Biopeptides in Milk

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

ii

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 (R. Balaji Raja)

Date: 12.8.11

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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 anti-

genotoxic 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

v

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)

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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. non-

fermented milk was found to be 7.05. The mean titratable acidity of the fermented milk

by commercial curd Dodla was 91.49º T (Toerner’s degree), commercial curd Aavin was

91.27º T, Lactobacillus acidophilus was 92.11º T and Lactobacillus bulgaricus was

93.52º T. The titratable acidity of the control sample, i.e. non-fermented milk was 50.78º

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 SDS-

PAGE (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

viii

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.

ix

TABLE OF CONTENTS

CHAPTER NO. TITLE PAGE NO.

1. INTRODUCTION

1.1 MILK CONSUMPTION AND COMPOSITION 1

1.2 COMPONENTS IN MILK AND THEIR HEALTH EFFECTS

1.2.1 Protein 2

1.2.2 Branched chain amino acids and other amino acids

1.2.2.1 Taurine 4

1.2.2.2 Glutathione (GSH) 4

1.2.3 Lipids

1.2.3.1 Fatty acids 5

1.2.3.2 Saturated fatty acids 5

1.2.3.3 Unsaturated fatty acids 5

1.2.3.4 Trans vaccenic acid (VA) 5

1.2.4 Phospholipids and glycosphingolipids 5

1.2.5 Minerals, vitamins and antioxidants

1.2.5.1 Calcium in milk 6

1.2.5.2 Selenium in milk 6

x

1.2.5.3 Iodine in milk 6

1.2.5.4 Magnesium in milk 7

1.2.5.5 Zinc in milk 7

1.2.5.6 Vitamin E in milk 7

1.2.5.7 Vitamin A in milk 7

1.2.5.8 Folate in milk 7

1.2.5.9 Riboflavin in milk 8

1.3.5.10 Vitamin B12 in milk 8

1.4 BACTERIAL FLORA OF MILK 8

1.5 INTOLERANCE TO MILK COMPONENTS 8

1.6 INTOLERANCE TO MILK PROTEINS 9

1.7 PHYSIOLOGICALLY ACTIVE MILK PEPTIDES

1.7.1 Antihypertensive Peptides (ACE Inhibitors) 9

1.7.2 Antithrombotic Peptides 10

1.7.3 Caseinophosphopeptides (CPP) 11

1.7.4 Immunomodulatory Peptides 12

1.7.5 Opioid Milk Peptides 13

1.7.6 Miscellaneous Peptides 16

1.8 Global Probiotic food market in industrialized nations 17

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1.9 Indian probiotic market 17

1.10 Current players in Indian Probiotic Market

1.10.1 Yakult danone 18

1.10.2 Amul 19

1.10.3 Nestle 19

1.10.4 Mother Dairy 19

1.11 Fermented milk 20

1.12 Motivation and Problem statement 21

2 REVIEW OF LITERATURE

. 2.1 Milk and milk-derived products 22

2.2 Bioactive peptides

2.2.1 Definition 23

2.2.2 Mechanisms of production of bioactive peptides 24

2.2.3 Mechanisms of action of bioactive peptides 26

2.2.4 Bioactive peptide based commercial dairy products 28

2.3 Digestion of bioactive peptides 30

2.4 Bioactive peptide absorption

2.4.1 Physiology of the digestion of proteins and peptides 32

2.4.2 Physical and chemical characteristics of potentially 33

absorbable bioactive peptides

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2.5 Bioactivities of milk and fermented milk peptides

2.5.1 ACE-inhibition 35

2.5.2 Immunomodulation

2.5.2.1 Immunomodulatory peptides from milk 36

2.5.2.2 Microorganisms for the production of fermented 38

milk with immunomodulatory activity

2.5.2.3 Two examples of immunomodulatory Peptides 40

derived from milk proteins

2.3.2.4.1 YGG peptide 41

2.3.2.4.2 β-CN (193-209) peptide 42

2.6 Prebiotics and Probiotics 42

2.7 Fermentations and microorganisms 43

2.8 Probiotics and their role in the human health

2.8.1 GI tract and its Pathogens 46

2.8.1.1 Salmonella infection in human 47

2.8.1.2 Salmonella enterica serotype enteritidis 47

2.8.2 Therapeutic effects of probiotics 48

2.8.2.1 Acute gastroenteritis 49

2.8.2.2 Inflammatory bowel disease 49

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2.9 Allergic diseases 50

2.10 Lactic acid bacteria and the immune system 50

2.10.1 Role of cytokines in the immune response 51

2.11 Interactions between epithelial cells and intestinal microflora 51

2.12 Casein Phosphopeptide and its uses 52

2.13 Anti-genotoxic role

2.13.1 Classification of radioprotective agent 55

2.13.2 Milk and fermented milk as an anti-genotoxic agent 55

2.13.3 Enzymes and their anti-genotoxic mechanism 57

3 OBJECTIVES 58

4 MATERIALS AND METHODS

4.1 Selection of milk brands and culture sources 59

4.1.1 Production of fermented milk using different sources 59

of bacterial cultures

4.1.2 Initial Standardization 59

4.1.2.1 pH 60

4.1.2.2 Titratable acidity 60

4.1.2.3 Viscosity 60

4.1.3 Microbiological analysis of fermented milk 60

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4.1.4 Isolation of CPP from fermented milk 60

4.1.5 Characterisation of four isolated CPPs

4.1.5.1 Antimicrobial activity of CPP 62

4.1.5.2 HPLC Analysis of CPP 62

4.1.5.3 FTIR Analysis of CPP 62

4.1.5.4 Molecular weight determination by SDS PAGE 63

4.2 Animal studies

4.2.1 Effect of CPP on weight loss and mortality rate in mice 63

challenged with GUT Pathogens

4.2.1.1 Acclimatization of animals 63

4.2.1.2 Feeding with CPP 64

4.2.1.3 Challenging with GI Tract Pathogens 64

4.2.1.4 Pathogen count determination in visceral organs 65

4.2.1.5 Histopathological studies 65

4.2.2 Immunomodulatory role 65

4.3 Anti-genotoxic studies using CPP

4.3.1 Gamma irradiation Experiment with Animals 66

4.3.1.1 Experimental set up for mice 66

4.3.1.2 Experimental set up for fish 66

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4.3.1.3 Irradiation with Co60 source 67

4.3.2 Micronucleus assay 68

4.3.3 Enzymatic assays

4.3.3.1 Oxidase test 69

4.3.3.2 Catalase test 69

5 RESULTS

5.1 Isolation and characterization

5.1.1 Initial Standardization 70

5.1.1.1 pH 70

5.1.1.2 Titratable acidity 74

5.1.1.3 Viscosity 75

5.1.2 Microbiological analysis of fermented milk 76

5.1.3 Yield and production cost of CPP 76

5.1.4 Antimicrobial activity of CPP 77

5.1.5 HPLC Analysis of CPP 79

5.1.6 FTIR Analysis of CPP 84

5.1.7 Molecular weight determination by SDS PAGE 90

5.2 Animal Studies

5.2.1 Challenging with GI Tract Pathogens 91

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5.2.2 Post infection studies 108

5.2.3 Determination of pathogen count in visceral organs 132

5.2.4 Histopathological studies 138

5.3 Determination of Immunomodulatory activity 146

5.4 Anti-genotoxic role of CPP

5.4.1 Micronucleus assay 154

5.4.2 Enzymatic assays

5.4.2.1 Oxidase enzyme test 166

5.4.2.2 Catalase enzyme test 166

6 DISCUSSION

6.1 General consideration 168

6.2 Initial standardization 168

6.3 Microbiological and SEM analysis 169

6.4 Anti-microbial activity 170

6.5 HPLC and FTIR analysis of CPP 171

6.6 Molecular weight determination by SDS-PAGE 171

6.7 Animal studies

6.7.1 Gastroprotective action against E. coli 173

6.7.2 Gastroprotective action against Salmonella sp. 173

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6.7.3 Gastroprotective action against Shigella sp. 174

6.8 Pathogen count determination 174

6.9 Histopathological studies 176

6.10 Immunomodulatory activity 176

6.11 Anti-genotoxic role of CPP 178

6.12 Application to human model 179

7 CONCLUSION 180

8 FUTURE PROSPECTS OF OUR WORK

8.1 Current status of probiotics in India 183

8.2 Factors favoring Indian probiotic market and its players 184

8.3 Challenges to be considered 186

9 LIST OF REFERENCES 187

10 LIST OF PUBLICATIONS 216

11 PATENT FILED 217

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LIST OF TABLES

NUMBER TITLE PAGE NO.

2.1 Bioactive peptides from milk proteins 27

2.2 Studies which have established the occurrence of peptides in 28

various fermented milk products

2.3 Examples of ACE-inhibitory peptides derived from milk 35

2.4 Immunomodulatory peptides derived from milk proteins 37

2.5 List of microorganisms producing immunomodulatory 38

activity from fermented milk

5.1 pH values of the fermented milk with time after added with 71

Dodla dairy curd


Aavin dairy curd


Lactobacillus acidophilus culture

5.4 pH values of the fermented milk with time after added 73

with Lactobacillus bulgaricus culture

5.5 The pH changes for milk fermented using commercial curds Dodla, 73

Aavin, Lactobacillus acidophilus and Lactobacillus bulgaricus.

xix

5.6 The titratable acidity values for milk fermented using commercial 74

curds Dodla, Aavin, Lactobacillus acidophilus and

Lactobacillus bulgaricus

5.7 The viscosity changes for milk fermented using commercial curds 75

Dodla, Aavin, Lactobacillus acidophilus and Lactobacillus bulgaricus

5.8 Zone of inhibition formed by CPP against Escherichia 78

coli and Pseudomonas sp.

5.9 Effect of AAVIN CPP on body weight of albino mice 92

after feeding for 10 days

5.10 Effect of DODLA CPP on body weight of albino mice 93


5.11 Effect of L. acido. CPP on body weight of albino mice 94


5.12 Effect of L. bulg. CPP fed on body weight of albino mice 95


5.13 Body weight of albino mice after feeding normal feed alone 96

without CPP for 10 days

5.14 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 97

on the body weight of albino mice after feeding for 10 days

5.15 The effect of AAVIN CPP on body weight in albino mice after 100

feeding for 15 days

xx

5.16 The effect of AAVIN CPP on body weight in albino mice after 101

feeding for 15 days

5.17 The effect of L. acido. CPP on body weight in albino mice after 102

feeding for 15 days

5.18 The effect of L. acido. CPP on body weight in albino mice after 103

feeding for 15 days

5.19 Body weight of albino mice after feeding normal feed alone 104

without CPP for 10 days

5.20 Comparison of the effect of AAVIN CPP, DODLA CPP, L. acido. 105

CPP and L. bulg. CPP on the body weight of albino mice after

feeding for 15 days

5.21 Comparison between the percentage increase in body weight of 106

AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP fed mice

for a feeding period of 10 and 15 days

5.22 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP 110

on the body weight of albino mice after feeding for 10 days

and infected with Escherichia coli.

5.23 Percentage of body weight loss in albino mice infected with 111

Escherichia coli fed with AAVIN CPP, DODLA CPP, L. acido.

xxi

CPP and L. bulg. CPP for 10 days

5.24 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and 113

L. bulg. CPP on the body weight of albino mice after feeding for

15 days and infected with Escherichia coli.





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 117

Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido.



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 120



xxii


L. bulg. CPP on the body weight of albino mice after feeding for

10 days and infected with Shigella sp. for 10 days


Shigella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP

and L. bulg. CPP for 10 days


L. bulg. CPP on the body weight of albino mice after feeding

for 15 days and infected with Shigella sp.


Shigella sp. fed with AAVIN CPP, DODLA CPP, L. acido.



L. bulg. CPP 15 days feeding on GUT pathogen count of

albino mice visceral organs after infected with Escherichia coli



albino mice visceral organs after infected with Salmonella sp.

xxiii

5.36 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. 136

CPP 15 days feeding on GUT pathogen count of albino mice

visceral organs after infected with Shigella sp.


CPP on IgA secretary cell production in albino mice fed with

CPP for 10, 15 days and infected with Escherichia coli


CPP 10, 15 days feeding on IgA secretary cell production in

albino mice after infected with Salmonella sp.


CPP 10, 15 days feeding on IgA secretary cell production

in albino mice fed after infected with Shigella sp.

5.40 Quantification of micro, bi and multi-nucleated cells of mice 161

fed with CPP for 10 days and subjected to Co60 irradiation

at LD50 value (1.9 Gy)



at LD50 value (1.9 Gy)

xxiv


not fed with CPP and subjected to Co60 irradiation at

LD50 value (1.9 Gy)

5.43 Quantification of micro, bi and multi-nucleated cells of fish 164


at LD50 value (135 Gy)





not fed with CPP and subjected to Co60 irradiation


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LIST OF PHOTOS


2.1 Scheme of the mechanisms by which bioactive peptides can 24

be released from the precursor proteins by microbial

fermentation and/or gastrointestinal digestion

2.2 The anatomic structure of the human stomach 30

2.3 Different pathways for intestinal absorption of a compound 32

2.4 Invasion of S. enteritidis 857 to Caco-2 cells 48

2.5 Gut microflora in inflammation 49

4.1 Casein isolated from fermented milk by 61

enzymatic hydrolysis

4.2 Gamma irradiator used in anti-genotoxic studies 68

(External view)

4.3 Gamma irradiator used in anti-genotoxic studies 68

(External view)

5.1 Fermented milk after the addition of commercial Aavin curd 70

5.2 Fermented milk after the addition of the culture 71


5.3 Scanning Electron microscopic image of Lactobacillus 77

Species

5.4 Zone of inhibition produced by CPP isolated from 79

commercial curd Aavin

xxvi

5.5 Molecular weight determination of CPP 90

5.6 Photograph showing the histopathological studies of 15 days 138

CPP fed albino mice liver cells infected with Escherichia coli

on seven days post infection staining with Methylene blue


CPP fed albino mice liver cells infected with Escherichia coli



CPP fed albino mice spleen cells infected with Escherichia coli



CPP fed albino mice small intestine cells infected with Escherichia

coli on seven days post infection staining with crystal violet

5.10 IgA secretary cell production in albino mice not fed with CPP, 152

fed only with normal feed for 15 days and infected with

Escherichia coli

5.11 Effect of AAVIN CPP on IgA secretary cell production in albino 153

mice fed with it for 10 days and infected with Escherichia coli

xxvii

5.12 Effect of AAVIN CPP on IgA secretary cell production in albino 153

mice fed with it for 15 days and infected with Escherichia coli

infected with Escherichia coli

5.13 Control fish fed with normal feed for 15 days and irradiated with 155

Co60 irradiation for 50 Gy for 940 seconds showing micronucleus

formation in the erythrocyte cells

5.14 Test fish fed for 15 days and irradiated with Co60 irradiation for 155

50 Gy for 940 seconds showing absence of micronucleus formation

in the erythrocyte cells





100 Gy for 1880 seconds showing absence of micronucleus



Co60 irradiation for 135 Gy for 2538 seconds showing nuclear

retraction and karyolysis in the erythrocyte cells

xxviii


135 Gy for 2538 seconds showing micronucleus formation in the

erythrocyte cells

5.19 Control mice fed with normal feed for 15 days and irradiated with 158



5.20 Test mice fed for 15 days and irradiated with Co60 irradiation for 158




Co60 irradiation for 1.9 Gy for 34 seconds showing micronucleus,

bi and multi nuclear formation in the erythrocyte cells


1.9 Gy for 34 seconds showing absence of micronucleus formation



Co60 irradiation for 5 Gy for 94 seconds showing karyolysis

and nuclear retraction in the erythrocyte cells

xxix




5.25 Oxidase enzyme test for albino mice fed with CPP for 15 days, fish 167

fed with CPP for 15 days and control batch, not fed with CPP, only

with standard feed for 15 days

5.26 Oxidase enzyme test for albino mice fed with CPP for 15 days and 167

control batch, not fed with CPP, only with standard feed for 15 days

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LIST OF FIGURES


5.1 The pH changes for milk fermented using commercial curds 74

Dodla, Aavin, Lactobacillus acidophilus and Lactobacillus

bulgaricus

5.2 The titratable acidity values for milk fermented using commercial 75



5.3 The viscosity changes for milk fermented using commercial 76



5.4 Anti-microbial activity of AAVIN CPP, DODLA CPP, L. acido. 78

CPP and L. bulg. CPP against Escherichia coli and Pseudomonas sp.

5.5 HPLC spectrum of non fermented control milk 80

5.6 HPLC spectrum of CPP isolated from milk fermented by 81

commercial curd Dodla

5.7 HPLC spectrum of CPP isolated from milk fermented by 82


xxxi

5.8 HPLC spectrum of CPP isolated from milk fermented 83

by the culture Lactobacillus acidophilus

5.9 HPLC spectrum of CPP isolated from milk fermented 84

by the culture Lactobacillus bulgaricus

5.10 FTIR spectrum of non fermented control milk 85

5.11 FTIR spectrum of CPP isolated from milk fermented by 86



commercial curd Dodla


the culture Lactobacillus acidophilus


the culture Lactobacillus bulgaricus


CPP on the percentage increase in body weight of albino mice

after fed for 10 days


CPP on the percentage increase in body weight of albino mice


xxxii




5.18 Percentage mortality rate in albino mice infected with Escherichia 112

coli fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.

CPP for 10 days

5.19 Body weight loss in albino mice infected with Escherichia coli 112

fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.

CPP for 10 days




5.21 Percentage mortality rate in albino mice infected with Escherichia 115

coli fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.

CPP for 15 days

5.22 Body weight loss in albino mice infected with Escherichia coli 115


CPP for 10 days

xxxiii




5.24 Percentage mortality rate in albino mice infected with Salmonella sp. 118


CPP for 10 days

5.25 Body weight loss in albino mice infected with Salmonella sp. 118


CPP for 10 days




5.27 Percentage mortality rate in albino mice infected with Salmonella 121

sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and

L. bulg. CPP for 15 days

5.28 Body weight loss in albino mice infected with Salmonella sp. 121


CPP for 15 days

xxxiv


Shigella sp. fed with AAVIN CPP, DODLA CPP, L. acido.


5.30 Percentage mortality rate in albino mice infected with Shigella sp. 126


CPP for 10 days

5.31 Body weight loss in albino mice infected with Shigella sp. fed 126

with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.

CPP for 10 days

5.32 Percentage of body weight loss in albino mice infected with Shigella 128

sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.

CPP for 15 days

5.33 Percentage mortality rate in albino mice infected with Shigella sp. 129


CPP for 15 days

5.34 Body weight loss in albino mice infected with Shigella sp. fed 129

with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.

CPP for 15 days

xxxv



albino mice visceral organs after infected with Escherichia coli


CPP 15 days feeding on GUT pathogen count of albino mice

visceral organs after infected with Salmonella sp.


CPP on GUT pathogen count of albino mice visceral organs fed

with CPP for 15 days and infected with Shigella sp.


CPP 10 days feeding on IgA secretary cell production in albino

mice fed with CPP for 10 days after infected with Escherichia coli.

5.39 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. 148

CPP fed for 15 days on IgA secretary cell production in albino mice

after infected with Escherichia coli.

5.40 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. 149

CPP 10 days feeding on IgA secretary cell production in albino mice

after infected with Salmonella sp.

xxxvi



mice after infected with Salmonella sp.



mice after infected with Shigella sp.



mice after infected with Shigella sp.

xxxvii

LIST OF SYMBOLS AND ABBREVIATIONS

CDC Centre for Disease Control (USA)

HPLC High Performance Liquid Chromatography

FTIR Fourier Transform Infra Red spectroscopy

60CO Cobalt-60

mm Millimeter

CPP Casein Phospho Peptide

TANUVAS Tamilnadu Veterinary & Animal Sciences University

IMTECH Institute of Microbial Technology

CFU Colony Forming Unit

S.D Standard Deviation

SAR Structure Activity Relationship

ºT Toerner’s degree

mPa.s Millipascal second

IgA Immunoglobulin A

LAB Lactic Acid Bacteria

Gm Gram

1

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,

2

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, 1-

antitrypsin, β-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

3

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 beta-

casein 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.

4

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: 46–91 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].

5

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

6

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.

7

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 700–900 µg/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 µg folate/l. Recommended intake of folate is 400 µg/day

for adults. Many scientists believe that folate deficiency is the most prevalent of all

vitamin deficiencies.

8

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 µg/l. The daily

recommendation is 2.4 µg. 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.

9

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 classifications 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 identified within the sequences of human β-

and κ-CN [26]. They are also generated by tryptic digestion of bovine αs1- and β-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

10

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 αs1- and β-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 β-CN hydrolysate, exhibited potent antihypertensive activity in these rats over an

8-h interval after oral administration [33]. A third subclass, β-lactorphins, are sequestered

within the primary amino acid sequence of bovine β-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 β-LG and α-LA.

1.7.2 Antithrombotic Peptides:

Antithrombotic peptides are present in milk. Early on, it was learned that the

mechanisms involved in milk clotting, defined by the interaction of κ-Casein (CN) with

chymosin and blood clotting processes, defined by the interaction of fibrinogen with

thrombin, were comparable. In this regard, the C-terminal dodecapeptide of human

fibrinogen γ-chain (residues 400 to 411) and the undecapeptide (residues 106 to 116)

from bovine κ-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 fibrinogen λ-chain to its

receptor region on the platelets’ surface [35]. A smaller κ-CN fragment (residues 106

to110), casopiastrin, was obtained from trypsin hydrolysates and exhibited antithrombotic

activity by inhibiting fibrinogen binding [36]. A second segment of the trypsin κ-CN

fragment, residues 103 to 111, inhibitedplatelet aggregation but did not affect fibrinogen

binding to the platelet receptor [37]. Later, it was reported that biologically active

peptides, isolated from both casein and lactotransferrin, inhibited platelet function [38].

11

Antithrombotic peptides have also been derived from κ-caseinoglycopeptides that

were isolated from several animal species. Bovine κ-caseinoglycopeptide, the C-terminal

end of κ-CN (residues 106 to 169), inhibited von Willebrand factor-dependent platelet

aggregation [39]. Two antithrombotic peptides, derived from human and bovine κ-

caseinoglycopeptides, have been identified in the plasma of 5-d-old newborns after

breast-feeding and ingestion of cow’s milk based formula, respectively [40]. The C-

terminal residues (106 to 171) of sheep κ-casein, or κ-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 identified after trypsin release from αs1-

, αs2-, and β-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, αs2-

CN > αs1-CN > β-CN > κ-CN; however, the distribution of their phosphoserine clusters

is not uniform. It was further demonstrated that the specific amino acid composition

associated with the phosphorylated binding site also influences 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

12

soy-based diets [48]. This passive transport system is the primary means of calcium

absorption under physiological conditions and provides calcium required for bone

calcification. Caseinophosphopeptides also inhibit caries lesions through recalcification

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, β-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 β-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 α-LA (dipeptide) and κ-CN (tripeptide),

significantly increased proliferation of human peripheral blood lymphocytes [100], while

the C-terminal sequence of bovine β-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 milk-

derived peptides exert either their immunopotentiating effects or influence proliferative

responses are not currently known; however, one example suggests that the opioid milk

13

peptide, β-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 β-CN, called β-casomorphins, due to

their exogenous origin and morphine-like properties; however, they have also been

obtained from pepsin hydrolysis of bovine αs1-CN fractions (43, 54, 66, and 102).

Similar peptides have been reported from human β-CN fractions (24, 98), and the Y-P-F

sequence, which is common to bovine β-casomorphin, was also found to be present in the

primary structure of human β-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 affinity for their receptor [51]. Schuster and co-workers in 1980 reported opioid

activity from synthetic tetra- and pentapeptide fragments of human β-casein [52]. Opioid

peptides have been generated in vitro by enzymatic digestion of β-caseins from cows,

water buffalo, and sheep. In general, the α- and β-CN fragments produce agonist

responses, while those derived from κ-CN elicit antagonist effects. Opioid peptides may

be further subdivided into classifications according to the specific 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 definite N-terminal sequence Y-G-

G-F. Milk derived peptides, generated by hydrolysis of various precursor proteins such as

α- and β-CN, α-LA, and β-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 defined above. With the exception of αs1-CN, most share a common sequence

14

feature, defined 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 fits well into the binding

pocket of the opioid receptor. One of the most potent milk-derived opioid peptides, β-

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 α-

casein fractions as well. In general, their structures differ considerably from those of β-

caseinomorphins. Active fractions were shown to be a mixture of two separate peptides

derived from α-casein fragments #90–95 and #90–96. 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

β-Casein peptides were among the first reported opioid peptides. β-Casomorphins

are fragments corresponding to the 60 to 70th

amino acid residues of bovine β-CN,

considered the “strategic zone,” and are classified as µ-type receptor ligands [54]. Three

exorphins, derived from bovine αs1-CN, were shown to be selective for δ-receptors.

Certain proteolytic bacteria, such as Pseudomonas aeruginosa and Bacillus cereus, also

produce high levels of β-casomorphins when inoculated and grown in milk. β-

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 influences 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 β-

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 β-casomorphin was

15

reported in the plasma of newborn calves and infants after ingestion of bovine milk. In

pregnant or lactating women, β-casomorphins originate in the milk pass through the

mammary tissue, and possibly influence the release of prolactin and oxytocin. More

recently, it was shown that many peptides derived from αs1-,β-, or κ-CN, and κ-caseino-

glycomacropeptide 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. Opioid-

like 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 β-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, β-casomorphin1-7

andenterostatin may interact with the same low affinity 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

κ-CN bound to opioid receptors of rat brain. The peptide was methylated at the C-

terminal end and exhibited antagonist effects selective for the µ- and κ-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 κ-

CN. Casoxin C is an opioid antagonist, obtained from tryptic digests of bovine κ-CN, that

also functions as an agonist for C3a receptors. Lastly, casoxin D, purified from human

αs1-CN fractions, elicits an opioid antagonist response. In general, the chemically

16

modified 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 purified from a methyl-esterified fraction were significantly more potent. HPLC

analyses resulted in purification of three separate active fractions designated lactoferroxin

A, B, and C, respectively. It was determined that the α-carbonyl group of each was

methyl esterified based on comparison of bioactivity measurements and HPLC retention

times to those of corresponding synthetic peptides. Like casoxins, the chemically

modified peptides may not actually exist in vivo. Lactoferroxin A, residues 318 to 323,

showed a preference for µ-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

κ-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 non-

ruminants, while caseinomacropeptide, a 64-amino acid glycopeptide released from κ-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 β-LG, was found to exert its effect on smooth

muscle.

17

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 world’s highest milk producer.

18

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 Japan’s

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

19

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 test-

marketing 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, b-

20

Activ Probiotic Lassi, b-Activ Curd and Nutrifit (Strawberry & Mango) are the

company’s 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 Toll-

like 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

21

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.

22

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

23

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.

24

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

25

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].

26

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 αs2-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 ACE-

inhibitory peptides and one antihypertensive peptide were isolated from the hydrolysate.

Kunio et al., 2000 [92] hydrolyzed casein using the same proteinase and identified a β-

casein-derived antihypertensive peptide, the fragment β-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

27

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 [103-

107] 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 Precursor protein Bioactivity

Casomorphins

α-lactorphin β-lactorphin

Lactoferroxins

Casoxins

Casokinins

Lactokinins

Immunopeptides

Lactoferricin

Casoplatelins

- CN, - CN

-LA -LG

LF

-CN

- CN, - CN

-LA, -LG

- CN, - CN

LF

Opioid agonist

Opioid agonist

Opioid agonist

Opioid antagonist

Opioid antagonist

ACE-inhibitory

ACE-inhibitory

Immunomodulatory

Antimicrobial

28

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 Example of

identified peptide

Bioactivity

Cheese type Parmigiano-

Reggiano

Cheddar

-CN (8-16),

-CN (58-77),

s2-CN(83-33)

s1-CN fragments

Phosphopeptides,

precursor of

-casomorphin

Several

Phosphopeptides

-CN, Transferrin

- CN, - CN

Antitrombotic

Mineral binding,

Anticariogenic

29

Italian varieties:

Mozzarella, Crescenza,

Gogonzola,

Italico Gouda

Festivo

Emmental

Manchengo

Fermented milks

Sour milk

Yogurt

Dahi

-CN fragments

-CN (58-72)

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

s1-CN (1-9),

s1-CN (1-7), s1-CN (1-6)

s1-CN fragments

-CN fragments

Ovine s1-CN,

s2-CN,

-CN fragments

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

-CN (108-111)

Active peptides not

identified

SKVYP

phosphopeptides

ACE-inhibitory

ACE-inhibitory

ACE-inhibitory

Immunostimulatory,

antimicrobial

ACE-inhibitory

Antihypertensive

Weak ACE-

inhibitory

ACE-inhibitory

30

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

31

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 ACE-

inhibitory 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

32

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.

33

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

34

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 casein-

derived 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, ACE-

35

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 Fragment IC50 (µmol/L)

VAP s1-CN (25-27) 2

FFVAP s1-CN (23-27) s1- 6

FFVAPPFPEVFGK CN (23-34) s1-CN 77

FPEVFGK (28-34) s1-CN 140

FGK (32-24) 160

YKVLPQL s1-CN (104-109) s1- 22

36

LAYFYP CN (142-147) s1-CN 65

DAYPSGAW (157-164) s1-CN 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 β-casein and residues 194-199 of αs1-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 β-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 αs1-casein) can either stimulate or inhibit

37

lymphocyte proliferation depending upon the concentration used, while some whey-

derived peptides can affect cytokine production from leucocytes [120].

Table 2.4 Immunomodulatory peptides derived from milk proteins

Protein sequence Fragment Activity

Bovine s1-CN

Bovine s1-CN

Bovine s1-CN

Bovine s1-CN

Bovine s1-CN

s1-CN (1-23)

s1-CN (23-34)

s1-CN (90-96)

s1-CN (90-95)

s1-CN (194-199)

Stimulation of phagocytosis

and immune responses

against bacterial infections




Stimulation effect on

lymphocytes proliferation, NK activity

and neutrophil

locomotion

Stimulation effect on

lymphocytes proliferation, NK activity

and neutrophil

locomotion




38

Bovine s2-CN

Bovine -CN

s2-CN (1-32)

-CN (1-28)

Stimulatory effect on spleen

cells

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 Protein source

L. helveticus 5089

L. helveticus R389

Caseins

Milk

39

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 β-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

β-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-α 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

40

[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 discussed reports strongly support the fact that

immunomodulatory peptides released in fermented milk contribute to the

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 k-

casein-derived peptide (β-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 β-LA (amino acid sequences 18-20) (a tri-

peptide named YGG) and to the long fragment β-CN (amino acid sequences 193-209)

41

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 Met-

enkephalin 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 β- 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-α 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

42

should be noted that YGG is contained several times in the primary structure of bovine β-

casein and α-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 β-CN (193-209) peptide with immunomodulatory activity:

The β-CN (193-209) peptide is released from the C-terminal end of β-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 β-CN (193-209), corresponding to the amino acid sequence Gly-Pro-Val-Arg-Gly-Pro-

Phe-Pro-Ile-Ile, displayed ACE-inhibitory activity, further supporting the concept that

ACE-inhibitors may also act as immunomodulatory peptides by acting as bradykinin-

potentiating peptides [147]. Interestingly, the presence of 4 proline residues within the

sequence can protect the long peptide β-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

43

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 non-

digestible 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

44

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 22ºC 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.,

45

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 1010

-

1011

/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

46

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

47

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

48

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)

49

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.

50

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

51

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, IL-

10, 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 surface-

layered proteins of lactobacilli play an important role in the adhesion to receptors such as

glycoproteins on the surface of intestinal epithelial cells.

52

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

53

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

54

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.

55

2.13 Anti-genotoxicity of CPP:

2.13.1 Classification of radioprotective agent:

Radioprotective agents can be classified as:

(i) chemical radioprotectors,

(ii) adaptogens, and

(iii) 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 Élie

Metchnikoff observed high life expectancy in Bulgarian persons who ate large amounts

56

of fermented-milk products. One hundred years later, the consumption of fermented-

milk 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 carcinogen-

induced 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.

57

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 anti-

genotoxic 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.

58

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

59

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.

60

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 Toerner‟s degree (ºT) which could also be expressed as ºT 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 25°C 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 37º C

for 48 hours [199]. The organisms were also identified by gram‟s 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

61

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 37ºC 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 CPP’s:

CPP‟s 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

62

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 100µl of all the four CPP‟s were added along with same

volume of streptomycin as the control. The plates were incubated at 37ºC 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 100µl, flow rate of 10µl/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

63

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,

64

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 ± 2°C], 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-2010-

027).

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.

65

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 37ºC. 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 50µl of 1/50 dilution of α-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

66

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 ± 2°C and a 12-

hours 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

67

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 ± 2°C 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 - (Ʃa*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.

68

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

69

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.

70

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 Toerner’s degrees (ºT). 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

71

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)

pH

(Mean ± S.D)

0 7.050 ± 0.010

1 6.175 ± 0.154

2 6.038 ± 0.182

3 5.927 ± 0.196

4 5.790 ± 0.218

5 5.657 ± 0.183

6 5.463 ± 0.166

7 5.255 ± 0.126

72

Table 5.2 pH values of the fermented milk with time after added with Aavin dairy

curd.


measured every hour using a pH meter under standard conditions, number of hours n = 7.

Time

(in hours)

Mean ± S.D

0 7.050 ± 0.010

1 6.380 ± 0.092

2 6.447 ± 0.151

3 6.237 ± 0.108

4 6.117 ± 0.106

5 5.792 ± 0.155

6 5.460 ± 0.107

7 5.282 ± 0.185

Table 5.3 pH values of the fermented milk with time after added with Lactobacillus

acidophilus culture.



Time (in hours) Mean ± S.D

0 7.050 ± 0.010

1 6.028 ± 0.108

2 6.053 ± 0.112

3 6.008 ± 0.135

4 5.910 ± 0.133

5 5.733 ± 0.191

6 5.420 ± 0.212

7 5.297 ± 0.091

73

Table 5.4 pH values of the fermented milk with time after added with Lactobacillus

bulgaricus culture.


measured for every hour using a pH meter under standard conditions.

Time (in hours) Mean ± S.D

0 7.050 ± 0.010

1 6.213 ± 0.107

2 5.982 ± 0.068

3 5.705 ± 0.083

4 5.705 ± 0.083

5 5.357 ± 0.095

6 5.238 ± 0.082

7 5.155 ± 0.095

Table 5.5 The pH changes for milk fermented using commercial curds Dodla, Aavin,

Lactobacillus acidophilus and Lactobacillus bulgaricus.



Time

(in hours)

Control Aavin Curd Dodla Curd L. acidophilus L. bulgaricus

0 7.050 ± 0.010 7.050 ± 0.010 7.050 ± 0.010 7.050 ± 0.010 7.050 ± 0.010

1 7.050 ± 0.010 6.380 ± 0.092 6.175 ± 0.154 6.028 ± 0.108 6.213 ± 0.107

2 7.050 ± 0.010 6.447 ± 0.151 6.038 ± 0.182 6.053 ± 0.112 5.982 ± 0.068

3 7.050 ± 0.010 6.237 ± 0.108 5.927 ± 0.196 6.008 ± 0.135 5.705 ± 0.083

4 7.050 ± 0.010 6.117 ± 0.106 5.790 ± 0.218 5.910 ± 0.133 5.705 ± 0.083

5 7.050 ± 0.010 5.792 ± 0.155 5.657 ± 0.183 5.733 ± 0.191 5.357 ± 0.095

6 7.050 ± 0.010 5.460 ± 0.107 5.463 ± 0.166 5.420 ± 0.212 5.238 ± 0.082

7 7.050 ± 0.010 5.282 ± 0.185 5.255 ± 0.126 5.297 ± 0.091 5.155 ± 0.095

74

Figure 5.1 The pH changes for milk fermented using commercial curds Dodla, Aavin,

Lactobacillus acidophilus and Lactobacillus bulgaricus.



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 Sample Titratable acidity

1 Control Milk 50.78 ± 2.91

2 Aavin curd 91.27 ± 3.89

3 Dodla Curd 91.49 ± 4.03

4 Lactobacillus acidophilus 92.11 ± 4.42

5 Lactobacillus bulgaricus 93.52 ± 4.67

75

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.

5.1.1.3 Viscosity:

Table 5.7 The viscosity changes for milk fermented using commercial curds Dodla,



measured using a viscometer under standard conditions and given with ±SD

S.No Sample Viscosity

1. Aavin curd 8.01 ± 1.93

2. Dodla Curd 7.72 ± 1.16

3. Lactobacillus acidophilus 7.12 ± 1.02

4. Lactobacillus bulgaricus 7.13 ± 1.04

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

Type of fermented milk

L. acido. CPP

L. bulg. CPP

76

Figure 5.3 The viscosity changes for milk fermented using commercial curds Dodla,



measured using a viscometer under standard conditions and given with ±SD.

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

Dodla curd Aavin curd L. acidophilus L. bulgaricus

Type of fermented milk

L. acidophilus

L.bulgaricus

77

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).

78

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)

Zone of inhibition

against Pseudomonas

sp. (mm)

1 Streptomycin (control) 12 13

2 Aavin CPP 14 16

3 Dodla CPP 13 15

4 L. acido. CPP 16 15

5 L. bulg. CPP 12 15

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

Type of Fermented Milk

L. acido. CPP L. bulg. CPP

79

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.

80

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.

81

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.

82

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



83

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



84

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.

85

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.

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0

0.00

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.80

cm-1

A

C1

3286

2929

1653

1543

1404

1243

1098

544

Ab

sorb

ance

cm

86

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.

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0

0.00

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.80

cm-1

A

C2

3726

3298

2971

2878

2761

1799

1654

1534

1403

1256 1098

977

688 555

Ab

sorb

ance

cm

87

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.

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0

0.00

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.80

cm-1

A

B

3700

3667

3575

3048

2881

2808

2060

1824

1659

1534

1403

1337

1182

1131

976

834

688

526

Ab

sorb

ance

cm

88

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0

0.00

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.80

cm-1

A

A

3916

3776

3697

3574

2969

2897

2872

2808

2352

2318

2051

1827

1654

1543 1405

1357

1308

1183

1130

1099

975

909

701

528

Figure 5.13 FTIR spectrum of CPP isolated from milk fermented by the culture Lactobacillus acidophilus.



Ab

sorb

ance

cm

89

Figure 5.14 FTIR spectrum of CPP isolated from milk fermented by the culture Lactobacillus bulgaricus.



4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0

0.00

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.80

cm-1

A

C

3715

3032

2879

2808

2351

2317 1831

1651

1542

1403

1127

1098

976

928

834

700 531

Ab

sorb

ance

cm

90

5.1.7 Determination of Molecular weight of CPP by Sodium Dodecyl Sulphate


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

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

bulgaricus)

Bands coinciding with the

test samples

91

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.

92

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

1 25 28 + 3

2 34 38 + 4

3 36 37 + 1

4 27 31 + 4

5 30 31 + 1

6 31 36 + 5

7 32 33 + 1

8 30 31 + 1

9 35 35 0

10 29 31 + 2

11 27 30 + 3

12 33 34 + 1

13 34 37 + 3

14 29 31 + 2

15 31 35 + 4

16 33 33 0

17 30 30 0

18 37 38 + 1

Mean 31.23 ± 3.27 33.23 ± 3.08 2 ± 0.57

93

Table 5.10 Effect of DODLA CPP on body weight in albino mice after injection

for 10 days.




Before injection with

CPP

After injection with CPP Increase

1 31 32 + 1

2 33 37 + 4

3 30 31 + 1

4 34 36 + 3

5 32 34 + 2

6 27 28 + 1

7 31 32 + 1

8 33 33 0

9 30 31 + 1

10 34 33 -1

11 32 32 0

12 27 28 + 1

13 33 39 + 6

14 37 41 + 4

15 34 40 + 6

16 30 34 + 4

17 28 33 + 5

18 31 36 + 5

Mean 31.5 ± 2.62 33.9 ± 3.69 2.59 ± 0.15

94

Table 5.11 Effect of L. acido. CPP on body weight in albino mice after injection

for 10 days.




Before injection with

CPP

After injection with CPP Increase

1 29 32 + 3

2 27 28 + 1

3 31 33 + 2

4 30 33 + 3

5 33 37 + 4

6 37 39 + 2

7 33 35 + 2

8 35 38 + 3

9 32 33 + 1

10 36 37 + 1

11 28 30 + 2

12 29 32 + 3

13 37 37 0

14 27 32 + 5

15 35 36 + 1

16 32 35 + 3

17 31 33 + 2

18 36 38 + 2

Mean 32.11 ± 3.36 34.33 ± 3.03 2.22 ± 0.22

95

Table 5.12 Effect of L. bulg. CPP on body weight in albino mice after injection

for 10 days.





1 28 30 + 2

2 37 38 + 1

3 35 38 + 3

4 33 33 0

5 30 34 + 4

6 32 33 + 1

7 31 33 + 2

8 30 33 + 3

9 34 36 + 2

10 29 30 + 1

11 32 34 + 2

12 36 39 + 3

13 28 30 + 2

14 31 34 + 3

15 33 35 + 2

16 30 33 + 3

17 36 39 + 3

18 29 33 + 4

Mean 31.89 ± 2.83 34.12 ± 2.87 2.28 ± 0.71

96

Table 5.13 Body weight of albino mice after injection normal feed alone without

CPP for 10 days.





1 30 30 0

2 28 29 + 1

3 31 31 0

4 34 34 0

5 32 32 0

6 35 34 -1

7 33 33 0

8 31 30 -1

9 33 35 + 2

10 28 28 0

11 32 32 0

12 34 34 0

13 33 33 0

14 27 27 0

15 35 35 0

16 32 33 -1

17 29 29 0

18 31 32 +1

Mean 31.56 ± 2.41 31.72 ± 2.42 0.16 ± 0.03

97

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 (%)

1 Control 31.56 ± 2.41 31.72 ± 2.42 0.50

2 AAVIN CPP 31.23 ± 3.27 33.23 ± 3.08 6.02

3 DODLA CPP 31.5 ± 2.62 33.9 ± 3.69 7.22

4 L. acido. CPP 32.11 ± 3.36 34.33 ± 3.03 6.47

5 L. bulg. CPP 31.89 ± 2.83 34.12 ± 2.87 6.54

98

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

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

Source of CPP


99

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.

100

Table 5.15 The effect of AAVIN CPP on body weight in albino mice after

injection for 15 days.




Before injection with CPP After injection with

CPP

Increase

1 32 38 + 6

2 29 32 + 3

3 34 40 + 6

4 31 34 + 3

5 28 31 + 3

6 30 34 + 4

7 28 32 + 4

8 31 33 + 2

9 33 34 + 1

10 30 33 + 3

11 36 38 + 2

12 29 31 + 2

13 34 38 + 4

14 29 34 + 5

15 31 36 + 5

16 37 43 + 6

17 32 35 + 3

18 31 33 + 2

Mean 31.39 ± 2.59 34.94 ± 3.28 3.56 ± 0.54

101

Table 5.16 Effect of DODLA CPP on body weight in albino mice after injection

for 15 days.





1 31 34 + 3

2 33 38 + 5

3 30 35 + 5

4 28 32 + 4

5 32 37 + 5

6 34 39 + 5

7 28 32 + 4

8 32 34 + 2

9 35 40 + 5

10 29 32 + 3

11 33 36 + 3

12 29 33 + 4

13 33 39 + 6

14 37 41 + 4

15 34 40 + 6

16 30 34 + 4

17 28 33 + 5

18 31 36 + 5

Mean 31.51 ± 2.62 35.83 ± 3.05 4.33 ± 1.08

102

Table 5.18 Effect of L. acido. CPP on body weight in albino mice after injection

for 15 days.





1 33 36 + 3

2 35 39 + 4

3 38 40 + 2

4 32 37 + 5

5 27 30 + 3

6 31 36 + 5

7 31 34 + 3

8 36 38 + 2

9 34 38 + 4

10 35 38 + 3

11 30 32 + 2

12 29 33 + 4

13 31 35 + 4

14 36 42 + 6

15 34 40 + 6

16 35 40 + 5

17 30 35 + 5

18 29 34 + 4

Mean 32.56 ± 2.99 36.50 ± 3.19 3.94 ± 1.30

103

Table 5.18 Effect of L. bulg. CPP on body weight in albino mice after injection

for 15 days.





1 34 37 + 3

2 30 32 + 2

3 31 33 + 2

4 29 34 + 5

5 27 30 + 3

6 32 34 + 2

7 27 28 + 1

8 29 32 + 3

9 26 30 + 4

10 24 25 + 1

11 28 30 + 2

12 32 36 + 4

13 33 37 + 4

14 35 38 + 3

15 32 38 + 6

16 36 41 + 5

17 28 32 + 4

18 29 32 + 3

Mean 30.11 ± 3.25 33.28 ± 4.03 3.17 ± 1.38

104

Table 5.19 Body weight of albino mice after injection with normal feed alone

without CPP for 15 days.





1 29 31 + 2

2 27 28 + 1

3 32 33 + 1

4 30 32 + 2

5 31 32 + 1

6 31 33 + 2

7 29 31 + 2

8 35 37 + 2

9 32 34 + 2

10 28 30 + 2

11 31 32 + 1

12 34 35 + 1

13 33 34 + 1

14 29 31 + 2

15 32 33 + 1

16 31 33 + 2

17 34 35 + 1

18 33 35 + 2

Mean 31.12 ± 2.20 32.72 ± 2.42 1.56 ± 0.51

105

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 (%)

1 Control 31.12 ± 2.41 32.72 ± 2.42 4.89

2 AAVIN CPP 31.39 ± 2.59 34.94 ± 3.28 10.16

3 DODLA CPP 31.51 ± 2.62 35.83 ± 3.05 12.06

4 L. acido. CPP 32.56 ± 2.99 36.50 ± 3.19 10.79

5 L. bulg. CPP 30.11 ± 3.25 33.28 ± 4.03 9.53

106

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.

S.No Source of CPP

Increase in body weight

after 10 days of injection

(%)

Increase in body weight

after 15 days of

injection (%)

1 Control 0.50 4.89

2 AAVIN CPP 6.02 10.16

3 DODLA CPP 7.22 12.06

4 L. acido. CPP 6.47 10.79

5 L. bulg. CPP 6.54 9.53

107

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

Source of CPP


108

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.

109

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


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%.



days with CPP and infected with Escherichia coli. Table 5.25 and Figure 5.20 gave



Escherichia coli. Figure 5.21 depicted the effect of AAVIN CPP, DODLA CPP, L.


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.

110

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

Source of

CPP

No. of

mice

Decrease in body weight (in grams)

Mice

mortality

Mortality

Rate Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7

Control 6 29.8 ± 3.07 27.5 ± 2.56 21.2 ± 2.05 19.7 ± 2.02 17.3 ± 1.43 16.7 ± 1.59 14.7 ± 1.72 6 100%

AAVIN

CPP

6 34.7 ± 2.50 35.5 ± 2.71 36.7 ± 2.83 32.3 ± 2.21 33.7 ± 3.54 32.2 ± 3.85 30.8 ± 2.31 2 33%

DODLA

CPP 6 30.8 ± 3.12 31.0 ± 2.03 28.0 ± 2.45 27.8 ± 3.71 27.2 ± 2.93 25.0 ± 2.55 23.7 ± 2.33 1 17%

L. acido.

CPP

6 30.8 ± 3.10 31.5 ± 3.59 31.2 ± 2.36 27.0 ± 2.71 26.5 ± 2.56 25.2 ± 2.32 23.3 ± 2.80 2 33%

L. bulg. CPP 6 32.5 ± 3.24 33.2 ± 3.68 33.7 ± 3.25 33.0 ± 3.18 32.3 ± 3.16 30.5 ± 2.92 25.0 ± 2.06 1 17%

111

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 Percentage of body weight loss

Control 50.67%

AAVIN CPP 11.24%

DODLA CPP 23.05%

L. acido. CPP 24.35%

L. bulg. CPP 23.08%

Figure 5.17 Percentage of body weight loss in albino mice infected with


CPP for 10 days

Source of CPP


112

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

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

Source of CPP


L. acido. CPP

L. bulg. CPP

113

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.


Source of

CPP

No. of

mice

Decrease in body weight (in grams)

Mice

mortality

Mortality

Rate Day 1 Day 2 Day 3 Day 4 Day 5` Day 6 Day 7

Control 6 31.5 ± 1.96 25.8 ± 2.14 24.3 ± 2.18 19.7 ± 1.67 19.0 ± 1.55 18.2 ± 1.48 17.5 ± 1.36 5 83%

AAVIN

CPP

6 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 2 33%

DODLA

CPP

6 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 2 33%

L. acido.

CPP

6 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 1 17%

L. bulg.

CPP

6 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 1 17%

114

Table 5.25 Percentage of body weight loss in albino mice infected with


CPP for 15 days


Control 18.60%

AAVIN CPP 5.29%

DODLA CPP 1.27%

L. acido. CPP 8.26%

L. bulg. CPP 7.17%



CPP for 15 days.

Treatment Source of CPP


115

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

Figure 5.22 Body weight loss in albino mice infected with Escherichia coli fed


Source of CPP


L. acido. CPP

L. bulg. CPP

116

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.


Source

of CPP

No. of

mice

Decrease in body weight ( in grams) Mice

mortality

Mortality


Control 6 31.7 ± 2.67 30.8 ± 2.49 29.7 ± 2.32 28.3 ± 2.60 27.3 ± 2.33 22.5 ± 1.78 17.3 ± 1.45 4 100%

AAVIN

CPP

6 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 1 17%

DODLA

CPP

6 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 0%

L. acido.

CPP

6 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 2 33%

L. bulg.

CPP

6 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 1 17%

117

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 Percentage body weight loss

Control 45.43%

AAVIN CPP 26.36%

DODLA CPP 20.19%

L. acido. CPP 9.38%

L. bulg. CPP 6.88%


Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.

CPP for 10 days

Source of CPP


118

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

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

Source of CPP


L. acido. CPP

L. bulg. CPP

119

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.


Source of

CPP

No. of

mice

Decrease in body weight (in grams) Mice

mortality

Mortality


Control 6 30.7 ± 3.16 28.7 ± 2.89 23.5 ± 1.72 19.0 ± 1.48 18.1 ± 1.37 17.3 ± 1.24 17.2 ± 1.14 7 100%

AAVIN

CPP

6 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 2 33%

DODLA

CPP

6 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 1 17%

L. acido.

CPP

6 32.0 ± 2.94 32.2 ± 2.62 32.5 ± 2.67 31.3 ± 2.44 29.7 ± 2.17 27.5 ± 2.04 25.7 ± 1.93 3 33%

L. bulg.

CPP

6 27.7 ± 2.04 29.3 ± 2.56 29.7 ± 2.59 29.8 ± 2.76 29.3 ± 2.80 27.7 ± 2.61 25.7 ± 2.36 2 33%

120

Table 5.29 Percentage body weight loss in albino mice infected with Salmonella


days


Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.

CPP for 15 days

Source of CPP Percentage body weight loss

Control 43.97%

AAVIN CPP 16.27%

DODLA CPP 4.73%

L. acido. CPP 19.69%

L. bulg. CPP 7.22%

Source of CPP


121

Figure 5.27 Percentage mortality rate in albino mice infected with Salmonella sp.


Figure 5.28 Body weight loss in albino mice infected with Salmonella sp. fed with


Source of CPP


L. acido. CPP

L. bulg. CPP

122

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



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



seventh day with a mortality rate of 33%. In L. bulg. CPP, 10 days fed, Salmonella sp.


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%.



days with CPP and infected with Salmonella sp. Table 5.27 and Figure 5.23 depicted



Salmonella sp. Figure 5.24 depicted the effect of AAVIN CPP, DODLA CPP, L.


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.

123

As given in the table 5.28 in case of Salmonella sp. infected control mice, there


of 34 %. But in AAVIN CPP, 15 days fed, Salmonella sp. infected mice there was a



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



seventh day with a mortality rate of 33%. In L. bulg. CPP, 15 days fed, Salmonella sp.


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%.



days with CPP and infected with Salmonella sp. Table 5.29 and Figure 5.26 depicted



Salmonella sp. Figure 5.27 depicted the effect of AAVIN CPP, DODLA CPP, L.


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.

124


after injection for 10 days and infected with Shigella sp.


Batch No. of

mice


mortality

Mortality


Control 6 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 5 83%

AAVIN

CPP

6 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 1 17%

DODLA

CPP

6 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 1 17%

L. acido.

CPP

6 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 0%

L. bulg.

CPP

6 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 2 33%

125

Table 5.31 Percentage of body weight loss in albino mice infected with Shigella


days


Control 38.27%

AAVIN CPP 18.92%

DODLA CPP 8.99%

L. acido. CPP 5.56%

L. bulg. CPP 2.22%

Figure 5.29 Percentage of body weight loss in albino mice infected with Shigella


days


Source of CPP

126

Figure 5.30 Percentage mortality rate in albino mice infected with Shigella sp.


Figure 5.31 Body weight loss in albino mice infected with Shigella sp. fed with


Source of CPP


L. acido. CPP

L. bulg. CPP

127


after injection for 15 days and infected with Shigella sp.


Batch No of

mice


mortality

Mortality


Control 6 31.5 ± 2.80 31.0 ± 2.54 29.9 ± 1.94 27.3 ± 1.75 26.1 ± 1.67 23.8 ± 1.56 22.4 ± 1.39 6 100%

AAVIN

CPP

6 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 2 33%

DODLA

CPP

6 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 3 50%

L. acido.

CPP

6 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 1 17%

L. bulg.

CPP

6 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 2 33%

128

Table 5.33 Percentage of body weight loss in albino mice infected with Shigella


days


Control 28.64%

AAVIN CPP 7.01%

DODLA CPP 6.86%

L. acido. CPP 6.23%

L. bulg. CPP 10.89%

Figure 5.32 Percentage of body weight loss in albino mice infected with Shigella


days

Source of CPP


129

Figure 5.33 Percentage mortality rate in albino mice infected with Shigella sp. fed


Figure 5.34 Body weight loss in albino mice infected with Shigella sp. fed with


Source of CPP


L. acido. CPP

L. bulg. CPP

130

As given in the table 5.30 in case of Shigella sp. infected control mice, there


of 33%. But in AAVIN CPP, 10 days fed, Shigella sp. infected mice there was a



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


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%.



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


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.

131

As given in the table 5.32 in case of Shigella sp. infected control mice, there


of 100%. But in AAVIN CPP, 15 days fed, Shigella sp. infected mice there was a



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


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%.



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


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.

132

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

S.No

Treatment

Pathogen count in Visceral organs ( x 103)

Liver Kidney

Spleen

Small intestine

1 Control 51 67 78 62

2 AAVIN CPP 21 13 13 11

3 DODLA CPP 18 19 15 6

4 L. acido. CPP 14 16 4 10

5 L. bulg. CPP 17 8 18 19

133



after infected with Escherichia coli

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.

L. acido.

L. bulg.

134

Table 5.35 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.


after infected with Salmonella sp.

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.

S.No

Treatment

Pathogen count in visceral organs (x 103)

Liver Kidney Spleen Small intestine

1 Control 79 81 61 45

2 AAVIN CPP 27 24 13 7

3 DODLA CPP 21 20 23 11

4 L. acido. CPP 16 26 9 4

5 L. bulg. CPP 23 19 15 14

135

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).


15 days injection on GUT pathogen count of albino mice visceral organs after

infected with Salmonella sp.

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).

L. acido.

L. bulg.

136


15 days injection on GUT pathogen count of albino mice visceral organs after

infected with Shigella sp.

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.

S.No Treatment

Pathogen count in visceral organs (x 103)

Liver Kidney Spleen Small intestine

1 Control 93 57 68 59

2 AAVIN CPP 15 6 16 8

3 DODLA CPP 20 13 19 13

4 L. acido. CPP 24 18 12 24

5 L. bulg. CPP 11 10 22 17

137

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).


on GUT pathogen count of albino mice visceral organs fed with CPP for 15 days

and infected with Shigella sp.

L. acido.

L. bulg.

138

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

139

(c) Dodla CPP 15 days fed mice

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

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

140

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.



141




142

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.



143




144

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



145




146

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).

147


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


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

S.No Source of CPP IgA secretary cells/10 fields of vision

10 days 15 days

1 Control 8 ± 2 6 ± 2

2 AAVIN CPP 35 ± 4 82 ± 4

3 DODLA CPP 31 ± 4 91 ± 4

4 L. acido. CPP 42 ± 4 78 ± 4

5 L. bulg. CPP 29 ± 4 95 ± 4

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

Source of CPP

L. acido. CPP

L. bulg. CPP

148


CPP fed for 15 days on IgA secretary cell production in albino mice after infected

with Escherichia coli and given with ±SD


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


10 days 15 days

1 Control 10 ± 2 14 ± 2

2 AAVIN CPP 28 ± 4 93 ± 4

3 DODLA CPP 37 ± 4 88 ± 4

4 L. acido. CPP 45 ± 4 96 ± 4

5 L. bulg. CPP 53 ± 4 102 ± 4

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

L. bulg. CPP

L. acido. CPP

Source of CPP

149


CPP 10 days injection on IgA secretary cell production in albino mice after

infected with Salmonella sp. and given with ±SD


15 days injection on IgA secretary cell production in albino mice after infected

with Salmonella sp. and given with ±SD


L. bulg. CPP

L. acido. CPP


L. bulg. CPP

L. acido. CPP

Source of CPP

Source of CPP

150

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.

151


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



with Shigella sp. and given with ±SD


10 days 15 days

1 Control 5 ± 2 11 ± 2

2 AAVIN CPP 39 ± 4 72 ± 4

3 DODLA CPP 26 ± 4 64 ± 4

4 L. acido. CPP 30 ± 4 87 ± 4

5 L. bulg. CPP 49 ± 4 92 ± 4

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

L. acido. CPP

L. bulg. CPP

Source of CPP

152



with Shigella sp. and given with ±SD

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.

Control milk AAVIN CPP DODLA CPP L. acido. CPP

L. acido. CPP

L. bulg. CPP

L. bulg. CPP

Source of CPP

153

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.

154

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


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


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


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

155

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

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


Micronucleus

Normal cells

156

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).


irradiation for 100 Gy for 1880 seconds showing micronucleus formation in the





Micronucleus

Normal cells

157

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).


irradiation for 135 Gy for 2538 seconds showing nuclear retraction and

karyolysis in the erythrocyte cells given in magnification of 100x per 10 fields


Gy for 2538 seconds showing micronucleus formation in the erythrocyte cells

Nuclear

retraction

Karyolysis

Micronucleus

158

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


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

Micronucleus

Normal cells

159

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).


Co60 irradiation for 1.9 Gy for 34 seconds showing micronucleus, bi and multi

nuclear formation in the erythrocyte cells

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

Micronucleus

Multinucleated erythrocyte

Normal cells

Binucleated

erythrocyte

160

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).


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

Photo 5.24 Test mice fed for 15 days and irradiated with Co60 irradiation for 5



Nuclear retraction

Karyolysis

Normal cells

161

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 5 16 3

48 hours 1000 9 20 5

72 hours 1000 13 21 8

96 hours 1000 15 23 8

162

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 1 12 1

48 hours 1000 4 13 1

72 hours 1000 7 17 4

96 hours 1000 7 19 6

163

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 7

48 hours 1000 31 35 13

72 hours 1000 38 42 15

96 hours 1000 53 45 18

164

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)

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

Time No. of

erythrocytes

No. of micro

nucleated

erythrocytes

No. of

binucleated

erythrocytes

No. of multi

nucleated

erythrocytes

24 hours 1500 8 31 7

48 hours 1500 11 39 9

72 hours 1500 14 43 9

96 hours 1500 16 48 12

165

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

No. of micro

nucleated

erythrocytes

No. of

binucleated

erythrocytes

No. of multi

nucleated

erythrocytes

24 hours 1500 4 19 13

48 hours 1500 5 22 16

72 hours 1500 8 22 17

96 hours 1500 11 25 20

166

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

No. of micro

nucleated

erythrocytes

No. of

binucleated

erythrocytes

No. of multi

nucleated

erythrocytes

24 hours 1500 23 14 11

48 hours 1500 27 26 15

72 hours 1500 35 32 18

96 hours 1500 39 51 19

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).

167

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

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

Test mice Test fish

Control

Control Test

168

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 CPP’s 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).

169

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

170

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 physico-

chemical 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 anti-

microbial 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.

171

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


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.

172

6.7 Animal studies:

CPP’s 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 CPP’s 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 CPP’s 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].

173

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 CPP’s

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

174

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

CPP’s 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

175

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.

176

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

177

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

178

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,

179

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

181

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, Peyer’s

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 milk-

182

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 anti-

genotoxic effect on the low ionizing background radiation, thus acting as a anti-

genotoxic 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

184

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 people’s money in health benefiting sectors. Increasing 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.

185

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

186

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.

187

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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).

217

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

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 University‟s 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

Who‟s 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).

Biopeptides in Milk

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