CHARACTERIZATION OF SQUID PROTEIN HYDROLYSATES FOR ANTIOXIDATIVE ...

101 Pages·2017·2.23 MB·English

CHARACTERIZATION OF SQUID PROTEIN HYDROLYSATES FOR
ANTIOXIDATIVE PROPERTIES

Thesis submitted in part fulfillment of the requirements for the Degree of
Master of Fisheries Science in Fish Quality Assurance and Management
to the Tamil Nadu Fisheries University, Nagapattinam

B. SIVARAMAN, B.F.Sc.
[MFT 12024 (FQM)]

DEPARTMENT OF FISH QUALITY ASSURANCE AND MANAGEMENT
FISHERIES COLLEGE AND RESEARCH INSTITUTE
TAMIL NADU FISHERIES UNIVERSITY
THOOTHUKUDI- 628 008

2014
CERTIFICATE
This is to certify that the thesis entitled “Characterization of squid protein
hydrolysates for antioxidative properties” submitted in part fulfillment of the
requirements for the award of the degree of Master of Fisheries Science in Fish
Quality Assurance and Management to the Tamil Nadu Fisheries University,
Nagapattinam is a record of bonafide research work carried out by B. Sivaraman
under my supervision and guidance and that no part of this thesis has been submitted
for any other degree, diploma, fellowship or similar titles.

Place : Thoothukudi Dr. R. JEYA SHAKILA
Date : Chairman
Place : Mangalore
Date :

EXTERNAL EXAMINER
APPROVED BY:

Chairman : Dr. R. JEYA SHAKILA

Professor

Members : 1. Dr. G. JEYASEKARAN

Professor& Head

2. Dr. D. SUKUMAR

Professor

Place : Thoothukudi
Date :
ACKNOWLEDGEMENTS
This thesis would not have been possible without the guidance
and the help of several individuals. First and foremost I offer my
sincerest gratitude to my supervisor, Dr. R. Jeya Shakila, who has
supported me throughout this research with her patience, knowledge,
encouragement. Without her enthusiastic guidance this thesis would
not have been completed or written. The knowledge, skills, and
confidence gained from her will always play a key role in my
professional life.
I would like express profound gratitude to my co-advisor
Dr. G. Jeyasekaran, for his invaluable suggestion and lessons for my
academic knowledge and personal life. His determination and
perseverance to train me to be a good researcher with responsibility,
vigilance and honesty are always acknowledged.
I would also like to thank my other co-advisor, Dr. D. Sukumar,
for accepting to be in my advisory committee and his suggestions and
ideas.
I sincerely thank Dr. G. Sugumar, Dean-in-charge, Fisheries
College and Research Institute for the facilities provided by him.
Special thanks to Assistant Professor Mrs. R. Shalini for her
kindness, care and encouragement when I needed in time.
My hearty thanks and everlasting love to Ulagammal, Renuka
and Elan, who were always there for me in every step of the way to
come this far, for their patience, kindness, and love throughout my
graduate experience. I am indebted to my wonderful friends
Elavarasan, Murali, Pari, Ravikumar and Gomathi for always
supporting me and for framing me what I am today. Without them this
is not possible to me.
I would like to thank my best friends, Mani and Mathi, for their
constant support, friendship, and humor. Many thanks to my friends
Subburaj, Karthy, Viji, Reddy, Rajesh, kannan, Innocen, Siva,
Faizullah, Selva, Santhiya, Kody and Jay who shared the hard time
with me and supporting me throughout all my days and stay at
FC & RI.
I record my heartfelt thanks to F. Parthiban, M. Palani kumar,
A. Varatharajakumar, P. Senthilkumar, G. Arunkumar and B. Edwin
Raj for their unlimited help throughout the research work. I also thank
Mr. R. Kumaresan, Mrs. R. Ezhilrani and Mrs. Indirani for having
provided me necessary support with regard to literature collection for
my study.
I have great pleasure in expressing my gratitude to Tamil
Nadu Fisheries University, Nagapattinam and TANUVAS, Chennai
for providing PG Merit Scholarship during the tenure of my study.

(B.Sivaraman)
To all,
who have behind in my science journey….
ABSRACT
Title : Characterization of squid protein hydrolysates for
antioxidative properties
Name : B. Sivaraman
Degree : M.F.Sc. (FQM)
Chairman : Dr. R. Jeya Shakila
Department : Fish Quality Assurance and Management
College : Fisheries College and Research Institute
Year & University : 2014, Tamil Nadu Fisheries University

Squid protein hydrolysates (SPH) were prepared using different
enzymes viz. alcalase, papain, trypsin and pepsin from Indian squid, Loligo duvauceli
to examine their antioxidative properties. Proximate composition showed that the
squid contained 15.31% protein and low fat content. Response surface methodology
(RSM) was used to optimize the hydrolysis conditions, such as temperature, time and
enzyme to substrate (E/S) ratio. DPPH radical scavenging activity was taken as a
response to evaluate the optimum conditions for SPH. Among the SPH, alcalase SPH
had the higher DPPH activity (78.37%) and further experiment carried out based on
optimized condition proved it. The extent of hydrolysis of SPH measured by degree of
hydrolysis (DH), showed that papain SPH had the highest DH. Peptide profiles of all
SPH were studied using Tricine- SDS- PAGE to determine the molecular weight of
the peptides. Amino acid profiles of all SPH were studied to know the role of amino
acids in antioxidative activities of hydrolysates. Different in vitro antioxidative
assays, including reducing power, metal chelating, ABTS radical scavenging,
hydroxyl radical scavenging and superoxide anion radical scavenging assays were
used to determine the antioxidative properties of SPH at different protein
concentrations viz. 0.5, 1.0, 1.5, 2 and 2.5 mg/ml, prepared under optimized condition.
Alcalase SPH had the highest metal chelating activity, papain SPH exhibited good
superoxide anion radical scavenging activity and trypsin had more reducing power.
Meanwhile alcalase and papain SPH exhibited similar ABTS and hydroxyl
scavenging activities. In vivo antioxidant activity determined using sardine mince
model system showed that papain and alcalase SPH reduced the formation of
secondary oxidation product, thiobarbituric acid reactive substances (TBARS) by
40%, which is similar to the reduction brought in by widely used antioxidant, ascorbic
acid. This in vivo result support SPH prepared from papain and alcalase could be used
as potential antioxidant in food processing industries.
ABBREVIATIONS

ABTS - 2,2`-Azino-bis (3-ethylbenzothiazoline-6- sulfonic acid) diammonium salt
ACE - Angiotensin Converting Enzyme
BBD - Box-Behnken Design
BHA - Butylated Hydroxy Anisole
BHT - Butylated Hydroxy Toluene
DH - Degree of Hydrolysis
DPPH - 2,2-Diphenyl-1-picrylhydrazyl
E/S ratio - Enzyme to Substrate ratio
EDTA - Ethylene diamine tetra acetic acid
ET - Electron Transfer
FPC - Fish Protein Concentrate
FPH - Fish Protein Hydrolysates
HAT - Hydrogen Atom Transfer
kDa - Kilodalton
NADH - β-Nicotinamide adenine dinucleotide
NBT - Nitroblue tetrazolium chloride
PG - Propyl Gallate
PMS - Phenazine Metho Sulfate
PUFA - Polyunsaturated Fatty Acids
ROS - Reactive Oxygen Species
RSM - Response Surface Methodology
SPH - Squid Protein Hydrolysates
TBARS - Thiobarbituric Acid Reactive Substances
TBHQ - Tert-Butyl Hydro Quinon
TCA - Trichloroacetic acid
TEMED - N,N,N’,N’-Tetramethylethylene diamine
TNBS - 2,4,6-TriNitroBenzene Sulfonic acid
Tricine-SDS - PAGE - Tricine-Sodium-Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis
UPLC - Ultra Performance Liquid Chromatography

LIST OF TABLES
Table Page
Title
No. No.

1 Commercial proteases used for hydrolysing fish proteins 8

2 Bioactive properties associated with fish protein hydrolysates 11

3 Antioxidative peptides derived from FPH 13

4 In vitro antioxidative methods and their mechanism 18

5 Box–Behnken design for optimizing hydrolysis conditions for 26
alcalase SPH

6 Box–Behnken design for optimizing hydrolysis conditions for 26
papain, trypsin and pepsin SPH

7 Binary solvent manager parameter values 30

8 Box–Behnken design for optimizing hydrolysis conditions and 38
response values for alcalase SPH

9 Box–Behnken design for optimizing hydrolysis conditions and 38
response values for papain, trypsin and pepsin SPH

10 Analysis of variance for the response of antioxidative activity in 39
alcalase SPH

11 Analysis of variance for the response of antioxidative activity in 39
papain SPH

12 Analysis of variance for the response of antioxidative activity in 40
trypsin SPH

13 Analysis of variance for the response of antioxidative activity in 40
pepsin SPH

14 Optimum conditions, expected and experimental DPPH activity 41
of hydrolysates prepared from different enzymes

15 DH of different SPH 44

16 Amino acid profiles (% of total composition) of raw squid and 46
different SPH
LIST OF FIGURES
Figure Page
Title
No. No.

1 Steps involved in the preparation of FPH by using enzymes 9

2 Experimental design 28

3 Proximate composition of Indian squid muscle 36

4 2D contour plots and 3D response surface plots of alcalase SPH 42

5 2D contour plots and 3D response surface plots of papain SPH 43

6 2D contour plots and 3D response surface plots of trypsin SPH 43

7 2D contour plots and 3D response surface plots of pepsin SPH 44

8 Tricine-SDS-PAGE peptide profile of SPH prepared from 45
different enzymes

9 Reducing power of SPH prepared using different enzymes 48

10 Metal chelating activity of SPH prepared using different 48
enzymes

11 ABTS radical scavenging activity of SPH using different 49
enzymes

12 Hydroxyl radical scavenging activity of SPH using different 50
enzymes

13 Superoxide anion radical scavenging activity of SPH using 51
different enzymes

14 Effect of different SPH on TBARS formation in oil sardine 52
minced meat model system

15 Different antioxidative mechanisms exhibited by different SPH 65