Physico-Chemical Properties of Fish Proteins Recovered ...epubs.surrey.ac.uk/841238/1/Final Thesis-NR Mohamed Zam 310517.pdfDalysa, parents and family in Malaysia to whom this thesis

Physico-Chemical Properties of Fish Proteins

Recovered from Waste Streams

Nor Raihana Mohamed Zam

Submitted for the Degree of Doctor of Philosophy of the University of Surrey

Department of Nutritional Sciences

Faculty of Health and Medical Sciences

University of Surrey

Guildford, Surrey GU2 7XH, UK

November 2016

Declaration of originality

This thesis and the work to which it refers are the results of my own efforts. Any

ideas, data, images or text resulting from the work of others (whether

published or unpublished) are fully identified as such within the work and

attributed to their originator in the text, bibliography or in footnotes. This

thesis has not been submitted in whole or in part for any other academic

degree or professional qualification. I agree that the University has the right

to submit my work to the plagiarism detection service TurnitinUK for

originality checks. Whether or not drafts have been so-assessed, the University

reserves the right to require an electronic version of the final document (as

submitted) for assessment as above.

i

Abstract

Food security can be improved by reducing post-harvest losses in sustainable food

product chains. About 50% of fish is lost during filleting, including significant levels of

high quality protein (10–23% (w/w)), which may be a source for biofunctional peptides.

The fish processing waste protein hydrolysate can be a potential solution for minimizing

the environmental issues related to marine processing products, and act as an alternative

to producing value added fish processing by products. The main aim of this study was to

investigate the physicochemical properties of fish processing waste streams from

Atlantic Mackerel (Scomber scombrus) and mixed fish by membrane separation.

Protein hydrolysates of Atlantic Mackerel (Scomber scombrus) and mixed fish from fish

waste streams were prepared by enzymatic hydrolysis using pepsin and pancreatin and

measured for their antioxidant and functional properties. The chemical composition

(moisture, protein, total lipids and ash) of the Atlantic mackerel and Nile Perch (Lates

niloticus) fish fillets (FF) compared to fish waste (FW) was also investigated.

Protein fractions from the fish waste samples were separated by using the TFF cogent

µscale ultrafiltration system from Millipore, and parameters including transmembrane

pressure (TMP) on flux excursion, protein performance scalability, mass transfer

analysis, as well as hold up volume were studied. The mechanism of antioxidant activity

was studied by DPPH, FRAP, FTC and TBARS assays. It was demonstrated that fish waste

water protein hydrolysate especially for Atlantic Mackerel showed good antioxidant

activities by the Ferric Thiocyanate (FTC) and Thiobarbituric acid Reactive substances

(TBARS) methods and compared well with other antioxidants (BHA, ascorbic acid and

trolox). There was significant difference (p<0.05) between samples and negative control

(no antioxidant). DPPH scavenging activity increased with the extract concentration in

ii

the range of 1.5-26 %. In FRAP assay, both sample showed that there was an increase in

absorbance with an increase in concentration.

Structural and thermodynamic changes in fish waste samples were also determined by

FT-Raman spectroscopy, differential scanning calorimetry (DSC) and small deformation

rheology respectively. The DSC thermograms from the samples indicated that fish waste

samples may be comparable. Moreover in fish waste sample hydrolysates, the same

trends were obtained in 10 kDa AM and MF fractions. Fish waste samples especially

hydrolysed fish waste samples, showed different protein denaturation transition peaks,

indicating that enzymatic hydrolysis can affect the thermodynamic and functional

properties of protein samples. Similarly, the rheological properties were different for

different fish samples (AM and MF) with AM showing higher G’ or elastic modulus values

(p<0.05). The proteins in mackerel and mixed fish waste stream were characterised by

FT-Raman spectroscopy and showed significant differences in their respective spectra

and most of the assigned peaks (p<0.05).

iii

Acknowledgments

There have been many people who have walked alongside me during the last four years.

I am lucky enough to have been given supportive gift of amazing people in my life, without

all of whom, this work would not have been completed. I would like to thank each and

every one of them.

Without hesitation I would like to thank my husband Mohd Hanif Yusoff, baby girl Qayla

Dalysa, parents and family in Malaysia to whom this thesis is dedicated for your belief in

me.

I am indebted to Professor Nazlin Howell for her excellent and dedicated research

supervision and for creating many opportunities for me.

I would like to thank Dr Barbara Fielding and Dr Farah Badii for supervision and support.

I would also like to thank Malaysia government especially MARA and UniKL for the

lecturership awarded to me during my four years in Surrey.

My bestfriend Masmunira Rambli and family, Dr Marwa Yousr, also our small Malaysian

family in Surrey.

My thanks extend to colleagues in Nutritional laboratory, and several people who

provided assistance, support and friendship during my 7 years in Surrey.

“Verily, after hardship comes ease” (94:6). Alhamdulillah, Thank you Allah.

iv

Contents

Abstract .................................................................................................................................................. i

Acknowledgments .............................................................................................................................. iii

Contents ............................................................................................................................................... iv

List of Figures .................................................................................................................................... viii

List of Tables ......................................................................................................................................xiv

Glossary of Terms ............................................................................................................................ xvii

1 Introduction .................................................................................................................................... 21

1.1 Proteins .................................................................................................................................... 21

1.1.1 Amino Acid Structure ....................................................................................................... 21

1.1.2 Amino Acid Classification ................................................................................................ 22

1.1.3 Molecular Forces Governing Protein Interactions ..................................................... 23

1.1.4 Protein Synthesis .............................................................................................................. 25

1.1.5 Protein Structure .............................................................................................................. 25

1.2 Fish and Shellfish Protein ..................................................................................................... 29

1.2.1 Current World Fisheries Statistics ................................................................................. 32

1.2.2 Fish Waste and Fish Waste Streams .............................................................................. 36

1.2.3 Fish Protein Hydrolysates ............................................................................................... 39

1.3 Nutritional Properties of Proteins in Fish ......................................................................... 40

1.3.1 Bioactive Hydrolysates and Peptides from Enzymatic Hydrolysis of Fish and

Shellfish Waste .................................................................................................................................. 40

1.3.2 Endogenous Bioactive Peptides ..................................................................................... 42

1.3.3 Bioactive Proteins Produced by Fish............................................................................. 49

1.3.4 Bioactive Amino Acids Present in Fish and Seafood .................................................. 50

1.4 Functional Properties of Fish Proteins .............................................................................. 51

1.4.1 Rheological Measurements ................................................................................................... 52

1.5 Protein Recovery .................................................................................................................... 54

1.5.1 The Recovery of Protein by Membrane Separation Technique ..................................... 54

1.5.2 Use of membrane separation process in wastewaters treatment ................................. 58

v

1.5.3 Membrane Separation Configuration ........................................................................... 59

1.5.4 Pre Treatment method ........................................................................................................... 60

1.6 Aims and objectives of the study ......................................................................................... 61

1.6.1 Aims of the study ............................................................................................................... 61

1.6.2 Objectives of the study ..................................................................................................... 61

1.6.3 Hypothesis of the study .................................................................................................... 62

2 Chemical Composition of Atlantic Mackerel (Scomber scombrus), Nile Perch (Lates

niloticus) fillets and Fish waste (FW) ............................................................................................ 65

2.1 Introduction............................................................................................................................. 65

2.1.1 Fish in general .......................................................................................................................... 65

2.1.2 Atlantic Mackerel Fish (Scomber scombrus) ...................................................................... 66

2.1.3 Nile Perch Fish (Lates niloticus) ........................................................................................... 67

2.1.4 Fish waste ................................................................................................................................. 70

2.2 Materials and Methods .......................................................................................................... 71

2.2.1 Samples and Materials ........................................................................................................... 71

2.2.2 Methods for proximate analysis of fish fillet (FF) and fish waste (FW) composition 72

2.2.3 Statistical analysis ................................................................................................................... 75

2.3 Results and Discussion .......................................................................................................... 76

2.3.1 Proximate analysis of fish fillets; Atlantic Mackerel (Scomber scombrus) and Nile

Perch (Lates niloticus) and fish waste ........................................................................................... 76

2.3.2 Composition of lipids present in Fish waste (FW) sample .............................................. 79

2.3.3 Minerals composition in Atlantic Mackerel (Scomber scombrus) and Nile Perch

(Lates niloticus) and fish waste (FW) based on literature. ....................................................... 81

2.4 Conclusion ................................................................................................................................ 82

3 Production of hydrolysate and ultrafiltration studies to obtain water soluble proteins

fraction (3 and 10 kDa) from fish waste stream ......................................................................... 84

3.1 Introduction............................................................................................................................. 84

3.2 Materials and Methods .......................................................................................................... 90

3.2.1 Production of Hydrolysate .................................................................................................... 90

3.2.2 The effect of Transmembrane pressure (TMP) on Flux Excursion ............................... 91

3.2.3 Protein Performance Scalability Study ............................................................................... 92

3.2.4 Hold up volume of Membrane............................................................................................... 93

vi

3.3 Results and Discussion .......................................................................................................... 94

3.3.1 Production of Hydrolysate .................................................................................................... 94

3.3.2 The effect of Trans membrane pressure (TMP) on Flux excursion ............................... 95

3.3.3 Protein Performance Scalability Study ............................................................................... 96

3.4 Hold up volume of Membrane ............................................................................................ 108

3.4.1 Conclusion .............................................................................................................................. 110

4 Antioxidant properties by lipid peroxidation inhibition (FTC and TBARS) and radical

scavenging activity (2, 2- Diphenyl-1 picryhydrazyl DPPH radical scavenging and ferric

reducing antioxidant power assay) in the fish from waste streams (Atlantic mackerel and

mixed fish) ........................................................................................................................................ 112

4.1 Introduction........................................................................................................................... 112

4.2 Materials and methods ........................................................................................................ 115

4.3 Results and Discussion ........................................................................................................ 119

4.3.1 Antioxidant Activity of Water Soluble Fish Protein Hydrolysate (Atlantic Mackerel

fish) .................................................................................................................................................... 119

4.3.2 Measurement of Lipid Oxidation Inhibition Activity ..................................................... 119

5 DSC and rheological properties of the fish waste (Atlantic Mackerel, mixed fish, 10 kDa

Mackerel and mixed fish protein hydrolysates) ....................................................................... 134

5.1 Introduction........................................................................................................................... 134

5.1.1 Rheology Small Deformation Testing ................................................................................ 137

5.2 Materials and method .......................................................................................................... 138

5.2.1 Materials ................................................................................................................................. 138

5.2.2 Methods ................................................................................................................................... 138

5.2.3 Sample preparation .............................................................................................................. 138

5.3 Results and Discussion ........................................................................................................ 142

5.3.1 Differential scanning calorimetry (DSC) .......................................................................... 142

5.3.2 Rheological Analysis (Small deformation testing) ......................................................... 147

5.4 Conclusion .............................................................................................................................. 151

6 FT-Raman spectroscopy characterisation of fish proteins from Mackerel and Mixed fish

waste streams .................................................................................................................................. 153

6.1 Introduction ..................................................................................................................... 153

6.1.1 Principle of Raman Spectroscopy ................................................................................ 153

6.1.2 Instrumentation .................................................................................................................... 155

vii

6.1.3 Application of Raman Analysis in food ............................................................................. 156

6.2 Materials and Methods ........................................................................................................ 159

6.2.1 Materials ................................................................................................................................. 159

6.2.2 Methods ................................................................................................................................... 159

6.3 Statistical analysis ................................................................................................................ 160

6.4 Results and discussion ........................................................................................................ 161

6.5 Conclusion .............................................................................................................................. 165

7 General Discussion ............................................................................................................. 167

7.1 Aims and Objectives ....................................................................................................... 167

7.2 Fish Waste Stream Quality .................................................................................................. 167

7.3 Production of hydrolysate and ultrafiltration studies to obtain water soluble

protein fraction (3 and 10 kDa) from fish waste stream .................................................... 169

7.4 Antioxidant properties by lipid peroxidation inhibition (FTC and TBARS) and

radical scavenging activity (2, 2- Diphenyl-1 picryhydrazyl DPPH radical scavenging

and ferric reducing antioxidant power assay) in the fish from waste streams (Atlantic

mackerel and mixed fish) .......................................................................................................... 171

7.5 DSC and rheological properties of the fish waste (Atlantic mackerel, Mixed fish,

10KD Atlantic mackerel and mixed fish protein hydrolysates) ........................................ 173

7.6 FT-Raman spectroscopy characterisation of fish proteins from Mackerel and Mixed

fish waste streams ...................................................................................................................... 175

7.7 General conclusions ............................................................................................................. 177

7.8 Future work ........................................................................................................................... 178

Bibliography ..................................................................................................................................... 179

Appendix ........................................................................................................................................... 212

viii

List of Figures

Figure 1.1 Structure of amino acid (Nelson and Cox, 2005) 22

Figure 1.2 Classification of amino acids (Nelson and Cox, 2005) 23

Figure 1.3 Binding of amino acids via peptide bond (Fennema et al., 2008) 25

Figure1.4 Four levels of protein structure (Fennema et al., 2008) 26

Figure 1.5 Cell section of various structures including myofibrils (Bell et al, 1976

adapted from http://www.fao.org/docrep/v7180e/v7180e04.htm)

30

Figure 1.6 World capture fisheries and Aquaculture production (FAO, 2014) 33

Figure 1.7 World Fish Utilization and Supply (FAO, 2014) 34

Figure 1.8 Resource Maps for Fish across Retail and Wholesale Supply Chains,

WRAP (2011)

35

Figure1.9 Average proportion of fish by- products (Dumay, 2006) 37

Figure 1.10 Products Utilization of fish cod waste (adapted from World fishing and

Aquaculture, website: http://www.worldfishing.net/news101/icelandic-

fisheries-exhibition/icefish-conference-fish-waste-for-profit )

38

Figure 1.11 The generation methods of peptides from food proteins (Najafan and

Babji, 2012)

41

http://www.fao.org/docrep/v7180e/v7180e04.htm

http://www.worldfishing.net/news101/icelandic-fisheries-exhibition/icefish-conference-fish-waste-for-profit


ix

Figure 1.12 Steps of the autoxidation process and the action of antioxidant (Najafan

and Babji, 2012)

43

Figure 1.13 In vitro chemical analysis for measuring the antioxidative capacity of

protein hydrolysates and peptides (Samaranayaka, 2010)

44

Figure 1.14 A separation spectrum (Dunwell, 2005) 55

Figure 2.1 The muscles types of white and fatty fish adapted from fao.org 66

Figure 2.2 Atlantic Mackerel (Scomber scombrus). Image adapted from EAA website 67

Figure 2.3 Nile Perch Fish (Lates niloticus) adapted from Peche Food Company

(www.Peche foods.com)

68

Figure 2.4 Fish waste in fish processing (filleting) 70

Figure 2.5 The chemical composition of fish waste (FW) 78

Figure 3.1 Normal Flow filtration and Tangential Flow Filtration 87

Figure 3.2 The cogent µscale TFF system used for separation process in this study 89

Figure 3.3 Water-soluble fish protein hydrolysate of Atlantic Mackerel (on the left)

and mixed fish (on the right)

94

Figure 3.4 TMP Excursion at two feed flows (Merck Millipore, 2014) 95

x

Figure 3.5 General diagram of the system adapted from the Merck Millipore

Membrane Performance Guide, 2014

96

Figure 3.6 Permeate flux of 3 kDa Pellicon 3 Ultracel membrane at different

transmembrane pressures in 10, 20, and 40 g/L BSA solution

97



98


transmembrane pressures in10, 20, and 40 g/L FWS protein solution

101

Figure 3.9 Permeate flux of 10 kDa membrane at different transmembrane

pressures in 10, 20, and 40 g/L FWS protein solution

103

Figure 4.1 Antioxidant activity of fish waste water (Atlantic mackerel) protein

hydrolysate. The activity was assessed by evaluating the degree of

linoleic acid oxidation using ferric thiocyanate method. Peroxide values

were measured at 24 hour intervals. A negative control was used without

sample or antioxidant. Trolox, ascorbic acid and butylated

hydroxylanisole (BHA) were used as positive controls.

120

Figure 4.2 Percentage of lipid oxidation inhibition activity of water-soluble fish

waste (Atlantic mackerel) protein hydrolysate as measured using ferric

thiocyanate method

121

xi

Figure 4.3 Antioxidant activity of water-soluble fish waste (mixed fish) protein

hydrolysate. The activity was assessed by evaluating the degree of

linoleic acid oxidation using ferric thiocyanate method. Peroxide value

was measured at 24 hours intervals. A negative control was used without

the sample. Trolox, ascorbic acid and butylated hydroxylanisole (BHA)

were used as positive controls.

122


waste (mixed fish) protein hydrolysate as measured by the ferric

thiocyanate method

123

Figure 4.5 Antioxidant activity of water-soluble fish waste (Atlantic Mackerel)

protein hydrolysate. The activity was assessed by the degree of linoleic

acid oxidation by measuring malondialdehyde (µg/ml) at 24 hours

intervals. The negative control contained mili-Q water without the

sample. Trolox, ascorbic acid and butylated hydroxylanisole (BHA) were

used as positive controls

124


waste (Atlantic mackerel) protein hydrolysate. The method was

measured using Thiobarbituric reactive substance method

125

Figure 4.7 Antioxidant activity of water-soluble fish waste protein (Mix fish)

hydrolysate. The activity was assessed by the degree of linoleic acid

oxidation by measuring malondialdehyde (ug/ml) at 24 hours intervals.

The negative control was used containing Mili-Q water without the

126

xii

sample. Trolox, ascorbic acid and butylated hydroxylanisole (BHA) were

used as positive controls


protein (mixed fish) hydrolysate as measured by Thiobarbituric reactive

substance method

127

Figure 4.9 Reducing power of Atlantic Mackerel fish waste protein hydrolysate and

mix fish waste protein hydrolysate

131

Figure 5.1 Differential Scanning Calorimetry (DSC) instrument and sample

preparation used in the experiment

139

Figure 5.2 Rheometrics constant stress rheometer used for the small deformation

testing of Atlantic Mackerel and Mixed fish waste protein fractions

140

Figure 5.3 DSC thermogram of Atlantic Mackerel water-soluble fish protein 142

Figure 5.4 DSC thermogram of Atlantic Mackerel (AM) fish waste sample 145

Figure 5.5 DSC thermogram of mixed fish (MF) waste sample 145

Figure 5.6 DSC thermogram of 10 kDa Atlantic Mackerel (AM) fish waste

hydrolysate sample

146

Figure 5.7 DSC thermogram of 10 kDa mixed fish (MF) waste hydrolysate sample 146

Figure 5.8 (a) Temperature sweep and rheological properties (G’ and G’’) of water

soluble fish processing waste sample; (b) Temperature sweep and

147

xiii

rheological properties (G’ and G’’) of salt soluble actomyosin fish

processing waste sample and (c) Temperature sweep and rheological

properties (G’ and G’’) of fresh Atlantic Mackerel fish processing waste

sample

Figure 5.9 Temperature sweep and rheological properties (G’ and G’’) of (A) water

soluble, and (B) actomyosin fish processing waste stream sample of

mixed fish

149

Figure 6.1 Energy level diagram of Rayleigh and Raman scattering adapted from

Boyaci et al., 2015

154

Figure 6.2 A schematic diagram of a typical Fourier transform (FT) - Raman

spectrometer where D= detector; L= laser; MI= Michelson

interferometer; O= objective lens; RF= Rayleigh filter; S= sample adapted

from Li et al., 2014

155

Figure 6.3 FT-Raman spectrophotometer used for the assignments and

quantification of the bands in the protein

155

Figure 6.4 FT-Raman spectra in freeze dried Atlantic Mackerel and mixed fish waste 161

xiv

List of Tables

Table 1.0 Essential amino-acids (percentage) in various proteins (FAO, 2014) 31

Table 1.1 World fisheries and aquaculture production and utilization (FAO,

2014)

34

Table 1.2 The antioxidative protein hydrolysates and amino acids derived

from fish

46

Table 1.3 Summary of recent endogenous bioactive peptides properties from

various seafood

48

Table 1.4 Comparisons of Process-Related Characteristics for Membrane

Module Configurations (Singh and Heldman, 2009)

59

Table 2.1 Nutrient composition of Nile perch fish after various processing

methods (adapted from Kabahenda et al., 2011)

69

Table 2.2 Nutritional and mineral composition of fish waste (FW), mean ±

standard deviation on a dry matter basis (Esteban et al., 2007)

70

Table 2.3 The chemical composition of fish fillet (FF) Atlantic Mackerel

(Scomber scombrus) and Nile Perch (Lates niloticus) obtained from

study compared to fish samples by standard method adapted from

Murray et al., in FAO ( 2001) and Okeyo et al., 2009

76

xv

Table 2.4 Proximate analysis of fish waste (FW) from fish processing industry.

The values are the means of three samples ± standard deviation.

Values in (%) on a wet matter basis, each values is expressed as

mean ± SD (n=3) of triplicate measurements

78

Table 2.5 Composition of lipids present in fish waste (FW) 80

Table 3.1 The subdivision of tangential flow filration processes 87

Table 3.2 Permeate flux of 3 kDa Pellicon 3 Ultracel membrane at different


97



99


transmembrane pressures in10, 20, and 40 g/L FWS protein

solution

102

Table 3.5 Permeate flux of 10 kDa membrane at different transmembrane

pressures in 10, 20, and 40 g/L FWS protein solution

103

Table 3.6 Limiting flux performance (LMH) at 60 psi of 88 cm2 Pellicon 3

membrane with 3 and 10 kDa cut-off

106

Table 3.7 Mass transfer coefficients for 3 and 10 kDa cut off membranes in

BSA of 88 cm2 Pellicon 3

107

xvi

Table 3.8 The Measured hold up volumes of 3 and 10 kDa fish processing

waste protein. Calculation based on 1g of water = 1 ml water. The

area of device is 88 cm

109

Table 4.1 DPPH scavenging activity in fish waste protein hydrolysates from

mixed fish and Atlantic Mackerel

129

Table 5.1 Transition temperature and enthalpy (J/g) for fish waste (FW)

samples in Atlantic Mackerel (AM), mixed fish (MF), 10 kDa AM and

10 kDa MF

144

Table 5.2 The G’ and G’’ point with the temperature of fish processing waste

streams of water soluble, salt soluble and fresh Atlantic Mackerel

fish protein sample

148

Table 5.3 The G’ and G’’ point with the temperature of fish processing waste

streams of water soluble, salt soluble and mixed fish protein sample

149

Table 6.1 Infrared and Raman characteristic group frequencies (Nauman et

al., 1991 and Piot et al., 2000) adapted from Thygesen et al., 2003

157

Table 6.2 Relative peak intensity values of Raman spectra of Atlantic Mackerel

and Mixed Fish waste streams

162

xvii

Glossary of Terms

ACE Angiotensin converting enzyme

AM Atlantic Mackerel

AOAC The Association of Analytical communities

AOS Active oxygen species

BHA Tert-butyl-4-hydroxyanisol

BHT 2, 6-Di-tert-butyl-4-methylphenol

BSA Bovine serum albumin

CNS Central nervous system

COD Chemical oxygen demand

DAF Dissolved air floatation

DF Diafiltration

DHA Docosahexaenoic acid

DPPH 2, 2- Diphenyl-1 picryhydrazyl

DSC Differential Scanning Calorimetry

EAA Energy and Environmental Affairs

EPA Eicosapentaenoic acid

ESR Electron spin resonance

xviii

ET Electron transfer

FAO The Food and Agriculture Organization

FF Fish fillet

FOSHU Food for Specified Health Use

FRAP Ferric Reducing Antioxidant power

FTC Ferric Thiocyanate

FW Fish waste

FWS Fish waste stream

G’ Storage modulus

G’’ Loss modulus

GABA Gamma-amino butyric acid

GF Gel filtration

HAT Hydrogen atom transfer

HIV-1 Human immunodeficiency virus

kDa Kilo Dalton

LAB Lactic acid bacteria

LIFDCs Low-income food-deficit countries

LMH L/hr/m2

LOOH Lipid hydroperoxide

MDA Malonaldehyde

xix

MF Microfiltration

MF Mixed fish

MTBE Tert-butyl methy ether

MUFA Monounsaturated fatty acids

MW Molecular weights

NF Nanofiltration

NFF Normal Flow Filtration

ORAC Oxygen radical absorbance capacity

PUFA Polyunsaturated fatty acids

RO Reverse Osmosis

ROS Reactive oxygen species

SDI Silt density index

SFA Saturated fatty acids

TBARS Thiobarbituric Acid Reactive Substances

TEAC Trolox equivalent antioxidant capacity

TFF Tangential Flow Filtration

TMP Transmembrane Pressure

TVBN Total volatile basic nitrogen

UF Ultrafiltration

20

Chapter 1

21

1 Introduction

1.1 Proteins

Proteins are polymers of amino acids, joined by peptide bonds (Bender, 2009). The name

was coined by the Dutch chemist Gerard Johann Mulder in 1838, meaning ‘of the first

importance’. There are twenty main amino acids in proteins. A protein may contain

hundreds amino acids giving rise to different proteins in food and in the human body.

Generally a polymer of relatively few amino acids is referred to as a peptide (e.g. di-, tri-,

and tetrapeptides); oligopeptides contain up to about 50 amino acids; larger molecules

are polypeptides or proteins. The sequence of the amino acids in a protein determines its

overall structure and function: many proteins are enzymes; others are structural (e.g.

collagen in connective tissue and keratin in hair and nails); many hormones are

polypeptides. Proteins are constituents of cells and are essential nutritionally. Proteins

are different from fats and carbohydrates as they contain nitrogen in addition to carbon,

hydrogen, oxygen, sulphur, and occasionally phosphorus ((Nelson and Cox, 2005)

1.1.1 Amino Acid Structure

The total number of common amino acids is 20 and their active form is L-α. These amino

acids are similar in the main structure but differ in the R-group. The central carbon atom

which is known as a α-carbon, holds carboxyl group (C-terminus), amino group (N-

terminus) and variable side chain (R) (Figure 1.1)

22

Figure 1.1. Structure of amino acid (Nelson and Cox, 2005)

Amino acids are amphoteric compounds because they carry both a negatively charged

carboxyl group and positively charged amino group in the same molecule; this property

results in an ability to react either as an acid (proton donor) or as a base (proton

acceptor), depending on the pH of the medium. Although the number of amino acids is

limited, the properties and functions of a particular protein depend mainly on the type

and the precise sequence of its amino acid which is unique to that protein. Moreover, the

variability of the structure and the size of R-groups play a fundamental role in the

physical properties of the protein (Fennema et al., 2008)

1.1.2 Amino Acid Classification

Nelson and Cox,(2005) reported that there are various approaches in the classification

of amino acids, but the most suitable one is in terms of the properties of their side-chain

rather than their chemical structure as indicated in figure 1.2.

23

Figure 1.2. Classification of amino acids (Nelson and Cox, 2005)

1.1.3 Molecular Forces Governing Protein Interactions

The molecular forces in the protein structure can influence their functional properties

especially after being exposed in a polar or non-polar environment. Intramolecular forces

are the attraction forces that exist between atoms within a molecule while intermolecular

forces are the attraction between molecules in a compound. Electrostatic interactions are

24

non-covalent and depend on the electrical charge of the protein molecules and happen

over great distances and the intensity varies inversely to distance. The force is repulsive

for like charges and attractive for opposite charges. Other non-covalent bonds include

hydrogen bonds, dipole-dipole bonds and London dispersion forces as well as

hydrophobic interactions (Alberts et al., 2002)

Hydrogen bonds are electrostatic interaction forces between polar molecules where two

atoms with partial negative charges share a partial positively charged hydrogen. This

interaction can happen between acid N-H and base C=O groups in either alpha helices or

beta sheets (Howell, 1992). Dipole-dipole electrostatic interactions occur when there is

a large difference in electro negativity between two atoms bonded together in a covalent

bond. This results in an unequally shared electron between pair of electrons (Fennema et

al., 2008)

The London dispersion force or Van der Waals forces are temporary weak attractions

that result when the electrons in two nearby atoms occupy positions that allow

temporary dipoles. Hydrophobic interactions exist when non-polar substances are added

to a polar aqueous environment. With the absence of hydrogen bonding, there are neither

attracting nor repelling forces between both substances. An example for this is the fact

that oil does not mix with water (Tartar, 1955).

Covalent bonds are the strongest type of intramolecular force. When two atoms share a

pair of electrons; some covalent bonds have dissociation energy of 400Kcal/mol which

depends upon the molecules involved. Therefore, materials that possess covalent bonds

in its structure may have very high melting points and are generally insoluble. Another

type of covalent bond includes the disulphide bond, but this bond is not as strong as the

peptide bond (Damodaran, 2008).

25

1.1.4 Protein Synthesis

To form a protein molecule, amino acids link together by a peptide bond, where the basic

amino group links to the carboxyl group of another amino acid, eliminating a molecule of

water (Figure 1.3).

Figure 1.3. Binding of amino acids via peptide bond (Fennema et al., 2008)

Amino acids with side-chains possessing carboxyl groups are known as acidic amino

acids; those with side chains possessing amino groups are known as basic amino acids,

the remaining amino acids are referred to as neutral amino acids. Two joined amino acids

form a dipeptide and a chain of up to 100 amino acids is called polypeptide. Ways in

which these amino acids are arranged leads to a variety of different proteins e.g. collagen,

albumin or haemoglobin and properties (Belitz, 2009; Damodaran, 2008)

1.1.5 Protein Structure

There are four distinct levels of protein structure, and each protein has a unique three-

dimensional structure as illustrated in figure 1.4.

26

Figure1.4. Four levels of protein structure (Fennema et al., 2008)

1.1.5.1 Primary Structure

The sequence of amino acids in the polypeptide chain is illustrated as a linear

arrangement with C-terminus at one end and N-terminus on the other side. In this

structure, the peptide bond places some restrictions on the shape of the molecule, as free

rotation does not occur around the covalent bond (Belitz, 2009)

1.1.5.2 Secondary Structure

This structure is represented by the coiling of the long chain, which is held in a more rigid

position by weak bonds (chiefly hydrogen bonds). This side chains (R groups) are

extended out of the backbone structure. Secondary structure includes two configurations.

The most common one is the α-helix that occurs when intra-chain hydrogen bonds

27

connect different parts of the same chain, leading to a very stable rod like cylinder. The

second type of conformation, which is known as β-sheet or pleated sheets, occurs when

the hydrogen bonds are inter-chain, leading to strands lining up in a parallel or anti-

parallel fashion; this zigzag shape helps to strengthen the polypeptide chain to a great

extent as compared to α-helix form (Belitz et al., 2009)

1.1.5.3 Tertiary Structure (Fold)

The tertiary structure governs the shape of each protein molecule, which results from

folding the secondary structure of the polypeptide chain. This structure is stabilized by

non-covalent hydrogen bonds (that link various amino residues), hydrophobic forces

(that link non-polar side groups), electrostatic interactions and covalent disulfide bridges

(which link cysteine amino acids at various places in the polypeptide chain) (Damodaran,

2008).

Non-polar hydrocarbon side groups are found in the inside of the molecule where they

can form hydrophobic bonds with one another. In contrast, the polar side chains tend to

move towards the exterior of the molecule, and give water-dispersible properties to

proteins by either forming hydrogen bonds with water molecules or with other parts of

the chain. Therefore, the tertiary structure determines the basic function of protein

(Belitz et al., 2009)

28

1.1.5.4 Quaternary Structure

The quaternary structure represents the non-covalent associations that occur when two

or more proteins in the form of tertiary structure are merged to form a multi-subunits

protein. This structure exists in proteins with complex functions. The most common

example is the haemoglobin molecule which comprises four peptide chains to form α2β2

(Belitz et al., 2009)

29

1.2 Fish and Shellfish Protein

The proteins in fish muscle tissue can be divided into three groups. First, the structural

proteins (actin, myosin, tropomyosin and actomyosin) which constitute 70-80 percent of

the total protein content (compared with 40 percent in mammals). These proteins are

soluble in neutral salt solutions of fairly high ionic strength (0.5 M). The sarcoplasmic

proteins (myoalbumin, globulin and enzymes) are soluble in neutral salt solutions of low

ionic strength (<0.15 M). This fraction constitutes 25-30 percent of the protein. The third

group comprises connective tissue proteins (collagen), which constitute approximately

3 percent of the protein in teleostei and about 10 percent in elasmobranchii, compared

with 17 percent in mammals) ( FAO, 2014).

Fish muscle tissue consists of striated muscle. The muscle cell contains sarcoplasma made

up by nuclei, glycogen grains, mitochondria, and up to 1000 myofibrils. The cell is

surrounded by a sheath of connective tissue called the sarcolemma. Besides that, the

myofibrils contain the contractile proteins, actin and myosin. These proteins or filaments

are arranged in a characteristic alternating system making the muscle appear striated

upon microscopic examination (Berge, 2013) (Figure 1.5)

30

Figure 1.5. Cell section of various structures including myofibrils (Bell et al, 1976

adapted from http://www.fao.org/docrep/v7180e/v7180e04.htm )

Contractile tissue for muscle movement is made up by the structural proteins. Amino acid

composition of fish muscle is usually similar to that of mammalian muscle, while small

differences are apparent in the physical properties. Once proteins are denatured by

processing, their functional properties are useful in product development e.g. surimi

production requires the good gelation properties of the myofibrillar proteins, addition

of salt to solubilise the myofibrillar proteins and proper heating and cooling process to

create a very strong gel (Ghaly et al., 2013).

In most sarcoplasmic proteins, enzymes are known to take part in the metabolism of cell,

for example glycolysis. If the muscle cells are broken, this protein fraction may also

consist of the metabolic enzymes (Berge, 2013). This may be used to monitor changes in

storage and processing.

The proteins in the sarcoplasmic fraction are very well suited to distinguishing fish

species, as each species has a characteristic band pattern when separated by the

http://www.fao.org/docrep/v7180e/v7180e04.htm

31

electrophoresis including the isoelectric focusing method (Le Fresne et al., 201; Huss,

1995)

The chemical and physical properties of collagen proteins are different in tissues such as

skin, swim bladder and the myocommata in muscle. In general, collagen fibrils form a

delicate network structure with varying complexity in the different connective tissues in

a pattern similar to that found in mammals. However, the collagen in fish is much more

thermolabile and contains fewer, but more labile, cross-links than collagen from warm-

blooded vertebrates. Different fish species contain varying amounts of collagen in body

tissues. This has led to a theory that the distribution of collagen may reflect the swimming

behaviour of the species. Furthermore, the varying amounts and varying types of collagen

in different fishes may also have an influence on the textural properties of fish muscle

(Pires and Batista, 2013 as cited in Berge, 2013).

Fish proteins contain all the essential amino-acids and like milk, eggs and mammalian

meat proteins, have a very high biological value as shown in Table 1.0

Table 1.0: Essential amino-acids (percentage) in various proteins (FAO, 2014)

Amino acid Fish Milk Beef Eggs

Lysine 8.8 8.1 9.3 6.8

Tryptophan 1.0 1.6 1.1 1.9

Histidine 2.0 2.6 3.8 2.2

Phenylalanine 3.9 5.3 4.5 5.4

32

Leucine 8.4 10.2 8.2 8.4

Isoleucine 6.0 7.2 5.2 7.1

Threonine 4.6 4.4 4.2 5.5

Methionine-

cystine

4.0 4.3 2.9 3.3

Valine 6.0 7.6 5.0 8.1

1.2.1 Current World Fisheries Statistics

The Food and Agriculture Organization (FAO) Fisheries Department is the responsible

body to provide the statistics of global fish capture and aquaculture production as well as

the data for by-products. World fish production has increased steadily from year 2009 to

2014 (Figure 1), around 3.2 percent average annual rate of food fish supply, over the

demand of world population of 1.6 percent.

33

Figure 1.6: World capture fisheries and Aquaculture production (FAO, 2014)

In general, fish is produced from the capture fisheries and aquaculture (FAO, 2014). The

world per capita food fish supply increased from an average of 17.6 to 19.2 kg in 2007 to

2012. This has been driven by a combination of population growth, rising incomes and

urbanization, and facilitated by the increase in fish production and more efficient

distribution channels.

China has extensive fish production, particularly from aquaculture and increased fish

consumption, 35.1 kg in 2010. Annual per capita fish supply in the rest of the world was

about 15.4 kg in 2010 fish consumption in developing regions rise from 5.2 kg in 1961 to

17.8 kg in 2010) and low-income food-deficit countries (LIFDCs) (from 4.9 to 10.9 kg).

However, developed regions still have higher levels of consumption. Fish consumed in

developed countries consists of import, because of the steady demand and reduced

production. People in developing countries, consume fish caught locally. However, rising

incomes and wealth, consumers in emerging economies are importing varied types of fish

(FAO, 2014)

34

Table 1.1: World fisheries and aquaculture production and utilization (FAO, 2014)

Figure 1.7: World Fish Utilization and Supply (FAO, 2014)

35

Approximately, half of an adult’s daily protein requirement can be provided from a

portion of 150 g of fish. Based on the statistics given, in 2010, world population fish made

up 16.7 percent of the intake of animal protein, and 6.5 percent of total protein consumed.

According to FAO 2014, fish provided more than 2.9 billion people with almost 20 percent

of their intake of animal protein, and 4.3 billion people with about 15 percent of such

protein. Fish proteins benefit densely populated countries nutritionally their protein

intakes are low.

78% of the total fish catch in developed and developing countries is used in human food

whilst, 21% goes to non-food uses (Vannuccini, 2004). Fish processing like filleting leads

to fish waste (e.g., skin, bones, and fins), that is discarded (7.3 million tons/year)

(Kelleher 2005).

A summary of material flows in the UK supply chain for an individual species of fish, as

example of cod is shown in Figure 1.8

Figure 1.8: Resource Maps for Fish across Retail and Wholesale Supply Chains, WRAP

(2011)

36

Economically beneficial conversion of the raw aquatic foods for human food products

instead of animal feeds can improve food security. Processing of by-products can lead to

value-added food products.

1.2.2 Fish Waste and Fish Waste Streams

Since the International Convention of Bâle, EU, waste is considered as “any substance or

object the holder discards, intends to discard or is required to discard” [European

Directive (WFD) 2006/12/EC]. This is a negative definition implying there is no added-

value for the raw materials. Therefore, “waste” is still widely used to define by- or co-

product. The term “co-product” is defined as products arising from fish product

manufacture e.g. filleting that are also used for human consumption e.g. liver cheek meat,

and are governed by the same regulations.

Fish by-products represent the remainder of the products after processing that is not

used as human food and are separated according to their potential risk to human health,

animal health and the environment as cited by Penven and Galvez (2013); and Berge

(2013). The set of materials derived from the production of finished products are

considered as by-products. The by-products are then separated into three categories

based on their potential risk to human, animal and environment well- beings (Rubio-

Rodriguez et al., 2012)

Seafood may undergo various processing steps such as, filleting, heading, gutting,

skinning or cutting. Fish by-products comprise heads, viscera, trimmings, fish bones or

cartilage, hides, tails, and eggs; the proportions are shown in Figure 1.9

37

Figure1.9: Average proportion of fish by- products (Dumay, 2006)

By-products are generated by fish trade wholesalers, and canning (sardines, mackerel,

herring and tuna) and smoking (salmonids, salmon and trout) industries (Berge, 2013).

Product utilization from cod fish involves only fillet (43%) and liver and the rest is usually

discarded. There is potential for dried cod products, e.g. in Iceland the dried head and

bones are exported. Cod skin and internal organs as well as the flesh (Figure 1.10) may

be used for pet food, fish leather and potentially other products. However, in order to use

potential discards at sea, chilling and storage facilities on board fishing vessels are

important.

38

Figure 1.10: Products Utilization of fish cod waste (adapted from World fishing and

Aquaculture, website: http://www.worldfishing.net/news101/icelandic-fisheries-

exhibition/icefish-conference-fish-waste-for-profit )

Waste waters from fish processing contains valuable proteins resulting in a high organic

load. This should not be discharged directly to the sea without suitable treatment, in

order to avoid pollution and undesired environmental impact (Yeo et al., 2004).

Fish food industry is one of the important industries worldwide. There are several

effluents that are produced during the fish processing as reviewed by Afonso and

Borquez (2002). This include pumped seawater with fish during ship unloading which

have a high chemical oxygen demand (COD=7-49g O2/L) and usually flow straight into

the sea without treatment. In addition, waste water produced during fish meal

production involves the total flow rate and COD becoming 1000-1200 m3/h and 3-9 g

O2/L respectively. After filtration and recovery of large pieces of fish, the rest of the water

is discarded into the sea.



39

Surimi production leads to substantial wastewater. The protein concentrate is

incorporated into various Japanese food products. Large amounts of fresh water, 27m3

per ton surimi is used and results in a high organic load of these waste waters. The waste

water has high turbidity, strong greenish yellow colour and strong odour, but does not

contain toxic or carcinogenic substances (Afonso and Borquez, 2003; 2002)

The common feature of the fish waste streams is their diluted protein content, which can

benefit from concentration to recover the valuable raw materials including proteins for

use in animal feed or human consumption and reuse the water by recirculation to the

process (Yeo et al., 2004)

1.2.3 Fish Protein Hydrolysates

1.2.3.1 Commercial Development of Marine-Derived Protein Hydrolysates and Peptides

Isolation of bioactive peptides from seafood protein hydrolysates include membrane

separation like nanofiltration (NF), ultrafiltration (UF) and electro-membrane filtration;

can be used to concentrate peptides molecular weight ranges, mostly 1-10 kDa.

Furthermore, membrane bioreactor technology combining enzymatic protein hydrolysis

and separation of peptides by ultrafiltration may be used (Kim &Wijesekara, 2010).

Peptides with specific activity may be obtained by an expensive in vitro bioactivity assay

i.e. purification and chromatography (Pauliot, Gauthier, & Groleau, 2006).

Several Food for Specified Health Use (FOSHU) products containing fish protein

hydrolysates and peptides as food ingredients have been approved in Japan. Many of

these products claim to be suitable for consumption by individuals with mild

hypertension (Harnedy and Fitzgerald, 2012; Hartmann and Meisel, 2007).

40

1.3 Nutritional Properties of Proteins in Fish

The fish waste either by-catch or fish by-products generated during processing contains

valuable nutritional proteins but they are usually used as a fertilizer, animal feed due to

poor functionality. Hygienic conditions are needed for the collection of fish mince and

separation steps to remove solids, oil, and water by boiling, pressing, evaporation and

dehydration.

Seafood such as fish and shellfish, contain rich biologically active components including

proteins, peptides and amino acids and bioactive peptides. Bioactive peptides have health

benefits and are functional food ingredients. Many studies on the activities including

antihypertensive, anti-oxidative, anticoagulant, antimicrobial, anti-diabetic and anti-

cancer properties. High quality protein fish waste protein (10-23% (w/w)), may be useful

as biofunctional peptides (Harnedy and Fitzgerald, 2012).

1.3.1 Bioactive Hydrolysates and Peptides from Enzymatic Hydrolysis of Fish and

Shellfish Waste

Bioactive peptides are defined as specific protein fragments that have many potential

physiological functions within the body in addition to the usual source of nitrogen and

amino acids (Singh et al, 2014; Nagpan et al., 2011). The physiological functions include

opioid, immunomodulatory, antibacterial, antithrombotic and antihypertensive activity.

Bioactive peptides comprising 2-20 amino acid residues and are obtained from protein

during food processing or digestion in the intestine (Murray and Fitzgerald, 2007;

Najafan and Babji, 2012)

41

Each year significantly large amounts of marine processing by-products are generated.

These include seafood waste: fish muscles, viscera, heads, skins, fins, frames, trimmings,

shellfish, and shell waste. Some by-products are either discarded or may be used as

animal feed or fertilizers. Utilisation of marine by products into functional ingredients

may give a good solution when dealing with the legal restrictions, high cost and

environmental problems related to waste material (Pfeiffer, 2003).

Methods to produce bioactive peptides include: solvent extraction, enzymatic hydrolysis

and microbial fermentation of food proteins (Figure 1.11). Laboratory-scale solvent

extraction systems have several drawbacks like low selectivity, low extraction efficiency,

solvent residue, and are non-environmentally friendly. However, enzymatic hydrolysis

systems e.g. lactic acid bacteria (LAB) for fermentation of milk and meat products and are

commonly used in the food and pharmaceutical industries. (Najafan and Babji, 2012)

Figure 1.11 The generation methods of peptides from food proteins (Najafan and

Babji, 2012)

42

1.3.2 Endogenous Bioactive Peptides

1.3.2.1 Antihypertensive Activity

Marine derived peptides are reported to have potent ACE inhibition activities expressed

as the ACE inhibitor concentration leading to 50% inhibition of ACE activity (IC50 value).

Competitive ACE inhibitory peptides, determined via Lineweaver-Burk plots, are known

to bind to an active site to block it or to the inhibitor binding site which is away from the

active site resulting in an alteration of enzyme conformation preventing the substrate

from binding with the active site. Non-competitive binding mechanism has also been

identified in some peptides. Some in vivo studies with marine-derived peptides in

spontaneously hypertensive rats also show peptides ACE inhibitory activity, possibly due

to higher tissue affinities by the peptides compared with captopril as suggested by Kim

and Wijesekara, (2010).

1.3.2.2 Antioxidant Activity

The antioxidant activity can be analysed in variety of ways. According to Najafan and

Babji (2012) methods to observe antioxidant capacity are based on hydrogen atom

transfer (HAT) reactions and electron transfer (ET) reactions. HAT-based assays

measures the ability of an antioxidant to donate a hydrogen atom. The ET methods

quantify the reducing capacity and depend on a colour change. An example of a HAT assay

is the inhibition of linoleic acid autoxidation by antioxidants (ORAC assay). Figure 1.12

shows lipid autoxidation pathways that are initiated by an azo compound, inhibition step

and termination by antioxidants

43

Figure 1.12 Steps of the autoxidation process and the action of antioxidant (Najafan

and Babji, 2012)

The ET-based reaction includes the trolox equivalent antioxidant capacity (TEAC) assay

as well as the FRAP assay. In addition, the scavenging capacity of individual ROS, such as

superoxide anion, singlet oxygen, hydrogen peroxide, hydroxyl radical, and peroxynitrite

are also used. Electron spin resonance (ESR) spectrometry, can monitoring free radicals

directly and the effect of antioxidants on free radical scavenging (Howell and Saeed,

1999). The chemical assays for measuring the antioxidative capacity of protein

hydrolysates and peptides from fish are shown in Figure 1.13.

44

Figure 1.13 In vitro chemical analysis for measuring the antioxidative capacity of

protein hydrolysates and peptides (Samaranayaka, 2010)

Marine protein hydrolysates from jumbo squid, oyster, blue mussel, hoki, tuna, cod,

pacific hake, capelin, scad, mackerel, Alaska Pollack, conger eel, yellow fin sole, yellow

stripe trevally, and microalgae and peptides demonstrate good antioxidant activity (Kim

and Wijesekara, 2010).

45

Marine bioactive peptides scavenge free radicals or interrupting the lipid peroxidation

chain reaction of (Rajapakse et al., 2005). The bioactive peptide, isolated from jumbo

squid had higher activity compared with α –tocopherol, similar to that of BHT as

determined by a linoleic acid model system, mainly due to the hydrophobic groups

(Mendis et al., 2005).

Other studies also show that some marine peptides have greater antioxidants properties

than α-tocopherol in different oxidative systems, but the exact mechanism is not clearly

proven (Jun et al., 2004; Howell and Kasase, 2010 ); however, aromatic amino acids and

histidine are known to play an important role. Thus, marine peptides with antioxidant

properties could potentially be used as nutraceuticals and pharmaceuticals or used as a

substitute for synthetic antioxidants. Hydrophobic amino acids in protein hydrolysates

may have to be altered with debittering enzymes (Moller et al., 2008).

A summary of current fish antioxidant peptides, the name of the organism producing

the peptide and the peptide the sequence are shown in Table 1.2 (Halim et al., 2016;

Najafan and Babji, 2012; Howell and Kasase, 2010)

46

Table 1.2 The antioxidative protein hydrolysates and amino acids derived from fish

Type of seafood Peptides sequences References

Thornback ray gelatin (Ala-Val-Gly-Ala-Thr, Gly-Gly-Val-Gly- Arg, Gly-Pro-Ala-Gly-Phe-Ala, Gly-Glu-Pro-Gly-Ala-Pro)

Lassoued, Mora, Barkia et al. (2015)

Horse Mackerel (Magalaspis cordyla) and croaker skin

(Asn-His-Arg-Tyr-Asp-Arg, Gly-Asn-Arg-Gly-Phe- Ala-Cys-Arg-His-Ala)

Kumar et al. (2011)

Nile tilapia (Oreochromis niloticus) scale gelatin

(Asp-Pro-Ala-Leu-Ala-Thr-Glu-Pro-Asp- Pro-Met-Pro-Phe)

Ngo et al. (2010)

Royal jelly protein (Lys-Tyr, Arg-Try, and Tyr-Tyr)

Guo et al. (2009)

Hoki frame protein Glu-Ser-Thr-Val-Pro-Glu-Arg-Thr- His-Pro-Ala-Cys-Pro-Asp-Phe-Asn

Kim et al. (2007)

Conger eel muscle (Conger myriaster)

(Leu-Gly-Leu-Asn-Gly-Asp- Asp-Val-Asn)

Ranathunga et al. (2006)

Alaska Pollack skin gelatin (G-E-Hyp-G-P-Hyp-G-P-Hyp- G-P-Hyp-G-P-Hyp-G and G-P-Hyp-G-P-Hyp-G-P-Hyp-G-P-Hyp-G)

Kim et al. (2001)

1.3.2.3 Anticoagulant Activity

Anticoagulant proteins have been purified from blood ark shell, and yellow fin sole and

the coagulant activity was compared with heparin, the commercial anticoagulant; they

may also have potential use in nutraceuticals or pharmaceuticals (Kim and Wijesekara,

2010).

1.3.2.4 Antimicrobial Activity

Marine peptides have shown antibacterial, antiviral, antifungal, immunomodulatory, and

antitumor properties. Achour et al., (1997) reported that oyster protein could enhance

47

proliferation of immunocytes in human immunodeficiency virus (HIV-1). Marine

antimicrobial peptides potentially have a broad spectrum antimicrobial activity.

Hepcidin TH1-5, an antimicrobial peptide, synthesized from tilapia, showed antitumor

activity against several tumour cell lines. It was shown that TH1-5 inhibited the

proliferation. These antimicrobial peptides can be used in the food as well as in the

pharmaceutical industry (Najafian and Babji, 2012).

1.3.2.5 Other Biological Activities

Marine-derived bioactive peptides are reported to reduce the risk of cardiovascular

diseases by lowering plasma cholesterol level and anti-cancer activity by reducing cell

proliferation (Picot et al., 2006). Moreover, (Jung and Kim, 2007) reported calcium-

binding peptides derived from pepsin hydrolysates of Alaska Pollack (Theragra

chalcogramma). Hoki (Johnius belengerii) frame peptides can benefit people with lactose

indigestion or intolerance and calcium rich non-dairy foods. A high affinity to calcium

may help reduce the risk of osteoporosis. Small peptides (di- and tripeptides) can be

absorbed across the brush border membrane by a specific peptide transport system and

result in biological effects. A summary of endogenous bioactive peptide is as shown in

Table 1.3.

48

Table 1.3 Summary of recent endogenous bioactive peptides properties from various

seafood

Bioactive properties Types of seafood References

Antioxidant activity thornback gelatin

horse mackerel

croaker skin

tilapia scale gelatin

Lassoued, Mora, Barkia, et al. (2015)

Sampath Kumar et al. (2011)

Ngo, Qian, Ryu, Park, and Kim (2010)

Antihypertensive ACE inhibitory activity

Monopterus sp

thornback ray

goby

lizard fish

grass carp

Channa striata

Alaska Pollack, big eye tuna, yellow fin sole, sardine, shark and sea bream

Baharuddin, Halim, and Sarbon (2016).

Lassoued, Mora, Barkia, et al. (2015), Lassoued, Mora, Nasri, Aydi,et al. (2015)

Nasri et al. (2013)

Wu et al. (2012)

Chen, Wang, Zhong, Wu, and Xia (2012)

Ghassem et al. (2011)

Wijesekara and Kim (2010)

Anticancer properties oyster

tuna dark muscle

half-fin anchovy

Umayaparvathi et al. (2014)

Hsu, Li-Chan, & Jao, (2011)

Song et al. (2011)

49

1.3.3 Bioactive Proteins Produced by Fish

While no studies have been carried out on humans, conflicting results in relation to the

hypocholesterolemic action of fish (cod) proteins in rats and rabbits have been reported.

Rats fed on high cholesterol diets containing cod proteins showed a significant decrease

in serum, plasma and liver cholesterol levels compared to those fed on high cholesterol

levels compared to those fed on high cholesterol diets containing casein proteins. In

contrast the same diet fed to rabbits resulted in an increase in serum cholesterol

compared to casein (Nagaoka, 2006). However, to date no mechanistic studies have been

performed on the hypocholesterolemic activity of marine proteins.

The effectiveness of nutraceutical products are known in preventing the bioavailability

of the active ingredients. Research has shown that compared to peptides with 2-6 amino

acids, proteins are less readily absorbed across the gastrointestinal tract. Limitations

such as intrinsic physical, chemical and biological properties, including limited

permeation through membranes due to molecular size, physical and chemical instability,

and degradation by intrinsic proteolyic enzmes, aggregation, adsorption and

immunogenicity may affect absorption. Therefore, in vivo studies are necessary prior to

using bioactive peptides (Harnedey and FitzGerald 2012).

50

1.3.4 Bioactive Amino Acids Present in Fish and Seafood

Taurine is a naturally occurring B-aminosulphonic acid, and essential for many functions

including bile acid conjugation, cell membrane stabilisation, development of the central

nervous system (CNS) and retina, osmoregulation, modulation of cellular calcium levels,

and immune function. Moreover, Taurine has also demonstrated antihypertensive,

hypocholesterolemic and antioxidant properties (Zhang et al., 2004).

Taurine can be found at high concentrations in seafood especially molluscs and

crustaceans. Taurine levels of 151 and 31mg/100g wet weight have been reported for

raw white fish and frozen cod, respectively. Raw mussel, fresh oysters and fresh clams

have reported levels of 655, 70 and 240mg/100g wet weight, respectively. Furthermore,

Atlantic salmon, Coho Salmon, Alaska Pollack and southern blue whiting extracts and

their hydrolysates have also been reported to contain 27, 19.5, 15.2 and 149 mg of

taurine/100g dry weight, respectively (Lourenco and Camilo, 2002)

GABA, an amino acid like molecule is synthesised from glutamate by two glutamic acid

decarboxylase enzymes; it is present all central nervous systems and is the most common

inhibitory neurotransmitter in vertebrates. It is reported to be involved in the regulation

of nearly all main development steps from cell proliferation. More recently GABA was

found to reduce blood pressure in spontaneously hypertensive rats (SHR) and in mildly

hypertensive human subjects. The activity of GABA as an inhibitory neurotransmitter was

first reported in crustacean neuromuscular junctions and stretch receptors. In addition

to taurine, GABA has been observed as one of the most abundant components of the

intracellular amino acid pool in erythrocytes of flounder, plaice and dab. While marine-

derived GABA and taurine may be source of functional food ingredients, industrial scale

51

production from marine sources may not be able to compete economically with the

production of GABA via fermentation using lactic acid bacteria or by the organic synthesis

of taurine (Fugelli, 1970).

1.4 Functional Properties of Fish Proteins

The term functional properties of proteins which comprise gelation, foaming and

emulsification that occur during food (processing, storage and consumption) relates to

physicochemical properties. In native proteins, any changes in physicochemical

properties are associated with changes in the primary structure comprising a linear chain

of amino acids, secondary structure (alpha helix, beta-sheet and random coil) as well as

the tertiary structure. Furthermore, free amino acids and peptides which are the

products resulting mainly from enzymatic hydrolysis (Chalamiah et al., 2012) or chemical

modification may also contribute to enhancement of functional properties. Smaller

polypeptides can improve the functional properties, particularly foaming and

emulsification that can be used in different applications. Therefore, enzymatic hydrolysis

has been used successfully to obtain useful peptides from fish and fish by-products

(Howell and Kasase, 2010, Halim et al., 2016)

According to Badii and Howell (2002), fish proteins are known to be exposed to

aggregation and denaturation during storage time as well as temperature which affects

its texture and nutritional values. Protein denaturation can be measured by the reduction

in extractable myofibrillar proteins; several studies have reported that during frozen

storage the myosin heavy chain fraction of the myofibrillar proteins is sensitive to freeze

denaturation (Del Mazo et al., 1999, Badii and Howell, 2002).

52

To date, whilst there are several papers published on the physicochemical properties of

proteins in different types of fish under several conditions (freezing, drying and heating),

only a few reports exist on fish waste protein fractions (Filho et al., 2011). Most papers

on waste recovery from fish processing report functional properties related to fish oil.

There are several studies that investigate the functionality (solubility, emulsion and foam

capacity and stability, as well as water and fat binding) of fish protein hydrolysates

especially from the muscle, viscera, skin and roe (Halim et al., 2016)

1.4.1 Rheological Measurements

Rheology is classified under functional properties that can be divided into those that

induce small or large deformations. These two types of tests give complementary

information but need not be correlated (Yada et al., 1994).

Small deformation tests viscoelastic parameters. These parameters are commonly

derived by dynamic oscillatory testing in the linear viscoelastic region of the test material.

Dynamic measurements can be to be monitored easily since the induced deformation is

very small and the effect on structure is minimal (Ross-Murphy, 1984). Two parameters

are obtained from dynamic measurements: the storage modulus (G’) measures the

energy that is stored elastically in the structure and the loss modulus (G’’) measures

energy loss or the viscous response. The phase angle, δ measures of the stress /strain

ratio. The phase angle is 0° for a completely elastic material and 90° for a purely viscous

fluid as stated below:

Tan δ = G’’/G’

53

Large deformation tests measure stress, strain and breakdown properties of materials

tested. Tests on gels can be by of biopolymer gels can be more commonly used

compression and tension. The total stress during compression involves both tensile and

shear stresses, whereas the shear stresses are minimal in in tensile tests (Yada et al.,

1994).

54

1.5 Protein Recovery

The properties of fish waste protein hydrolysate can be further analysed by purification

process, thus the pressure-driven membrane separation can be employed as they are

known to increase the efficacy of specific activities as reported by Bourseau et al. (2009).

The separation method namely ultrafiltration (UF), nanofiltration (NF) as well as gel

filtration (GF) will separate hydrolysate, increase their specific activity in order to

produce bioactive products for human and animal feed (Picot et al., 2010).

1.5.1 The Recovery of Protein by Membrane Separation Technique

Membrane filtration is a technique that uses physical barrier or filter to separate particles

in a fluid. These particles are separated on basis of their size and shape with the use of

pressure and specially designed membrane with difference pore size (Sutherland &

Chase, 2011 and Cheryan, 1998).

The success of a membrane application or process is related to the main membrane

properties. Interactions between membrane surface, environmental and solute

conditions affect membrane performance including on fouling properties, and membrane

compatibility and selectivity (Huang, 2012).

For membrane separation, liquids with more than two components makes contact with

a membrane that allows specific components like water to permeate more readily than

other solutes. Membrane pore size and pore size distribution influence the separation of

liquids and solutes. In reverse-osmosis, under very pressures, water passes through the

membrane selectively against osmotic pressure whereas dissolved salts and sugars are

rejected. In ultrafiltration separation is achieved by fractionating components based on

the molecular weights (MW), using different MW cut off membranes e.g. 2, 5, 10 kDa. In

microfiltration, the membranes have very small pores that separate very small particles

55

e.g. bacteria (Singh and Heldman, 2009). The application of each of the membrane for

separating different materials is as shown in Figure 1.14.

Figure 1.14. A separation spectrum (Dunwell, 2005)

Membrane is a special designed thin film, that allows certain sized matter to pass

through whilst others are rejected, and this is also affected the permeability of

membrane (Luo et al., 2014 and Lee et al., 2002). Membrane technology allows high

efficiency in purification process, for example bacteria and viruses can be filtered out a

contaminated water (Das et al., 2014); thus membrane technology and specific

pressure is applied as the main principle of reverse osmosis which are used in the sea

water desalination process.

56

The main principle of filtration spectrum are as follows:

Microfiltration

Microfiltration is also known as micro particle range especially for bacteria, with pore

size of 0.2 microns. The particle size is usually between 0.1-1.5 microns, and this type

of membrane are used to remove suspended solids, bacteria or other impurities. On the

other hand, the effluent is free of pollen, bacteria, pigment and yeasts (Hilal et al., 2006).

Ultrafiltration

The membrane is a macro molecular range for example viruses, with pore size of 0.1-

0.01µm (Arvanitoyannis et al., 2008). With particle sizes between 0.005-0.1 microns, it

is capable of removing salts, proteins and other impurities (Hilal et al., 2006). Due to

increased pore size, lower driving force pressure on the membrane, it is not possible to

separate dissolve substances unless they are absorbed or coagulated (Shirazi et al.,

2010). The effluents are free of emulsions, viruses, starches, gelatines and proteins.

Nanofiltration

The particle size for this type of membrane is around 0.0001-0.005 microns, while the

pore size is around 0.0001-0.01µm (Schafer et al., 2005). It can produce hard water

because it removes all organic matters, a range of salts and divalent ions, and the

effluents are free of emulsion, herbicides, pesticides, sugars, and synthetic dyes (Van

der Bruggen et al., 2008 and Martin-Orue et al., 1998).

57

Reverse osmosis

Reverse osmosis is the finest membrane of all, with the particle size up to 0.0001

microns and membrane pore size between 0.0001-0.001 µm (Nath, 2017). The effluent

are free of aquous salt, metal ions, atomic radius as well as acids. The simple

phenomena involving reverse osmosis is when two identical fluids by nature wants to

mix and dilute until reach the equilibrium state, if separation by semi permeable

membrane, the exchange will continuing until the osmotic pressure on both sides are

equal (Borg, 2003). By applying enough pressure on one side of the membrane, natural

osmotic tendency to reach equilibrium can be reversed, hence substances can be

separated (Brown et al., 2017 and Borg, 2003). This technique is usually applied in the

desalinate seawater or brackish into fresh water for human consumption.

Osmosis is a natural process when two solutions with different concentrations of solute

are separated by semipermeable membrane to a region of low solute concentration

(high water potential) to a high solutes concentrations (low water potential) to reach

chemical potential equilibrium (Cath et al., 2006). Reverse osmosis on the other hand,

is a process of forcing a solvent from a region of high concentration of solutes into a

low concentration of solutes through a semipermeable membrane by applying

pressure in excess of osmotic pressure (Borg, 2003). The example is in the drinking

water purification, especially in a separation of pure water from seawater and brackish

water.

There are major difference between reverse osmosis and filtration. In filtration, it is

predominant removal mechanism used in straining or size exclusion, thus intended

process can theoretically achieve perfect efficiency regardless the parameters of

solutions pressure and concentration; whereby reverse osmosis involves diffusion,

58

making the process dependent on pressure, flowrate and other conditions(Lee et al.,

2010).

1.5.2 Use of membrane separation process in wastewaters treatment

Afonso and Borquez (2002) cited that “The term “wastewaters” comprises all liquid

effluents from the processing of fish, shellfish and crustaceans, usually washing, cooking

or pressing waters”. Fish waste processing streams which contain diluted protein, can be

recovered by concentrating using suitable recirculation system. However, they are

known for economically low to be recovered by classical process e.g. by centrifugation,

drum dying or evaporation method. Method by dissolved air floatation (DAF) or

coagulation/flocculation are not suitable since the flow rates are too high to be handled.

Moreover, the usage of enormous reagents and equipment despite disposal of sludge and

transportation, can be costly and reduced the protein quality of the streams (Afonso and

Borquez, 2002).

Therefore, the use of membrane separation process in particular ultrafiltration (UF) and

nanofiltration (NF) is the great alternative. The advantages when using the processes are

good quality of permeate produced, to be reused or directly disposed to environment.

Without the usage of heat or chemicals, they are also susceptible to sensitive biological

substances of high added value during recovering process includes proteins, enzymes

and hormones. Thus the use of membrane separations is suitable for the treatment of the

waste waters since the end product which recovered from this process has been directly

come in contact with fish parts (Van der Bruggen et al., 2003).

59

1.5.3 Membrane Separation Configuration

Membrane module configuration used for reverse osmosis and ultrafiltration systems

include plate-and-frame, tubular, spiral-wound, and hollow fibre membrane systems.

Table 1.4 compares the four types of devices.

Table 1.4 Comparisons of Process-Related Characteristics for Membrane Module

Configurations (Singh and Heldman, 2009)

Module type

Characteristic

Plate and

Frame Spiral – wound

Tube-in-

shell Hollow Fibre

Packing density (m2/m3) 200-400 300-900 150-300 9000-30000

Permeate flux (m3/ [m2-

day]) 0.3-1.0 0.3-1.0 0.3-1.0 0.004-0.08

Flux density (m3/[m3 day]) 60-400 90-900 45-300 36-2400

Feed channel diameter

(mm) 5 1.3 13 0.1

Method of replacement As sheets

As module

assembly As tubes

As entire

module

Replacement labour High Medium High Medium

Pressure drop

Product side Medium Medium Low High

Feed side Medium Medium High Low

Concentration polarization High Medium High Low

Suspended solids build-up Low/medium Medium/high Low High

60

1.5.4 Pre Treatment method

Traditional filtration which is mainly known as mechanical treatment includes media

filters, cartridge filters that involve substantial chemical treatment (Bruggen et al., 2002).

Pre-treatment mainly includes coagulation, flocculation, acid treatment, pH adjustment,

addition of anti-scalant and media filtration.

Problems associated with these methods are corrosion and corrosion products that will

damage and scaling of equipment surface (Al-Ahmad and Aleem, 1993); it is also labour

intensive and consumes space.

It is advised to use membrane filtration to remove microorganisms and bacteria from

water since membrane is a physical barrier and passage through is impossible

(Lingireddy, 2002 and Mallevialle et al., 1996). Most substitute filtration methods are

prone to uncertainty of effluent water quality due to channelling clogging saturation of

filter medium (Escobar et al., 2009).

Thus the preferable separation methods are membrane processes like Microfiltration

(MF), ultrafiltration (UF) and Nanofiltration (NF). The MF can remove suspended solid

and lower the silt density index (SDI) while in UF system, suspended solutes, colloids,

small and large bacteria that are concentrated.

61

1.6 Aims and objectives of the study







1.6.1 Aims of the study

The aims of this study were to investigate the physico-chemical properties of fish

(Atlantic Mackerel, Scrombus scromber and mixed fish) waste water from filleting plants;

produce hydrolysates, and separate peptides by ultrafiltration, measure the nutritional,

antioxidant and functional activity to improve the utilisation of fish waste protein as

value added protein for food ingredients and pharmaceuticals.

1.6.2 Objectives of the study

The overall objectives of this study were:

I. To analyse the composition of the Mackerel and mixed fish waste streams

(mackerel, Nile Perch fish, and fish waste stream): protein, moisture, ash, amino

acids fatty acids

II. To produce fish waste protein hydrolysates and study the ultrafiltration

parameters to obtain water-soluble proteins fraction (3 and 10 kDa) from the

Mackerel and mixed fish waste stream).

62

III. To study the antioxidant properties by lipid peroxidation inhibition (FTC and

TBARS); radical scavenging activity (2, 2- Diphenyl-1 picryhydrazyl DPPH radical

scavenging and ferric reducing antioxidant power assay) in the Atlantic mackerel

and mixed fish waste streams.

IV. To study the thermodynamic properties by differential scanning calorimetry

(DSC) and rheological properties of fish waste (mackerel, mixed fish, 10 kDa

mackerel fraction, 10 kDa mixed fish fraction) and

V. To characterize protein structure of mackerel and mixed fish waste stream

proteins using FT-Raman spectroscopy.

1.6.3 Hypothesis of the study

The physicochemical properties of fish processing waste streams from Atlantic Mackerel

(Scomber scombrus) and mixed fish can be investigated by their production of

hydrolysates, analysing each chemical composition of protein, moisture, ash, amino acids

and fatty acids; ultrafiltration studies by investigating its processing parameters;

determine its antioxidant properties of lipid peroxidation inhibition and radical

scavenging activity; thermodynamic properties using differential scanning calorimetry

(DSC) and rheological properties; and protein structure characterization.

I. The proximate analysis of fish fillet (Atlantic Mackerel and Nile Perch) as well as

the fish waste stream quality can be investigated. Fish waste may contain higher

amount of moisture, ash, lipids and protein content. Higher amount of ash content

may obtained in fish waste than in fish fillet since there is a presence of inorganic

components in waste water. Fish fillet samples of Atlantic Mackerel and Nile Perch

are also comparable .Oily fish such as Atlantic Mackerel may contain significantly

higher amount of lipid compare to Nile Perch, also contributed to higher amounts

63

of polyunsaturated fatty acids compared to Nile perch sample. The most abundant

fatty acid in most fish will be palmitic acid.

II. Protein hydrolysates from fish waste streams can be obtained by enzymatic

digestion by pepsin and pancreatin and the processing parameters for

ultrafiltration studies are in agreement with studies by the manufacturer

(Millipore) since the process are very straight forward especially in the process of

separation of desired products from the UF TFF microcogent scale by using 3 and

10 kDa membrane.

III. Significant difference (P<0.05) between samples and negative control (no

antioxidant) of fish waste water soluble hydrolysates can be observed in both

Atlantic Mackerel and mixed fish in the measurement of lipid oxidation inhibition

activity by FTC and TBARS, thus there will be increase in DPPH scavenging activity

with the extract concentration for both samples. Reducing power and antioxidant

activity is proportional to one another.

IV. The DSC thermograms of fish waste samples (Atlantic Mackerel, mixed fish, 10kDa

Atlantic Mackerel and mixed fish protein hydrolysates) can be compared. There is

a significant difference (P<0.05) between the samples in terms of enthalpy

changes in myosin and actin. The G’ values (energy stored elastically in structured)

shows tougher samples among samples.

V. There will be a significant difference (P<0.05) of proteins in mackerel and mixed

fish waste streams when characterised by Raman spectroscopy in most of their

spectra and assigned peaks.

64

Chapter 2

65

2 Chemical Composition of Atlantic Mackerel (Scomber

scombrus), Nile Perch (Lates niloticus) fillets and Fish waste

(FW)

2.1 Introduction

2.1.1 Fish in general

Fish is an aquatic organism which has fins and breathes using its gills that are located on the

sides of its pharynx. Fish comes in different shapes and sizes. The nutritional content of fish

also depends on intrinsic and extrinsic factors like the living environment, size, age, nutrient

status, and season. The nutrient composition of fish includes protein, fat and oil,

carbohydrate, vitamins, minerals and water.

Fish protein is a high quality protein with a full range of amino acids, and it is therefore

regarded as an important source in human diet. Fish has a high moisture content of about

75- 80 % and enzymes that make it susceptible to biochemical and microbial spoilage

(Makanjuola, 2012)

Two classes of fish are commonly known as fatty and white fish. Fatty fish like salmon,

mackerel, herring and sardines has a high amount of fat that is considered to be beneficial

for human health, as it is high in eicosapentaenoic acid (EPA) and docosahexaenoic acid

(DHA). These polyunsaturated fatty acids also known as omega-3 fatty acids. White fish also

called lean fish e.g. haddock and cod, contains a small amount of fat which includes PUFA.

66

There are two types of fish muscles, known as light and dark muscle. The proportions of

dark muscle is higher in fatty fish compared to the white fish. Lean fish also contains more

light muscle approximately 18-23 % of proteins (Figure 2.1)

(http://www.fao.org/wairdocs/tan/x5916e/x5916e01.htm).

Figure 2.1. The muscles types of white and fatty fish adapted from fao.org

2.1.2 Atlantic Mackerel Fish (Scomber scombrus)

Atlantic Mackerel (Scomber scombrus) are a pelagic species of fish that usually stay offshore.

The young ones usually live in the area of the water surface down to 150 – 180 ft. They live

in mainly two areas of Mid Atlantic Bight Ocean and Gulf of St Lawrence, in Western North

Atlantic Ocean. They move in a large shoals or schools, and the life span is up to 20 years old.

Atlantic mackerel usually eat small fish and squid, also planktons and crustaceans like

shrimp and krill (Studholme at al., 1999).

67

Atlantic mackerel are known for their distinct strong flavour; they are a grey and oily flesh,

and will turn white moist and flaky when cooked. The size of this fish is around 16 inches

long and weight up to 2 pounds. The skin is metallic blue- green with silver sides, large head,

mouth and fins as shown in Figure 2.2.

Figure 2.2 Atlantic Mackerel (Scomber scombrus). Image adapted from Energy and

Environmental Affairs (EAA) website:

(http://www.mass.gov/eea/agencies/dfg/dmf/recreational-fishing/atlantic-mackerel.html)

2.1.3 Nile Perch Fish (Lates niloticus)

Nile Perch Fish (Lates niloticus), a large sized palatable bone-free white flesh freshwater fish

species can be found in most lakes and rivers of central Africa. They are silver with blue

tinge in colour, and have distinctive dark eyes and bright yellow outer ring. This type of fish

can reach more than two metres and, weigh approximately 200 Kg, and are known as the

largest freshwater fish. They feed on fish, crustaceans and insects, as well as zooplankton

(Azeroual et al., 2010)

68

The processing of this fish must meet the requirements specified by the EU hygiene directive

(91/493/ EEC). After washing and sorting, they are kept chilled in ice for further processing.

They are then filleted manually by hand without gutting, followed by filleting, skinning and

trimming. The fillet sizes may vary from 300 g to 1500 g. Large fillets are usually portioned

and kept frozen because of quality assurance for premium fresh market.

By-products of Nile Perch form approximately 50 % of total fish mass. The fish usually grow

very quickly after hatching, and they are caught for food or eaten by other fish before they

grow bigger in size. The health benefits of this fish includes high omega 3 fatty acids, no

saturated fat, and low sodium and sugar content (Peche foods, 2014)

Figure 2.3 Nile Perch Fish (Lates niloticus) adapted from Peche Food Company website:

(www.Peche foods.com)

69

Table 2.1 Nutrient composition of Nile perch fish after various processing methods

(Kabahenda et al., 2011)

70

2.1.4 Fish waste

Solid fish waste is a by-product of the fish processing industry and consist of the head, tails,

skin, gut, fins and frames and can be a good source of value added proteins, amino acids,

gelatin and enzymes. Dried fish waste has high protein content (58 %), 19 % of fat and

minerals (Ghaly et al., 2013).

Figure 2.4 Fish waste in fish processing (filleting)

Table 2.2 Nutritional and mineral composition of fish waste (FW), mean ± standard

deviation on a dry matter basis (Esteban et al., 2007).

Nutrient (% or ppm)

Crude protein 57.92 ± 5.26

Ether extract 19.10 ± 6.06

Crude fibre 1.19 ± 1.21

Ash 21.79 ± 3.52

Ca 5.80 ± 1.35

P 2.04 ± 0.64

K 0.68 ± 0.11

Na 0.61 ± 0.08

Mg 0.17 ± 0.04

Fe (ppm) 100 ± 42

Zn (ppm) 62 ± 12

Mn (ppm) 6 ± 7

Cu (ppm) 1 ± 1

71

The main aim of this study is to evaluate the chemical composition of fish fillets Atlantic

Mackerel Fish (Scomber scombrus) and Nile Perch Fish (Lates niloticus), and compare their

nutritional value to the fish waste obtained from the fish processing industry.

2.2 Materials and Methods

2.2.1 Samples and Materials

Fish fillets (Atlantic Mackerel) and fish waste (FW) samples from the fish industry

processing were purchased from The Fish Society, Wormley, Surrey GU8 5TG, UK. Fish

fillet of Nile Perch was supplied from Peche foods, Kenya. The fish samples were

despatched in ice directly to the Food Science laboratory. The fish waste samples were

processed upon arrival to the laboratory, weighed and homogenized in an Omni Mixer-

CAMLAB) at 6000 rpm for 30 seconds in ice. All chemicals used were bought from Fisher

Scientific, Loughborough, UK and Sigma Aldrich, Poole, UK. Sodium hydroxide and

hydrochloric acid; also all reagents used for experiments were of analytical grade.

2.2.1.1 Heat Treatment

FW samples were treated with heat at 65 °C in a water bath for 20 minutes to achieve

microbiological quality and avoid the existence of any endogenous enzymes.

72

2.2.2 Methods for proximate analysis of fish fillet (FF) and fish waste (FW)

composition

2.2.2.1 Determination of protein content by Kjeldahl method

The protein content analysis was based on the standard AOAC Kjeldahl procedure (AOAC,

2006), which measures the nitrogen content. The procedure consists of three basic steps.

The first is the digestion of sample in concentrated sulphuric acid, which converts

nitrogen to ammonia using concentrated NaOH, followed by the distillation of ammonia

into a trapping solution, and finally is the titration of nitrogen with a standard dilute acid.

The crude protein was then calculated using the following equation where 𝑁 × 6.25

where N is the nitrogen content.

Fish sample (1 g) was digested in 20 mL of concentrated sulphuric acid (96 %) with two

selenium catalyst tablets (5g K2SO4: 0.15 g CUSO4.5H2O; 0.15 g TiO2) and 1 Kg K2 SO4 by

boiling the mixture in a Tecator Kjeltec apparatus for 2 hours. The digestion of the fish

sample was monitored by the presence of a clear solution, and the sample tubes were left

to cool up to 15 minutes upon completion. During the digestion step, the presence of

nitrogen would be converted to ammonia through a conversion to ammonium sulphate.

0.5 M sodium Hydroxide (20 mL) was added to allow for the release of ammonia by a

distillation step, and the product was collected in a 25 mL of 4 % w/v of boric acid. In the

titration step, the samples collected were titrated against the 0.05 M Hydrochloric acid

and methylene red as an indicator. The total nitrogen (%) and protein content were then

determined using the following equation:

73

𝑵 % =100 × ( 𝑚𝐿 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑡𝑖𝑡𝑟𝑎𝑛𝑡 − 𝑚𝐿 𝑜𝑓 𝑏𝑙𝑎𝑛𝑘 𝑡𝑖𝑡𝑟𝑎𝑛𝑡 ) × 0.0007

𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒

∴ 𝑷 % = 𝑁 × 𝐾 , 𝑤ℎ𝑒𝑟𝑒 𝐾 = 6.25 𝑖𝑠 𝑡ℎ𝑒 𝑓𝑎𝑐𝑡𝑜𝑟 𝑓𝑜𝑟 𝑠𝑒𝑎𝑓𝑜𝑜𝑑 𝑎𝑛𝑑 𝑚𝑒𝑎𝑡 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑠

2.2.2.2 Lipid extraction

2.2.2.2.1 Measurement of Total Lipid by Soxhlet extraction

Lipid content of the fish sample was determined by the Soxhlet extraction method

adapted from Lee et al. (1996). Three portions, each 5 g, of minced fish samples were

placed in a thimble and defatted cotton wool plug was placed on the top of the sample.

The thimbles were then placed into condensers, and set into a boiling position. Boiling

chips were placed in six extraction cups, followed by an addition of 30 mL petroleum

ether (60-80 °C), and boiled for 3 hours. The setting was then changed to a rinsing

position, and the remaining solvent was then collected into a condenser. The weight of

the lipids was determined after the weight of thimbles and cups were recorded.

2.2.2.2.2 Lipid extraction and optimization process of fish oil in fish waste

Lipids were extracted using the modified Bligh and Dyer method (Bligh and Dyer, 1959)

of Norziah et al., (2009). 50 g portions of FW sample was thawed and initially

homogenized with the solvent mixture ratio of chloroform, methanol and water (2:4:1)

for 15 minutes. The homogenized mixture was centrifuged at 3000 x g for fifteen minutes.

74

The fish sample was re- extracted with the same amount of chloroform to obtain higher

recovery of oil. The final mixture was transferred to a separating funnel and the lower

layer containing lipids was collected. The solvent was evaporated off in a rotary

evaporator at 25 °C. The resulting oil was collected and stored in a screw capped bottle,

flushed with nitrogen gas and stored at -80°C for further tests.

2.2.2.2.3 Derivation of fatty acid methyl esters (transesterification) in fish waste sample

The method used for this study was adapted from Saeed and Howell (1999). Fish oil

(100mg) was transferred into 10mL glass fitted screw cap and 2 mL of sodium methylate

diluted with tert-butyl methy ether (MTBE) in 4:6 v/v ratio was added and vortexed for

1 minute. The tube mixture was then wrapped using aluminium foil and left in a dark

room for one hour. 2 ml of distilled water and 5 ml chloroform were added and vortexed

for a minute, centrifuged at 3000 x g for 5 minutes. The mixture was left to stand and the

upper layer was removed and discarded using a Pasteur pipette. 2 ml of 1 % citric acid

solution was added to the bottom layer, in order to neutralize the alkali. The mixture was

vortexed for 1 minute and centrifuged for 5 minutes at 3000 x g to allow for phase

separation. The upper aqueous phase was then removed using a Pasteur pipette and the

lower chloroform layer was evaporated under a stream of nitrogen in a warm water bath

set at 37 °C. The sample preserved for further analysis.

2.2.2.3 Determination of moisture content

The moisture content was determined by measuring the weight of sample before and

after drying by oven, until a constant values obtained (AOAC, 2006). A moisture dish was

75

oven dried at 100 °C for 30 minutes and cooled in a desiccator, the mass of the dish was

then recorded. 5g of fish sample was then placed on the dried moisture dish and was kept

in an oven overnight at 100 °C. The moisture dish was then removed from the oven,

allowed to cool in the desiccator, and the mass was recorded. This method was done in

triplicate. The moisture content was calculated using the equation as below:

% 𝑴𝒐𝒊𝒔𝒕𝒖𝒓𝒆 =𝑊𝑒𝑖𝑔ℎ𝑡 𝑙𝑜𝑠𝑠 𝑜𝑛 𝑑𝑟𝑦𝑖𝑛𝑔 𝑎𝑡 100℃

𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 × 100

2.2.2.4 Measurement of ash

Ash content was determined by measuring the weight of a dried sample before and after

heating process in a muffle furnace (AOAC, 2006). The ash was measured by placing

approximately 6 g of fish muscle into a silica dishes in triplicate. Samples were heated on

a hot plate for 10 minutes to remove moisture and heated in a muffle furnace overnight

at 550 °C. The samples were kept in a desiccator and allowed to cool. The masses were

recorded, and the total ash content was calculated using the equation below:

𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝒂𝒔𝒉 (𝒈) = (𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑖𝑙𝑖𝑐𝑎 𝑑𝑖𝑠ℎ 𝑤𝑖𝑡ℎ 𝑎𝑠ℎ ) − (𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑖𝑙𝑖𝑐𝑎 𝑑𝑖𝑠ℎ)

2.2.3 Statistical analysis

All analyses were conducted on triplicate samples. Values are reported as the average

and standard deviation of measurements on fish fillet and fish waste samples.

Statistical test were performed by SPSS package version 16 to analyse data. One way

analysis of variance (ANOVA) was carried out. Differences between pairs of means were

assessed on the basis of confidence intervals by using Least Significant Difference (LSD)

test, followed by the T-test. The level of significance was considered at (P <0.05)

76

2.3 Results and Discussion

2.3.1 Proximate analysis of fish fillets; Atlantic Mackerel (Scomber scombrus) and

Nile Perch (Lates niloticus) and fish waste

Table 2.3 The chemical composition of fish fillet (FF) Atlantic Mackerel (Scomber

scombrus) and Nile Perch (Lates niloticus) obtained from study compared to fish samples

by standard method adapted from Murray et al., in FAO ( 2001) and Okeyo et al., 2009.

Fish Type (fillet) Moisture

(%)

Total

lipids (%)

Protein

(%)

Ash (%)

Atlantic Mackerel

(S.scrombus)

70.3 ± 1.03 11.3±0.18 19.3±0.35 1.1±0.25

Nile Perch ( L. niloticus) 80.4 ± 1.07 1.9±0.21 16.3±0.55 0.5±0.33

Fish proximate analysis by standard method Murray et al., in FAO ( 2001)

and Okeyo et al., 2009

Atlantic Mackerel

(S.scrombus)

60-74

(±1.8)

1.0-23.5

(±1.6)

16-20

(±0.5)

1.1

(±0.1)

Nile Perch ( L. niloticus) 79 ±0.37 -

80±0.21

0.59 ±0.01

-0.63±0.05

17.6±0.2-

19±0.2

0.55±0.05-

0.63±0.05

% on wet matter basis. Each values is expressed as mean ± SD (n=3) of triplicate measurements.

From the results obtained in fish fillet, although Nile Perch is known as a low value fish

product, the chemical composition shows that it contains 80.4 % moisture a slightly

higher values compared to the Atlantic Mackerel with 70.3 % moisture as well as 1.9 %

of total lipids, and 16.3 % of protein and 0.5 % of ash. Atlantic Mackerel from this study

has higher total of lipids (11.3 %), protein (19.3 %) and ash (1.1 %) compared in Nile

Perch.

77

The decreased in total crude protein content of fish flesh is usually associated with the

decrease in the water and salt soluble protein. Besides that, it also could possibly due to

an autolytic deterioration associated with the actions of endogenous enzymes and

bacteria (Hultman et al., 2004). Further biochemical analysis can be conducted such as

pH and Total volatile basic nitrogen (TVBN) and determination of bacterial count. The

increase in total bacterial count is usually due to the proteolytic breakdown of protein

molecules to release volatile nitrogen compounds determined as TVBN (Okeyo et al.,

2009).

Atlantic Mackerel is known as an oily fish and this was confirmed with the high % lipid

content (11.3 %) compare to the Nile Perch (1.9 %). Difference fish compositions usually

depend on the type of fish, sex, age, nutritional state, time of year as well as health

conditions (Ghaly et al., 2013).

Food and Agriculture Organization of the United Nations (FAO) summarized the chemical

composition of fish flesh of Atlantic Mackerel and Perch fish as shown in Table 2.3. The

Nile Perch fish proximate analysis was adapted from Okeyo et al., 2009. The proximate

analysis of Atlantic Mackerel obtained in this study shows that the values were in the

range obtained in previous studies.

78

Table 2.4 Proximate analysis of fish waste (FW) from fish processing industry. The

values are the means of three samples ± standard deviation. Values in (%) on a wet matter

basis, each values is expressed as mean ± SD (n=3) of triplicate measurements.

Figure 2.5. The chemical composition of fish waste (FW) on a % wet matter basis

As for the fish waste (FW) composition, the moisture content obtained was 60.13 % ±

2.15. Higher percentage of total lipids (6.12 %), protein (24.31 %) and ash (9.74 %)

obtained compared to in the Atlantic Mackerel and Nile Perch FF

Proximate Analysis (%)

Total lipids 6.12 ±3.06

Protein 24.31±3.26

Ash 9.74 ±3.51

Moisture 60.13 ± 2.15

79

Ash content was higher in FW (9.74 %) compared to FF may due to the presence of

inorganic components of the sample e.g. bone. The solid fish waste usually consist of by

products such as; heads, frames, tails and some flesh. FW is also known for its value added

source as it contains high protein, minerals, and essential fatty acids (Moini et al., 2012).

The chemical content obtained in FW were in agreement with the previous study and

experiments using similar FW samples (Esteban et al., 2007 and Grande, 1998).

2.3.2 Composition of lipids present in Fish waste (FW) sample

Almost 50 % of the body weight which generated as fish waste by fish processing. FW can

either be used for human consumptions or in biodiesel production as it consists of 2-30

% good quality fish oil. Fish oil obtained from a fish processing by products generally

depends on the fat content of specific fish species (Ghaly et al., 2013). Omega-3 fatty acids

eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), beneficial for health, can

be found mainly in marine animals that have high polyunsaturated fatty acids (PUFA)

content (Zuta et al., 2003)

Fatty acids composition of the fish waste sample is presented in Table 2.5.

Monounsaturated fatty acids (MUFA), also beneficial for health, constitute the majority of

fatty acids pool (40.75%), followed by saturated fatty acids (SFA) with 31.56 % and 21.18

% polyunsaturated fatty acids (PUFA). Esteban at al., 2007 and FEDNA, 1999 found that

the most abundant fatty acids in FW were from the oleic acid C18:1 and palmitic acid

C16:0. The same trend of fatty acids components was also obtained in this study of fish

waste sample where oleic acid constituted 24.7 % followed by 21.8 % of palmitic acid.

14.35 % of fatty acids came from docosahexaenoic acid (DHA) after oleic acid and

palmitic acid; was in agreement with previous studies reported by Khoddami et al. (2012)

80

on tuna fish waste sample; Garcia-Arias et al. (2003) on sardine fish fillet sample; and

Osman et al. (2001) on lean fish sample. Thus FW could be used as an alternative source

to extract the fish lipid, and the advantages of the lipid from the fish waste will be a much

cheaper fish oil supply, compared with the lipid extraction from flesh.

Table 2.5 Composition of lipids present in fish waste (FW)

Assignment Peak FW (% of fat)

Lauric acid C12:0 0.07 (± 0.01)

Myristic acid C14:0 5.38 (± 0.26)

Palmitic acid C16:0 21.83 (± 1.07)

Stearic acid C18:0 3.9 ± (0.51)

Arachidic acid C20:0 0.38 ± (0.02)

Total saturated fatty acid = 31.56

Palmitoleic acid C16:1 7.22 (± 0.46)

Margaric acid C17:0 0.76 (± 0.1)

Magroleic acid C17:1 0.52 (± 0.005)

Oleic acid C18:1 24.73 (± 0.47)

Gadoleic acid C20:1 7.52 (± 0.042)

Total mono unsaturated fatty acid = 40.75

Linoleic acid C18:2 4.03 (± 0.51)

Linolenic acid C18:3 1.37 (± 0.83)

Eicosapentaenoic

acid (EPA)

C20:5 1.43 (±0.05)

Docosahexaenoic

acid (DHA)

C22:6 14.35 (±0.92)

Total polyunsaturated fatty acid = 21.18

Each values is expressed as mean ± SD (n=3) of triplicate measurements.

81

In comparison with fish fillets sample such as Atlantic Mackerel and Nile Perch, PUFA

constitute most of fatty acids in Atlantic Mackerel fish followed by SFA, and MUFA.

Palmitic acid (C16:0) was the major fatty acid followed by stearic acid (C18:0) and

myristic acid (C14:0). PUFA content contributed the biggest part in Mackerel lipid profile

with EPA (C20:5) and DHA (C22:6) (Guizani et al., 2015). A study on the fatty acid

composition of Nile perch by Kwetegyeka et al., 2006 found that palmitic acid (C16:0),

stearic acid (C18:0) and oleic acid (C18:1n9) were the most abundant individual fatty

acids detected from the muscle and heart tissues of Nile perch. From both fish type as

well as fish waste, palmitic acid was among the most abundant fatty acids and this is

because palmatic acid is known to be the key metabolite in fish species (Guizani et al.,

2015).

2.3.3 Minerals composition in Atlantic Mackerel (Scomber scombrus) and Nile

Perch (Lates niloticus) and fish waste (FW) based on literature.

Fish is one of the sources of the diet, which provide a well-balanced supply of essential

minerals in a readily usable form.

A high amount of ash content in fish waste may due to a largely number of different

minerals from bone in the FW sample thus indicating that fish waste is a rich source of

minerals. A study on minerals content of fish waste against fruit-vegetable waste (FVW)

reported Ca, P, K, Na and Mg as well as Fe, Zn, Mn, and Cu minerals; FW was a richer

source of minerals than the FVW (Table 2.2). The most abundant minerals were P

(2.04%), Fe (100 ppm), Zn (62 ppm) and Ca (5.8%) (Esteban et al., 2007).

82

As for a minerals composition in Atlantic Mackerel (Scomber scombrus), a similar

distribution was reported in flesh and its body parts except for the calcium is usually

highly concentrated in the bones (Makanjuola, 2012).

In contrast, the chemical composition of Nile perch (Lates niloticus) in Table 2.1, fillet was

reported to contain lowest amount of iron (1.06 mg per 100g dry matter), zinc (0.72 mg/

100 g) and calcium (134.24 mg/100 g dry matter) although the zinc and calcium amounts

were not significantly different from other low –value fish products.

2.4 Conclusion

Both Atlantic mackerel and Nile Perch contain substantial amounts of protein, lipids and

minerals which make them very desirable food sources. FW contains high protein and

fat, as well minerals and could be used as an alternative to fish flesh for the supply of oil

and valuable protein. However, the heat treatment is a necessary step in this study to

avoid microbial growth, and maintain the nutritional quality and safety of FW (Sancho et

al., 2004).

83

Chapter 3

84

3 Production of hydrolysate and ultrafiltration studies to

obtain water soluble proteins fraction (3 and 10 kDa) from

fish waste stream

3.1 Introduction

There are three main fish components known as muscle (flesh), skin and waste (roe,

trimmimgs, head, fins, viscera, and frames) which known as a major sources of fish

protein hydrolysate. Fish waste are usually untreated without many people knowing that

they contain high protein components which can be converted into fish protein

hydrolysates thus benefited in producing high added value food products (Halim et al.,

2016; Benhabiles et al., 2012)

Fish waste protein hydrolysate can be produced by various different methods such as;

enzymatic hydrolysis, thermal hydrolysis, bacterial fermentation and autolysis (Halim et

al., 2016). The most common method used in producing fish protein hydrolysate is by the

enzymatic hydrolysis. This method break down fish protein by specific enzymes

producing soluble and insoluble fractions a reported by Molla and Hovannisyan (2011)

as cited in Halim et al. (2016). The first step in enzymatic hydrolysis is homogenizing and

heating sample at 85-90° C for 20 minutes in order to remove the activity of endogenous

enzyme (da Rosa Zavareze et al., 2014 and Wang et al., 2013). Parameters such as enzyme

concentration, temperature, pH value and time are the key components during

hydrolysis, and they can affect the properties of hydrolysate produce (Srichanun et al.,

2014). Jamil et al. (2016) reported that as the numbers of broken peptide bonds

increased, the enzyme concentration and temperature will also increase until an

85

optimum point when the enzyme starts to denature. Therefore, a pH value will be

adjusted using 1N or 2N sodium hydroxide (NaOH) or hydrochloric acid (HCl) for the

unfolding conformation of native protein in order to protect its specific sites from

enzymatic hydrolysis. The enzyme activity is terminated by heating process at 85-95°C

for 5-20 minutes, and this marked the end of hydrolysis process (Intarasirisawat et al.,

2014).







The main principle of Ultrafiltration (UF) is to concentrate large molecular weight

components without the application of heat or a change of phase. Those large MW

components will be rejected by the membrane, while the low molecular weight

components needed (permeate) will produce a concentration the same as in the feed.

UF is known as one of a membrane separation method which uses pressure-activated

process in the range between 1-5 bars. Besides concentrating macromolecules such as

proteins and starches, UF is also used in the separation of components in food processing

waste streams and broth fermentation for example in dairy, fish and seafood industries.

However, a major problem is concentration polarisation and membrane fouling that can

limit effective UF processing (Lewis, 1996)

86

3.1.1 The Recovery of Protein by Membrane Separation Technique

Filtration is one of separation processes which is pressure driven and separates liquid

solution or suspension components via membrane based on their size and differences in

charge. There are two types of filtration in general, known as Normal Flow Filtration

(NFF) and Tangential Flow Filtration (TFF) (Merck Millipore, 2014). The subdivision of

tangential flow filtration processes is also described in Table 1.1.

The Normal Flow Filtration (NFF) works on the basic filtration process where the

pressure is applied directly to the fluids which then move towards the membrane. Large

particles that are too large to pass the pores of membrane will accumulate at the surface

of the membrane while smaller molecules will pass through to the downstream side. This

separation method is also known as dead-end filtration. In other words, it is known as

NFF since the fluid flow occurs in the direction normal to the membrane surface, so NFF

is a more descriptive and preferable name. Applications for this type of filtration includes

sterile filtration of clean streams, clarifying pre-filtration, and virus / protein separations

(Dalwadi et al., 2005 and Merck Millipore, 2014).

As the name implies, Tangential Flow Filtration (TFF) occurs when the fluid is pumped

tangentially along the membrane surface. Applied pressure is used to force a portion of

the fluid through the membrane to the filtrate side. The principle after that is just the

same as NFF but differs as the retained components do not build up at the surface of the

membrane. They will be swept along by the tangential flow. This feature therefore makes

it a perfect process for linear sized based separations. TFF is also known as cross-flow

filtration, whereby the tangential flow describe the direction of the fluid flow relative to

the membrane (Dalwadi et al., 2005).

87

Figure 3.1. Normal Flow filtration and Tangential Flow Filtration (Merck Millipore,

2014)

Table 3.1.The subdivision of tangential flow filtration processes (Merck Millipore,

2014)

88

3.1.2 Microfiltration (MF)

Microfiltration (MF) has membrane cut offs which falls in the range of 0.05 µm to 1.0 µm

(Merck Millipore, 2014). The separation involves intact cells and some cell debris in the

feed tank. Either the retained cells or the clarified filtrate can be the product stream

(Russell et al., 2006 and Shukla et al., 2006).

3.1.3 Ultrafiltration (UF)

Ultrafiltration (UF) is also used regularly in a TFF form, and separates proteins from

buffer components for buffer exchange, desalting, or concentration (Rathore et al., 2011).

The range for this type of membrane usually depends on the retained protein thus 1 kDa

to 1000 kDa are used (Merck Millipore, 2014).

3.1.4 Nanofiltration (NF)/ Reverse Osmosis (RO)

Reverse Osmosis (RO) and Nanofiltration (NF) are type of TFF that use very tight small

pore membranes in order to separate salts and small molecules (Merck Millipore, 2014).

A molecular mass which is lower than 1500 Da is also required for water and other

solvents separation. Thus ≤ 1 kDa cut offs membranes are used (Walter et al., 2011)

3.1.5 Diafiltration (DF)

To promote a product yield or purification, diafiltration can be implemented with any

other types of separation (Jungbauer, 2013 and Dalwadi et al., 2005). In the process of

diafiltration, removal of filtrate will take place together with a recirculation of buffer. In

the case of the retentate, components wash off from the product by the DF, into the

89

filtrate, so that buffer exchange occurs, thus lowering the chance of concentrating of

unwanted substances. As the product attaches to the filter DF washes it down the

membrane into a collection vessel (Dalwadi et al., 2005; Campbell, 2012 and Walter et al.,

2011).

The aim of this study was to obtain soluble proteins from fish waste by ultrafiltration

using the cogent µscale TFF system of UF unit shown in Figure 3.2.

Figure 3.2. The cogent µscale TFF system used for separation process in this study

90


3.2.1 Production of Hydrolysate

3.2.1.1 Materials

Pepsin from porcine gastric mucosa and pancreatin from porcine pancreas and all

chemicals used were bought from Fisher Scientific, Loughborough, UK and Sigma Aldrich,

Poole, UK. Sodium hydroxide and hydrochloric acid; also all reagents used for

experiments were of analytical grade.

Water soluble fish protein was obtained from The Fish Society, Wormley, Surrey GU8

5TG, UK. The fish waste samples were usually despatched in ice directly to the Food

Science laboratory, processed upon arrival to the laboratory, weighed and homogenized

in an Omni Mixer-CAMLAB) at 6000 rpm for 30 seconds in ice.

3.2.1.2 Method

The method used was according to Megias et al., (2004), the powder form of the water

soluble fish protein was mixed with mili-Q water (ultrapure water; 0.22 µm membrane

filter from Merck Millipore Corporation, USA) in the ratio (1:10 w/v) and homogenized

(omni mixer homogenizer, USA). The pH was adjusted to pH 2.0 using 1M HCl. Then,

pepsin was added to the suspension with substrate/enzyme ratio (20:1). After 3 hours,

the pH of the mixture was adjusted again to pH 7.5 using 1M NaOH. Subsequently,

pancreatin was added using the same ratio and time of digestion as described for pepsin.

In order to deactivate the reaction of enzymes, the mixture was submerged in boiling

water for 20 minutes. Then, after cooling, the sample was filtered and lyophilized for

further study.

91

3.2.2 The effect of Transmembrane pressure (TMP) on Flux Excursion

The method for measuring the effect of Transmembrane pressure (TMP) on the flux

Excursion was adapted from Merck Millipore, 2014. First, mili-Q water (Merck Millipore

Corporation, USA) was used to run the cassette for the first time and the pump was set to

be 30 RPM (0.5 Hertz), follow by 25 RPM (0.42 Hertz). The pinch tube was carefully open

entirely and the transmembrane pressure was carefully maintained (pressure drop was

monitored to always be between 5 psi -14 psi (34.47-96.52 kPa). A few drops of permeate

was carefully monitored and the pinch was closed but not entirely, while the pump was

increased to 32 RPM (0.53 Hertz). The concentration of feed was measured, and both

retentate and permeate was used to measure protein concentration by

spectrophotometer at 595nm by Bradford method, which the measurement was based

on the absorbance values of dye coomassie blue under acidic condition(red form) to turn

into blue (the binding to preotein which been assayed). The steps were then repeated in

following order:

First with 100 ml of water, followed by the product (fish sample), once again with 50 ml

water, 50 ml of 0.5 N Sodium hydroxide in both retentate and permeate, water was then

flushed out the device tank and membrane until neutral pH obtained and finally the tank

was filled with 25 ml of 0.1 N sodium hydroxide for storage.

In order to ensure a continuous mixing, mixing step and pressure were monitored

throughout the process.

92

3.2.3 Protein Performance Scalability Study

The method of this experiment is adapted from the standard method of Protein

Performance Scalability by the Millipore Company. Process parameters in this study refer

to key process variables; solution temperature; inlet, outlet and permeate pressures; as

well as permeate and retentate flow rates. The membrane device used in this study is

Pellicon 3 Ultracel 3 kDa C Screen, and active membrane area of used in this study is

88cm2

Process parameters were first measured alongside with buffer recirculation in a 10 psi

(68.94 kPa) retentate pressure and a sequence feed side pressure drops of 3, 6, 12, and

18 psi (20.68, 41.37, 82.74, and 124.12 kPa). A retentate pressure of 20 psi (137.9 kPa)

with a 295 LMH mean cross flow rate were used for the recirculation of the maximum

concentration of protein solution, and the process parameters were recorded at regular

intervals of time. The performance scalability was maintained throughout the study by

the recirculation step. Flux vs TMP graph curves were then generated for 10, 20 and 40

g/L fish waste and BSA solutions. Average cross flow rate was maintained at 5 l/min/ m2

during the run. To ensure a uniform mixing, diluted solution was formed and allowed to

recirculate in a minimum of 30 minutes between the TMP excursions. Feed and permeate

samples obtained during each run were analysed using UV spectrophotometry. The

system was then drained, followed by the analysis of fouled hydraulic performance if any,

compared to the membrane’s initial cleaned condition. A cleaning routine was done in a

final step by recirculating 0.1 N of NaOH for 30 minutes, drained, flushed and loaded with

buffer.

93

3.2.4 Hold up volume of Membrane

3.2.4.1 Material

Fish waste protein, Pellicon 3 cassettes with C screen in both 3 and 10 kDa Ultracel

membrane, with 88 cm2 size.

3.2.4.2 Method

The method was adapted from the Standard Method of Hold up Volume of Cassettes by

Merck Millipore. The Membrane device was torqued according to Merck Millipore

specifications at 190 in-lb (21.47 newton-meter), before flushed with RO water. The

device was then filled with water, and removed from the holder to weigh. The feed

channel was drained and residual water was blown down at 5 psi (34.47 kPa) for 10

minutes. The device was removed from the holder and weighed, while the permeate

channel was drained with blowing down of residual water at 5psi (34.47 kPa) for 10

minutes. After the permeate channel was flushed down with water, the device was

removed from the holder and weighed. The steps were repeated using the protein

samples instead of water.

94


3.3.1 Production of Hydrolysate

Enzymatic hydrolysis of protein is one of the most important options to modify the

functional and nutritional properties of proteins and peptides (Korhonen et al., 1998).

Protein hydrolysates for both samples were produced by treatment with enzymatic

digestion using pepsin and pancreatin in order to mimic the digestive process in the

human body and achieve a high degree of hydrolysis that is greater than that produced

when using either pepsin or pancreatin alone. It is recognized that the degree of

hydrolysis determines the application of protein hydrolysates.

Figure 3.3 Water-soluble fish protein hydrolysate of Atlantic Mackerel (on the left) and

mixed fish (on the right)

95

3.3.2 The effect of Trans membrane pressure (TMP) on Flux excursion

Figure 3.4 TMP Excursion at two feed flows (Merck Millipore, 2014)

This work was undertaken to examine the effect of TMP on flux excursion. Figure 3.4

shows that the flux rate increased as the TMP increased thus affecting the feed flux (pump

speed). The optimum point was obtained by calculating the optimum feed flow against

the required pump rate at each of the two feed flow conditions. This is because the

appropriate combination of feed flow rate and TMP maximizes the impact of pumping

and shear on the product, thus minimizing the processing time and/or membrane area

(Ng et al., 1976). This method is usually used for maintenance purposes in order to

determine fouling of membrane before replacement.

96

3.3.3 Protein Performance Scalability Study

The main objective of the study was to measure the scalability of Pellicon 3 cassete using

a selected membrane cut off (3 and 10 kDa) and in different protein stream

concentrations (10, 20 and 40 g/L fish waste stream (FWS) and BSA protein solutions

(model system).

The study was evaluated using sulfhydryl modified bovine serum albumin in pH 7.1

phosphate buffer saline, considering protein flux performance and mass transfer

comparability. The protein performance study consisted of transmembrane protein

excursions with three protein concentrations.

Figure 3.5. General diagram of the system adapted from the Merck Millipore Membrane

Performance Guide, 2014

97

3.3.3.1 Protein Flux Performance





40 g/L BSA

TMP (psi) Flux (LMH)

10 37.86±0.02

20 70.12±0.01

30 103.71±0.01

40 111.24±0.01

50 128.45±0.02

60 136.12±0.01

70 136.11±0.04

0

50

100

150

200

250

10 20 30 40 50 60 70

FL

UX

(L

MH

)

TMP (PSI)

Permeate f lux (LMH) vs Transmembrane pressure (psi) in 3

Kda Membrane with BSA

40 g/L BSA 20 g/L BSA 10 g/L BSA

98

20 g/L BSA


10 47.01±0.12

20 96.81±0.10

30 103.44±0.13

40 141.73±0.01

50 149.32±0.21

60 161.27±0.13

70 161.2±0.02

10 g/L BSA


10 63.22±0.13

20 101.32±0.22

30 141.21±0.14

40 163.44±0.23

50 197.11±0.21

60 205.46±0.16

70 205.4±0.03




0

50

100

150

200

250

0 10 20 30 40 50 60 70 80

FL

UX

(L

MH

)

TMP (PSI)

Permeate f lux (LMH) vs Transmembrane Pressure (psi) in 10

Kda Membrane with BSA

40 g/L BSA 20 g/L BSA 10 g/L BSA

99



40 g/L BSA

TMP (psi)

Flux (LMH)

10 45.73±0.11

20 83.52±0.04

30 110.62±0.14

40 124.33±0.12

50 137.42±0.08

60 142.29±0.06

70 142.3±0.03

20 g/L BSA

TMP (psi)

Flux (LMH)

10 48.52±0.04

20 95.67±0.12

30 112.89±0.12

40 150.2±0.22

50 149.2±0.25

60 173.42±0.12

70 173.41±0.22

10 g/L BSA

TMP (psi)

Flux (LMH)

10 73.22±0.15

20 110.32±0.14

30 151.21±0.14

40 173.44±0.03

50 200.11±0.16

60 223.46±0.38

70 223.4±0.52


Figure and table above shows the permeate flux of Pellicon 3 Ultracell of 3 and 10 kDa

membrane through series of different BSA protein concentrations where different TMP

applies. The BSA was used as a protein model system as recommended by the Millipore

Merck company for evaluating the flux performance. The permeate flux increased along

100

with the TMP when the concentration of protein was reduced. The standard error for all

three trend lines showed a good agreement.

For both different cut off membranes, full polarization occurred in 20 and 40 g/L of BSA

solutions, compared to the 10 g/L of solution. Although the lowest concentration showed

lower polarization, the data results can be used for a further mass transfer analysis.

Smaller membrane cut off gave smaller flux rate, thus higher concentration polarization

because of the accumulation of retained solutes on the membrane wall (Berg and

Smolders, 1990). Besides that, membrane fouling might have occurred, which resulted in

the low flow rate using smaller membrane cut off.

These observations resulted in membranes size performance. A smaller cut off (3 kDa)

resulted in a lower flux rate, and higher limiting flux performance at series of TMP

excursions, compared to the 10 kDa membrane. This is because the limiting flux is known

to occurs when an increase in TMP permeate flux is not significantly affected by the

difference in pressure, resulting the levels off to almost constant values (Wijmans et al.,

1984).

The protein performance in both membranes also showed a good consistency. Since the

membrane used in this study was 88 cm2 , it is also known for the variability within a

range of ±10 % range comparing to other type of membrane because of a single feed

channel known for incompetency of equate multiple channel cross flows ( Millipore,

2014).

A constant pressure recirculation and also series of TMP runs are two main important

parameters to observe in this study. The mean cross flow rate was controlled to 295 LMH

(5 L/min/m2), and each run operated at a temperature of 25°C after limiting flux was

normalized. This study evaluated the inclusive protein performance thoroughly in Flux

101

against TMP curve lines, no data corrections were tabulated in order to standardize the

points around specific TMP.

This trend of results in all three concentrations agree with the standard protein Flux

performance analysis, and proves that the device used for the study meets the

requirement set by the manufacturer. The standard method used a ± 10 % range and a

0.11 m2 device as a baseline.



0

50

100

150

200

250

10 20 30 40 50 60 70

FL

UX

(L

MH

)

TMP (PSI)

Permeate Flux (LMH) vs Transmembrane pressure (psi) in 3Kda membrane with

FWS protein

40 g/L Fish waste protein 20 g/L of fish waste protein 10 g/L fish wate protein

102



40 g/L BSA


10 45.77±0.53

20 81.97±0.44

30 106.31±0.13

40 114.26±0.32

50 140.42±0.15

60 138.12±0.15

70 136.14±0.75

20 g/L

BSA


10 36.17±0.75

20 87.56±0.52

30 91.53±1.32

40 142.43±0.13

50 137.42±0.55

60 165.79±0.74

70 155.87±0.33

10 g/L

BSA


10 66.32±1.53

20 101.64±0.12

30 145.3±0.02

40 162.12±0.24

50 193.35±0.13

60 214.19±0.11

70 214.12±0.09


103

Figure 3.9 Permeate flux of 10 kDa membrane at different transmembrane pressures in

10, 20, and 40 g/L FWS protein solution

Table 3.5 Permeate flux of 10 kDa membrane at different transmembrane pressures in

10, 20, and 40 g/L FWS protein solution

40 g/L BSA

TMP (psi)

Flux (LMH)

10 46.55±0.55

20 82.13±0.12

30 108.74±0.15

40 124.22±0.12

50 136.12±0.01

60 142.11±0.01

70 146.32±0.05

20 g/L BSA

TMP (psi)

Flux (LMH)

10 47.65±0.11

20 94.13±0.03

30 113.76±0.05

40 151.22±0.09

50 149.73±0.03

60 175.21±0.01

70 175.07±0.11

050

100150200250

10 20 30 40 50 60 70FL

UX

(L

MH

)

TMP (PSI)

Permeate Flux (LMH) vs Transmembrane pressure (psi) in 10 Kda membrane with

FWS protein

40 g/L Fish waste protein 20 g/L fish waste protein 10 g/L fish waste protein

104

10 g/L BSA

TMP (psi)

Flux (LMH)

10 74.12±1.04

20 110.32±0.13

30 154.23±0.02

40 173.44±0.16

50 201.23±0.66

60 225.67±0.03

70 224.13±0.02


Based on the results obtained in Figure 3.8 and 3.9, same trend was observed in both 3

kDa and 10 kDa of fish processing waste streams (FWS) protein, where the flux increased

along the difference in Trans membrane pressures, in three difference concentrations

(10, 20 and 40 g/L protein samples). The limiting flux was observed at 60 psi.

Comparing the size of the membrane cut off, 3 kDa membrane showed a smaller flow rate

compared to the 10 kDa membranes; both membranes were treated with the same three

concentrations. Full polarization trends were evaluated as in the BSA protein model

systems but the values for all data were significantly lower in fish waste proteins

compared to BSA.

Furthermore, the flow rate of FWS samples through both membrane were higher than in

BSA sample solutions. The same operating temperature of 25°C after limiting flux was

normalized and mean cross flow rate was controlled to 295 LMH (5 L/min/m2).

From the results obtained, although higher values of flux were obtained compared to the

BSA solutions, fish processing waste streams protein solutions also showed a potential

performance scalability. Moreover, the membranes used in this study showed good

performance and membrane fouling slowly increased through the run when exposed to

the proteins. Overall, since the recirculation was normalized at a retentate pressure of 20

105

psi for one hour, this step ensured that the membrane fouling did not affect the

performance of membrane.

As stated by Merck Millipore, the manufacturer recommended the importance of micros

to screen and examine the initial process parameters, as long as the performance has

been confirmed on 0.11m2 scale prior to full scale up.

Both BSA and FWS protein solutions showed the same trend of results obtained as in the

Merck Millipore standard method of protein performance scalability. An appropriate

membrane cut-off in a different protein stream indicates that the ability of the Pellicon 3

membrane and ultrafiltration device used in this study shows a good agreements of

protein performance.

From the study, FWS protein has a very good potential to be used in different types of

ultrafiltration studies for the isolation of valuable proteins from fish waste streams.

Further experiments should be conducted to discover more benefits of membrane

separation of this type of protein and a long term applications to the field.

3.3.3.2 Mass Transfer Analysis

Mass transfer coefficient is one of the parameters which represents the protein flow rate

from a polarized layer back into the bulk solution. This occurs whenever the

transmembrane pressure builds up a concentration polarization, osmotic pressure which

occurs from the differences in concentrations, leads the protein to the bulk fluid.

The Mass Transfer coefficient (K) in this study was adapted from the combined osmotic

pressure/ stagnant film model. Limiting flux data in different concentrations plus the

experimentally measured process/ fouled permeability were fit using the following

equation:

106

𝐽 = 𝐿𝑓𝑚 (𝑇𝑀𝑃 − 𝛼 ∙ 𝐶𝑏 exp [𝐽

𝐾] − 𝛽 (𝐶𝑏 exp

𝐽

𝐾))

2

𝑘 = Mass transfer coefficient

𝐶𝑏 = Concentration of the bulk solution

𝑇𝑀𝑃 = Trans membrane pressure

𝐿𝑓𝑚 = fouled water membrane permeability

𝐽 = Permeate flux

𝛼 𝑎𝑛𝑑 𝛽 virial expansion coefficient for the protein

Table 3.6 Limiting flux performance (LMH) at 60 psi of 88 cm2 Pellicon 3 membrane

with 3 and 10 kDa cut-off

Protein concentration (g/L)

Flux (L/min/m2) at 60 psi TMP

BSA FWS

3 KDa 10 KDa 3 KDa 10 KDa

40 136 ±1.1aA 143±0.3aB 138 ±0.3cC 142±0.1cD

20 161±1.4bA 173 ±0.1bB 165 ±1.1dC 175±0.4dD

10 205 ±1.1cA 223±0.2cB 214 ±1.4eC 225±0.6eC


a-b Means within a row with different letters are significantly different (P<0.05) on BSA

A-B Means within a column with different letters are significantly different (P<0.05) on BSA

c-e Means within a row with different letters are significantly different (P<0.05) on FWS

C-D Means within a column with different letters are significantly different (P<0.05) on FWS

ANOVA single factor was carried out by using Least Significant Difference (LSD) test, followed by T-test

107

Table 3.7 Mass transfer coefficients for 3 and 10 kDa cut off membranes in BSA of 88

cm2 Pellicon 3

Membrane size cut off

mass transfer

coefficient (k)

(L/min/m2)

3 kDa 35.6 ±1.2a

10 kDa 38.0 ±0.4b


a-b means with different letters are significantly different (P<0.05)

ANOVA single factor was carried out by using Least Significant Difference (LSD) test, followed by T-test

The results obtained show that in general, the mean mass transfer coefficients obtained

in both membranes were within one standard deviation of each other. The virial

expansion coefficients of α and β are the same for both membrane size which is α = 3.81

x 107 and β = 9.99 x 105 which are the standard values for the BSA protein.

Better standard deviation values in 10 kDa membrane compared to 3 kDa were obtained,

showing that the polarized protein flux movements back into the bulk solution of 10 kDa

is higher than in 3 kDa; this reflects the transmembrane pressures which help in a build-

up of concentration in the surface of membrane, thus allowing the osmotic pressure to

send the protein back to the bulk fluid. The standard method suggest that the acceptance

evaluation requires that the population means to fall in the range of <10 % difference

among the difference scales. The results obtained also fall in the acceptance criteria to

have less than 10 % mean difference.

108

The calculations were done for both 3 and 10 kDa membrane cut off in BSA protein

solutions only since no standard virial expansion coefficient for the fish waste proteins

has been done for this study. The mass transfer coefficient obtained in the study of BSA

protein solutions has been referred to the standard manufacturing procedures which use

the BSA as a model system. Future study will be undertake to measure the virial

expansion coefficient for FWS protein as well as the mass transfer coefficient.

3.3.3.3 Conclusion

The 3 kDa and 10 kDa membrane of Pellicon 3 cassetes with Ultracel shows good

performance in both types of proteins (Sulfhydryl Modified BSA and Fish Processing

waste streams proteins). The results obtained have met the acceptance criteria for

protein performance scalability study as well as in limiting flux and mass transfer

analysis. Future work should undertake the mass transfer study of fish waste streams

protein.

3.4 Hold up volume of Membrane

The objective of this study was to determine the hold-up volume in the feed and permeate

in Pellicon 3 88 cm2 (3 and 10 kDa) cassettes using fish processing waste protein. Weights

were calculated to volumes as shown in Table using the formula as shown below. The

volumes for both cut off were averaged and presented in Table 3.4

𝐻𝑜𝑙𝑑 𝑢𝑝 𝑉𝑜𝑙𝑢𝑚𝑒(𝑓𝑒𝑒𝑑) = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑓𝑖𝑙𝑙𝑒𝑑 𝑑𝑒𝑣𝑖𝑐𝑒 − 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝐹𝑒𝑒𝑑

𝐻𝑜𝑙𝑑 𝑢𝑝 𝑉𝑜𝑙𝑢𝑚𝑒(𝑃𝑒𝑟𝑚𝑒𝑎𝑡𝑒) = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑓𝑒𝑒𝑑 𝑐ℎ𝑎𝑛𝑛𝑒𝑙 − 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑃𝑒𝑟𝑚𝑒𝑎𝑡𝑒

109

Table 3.8 The Measured hold up volumes of 3 and 10 kDa fish processing waste protein.

Calculation based on 1g of water = 1 ml water. The area of device is 88 cm2

MW Cut off (kDa) Feed Channel

(ml)

Permeate channel (ml)

3 14.5 25.2

10 14.5 33.4

Based on the calculated values obtained from this study, the hold-up volumes determined

the minimum working volume for the system used, and the values obtained are in

agreement with the standard values given by the manufacturer. The difference in

membrane size indicates that the smaller molecular cut off (3 kDa) gives smaller values

in the permeate channel, likewise the 10 kDa membrane. The values also gave some

information for the system used to estimate the volumes of samples needed and expected

feed and permeate volume obtained when using different membrane sizes for different

samples. In this study, there was a slight membrane fouling phenomena when using

protein samples, compared to using just water, in the determination of the hold-up

volumes. Larger molecules could not pass through smaller membrane size, thus created

the membrane fouling and limiting the volume of permeate, but allowing more volume of

retentate.

110

3.4.1 Conclusion

Ultrafiltration was found to be a mild and efficient process for separating valuable

proteins from fish waste streams, and compared will to the model BSA protein.

The potential for this technique in recovering cheap waste proteins with a valuable

nutritional and functional quality in many different food and pharmaceutical products is

great.

111

Chapter 4

112

4 Antioxidant properties by lipid peroxidation inhibition (FTC and

TBARS) and radical scavenging activity (2, 2- Diphenyl-1 picryhydrazyl

DPPH radical scavenging and ferric reducing antioxidant power assay)

in the fish from waste streams (Atlantic mackerel and mixed fish)

4.1 Introduction

Over 60 % of raw materials from fish processing industries end up as processing waste

or by-products. The raw materials include head, frames, skin, viscera, bones, roes and

trimmings (Chalamiah et al., 2012). In most developing countries, this type of seafood

waste is usually processed for disposal or enhanced to be used as fish meal, fertilizer and

animal feed (Kristinsson and Rasco, 2000). For this reason, it is important to utilize

available resources sustainably. The application of enzymes for the recovery of proteins

is cheap and feasible. Furthermore, enzyme hydrolysis results in protein hydrolysates

that are a combination of peptides which have been studied and researched recently for

their nutritional and functional properties (Yousr and Howell, 2015).

Purification is a crucial step to improve fish hydrolysate properties. Ultrafiltration

membrane separation processes can increase the efficacy of bioactivity (Bourseau et al.,

2009). The membrane separation techniques include ultrafiltration (UF), nanofiltration

(NF), and gel filtration (GF) which could be used to fractionate hydrolysates and

maximize the utilisation of seafood waste products to exhibit the bioactive components

for human and animal used (Picot et al., 2010). UF and NF peptide hydrolysates can be

applied in various products (Bourseau, 2009). NF can concentrate hydrolysates whereas

UF membranes with high molecular weight cut off around 20-100 kDa) are useful for

separating peptides from the parent proteins or enzymes. UF with membrane cut off in a

113

range of 4-8 kDa is also applied in the fractionation of hydrolysate. The example of

activities determined for fractionated hydrolysates include antioxidant and functional

properties.

4.1.1 Antioxidant activity

Recent publications on peptides derived from enzymatic fish protein hydrolysis and their

antioxidant activity include those by Halim, Gajanan et al., (2016), Lassoued et al., (2015)

and Howell and Kasase (2010). Most studies shown significant activities of various

oxidation systems. The most popular antioxidative assays are DPPH, free radical

scavenging, hydroxyl radical scavenging, Thiobarbituric acid reactive substance (TBARS),

and ferric reducing antioxidant power (FRAP), lipid peroxidation inhibition activity, and

linoleic acid peroxidation inhibition.

4.1.2 Lipid oxidation in fish

Lipid oxidation products in fish and seafood are potentially harmful for human

consumption, as the toxic oxidation products can lead to cancer, coronary heart disease

and Alzheimer’s disease (Saeed and Howell, 2002 and Zou et al., 2016). Fish proteins or

peptide hydrolysates are able to decrease the lipid oxidation process during food storage

and processing steps (Leong, 2015; Howell and Kasase, 2010 and Nasri et al., 2013).

Although some synthetic antioxidants like BHT can be effective than known food protein-

derived peptides, for long term usage, natural food antioxidants and food derived

peptides may be safer to use. Antioxidant peptides are known to be produced from

various fish processing by- products such as mackerel and tuna backbone protein

hydrolysate (Sheriff et al., 2014; Gajanan et al., 2016).

114

4.1.3 Radical Scavenging activity

The active form of oxygen can be in a form of two different species, known as active

oxygen species (AOS) and reactive oxygen species (ROS). Free radicals i.e. superoxide

ions (O2-) and hydroxyl radicals (OH-), in addition to non-free radical species such as

hydrogen peroxide (H2O2) are included in ROS. ROS is also involved in the pathological

processes in coronary heart disease, cancers, aging, Alzheimer’s disease, cataracts,

inflammations and atherosclerosis (Padmanabhan and Jangle, 2012). The term

antioxidant is defined as the activity of vitamins, minerals, as well as phytochemicals

react as the defence against damage caused by the ROS. Antioxidants can act as

scavenging free radicals, free metal chelators, and electron donors that interrupt the

oxidative processes (Gulcin et al., 2005). The free radicals are formed when cells use the

oxygen to form energy; the products include ROS (superoxide anion, hydroxyl radical and

hydrogen peroxide) that resulted from cellular redox process.

The objective of this study is to determine the level of oxidation in fish waste water (FW)

from Atlantic Mackerel and mixed fish to protein hydrolysates by enzymatic digestion

(pepsin and pancreatin). Secondly, evaluating the antioxidant properties by lipid

peroxidation inhibition (FTC and TBARS) and radical scavenging activity (DPPH and

ferric reducing antioxidant power assay)

115

4.2 Materials and methods

4.2.1 Materials

Fish waste water from Atlantic Mackerel (Scomber scombrus) and mixed fish samples

were obtained from The Fish Society, Wormley, Surrey GU8 5TG, UK. The fish waste

samples were dispatched in ice, and processed upon the arrival to the lab, weighed and

homogenized in Omni Mixer-CAMLAB at 6000 rpm for 30 seconds in ice. All chemicals

used included Pepsin from porcine gastric mucosa and pancreatin from porcine pancreas

were purchased from Fisher Scientific, Loughborough, UK and Sigma Aldrich, Poole, UK.

Sodium hydroxide and hydrochloric acid; also all reagents used for experiments were of

analytical grade

4.2.2 Sample preparation:

4.2.2.1 Fish protein hydrolysates by enzymatic digestion

The method used was according to Megias et al., (2004), the powder form of the water

soluble fish waste protein was mixed with milli-Q water in the ratio (1:10 w/v) and

homogenized (Omni mixer homogenizer, USA). The pH was adjusted to pH 2.0 using 1M

HCl. Then, pepsin was added to the suspension with substrate/enzyme ratio (20:1). After

3 hours, the pH of the mixture was adjusted again to pH 7.5 using 1M NaOH. Subsequently,

pancreatin was added using the same ratio and time of digestion as described for pepsin.

In order to deactivate the reaction of enzymes, the mixture was immersed in boiling

water for 20 minutes. Then, after cooling, the sample was filtered and lyophilized for

further study.

116

4.2.3 Antioxidant properties

4.2.3.1 Measurement of lipid oxidation inhibition activity on water soluble fish

waste hydrolysate fractions

4.2.3.1.1 Materials

Water soluble fish waste protein hydrolysate of fish was prepared as above. Absolute

ethanol, linoleic acid, sodium phosphate, hydrochloric acid, butylated hydroxyl toluene

(BHT), Trolox, sodium dodecyl sulphate, acetic acid, 2-thiobarbituric acid (TBA),

ammonium thiocyanate and ferrous chloride obtained from Sigma-Aldrich, Poole, UK. All

reagents used for experiments were of analytical grade.

4.2.3.1.2 Methods

Lipid peroxidation inhibition activity of peptides was measured using a oxidising linoleic

acid model system, following the method of Osawa and Namiki, (1985) with some

modification. This method was applied for water soluble fish waste protein for both

mackerel and mixed fish

117

4.2.3.1.3 Preparation of reaction mixtures for lipid oxidation assays

Each dried fraction (100g) was dissolved individually with 10ml absolute ethanol, 0.13ml

linoleic acid, 4.87ml distilled water and 10ml of 50 mM phosphate buffer (pH 7) in glass

tubes. Samples were homogenized by a sonicator (Labsonic® M, B. Braun Biotech

International, Germany). After sealing tightly with silicone rubber caps, the tubes were

kept in the dark at 60°C in an oven (Raven oven, LTE Scientific Ltd,Great Britain). Aliquots

for FTC and TBARS were taken from the samples daily to measure the activity over 7 days.

A negative control without protein or peptides was used. 0.2mg/ml of butylated

hydroxylanisole (BHA), trolox and ascorbic acid (highest permitted limit commercially)

were used as a positive controls.

4.2.3.2 Thiobarbituric Acid Reactive Substances Method (TBARS)

The method was adapted from Ohkawa et al., (1979) with some modifications. 50µl of

aliquots were micro-syringed at 24 hour intervals from each reaction mixture and added

to test tube containing (0.8ml) of distilled water, (0.2) ml of 8.1% sodium dodecyl

sulphate (w/v), (1.5 ml) 20% (w/v) acetic acid (pH 3.5) and (1.5ml) 0.8% 2-

thiobarbituric acid (TBA) solution in water (w/v). The mixture was heated at 100°C for

60 minutes and cooled prior to centrifugation at 4300 x g for 10minutes. The absorbance

of the upper layer was measured at 532nm. The % of antioxidant activity was calculated

according to the following formula:

Antioxidant activity (%) = [(A control- A sample)/A control] x 100

118

4.2.3.3 Ferric Thiocyanate Method (FTC)

The method of FTC was according to Mitsuda et al., (1966). 100 µl aliquots were micro-

syringed from each reaction mixtures at 24 hour intervals and added to a test tube

containing (4.7ml) 75% ethanol (v/v), (100µl) and 30% ammonium thiocyanate )(w/v).

100µl of 20mM ferrous chloride solution was then added and after 3 minutes precisely

the thiocyanate value was measured by reading the absorbance at 500 nm. The % of lipid

oxidation inhibition was calculated according to the following formula:

Antioxidant activity (%) = [(A control- A sample)/A control] x 100

4.2.3.4 2, 2- Diphenyl-1 picryhydrazyl (DPPH) radical scavenging

DPPH radical scavenging in fish waste water hydrolysate was determined according to

Yen and Wu (1999). 2 mL of 0.5 mM DPPH solution was added to 0.2 mL of sample extract

diluted in methanol. Absorbance was measured after 30 minutes at 517 nm by

spectrophotometer. The activity was calculated using following equation:

𝐷𝑃𝑃𝐻 𝑓𝑟𝑒𝑒 𝑟𝑎𝑑𝑖𝑐𝑎𝑙 𝑠𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (%) = 1 − 𝐴𝑏𝑠𝑠𝑎𝑚𝑝𝑙𝑒

𝐴𝑏𝑠𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100

4.2.3.5 Ferric reducing antioxidant power assay

The ferric reducing antioxidant power of fish waste hydrolysate was conducted as

described by Oyaizu (1986) at a protein concentration of 10 mg/ml. The study was

carried out in triplicate with the mean values and standard deviation.

4.2.3.6 Statistical Analysis

Statistical analysis was performed using Microsoft Excel and SPSS. All values represent

means of triplicate analysis and are given with standard deviations and those at p<0.05

were considered significant.

119


4.3.1 Antioxidant Activity of Water Soluble Fish Protein Hydrolysate (Atlantic

Mackerel fish)

Enzymatic hydrolysis of protein is one of the most important options to modify the

functional and nutritional properties of proteins and peptides (Korhonen et al., 1998).


digestion using pepsin and pancreatin in order to mimic the human digestive process and

achieve a higher degree of hydrolysis than that achieved with either pepsin or pancreatin

alone. It is recognized that the degree of hydrolysis determines the application of protein

hydrolysates.

4.3.2 Measurement of Lipid Oxidation Inhibition Activity

There were 2 methods used to analyse the antioxidant activity of the fish waste water-

soluble waste hydrolysates, namely FTC and TBARS. FTC was the method used to monitor

the formation of peroxides (as primary products from lipid oxidation) while TBARS was

used to monitor the carbonyl compounds (as secondary products from lipid oxidation).

The antioxidant activity of hydrolysates were investigated and compared with that of

butylated hydoxyanisole (BHA), trolox and ascorbic acid that are well-known

antioxidants used as food additives by food manufacturers. The sample absorbance was

measured over 8 days at 500 nm. Peroxide value or TBARS were measured when the

control (no antioxidant present) reached the highest peak absorbance.

120

4.3.2.1 Ferric Thiocyanate (FTC) Method

4.3.2.1.1 Antioxidant activity of Atlantic mackerel fish waste Water Protein Hydrolysate

with Pepsin and Pancreatin

The antioxidant activity of Atlantic mackerel fish waste water soluble protein hydrolysate

sample demonstrated the third highest antioxidant activity after BHA and Trolox, while

ascorbic acid showed the lowest antioxidant activity by the FTC method.

The statistical data for all samples were significantly distributed (p<0.05) as indicated in

the appendix for statistical analysis during the 8 days.

Figure 4.1 Antioxidant activity of fish waste water (Atlantic mackerel) protein

hydrolysate. The activity was assessed by evaluating the degree of linoleic acid

oxidation using ferric thiocyanate method. Peroxide values were measured at 24 hour

intervals. A negative control was used without sample or antioxidant. 0.2 mg/ml of

trolox, ascorbic acid and butylated hydroxylanisole (BHA) were used as positive

controls.

121

The results were then compared at day 6 when the values peaked, as shown in Figures

4.1 and 4.2; BHA shows the highest percentage of lipid oxidation inhibition activity of

33.8% followed by Trolox (33.7%), water soluble fish sample (33.1 %) and ascorbic acid

(31.7%). (See appendix for statistical data).

All values represent means of triplicate analysis and are given with standard deviations and those at p<0.05 were considered significant.

Figure 4.2 Percentage of lipid oxidation inhibition activity of water-soluble fish waste

(Atlantic mackerel) protein hydrolysate as measured using ferric thiocyanate method.

122

4.3.2.1.2 Antioxidant Activity of Mixed fish waste Water Soluble Protein Hydrolysate with

Pepsin and Pancreatin

The mixed fish waste water-soluble protein hydrolysate demonstrated the highest value

of antioxidant activity compared to other antioxidants when treated with pepsin and

pancreatin.

The statistical data for all samples were significantly distributed (p<0.05) as indicated in

the appendix for statistical analysis during the 8 days.

Figure 4.3 Antioxidant activity of water-soluble fish waste (mixed fish) protein

hydrolysate. The activity was assessed by evaluating the degree of linoleic acid

oxidation using ferric thiocyanate method. Peroxide value was measured at 24 hours

intervals. A negative control was used without the sample. 0.2 mg/ml of trolox, ascorbic

acid and butylated hydroxylanisole (BHA) were used as positive controls.

123

The results were compared at day 6 as a general trend as shown in Figure 4.4 ; Mixed fish

waste water soluble protein hydrolysate showed the highest percentage of lipid

oxidation inhibition activity of 30.4 %, followed by BHA (29.3%), Trolox (29.0%) and

ascorbic acid (27.4%).


(mixed fish ) protein hydrolysate as measured by the ferric thiocyanate method

124

4.3.2.2 Thiobarbituric Acid Reactive Substances (TBARS) Method

4.3.2.2.1 Antioxidant Activity of Atlantic Mackerel fish waste Water Soluble Protein

Hydrolysed with Pepsin and Pancreatin

The antioxidant activity by TBARS was observed for 8 days (Figure 4.5). The statistical

data shows that the data were significantly distributed in most of the samples (p<0.05)

during the 8 days.

Figure 4.5 Antioxidant activity of water-soluble fish waste (Atlantic Mackerel) protein

hydrolysate. The activity was assessed by the degree of linoleic acid oxidation by

measuring malondialdehyde (µg/ml) at 24 hours intervals. The negative control

contained mili-Q water without the sample. 0.2 mg/ml of trolox, ascorbic acid and

butylated hydroxylanisole (BHA) were used as positive controls

The results was then compared at day 1, when the level peaked, as shown in Figure 4.6;

BHA showed the highest percentage of lipid oxidation inhibition activity of 17.4 %,

followed by ascorbic acid (13.0 %), Atlantic Mackerel fish waste water soluble protein

hydrolysate (12.4 %) and Trolox (11.8 %).

-0.010

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0 1 2 3 4 5 6 7

MD

A (

µg/

ml)

Time (Days)

Antioxidant activity using (TBARS)sample

blank

control

trolox

BHA

Ascorbic acid

125


(Atlantic mackerel) protein hydrolysate. The method was measured using

Thiobarbituric reactive substance method

126

4.3.2.2.2 Antioxidant Activity of Mix fish waste Water Soluble Protein Hydrolysate with

Pepsin and Pancreatin

The antioxidant activity by TBARS was observed for 8 days (Figure 4.7). The statistical

data shows that the data were significantly distributed in most of the samples (p<0.05)

during the 8 days.

Figure 4.7 Antioxidant activity of water-soluble fish waste protein (Mix fish)

hydrolysate. The activity was assessed by the degree of linoleic acid oxidation by

measuring malondialdehyde (ug/ml) at 24 hours intervals. The negative control was

used containing Mili-Q water without the sample. 0.2 mg/ml of trolox, ascorbic acid and

butylated hydroxylanisole (BHA) were used as positive controls

The results was then compared at day 1 when the values peaked, as shown in Figure 4.8;

Ascorbic acid and BHA show the highest percentage of lipid oxidation inhibition activity

of 38.5 % and 34.3 %, followed by Trolox (32.4 %) and Mix fish waste water soluble

protein hydrolysate (31.5 %).

-0.010

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0 2 4 6 8

MD

A (

µg/

ml)

Time (Days)

Antioxidant activity using TBARS

SAMPLE

BLANK

CONTROL

TROLOX

BHA

ASCORBIC ACID

127

Figure 4.8 Percentage of lipid oxidation inhibition activity of water-soluble fish protein

(mixed fish) hydrolysate as measured by Thiobarbituric reactive substance method

Fish waste water protein hydrolysate especially for Atlantic Mackerel showed good

antioxidant activities by the Ferric Thiocyanate (FTC) and Thiobarbituric acid Reactive

substances (TBARS) methods and compared well with other antioxidants (BHA, Ascorbic

acid and trolox). There was significant difference (p<0.05) between samples and negative

control (no antioxidant).

The FTC method is good especially to determine peroxide value at early stages of

oxidation. However, this method is highly empirical and the results sometimes vary with

the method and temperature. On the other hand, TBARS which measures the secondary

products of lipid oxidation (malonaldehyde) is known to be a good to measure samples

with a single component at different states of lipid oxidation, but sometimes can be

insensitive especially when measuring a very low level of malonaldehyde which can react

128

with protein in the oxidation system. In addition, both methods require careful

monitoring as products change from primary to secondary and then finally tertiary

products.

4.3.3 DPPH scavenging activity

Table 4. Illustrates the DPPH activities in both mixed fish and Atlantic Mackerel fish waste

hydrolysates. The samples were dissolved with different concentrations of methanol

which are 500, 1000 and 10000 µg/mL.

The radical scavenging activity measurement is generally done through a DPPH method

which is fast, reliable and reproducible. The DPPH scavenging activity has been widely

used to study the ability of various components as free radical chelating scavengers, and

as hydrogen donors.

From Table 4.1, it can be concluded that the DPPH scavenging activity increased with the

extract concentration in the range of 1.5-26 %. The DPPH scavenging activity in mixed

fish waste hydrolysate was found to be slightly higher than in Atlantic Mackerel fish waste

hydrolysate as the sample increased in concentration. The statistical data however shows

that there were no significant difference in the two samples (p>0.05)

129

Table 4.1 DPPH scavenging activity in fish waste protein hydrolysates from mixed fish

and Atlantic Mackerel

Fish waste hydrolysate

Percentage (%)

10000 µg/mL

Methanol

1000 µg/mL

Methanol

500 µg/mL

Methanol

Mixed fish

46.04 ± 0.003

5.39 ± 0.005

1.78 ±0.005

Atlantic Mackerel

25.45 ± 0.005

4.73 ± 0.003

1.56 ± 0.004

Recent studies on the DPPH activity of fish hydrolysates including those on yellow stripe

trevally hydrolysates (Klompong et al.,2007); sardinelle hydrolysates (Bougatef et al.,

2010) and on Threadfin bream frames hydrolysate (Gajanan et al., 2016) showed a higher

activity of DPPH free radical scavenging as the degree of hydrolysis increased. These

studies showed that their fish hydrolysate samples contained peptides in the hydrolysate

which could donate electron/ proton, and thus scavenge the DPPH free radical.

The DPPH assay is categorized under the H-atom transfer reaction and electron transfer

reaction based on the major antioxidant activity (Prior et al., 2005 in Gajanan et al., 2016).

When there is a presence of peptides with different sizes at different degree of hydrolysis,

a greater number of smaller peptides will be increased as the higher degree of hydrolysis

increases.

130

The antioxidative properties in protein hydrolysates depends on the sequence of the

amino acid residue that is released by peptides. Thus, the antioxidant activity depends on

the specific enzymes used and the conditions during hydrolysis (Elavarasan et al., 2014).

Although a small level of DPPH activity for the fish waste water protein hydrolysates

obtained in this study, it shows that the samples scavenged free radicals. With further

analysis and specific selection of proteases and degree of hydrolysis conditions, the fish

processing waste water protein hydrolysates may be considered as one of the natural

antioxidants that could be used to preserve food products.

4.3.4 Ferric-reducing antioxidant power (FRAP)

The ferric-reducing antioxidant power is also a method known as a reduction method as

stated by Jayaprakash et al. (2001). In general, the principle is based on the increasing

values in absorbance of reaction mixtures. Absorbance is directly related to the

concentration of antioxidants, thus an increase in absorbance determines the increase in

antioxidant activity. A coloured complex of the fish waste hydrolysate samples and

reagents potassium ferricyanide, trichloro-acetic acid and ferric chloride was formed.

The complex was diluted in deionised water and its absorbance was measured using

spectrophotometer at the wavelength of 700 nm. An increase in the absorbance indicated

the reducing power of the samples.

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Figure 4.9: Reducing power of Atlantic Mackerel fish waste protein hydrolysate and

mix fish waste protein hydrolysate

Figure 4.9 illustrates the reducing power of mixed fish waste protein hydrolysate and

Atlantic Mackerel fish waste hydrolysate. Both sample showed an increase in absorbance

with an increase in concentration. Mixed fish FW showed an increase in absorbance with

concentrations up to 1000 µg/mL, while Atlantic Mackerel FW showed increases in

absorbance for up to 750 µg/mL followed by a decrease then remains at a constant value.

The difference might be because of specific peptide/ amino acid composition and the

nature of enzyme used as describe by Gajanan et al., (2016). Jayaprakash et al. (2001)

stated that the relation between reducing power and antioxidant activity is proportional.

Thus, it is clear that the antioxidant activity of mixed fish waste protein hydrolysate is

higher than in Atlantic Mackerel fish waste protein hydrolysate.

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4.4 Conclusion

The protein hydrolysates obtained from Atlantic Mackerel (Scomber scombrus) fish waste

streams and mixed fish waste streams using pepsin and pancreatin enzymes show good

antioxidant potential as measured by several different methods.

As hydrolysates are widely used as food functional ingredients, future work on this low

value added fish protein could be converted to bioactive peptides for the sustainable

utilization of fisheries by products.

Future work should include the study on the efficiency of hydrolysis using other enzymes

such as papain to study antioxidant activity further as well as explore new applications

in the field of bioactivity e.g. on the production of natural ACE inhibitors.

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Chapter 5

134

5 DSC and rheological properties of the fish waste (Atlantic

Mackerel, mixed fish, 10 kDa Mackerel and mixed fish protein

hydrolysates)

5.1 Introduction

In a living system, proteins are found in cell membranes, aqueous solutions as well as in

lipid-protein complexes. Proteins denaturation is usually associated with changes in

molecular state. The denaturation process may include dimerization due to weak bonds

that occur at specific sites of interaction. Extensive protein- protein interactions, can lead

to aggregation or polymerization, precipitation, coagulation and flocculation, which

results in a higher molecular weights with larger complexes. Gelation is a process

involving native or partially denatured proteins. The exposed reactive groups in the

unfolded protein molecules interact systematically to form aggregates, via balanced

protein- protein and protein- solvent interactions. This process can result in a three-

dimensional protein matrix with water holding capabilities (Messens et al., 1997 and

Howell, 1992).

According to Shahidi (1994), fish processing of by-products using appropriate enzymatic

hydrolysis is known to enhance the quality as well as functional properties of the fish

products. Physicochemical, functional, as well as nutritional and sensory properties of

proteins in its original state can be improved by the enzymatic hydrolysis. Thus enzyme

hydrolysis is often used to enhance the applications of low-value food proteins (Althouse

et al, 1995; and Kuipers et al., 2005).

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Very few papers on hydrolysates from fish processing by products have been published;

these include, for example, fish processing of viscera (Ovissipour et al., 2009), fish skin

(Mendis et al., 2005), wastes from cod (Gadus morhua), shrimp (Pandalus borealis) and

sardine (Sardina pilchardus) and processing industries by products (Bordenave et al.,

2002 and Yin et al., 2010). Hydrolysates obtained from fish proteins are highly affected

by the source of protein, enzymes used, degree of hydrolysis, buffer pH and the reaction

temperature and time (Feng et al., 2003). Example of fish hydrolysates with useful

functional properties include those from the skin Nile Perch (Lates niloticus), Grass carp

(Ctenopharyngodon idella) and Nile tilapia (Oreochromis niloticus) (Wasswa et al., 2008).

Protein denaturation can be measured thermodynamically and biochemically. The

thermodynamic measurement can be performed by Differential Scanning calorimetry

(DSC analysis), which describes the changes of free energy enthalpy (∆H) in folded and

unfolded states after heating.

Molecules in solution exist in equilibrium as native (folded) and denatured (unfolded)

conformations. On heating, the term thermal transition midpoint (Tm) is used when 50%

of molecules are unfolded; a high Tm denotes a more stable molecule. In addition, the

change in heat capacity (∆Cp), primarily due to changes in hydration of side chains that

were buried in the native state, but become solvent exposed in the denatured state (Badii

and Howell, 2006) can be also be measured by DSC.

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The biochemical separation of protein can be performed by gel electrophoresis. The

protein denaturation can be defined as changes in secondary, tertiary or quaternary

conformation without interrupting the primary structure of protein. When denaturation

of protein occurs, the structure is changed and results in a loss of its biological active

form; the changes can be reversible or irreversible depending on the molecular structure

and extent of processing. Protein denaturation can be affected by temperature and

pressure or chemically by the employment of strong acid or base, concentrated inorganic

salts as well as organic solvents (Alberts et al., 1994; Howell and Li-Chan, 1996) on

chemical and enzymatic modification of proteins (Stryer L, 1988).

Denaturation changes affect the functional properties like gelation. Gelation properties

of proteins are generally measured by rheological methods. Rheology is a term used to

determine the flow and deformation of materials that affects the texture and consumer

acceptance of products, especially in food products. Rheology is also regarded as a key

method for monitoring in food processing, quality control as well as shelf life of food

materials (Ötles, 2011). Food materials display elastic and viscous behaviour, which is

measured by viscosity and oscillatory shear, as well as a wide range of small oscillatory

and large deformation rheological tests that are commonly used to characterise food gels.

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5.1.1 Rheology Small Deformation Testing

Small deformation testing measures viscoelastic parameters, storage modulus (G’)

indicates the amount of energy stored elastically in the structure, and the loss modulus

(G’’) that measures the energy loss /viscous response. G’ and G’’ values are obtained by

dynamic oscillatory testing in the linear viscoelastic region of the sample. The G’/G’’

crossover point indicates the gelling point (Murata, 2012).

Rheological properties of gels and emulsions can give very interesting information that

can be used to assess storage stability, sensory evaluation, texture and quality control of

products such as in mayonnaise and salad dressing (Davis, 1973).

The main objective of this study was to measure the differences in the physicochemical

properties of fish waste proteins and the fish protein hydrolysates obtained from Atlantic

Mackerel and mixed fish waste by ultrafiltration (10 kDa protein fractions).

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5.2 Materials and method

5.2.1 Materials

Fish waste (FW) from Atlantic Mackerel (AM) and mixed fish (MF) samples from the fish

industry processing, were bought from The Fish Society, Wormley, Surrey GU8 5TG, UK.

The fish samples were usually despatched in ice directly to the Food Science laboratory.

The fish waste samples were processed upon the arrival to the lab, weighed and

homogenized in Omni Mixer-CAMLAB) at 6000 rpm for 30 seconds in ice. All chemicals

used were mostly bought from Fisher Scientific, Loughborough, UK and Sigma Aldrich,

Poole, UK.

5.2.2 Methods

5.2.2.1 Heat treatment

See section 2.2.1.1

5.2.2.2 Fish protein hydrolysate

See section 3.2.1

5.2.3 Sample preparation

5.2.3.1 Fish protein hydrolysate

See section 3.2.1

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5.2.3.2 Different Scanning Calorimetry (DSC)

Protein Denaturation was measured by using a Setaram Micro DSC VII differential

scanning calorimeter (Setaram, Lyons, France) (Figure 5.1).

Figure 5.1 Differential Scanning Calorimetry (DSC) instrument and sample

preparation used in the experiment.

The fish sample (0.8-1 g wet weight) and reference (distilled water) were scanned at 1 oC

min-1 over the range 10-90 °C and cooled to 10 °C (first scan). The sample was rescanned

(second scan); this was subtracted from the first scan to obtain a heat flow-corrected

signal as described in the Setaram DSC handbook and Setsoft software. For each peak a

linear baseline was constructed by joining the first point at the peak start and the last

140

selected point at the end of the peak, according to the Setsoft software. The total energy

required to denature the protein, i.e. the change in enthalpy (∆H), was estimated by

integrating the area under each peak. The temperature reached when half of the protein

is denatured is the transition temperature (Tm), which was measured at the tip of the

peak (Setaram DSC handbook and Setsoft software). Three replicates of each sample were

tested.

5.2.3.3 Small deformation rheology test

Small deformation rheological analysis of fish water soluble and salt soluble protein

concentrations of Atlantic Mackerel and mix fish was determined on a Rheometrics

(Leatherhead, Surrey, UK) controlled stress 200 rheometer using a 40 mm parallel plate

geometry and a 0.3 mm gap. A temperature sweep, with a stress of 0.1 Pa and a frequency

of 1rad/s was used. . Evaporation during heating was minimised by using Silicone oil.

Figure 5.2 Rheometrics constant stress rheometer used for the small deformation testing of

Atlantic Mackerel and Mixed fish waste protein fractions

141

The temperature of Peltier plate was programmed to ramp, at a rate of 2°C min -1, from

20 to 90°C, was then cooled to 20°C at the rate of 2°C min-1. Changes in the elastic or

storage modulus (G’) and loss modulus (G’’) were recorded and G’/G’’ crossover point

were recorded.

5.2.3.4 Statistical Analysis

Statistical analysis by t-test was performed using Microsoft Excel. ANOVA single factor

was carried out by Least Significant Difference (LSD) test, followed by the T-test. All

values represent means of triplicate analysis and are given with standard deviations and

those at P < 0.05 were considered significant

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5.3.1 Differential scanning calorimetry (DSC)

5.3.1.1 Water soluble fish protein (Atlantic Mackerel)

The DSC thermogram analysis parameters include; heat energy uptake (Cp endotherm),

transition peak (Tm ) as well as calorimetric enthalpy change (∆Hcal) (Figure 5.3)

Figure 5.3 DSC thermogram of Atlantic Mackerel water-soluble fish protein

On heating the sample at a constant rate, heat change was detected due to thermal

denaturation. The main transition temperature (Tm) was 48.57 °C, which is the point

where water soluble proteins started to denature.

The fish proteins in aqueous solution were in equilibrium between the native and

denatured (unfolded) conformation. The native state remains stable depending on the

size of the Gibbs free energy (∆G) and the thermodynamic interplay between enthalpy

(∆H) and entropy (∆S) changes. Therefore, a positive ∆G is characteristic of a stable the

143

native state rather than the denatured state. Proteins denature and unfold when covalent

and non-covalent bonds are broken. When conformational entropy overcomes the

bonding forces, the protein is able to unfold on heating to temperatures where entropy

becomes the main factor (Johnson, 1995).

5.3.1.2 Fish Waste samples (Atlantic Mackerel and Mixed fish)

The DSC analysis was undertaken on waste obtained from Atlantic Mackerel and mixed

fish as well as hydrolysates samples prepared from fish waste (10 kDa of Atlantic

Mackerel and 10 kDa mixed fish). The DSC thermograms from the samples indicated that

they may be comparable. By investigating the denaturation peaks enthalpies (J/g) and

denaturation temperatures (°C) (Table 5.1), the t-test of significance shows that there is

no significant differences between the two types of fish waste samples (p> 0.05).

The denaturation enthalpy for myosin in Atlantic mackerel (0.349 J/g) is higher at the

transition temperature (TM) of 49.3 °C than mixed fish (0.286 J/g at 47.4 °C). For the

actin denaturation enthalpies, mixed fish has the highest enthalpy change values (0.12

J/g) compared to the Atlantic Mackerel (0.055 J/g) and there is no significant difference

between samples (p>0.05). Sarcoplasmic proteins in mixed fish waste sample have

denatured more readily with a lower enthalpy change value than in Atlantic Mackerel fish

waste. Different habitats and type of fish have different muscle fibre composition, i.e.

Mixed fish has more white muscle fibre than Atlantic mackerel. White muscle fibres

comprise myofibrils, and a few lipid droplets. The fish fat content may contribute to

different heat conductivity, which is also different between the species.

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For the DSC thermograms of fish waste sample hydrolysates, the same trends were

obtained in 10 kDa AM and MF. Atlantic Mackerel showed higher denaturation enthalpy

changes for myosin (0.69 J/g) and lower denaturation of actin (0.024 J/g) than mixed fish

(0.202 J/g for myosin and 0.12J/g in actin) shows that there is significant difference

(p<0.05) between the samples.

The controlled enzymatic hydrolysis of fish waste is known to affect the functional

characteristics like foaming and emulsification because of the production of smaller

polypeptides (Chalamiah et al., 2012 in Halim et al., 2016). In addition, the small peptides

are reported to have several therapeutic properties including anti-hypertensive and anti-

cancer properties (Yousr and Howell, 2015).

Table 5.1 Transition temperature and enthalpy (J/g) for fish waste (FW) samples in

Atlantic Mackerel (AM), mixed fish (MF), 10 kDa AM and 10 kDa MF


a-b Means within a column with different letters are significantly different (P<0.05)

ANOVA single factor was carried out by Least Significant Difference (LSD) test, followed by the T-test.

FW Sample AM MF 10KD AM 1O kDa MF

Peak 1 (°C) 49.3 ± 0.2a 47.4 ± 0.5a 48.1 ± 0.5a 47.1 ± 0.1a

Enthalpy (J/g) 0.349 ± 0.06a 0.286 ± 0.050a 0.69 ± 0.005a 0.202 ± 0.0080a

Peak 2 (°C) 60.1 ± 0.3a -- 59.7 ± 0.7a --

Enthalpy (J/g) 0.001 ± 0.001a -- -- --

Peak 3 (°C) 71.2 ± 0.2a -- 70.1 ± 0.6a --

Enthalpy (J/g) 0.007 ± 0.002a -- 0.007 ± 0.001a --

Peak 4 (°C) 80 ± 0.3a 76.7 ± 0.1a 79.4 ± 0.003a 75.2 ± 0.001a

Enthalpy (J/g) 0.055 ± 0.002a 0.120 ± 0.030a 0.024 ± 0.002b 0.120 ± 0.020b

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Figure 5.4 DSC thermogram of Atlantic Mackerel (AM) fish waste sample

Figure 5.5 DSC thermogram of mixed fish (MF) waste sample

146

Figure 5.6 DSC thermogram of 10 kDa Atlantic Mackerel (AM) fish waste hydrolysate sample

Figure 5.7 DSC thermogram of 10 kDa mixed fish (MF) waste hydrolysate sample

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5.3.2 Rheological Analysis (Small deformation testing)

5.3.2.1 Rheology small deformation results for Atlantic Mackerel Fish and mixed fish

proteins.

(a) water soluble fish (b) salt soluble actomyosin fish

(c) fresh Atlantic Mackerel fish

Figure 5.8 (a) Temperature sweep and rheological properties (G’ and G’’) of water soluble fish

processing waste sample; (b) Temperature sweep and rheological properties (G’ and G’’) of salt

soluble actomyosin fish processing waste sample and (c) Temperature sweep and rheological

properties (G’ and G’’) of fresh Atlantic Mackerel fish processing waste sample

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Table 5.2 The G’ (storage modulus) and G’’(loss modulus) point with the temperature of fish

processing waste streams of water soluble, salt soluble and fresh Atlantic Mackerel fish protein

sample.

ATLANTIC MACKEREL

FISH

Temperature (°C)

G' / G'' CROSSOVER POINT

(°C) WATER SOLUBLE

PROTEINS 20 90 20 57.01

G' (Pa) 0.02 7.62 36.82

G'' (Pa) 0.03 0.91 10.21

ACTOMYOSIN SALT

SOLUBLE PROTEINS

Temperature (°C) 70.83

20 90 20

G' (Pa) 0.01 6.15 70.93

G'' (Pa) 0.01 0.72 19.74

FRESH FISH Temperature (°C) 53.94

20 90 20

G' (Pa) 5163 18399 1.02E+05

G'' (Pa) 1605 2214 24878

Fish samples were analysed in triplicate, values presented based on the optimum crossover point (interception of G’ and G’’) from the best curve obtained.

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A B

Figure 5.9 Temperature sweep and rheological properties (G’ and G’’) of (A) water soluble, and

(B) actomyosin fish processing waste stream sample of mixed fish

Table 5.3 The G’ and G’’ point with the temperature of fish processing waste streams of water

soluble, salt soluble and mixed fish protein sample.

MIX FISH Temperature (°C) G' / G'' CROSSOVER

POINT (°C) WATER SOLUBLE

20 90 20

56.11 G' (Pa) 0.011 3.68 17.63

G'' (Pa) 0.007 0.43 3.94

ACTOMYOSIN SALT

SOLUBLE

Temperature (°C)

75.3 20 90 20

G' (Pa) 0.01 5.03 215.97

G'' (Pa) 0.01 0.62

124.28

Fish samples were analysed in triplicate, values presented based on the optimum crossover point (interception of G’ and G’’) from the best curve obtained.

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The results obtained for both water soluble fish protein fractions shows that prior to

heating, the storage modulus (G’) was greater than the loss modulus (G’’), demonstrating

that the samples had a gel-like structure as shown in Figure 5.7 and Figure 5.8. The

viscous and loss modulus of both Atlantic Mackerel and mixed fish increased with time

and temperature after storage at -80°C.

The G’ (storage modulus) of Atlantic Mackerel fish sample were considerably higher at

90°C (18399 Pa) and cooled to 20oC (G’=1.02E+05 Pa) compared with water and salt

soluble sample at 90°C (G’= 7.62 and 6.15 Pa respectively) and 36.82, 70.93 Pa

respectively when cooled at 20°C (p<0.005).

Meanwhile, the G’ (storage modulus) of salt soluble mixed fish processing waste streams

protein was higher at 90°C= 5.0268 Pa and cooled to 20°C (G’=215.97 Pa) compared to

the water soluble sample at 90° C (G’= 3.6774 Pa) and 17.629 when cooled at 20°C

(p<0.05).

As G’ is the energy stored elastically in the structure, this shows that Atlantic Mackerel

sample was tougher than the mixed fish. The difference between the G’ values of the

water-soluble (small proteins, peptides) and salt soluble proteins high MW myosin and

actin that can form 3D gel networks influences the rheological properties.

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5.4 Conclusion

Fish waste samples especially hydrolysed fish waste samples, showed different protein

denaturation transition peaks, indicating that enzymatic hydrolysis can affect the

thermodynamic and functional properties of protein samples. Similarly, the rheological

properties were different for different fish samples (AM and MF) with AM showing higher

G’ or elastic modulus values. The salt soluble actomyosin fraction had higher G’ values

than the water soluble fraction (p <0.05)

Changes in myofibrillar as well as sarcoplasmic proteins of fish wastes can be exploited

in the future to discover the effect of freezing and heat in storage as well as the effect of

antioxidants to the fish waste samples.

Further work to be taken into consideration includes measurement of the amino acid

composition by HPLC in order to see if the different amino acid content in both Atlantic

Mackerel and mixed fish processing waste protein contributed to the gel strength

Few papers report changes in water holding capacity of fish due to surface dehydration

of proteins as well as the relationship between protein solubility of muscle and

toughening of tissue during frozen storage fish (Badii and Howell, 2002). Thus, further

work can be done on moisture content and protein solubility of fish proteins (myofibrillar

protein and other mixture of protein components) alongside the small deformation

testing.

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Chapter 6

153

6 FT-Raman spectroscopy characterisation of fish proteins

from Mackerel and Mixed fish waste streams

6.1 Introduction

6.1.1 Principle of Raman Spectroscopy

Raman spectroscopy is a vibrational spectroscopy method that can be used to study

processing effects and variation in the properties of food components. It is a rapid method

and has been used to determine chemical and molecular changes that contribute to

physical properties of food and pharmaceutical products (Scotter, 1997).

When a monochromatic source of light i.e. such as a laser beam irradiates materials,

photons will be scattered. A major fraction of scattered light that has the e same

wavelength, as the laser light is known as Rayleigh scattering. Raman scattering results

from a wavelength of scattered change of photons, due to inelastic collision between the

sample and the incident photons (Figure 1). Photons shift to a longer wavelength (Stokes

line) when molecules gain energy in Raman scattering, while shorter wavelengths (Anti-

Stokes line) result when molecules lose energy. The properties such as vibrations,

arrangements of atoms in a molecule, and strength of chemical bonds between atoms

result in a vibrational spectrum for a given molecule (Boyaci et al., 2015)

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Figure 6.1. Energy level diagram of Rayleigh and Raman scattering adapted from

Boyaci et al., 2015

In Figure 6. 1, scattering intensity (photons per second) is shown against wavelength in

nanometres or Raman shift in reciprocal centimetres. The bands in the spectrum describe

a Raman shift due to light energy (McCreery, 2001). In other words, the bands are known

to correspond with the specific vibrations of molecular bonds as well as chemical groups.

Thus, a fingerprint of a molecule can be determined with Raman spectroscopy, using a

single method. The intensity of the band at a particular wavenumber is proportional to

the concentration of the molecular groups in the sample. By using normalisation methods

and standards, a quantitative analysis can also be studied by Raman spectroscopy (Yang

et al., 2011).

155

6.1.2 Instrumentation

6.1.2.1 Fourier Transform Raman (FT-Raman) spectroscopy

Figure 6.2 A schematic diagram of a typical Fourier transform (FT) - Raman

spectrometer where D= detector; L= laser; MI= Michelson interferometer; O= objective

lens; RF= Rayleigh filter; S= sample adapted from Li et al., 2014

Figure 6.3 FT-Raman spectrophotometer used for the assignments and quantification

of the bands in the protein

156

Fourier transform (FT) - Raman spectrometer uses a near infrared excitation source

and was first commercially available in 1987. A typical FT Raman instrument is

illustrated as in Figure 6.2. The beam from laser (L) is focused onto sample (s) by 180°

backscatter geometry. After passing through lens (O) The scattered radiation goes

through the Michelson interferometer (MI). The output is focused on either a liquid

nitrogen cooled or room temperature as detector (D). The Raman scattered light must

be optically filtered (RF) before reaching the detector, to reduce the Rayleigh

scattering. The FT-Raman spectrometer used in these experiments used a 1064 nm

excitation of the laser which provided a very good, spectral resolution and accurate

shifts in wavelength.

6.1.3 Application of Raman Analysis in food

There are several ways to study molecular alterations of food components. These studies

are necessary is due to the importance of determining food composition which

contributes to the eating quality, nutritional, and safety properties and economic value of

the end products. It is important to monitor changes in food components and detect any

problems that occur during the processing and production of food. Therefore, there has

been a quite demand in the use of methods such as Infra-Red and Raman spectroscopy

for quantitative and qualitative assessment of food ingredient and products.

Many food protein studies report the application of Raman spectroscopy in the

characterisation of hydrophobic, disulphide and amide groups of proteins. Specifically,

the study of secondary structure of proteins has received much attention (Howell and Li-

Chan 1996, Badii and Howell, 2002). This determination of secondary structure includes

157

the quantification of amide I (1645-1685 cm-1) and amide III (1200-1350 cm-1) bands.

The secondary structure namely α-helix, β-sheets, and turns as well as random coil can

been characterised through bands assigned to these structures. The characteristic

absorbance bands in food system are as shown in Table 6.1

Table 6.1. Infrared and Raman characteristic group frequencies (Nauman et al., 1991 and

Piot et al., 2000) adapted from Thygesen et al., 2003

Raman spectroscopy also has been used to monitor the reactions that occurs in a food

matrix in different processing techniques. This includes studies on the heat gelation

process of whey proteins (Nonaka, Li-Chan and Nakai 1993; Howell and Li-Chan 1996).

A study on rheological changes and interactions of egg albumin and whey protein also

found increase in beta-sheet and a decrease in alpha-helix with heat processing and gel

formation. Changes in hydrophobic and disulphide bands were also noted (Howell and

158

Li-Chan 1996; Ngarize et al., 2004). FT-Raman spectroscopy has also been used in a study

by Sanchez-Gonzalez et al., (2008) to determine the structural changes in proteins and

water during gelation of fish surimi.

The changes was detected by the monitoring of Amide bands and amino acid residue

bands. Several studies have been reported by Badii and Howell (2002); Sarkardei and

Howell (2007); Badii and Howell (2003) on the changes in secondary structure and

hydrophobic groups and amino acids due to biochemical changes during processing and

storage in frozen and dried fish.

Other application of Raman spectroscopy in food components, other than proteins

includes, characterisation of carbohydrate (Delfino et al., 2011; Ilaslan et al., 2015;

Mutungi et al., 2012; and Roman et al., 2011 ), lipid (Sadeghijorabchi et al., 1990; Silveira

et al., 2009 ), vitamins and mineral in (Rimai et al., 1971; Tsai and Morris, 1975); food

adulteration, food additives, toxins and chemicals (Brandt et al., 2005; Sortur et al., 2006)

as well as studies of micro-organisms and viruses (Rosch et al., 2003; Yang and

Irudayaraj, 2003).

The aim of this study was to characterise the proteins resulting from filleting waste from

Mackerel and for mixed fish streams.

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6.2.1 Materials

Atlantic Mackerel and mixed fish waste (FW) samples were purchased from the fish

industry processing plant from The Fish Society, Wormley, Surrey, GU8 5TG, UK. The fish

samples were despatched in ice directly to the Food Science laboratory. The fish waste

samples were processed upon the arrival to the lab, weighed and homogenized in an

Omni Mixer-CAMLAB) at 6000 rpm for 30 seconds in ice. All chemicals used were bought

from Fisher Scientific, Loughborough, UK and Sigma Aldrich, Poole, UK.

6.2.2 Methods

6.2.2.1 Sample preparation for FT Raman spectroscopy

Freeze-dried sample stored at 40°C were placed in 7 mL glass containers (FBG- Anchor,

Cricklewood, London) in a Perkin- Elmer System 2000 FT-Raman spectrophotometer

with excitation from a Nd: YAG (Neodymium doped yttrium aluminium garnet ) laser at

1064 nm. The instrument was calibrated for frequency using the sulphur line at 217 cm-

1. The average of 64 scans was used and the baseline was corrected; scans were smoothed

and normalised to the intensity of the phenylalanine band at 1004 cm-1 (Howell et al.,

1999b; Li-Chan et al., 1994). The spectra were analysed using Grams 32 (Galactic

Industries Corp., Salem, NH).

Using literature values, assignments of the bands in the spectra to protein vibrational

modes were made (Howell et al., 1999; Careche et al., 1999; Li-Chan et al., 1994).

160

Since the intensity of phenylalanine shows a strong band at 1004 cm-1 and it is known to

be unaffected by changes in the microenvironment, this band was used as an internal

standard for normalisation (Tu, 1986). The results are presented as mean ± standard

deviation for relative peak intensity of the spectra bands.

6.3 Statistical analysis

Statistical analysis by one way analysis of variance (ANOVA) was carried out. Differences

between pairs of means were assessed on the basis of confidence intervals by using Least

Significant Difference (LSD) test, followed by the t-test was performed using the SPSS

package version 16. All values represent means of triplicate analysis and are given with

standard deviations and those at p<0.05 were considered significant.

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6.4 Results and discussion

Atlantic Mackerel and mixed fish waste protein were prepared and analysed by Raman

spectroscopy in triplicate gave rise to spectra that are displayed and presented as mean

± standard deviation for relative peak intensity of the spectral bands (Table 6.2).

According to Tu 1986, the aromatic amino acid phenylalanine showed a strong band at

1007 cm-1. Since the intensity of the band is not affected by external factors, it was used

for normalising the spectrum.

Figure 6.4 FT-Raman spectra in freeze dried Atlantic Mackerel and mixed fish waste

162

Table 6.2 Relative peak intensity values of Raman spectra of Atlantic Mackerel and Mixed

Fish waste streams

Peak assignment (wavenumber ±2cm-1)

Relative Peak Intensity of

FWS

Atlantic

Mackerel

Mixed fish

Trp (760) 0.35 ±0.06a 0.53±0.11a

Tyr (830, 855) 0.53± 0.01a 0.70±0.03a

Helix C-C stretch, CH3 Symmetric stretch (937) 0.49±0.08a 0.93±0.05a

B-sheet type structure (990) 0.35±0.19a 0.49±0.12b

Phe, ring band (1034) 0.55±0.12a 0.94±0.13a

Isopropyl ant symmetric stretch CH stretch back

bone (1128)

0.53±0.13a 0.71±0.005a

CH3 ant symmetric (aliphatic) CH3 rook (aromatic)

(1160)

0.28±0.20a 0.71±0.01a

B-sheet type (1239) 0.52±0.19a 2.28±0.19a

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Amide III (random coil) (1245) 0.52±0.18a 2.65±0.06a

Amide III (1264) 0.73±0.1278a 3.13±0.63a

Amide II (1320) 0.96±0.09a 2.42±0.12a

H band double from trp (1340) 0.96±0.12a 2.40±0.31a

(sh*, residue vibration) asp, glu, lys (1425) 0.68±0.20a 2.90±0.13b

Aliphatic groups, CH bend (1451) 1.90±0.39a 4.95±0.06b

Trp (1554) 0.31±0.11a 1.20±0.16a

Amide I (1660) 0.69±0.29a 4.15±0.04a

CH stretch, aliphatic (2940) 5.64±1.95a 9.78±1.01a

Shoulder (2888) 2.54±1.01a 4.38±0.42a

Shoulder (2976) (2969) 3.64±1.23a 5.71±0.06a

Each values is a mean of three replicate determinations and is reported with its standard deviation.

a-b Means within a column with different letters are significantly different (P<0.05)

From the results obtained, both Atlantic Mackerel and Mixed fish show significantly different spectra as the mixed fish contained different species (p< 0.05).

Sh* Shoulder

Protein structure of fish wastes were investigated by monitoring the Tyrosine doublet

bands at 830 and 850cm-1. There were differences in the tyrosine residue vibrations

which are related to hydrogen bond formation. They are useful in monitoring external

factors around tyrosine residues, and a high ratio I855/I830 of 0.90-1.45 indicates that the

164

tyrosine residue is exposed whereas a low ratio indicates strong hydrogen bonding (Li-

Chan et al., 1994). Relative peak intensity showed ratio I855/I830 values of 0.53 and 0.70

for Atlantic Mackerel and mixed fish respectively, indicating strong hydrogen bonding in

both samples. This has been reported by Badii and Howell (2002) and Shih et al., (2003),

if the ratio range of 0.5-1.0, it indicates there is a participation of H-bonding of hydroxyl

group in tyrosine residues which may be due to protein denaturation.

Both fish waste protein sample also showed a strong band around 1425cm-1 which are

0.68 in Atlantic Mackerel and 2.90 for Mixed fish respectively (P<0.05); assigned to the

ionized carboxyl group (COO-) vibration of aspartic and glutamic acids.

A huge difference was observed in the aliphatic CH2 and CH3 groups, which are the

differences in the hydrophobic groups. The CH stretching bands of aliphatic amino acids

were located at 1451 cm-1 with relative intensity of 1.90 and 4.95 for Atlantic Mackerel

and mixed fish waste protein respectively, indicating greater hydrophobic groups in the

mixed fish sample.

The bands around 760 and 1554 cm-1 come from indole-ring vibrations of tryptophan

residues and showed relative intensities of (0.35, 0.31) and (0.53, 1.20) for Atlantic

Mackerel and mixed fish waste sample respectively. Atlantic Mackerel sample result

indicates that exposure of the hydrophobic groups and shows the presence of

hydrophobic interactions in the sample.

Frushour and Koening (1975) stated that the location of the Amide I band is related to

hydrogen bonding and protein conformation. In this study, the relative peak intensities

for Amide I and III bands showed differences in the secondary structure of proteins,

Amide I band typically shows alpha helix, beta sheet and coil structures as shoulders in a

broad peak that can be convoluted to quantify the exact amount of each component. From

165

the results obtained in the study, there was a strong peak around 1660 cm-1 especially in

mixed fish sample, with the intensity of 4.15 compared to 0.69 in the Atlantic Mackerel

sample.

6.5 Conclusion

The proteins in mackerel and mixed fish waste stream were characterised by Raman

spectroscopy and showed significant differences in few of their respective spectra and

assigned peaks (P<0.05). The FT- Raman spectroscopy spectra shows that there were

substantial changes in the protein structure of fish waste samples. For example, a tyrosine

ratio of 0.5 to 1.0 indicates that hydroxyl groups are distributed in a strong hydrogen

bond which defines cross-linking and protein denaturation. In addition, the results

indicated hydrophobic interactions that could also result in protein denaturation. The

denaturation may be caused by the time taken for filleting and processing; freeze drying

and a storage temperature of 40 °C that can lead to changes in protein structures (Badii

and Howell, 2001). In order to maintain the quality of fish muscle proteins, antioxidants

can be added in order to avoid denaturation of protein structure and subsequent effect

on the nutritional and eating quality.

Future work can be employed in order to study the chemical composition of fish waste

protein as well as lipid using Raman spectroscopy. The study include the effect of

antioxidants on the protein and lipid structure of fish waste during frozen storage.

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Chapter 7

167

7 General Discussion

7.1 Aims and Objectives

The objectives of this study were to recover proteins from Atlantic Mackerel (Scomber

scombrus) and mixed fish filleting waste streams recovered by membrane separation, and

to study their physico-chemical properties. Several ultrafiltration processing parameters

including Trans membrane pressure (TMP) on flux excursion, protein performance

scalability study, and mass transfer analysis and hold up volume of membrane were

studied. The nutritional properties of the waste stream products were determined. Fish

waste protein samples were hydrolysed enzymatically using pepsin and pancreatin, and

the antioxidant properties were determined for lipid oxidation inhibition activity and

radical scavenging activities by DPPH and FRAP assays. The rheological and

thermodynamic properties of fish waste proteins were analysed by small deformation

rheology and differential scanning calorimetry methods respectively. The proteins in

mackerel and mixed fish waste streams were characterised by FT-Raman spectroscopy.

The above aims were fulfilled and are discussed in detail below.

7.2 Fish Waste Stream Quality

In this study, the nutritional and functional properties of fish waste, obtained from a local

filleting plant, were examined. Proximate studies on fish waste (FW) samples compared

to the Atlantic mackerel (Scomber scombrus) and Nile perch fish (Lates niloticus) fish fillets

(FF) showed that ash content was higher in FW (9.74 %) compared to FF probably due

to the presence of larger amounts of inorganic components e.g. bone in the waste stream.

A high percentage of total lipids (6.12 %), protein (24.31 %) was also obtained for FW

168

compared to Atlantic Mackerel and Nile Perch FF Fish waste (FW), moisture content was

60.13 %.

In contrast, Nile Perch FF chemical composition showed 80.4 % moisture, a higher value

compared to the Atlantic Mackerel with 70.3 % moisture. Nile perch FF comprised 1.9 %

total lipids, 16.3 % protein and 0.5 % of ash. Atlantic Mackerel in this study had higher

(p<0.05) total lipids (11.3 %), protein (19.3 %) and ash (1.1 %) compared to Nile Perch.

Atlantic mackerel is considered to be an oily fish and this was confirmed with the high

lipid content (11.3 %) compared to the Nile Perch (1.9 %). The proximate analysis of

Atlantic mackerel, Nile Perch and fish waste streams obtained in this study showed that

the values were in the range obtained in previous studies (Okeyo et al., 2009; FAO, 2014

and Esteban et al., 2007).

Fatty acids composition of the fish waste sample was high in monounsaturated fatty acids

(MUFA) (40.7%), followed by saturated fatty acids (SFA) (31.6 %) and polyunsaturated

fatty acids (PUFA) (5.4 %). In Atlantic Mackerel fish fillets, PUFA constituted most of the

fatty acids, followed by SFA, and MUFA. Palmitic acid (C16:0) was the major fatty acid

followed by stearic acid (C18:0) and myristic acid (C14:0). PUFA (mainly EPA (C20:5) and

DHA (C22:6) comprised the biggest fraction in the mackerel lipid profile, confirming

previous reports (Guizani et al., 2015). For both Atlantic mackerel and Nile perch FF as

well as fish waste, palmitic acid was among the most abundant fatty acid and this is

because palmatic acid is known to be a key metabolite in fish species. Fish oil is very

important in the human diet and has been reported to reduce the risk of cardiovascular

disease (Simopoulos, 2008; Kris-Etherton et al., 2002).

169

7.3 Production of hydrolysate and ultrafiltration studies to obtain

water soluble protein fraction (3 and 10 kDa) from fish waste stream


digestion using pepsin and pancreatin in order to mimic the digestive process in the

human body and achieve a high degree of hydrolysis that is greater than that produced

when using either pepsin or pancreatin alone(Mäkinen et al., 2012 and Ena et al.,1995).

A study to examine the effect of Transmembrane pressure (TMP) on flux excursion shows

that the flux rate increased as the TMP increased thus affecting the feed flux (pump

speed). The optimum point was obtained by calculating the optimum feed flow against

the required pump rate at each of the two feed flow conditions. A study by Kumar et al.

(2004) on ultrafiltration of soy protein concentrate shows that the effect of TMP and

crossflow velocity on flux for tubular module was with 5% soy flour. Other study by

Casey et al.(2011) used a single –pass tangential flow filtration (SPTFF) process for

concentrating their protein sample in continuous mode of operation, produced cycles

with high concentration, recovery of product as well as cleaning for storage; all of it

requires carefully observation on TMP against flux excursion.

In protein performance scalability study, in permeate flux of Pellicon 3 Ultracell of 3 and

10 kDa membrane through different BSA protein concentrations, full polarization

occurred in 20 and 40 g/L of BSA solutions, compared to the 10 g/L of solution. A smaller

cut off (3 kDa) resulted in a lower flux rate, and higher limiting flux performance at series

of TMP excursions, compared to the 10 kDa membrane. The protein performance in both

membranes also showed a good consistency. This trend of results for all three

concentrations agree with the standard protein Flux performance analysis, and proves

170

that the device used for the study meets the requirement set by the manufacturer. The

standard method used a ± 10 % range and a 0.11 m2 device as a baseline.

The same trend was observed in both 3 kDa and 10 kDa fractions of fish processing waste

streams (FWS) protein, where the flux increased along the difference in trans membrane

pressures, in three difference concentrations (10, 20 and 40 g/L) protein samples. The

limiting flux was observed at 60 psi. Comparing the size of the membrane cut off, 3 kDa

membrane showed a smaller flow rate compared to the 10 kDa membranes; both

membranes were treated with the same three concentrations. Full polarization trends

were evaluated as in the BSA protein model systems but the values for all data were

significantly lower in fish waste proteins compared to BSA. Furthermore, the flow rate of

FWS samples through both membrane were higher than in BSA sample solutions. The

same operating temperature of 25°C after limiting flux was normalized and mean cross

flow rate was controlled to 295 LMH (5 L/min/m2).

As for the mass transfer analysis, the mean mass transfer coefficients obtained in both

membranes were within one standard deviation of each other. The virial expansion

coefficients of α and β are the same for both membrane size which is α = 3.81 x 107 and

β = 9.99 x 105 which are the standard values for the BSA protein. Better standard

deviation values in 10 kDa membrane compared to 3 kDa were obtained, showing that

the polarized protein flux movements back into the bulk solution of 10 kDa is higher than

in 3 kDa cut-off membrane; this reflects the transmembrane pressures which help in a

build-up of concentration at the surface of membrane, thus allowing the osmotic pressure

to send the protein back to the bulk fluid.

In the hold-up volume study, the values obtained are in agreement with the standard

values given by the manufacturer (Merck Millipore, 2014). The difference in membrane

171

size indicates that the smaller molecular cut off (3 kDa) gives smaller values in the

permeate channel, likewise the 10 kDa membrane. The values also gave some

information for the system used to estimate the volumes of samples needed and expected

feed and permeate volume obtained when using different membrane sizes for different

samples. In this study, there was a slight membrane fouling phenomenon when using

protein samples, compared to using just water, in the determination of the hold-up

volumes. Larger molecules could not pass through smaller membrane size, thus creating

membrane fouling and limiting the volume of permeate, but allowing more volume of

retentate. This fouling was removed by thorough cleaning of the membranes after UF

(Nataraj et al., 2008).

7.4 Antioxidant properties by lipid peroxidation inhibition (FTC and

TBARS) and radical scavenging activity (2, 2- Diphenyl-1 picryhydrazyl

DPPH radical scavenging and ferric reducing antioxidant power assay)

in the fish from waste streams (Atlantic mackerel and mixed fish)

The measurement of lipid oxidation inhibition activity by FTC and TBARS to analyse the

antioxidant activity of the fish waste water-soluble waste hydrolysates. FTC was the

method used to monitor the formation of peroxides (as primary products from lipid

oxidation) while TBARS was used to monitor the carbonyl compounds (as secondary

products from lipid oxidation). Fish waste water protein hydrolysate especially for

Atlantic Mackerel showed good antioxidant activities by the Ferric Thiocyanate (FTC) and

Thiobarbituric acid Reactive substances (TBARS) methods and compared well with other

172

antioxidants (BHA, ascorbic acid and trolox). There was significant difference (p<0.05)

between samples and negative control (no antioxidant).

DPPH scavenging activity increased with the extract concentration in the range of 1.5-26

%. The DPPH scavenging activity in mixed fish waste hydrolysate was slightly higher than

in Atlantic Mackerel fish waste as there sample increased in concentration. The statistical

data however shows that there was no significant difference between two samples

(p>0.05). The antioxidative properties of protein hydrolysates depended on the sequence

of the amino acid residue released by enzymess. Thus, the specific enzymes used and the

condition during hydrolysis (Elavarasan et al., 2014) is important. Although DPPH

activity of the fish waste water protein hydrolysates was low, this study shows that the

samples may scavenge free radicals and with further analysis and specific selection on

proteases and control of the degree of hydrolysis, fish processing waste water protein

hydrolysates may be considered as natural antioxidants.

In the FRAP assay, both samples showed that absorbance increased with concentration.

Mixed fish FW showed an increase in absorbance with concentrations up to 1000 µg/mL,

while Atlantic Mackerel FW showed an increase in absorbance up to 750 µg/mL followed

by a decrease which then remained constant. The difference might be because of specific

peptide/ amino acid composition and the nature of enzyme used (Gajanan et al., (2016).

Jayaprakash et al. (2001) stated that the relation between reducing power and

antioxidant activity is proportional; it is clear that the antioxidant activity of mixed fish

waste protein hydrolysate is higher than in Atlantic Mackerel fish waste protein

hydrolysate.

Recent publications on peptides derived from enzymatic fish protein hydrolysis and their

antioxidant activity include those by Halim, Gajanan et al., (2016), Lassoued et al., (2015)

173

and Howell and Kasase (2010). Most studies shown significant activities of various

oxidation systems. The most popular antioxidative assays are DPPH, free radical

scavenging, hydroxyl radical scavenging, Thiobarbituric acid reactive substance (TBARS),

and ferric reducing antioxidant power (FRAP), lipid peroxidation inhibition activity, and

linoleic acid peroxidation inhibition.

7.5 DSC and rheological properties of the fish waste (Atlantic mackerel,

Mixed fish, 10KD Atlantic mackerel and mixed fish protein hydrolysates)

Protein denaturation can be affected by temperature and pressure or chemically by the

employment of strong acid or base, concentrated inorganic salts as well as organic

solvents (Alberts et al., 1994; Howell and Li-Chan, 1996) on chemical and enzymatic

modification of proteins (Stryer L, 1988).

DSC analysis was undertaken on waste obtained from Atlantic Mackerel and mixed fish

as well as hydrolysates samples prepared from fish waste (10 kDa fraction of Atlantic

Mackerel and 10 kDa fraction mixed fish). The DSC thermograms from the samples

indicated that they were comparable. By investigating the denaturation peaks enthalpies

(J/g) and denaturation temperatures (°C) (Table 5.1), the t-test of significance shows that

there was no significant difference between the two types of fish waste samples (p> 0.05).

The denaturation enthalpy for myosin in Atlantic mackerel (0.349 J/g) was higher at the

transition temperature (TM) of 49.3 °C than mixed fish (0.286 J/g at 47.4 °C). For the actin

denaturation enthalpies, mixed fish had the highest enthalpy change values (0.12 J/g)

compared to the Atlantic Mackerel (0.055 J/g) and there was no significant difference

between samples (p>0.05). Sarcoplasmic proteins in mixed fish waste sample denatured

174

more readily with a lower enthalpy change value than Atlantic Mackerel fish waste

protein. For the DSC thermograms of fish waste sample hydrolysates, the same trends

were obtained in 10 kDa AM and MF. Atlantic Mackerel showed higher denaturation

enthalpy changes for myosin (0.69 J/g) and lower denaturation of actin (0.024 J/g) than

mixed fish (0.202 J/g for myosin and 0.12J/g in actin) shows that there is significant

difference (p<0.05) between samples.

Rheology is regarded as a key method for monitoring in food processing, quality control

as well as shelf life of food materials (Ötles, 2011). Rheological properties of gels and

emulsions can give very interesting information that can be used to assess storage

stability, sensory evaluation, texture and quality control of products such as in

mayonnaise and salad dressing (Davis, 1973).

The G’ (storage modulus) of Atlantic Mackerel fish sample were considerably higher at

90°C (18399 Pa) and when cooled to 20oC (G’=1.02E+05 Pa) compared with water and

salt soluble samples at 90°C (G’= 7.62 and 6.15 Pa respectively) and 36.82, 70.93 Pa

respectively when cooled at 20°C (p<0.005). Meanwhile, the G’ (storage modulus) of salt

soluble mixed fish processing waste streams protein was higher at 90°C= 5.0268 Pa and

when cooled to 20oC (G’=215.97Pa) compared to the water soluble sample at 90° C (G’=

3.67Pa) and 17.63 when cooled at 20°C (p<0.05). As G’ is the energy stored elastically in

the structure, this shows that Atlantic Mackerel sample was tougher than the mixed fish.

The difference between the G’ values of the water-soluble (small proteins, peptides) and

salt soluble proteins (high MW myosin and actin) that can form 3D gel networks

influences the rheological properties.

Few papers report changes in water holding capacity of fish due to surface dehydration

of proteins as well as the relationship between protein solubility of muscle and

175

toughening of tissue during frozen storage fish (Badii and Howell, 2002). Thus, further

work can be done on moisture content and protein solubility of fish proteins (myofibrillar

protein and other mixture of protein components) alongside the small deformation

testing.

7.6 FT-Raman spectroscopy characterisation of fish proteins from Mackerel

and Mixed fish waste streams

The applications of Raman spectroscopy in food components, other than proteins

includes, characterisation of carbohydrate (Delfino et al., 2011; Ilaslan et al., 2015;

Mutungi et al., 2012; and Roman et al., 2011 ), lipid (Sadeghijorabchi et al., 1990; Silveira

et al., 2009 ), vitamins and mineral in (Rimai et al., 1971; Tsai and Morris, 1975); food

adulteration, food additives, toxins and chemicals (Brandt et al., 2005; Sortur et al., 2006)

as well as studies of micro-organisms and viruses (Rosch et al., 2003; Yang and

Irudayaraj, 2003).

Protein structure changes after freeze drying fish wastes were investigated by FT Raman

spectroscopy. In particular, by monitoring the tyrosine doublet bands at 830 and 850 cm-

1. There were differences in the tyrosine residue vibrations which are related to hydrogen

bond formation. They are useful in monitoring external factors around tyrosine residues,

and a high ratio I855/I830 of 0.90-1.45 indicates that the tyrosine residue is exposed

whereas a low ratio indicates strong hydrogen bonding (Li-Chan, 1994). Relative peak

intensity showed ratio I855/I830 values of 0.53 and 0.70 for Atlantic Mackerel and mixed

fish respectively, indicating strong hydrogen bonding in both samples. This has been

reported by Badii and Howell (2002) and Shih et al., (2003), if the ratio range is 0.5-1.0,

176

it indicates that there is participation of H-bonding of hydroxyl groups in tyrosine

residues which may be due to protein denaturation.

Both fish waste protein samples also showed a strong band around 1425 cm-1 assigned

to the ionized carboxyl group (COO-) vibration of aspartic and glutamic acids.

A large difference was observed in the aliphatic CH2 and CH3 groups, which are the

differences in the hydrophobic groups. The CH stretching bands of aliphatic amino acids

were located at 1451 cm-1 with relative intensity of 1.90 and 4.95 for Atlantic Mackerel

and mixed fish waste protein respectively, indicating greater hydrophobic groups in the

mixed fish sample.

The bands around 760 and 1554 cm-1 come from indole-ring vibrations of tryptophan

residues and showed relative intensities of (0.35, 0.31) and (0.53, 1.20) for Atlantic

Mackerel and mixed fish waste sample respectively. Atlantic Mackerel sample result

indicates that exposure of the hydrophobic groups and shows the presence of

hydrophobic interactions in the sample.

Frushour and Koening (1975) stated that the location of the Amide I band is related to

hydrogen bonding and protein conformation. In this study, the relative peak intensities

for Amide I and III bands showed differences in the secondary structure of proteins,

Amide I band typically shows alpha helix, beta sheet and coil structures as shoulders in a

broad peak that can be convoluted to quantify the exact amount of each component. From

the results obtained in the study, there was a strong peak around 1660 cm-1 especially in

mixed fish sample, with the intensity of 4.15 compared to 0.69 in the Atlantic Mackerel

sample.

177

Several studies have also been reported by Badii and Howell (2002); Sarkardei and

Howell (2007); Badii and Howell (2003) on the changes in secondary structure and

hydrophobic groups and amino acids due to biochemical changes during processing and

storage in frozen and dried fish.

7.7 General conclusions

Both Atlantic mackerel and Nile Perch contain substantial amounts of protein,

lipids and minerals which make them very desirable food sources. FW contains

high protein and fat, as well minerals and could be used as an alternative to fish

flesh for the supply of oil and valuable protein.

Ultrafiltration was found to be a mild and efficient process for separating valuable

proteins from fish waste streams, and compared well to the model BSA protein.

The potential for this technique in recovering cheap waste proteins with a

valuable nutritional and functional quality in many different food and

pharmaceutical products is very good, although it is expensive.

The protein hydrolysate obtained from Atlantic mackerel (Scomber scombrus) fish

waste streams and mixed fish waste streams using pepsin and pancreatin

enzymes shows antioxidant potential.

Fish waste samples especially hydrolysed fish waste samples, showed different

protein denaturation transition peaks, indicating that enzymatic hydrolysis can

affect the thermodynamic and functional properties of protein samples. Similarly,

the rheological properties were different for different fish samples (AM and MF)

with AM showing higher G’ or elastic modulus values. The salt soluble actomyosin

fraction had significantly higher G’ values than the water soluble fraction (p

<0.05). As G’ is the energy stored elastically in the structure, this shows that

Atlantic Mackerel sample was tougher than the mixed fish.

178

The proteins in mackerel and mixed fish waste streams were characterised by

Raman spectroscopy and showed significant differences in their respective

spectra and most of the assigned peaks (p<0.05).

7.8 Future work

Determination of the mass transfer study of fish waste streams protein,

measurement of the virial expansion coefficient for FWS protein as well as the

mass transfer coefficient in order to master the principle of mass transfer in

seafood proteins and the parameters so that the values can be used as future

references especially in membrane separation of seafood protein.

Discover more benefits of membrane separation of fish waste protein as well as

bioactive peptides recovered and long term applications to the body of knowledge.

Study the efficiency of protein hydrolysis using other enzymes such as papain, and

study other bioactive properties of hydrolysates such as natural ACE inhibitors

and antioxidants to broaden the types of analysis and determine novel bioactive

peptide from waste of other marine species especially local fish and seafood waste.

Measurement of the amino acid composition analysis by HPLC in order to see if

the different amino acid content in both Atlantic Mackerel and mixed fish

processing waste protein contributed to the gel strength.

Study the moisture content and protein solubility of fish proteins (myofibrillar

protein and other mixture of protein components) alongside the small

deformation testing.

Study the chemical composition of fish waste protein as well as lipids using Raman

spectroscopy. The study could include the effect of peptide antioxidants to

minimise oxidative damage of protein and lipid in fish waste during frozen storage

and drying.

179

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Appendix

213

PAIRED SAMPLE T-TEST

ATLANTIC MACKEREL

FTC

Paired Samples Test

Paired Differences t df Sig. (2-

tailed) Mean Std.

Deviation

Std. Error

Mean

95% Confidence Interval

of the Difference

Lower Upper

Pair

1

CONTROL -

SAMPLE

.1812042 .1335785 .0472271 .0695298 .2928786 3.837 7 .006

Pair

2

CONTROL -

TROLOX

.1819583 .1366824 .0483245 .0676890 .2962277 3.765 7 .007

Pair

3

CONTROL -

BHA

.1839650 .1244123 .0439864 .0799537 .2879763 4.182 7 .004

Pair

4

CONTROL -

AA

.2081708 .1072473 .0379177 .1185098 .2978318 5.490 7 .001

214

ATLANTIC MACKEREL

TBARS

Paired Samples Test


tailed) Mean Std.

Deviation

Std. Error

Mean


of the Difference

Lower Upper

Pair

1

CONTROL -

SAMPLE

.0041501 .0058942 .0020839 -.0007776 .0090778 1.992 7 .087

Pair

2

CONTROL -

TROLOX

.0057483 .0043880 .0015514 .0020799 .0094168 3.705 7 .008

Pair

3

CONTROL -

BHA

.0052608 .0038977 .0013780 .0020023 .0085194 3.818 7 .007

Pair

4

CONTROL -

AA

.0058263 .0034891 .0012336 .0029093 .0087432 4.723 7 .002

215

MIXED FISH

FTC

Paired Samples Test


tailed) Mean Std.

Deviation

Std. Error

Mean


of the Difference

Lower Upper

Pair

1

CONTROL -

SAMPLE

.1712042 .1189539 .0420565 .0717562 .2706521 4.071 7 .005

Pair

2

CONTROL -

TROLOX

.1605725 .1158016 .0409420 .0637599 .2573851 3.922 7 .006

Pair

3

CONTROL -

BHA

.1625500 .1028627 .0363674 .0765547 .2485453 4.470 7 .003

Pair

4

CONTROL -

AA

.1906375 .0829017 .0293102 .1213299 .2599451 6.504 7 .000

216

MIXED FISH

TBARS

Paired Samples Test


tailed) Mean Std.

Deviation

Std. Error

Mean


of the Difference

Lower Upper

Pair

1

CONTROL -

SAMPLE

.0024299 .0061734 .0021826 -.0027312 .0075909 1.113 7 .302

Pair

2

CONTROL -

TROLOX

.0050854 .0078006 .0027579 -.0014361 .0116069 1.844 7 .108

Pair

3

CONTROL -

BHA

.0051862 .0074117 .0026204 -.0010102 .0113826 1.979 7 .088

Pair

4

CONTROL -

AA

.0051521 .0072983 .0025803 -.0009494 .0112536 1.997 7 .086

217

STATISTICAL DATA

DPPH

DSC

AM VS MF

t-Test: Two-Sample Assuming Equal Variances

Variable 1 Variable 2

Mean 17.73666667 10.58

Variance 604.0670333 168.3499

Observations 3 3

Pooled Variance 386.2084667

Hypothesized Mean Difference 0

df 4

t Stat 0.446010953

P(T<=t) one-tail 0.339333746

t Critical one-tail 2.131846786

P(T<=t) two-tail 0.678667492

t Critical two-tail 2.776445105

t-Test: Two-Sample Assuming Unequal Variances


Mean 0.103 0.1015

Variance 0.02748 0.018329

Observations 4 4


df 6

t Stat 0.014016702

P(T<=t) one-tail 0.494635554


P(T<=t) two-tail 0.989271107


218

AM VS 10 kDa AM

MF VS 10 kDa MF

t-Test: Paired Two Sample for Means


Mean 0.103 0.18025

Variance 0.02748 0.115588

Observations 4 4

Pearson Correlation 0.993128893


df 3

t Stat -0.875747762

P(T<=t) one-tail 0.222815664


P(T<=t) two-tail 0.445631328


t-Test: Paired Two Sample for Means


Mean 0.1015 0.0805

Variance 0.018329 0.009761

Observations 4 4

Pearson Correlation 0.984097978


df 3

t Stat 1

P(T<=t) one-tail 0.195501109


P(T<=t) two-tail 0.391002219


219

10 kDa AM VS 10 kDa MF

t-Test: Two-Sample Assuming Equal Variances


Mean 0.18025 0.0805

Variance 0.11558825 0.009761

Observations 4 4

Pooled Variance 0.062674625


df 6

t Stat 0.563484573

P(T<=t) one-tail 0.296761421


P(T<=t) two-tail 0.593522842




Mean 0.18025 0.0805

Variance 0.11558825 0.009761

Observations 4 4


df 4

t Stat 0.563484573

P(T<=t) one-tail 0.301597315


P(T<=t) two-tail 0.60319463


220

FT RAMAN



Mean 1.167057895 2.681315789

Variance 1.920762311 5.538571827

Observations 19 19


df 29

t Stat -2.416721558

P(T<=t) one-tail 0.011090355


P(T<=t) two-tail 0.02218071


Physico-Chemical Properties of Fish Proteins Recovered ...epubs.surrey.ac.uk/841238/1/Final Thesis-NR Mohamed Zam 310517.pdfDalysa, parents and family in Malaysia to whom this thesis

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