Invasive SIlver Carp (Hypophthalmichthys molitrix) Protein

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Invasive SIlver Carp (Hypophthalmichthysmolitrix) Protein Hydrolysates- A Potential Sourceof Natural AntioxidantSravanthi Priya MalaypallyPurdue University

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Recommended CitationMalaypally, Sravanthi Priya, "Invasive SIlver Carp (Hypophthalmichthys molitrix) Protein Hydrolysates- A Potential Source of NaturalAntioxidant" (2013). Open Access Dissertations. 85.https://docs.lib.purdue.edu/open_access_dissertations/85

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PURDUE UNIVERSITY GRADUATE SCHOOL

Thesis/Dissertation Acceptance

This is to certify that the thesis/dissertation prepared By Entitled For the degree of Is approved by the final examining committee: Chair To the best of my knowledge and as understood by the student in the Research Integrity and Copyright Disclaimer (Graduate School Form 20), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material.

Approved by Major Professor(s): ____________________________________

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Approved by: Head of the Graduate Program Date

Sravanthi Priya Malaypally

INVASIVE SILVER CARP (Hypophthalmichthys molitrix) PROTEIN HYDROLYSATE - A POTENTIAL SOURCE OF NATURAL ANTIOXIDANT

Doctor of Philosophy

Liceaga, Andrea M. Kim, Kee-Hong

San Martin, Fernanda

Ferruzzi, Mario

Goforth, Reuben R.

Liceaga, Andrea M.

Rengaswami Chandrasekaran 12/03/2013

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INVASIVE SILVER CARP (Hypophthalmichthys molitrix) PROTEIN

HYDROLYSATES- A POTENTIAL SOURCE OF NATURAL ANTIOXIDANT

A Dissertation

Submitted to the Faculty

of

Purdue University

by


In Partial Fulfillment of the

Requirements for the Degree

of

Doctor of Philosophy

December 2013

Purdue University

West Lafayette, Indiana

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Dedicated to Almighty and my loved ones. Fiancé, Parents, Family and Friends

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ACKNOWLEDGEMENTS

I would like to take this opportunity to thank many people who helped me

in completing my research and thesis. First and foremost, I would like to express my

deepest appreciation to Dr. Andrea Liceaga for her invaluable guidance and being

supportive during my graduate studies. I would also like to thank my committee members

- Dr.Mario Ferruzzi, Dr. Reuben R. Goforth, Dr. Kee-Hong Kim and Dr. Fernanda San-

Martin for their suggestions and help. Without their guidance and persistent help this

research would not have been possible. I would also want to thank the faculty and staff of

Department of Food Science at Purdue University for making my graduate school learning

experience valuable.

A thank you to Inerowicz Halina Dorota and Bindley Bioscience Center (Discovery

Park, Purdue University) for the technical expertise in peptide sequencing and amino acid

analysis. Help from Dr. Chih-Yu Chen and Dr. Choon Young Kim during Caco-2 cell

assays, Benjamin Redan during MTT assay and David Petros during size exclusion

chromatography is greatly appreciated. I must thank my friends and fellow lab students in

the department, especially Veronica Rodriguez, Eileen Duarte Gomez, Titiksha Dikshit,

and Gulsha Bakir for making my stay at Purdue so delightful. I would also convey my

gratitude to my friends from Chicago (it’s a big list) for their love and for always being

there for me.

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I can’t express my gratitude enough to my parents Mr. Sree Ramulu, Mrs.

Shiromani and my fiancé Srikanth Bangari who are my inspiration and all time support

system.

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TABLE OF CONTENTS

Page

LIST OF TABLES .............................................................................................................. x LIST OF FIGURES ........................................................................................................... xi

ABSTRACT ............................................................................................................ xxi CHAPTER 1. REVIEW OF LITERATURE ................................................................ 1

1.1 Introduction ............................................................................................... 1

1.2 Silver Carp and its Introduction to the United States ................................ 3

1.2.1 Strategies to Minimize the Spread of Invasive Silver Carp ............... 4

1.2.2 Fish Protein-Potential Application as Food Ingredient ...................... 5

1.2.2.1 Enzymatic Hydrolysis Variables ............................................. 8

1.2.2.2 Improved Nutritional Quality of FPH ..................................... 9

1.2.2.3 Improved Functional Properties of FPH ............................... 10

1.2.2.4 Bioactive Properties of FPH .................................................. 13

1.3 Fish Bioactive Peptides as Antioxidants ................................................. 17

1.3.1 Antioxidants ..................................................................................... 17

1.3.2 Antioxidants Application in Food Systems ...................................... 18

1.3.3 Antioxidants and Human Health ...................................................... 19

1.3.3.1 Oxidative Stress and Diseases ............................................... 21

1.3.4 Endogenous Antioxidant Defense System ....................................... 24

1.3.5 Natural Antioxidants in Food and Biological Systems .................... 25

1.3.6 Fish Protein Hydrolysates as a Source of Antioxidants ................... 27

1.3.7 Mechanism of Antioxidant Peptides ................................................ 28

1.3.7.1 Molecular Weight of the Peptide ........................................... 30

1.3.7.2 Hydrophobicity ...................................................................... 31

1.3.7.3 Amino Acid Composition ..................................................... 32

1.3.7.4 Amino Acid Sequence ........................................................... 33

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1.3.8 Interaction of Food Derived Antioxidant Peptides and Endogenous

Antioxidant System ............................................................................................... 34

1.3.9 Bioavailability of Antioxidant Peptides ........................................... 36

1.3.10 Studying Antioxidants on Intestinal Cell Line ................................. 37

1.3.11 In Vitro Measurement of Antioxidant Activity ................................ 38

1.3.11.1 Chemical Based Assays ......................................................... 39

1.3.11.2 In Vitro Cell Culture Model .................................................. 42

1.3.12 Research Hypothesis and Objectives ............................................... 44

CHAPTER 2. ANTIOXIDANT PROPERTIES AND FREE AMINO ACIDS

COMPOSITION OF SILVER CARP (HYPOPHTHALMICHTHYS MOLITIX)

PROTEIN HYDROLYSATES ......................................................................................... 47

2.1 Abstract ................................................................................................... 47

2.2 Introduction ............................................................................................. 48

2.3 Materials and Methods ............................................................................ 49

2.3.1 Materials ........................................................................................... 49

2.3.2 Preparation of Silver Carp Protein Hydrolysates (SPH) .................. 50

2.3.3 Degree of Hydrolysis ....................................................................... 51

2.3.4 Proximate Analysis and Amino Acid Composition ......................... 52

2.3.5 DPPH Scavenging Activity .............................................................. 53

2.3.6 Trolox Equivalence Antioxidant Property ....................................... 53

2.3.7 Ferric Ion Reducing Antioxidant Capacity ...................................... 54

2.3.8 Lipid Peroxidation Inhibition Assay ................................................ 55

2.3.9 Statistical Analysis ........................................................................... 55

2.4 Results and discussion ............................................................................. 56

2.4.1 Effect of Enzyme and Enzymatic Conditions on Degree of

Hydrolysis (%DH) ................................................................................................ 56

2.4.2 Proximate Analysis and Free Amino Acid Composition ................. 56

2.4.3 Antioxidant Properties of SPH ......................................................... 58

2.4.4 DPPH Radical Scavenging Activity ................................................. 58

2.4.5 TEAC Value ..................................................................................... 60

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2.4.6 Ferric Ion Reduction Capacity ......................................................... 61

2.4.7 Inhibition of Lipid Peroxidation by SPH ......................................... 63

2.4.8 Overall Antioxidant Activity Among SPH-A and SPH-F Sample .. 64

2.4.9 Conclusion ........................................................................................ 65

CHAPTER 3. EFFECT OF MOLECULAR WEIGHT AND PEPTIDE

SEQUENCE OF HYDROLYSATE FRACTION ON CELL BASED ANTIOXIDANT

ACTIVITY OF SILVER CARP (HYPOPHTHALMICHTHYS MOLITRIX) PROTEIN

HYDROLYSATES ........................................................................................................... 75

3.1 Abstract ................................................................................................... 75

3.2 Introduction ............................................................................................. 76

3.3 Materials and Methods ............................................................................ 78

3.3.1 Materials ........................................................................................... 78

3.3.2 Preparation of SPH ........................................................................... 78

3.3.3 Degree of Hydrolysis ....................................................................... 79

3.3.4 Molecular Weight Profile of F-30 and A-60 Samples ..................... 79

3.3.5 Fractionation of Hydrolysates According to Molecular Weight ...... 80

3.3.6 Size-Exclusion Chromatography ...................................................... 80

3.3.7 Ultrafiltration .................................................................................... 80

3.3.8 Molecular Weight Determination ..................................................... 81

3.3.9 LC-MS/MS (ESI) ............................................................................. 81

3.3.10 Caco-2 BBe Cell Culture Treatment ................................................ 82

3.3.11 Cell Based Antioxidant Activity ...................................................... 83

3.3.12 Statistical Analysis ........................................................................... 84

3.4 Results and Discussion ............................................................................ 84

3.4.1 Choice of Hydrolysates .................................................................... 84

3.4.2 Molecular Weight Distribution of F-30 and A-60 ........................... 85

3.4.3 Fractionation of F-30 and A-60 Samples According to Molecular

Weight .......................................................................................................... 85

3.4.4 Peptide Sequencing of F-30<3 and A-60<3 Samples ...................... 86

3.4.5 Intracellular Antioxidant Activity of Different Molecular Weight

Peptides .......................................................................................................... 87

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3.4.6 Inhibition of Cellular Oxidation by Different Molecular Weight

Peptides Under Stress and Non-Stress Conditions ............................................... 89

3.4.7 Conclusion ........................................................................................ 92

CHAPTER 4. EFFECT OF SIMULATED GASTROINTESTINALDIGESTION

ON IN VITRO CELL BASED ANTIOXIDANT ACTIVITY OF SILVER CARP

(HYPOPHTHALMICHTHYS MOLITRIX) PROTEIN .................................................... 110

4.1 Abstract ................................................................................................. 110

4.2 Introduction ........................................................................................... 112

4.3 Materials and Methods .......................................................................... 114

4.3.1 Materials ......................................................................................... 114

4.3.2 Preparation of Silver Carp Protein Hydrolysates (SPH) ................ 115

4.3.3 Preparation of Unhydrolyzed Silver Carp Protein (SPU) .............. 115

4.3.4 Simulated Gastrointestinal Digestion Silver Carp Protein

Hydrolysates (SPH-GI) ....................................................................................... 116

4.3.4.1 Protocol 1:- Using Lower GI Enzyme Activity ................... 116

4.3.4.2 Protocol-2:--Using Extreme GI Conditions ........................ 117

4.3.5 Molecular Weight Distribution ...................................................... 117

4.3.6 MTT Assay ..................................................................................... 118

4.3.7 Caco-2 BBe Cell Culture Treatment .............................................. 119

4.3.8 Cell Based Antioxidant Activity .................................................... 119

4.3.9 Statistical Analysis ......................................................................... 119

4.4 Results and Discussion .......................................................................... 119

4.4.1 Choice of Enzymes and Enzyme Activity used to Simulated

Gastrointestinal Digestion ................................................................................... 119

4.4.2 Effect of Gastrointestinal Digestion on Molecular Weight of F-30

and A-60 Samples ............................................................................................... 121

4.4.3 Viability of Cells in the Presence of Gastrointestinal Digested

Samples ........................................................................................................ 122

4.4.4 Effect of Gastrointestinal Digestion of F-30 and A-60 Samples on its

Antioxidant Activity ........................................................................................... 123

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4.4.5 Intracellular Antioxidant Activity of Simulated GI Digest of F-30

and A-60 Samples Under Stress and Non-Stress Conditions ............................. 125

4.4.6 Intracellular Antioxidant Activity of Simulated GI Digest of

Unhydrolyzed Pasteurized Silver Carp Protein .................................................. 127

4.5 Conclusion ............................................................................................. 129

CHAPTER 5. CONCLUSIONS AND FUTURE DIRECTIONS ............................ 151

5.1 Conclusions ........................................................................................... 151

5.2 Future Direction .................................................................................... 153

5.2.1 Effect of GI Digestion on Lower Molecular Weight Peptide

Fractions (F-30<3 and A-60<3) .......................................................................... 153

5.2.2 Identification and Characterization of Antioxidant Peptides Present

in Silver Carp Protein Hydrolysates ................................................................... 154

5.2.3 Further Assays to Confirm the Antioxidant Mechanisms of the

Peptides Fractions ............................................................................................... 155

5.2.4 Sensory Studies on Antioxidant SPH ............................................. 157

BIBLIOGRAPHY ........................................................................................................... 158

VITA ............................................................................................................. 56

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LIST OF TABLES

Table .............................................................................................................................. Page

Table 1-1. In vitro antioxidative chemical assays. Adapted from Huang, D., et

al.,(2005)(Dejian Huang, Ou, & Prior, 2005) ................................................................... 40

Table 2-1. Protein content of SPH-A and SPH-F samples. .............................................. 66

Table 2-2. Free amino acid composition of SPH and their control samples ..................... 67

Table 2-3. Summary of results for the antioxidant properties of silver carp protein

hydrolysates prepared using Alcalase® (SPH-A) and Flavourzyme® (SPH-F) at different

hydrolysis times (15 min, 30 min, 45 min and 60 min). ................................................... 69

Table 3-1. The percent molecular weight distribution of Flavourzyme® and Alcalase®

hydrolyzed silver carp protein at 30 and 60 min hydrolysis time respectively (F-30 and A-

60) samples calculated using Sephadex G-25 gel filtration chromatogram and calibration

curve .................................................................................................................................. 94

Table 3-2. IC50 values (mg/ mL) of F-30, F-30<3, A-60 and A-60 <3 samples at 30 and

120 min incubation times under both stress and non-stress conditions ............................ 95

Table 4-1. IC50 values (mg/ mL) of F-30 and F-30#1 samples at 30 and 120 min

incubation times under both stress and non-stress conditions ........................................ 130

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LIST OF FIGURES

Figure ............................................................................................................................. Page

Figure 1-1.General protocol for the production of FPH ..................................................... 7

Figure 1-2. Reaction between TNBS and primary amines (Alder-Nissen, 1979) .............. 9

Figure 1-3. Schematic diagram illustrating both chemical and physical antioxidant

mechanism by 1. Metal chelating ability, 2. Radical scavenging activity and 3. Lipid

peroxidation ability of antioxidant peptides (Xiong, 2010) .............................................. 29

Figure 2-1. Degree of hydrolysis (%) of Silver carp (Hypophthalmichthys molitrix) by

Flavourzyme® and Alcalase® enzymes. ............................................................................ 71

Figure 2-2. The percentage scavenging activity of DPPH radical (0.02 %) of (a)

Alcalase® hydrolyzed silver carp protein hydrolysates (SPH-A) and (b) Flavourzyme®

hydrolyzed silver carp protein hydrolysates (SPH-F) at different concentrations (mg/mL)

after 60 min incubation in dark. Ethanol (99.5 %) was used as an assay media. Analysis

was done in triplicate. ....................................................................................................... 72

Figure 2-3. Lipid peroxidation values (Abs @ 500 nm) evaluated in linoleic acid

emulsion system in presence of (A) Alcalase® hydrolyzed silver carp protein (SPH-A)

(0.052 mg/ mL) and (B) Flavourzyme® hydrolyzed silver carp protein (SPH-F) (0.052

mg/ mL) incubated in dark at room temperature (25 °C) at different incubation times (0,

40, 72, 156 and 168 h). Analysis was done in triplicate. .................................................. 74

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Figure ............................................................................................................................. Page

Figure 3-1. Sephadex G-25 gel filtration Chromatogram of Flavourzyme® and Alcalase®

hydrolyzed Silver carp protein at 30 and 60 min hydrolysis time respectively (F-30 and

A-60). The protein content was analyzed using BCA assay ............................................. 96

Figure 3-2. Standard curve of Sephadex G25 column prepared using Insulin chain B,

oxidation (3495.89 Da) and Vitamin B12 (1355 Da). Molecular weight of 3000 Da

occurs at 129.88 mL elution volume, which occurs nearly at 22nd tube. .......................... 97

Figure 3-3. Standard curve of Sepax zenix SEC-100, 3 µm, 100 A°, 4.6 X300mm (100-

100,000 Da) column prepared using Carbonic anhydrase (29 KDa), Aprotinin (6511.44

Da), Vitamin B12 (1355.37 Da) and Cytidine (243.22 Da). Molecular weight of 3000 Da

occurs at 8.63 minute. ....................................................................................................... 98

Figure 3-4. Molecular weight profile of < 3 KDa peptide fractions of A-60 (A-60<3) ran

on Waters e2695 Separation Module HPLC system equipped with UV/Vis detector and

Sepax zenix SEC-100, 3µm, 100 A°, 4.6 × 300 mm2 column. Phosphate buffer (150 mM,

pH 7.0) was used as solvent phase at 0.35 mL/ min flow rate. The retention time of 3

KDa peptide occurs at 8.63 min. ....................................................................................... 99

Figure 3-5. Molecular weight profile of < 3 KDa peptide fractions of F-30 (F-30<3) ran




KDa peptide occurs at 8.63 min ...................................................................................... 100

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Figure ............................................................................................................................. Page

Figure 3-6 Molecular weight profile of <10 and >3 KDa peptide fractions of A-60 (A-

60>3) and ran on Waters e2695 Separation Module HPLC system equipped with UV/Vis

detector and Sepax zenix SEC-100, 3µm, 100 A°, 4.6 × 300 mm2 column. Phosphate

buffer (150 mM, pH 7.0) was used as solvent phase at 0.35 mL/ min flow rate. The

retention time of 3 KDa peptide occurs at 8.63 min ....................................................... 101

Figure 3-7 Molecular weight profile of <10 and >3 KDa peptide fractions of F-30 (F-

30>3) and ran on Waters e2695 Separation Module HPLC system equipped with UV/Vis

detector and Sepax zenix SEC-100, 3µm, 100 A°, 4.6 × 300 mm2 column. Phosphate

buffer (150 mM, pH 7.0) was used as solvent phase at 0.35 mL/ min flow rate. The

retention time of 3 KDa peptide occurs at 8.63 min ....................................................... 102

Figure 3-8. Inhibition of cellular oxidation in presence of molecular weight fraction of

(A) F-30 under non-stress; (B) A-60 under non-stress; (C) F-30 under oxidation stress:

and (D) A-60 under oxidative stress during 120 min of incubation. Assay was conducted

in duplicate and analysis was done in triplicate. ............................................................. 103

Figure 3-9. Percentage intracellular oxidation measured in Caco-2 BBe cells in the

presence of F-30, its fractions (F-30 <3 and F-30 >3) and positive control (Vitamin C)

measure at different incubation times under both non-stressed (no AAPH added) and

stressed (AAPH, 0.1 mM, 1h at 37 °C). Intracellular oxidation of negative control (no

sample added) at a given condition was used as a reference (100 % oxidation). ........... 106

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Figure ............................................................................................................................. Page


presence of A-60, its fractions (A-60 <3 and A-60 >3) and positive control (Vitamin C)



sample added) at a given condition was used as a reference (100 % oxidation). Values are

mean ± SE, n = 3. ............................................................................................................ 109

Figure 4-1 Molecular weight profile of F-30 (20 µL, 2.5 mg/ mL) ran on Waters e2695

Separation Module HPLC system equipped with UV/Vis detector and Sepax zenix SEC-

100, 3µm, 100 A°, 4.6 × 300 mm2 column. Phosphate buffer (150 mM, pH 7.0) was used

as solvent phase at 0.35 mL/ min flow rate. ................................................................... 131

Figure 4-2 Molecular weight profile of F-30#1 (GI digest of F-30 using protocol 1) (20

µL, 2.5 mg/ mL) ran on Waters e2695 Separation Module HPLC system equipped with

UV/Vis detector and Sepax zenix SEC-100, 3µm, 100 A°, 4.6 × 300 mm2 column.

Phosphate buffer (150 mM, pH 7.0) was used as solvent phase at 0.35 mL/ min flow rate.

......................................................................................................................................... 132

Figure 4-3. Molecular weight profile of F-30#2 (GI digest of F-30 using protocol 2) (20




......................................................................................................................................... 133

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Figure ............................................................................................................................. Page

Figure 4-4 Molecular weight profile of A-60 (20 µL, 2.5 mg/ mL) ran on Waters e2695



as solvent phase at 0.35 mL/ min flow rate. ................................................................... 134

Figure 4-5 Molecular weight profile of A-60#1 (GI digest of A-60 using protocol 1) (20




......................................................................................................................................... 135

Figure 4-6 Molecular weight profile of A-60 #2 (GI digest of A-60 using protocol 2) (20




......................................................................................................................................... 136

Figure 4-7 Molecular weight profile of SPU #1 (GI digest of SPU using protocol 1) (20




......................................................................................................................................... 137

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Figure ............................................................................................................................. Page

Figure 4-8 Molecular weight profile of SPU #2 (GI digest of SPU using protocol 2) (20




......................................................................................................................................... 138

Figure 4-9. Viability of Caco-2/TC7 clone cells in the presence of GI digestion blanks

(Blk#1 and Blk#2) and SPH-GI samples (F-30#1, F-30#2, A-60#1 and A-60#2) measured

using MTT assay after 2 h of incubation. The SPH-GI samples are at a concentration of 5

mg/ mL and blank samples were diluted to obtain protease content similar to that present

in SPH-GI samples .......................................................................................................... 139

Figure 4-10 Inhibition of cellular oxidation in presence of F-30, GI digest of F-30 (F-

30#1 and F-30#2) and blank sample (Blk#1 and Blk#2) under non-stress (A) and

oxidative stress (C); A-60, GI digest of A-60 (A-60#1 and A-60#2) and blank sample

(Blk#1 and Blk#2) under non-stress (B) and oxidation stress (D) during 120 min of

incubation at 5 mg/ mL concentration. Assay was conducted in duplicate and analysis

was done in triplicate. ..................................................................................................... 142

Figure 4-11 Inhibition of cellular oxidation in presence of Blk#1 and Blk#2 under Non-

stress and oxidative stress condition under different dilutions. Blk#1 and Blk#2 samples

were diluted to obtain similar concentration of protease content present in 0.625 mg/ mL

(A) and (D); at 1.25 mg/ mL (B) and (E); and at 5 mg/ mL (C) and (F) of SPI-GI sample

and incubated for 120 min. Assay was conducted in duplicate and analysis was done in

triplicate. ......................................................................................................................... 144

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Figure ............................................................................................................................. Page


presence of F-30 and its simulated GI digested samples prepared using protocol 1 (F-

30#1) and protocol 2 (F-30#2) measured at different incubation times under both non-

stressed (no AAPH added) and stressed (AAPH, 0.1 mM, 1h at 37 °C). Intracellular

oxidation of negative control (no sample added) at a given condition was used as a

reference (100 % oxidation). Values are mean ± SE, n = 3. ........................................... 146


presence of A-60 and its simulated GI digested samples prepared using protocol 1 (A-

60#1) and protocol 2 (A-60#2) measured at different incubation times under both non-

stressed (no AAPH added) and stressed (AAPH, 0.1 mM, 1h at 37 °C). Intracellular



Figure 4-14 Percentage intracellular oxidation measured in Caco-2 BBe cells in the

presence of unhydrolyzed silver carp protein after simulated GI digestion using protocol

1 (SPU#1) and protocol 2 (SPU#2) measured at different incubation times under both

non-stressed (no AAPH added) and stressed (AAPH, 0.1 mM, 1h at 37 °C). Intracellular



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LIST OF ABBREVIATION

SPH Silver carp protein hydrolysates

BHA Butylated hydroxyanisole

BHT Butylated hydroxytoluene

TBHQ Tert-butylhydro-quinone

FPH Fish protein hydrolysates

%DH Percentage degree of hydrolysis

CVD Cardiovascular disease

ACE Angiotensin-I converting enzyme

APTT Activated partial thromboplastin

PT Prothrombin

TP Thrombin

ROS Reactive oxygen species

FA Fatty acid

LDL Low density lipoprotein

Ox-LDL Oxidized low density lipoprotein

AD Alzheimer’s disease

PD Parkinson’s disease

ALS Amytrophic lateral sclerosis

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HNE 4-Hydroxy-2,3-nonenal

SOD Superoxide dismutase

CAT Catalase

GSH-Px Glutathione peroxidase

GSH Glutathione

QSAR Quantitative structure activity relationship

SS Szeto-Schiller

γ-GCL γ-Glutamyl cysteine ligase

HAT Hydrogen atom transfer

ET Electron transfer

DPPH 2,2-Diphenyl-1-picrylhydrazyl

ABTS 2,2'-Azinobis(3-ethylbenzothiaazoline-6-sulphonic

acid

ORAC Oxygen radical absorbance capacity assay

ESR Electro spin resonance

AAPH 2,2’-Azobis-2-methyl-propaninidamine dyhydro

chloride

MTT 3-(4,5-Dimethyl thiazol-2-yl)-2,5-diphenyl tetra

zolium

DCFH-DA Dichlorofluorescein diacetate

DCFH Dichlorofluorescein

GI Gastrointestinal

LP-IA Lipid peroxidation inhibition activity

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FI-RC Ferric ion reducing capacity

SPH-F Flavourzyme® hydrolyzed silver carp protein

hydrolysates

SPH-A Alcalase® hydrolyzed silver carp protein

hydrolysates

SPU Unhydrolyzed silver carp protein

SPH-GI Simulated gastrointestinal digests of Silver carp

protein hydrolysates

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ABSTRACT

Malaypally, Sravanthi P. Ph.D., Purdue University, December 2013. Invasive Silver Carp

(Hypophthalmichthys molitrix) Protein Hydrolysates – A Potential Source of Natural

Antioxidant. Major Professor: Andrea Liceaga.

Invasive silver carp (Hypophthalmichthys molitrix), continue to spread over the

Mississippi River causing a great concern for the river ecosystem due to their impact on

native fish species. To minimize the negative effects of silver carp, many strategies were

implemented including using it for animal feed, as fertilizers or simply discarding them

into waste. However, these fish are high in protein content, making them excellent starting

material for protein-derived by-products. One alternative is to recover and modify the

protein in the fish into bioactive food ingredient. In the functional/ nutraceutical food

industry, there is high demand for antioxidant agents. In this study, silver carp hydrolysates

(SPH) were evaluated for their antioxidant activity using in vitro assays which simulate

food and in vivo cell model. This study also attempts to understand the factors affecting

antioxidant activity of SPH.

The general goals of this study were to (1) evaluate the antioxidant potential of

SPH prepared using two different commercial enzymes (Flavourzyme®® and Alkalase)

and to characterize the amino acid composition of the SPH in order to understand the

influence of free amino acid composition on different antioxidant mechanisms; (2) to

evaluate antioxidant activity of SPH using a Caco-2 cell based model and to determine the

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relationship between molecular weight of the peptide fractions and antioxidant activity (3)

to determine the effect of simulated gastro intestinal digestion of SPH on its cell based

antioxidant activity.

The results showed that most of the SPH samples displayed higher or comparable

antioxidant capacity than commercial antioxidants (BHA, BHT and α-tocopherol). Further,

the antioxidant activity of the SPH was shown to depend on the presence of specific amino

acids which include acidic amino acids and hydrophobic amino acids. In addition, peptide

fractions with low molecular weight (<3 KDa) improved cell based antioxidant activity

even after longer incubation time under both oxidative stress and non-stress conditions.

Dose dependent cell based antioxidant activity was observed in the low molecular weight

peptide fractions. Lower molecular weight peptide fraction of Flavourzyme® SPH showed

higher activity than Alcalase® SPH due to presence of high number of peptides with active

sequence in former than later. In addition, simulated gastrointestinal digestion of

Flavourzyme® SPH using a lower digestion enzyme activity was able to retain its activity

and also was able to show dose dependent activity. Overall, SPH displayed potential

antioxidant capacity both in chemical based and cell based assays thus providing

alternatives for using an invasive fish in the form of potential functional, bioactive

ingredients.

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CHAPTER 1. REVIEW OF LITERATURE

1.1 Introduction

Oxidation in foods is one of the major concerns in food industry since it causes

deterioration of quality and compromise food safety. Oxidation causes rancidity of lipids,

production of toxic oxidation compounds, causes unpleasant flavor, color and texture in

the food matrix. To prevent oxidation, many synthetic antioxidants such as buthylated

hydroxyanisole (BHA), buthylated hydroxytoluene (BHT), tert-butylhydro-quinone

(TBHQ) and propyl gallate are commonly used. However their use in food systems is

restricted by strict regulations or prohibition in certain countries due to their adverse health

effects (Velioglu, Mazza, Gao, & Oomah, 1998). This resulted in great interest in the

development of natural alternative as effective as synthetic antioxidants.

Many scientific studies have indicated that increase in reactive oxygen species and

free radicals in the human body results in oxidative stress and causes various chronic

diseases such as cardiovascular disease, cancer and neurodegenerative diseases (Barnham,

Masters, & Bush, 2004; Lakshmi, Padmaja, Kuppusamy, & Kutala, 2009; Reuter, Gupta,

Chaturvedi, & Aggarwal, 2010). In order to combat the onset of oxidative stress,

endogenous antioxidant system plays an important role. However during oxidative stress,

external antioxidant source can attenuate the condition and/ or boost the endogenous

antioxidant system.

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Natural antioxidants including vitamin E, vitamin C and few flavonoids obtained

from plant source have shown a promising alternative. However, over exploitation of these

limited biological resources increases the concern of depleting them (T. Wang, Zhao, &

Wang). Therefore a need to explore an alternative unused or underutilized source for the

natural antioxidant.

Research on the use of fish protein hydrolysates (FPH) as a source of antioxidant

peptides has increased during last decade. These antioxidant peptides have potential use in

pharmaceutical/ nutraceutical and food applications. However this research is relatively

new and requires further work to identify FPH with antioxidant properties using

appropriate enzyme and substrate (underutilized fish / fish by-product), understanding

structure-activity relationship, scaling up the hydrolysis process commercially and testing

efficacy of these peptides in biological systems as well as food systems (Samaranayaka,

2010).

Many studies have emphasized the use of fish processing by-products as a source

of these FPH and their bioactive peptides. Apart from by-products, large amounts of fish

catch remain unused due to problems related to unacceptable color, flavor, texture or

simply being an unconventional species. In recent years the spreading of unconventional

carp species such as Bighead Carp (Hypophthalmichthys nobilis) and Silver Carp

(Hypophthalmichthys molitrix) has increased in the Mississippi River System causing

ecological imbalance leading to depletion of native species (Rogowski, Soucek, Chick,

Dettmers, Pegg, Johnson, et al., 2005). To alleviate the negative impact of these invasive

carps, efforts are being implemented to reduce carp population and to minimize their

opportunity to enter the Great Lakes basin (Bowzer, Trushenski, Rawles, Barrows, &

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Gaylord, 2013). To accomplish this, most of these carps are harvested to simply be

discarded into waste causing environmental pollution. However these two species are

considered as highly nutritious (Kolar, Chapman, Courtenay Jr, Housel, Williams, &

Jennings, 2005) and recovery of this highly nutritious “waste” to produce antioxidant

peptides for human consumption will be a great alternative. To present knowledge, no

attempts have been made to utilize the proteins from these invasive carps, obtained from

the U.S. Mississippi River System to make food ingredients available for human

consumption.

1.2 Silver Carp and its Introduction to the United States

Silver carp (Hypophthalmichthys molitrix), one of the Asian carp species, is a fresh

water fish native to north and northeast Asia. They are voracious filter feeders and feed on

variety of planktonic biomass (Cremer & Smitherman, 1980; Kolar, Chapman, Courtenay

Jr, Housel, Williams, & Jennings, 2005). Because of their feeding habits, they have been

introduced in many countries beyond their native ranges as a biological aid to control

plankton in sewage treatment lagoons, aquaculture tanks and reservoirs. Asian carp,

including silver carp, were first introduced in the United States in early 1970’s for the

above benefits. However, during flooding events they escaped confinements and now they

are well established throughout much of the middle and lower Mississippi River System in

the Midwestern U.S. (Chick & Pegg, 2001). Their continuing spread and increasing local

densities are of great concern from both natural ecosystem and human safety perspectives

and there is evidence that they have the potential to become established in wider variety of

novel habitats than previously thought (Coulter, Keller, Amberg, Bailey, & Goforth, 2013).

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Direct and indirect competition for food of the Asian carp with native fish can have

significant impacts on freshwater biodiversity (Alam, Khan, Hussain, Moumita, Mazlan,

& Simon, 2012; Calkins, Tripp, & Garvey, 2012; Irons, Sass, McClelland, & Stafford,

2007). In addition, the tendency of silver carp to jump out of the water when disturbed has

resulted in many human injuries from collisions with the airborne fish. According to recent

survey, the standing biomass of the Asian carp in the Mississippi River was about 1.4

million kg, with silver carp alone contributing 63% of the total invasive and native fish

biomass (Roth & Secchi, 2012). Because silver carp replaced many of the native fish

species, there is a huge negative impact on the fishing industry, recreational sport fishing

and related industries. The term ‘invasive’ can be applied to silver carps since they are non-

native species that affects the native habitat by disrupting the ecological balance (Kolar,

Chapman, Courtenay Jr, Housel, Williams, & Jennings, 2005).

1.2.1 Strategies to Minimize the Spread of Invasive Silver Carp

To minimize the alarming spreading of Asian carps including silver carps in

Mississippi River System and also to minimize their opportunity to enter the Great Lakes

basin, a variety of management strategies are being implemented. The strategies include –

physical barriers (e.g. floating curtains, vertical drops), behavior barriers (e.g. electrical

barrier, acoustic deterrents, strobe light) and chemical barriers (e.g. low oxygen, poisoned

baits) (Bowzer, Trushenski, Rawles, Barrows, & Gaylord, 2013). In addition to the above

strategies, harvesting huge quantities of carps is also being encouraged to reduce their

population. Since carps are not a favored food fish in the USA, the harvested carps are

simply discarded as waste (Bowzer, Trushenski, Rawles, Barrows, & Gaylord, 2013; Kolar,

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Chapman, Courtenay Jr, Housel, Williams, & Jennings, 2005). Disposal into the waste

system causes pollution and also emits offensive odors. However these species are

considered as highly nutritious and widely consumed as a common food fish in Asian,

European and African countries. Therefore, invasive Asian carps have been exported from

Illinois (USA) to China for human consumption. However the huge transportation cost,

logistics and increase in carbon foot print have limited this approach (Varble & Secchi,

2013).

Hence, there is a need to utilize these protein-rich underutilized species. At present

in United States of America, huge amounts of harvested carps are used as fertilizers or

livestock feeds (Kristinsson and Rasco, 2000). In addition, very recently a new rendering

company called Heartland Processing in Havana, IL, started to extract Omega 3 fatty acids

from Asian carps and by-product containing proteins was used as animal feed. One

alternative is to recover and modify protein rich fish meat into different value added food

ingredients available for human consumption.

1.2.2 Fish Protein-Potential Application as Food Ingredient

The composition of fish protein depends on many factors such as species, size,

maturity, sex, specimen and environmental conditions of its habitat. In general there are

three major fish muscle tissue proteins: structural (myofibrillar) proteins, sarcoplasmic

proteins and connective tissue (stromal) proteins. Approximately, 70-80 % of the total

proteins correspond to structural proteins consisting of actin, myosin, tropormyosin and

actomyosin. Sarcoplasmic proteins such as myoalbumin, globulin and enzymes constitute

to 25–30 % of the total. The remaining 3-10 % corresponds to connective tissue proteins

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(B. N. Ahn & Kim, 2013). The main component of muscle proteins myosin and actin are

insoluble in water. The poor solubility of fish proteins limits their use as a food ingredient.

For the high utilization of fish protein, attempts are being made to solubilize the protein

using proteolytic enzymes and acid hydrolysis (Fonseca, Cachaldora, & Carballo, 2013).

Present research on protein hydrolysates mostly uses enzymatic hydrolysis because

proteolytic enzymes can cleave proteins at specific peptide bonds resulting in formation of

uniform and highly functional, nutritional as well as bioactive hydrolysates (Mullally et al.,

1994).

The main step involved in the production of FPH using exogenous enzymes is

outlined in the Figure 1-1. Fish fillets are homogenized in distilled water, the pH and/or

temperature of the slurry adjusted to optimum conditions and hydrolysis is initiated by

adding the enzyme. The hydrolysis reaction is allowed to continue for a period ranging

from few minutes to several hours depending on the target molecular weight range of the

peptide, enzyme activity and other factors.

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Figure 1-1.General protocol for the production of FPH

Fish muscle

(or underutilized by-product)

Homogenization in water

Temperature and/ or pH

adjustment

Enzyme addition

Enzyme inactivation

Supernatant collection and

hydration

Fish protein hydrolysate

Degree of hydrolysis

measurement

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The primary drawback of protein hydrolysates use in food industry is their potential

for giving a bitter taste due to release of bitter hydrophobic peptides (Szente & Szejtli,

2004).

1.2.2.1 Enzymatic Hydrolysis Variables

The functional and bioactive properties of FPH from enzymatic hydrolysis depend

on the extent of hydrolysis. The extent of hydrolysis in turn depends on various factors

applied during enzymatic reactions such as temperature, pH, enzyme to substrate ratio (%

E/S) and substrate concentration (S) (Cinq-mars, 2002; Adler-Nissen 1986). Apart from

these factors, the extent of hydrolysis depends on number of other factors such as

homogenization step which influences enzyme-substrate access, the choice of substrate

(fish species used), and protease used (Dalton, 1995; Kristinsson and Rasco, 2000). The

choice of substrate and enzyme specificity are important factors influencing amino acid

sequence of peptide or amino acid residues leading to different functional and bioactive

properties. The enzymes can be either endoprotease or exoprotease resulting in peptides or

free amino acids respectively yielding very different hydrolysate properties.

The extent of hydrolysis in a proteolytic reaction, which is equivalent to degree of

hydrolysis, can be measured using the method described by Adler-Nissen (1979). In this

method, degree of hydrolysis is defined as the percentage of ratio of the number of cleaved

peptide bonds to total number of bonds per unit weight of the protein (htot = meq/ g protein),

calculated from amino acid composition of substrate (Adler-Nissen, 1979).

%DH = h/ htot * 100 %

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In this method the hydrolysis equivalence is determined by quantifying the

chromophore formed by the reaction of Trinitro benzenesulfonic acid (TNBS) with primary

amines (Figure 1-2) using spectrophotometer at 340 nm. The reaction takes place under

alkaline conditions and is terminated as the pH is lowered (Alder-Nissen, 1979).

Figure 1-2. Reaction between TNBS and primary amines (Alder-Nissen, 1979)

1.2.2.2 Improved Nutritional Quality of FPH

High quality protein with high solubility that can replace milk is required in animal

feeding especially young animals. Young animal are raised on variety of protein sources

to substitute partially or completely for their mother’s milk. However most of the alternate

protein sources are poorly digested compared to casein. Studies have shown that FPH fed

to animals exhibited excellent digestibility and also good amino acid profile (Rebeca, Pena‐

Vera, & Diaz‐Castaneda, 1991). Nutritional studies on calves have shown no negative

effect on the growth when two third portion of milk protein is replaced with FPH (Diaz-

Castaneda & Brisson, 1987). In another study, addition of FPH to the diet of laboratory

rats showed an increased growth rate and higher body weight compared to rats fed on a

diet containing equal amounts of casein (Atia and Shekib, 1992). Anggawati et al., (1990)

(Anggawati, Murtini, & Heruwati, 1990) found that 3 % replacement of fishmeal with FPH

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was enough to improve the weight of Penaeusmonodon shrimp. Based on the positive

results of FPH on the animals, it is expected to have positive results on the humans as well.

Apart from improving the growth rate and digestibility, the protein hydrolysis of

fish muscle resulted in high availability of essential amino acids. Since the essential amino

acids have high probability of absorption when protein is predigested (Boza et al., 1995).

In fact, the lysine content which is deficient in many cereal diets (2.6 – 3.8 g/ 100 g protein)

is found to be higher in fish protein (8.5 g/ 100 g protein) where the minimum requirement

of lysine for an average adult is about 4.5 g/ 100 g protein (Liceaga-Gesualdo and Li-Chan,

1999; Pellett & Ghosh, 2004). Including FPH in a diet containing cereals may fulfill the

amino acid requirements that is easy to absorb and this could have application for the

patients with digestive disorder or for young individuals with limited digestibility.

1.2.2.3 Improved Functional Properties of FPH

Apart from the nutritional point of view, the overall behavior and performance

(functional property) of the protein in a food system during processing, storage and

consumption quality are very important factors to be considered by food processors. FPH

from enzymatic hydrolysis are found to have several functional properties such as: high

solubility (Pacheco-Aguilar et al., 2008; Sugiyama et al., 1991), water holding capacity

(Cheung et al., 2009; Slizyte et al., 2009), emulsifying capacity and stability (Slizyte et al.,

2009; Pacheco-Aguilar et al., 2008; Liceaga-Gesualdo and Li-Chan 1999), and foaming

capacity and stability (Pacheco-Aguilar et al., 2008; Liceaga-Gesualdo and Li-Chan 1999).

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Solubility is the most the important functional property of FPH because it will

influence other functional properties such as emulsification and foaming (Wilding et al.,

1984). Solubility of a protein is influenced by hydrophobic and hydrophilic groups.

Generally, most of the native proteins have hydrophobic amino acids in the interior of their

tertiary structure. During hydrolysis, enzymes cleave the peptide bonds in the protein and

release the hydrophobic groups. In addition, there is an increase in hydrophilic ionic

residues of amino and carboxyl groups at the cleavage sites. These ionic residues produce

electrostatic repulsion between the peptides and increases solubility (Kinsella, 1982).

The ability of proteins to bond water and retain it against gravitational force or from

evaporation within a protein matrix, such as protein gels, beef, fish muscle, is referred to

as Water–holding capacity (WHC) or Water–binding capacity (WBC) of a protein. Water-

holding capacity is one of the important properties of proteins which influence the texture

and integrity of the food products like frozen fish fillets or meat (Cheung et al., 2009).

WHC in a food system depends on protein-protein and water–protein interactions

(Kristinsson and Rasco, 2000). Since FPH are highly hydrophilic when compared to intact

fish proteins they have a tendency to react with water molecules in addition to proteins

(Kristinsson and Rasco, 2000; Buinov et al., 1977).

A good emulsifier should effectively lower the interfacial tension between

hydrophobic and hydrophilic components in a food system (Zayas, 1997). Therefore, in

order to have emulsifying capacity, an emulsifier should have both hydrophilic and

hydrophobic parts that orient towards oil and water phases respectively. Other than

emulsifying capacity, a good emulsifying agent should also stabilize the created emulsion.

In addition, the formed emulsifier film around the oil-water interface should withstand

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Vander Waals forces between droplets to avoid agglomeration for emulsion stability

(Zayas, 1997). In most of the intact proteins, the denaturing step helps the proteins to unfold

their hydrophobic interior and results in a hydrophobic-hydrophilic equilibrium. However,

bulkiness of some intact proteins results in less solubility in water and thereby inefficient

emulsifying activity. Enzymatic hydrolysis of intact proteins results in release of amino

acids containing hydrophobic groups as well as increase in hydrophilic COOH and NH2

groups. Apart from releasing both hydrophobic and hydrophilic groups, enzymatic

hydrolysis results in smaller peptide chains with high solubility that can orient at the

interface of the emulsion very rapidly. The extent of hydrolysis of fish protein also has an

effect on its emulsifying property. For example, very smaller peptides cannot reorient

completely at the interface, this causes interfacial tension and ultimately leading to

emulsion instability. Therefore, hydrolysis of proteins should be carefully controlled to

yield maximum emulsifying properties. It is generally accepted that for good emulsifying

and interfacial properties, a peptide should have a minimum length of 20 amino acid

residues (Gauthier et al., 1993).

Foaming properties rely on the surface properties of proteins similar to those

described for emulsifying properties. Food foams are made up of air bubbles dispersed in

liquid, where a foaming agent or surfactant lowers the surface or interfacial tension at the

liquid-air interface in order to stabilize foam (Kinsella 1976). In a foam matrix the

hydrophobic group of the protein extends into the air and hydrophilic groups into the

aqueous phase, which in turn is responsible for lowering interfacial tension between the

two phases (Kristinsson and Rasco, 2000). This is attributed to release of hydrophobic side

chains amino acids and also formation of free hydrophilic groups (COOH and NH2) upon

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hydrolysis. Apart from this enzyme hydrolysis results in smaller peptide, which helps to

absorb rapidly on the air-water interface very rapidly compared to intact proteins.

1.2.2.4 Bioactive Properties of FPH

Bioactive peptides are peptides that have physiological function within the body

apart from having nutritional value. Some bioactive peptides are naturally present in the

foods and the majority are integrated in the proteins, which are released during in-vitro

digestion enzyme (Korhonen & Pihlanto, 2003).

Bioactive peptides are peptides that have physiological function within the body

beyond basic nutritional value. Some bioactive peptides are naturally present in the foods

and the majority are integrated in the proteins, which are formed during gastrointestinal

digestion or food processing enzyme (Korhonen & Pihlanto, 2003). Based on structural

properties, amino acid composition and peptide sequence, FPH exhibits different

bioactivity such as antimicrobial, antioxidant, anticarcinogenic, anticoagulation and

antihypertensive (Kitts & Weiler, 2003; Korhonen & Pihlanto, 2003).

Cardiovascular disease (CVD) is leading problem in developed countries and high

blood pressure is one of the risk factor for CVD onset. Angiotensin-I converting enzyme

(ACE) plays an important role in regulating blood pressure and it converts inactive

angiotensin-I into its active form angiotensin-II, which is a vascoconstrictor, resulting high

blood pressure. Studies have shown that several food proteins and peptides may have

potential ACE inhibitory activity (Pihlanto-Leppälä, 2000). Most of these peptides are

derived from casein (FitzGerald & Meisel, 2000; Maeno, Yamamoto, & Takano, 1996;

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Yamamoto, Akino, & Takano, 1994); Soy (Kuba, Tanaka, Tawata, Takeda, & Yasuda,

2003; J. Wu & Ding, 2002), egg (Miguel, Recio, Gómez-Ruiz, Ramos, & López-Fandiño,

2004; Yoshii, Tachi, Ohba, Sakamura, Takeyama, & Itani, 2001), and fish protein

(Bougatef, Nedjar-Arroume, Ravallec-Plé, Leroy, Guillochon, Barkia, et al., 2008; Byun

& Kim, 2001; Ichimura, Hu, Aita, & Maruyama, 2003). In recent years many studies

concentrated on aquatic food-derived peptides and their ACE inhibitory activity. Raghavan

& Kristinsson (2009) (Raghavan & Kristinsson, 2009) found that Tilapia protein

hydrolysates with 25% DH prepared using Cryotin and Flavourzyme® enzyme have an

optimum ACE inhibitory activity in vitro. In addition, they also found that low molecular

weight peptides showed higher ACE inhibitory activity when compared to high molecular

weight peptides. Jung et al., (2006) (Jung, Mendis, Je, Park, Son, Kim, et al., 2006) also

found that Yellow fin sole protein hydrolysates with molecular weight <5 kDa are more

effective in inhibiting ACE activity in vitro than high molecular weight hydrolysates. In

vivo studies were also conducted to study efficiency of fish protein hydrolysates to inhibit

ACE activity. Fehmi et al., (2004) (Fahmi, Morimura, Guo, Shigematsu, Kida, & Uemura,

2004) found a significant drop in the blood pressure of hypertensive rats when 300 mg of

purified peptide from sea bream scale hydrolysates were fed and purified peptides showed

higher inhibition than the commercial hypertension drug, enalapril maleate. Je et al., (2004)

(J.-Y. Je, Park, Kwon, & Kim, 2004) purified a novel ACE inhibitory peptide with

maximum inhibition (or minimum IC50) of all the peptides obtained during pepsin

hydrolyzed Alaska Pollack protein and found that ACE inhibition pattern of the peptide

was found to be non-competitive inhibition.

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Peptides from various sources such as egg whites(K. Watanabe, Tsuge,

Shimoyamada, Ogama, & Ebina, 1998), soy proteins (S. E. Kim, Kim, Kim, Kang, Woo,

& Lee, 2000) and broccoli (Matusheski, Swarup, Juvik, Mithen, Bennett, & Jeffery, 2006)

have indicated to have anticancer activity. However, studies on anticancer activity of FPH

are very limited. Peptide fractions from anchovy sauce induced apoptosis in a human

lymphoma cell lines and those peptide are mostly composed of Ala and Phe in their peptide

sequence. Picot et al., (2006) has evaluated antiproliferation activity of 18 commercial fish

protein hydrolysates on two human breast cancer cells. Among them blue whiting, cod,

plaice and salmon hydrolysates have shown to inhibit growth of two cancer cell lines (Picot,

Bordenave, Didelot, Fruitier-Arnaudin, Sannier, Thorkelsson, et al., 2006). Papain

hydrolyzed tuna dark muscle proteins were tested for their antiproliferation activity on the

human breast cancer cell line and found that peptide fractions with molecular weight

ranging from 390 to 1400 Da (Hsu, Li-Chan, & Jao, 2011).

Natural antimicrobial agents that can replace current artificial antimicrobial agents

is an urgent need to food industry. Additionally, antimicrobial peptides are more preferred

to conventional bactericidal antibiotics as they can kill bacteria faster and are unaffected

by antibiotic resistance mechanism (Shahidi & Zhong, 2008). At present natural

antimicrobial agents such as lactoperoxidase (milk), lysozymes (egg white, figs),

lactoferricin (milk), nisin (lactic acid bacteria), chitosan (shrimp shells) and other

bacteriocins are being investigated extensively (Devlieghere, Vermeulen, & Debevere,

2004). Most of the documented antimicrobial peptides derived from marine source are

naturally secreted in the form of fish exudes as a host defense mechanism (Najafian &

Babji, 2012; J.-G. Zhou, Wei, Xu, Cui, Yan, Ou-Yang, et al., 2011). Very few studies have

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been focused on studying antimicrobial peptides derived from fish proteins by enzyme

hydrolysis. The work done by Liu et al., (2008) (Z. Liu, Dong, Xu, Zeng, Song, & Zhao,

2008) is one of the first reference to isolate antimicrobial peptides from Oyster protein

hydrolysates. They found that cysteine rich peptides are more effective against gram

positive bacteria. In another study, the presence of peptides from oyster hydrolysates

prepared using thermolysin inhibited HIV-I protease (T.-G. Lee & Maruyama, 1998).

Peptides from half-fin anchovy has displayed broad in vitro antibacterial activity. Further

Song et al., (2012), have isolated and characterized peptides from the peptic hydrolysis of

half-fin anchovy with antibacterial activity against Escherichia coli.

Blood coagulation is a process of stopping the blood flow whenever an abnormal

vascular condition or when vascular injury occurs. An external or endogenous

anticoagulant is used as a medication to prolong/stop the blood coagulation (Jung, Je, Kim,

& Kim, 2002). Heparin is widely used commercial anticoagulant used during

thromboembolic disorders. However, the use of heparin is associated with several side

effects and there is a need to develop an alternative (Pereira, Melo, & Mourão, 2002). Not

many studies are done on marine peptides as a source of anticoagulant. In some of the

studies marine derived anticoagulant peptides have been isolated from yellow fin sole

(Rajapakse, Jung, Mendis, Moon, & Kim, 2005), blood ark shell(Jung, Je, Kim, & Kim,

2002), starfish (Koyama, Noguchi, Aniya, & Sakanashi, 1998) and blue mussels(Jung &

Kim, 2009). Most of the above peptides have shown to prolong the activity time of

coagulating factors such as, activated partial thromboplastin (APTT), prothrombin (PT)

and thrombin (TP). The activity was comparable to heparin, for example, purified peptides

from blue mussels shown to prolongate the clotting time of APTT and TP from 35.3±0.5

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seconds to 321±2.1 seconds and 11.6±0.4 seconds to 81.3±0.8 seconds respectively(Jung

& Kim, 2009). In addition to anticoagulant of these marine peptides, they are also non-

toxic at usage level unlike Heparin.

Among all bioactive properties, the antioxidant property of FPH has received great

interest in the pharmaceutical and health-food industries. Due to increasing necessity to

utilize proteins from fish by-products and under-utilized fish, researchers have started

concentrating on FPH as a source of antioxidants in food system as well as living systems

(Raghavan, Kristinsson, Thorkelsson, & Johannsson, 2010).

1.3 Fish Bioactive Peptides as Antioxidants

1.3.1 Antioxidants

Antioxidants are compounds or systems that delay oxidation by inhibiting or

interrupting the propagation of free radicals by one or more of several mechanisms

including – 1. Scavenging radical species that propagate peroxidation, 2. Chelate metal ion

such that they cannot generate reactive species, 3. Quenching ROS preventing formation

of peroxides, 4. Breaking the autoxidation chain reaction, and 5. Reducing localized

oxygen content (Fennema, Damodaran, & Parkin, 2008). For efficiency of antioxidants,

apart from chemical potency, its accessibility to radicals is required for effectiveness of its

activity (Y. Watanabe, Nakanishi, Goto, Otsuka, Kimura, & Adachi, 2010). Antioxidants

are used in the food systems as well as to combat oxidative stress related disease conditions.

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1.3.2 Antioxidants Application in Food Systems

Oxidation in food systems has many deleterious effects primarily by decreasing

sensorial and nutritional value of the product. In addition, consumption of oxidized food

pose health hazards by increasing reactive oxygen species (ROS) in the body. The factors

effecting the oxidative rancidity include: fatty acid (FA) composition, surface area in

contact with oxygen, light, temperature, irradiation, water activity, and presence of pro-

oxidants such as metal ions (Kofakowska, 2010). Oxidation in food is contributed by lipid

oxidation, protein oxidation and oxidation of lipid soluble minor food components. Lipid

oxidation is the main form of oxidation in lipid or emulsion based food systems. In meat

products, apart from lipid oxidation, protein oxidation is also prominent.

Extensive research has been done to study lipid oxidation and their implications in

food systems. Food lipids undergo both enzymatic and non-enzymatic rancidity (Loliger,

1991). Non-enzymatic oxidation of lipid pathways includes autoxidation, singlet oxygen-

mediated mechanisms, or photo-oxidation. Whereas, lipid oxygenases catalyzed oxidation

of lipids contribute to enzymatic oxidation (Choe & Min, 2005; Min & Boff, 2002). Lipid

oxidation is a complex chain reaction which give rise to oxidation products such as ketones,

aldehydes, epoxides, hydroxyl compounds, oligomers and polymers. These oxidative

products have undesirable sensorial and biological effects (Barriuso, Astiasarán, &

Ansorena, 2013; Márquez-Ruiz, Holgado, Garcia-Martinez, & Dobarganes, 2007).

The significance of protein oxidation in deterioration of food quality has been

largely unexplored (M. N. Lund, Heinonen, Baron, & Estevez, 2011). However, it is known

that ROS react with proteins and peptides altering their structure by cleaving peptide bonds,

modifying amino acid side chains or forming intermolecular cross linked protein

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derivatives (Davies, 2003; M. Lund, Luxford, Skibsted, & Davies, 2008). All these

modifications can influence their functionality and therefore affect the sensory properties

of the food (Decker, Faustman, & Lopez-Bote, 2000).

Addition of antioxidants in foods improves quality and sensory attributes such as

flavor, taste, color and texture of the product (Barnham, Masters, & Bush, 2004) . Usually,

commercially available synthetic antioxidants such as butylated hydroxyanisole (BHA),

butylated hydroxytoluene (BHT) etc., are added in small quantities. However the usage of

BHA and BHT has become a controversial issue due to their possible adverse toxicological

data (Madhavi, Deshpande, & Salunkhe, 1995). However, the publication on toxicological

data of these compounds is still contradicting (Carocho & Ferreira, 2012).

1.3.3 Antioxidants and Human Health

Normal biological processes in the human body, including aerobic respiration,

involve production of ROS, by-products of oxidative metabolism and free radicals such as

hydroxyl radicals (•OH), peroxyl radicals (•OOR), Super oxide anion (O2-•), and

peroxynitrite (ONOO-). Apart from the endogeneous radicals, radicals derived from

external or environmental origin such as tobacco, radiation, toxins, excessive minerals and

air pollution can increase ROS in the body. All the aerobic organisms. including humans,

through evolution have developed endogenous antioxidant defense systems to protect

against oxidative damage. The endogenous defense systems are very dynamic and depends

on many factors such as composition of diet, emotional state that influence rate of

antioxidant synthesis and secretion. In a healthy individual an equilibrium between

oxidative species and endogenous antioxidant defense systems is maintained (Power,

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Jakeman, & FitzGerald, 2013). However this antioxidant – pro-oxidant balance in the body

can be altered with the progression of age and also due to other factors such as exposure to

environmental pollution, fatigue, high fat diet, and excessive caloric intake. Age

advancement will gradually diminish absorption of supplied nutrients as well as reduces

plasma and cellular antioxidant potential (Rizvi et al., 2006; Elmadfa and Meyer 2008).

Further, environmental factors mentioned above can weaken the body’s immunity and will

make it vulnerable to oxidative stress. Oxidative stress may damage cell membranes,

oxidation of proteins, membrane lipids and also damage DNA/ RNA (Powers & Jackson,

2008). Over time, oxidative stress compromises normal cellular function and may lead to

many diseases including – cardiovascular diseases (Lakshmi, Padmaja, Kuppusamy, &

Kutala, 2009; Rochette, Lorin, Zeller, Guilland, Lorgis, Cottin, et al., 2013),

neurodegenerative disorder (Barnham, Masters, & Bush, 2004; Katzman & Saitoh, 1991)

and cancer (Reuter, Gupta, Chaturvedi, & Aggarwal, 2010). Consumption of exogenous

dietary antioxidants will potentially promote health benefits by increasing the body’s

antioxidant load (Samaranayaka, 2010). Over the past two decades, the use of dietary

antioxidant supplements has expanded exponentially. Currently used antioxidant

supplements contain α-tocopherol, vitamin C, or plant derived antioxidant compounds such

as phytochemicals and extracts such as isoflavones, lutein, lycopene, green tea and grape

seed extracts. Addition of antioxidant peptides or protein hydrolysates to these supplements

is a new trend and use of these peptides into food products or natural health foods is

expected to have a huge market (Samaranayaka, 2010).

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1.3.3.1 Oxidative Stress and Diseases

1.3.3.1.1 Cardiovascular Diseases

The link between oxidative stress and cardiovascular disease symptoms including

atherosclerosis, hypertension and heart failure is now widely accepted (Takahashi & Hara,

2003). The above symptoms are mainly initiated by endothelial dysfunction which is

manifested by several pathological conditions including decrease in barrier properties,

altered anti-inflammatory properties and anticoagulant of the endothelium (Cai & Harrison,

2000). The endothelium layer, the inner layer of blood vessels secretes nitric oxide (NO),

which is potent vasodilator, decreases permeability, anti-proliferative and has anti-

inflammatory properties. ROS produced in the body can decrease NO bioavailability by

decreasing endothelial cell NO synthase expression (eNOS) (Wilcox, Subramanian,

Sundell, Tracey, Pollock, Harrison, et al., 1997), limiting the substrate or cofactor for

eNOS (Pou, Pou, Bredt, Snyder, & Rosen, 1992), altering the cellular signal (Shimokawa,

Flavahan, & Vanhoutte, 1991) or simply degrading NO. Decrease in NO bioavailability

causes vasoconstriction, increases permeability of leukocyte into the subendothelial layer

causing atherogenesis. In addition, ROS readily oxidize low density lipoprotein (LDL) to

produce oxidized–LDL (Ox-LDL). Ox-LDL are readily absorbed by sub-endothelial

leukocyte-derived macrophage, initiate formation of fatty cells and contribute to the

formation of atheroscelorotic plaque. Both inactivation of NO and formation of OX-LDL

by ROS increases hypertension that characterize disease progression (Lakshmi, Padmaja,

Kuppusamy, & Kutala, 2009). Research indicates that consumption of PUFA in

combination with dietary antioxidants can reduce atherosclerosis in animal models

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(Eilertsen, Mæhre, Cludts, Olsen, & Hoylaerts, 2011; Eilertsen, Mæhre, Jensen, Devold,

Olsen, Lie, et al., 2012; Verschuren, Wielinga, van Duyvenvoorde, Tijani, Toet, van

Ommen, et al., 2011).

1.3.3.1.2 Neurodegenerative Diseases

Neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s

disease (PD) and amytrophic lateral sclerosis (ALS) are associated with protein aggression

and loss of neuronal cell population. These diseases are often associated with extensive

oxidative stress causing dysfunction or death of neuronal cells and leads to disease

pathogenesis (Barnham, Masters, & Bush, 2004). The brain is very vulnerable to oxidative

stress since it uses high amount of oxygen, pro-oxidative transitional minerals and lower

transfer of dietary antioxidants. The oxidative damage of DNA/RNA results in

hydroxylations and significant protein carboxylation and nitration in brains (Butterfield,

Castegna, Pocernich, Drake, Scapagnini, & Calabrese, 2002; Elmadfa & Meyer, 2008).

Such oxidation stress will lead to neurodegenerative disease and neuronal cell death. High

concentration of 4-Hydroxy-2,3-nonenal (HNE), a lipid peroxidation chain reaction

product, has been observed in PD and AD patient’s brain tissues (Allan Butterfield,

Castegna, Lauderback, & Drake, 2002; Dexter, Carter, Wells, Javoy‐Agid, Agid, Lees, et

al., 1989; Selley, Close, & Stern, 2002). The lipid peroxide products acrolein and HNE

induce crosslinking of lysine, cysteine and histidine via Michael addition causing toxicity.

This modification of proteins impair endogeneous antioxidant enzymes and result in

increased ROS content (Tamagno, Robino, Obbili, Bardini, Aragno, Parola, et al., 2003).

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The increased ROS concentration results in further DNA/RNA and protein mutation. Diet

derived antioxidants may delay or prevent the onset or progression of these diseases

(Butterfield, Castegna, Pocernich, Drake, Scapagnini, & Calabrese, 2002).

1.3.3.1.3 Cancer

Normal (somatic) cells multiple when needed, die eventually and are replaced by

new cells. Cancer is caused by uncontrolled growth of abnormal cells in the body (Moscow

& Cowan, 2007). The disease development is characterized by three phases – initiation,

promotion and progression. During initiation phase, the normal cell activity is challenged

by exogenous and endogenous factors (carcinogens). Various carcinogens are believed to

generate ROS in the body that can damage significantly cell structure and function forming

cancerous cells (Jensen, Eilertsen, Mæhre, Elvevoll, & Larsen, 2013; Waris & Ahsan,

2006). In the promotion phase, the cancerous cell surrounded by normal cells will grow

tremendously than other cells and causing inadequate supply of nutrients to other cells

(Brannon-Peppas & Blanchette, 2012). The progression step is characterized by rapid cell

division of cancerous cells, increased invasiveness and ultimately causing metastasis. In

addition, ROS induced DNA mutation can inactivate organism detoxification systems and

DNA repair systems in the body that will try to neutralize the initialization and progression

phases (Visconti & Grieco, 2009).

Many institutions such as National Research Council and American Cancer Society,

emphasize the importance of healthy food habits and dietary factors in prevention of cancer

(Marmot, Atinmo, Byers, Chen, Hirohata, Jackson, et al., 2007). Some of the food

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components that have radical scavenging activity act as antioxidants and are suggested to

have cancer prevention properties. This assumption is still controversial with few studies

showing no correlation between food derived antioxidant and cancer prevention (Annema,

Heyworth, McNaughton, Iacopetta, & Fritschi, 2011; Aune, Lau, Chan, Vieira, Greenwood,

Kampman, et al., 2011; Boffetta, Couto, Wichmann, Ferrari, Trichopoulos, Bueno-de-

Mesquita, et al., 2010; Büchner, Bueno‐de‐Mesquita, Ros, Kampman, Egevad, Overvad,

et al., 2011; Goodman, Bostick, Kucuk, & Jones, 2011)

1.3.4 Endogenous Antioxidant Defense System

In a healthy individual there is a balance between ROS and the endogenous

antioxidant defense system. The endogenous antioxidant system includes some

endogenous enzymes such as superoxide dismutase (SOD), catalase (CAT), and

glutathione peroxidase (GSH-Px) as well as various endogeneous amino acid or peptides

(Decker and Xu 1998). Endogenous peptides such as glutathione (γ-Glu-Cys-Gly) (GSH),

carnosine (β-alanyl-L-histidine), arserine (β-alanyl-L-1-methylhistidine) and ophidine (β-

alanyl-L-3-methylhistidine) are produced naturally in muscles and can act as antioxidants

(Chan and Decker 1994; Babizhayev et al., 1994).

In cellular metabolism, oxygen and its radicals are reduced to water molecule after

forming intermediates by endogenous antioxidant systems. The conversion of oxygen to

water occurs in mitochondria by enzymatic antioxidants and all the intermediates during

reduction will not leave mitochondria until converted to water. During cellular metabolism,

oxygen is converted into superoxide by cellular superoxide production mechanism in the

mitochondria (Freeman & Crapo, 1982; Roede & Jones, 2010). The formed superoxide is

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converted to hydrogen peroxide (H2O2) by SOD. CAT, GSH-Px and other peroxidase

enzymes convert H2O2 to water. However, these conversions may not occur as expected

and lead to highly potent oxidants. Superoxide reacts with H2O2 to form hydroxyl free

radicals which are more reactive than superoxide anions. H2O2 is stable in the absence of

metal ions, however in presence of transition metals it contribute to the formation of singlet

oxygen, other ROS (Rodrigo, Miranda, & Vergara, 2011; Webster & Nunn, 1988).

Apart from enzymatic antioxidants, endogenous antioxidant peptides and non-

enzymatic/ non-protein antioxidants also contribute overall antioxidant capacity in cells.

GSH, an electron donor, protect cell from free radicals by reducing lipid peroxides in

presence of catalyst GSH-Px (Bray & Taylor, 1994; Chan, Decker, & Feustman, 1994;

Christophersen, 1969). Carnosine, anserine and ophidine are histidine containing

dipeptides occur in the skeleton muscle of most vertebrates. Since they share similar

structure, they possibly have similar biological role (Boldyrev, 1986). Carnosine and

anserine have been postulated to have free radical scavenging activity as well as metal ion

chelating ability (Kang, Kim, Choi, Kwon, Won, & Kang, 2002). They demonstrated

antioxidant activity in vivo and in vitro in rat skeletal muscle under oxidative stress

(Nagasawa, Yonekura, Nishizawa, & Kitts, 2001).

1.3.5 Natural Antioxidants in Food and Biological Systems

Addition of antioxidants in food products shown to increase shelf life by preserving

quality and safety of the product. Synthetic antioxidants (BHA, BHT and propyl gallate)

are extensively used as food additives but their safety is still questioned (M. Brewer &

Rojas, 2008; M. S. Brewer, Sprouls, & Russon, 1994). Additionally, one of the top trends

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for the food industry is the use of clean labeling (Hillman, 2010), with natural food

ingredients and additives that are perceived as healthy (Joppen, 2006). Furthermore,

increasing attention has been paid to the role of diet in human health. This stimulated

growing interest in replacing synthetic antioxidants with natural antioxidants from food

sources for the potential health benefits and food safety. Many naturally occurring

antioxidants from herbs and spices have been extensively studied (M. Brewer, 2011;

Maestri, Nepote, Lamarque, & Zygadlo, 2006; Y. Zhang, Yang, Zu, Chen, Wang, & Liu,

2010). In food systems, rosemary extracts and different forms of vitamin C have been

widely used. Several epidemiological studies have indicated that antioxidants from fruits

and vegetables (vitamin C, vitamin E, carotenoids and phenolic compounds) is associated

with prevention of chronic diseases that are caused by oxidative stress (Gosslau & Chen,

2004; Martínez, Penci, Ixtaina, Ribotta, & Maestri, 2012; Podsędek, 2007). Most of the

dietary based antioxidants such as selenium, α-tocopherol and vitamin C are also essential

to minimize ROS content in the body. In addition to plant derived natural antioxidants,

peptides from plant and animal sources such as soybean (Peñta‐Ramos & Xiong, 2002),

casein (Suetsuna, Ukeda, & Ochi, 2000), whey protein (Peng, Xiong, & Kong, 2009),

wheat protein (E. Y. Park, Imazu, Matsumura, Nakamura, & Sato, 2012), porcine (Q. Liu,

Kong, Xiong, & Xia, 2010), and egg protein (Chay Pak Ting, Mine, Juneja, Okubo,

Gauthier, & Pouliot, 2011) have demonstrated antioxidant activity. Recently antioxidant

peptides derived from different fish proteins have been studied.

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1.3.6 Fish Protein Hydrolysates as a Source of Antioxidants

Hydrolysis of protein from plant sources (soybean, rice, canola, quinoa seeds) and

animal sources (milk, eggs, porcine, marine) have shown to possess antioxidant properties.

Owing to limited biological resources, concerns of environmental pollution and in an

attempt to use fish by-products/ underutilized fish, research emphasis in utilizing marine

proteins as alternative antioxidant source is increasing. Many studies have been performed

to evaluate antioxidant properties of protein hydrolysates from different fish species. One

of the first studies demonstrating antioxidant activity from marine sources was published

in 1990 using sardine protein (Hatate, Numata, & Kochi, 1990). They found that sardine

protein hydrolysates and commercial antioxidants synergistically inhibit oxidation of

linoleic acid. Recently, many researchers emphasize the potential application of FPH as an

alternative source of antioxidants. Potent antioxidant peptides have been identified from

many seafood sources such as oyster (Qian, Jung, Byun, & Kim, 2008), blue mussel (Jung,

Rajapakse, & Kim, 2005), hoki frame proteins (S.-Y. Kim, J.-Y. Je, & S.-K. Kim, 2007),

Pacific hake (A. G. Samaranayaka & E. C. Li-Chan, 2008), tuna (J.-Y. Je, Z.-J. Qian, H.-

G. Byun, & S.-K. Kim, 2007), Atlantic cod (Ngo, Ryu, Vo, Himaya, Wijesekara, & Kim,

2011), and grass carp (Ren, Zhao, Shi, Wang, Jiang, Cui, et al., 2008). These peptides have

shown greater potential as natural antioxidant in both food systems and in nutraceutical or

pharmaceutical industries. Few studies were performed on antioxidant activity of silver

carp protein hydrolysates (SPH) obtained from China (S. Dong, M. Zeng, D. Wang, Z. Liu,

Y. Zhao, & H. Yang, 2008; Zhong, Ma, Lin, & Luo, 2011). Promising radical scavenging

and lipid peroxidation activities was shown by silver carp protein hydrolysates (S. Dong,

M. Zeng, D. Wang, Z. Liu, Y. Zhao, & H. Yang, 2008). However to date multifaceted

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antioxidant activity using different chemical based assays, as well as influence of peptide

size, peptide molecular weight and peptide sequence has not been fully explored for silver

carp protein hydrolysates (SPH) from the Mississippi River System.

1.3.7 Mechanism of Antioxidant Peptides

The mechanism of bioactive peptides with respect to their structure is not fully

understood. Most of the proposed mechanisms were based on some common

characteristics of most antioxidant peptides or using studies done on synthetic peptides.

Quantitative structure activity relationship (QSAR) is a statistical modeling software is

generally used to study relationship between structure of synthetic peptide and its activity

(T. Wang, Zhao, & Wang). The schematic description of different antioxidant mechanism

used by antioxidant peptides is shown in Figure 1-3. The most common characteristics that

are responsible for the different mechanisms include molecular weight of the peptide,

hydrophobicity, amino acid composition and amino acid sequence.

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Figure 1-3. Schematic diagram illustrating both chemical and physical antioxidant

mechanism by 1. Metal chelating ability, 2. Radical scavenging activity and 3. Lipid

peroxidation ability of antioxidant peptides (Xiong, 2010)

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1.3.7.1 Molecular Weight of the Peptide

Peptide molecular weight is considered as a crucial factor in determining its

bioactivity. In general, most of the antioxidant peptides have 2-20 amino acids and are

more potent than their parent protein molecule (20 to 50 amino acids or more) (Chen,

Suetsuna, & Yamauchi, 1995; Ranathunga, Rajapakse, & Kim, 2006; H.-C. Wu, Chen, &

Shiau, 2003). Low molecular weight peptides are believed to have easier accessibility to

lipid radicals than their bulky parent proteins or larger peptides and can inhibit free radical

mediated lipid peroxidation (Ranathunga, Rajapakse, & Kim, 2006). For example, among

the four molecular fractions (5-10, 3-5, 1-3 and <1 kDa) of Hoki frame protein hydrolysates,

the lower molecular weight fractions (1-3 kDa) showed higher antioxidant activity.

However, the lowest molecular weight peptide fraction (<1 kDa) from Hoki frame protein

hydrolysate exhibited weakest antioxidant activity (Kim, J.-Y. Je, & S.-K. Kim, 2007).

Similarly, the radical scavenging activity of porcine protein hydrolysates increased with

degree of hydrolysis (DH) initially but decreased when the DH reached certain value (85%)

(B. Li, Chen, Wang, Ji, & Wu, 2007). During hydrolysis of proteins, the peptide fractions

with superior antioxidant activity are released from inactive parent proteins. Further

hydrolysis will result in intense degradation of formed peptides thereby lowering their

antioxidant activity (Moure, Dominguez, & Parajo, 2006; X. Zhou, Wang, & Jiang, 2012).

Therefore extent of hydrolysis should be optimized to release antioxidant peptides by

carefully controlling hydrolysis conditions.

In food systems, antioxidant peptides can form a protective membrane around the

lipid droplets in the emulsion system, prevent penetration or diffusion of radicals and

prevent lipid oxidation. In order to function as protective membrane, peptides should have

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structural integrity and extreme hydrolysis will result in free amino acids or very small

peptides which cannot form this protective membrane (Kong & Xiong, 2006; Peña‐Ramos,

Xiong, & Arteaga, 2004).

1.3.7.2 Hydrophobicity

Several studies indicate that peptide fractions with hydrophobic amino acids

showed higher radical scavenging activity, metal chelating activity and inhibited lipid

peroxidation. Hydrophobic–hydrophilic balance in peptides will help in facilitating water-

lipid interface and particularly hydrophobic amino acids scavenge lipid soluble radicals in

the food matrix (Figure1-2) (Chen, Suetsuna, & Yamauchi, 1995; Rajapakse, Mendis,

Byun, & Kim, 2005; Q. Sun, Luo, Shen, Li, & Yao, 2012). In addition, hydrophobic amino

acids in the peptide may also help in scavenging hydrophobic cellular oxidation targets

such as long chain fatty acids (H.-M. Chen, Muramoto, Yamauchi, Fujimoto, & Nokihara,

1998; Mendis, Rajapakse, Byun, & Kim, 2005). Further, hydrophobic-hydrophilic

equilibrium of the peptide may aid in the penetration of cell membrane (Henriques, Melo,

& Castanho, 2006) and may subsequently scavenge radicals to prevent oxidation in

mitochondria.

Histidine has been recognized for its metal chelating activity, lipid peroxide radical

scavenging, and hydrogen donating capacity (Chan, Decker, & Feustman, 1994). However,

the antioxidant activity of histidine depends on its solubility in the physical environment.

Many studies showed that efficiency in scavenging hydrophobic peroxyl radicals by

histidine improves when attached to hydrophobic amino acid residues such as N-(long-

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chain-acyl) group (S.-Y. Kim, J.-Y. Je, & S.-K. Kim, 2007; Murase, Nagao, & Terao, 1993;

Ranathunga, Rajapakse, & Kim, 2006). Increased hydrophobicity caused by N-(long-

chain-acyl) group in N-(long-chain-acyl)- histidine helps histidine interact with peroxyl

radicals (Murase, Nagao, & Terao, 1993). Sequence analysis of most antioxidant peptides

revealed the presence of hydrophobic amino acid (glycine, leucine and alanine)

representing more than 40 % of the peptide sequence.

1.3.7.3 Amino Acid Composition

Apart from molecular weight and hydrophobicity of the peptides, the presence of

some important amino acid residues plays an important role in its antioxidant activity.

Aromatic amino acids (Tyrosine, phenylalanine, thryptophan & Histadine) are considered

as effective free radical scavengers. Presence of hydroxyl group on aromatic ring of

tyrosine believed to act as a chain breaking antioxidant by hydrogen atom transfer

mechanism (HAT). In addition, its antioxidant activity remains stable via resonance

structure (Ou, Huang, Hampsch-Woodill, Flanagan, & Deemer, 2002; Rajapakse, Mendis,

Jung, Je, & Kim, 2005).

It is well known that the presence of acidic and basic amino acids can chelate Fe2+

and Cu2+ ions (Figure 1-3.). The carboxyl and amino group of acidic and basic amino acids

respectively are responsible for metal-chelating capacity. The carboxyl group in acidic

amino acid can interact with charged residues of metal ions and inhibit their pro-oxidant

activity of metal ion (Saiga, Tanabe, & Nishimura, 2003). Basic amino acid residues

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(Histadine & Lysine) act as electron acceptors and quench free radicals formed during

oxidation reaction and terminate the reaction (Chan, Decker, & Feustman, 1994).

In addition, the presence of glycine and proline also contribute to antioxidant

activity of some peptides. The simple structure of glycine with a single hydrogen atom as

side chain, contributes more flexibility in the peptide structure than other amino acids.

Peptide flexibility helps to form a membrane around the lipid drop in the emulsion system

and inhibits lipid oxidation (Figure 1-3). In biological systems, the flexibility of the peptide

will enhance membrane penetration and increase accessibility to oxidation sites. The cyclic

nature of the rigid pyrolidine ring of proline has an steric effect, which can act as free

radical scavenger (Alemán, Giménez, Pérez-Santin, Gómez-Guillén, & Montero, 2011; C.

Chen, Chi, Zhao, & Lv, 2012; N. Rajapakse, E. Mendis, H. G. Byun, & S. K. Kim, 2005).

1.3.7.4 Amino Acid Sequence

In addition to the presence specific amino acids, the location of the amino acid with

in the sequence of the peptide is also crucial (H.-M. Chen, Muramoto, Yamauchi, &

Nokihara, 1996; Mendis, Rajapakse, & Kim, 2005). The amino acid sequence of the

peptide depends on protein source, protease enzyme specificity, extent of hydrolysis and

hydrolytic conditions. The amino acids at N & C terminal has huge impact on its

antioxidant activity. For example, hydrophobic amino acid at N-terminal has shown to

increase the antioxidant activity of the peptide (Elias, Kellerby, & Decker, 2008; P.-J. Park,

Jung, Nam, Shahidi, & Kim, 2001). The net charge of C-terminal amino acids is also an

important prediction of antioxidant activity. In most of the antioxidant peptides C-terminal

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amino acids are often occupied by tryptophan, glutamic acid, leucine, isoleucine,

methionine, valine, and tyrosine (Power, Jakeman, & FitzGerald, 2013).

The location of the amino acid present in the protein sequence determines its ability

to reach relevant cellular sites (e.g. mitochondria) where free radicals are produced in a

cell. For example, Szeto-Schiller (SS) peptides are reported to be cell permeable,

mitochondria targeted and also can protect mitochondria from cellular oxidation. This

activity of SS peptides is attributed to their unique structural motif containing alternative

aromatic and basic amino acids (Zhao, Zhao, Wu, Soong, Birk, Schiller, et al., 2004). This

ability of some antioxidant peptides make them more efficient cell based antioxidants than

other lipophilic antioxidants such as vitamin E that remains on the cell surface (Hazel H

Szeto, 2008).

1.3.8 Interaction of Food Derived Antioxidant Peptides and Endogenous Antioxidant

System

Well established antioxidants such as ascorbic acid and α-tocopherol under certain

conditions act as pro-oxidants and promote oxidative reaction limiting their therapeutic

efficacy. A novel therapeutic approach includes using external antioxidants to up-regulate

endogenous antioxidant systems. Some food derived antioxidants such as flavonoids, olive

oil phenols and curcumin have shown to positively influence endogenous antioxidant

enzymes. In addition, studies shows that protein hydrolysates from egg (Katayama,

Ishikawa, Fan, & Mine, 2007; Young, Fan, & Mine, 2010), milk protein (Bounous, Batist,

& Gold, 1989; McIntosh, Regester, Le Leu, Royle, & Smithers, 1995), and plant (S.-J. Lee,

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Oh, Ko, Lim, & Lim, 2006; Oh & Lim, 2006) have shown to increase intracellular

antioxidant enzyme/peptides during oxidative stress conditions.

Endogenous antioxidant peptide GSH, a tri-peptide (γ-glutamyl-cysteinyl-glycine),

is of greater importance that protect cell against oxidative damage. The synthesis of GSH

is not only regulated by its synthesizing enzyme but also by the supply and metabolism of

sulfur containing amino acids (cysteine and methionine) (Kim, Kim, Kwon, Park, Kim,

Kim, et al., 2003). In particular, cysteine is the limiting amino acid in GSH synthesis and

also serves as a precursor in the synthesis of taurine, an antioxidant organic acid (Kim, et

al., 2003; Parcell, 2002). In addition, cysteine can also restore activity of γ-glutamyl

cysteine ligase (γ-GCL), which is a rate-limiting enzyme for GSH synthesis (Kim, et al.,

2003).

Carnosine (β-alanyl-L-histidine) and anserine (β-alanyl-L-1-methylhistidine) are

two histidine based antioxidant dipeptides that are formed in skeletal muscles. For the

synthesis of both the peptides, β-alanine and histidine are demonstrated as precursors and

their concentration increases when these amino acids are supplemented through the diet

(Chan, Decker, & Feustman, 1994; Rosario, Wood, & Johnson, 1981).

To date, no study has been conducted to compare the efficiency in influencing

endogenous antioxidant systems by sulfur containing amino acids or histidine when present

in its free form and in a peptide. This will help to understand if bioactive peptides with

these amino acids have any influence. However, Bounous et al., (1989) (Bounous, Batist,

& Gold, 1989) showed that dietary cysteine present in whey protein improved the GSH

synthesis more efficiently compared to free cysteine or from casein protein in mice. This

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observation highlights that these amino acids when present in specific sequence in a peptide

can up-regulate endogenous antioxidant systems.

In conclusion, incorporation of exogenous peptides that has a combination of

radical scavenging activity and that up-regulates endogenous antioxidants would be an

ideal therapy for oxidative stress.

1.3.9 Bioavailability of Antioxidant Peptides

Well established antioxidants such as ascorbic acid and α-tocopherol under certain

conditions act as pro-oxidants and promote oxidative reaction limiting their therapeutic

efficacy. A novel therapeutic approach includes using external antioxidants to up-regulate

endogenous antioxidant systems. Some food derived antioxidants such as flavonoids, olive

oil phenols and curcumin have shown to positively influence endogenous antioxidant

enzymes. In addition, studies shows that protein hydrolysates from egg (Katayama,

Ishikawa, Fan, & Mine, 2007; Young, Fan, & Mine, 2010), milk protein (Bounous, Batist,

& Gold, 1989; McIntosh, Regester, Le Leu, Royle, & Smithers, 1995), and plant (S.-J. Lee,

Oh, Ko, Lim, & Lim, 2006; Oh & Lim, 2006) have shown to increase intracellular

antioxidant enzyme/peptides during oxidative stress conditions.

Endogenous antioxidant peptide GSH, a tri-peptide (γ-glutamyl-cysteinyl-glycine),

is of greater importance that protect cell against oxidative damage. The synthesis of GSH

is not only regulated by its synthesizing enzyme but also by the supply and metabolism of

sulfur containing amino acids (cysteine and methionine) (Kim, et al., 2003). In particular,

cysteine is the limiting amino acid in GSH synthesis and also serves as a precursor in the

synthesis of taurine, an antioxidant organic acid (Kim, et al., 2003; Parcell, 2002). In

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addition, cysteine can also restore activity of γ-GCL, which is a rate-limiting enzyme for

GSH synthesis (Kim, et al., 2003).

Carnosine (β-alanyl-L-histidine) and anserine (β-alanyl-L-1-methylhistidine) are

two histidine based antioxidant dipeptides that are formed in skeletal muscles. For the

synthesis of both the peptides, β-alanine and histidine are demonstrated as precursors and

their concentration increases when these amino acids are supplemented through the diet

(Chan, Decker, & Feustman, 1994; Rosario, Wood, & Johnson, 1981).

To date, no study has been conducted to compare the efficiency in influencing

endogenous antioxidant systems by sulfur containing amino acids or histidine when present

in its free form and in a peptide. This will help to understand if bioactive peptides with

these amino acids have any influence. However, Bounous et al., (1989) (Bounous, Batist,

& Gold, 1989) showed that dietary cysteine present in whey protein improved the GSH

synthesis more efficiently compared to free cysteine or from casein protein in mice. This

observation highlights that these amino acids when present in specific sequence in a peptide

can up-regulate endogenous antioxidant systems.

In conclusion, incorporation of exogenous peptides that has a combination of

radical scavenging activity and that up-regulates endogenous antioxidants would be an

ideal therapy for oxidative stress.

1.3.10 Studying Antioxidants on Intestinal Cell Line

For an antioxidant to display its properties in the human body, it is important for it

to be available to the target site without losing its activity. However, validating the

absorption and bioavailability of these bioactive peptides is very difficult task. Before

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studying its absorption and its transport to the target tissue in the body it is important to

evaluate its effect on intracellular oxidation inhibition in intestinal cells.

The intestine serves as the interface between the organisms in the gut lumen and

the blood stream; therefore it acts as a crucial defense barrier against luminal toxic agents.

In addition, it is constantly exposed to diet-derived oxidants, mutagens, carcinogens as well

as ROS from the diet (Bruce N Ames, 1984). In order to preserve the integrity of the

intestinal membrane, the intestine possess many defense systems such as the ability to up-

regulate antioxidant enzymes systems, maintain high endogenous antioxidant content, as

well as to induce cell death and dispose of injured enterocytes (Aw, 1999). However, with

age and other environmental factors, previously mentioned this defense mechanism can be

damaged and lead to entry of pathogens.

Therefore, it is important to also study the antioxidant properties of FPH as

potential inhibitors of intracellular oxidation in intestinal cells.

1.3.11 In Vitro Measurement of Antioxidant Activity

No specific assays have been developed to measure the antioxidant capacity of

peptides; however the assays usually used for other antioxidants are commonly applied. In

vitro chemical assays are widely used to quantify antioxidant capacity of FPH in food

systems and sometimes for biological systems. However to completely study intracellular

oxidation inhibition of FPH, fate of peptides during gastrointestinal digestion, their ability

to penetrate through cellular membrane as well as its stability in vivo and its bioavailability

are required. Usually in vitro cell models are used as preliminary tool to study bioactivity

of the peptides instead of expensive animal based or human based studies.

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1.3.11.1 Chemical Based Assays

Food oxidation is very complex process involving different oxidative processes.

Therefore evaluating all the mechanism of an antioxidant in a single method is not an easy

task and requires a variety of analytical techniques (Samaranayaka, 2010). Based on the

mechanisms of the assays, they are divided into two groups: (1) hydrogen atom transfer

reaction (HAT) assays, which quantify H-atom donating ability and (2) electron transfer

(ET) based assays, which measure the reducing capacity of the antioxidant (Dejian Huang,

Ou, & Prior, 2005). In both of the mechanisms the end result would be stabilizing free

radicals; however the reaction kinetics can vary. In presence of an antioxidant peptide these

mechanism can happen simultaneously, but dominating mechanism depends on the

structure of the peptide, its solubility in the system, ionization potential, and bond

dissociation energy (Prior, Wu, & Schaich, 2005). A summary of the main analytical in

vitro chemical based assays used to evaluate food antioxidant peptides is provided in Table

1-1.

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Table 1-1. In vitro antioxidative chemical assays. Adapted from Huang, D., et

al.,(2005)(Dejian Huang, Ou, & Prior, 2005)

Reaction mechanism Assays

Hydrogen atom transfer reaction:

ROO• + AH ROOH + A•

Inhibition of linoleic acid oxidation

ROO• + LH ROOH + L• TRAP (total radical trapping

ORAC (oxygen radical absorbance capacity)

IOU (inhibited oxygen uptake)

Inhibition of LDL oxidation

Electron-transfer reaction

M(n) + e(from AH) AH•+ +

M(n-1)

ABTS(2,2'-Azinobis(3-

ethylbenzothiaazoline-6-sulphonic acid)

DPPH (2,2-Diphenyl-1-picrylhydrazyl)

FRAP (ferric ion reducing antioxidant

parameter)

Other assay TOSC (total oxidant scavenging capacity)

Inhibition of Briggs – Rauscher oscillation

reaction

Chemiluminescence

Electrochemiluminescence

AH = Antioxidant, LH = Substrate

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ET mechanism is non-competitive redox reaction where the test antioxidant transfer

electron to reduce an oxidisable probe and stops the oxidation reaction cycle. In most of

the ET assays the change in spectrophotometric absorbance is used to quantify the reducing

capacity of the antioxidant. The 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging

activity is the most commonly used ET based assay (Ak & Gülçin, 2008). DPPH is a stable

free radical with one unpaired electron at its nitrogen bridge and its deep violet color is

used to monitor the radical scavenging activity of an antioxidant. The decolorization during

the assay is an indication of antioxidant ability to reduce DPPH radical (Brand-Williams,

Cuvelier, & Berset, 1995). Similarly, the 2,2'-Azinobis(3-ethylbenzothiaazoline-6-

sulphonic acid (ABTS) radical scavenging activity mechanism is based on scavenging of

ABTS radical cation by an antioxidant. The ferric ion reducing antioxidant activity

measures the ability to reduce the ferric ion complex [Fe(III)-(TPTZ)2]3+ to the ferrous

complex [Fe(II)-(TPTZ)2]2+ by the antioxidant at pH 3.6 (Benzie & Strain, 1996).

In HAT based assays the ability of the antioxidant to donate hydrogen atom in order

to quench the free radical and inhibit the oxidation of the probe is monitored. It is a

competitive reaction between the test antioxidant, free radical generator and an oxidisable

probe (Wayner, Burton, Ingold, & Locke, 1985). Inhibition of lipid peroxidation in linoleic

acid emulsion assay follows the HAT mechanism. The major steps during inhibition of

lipid involve: inhibition by an azo-compound, inhibition by the antioxidant by donating a

H-atom, and termination of radical generation. The extent of lipid peroxidation is evaluated

using Ferric theocyanate assay. The formed lipid peroxide radical reacts with Fe2+ of

ferrous theocyante to Fe3+ to produce red colored ferrous theocyanate complex that can be

measured using spectrophotometrically (Wayner, Burton, Ingold, & Locke, 1985). The

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oxygen radical absorbance capacity assay (ORAC) also comes under the HAT reaction

mechanism. In this assay, peroxyl radicals react with the fluorescent probe 2,2’-azobis-2-

methyl-propaninidamine dyhydro chloride (AAPH) and form a non-fluorescent product

thereby decreasing fluorescence. The antioxidant protects the fluorescent probe by

inhibiting oxidation and maintaining the fluorescence value (Dejian Huang, Ou, Hampsch-

Woodill, Flanagan, & Prior, 2002; Ou, Hampsch-Woodill, & Prior, 2001).

The electron spin resonance (ESR) spectroscopy method involves interaction

between an applied magnetic field and chemical species with an unpaired electron such as

free radicals and transmission metal ions. ESR has been successfully used to evaluate the

DPPH radical scavenging activity by marine protein hydrolysate peptides (Rajapakse,

Mendis, Byun, & Kim, 2005). ESR is a very sensitive technique and free of interferences

from sample matric components such as pigments (Peng, Xiong, & Kong, 2009).

The antioxidant assay mechanisms are different from one assay to another.

Antioxidant activity of a compound can give different results depending on the assay used.

Therefore the total antioxidant activity of the compound is evaluated by integrating the

results from different in vitro chemical assays (Sun & Tanumihardjo, 2007).

1.3.11.2 In Vitro Cell Culture Model

In vitro cell models serve as rapid and inexpensive and preliminary tool to evaluate

the potential health effects of Antioxidant peptides or hydrolysates. They should be applied

prior to any animal or human clinical trials. The cell based assays can be used to estimate

bioavailability metabolism and biological activity of antioxidants. Different cell types are

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used as cell models to perform antioxidant assay. The human adenocarcinoma colon cancer

cells monolayer (Caco-2) cells are most commonly used and are widely accepted due to

their similarities with intestinal endothelium cells. These Caco-2 cells are predominantly

used to study paracellular and transcellular movement of an antioxidant compound. When

the cells are cultured under specific conditions, they form a continuous monolayer with

structural arrangement that serves as model for intestinal permeability assay (Irvine,

Takahashi, Lockhart, Cheong, Tolan, Selick, et al., 1999).

The toxicity of antioxidant is very important to determine/test using cell based

assays prior to their clinical testing. The 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetra

zolium (MTT) assay is commonly used to evaluate the viability of cells in presence of the

test antioxidant. The assay evaluates the reduction reaction catalyzed by the functional

mitochondria. Since the reaction only proceeds in the presence of alive cells, giving an

indication of cell viability (Rajapakse, Mendis, Jung, Je, & Kim, 2005).

In intracellular antioxidant activity models, cells are pretreated with the test

compound and addition of AAPH, a peroxyl radical generator, or H2O2 can induce

oxidation of cells. A probe dichlorofluorescein diacetate (DCFH-DA) is added and during

cellular activity the probe will diffuse in the cell to DCFH by intracellular hydrolysis

processes. The formed cellular product will undergo oxidation by peroxyl radicals to form

a fluorescence compound (DCF). In the presence of antioxidant, the oxidation reaction of

DCFH is inhibited and fluorescence intensity is reduced since DCF is not formed (Y. Wang,

Zhu, Chen, Han, & Wang, 2009)

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1.3.12 Research Hypothesis and Objectives

This study aims to investigate potential utilization of silver carp protein as a source

of antioxidant food ingredient and also to evaluate factors effecting the activity. The overall

hypotheses and objectives were

STUDY 1: ANTIOXIDANT PROPERTIES AND FREE AMINO ACIDS

COMPOSITION OF SILVER CARP (Hypophthalmichthys molitrix) PROTEIN

HYDROLYSATES

Hypotheses

The type of commercial enzyme and hydrolysis conditions used to produce SPH

has an influence on their multifaceted antioxidant activity measured using different

assays

Free amino acid composition of the SPH may have an influence on their

multifaceted antioxidant activity measured using different assays

Objectives

To produce SPH samples using two commercially available proteases

(Flavourzyme®® and Alacalase®) at different hydrolysis conditions

To study the multifaceted antioxidant activity of the SPH samples using different

in vitro chemical based assays

To investigate the influence of free amino acid composition on different antioxidant

mechanism

To identify one or more SPH samples with promising chemical based antioxidant

activity

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STUDY 2: EFFECT OF MOLECULAR WEIGHT AND PEPTIDE SEQUENCE OF

HYDROLYSATE FRACTIONS ON CELL BASED ANTIOXIDANT ACTIVITY

OF SILVER CARP

Hypotheses

Both molecular weight and sequence of peptide will have an influence on

antioxidant activity

Peptide fraction of SPH can demonstrate antioxidant activity under oxidative stress

condition and can retain its activity for long incubation time

Objectives

To fractionate potential SPH samples from study 1 according to according to

different molecular weight using ultrafiltration and chromatographic methods

To measure the in vitro cell based antioxidant activity of different molecular weight

fractions using Caco-2 cells under both oxidation stressed and non-stressed

conditions

To determine the influence of molecular weight of peptide fractions on its cellular

antioxidant activity

To determine amino acid sequence of peptide fractions and to identify active

peptide sequence responsible for antioxidant activity

Study 3: EFFECT OF SIMULATED GASTROINTESTINAL DIGESTION ON IN

VITRO CELL BASED ANTIOXIDANT ACTIVITY OF SILVER CARP

(Hypophthalmichthys molitrix) PROTEIN

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Hypotheses

Resistance towards simulated GI digestion of SPH depends on the enzyme used

during the production of SPH

SPH samples can retain their antioxidant activity even after simulated GI digestion.

Objectives

To perform simulated GI digestion on potential SPH sample from study 1 using

two protocols with varying GI digestion enzymes and activities

To study effect of simulated GI digestion on cellular antioxidant activity of

potential SPH samples from study 1.

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CHAPTER 2. ANTIOXIDANT PROPERTIES AND FREE AMINO ACIDS

COMPOSITION OF SILVER CARP (HYPOPHTHALMICHTHYS MOLITIX)

PROTEIN HYDROLYSATES

2.1 Abstract

Invasive silver carp protein hydrolysates (SPH) were prepared using Flavourzyme®

(F-15 to F-60) and Alcalase® (A-15 to A-60) at 15, 30, 45 and 60 min, respectively.

Samples were analyzed for amino acid composition and by: 1,1-diphenyl-2-picrylhydrazyl

scavenging activity (DPPH-SA), lipid peroxidation inhibition (LP-IA), Trolox equivalent

antioxidant (TEAC) and ferric ion reducing (FI-RC) capacities. F-30 and F-60 samples,

with higher valine and leucine content, revealed higher (P<0.05) DPPH-SA than BHT and

α-tocopherol. All SPH samples showed higher TEAC values than BHA; while A-60 with

higher content of aspartic and glutamic acid, exhibited higher FI-RC. These acidic amino

acids are known to reduce Fe3+ to Fe2+. All hydrolysates also showed significantly higher

(P<0.05) LP-IA than natural α-tocopherol. Overall, SPH displayed higher or comparable

antioxidant capacity than commercial antioxidants, thus providing alternatives for using an

invasive fish that can be exploited for environmental and economic gain in the form of

potential functional, bioactive ingredient.

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2.2 Introduction

In recent years the spreading of unconventional carp species, such as silver carp

(Hypophthalmichthys molitrix), has been increasing in the U.S. Mississippi River system.

Due to their invasive characteristics that include competition for food and habitat with

native fish species, silver carp have a significant impact on the ecosystem processes

(Calkins, Tripp, & Garvey, 2012; Coulter, Keller, Amberg, Bailey, & Goforth, 2013).

Apart from this, reports of large sized Silver carp jumping resulting in severely injuring

boaters and water skiers and damaging water crafts are becoming more frequent (Interests,

2012; Kolar, Chapman, Courtenay Jr, Housel, Williams, & Jennings, 2005). To alleviate

the negative impacts of these invasive carps, efforts are being made to decrease their

population and minimize their opportunity to enter the Great Lakes basin (Rogowski, et al.,

2005). Currently, most of these fish are used as fertilizers, livestock feeds or simply

discarded without making an attempt to use them for human consumption. One alternative

is to recover and modify the protein-rich fish meat into different value added alternatives

suitable for human consumption.

It is well recognized that hydrolysates of fish protein from various sources showed

improved functional and bioactive properties than its parent protein. In particular,

antioxidant properties of FPH in species such as Tilapia (Foh, Amadou, Foh, Kamara, &

Xia, 2010; Raghavan, Kristinsson, & Leeuwenburgh, 2008), Pacific hake (Cheung, Cheung,

Tan, & Li-Chan, 2012; A. G. P. Samaranayaka & E. C. Y. Li-Chan, 2008), Herring

(Sannaveerappa, Westlund, Sandberg, & Undeland, 2007), Tuna (J. Y. Je, Z. J. Qian, H. G.

Byun, & S. K. Kim, 2007; J. Y. Je, Qian, Lee, Byun, & Kim, 2008; Nalinanon, Benjakul,

Kishimura, & Shahidi, 2011), Alaskan Pollack (Jia, Zhou, Lu, Chen, Li, & Zheng, 2010),

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Hoki (S. Y. Kim, J. Y. Je, & S. K. Kim, 2007), and Scad (Y. Thiansilakul, S. Benjakul, &

F. Shahidi, 2007) is well documented. A few studies have been done on antioxidant activity

of silver carp protein hydrolysates in countries like China, where this fish is commonly

consumed (S. Dong, M. Zeng, D. Wang, Z. Liu, Y. Zhao, & H. Yang, 2008; Zhong, Ma,

Lin, & Luo, 2011). Silver carp protein hydrolysates showed promising radical scavenging

and lipid peroxidation activities (S. Dong, M. Zeng, D. Wang, Z. Liu, Y. Zhao, & H. Yang,

2008). However, multifaceted antioxidant activity of silver carp protein hydrolysates using

different antioxidant assays as well as the effect of free amino acid composition has not

been fully explored. All the above factors indicate that there is need to investigate

antioxidant activity with respect to amino acid composition of invasive silver carp protein

hydrolysates derived from the U.S. Mississippi River system. These results will serve as

an initial step to develop potential natural antioxidants from these invasive fish species,

which would be an excellent alternative to their otherwise disposal into the environment.

The objective of the study was to evaluate the antioxidant potential of silver carp

protein hydrolysates prepared using two different commercial enzymes (Flavourzyme® and

Alcalase®), and to evaluate the free amino acid composition of the hydrolysates, in order

to understand the influence of amino acid composition on antioxidant activity of

hydrolysates.

2.3 Materials and Methods

2.3.1 Materials

Fresh silver carp (Hypophthalmichthys molitrix) were harvested from the Wabash

River (Lafayette, IN, USA). Within 24 hours, the fish were transported on ice to the food

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science department where they were beheaded, eviscerated and immediately frozen at -

20 °C until used. The enzymes Alcalase® 2.4 L (EC 3.4.21.14, P - 4860, 2.4 U/G) and

Flavourzyme® (EC 232-752-2, P-6110, 500 U/G), ʟ-leucine, 2,4,6-

trinitrobenzenesulphonic acid (TNBS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-

carboxylic acid (Trolox), 2,2ʹ-azinobis-(3-ethylbenzothiazolin-6-sulphonic acid) (ABTS),

1,1-diphenyl-2-picrylhydrazyl (DPPH), potassium ferricyanide, ferrous chloride, linoleic

acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and α-tocopherol,

were purchased from Sigma-Aldrich, (St. Louis, MO, USA).

2.3.2 Preparation of Silver Carp Protein Hydrolysates (SPH)

SPH were prepared according to (Liceaga‐Gesualdo & Li‐Chan, 1999) with few

modifications. Fish fillets (500 g) were thawed at 4°C overnight, skin was removed and

rinsed once using cold distilled water. The fillets were minced using a meat grinder

(Cabela’s, Hammond, IN, USA) and homogenized in a blender with two volumes of water

(w/v). The slurry was hydrolyzed using Alcalase® (0.072 U/ g of mince protein) or

Flavourzyme® (15 U/ g of mince protein), respectively, under optimum temperature (50°C)

at different incubation times (15, 30, 45 and 60 min). The enzyme choice was made to

investigate the effect of endopeptidase and exopeptidase activity on the antioxidant activity

of protein hydrolysates respectively. The pH of the slurry was measured to ensure it was

within optimum pH range of the enzymes (6.5-8.5 and 5.5-7.5, respectively) (Cinq-Mars,

2006). After incubation with enzyme, samples were immediately pasteurized (90 °C, 15

min) to inactivate the enzymes and resulting slurry was centrifuged at 16,300 × g for 15

min. The resultant supernatant was freeze-dried to yield SPH using Alcalase® (SPH-A) and

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SPH using Flavourzyme® (SPH-F). SPH-A and SPH-F samples from different hydrolysis

time (15, 30, 45 and 60 min) were denoted by A-15, A-30, A-45, A-60 and F-15, F-30, F-

45, F-60, respectively. For control sample, mince slurry was pasteurized immediately

without any enzyme addition and incubation. Freeze dried samples were stored at -20 ±

3°C until used for antioxidant assay.

2.3.3 Degree of Hydrolysis

Degree of hydrolysis of the SPH was measured using the trinitrobenzene sulfonic

acid (TNBS) adapted from (J. Adler-Nissen, 1979) with modifications by (Liceaga‐

Gesualdo & Li‐Chan, 1999). Triplicate aliquots (0.5 mL) from each hydrolysis time were

diluted 10-fold in d-d water, mixed with 5 mL of 24% TCA (trichloroacetic acid) and

centrifuged at 12,100 × g for 5 min. The supernatant (0.2 mL) was mixed with 2 mL of 0.2

M sodium borate buffer (pH 9.2) and 1 mL of 4.0 mM of TNBS. The solution was vortexed

and immediately incubated in the dark at room temperature for 30 min followed by addition

of 1 mL of 2 M NaH2PO4 containing 18 mM Na2SO3. Absorbance of the solution was

measured at 420 nm using UV-Visible spectrophotometer (Beckmann, Irvine, CA, USA).

Degree of hydrolysis (%DH) was defined as percent ratio of the number of peptide bonds

broken (h) to the total number of bonds present per unit weight (htot) and calculated using

equation [1].

% Degree of hydrolysis (DH) = [ℎ

ℎ𝑡𝑜𝑡] × 100 [1]

For fish proteins the total number of peptide bonds htot was assumed to be 8.6 meq/g

(Adler‐Nissen, 1984). Hydrolysis equivalents (h) were calculated by the increase in

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released amino groups and can be calculated using lysine equivalence from leucine

equivalence (Leu-NH2) standard curve as shown below:

ℎ = ((Leu−NH2)−a)

𝑏 [2]

In the above equation, a = 0.4 and b=1 for meat proteins (Jens Adler-Nissen,

1986).

2.3.4 Proximate Analysis and Amino Acid Composition

Silver carp fish mince was analyzed for moisture, lipid and ash content using standard

methods AOAC 948.12, 960.39 and 942.05, respectively. Crude protein content of fish

mince and all SPH was analyzed by the combustion method, AOAC 992.15 using a Dumas

Nitrogen analyzer (Perkin Elmer Series II NA 2410, MA, USA) calibrated with

ethylenediaminetetraacetic acid (EDTA, 9.58% N). The protein content was calculated

using the conversion factor (N = 5.88) as described by Sosulski & Imafidon (1990)

((Sosulski & Imafidon, 1990). Analysis was performed in triplicate and calculated on a dry

basis (except moisture content). Total carbohydrate content was calculated by subtracting

the sum of % weight of moisture, protein, lipid and ash content from 100 g of fish.

The free amino acid composition of SPH and control sample was performed by the

Bindley Discovery Park Proteomics Facility at Purdue University. The analysis was

performed using a Beckman HPLC (126 pump, 166 Detector, 507 autosampler, and

“System Gold” data system) with a Waters AccQ Tag amino acid analysis column and UV

detector. Analysis was done at 37°C and amino acid standards (Pierce, Rockford, IL,

U.S.A) were used for calibration.

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2.3.5 DPPH Scavenging Activity

DPPH radical-scavenging activity of the SPH was determined as outlined (A.

Bougatef, M. Hajji, R. Balti, I. Lassoued, Y. Triki-Ellouz, & M. Nasri, 2009). Different

concentrations (3, 2, 1.5, 1 and 0.5 mg/mL of water) of SPH or control sample solution

(500 μL) were mixed with 99.5% ethanol (500 μL) and 0.02% DPPH in 99.5% ethanol

(125 μL). The solution was incubated in the dark at room temperature for 60 min.

Following incubation, absorbance at 517 nm was measured. An assay control (Absassay ctl)

was included, except distilled water was used instead of sample solution. In addition,

sample controls (Abssample ctl) were also prepared for each sample by mixing sample

solution (500 μL) with 99.5% ethanol solution (625 μL). DPPH radical scavenging capacity

of the sample was calculated as follows:

% DPPH radical scavenging activity

= [Absassay ctl – (Abssample – Abssample ctl)

Absassay ctl ] × 100

[2]

The lower the absorbance of the sample, the higher its DPPH radical scavenging

activity. BHA, BHT and α-tocopherol were used as positive controls.

2.3.6 Trolox Equivalence Antioxidant Property

Trolox equivalent antioxidant capacity (TEAC) of a sample was calculated using

the ABTS radical scavenging assay (Re, Pellegrini, Proteggente, Pannala, Yang, & Rice-

Evans, 1999). A stock solution was prepared by mixing 5 mL of 7 mM of ABTS solution

with 88 μL of 140 mM of potassium persulfate, and incubated in the dark at room

temperature for 16 h. The prepared stock solution was diluted in 5 mM phosphate buffer

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(pH 7.4) containing 0.15 M NaCl to obtain a working solution of ABTS radicals with

absorbance at 734 nm of 0.70 ± 0.02. Then, a 65 μL aliquot of SPH was dissolved in 5 mM

phosphate buffer containing 0.15 M NaCl (pH 7.4) at different final concentrations or only

buffer was used for assay control. The sample solution was mixed with 910 μL ABTS

working solution, incubated in the dark for 8 min at room temperature and absorbance was

read at 734 nm. The % reduction of ABTS+ to ABTS was calculated according to the

following equation:

% ABTS radical scavenging capacity = (1 − (Abs𝑠𝑎𝑚𝑝𝑙𝑒

𝐴𝑏𝑠𝐶𝑡𝑙)) × 100 [3]

TEAC of a sample, the concentration of sample that gives same % ABTS radical

scavenging capacity as 1 mM Trolox, was determined.

2.3.7 Ferric Ion Reducing Antioxidant Capacity

Ferric ion reducing antioxidant capacity of SPH samples was done according to

method described by (A. G. P. Samaranayaka & E. C. Y. Li-Chan, 2008). SPH Samples

(18 mg) were dissolved in 2 mL of 0.2 M phosphate buffer (pH 6.6). Solutions were further

mixed with 2 mL of the same buffer and 2 mL of 1% (w/v) potassium ferricyanide to final

concentration of 3 mg/mL of SPH solution. The resultant solution was incubated at 50°C

for 20 min, 2 mL of 10% trichloroacetic acid added and mixed well. The solution (2 mL)

was mixed with 2 mL of d-d water and 0.4 mL of 0.1% (w/v) ferric chloride. After 10 min

of incubation in dark at room temperature, absorbance at 700 nm was measured as an

indication of reducing power of the sample.

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2.3.8 Lipid Peroxidation Inhibition Assay

The Lipid peroxidation inhibition activity in a linoleic acid emulsion system for the

SPH was measured as outlined in (J. Y. Je, Z. J. Qian, H. G. Byun, & S. K. Kim, 2007).

Freeze dried SPH (1.3 mg) were dissolved in 10 mL of 50 mM phosphate buffer (pH 7.0),

which was mixed with 0.13 mL linoleic acid and 99.5% ethanol (10 mL). The solution was

adjusted to a final volume of 25 mL using d-d water. The mixture was incubated in a 50

mL amber bottle at 40 ± 1°C in the dark and degree of oxidation was evaluated at different

time intervals (40, 72, 156 and 168 h) using ferric thiocynate. An assay control (Absctl) was

included, except phosphate buffer (50 mM, pH 7.0) was used instead of sample. The

incubated solution (100 μL) was mixed with 75% ethanol (4.7 mL), 30% ammonium

thiocyanate (0.1 mL), 20 mM ferrous chloride solution in 3.5% HCl (0.1 mL) and incubated

for 3 min in the dark. After incubation, absorbance at 500 nm was read which corresponds

to the thiocyanate value. The % lipid inhibition was calculated as follows:

% Lipid peroxidation inhibition = (1 − (Abs𝑠𝑎𝑚𝑝𝑙𝑒

𝐴𝑏𝑠𝑐𝑡𝑙)) × 100 [4]

2.3.9 Statistical Analysis

Analysis of variance (ANOVA) using a General Linear Model with Tukey’s

pairwise comparison of means (P < 0.05) was used to determine statistical significance of

observed difference among means. The statistical software program MINITAB® Version

16.0 (Minitab Inc, State College, PA) was used. All antioxidant assays were conducted in

duplicate and analyses done in triplicate.

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2.4 Results and Discussion

2.4.1 Effect of enzyme and enzymatic conditions on degree of hydrolysis (%DH)

Two different commercially available enzymes Flavourzyme® (exo- and endo-

peptidase) and Alcalase® (endopeptidase) were used for the production of silver carp

protein hydrolysates (SPH-F and SPH-A). The enzyme choice was made to investigate the

effect of endopeptidase and exopeptidase activity on the antioxidant activity of protein

hydrolysates. The pH of the minced fish slurry at the beginning of hydrolysis was 6.9,

which is within the optimum pH range of both enzymes (Cinq-Mars, 2006). The enzyme

efficiency in cleaving proteins is quantified through %DH. The %DH pattern of both the

enzymes is shown in Figure 2-1.

For SPH-A, there was a gradual increase in %DH with increased hydrolysis time.

SPH-F showed a similar trend. However, during hydrolysis using Flavourzyme®, from 30

min to 45 min there was a slow increase in %DH followed by a sharp increase in up to the

60 min. Alcalase® showed higher %DH than Flavourzyme® throughout hydrolysis. During

60 min of hydrolysis time for both enzymes, the stationary phase was not reached.

The %DH value obtained for the A-60 sample was very similar to the %DH reported for

different protein sources prepared using Alcalase® under similar hydrolysis conditions

(Hoyle & Merritt, 2006; Liceaga‐Gesualdo & Li‐Chan, 1999).

2.4.2 Proximate Analysis and Free Amino Acid Composition

Proximate analysis of silver carp minced meat consisted of 82.3% moisture. On a wet

basis, the protein, ash and lipid contents were 14.6%, 1.1% and 1.6%, respectively. Based

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on the protein and lipid contents, silver carp can be classified as a high-protein, lean fish

(Abimbola, Kolade, Ibrahim, Oramadike, & Ozor, 2010)

The protein content of all the SPH-A and SPH-F samples is shown in Table 2-1,

indicating the protein of all the hydrolysate samples was higher than the control (non-

hydrolyzed) sample. This higher protein content of SPH is attributed to an increase in

solubility of smaller peptides when compared to the native protein in the supernatant during

the centrifugation step of the hydrolysate preparation. The protein content of hydrolysates

is also within the range reported by several researchers using different fish species (Choi,

Hur, Choi, Konno, & Park, 2008; Khantaphant, Benjakul, & Kishimura, 2011; Ovissipour,

Safari, Motamedzadegan, & Shabanpour, 2012; Pacheco-Aguilar, Mazorra-Manzano, &

Ramírez-Suárez, 2008).

The composition of free amino acids can assist in providing information about the

antioxidant activity of the hydrolysates (H.-C. Wu, Chen, & Shiau, 2003). Free amino acid

composition is given in Table 2-2. In general, with the increase in hydrolysis time there

was an increase in concentration of free amino acids (mg/mL) in both SPH-A and SPH-F

samples. However, free amino acid content in samples incubated with Alcalase®, an

endopeptidase, was lower than samples hydrolyzed by Flavourzyme® which is both endo

as well as exopeptidase. In addition, in both the SPH-A and SPH-F there was an increase

in the hydrophobic amino acids fraction. In particular, there was an increase in branched

chain amino acids such as tyrosine, valine, leucine, isoleucine and phenylalanine. This

increase can be attributed to the exposure of internal hydrophobic amino acids due to

endopeptidase activity by both Alcalase® and Flavourzyme®, which break peptide bonds

within the protein molecule, contributing to refold and reorientation of the protein structure

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and exposure of internal hydrophobic amino acids (Kinsella, 1982; Malaypally & Ismail,

2010).

2.4.3 Antioxidant Properties of SPH

In order to test antioxidant properties of SPH, different antioxidant assays were

performed owing to the multifaceted nature of antioxidants. The overall antioxidant

activity of the hydrolysates depends on many factors such as free amino acid composition

(Li, B. Jiang, T. Zhang, W. Mu, & J. Liu, 2008; H.-C. Wu, Chen, & Shiau, 2003), size of

the peptides (Moure, Dominguez, & Parajo, 2006; Peng, Xiong, & Kong, 2009), amino

acid sequence of the peptide (Klompong, Benjakul, Kantachote, Hayes, & Shahidi, 2007;

Peña‐Ramos, Xiong, & Arteaga, 2004) as well as solubility in assay medium A summary

of the results for all the antioxidant assays is given in Table 2-3. In the table, % DPPH

radical scavenging activity and ferric ion reducing capacity of the hydrolysates at a

concentration of 3 mg/ mL is shown. Ferric ion reducing capacity of the hydrolysates was

measured in terms of absorbance value at 700 nm. ABTS radical scavenging activity of

SPH samples in terms of Trolox equivalence (mMol trolox/ g of protein) was calculated

(Table 2-3). In addition, the % lipid oxidation inhibition of the hydrolysates after 7 days

incubation in dark is also shown. All the assays were analyzed in triplicates and statistical

significance among the means was calculated.

2.4.4 DPPH Radical Scavenging Activity

DPPH radical scavenging activity is widely used because the DPPH radical is one of

the few stable, commercially available organic-nitrogen radicals and its reduction can be

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readily quantified using a spectrophotometer (D. Huang, Ou, & Ronald, 2005). Stable

DPPH radicals have a maximum absorbance at 517 nm and when a proton-donating

substance or antioxidant reduces the DPPH radical, the absorbance is decreased (Shimada,

Fujikawa, Yahara, & Nakamura, 1992). The percent decrease of absorbance is taken as a

measure for radical scavenging capacity.

Dose dependent radical scavenging activity of SPH-A or SPH-F samples are displayed

in Figures 2-2. In general, with increasing sample concentration, there was an increase in

DPPH radical scavenging properties. In both SPH-A and SPH-F samples, the hydrolyzed

samples (20 – 87 %) have higher radical scavenging activity than the control non-

hydrolyzed counterpart (10–53.61 %) at all concentrations. This increase in scavenging

capacity after protein hydrolysis can be caused by the increase in solubility of the smaller

peptides, formation of active peptides and release of free amino acids. Previous studies

show that protein hydrolysates from different sources with high scavenging properties have

higher concentration of hydrophobic amino acids (Y. Li, B. Jiang, T. Zhang, W. Mu, & J.

Liu, 2008; Pownall, Udenigwe, & Aluko, 2010). The release of hydrophobic amino acids

during hydrolysis in both SPH-F and SPH-A can be contributing to its DPPH radical

scavenging activity. The increase in aromatic amino acids such as phenylalanine and

tyrosine can also be contributing to the hydroxyl radical scavenging activity since they can

form stable para-, meta- and ortho-substituted derivatives on the aromatic ring (M. Li,

Carlson, Kinzer, & Perpall, 2003; Y.-W. Liu, Han, Lee, Hsu, & Hou, 2003; Yan, Suzuki,

Ohnishi-Kameyama, Sada, Nakanishi, & Nagata, 1999).

The DPPH scavenging capacity of SPH at 3.0 mg/mL is given in Table 2-3. At this

concentration, A-15 to A-60 and F-15 to F-60 had similar DPPH scavenging activity to the

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synthetic antioxidants (BHT and BHA) as well as the natural antioxidant (α-tocopherol).

Interestingly, SPH-F samples showed overall higher (P<0.05) scavenging activity than

SPH-A samples. A similar trend was observed in round scad muscle (Yaowapa

Thiansilakul, Soottawat Benjakul, & Fereidoon Shahidi, 2007), yellow stripe trevally

(Klompong, Benjakul, Kantachote, Hayes, & Shahidi, 2007), and canola (Cumby, Zhong,

Naczk, & Shahidi, 2008) protein hydrolysates using Alcalase® and Flavourzyme®. These

observations can be attributed to higher free amino acid content released in SPH-F samples

compared to SPH-A samples. The released free amino acids have higher solubility and may

have more accessibility to the DPPH radical.

All the SPH-F (F-15 to 60) samples had the same DPPH scavenging capacity (P > 0.05)

when compared to BHA (80.81%). In addition, F-30 and F-60 showed higher (P < 0.05)

scavenging capacity than BHT (75.05%) and α-tocopherol (75.25%); whereas only the

Alcalase hydrolysates A-60 and A-45 showed similar (P> 0.05) radical scavenging

capacity compared to BHA, BHT and α-tocopherol. The higher scavenging activity of the

samples coincides with higher valine, leucine and alanine fractions, which are believed to

be responsible for DPPH radical scavenging activity (Rajapakse, Mendis, Byun, & Kim,

2005)

2.4.5 TEAC Value

Trolox equivalence antioxidant capacity was measured using the ABTS radical

scavenging assay. In order to calculate TEAC values, different concentration of samples

were evaluated for ABTS scavenging activity and their corresponding TEAC values were

calculated using a Trolox standard curve. TEAC values of all the hydrolysates were

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significantly higher (P < 0.05) than the control (Table 2-3), suggesting that there is

improved antioxidant capacity upon hydrolysis of the protein. All of the SPH samples,

except A-45, showed TEAC values comparable to BHA (32.52 mMol/g of BHA). The

TEAC values of all the SPH and even the control sample were higher than those of BHT

(0.46 mMol/g of BHT). This was attributed to the poor solubility of BHT in the aqueous

ABTS assay media (Chang & Maurey, 1985). However, a higher TEAC value for BHT

(3.35 mMol/g) was obtained when the ABTS assay was done in ethanol (Tachakittirungrod,

Okonogi, & Chowwanapoonpohn, 2007) instead of phosphate buffer. Difference in

solubility of hydrolysates in different assay media and their affinity towards different

radicals can explain the difference in DPPH and ABTS radical scavenging activity trends.

2.4.6 Ferric Ion Reduction Capacity

Ferric ion reduction capacity was used to measure the ability of peptide molecules to

reduce the ferric state (Fe3+ -ferricyanide complex) to ferrous (Fe2+) form (Ali Bougatef,

Mohamed Hajji, Rafik Balti, Imen Lassoued, Yosra Triki-Ellouz, & Moncef Nasri, 2009).

The reduction capacity of the sample is directly proportional to its absorbance at 700 nm.

At 3.0 mg/mL, SPH-A samples (A-30 to A-60) (Abs700 : 0.61 to 0.65) showed higher (P <

0.05) reduction capacity than the control (Abs700 : 0.56), while no significant difference

was observed among the SPH-A samples (Table 2-3). Amongst SPH-F samples, F-30

(Abs700 = 0.63) and F-60 (Abs700 = 0.62) showed the highest reducing capacity (P < 0.05)

than control (Abs700 : 0.56). With increase in %DH, an increase in ferric ion reduction was

observed in SPH-A samples similar to the observation made by Dong et al. (2008) also

using silver carp hydrolysates from China origin (Shiyuan Dong, Mingyong Zeng,

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Dongfeng Wang, Zunying Liu, Yuanhui Zhao, & Huicheng Yang, 2008). However, this

trend was not observed with our SPH-F samples.

In other studies, (Xie, Huang, Xu, & Jin, 2008) reported the highest reduction capacity

of purified (<3 kDa) alfalfa leaf peptide with an Abs700 = 0.4 at a concentration of 2.5 mg

of sample/mL. Autolyzed Pacific hake protein hydrolysates displayed a reduction capacity

ranging from Abs700 = 0.3 to 0.6 at 3 mg/mL sample concentration (A. G. Samaranayaka

& E. C. Li-Chan, 2008). In the present study, both SPH-A and SPH-F exhibited higher

reduction capacity ranging from Abs700 = 0.56 to 0.65 and 0.56 to 0.63, respectively (Table

2-3). It has been suggested that the presence of specific amino acids contributes to the ferric

ion reduction capacity (Pownall, Udenigwe, & Aluko, 2010). For example, it has been

reported (Martínez Ayala, 2013; You, Zhao, Cui, Zhao, & Yang, 2009) that peptides

containing histidine have a higher reducing power. Interestingly, we found that the control

sample has higher histidine + glutamine content (45.6%) that may contribute to its high

initial reduction capacity (Abs700 = 0.56) when compared to the results presented by other

researchers. Apart from histidine, acidic amino acids (aspartic and glutamic acid) exhibit

reducing capacity as they can readily reduce or chelate Fe3+ to Fe2+.49 Sample A-60 had the

highest percent of the above amino acids when compared to other SPH-A samples

respectively, which could explain some of its overall higher reduction capacity (Abs700 =

0.63).

Explaining reducing capacity of SPH with respect to hydrophobic amino acids is

difficult since relation between reducing capacity and the amount of hydrophobic amino

acids is contradictory in available literature. For example, rapeseed fractions containing

high hydrophobic amino acids showed higher reducing power (S. B. Zhang, Wang, & Xu,

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2007, 2008). While in contrast, pea protein hydrolysate fractions showed lower reducing

power even though they had high content of hydrophobic amino acids (Pownall, Udenigwe,

& Aluko, 2010). In the present study, we noticed that SPH-A and SPH-F samples with

higher hydrophobic amino acid content did show higher ferric ion reduction capacity.

2.4.7 Inhibition of Lipid Peroxidation by SPH

The lipid peroxidation assay was conducted to evaluate the efficiency in quenching

peroxide radicals. In absence of inhibitor or antioxidant, lipid oxidation products react with

ferrous iron to produce ferric iron, which generates a colored solution after reacting with

an ammonium thiocyanate solution. It is expected that in presence of an antioxidant, the

above reaction is inhibited and absorbance at 500 nm is decreased. Thus, the lower the

absorbance at 500 nm implies higher antioxidant capacity (Jayaprakasha, Singh, &

Sakariah, 2001).

Lipid peroxidation values of SPH-A and SPH-F samples in comparison with synthetic

antioxidants (BHA and BHT) and natural antioxidant (α-tocopherol) during 168 h (7 days)

of incubation are shown in Figures 2-3. During 168 h of incubation, both SPH-A and SPH-

F showed inhibitory activity towards lipid peroxidation when compared to the control. In

general, for all the samples, with increased incubation time there was an increase in lipid

peroxidation value. It is known that enzymatic hydrolysis results in smaller peptide chains

with hydrophobic-hydrophilic equilibrium as well as high solubility that can orient at the

interface of the lipid droplet very rapidly. The formed interfacial layer protects the lipid

molecules from aqueous phase pro-radicals such as transition metal ions (Xiong, 2010).

The increase in free hydrophobic amino acid in SPH samples serves as an indicator of the

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released hydrophobic interior of the peptide, which indirectly inhibits peroxidation.

Hydrophobic amino acids are believed to have strong inhibition capacity to lipid oxidation

due to their ability to interact with the lipid peroxides. The increase in hydrophobic amino

acids in SPH-A and SPH-F resulted in higher (P < 0.05) lipid peroxide inhibition than the

control samples.

In addition, all SPH-A samples (A-15 to A-60) showed significantly higher (P< 0.05)

inhibition than BHA and α-tocopherol. Similarly, except for F-45, all other SPH-F samples

(F-15, F-30 and F-60) showed higher (P < 0.05) lipid oxidation inhibition compared to

BHA and α-tocopherol (Table 2-3). It is interesting that even after seven days of

incubation, all the silver carp hydrolysates (SPH-A and SPH-F) exhibited higher (P < 0.05)

inhibition of lipid peroxidation compared to α-tocopherol. Similar results were reported for

pacific hake protein hydrolysates, obtained after 1 h autolysis, which showed higher lipid

oxidation inhibition even after 7 d of incubation, whereas α-tocopherol activity diminished

during the incubation (A. Samaranayaka & E. Li-Chan, 2008).

2.4.8 Overall Antioxidant Activity Among SPH-A and SPH-F Sample

Overall antioxidant activity of SPH-A and SPH-F samples showed higher activity

than unhydrolyzed control sample. Among SPH-F samples, F-30 sample showed higher

lipid peroxidation inhibition capacity and second highest DPPH and ABTS radical

scavenging activity and ferric ion reduction capacity. Similarly, A-60 showed relatively

pronounced antioxidant capacity among SPH-A samples. These two samples were used to

perform further experiments.

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2.4.9 Conclusion

This study shows that invasive silver carp protein hydrolysates exhibit comparable

or higher antioxidant capacity than synthetic antioxidants (BHA and BHT) as well as

natural antioxidant (α-tocopherol). This study also emphasizes that antioxidant activity of

the hydrolysates determined by different assays could depend on the presence of specific

free amino acids. However, evaluating the molecular weight distribution and amino acid

sequence of the formed peptides will assist in better understanding of various antioxidant

activities of the hydrolysates. TEAC values of hydrolysates (SPH-A and SPH-F) are nearly

equal to the TEAC value of BHT and higher than the BHA TEAC value. The effect of

silver carp hydrolysates against linoleic acid oxidation suggests that they have a potential

role as in vivo antioxidants and their effects on chronic diseases that are related to cellular

oxidation stress should be further investigated. This study has allowed us to explore using

an underutilized, invasive fish for environmental and economic gain in the form of

potential bioactive ingredients.

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Table 2-1. Protein content of SPH-A and SPH-F samples.

Sam

ple

s

Protein content (%)A

SPH-AB SPH-Fc

CtlD 66.99 (±1.5) aE 66.99 (±1.5) a

15 min 86.78 (±0.99) ab 71.77 (±0.89) ab

30 min 82.01 (±4.42) ab 75.04 (±0.65) b

45 min 92.18 (±6.54) b 81.22 (±2.06) c

60 min 87.75 (±0.55) ab 86.95 (±2.20) d

A Protein content of samples on wet basis.

B SPH-A = hydrolysate samples obtained from Alcalase®

.

C SPH-F = hydrolysate samples obtained from Flavourzyme®

.

DCtl = non-hydrolyzed muscle meat;

E Different letters (a,b,c,d) indicate significant difference (p <0.05) within means in each column.

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Table 2-2. Free amino acid composition of SPH and their control samples

Amino acid

Amino acid composition mg/ mLA (mg/ 100 mg, %)B

CtlC F-15 F-30 F-45 F-60 A-15 A-30 A-45 A-60

ASP 0.0(0.0) 2.3(4.6) 1.8(4.3) 1.9(3.9) 2.5(4.4) 0.0(0.0) 0.3(1.7) 0.3(2.0) 0.5(2.5)

SER + ASND 0.3(2.1) 1.9(3.9) 1.5(3.6) 1.8(3.6) 2.1(3.6) 0.4(2.7) 0.5(2.9) 0.5(3.3) 0.7(3.4)

GLU 0.5(3.0) 1.8(3.8) 2.1(5.1) 1.9(3.9) 2.4(4.1) 0.5(3.3) 0.6(3.4) 0.6(4.0) 0.8(4.3)

GLY 2.5(15.8) 1.7(3.4) 1.1(2.5) 1.5(3.0) 1.4(2.4) 2.1(13.1) 2.2(12.8) 2.0(11.2) 2.3(11.9)

HIS + GLND 7.3(45.6) 7.8(15.7) 6.7(16.1) 6.9(14.0) 6.8(11.9) 2.9(18.7 3.2(18.3) 2.6(16.7) 3.1(16.4)

ARG 0.2(1.3) 5.2(10.5) 4.7(11.3) 5.3(10.6) 6.3(11.1) 0.6(4.0) 0.9(5.1) 0.8(5.0) 0.6(3.1)

THR 2.0(10.7) 1.9(3.8) 1.0(2.4) 1.8(3.7) 2.1(3.7) 0.2(1.6) 0.4(2.3) 0.4(2.2) 0.3(1.7)

ALA 0.8(4.9) 2.2(4.5) 1.8(4.3) 2.1(4.3) 2.5(4.4) 0.8(5.2) 1.0(5.5) 0.9(5.6) 1.2(6.1)

PRO 0.8(5.3) 0.7(1.4) 0.4(1.0) 0.6(1.3) 0.7(1.2) 0.2(1.4) 0.2(1.3) 0.2(1.2) 0.2(1.2)

Cys/2 0.0(0.0) 0.2(0.4) 0.2(0.4) 0.2(0.4) 0.3(0.5) 0.1(0.6) 0.2(1.3) 0.1(0.7) 0.1(0.7)

TYR 0.0(0.0) 1.7(3.4) 1.2(3.0) 1.5(3.1) 2.1(3.7) 0.6(3.6) 0.6(3.7) 0.6(4.0) 0.6(3.0)

VAL 0.2(1.3) 2.5(5.0) 1.9(4.7) 2.7(5.4) 3.3(5.7) 0.7(4.5) 0.8(4.6) 0.8(5.0) 1.0(5.0)

MET 0.0(0.0) 2.0(4.1) 1.7(4.2) 2.0(4.0) 2.4(4.3) 0.9(5.9) 0.9(5.1) 0.8(5.0) 1.0(5.0)

LYS 1.0(6.3) 6.6(13.4) 5.0(12.0) 6.2(12.4) 7.1(12.4) 2.3(14.7) 1.9(11.3) 1.9(12.1) 2.1(11.2)

ILE 0.1(0.4) 1.8(3.7) 1.6(3.9) 2.0(4.1) 2.4(4.3) 0.4(2.7) 0.4(2.7) 0.4(2.6) 0.6(3.0)

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LEU 0.3(1.9) 5.9(12.0) 4.6(11.1) 6.2(12.4) 7.3(12.9) 2.0(12.5) 2.1(12.3) 2.0(12.7) 2.9(14.9)

PHE 0.0(0.0) 2.9(6.0) 2.6(6.2) 4.7(9.4) 5.3(9.4) 0.9(5.4) 1.0(5.5) 1.0(6.2) 1.1(5.9)

AConcentration of free amino acid in the samples (mg/ mL); BPercent free amino acid composition of samples (mg/ 100 mg):

CCtl = control sample, SPH-A (A-15 to A-60), SPH-F (F-15 to F-60). Cysteine was underestimated, since pre-derivatization was not performed; Tryptophan

was not determined.

DSER and ASN eluted at same time; HIS and GLN eluted at same time.

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Table 2-3. Summary of results for the antioxidant properties of silver carp protein hydrolysates prepared using Alcalase® (SPH-A)

and Flavourzyme® (SPH-F) at different hydrolysis times (15 min, 30 min, 45 min and 60 min). S

ample

DPPH radical

scavenging (%)A

TEAC values

(mMol trolox/g

prot)B

Ferric ion reduction

capacity (Abs700)C

% Lipid peroxidation

inhibition D

SPH-A SPH-F SPH-A SPH-F SPH-A SPH-F SPH-A SPH-F

CtlE 53.61 dxF 53.61 cx 12.64 ax 12.64 ax 0.56 ax 0.56 ax 13.99 ax 13.99 ax

15 min 71.29 cx 80.81 aby 25.53 bx 27.75 by 0.59 abx 0.56 ax 88.88 bx 88.88 bdx

30 min 70.25 cx 84.85 ay 27.28 cx 25.97 cy 0.61 bcx 0.63 bx 89.23 bx 91.67 by

45 min 77.70 abx 79.98 aby 17.33 dx 23.95 dy 0.62 bcx 0.59 abx 87.50 bx 61.46 cy

60 min 79.01 abx 86.87 ay 23.81 ex 23.28 dx 0.65 cx 0.62 bx 92.95 cx 88.19 dy

BHT 75.05 bcx 75.05 bx 0.46 gx 0.46 fx 0.08 dx 0.08 cx 95.13 cx 95.13 ex

BHA 80.81 ax 80.81 abx 32.52 fx 32.52 ex -- -- 86.46 bx 86.46 dx

ATG 75.25 bcx 75.25 bx -- -- -- -- 33.68 dx 33.68 fx

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ADPPH radical scavenging activity of SPH-A and SPH-F at sample concentration of 3 mg/mL. BTrolox equivalence antioxidant capacity (TEAC) of FPH-A

and FPH-F in terms of mMol of Trolox/ g of protein.

CFerric ion reduction capacity of SPH-A and SPH-F (at 3 mg/mL) measured as absorbance at 700 nm.

DLipid peroxidation inhibition (%) values of SPH-A and SPH-F measured after 168 h (7 days) of incubation.

ECtl = non-hydrolyzed muscle meat;

FDifferent letters (a, b, c,….) indicate significant difference (p <0.05) in means within each column for each individual assay. Different letters (x, y) indicate

significant difference (p <0.05) among the means within each row for each antioxidant assay.

GAT = alpha Tocopherol

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Figure 2-1. Degree of hydrolysis (%) of Silver carp (Hypophthalmichthys molitrix) by

Flavourzyme® and Alcalase® enzymes.

0

5

10

15

20

25

30

35

40

0 10 20 30 40 50 60 70

Deg

ree

of

hydro

lysi

s %

Time (min)

Flavourzyme Alcalase

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Figure 2-2. The percentage scavenging activity of DPPH radical (0.02 %) of (a) Alcalase®

hydrolyzed silver carp protein hydrolysates (SPH-A) and (b) Flavourzyme® hydrolyzed

silver carp protein hydrolysates (SPH-F) at different concentrations (mg/mL) after 60 min

incubation in dark. Ethanol (99.5 %) was used as an assay media. Analysis was done in

triplicate.

-20

-10

0

10

20

30

40

50

60

70

80

90

0.5 1 1.5 2 3

% D

PP

H s

cavengin

g a

ctivity

Concentration of SPH-A (mg/ mL)

-20

-10

0

10

20

30

40

50

60

70

80

90

100

0.5 1 1.5 2 3

% D

PP

H S

cavengin

g a

ctivity

Concentration of SPH-F (mg/ mL)

Control 15 min 30 min 45 min

60 min BHA BHT

(A)

(B)

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73

-0.1

0.4

0.9

1.4

1.9

2.4

2.9

0 40 72 156 168

Ab

so

rban

ce @

500 n

m

Time (h)

Assay Ctl Ctl A-15 A-30 A-45

A-60 BHA BHT Tocopherol

(A)

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Figure 2-3. Lipid peroxidation values (Abs @ 500 nm) evaluated in linoleic acid emulsion

system in presence of (A) Alcalase® hydrolyzed silver carp protein (SPH-A) (0.052 mg/

mL) and (B) Flavourzyme® hydrolyzed silver carp protein (SPH-F) (0.052 mg/ mL)

incubated in dark at room temperature (25 °C) at different incubation times (0, 40, 72, 156

and 168 h). Analysis was done in triplicate.

-0.1

0.4

0.9

1.4

1.9

2.4

2.9

0 40 72 156 168

Ab

so

rba

nc

e @

50

0 n

m

Time (hrs)Assay Ctl Ctl F-15 F-30 F-45

F-60 BHA BHT Tocopherol

(B)

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CHAPTER 3. EFFECT OF MOLECULAR WEIGHT AND PEPTIDE SEQUENCE OF

HYDROLYSATE FRACTION ON CELL BASED ANTIOXIDANT ACTIVITY

OF SILVER CARP (HYPOPHTHALMICHTHYS MOLITRIX) PROTEIN

HYDROLYSATES

3.1 Abstract

Invasive silver carp protein hydrolysates prepared using Flavourzyme® and

Alcalase® at 30 min and 60 min, respectively (F-30 and A-60) showed highest chemical

based antioxidant activity. F-30 and A-60 samples were tested for in vitro cell based

antioxidant activity using human colon cells (Caco-2) under oxidative stress (AAPH, 0.1

mM, 1h at 37 °C) and non-stress (no AAPH added). Both samples have shown to inhibit

cellular oxidation and exhibited in vitro antioxidant activity in Caco-2 cells. Furthermore

peptides <3 KDa from F-30 and A-60 (F-30<3 and A-60<3) samples showed improved

cell based antioxidant activity even after longer incubation time under both stress and non-

stress conditions. Dose dependent antioxidant activity was observed in F-30, A-60, F-30<3

and A-60<3 samples from 0.625 to 5 mg/ mL concentration under both conditions.

However, peptide fractions larger than 3 KDa (F-30>3 and A-60>3) did not show any dose

dependent antioxidant and showed very low cellular oxidation inhibition capacity.

Additionally, IC50 values of F-30<3 fraction (1 to 3 mg/ mL) was lower than A-60<3

fractions (4 to 12 mg/ mL), indicating higher cellular antioxidant activity of F-30<3

fractions compared to A-60<3 under all conditions. Higher number of active peptides with

desired amino acid sequence of F-30<3 sample compared to A-60<3 may have contributed

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to its higher cellular antioxidant activity. The preliminary results shows that F-30<3 sample

may have promising pharmaceutical or nutraceutical application.

3.2 Introduction

Physiological activity of dietary peptides in improving human health is being

increasingly acknowledged. These bioactive peptides are inactive in parent proteins and

will release upon hydrolysis through digestive enzymes, microbial digestion or by using

commercial enzymes obtained from plant, animals or microorganisms (Korhonen &

Pihlanto, 2006; Pihlanto & Korhonen, 2003). In particular, antioxidant properties of

peptides has become of great interest to pharmaceutical, health foods/ nutraceutical

industries. In recent years, the antioxidant peptides from fish proteins has gained much

attention (Hagen & Sandnes, 2004; Ngo, Wijesekara, Vo, Van Ta, & Kim, 2011). Aquatic

products and by-products from many marine sources such as pacific hake (Merluccius

productus) (Samaranayaka, 2010), squid (Teuthida) gelatin (Alemán, Pérez-Santín,

Bordenave-Juchereau, Arnaudin, Gómez-Guillén, & Montero, 2011), and tilapia

(Oreochromis niloticus) (Raghavan, Kristinsson, & Leeuwenburgh, 2008) have been

studied for their antioxidant activity in biological systems. However to the best of our

knowledge, no attempt has been done to evaluate the bioactive properties of invasive,

under-utilized silver carp (Hypophthalmichths molitrix) protein hydrolysates for its

pharmaceutical or nutracuetical use.

Bioactivity of a peptide depends on its molecular weight, amino acid composition

and sequence, and its structural properties. Especially smaller peptides (2-20 amino acids)

with active amino acid sequence are believed to be cell permeable, can scavenge

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intracellular oxygen species and may also up-regulate the endogenous antioxidant system

(Hazel H Szeto, 2008; Zhao, et al., 2004). In biological systems, antioxidant peptide

operates by complex mechanism, and simple chemical assays cannot accurately measure

bioactivity (R. H. Liu, 2004; Wolfe & Liu, 2007). Chemical based assays do not take

antioxidant peptide interactions with intracellular oxidation species into consideration and

cannot measure its true antioxidant activity in biological systems. To truly evaluate the

bioactivity, animal or human clinical studies are ideal. However, these studies are very

expensive and time consuming for preliminary screening. Cell based antioxidant assays

which are inexpensive and rapid are usually performed for preliminary screening

(Eiamphungporn, Prachayasittikul, Isarankura-Na-Ayudhya, & Prachayasittikul, 2012; R.

H. Liu & Finley, 2005). For this study, hydrolysates with high chemical based antioxidant

activity are tested using human heterogeneous epithelial colorectal adenocarcinoma cells

(Caco-2 cells) as model. Moreover, effect of peptide molecular weight and peptide

sequence on cell based antioxidant activity was also evaluated using cell-based assay. In

this study, two silver carp protein hydrolysate samples prepared using Flavourzyme® and

Alcalase® enzymes with highest chemical based antioxidant activity were selected for the

experiment.

The objective of the study was to test hydrolysates with high chemical based

antioxidant activity using human heterogeneous epithelial colorectal adenocarcinoma cells

(Caco-2 cells) as a model under oxidative stress and non-stress conditions. In addition, the

effect of peptide molecular weight and peptide sequence on intracellular antioxidant

activity was also evaluated using cell-based assay. Combined, these results will serve as

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an initial step to develop potential natural nutraceutical antioxidants from invasive silver

carp as an excellent alternative to wasteful disposal into the environment.


3.3.1 Materials





Flavourzyme® (EC 232-752-2, P-6110, 500 U/G), ʟ-leucine and 2,4,6-

trinitrobenzenesulphonic acid (TNBS) were purchased from Sigma-Aldrich, (St. Louis,

MO, USA). Sodium Pyruvate 100mM, Penicillin-Streptomycin (100X), Fetal Bovine

Serum, Trypsin EDTA (0.25%), 96 well culture plates and culture dishes were purchased

from VWR (Radnor, PA, USA). Dulbecco’s modified eagle medium (DMEM)/High w/ L-

Glutamine/With Sodium Pyruvate (1000mL), Phosphate Buffered Saline (1X, 500ml), 2,2-

azobios-(2-amidinopropane) dihydrochloride (AAPH), 2,7-dichlorodihydrofluorescein

diacetate and DMEM/ high modified no phenol red were purchased from Fisher Scientific

(Pittsburgh, PA, USA).

3.3.2 Preparation of SPH

Silver carp protein hydrolysis was performed using Flavourzyme® and Alcalase®

at 30 min and 60 min hydrolysis times. The hydrolysates were prepared under optimum

temperature (50 °C), pH (Flavourzyme®: 6.5 to 8.5; Alcalase®: 5.5 to 7.5) and desired

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hydrolysis times to obtain F-30 and A-60 using Flavourzyme® and Alcalase® respectively.

F-30 and A-60 sample were prepared according to (Liceaga‐Gesualdo & Li‐Chan, 1999)

as outlined in Chapter 2, section 2.3.2.

3.3.3 Degree of Hydrolysis

Degree of hydrolysis of the SPH was measured using the Trinitrobenzene sulfonic

acid (TNBS) adapted from (J. Adler-Nissen, 1979) with modifications by (Liceaga‐

Gesualdo & Li‐Chan, 1999) as outlined in Chapter 2, section 2.3.3.

3.3.4 Molecular Weight Profile of F-30 and A-60 Samples

Molecular weight profile of A-60 and F-30 peptide fractions was determined using

low pressure size exclusion chromatography. Sephadex G25 fine column (Pharmacia

Biotec Inc, Piscataway, NJ, USA) with dimensions of 1.7 cm (diameter) × 90 cm (height)

and with a void volume of 109 mL was used. Sample (2.5 mg/mL) was dissolved in 5 mM

phosphate buffer (pH 7) and filtered through 25 mm syringe filter with 0.2 μm membrane

(Fisher Scientific, Pittsburgh, PA, USA). Purified solution (5 mL) was injected into column

and eluted using distilled water with a flow rate of 1.5 ml/ min. Peptide fractions were

collected every 4 min using Bio-Rad model 2110 fraction collector (Bio-Rad, Hercules,

CA, USA). Protein content in each tube was calculated using bicichoninic acid (BCA)

assay and expressed as mg/ mL using user instructions. Molecular weight profile of the

samples was determined using calibration curve generated using different protein standards

(Insulin (~3.5 kDa) and vitamin B12 (1355 KDa)). A plot between protein content and

elution volume of A-60 and F-30 samples was used to determine molecular weight profile.

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3.3.5 Fractionation of Hydrolysates According to Molecular Weight

F-30 and A-60 samples were fractionated according to molecular weight into two

fractions: <10 to >3 kDa (F-30 >3 and A-60>3 respectively) and <3 kDa (F-30 <3 and A-

60 <3 respectively). All the molecular weight fractions of F-30 and A-60 samples were

freeze-dried and stored at -20 °C until used.

3.3.6 Size-Exclusion Chromatography

The A-60 and F-30 peptide fractions with molecular weight <3 kDa were

fractionated using low pressure size exclusion chromatography using a sephadex G25 fine

column (Pharmacia Biotec Inc, Piscataway, NJ, USA) as outlined in section 3.3.4. Peptide

fractions were collected every 4 min using Bio-Rad model 2110 fraction collector (Bio-

Rad, Hercules, CA, USA) and molecular weight of a group of five test tube fractions were

analyzed using HPLC as outlined in the section 3.3.8.

3.3.7 Ultrafiltration

The peptide fractions ranging from 10 to 3 kDa of F-30 and A-60 samples were

fractionated using ultrafiltration. Two different filters with molecular weight cut off of 10

and 3 kDa was used as outlined by Centricon® centrifugal filter devices user manual.

Peptides that can pass through 10 kDa and cannot pass through 3 kDa MWCO filters have

molecular weight between 10 kDa and 3 kDa. The fractionated samples with molecular

weight ranging from 10 to 3 kDa were freeze-dried and their molecular weight profile was

confirmed using HPLC as outlined above.

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3.3.8 Molecular Weight Determination

The molecular weight range of a sample was determined using High performance

liquid chromatography (HPLC) as outlined below. A 20 μL of sample (2.5 mg/ mL) was

injected to Waters e2695 Separation Module HPLC system (Waters Corporation, Milford,

MA, USA) equipped with an auto injector and UV/Vis detector. Sepax zenix SEC-100,

3µm, 100 A°, 4.6 × 300 mm2 column and a guard column (2.1 × 50 mm2) with same

material was used to separate samples according to size at 215 nm absorbance. Phosphate

buffer (150 mM, pH 7.0) was used as solvent with a flow rate of 0.35 mL/ min and

separation was performed under ambient temperature (23 °C). Molecular weight range of

the samples was obtained using calibration curve generated using different protein

standards (Carbonic anhydrase (29 kDa), Aprotinin (6.5 kDa), and Cytidine (0.243 kDa)).

All the molecular weight fractions of F-30 and A-60 samples were freeze-dried and stored

at -20 °C until used.

3.3.9 LC-MS/MS (ESI)

Peptides were separated on a nano LC system (1100 Series LC , Agilent Technologies,

Santa Clara, CA). The peptides were loaded on the Agilent 300SB-C18 enrichment column

for concentration and the enrichment column was switched into the nano-flow path after 5

min. Peptides were separated with the C18 reversed phase ZORBAX 300SB-C18

analytical column (0.75 μm × 150 mm, 3.5um) from Agilent. Column was connected to

the emission tip from New Objective and coupled to the nano-electrospray ionization (ESI)

source of the high resolution hybrid ion trap mass spectrometer LTQ-Orbitrap LX (Thermo

Scientific). The peptides were eluted from the column using the acetonitrile /0.1% formic

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acid (mobile phase B) linear gradient. For the first 5 min, the column was equilibrated with

95% H2O /0.1% formic acid (mobile phase A) followed by linear gradient of 5% B to 40%

B in 50 min. at 0.3ul/min, then from 40% B to 100% B in additional 10 minutes. Column

was washed with 100% of ACN/0.1%FA and equilibrated with 95% of H2O/0.1%FA

before next sample was injected. A blank injection was run between samples to avoid

carryover.

The LTQ-orbitrap mass spectrometer was operated in the data-dependent positive

acquisition mode in which each full MS scan (30.000 resolution) was followed by six

MS/MS scans where the six most abundant molecular ions were selected and fragmented

by collision induced dissociation (CID) using a normalized collision energy of 35%. Raw

data were collected by Xcalibur (v 2.0.7).

Database search analysis was done using Spectrum Mill A.03.02.060 software package

(Agilent Technologies). The search was performed against NCBI database, species: Fish

Reptiles. The search parameters were: precursor mass tolerance 0.05Da, fragment mass

tolerance 0.7 Da, no enzyme was specified. For peptides identification only peptides with

a Spectrum Mill score of 5 or higher and Spectrum Mill scored Peak Intensity (SPI) of 70

or higher were considered positive.

3.3.10 Caco-2 BBe Cell Culture Treatment

Intracellular antioxidant assay of F-30, A-60 and their fractions was done using

the Caco-2 BBe cell line. In this experiment, Caco-2 BBe cells (passage: 65-69) were

used as mammalian cell model but not to simulate highly differentiated intestinal cell

model. Caco-2 BBe cell line were incubated in DMEM, supplemented with 10% (v/v)

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fetal bovine serum, penicillin (100 unit/ mL), streptomycin (100 μg/ mL) and sodium

pyruvate (0.11 g/ L). Cells were incubated in a fully humidified environment under 5 %

CO2 at 37 °C and were sub-cultured at 2-3 days interval before reaching 70-80%

confluence.

3.3.11 Cell Based Antioxidant Activity

Cell based antioxidant activity assay was performed as outlined by (C. Y. Kim, Kim,

Wiacek, Chen, & Kim, 2012). Caco-2 cells were seeded in 96 well-plates at a density of

20,000 cells/ cm2 and incubated for 24 h under 5 % CO2 at 37 °C. Sample solutions with

different concentrations (0.625, 1.25 and 5 mg of protein / mL) were prepared by diluting

in DMEM/ high modified phenol red free and were added to cells. In one set of treatment

0.1 mM of 2,2-azobios-(2-amidinopropane) dihydrochloride (AAPH) was added which

serves as an oxidative stress (stressed) condition and another set without addition of AAPH

serves as normal physiological (non-stressed) condition. Cells with the sample/ sample +

AAPH were incubated for 1 h at 37°C and probe 2,7-dichlorodihydrofluorescein diacetate

(DCFH-DA) to make final concentration of 20 μM. After addition of probe, samples were

incubated in the dark for 30 min at 37 °C and fluorescence signal (λexcitation = 485 nm,

λemission = 538 nm) was measured at different time intervals while keeping the cells in dark.

Reactive oxygen species (ROS) generated during cellular oxidation is known to oxidize

DCFA, cellular activity by-product DCFA-DA, to the fluorescent compound DCF.

Therefore increase in fluorescence implies increase in cellular oxidation. A control sample

with DMEM/ high modified no phenol instead of sample was performed which acts as a

negative control. A positive control (Vitamin C) at different concentrations (40, 20 and 10

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µM) were also analyzed. Both the controls were tested at stressed and non-stressed

conditions with and without AAPH respectively. The percentage cellular oxidation in

presence of a sample was calculated by considering cellular oxidation of negative control

as 100 % at a particular incubation time.

In order to measure the effect of incubation on reduction of cellular oxidation, IC50

values of the sample (minimum concentration of the sample required to inhibit 50 % of

cellular oxidation) were determined at 30 min and 120 min of incubation. IC50 values of

the samples were calculated using graph between concentration of the sample and %

cellular oxidation in presence of the sample.








3.4.1 Choice of Hydrolysates

Overall among SPH-F and SPH-A samples F-30 and A-60 sample respectively showed

higher antioxidant activity. Therefore they were further fractionated according to molecular

weight and screened for their antioxidant activity using in vitro cell based assay.

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3.4.2 Molecular Weight Distribution of F-30 and A-60

Molecular weight profile of F-30 and A-60 samples when ran on Sephadex G-25 gel

filtration column is shown in Figure 3-1. Using molecular weight profile and calibration

curve (Figure 3-2), molecular weight distribution of F-30 and A-60 samples was calculated,

which is shown in Table 3-1. Hydrolysis of silver carp using Alcalase® for 60 minutes

resulted in high percent of lower molecular weight peptides (< 5 KDa = 57.56 %) compared

to Flavourzyme® hydrolysis for 30 min (< 5 KDa = 47.94 %) (Table 3-1). This observation

is in accordance with the higher % degree of hydrolysis of A-60 (36.43) than F-30 (14.47).

3.4.3 Fractionation of F-30 and A-60 Samples According to Molecular Weight

During Sephadex G-25 gel filtration eluted sample was collected for every 4 minute

interval and molecular weight was analyzed using Sepax zenix size exclusion

chromatography. The sample tubes with molecular weight <3 KDa were pooled for both

F-30 and A-60 from 28-38 and 28-35 tubes respectively. According to the standard curve

of Sepax zenix column (Figure 3-3), the peptide fractions with molecular weight <3 kDa

occurs after 8.63 min. The molecular profile of peptide fractions F-30 <3 and A-60<3

shows that more than 80% of the sample is eluted after 8.63 min, confirming the purity of

peptides <3 kDa (Figure 3-4 and 3-5). The protein content of the freeze dried peptides <3

kDa fractionated from F-30 and A-60 samples was 84.2 and 82.9 %, respectively.

The peptide fractions greater than 3 kDa was separated by ultrafiltration with

molecular weight cut-off of 10 and 3 kDa. The molecular weight profile of these peptide

fraction collected was tested using Sepax zenix column and more than 70 % of the sample

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was eluted before 8.63 min, showing that the peptides > 3 kDa was fractionated (Figure 3-

6 and 3-7).

3.4.4 Peptide Sequencing of F-30<3 and A-60<3 Samples

Peptide sequencing of F-30<3 and A-60<3 samples was performed using LC-

MS/MS (ESI). The amino acid sequence of peptides which match with the common

characteristics of most antioxidant peptides in the literature were identified. It is well

recognized that hydrophobic amino acid at N-terminal has shown to increase the

antioxidant activity of the peptide (Elias, Kellerby, & Decker, 2008; P.-J. Park, Jung, Nam,

Shahidi, & Kim, 2001) and more than half of the antioxidant peptides identified have

hydrophobic amino acid at N terminal (Power, Jakeman, & FitzGerald, 2013). The higher

electronic property of C-terminal amino acids is also an important prediction of antioxidant

activity. In most of the antioxidant peptide C-terminal amino acids are often occupied by

Trp, Leu, Glu, Ile, Met, Val, and Tyr (Y. W. Li, Li, He, & Qian, 2011; Power, Jakeman, &

FitzGerald, 2013). According to Li et al., (2011) (Y. W. Li, Li, He, & Qian, 2011) the

second amino from C terminal is a major contributor to antioxidant activity and basic or

acidic amino acids (Asp, Glu, His, Arg, Lys) or hydrophilic amino acids (Ser, Thr) are

preferred. Most of the potent antioxidants derived from casein and β-lactoglobulin typically

have Glu, Agr, Asp, Pro and Leu in this position. The peptides with above characteristic

were pooled from both F-30<3 and A-60<3 samples.

Apart from N and C terminal amino acids, presence of antioxidant amino acids such

as Tyr, Met, His, Cys and Trp in the sequence can contribute to the antioxidant activity of

the peptide (You, Zhao, Cui, Zhao, & Yang, 2009). Therefore, the peptide sequence with

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desired N and C terminal amino acids were further screened for the above antioxidant

amino acids. The total number of peptides with desired N and C terminal amino acids and

with three of the above antioxidant amino acid from F-30 <3 and A-60<3 samples were 7

and 4 respectively. In addition two of the peptides, (V)NCSQNLMNYSQHTKIS(I) and

(F)YMGKSHCHIFHSIMS(I), from F-30<3 sample has combination of Tyr, Met, Cys, and

His. These two peptide have high potential to contribute antioxidant activity.

Interestingly, the total number of peptides containing sulphur containing amino

acids acids such as cysteine and methionine is higher in F-30<3 (49 peptides) compared to

A-60<3 (36 peptides).

3.4.5 Intracellular Antioxidant Activity of Different Molecular Weight Peptides

Intestine serves as the interface between gut lumen and blood stream, therefore it

acts as a crucial defense barrier against luminal toxic agents. In addition, it is constantly

exposed to diet-derived oxidants, mutagens, carcinogens as well as reactive oxygen species

from diet (B.N. Ames, 1983). Therefore it is crucial to protect gastro intestinal cells from

cellular oxidation using additional external antioxidants and justifying the choice of Caco-

2 cells for the assay. In this assay, antioxidant activity of different molecular weight

fractions of F-30 and A-60 samples was evaluated under AAPH- induced oxidative stress

condition as well as under normal cellular conditions without any external oxidative stress

(non-stress). The oxidation in the cell is measured in terms of fluorescence reading.

Oxidation of cellular activity by-product DCFA results in production of DCF, which has

fluorescence at λExcitation

= 485 nm and λEmission

= 527 nm. Therefore increase in

fluorescence implies increase in cellular oxidation.

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Inhibition of intracellular oxidation in presence of different molecular weight

peptide fractions of F-30 and A-60 samples at 5 mg/ mL concentration during 150 min

incubation under both oxidation stress (AAPH, 0.1 mM, 1h at 37 °C) and non-stress (no

AAPH added) conditions in a Caco-2 human intestinal cell model is shown in the Figure

3-8. This model was chosen based on its common use as an intestinal cell model (Albersen,

Bosma, Knoers, de Ruiter, Diekman, de Ruijter, et al., 2013; Thompson, Al Mutairi, Sharp,

Elliott, & Fairweather-Tait, 2010) and the exposure of the gut to oxidants and reactive

oxygen species derived from the diet and/or generated from digestion and normal

metabolic function (Ames, 1983). Addition of AAPH, a oxidative stress inducer, results in

diminishing of antioxidant reserves of the cell and increase oxidative damage (Razinger,

Drinovec, & Zrimec, 2010). The pronounced increase in cellular oxidation due to AAPH

is evident in negative control samples under stress when compared to that in non-stress

conditions (Figure 3-8). Under both stress and non-stress conditions, F-30 <3 sample

showed decreased cellular oxidation with increase in incubation time compared to F-30. A

similar observation was shown in A-60 <3 sample only under stress condition. Conversely,

peptides with molecular weight >3 KDa (F-30 >3 and A-60 >3) did not show any improved

antioxidant activity compared to F-30 and A-60. Further, both F-30 >3 and A-60 >3

samples increased cellular oxidation than negative control after 120 min incubation under

non-stress condition. The results emphasize that the antioxidant activity of peptides is

highly dependent on molecular weight and this observation is supported by many studies

(C.-B. Ahn, Je, & Cho, 2012; J.-Y. Je, Park, & Kim, 2005; W.-S. Lee, Jeon, & Byun, 2011)

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3.4.6 Inhibition of Cellular Oxidation by Different Molecular Weight Peptides Under

Stress and Non-Stress Conditions

Dose-dependent inhibition of cellular oxidation of different fraction of F-30 and A-

60 samples as well as vitamin C under non-stressed (no AAPH added) and stressed (AAPH,

0.1 mM, 1h at 37 °C) conditions is shown in Figure 3-9 and 3-10. The positive control

(vitamin C) failed to show dose dependency in inhibiting cellular oxidation. Comparable

results were observed by Intra. J. and Kuo. S-M. (2007) (Intra & Kuo, 2007) at similar

concentration of vitamin C in human Caco -2 cell model. One possible reason is that many

antioxidants such as vitamins can have pro-oxidant effect besides antioxidant activity,

especially at higher doses and prolonged incubation (Brown, Morrice, & Duthie, 1997;

Intra & Kuo, 2007; Versari, Daghini, Rodriguez-Porcel, Sattler, Galili, Pilarczyk, et al.,

2006).

Under both conditions, with the increase in concentration from 0.625 to 5 mg/ mL

there was an increase in inhibition of cellular oxidation in the presence of F-30, F-30 <3,

A-60 and A-60 <3 samples. However, dose-dependent inhibition in cellular oxidation was

not demonstrated by peptide fractions greater than 3 kDa samples (F-30 >3 and A-60 >3)

under the non-stress condition. Interestingly, under the stress condition there was a

significant increase (P<0.05) in cellular oxidation inhibition by F-30 >3 sample at its

highest concentration (5 mg/ mL) compared to lower concentrations (0.625 and 1.25 mg/

mL). The reason for this improvement in its antioxidant activity is unknown and further

evaluation is required. The above results emphasize that the antioxidant activity of peptides

is highly dependent on molecular weight and this observation is supported by many studies

(C.-B. Ahn, Je, & Cho, 2012; J.-Y. Je, Park, & Kim, 2005; W.-S. Lee, Jeon, & Byun, 2011).

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Overall, IC50 values of F-30 fractions are lower than A-60 fractions, indicating higher

cellular antioxidant activity of F-30 fractions compared to A-60 fractions (Table 3-2) under

both conditions. This is in accordance with the higher chemical based radical scavenging

activity of F-30 sample compared to the A-60 sample.

A large part of the cellular oxidation occurs in mitochondria because they are

constantly exposed to high ROS levels during cellular activity. Therefore an ideal

antioxidant should be able to permeate through the cell, target mitochondria and should

protect mitochondria against oxidative stress (H.H. Szeto, 2008). Some small peptides with

active amino acid sequence have shown to penetrate the cell membrane freely and inhibit

mitochondrial oxidation (Rocha, Hernandez-Mijares, Garcia-Malpartida, Bañuls, Bellod,

& M Victor, 2010; H.H. Szeto, 2008). At all concentrations of peptide fractions with

molecular weight greater than 3 kDa (F-30>3 and A-60>3) there was a significant increase

in cellular oxidation with increase in incubation time (Figure 3-9 and 3-10). Similarly,

under non-stress conditions, the overall antioxidant activity of F-30 and A-60 samples

decreased from incubation periods of 30 min (at 30 min: IC50 of F-30 = 4.20 mg; IC50 of

A-60 = 5.87 mg) to 120 min (at 120 min: IC50 of F-30 = 5.31 mg; IC50 of A-60 = 8.88 mg).

This observation may be attributed to their poor cell permeability and inability to inhibit

mitochondrial oxidation. However, F-30 <3 was able to inhibit cellular oxidation rapidly

within 30 min of incubation at all concentrations and was also able to retain its activity

throughout 120 min of incubation (Figure 3.9). This observation may suggests the rapid

uptake of F-30 <3 into the cell interior and targeting mitochondrial oxidation. However,

further confirmation by studying cellular and mitochondrial uptake of peptides should be

performed. On the other hand, under both stress and non-stress conditions, A-60 <3

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samples showed very low antioxidant activity at 30 min but with further incubation (120

min) its antioxidant activity significantly improved (P<0.05). This antioxidant activity

improvement is evident with the significant decrease (P<0.05) in its IC50 value from 30 min

to 120 min incubation. This delayed inhibition of cellular oxidation of A-60 <3 sample can

be attributed to many factors such as a delay in cellular permeation or delayed in targeting

cellular mitochondria of A-60 <3. Since amino acid sequence of a peptide is a crucial factor

that influences its cell permeability (V. H. Lee & Yamamoto, 1989; H.H. Szeto, 2008),

peptides obtained from Alcalase® hydrolysis of silver carp protein may not have amino

acid sequences “active” for rapid cellular uptake unlike peptides from Flavourzyme® (an

endo- and exo-peptidase) hydrolysis. Another reason may be due to higher number of

active peptides with desired amino acid sequence of F-30 <3 sample compared to A-60 <3

may have contributed to its higher cellular antioxidant activity during initial incubation

times (Section 3.4.4.). However, with increased incubation time of A-60 <3 peptide might

have generated more potent antioxidant peptides with the action of membrane proteases.

In addition, the higher number peptides with sulphur containing amino acids

and free sulphur containing amino acids (Chapter 2, table 2.2) (Cys and Met) present in

the F-30<3 KDa fractions compared to A-60<3 KDa may also contribute to its higher

antioxidant activity. The presence of sulphur containing amino acids such as cysteine and

methionine can enhance glutathione synthesis by activating γ-glutamyl cysteine ligase

enzyme (Kim, et al., 2003). Furthermore, the efficiency of dietary cysteine in the form of

peptide showed enhanced glutathione synthesis in spleen cells when compared to free form

(Bounous, Batist, & Gold, 1989). Glutathione, a major component of cellular antioxidant

system, plays a crucial role in reducing ROS and free radicals in the cell. It is a substrate

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for glutathione transferases and peroxidases enzymes that detoxify xenobiotics and ROS

in the cell (Fang, Yang, & Wu, 2002; Parcell, 2002). Therefore presence of sulphur

containing amino acids may increase the concentration of glutathione and reduce the

vulnerability of the cell to oxidative stress (Atmaca, 2004).

At all incubation conditions, F-30 <3 sample showed higher antioxidant activity

than rest, which is evident by the lower IC50 values of F-30 <3. Moreover, under the stress

condition F-30<3 shown to improve cellular antioxidant activity compared to the non-

stress condition. This preliminary findings suggest that F-30 <3 peptide fractions may have

potential to protect mitochondria against oxidative damage even under stress conditions.

However, further pharmacological studies to test its stability towards digestion and also

clinical studies using animal model are crucial for its successful nutraceutical application.

In addition, further purification of the most potent antioxidant peptide from the fraction

and studying its amino acid sequence helps to understand the mechanism of its antioxidant

activity.

3.4.7 Conclusion

Invasive silver carps are spreading into Mississippi great lake region causing

negative impact on ecological balance and economic. These species are not used for human

consumption owing to their non-conventional and are used as pet food or simply discarded

into waste. These under-utilized silver carp protein can serve as promising natural

resources that can be used in health food industries or nutracuetical industries as a health

benefit ingredient. In this study, silver carp protein hydrolysates from Flavourzyme® and

Alcalase® under certain hydrolysis conditions have shown Caco-2 cell based antioxidant

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activity which is higher than vitamin C. In addition, peptide fractions <3 KDa of F-30 and

A-60 samples purified using chromatographic method have shown increased cell based

antioxidant activity. Particularly, <3 KDa peptide fraction of F-30 sample with peptides

with higher active amino acid sequence showed higher antioxidant activity and activity

was persistent during long incubation time. F-30<3 sample might be a promising food

additive or pharmaceutical agent. Further investigating the influence of amino acid

sequence of the peptide fractions on their cellular uptake and antioxidant machinery in the

cell is essential to clearly understand their antioxidant mechanism. Studies on factors

effecting bioavailability such as effect of gastrointestinal digestion and cell permeability

of these antioxidant hydrolysates and peptide fractions is needed.

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Table 3-1. The percent molecular weight distribution of Flavourzyme® and Alcalase®

hydrolyzed silver carp protein at 30 and 60 min hydrolysis time respectively (F-30 and A-

60) samples calculated using Sephadex G-25 gel filtration chromatogram and calibration

curve

Sample

Molecular weight distribution (%)

10-5 KDa 5-3 KDa <3KDa

F-30 41.49 10.72 37.22

A-60 35.18 11.51 46.05

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Table 3-2. IC50 values (mg/ mL) of F-30, F-30<3, A-60 and A-60 <3 samples at 30 and

120 min incubation times under both stress and non-stress conditions

Samples Non-stress Stress

30 min 120 min 30 min 120 min

F-30 4.20 AxyA 5.31 Ax 3.08 Ay 4.54 Ax

F-30 <3 3.02 Ax 2.62 Bx 1.97 Axy 1.19 By

A-60 5.87 Bx 8.88 Cy 4.74 Bx 4.72 Ax

A-60 <3 8.77 Cx 5.05 Az 11.62 Cy 4.60 Az

ADifferent letters (A, B, C,….) indicate significant difference (p <0.05) in means within each column for each

individual incubation time. Different letters (x, y, z…) indicate significant difference (p <0.05) among the

means within each row for stress or non-stress condition, respectively.

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Figure 3-1. Sephadex G-25 gel filtration Chromatogram of Flavourzyme® and Alcalase®

hydrolyzed Silver carp protein at 30 and 60 min hydrolysis time respectively (F-30 and

A-60). The protein content was analyzed using BCA assay

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0 50 100 150 200 250 300 350 400

Pro

tein

conte

nt

(mg/

mL

)

Volume (ml)

Chromatogram using BCA

F-30

A-60

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Figure 3-2. Standard curve of Sephadex G25 column prepared using Insulin chain B,

oxidation (3495.89 Da) and Vitamin B12 (1355 Da). Molecular weight of 3000 Da occurs

at 129.88 mL elution volume, which occurs nearly at 22nd tube.

124, 3495.89

163.5, 1355

129.88, 3000

y = -52.398x + 9907

0

500

1000

1500

2000

2500

3000

3500

4000

110 120 130 140 150 160 170

Log (

mol

wt)

Elution Volume (mL)

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Figure 3-3. Standard curve of Sepax zenix SEC-100, 3 µm, 100 A°, 4.6 X300mm (100-

100,000 Da) column prepared using Carbonic anhydrase (29 KDa), Aprotinin (6511.44

Da), Vitamin B12 (1355.37 Da) and Cytidine (243.22 Da). Molecular weight of 3000 Da

occurs at 8.63 minute.

10.77, 2.39

7.82, 3.81

6.87, 4.46

8.63, 3.48

y = -0.5204x + 7.9699

2

2.5

3

3.5

4

4.5

5

5 6 7 8 9 10 11 12

Log (

Mw

t)

Retention time (min)

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Figure 3-4. Molecular weight profile of < 3 KDa peptide fractions of A-60 (A-60<3) ran




KDa peptide occurs at 8.63 min.

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Figure 3-5. Molecular weight profile of < 3 KDa peptide fractions of F-30 (F-30<3) ran on

Waters e2695 Separation Module HPLC system equipped with UV/Vis detector and Sepax

zenix SEC-100, 3µm, 100 A°, 4.6 × 300 mm2 column. Phosphate buffer (150 mM, pH 7.0)

was used as solvent phase at 0.35 mL/ min flow rate. The retention time of 3 KDa peptide

occurs at 8.63 min

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Figure 3-6 Molecular weight profile of <10 and >3 KDa peptide fractions of A-60 (A-60>3)

and ran on Waters e2695 Separation Module HPLC system equipped with UV/Vis detector

and Sepax zenix SEC-100, 3µm, 100 A°, 4.6 × 300 mm2 column. Phosphate buffer (150

mM, pH 7.0) was used as solvent phase at 0.35 mL/ min flow rate. The retention time of 3

KDa peptide occurs at 8.63 min

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Figure 3-7 Molecular weight profile of <10 and >3 KDa peptide fractions of F-30 (F-30>3)

and ran on Waters e2695 Separation Module HPLC system equipped with UV/Vis detector

and Sepax zenix SEC-100, 3µm, 100 A°, 4.6 × 300 mm2 column. Phosphate buffer (150

mM, pH 7.0) was used as solvent phase at 0.35 mL/ min flow rate. The retention time of 3

KDa peptide occurs at 8.63 min

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0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120 140 160

Ca

llu

lar

Ox

ida

tio

n (

Flu

ore

sce

nce

re

ad

ing

)

Non-stress condition (5 mg/ mL)

F-30 F-30 <3 F-30 >3 Negative control

0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120 140 160

Non-stress conditions (5 mg/ mL)

A-60 A-60 <3 A-60 >3 Negative control

`

0

500

1000

1500

2000

2500

3000

3500

4000

4500

0 20 40 60 80 100 120 140 160

Ce

llu

lar

Ox

ida

tio

n (

Flu

ore

sce

nce

re

ad

iin

g)

Min

Oxidation stress (5 mg/ mL)

F-30 F-30 <3 F-30 >3 Negative control

0

500

1000

1500

2000

2500

3000

3500

4000

4500

0 20 40 60 80 100 120 140 160

min


A-60 A-60 <3 A-60 >3 Negative control

Figure 3-8. Inhibition of cellular oxidation in presence of molecular weight fraction of (A)

F-30 under non-stress; (B) A-60 under non-stress; (C) F-30 under oxidation stress: and (D)

A-60 under oxidative stress during 120 min of incubation. Assay was conducted in

duplicate and analysis was done in triplicate.

(A) (B)

(C)

(A) (B)

(C) (D)

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Aax Aax Aax

Aax

BaxBax

Aax

Aax

Cax

Cax

BaxBax

0

10

20

30

40

50

60

70

80

90

100

110

30 NS 30 S 120 NS 120 S

Ce

llula

r o

xid

atio

n %

(-v

e c

on

tro

l = 1

00

%)

Concentration (mg/ mL)

F-30 0.625

1.25

5

AaxAbx

Aax

AbxBax

BbxAax

Bbx

Cax Cax Bax

Cby

0

10

20

30

40

50

60

70

80

90

100

110

30 NS 30 S 120 NS 120 S

Ce

llula

r o

xid

atio

n %

(-v

e c

on

tro

l = 1

00

%)


F-30<3 0.625

1.25

5

105

105

Aax Aax

Aay

Aby

Aax Aax

ABay

Aby

Aax

Bbx

Bay

Bby

0

10

20

30

40

50

60

70

80

90

100

110

120

130

30 NS 30 S 120 NS 120 S

Ce

llula

r o

xid

atio

n %

(-v

e c

on

tro

l = 1

00

%)


F-30>3 0.625

1.25

5

Aax

Abx

Aay

Abx

Aax

Bbx

Aay

AaxAax

Bbx

AaxAax

0

10

20

30

40

50

60

70

80

90

100

110

30 NS 30 S 120 NS 120 S

Ce

llula

r o

xid

atio

n %

(-v

e c

on

tro

l = 1

00

%)


Vitamin C10 µM

20 µM

40 µM

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106


presence of F-30, its fractions (F-30 <3 and F-30 >3) and positive control (Vitamin C)



sample added) at a given condition was used as a reference (100 % oxidation).

Values are mean ± SE, n = 3. 30 NS and 30 S = after 30 min incubation under non-stressed

and stressed conditions; 120 NS and 120 S = after 120 min incubation under non-stressed

and stressed conditions.

Different upper case letters (A, B, C…) indicate significant different among the means at

different concentration within a condition (30 NS, 30 S, 120 NS and 120 S). Different lower

case letter (a, b, c…) represent significant difference among the means of stressed and non-

stressed condition under similar concentration and incubation time. Different lower case

letters (x, y, z…) represent significant difference among the means at different incubation

times under similar concentration and condition (stressed and non-stressed). Assay was

conducted in duplicate and analysis was done in triplicate.

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107

Aax

AbxAax

Aby

Aax

Bbx

Aax

Abx

Bax

Cbx

Bay

Bbx

0

10

20

30

40

50

60

70

80

90

100

110

30 NS 30 S 120 NS 120 S

Ce

llula

r o

xid

atio

n %

(-v

e c

on

tro

l = 1

00

%)


A-60 0.625

1.25

5

AaxAax

AaxAay

AaxBax

BayBby

Bax

Bax

Cay Cay

0

10

20

30

40

50

60

70

80

90

100

110

30 NS 30 S 120 NS 120 S

Ce

llula

r o

xid

atio

n %

(-v

e c

on

tro

l = 1

00

%)


A-60<3 0.625

1.25

5

108

108

Aax

Aax

Aay

AbyBax

Aax

Aay

Aby

ABax Aax

Aay

Aby

0

10

20

30

40

50

60

70

80

90

100

110

120

130

30 NS 30 S 120 NS 120 S

Ce

llula

r o

xid

atio

n %

(-v

e c

on

tro

l = 1

00

%)


A-60>3 0.625

1.25

5

Aax

Abx

Aay

Abx

Aax

Bbx

Aay

AaxAax

Bbx

AaxAax

0

10

20

30

40

50

60

70

80

90

100

110

30 NS 30 S 120 NS 120 S

Ce

llula

r o

xid

atio

n %

(-v

e c

on

tro

l = 1

00

%)


Vitamin C10 µM

20 µM

40 µM

109

109


presence of A-60, its fractions (A-60 <3 and A-60 >3) and positive control (Vitamin C)



sample added) at a given condition was used as a reference (100 % oxidation). Values are

mean ± SE, n = 3.

30 NS and 30 S = after 30 min incubation under non-stressed and stressed conditions; 120

NS and 120 S = after 120 min incubation under non-stressed and stressed conditions.








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CHAPTER 4. EFFECT OF SIMULATED GASTROINTESTINALDIGESTION ON IN

VITRO CELL BASED ANTIOXIDANT ACTIVITY OF SILVER CARP

(HYPOPHTHALMICHTHYS MOLITRIX) PROTEIN

4.1 Abstract

Invasive silver carp protein hydrolysates (F-30 and A-60) with higher cellular

antioxidant activity and pasteurized control (unhydrolyzed) (SPU) was digested using two

simulated gastrointestinal (GI) digestion protocols: Protocol 1: Low digestive enzyme

activity (F-30#1, A-60#1 and SPU#1); Protocol 2: High digestive enzyme activity (F-30#2,

A-30#2 and SPU#2). Using both the protocols, the final GI digestion of F-30 and A-60

samples showed an increase in short chain peptides fraction (<500 Da). In presence of GI

digest samples of F-30 (F-30#1, F-30#2) and A-60 (A-60#1 and A-60#2), the cell viability

was higher than 78 %, showing no cytotoxic effects at its highest concentration (5 mg/ mL)

and even after 2 h of incubation. GI digestion of F-30 and A-60 using protocol with high

digestive enzyme activity have resulted in the destruction of their cellular antioxidant

activity. However, GI digestion of F-30 using protocol with low enzyme activity (F-30#1)

was able to retain its antioxidant activity to an extent and also showed dose dependent

antioxidant activity. GI digestion of SPU using high enzyme activity (SPU#2) only showed

cellular antioxidant activity. However, SPU#2 did not show any dose dependent activity

and could not retain its activity for longer incubation times (120 min). The results shows

that extreme GI digestion of F-30 and A-60 samples would result in complete annihilation

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111

of their antioxidant peptides. Limited GI digestion of SPU sample did not release

antioxidant peptides and could not show any cellular antioxidant activity. F-30 sample

showed more resistant towards GI digestion, could retain its activity at limited GI digestion

conditions.

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112

4.2 Introduction

Hydrolysis of food derived proteins can release peptides with certain bioactive

properties (Korhonen & Pihlanto, 2003). Depending on structural aspects, molecular

weight, amino acid composition and sequence of the peptide, it may have antioxidant,

antimicrobial, antihypertensive, anti-inflammatory activity (Kitts & Weiler, 2003;

Korhonen & Pihlanto, 2003). In recent years, peptide fractions with antioxidant activity

have received special interest in health foods, nutraceutical and pharmaceutical industries.

Antioxidant peptides from natural resources are believed to prevent many chronic diseases

caused by oxidative stress in the body. Protein hydrolysates derived from both plant and

animal sources including milk (Pihlanto, 2006), soy (Peñta‐Ramos & Xiong, 2002), eggs

(Chay Pak Ting, Mine, Juneja, Okubo, Gauthier, & Pouliot, 2011), alfalfa leaf (Xie, Huang,

Xu, & Jin, 2008), peanuts (G. t. Chen, Zhao, Zhao, Cong, & Bao, 2007), porcine (Q. Liu,

Kong, Xiong, & Xia, 2010) and maize (X. x. Li, Han, & Chen, 2008) have shown to have

antioxidant activity. In addition, numerous studies have evaluated antioxidant activity of

peptides derived from marine origin (Ali Bougatef, Mohamed Hajji, Rafik Balti, Imen

Lassoued, Yosra Triki-Ellouz, & Moncef Nasri, 2009; J.-Y. Je, Park, & Kim, 2005; J. Y.

Je, Qian, Lee, Byun, & Kim, 2008; Mendis, Rajapakse, & Kim, 2005). However, most of

these studies did not attempt to evaluate stability during gastrointestinal digest and

intracellular antioxidant activity of antioxidant peptides. The above factors affect

bioavailability and bioaccessibility of these antioxidant peptides to the target site of action.

Gastrointestinal (GI) track is constantly exposed to diet derived oxidants including iron,

copper, lipid peroxides and aldehydes. This leads to generation of reactive oxygen species

in the stomach, causing disruption of intestinal cell line and compromising defense barrier

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113

against luminal toxic agents (Bruce N Ames, 1984). It is important that natural antioxidant

sources such as peptides from diet is necessary to preserve the integrity of intestinal mono

layer. Antioxidant peptides must be resistant various GI digestion enzymes and should

retain their activity in order to minimize the oxidation in the GI-track. Thus there is a need

to study the stability of antioxidant peptides during GI digestion.

Apart from food processing protease enzymes, bioactive peptides can be formed from

inactive protein during gastrointestinal digestion. Release of bioactive peptide through

simulated in vitro GI digestion was demonstrated in many studies and the possible

antioxidant activity of peptides derived from simulated GI digestion is worth evaluating

(Ali Bougatef, Mohamed Hajji, Rafik Balti, Imen Lassoued, Yosra Triki-Ellouz, & Moncef

Nasri, 2009; Kim, J.-Y. Je, & S.-K. Kim, 2007; Li, B. Jiang, T. Zhang, W. Mu, & J. Liu,

2008).

Different in vitro methods to simulate human GI digestion have been evaluated

extensively. These methods have varying combination of protease enzymes and enzyme

activity to represent gastric and intestinal digestion (Hur, Lim, Decker, & McClements,

2011). These methods have been used to evaluate the fate of many bioactive agents under

simulated GI digestive conditions.

In the present study, silver carp protein hydrolysates (F-30 and A-60) prepared

using Flavourzyme® and Alcalase® under certain hydrolysis conditions have been

identified as potential antioxidant peptide source based on Caco-2 cell based antioxidant

assay (Chapter 2). However, before confirming its efficiency under in vivo conditions, it is

important to evaluate the effect of simulated GI digestion of antioxidant peptide. Two

different GI digestion protocols with different enzyme combinations and activity were

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114

applied on F-30 and A-60 samples to study any possible effects on their antioxidant activity.

Further cell cytotoxicity in presence of GI digest samples was also evaluated. In addition,

cellular antioxidant activity of peptides formed during simulated GI digestion of

unhydrolyzed silver carp protein (SPU) was also evaluated.


4.3.1 Materials





Flavourzyme® (EC 232-752-2, P-6110, 500 U/G), ʟ-leucine, 2,4,6-

trinitrobenzenesulphonic acid (TNBS), Pepsin from porcine gastric mucosa (EC 232-629-

3, P7000, ≥ 250 U/ mg), pancreatin from porcine pancreas (EC 232-468-9, P7545, 8 X

USP), trypsin from porcine pancreas (EC 232-650-8, T0303, 14000 U/ mg), α-

chymotrypsin from bovine pancreas (EC 232-671-2, C4129, ≥ 40 U/ mg), Bile extract

porcine (B8631) were purchased from Sigma-Aldrich, (St. Louis, MO, USA). Sodium

Pyruvate 100mM, Penicillin-Streptomycin (100X), Fetal Bovine Serum, Trypsin EDTA

(0.25%), 96 well culture plates and culture dishes were purchased from VWR (Radnor,

PA, USA). Dulbecco’s modified eagle medium (DMEM)/High w/ L-Glutamine/With

Sodium Pyruvate (1000mL), Phosphate Buffered Saline (1X, 500ml), 2,2-azobios-(2-

amidinopropane) dihydrochloride (AAPH), 2,7-dichlorodihydrofluorescein diacetate and

DMEM/ high modified no phenol red were purchased from Fisher Scientific (Pittsburgh,

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115

PA, USA). Caco-2 BBe (brush border expressing) line and Caco-2/TC7 clone cell line were

obtained from American Type Culture Collection. 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) and anhydrous dimethyl sulfoxide (DMSO) was

purchased from Biotium (Biotium Inc., Hayward, CA).

4.3.2 Preparation of Silver Carp Protein Hydrolysates (SPH)

Silver carp protein hydrolysis was performed using Flavourzyme® and Alcalase®

at 30 min and 60 min hydrolysis times. The hydrolysates were prepared under optimum

temperature (50 °C), pH (Flavourzyme®: 6.5 to 8.5; Alcalase®: 5.5 to 7.5) and desired

hydrolysis times to obtain F-30 and A-60 using Flavourzyme® and Alcalase®, respectively.

F-30 and A-60 sample were prepared according to (Liceaga‐Gesualdo & Li‐Chan, 1999)

as outlined in Chapter 2, section 2.2.2.

4.3.3 Preparation of Unhydrolyzed Silver Carp Protein (SPU)

Silver carp fillets were minced using a meat grinder (Cabela’s, Hammond, IN, USA)

and homogenized in a blender with two volumes of water (w/v). The slurry were

immediately pasteurized (90 °C, 15 min) and resulting slurry was centrifuged at 16,300 ×

g for 15 min. The resultant supernatant was freeze-dried to yield SPU. Freeze dried samples

were stored at -20 ± 3°C until used for cell-based antioxidant assay.

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4.3.4 Simulated Gastrointestinal Digestion Silver Carp Protein Hydrolysates (SPH-GI)

Two protocols with different enzyme combination and activities were used to

perform in vitro gastrointestinal (GI) digestion of the samples. Both the protocols were

performed in two stages – gastric digestion, intestinal digestion.

4.3.4.1 Protocol 1:- Using Lower GI Enzyme Activity

Samples (F-30, A-60 and SPU) concentration of 200 mg of protein/ 10 mL were prepared

after adjusting the pH to 1.7 using HCl (1N). Pepsin solution (3 mL) with a final

concentration of 20 U/ g of sample protein was added to the sample solution and incubated

at 37 °C for an hour. After incubation the pH of the solution was increased immediately to

5.3 to stop pepsin digestion using 2M NaHCO3. Intestinal digestion was carried out by

adding 2.25 mL of Bile salt (24 mg of bile extracts/ 1 mL of 0.1 NaHCO3) and 1 mL of

pancreatin in 0.1 NaHCO3 with a final concentration of 0.0624 U/ mg of sample protein.

The pH of the sample was increased to 7 using 2N NaOH and was incubated at 37 °C for

1 h. After incubation the samples were immediately stored at -80 °C to stop the digestion.

The GI digest products from F-30, A-60 and SPU samples using protocol 1 were

represented by F-30#1, A-60#1 and SPU#1 respectively. A blank sample (Blk#1) using

above protocol with same amount of digestive enzymes but without any sample was also

prepared.

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4.3.4.2 Protocol-2:--Using Extreme GI Conditions

Samples (F-30, A-60 and SPU) concentration of 200 mg of protein/ 10 mL were

prepared after adjusting the pH to 1.7 using concentrated HCl. Pepsin solution (3 mL) with

a final concentration of 182000 U/ g of sample protein was added to the sample solution

and incubated at 37 °C for an hour. After incubation the pH of the solution was increased

to 5.3 to stop pepsin digestion using 2M NaHCO3. Intestinal digestion was carried out by

adding trypsin (34500 U/ g of sample protein in 0.1M NaHCO3) and Chymotrypsin (400

U/ g of sample protein in 0.1 NaHCO3). The pH of the sample was increased to 7 using 2N

NaOH and was incubated at 37 °C for 2 h. After incubation the samples were immediately

stored at -80 °C to stop the digestion. F-30#2, A-60#2 and SPU#2 represent the final

product of GI digestion of F-30, A-60 and SPU samples using protocol 2. Similar to

protocol 1, a blank sample (Blk#2) was also prepared using protocol 2 but without any

sample and with same amount of GI enzymes that are present in the sample.

4.3.5 Molecular Weight Distribution

The molecular weight range of a sample was determined using high performance

liquid chromatography (HPLC) as outlined below. A 20 μL of sample (2.5 mg/ mL) was

injected to Waters e2695 Separation Module HPLC system (Waters Corporation, Milford,

MA, USA) equipped with an auto injector and UV/Vis detector. Sepax zenix SEC-100,

3µm, 100 A°, 4.6 × 300 mm2 column and a guard column (2.1 × 50 mm2) with same

material was used to separate samples according to size at 215 nm absorbance. Phosphate

buffer (150 mM, pH 7.0) was used as solvent with a flow rate of 0.35 mL/ min and

separation was performed under ambient temperature (23 °C). Molecular weight range of

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the samples was obtained using calibration curve generated using different protein

standards (Carbonic anhydrase (29 kDa), Aprotinin (6.5 kDa), and Cytidine (0.243 kDa)).

All the molecular weight fractions of F-30 and A-60 samples were freeze-dried and stored

at -20 °C until used.

4.3.6 MTT Assay

Caco-2/TC7 clone cells were seeded on a 24-well plate at a density of 150,000

cell/mL (n = 3). Cells were maintained in DMEM/10% FBS (supplemented with 10 mM

HEPES, 100 µM non-essential amino acids, 50 µg/L gentamycin sulfate, and 100 U/L

penicillin/streptomycin) and were incubated at 37°C in a 5% CO2 atmosphere until

confluent. Media was aspirated, and cell monolayers were treated with a 5 mg/mL

concentration of sample (F-30#1, F-30#2, A-60#1 and A-60#2) in DMEM solution for 2 h.

Blk#1 and Blk#2 samples were also diluted in DMEM to obtain same protease

concentration present in GI digests from protocol 1 (F-30#1 and A-60#1) and protocol 2

(F-30#2 and A-60#2) respectively at 5 mg/ ml concentration and were also tested. The

solutions were aspirated, and cell monolayers were washed with PBS. MTT reagent

(Biotium Inc., Hayward, CA) was added at 5 µL per well, diluted in 300 µL DMEM

without phenol red, and left to incubate for 4 h. Formazan crystals were dissolved in 300

µL DMSO and then loaded onto a 96-well plate (Corning). Absorption was measured at

570 nm – 630 nm. SDS (1%) was used as a toxic control.

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4.3.7 Caco-2 BBe Cell Culture Treatment

Intracellular antioxidant assay of GI digests of F-30, A-60 and SPU samples was

done using Caco-2 BBe line. For this experiment, the cells were treated as outlined in

Chapter 3, section 3.3.9 to reach 70-80 % confluence.

4.3.8 Cell Based Antioxidant Activity

Cell based antioxidant activity assay was adopted from Kim et al.,(2012) (C. Y. Kim,

Kim, Wiacek, Chen, & Kim, 2012) and performed as outlined in Chapter 3, section 3.3.10.

The percentage cellular oxidation in presence of a sample was calculated by considering

cellular oxidation of their corresponding blank sample (Blk#1 and Blkl#2) as 100 % at a

particular incubation time.








4.4.1 Choice of Enzymes and Enzyme Activity Used to Simulated Gastrointestinal

Digestion

Several in vitro gastrointestinal digestion assays have been evaluated to simulate

true gastrointestinal digestion in human body. Wide range of digestive enzymes were used

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120

sequentially rather than all together, to simulate the different digestion processes (gastric

and intestinal digestion). Huge variation in the activity of digestive enzymes was found in

many in vitro studies simulating in vivo gastrointestinal digestion in the literature (Hur,

Lim, Decker, & McClements, 2011). The reason behind inconsistent enzyme levels can be

attributed to variability in concentration and volume of secreted digestive enzymes from

individual to individual. The individual variation depends on mental state, age, amount and

type of food, time of day food is consumed and health conditions (Hur, Lim, Decker, &

McClements, 2011; Rothman, 1976).

In the current study, hydrolysate digestion was performed using two in vitro

methods – Protocol 1. Two enzyme system (pepsin and pancreatin) + bile salt; Protocol 2.

Three enzyme system (pepsin, trypsin and chymotrypsin). In protocol 1, the activity of

pepsin and pancreatin used is about 20 U/ g of protein and 62.4 U/ g of protein, respectively.

Similar activity of pepsin and pancreatin was observed in gastric and duodenal juice

collected from 18 healthy individuals (Furlund, Kristoffersen, Devold, Vegarud, &

Jonassen, 2012). The GI digest product of F-30, A-60 and SPU samples using protocol 1

is represented as F-30 #1, A-60 #1 and SPU #1, respectively. It is believed that the

efficiency of in vivo digestion enzymes is more than in vitro digestion enzyme under same

activity (Hur, Lim, Decker, & McClements, 2011). Therefore in most of the in vitro GI

digestion assays, extreme digestion conditions such as high enzymatic activity and/ or

higher incubation time are generally employed. In addition, the effect of GI digestion on

the activity of antioxidant peptides is effectively studied using two GI digestion protocols

with different efficiencies. In protocol 2, three enzyme systems which are specific to

protein hydrolysis and at higher activity were used to produce extreme protein digestion

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121

conditions. The GI digest product of F-30, A-60 and SPU sample using protocol 2 is

represented by F-30 #2, A-60 #2 and SPU #2 respectively. Blank samples without any

protein substrate, with only digestive enzymes was prepared using both protocol and are

represented by Blk #1 and Blk #2.

4.4.2 Effect of Gastrointestinal Digestion on Molecular Weight of F-30 and A-60

Samples

The molecular weight profile of GI digest samples of SPU (SPU#1 and SPU#2)

obtained using gel filtration chromatography at similar concentration (20 µL, 2.5 mg/ mL)

is shown in the Figure 4.7 and 4.8. Similarly, the chromatograms of F-30 and A-60 and

their GI digest products (F-30#1, F-30#2, A-60#1 and A-60#2) at similar concentration is

shown in the Figure 4.1, 4.2, 4.3, 4.4, 4.5, and 4.6. Most of the peptide peaks of F-30

sample correspond to molecular weight of >1000 Da (retention time < 9.54 minutes).

However after GI digestion using both protocol 1 and 2, F-30 sample was degraded to more

smaller peptides which is obvious with the increase in peptide peaks with molecular weight

< 500 Da (retention time > 10.12 minutes). Among both F-30#1 and F-30#2, F-30#2

sample showed increased concentration of peptides < 1000 Da which is evident with

increase in peak areas of those peptides. The chromatogram of A-60 samples shows smaller

peptides when compared to F-30 sample with the highest peptide peak is found to have

molecular weight between 500 to 100 Da (retention time between 10.12 to 11.46 minutes)

(Figure 4.1 and Figure 4.4). This is accordance to the higher %DH of A-60 (36.43 %)

compare to that of F-30 (14.47 %). Similar to F-30 samples, GI digest of A-60 using

protocol 1 and 2 (A-60#1 and A-60#1) also resulted in increased peptide peaks with lower

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molecular weight and in A-60#2 chromatogram further shows additional smaller peptide

peaks (retention time > 17.74 minutes) than A-60#1 (Figure 4.5 and 4.6).

Since the digestive enzyme activity in protocol 2 is higher than in protocol 1,

simulated GI digestion using protocol 2 (SPU#2, F-30#2 and A-60#2) resulted in higher

concentration of smaller peptides compared to Simulated GI digestion using protocol 1

samples (SPU#1, F-30#1 and A-60#1). Chromatogram from SPU#1 shows that most of the

peptides (73 %) have retention time below 7.5 min, which corresponds to molecular weight

>11,500 Da. Simulated GI digestion of SPU using protocol 2 (SPU#2) resulted in further

breakdown of peptides and the percent peptide with molecular weight >11,500 Da is

reduced in SPU#2 (63 %) compared to SPU#1 (73 %). Simulated GI digestion of

unhydrolyzed sample (SPU) did not released smaller peptides as compared to hydrolyzed

samples (F-30 and A-60).

4.4.3 Viability of Cells in the Presence of Gastrointestinal Digested Samples

Viability of Caco-2/TC7 clone cells of all simulated gastrointestinal digested

samples of F-30 and A-60 (except SPU) at 5 mg/ mL concentration was performed.

Viability of cells was calculated using MTT assay which measures the ability of

mitochondrial dehydrogenase enzyme to produce purple colored formazan precipitate from

MTT. The formed formazam is measured colorimetrically which corresponds to functional

mitochondria of living cells (Y. Liu, Peterson, Kimura, & Schubert, 1997).

Measuring cell viability in presence of an antioxidant is very crucial since it affects

the cellular antioxidant assay results. Therefore, the possible toxic effect caused by GI

digest samples at highest concentration used for antioxidant assay was measured using this

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assay. Incubation of GI digest samples of F-30 (F-30#1 and F-30#2) and A-60 (A-60#1

and A-60#2) for 2 h at 5 mg/ mL sample concentration showed >78 % of cell viability. In

addition both the blank samples (Blk#1 and Blk#2) also showed cell viability greater than

87 % (Figure 4.9). The Donryu rat liver cell viability in presences of α-tocopherol (0.2 mg/

mL) measured after 2.5 h is very similar (80 %) to the SPH in this study (S.-K. Kim, Kim,

Byun, Nam, Joo, & Shahidi, 2001). Pacific hake protein hydrolysates and their GI digests

also showed no cytotoxicity (cell viability = 80-100%) when tested on Caco-2 cells under

similar conditions (Samaranayaka, 2010) Therefore, the samples did not show any

cytotoxic effect at its highest concentration used for evaluation of cellular antioxidant

activity.

4.4.4 Effect of Gastrointestinal Digestion of F-30 and A-60 Samples on its Antioxidant

Activity

Antioxidant activity of different concentration of F-30#1, F-30#2, A-60#1 and A-

60#2 was determined using Caco-2 BBe cells under non-stressed (no AAPH added) and

stressed (AAPH, 0.1 mM, 1h at 37 °C) conditions. Apart from GI digests of F-30 and A-

60, peptides from proteases used in GI digestion may also contribute to the antioxidant

activity. Therefore, intracellular antioxidant activity of Blk#1 and Blk#2 containing only

protease enzymes prepared using protocol 1 and 2 respectively were also studied.

Intracellular antioxidant activity of GI digest of F-30 and A-60 samples at 5 mg/

mL during 150 min incubation under non-stressed (no AAPH added) and stressed (AAPH,

0.1 mM, 1h at 37 °C) conditions is shown in Figure 4.10. Under both stress and non-stress

conditions, the samples digested using protocol 2 (F-30#2 and A-60#2) failed to inhibit

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cellular oxidation but instead they increased cellular oxidation. However under oxidative

stress condition, F-30#2 sample showed slight inhibition to cellular oxidation than its

corresponding blank sample (Blk#2) during initial incubation time (< 75 minutes). GI

digest of A-60 sample using lower protease (pepsin and pancreatin) activity (protocol 1)

also failed to show antioxidant activity. This observation can be attributed to destruction

of bioactive peptides by gastrointestinal digestion enzymes to further inactive small

peptides (Kitts & Weiler, 2003). Significant reduction of bioactivity of peptides after GI

digestion was observed in previous studies (Pasini, Simonato, Giannattasio, Peruffo, &

Curioni, 2001; Qian, Jung, Byun, & Kim, 2008; A. G. Samaranayaka, Kitts, & Li-Chan,

2010). Nevertheless, limited GI digestion of F-30 (F-30#1) sample showed cellular

oxidation inhibition than its corresponding blank sample (Blk#1). In addition, under stress

condition F-30#1 sample showed higher antioxidant activity than assay control during

initial incubation times. This observation indicates that either F-30 sample is able to resist

limited GI digestion and retain its activity or further digestion of F-30 resulted in peptide

with antioxidant activity.

Figure 4.11 shows that intracellular oxidation in the presence of different dilutions

of Blk#1 and Blk#2. Blank samples were diluted to obtain similar amount of protease

enzymes that are present in 0.25, 1.25 and 5 mg/ mL of SPH-GI samples. Interestingly

under both stressed and non-stress conditions, Blk#1 sample showed inhibition of cellular

oxidation at its lowest concentration and with the increase in its concentration there was a

decrease in its antioxidant activity. The reason may be presence of peptides from pepsin or

pancreatin enzyme caused due to incomplete self-hydrolysis at lower concentration may

have contributed to its antioxidant activity (Figure 4.11).

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4.4.5 Intracellular Antioxidant Activity of Simulated GI Digest of F-30 and A-60

Samples Under Stress and Non-Stress Conditions

Dose dependent antioxidant activity of GI digested samples (F-30#1, F-30#2, A-

60#1 and A-60#2) is shown in the Figure 4.12 and 4.13. Under both stress and non-stress

condition both F-30 and F-30#1 showed increased antioxidant activity with the increased

in its concentration. Under non-stressed conditions, cellular oxidation inhibition capacity

of F-30#1 at its highest concentration (5 mg/ mL) was significantly higher when compared

to its lower concentration (0.625 and 1.25 mg/ mL). With increased incubation time from

30 min to 120 min, the antioxidant activity of F-30#1 sample decreased which is evident

with increase in its IC50 value from 30 min (IC50 (stress) = 6.79 mg; IC50 (non-stress) =

6.33 mg) to 120 min (IC50 (stress) = 9.54 mg; IC50 (non-stress) = 7.39 mg) incubation

(Table 4.1). However the decrease in antioxidant activity of F-30#1 sample from 30 min

to 120 min incubation was not significant under non-stress condition but was significantly

different under stressed condition (Table 4.1). Apart F-30#1, all the other GI digest

samples (F-30#2, A-60#1 and A-60#2) did not show any dose dependent antioxidant

activity (Figure 4.12 and 4.13). In addition, protocol 2 digestion sample (F-30#2 and A-

60#2) including Blk#2 have shown to increase cellular oxidation. This increase in cellular

oxidation in the presence of F-30#2, A-60#2 and Blk#2 is not certain, but could be due to

proteolytic cleavage of endogenous antioxidant peptides by GI enzymes which are present

at higher concentration. Overall, GI digestion of F-30 and A-60 using both protocols did

not improve its antioxidant activity rather the activity was significantly diminished. During

GI digestion antioxidant peptides present in F-30 and A-60 samples might be broken down

to smaller peptides or free amino acids with no or less antioxidant activity.

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In a healthy individual during fasting state the pH varies from 1.0 to 3.5

(Fallingborg, 1999) while during a meal the pH is further increased with a median between

6 and 7 (Dressman, Berardi, Dermentzoglou, Russell, Schmaltz, Barnett, et al., 1990).

Further during gastro-oesophageal reflux disease, the patient is frequently treated with

proton pump inhibitor to protect oesophageal injury which leads to constant gastric pH >

4.0 (Hunt, Armstrong, James, Chowdhury, Yuan, Fiorentini, et al., 2005). The activity of

pepsin is highly dependent on pH of the gastric juice and with the pH increase there is

gradual decrease in pepsin activity (Roberts, 2006). In most of the in vitro GI digestion

assays the gastric digestion is carried out at a constant pH between 1.0 and 2.0

(Ekmekcioglu, 2002; S. Kim, Ki, Khan, Lee, Lee, Ahn, et al., 2007; Schmelzer, Schöps,

Reynell, Ulbrich-Hofmann, Neubert, & Raith, 2007; Thomas, Aalbers, Bannon, Bartels,

Dearman, Esdaile, et al., 2004). Therefore the possibility of lower activity of pepsin during

in vivo gastric digestion resulting in high amount of intact antioxidant peptide is ruled out

during an in vitro gastro digestion model. In addition consumption of bioactive peptides

with complex food matrices can also protect the peptide from adverse effects of GI

digestive enzymes. In the present study, after GI digestion, the F-30 sample using protocol

1, was shown to retain its antioxidant activity to an extent. Therefore, it is assumed that

under in vivo digestion conditions, F-30 sample may retain its antioxidant activity more

than in vitro digestion models.

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4.4.6 Intracellular Antioxidant Activity of Simulated GI Digest of Unhydrolyzed

Pasteurized Silver Carp Protein

Simulated gastrointestinal digestion of unhydrolyzed silver carp protein was

performed and its intracellular antioxidant activity usins Caco-2 cells was evaluated.

Considering dynamic nature of in vivo protein digestion, two GI digestion protocols were

used. Pasteurization step was performed before in vitro gastrointestinal digestion of SPU

to simulate protein denaturation that occurs during cooking process.

Intracellular antioxidant activity of simulated GI digested samples of unhydrolyzed

silver carp protein (SPU#1 and SPU#2) at 0.625 and 1.25 mg/ mL concentration are shown

in Figure 4.14. GI digestion of SPU sample with lower GI protease (pepsin and pancreatin)

activity (protocol 1) did not improve its antioxidant activity under both stressed (AAPH,

0.1 mM, 1h at 37 °C) and non-stressed (no AAPH) conditions. Most of the potent

antioxidant peptides found in literature have 2 – 20 amino acids (Chen, Suetsuna, &

Yamauchi, 1995; Ranathunga, Rajapakse, & Kim, 2006; H.-C. Wu, Chen, & Shiau, 2003)

and therefore SPU#1 sample with higher molecular weight (>11,500 Da) peptides did not

show any antioxidant activity. However, extreme GI digestion (protocol 2) of SPU sample

showed cellular oxidation inhibition during initial incubation period (30 min) under both

stressed and non-stressed conditions. Interestingly under non-stressed condition, SPU#2

showed dose dependent antioxidant activity. Conversely, dose dependent antioxidant

activity of SPU#2 is not shown during stressed condition.

Extreme GI digestion of SPU (SPU#2) resulted in increased peptides fractions with

molecular weight < 1055 Da (retention time > 9.5 min) when compared to limited GI

digestion (SPU#1). In hoki (Macruronus novaezelandiae) frame protein hydrolysates, the

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peptide fraction with molecular weight between 1-3 kDa showed highest antioxidant

activity (Kim, J.-Y. Je, & S.-K. Kim, 2007). The increase in lower molecular weight

peptide in SPU#2 may have contributed to its antioxidant activity, which is in accordance

with previous observation in Chapter 3.

Among all simulated GI digests of SPU, F-30 and A-60 samples using protocol 2,

SPU#2 sample showed higher antioxidant activity. This observation can be attributed to

degradation of antioxidant peptides of F-30 and A-60 sample by extreme GI digest sample.

On other hand, extreme GI digestion of SPU resulted in release of antioxidant peptides. At

0.625 mg/ mL of SPU#2 sample and 30 min incubation under stress condition showed

higher cellular oxidation inhibition (cellular oxidation = 75.46 %). Similar cellular

antioxidant activity was exhibited by F-30#1 sample at similar conditions. However,

limited GI digested F-30 sample (F-30#1) showed dose dependent antioxidant activity and

retained its activity even with the increased incubation time (120 min). This observation

emphasize that consumption of silver carp protein hydrolysates prepared using external

protease (Flavourzyme®) is more beneficial. However, simulated GI digestion of F-30 and

A-60 resulted in reduction of their antioxidant activity (Figure 4.12 and 4.13). Therefore,

strategies including encapsulation may protect antioxidant peptide of F-30 and A-60

samples and can also control release in the intestine for them to absorb (Mozafari,

Khosravi-Darani, Borazan, Cui, Pardakhty, & Yurdugul, 2008; Sessa, Tsao, Liu, Ferrari,

& Donsì, 2011). Studies on ability of encapsulation technique to enhance in vivo

antioxidant activity of fish protein hydrolysates samples are limited.

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4.5 Conclusion

In this study simulated gastrointestinal (GI) digestion of unhydrolyzed silver carp

protein (SPU), F-30 and A-60 samples using two protocol with varying enzyme activity

was performed. Results show that GI digestion of F-30 and A-60 using two protocols

resulted in increased short chain peptide fractions with molecular weight <500 Da.

Compared to F-30 and A-60 samples, the final GI digest product using both protocols

resulted in reduced cell based antioxidant activity. Nevertheless, limited GI digestion of F-

30 samples (F-30#1) still could retain its cell based antioxidant activity for longer

incubation times and also showed dose dependent activity. Limited GI digestion of SPU

with high molecular weight peptide fractions did not show any cell based antioxidant. On

the other hand, GI digestion of SPU using extreme condition exhibited cellular antioxidant

activity but did not show any dose dependency. This result demonstrate that consuming F-

30 sample with complex food matrix where digestive enzyme activity is hindered may help

to retain its activity in vivo.

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Table 4-1. IC50 values (mg/ mL) of F-30 and F-30#1 samples at 30 and 120 min

incubation times under both stress and non-stress conditions

Samples Non-stress Stress

30 min 120 min 30 min 120 min

F-30 4.20 AxyA 5.31 Ax 3.08 Ay 4.54 Ax

F-30#1 6.33 Bx 7.39 Bx 6.79 Bx 9.54 By

ADifferent letters (A, B, C,….) indicate significant difference (p <0.05) in means within each column for

each individual incubation time. Different letters (x, y, z…) indicate significant difference (p <0.05) among

the means within each row for stress or non-stress condition.

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Figure 4-1 Molecular weight profile of F-30 (20 µL, 2.5 mg/ mL) ran on Waters e2695



as solvent phase at 0.35 mL/ min flow rate.

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Figure 4-2 Molecular weight profile of F-30#1 (GI digest of F-30 using protocol 1) (20 µL,

2.5 mg/ mL) ran on Waters e2695 Separation Module HPLC system equipped with UV/Vis

detector and Sepax zenix SEC-100, 3µm, 100 A°, 4.6 × 300 mm2 column. Phosphate buffer

(150 mM, pH 7.0) was used as solvent phase at 0.35 mL/ min flow rate.

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Figure 4-3. Molecular weight profile of F-30#2 (GI digest of F-30 using protocol 2) (20




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Figure 4-4 Molecular weight profile of A-60 (20 µL, 2.5 mg/ mL) ran on Waters e2695



as solvent phase at 0.35 mL/ min flow rate.

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Figure 4-5 Molecular weight profile of A-60#1 (GI digest of A-60 using protocol 1) (20




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Figure 4-6 Molecular weight profile of A-60 #2 (GI digest of A-60 using protocol 2) (20




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Figure 4-7 Molecular weight profile of SPU #1 (GI digest of SPU using protocol 1) (20 µL,




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Figure 4-8 Molecular weight profile of SPU #2 (GI digest of SPU using protocol 2) (20 µL,




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Figure 4-9. Viability of Caco-2/TC7 clone cells in the presence of GI digestion blanks

(Blk#1 and Blk#2) and SPH-GI samples (F-30#1, F-30#2, A-60#1 and A-60#2) measured

using MTT assay after 2 h of incubation. The SPH-GI samples are at a concentration of 5

mg/ mL and blank samples were diluted to obtain protease content similar to that present

in SPH-GI samples

BC

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Figure 4-10 Inhibition of cellular oxidation in presence of F-30, GI digest of F-30 (F-30#1

and F-30#2) and blank sample (Blk#1 and Blk#2) under non-stress (A) and oxidative stress

(C); A-60, GI digest of A-60 (A-60#1 and A-60#2) and blank sample (Blk#1 and Blk#2)

under non-stress (B) and oxidation stress (D) during 120 min of incubation at 5 mg/ mL

concentration. Assay was conducted in duplicate and analysis was done in triplicate.

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Figure 4-11 Inhibition of cellular oxidation in presence of Blk#1 and Blk#2 under Non-

stress and oxidative stress condition under different dilutions. Blk#1 and Blk#2 samples

were diluted to obtain similar concentration of protease content present in 0.625 mg/ mL

(A) and (D); at 1.25 mg/ mL (B) and (E); and at 5 mg/ mL (C) and (F) of SPI-GI sample

and incubated for 120 min. Assay was conducted in duplicate and analysis was done in

triplicate.

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Aax Aax Aax

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presence of F-30 and its simulated GI digested samples prepared using protocol 1 (F-30#1)

and protocol 2 (F-30#2) measured at different incubation times under both non-stressed

(no AAPH added) and stressed (AAPH, 0.1 mM, 1h at 37 °C). Intracellular oxidation of

negative control (no sample added) at a given condition was used as a reference (100 %

oxidation). Values are mean ± SE, n = 3.










AaxAax

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presence of A-60 and its simulated GI digested samples prepared using protocol 1 (A-60#1)

and protocol 2 (A-60#2) measured at different incubation times under both non-stressed

(no AAPH added) and stressed (AAPH, 0.1 mM, 1h at 37 °C). Intracellular oxidation of












Aax Aax AaxAay

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Figure 4-14 Percentage intracellular oxidation measured in Caco-2 BBe cells in the presence

of unhydrolyzed silver carp protein after simulated GI digestion using protocol 1 (SPU#1)

and protocol 2 (SPU#2) measured at different incubation times under both non-stressed (no

AAPH added) and stressed (AAPH, 0.1 mM, 1h at 37 °C). Intracellular oxidation of












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CHAPTER 5. CONCLUSIONS AND FUTURE DIRECTIONS

5.1 Conclusions

Spreading of invasive silver carps in the Mississippi River Systems is increasing

and resulting in depletion of native fish species. At present the harvested silver carps are

used as fertilizers, as animal feed or simply discarded. Alternatively for environmental and

economic gain, this highly nutritious protein source is to recover and modify to produce

value added food ingredients such as bioactive peptides with antioxidant activity.

Antioxidant peptides have potential use in pharmaceutical and functional food industry.

These under-utilized silver carp protein may serve as promising natural resources that can

be used in health food industries or nutracuetical industries as a health benefit ingredient.

In this study SPH were prepared using two proteases from microbial origin

(Flavourzyme® and Alcalase®) at different hydrolysis times (15, 30, 45 and 60 min).

Antioxidant activity of these hydrolysates was evaluated using different chemical based

antioxidant assay: 1,1-diphenyl-2-picrylhydrazyl scavenging activity (DPPH-SA), lipid

peroxidation inhibition (LP-IA), Trolox equivalent antioxidant (TEAC) and ferric ion

reducing (FI-RC) capacities. All the hydrolysates exhibit comparable or higher antioxidant

capacity than synthetic antioxidants (BHA and BHT) as well as natural antioxidant

(α-tocopherol). For example, TEAC values of hydrolysates (SPH-A and SPH-F) was

similar to the TEAC value of BHT and higher than the BHA TEAC value. Further, the

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presence of specific amino acids showed an influence on the antioxidant activity of the

hydrolysates determined by different assays. Most of the silver carp hydrolysates was able

to inhibit more than 85 % of linoleic acid peroxidation during seven days of incubation.

This observation suggests that they may have potential role as in vivo antioxidants and their

effects on chronic diseases that are related to cellular oxidation stress should be further

investigated. Therefore, F-30 and A-60 samples from SPH-F and SPH-A samples

respectively was further evaluated for their preliminary cell based antioxidant activity.

Both F-30 and A-60 samples have shown Caco-2 cell based antioxidant activity which

is higher than vitamin C under both oxidative stressed and non-stressed conditions. F-30

and A-60 samples were further fractionated into two molecular weight fractions: < 3 kDa

(F-30<3 and A-60<3) and < 10 to > 3 kDa (F-30>3 and A-60>3). Peptide fractions with

molecular weight <3 kDa showed improved Caco-2 cell based antioxidant activity. In

particular, F-30<3 sample showed higher antioxidant activity than A-60<3 sample and its

activity was persistent during long incubation time. The higher cellular antioxidant activity

of F-30<3 sample is may be due to higher number of peptides with active amino acid

sequence than A-60<3. Result of this study emphasize that F-30<3 sample might be a

promising food additive or pharmaceutical agent.

It is important for the potent antioxidant peptides to resist various GI digestion enzymes

and retain its activity in order to minimize the oxidation stress at GI track. In Chapter 4

simulated gastrointestinal (GI) digestion of F-30 and A-60 samples using two protocol with

varying GI enzymes and enzyme activity was performed. GI digestion of F-30 and A-60

samples using two protocols resulted in smaller molecular weight peptides (< 500 Da). GI

digestion of F-30 and A-60 sample using pepsin, trypsin and chymotrypsin enzymes at

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higher activity completely destroyed cellular antioxidant activity. However, limited GI

digestion of F-30 sample (F-30#1) was able to retain its cellular antioxidant activity to an

extent. F-30#1 sample showed cellular antioxidant activity even after longer incubation

time and also showed dose dependent antioxidant activity. Cell based antioxidant activity

of GI digested unhydrolyzed silver carp protein (SPU) was also tested. Molecular weight

profile of limited GI digestion of SPU (SPU#1) still contains greater percent of high

molecular weight peptide fractions (>11500 Da) and did not show any cell based

antioxidant. On the other hand, extreme GI digestion of SPU exhibited cellular antioxidant

activity but could not retain its activity for longer period of incubation and also did not

show any dose dependency. This result demonstrate that consuming F-30 sample with

complex food matrix where digestive enzyme activity is hindered may help to retain its

activity in vivo.

5.2 Future direction

5.2.1 Effect of GI Digestion on Lower Molecular Weight Peptide Fractions (F-30<3

and A-60<3)

The two hydrolysates F-30 and A-60 samples were fractionated according to

different molecular weight fractions. The lower molecular weight samples (F-30<3 and A-

60<3) have shown improved cell based antioxidant activity and can be a potential

functional foods ingredient or a nuetracuetical. To ensure its in vivo activity at the target

site, after ingestion of the peptide, they should resist GI digestion and maintain their

activity (Hur, Lim, Decker, & McClements, 2011). Therefore it is important to study the

influence of GI digestion on F-30<3 and A-60<3. In this study, the effect of simulated GI

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digestion antioxidant activity of F-30 and A-60 sample was demonstrated in the Chapter 4.

The type of GI digestion enzyme and enzyme activity levels during GI digestion of F-30

and A-60 samples has huge impact on their cellular antioxidant activity.

In the present study, gel filtration column of 1.7 cm (diameter) × 90 cm (height)

using Sephadex G-25 was used to prepare <3 kDa sample. Each run using this column take

approximately 4 h and <3 kDa sample collected during each run is very minute amounts.

Therefore in order to perform simulated GI digestion studies on F-30<3 and A-60<3

samples, a large scale gel filtration preparation columns is required.

5.2.2 Identification and Characterization of Antioxidant Peptides Present in Silver Carp

Protein Hydrolysates

Present study attempted to show the influence of amino acid composition,

molecular weight of peptide fraction and peptide sequence on antioxidant activity of Silver

carp protein hydrolysates. However, identification and characterization of peptides from

SPH responsible for antioxidant activity was not attempted. Two hydroysate samples F-30

& A-60, were fractionated into two different molecular weight fractions and lower

molecular weight fractions showed higher activity. Further fractionations using different

chromatographic techniques such as 1) Reverse phase - high pressure liquid

chromatography ((RP)-HPLC) for separating according to hydrophobicity/hydrophilicity

of peptides and 2) Ion pair reverse phase-high pressure liquid chromatography to separate

based on the charge of peptides should be used for identification. For identification of

potent antioxidant peptide, these chromatography technics can be used sequentially by

choosing the potent antioxidant fraction

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(RP)-HPLC can be done as outlined by (J. Wu, Aluko, & Muir, 2008). The potent

antioxidant peptide fraction can be analyzed on HPLC (Hewlett Packard 1100, CA, U.S.A)

equipped with C18 column protected by a 100 X 4 mm guard column. A binary gradient

using solvent A (water + 10% acetic acid) and solvent B (methanol + 10 % acetic acid)

will be developed at constant temperature. The absorbance profile at 214 nm will be

analyzed, peak fractions will be collected. This procedure will be replicated to obtain

appropriate volume of peak fractions and freeze dried for further antioxidant analysis. Ion-

Pair RP-HPLC is used to separate the peak fraction with highest activity in to individual

peptides. The analysis is done on RP-HPLC column using solvents containing 0.10 %

heptafluorobutyric acid and 0.05 % trifluoroacetic acid. These acids form organic salts with

peptides and maximize trapping efficiency which will help separate of individual peptides

(Lasaosa, 2008). After potent antioxidant peptide is identified, amino acid sequence of the

peptides is determined using LC-MS. LC-MS will be done using Nano LC-high resolution

hybrid ion trap (Thermo Orbitrap, CA, U.S.A) which is available at Bindley Bioscience

center, Purdue University.

5.2.3 Further Assays to Confirm the Antioxidant Mechanisms of the Peptides Fractions

In chapter 3, the low molecular weight peptide fractions of F-30 & A-60 samples

(F-30<3 and A-60<3) showed higher cell based antioxidant capacity. However, F-30<3

samples have inhibited cellular oxidation significantly even at the initial incubation time

and was able to retain its antioxidant activity even at higher incubation times when

compared to A-60<3. It is speculated that higher sulphur containing amino acids and

peptides containing sulphur amino acids of F-30<3 compared to A-60<3 may have

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stimulated the endogenous antioxidants such as glutathione synthesis in the cells (Kim, et

al., 2003). Measurement of intra cellular glutathione levels in the presence of peptide

fraction will help to demonstrate the importance of the sulphur containing amino acids on

antioxidant activity.

Permeability of antioxidant peptides through intestinal cell mono layer is one of the

important factor effecting antioxidant activity of peptide fractions at the target site (Dunn,

2002; Ekmekcioglu, 2002; Scheele, Bartelt, & Bieger, 1981). Most commonly, mono layer

of human adenocarcinoma colon cells (Caco-2) are used for studying intestinal

permeability of bio-active compounds as they are similar to intestinal endothelium colon

cells (R. H. Liu & Finley, 2005; Vermeirssen, Augustijns, Morel, Van Camp, Opsomer, &

Verstraete, 2005). Caco-2 cells are differentiated on semi-permeable plastic support to

form tight junction between cells to serve as an intestinal model. The test components are

added to one side of the mono layer and incubated at different time intervals. After desired

incubation time, the buffer solutions in the opposite chamber was tested for permeated test

sample concentration to calculate the permeability of peptide fraction (van Breemen & Li,

2005). Studying the influence of different concentrations of the test peptide fraction and its

permeability may help to predict permeation mechanism of the test peptide fraction.

Further, the different molecular weight samples were not tested using chemical

based antioxidant assays. This will allow us to correlate the effect of molecular weight on

different antioxidant mechanisms (Metal Chelating, radical scavenging and lipid

peroxidation inhibition)

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5.2.4 Sensory Studies on Antioxidant SPH

One of the possible problems with low molecular weight bioactive peptides is the

bitter taste associated with it. It is a major limitation to the utilization of bioactive peptides

in various functional food applications. During protein hydrolysis interior hydrophobic

amino acids are released which are responsible of bitter taste. Usually bioactive peptides

with lower molecular weights and containing hydrophobic residues are more prone to be

bitter (Aluko, 2012).

Sensory studies are necessary to evaluate bitterness associated with antioxidant

peptide fractions especially with molecular weights less than 3 kDa. Strategies such as

using debittering ingredients including cyclo dextrins or use of active char coal and enzyme

technology should be evaluated. Hydrophobic bitter peptides will absorb to the active

charcoal and will reduce bitterness in the peptide hydrolysates. Apart from the presence of

hydrophobic amino acids, their location in the peptides also effect the intensity of bitterness.

For example, peptide containing at least two hydrophobic amino acids at the C-terminal

found to be bitter (Raksakulthai & Haard, 2003). Therefore using enzyme technology to

hydrolyze the bitter peptides can also reduce the overall bitterness in the peptide fractions.

However employing these strategies may inactivate or remove hydrophobic antioxidant

activity and may result in reduction of antioxidant activity. More studies are required to

overcome the bitterness of bioactive peptides without losing intended activity.

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VITA

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VITA


Graduate School, Purdue University

Education

Doctor of Philosophy, Food Science

Purdue University, West Lafayette, IN Anticipated in December 2013

Masters of Science, Food Science

University of Minnesota, Twin Cities, MN May 2009

Bachelors of Technology, Food Technology

Osmania University, Hyderabad, India May 2007

Professional Experience

Product Development Intern, Godiva Chocolatier January 2013-June 2013

Responsible for testing new ingredients on bench scale, communicate results with

PD team and propose potential changes to meet or exceed quality standard of

product

Supported full scale manufacturing trails of new products to be launched

Developed experimental design and executed testing to demonstrate the effect of

temperature on textural and visual properties of filled chocolates

Developed analytical testing protocols to evaluate product quality

Documented all the observations and suggestions in the form of reports for future

references

Provided technical assistance in the development of a new non-chocolate product

platform

Teaching Assistant, Sensory Science, Purdue University Spring 2012

Helped in designing and conducting different sensory evaluation techniques

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Statistically analyzed data and interpret results

Evaluating students output

Quality Control Intern Creamline Dairy Products Ltd May 2005-July 2005

Analyzed chemical and microbiological aspects of milk and milk products

Assisted in material balances at different operation levels

Collaborated closely with co-workers during processing of milk, production and

packaging

Learned practical aspects of HACCP and GMP principles at industrial level

Research Experience

Research Assistant, Purdue University, West Lafayette IN, January 2010-December

2013 Project: Antioxidant and Angiotensin converting enzyme (ACE) inhibitory activity of Silver

carp protein hydrolysates

Skills:

Expert in various functional and bioactive assays (including cell based assays)

Developed new and improved assays for functional / bioactive properties

Expert in various protein analysis

Developed protocols for different instruments/ techniques and training new lab

members

Research Assistant, University of Minnesota, Twin Cities MN, August 2007-May 2009

Project: Effect of protein content and denaturation on extractability and stability of

isoflavones in different soy systems

Skills:

Expert in various extraction techniques

Running and maintenance of Reverse Phase High Performance Liquid Chromatography to

detect isoflavone content

Running and maintenance of Leco Nitrogen Analyzer (Dumas) to measure protein content

in samples

Gel electrophoresis techniques to visualize protease activity

Degree of hydrolysis using O-phthaldialdehyde method (OPA) and TNBS method to

quantify protease activity

Bicinchoninic Acid (BCA) assay for protein determination

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Differential Scanning Colorimeter (DSC) for comparing protein denaturation state in

samples

Achievements

Participated in Aseptic Processing and Packaging workshop from May 14-17, 2012

Won 1st prize for Developing Solutions for Developing Countries competition at IFT-2009,

Anaheim, CA

Member of The Golden Key International Honour Society

Participated in General Mills Leadership and Professional Development seminar series

Affiliations

Institute of Food Technologists (IFT), Chicago, IL, USA, Student Member

Publications

Malaypally, S.P., and Liceaga, A. (2013). ‘Influence of free amino acid

composition on antioxidant properties of Silver carp (Hypophthalmichthys

molitrix) protein hydrolysates. (In preparation)

Malaypally, S.P., and Ismail, B. (2010). Effect of protein content and denaturation

on the extractability and stability of isoflavones in different soy systems. Journal

of Agricultural and Food Chemistry, 58. 8958 – 8965

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Presentations

Oral presentation titled ‘Antioxidant properties and free amino acid

composition of Silver carp (Hypophthalmichthys molitrix) protein

hydrolysates’ in Canadian Institute of Food Science and Technology (CIFST)-

12, Niagara falls, Canada

Poster presentation titled ‘Optimization of Isoflavone Extraction Protocol

based on Protein Content and Denaturation State’ in Institute of Food

technologist (IFT)-09 annual meeting, California, USA

7th June 2009

Poster presentation titled ‘Effect of Protein Content on Extractability of

Isoflavones from Complex Soy Systems’ in Institute of Food Technologists

(IFT)-08 annual meeting + Food Expo, New Orleans, USA

29th June 2008

Poster presentations on ‘Food Bio packaging’, ‘Pulsed Electric Field-A Non

Thermal Process’ and ‘Disease resistance through U V Hormesis’ in

international conference FOOD PLUS 2005 held at Bangalore, India. Organizer:

Association of Food Scientists and Technologists of India (AFSTI), India

9-10 December 2005

Attended workshop of FOOD PLUS 2005 on “Value Addition to Foods-Fruits

& Vegetables” held at Taj Residency, Hyderabad. Organizer: Association of

Food Scientists and Technologists of India

1st October 2005

Poster presentations titled ‘Fat replacers’, ‘Natural Foods Vs. Processed Foods’ in

National Conference on Innovations & Challenges in Food Technology Organizer:

University College of Technology, Osmania University, India

23rd March 2005

Activities

Served as fund raising director for Food Science Graduate Students Association

(FSGSA), Purdue University May 2011-Januray 2012

Served as descriptive analysis panelist Spetember 2011-November 2011

University of Minnesota FSN Student Association Club Member August 2007-

May 2009

Coordinator for the Intra College Food Science Club CALORIES (Call for the

Individual Excellence) September 2005-May 2007

Organized national level conferences like NCETFT (National Conference on

Emerging Trends in Food Technology) 2003, NHFT (New Horizons in Food

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Technology) 2004, and NCICFT (National Conference on Innovations and

Challenges in Food Technology) 2005

Member of organizing committee of CATALYSIS 2005 – the annual college

festival at Osmania University, India May, 2005

Attended Entrepreneurship Awareness Camp organized by EDC Entrepreneurship

Development Cell), Osmania University February, 2007

Volunteered for environment cleaning program held at Osmania University, under

the guidance of National Service Scheme January, 2005

Documents

Invasive SIlver Carp (Hypophthalmichthys molitrix) Protein