ISOLATION AND CHARACTERIZATION OF A NATIVE ISOLATE OF LEUCONOSTOC FOR FUNCTIONAL ATTRIBUTES A Thesis Submitted to University of Mysore, Mysore for the Degree of DOCTOR OF PHILOSOPHY IN MICROBIOLOGY By SHOBHA RANI. P Under the guidance of Dr. RENU AGRAWAL DEPARTMENT OF FOOD MICROBIOLOGY CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE-20 2008 ACKNOWLEDGEMENT With great reverence, I extent my deep sense of gratitude to my respected guide and teacher Dr. Renu Agrawal for her advice and able guidance, constant supervision, co-operation, inspiration, constructive criticism and novel suggestions throughout the investigation without whose initiative and enthusiasm this study would not be completed. I remain indebted to her for being there for me as a personnel supporter and career builder. My thanks are due to Dr. Prakash,V., Director, CFTRI for providing the necessary facilities to carryout research work in the institute. I take this opportunity to thank Dr. Umesh Kumar S, Head, Food Microbiology Department, CFTRI for his support and encouragement during the course of the study. I sincerely thank Dr. Baskaran, V., Department of Biochemistry and Nutrition, Dr. Manjunath, M.N., Food Safety and Analytical Quality Control Laboratory and Dr. Ramesh, B.S., TTBD Department for their suggestions and advice. I am also thankful to staff of animal house for their assistance and special thanks to Dr. Saibaba, P., for his help and cooperation during the work. I will always cherish my memorable time that I have spent in the lab. I wish to express my sincere thanks to all my Scientists and fellow colleagues in the department both past and present for cooperation and ambient atmosphere all through my research period. Prathibha. DV deserves special thanks for having been there all the time as a great source of moral support. I had an opportunity to interact with many research fellows and Scientists from various department and they always assisted me every time. I thank all of them. It is my sincere duty to acknowledge the help rendered by the staff of Central Instrumentation Facilities, computer center, library and administration. University of Mysore is greatly acknowledged for the financial support in the form of fellowship. Finally I am deeply indebted to my parents and brothers who were a constant source of support and encouragement in this endeavor. Date: Place: Shobha Rani. P CONTENTS Page No. Acknowledgement Abbreviations List of tables List of figures Review of literature 1 Aim and Scope of present investigation 40 Chapter - 1 Isolation and screening of lactic acid bacteria from 42 milk and milk products Chapter – 2 Probiotic functional properties of culture isolate 66 Chapter – 3 Leuconostoc as a source for β-galactosidase enzyme 104 Chapter – 4 Enhancement of culture shelf life on storage 125 Chapter – 5 Functional food with Leuconostoc: a native isolate 143 Chapter – 6 Preservation of fermented milk over shelf storage 160 Chapter – 7 In-vivo studies using Leuconostoc for functional 183 attributes Summary and Conclusions 204 Bibliography 205 Outcome of the present work. LIST OF TABLES Table Page Title No. No. 1 Characteristic features of lactic acid bacteria 4 2 Antagonistic activity caused by lactic acid bacteria 13 3 Bacteriocins of lactic acid bacteria 14 4 The protective effect of LAB in human and animal health 19 5 Action of probiotic culture 20 6 Presumptive identification of Leuconostoc by phenotypical 28 tests 7 Species included in the genus Leuconostoc 29 8 Fermented foods that involve Leuconostoc 32 9 Incidence of lactose intolerance in different population 35 group around the world 10 Diagnostic tests for lactose intolerance 36 11 List of organisms that produce lactase 38 1.1 Composition of MRS media (deMann Rogosa Sharpe 45 Media) 1.2 Preliminary characterization of isolated cultures 52 1.3 Adaptation of isolated cultures to low pH and high bile salt 55 mix concentration 1.4 Physiology and biochemical identification of culture 58 isolates 2.1 Antimicrobial activity of L. mesenteroides (PLsr-1 and 79 (W) Lsr-1 ) in comparison with standard cultures (W) 2.2 Antimicrobial activity of heat killed and neutralized culture 81 supernatant of PLsr-1 (W) 2.3 Antibiotic susceptibility of PLsr-1 83 (W) 2.4 Minimum inhibitory concentration of antibiotics tested 84 against probiotic L. mesenteroides (PLsr-1 ) (W) 2.5 Inhibition of ascorbate autooxidation by intracellular cell 86 free extract of L. mesenteroides (PLsr-1 and Lsr-1 ) (W) (W) 2.6 Ferrous ion chelating ability of intracellular cell free extract 87 of PLsr-1 and Lsr-1 (W) (W) 2.7 DPPH scavenging activity of intracellular cell free extract 87 2.8 Cholesterol assimilation ability of PLsr-1 88 (W) 2.9 Cell hydrophobicity to different hydrocarbons 93 2.10 Yield of volatile compounds at different time intervals 98 2.11 Cellular fatty acids composition of PLsr-1 and Lsr-1 101 (W) (W) 3.1 Strain improvement by chemical mutation using EMS 115 3.2 Strain improvement by UV-irradiation 115 3.3 β-galactosidase activity on permeabilization of 116 M7-PLsr-1 (W) 3.4 Effect of lactose (%), temperature(°C) and pH on 119 β-galactosidase activity in M7-PLsr-1 (W) 3.5 Enhancement of β-galactosidase production 121 3.6 Effect of pH on the β-galactosidase activity of 121 M7-PLsr-1 (W) 3.7 Effect of temperature on the β-galactosidase activity of 122 M7-PLsr-1 (W) 4.1 Viability of the culture L. mesenteroides M7-PLsr-1 on 132 (W) freeze drying 4.2 Effect of varying concentration of cryoprotectants on the 133 viable cell count of M7-PLsr-1 on freeze drying (W) 4.3 Effect of cryoprotectant on cell viability of M7-PLsr-1 133 (W) on freeze drying 4.4 Cellular fatty acid profile of freeze dried culture 135 4.5 Antimicrobial activity of freeze dried cells 140 5.1 Chemical and microbial composition of fermented milk 150 beverage 5.2 Effect of adjuvant supplementation on cell viability 152 5.3 Protein content of fermented milk on adjuvant 153 supplementation during storage 5.4 Effect of adjuvant supplementation on titrable acidity of 154 fermented milk during storage 5.5 Effect of adjuvants on total sugar content of fermented milk 155 5.6 Effect of adjuvant supplementation on fat content of 156 fermented milk 5.7 Effect of adjuvant supplementation of soluble mineral 157 content of fermented milk 5.8 Effect of fatty acid composition of fermented milk on 157 adjuvant supplementation 6.1 Characteristics of spoilage bacterial culture 168 6.2 Effect of 2(5H)-furanone on Pseudomonas sp growth in 180 fermented milk 6.3 Preservation of fermented milk with 2(5H)-furanone for 181 longer shelf life 7.1 Toxicity of M7-PLsr-1 in experimental rats 190 (W) 7.2 Glucose and Urea in serum of experimental rats fed with 191 M7-PLsr-1 (W) 7.3 Caecum analysis of experimental rats 193 7.4 β-galactosidase activity in caecum on feeding 194 L. mesenteroides (M7-PLsr-1 ) (W) 7.5 Weight range of experimental rats on probiotic feeding 196 7.6 pH change during probiotic feeding for 90 days 197 7.7 Glucose and urea concentration in serum and urine of M7- 200 PLsr-1 fed rats (W) LIST OF FIGURES Figure Title Page No. No. 1.1 Distribution of isolated cultures from milk and milk 51 products 1.2 Survival of isolated cultures under simulated intestinal 57 conditions 1.3 SEM photograph of selected cultures 58 1.4 PCR product obtained by amplification of 16srDNA 59 1.5 (a) 16srDNA sequence data of Lsr-1 60 (W) (b) 16srDNA sequence data of Lsr-12 (Cu) 1.6 Phylogenetic tree of (A) Lsr-1 : Leuconostoc 61 (W) mesenteroides (B) Lsr-12 : Lactobacillus plantarum (Cu) 1.7 Tolerance of the selected cultures to digestive enzymes 64 1.8 Growth of curve Lsr-1 in MRS broth at 37°C 65 (W) 2.1 Standard graph of cholesterol estimation 72 2.2 Antimicrobial activity of L. mesenteroides (PLsr-1 ) 81 (W) 2.3 Antibiotic susceptibility test of PLsr-1 83 (W) 2.4 Qualitative assay for β-galactosidase activity using ONPG 89 disc 2.5 Effect of carbon source on β-galactosidase activity of 90 PLsr-1 (W) 2.6 Adhesion of L. mesenteroides (PLsr-1 ) to the intestinal 93 (W) epithelium of the rat 2.7 SDS PAGE analysis of S-layer protein of PLsr-1 95 (W) 2.8 Mass spectra of isolated volatile compounds 97 2.9 SDS-PAGE showing the protein pattern of L. 102 mesenteroides (PLsr-1 and Lsr-1 ) (W) (W) 3.1 Standard graph for O-nitrophenol 108 3.2 Standard graph for protein 108 3.3 Effect of sonication on enzyme release 117 3.4 Effect of incubation time on release of enzyme from 117 cellular acetone powder 3.5 Effect of solvents on enzyme release 117 3.6 RSM study to optimize the condition for maximum 120 β-galactosidase activity in M7-PLsr-1 (W) 3.7 SDS PAGE analysis of β-galactosidase enzyme in 121 M7-PLsr-1 (W) 3.8 Effect of substrate concentration 122 3.9 Effect of enzyme concentration 122 3.10 Effect of enzyme inhibitors/ modulators on enzyme 123 activity 3.11 Effect of metal ions on enzyme activity 123 4.1 Comparison of preservative methods for stability of 131 M7-PLsr-1 (W) 4.2 GC profile of cellular fatty acid 136 4.3 Cellular protein profile of M7-PLsr-1 137 (W) 4.4 Storage stability of L. mesenteroides M7-PLsr-1 after 138 (W) freeze drying. (a) Viability at 30°C (b) Viability at 4°C (c) Viability at -20°C 4.5 Tolerance of M7-PLsr-1 (A) to acidic pH 2.0 (B) to 139 (W) high bile salt concentration (4%) 4.6 (a) SEM of L. mesenteroides M7-PLsr-1 before freeze 141 (W) drying in skim milk (b) SEM of L. mesenteroides M7-PLsr-1 after freeze (W) drying in skim milk 5.1 Standard graph for the estimation of total sugars 147 6.1 Standard graph for rhamnose estimation 165 6.2 SEM of Pseudomonas sp 168 6.3 TLC Overlay assay for identification of signal molecule 170 6.4 GC chromatograph of (A) standard HHSL (B) standard 171 BHSL and (C) Pseudomonas sp extract (D) Pseudomonas sp extract spiked with standard HHSL 6.5 GCMS fragmentation pattern of AHL produced by 173 Pseudomonas sp 6.6 Effect of furanone on Pseudomonas sp growth in culture 176 media 6.7 Effect of furanone on rhamnolipid content of 177 Pseudomonas sp 6.8 Inhibition of Pseudomonas sp motility by 2(5H)-furanone 178 6.9 Effect of furanone on exoprotease activity of 179 Pseudomonas sp 7.1 Serum glucose concentration and β-galactosidase activity 192 in caecum of M7-PLsr-1 fed rats (W) 7.2 Effect of L. mesenteroides (M7-PLsr-1 ) on serum 193 (W) cholesterol of experimental rats 7.3 pH change and LAB count in caecum on feeding 194 L. mesenteroides (M7-PLsr-1 ) (W) 7.4 SEM photograph of probiotic fed rat intestine showing 194 adhesion of probiotic culture 7.5 Probiotic influence on E. coli in (A) Caecum, (B) Feces, 198 (C) Large intestine 7.6 SEM photograph showing adhesion of probiotic culture to 199 large intestine (LI) and caecum of experimental rats 7.7 Serum glucose concentration and the enzyme activity in 201 the caecum of experimental rats 7.8 β-galactosidase activity in caecum and small intestine of 202 experimental rats