evaluation of probiotic properties of lactobacillus reuteri strain using animal model 2012

180 Pages·2012·8.36 MB·English

EVALUATION OF PROBIOTIC PROPERTIES OF
LACTOBACILLUS REUTERI
STRAIN USING
ANIMAL MODEL

THESIS SUBMITTED TO THE
NATIONAL DAIRY RESEARCH INSTITUTE, KARNAL
(DEEMED UNIVERSITY)
IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF

MASTER OF TECHNOLOGY
IN
DAIRY MICROBIOLOGY

BY
KATKAMWAR SNEHAL GOPALRAO
B.Tech.(Dairy Technology)

DIVISION OF DAIRY MICROBIOLOGY
NATIONAL DAIRY RESEARCH INSTITUTE
(DEEMED UNIVERSITY)
KARNAL - 132001 (HARYANA), INDIA
2012

Regn. No. 2011006
Dedicated
To
My Lovely Family
and Guide

DAIRY MICROBIOLOGY DIVISION
NATIONAL DAIRY RESEARCH INSTITUTE
(DEEMED UNIVERSITY)
KARNAL-132001 (HARYANA), INDIA

Dr. R.K. Malik
Principal Scientist

CERTIFICATE

This is to certify that the thesis entitled, “EVALUATION OF PROBIOTIC
PROPERTIES OF LACTOBACILLUS REUTERI STRAIN USING
ANIMAL MODEL ” submitted by Miss Katkamwar Snehal Gopalrao
towards the partial fulfilment of the award of the degree of Master of
Technology (Dairy Microbiology) of the National Dairy Research Institute
(Deemed University), Karnal (Haryana), India, is a bonafide research work
carried out by her under my supervision, and no part of the thesis has been
submitted for any other degree or diploma.

Dated:
Acknowledgements…..
“We think of the effective teachers we have had over the years with a sense of
recognition, but those who have touched our humanity we remember with a deep
sense of gratitude’.
This was much more than a blessing God bestowed upon me when I was obliged to work under the
guidance of my major advisor Dr. R.K. Malik, Principal Scientist, Dairy Microbiology Division, NDRI,
Karnal, who as a compass activated the magnets of curiosity, knowledge, and wisdom in me. Words fail to
elucidate my profound sense of veneration and indebtness for him for the constant encouragement,
sagacious guidance, and inspiration for making right decisions throughout the study.
I gratefully acknowledge the valuable suggestions given from time to time, by the members of my advisory
committee: Dr. Sunita Grover, Dr. S. K. Tomar, Dr. Latha Sabikhi, and Dr. Suman Kapila. Their
critical evaluation along with prolific ideas and suggestions helped me in the successful execution of my
work.
I also offer my sincere thanks to Dr T. K. Mohanty, Dr A. K Puniya, Dr. Sandip Khokhar, Dr.
Sachidanad De, Dr. R. P. Singh, Dr. Malhotra, Dr. Gyan Singh and other faculty members of the
Division for their unconditional help without any reservation throughout the study.
My sincere thanks are humbly submitted to Dr. A.K.Srivastava, Director, NDRI, Karnal and Dr.
Rameshwar Singh, Head, Dairy Microbiology Division, for providing all the necessary facilities to carry
out the research.
Financial assistance from ICAR in the form of institute fellowship is gratefully acknowledged.
A formal line of appreciation would hardly meet the end of justice to record my gratitude for Purti ji for
his fatherly affection, blessings and the technical assistance. My thanks are also extended to Randhir ji
and Pravin Bhaiya for constant help without any reservation
Words fail to express my deepest sense of gratitude and genuine love to the pillars of my strength,
Santosh sir, T. P. Sir, Diksha, Neha mam, Renuka didi, Satvinder sir, Preeti di, Garima di,
Deepika mam, Geetika di, Neha Tanwar di, and Gurpreet mam for having stood by me for the past
two years during my good and bad times. Their inevitable love, care and critical but positive friendly
suggestions made me a better human being. Special thanks to all of my seniours and juniours. I am also
thankful from the very core of my heart to my friend Bhawna, Vandana, Vaibhao, Prity di, Naren and
Renuka di for their wonderful support, company, friendship and immense help in the final drafting of this
piece of work. My thanks are also extended to Samridhi, Ritu di, Kesha, Prachi, Pranoti, Pritee,
Bulbul di, Sneha for appreciating my achievements, enthusiasm and helping me in all possible ways.
A special note of acknowledgement is presented with courtesy to my Classmates Vaibhao, Narendra,
Shashank, Sukirti, Dewashish, Bhanu, Vandna, Vaishali, Upasana, Ayesha and Bhawna again for
their utmost help, care and concern. I express my heartfelt appreciation and thanks to my best friends
Ashish G, Vinu Dada, Sarang, Nilay, Nishant for their friendly help and required cooperation.
I feel much indebted to technical staff of our Division R. P. Singh sir for availing me the instrument
facility. Thanks are also due to staff of animal house for providing the required facilities.
I honorably express my deepest gratefulness towards my parents and my whole family for their godly
influence upon my life and their countless blessings. I gratefully acknowledge the concern, affection and
understanding of my brothers, which can only be felt and not expressed in words.
Last but not the least, my humble and absolute obeisance to Almighty God who has guided me through all
good and bad times and also for blessing me abundantly. Thanks a lot!!!
Dated: July, 2012

Place: Karnal
ABSTRACT
Probiotics are live organisms which when administered in adequate amount
confers health benefit to host by maintaining gut homeostasis. Probiotics
exerts their beneficial effects beyond gastrointestinal health, as they have
been found to improve certain inflammatory metabolic disorders such as
hypertension, cardiovascular diseases, diabetes etc. there by reducing the
biological risk factors associated with these life-style diseases. In the present
investigation, based on a previous in vitro study in our lab, a total number of
8 Lactobacillus reuteri isolates were selected and reconfirmed as L. reuteri on
the basis of physiological, biochemical and molecular characterization. Most
of the isolate precipitated sodium taurocholate and sodium tauroglycholate
indicating good bile salt hydrolase activity. Molecular identification revelaed
the presence of bsh gene in one isolate i.e. L. reuteri LR 6. LR 6 was also
found to exhibit maximum hydrophobicity for xylene (48.83%) and n-octane
(36.37%). All the isolates exhibited good spectrum of antimicrobial activity
against selected pathogens and have moderate antioxidative activity. In in
vitro cholesterol assimilation studies, LR 9 exhibited maximum cholesterol
assimilation followed by LR 6. Hypocholesterolemic activity of LR 6 was
studied in vivo using rats as an animal model. After 60 days of feeding,
significant reduction in serum cholesterol level was noticed in probiotic fed
group, with reduction in total cholesterol by 33.36 % (79.24mg/dl),
enhancement of HDL cholesterol by 29.30% (46.33mg/dl), reduction in
triglycerides level by 25.25% (59.24mg/dl), reduction in LDL cholesterol by
68% (21.06mg/dl), reduction of VLDL by 25.23% (11.85mg/dl) and
reduction in Atherogenic index by 79.93% (0.45). during immunomodulatory
studies feeding of LR 6 caused significant increase in IgA concentration on
5th day. Maximum decrease in S. typhi counts was noticed in group IV on 8th
day in different organs like liver, spleen and large intestine. The overall
investigation shows that LR 6 can be used to develop functional foods to
control hypercholesterolemia.
र र
| र र र
र | : र , र ,
र , र र र
| र 8
र र | र
र र र र र |
र र र
र |
र र र र 6
| र
र र और | र र
र र र 9 र र र 6
र | र र र 6
| 60 , र
र | 33.36 % ,
29.30% , 68%
, र 25.25% , 25.23% और
79.93% | र र
र र र र 6 5
| 8 र , , और
| र
6 र र
|
CONTENTS

Page
Chapter Title
No.
1.0 INTRODUCTION 1-4
2.0 REVIEW OF LITERATURE 5-36
2.1 Cardiovascular Diseases (CVD) 5
2.2 Cholesterol and Human Health 6
2.3 Background on Probiotics 8
2.3.1 History of Probiotic Organism 8
2.3.2 Definition of probiotics 9
2.4 Selection of Probiotic Strain 11
2.5 Proposed Mechanism of Action of Probiotics 12
2.6 Lactobacilli as Probiotics 13
2.6.1 Lactobacilli – General Characteristics 14
2.7 Suitability of Lactobacilli as Probiotics: 16
Selection Criteria
2.7.1 Acid and Bile Salt Tolerance 16
2.7.2 Antimicrobial Action 17
2.7.3 Adherence Properties 19
2.7.4 BSH Activity of Probiotics 21
2.8 Anticholesterolemic Activity of Probiotic 22
Organisms
2.9 Lactobacillus reuteri as a Probiotics 26
2.9.1 Historical Background of L. reuteri 27
A Morphology 27
B Ecology 27
C Biochemistry 27
2.9.2 Probiotic Attributes of L. reuteri 28
2.9.3 L. reuteri: Safety Aspects 29
2.9.4 Applications of Lactobacillus reuteri 31
2.10 Immunomodulatory Activity of Lactobacillus 32
Reuteri
2.11 Hypocholesterolemic Activity of Lactobacillus 35
Reuteri
3. MATERIALS AND METHODS
3.1 Bacterial Cultures 37
3.2 Maintenance and Propagation of Cultures 38
3.3 Purity and Confirmation of Cultures 39
3.4 Selection of L. Reuteri Isolates 39
3.4.1 Identification and Characterization of 39
Lactobacilli
3.4.1.1 Microscopic examination 39
3.4.1.2 Catalase test 39
3.4.1.3 Physiological 40
characterization
a Growth at 15 and 40
450C
3.4.1.4 Biochemical 40
characterization of
lactobacilli
a Gas from glucose 40
b Argininie hydrolysis 40