Contents

Contents
Page No.
List of Figures i-ii
List of Tables iii-iv
Symbols and Abbreviations v-vii
Chapter 1: Introduction 1-3
Chapter 2: Review of Literature 4-24
2.1. Fermented foods 4
2.2. Fermentation 5
2.3. Lactic-acid fermentation 6
2.4. Culture dependent methods for the isolation of microbiota 7
2.5. Microbiota responsible for fermentation 8
2.6. Fermented foods and health 9
2.7. Fermented brine mango pickle 10
2.8. Probiotics 12
2.9. Probiotics in human health 18
2.10 Antioxidants Properties 18
2.11 Anti-tumor properties 22
Chapter 3: Screening and Probiotic characterization of Non-
hemolytic bacteria from fermented mango pickle 25-49
3.1 Introduction 25
3.2. Materials and Methods 26
3.2.1. Sample Collection 26
3.2.2. Bacterial Isolates and growth conditions 26
3.2.3. Isolation of bacteria from fermented pickle 26
3.2.4. Hemolytic activity 27
3.2.5. Morphological characterization of the isolates 27
3.2.6. Probiotic characterization 27
3.2.6.1. Acid and bile tolerance 27
3.2.6.2. Salt tolerance assay 27
3.2.6.3. Lysozyme tolerance 28
3.2.6.4. Cholesterol Removal 28
3.2.6.5. Antimicrobial activity 28
3.2.6.6. Antibiotic susceptibility test 28
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3.2.6.7. β-Galactosidase activity 29

3.2.6.8. Screening for exopolysaccharide (EPS) 29
production
3.2.6.9. Bacterial adhesion to hydrocarbons (BATH 29
assay)
3.2.6.10. Auto – aggregation 29
3.2.6.11. Adhesion to human adenocarcinoma cell 30
line HT-29
3.2.7. Molecular characterization of the isolates 30
3.2.7.1. Amplification of 16S rDNA of selected 30
isolates
3.2.7.2. DNA Sequencing and phylogenetic analysis 30
3.2.8. Genetic Diversity of Bacterial Isolates 31
3.2.8.1. Randomly Amplified Polymorphic DNA 31
(RAPD)-Polymerase Chain Reaction
3.2.8.2. Analysis of RAPD band 31
3.2.9. Statistical analysis 32
3.3. Results and Discussion 32
3.3.1. Hemolytic activity of isolates 32
3.3.2. Morphological and biochemical
Characterization 33
3.3.3. Probiotic characterization 34
3.3.3.1. Acid and bile tolerance 34
3.3.3.2. Salt tolerance assay 35
3.3.3.3. Lysozyme tolerance 35
3.3.3.4. Cholesterol-Removal Ability 36
3.3.3.5. Antimicrobial activity 38
3.3.3.6. Antibiotic resistance 38
3.3.3.7. β -Galactosidase activity 39
3.3.3.8. Exopolysaccharide production 40
3.3.3.9. Bacterial adhesion to hydrocarbons (BATH 40
assay)
3.3.3.10. Auto-aggregation 41
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3.3.3.11. Adhesion to human adenocarcinoma cell

line HT-29 42
3.3.4. Species identification by 16S rDNA sequencing 43
3.3.5. RAPD- Band Analysis 44
3.3.6. Principal component analysis 47
3.4. Conclusions 49
Chapter 4: Functional characterization of selected isolates from
fermented mango pickle 50-69
4.1 Introduction 50
4.2. Materials and Methods 51
4.2.1. Bacterial isolates and growth conditions 51
4.2.2. Cell culture 51
4.2.3. Preparation of cell-free supernatants (CFS), intact-
cells (IC) and intracellular cell-free extracts (CFE) 52
4.2.4. Antioxidative properties 52
4.2.4.1. DPPH•-(α, α-diphenyl-b-picrylhydrazyl)
scavenging activity 52
4.2.4.2. ABTS•+- Scavenging activity 52
4.2.4.3. Hydroxyl-Radical OH•− Scavenging
activity 53
4.2.4.4. Linoleic acid peroxidation inhibition
capacity 53
4.2.4.5. Reducing-Activity 53
4.2.4.6. Superoxide Anion Radical (O 2•−)
Scavenging Activity 54
4.2.4.7. Plasma Lipid Peroxidation 54
4.2.5. Antidiabetic property 54
4.2.5.1. α -amylase inhibition assay 54
4.2.5.2. α-glucosidase inhibitory activity 55

4.2.6. Tyrosinase Inhibitory activity 55

4.2.7. Anticancer Activity 55
4.2.7.1. Cytotoxic assessment using (3-(4,5- dime
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thylthazolk-2-yl)-2,5-diphenyl tetrazolium
bromide) MTT assay 55
4.2.7.2. Crude ethyl acetate extracts preparation 56
4.2.7.3. Dual staining 56
4.2.7.4. DNA fragmentation assay 56
4.2.8. Statistical Analysis 56
4.3. Results and Discussion 57
4.3.1. Antioxidant activity of probiotic isolates 57
4.3.1.1. DPPH• scavenging activity 57
4.3.1.2. ABTS •+ radical cation scavenging ability
of isolates 57
4.3.1.3. Scavenging of Hydroxyl-Radicals 58
4.3.1.4. Linoleic-acid peroxidation inhibitory
activity 58
4.3.1.5. Reducing activity 58
4.3.1.6. Superoxide anion radical scavenging
activity 59
4.3.1.7. Inhibition of plasma lipid peroxidation 59
4.3.2. Antidiabetic and tyrosinase inhibitory effect of
isolates 62
4.3.2.1. α-amylase and α-glucosidase inhibition
activity 62
4.3.2.2. Tyrosinase inhibitory activity 62
4.3.3. Anticancer activity of bacterial isolates 64
4.3.3.1. Cytotoxicity of bacterial isolates 64
4.3.3.2. Morphological observations and DNA
fragmentation assay 67
4.4. Conclusions 69
Chapter 5: Technological and safety assessments of selected isolates
from fermented mango pickle 70-85
5.1 Introduction 70
5.2. Materials and Methods 71
5.2.1. Bacterial isolates and growth conditions 71
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5.2.2. Technological Properties 71

5.2.2.1. Carbohydrate fermentation and gas
production 71
5.2.2.2. Heat tolerance 72
5.2.2.3. Proteolytic activity 72
5.2.2.4. Amylolytic activity 72
5.2.2.5. Lipolytic activity 72
5.2.2.6. Autolysis activity 72
5.2.3. Safety investigation 73
5.2.3.1. Hydrogen Peroxide Production 73
5.2.3.2. Production of histamine 73
5.2.3.3. DNase activity 73
5.2.3.4. PCR detection of virulence factor genes in
bacterial isolates 73
5.2.4. Statistical Analysis 75
5.3. Results and Discussion 75
5.3.1. Technological Properties 75
5.3.1.1. Carbohydrate fermentation test 75
5.3.1.2. Assay of bacterial heat resistance 76
5.3.1.3. Proteolytic Amylolytic and Lipolytic
activity 76
5.3.1.4. Autolysis activity 80
5.3.2. Safety investigation 80
5.3.2.1. Hydrogen Peroxide production 80
5.3.2.2. Production of histamine and DNase
activity 81
5.3.2.3. Detection of virulence factor genes among
test isolates 81
5.4. Conclusions 85
Chapter 6: In situ evaluation of probiotic candidate isolates on
physicochemical properties of gamma irradiated mango
pickle 86-99
6.1 Introduction 86
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6.2. Materials and Methods 87

6.2.1. Mango pickle samples 87
6.2.2. Sterilization of fermented mango pickle sample by
gamma-irradiation 87
6.2.3. Bacterial isolates and growth conditions 87
6.2.4. Preparation and inoculation of probiotic test isolates
into mango pickle samples 87
6.2.5. pH 88
6.2.6. Water activity 88
6.2.7. Determination of fat content 88
6.2.8. Determination of the acidity of the extracted fat 89
6.2.9. Determination of protein by Kjeldahl method 89
6.2.10. Determination of Ash 90
6.2.11. Determination of moisture content 90
6.2.12. Enumeration of probiotic bacteria 90
6.2.13 Statistical Analysis 91
6.3. Results and Discussion 91
6.3.1. Sterilization of mango pickle sample by gamma-
irradiation 91
6.3.2. Viability of incorporated bacteria during storage 91
6.3.3. pH 93
6.3.4. Water activity 94
6.3.5. Fat content 95
6.3.6. Titrable acidity (TA) 96
6.3.7. Total protein content 96
6.3.8. Ash content 97
6.3.9. Moisture content 98
6.4. Conclusions 99
Chapter 7: Summary and Conclusion 100-104
Bibliography 105-136
List of Publications 137