isolation and characterization of antifungal compounds from lactic acid bacteria

200 Pages·2015·4.16 MB·English

ISOLATION AND CHARACTERIZATION OF
ANTIFUNGAL COMPOUNDS FROM LACTIC ACID
BACTERIA

THESIS SUBMITTED TO
THE UNIVERSITY OF KERALA
FOR THE AWARD OF THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN
BIOTECHNOLOGY
UNDER THE FACULTY OF APPLIED SCIENCE

BY
VARSHA K K

Under the Supervision of Dr. K Madhavan Nampoothiri

Biotechnology Division
CSIR-National Institute for Interdisciplinary Science and Technology
(CSIR-NIIST), Thiruvananthapuram – 695 019
Kerala, India
JULY 2015
Dedicated to my parents and brother
DECLARATION

I hereby declare that the PhD thesis entitled “Isolation and characterization of
antifungal compounds from lactic acid bacteria” is an independent work
carried out by me under the supervision of Dr K Madhavan Nampoothiri,
Biotechnology Division, CSIR-National Institute for Interdisciplinary Science and
Technology, Thiruvananthapuram-695 019, India, and it has not been submitted
anywhere else for any other degree, diploma or title.

Varsha K K
Thiruvananthapuram
National Institute for Interdisciplinary Science
&Technology (NIIST)
Council of Scientific and Industrial Research, CSIR
Trivandrum - 695019, Kerala, India

Dr. K.Madhavan Nampoothiri Tel: +91 - 471-2515366, 2490674; Fax: + 91- 471-2491712, 2490186

Principal Scientist E.mail: [email protected],
[email protected]
Biotechnology Division

CERTIFICATE
This is to certify that the work embodied in the thesis entitled “Isolation and
characterization of antifungal compounds from lactic acid bacteria” is based
on the original research work carried out by Ms.Varsha KK under my supervision
in the Biotechnology Division of CSIR-NIIST, Thiruvananthapuram and that no
part of this work has been submitted previously anywhere for the award of any
degree.

Place: Trivandrum Dr. K. Madhavan Nampoothiri
Date : Research Supervisor
ACKNOWLEDGMENTS

This thesis would not have been possible without the kind support, help,
criticism and encouragement of many individuals and organization. I would like
to extend my sincere thanks to all of them.
First of all I thank Council of Scientific and Industrial Research, New
Delhi for providing me the research fellowship to carry out this study.
With gratitude and pleasure I thank my research supervisor Dr. K.
Madhavan Nampoothiri, for giving me the opportunity to work with his research
group and for allowing me to choose this topic for my research. He always
provided the freedom to work independently and showed support and confidence
during my entire study for which I will always remain grateful to him.
I am deeply indebted to Prof. Ashok Pandey, HOD of our division for
always being supportive and encouraging during my work. His suggestions
helped me to overcome the hard times of my research for which I will stay
thankful to him.
I place on record, my thanks to Director, CSIR-NIIST for providing me
the necessary facilities and infrastructure of the institute for carrying out this
work.
My heartfelt thanks for the helps received for bioactive structure analysis
from Dr. Leena Devendra, without whose support I would never have completed
this work. I gratefully acknowledge her for the encouragement and fruitful
discussions which enabled me to achieve my target.
I deeply appreciate Dr. Priya S and Ms. Shilpa G from Agroprocessing
Division, CSIR-NIIST for the help and guidance provided for cell-line studies that
added much significance to my work.
I thank Dr. N. Ramesh Kumar for carrying out DNA sequencing and also
for his valuable advices and suggestions which inspired me to explore more
domains of my work.
i
I extend my thanks to Dr. Binod Parameswaran, Dr. Rajeev K Sukumaran,
Dr. Muthu Arumugam, Dr. Sindhu R and Mr. Kiran Kumar for their helps,
support and encouragement.
My completion of this work could not have been accomplished without
the support and help of Anusree, Nishant, Kiran, Rajasree and Kuttiraja. I am
deeply indebted to each one of you for sparing your valuable time to find
solutions for the troubles I encountered during my work. The suggestions and
advices provided by them helped me focus on various possibilities to reach my
goal. My sincere thanks to Sneha for the helps received for aminopeptidase
inhibition studies.
I deeply acknowledge the helps received from Dr. Neetha Joseph and Dr.
Venkata Ramana Vemuluri, Microbial culture collection, National Centre for Cell
Science for performing the FAME analysis and DDH experiment.
I thank Mr. Saravana Kumar and Mr. Arun Kumar from Mass
spectrometry and Proteomic core facility of Rajiv Gandhi Centre for
Biotechnology for performing MALDI-TOF and Peptide Mass Fingerprinting
analyses.
I am grateful to the faculty of Analytical facility unit especially Mass and
NMR spectrometry Units, CSIR-NIIST for performing my analyses. I extend my
thanks to Dr. Sinu C R, Chemical Science and Technology for performing the
FTIR analysis.
I sincerely thank my respectful seniors and colleagues Dr. Vipin Gopinath
, Dr. Arya Nandan , Dr. Dhanya Gangadharan, Dr. Shyam Krishna, Dr. Raji, Dr.
Nisha, Dr. Bindumol, Dr. Sai shyam, Dr. Vidya J, Dr. Ushasree M V, Deepthi
Alex, Deepa and Dr. Abraham Mathew for giving support and motivation during
my stay in lab.
I extend my thanks to my colleagues Aravind, Ramya, Divya, Nimisha,
Sabeela, Sajna, Preeti and Lalitha for their help and support at various stages of
my work along with my gratitude to my lab mates Dilna, Vini, Surya, Ram
ii
Kumar, Niyas, Karthik, Soya, Koyna, Vani, Leya, Anju, Dhanya, Sowmya, Seeta,
Rahul and Vivek.
I deeply acknowledge the technical support provided by the technical staff
of our division. I am thankful to Mrs Radhamony, Mrs Sasikala and Mrs
Rajalekshmi for all their support and would like to extend my gratitude to the
administration, library and all other non-scientific staff members of CSIR-NIIST
for the technical guidance.
Finally, to my loving, caring and encouraging family: my deepest
gratitude. It‟s their faith and support that give me the strength and inspiration to
pursue my dreams.

Varsha K K

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CONTENTS
Page No
List of tables xiv
List of figures xv-xviii
List of abbreviations xix-xxii
CHAPTER 1 Intoduction 1-3
1.1 Introduction 1
1.2 Objectives of the study 2
CHAPTER 2 Review of literature 4-36
2.1 Introduction 4
2.2 Lactic acid bacteria 4
2.2.1 Phylogenetic characterization of LAB 6
2.3 LAB as probiotics 7
2.3.1 Probiotic market 10
2.3.2 Health benefits of probiotics 10
2.4 Synbiotics, nutraceuticals and functional foods 14
2.5 Lactic acid bacteria and biopreservation 17
2.5.1 Fungal spoilage of food and feed 18
2.5.2 Control of food spoilage fungi 18
2.5.3 Antifungal LAB 20
iv
2.5.4 Antifungal compounds produced by LAB 21
2.5.4.1 Organic acids 21
2.5.4.2 Proteinaceous compounds 23
2.5.4.3 Low molecular weight compounds 26
2.6 Purification and identification of antifungal 28
compounds
2.7 Application of LAB as protective culture 32
2.8 Conclusion 35
CHAPTER 3 Materials and Methods 37-44
3.1 Materials 37
3.1.1 Chemicals and reagents 37
3.1.2 Microorganisms, cell lines and culture media 38
3.2 General instruments 39
3.3 General methods 39
3.3.1 Methods in general microbiology 39
3.3.1.1 Gram staining 39
3.3.1.2 Microorganisms and cell lines maintenance 39
3.3.1.3 Inoculum preparation 40
3.3.4 Analytical methods 40
3.3.4.1 Bicinchoninic acid (BCA) assay for protein 40
v
estimation
3.3.4.2 Thin layer chromataography 40
3.3.5 Molecular methods 40
3.3.5.1 Extraction of genomic DNA 40
3.3.5.2 Determination of 16S rRNA gene sequence 42
3.3.5.3 Electrophoretic techniques 42
3.3.5.3.1 Agarose gel electrophoresis 42
3.3.5.3.2 Tricine-SDS-PAGE 42
3.3.5.3.2a Coomassie staining 43
3.3.5.3.2b Silver staining 44
3.4 Conclusion 44
CHAPTER 4 Isolation and probiotic characterization of 45-57
antifungal lactic acid bacteria
4.1 Introduction 45
4.2 Materials and methods 46
4.2.1 Microorganisms and growth conditions 46
4.2.2 Isolation of lactic acid bacteria 46
4.2.3 Antifungal activity assay 46
4.2.4 Identification of selected strains by 16S rDNA 47
sequencing
4.2.5 Probiotic characterization of selected antifungal 47
LAB isolates
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