Extraction and characterization of fish protein isolate from yellowfin (thunnus albacares) dark muscle using ph shift method

MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY LAURINE MULE MUENI 60CH300 EXTRACTION AND CHARACTERIZATION OF FISH PROTEIN ISOLATE FROM YELLOWFIN Thunnus albacares DARK MUSC

MINISTRY OF EDUCATION AND TRAINING

NHA TRANG UNIVERSITY

LAURINE MULE MUENI

60CH300

EXTRACTION AND CHARACTERIZATION

OF FISH PROTEIN ISOLATE FROM YELLOWFIN (Thunnus albacares)

DARK MUSCLE USING pH-SHIFT METHOD

MASTER THESIS

KHANH HOA – 2020

MINISTRY OF EDUCATION AND TRAINING

NHA TRANG UNIVERSITY

LAURINE MULE MUENI

60CH300

EXTRACTION AND CHARACTERIZATION

OF FISH PROTEIN ISOLATE FROM YELLOWFIN (Thunnus albacares)

DARK MUSCLE USING pH-SHIFT METHOD

MASTER THESIS

Decision on establishing the Committee:

UNDERTAKING

I undertake that the thesis entitled: “Extraction and characterization of fish

protein isolate from Yellowfin (Thunnus albacares) dark muscle using pH-shift method” is my own work The work has not been presented elsewhere for assessment

until the time this thesis is submitted

26 September 2020

Laurine Mule Mueni

FUNDING

This research was funded by National Foundation for Science and Technology Development - Ministry of Science and Technology of Vietnam in the Nafosted project with No 106.99-2018.42

In addition, I would like to thank the National Foundation for Science and Technology Development - Ministry of Science and Technology of Vietnam in the Nafosted project with No 106.99-2018.42 for financial support to do the research

My special and deep appreciations go to my supervisors Dr Nguyen Trong Bach and Dr Bui Tran Nu Thanh Viet for their continuous support of my master‟s studies and research, for their patience, motivation, enthusiasm, and immense knowledge Their guidance helped me in all the time of research and writing of this thesis

Last but not the least; I would like to thank my family: my guardian, my friends, classmates and colleagues for supporting me spiritually, mentally and physically throughout writing this thesis

26 September 2020, Nha Trang

Laurine Mule Mueni

TABLE OF CONTENTS

UNDERTAKING iii

FUNDING iv

ACKNOWLEDGMENT v

TABLE OF CONTENTS vi

LIST OF ABBREVIATIONS ix

LIST OF TABLES x

LIST OF FIGURES xi

ABSTRACT xiii

GENERAL INTRODUCTION 1

PROBLEM STATEMENT 2

CHAPTER 1 : BACKGROUND 4

1.1 Tuna fish and its by-products 4

1.1.1 Tuna productions 4

1.1.2 The composition of yellowfin dark muscle 5

1.1.2.1 Myofibrillar proteins 7

1.1.2.2 Sarcoplasmic proteins 8

1.1.2.3 Lipids 8

1.1.3 Tuna by-product 10

1.2 Factors affecting the utilization of TDM 11

1.2.1 The dark colour and Myoglobin oxidation 11

1.2.2 Lipid and protein oxidation 13

1.2.3 Histamine 15

1.3 Protein extraction techniques used and FPI utilization 16

1.3.1 pH –shift method 17

1.3.2 Protein recovery 19

1.3.3 By-products and FPI utilization 20

1.3.4 Fish protein isolate characterization 21

CHAPTER 2 : MATERIALS AND METHODS 24

2.1 Materials collection and preparation 24

2.1.1 Dark muscle 24

2.1.2 Chemicals 24

2.1.3 FPI extraction by pH-shift method 24

2.2 Methods of characterization 26

2.2.1 Fourier transform infrared (FTIR) spectroscopic analysis 27

2.2.2 Zeta potential 28

2.2.3 Light scattering 29

2.2.4 Turbidity measurements 31

2.2.5 Determination of the protein concentration with UV-Visible spectroscopy 31

2.2.6 Confocal laser scanning microscopy 32

2.2.7 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS- page) 33

2.2.8 Viscosity measurement 36

2.2.9 Data analysis 37

CHAPTER 3: RESULTS AND DISCUSSIONS 38

3.1 FPI extraction 38

3.2 Characterization of TDMPI 39

3.2.1 Amino acids 39

3.2.2 Fourier-Transform Infrared Spectroscopy 41

3.2.3 Size-Exclusion Chromatography Analysis of TDMPI 43

3.3 Structure and physical properties of TDMPI solution 44

3.3.1 Protein solubility 44

3.3.2 Light scattering 46

3.3.3 Micro-structure 47

3.3.4 SDS-PAGE 50

3.3.5 Zeta Potential of TDMPI solution 51

3.3.6 Viscosity of TDMPI solution 54

CONCLUSION AND OUTLOOK 57

REFERENCES 59 APPENDICES I Appendix 1: Test report of Amino acid in TDM II Appendix 2: Test report of Amino acid in TDM III Appendix 3: Test report of Amino acid in TDMPI IV Appendix 4: Test report of Amino acid in TDMPI V Appendix 5: Determination of histamine in TDM VI Appendix 6: Determination of histamine in TDMPI VII Appendix 7: Effect of pH (using NaOH) VIII

ix

LIST OF ABBREVIATIONS

AA : Amino acid APS : Ammonium persulfate

Cp : Protein concentration

DI : Deionised water DLS : Dynamic Light Scattering EAA : Essential amino acid FPI : Fish protein isolate HMW : High molecular weight LMW : Low molecular weight LOD : Limit of detection

LS : Light scattering MHC : Myosin heavy chain MLC : Myosin light chain

MW : Molar mass NEAA : Non-essential amino acid

pI : Isoelectric point

Rg : Radius of gyration

Rh : Hydrodynamic radius

RI : Refractive index RPM :Revolutions per minute SLS : Static Light Scattering TAA : Total amino acid TEMED : Tetramethylethylenediamine TDM : Tuna dark muscle

TDMPI : Tuna dark muscle protein isolate TWM : Tuna white muscle

UV : Ultraviolet absorbance

x

LIST OF TABLES

Table 1.1: Proximate composition of the muscle tissue of yellowfin tuna (%, wet basis) 9Table 1.2: Protein recovery yield from processing trout by-products by the isoelectric solubilisation/precipitation technology 20Table 2.1: Recipe for stocking gel and resolving gel 35Table 3.1: Proximate composition of TDM and TDMPI 39Table 3.2: Amino acid composition of TDMPI powder, and reference proteins (dry basis) 40Table 3.3: Effect of salt on pI of TDMPI in different ionic strengths 53

xi

LIST OF FIGURES

Figure 1.1: Yellowfin tuna (Thunnus albacares (Bonnaterre, 1788) 4

Figure 1.2: Raw material production at Hai Vuong Corp (Nha Trang, Khanh Hoa) 5

Figure 1.3: Image of yellowfin tuna muscle 6

Figure 1.4: Reaction of O2 species generated by Fenton reaction (Hultin, 1992) 12

Figure 1.5: The proportion of each fish causing histamine fish poisoning reactions, 1998–2012 (CDC Food Outbreak Online Database) 16

Figure 1.6: pH-shifts process for production fish protein isolate 17

Figure 1.7: Minced tuna dark muscle before treatment (A), after treatment (B) and after heating (C) 22

Figure 2.1: Block (left) and minced TDM (right) 24

Figure 2.2: Freeze dryer Lyobeta 35 (Spain) 25

Figure 2.3: Flow chart of yellowfin tuna dark muscle protein isolation 26

Figure 2.4: TDMPI characterization chart 27

Figure 2.5: FT-IR spectrometer Model Alpha, S/N 201418 (Germany) 28

Figure 2.6: Zeta Nanosizer (Model SZ-100Z_Horiba, Japan) 29

Figure 2.7: ALV-5000, ALV-Langen, Germany 30

Figure 2.8: UV-Visible Spectrometer Libra S50 Bio (Cambridge, England) 31

Figure 2.9: Centrifuge Hettich D-78532 Tuttlingen (Germany) 32

Figure 2.10: Schematic diagram of the optical pathway and principal components in a modern confocal laser scanning microscope 32

Figure 2.11: Confocal microscopy (Zeiss LSM800, Germany) 33

Figure 2.12: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS- page) 36

Figure 2.13: Rheometer Kinexus Pro 50N_ Malvern, (England) 36

Figure 3.1: TDMPI was extracted by pH-shift method 38

xii

Figure 3.2: Fourier Transform Infrared Spectroscopy (FTIR) spectra of TDMPI powder 42Figure 3.3: Size-Exclusion Chromatography spectra of TDMPI with light scattering signal (blue), UV signal (green) and refractive index signal (pink) 44Figure 3.4: TDMPI solution at different pH 44Figure 3.5: Turbidity of TDMPI solutions at different pH 45Figure 3.6: Fraction of protein in the supernatant from the solution of 10g/L TDMPI at different pH after centrifugation in the absence of salt (black), in the presence of 10

mM NaCl (red) and of 10 mM CaCl2 (blue) 45Figure 3.7: q-Dependence of hydrodynamic radius Rh (top left) and molar mass Mw(top right) of TDMPI solutions (5 g/L) at different pHs and Rh, Mw taken at low q-values (took at plateau regime) as a function of pH values (middle) 47Figure 3.8: CLSM images of solution containing 10 g/L TDMPI at different pH The protein aggregates were formed without heating 48Figure 3.9: CLSM images of solution containing 10 g/L TDMPI at different pH The protein aggregates were formed by heating at 80 °C overnight 49Figure 3.10: SDS-PAGE patterns of TDMPI M: Molecular weight standards

(Marker) Lanes 2-12 represent proteins of 10 g/L without salt (a), with added 10 mMNaCl (b), and with added 10 mM CaCl2 (c) at different pH 51

Figure 3.11: Zeta potential of TDMPI as a function of pH 52Figure 3.12: Variation of zeta potential of TDMPI in the presence of 0.005M (a) and 0.01 M (b) salt as a function of pH 53Figure 3.13: Viscosities of different TDMPI solutions at pH 2, pH 7 and pH 12 as a function of shear rate 55Figure 3.14: Viscosities of TDMPI solution at 10 g/L as a function of shear rate at different pH 55Figure 3.15: Viscosities of different TDMPI solutions in the presence of 10 mM CaCl2

as a function of shear rate at neutral pH 56

xiii

ABSTRACT

Yellowfin tuna processing results in about 42.49% by-products of its original weight with tuna dark muscle (TDM) contributing around 6.40% The TDM consists

of protein-enrich amounts However, its use is limited due to the colour, susceptibility

to oxidation and off-flavour contributing to environmental pollution due to its low economic value

Despite the high protein content in TDM, the greatest challenge has been on how to improve the by-product In recent studies, crude protein from TDM has been extracted by physicochemical and biological (use of enzymes) methods, with the aim

of providing high-quality protein sources for humans The focus of the study was therefore to extract tuna dark muscle protein isolates (TDMPI) using alkali-pH-shift and characterizing the isolates in order to develop and diversify products from TDM

to increase the economic and nutritional value of these by-products with elimination

of detrimental properties or ingredients (discolouration, odour, or allergy) Proximate and amino acid composition done to determine the nutritional properties of protein isolates found that TDMPI exhibited low fat and high protein contents (0.66 wt % and 20.10 wt % respectively), and met all essential amino acids (EAA) requirements, with glutamic being predominate17.58 g/100 g protein) Protein secondary structures present were observed by Fourier-Transform Infrared Spectroscopy (FT-IR) The proteins showed low solubility and high turbidity as the pH approached the isoelectric point (pI)(5.5) Actin protein was observed at 42kDa by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-Page) while the zeta potential of the proteins decreased with increasing ionic strength To determine the stability of the protein solution, viscosity measurements were carried out for various pH values, ionic

strength and protein concentration (Cp) It was also noted that the pH of the solution

and the temperature affected the microstructure of protein isolates The results obtained showed that the TDMPI is a promising tool that could successively be used

in value-added food products for human consumption

Key words: pH-shift process, fish protein isolates, Light Scattering, Confocal

Microscopy, SDS-page, zeta potential, viscosity

1

GENERAL INTRODUCTION

Yellowfin tuna (Thunnus albacares, Bonnaterre, 1788) is one of the major tuna

species that is widely distributed in epipelagic waters of the major oceans It is one of the most nutritious and economically important species in the fisheries world that are caught in large quantities Tuna flesh is processed in raw form and is often marketed

as loins/steaks or canned products Besides, tuna products are much diversified; such

as sashimi, frozen, canned or smoked products

Protein-rich by-products from tuna processing such as dark meat have limited use because of their colour, susceptibility to oxidation and off-flavour However, there

is limited recognition of biological resources while the increasing environmental pollution has emphasized the need for better utilization of by-products from fisheries The proximate analysis of crude protein content in tuna dark muscle (TDM) is higher than in tuna white muscle (TWM) with TDM of 28% while TWM consists of 26% This makes TDM a potential material that can be exploited to provide high-quality protein sources for humans

Fish protein isolate (FPI) can be extracted by a pH-shift method where proteins are solubilised at extreme pH values separating soluble proteins, connective tissue, bone, skin, neutral lipids and cellular membranes through a centrifugation step minimizing the risk of lipid oxidation The process employs solubilisation of the proteins using acid and alkali followed by a precipitation process at their isoelectric

pH to recover functional and stable protein isolates from the underutilized tuna products Recently, research has focused on recovering and characterizing protein from TDM which eliminated detrimental properties/ingredients (discolouration, odour, and allergy) The research is to develop and diversify products from TDM so

by-as to increby-ase the economic and nutritional value of these by-products The studies used advanced techniques such as light scattering, SDS-PAGE, confocal microscopy, rheology, zeta-analysis to evaluate morphology, denaturation and electrical property

of polypeptides in native or heat-induced formation

2

PROBLEM STATEMENT

Given the rapid increase in aquaculture in the last decade, it is expected that 99.33 million tonnes of by-products will be generated annually by 2022 (Neves, 2015) Dark tuna muscle is an important edible material when treated well as it contains a great amount of beneficial amino acids for the human body In tuna processing plants, there are high yields of the by-product but due to its low commercial value , it is often ineffective if used directly because some of its ingredients lose sensory value (due to

Mb, lipid oxidation), allergy (due to histamine content) or alteration of quality of materials and final products during processing and storage (due to lipid oxidation).With yellowfin tuna by-products generating about 42.49% of its weight and with TDM representing 6.40% (Bao, 2013) of that makes it susceptible to histamine formation When histamine is formed, the quality of meat drops and it becomes a

problem (Shahidi et al., 2005; Herpandi et al., 2011) This makes the majority TDM be

utilized in the processing of animal feed or pet food Gildberg (2002) sustains that the value of components increases about five times when they are recovered from processing by-products and used in the development of human food Therefore, the utilization of these by-products not only improves the economic aspect but also

recovers nutrients that are beneficial to human beings (Lee et al., 2016a) Study results

will be used to solve the socio-economic problem of the increasing value of tuna dark muscle and also reduce the source of soluble protein waste into the environment by adding into low-value fish powder production Furthermore, the results of the study are

a scientific basis to apply for the dark meat of many other seawater species

Significance of the study

The growing population of the earth to about 10 billion people by 2050 makes

it fundamental to obtain sufficient and nutritious food Global seafood consumption has more than doubled in the past 50 years to over 20 kg per capita per year in 2018

In the EU, seafood consumption as observed in Portugal is 61.5 kg per head while outside the EU, with Korea as the top consumers are 78.5 kg per head followed by Norway 66.6 kg per head The global per capita seafood consumption is estimated at 22.3 kg (FAO, 2018)

3

Sustainability of fish stock becomes an ever more pressing issue, as demand for

seafood rises (Tahergorabi et al., 2012) This trend is particularly relevant in light of a

growing human population and recent forecasts indicating that some of the current

fisheries may collapse by mid-century if they are not managed properly (Kristinsson et

al., 2006) Therefore, the discarded protein-rich fish (TDM) by-products can be used to

extract proteins and be used for human food (products) to meet the increasing demand

of fish proteins while eliminating the pollution caused by the tuna by-product Therefore the target to extract proteins from these dark muscles (TDM) for better utilization as human food: as most proteins extracted are hampered with very limited success in developing functional and acceptable products for consumers

Objectives of the study

General objective

The overall aim of the work was to characterize the various pH-treated fish protein isolates extracted by pH-shift method from yellowfin dark tuna muscle

Specific objectives

 To extract protein isolates from yellowfin tuna dark muscle

 To determine the charge of the extracted protein isolates

 To determine the size and molecular weight of the extracted protein isolates

 To evaluate the microstructure of extracted protein isolates

 To determine the molecular distribution of different extracted protein isolates

 To establish viscosity of protein solution

This thesis consists of three chapters and a general conclusion:

Chapter 1 gives a review of literature on objects of research

Chapter 2 describes the materials and methods used in the research

Chapter 3 presents results and discussions

General conclusion and outlook

Figure 1.1: Yellowfin tuna (Thunnus albacares ( Bonnaterre, 1788)

(Source: https://fishider.org/en/guide/osteichthyes/scombridae/thunnus/thunnus-albacares)

With current fishing production, there is an inadequate supply of raw materials for tuna processing factories For example, for Hai Vuong Corporation (the leading unit in tuna processing and import-exporting in Vietnam), the amount of raw materials imported in the last 6 years (Figure 1.2) shows that domestic caught tuna only supply less than 10% of the raw material requirements of the production in tuna factories

5

Figure 1.2: Raw material production at Hai Vuong Corp (Nha Trang, Khanh Hoa)

About a third of the white meat (TWM) of the whole fish is used during the

cunning (Herpandi et al., 2011) At present, tuna raw materials mainly use meat to

produce valuable products while parts like bone, skin, fin, dark meat are considered as

a by-product of low economic value (Peng et al., 2013; Swatland, 2012) constituting

up to 42.49% of the original material ( Bao, 2013) The dark muscle is a group of dark meat that lies beneath the skin throughout the body; in tuna, this meat is located near the backbone The relationship between light and dark muscle, however, varies with the fish activity where fatty fish contains a higher percentage of dark muscle

(Sánchez-Zapata et al., 2011) Bao (2013) found that dark muscle accounts for 6.4%

of total tuna weight; while Herpandi et al (2011) found about 12% is dark/red meat

However, at fishery processing factories, these by-products are mainly used for animal feed or fertilizer, as they are considered as low-value resources with insignificant market value Panggat (2003) stated that TDM is very slimy, fatty and has strong odour and taste which has made it not used presently for any value-added purposes

1.1.2 The composition of yellowfin dark muscle

In fish, the white muscle fibres are separated from the red (dark) muscle fibres,

unlike in most homeotherms, where the muscle cells are mixed (Park et al., 2013) In

addition, the relationship between the light and the dark muscle varies with the activity

6

of the fish In yellowfin tuna, the dark muscle is a band of dark tissue that lies beneath

the skin throughout the body; and is located near the backbone (Sánchez-Zapata et al.,

2011) (Figure 1.3) Moreover, the yellowfin tuna contains 6.40% dark muscle (Bảo, 2013) The dark muscle is designed for long-term exercise and is used by migrating species that travel great distances with a high content of oil in the muscle where in lean fish it is contained mainly in the liver Consequently, fatty fish like yellowfin tuna needs more fat, glycogen and myoglobin for their long journeys This, therefore, makes the fish (dark muscle) depend on the oxidative metabolism of lipid as its

principal source of energy (Herpandi et al., 2011; Park et al., 2013; Sánchez-Zapata et

al., 2011) The dark muscle is rich in lipids, lipases, blood capillaries and myoglobin

(Mb) The white muscle on the other hand contains very little Mb even in dark-fleshed species (Richards & Hultin, n.d.)

Figure 1.3: Image of yellowfin tuna muscle

Source: Herpandi et al., (2011)

In fish, there are three main groups of proteins: the myofibrillar, the sarcoplasmic and the stroma The myofibrillar, the most abundant proteins making up between 70-80% of the total proteins are the structural proteins that build up the contractile unit (sarcomere) of the myofibril (the muscle fibre) The sarcoplasmic protein includes the myoalbumin, globulin, heme proteins, parvalbumin and enzymes

7

It makes up about 20-30% of the total proteins The stroma proteins are mostly collagen and other connective tissue proteins that constitute only about 3% of the muscle proteins (Love, 1970) The protein groups are separated by their different solubility properties in salt solution While sarcoplasmic proteins are soluble in pure water and at low ionic strengths at a neutral pH, the myofibrillar proteins on the other hand are soluble in pure water and at higher salt concentrations (0.5-1 M) (Stefansson

& Hultin, 1994) while the stroma proteins are insoluble regardless of ionic strength

1.1.2.1 Myofibrillar proteins

Myofibrillar proteins are made up of many different proteins, with myosin and actin being the two major ones In structural proteins, myosin is the most abundant and

is the most important protein in gel formation and water holding (Kristinsson et al.,

2006) It comprises two large subunits (myosin heavy chains, MHC, around 200 kDa) and four smaller (light chains, about 17-20 kDa) The MHC has one globular “head” and one fibrous “rod” each, and the rods are bound together in a coiled-coil structure (Frederiksen & Holtzer, 1968) The heads contain binding sites for actin and active

sites for ATPase (Nano et al., 2018; Sikorski, 1994) In the sarcomere, myosin

molecules are bound together forming the thick filaments Actin monomers are globular (G-actin), and the monomers can build up filaments (F-actin), as in the thin

filaments of the sarcomere (Alberts et al, 2008) When the fish is alive, the myosin

head binds the actin filaments and uses ATP to bend its head and contract the

sarcomere (Alberts et al,2008) Post mortem, actin and myosin bind tightly to each other and form actomyosin (Kristinsson et al., 2006) Tropomyosin together with

troponin are important proteins for the regulation of sarcomere contraction and

relaxation (Kristinsson et al., 2006) Other important structural proteins in the muscle

are titin, a very large protein that is important for the structural arrangement of thin and thick filaments; nebulin, which is found in association with the thin filaments; α-actinin, which is part of the Z-disc and acts as an actin-binding protein; desmin, which

is part of the Z-disc; and myomesin which acts as a scaffolding protein for actin and myosin (Alberts, 2008)

8

1.1.2.2 Sarcoplasmic proteins

Sarcoplasmic proteins consist mostly of the enzymes involved in different parts

of the cell metabolism These enzymes include proteinases and peptidases, such as the cathepsins, which are important during fish processing since they can cause protein

degradation and for instance, softening of the fish tissue (Ladrat et al., 2002)

Myoglobin (Mb) and parvalbumin are the two sarcoplasmic proteins that affect the fish quality and human health Mb, an iron-containing protein binds oxygen and functions

by receiving oxygen from the blood and storing it for use in aerobic respiration (Baron

& Andersen, 2002) Parvalbumin is a small heat-stable protein (~12 kDa) involved in calcium-signalling It is what most people who are allergic to fish are sensitive to it

(Colombo et al., 2018; Taylor et al., 2004) Sarcoplasmic proteins have long been

thought to act adversely on protein gel formation (Jafarpour & Gorczyca, 2012) However, the field is now divided since several researchers have shown that sarcoplasmic proteins instead increase gel strength (Yongsawatdigul, 2007) and the topic has recently been reviewed without solving the question (Jafarpour & Gorczyca, 2012) Comparing to TWM, TDM has higher lipid contents, less stable proteins, greater concentrations of heme proteins, lower ultimate pH values, higher concentrations of sarcoplasmic proteins contributing to the difficulties in its

industrialization and making high-quality products from raw material (Park et al., 2013; Sánchez-Zapata et al., 2011)

1.1.2.3 Lipids

In fish, there are two types of lipids: neutral lipids and polar lipids The neutral lipids, mostly triglycerides, are generally found as depot fat while the polar lipids, mostly phospholipids, are the main constituents of the cell membranes (Ashton, 2002) All fish have more lipids in their dark muscle than in their white muscle According to

Sánchez-Zapata et al (2011), there are many differences in the chemical composition

between dark and white muscle, but the most important is the high fat and haem pigments content in the dark muscle The fish lipids are richer in long-chain polyunsaturated fatty acids (LC-PUFA), especially from the omega-3 (n-3) family The two most common (n-3) PUFA in fish are EPA (20:5 n-3) and DHA (22:6 n-3)

9

(Matak et al., 2015) TDM could give a high nutritive value, even higher than that of

light muscle as it contains a higher amount of lipid content These fatty acids have various bioactive functions, such as anti-cancer activity, recovery from heart failure,

attenuation of cerebrovascular disease, and anti-arteriosclerosis action (Kristinsson et

al., 2006; Nakamura et al., 2007)

Table 1.1: Proximate composition of the muscle tissue of yellowfin tuna (%, wet basis)

Moisture 73.57 ± 0.55 67.03 ± 0.97 Crude protein 23.52 ± 0.61 26.92 ± 0.27 Crude fat 1.93 ± 0.13 4.87 ± 0.74 Crude ash 1.54 ± 0.06 0.97 ± 0.05

SOURCE: Parameter (g/100 g of muscle) (Lee et al., 2016b; Sánchez-Zapata et al., 2011)

Due to their properties (high nutritional quality, functional properties, high protein level and low content of antinutritional factors), protein concentrates are

widely used as ingredients in the food industry (Lee, et al., 2016b) Thomas (2010)

reported that the moisture content of Yellowfin was in the range of 70-71% where the TDM and TWM did not show much variation in the moisture content Crude protein content was found to be between 26-28% TDM of Yellowfin showed a slightly higher amount of protein (28%) while the white meat contained 26% Crude fat in the samples was found to be between 0.39 and 1.28%, the higher amount being shown by

white meat of yellowfin tuna However, Lee et al (2016b) found that the fish

consisted of 72.89 –73.57%, 23.52 – 23.72%,1.93 – 2.06% and 1.54 –1.77% moisture,

protein, fat and ash respectively Sánchez-Zapata et al (2011) while experimenting on

quality characteristics of TDM also found that the muscle proximate composition was 67.03 ± 0.97 moisture, 26.92 ± 0.27 protein, 4.87± 0.74 fat, and 0.97 ± 0.05 ash Therefore, the recovery of the proteins from TDM would be of great value as it is high

in proteins The use of fish proteins in powder form presents some advantages as they

do not require special storage conditions and they can also easily be used as an ingredient in foods

10

1.1.3 Tuna by-product

In the wild, several fish species with dark muscle may account for 40-50% of

the total number of caught species (Undeland et al., 2002), whereby, dark meat can account for up to 20% of the total fish weight (Gamarro et al., 2013) Tuna is mainly marketed in a fresh, chilled, frozen or canned form (Gamarro et al., 2013) It is an

important source of global tuna fisheries and one of the major target species for the tuna fishery and a popularly caught marine fish with an estimated annual availability

of 14,000 metric tons (NOAA fisheries 2018; Lee et al., 2016a) Vietnamese tuna

fisheries primarily occur within three provinces: Binh Dinh, Phu Yen and Khanh Hoa

Yellowfin tuna is extensively used in raw cuisines such as sushi and sashimi, but by-products (scales, heads, skin, fat, red meat (dark muscle), visceral, and roe) are

generated progressively and discarded as waste (Swatland, 2012; Peng et al,

2013).Since the production of sashimi and sushi demands a prime quality raw material

of tuna, any deviations from specified quality standards could result in detention or rejection, which subsequently may increase the volume of wastes The quantity of by-products produced during fish processing can exceed 75% of the total weight of fish

(Herpandi et al., 2011) With above 50% of by-products waste recorded, only about

35% are processed (Panggat, 2003) Considering the large amount of unprocessed products, this creates an opportunity for improvement in the utility of fish proteins

by-Protein-enrich by-products from the canning industry such as dark muscle have limited use because of their colour, susceptibility to oxidation and off-flavour

(Gamarro et al., 2013) However, there is limited recognition of biological resources

while the increasing environmental pollution has emphasized the need for better

utilization of by-products from fisheries (Herpandi et al., 2011; Shahidi et al., 2005)

Remarkably, the proximate analysis of crude protein content in tuna dark muscle (TDM) is higher than in tuna white muscle (TWM) with TDM having 28%

while TWM contains 26% (Kristinsson et al., 2006; Bảo, 2013) This makes TDM a

potential material that can be exploited to provide high-quality protein source for humans after technical treatment to make useful peptides applying for antioxidant,

antihypertensive (Qian et al., 2007) and antimicrobial (Cheung et al., 2015)

By-11

products from TDM have been used in the production of collagen, chondroitin sulfate

and gelatin which have indirect environmental benefits (Sánchez-Zapata et al., 2011)

Efficient recovery and use of such by-products (TDM) are very important to reduce

environmental problems and to maximize economic benefits (Lee et al., 2016; Gamarro et al., 2013) Moreover, with the advent of surimi and other advanced

technologies, these materials may be transformed into high-end value-added products while minimizing the loss of valuable proteins(Gamarro et al., 2013)

Several factors affect the quality of fish muscle such as fat, soluble muscle protein (myoglobin (Mb), haemoglobin (Hb), etc.) Tuna dark muscle (TDM) as a by-product has high nutritional value because of high levels of essential amino acids Therefore, scientists in the developed countries that thrive on seafood (USA, Iceland, France, Japan, China, and Korea) have researched on chemical composition, extracting methods, or functional properties of peptides that were hydrolysed from dark muscle The studies focused on the muscle of mackerel, herring, sardine, big eye tuna, skipjack tuna, yellowfin tuna, yellow stripe, and pony fish The low-value dark muscle can be

treated by chemical method (Gamarro et al., 2013; Kristinsson & Liang, 2006; Undeland et al., 2002) or use hydrolysis enzyme (Gamarro et al., 2013; Je et al., 2009;

Kristinsson & Liang, 2006) to extract the protein as raw material for the surimi

production (Gamarro et al., 2013; Shaviklo, 2008; Undeland et al., 2002) Hultin and

colleagues registered intellectual property in the United States for dark muscle protein treated by pH adjustment (Hultin & Kelleher, 1999) Protein isolates extracted from the muscle of a terrestrial animal or fish muscle was treated at different pH (9.05 to11.5) obtained protein content from 3.37 to 99.9% and this protein source was used

to produce surimi products

1.2 Factors affecting the utilization of TDM

1.2.1 The dark colour and Myoglobin oxidation

The colour of a fish product is an important factor for the consumer and affects the price Whitefish achieves the highest prices on the world market together with some coloured fish species such as tuna and salmon TDM contains more pigments

than fillets (Park et al., 2013) In addition, tuna produces its energy for dark muscle

12

using oxygen which is transported throughout the body by the blood In dark muscle, the diameter of the fibres (cell) is smaller compared to that of the white muscle, but has the same number of capillary vessels surrounding each one, giving dark muscle up

to 10 times more capillaries than the white muscle Hence, the blood supply to the dark fibres is correspondingly higher than to the white fibres Therefore, a higher amount of haemoglobin present is due to the higher amount of blood Moreover, high concentrations of myoglobin are required to bind and transport oxygen within the

muscle cell, thus, the high concentrations of the (red) in the dark muscle (Nishioka et

al., 2007; Park et al., 2013)

Myoglobin is a globular heme protein known to be a major contributor to the colour of muscle, depending upon its redox state and concentration Myoglobin is made up of a single polypeptide chain, globin, consisting of 153 amino acids and a

prosthetic heme group, an iron (II) protoporphyrin-IX complex (Hayashi et al., 1998;

Pegg & Shahidi, 1997) This heme group gives myoglobin and its derivatives their

distinctive colour (Pegg & Shahidi, 1997; Peng et al., 2013)

The structure and chemistry of the iron atom have an impact on the reactions and colour changes that myoglobin undergoes The oxidation of ferrous-oxymyoglobin (Fe2+) to ferric-metmyoglobin (Fe3+) is responsible for the discolouration of meat during storage Ferrous iron (Fe2+) can react with molecular oxygen to produce superoxide anion (O2-) with concomitant oxidation to ferric iron (Fe3+) Hydrogen peroxide (H2O2), which may be produced by dismutation of O2-, can react with Fe2+ to produce hydroxyl radical (OH) (Hultin, 1992) Figure 1.4 shows the reaction termed the Fenton reaction which is the principal mechanism for myoglobin oxidation

13

Fish myoglobin is more readily oxidized hence; the discolouration of tuna meat during frozen storage is associated with the formation of metmyoglobin Chaijan(2008) demonstrated that sardine myoglobin was prone to oxidation and denaturation at a temperature above 40°C and very acidic or alkaline pHs as evidenced

by the formation of metmyoglobin, the changes in tryptophan fluorescence intensity as well as the disappearance of Soret absorption Besides, the rate of myoglobin autoxidation was related to oxygen concentration Atmospheres enriched in carbon dioxide (CO2) are effective in delaying spoilage of meat; however, one problem is that carbon dioxide can promote the oxidation of oxymyoglobin to metmyoglobin, thereby causing the discolouration (Haard, 1992)

1.2.2 Lipid and protein oxidation

Rancidity is one of the main reasons why fish and other seafood can become

unacceptable to the consumer (Tahergorabi et al., 2012) Fatty fish is particularly

susceptible to rancidity since it contains high amounts of n-3 LC-PUFA with up to six double bonds The latter makes PUFA more susceptible to oxidation than more saturated lipids

Reactive oxygen species (ROS) such as superoxide (O2¯) hydroxyl (OH), peroxyl (RO2), alkoxyl (RO) and hydrogen peroxide (H2O2), in food and biological systems, are

naturally generated and can react with biological molecules(Sánchez-Zapata et al., 2011;

Tokur & Korkmaz, 2007) Excessive production of ROS could damage complex cellular molecules like fats, proteins, or DNA They create anti-oxidative defence mechanisms which neutralize the production and adverse effects of reactive oxygen species in living organisms However, the mechanisms do not work after fish death This can cause an accumulation of ROS in muscles, which leads to lipid and protein modifications Lipid oxidation process follows a free radical mechanism and therefore, once started, it

propagates and catalyses further oxidation of lipids and proteins, including Hb (Lund et

al., 2011) The reaction takes three main steps

i Initiation– Where radicals are formed, often by the interaction of lipids with different active oxygen forms or with transition metals

14

ii Propagation – Where the lipid-free radical reacts with oxygen to form a peroxy radical which then reacts with an intact fatty acid, producing new lipid radicals and lipid hydroperoxides After this step, chain-breaking can happen, where lipid hydroperoxides are cleaved to volatiles and new radicals by low molecular weight (LMW) metals or heme The most important metal ions involved in lipid oxidation are

Cu2+, Fe2+ and Fe3+

iii Termination – This is the step where two radicals are combined into a new stable compound

During storage a major problem experienced in fatty fish is lipid oxidation in

dark muscle According to Pazos et al (2005), a possible reason behind this

vulnerability is the amounts of pro-oxidants, such as heme proteins (Hb), myoglobin, low molecular weight (LMW) transition metal complexes, and lipoxygenases, in their dark muscle Chaijan (2008) reported that lipid oxidation and myoglobin oxidation concurrently occurs in muscle foods as each process appears to enhance the other Superoxide anion and hydrogen peroxide produced during oxymyoglobin oxidation, react with iron to produce hydroxyl radical This hydroxyl radical can penetrate the hydrophobic lipid region and hence facilitate lipid oxidation (Chaijan, 2008; Chaijan

& Panpipat, 2009) Lipid oxidation is also affected by temperature, light; water activity, pH and chemical environment, such as the level of oxygen present (Ashton, 2002) At a lower pH, Hb de-oxygenation (the Bohr Effect) and Hb autoxidation that leads to the formation of metHb (contains Fe3+ instead of Fe2+) are increased (Richards

& Hultin, n.d.) Deoxygenated Hb and MetHb are more prone to induce oxidation than

reduced oxy-Hb (Richards et al., 2005) MetHb also more easily loses its heme-groups

which are hydrophobic and easily enter into the lipid-membranes where they can

catalyse the oxidation(Richards et al., 2005)

The oxidizing of proteins and their damage in biological systems has been studied over the past two decades Results of these studies showed that ROS lead to oxidation of amino acid side chains, the formation of protein-protein cross-linkages,

and oxidation of protein backbone, resulting in protein fragmentation (Pazos et al., 2005) Richards et al (2002a) stated that the process of oxidation in muscles leads to

15

discolouration, drip losses, off-odour and off-flavour development, texture defects and the production of potentially toxic compounds Additionally, the characteristics of yellowfin TDM makes it not acceptable for these industries due to its strong dark colour and highly susceptible to lipid oxidation speeding up its deterioration (Nishioka

et al., 2007)

2.2.3 Histamine

TDM is restricted to direct use due to colour, odour, fat content as well as a significant amount of histamine that causes allergies, making the value of this by-product is very low Tuna fish species have a high level of histidine in their muscle

(García-Tapia et al., 2013) According to Feng et al (2016), histamine fish

poisoning/scombroid poisoning results from ingestion of histamine contaminated fish

in the Scombridae and Scomberesocidae families (e.g tuna) While Akbari-Adergani

et al (2012) described it as an acute allergy-like food poisoning mainly caused

consumption of fish containing high levels of histamine and is one of the most frequent intoxications related to seafood, and is the most common cause of

ichythyotoxicosis worldwide (Feng et al., 2016)

Decarboxylative converts histidine to histamine during fermentation of

enterobacteriaceae lactic acid bacteria, and phytobacteria (Akbari-Adergani et al.,

2012; Tao et al., n.d.) Histamine is resistant to thermal processes (freezing, cooking,

canning, etc.) and the only way to prevent its accumulation is storing fish below 4◦C Rapid removal of viscera and washing fish can significantly reduce the production of histamine in bacteria and it can be regarded as an effective approach for lowering histamine levels in fish In addition, tuna gut contains psychotropic bacteria growing, which can contaminate the flesh during the gutting process and can increase in numbers if temperature abuse occurs during processing Therefore, if decarboxylating microorganisms are present in the flesh, they can induce the formation of high levels

of histamine in the final product, and the level of histamine formed is affected by the

combination of both time and temperature (García-Tapia et al., 2013; Kannaiyan et al., 2019; Tao et al., n.d.) Different fish species cause histamine fish poisoning (Figure

1.5)

16

Figure 1.5: The proportion of each fish causing histamine fish poisoning reactions,

1998–2012 (CDC Food Outbreak Online Database)

1.3 Protein extraction techniques used and FPI utilization

Extraction of fish protein isolate (FPI) can be done by a pH-shift method where proteins are solubilised at extreme pH values separating soluble proteins, connective tissue, bone, skin, neutral lipids and cellular membranes through a centrifugation step (Surasani, 2018) minimizing the risk of lipid oxidation The process employs solubilisation of the proteins using acid and alkali followed by a precipitation process

at their isoelectric pH to recover functional and stable protein isolates from the

underutilized tuna by-products (Kristinsson et al., 2006) During solubilisation and

precipitation, changes can occur in the three dimensional structure of myosin and predominantly on myosin heavy chain, which affect the functional properties of the

proteins recovered (Tahergorabi et al., 2012) Shaviklo et al (2017) deposed that the

method has high recovery yield (the sarcoplasmic proteins), economically feasible, and has improved functionalities of the recovered proteins The partially unfolded/folded structure of proteins is more flexible, allowing the ability to form better protein

networks (on heating) (Taktak et al., 2018) Therefore, structural changes (folding

phenomena) occur during the pH-shift method that can cause an increase of surface hydrophobicity and reactive–SH groups in the produced proteins which may contribute

to their technologically useful functionalities (Undeland et al, 2010)

17

1.3.1 pH –shift method

Acid and alkaline aided solubilisation or pH shift method is a technology that efficiently recovers functional and nutritious protein isolates from sources difficult to process, through conventional means The simple outline of the pH-shift method is simple Hultin &Kelleher (1999) opined that the most important steps of pH method (Figure 1.6) are:

i Solubilisation of the proteins at low or high pH;

ii Removing fat and impurities by using a high-speed centrifuge, and lastly iii Precipitation of the proteins at their isoelectric point

Fish muscle

Solubilization in alkali or acid

Supernatant (soluble protein) centrifugation

Figure 1.6: pH-shifts process for production fish protein isolate

(Adopted from Hultin et al., 2005)

In the pH-shift method, protein is subjected to extreme pH conditions followed

by a recovery step at an isoelectric pH The pH-shift method induces protein unfolding and refolding, which modifies protein conformation and allows for more hydrophobic

to Kristinsson et al (2006), the removal of the undesirable materials like skin, bones,

microorganisms, cholesterol, membrane lipids, and other contaminants during the first centrifugation stage is one of the most important technical issues in the pH-shift method However, the bones may be removed during mechanical deboning The pH- shift method maximizes the protein recovery of surimi products compared to the washing method which gives low yield due to the multiple washing steps

Protein isolate made from an acid-aided process differs from a protein isolated with the alkali-aided process In a comparative study between the acid and alkaline methods and surimi processing for recovery of proteins from channel catfish muscles,

Kristinsson et al (2006)found that the alkali process gives better attributes such as gel

strength to FPI than the acid-aided process However, the latter process generally results

in higher protein yields compared to the former process Solubilised proteins collected and recovered by isoelectric precipitation to give a highly functional and stable protein isolate

(Hultin & Kelleher, 1999; Kristinsson & Liang, 2006; Park et al., 2013)

The method provides several advantages such as higher yield, lower waste, greater protein quality, and effective removal of insoluble materials Even so, not every species has demonstrated greater gel texture values when fish proteins were

extracted at alkaline pH Kim et al (2003) preliminary study, using rockfish, indicated

that gels prepared from solubilised proteins at alkaline pH (10–11) exhibited better gel quality than those prepared from the acid-aided or conventional process whereas Atlantic menhaden demonstrated higher gel quality when using the conventional-wash method compared to alkaline extraction (Pérez-Mateos and Lanier, 2006) The alkali-aided process was hailed to provide a more oxidative stable protein isolate than the

acid-aided process (Thorkelsson et al., 2008) and according to Kristinsson et al

(2006), the method is sometimes more stable than surimi Heme proteins are denatured

19

and co-precipitated in the acid process and make the product less stable and darker

(Kristinsson et al., 2006; Park et al., 2012) Moreover, Julio et al (2013) found that

protein concentrates from the pH-shift process had a good balance of amino acids compared to enzymatic hydrolysis

According to Kim et.al (2003), proteins of higher whiteness are recovered from

the alkali-aided process The acid-aided process on the other hand results in a higher yellowness than the alkali-aided protein isolate and surimi made from catfish muscle Scientists generally agree that alkaline extraction provides superior gel strength (Park

et al., 2012) Kristinsson & Liang, (2006) reported that 68.4% of lipid was removed

using the alkali-aided process compared to only 16.7% removal using the conventional surimi process Following up these previously done studies, the application of alkaline

pH for the solubilisation of yellowfin TDM proteins was, therefore, needed

1.3.2 Protein recovery

Parke and Lin (2005) who worked on surimi processing reported that protein recovery processing depends on fish freshness, the water/meat ratio, washing time,

washing cycle, and pH of the washing solution Torres et al.(2006) studied protein

recovery from processing trout by-products by the pH-shift technology and reported that protein recovery depended on the pH of both solubilisation and precipitation steps

Similar findings were also reported by Hultin et al (2005) who reported more than

85% yields from the fillets in the pH-shift process compared to 55-70% from the

surimi process Kristinsson et al (2006) and Tahergorabi et al (2012) reported 62.5%

protein recovery for channel catfish surimi and 71.5% and 70.3% protein recovery for cat-fish protein isolates made from acid-aided and alkali-aided processes in lab-scale

respectively Kristinsson et al (2006) in another work, reported 57.7% protein

recovery for Atlantic Croaker and 78.7% and 65.0% protein recovery for Croaker

protein isolate made from acid-aided and alkali-aided processes respectively In Julio

et al (2013) protein recovery by auto-hydrolysis and pH-shift was 83.3% (sum of two

fractions) and 87.5 respectively applied on shrimp waste

Elizondo-Garza et al (2016)who studied protein recovery from skipjack tuna

using wash water with different pH and temperature combinations reported that the best processing conditions for protein precipitation were 4°C and pH 4.5, 5.5 or 6.5, with protein recoveries of about 92% which represented an average of 60% of original skipjack flesh, and around 50% of the initial solids The maximum protein extraction was achieved at 4°C, a temperature recommended to minimize microbial spoilage and keep protein functionality

1.3.3 By-products and FPI utilization

Yellowfin tuna is a large epipelagic species widely distributed in the tropical

and subtropical waters of the major oceans (Collette and Nauen 1983; Zudaire et al.,

2013) Due to its high demand, yellowfin is harvested widely, and many types of fishing gear are used The fish is widely used in raw fish dishes and therefore

generates about 75% of its weight as products (Herpandi et al., 2011) This figure

means that the waste of high-quality protein becomes a problem as it elevates

21

environmental burden (Shahidi et al., 2005; Herpandi et al., 2011) Concentration of

solids in the discarded water from fish processing reaches up to 40 to 50 g per every

100 grams of processed flesh tissue Most of the recovered proteins are mainly used as animal feed or pet food Gildberg (2002) sustains that when components are recovered from processing by-products and they are used in the development of human foods, their value increases about five times Fish by-products and under-utilized fish species that usually are not used directly for human food can be utilized in the pH-shift

process (Hultin etal., 2005) Besides, the recovery of water-soluble proteins is required

to reduce the environmental impact of food processing plants(Gamarro et al., 2013)

By-products utilization will improve the economic aspects of the processing industry and further their nutritional beneficiation through valuable essential amino

acid and fatty acid components (Lee et al., 2016b) It is estimated that the value

addition of human food developed from the by-product will increase significantly in

the future (Kristinsson et al., 2006; Tahergorabi et al., 2012) Tahergorabi et al (2012) and Shaviklo et al (2017) reported that fish protein isolates could also be used

to formulate several food products such as fish balls, functional fish protein gels, and fish burgers Due to its high nutritional and economic value, the recovery of TDM protein isolates at high concentrations would, therefore, maximize the fishery resources and help in reducing the environmental pollution

1.3.4 Fish protein isolate characterization

Yellowfin tuna dark meat is yet to be extensively studied Most studies have focused on optimizing the extraction process, consideration of some basic characteristics such as the degree of hydrolysis, solubility, water-holding capacity, or antioxidant capacity of peptides However, no studies on rheological analysis, molecular weight determination for polypeptide or separation, and characterization of functional properties of polypeptides with different sizes have been conducted Despite the low studies on yellowfin tuna, some authors have studied the physico-chemical properties (rheology, structure, etc.) of protein extracted from the muscle of some fish

species by alkali or acid-soluble method (Brenner et al., 2009) The authors studied the

size; nanostructures, using light scattering techniques; or the morphological change of the protein gel extracted from the cod muscle by confocal laser scanning microscopy;

22

or rheological properties by deformation surveys, determining the elastic modulus of a

structural break Moosavi-Nasab et al (2013) investigated the physico-chemical

properties of fish protein isolate (FPI) by adjusting the pH method and surimi prepared directly from silver carp meat The results indicated that the hardness and elasticity of the FPI sample were higher than the surimi one Moreover, Chaijan (2004) studied the mechanical and colouring properties of the extracted protein by pH shift method, the results showed that the colour, structure, and gelation of surimi that was prepared from

sardine meat is better than from herring meat Ingadottir et al (2011) investigated the

gel possibility of protein extracted from tilapia meat that prepared by pH adjustment method in the presence and absence of NaCl, results indicated hardness and elasticity increased in the presence of 2% NaCl, the pH and heat-inducing significant dependence on the elastic modulus (G') of protein gelation was also investigated by

these authors Most recently, Felix et al (2017) studied the rheological properties of

soluble protein from crayfish meat, the results showed that elastic modulus (G‟) of 12% protein solution heated at 90 °C for 30 minutes was the highest at pH 6.5 and the lowest at pH 8, and at pH 2, G' was intermediate

In tuna by-products, researches have been limited and focused on the optimal

process of hydrolysis for head, tail, and viscera of tuna (Nguyen et al., 2011), for tuna

dark muscle is only the first step in extracting protein using the adjusting pH method

by (Bảo, 2013) This is the basis for our specific studies on physico-chemical properties of this protein for flexible application of simulated and functional products

From the experimental research, yellowfin tuna dark muscle was provided by Hai Vuong Corporation (Khanh Hoa, Vietnam), the research team had the positive results on sensory after some preliminary processing steps (Figure 1.7)

Figure 1.7: Minced tuna dark muscle before treatment (A), after treatment (B) and

after heating at 80 °C for 20 minutes (C)

B

23

The tuna dark muscle before treatment was dark on colour due to the myoglobin present in it After treatment, the colour appeared to be brighter than before treatment TDMPI was then heated to observe the how the protein would behave since most food application of protein involve heating

Part of the by-products is used to produce fish sauces and food products such as dry-salted roe while the rest is used in production of animal feeds The amount of hazardous waste produced from fish processing has tended to increase annually

(Sánchez-Zapata et al., 2011) The heads, gills and entrails; and black meat provide the

highest share at the respective levels of 28.4 % and 22% The skin which is about 5.8% is used for making chi Charon or cracklings while the brown meat, which is about 3.5% is mostly used in making value-added tuna products (Panggat, 2003; Solidarism, 2010)

24

Chapter 2 : MATERIALS AND METHODS

2.1 Materials collection and preparation

2.1.1 Dark muscle

Yellowfin (T albacare) dark muscles were collected with blocks of 5 kg from

Tuna Factory, Hai Vuong Group, Vietnam Then they were frozen at -45 °C to reach at

a centre temperature of -18 °C before being stored at -20 °C ±2 The frozen TDM were packed in a polystyrene box and transported quickly to the laboratory in the frigorific vehicle at (-20 °C) They were continuously stored at -20°C ±2 for experiments for a period of 1 month maximum

Figure 2.1: Block (left) and minced TDM (right) 2.1.2 Chemicals

Chemicals were used in the research that was purchased from Sigma or Merck

2.1.3 FPI extraction by pH-shift method

Fish protein isolate was produced by pH-shift processing following the method

of alkaline solubilisation process described by Undeland et al (2002)with some slight

modifications The frozen TDM blocks were cut into rectangular (3x10x3cm) chunks, minced using a meat grinder with a small pore diameter of 2.5 mm to facilitate the solubility of the proteins in basic solutions The mince was washed twice by deionized water at 4°C (3:1 ratio) to remove the dark colour of meat To recover the slurry, excess water was removed from the precipitates, by wrapping in a filter bag with a pore-size of 50µm and manually pressed Then they were solubilized in a basic solution with a ratio of minced dark muscle to alkali solution (1:5) at pH 12 The

25

mixture was homogenized at 4 °C for 2 min at speed 3500 rpm using an Ultra-turrax (model: IKA T18) NaOH 2 N was used to adjust to pH 12 after homogenization The mixture was stirred continuously for 3 hours at room temperature (20 °C) using Orbital shaker (model: NB-101M) then centrifuged (MF600, Labentech Co., Ltd.) at 5000 rpm for 30 min at 4 °C, separating the homogenate into three layers The top layer contained neutral lipids, the middle layers contained soluble proteins, while the bottom layer contained insoluble fragments The top and middle layers were decanted in a filter bag with a pore-size of 5µm to remove the lipid fraction and the insoluble fragments The resulting filtered fraction of soluble protein (supernatant) was

precipitated using 2 N HCl to pH 5.5 (pI of most Proteins) (Cha et al., 2002)

Precipitated proteins (FPI) collected after thoroughly washing in cold distilled water (4

°C) until neutral, to remove the salt Excess water was removed by manually wrapping and squeezing protein isolate in cheesecloth (filter bag with a pore size of 5 mm) Finally, fresh FPI was dehydrated by a freezing drier at -50 °C by Lyobeta 35, Spain (Figure 2.2) Dried FPI powder was stored at -20 °C ± 2 for study Figure 2.3 shows a flow chart of protein isolation

Figure 2.2: Freeze dryer Lyobeta 35 (Spain)

Figure 2.4: TDMPI characterization chart

Some measurements of the effect of salt on properties of TDMPI solution were studied Different concentrations of (NaCl and CaCl2) salts were incorporated into the solutions before the measurements

Proximate composition and amino acid composition were determined by Test method (AOAC 2005) and TCVN 8764: 2012 (ISO13903:2005) respectively

2.2.1 Fourier transform infrared (FTIR) spectroscopic analysis

Fourier transform infrared (FTIR) spectroscopy has emerged as a useful tool for the characterization of protein secondary structure with a precision lying between that

of the purely predictive and the molecular coordinate approaches FTIR is one of the earliest experimental methods for estimating the secondary structure of polypeptides and proteins That IR spectroscopy could give us information related to the secondary

structure of proteins (Bunaciu et al., 2014; Tatulian, 2019)

Data acquisition was performed using an FT-IR spectrometer Model Alpha, S/N 201418 (Germany) Figure 2.5 FTIR spectra of the TDMPI samples were obtained using an FTIR spectrometer equipped with a deuterated L-alanine tri-glycine Sulphate (DLATGS) detector A horizontal attenuated total reflectance accessory (HATR) was mounted into the sample compartment The internal reflection crystal was made of zinc selenide, and had a 45° angle of incidence to the IR beam The spectra in the