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Milk bio-peptides
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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
iii
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
iv
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)
vi
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
vii
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
xi
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
xii
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
xiii
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
xiv
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
xv
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
xvi
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
xvii
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
xviii
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
5.2 pH values of the fermented milk with time after added with 72
Aavin dairy curd
5.3 pH values of the fermented milk with time after added with 72
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
after feeding for 10 days
5.11 Effect of L. acido. CPP on body weight of albino mice 94
after feeding for 10 days
5.12 Effect of L. bulg. CPP fed on body weight of albino mice 95
after feeding for 10 days
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.
5.25 Percentage of body weight loss in albino mice infected with 114
Escherichia coli fed with AAVIN CPP, DODLA CPP, L. acido.
CPP and L. bulg. CPP for 15 days
5.26 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and 116
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.
CPP and L. bulg. CPP for 10 days
5.28 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and 119
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
Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido.
CPP and L. bulg. CPP for 15 days
xxii
5.30 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and 124
L. bulg. CPP on the body weight of albino mice after feeding for
10 days and infected with Shigella sp. for 10 days
5.31 Percentage of body weight loss in albino mice infected with 125
Shigella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP
and L. bulg. CPP for 10 days
5.32 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and 127
L. bulg. CPP on the body weight of albino mice after feeding
for 15 days and infected with Shigella sp.
5.33 Percentage of body weight loss in albino mice infected with 128
Shigella sp. fed with AAVIN CPP, DODLA CPP, L. acido.
CPP and L. bulg. CPP for 15 days
5.34 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and 132
L. bulg. CPP 15 days feeding on GUT pathogen count of
albino mice visceral organs after infected with Escherichia coli
5.35 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and 134
L. bulg. CPP 15 days feeding on GUT pathogen count of
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.
5.37 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. 147
CPP on IgA secretary cell production in albino mice fed with
CPP for 10, 15 days and infected with Escherichia coli
5.38 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. 148
CPP 10, 15 days feeding on IgA secretary cell production in
albino mice after infected with Salmonella sp.
5.39 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. 151
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)
5.41 Quantification of micro, bi and multi-nucleated cells of mice 162
fed with CPP for 10 days and subjected to Co60 irradiation
at LD50 value (1.9 Gy)
xxiv
5.42 Quantification of micro, bi and multi-nucleated cells of mice 163
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
fed with CPP for 10 days and subjected to Co60 irradiation
at LD50 value (135 Gy)
5.44 Quantification of micro, bi and multi-nucleated cells of fish 165
fed with CPP for 15 days and subjected to Co60 irradiation
at LD50 value (135 Gy)
5.45 Quantification of micro, bi and multi-nucleated cells of fish 166
not fed with CPP and subjected to Co60 irradiation
at LD50 value (135 Gy)
xxv
LIST OF PHOTOS
NUMBER TITLE PAGE NO.
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
Lactobacillus bulgaricus
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
5.7 Photograph showing the histopathological studies of 15 days 140
CPP fed albino mice liver cells infected with Escherichia coli
on seven days post infection staining with Methylene blue
5.8 Photograph showing the histopathological studies of 15 days 142
CPP fed albino mice spleen cells infected with Escherichia coli
on seven days post infection staining with Methylene blue
5.9 Photograph showing the histopathological studies of 15 days 144
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
5.15 Control fish fed with normal feed for 15 days and irradiated with 156
Co60 irradiation for 100 Gy for 1880 seconds showing micronucleus
formation in the erythrocyte cells
5.16 Test fish fed for 15 days and irradiated with Co60 irradiation for 156
100 Gy for 1880 seconds showing absence of micronucleus
formation in the erythrocyte cells
5.17 Control fish fed with normal feed for 15 days and irradiated with 157
Co60 irradiation for 135 Gy for 2538 seconds showing nuclear
retraction and karyolysis in the erythrocyte cells
xxviii
5.18 Test fish fed for 15 days and irradiated with Co60 irradiation for 157
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
Co60 irradiation for 1 Gy for 19 seconds showing micronucleus
formation in the erythrocyte cells
5.20 Test mice fed for 15 days and irradiated with Co60 irradiation for 158
1 Gy for 19 seconds showing absence of micronucleus formation
in the erythrocyte cells
5.21 Control mice fed with normal feed for 15 days and irradiated with 159
Co60 irradiation for 1.9 Gy for 34 seconds showing micronucleus,
bi and multi nuclear formation in the erythrocyte cells
5.22 Test mice fed for 15 days and irradiated with Co60 irradiation for 159
1.9 Gy for 34 seconds showing absence of micronucleus formation
in the erythrocyte cells
5.23 Control mice fed with normal feed for 15 days and irradiated with 160
Co60 irradiation for 5 Gy for 94 seconds showing karyolysis
and nuclear retraction in the erythrocyte cells
xxix
5.24 Test mice fed for 15 days and irradiated with Co60 irradiation for 160
5 Gy for 904 seconds showing absence of micronucleus formation
in the erythrocyte cells
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
xxx
LIST OF FIGURES
NUMBER TITLE PAGE NO.
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
curds Dodla, Aavin, Lactobacillus acidophilus and
Lactobacillus bulgaricus
5.3 The viscosity changes for milk fermented using commercial 76
curds Dodla, Aavin, Lactobacillus acidophilus and
Lactobacillus bulgaricus
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
commercial curd Aavin
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 Aavin
5.12 FTIR spectrum of CPP isolated from milk fermented by 87
commercial curd Dodla
5.13 FTIR spectrum of CPP isolated from milk fermented by 88
the culture Lactobacillus acidophilus
5.14 FTIR spectrum of CPP isolated from milk fermented by 89
the culture Lactobacillus bulgaricus
5.15 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. 98
CPP on the percentage increase in body weight of albino mice
after fed for 10 days
5.16 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. 107
CPP on the percentage increase in body weight of albino mice
after feeding for 15 days
xxxii
5.17 Percentage of body weight loss in albino mice infected with 111
Escherichia coli fed with AAVIN CPP, DODLA CPP, L. acido.
CPP and L. bulg. CPP for 10 days
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
5.20 Percentage of body weight loss in albino mice infected with 114
Escherichia coli fed with AAVIN CPP, DODLA CPP, L. acido.
CPP and L. bulg. CPP for 15 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
fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.
CPP for 10 days
xxxiii
5.23 Percentage of body weight loss in albino mice infected with 117
Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido.
CPP and L. bulg. CPP for 10 days
5.24 Percentage mortality rate in albino mice infected with Salmonella sp. 118
fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.
CPP for 10 days
5.25 Body weight loss in albino mice infected with Salmonella sp. 118
fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.
CPP for 10 days
5.26 Percentage of body weight loss in albino mice infected with 120
Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido.
CPP and L. bulg. CPP for 15 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
fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.
CPP for 15 days
xxxiv
5.29 Percentage of body weight loss in albino mice infected with 125
Shigella sp. fed with AAVIN CPP, DODLA CPP, L. acido.
CPP and L. bulg. CPP for 10 days
5.30 Percentage mortality rate in albino mice infected with Shigella sp. 126
fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.
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
fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.
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
5.35 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and 133
L. bulg. CPP 15 days feeding on GUT pathogen count of
albino mice visceral organs after infected with Escherichia coli
5.36 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. 135
CPP 15 days feeding on GUT pathogen count of albino mice
visceral organs after infected with Salmonella sp.
5.37 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. 137
CPP on GUT pathogen count of albino mice visceral organs fed
with CPP for 15 days and infected with Shigella sp.
5.38 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. 147
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
5.41 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. 149
CPP 15 days feeding on IgA secretary cell production in albino
mice after infected with Salmonella sp.
5.42 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. 151
CPP 10 days feeding on IgA secretary cell production in albino
mice after infected with Shigella sp.
5.43 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. 152
CPP 15 days feeding on IgA secretary cell production in albino
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 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
Stimulation of phagocytosis
and immune responses
against bacterial infections
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.
The values given are mean ± Standard Deviation of triplicate samples. The values were
measured every hour using a pH meter under standard conditions, number of hours n = 7.
Time
(in hours)
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.
The values given are mean ± Standard Deviation of triplicate samples. The values were
measured every hour using a pH meter under standard conditions, number of hours n = 7.
Time (in hours) 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.
The values given are mean ± Standard Deviation of triplicate samples. The values were
measured for every hour using a pH meter under standard conditions.
Time (in hours) 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.
The values given are mean ± Standard Deviation of triplicate samples. The values were
measured every hour using a pH meter under standard conditions, number of hours n = 7.
Time
(in hours)
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.
The values given are mean ± Standard Deviation of triplicate samples. The values were
measured every hour using a pH meter under standard conditions, number of hours n = 7.
5.1.1.2 Titratable acidity:
Table 5.6 The titratable acidity values for milk fermented using commercial curds
Dodla, Aavin, Lactobacillus acidophilus and Lactobacillus bulgaricus. The values given
are mean ± Standard Deviation of triplicate samples. The values were measured using acid
base neutralization reaction under standard conditions and given with ±SD.
S.No 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,
Aavin, Lactobacillus acidophilus and Lactobacillus bulgaricus.
The values given are mean ± Standard Deviation of triplicate samples. The values were
measured using a viscometer under standard conditions and given with ±SD
S.No 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,
Aavin, Lactobacillus acidophilus and Lactobacillus bulgaricus.
The values given are mean ± Standard Deviation of triplicate samples. The values were
measured using a viscometer under standard conditions and given with ±SD.
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
milk peptides. The area under peak had significance less than 0.005 (P < 0.005) with
number of peaks, n=8.
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
milk peptides. The area under peak had significance less than 0.005 (P < 0.005) with
number of peaks, n=8.
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.
Peaks at 2808 cm, 1654 cm, 1130 cm, 975 cm and 909 cm1 showed the characteristic features of fermented milk peptides. The area
under peak had significance less than 0.002 (P < 0.002) with number of peaks, n=24.
Ab
sorb
ance
cm
89
Figure 5.14 FTIR spectrum of CPP isolated from milk fermented by the culture Lactobacillus bulgaricus.
Peaks at 2808 cm, 1651 cm, 1127 cm, 976 cm and 834 cm1 showed the characteristic features of fermented milk peptides. The area
under peak had significance less than 0.002 (P < 0.002) with number of peaks, n=24.
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
Polyacrylamide Gel Electrophoresis (SDS PAGE):
The CPP isolated from the fermented milk by the cultures of L. acidophilus and
L. bulgaricus were run along with CPP from the fermented milk by two commercial
curds in SDS PAGE along with the standard markers. The molecular weight of the
four isolated CPPs was found to be in the range of 1.5 – 3.5 kD (Photo 5.5).
Photo 5.5 Molecular weight determination of CPP isolated from milk fermented
by commercial curd Aavin, commercial curd Dodla, Lactobacillus acidophilus
and Lactobacillus bulgaricus using Sodium Dodecyl Sulphate - Polyacrylamide
Gel Electrophoresis (SDS-PAGE) having 2.5% of acrylamide and run at 250 V
constant for about 30 minutes
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.
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 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.
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 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.
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 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.
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 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
L. acido. CPP L. bulg. 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.
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 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.
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 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.
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 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.
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 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.
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 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
L. acido. CPP L. bulg. 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
infected mice there was a steady increase in the body weigh from the day one of post
infection till the fifth day. Starting from the sixth day there was a decline in the body
weight of the mice till the seventh day with a mortality rate of 17%. In L. bulg. CPP,
15 days fed, E.Coli infected mice there was a steady increase in the body weigh from
the day one of post infection till the fourth day. Starting from the fifth day there was a
decline in the body weight of the mice till the seventh day with a mortality rate of
17%.
Table 5.24 summarised the effect of AAVIN CPP, DODLA CPP, L. acido.
CPP and L. bulg. CPP on the body weight loss percentage of albino mice fed for 15
days with CPP and infected with Escherichia coli. Table 5.25 and Figure 5.20 gave
the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body
weight loss percentage of albino mice fed for 10 days with CPP and infected with
Escherichia coli. Figure 5.21 depicted the effect of AAVIN CPP, DODLA CPP, L.
acido. CPP and L. bulg. CPP on the mortality percentage of albino mice fed for 15
days with CPP and infected with Escherichia coli. Figure 5.22 summarised the effect
of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of
albino mice fed for 15 days with CPP and infected with Escherichia coli.
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
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
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
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.
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 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
Escherichia coli fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.
CPP for 15 days
Source of CPP Percentage of body weight loss
Control 18.60%
AAVIN CPP 5.29%
DODLA CPP 1.27%
L. acido. CPP 8.26%
L. bulg. CPP 7.17%
Figure 5.20 Percentage of body weight loss in albino mice infected with
Escherichia coli fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.
CPP for 15 days.
Treatment Source of CPP
L. acido. CPP L. bulg. 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
with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days
Source of CPP
L. acido. CPP L. bulg. 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.
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 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%
Figure 5.23 Percentage of body weight loss in albino mice infected with
Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.
CPP for 10 days
Source of CPP
L. acido. CPP L. bulg. 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
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.
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 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
sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15
days
Figure 5.26 Percentage of body weight loss in albino mice infected with
Salmonella sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.
CPP for 15 days
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
L. acido. CPP L. bulg. CPP
121
Figure 5.27 Percentage mortality rate in albino mice infected with Salmonella sp.
fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days
Figure 5.28 Body weight loss in albino mice infected with Salmonella sp. fed with
AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days
Source of CPP
L. acido. CPP L. bulg. 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
steady increase in the body weigh from the day one of post infection till the fourth
day. Starting from the fifth day there was a decline in the body weight of the mice till
the seventh day with a mortality rate of 17%. In DODLA CPP, 10 days fed,
Salmonella sp. infected mice there was a steady increase in the body weigh from the
day one of post infection till the third day. Starting from the fourth day there was a
decline in the body weight of the mice till the seventh day with a mortality rate of 0%.
In L. acido. CPP, 10 days fed, Salmonella sp. infected mice there was a steady
increase in the body weigh from the day one of post infection till the third day.
Starting from the fourth day there was a decline in the body weight of the mice till the
seventh day with a mortality rate of 33%. In L. bulg. CPP, 10 days fed, Salmonella sp.
infected mice there was a steady increase in the body weigh from the day one of post
infection till the fourth day. Starting from the fifth day there was a decline in the body
weight of the mice till the seventh day with a mortality rate of 17%.
Table 5.26 summarised the effect of AAVIN CPP, DODLA CPP, L. acido.
CPP and L. bulg. CPP on the body weight loss percentage of albino mice fed for 10
days with CPP and infected with Salmonella sp. Table 5.27 and Figure 5.23 depicted
the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body
weight loss percentage of albino mice fed for 10 days with CPP and infected with
Salmonella sp. Figure 5.24 depicted the effect of AAVIN CPP, DODLA CPP, L.
acido. CPP and L. bulg. CPP on the mortality percentage of albino mice fed for 10
days with CPP and infected with Salmonella sp. Figure 5.25 summarised the effect of
AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of
albino mice fed for 10 days with CPP and infected with Salmonella sp.
123
As given in the table 5.28 in case of Salmonella sp. infected control mice, there
was a steep decline of body weight from first day till seventh day with a mortality rate
of 34 %. But in AAVIN CPP, 15 days fed, Salmonella sp. infected mice there was a
steady increase in the body weigh from the day one of post infection till the fourth
day. Starting from the fifth day there was a decline in the body weight of the mice till
the seventh day with a mortality rate of 33%. In DODLA CPP, 15 days fed,
Salmonella sp. infected mice there was a steady increase in the body weigh from the
day one of post infection till the third day. Starting from the fourth day there was a
decline in the body weight of the mice till the seventh day with a mortality rate of
17%. In L. acido. CPP, 15 days fed, Salmonella sp. infected mice there was a steady
increase in the body weigh from the day one of post infection till the third day.
Starting from the fourth day there was a decline in the body weight of the mice till the
seventh day with a mortality rate of 33%. In L. bulg. CPP, 15 days fed, Salmonella sp.
infected mice there was a steady increase in the body weigh from the day one of post
infection till the fourth day. Starting from the fifth day there was a decline in the body
weight of the mice till the seventh day with a mortality rate of 33%.
Table 5.28 summarised the effect of AAVIN CPP, DODLA CPP, L. acido.
CPP and L. bulg. CPP on the body weight loss percentage of albino mice fed for 15
days with CPP and infected with Salmonella sp. Table 5.29 and Figure 5.26 depicted
the effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body
weight loss percentage of albino mice fed for 15 days with CPP and infected with
Salmonella sp. Figure 5.27 depicted the effect of AAVIN CPP, DODLA CPP, L.
acido. CPP and L. bulg. CPP on the mortality percentage of albino mice fed for 15
days with CPP and infected with Salmonella sp. Figure 5.28 summarised the effect of
AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of
albino mice fed for 15 days with CPP and infected with Salmonella sp.
124
Table 5.30 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice
after injection for 10 days and infected with Shigella sp.
Number of mice n = 6 and the level of significance was not less than 0.005 (P ≤ 0.005) and given with ±SD
Batch 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 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
sp. 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 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
sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10
days
L. acido. CPP L. bulg. CPP
Source of CPP
126
Figure 5.30 Percentage mortality rate in albino mice infected with Shigella sp.
fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days
Figure 5.31 Body weight loss in albino mice infected with Shigella sp. fed with
AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 10 days
Source of CPP
L. acido. CPP L. bulg. CPP
L. acido. CPP
L. bulg. CPP
127
Table 5.32 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice
after injection for 15 days and infected with Shigella sp.
Number of mice n = 6 and the level of significance was not less than 0.005 (P ≤ 0.005) and given with ±SD
Batch 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 ± 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
sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15
days
Source of CPP Percentage of body weight loss
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
sp. fed with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15
days
Source of CPP
L. acido. CPP L. bulg. CPP
129
Figure 5.33 Percentage mortality rate in albino mice infected with Shigella sp. fed
with AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days
Figure 5.34 Body weight loss in albino mice infected with Shigella sp. fed with
AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP for 15 days
Source of CPP
L. acido. CPP L. bulg. CPP
L. acido. CPP
L. bulg. CPP
130
As given in the table 5.30 in case of Shigella sp. infected control mice, there
was a steep decline of body weight from first day till seventh day with a mortality rate
of 33%. But in AAVIN CPP, 10 days fed, Shigella sp. infected mice there was a
steady increase in the body weigh from the day one of post infection till the fourth
day. Starting from the fifth day there was a decline in the body weight of the mice till
the seventh day with a mortality rate of 17%. In DODLA CPP, 10 days fed, Shigella
sp. infected mice there was a steady increase in the body weigh from the day one of
post infection till the third day. Starting from the fourth day there was a decline in the
body weight of the mice till the seventh day with a mortality rate of 17%. In L. acido.
CPP, 10 days fed, Shigella sp. infected mice there was a steady increase in the body
weigh from the day one of post infection till the fourth day. Starting from the fifth day
there was a decline in the body weight of the mice till the seventh day with a mortality
rate of 0%. In L. bulg. CPP, 10 days fed, Shigella sp. infected mice there was a steady
increase in the body weigh from the day one of post infection till the fourth day.
Starting from the fifth day there was a decline in the body weight of the mice till the
seventh day with a mortality rate of 33%.
Table 5.30 summarised the effect of AAVIN CPP, DODLA CPP, L. acido.
CPP and L. bulg. CPP on the body weight loss percentage of albino mice fed for 10
days with CPP and infected with Shigella sp. Table 5.31 and Figure 5.29 depicted the
effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body
weight loss percentage of albino mice fed for 10 days with CPP and infected with
Shigella sp. Figure 5.30 depicted the effect of AAVIN CPP, DODLA CPP, L. acido.
CPP and L. bulg. CPP on the mortality percentage of albino mice fed for 10 days with
CPP and infected with Shigella sp. Figure 5.31 summarised the effect of AAVIN CPP,
DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice fed
for 10 days with CPP and infected with Shigella sp.
131
As given in the table 5.32 in case of Shigella sp. infected control mice, there
was a steep decline of body weight from first day till seventh day with a mortality rate
of 100%. But in AAVIN CPP, 15 days fed, Shigella sp. infected mice there was a
steady increase in the body weigh from the day one of post infection till the fourth
day. Starting from the fifth day there was a decline in the body weight of the mice till
the seventh day with a mortality rate of 33%. In DODLA CPP, 15 days fed, Shigella
sp. infected mice there was a steady increase in the body weigh from the day one of
post infection till the fifth day. Starting from the fifth day there was a decline in the
body weight of the mice till the seventh day with a mortality rate of 50%. In L. acido.
CPP, 15 days fed, Shigella sp. infected mice there was a steady increase in the body
weigh from the day one of post infection till the fourth day. Starting from the fifth day
there was a decline in the body weight of the mice till the seventh day with a mortality
rate of 17%. In L. bulg. CPP, 15 days fed, Shigella sp. infected mice there was a
steady increase in the body weigh from the day one of post infection till the fifth day.
Starting from the sixth day there was a decline in the body weight of the mice till the
seventh day with a mortality rate of 33%.
Table 5.32 summarised the effect of AAVIN CPP, DODLA CPP, L. acido.
CPP and L. bulg. CPP on the body weight loss percentage of albino mice fed for 15
days with CPP and infected with Shigella sp. Table 5.33 and Figure 5.32 depicted the
effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP on the body
weight loss percentage of albino mice fed for 15 days with CPP and infected with
Shigella sp. Figure 5.33 depicted the effect of AAVIN CPP, DODLA CPP, L. acido.
CPP and L. bulg. CPP on the mortality percentage of albino mice fed for 15 days with
CPP and infected with Shigella sp. Figure 5.34 summarised the effect of AAVIN CPP,
DODLA CPP, L. acido. CPP and L. bulg. CPP on the body weight of albino mice fed
for 15 days with CPP and infected with Shigella sp.
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
Figure 5.35 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.
CPP 15 days injection on GUT pathogen count of albino mice visceral organs
after infected with Escherichia coli
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.
CPP 15 days injection on GUT pathogen count of albino mice visceral organs
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).
Figure 5.36 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP
15 days injection on GUT pathogen count of albino mice visceral organs after
infected with Salmonella sp.
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
Table 5.36 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP
15 days injection on GUT pathogen count of albino mice visceral organs after
infected with Shigella sp.
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).
Figure 5.37 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP
on GUT pathogen count of albino mice visceral organs fed with CPP for 15 days
and infected with Shigella sp.
L. acido.
L. bulg.
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.
(a) Control batch of CPP unfed mice
(b) Aavin CPP 15 days fed mice
141
(c) Dodla CPP 15 days fed mice
(d) L. acido. CPP 15 days fed mice
(e) L. bulg. CPP 15 days fed mice
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.
(a) Control batch of CPP unfed mice
(b) Aavin CPP 15 days fed mice
143
(c) Dodla CPP 15 days fed mice
(d) L. acido. CPP 15 days fed mice
(e) L. bulg. CPP 15 days fed mice
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
(a) Control batch of CPP unfed mice
(b) Aavin CPP 15 days fed mice
145
(c) Dodla CPP 15 days fed mice
(d) L. acido. CPP 15 days fed mice
(e) L. bulg. CPP 15 days fed mice
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
Table 5.37 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP
on IgA secretary cell production in albino mice fed with CPP for 10, 15 days and
infected with Escherichia coli. Number of mice infected were, n = 6 and the level of
significance was not more than 0.002 (P ≥ 0.02) when calculated by ANOVA and
given with ±SD
Figure 5.38 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.
CPP 10 days injection on IgA secretary cell production in albino mice fed with
CPP for 10 days after infected with Escherichia coli and given with ±SD
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
Figure 5.39 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.
CPP fed for 15 days on IgA secretary cell production in albino mice after infected
with Escherichia coli and given with ±SD
Table 5.38 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP
10, 15 days injection on IgA secretary cell production in albino mice after
infected with Salmonella sp.
Number of mice infected were, n = 6 and the level of significance was not more than
0.002 (P ≥ 0.02) when calculated by ANOVA and given with ±SD
S.No Source of CPP IgA secretary cells/10 fields of vision
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
Figure 5.40 The effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg.
CPP 10 days injection on IgA secretary cell production in albino mice after
infected with Salmonella sp. and given with ±SD
Figure 5.41 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP
15 days injection on IgA secretary cell production in albino mice after infected
with Salmonella sp. and given with ±SD
Control milk AAVIN CPP DODLA CPP L. acido. CPP L. bulg. CPP
L. bulg. CPP
L. acido. CPP
Control milk AAVIN CPP DODLA CPP L. acido. CPP L. bulg. 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
Table 5.39 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP
10, 15 days injection on IgA secretary cell production in albino mice fed after
infected with Shigella sp.
Number of mice infected were, n = 6 and the level of significance was not more than
0.002 (P ≥ 0.02) when calculated by ANOVA and given with ±SD
Figure 5.42 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP
10 days injection on IgA secretary cell production in albino mice after infected
with Shigella sp. and given with ±SD
S.No Source of CPP IgA secretary cells/10 fields of vision
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
Figure 5.43 Effect of AAVIN CPP, DODLA CPP, L. acido. CPP and L. bulg. CPP
15 days injection on IgA secretary cell production in albino mice after infected
with Shigella sp. and given with ±SD
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
having 39 ± 4 IgA secretary cells/10 fields of vision at a magnification of 100 and test
mice fed with AAVIN CPP for 15 days and then infected with Shigella sp. was having
72 ± 4 IgA secretary cells/10 fields of vision at a magnification of 100x (Figure 5.42).
Test mice fed with DODLA CPP for 10 days and then infected with Shigella sp. was
having 26 ± 4 IgA secretary cells/10 fields of vision at a magnification of 100 and test
mice fed with DODLA CPP for 15 days and then infected with Shigella sp. was
having 64 ± 4 IgA secretary cells/10 fields of vision at a magnification of 100.
Test mouse fed with L. acido. CPP for 10 days and then infected with Shigella
sp. was having 30 ± 4 IgA secretary cells/10 fields of vision at a magnification of 100
and test mice fed with L. acido. CPP for 15 days and then infected with Shigella sp.
was having 87 ± 4 IgA secretary cells/10 fields of vision at a magnification of 100.
Test mouse fed with L. bulg. CPP for 10 days and then infected with Shigella sp. was
having 49 ± 4 IgA secretary cells/10 fields of vision at a magnification of 100 and test
mice fed with L. bulg. CPP for 15 days and then infected with Shigella sp. was having
92 ± 4 IgA secretary cells/10 fields of vision at a magnification of 100x (Figure 5.43).
The control mice not fed with CPP and infected with Shigella sp. had 11 ± 2 IgA
secretary cells/10 fields of vision at a magnification of 100, 15 days post infection
(Table 5.39).
5.4 Determination of the Anti-genotoxic acivity of the CPP:
5.4.1 Micronucleus assay:
The micronucleus assay results of the fish and albino mice blood cells showed
distinct features. The formation of micronucleus in the cell was taken as the major
parameter of distinguishment. Other features noted were the presence of bi or multi
nucleated cells and cell membrane disintegration, karyolysis and nuclear retraction.
These three parameters together were used to study the anti-genotoxic effect of CPP
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
erythrocyte cells given in magnification of 100x per 10 fields
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).
Photo 5.15 Control fish fed with normal feed for 15 days and irradiated with Co60
irradiation for 100 Gy for 1880 seconds showing micronucleus formation in the
erythrocyte cells given in magnification of 100x per 10 fields
Photo 5.16 Test fish fed for 15 days and irradiated with Co60 irradiation for 100
Gy for 1880 seconds showing absence of micronucleus formation in the
erythrocyte cells given in magnification of 100x per 10 fields
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).
Photo 5.17 Control fish fed with normal feed for 15 days and irradiated with Co60
irradiation for 135 Gy for 2538 seconds showing nuclear retraction and
karyolysis in the erythrocyte cells given in magnification of 100x per 10 fields
Photo 5.18 Test fish fed for 15 days and irradiated with Co60 irradiation for 135
Gy for 2538 seconds showing micronucleus formation in the erythrocyte cells
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
erythrocyte cells given in magnification of 100x per 10 fields
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).
Photo 5.21 Control mice fed with normal feed for 15 days and irradiated with
Co60 irradiation for 1.9 Gy for 34 seconds showing micronucleus, bi and multi
nuclear formation in the erythrocyte cells
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).
Photo 5.23 Control mice fed with normal feed for 15 days and irradiated with
Co60 irradiation for 5 Gy for 94 seconds showing karyolysis and nuclear
retraction in the erythrocyte cells given in magnification of 100x per 10 fields
Photo 5.24 Test mice fed for 15 days and irradiated with Co60 irradiation for 5
Gy for 904 seconds showing absence of micronucleus formation in the
erythrocyte cells given in magnification of 100x per 10 fields
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
Polyacrylamide Gel Electrophoresis (SDS PAGE):
Molecular weight determination was ascertained using SDS-PAGE and the
value was found to be 3.5 – 4 kilo Daltons (Photo 5.5). Molecular weight is always of
critical importance to bioactive components as it indirectly determines their mode of
delivery in to human body as a nutraceutical and therefore efficacy as stated by
Antonius et al., 2000 [106]. The bioactive components present in fermented milk
peptide of CPP could be broken in to smaller components for an improved
formulation and effective delivery. Production of these peptides at nano scale could be
pondered upon in the future which will open new vistas of fast acting nutraceutical.
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.
180
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).
183
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).