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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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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
Sravanthi Priya Malaypally
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
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 ...................................................................................... 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
Figure 3-10. Percentage intracellular oxidation measured in Caco-2 BBe cells in the
presence of A-60, its fractions (A-60 <3 and A-60 >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). 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
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.
......................................................................................................................................... 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
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. ................................................................... 134
Figure 4-5 Molecular weight profile of A-60#1 (GI digest of A-60 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.
......................................................................................................................................... 135
Figure 4-6 Molecular weight profile of A-60 #2 (GI digest of A-60 using protocol 2) (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.
......................................................................................................................................... 136
Figure 4-7 Molecular weight profile of SPU #1 (GI digest of SPU 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.
......................................................................................................................................... 137
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Figure ............................................................................................................................. Page
Figure 4-8 Molecular weight profile of SPU #2 (GI digest of SPU using protocol 2) (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.
......................................................................................................................................... 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
Figure 4-12. Percentage intracellular oxidation measured in Caco-2 BBe cells in the
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
Figure 4-13. Percentage intracellular oxidation measured in Caco-2 BBe cells in the
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 negative control (no sample added) at a given condition was used as a
reference (100 % oxidation). Values are mean ± SE, n = 3. ........................................... 148
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 negative control (no sample added) at a given condition was used as a
reference (100 % oxidation). Values are mean ± SE, n = 3. ........................................... 150
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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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4
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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6
(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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14
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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-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 Materials and Methods
3.3.1 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
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 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.
3.3.12 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.
3.4 Results and Discussion
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
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 3-5. 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
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1000
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3500
0 20 40 60 80 100 120 140 160
Ca
llu
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Non-stress condition (5 mg/ mL)
F-30 F-30 <3 F-30 >3 Negative control
0
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Non-stress conditions (5 mg/ mL)
A-60 A-60 <3 A-60 >3 Negative control
`
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Oxidation stress (5 mg/ mL)
F-30 F-30 <3 F-30 >3 Negative control
0
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3500
4000
4500
0 20 40 60 80 100 120 140 160
min
Oxidation stress (5 mg/ mL)
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
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30 NS 30 S 120 NS 120 S
Ce
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(-v
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%)
Concentration (mg/ mL)
F-30 0.625
1.25
5
AaxAbx
Aax
AbxBax
BbxAax
Bbx
Cax Cax Bax
Cby
0
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30 NS 30 S 120 NS 120 S
Ce
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(-v
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l = 1
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%)
Concentration (mg/ mL)
F-30<3 0.625
1.25
5
105
105
Aax Aax
Aay
Aby
Aax Aax
ABay
Aby
Aax
Bbx
Bay
Bby
0
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30 NS 30 S 120 NS 120 S
Ce
llula
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n %
(-v
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l = 1
00
%)
Concentration (mg/ mL)
F-30>3 0.625
1.25
5
Aax
Abx
Aay
Abx
Aax
Bbx
Aay
AaxAax
Bbx
AaxAax
0
10
20
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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)
Vitamin C10 µM
20 µM
40 µM
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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).
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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Aax
AbxAax
Aby
Aax
Bbx
Aax
Abx
Bax
Cbx
Bay
Bbx
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30 NS 30 S 120 NS 120 S
Ce
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(-v
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l = 1
00
%)
Concentration (mg/ mL)
A-60 0.625
1.25
5
AaxAax
AaxAay
AaxBax
BayBby
Bax
Bax
Cay Cay
0
10
20
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30 NS 30 S 120 NS 120 S
Ce
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(-v
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l = 1
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%)
Concentration (mg/ mL)
A-60<3 0.625
1.25
5
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108
Aax
Aax
Aay
AbyBax
Aax
Aay
Aby
ABax Aax
Aay
Aby
0
10
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30 NS 30 S 120 NS 120 S
Ce
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(-v
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l = 1
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%)
Concentration (mg/ mL)
A-60>3 0.625
1.25
5
Aax
Abx
Aay
Abx
Aax
Bbx
Aay
AaxAax
Bbx
AaxAax
0
10
20
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50
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30 NS 30 S 120 NS 120 S
Ce
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n %
(-v
e c
on
tro
l = 1
00
%)
Concentration (mg/ mL)
Vitamin C10 µM
20 µM
40 µM
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Figure 3-10. Percentage intracellular oxidation measured in Caco-2 BBe cells in the
presence of A-60, its fractions (A-60 <3 and A-60 >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). 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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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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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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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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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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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 Materials and Methods
4.3.1 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
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), 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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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.
4.3.9 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.
4.4 Results and Discussion
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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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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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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129
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
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 4-2 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
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 4-4 Molecular weight profile of A-60 (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 4-5 Molecular weight profile of A-60#1 (GI digest of A-60 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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136
Figure 4-6 Molecular weight profile of A-60 #2 (GI digest of A-60 using protocol 2) (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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137
Figure 4-7 Molecular weight profile of SPU #1 (GI digest of SPU 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 4-8 Molecular weight profile of SPU #2 (GI digest of SPU using protocol 2) (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 4-9. 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
BB
BC
C
B
A0
20
40
60
80
100
120
140
F30 #1 F30 #2 A60 #1 A60 #2 Blk #1 Blk #2 toxiccontrol
% V
iab
le c
ells
Sample concentration (5 mg/ mL)
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Non-stress condition (5 mg/ mL)
F-30 F-30 #1
F-30 #2 Blk #1
Blk #2 Negative control
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Incubation time (min)
Non-stress condition (5 mg/ mL)
A-60 A-60 #1 A-60 #2
Blk #1 Blk #2 Negative control
(A)
(B)
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y
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Oxidation stress (5 mg/ mL)
F-30 F-30 #1 F-30 #2
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Incubation time (min)
Oxidative stress (5 mg/ mL)
A-60 A-60 #1 A-60 #2
Blk #1 Blk #2 Negative control
(C)
(D)
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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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0
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1500
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2500
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0 50 100 150min
Stress condition (0.625 mg/ mL)
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0 50 100 150
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(1.25 mg/ mL)
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0 50 100 150min
Stress condition (1.25 mg/ mL)
0
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(5 mg/ mL)
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0
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Blk #1 Blk #2 Negative control
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Non-stress condition
(0.625 mg/ mL)
(A)
(B)
(C)
(D)
(E)
(F)
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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
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
Aax
Abx
Aax
Abx
Bax BaxAay
Aby
CaxCbx Bax
Abx
0
20
40
60
80
100
120
140
160
180
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#10.625
1.25
5
(A)
(B)
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Figure 4-12. Percentage intracellular oxidation measured in Caco-2 BBe cells in the
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.
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.
AaxAax
Aax
Aby
Aax AaxAax
Aby
Bax
Abx
Bax
Aax
0
20
40
60
80
100
120
140
160
180
200
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 #2 0.625
1.25
5
(C)
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147
Aax
AbxAax
Aby
Aax
Bbx
Aax
Abx
Bax
Cbx
Bay
Bbx
0
10
20
30
40
50
60
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90
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110
30 NS 30 S 120 NS 120 S
Ce
llula
r o
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n %
(-v
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on
tro
l = 1
00
%)
Concentration (mg/ mL)
A-60 0.625
1.25
5
Aax
Abx
Aay
AbyAax BaxBax
AaxAax
Cbx
Bax
Bbx
0
20
40
60
80
100
120
140
160
180
200
30 NS 30 S 120 NS 120 S
Ce
llula
r o
xid
atio
n %
(-v
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on
tro
l = 1
00
%)
Concentration (mg/ mL)
A-60 #1 0.625
1.25
5
(A)
(B)
(B)
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Figure 4-13. Percentage intracellular oxidation measured in Caco-2 BBe cells in the
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
negative control (no 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.
Aax Aax AaxAay
ABax Aax
Bax AayBax Bax
CayBay
0
20
40
60
80
100
120
140
160
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)
A-60 #20.625
1.25
5
(C)
149
149
Aax Aax
Aax
Abx
Bax
Bbx
Bax
Abx
0
50
100
150
200
250
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)
SPU #10.625
1.25
Aax
Abx
Aay
Aby
Bax Bax
BayAay
0
20
40
60
80
100
120
140
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)
SPU #2 0.625
1.25
150
150
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
negative control (no 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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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
Sravanthi Priya Malaypally
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