weight loss in albino mice infected with Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days Source of CPP Control AAVIN CPP DODLA CPP L. acido. CPP L. bulg. CPP Percentage body weight loss 45.43% 26.36% 20.19% 9.38% 6.88%

Figure 5.23 Percentage of body weight loss in albino mice infected with Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days

L. acido. CPP

L. bulg. CPP

Source of CPP
117

Figure 5.24 Percentage mortality rate in albino mice infected with Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days

L. acido. CPP

L. bulg. CPP

Source of CPP

Figure 5.25 Body weight loss in albino mice infected with Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days

L. acido. CPP L. bulg. CPP

118

Table 5.28 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 15 days injection on the body weight of albino mice infected with Salmonella sp. Number of mice n = 6 and the level of significance was not less than 0.005 (P 0.005) and given with SD

Decrease in body weight (in grams) Source of CPP

Control AAVIN CPP DODLA CPP L. acido. CPP L. bulg. CPP 6 27.7 2.04 6 32.0 2.94

No. of mice
6

Day 1
30.7 3.16

Day 2
28.7 2.89

Day 3
23.5 1.72

Day 4
19.0 1.48

Day 5`
18.1 1.37

Day 6
17.3 1.24

Day 7
17.2 1.14

Mice mortality
7

Mortality Rate
100%

33.2 3.19

34.3 3.34

35.2 3.41

35.2 3.39

32.3 3.01

30.7 2.94

27.8 2.72

33%

31.7 3.06

32.5 3.12

34.0 3.34

34.7 3.21

34.0 3.45

32.0 2.93

30.2 2.76

17%

32.2 2.62

32.5 2.67

31.3 2.44

29.7 2.17

27.5 2.04

25.7 1.93

33%

29.3 2.56

29.7 2.59

29.8 2.76

29.3 2.80

27.7 2.61

25.7 2.36

33%

119

Table 5.29 Percentage body weight loss in albino mice infected with Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days

Source of CPP Control AAVIN CPP DODLA CPP L. acido. CPP L. bulg. CPP

Percentage body weight loss 43.97% 16.27% 4.73% 19.69% 7.22%

Figure 5.26 Percentage of body weight loss in albino mice infected with Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days

L. acido. CPP L. bulg. CPP

Source of CPP
120

Figure 5.27 Percentage mortality rate in albino mice infected with Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days

L. acido. CPP

L. bulg. CPP

Source of CPP

Figure 5.28 Body weight loss in albino mice infected with Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days

L. acido. CPP L. bulg. CPP

121

As given in the table 5.26 in case of Salmonella sp. infected control mice, there was a steep decline of body weight from first day till seventh day with a mortality rate of 100 %. But in AAVIN CPP, 10 days fed, Salmonella sp. infected mice there was a steady increase in the body weigh from the day one of post infection till the fourth day. Starting from the fifth day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 17%. In DODLA CPP, 10 days fed, Salmonella sp. infected mice there was a steady increase in the body weigh from the day one of post infection till the third day. Starting from the fourth day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 0%. In L. acido. CPP, 10 days fed, Salmonella sp. infected mice there was a steady increase in the body weigh from the day one of post infection till the third day. Starting from the fourth day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 33%. In L. bulg. CPP, 10 days fed, Salmonella sp. infected mice there was a steady increase in the body weigh from the day one of post infection till the fourth day. Starting from the fifth day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 17%.

Table 5.26 summarised the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight loss percentage of albino mice fed for 10 days with CPP and infected with Salmonella sp. Table 5.27 and Figure 5.23 depicted the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight loss percentage of albino mice fed for 10 days with CPP and infected with Salmonella sp. Figure 5.24 depicted the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the mortality percentage of albino mice fed for 10 days with CPP and infected with Salmonella sp. Figure 5.25 summarised the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice fed for 10 days with CPP and infected with Salmonella sp.

122

As given in the table 5.28 in case of Salmonella sp. infected control mice, there was a steep decline of body weight from first day till seventh day with a mortality rate of 34 %. But in AAVIN CPP, 15 days fed, Salmonella sp. infected mice there was a steady increase in the body weigh from the day one of post infection till the fourth day. Starting from the fifth day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 33%. In DODLA CPP, 15 days fed, Salmonella sp. infected mice there was a steady increase in the body weigh from the day one of post infection till the third day. Starting from the fourth day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 17%. In L. acido. CPP, 15 days fed, Salmonella sp. infected mice there was a steady increase in the body weigh from the day one of post infection till the third day. Starting from the fourth day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 33%. In L. bulg. CPP, 15 days fed, Salmonella sp. infected mice there was a steady increase in the body weigh from the day one of post infection till the fourth day. Starting from the fifth day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 33%.

Table 5.28 summarised the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight loss percentage of albino mice fed for 15 days with CPP and infected with Salmonella sp. Table 5.29 and Figure 5.26 depicted the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight loss percentage of albino mice fed for 15 days with CPP and infected with Salmonella sp. Figure 5.27 depicted the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the mortality percentage of albino mice fed for 15 days with CPP and infected with Salmonella sp. Figure 5.28 summarised the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice fed for 15 days with CPP and infected with Salmonella sp.

123

Table 5.30 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice after injection for 10 days and infected with Shigella sp. Number of mice n = 6 and the level of significance was not less than 0.005 (P 0.005) and given with SD Decrease in body weight (in grams)
Day 1 Day 2 Day 3 Day 4 Day 5` Day 6 Day 7

Batch
Control AAVIN CPP DODLA CPP L. acido. CPP L. bulg. CPP

No. of mice
6

Mice mortality
5

Mortality Rate
83%

32.7 2.74 31.1 2.59 30.5 2.41 27.9 2.07 25.5 1.96 23.7 1.67 20.2 1.54 33.3 2.67 34.2 2.71 35.3 2.74 36.5 2.83 35.5 2.69 33.3 2.35 27.0 1.98

17%

34.5 2.45 35.8 3.20 37.0 2.96 36.1 2.82 35.2 2.65 33.9 2.68 32.4 1.91

17%

32.4 2.82 34.1 3.14 35.7 3.45 36.5 3.71 33.9 2.65 32.2 2.57 30.6 2.42

0%

31.6 2.15 32.9 3.04 34.4 2.78 35.7 3.15 33.9 3.26 32.5 2.98 30.9 2.87

33%

124

Table 5.31 Percentage of body weight loss in albino mice infected with Shigella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days Source of CPP Control AAVIN CPP DODLA CPP L. acido. CPP L. bulg. CPP Percentage of body weight loss 38.27% 18.92% 8.99% 5.56% 2.22%

Figure 5.29 Percentage of body weight loss in albino mice infected with Shigella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days

L. acido. CPP L. bulg. CPP

Source of CPP
125

Figure 5.30 Percentage mortality rate in albino mice infected with Shigella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days

L. acido. CPP

L. bulg. CPP

Source of CPP

Figure 5.31 Body weight loss in albino mice infected with Shigella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days

L. acido. CPP L. bulg. CPP

126

Table 5.32 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice after injection for 15 days and infected with Shigella sp. Number of mice n = 6 and the level of significance was not less than 0.005 (P 0.005) and given with SD Decrease in body weight (in grams)
Day 1 31.5 2.80 Day 2 31.0 2.54 Day 3 29.9 1.94 Day 4 27.3 1.75 Day 5` 26.1 1.67 Day 6 23.8 1.56 Day 7 22.4 1.39

Batch
Control AAVIN CPP DODLA CPP L. acido. CPP L. bulg. CPP

No of mice
6

Mice mortality
6

Mortality Rate
100%

32.8 3.28

34.9 3.67

35.8 3.78

36.1 3.81

34.7 2.98

33.2 3.21

30.5 2.94

33%

32.3 3.17

33.1 3.22

34.7 3.54

36.2 3.68

37.4 3.77

34.7 3.42

31.7 2.85

50%

33.0 2.65

34.3 2.72

34.9 3.27

36.3 3.40

36.2 3.42

34.6 3.16

32.8 2.61

17%

29.4 2.43

31.2 3.20

33.2 3.42

34.9 3.64

36.1 3.89

34.4 3.32

32.6 3.17

33%

127

Table 5.33 Percentage of body weight loss in albino mice infected with Shigella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days Source of CPP Control AAVIN CPP DODLA CPP L. acido. CPP L. bulg. CPP Percentage of body weight loss 28.64% 7.01% 6.86% 6.23% 10.89%

Figure 5.32 Percentage of body weight loss in albino mice infected with Shigella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days

L. acido. CPP L. bulg. CPP

Source of CPP

128

Figure 5.33 Percentage mortality rate in albino mice infected with Shigella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days

L. acido. CPP L. bulg. CPP

Source of CPP

Figure 5.34 Body weight loss in albino mice infected with Shigella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days

L. acido. CPP L. bulg. CPP

129

As given in the table 5.30 in case of Shigella sp. infected control mice, there was a steep decline of body weight from first day till seventh day with a mortality rate of 33%. But in AAVIN CPP, 10 days fed, Shigella sp. infected mice there was a steady increase in the body weigh from the day one of post infection till the fourth day. Starting from the fifth day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 17%. In DODLA CPP, 10 days fed, Shigella sp. infected mice there was a steady increase in the body weigh from the day one of post infection till the third day. Starting from the fourth day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 17%. In L. acido. CPP, 10 days fed, Shigella sp. infected mice there was a steady increase in the body weigh from the day one of post infection till the fourth day. Starting from the fifth day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 0%. In L. bulg. CPP, 10 days fed, Shigella sp. infected mice there was a steady increase in the body weigh from the day one of post infection till the fourth day. Starting from the fifth day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 33%.

Table 5.30 summarised the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight loss percentage of albino mice fed for 10 days with CPP and infected with Shigella sp. Table 5.31 and Figure 5.29 depicted the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight loss percentage of albino mice fed for 10 days with CPP and infected with Shigella sp. Figure 5.30 depicted the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the mortality percentage of albino mice fed for 10 days with CPP and infected with Shigella sp. Figure 5.31 summarised the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice fed for 10 days with CPP and infected with Shigella sp.

130

As given in the table 5.32 in case of Shigella sp. infected control mice, there was a steep decline of body weight from first day till seventh day with a mortality rate of 100%. But in AAVIN CPP, 15 days fed, Shigella sp. infected mice there was a steady increase in the body weigh from the day one of post infection till the fourth day. Starting from the fifth day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 33%. In DODLA CPP, 15 days fed, Shigella sp. infected mice there was a steady increase in the body weigh from the day one of post infection till the fifth day. Starting from the fifth day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 50%. In L. acido. CPP, 15 days fed, Shigella sp. infected mice there was a steady increase in the body weigh from the day one of post infection till the fourth day. Starting from the fifth day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 17%. In L. bulg. CPP, 15 days fed, Shigella sp. infected mice there was a steady increase in the body weigh from the day one of post infection till the fifth day. Starting from the sixth day there was a decline in the body weight of the mice till the seventh day with a mortality rate of 33%. Table 5.32 summarised the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight loss percentage of albino mice fed for 15 days with CPP and infected with Shigella sp. Table 5.33 and Figure 5.32 depicted the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight loss percentage of albino mice fed for 15 days with CPP and infected with Shigella sp. Figure 5.33 depicted the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the mortality percentage of albino mice fed for 15 days with CPP and infected with Shigella sp. Figure 5.34 summarised the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice fed for 15 days with CPP and infected with Shigella sp.

131

5.2.3 Determination of Pathogen count in visceral organs: The pathogen (E. coli) count present in liver of the albino mice fed with AAVIN CPP was 21 x 103. The pathogen (E. coli) count present in liver of the albino mice fed with DODLA CPP was 18 x 103. The pathogen (E. coli) count present in liver of the albino mice fed with L. acido. CPP was 14 x 103. The pathogen (E. coli) count present in liver of the albino mice that was fed with L. bulg. CPP was 17 x 103. The pathogen (E. coli) count present in liver of the albino mice that were not fed with CPP (control batch) was 51 x 103. The pathogen (E. coli) count present in kidney of the albino mice fed with AAVIN CPP was 13 x 103. The pathogen (E. coli) count present in kidney of the albino mice fed with DODLA CPP was 19 x 103. The pathogen (E. coli) count present in kidney of the albino mice fed with L. acido. CPP was 16 x 103. The pathogen (E. coli) count present in kidney of the albino mice fed with L. bulg. CPP was 8 x 103. The pathogen (E. coli) count present in kidney of the albino mice that were not fed with CPP (control batch) was 67 x 103 (Table 5.34). Table 5.34 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 15 days injection on GUT pathogen count of albino mice visceral organs after infected with Escherichia coli

Pathogen count in Visceral organs S.No Treatment Liver 1 2 3 4 5 Control AAVIN CPP DODLA CPP L. acido. CPP L. bulg. CPP 51 21 18 14 17 Kidney 67 13 19 16 8 Spleen 78 13 15 4 18

( x 103) Small intestine 62 11 6 10 19

132

Figure 5.35 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 15 days injection on GUT pathogen count of albino mice visceral organs after infected with Escherichia coli

L. acido. L. bulg.

As seen in figure 5.35, the pathogen (E. coli) count present in spleen of the albino mice fed with AAVIN CPP was 13 x 103. The pathogen (E. coli) count present in spleen of the albino mice fed with DODLA CPP was 15 x 103. The pathogen (E. coli) count present in spleen of the albino mice fed with L. acido. CPP was 4 x 103. The pathogen (E. coli) count present in spleen of the albino mice fed with L. bulg. CPP was 18 x 103. The pathogen (E. coli) count present in spleen of the albino mice not fed with CPP (control batch) was 78 x 103. The pathogen (E. coli) count present in liver of the albino mice fed with AAVIN CPP was 11 x 103. The pathogen (E. coli) count present in liver of the albino mice fed with DODLA CPP was 6 x 103. The pathogen (E. coli) count present in liver of the albino mice fed with L. acido. CPP was 10 x 103. The pathogen (E. coli) count present in liver of the albino mice fed with L. bulg. CPP was 19 x 103. The pathogen (E. coli) count present in liver of the albino mice not fed with CPP (control batch) was 62 x 103.

133

Table 5.35 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 15 days injection on GUT pathogen count of albino mice visceral organs after infected with Salmonella sp. Pathogen count in visceral organs (x 103) S.No Treatment Liver Kidney Spleen Small intestine

1 2 3 4 5

Control AAVIN CPP DODLA CPP L. acido. CPP L. bulg. CPP

79 27 21 16 23

81 24 20 26 19

61 13 23 9 15

45 7 11 4 14

The pathogen (Salmonella sp.) count present in liver of the albino mice fed with AAVIN CPP was 27 x 103. The pathogen (Salmonella sp.) count present in liver of the albino mice that was fed with DODLA CPP was 21 x 103. The pathogen (Salmonella sp.) count present in liver of the albino mice fed with L. acido. CPP was 16 x 103. The pathogen (Salmonella sp.) count present in liver of the albino mice fed with L. bulg. CPP was found to be 23 x 103. The pathogen (Salmonella sp.) count present in liver of the albino mice not fed with CPP (control batch) was 79 x 103. The pathogen (Salmonella sp.) count present in kidney of the albino mice fed with AAVIN CPP was 24 x 103. The pathogen (Salmonella sp.) count present in kidney of the albino mice fed with DODLA CPP was 20 x 103. The pathogen (Salmonella sp.) count present in kidney of the albino mice fed with L. acido. CPP was 26 x 103. The pathogen (Salmonella sp.) count present in kidney of the albino mice fed with L.bulg .CPP was 19 x 103.
134

The pathogen (Salmonella sp.) count present in liver of the albino mice not fed with CPP (control batch) was 81 x 103. The pathogen (Salmonella sp.) count present in spleen of the albino mice fed with AAVIN CPP was 13 x 103. The pathogen (Salmonella sp.) count present in spleen of the albino mice fed with DODLA CPP was 23 x 103. The pathogen (Salmonella sp.) count present in spleen of the albino mice fed with L. acido. CPP was 9 x 103. The pathogen (Salmonella sp.) count present in spleen of the albino mice fed with L. bulg. CPP was 15 x 103 (Table 5.35). Figure 5.36 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 15 days injection on GUT pathogen count of albino mice visceral organs after infected with Salmonella sp.

L. acido. L. bulg.

The pathogen (Salmonella sp.) count present in spleen of the albino mice not fed with CPP (control batch) was 61 x 103. The pathogen (Salmonella sp.) count present in small intestine of the albino mice fed with AAVIN CPP was 7 x 103. The pathogen (Salmonella sp.) count present in small intestine of the albino mice fed with DODLA CPP was 11 x 103. The pathogen (Salmonella sp.) count present in small intestine of the albino mice fed with L. acido. CPP was 4 x 103. The pathogen (Salmonella sp.) count present in small intestine of the albino mice fed with L. bulg. CPP was 14 x 10-3. The pathogen (Salmonella sp.) count present in small intestine of the albino mice not fed with CPP (control batch) was 45 x 103 (Figure 5.36).
135

Table 5.36 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 15 days injection on GUT pathogen count of albino mice visceral organs after infected with Shigella sp. Pathogen count in visceral organs (x 103) S.No Treatment Liver 1 2 3 4 5 Control AAVIN CPP DODLA CPP L. acido. CPP L. bulg. CPP 93 15 20 24 11 Kidney 57 6 13 18 10 Spleen 68 16 19 12 22 Small intestine 59 8 13 24 17

The pathogen (Shigella sp.) count present in liver of the albino mice fed with AAVIN CPP was 15 x 103. The pathogen (Shigella sp.) count present in liver of the albino mice fed with DODLA CPP was 20 x 103. The pathogen (Shigella sp.) count present in liver of the albino mice fed with L. acido. CPP was 24 x 103. The pathogen (Shigella sp.) count present in liver of the albino mice fed with L. bulg. CPP was 11 x 103. The pathogen (Shigella sp.) count present in liver of the albino mice not fed with CPP (control batch) was 93 x 103. The pathogen (Shigella sp.) count present in kidney of the albino mice fed with AAVIN CPP was 6 x 103. The pathogen (Shigella sp.) count present in kidney of the albino mice fed with DODLA CPP was 13 x 103. The pathogen (Shigella sp.) count present in kidney of the albino mice fed with L. acido. CPP was 18 x 103. The pathogen (Shigella sp.) count present in kidney of the albino mice fed with L. bulg. CPP was found to be 10 x 103. The pathogen (Shigella sp.) count present in liver of the albino mice not fed with CPP (control batch) was 57 x 103.
136

The pathogen (Shigella sp.) count present in spleen of the albino mice fed with AAVIN CPP was 16 x 103. The pathogen (Shigella sp.) count present in spleen of the albino mice fed with DODLA CPP was 19 x 103. The pathogen (Shigella sp.) count present in spleen of the albino mice fed with L. acido. CPP was 12 x 103. The pathogen (Shigella sp.) count present in spleen of the albino mice fed with L. bulg. CPP was 22 x 103. The pathogen (Shigella sp.) count present in spleen of the albino mice not fed with CPP (control batch) was 68 x 103 (Table 5.36). The pathogen (Shigella sp.) count present in small intestine of the albino mice fed with AAVIN CPP was 8 x 103. The pathogen (Shigella sp.) count present in small intestine of the albino mice fed with DODLA CPP was 13 x 103. The pathogen (Shigella sp.) count present in small intestine of the albino mice fed with L. acido. CPP was 24 x 103. The pathogen (Shigella sp.) count present in small intestine of the albino mice fed with L. bulg. CPP was 17 x 103. The pathogen (Shigella sp.) count present in small intestine of the albino mice not fed with CPP (control batch) was 59 x 103 (Figure 5.37). Figure 5.37 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on GUT pathogen count of albino mice visceral organs fed with CPP for 15 days and infected with Shigella sp.

L. acido. L. bulg.

137

5.2.4 Histopathological studies: Photo 5.6 showed the histopathological study results of 15 days CPP fed albino mice liver cells infected with Escherichia coli after seven days post infection period. Photo 5.7 showed the histopathological study results of 15 days CPP injection period on albino mice kidney cells infected with Escherichia coli after seven days post infection period. Photo 5.8 showed the histopathological study results of 15 days CPP injection period on albino mice spleen cells infected with Escherichia coli after seven days post infection period. Photo 5.9 showed the histopathological study results of 15 days CPP injection period on albino mice small intestine cells infected with Escherichia coli after seven days post infection period. Photo 5.6 Photograph showing the histopathological studies of 15 days CPP fed albino mice liver cells infected with Escherichia coli on seven days post infection staining with Methylene blue with 10 fields of vision at 100X magnification. (a) Control batch of CPP unfed mice

(b) Aavin CPP 15 days fed mice

138

(c) Dodla CPP 15 days fed mice

(d) L. acido. CPP 15 days fed mice

(e) L. bulg. CPP 15 days fed mice

139

Photo 5.7 Photograph showing the histopathological studies of 15 days CPP injection period on albino mice kidney cells infected with Escherichia coli on post infection period of seven days after staining with Methylene blue.

(a) Control batch of CPP unfed mice

(b) Aavin CPP 15 days fed mice

140

(c) Dodla CPP 15 days fed mice

(d) L. acido. CPP 15 days fed mice

(e) L. bulg. CPP 15 days fed mice

141

Photo 5.8 Photograph showing the histopathological studies of 15 days CPP injection period on albino mice spleen cells infected with Escherichia coli on post infection period of seven days after staining with Methylene blue with 10 fields of vision at 100X magnification. (a) Control batch of CPP unfed mice

(b) Aavin CPP 15 days fed mice

142

(c) Dodla CPP 15 days fed mice

(d) L. acido. CPP 15 days fed mice

(e) L. bulg. CPP 15 days fed mice

143

Photo 5.9 Photograph showing the histopathological studies of 15 days CPP injection period on albino mice small intestine cells infected with Escherichia coli on post infection period of seven days after staining with crystal violet with 10 fields of vision at 100X magnification (a) Control batch of CPP unfed mice

(b) Aavin CPP 15 days fed mice

144

(c) Dodla CPP 15 days fed mice

(d) L. acido. CPP 15 days fed mice

(e) L. bulg. CPP 15 days fed mice

145

5.3 Determination of Immunomodulatory activity: [169] To determine the effect of the isolated CPP on immunomodulatory activity of the IgA secretary cells in the mice intestine was done using direct

Immunofluorescence assay procedure described by Makino et al., 2006 [169]. The results showed that the control mice which was not fed with CPP but infected with Escherichia coli had 8 2 IgA secretary cells/10 fields of vision at a magnification of 100x (Photo 5.10) for 10 days post infection. Test mouse fed with AAVIN CPP for 10 days and then infected with Escherichia coli was having 35 4 IgA secretary cells/10 fields of vision at a magnification of 100x (Photo 5.11) and test mice fed with AAVIN CPP for 15 days and then infected with Escherichia coli was having 82 4 IgA secretary cells/10 fields of vision at a magnification of 100x (Photo 5.12). Test mouse fed with DODLA CPP for 10 days and then infected with Escherichia coli was having 31 4 IgA secretary cells/10 fields of vision at a magnification of 100 and test mice fed with DODLA CPP for 15 days and then infected with Escherichia coli was having 91 4 IgA secretary cells/10 field of vision at a magnification of 100x (Figure 5.36).

Test mice fed with L. acido. CPP for 10 days and then infected with Escherichia coli was having 42 4 IgA secretary cells/10 fields of vision at a magnification of 100 and test mice fed with L. acido. CPP for 15 days and then infected with Escherichia coli was having 78 4 IgA secretary cells/10 fields of vision at a magnification of 100x. Test mice fed with L. bulg. CPP for 10 days and then infected with Escherichia coli was having 29 4 IgA secretary cells/10 fields of vision at a magnification of 100 and test mice fed with L. bulg. CPP for 15 days and then infected with Escherichia coli was having 95 4 IgA secretary cells/10 fields of vision at a magnification of 100x (Figure 5.37). The control mice not fed with CPP and infected with Escherichia coli had 8 2 IgA secretary cells/10 fields of vision at a magnification of 100x after 10 days of post infection period (Table 5.37).

146

Table 5.37 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on IgA secretary cell production in albino mice fed with CPP for 10, 15 days and infected with Escherichia coli. Number of mice infected were, n = 6 and the level of significance was not more than 0.002 (P 0.02) when calculated by ANOVA and given with SD

IgA secretary cells/10 fields of vision S.No 1 2 3 4 5 Source of CPP 10 days Control AAVIN CPP DODLA CPP L. acido. CPP L. bulg. CPP 8 2 35 4 31 4 42 4 29 4 15 days 6 2 82 4 91 4 78 4 95 4

Figure 5.38 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 10 days injection on IgA secretary cell production in albino mice fed with CPP for 10 days after infected with Escherichia coli and given with SD

L. acido. CPP L. bulg. CPP

Control milk

AAVIN CPP

DODLA CPP

L. acido. CPP

L. bulg. CPP

Source of CPP
147

Figure 5.39 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP fed for 15 days on IgA secretary cell production in albino mice after infected with Escherichia coli and given with SD

L. acido. CPP L. bulg. CPP

Control milk

AAVIN CPP

DODLA CPP

L. acido. CPP

L. bulg. CPP

Source of CPP

Table 5.38 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 10, 15 days injection on IgA secretary cell production in albino mice after infected with Salmonella sp. Number of mice infected were, n = 6 and the level of significance was not more than 0.002 (P 0.02) when calculated by ANOVA and given with SD

IgA secretary cells/10 fields of vision S.No 1 2 3 4 5 Source of CPP 10 days Control AAVIN CPP DODLA CPP L. acido. CPP L. bulg. CPP 10 2 28 4 37 4 45 4 53 4
148

15 days 14 2 93 4 88 4 96 4 102 4

Figure 5.40 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 10 days injection on IgA secretary cell production in albino mice after infected with Salmonella sp. and given with SD

L. acido. CPP L. bulg. CPP

Control milk

AAVIN CPP

DODLA CPP

L. acido. CPP

L. bulg. CPP

Source of CPP

Figure 5.41 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 15 days injection on IgA secretary cell production in albino mice after infected with Salmonella sp. and given with SD

L. acido. CPP L. bulg. CPP

Control milk

AAVIN CPP

DODLA CPP

L. acido. CPP

L. bulg. CPP

Source of CPP
149

The control mice not fed with CPP and infected with Salmonella sp. had 10 2 IgA secretary cells/10 fields of vision at a magnification of 100x, 10 days post infection. Test mouse fed with AAVIN CPP for 10 days and then infected with Salmonella sp. was having 28 4 IgA secretary cells/10 fields of vision at a magnification of 100 and test mice fed with AAVIN CPP for 15 days and then infected with Salmonella sp. was having 93 4 IgA secretary cells/10 fields of vision at a magnification of 100x (Figure 5.40).

Test mouse fed with DODLA CPP for 10 days and then infected with Salmonella sp. was having 37 4 IgA secretary cells/10 fields of vision at a magnification of 100x and test mice fed with DODLA CPP for 15 days and then infected with Salmonella sp. was having 88 4 IgA secretary cells/10 fields of vision at a magnification of 100x (Table 5.38). Test mouse fed with L. acido. CPP for 10 days and then infected with Salmonella sp. was having 45 4 IgA secretary cells/10 fields of vision at a magnification of 100 and test mice fed with L. acido. CPP for 15 days and then infected with Salmonella sp. was having 96 4 IgA secretary cells/10 fields of vision at a magnification of 100x (Figure 5.41). Test mouse fed with L. bulg. CPP for 10 days and then infected with Salmonella sp. was having 53 4 IgA secretary cells/10 fields of vision at a magnification of 100 and test mice fed with L. bulg. CPP for 15 days and then infected with Salmonella sp. was having 102 4 IgA secretary cells/10 fields of vision at a magnification of 100. The control mice not fed with CPP and infected with Salmonella sp. had 14 2 IgA secretary cells/10 fields of vision at a magnification of 100, 15 days post infection.

150

Table 5.39 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 10, 15 days injection on IgA secretary cell production in albino mice fed after infected with Shigella sp. Number of mice infected were, n = 6 and the level of significance was not more than 0.002 (P 0.02) when calculated by ANOVA and given with SD

S.No 1 2 3 4 5

Source of CPP Control AAVIN CPP DODLA CPP L. acido. CPP L. bulg. CPP

IgA secretary cells/10 fields of vision 10 days 5 2 39 4 26 4 30 4 49 4 15 days 11 2 72 4 64 4 87 4 92 4

Figure 5.42 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 10 days injection on IgA secretary cell production in albino mice after infected with Shigella sp. and given with SD

L. acido. CPP L. bulg. CPP

Control milk

AAVIN CPP

DODLA CPP

L. acido. CPP

L.Control bulg. CPP milk

Source of CPP
151

Figure 5.43 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 15 days injection on IgA secretary cell production in albino mice after infected with Shigella sp. and given with SD

L. acido. CPP L. bulg. CPP

Control milk

AAVIN CPP

DODLA CPP

L. acido. CPP

L. bulg. CPP

Source of CPP
Photo 5.10 IgA secretary cell production in albino mice not fed with CPP, fed only with normal feed for 15 days and infected with Escherichia coli.

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Photo 5.11 Effect of AAVIN CPP on IgA secretary cell production in albino mice fed with it for 10 days and infected with Escherichia coli.

Photo 5.12 Effect of AAVIN CPP on IgA secretary cell production in albino mice fed with CPP for 15 days and infected with Escherichia coli.

153

The control mice not fed with CPP and infected with Shigella sp. had 5 2 IgA secretary cells/10 fields of vision at a magnification of 100, 10 days post infection. Test mouse fed with AAVIN CPP for 10 days and then infected with Shigella sp. was having 39 4 IgA secretary cells/10 fields of vision at a magnification of 100 and test mice fed with AAVIN CPP for 15 days and then infected with Shigella sp. was having 72 4 IgA secretary cells/10 fields of vision at a magnification of 100x (Figure 5.42). Test mice fed with DODLA CPP for 10 days and then infected with Shigella sp. was having 26 4 IgA secretary cells/10 fields of vision at a magnification of 100 and test mice fed with DODLA CPP for 15 days and then infected with Shigella sp. was having 64 4 IgA secretary cells/10 fields of vision at a magnification of 100.

Test mouse fed with L. acido. CPP for 10 days and then infected with Shigella sp. was having 30 4 IgA secretary cells/10 fields of vision at a magnification of 100 and test mice fed with L. acido. CPP for 15 days and then infected with Shigella sp. was having 87 4 IgA secretary cells/10 fields of vision at a magnification of 100. Test mouse fed with L. bulg. CPP for 10 days and then infected with Shigella sp. was having 49 4 IgA secretary cells/10 fields of vision at a magnification of 100 and test mice fed with L. bulg. CPP for 15 days and then infected with Shigella sp. was having 92 4 IgA secretary cells/10 fields of vision at a magnification of 100x (Figure 5.43). The control mice not fed with CPP and infected with Shigella sp. had 11 2 IgA secretary cells/10 fields of vision at a magnification of 100, 15 days post infection (Table 5.39).

5.4 Determination of the Anti-genotoxic acivity of the CPP: 5.4.1 Micronucleus assay: The micronucleus assay results of the fish and albino mice blood cells showed distinct features. The formation of micronucleus in the cell was taken as the major parameter of distinguishment. Other features noted were the presence of bi or multi nucleated cells and cell membrane disintegration, karyolysis and nuclear retraction. These three parameters together were used to study the anti-genotoxic effect of CPP
154

on gamma irradiated cells of albino mice and fish. Control batch of fish, subjected to 50 Gy of radiation had higher instance of micronucleus formation, cell wall disintegration and multi nucleated cells (Photo 5.13) whereas test batch of fish, subjected to 50 Gy of radiation had lower instance of micronucleus formation, cell wall disintegration and multi nucleated cells (Photo 5.14). Photo 5.13 Control fish fed with normal feed for 15 days and irradiated with Co60 irradiation for 50 Gy for 940 seconds showing micronucleus formation in the erythrocyte cells given in magnification of 100x per 10 fields

Micronucleus

Photo 5.14 Test fish fed for 15 days and irradiated with Co60 irradiation for 50 Gy for 940 seconds showing absence of micronucleus formation in the erythrocyte cells given in magnification of 100x per 10 fields

Normal cells
155

Control batch of fish subjected to 100 Gy of radiation had higher instance of micronucleus formation, cell wall disintegration and multi nucleated cells (Photo 5.15) whereas test batch of fish subjected to 100 Gy of radiation had lower instance of micronucleus formation, cell wall disintegration and multi nucleated cells (Photo 5.16). Photo 5.15 Control fish fed with normal feed for 15 days and irradiated with Co60 irradiation for 100 Gy for 1880 seconds showing micronucleus formation in the erythrocyte cells given in magnification of 100x per 10 fields

Micronucleus

Photo 5.16 Test fish fed for 15 days and irradiated with Co60 irradiation for 100 Gy for 1880 seconds showing absence of micronucleus formation in the erythrocyte cells given in magnification of 100x per 10 fields

Normal cells

156

Control batch of fish subjected to 135 Gy of radiation had higher instance of micronucleus formation, cell wall disintegration and multi nucleated cells (Photo 5.17) whereas test batch of fish, subjected to 135 Gy of radiation had lower instance of micronucleus formation, cell wall disintegration and multi nucleated cells (Photo 5.18). Photo 5.17 Control fish fed with normal feed for 15 days and irradiated with Co60 irradiation for 135 Gy for 2538 seconds showing nuclear retraction and karyolysis in the erythrocyte cells given in magnification of 100x per 10 fields

Nuclear retraction Karyolysis

Photo 5.18 Test fish fed for 15 days and irradiated with Co60 irradiation for 135 Gy for 2538 seconds showing micronucleus formation in the erythrocyte cells

Micronucleus

157

Control batch of albino mice, subjected to 1 Gy of radiation had higher instance of micronucleus formation, cell wall disintegration and multi nucleated cells (Photo 5.19) whereas test batch of albino mice subjected to 1 Gy of radiation had lower instance of micronucleus formation, cell wall disintegration and multi nucleated cells (Photo 5.20). Photo 5.19 Control mice fed with normal feed for 15 days and irradiated with Co60 irradiation for 1 Gy for 19 seconds showing micronucleus formation in the erythrocyte cells given in magnification of 100x per 10 fields

Micronucleus

Photo 5.20 Test mice fed for 15 days and irradiated with Co60 irradiation for 1 Gy for 19 seconds showing absence of micronucleus formation in the erythrocyte cells given in magnification of 100x per 10 fields

Normal cells

158

Control batch of albino mice subjected to 1.9 Gy of radiation had higher instance of micronucleus formation, cell wall disintegration and multi nucleated cells (Photo 5.21) whereas test batch of albino mice which was subjected to 1.9 Gy of radiation had lower instance of micronucleus formation, cell wall disintegration and multi nucleated cells (Photo 5.22). Photo 5.21 Control mice fed with normal feed for 15 days and irradiated with Co60 irradiation for 1.9 Gy for 34 seconds showing micronucleus, bi and multi nuclear formation in the erythrocyte cells

Multinucleated erythrocyte

Micronucleus

Binucleated erythrocyte

Photo 5.22 Test mice fed for 15 days and irradiated with Co60 irradiation for 1.9 Gy for 34 seconds showing absence of micronucleus formation in the erythrocyte cells given in magnification of 100x per 10 fields

Normal cells
159

Control batch of albino mice, subjected to 5 Gy of radiation had higher instance of micronucleus formation, cell wall disintegration and multi nucleated cells (Photo 5.23) whereas test batch of albino mice which was subjected to 5 Gy of radiation had lower instance of micronucleus formation, cell wall disintegration and multi nucleated cells (Photo 5.24). Photo 5.23 Control mice fed with normal feed for 15 days and irradiated with Co60 irradiation for 5 Gy for 94 seconds showing karyolysis and nuclear retraction in the erythrocyte cells given in magnification of 100x per 10 fields

Karyolysis

Nuclear retraction

Photo 5.24 Test mice fed for 15 days and irradiated with Co60 irradiation for 5 Gy for 904 seconds showing absence of micronucleus formation in the erythrocyte cells given in magnification of 100x per 10 fields

Normal cells

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The albino mice test batch, fed with CPP for 10 days had 1000 erythrocytes out of which 5 had micro nucleated erythrocytes, 16 had binucleated erythrocytes and 3 had multinucleated erythrocytes at the end of 24 hours after they were subjected to Co60 irradiation of 1.9 Gy which was its LD50. At the end of 48 hours, there were 9 had micro nucleated erythrocytes, 20 had binucleated erythrocytes and 5 had multinucleated erythrocytes. At the end of 72 hours, there were 13 micro nucleated erythrocytes, 21 had binucleated erythrocytes and 8 had multinucleated erythrocytes. At the end of 96 hours, there were 15 had micro nucleated erythrocytes, 23 had binucleated erythrocytes and 8 had multinucleated erythrocytes (Table 5.40). Table 5.40 Quantification of micro, bi and multi-nucleated cells of mice fed with CPP for 10 days and subjected to Co60 irradiation at LD50 value (1.9 Gy)

Time

No. of erythrocytes

No. of micro nucleated erythrocytes

No. of binucleated erythrocytes

No. of multi nucleated erythrocytes

24 hours

1000

16

48 hours

1000

20

72 hours

1000

13

21

96 hours

1000

15

23

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The albino mice test batch fed with CPP for 15 days had 1000 erythrocytes out of which 1 had micro nucleated erythrocyte, 12 had binucleated erythrocytes and 1 had multinucleated erythromycete at the end of 24 hours after they were subjected to Co60 irradiation of 1.9 Gy which was its LD50. At the end of 48 hours, there were 4 had micro nucleated erythrocytes, 13 had binucleated erythrocytes and had 1 multinucleated erythromycete. At the end of 72 hours, there were 7 micro nucleated erythrocytes, 17 had binucleated erythrocytes and 6 had multinucleated erythrocytes. At the end of 96 hours, there were 7 micro nucleated erythrocytes, 19 had binucleated erythrocytes and 6 had multinucleated erythrocytes (Table 5.41). Table 5.41 Quantification of micro, bi and multi-nucleated cells of mice fed with CPP for 15 days and subjected to Co60 irradiation at LD50 value (1.9 Gy)

Time

No. of erythrocytes

No. of micro nucleated erythrocytes

No. of binucleated erythrocytes

No. of multi nucleated erythrocytes

24 hours

1000

12

48 hours

1000

13

72 hours

1000

17

96 hours

1000

19

162

The albino mice control batch not fed with CPP had 1000 erythrocytes out of which 22 had micro nucleated erythrocytes, 30 had binucleated erythrocytes and 7 had multinucleated erythrocytes at the end of 24 hours after they were subjected to Co60 irradiation of 1.9 Gy which was its LD50. At the end of 48 hours, there were 31 micro nucleated erythrocytes, 35 had binucleated erythrocytes and 13 had multinucleated erythrocytes. At the end of 72 hours, there were 38 micro nucleated erythrocytes, 42 had binucleated erythrocytes and 15 had multinucleated erythrocytes. At the end of 96 hours, there were 53 micro nucleated erythrocytes, 45 had binucleated erythrocytes and 18 had multinucleated erythrocytes (Table 42). Table 5.42 Quantification of micro, bi and multi-nucleated cells of mice not fed with CPP and subjected to Co60 irradiation at LD50 value (1.9 Gy)

Time

No. of erythrocytes

No. of micro nucleated erythrocytes

No. of binucleated erythrocytes

No. of multi nucleated erythrocytes

24 hours

1000

22

30

48 hours

1000

31

35

13

72 hours

1000

38

42

15

96 hours

1000

53

45

18

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The test batch of fish, fed with CPP for 10 days had 1500 erythrocytes out of which 8 had micro nucleated erythrocytes, 31 had binucleated erythrocytes and 7 had multinucleated erythrocytes at the end of 24 hours after they were subjected to Co60 irradiation of 135 Gy which was its LD50. At the end of 48 hours, there were 11 micro nucleated erythrocytes, 39 had binucleated erythrocytes and 9 had multinucleated erythrocytes. At the end of 72 hours, there were 14 micro nucleated erythrocytes, 43 had binucleated erythrocytes and 9 had multinucleated erythrocytes. At the end of 96 hours, there were 16 micro nucleated erythrocytes, 48 had binucleated erythrocytes and 12 had multinucleated erythrocytes (Table 5.43). Table 5.43 Quantification of micro, bi and multi-nucleated cells of fish fed with CPP for 10 days and subjected to Co60 irradiation at LD50 value (135 Gy) Time No. of erythrocytes 1500 1500 1500 1500 No. of micro nucleated erythrocytes 8 11 14 16 No. of binucleated erythrocytes 31 39 43 48 No. of multi nucleated erythrocytes 7 9 9 12

24 hours 48 hours 72 hours 96 hours

The test batch of fish, fed with CPP for 15 days had 1500 erythrocytes out of which 4 had micro nucleated erythrocytes, 19 had binucleated erythrocytes and 13 had multinucleated erythrocytes at the end of 24 hours after they were subjected to Co60 irradiation of 135 Gy which was its LD50. At the end of 48 hours, there were 5 micro nucleated erythrocytes, 22 had binucleated erythrocytes and 16 had multinucleated
164

erythrocytes. At the end of 72 hours, there were 8 micro nucleated erythrocytes, 22 with binucleated erythrocytes and had 17 erythrocytes were multinucleated. At the end of 96 hours, 11 had micro nucleated erythrocytes, 25 had binucleated erythrocytes and 17 had multinucleated erythrocytes (Table 5.44).The control batch of fish, not fed with CPP had 1552 erythrocytes out of which 23 had micro nucleated erythrocytes, 14 had binucleated erythrocytes and 11 had multinucleated erythrocytes at the end of 24 hours after they were subjected to Co60 irradiation of 135 Gy which was its LD50. At the end of 48 hours, there were 27 micro nucleated erythrocytes, 26 had binucleated erythrocytes and 15 had multinucleated erythrocytes. At the end of 72 hours, there were 35 micro nucleated erythrocytes, 32 with binucleated erythrocytes and 18 had multinucleated erythrocytes. At the end of 96 hours, 39 had micro nucleated erythrocytes, 51 had binucleated erythrocytes and 19 had multinucleated erythrocytes (Table 5.45).

Table 5.44 Quantification of micro, bi and multi-nucleated cells of fish fed with CPP for 15 days and subjected to Co60 irradiation at LD50 value (135 Gy) Time No. of erythrocytes 1500 1500 1500 1500 No. of micro nucleated erythrocytes 4 5 8 11 No. of binucleated erythrocytes 19 22 22 25 No. of multi nucleated erythrocytes 13 16 17 20

24 hours 48 hours 72 hours 96 hours

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Table 5.45 Quantification of micro, bi and multi-nucleated cells of fish not fed with CPP and subjected to Co60 irradiation at LD50 value (135 Gy)

Time

No. of erythrocytes 1500 1500 1500 1500

No. of micro nucleated erythrocytes 23 27 35 39

No. of binucleated erythrocytes 14 26 32 51

No. of multi nucleated erythrocytes 11 15 18 19

24 hours 48 hours 72 hours 96 hours

5.4.2 Enzymatic assays: 5.4.2.1 Oxidase enzyme test: The test batch solution of both mice and fish produced blue colour immediately after the addition of them to oxidase reagent discs. The control batch solution did not produce any colour change when added with the oxidase reagent discs (Photo 5.25). 5.4.2.1 Catalase enzyme test: The test batch smears of both mice and fish produced air bubbles immediately after the addition of them to catalase reagent. The control batch solution did not produce any air bubble when added with the catalase reagent (Photo 5.26).

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Photo 5.25 Oxidase enzyme test for albino mice fed with CPP for 15 days, fish fed with CPP for 15 days and control batch, not fed with CPP, only with standard feed for 15 days

Control

Test mice

Test fish

Photo 5.26 Catalase enzyme test for albino mice fed with CPP for 15 days and control batch, not fed with CPP, only with standard feed for 15 days

Control

Test

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CHAPTER 6 DISCUSSION

6.1 General consideration: The fermented milk peptides play a natural role in many biochemical and immunological mechanisms in human body and they can be formulated as various oral supplements to bring out positive health effect for all age groups. Many reports are available for various Immunomodulatory activities, antihypertensive potential to lower the blood pressure and to be used as a sports medicine CPP [16]. Further work in the area can be performed to understand the mechanism of CPPs role as an Immunomodulatory agent. 6.2 Initial standardization: During the fermentation of milk by various lactic acid producing microorganisms, the pH reduced and becomes acidic in all the samples but not much difference in the ranges of pH was observed among the 4 fermenting agents, i.e. Aavin curd, Dodla curd, Lactobacillus acidophilus and Lactobacillus bulgaricus. Compared to control the titratable acidity was in uniform range for all the fermented milk taken up for study. The test samples intra comparison also proved that titratable acidity did not vary much among them. The lowest pH observed was in the case of milk

fermented using Lactobacillus bulgaricus (Table 5.4).

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Titratable acidity shown in table 5.6 acts as an indicator of acid volume produced in the fermented milk due to fermentation process and low level variation shows that the volume of acid formed in all the test samples of fermented milk is almost the same. The lowest titratable acidity was observed in the case of milk fermented by Lactobacillus bulgaricus and highest titratable acidity was observed in the case of milk fermented by commercial Dodla. As observed in table 5.7, not a substantial variation in viscosity was observed between the four fermented milk samples and the control, the non-fermented milk. Viscosity is directly related to the physical appearance and palatability of the fermented milk samples. If viscosity is high, it affects the shear stress of the fermented milk, thus making the flow a bit less smoother. The lowest viscosity was observed in the case of milk fermented by Lactobacillus acidophilus and highest viscosity was observed in the case of milk fermented by commercial Dodla (Figure 5.3). Excess increase of viscosity in fermented milk drastically affects the palatability of the fermented milk. All the test samples had their viscosity in the range which favoured high palatability of the fermented milk samples ensuring a good shear stress balance in accordance with Funian et al., 2004 [116]. 6.3 Microbiological and Scanning Electronic Microscopic (SEM) analysis: The presence of Lactobacillus sp. was confirmed by gram staining and SEM. Ness et al., 2000 [67] and Drago et al., 1997 [183] have stated that commercially produced fermented milks always contains mixed cultures including more than one bacterial culture and it corresponded with our findings. This mixed culture model
169

ensures speedy and effective fermentation of milk which is commercially economical for them. The test samples of milk fermented by Lactobacillus acidophilus and Lactobacillus bulgaricus contained a single strain. Although there is a variation in the cultures used and the texture of all the samples remained the same when examined physically indicating that mixed cultures produce fermentation in a quicker way than single culture samples but has no significant impact on texture and related physicochemical properties. The presence of Lactobacillus sp. as rod shaped bacillus in the fermented milk was demonstrated in photo 5.3 by SEM analysis. 6.4 Anti-microbial activity: The anti-microbial activity (photo 5.4) of our CPP isolate against common clinical pathogens such as Escherichia coli and Pseudomonas sp. corresponds to the mucosal secretions associated with milk peptides and fermented milk peptides and our findings correspond with Sun et al., 2002 and Pihlanto et al., 1999 [51, 37]. There was a slightly higher zone of inhibition formed by CPP against Pseudomonas sp. when compared with Escherichia coli. Pihlanto et al., 1999 [37] have reported that Lactoferrin is a milk peptide in that front towards which relatively higher time and studies had been devoted. This fact can be ascertained by the literature review of the milk and fermented milk peptides [134,151, 172, 174, 187]. Production of antimicrobial agents from fermented milk samples especially employing CPP will mark a new beginning in nutraceuticals. CPP will be a value addition to the existing food based nutrients available in the market.

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6.5

High Pressure Liquid Chromatography (HPLC) and Fourier Transform Infra Red (FTIR) spectroscopy analysis of CPP: HPLC analysis of the fermented milk samples of our study produced exclusive

peaks which were characteristic to fermented milk peptides [55, 154] and the peaks which corresponded to components produced during fermentation is seen in Figures 5.5 5.9. The HPLC analysis of the control sample, i.e. non-fermented milk did not produce the peaks which were produced by fermented milk test samples indicating the formation of new components in milk due to fermentation. FTIR analysis of the same test samples yielded similar results. The fermented milk samples exhibited peaks which are characteristic of the bioactive component which was absent in control sample. Both the HPLC, FTIR results confirm the above mentioned sentence of new bioactive peptides formation. 6.6 Molecular weight determination by Sodium Dodecyl Sulphate

Polyacrylamide Gel Electrophoresis (SDS PAGE): Molecular weight determination was ascertained using SDS-PAGE and the value was found to be 3.5 4 kilo Daltons (Photo 5.5). Molecular weight is always of critical importance to bioactive components as it indirectly determines their mode of delivery in to human body as a nutraceutical and therefore efficacy as stated by Antonius et al., 2000 [106]. The bioactive components present in fermented milk peptide of CPP could be broken in to smaller components for an improved formulation and effective delivery. Production of these peptides at nano scale could be pondered upon in the future which will open new vistas of fast acting nutraceutical.
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6.7 Animal studies: CPPs effect on the weight gain of albino mice was demonstrated by animal studies. The Dodla CPP isolated from milk fermented by Dodla culture produced the highest increase in weight in mice was 2.59 grams as seen in table 5.10. The least weight increase by a test batch was 2 grams by Aavin CPP, i.e. CPP which was isolated from milk fermented by commercial Aavin. When compared with the control batch (non-fermented milk) weight increase of 1.28 grams, all the four test batch of fermented milk CPPs produced substantial increase in the weight of albino mice in a injection period of 10 days (Figure 5.15), proving the point that continuous intake of fermented milk products contribute to uniform increase in body weight. As far as the weight increase over the 15 days injection period is concerned, the Dodla CPP produced the highest weight increase in mice which was 4.33 grams. The least weight increase by a test batch was 3.17 grams by Aavin CPP, i.e. CPP which was isolated from milk fermented by Lactobacillus bulgaricus. When compared with the control batch (non-fermented milk) weight increase was 1.56 grams (table 5.19), all the four test batch of fermented milk CPPs produced substantial increase in the weight of albino mice in a injection period of 15 days indicating that injection period was directly proportional to the weight increase as seen in the Figure 5.16. These results were in accordance with Mogensen et al., 1977 [137].

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6.7.1 Gastroprotective action against E. coli The mice mortality during post infection state indicated the gastroprotective effect of CPP. Gastroprotective nature of the fermented milk peptides has been already shown by Antonio et al., 2001 [97]. The highest gastroprotective effect against Escherichia coli infection was observed in the case of Dodla CPP and L. bulg. CPP which had the lowest percentage of mice mortality at 17%. Control mice had 100% mortality due to lack of CPP injection while only 33% mortality rate was observed in the 10 days CPP fed groups indicated their gastroprotective effect. In case of 15 days injection, the L. acido. CPP fed group showed the highest gastroprotective effect against Escherichia coli and L. bulg. CPP fed group showed the lowest percentage mortality as 17%. Due to the lack of CPP injection the control group mice showed 83% mortality. 6.7.2 Gastroprotective action against Salmonella sp. The highest gastroprotective effect against Salmonella sp. infection was observed in the case of Dodla CPP which showed 0% mortality. Control mice had 100% mortality due to lack of CPP injection. 17 - 33% mortality rate by other CPPs indicated their gastroprotective ability during the injection period of 10 days. In case of 15 days injection, the highest gastroprotective effect against Salmonella sp. infection was observed in Dodla CPP which was confirmed by 17% mortality where as in Control 100% mortality was observed due to lack of CPP injection. Rearrangements of cytoskeleton with the formation of membrane ruffles of the

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intestinal tissues is the possible mechanism by which gastroprotection was enabled and this has already been dealt by Benkerroum et al., 2002 [167] 6.7.3 Gastroprotective action against Shigella sp. The highest gastroprotective effect against Shigella sp. infection was observed in the case of L. acido. CPP which had the 0% percentage of mice mortality. Control mice had 83% mortality due to lack of CPP injection. 17 - 33% mortality rate by other CPPs indicated their gastroprotective ability during the injection period of 10 days. In case of 15 days injection period mice, the highest gastroprotective effect against Shigella sp. infection was observed in the case of L. acido. CPP which had 17% percentage mortality as seen in table 5.30. Control mice had 100% mortality due to lack of CPP feed. Increase in the injection period was found to be directly

proportional to the enhancement of gastroprotective effect on albino mice, drastically reducing the mortality percentage as indicated in figure 5.34. 6.8 Pathogen count determination Visceral Organs: The pathogen count present in visceral organs of albino mice after post infection by GIT pathogens such as Escherichia coli, Salmonella sp. and Shigella sp. was determined. The visceral organs in which pathogen count was determined were liver, spleen, kidney and small intestine. As observed in table 5.34, the lowest pathogen count in case of Escherichia coli infection was observed in the case of L. acido. CPP fed mice liver. The highest pathogen count in case of Escherichia coli infection was observed in the case of Aavin CPP whereas control mice in comparison
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had 51 x 103 pathogens due to lack of CPP injection (Figure 5.35). Similarly the test batch of mice had lower pathogen count in other visceral organs such as kidney, spleen and small intestine when compared with control batch, indicating the role of CPP in reducing the pathogen count in visceral organs during post infective period. The lowest pathogen count in Salmonella sp. infected mice was observed in the L. acido. CPP fed mice liver. The highest pathogen count was observed in Aavin CPP fed mice whereas control mice had 79 x 103 pathogens because there was no injection of CPP. Similarly the test batch of mice had lower pathogen count in other visceral organs such as kidney, spleen and small intestine when compared with control batch, indicating the role of CPP in reducing the pathogen count in visceral organs during post infective period. The lowest pathogen count was observed in Shigella sp. infected mice liver fed with L. bulg. CPP. The highest pathogen count was observed in Shigella sp. infected mice fed with L. acido. CPP whereas the control mice had 93 x 103 pathogens due to lack of CPP injection. Similarly the test batch of mice had lower pathogen count in other visceral organs such as kidney, spleen and small intestine when compared with control batch, indicating the role of CPP in reducing the pathogen count in visceral organs during post infective period. Ours is the first study to show the effect of CPP on the lowering of pathogen count in GI tract infected mice. CPP injection was able to effectively reduce the pathogen count in the infected mice when compared with control unfed mice.

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6.9 Histopathological studies: Histopathological studies of the small intestines from the albino mice infected with GIT pathogens like Escherichia coli, Salmonella sp. and Shigella sp. showed the disruption in cell organization when compared with the uniformly organized cell organization present in test batch mice fed with CPP for 10 and 15 days. The difference in cell structure was in line with the findings of Warensjo et al., 2004 [69]. The level of disorientation in cells was quite low in test mice batches when compared with control mice batches where the level of disorientation was relatively higher. Histopathology of albino mice spleen, kidney and liver cells infected with Escherichia coli, Salmonella sp. and Shigella sp. showed similar disruption in cell organization of control batch mice when compared with the uniformly organized cell organization present in test batch mice fed with CPP for 10 and 15 days. Histopathological studies proved the gastroprotective potential of CPP at the cellular and molecular level. CPP was able to bring down the cell attrition rate and preserve the cellular integration which was evident by its impact on test batches (Photos 5.6-5.9). 6.10 Immunomodulatory activity: The immunomodulatory roles of CPP on the GIT pathogen infection were determined by immunofluoresecence assay [189] using mice small intestine tissue samples. In E. coli infected mice the highest number of secretary IgA cells was produced in L. acido. CPP 10 days fed mice intestine and least was observed in Dodla CPP 10 days fed mice. Control batch had produced just 8 IgA cells (table 5.37). In case of 15 days fed group, the highest number of secretary IgA cells was produced by
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L. bulg. CPP and least secretary IgA cells being produced by L. acido. CPP. Control batch had produced only 6 IgA cells. As seen in Figure 53,the highest number of secretary IgA cells was produced by L. bulg. CPP in case of mice fed with CPP for 10 days and then subsequently infected using Salmonella sp. with the least secretary IgA cells being produced by Aavin CPP. Control batch had produced just 10 IgA cells. In case of 15 days (Figure 5.41) injection period test batches, highest number of secretary IgA cells was produced by L. bulg. CPP and least secretary IgA cells being produced by Dodla CPP. Control batch had produced 14 IgA cells. The results were in accordance with Rojas et al., 2002 [180]. In Shigella infected mice the secretary IgA cells produced was highest (table 5.39) by L. bulg. CPP 10 days fed mice and it was low in Dodla CPP 10 days fed mice. Control batch had produced just 5 IgA cells. In case of 15 days injection period batches, highest number of secretary IgA cells was produced by L. bulg. CPP and least secretary IgA cells being produced by Dodla CPP. Control batch had produced 11 IgA cells. All the fermented milk batches seem to induce a considerable immune enhancement in albino mice which is evident from the substantial increase in the number of IgA secretary cells by CPP. The immune enhancement potential of CPP had been proved beyond a doubt by the concurrent checking with all the four batches of CPP isolated from milk sources fermented by four different fermenting agents. The possible mechanism by which immune enhancement observed is shown by the increase in secretary IgA cells production as stated by various other studies [112,141,97]. This mechanism also explains the lower mortality rate, lesser percentage
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of weight loss in experimental mice group fed with CPP compared to control group which was not fed with CPP. Another possible mechanism for immune enhancement effect observed due to CPP enhances the production of IgA secreting cells. 6.11 Anti-Genotoxic role of CPP: Few works have been carried out to study the anti-genotoxic role of milk and fermented milk peptides [191,192]. The deleterious effect of gamma radiation on the albino mice and fish were determined using the number of micronuclei formed, number of binucleated cells seen and the number of multinucleated cells observed. Our experimental results (tables 5.40-5.45) proved the presence of anti-genotoxic component in all the four CPPs isolated from fermented milk. The number of micronuclei formed, number of binucleated cells seen and the number of multinucleated cells observed were all relatively lower in the CPP fed mice and CPP treated fish compared to control after exposed to radiation at different periods of time.(Photos 5.13-5.24). Lourens-Hattingh et al., 2001 [185] have stated that the possible mechanism by which fermented milk products could act as anti-genotoxic agents and the CPP, a class of fermented milk peptide bringing about anti-genotoxicity is in accordance with the same possible mechanism [125]. A number of radioprotectants of chemical nature are present today but a nutraceutical based anti-genotoxic agent could be of immense benefit. One possible mechanism of anti-genotoxicity was due to stimulation of enzymatic secretion in the small intestine cells by CPP which was confirmed by the positive oxidase and catalase enzyme tests carried out for test batches of albino mice,
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test batch fish, control batch of albino mice and control batch of fish. The presence of these enzymes could be the probable reason for the anti-genotoxic nature of CPP [170].

6.12. Application to human model: The concentration of CPP in 0.5 ml of crude sample isolated from fermented milk was found to be 65 mg. Assuming that an adult human consumes 100 ml of fermented milk through his/her regular diet on a daily basis, approximately 13000 mg or 13 grams of CPP would be intaken. This amount should be enough for the body to carry out all the necessary functions that the CPP seems to be effecting.

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CHAPTER 7 CONCLUSION The review of literature and the results obtained in the present thesis suggest that food microorganisms isolated from food matrices, in particular of bacterial origin, can act on the nutrients contained in the food. These microorganisms could thus generate functional foods enriched in specific components which can influence the important physiological processes in human especially in the intestinal system, immune response and anti-genotoxicity. In this view, the present work explored the possibility to produce fermented milk with commercial fermented milks, Lactobacillus acidophilus, Lactobacillus bulgaricus and obtain Casein

Phosphopeptide (CPP) with gastroprotective, immunomodulatory and anti-genotoxic activity. As a consequence, there is an increasing need to select the microorganism present in food matrices for their ability to produce functional food enriched in specific bioactivities on large scale. More research is thus needed to characterize the microorganisms and the associated bioactivities and to develop new methods for quantification of the bioactivity in the foodstuff and the identification of the food components responsible for such bioactivity. For example, it would be interesting to identify the presence of the peptides in milk fermented by L. bulgaricus to acquire better knowledge about the mechanisms determining the associated

immunomodulatory activity. With this purpose, the Experiment chapters 4.3 and 4.4 have been performed. We aimed to study the immunomodulatory activity of the milk
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fermented by two bacterial strains frequently found in dairy products of India and to explore the possible mechanism of action of a milk-derived peptide and compare with already documented immunomodulatory activity on lymphocytes, and considered as a model peptide. The results we got demonstrate that the in vitro methods manifest some limitations in the characterization of immunomodulatory bioactivity and that an exhaustive view of the action of immunomodulatory peptides could be achieved only by a multi-view approach that should take into account the complexity of the interactions between the bioactive peptides and the different components of the immune system in vivo. In fact, the Experiment 4.5 and the section 7.1.evidenced the lack of knowledge about the interaction of the immunomodulatory peptides derived from food and the immune system dispersed along the GI tract (as GALT, Peyers Patches, antigen-presenting cells) that could represent a potential target of immunomodulatory peptides, even before to be absorbed at gut level and circulate in the body. At the moment the interactions between food-derived peptides and the gut associated immune system have been explored to elucidate the mechanisms underlying allergies but it would be interesting to apply the same approach to evaluate the bioactivities, considering both allergies and bioactivities as properties that could be displayed by peptides. The present thesis focused also on the physiology of absorption of bioactive peptides and demonstrated for the first time that a long hydrophobic bioactive peptide crossed intact a Caco-2 cell monolayer, a well recognized in vitro model for the intestinal epithelium. In fact, the fermented milk181

derived immunomodulatory peptide CPP was demonstrated to be resistant to the digestion of gastrointestinal peptidases and to pass intact across Caco-2 cells. This interesting result permits to suggest that even large peptides could be absorbed in small quantities and that it cannot be excluded that at these concentrations the peptide CPP could interact with the gut-associated immune system, as explained before. The application of CPP isolated from milk fermented by Lactobacillus acidophilus and Lactobacillus bulgaricus as an active probiotic can be studied with human clinical trials which will yield immense health benefits and may be commercialized into a product readily available in the Indian market as an immune system booster. Fermented milk peptides seem to be having an effective antigenotoxic effect on the low ionizing background radiation, thus acting as a antigenotoxic agent. CPP functioned reasonably well with both albino mice and fish. Development of low cost, high efficient anti-genotoxic agent especially for low income radiation workers can be initiated using the fermented milk peptides as the base. The approximate cost of fermented milk required to provide CPP for each person would be only Rs.2 (market price of 100ml fermented milk).

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CHAPTER 8 FUTURE PROSPECTS OF THE WORK 8.1 Current status of probiotics in India: In India, probiotics are often used as animal feed supplements for cattle, poultry and piggery. This requirement is also met by importing probiotics from other countries. It is rarely used for human beings Sporolac, Saccharomyces boulardii and yogurt (L. bulgaricus + L. thermophillus) are the most common ones. Sporolac is manufactured using Sporolactobacilli. Lactobacilli solution is an example of a probiotic, usually given to paediatric patients in India. The latest and recent addition to the list of probiotics in India is ViBact (which is made up of genetically modified Bacillus mesentricus), which acts as an alternate to B-complex capsules. In India, only sporulating lactobacilli are produced and they are sold with some of the antibiotic preparations. India is a challenging market as it has not been exposed to probiotic products as have Western & other Asian countries. Countries like Japan, UK and some other countries in Asia have been part of the growing probiotic market since the early 1980s. But, in India, commercial probiotic foods only started cropping up on store shelves around 2007. Hence, it will be a while before we are able to overcome hurdles such as lack of awareness, retail mind set, lack of cold chain and such facilities. The global probiotic market today is $17 billion, whereas the market size in India is just about Rs 100 crores with a handful of players. While probiotics in the form of drugs
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are widely accepted, probiotic foods are still viewed with scepticism. Acceptance is growing slowly, but it will be a long while before people start consuming bacteria for breakfast. 8.2 Factors favouring indian probiotic market and its players: With India undergoing a rapid economic growth at a pace and with increasing number of Indian middle class population, there is a steady, increasing shift towards preventive therapies which did not exist before. People were spending only for post disease conditions out of compulsion. Increased money flow in the hands of Indian people is making to take a paradigm shift towards preventive therapies in which probiotics play a prominent role. Increase in disposable income of Indian population is another driving force which acts in favour of probiotic industry. Indian per capita income has risen to Rs.48,856 from Rs.22,792 in 1991 (Indian economic survey, 2010). When there is an increase in per capita income, it usually increases the dispensability of peoples money in health benefiting sectors. Increa sing shift towards self-medication is a factor which has a positive impact on Indian probiotic industry prospects. As probiotics are not purviewed under any health related law in India and with ICMR (Indian Council of Medical Research) still framing the guidelines for probiotic sales (ICMR status report on probiotics, 2009), probiotics face no hindrance from government health officials on its sales. Many elite and upper middle Indians view probiotics as self medication and their tendency to self medicate helps in the growth of Indian probiotic industry.

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Increase in healthcare spending is an associated factor with increase in per capita income and ease of money dispensability. Increase in healthcare expenditure also creates the scenario for an inclusive growth in Indian probiotic market. Next important factor is the ageing population of India. It is estimated that in India, there will be an increase of 18% in the number of people in the above 60 years category by the end of this decade (Indian bureau of statistics, 2008). Ageing population with increased income at hand will have an ideal setting for Indian probiotic companies which produce and market specialized probiotic products meant for geriatric patients. Pharma retail growth is the next factor touted to advance the probiotic market in India. Indian pharmaceutical industry is growing at a steadfast rate and is looking to diversify its products for catering domestic, foreign needs. Indian pharma industry is in compelling need to diversify due to the strict patent regime which came in to force on January 1st, 2005. The loss in the generic drug business has to be compensated in functional food business in which probiotics is the major class of products. With retail growth in pharma field going at a brisk pace, the ease of access in case of probiotics will also grow along with it. Favorable pricing environment is also becoming possible due to number of Indian and global players entering the probiotic market. As the field is nascent, the pricing is extremely competitive taking in to consideration the fact that every player in the market is trying to consolidate their consumer market base and build brand value. Any fluctuation in prices may turn away the first time consumers who are crucial for the sustained growth of the industry in a flourishing market like India where pricing plays an important role. These factors contribute to the
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competitive pricing which again is a factor working for Indian probiotic industry as a whole. 8.3 Challenges to be considered: Lack of standardization is a major challenge for the Indian probiotic industry. As the industry is in its initial stage, there is not a proper standardization parameters present. This scenario will improve with the entry of more established players entering the Indian market and bringing standardization along with them. Lack of awareness from the lower middle class population in urban areas and rural masses may provide a rocky platform for the companies in their expansion plans. A sustained television advertisement campaign with prominent faces being roped in to promote the product may help to counter this challenge to the farthest extent since the same strategy has proved to be useful for other products which were in the same league before. Marketing and distribution challenges exist in a country like India which is very diverse and presents a topography which requires specific case studies and temperaments. Region specific marketing strategies with local sales team being involved in the decision making process will help the business cause. Involving defined strategies with positive outlook can make a difference as far as this challenge is concerned. Launching the products with Indian consumer interests in mind and forming a team of Indian sales experts by the companies will reduce this challenge in a very effective way.

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LIST OF PUBLICATIONS

1. R. Balaji Raja and Kantha D. Arunachalam. Market potential for probiotic nutritional supplements in India. African Journal of Business Management (ISI indexed jounal ; Impact factor 1.105), (Accepted for publication and to be published in the issue of April 2011)

2. R. Balaji Raja and Kantha D. Arunachalam. Protective Effect of Casein Phosphopeptides derived from fermented milk on the mortality rate and weight loss of Albino mice infected with Escherichia coli. International Journal of Engineering Science and Technology. Vol. 2(3), 2010, 247-252.

3. Kantha D. Arunachalam and R. Balaji Raja. Isolation and characterization of CPP (casein phosphopeptides) from fermented milk. African Journal of Food Science Vol. 4(4), 2010, 167-175.

4. R. Balaji Raja and Kantha D. Arunachalam. Immunomodulatory effect of CPP (casein phosphopeptides) from fermented milk. Clinical and Developmental Immunology under review process. (ISI indexed jounal ; Impact factor 1.6).

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PATENT FILED

1. Title of the invention patented : A novel method to isolate Casein Phosphopeptides (CPP) from fermented milk Patent number : 1304/CHE/2010 Date filed : 10th May 2010

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Vitae
R.Balaji Raja was born in Vellore, Vellore district, Tamilnadu in 1984. He completed his U.G with first class as the batch topper from Pallavan College of Pharmacy, Kanchipuram in 2005. He secured All India 2nd in Vellore Institute of Technology Universitys M.Tech. 2005 entrance examination in Biotechnology stream. He completed his M.Tech. Degree in Biotechnology from VIT, Vellore in 2007 with first class, distinction. Mr. Balaji has started his doctoral work in 2008 under the guidance of Dr. Kantha D. Arunachalam, Professor and Coordinator, Centre for Environmental Nuclear Research, SRM University, Kattankulathur. He has worked as an Assistant Professor in the Department of Biotechnology, SRM University, Chennai for 3 years from July 2007 to May 2010 and in the Department of Biotechnology, National Institute of Technology (NIT), Raipur, Chhattisgarh from July 2010 to till date.

He has published 38 research articles in international journals and filed for 5 Indian patents at IPR branch, Chennai. He has written a book titled Let us know our Indian medicinal plants better published in April 2008. He has presented papers in 21 International and national conferences. He is an Associate editor in African Journal of Microbiology Research, Academic journals, USA (indexed in ISI web of science), Editorial board member of International Journal of Engineering Science and Technology, Engineering publishers, Singapore (indexed in scopus, DOAJ) and reviewer in CyTA Journal of food, Taylor and Francis group, London (indexed in ISI web of science), Scientific Research and Essays (indexed in ISI web of science), Journal of Medicinal Plants Research (indexed in ISI web of science). His name has been included in Marquis Whos Who in Medicine and Health care, USA, 2011 edition. He has attended 4 workshops, given 2 guest lectures in Engineering colleges about prospects of biotechnology and holds membership of 2 professional bodies, ISTE and SBTI (Society for Biotechnologists, India).