Development of bioprocess for generation of antimicrobial and antioxidative peptides from milk ...

173 Pages·2016·14.37 MB·English

DEVELOPMENT OF BIOPROCESS FOR
GENERATION OF ANTIMICROBIAL AND
ANTIOXIDATIVE PEPTIDES FROM MILK PROTEINS
By
SEEMARANA
THESIS SUBMITED TO THE
NATIONAL DArRY RESEARCH INSTITUTE, KARNAL
(DEEMED UNNERSITY)
IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN DAIRYING
(DAIRY CHEMISTRy)
Approved by:
~~ll1
..
P~ J.~'
( ) t RAJESH KUMAR
MAJOR ADVISOR & CHAIRMAN
EXTERNAL EXAMINER
(GUIDE)
Members of Advisory Committee
1. Dr. Bimlesh Mann
Principal Scientist, DC Division
2. Dr. Sumit Arora
Principal Scientist, DC Division
3. Dr. S. K. Tomar
Principal Scientist, OM Division
4. Dr. J. K. Kaushik
Principal Scientist, ABTC Division
5. Dr. A. K Singh
Senior Scientist, DT Division
DIVISION OF DAIRY CHEMISTRY
" ICAR-NATIONAL DAIRY RESEARCH INSTITUTE
(DEEMED UNIVERSITY)
"~rl KARNAL-132001 (HARYANA), INDIA
Dr. RAJESH KUMAR
Principal Scientist
CERTIFICATE
This is to certify that the thesis entitled "DEVELOPMENT OF BIOPROCESS
FOR GENERATION OF ANTIMICROBIAL AND ANTIOXIDATIVE PEPTIDES
FROM MILK PROTEINS" submitted by MS. SEEMA RANA towards the partial
fulfilment of the requirements for the award of the degree of DOCTOR OF
PHILOSOPHY in DAIRY CHEMISTRY of the ICAR-NATIONAL DAIRY
RESEARCH INSTITUTE (DEEMED UNIVERSITY), Kamal (Haryana), India, is a
bonafide research work carried out by him under my supervision and guidance
and no part of the thesis has been submitted for any other degree or diploma .
~
.
\"1
Dated: \ '1 \ \. \ (RAJESH KUMAR)
Major Advisor
(GUIDE)
Website: http://www.ndri.res.in, GRAMS: DAIRY RESEARCH,
FAX: [phone], TELEX: 0396-204, PHONE: [phone](0
Dedicated

To My

Beloved Parents
ACKNOWLEDGEMENT

This dissertation is the pinnacle of life long process of learning and growing
which is a consequence of patience, hard work, guidance, assistance and caring. There
were highs and lows during these last four years of my doctoral research endeavour,
when I was shaped, guided and rejuvenated by valuable ideas, criticism and suggestions
of a number of individuals. It was all dark in beginning, but dawn was brought in my
life by all my loved and respected ones, who helped me in immeasurable ways to not
only complete this thesis, but also amass many great and unforgettable memories and
here I hope that I have remembered you all.
First of all, I sincerely acknowledge my gratitude to Almighty God for his
compassion and bountiful of blessings, which made me to see this wonderful moment.
A formal statement of acknowledgement will hardly meet the ends of justice in
the form of expression of my deeply felt sincere and alligent gratitude to the major
advisor and chairman of my advisory committee Dr. Rajesh Kumar, Principal Scientist,
Dairy Chemistry Division, NDRI for his scholastic guidance, everlasting affection,
unstinted encouragement, keen interest, constructive criticism and untiring and help
extended to me throughout my research work. I also wish to thank him for his inspiring
guidance, for the precious time he has devoted for discussion. I hope my words of
thanks will find a small place on his busy desk.
I reverently and honestly acknowledge by obligation to members of my advisory
committee: Dr. (Mrs). Bimlesh Mann, Principal Scientist (DC Division). Dr. Sumit Arora,
Principal Scientist (DC Division), Dr. S.K.Tomar, Principal Scientist (DM Division), Dr.
Jai K Kaushik, Principal Scientist (Animal Biotechnology Centre) and Dr. A.K. Singh,
Senior Scientist (DT Division) for their useful suggestions, constructive criticism as well
as cordial assistance during the course of my investigation.
I am indeed highly indebted to Dr. A.K. Srivastava, Director, NDRI and Dr. G. R,
Patil, Joint Director (Academics) for providing me requisite infrastructure facilities that
enabled incessant compilation of this project.
I owe a profound sense of gratitude to Dr. (Mrs.) Bimlesh Mann, Head, Dairy
Chemistry Division, NDRI, Karnal, for providing me all the necessary infrastructural
facilities for carrying out this work with great ease and precision.
I am deeply indebted to all the Scientists of the Dairy Chemistry Division, for
their valuable suggestions, sincere encouragement, good wishes and cordial assistance
in different phases of my research work.
I am sincerely thankfull to all the members of NAIP for fundings and their
support for successful completion of research work.
I thank Mr. P.C.Singh, Technical Officers, Dairy Chemistry Division for his
valuable help during the study.
I would like to thank Shakuntala mam, Deepak ji and other administrative staff
for their invaluable co-operation throughout my study.
My deep sense of gratitude and warm regards to Kulvinder ji, Krishna ji,
chandrapal ji, Nafe singh ji, Jagath ji, Balavant ji for their valuable help and all possible
co-operation extended during the course of this study. A special thanks to M.S.Yadav ji
with whome I spent most of the time in the lab without whose moral support and help
this piece of work would not have been successful.
I express my heartful thanks to my classmates Ms. Neelima, Bhawna, Anamika
and my juniors Arvind, Kanta for their kind co-operation and help. Moments spend
with my dear juniors Richa, Priyanka, Anu, Meenakshi, Athira Rita, Dinesh, Kanta will
always be there in my fond memories.
Life is a journey. During this journey we come across many but few of them
leave a strong impression on us because of their rectitude and grandeur. In the very
outset, I feel over welcomed to thank Chaudhary family, Ms. Deepkia, Aunty, Uncle and
vicky whose moral support made easy to accomplish this golden piece of work.
My target cannot be accomplished without touching the feet of my mother, who
is my mentor, my willpower and inspiration. She provided me with the comfort zone by
new found optimism through dismantled barriers in my life.
But, no one deserves more thanks than my family, whose life-long love and
support, encouragement, patience and belief in me ultimately made this Ph.D possible.
Their silent prayers, aesthetic love and affection, support and steel belief in my
capabilities have enabled me to make this endeavor a successful one. Unending support
and moral boosting from my loving brother and sister Mohit and Anu is always
remembered through out my life.
My thanks are due to my new family, beloved parents, Balwinder ji, Pooja di,
Devinder bhaiya and Manvi for their moral support in thesis writing.
Financial assistance from National Dairy Research Institute in the form of
Institutional fellowship is duly acknowledged.

June 2014 (SEEMA RANA)
nw/k ls jksxk.kqjks/khs vkSj izfrvksfDldkjd isIVkbMl ds mRiknu ds fy;s ck;ksizkslSl dk fodkl

lkjka’k

X;kjg ySDVkscSflyl miHksn lhbZih xfrfof/k ds fy;s ij[ks x;s vkSj MRS ehfM;k dh
rqyuk esa MDM ehfM;k esa xfrfof/k (0.03-1.30units/mg) vf/kd ikbZ xbZA lhbZih xfrfof/k eykbZ
fudkyk nw/k esa nl xq.kk vf/kd (0.22-16units/mg) ikbZ xbZA lkr miHksnksa esa xSj jksxtud bZ-
dksykbZ ds fo:/k fu"ks/k tksu 10&20 fe0eh0 vkSj izfrvksfDldkjd (0.15-0.55 mM TEAC)
xfrfof/k ikbZ xbZA pkj miHksn ,y- ,flMksfQyl ,u- lh- Mh- lh- 14] ,y- Msyczdq h ,u- lh- Mh-
lh- 09] ,y- dstkbZ f'kjksVk] ,y- jgeuksll C6 isIVkbM mRiknu ds fy;s pqus x;sA fd.ou ds
izHkko ds vuqdwyu ds fy;s m"ek;u vof/k (12 ls 72 ?k.Vs), ek=k (1 ls 3 izfr’kr) vkSj
m"ek;u rkieku (37 ls 42 fMxzhs) dk ewY;kadu fd;k x;k FkkA lsy fodkl nj vf/kdre ,y-
Msycqdh ,u- lh- Mh- lh- 09 esa Log CFU/ml(9.5-10) vkSj izksVksyhfll vf/kdre (12 ls 27 mM
Leucine), ,y- ,flMksfQyl ,u- lh- Mh- lh- 14 (5-4&11- mM Leucine), ,y- jgeuksll C6 (2.2-
9.3 mM Leucine), ,y dstkbZ f’kjksVk (2.4-8 mM Leucine) esa ik;k x;kA cSp fd.ou ds vuqdwy
ifjfLFkfr;ksa ds ifj.kkeLo:i jksxk.kqjks/kh xfrfof/k (Log N/N eku) ,y- ,flMksfQYl ,u- lh- Mh-
0 f
lh- 14 esa 0.55, ,y- jgeuksle C6 e sa 0.49, ,y-Msyczqdh ,u-lh- Mh- lh- 09 esa 0.35 vkSj ,y-
dStkbZ f’kjksVk esa 0.25 ikbZ xbZA izfr vkksfDldkjd xfrfof/k ,y- ,flMksfQyl ,u-lh-Mh- lh- 14
vkSj ,y MSyczqdh ,u-lh- Mh-lh- 09 esa 3.8-4 µM TEAC/µg isIVkbM vkSj ,y- jgeuksll C6, ,y
dstkbZ f’kjksVk esa 2.2-3 µM TEAC/µg isIVkbM ikbZ xbZA ,y- ,flMksfQyl ,u- lh- Mh- lh- 14 d s
vkj- ih- ,p- ih- ,y lh- izksQkbZy esa 12 ihd ikbZ xbZA mPp jksxk.kq jks/kh xfrfof/k (Log N/N
0 f
eku) ihd QzSD’kal 2(1.5), 3(1.1) vkSj 8(1.7) esa vkSj MIC 38.3-48.3 µg/ml ikbZ xbaaaaA izfrvksfDldkjd
xfrfof/k 10-18µM TEAC/µg isIVkbM vkSj ihd 7 ds fy;s mPp ikbZ xbZA LC-MS ds ek/;e ls
51 isIVkbM vuqdze.k ik;s x;s ftuesa ls 16 isIVkbMl vuqdze mPp ghbksQksfcfdR; (35-40%) ds
Fks vkSj ukS isIVkbMl 1,+2 pktZ ds FksA l=g isIVkbZM igys fjiksVZ jksxk.kqjks/kh isIVkbM] β-CN f(199-
209) GPVRGPFPIIV vkSj αs1-CN f(24-34) FVAPFPEVFGK vkSj izfrvksfDldkjd isIVkbM β-CN
f(194-206) QEPVLGPVRGPFP f(17-24) NENLLRFF ds lkFk laHkkfor lk>k dj jgs gSA ,y-
jgeuksll dh mPpre Log N /N eku ihd QSzD’kal 10 (1.6), 6 (1.5) vkSj 3 (1.2) esa vkSj MIC 101-
0 f
390 µg/ml ikbZ xbZA izfrvksfDldkjd xfrfof/k 15-45µM TEAC/µg isIVkbM vkSj mPpre ihd QSzD’kal
9(43.9µM TEAC/µg isIVkbM) esa ikbZ xbZA X;kjg isIVkbM igys fjiksVZ jksxk.kqjks/kh]
SFSDIPNPIGSENSEK vkSj YQEPVLGPVRGPFPII ds lkFk laHkkfor lk>k dj jgs gS] ftuesa
ghMksQksfcfdR; (36-44%) ik;k x;kA ,y- Msyczqdh ,u-lh-Mh- lh 09 vkSj ,y- dstkbZ f’kjksVk ds
vkj ih gIyl izksQkbZy 11 vkSj 15 ihd Hkkxks esa ikbZ xbZA ,y Msyczzqdh ,u-lh-Mh- lh 09 ds
4 QSD’kal esa jksxk.kqjks/kh xfrfof/k 0.3-1.2 vkSj mPpre QSD’kla 1 (1.2) esa vkSj MIC 62.7-
270µg/ml ikbZ xbzZA izfrvksfDldkjd xfrfof/k ,y- Msyczqdh ,u-lh-Mh- lh 09 vkSj ,y- dstkbZ
f’kjksVk ds isIVkbM esa 9-50µM TEAC/µg isIVkbM vkSj 20-100µM TEAC/µg isIVkbM ikbZ xbZA
ABSTRACT

Eleven Lactobacillus strains were screened for CEP activity and was higher in Minimal
Defined Media (0.03-1.30Units/mg) as compared to MRS. CEP activity was observed to
be tenfold higher in skim milk (0.22-16 Units/ mg). Selected seven strains showed
antimicrobial activity against non-pathogenic E. coli showing zone of inhibition (10-
20mm) and antioxidant activity (0.15-0.55mM TEAC). Four strains, L. acidophilus
NCDC 14, L. delbrueckii NCDC 09, L. casei Shirota and L. rhamnosus C6 were selected
for optimization of conditions for peptide production. For optimization of fermentation
conditions effect of incubation period (12-72hrs), inoculation volume (1-3%) and
incubation temperature (37 & 42°C) was assessed. Cell growth rate (Log CFU/ml, 9.5-10)
was maximum for L. delbrueckii NCDC 09 leading to drop of pH to 3.9 within 12 hrs and
maximum extent of proteolysis (10-27mM Leucine) followed by L. acidophilus NCDC
14, (5.4-11.2mM Leucine), L. rhamnosus C6 (2.2-9.3mM Leucine) being lowest for L.
casei Shirota (2.4-8 mM Leucine). The batch fermentation under optimized conditions
resulted in highest antimicrobial activity (Log N /N value) for L. acidophilus NCDC
0 f
(0.55) followed by L. rhamnosus C6 (0.49), L. delbrueckii NCDC 09 (0.35) and L. casei
Shirota (0.25). Antioxidant activity was higher for L. acidophilus NCDC 14, L. casei
Shirota (3.8-4 µM TEAC/µg peptide) followed by L. delbrueckii NCDC 09. L. rhamnosus
C6, (2.2-3 µM TEAC/µg peptide). RP-HPLC profile of L. acidophilus NCDC 14 resolved
into 12 Peak fractions. Antimicrobial activity (Log N /Nf value) was higher in 2 (1.5), 3
0
(1.1) and 8 (1.7) peak fractions for which minimum inhibitory concentration ranged from
38.3-48.3 µg/ml and antioxidant activity from 10-18µM TEAC/µg peptide. Sequencing
through LC-MS identified 51 peptide sequences among which 16 novel peptides
sequence showed character related high hydrophobicity (35-40%). Nine peptides were
cationic with +1 to +2 charge. Seventeen peptide sequence shared sequence homology
with previously reported antimicrobial, antioxidant and ace-inhibitory peptides among
which β-CN f(199-209) GPVRGPFPIIV and αs -CN f(24-34) FVAPFPEVFGK are
1
potential antimicrobial peptides and β-CN f(194-206) QEPVLGPVRGPFP and f(17-24)
NENLLRFF are potential antioxidant peptides reported in literature previously. Similarly
Log N /N value of eleven peaks fractions of L. rhamnosus C6 ranged from (0.4-1.5)
0 f
being higher in 10 (1.6), 6 (1.5) and 3 (1.2) peaks and MIC ranged from 101-390µg/ml.
Antioxidant activity ranged from 15-45µM TEAC/µg peptide being highest in 9
(43.9µM TEAC/µg peptide) peak fraction. Thirteen novel peptides sequence were
obtained with high hydrophobicity (36-44%). Twenty peptide sequences shared sequence
homology with previously reported antimicrobial and antioxidant peptides among which
SFSDIPNPIGSENSEK and YQEPVLGPVRGPFPII are known antimicrobial peptides as
Caseicin C and Casecidin-17 respectively. For L. delbrueckii NCDC 09 and L. casei
Shirota 11and 15 Peak fractions were obtained respectively. Log N /N ranged from (0.3-
0 f
1.2) for 4 fractions was higher in fraction 1 (1.2) and MIC ranged from 62.7-270µg/ml.
Antioxidant activity ranged from 9-50µM TEAC/µg peptide and 20-100µM TEAC/µg
peptide for L. delbrueckii and L. casei Shirota respectively.
CONTENTS
Chapter Title Page No.
1.0 INTRODUCTION … 1-3
2.0 REVIEW OF LITERATURE … 4-38
2.1 Bioactive Peptides … 4
2.2 Milk proteins: Biologically active peptides source … 4
2.3 Proteolytic system of LAB … 6
2.4 Production of bioactive peptides … 9
2.4.1 Production of peptides by digestive enzymes … 9
2.3 2.4.2 Liberation of Bioactive peptides via fermentation by … 11
Lactic acid bacteria
2.5 Antimicrobial peptides … 15
2.5.1 Antimicrobial peptides from milk proteins by … 16
digestive proteases
2.5.1.1Casein derived antimicrobial peptides … 18
2.5.1.2 Whey proteins derived antimicrobial peptides … 23
2.5.2 Antimicrobial peptides via fermentation by Lactic … 26
acid bacteria
2.6 Antioxidative peptides … 27
2.6.1 Antioxidative peptides from milk proteins by … 30
digestive enzymes
2.6.1.1 Casein derived peptides … 30
2.6.2.2 Whey protein derived peptides … 34
2.6.2 Antioxidative peptides via milk fermentation by … 37
Lactic acid bacteria
3.0 MATERIALS AND METHODS … 39-54
3.1 Characterization of lactobacilli
3.1.1 Microbial cultures … 39
3.1.2 Morphological characterization … 39
3.1.3 Maintenance and preservation of cultures … 39
3.2 Culturing of lactobacilli … 40
3.3 Culturing of Test organism … 40