studies on probiotics and antimicrobial properties of lactic acid bacteria isolated from marine fish

224 Pages·2011·4.31 MB·English

STUDIES ON PROBIOTICS AND ANTIMICROBIAL
PROPERTIES OF LACTIC ACID BACTERIA ISOLATED FROM
MARINE FISH, SHRIMP AND SOUTH INDIAN FERMENTED
FOODS

Thesis Submitted to Pondicherry University for the Degree of
DOCTOR OF PHILOSOPHY
By
R. SATISH KUMAR, M.Sc.,

Department of Biotechnology
School of Life Sciences
Pondicherry University
Pondicherry 605014
INDIA
December 2011
PONDICHERRY UNIVERSITY
DEPARTMENT OF BIOTECHNOLOGY
SCHOOL OF LIFE SCIENCES
PONDICHERRY-605014
INDIA

DR. V. ARUL
Associate Professor
CERTIFICATE
Certified that this thesis entitled “Studies on probiotics and antimicrobial properties of lactic
acid bacteria isolated from marine fish, shrimp, and South Indian fermented foods” is a
record of research work done by the candidate Mr. R. Satish Kumar during the period of his
study in the Department of Biotechnology, School of Life Sciences, Pondicherry University,
under my supervision and that it has not previously formed the basis of the award of any degree,
diploma, associateship or fellowship.

Pondicherry
Date: (V. ARUL)

Phone: [phone] (Off.) Fax: [phone]
91-413-2357492 (Res.) Email: [email protected]
DECLARATION

I hereby declare that the work presented in this thesis has been carried out by me under the
guidance of Dr. V. Arul, Associate Professor, Department of Biotechnology, School of Life
Sciences, Pondicherry University, Pondicherry, and this work has not been submitted elsewhere
for any other degree.

Pondicherry

Date: R. SATISH KUMAR
DEDICATED TO MY
BELOVED PARENTS
CONTENTS
S.NO. CHAPTERS PAGE NO.
1 GENERAL INTRODUCTION 1
2 REVIEW OF LITERATURE 5
3 MATERIALS AND METHODS 29
4 ISOLATION AND SCREENING OF LACTIC ACID BACTERIA 59
FROM SOUTH INDIAN FERMENTED FOODS
5 PURIFICATION AND CHARACTERIZATION OF BACTERIOCIN 71
PRODUCED BY STREPTOCOCCUS PHOCAE PI80
6 PURIFICATION AND CHARACTERIZATION OF BACTERIOCIN 88
PRODUCED BY ENTEROCOCCUS FAECIUM MC13
7 PURIFICATION AND CHARACTERIZATION OF BACTERIOCIN 101
PRODUCED BY LACTOBACILLUS PLANTARUM AS1
8 IN VITRO CHARACTERIZATION OF LACTIC ACID BACTERIA 114
STRAINS FOR PROBIOTIC CHARACTERISTICS
9 LACTOBACILLUS PLANTARUM AS1 BINDS TO CULTURED 126
HUMAN INTESTINAL CELL LINE HT-29 AND INHIBITS CELL
ATTACHMENT BY ENTEROVIRULENT BACTERIUM VIBRIO
PARAHAEMOLYTICUS
10 LACTOBACILLUS PLANTARUM AS1 ISOLATED FROM SOUTH 140
INDIAN FERMENTED FOOD KALLAPPAM SUPPRESS 1, 2-
DIMETHYL HYDRAZINE (DMH) INDUCED COLORECTAL
CANCER IN MALE WISTAR RATS
11 SUMMARY 158
12 REFERENCES 161
13 PUBLICATIONS 200
ACKNOWLEDGEMENTS
Foremost, I would like to express my heartfelt gratitude to my supervisor Dr. V. Arul, Associate
Professor, Department of Biotechnology for the continuous support of my Ph.D., study and
research, for his patience, motivation, enthusiasm, and immense knowledge. His guidance helped
me throughout my research and writing thesis.
Besides my supervisor, I would like to thank my doctoral committee members: Prof. Priya
Davidar and Dr. N. Arumugham, for their encouragement, insightful comments, and hard
questions.
I express my sincere gratitude to Prof. N. Sakthivel, Head, Department of Biotechnology for
extending lab facilities and valuable suggestions.
My special thanks go to Dr. C. Thirunavukarasu, Assistant Professor, Department of
Biochemistry and Molecular Biology, for his suggestions and guidance in animal studies.
I am thankful to Prof. S. Jayachandran, Dr. Sudhakar, Dr. Hanna Rachel Vasanthi, Dr.
Prashant, Dr. Arun Kumar Dhayalan, Dr. Venkateswara Sarma, and Mr. V.
Balasubramanian who have been influential in all my academic pursuits.
I thank my fellow labmates: Dr. P. Kanmani, Mr. N. Yuvaraj, Mr. K.A. Paari, Mr. V.
Pattukumar and Mr. Venkatesh, for the stimulating discussions, immense co-operation,
emotional support, and for all the fun we had in the last four years.
I genuinely thank my seniors, Dr. A. Gopalakannan, Dr. S. Isaac Kirubakaran, Dr.
Krishnaveni, Dr. P. Ravindra Naik, Dr. S. Vaithinathan, Dr. N. Badrinarayanan and
colleagues Mr. G. Raman, Mr. Jean Cletus, Mr. Kennedy, Mr. Saranathan, Mr. Abhijith,
Mr. Aezaj, Mr. Wasim, Mr. Abid, Mr. Anand, Mr. Suresh, Mr. Manoj, Ms. M. Revathi,
Ms. P. Lalitha, Ms. Moushmi Priya, Ms. Asha, Ms. Pathma, Ms. Veena, Ms. Gokulapriya,
and Ms. Sonia for their immense co-operation and valuable support.
I also thank our office staffs, Mr. Ramalingam, Mr. C. Balakrishnan, Mr. Vadivel, Ms.
Sarala, Ms. Vinothini, Mr. Meiappan, and Ms. Muthammal for their co-operation and help.
I gratefully acknowledge the monetary support given by Indian Council of Medical Research,
New Delhi in the form of Junior/Senior Research Fellowship and Department of
Biotechnology, New Delhi for project funding.
I would like to thank Central Instrumentation Facility (CIF), Pondicherry University for
providing assistance in using necessary instruments.
I wish to thank my brother Mr. Senthil Kumar, sisters Ms. Kavita, Ms. Krishna Veni and their
kids Harshitha, Siddhanth for providing a loving environment for me.
I wish to thank my parents, Ms. Subbulakshmi Ramraj and Mr. K.S. Ramraj for showering
with love and care throughout my life. To them I dedicate this thesis.
Finally, I would like to thank everybody who was important to the successful realization of
thesis, as well as expressing my apology that I could not mention personally one by one.

Date: R. Satish Kumar
LIST OF ABBREVIATIONS
CFU Colony Forming Units
AU Arbitrary Unit
CFCS Cell Free Culture Supernatant
cm Centimeter
DNA Deoxyribose Nucleic Acid
dNTP 2’ deoxynucleotide 5’ triphosphate
EDTA Ethylene Diamine Tetra Acetic acid
MRS de Man Rogosa and Sharpe
SDS Sodium Dodecyl Sulfate
PBS Phosphate Buffered Saline
rpm Revolution Per Minute
TEMED N, N , N - Tetramethylethylenediamine
APS Ammonium Per Sulphate
PCR Polymerase Chain Reaction
UV Ultra Violet
bp Base pairs
g/l Gram per liter
h Hours
kDa Kilo Dalton
V Volt
μl Microliter
μM Micromolar
mg/l Milligram per liter
min Minutes
ml Milliliter
mM Millimolar
mm Millimeter
nm Nanometer
OD Optical Density
w/v Weight per volume
v/v Volume per volume
N Normality
s Second
CHAPTER 1
GENERAL INTRODUCTION