Study on initial proximate compositions and some physical parameters of yellowfin tuna; changes of total volatile basic nitrogen and sensory attributes of (super)chilled fish treated by liquid ice

MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY LE THIEN SA STUDY ON INITIAL PROXIMATE COMPOSITIONS AND SOME PHYSICAL PARAMETERS OF YELLOWFIN TUNA; CHANGES OF TOTAL VOLATILE BA

MINISTRY OF EDUCATION AND TRAINING

NHA TRANG UNIVERSITY

LE THIEN SA

STUDY ON INITIAL PROXIMATE COMPOSITIONS AND SOME PHYSICAL PARAMETERS OF YELLOWFIN TUNA; CHANGES OF TOTAL VOLATILE BASIC NITROGEN AND SENSORY ATTRIBUTES

OF (SUPER)CHILLED FISH TREATED BY LIQUID ICE

MASTER THESIS

KHANH HOA - 2020

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MINISTRY OF EDUCATION AND TRAINING

NHA TRANG UNIVERSITY

LE THIEN SA

STUDY ON INITIAL PROXIMATE COMPOSITIONS AND SOME PHYSICAL PARAMETERS OF YELLOWFIN TUNA; CHANGES OF TOTAL VOLATILE BASIC NITROGEN AND SENSORY ATTRIBUTES

OF (SUPER)CHILLED FISH TREATED BY LIQUID ICE

MASTER THESIS

Topic allocation Decision 583/ QĐ-ĐHNT dated 09/6/2020

Decision on establishing the Committee: 899/QĐ-ĐHNT dated 04/9/2020

Department of Graduate Studies:

KHANH HOA - 2020

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UNDERTAKING

I undertake that the thesis entitled: “Study on initial proximate compositions and

some physical parameters of yellowfin tuna; changes of total volatile basic nitrogen and

sensory attributes of (super)chilled fish treated by liquid ice” is my work

The data collection was an effort of a research team led by my supervisor, Dr Mai

Thi Tuyet Nga, who started the project KC.05.10/16-20 of Vietnam “Studying, designing,

and manufacturing a liquid ice production system for handling and preservation of ocean

tuna” since April 2018 before I began my MSc study I am luckily allowed to use the data

collected by myself and my teammates

The work has not been presented elsewhere for assessment until the time this

thesis is submitted 31st August 2020

I am responsible and the results of my research

Khanh Hoa, 31st August 2020

Author

Le Thien Sa

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ACKNOWLEDGEMENTS

Firstly, I would like to thank Nha Trang University and the VLIR project for giving

me the opportunity to participate in this course

I would like to express my deepest thanks to the lecturers of the Faculty of Food Technology, Nha Trang University, the professors from Can Tho University and Hue University for teaching and giving me too much useful knowledge, which will be applied

parameters of yellowfin tuna; changes of total volatile basic nitrogen and sensory attributes of (super)chilled fish treated by liquid ice”

Fourthly, I appreciate financial support from the project KC.05.10/16-20 of Vietnam

“Studying, designing, and manufacturing a liquid ice production system for handling and preservation of ocean tuna”

Finally, I would like to thank my family, my husband and children for their spiritual support and taking care of themselves while I was busy with the field trips or research

Khanh Hoa, 31st August 2020

Author

Le Thien Sa

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TABLE OF CONTENTS

UNDERTAKING iii

ACKNOWLEDGEMENTS iv

LIST OF SYMBOLS 3

LIST OF ABBREVIATIONS 4

LIST OF TABLES 5

LIST OF FIGURES 6

ABSTRACT 7

INTRODUCTION 8

1 Problem statement and purpose of the study 8

2 The objective of the study 9

3 Scientific and applications aspects of the topic 9

4 Research question 10

CHAPTER 1 LITERATURE REVIEW 11

1.1 Introduction a yellowfin tuna 11

1.2 Present status of the tuna fisheries in Viet Nam 13

1.3 Chemical composition of tuna 15

1.4 Freezing point of tuna meat 16

1.5 Enthalpy 16

1.6 Effect of temperature on the cooling process 17

1.7 Introduction of liquid ice and application on foods 18

1.8 Quality changes and shelf life of chilled fish 20

1.8.1 Autolytic changes 20

1.8.2 Bacterial changes 21

1.8.3 Lipid oxidation and hydrolysis 22

1.9 Methods of sensory evaluation 23

1.9.1 Quantitative Descriptive Analysis 23

1.9.2 Sensory Evaluation by Quality Index Method (QIM) 24

CHAPTER 2 MATERIALS AND METHODS 26

2.1 Materials 26

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2.1.1 Materials and their handling 26

2.1.2 Appliance and chemicals 27

2.2 Methods 28

2.2.1 General experimental design 28

2.2.2 Methods of determining the proximate composition of tuna flesh 30 2.2.3 Methods for determining physical parameters of tuna 32

2.2.4 Sensory evaluation 34

2.2.5 Determination of TVB-N content 36

2.2.6 Statistical analysis 36

CHAPTER 3 RESULTS AND DISCUSSION 37

3.1 Proximate composition of tuna flesh 37

3.2 Physical properties of tuna fish 40

3.3 Sensory changes of yellowfin tuna during storage 42

3.3.1 Sensory changes of 30 kg up tuna, stored in liquid ice of different salt and initial ice concentrations against crushed block ice, over time 42

3.3.2 Sensory changes of tuna of different sizes over storage time 44

3.3.3 Sensory changes of 30 kg up tuna, refrigerated and stored in different media, over time 46

3.4 Changes in total volatile base nitrogen of yellowfin tuna during chilled storage 49

3.4.1 Changes in TVB-N content of 30 kg up tuna, stored in liquid ice of different salt and initial ice concentrations against crushed block ice, over time 49 3.4.2 Changes in TVB-N level of tuna of different sizes over storage time 51

3.4.3 Changes in TVB-N of 30 kg up tuna, refrigerated and stored in different media, over time 52

CONCLUSIONS AND RECOMMENDATIONS 55

Conclusions 55

Recommendations 55

REFERENCES 56

APPENDICES 1

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LIST OF ABBREVIATIONS

TVB-N Total Volatile Basic Nitrogen

VASEP Vietnam Association of Seafood Exporters and Producers

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LIST OF TABLES

Table 1.1 Vietnam tuna products for exports in 2018 (US$) 14

Table 2 1 Quality index method (QIM) scheme for whole yellowfin tuna 34

Table 3.1 Major dry matter composition of yellowfin tuna flesh 38

Table 3.2 Physical properties of yellowfin tuna 40

Table 3.3 Sensory rejection time of 30 kg up tuna based on the control scheme 43

Table 3.4 Storage and sensory rejection time of tuna of various sizes based on the control scheme 45

Table 3.5 Sensory rejection time of 30 kg up tuna in various media based on the control scheme 47

Table 3.6 The linear relationship between quality index (QI) and storage time of yellowfin tuna 48

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LIST OF FIGURES

Figure 1 1 Yellowfin tuna 11

Figure 1.2 Global capture production for species (tonnes) 12

Figure 2.1 Raw materials at the port 26

Figure 2 2 Flow chart of the study 28

Figure 3 1 Moisture and dry matter contents of yellowfin tuna flesh 38

Figure 3.2 Changes in sensory quality of yellowfin tuna of size 30 kg up over storage time 42

Figure 3.3 Changes in sensory quality of yellowfin tuna of various sizes over storage time 44

Figure 3.4 Changes in sensory quality of 30 kg up yellowfin tuna in various media over storage time 46

Figure 3.5 Changes in TVB-N content of yellowfin tuna of size 30 kg up over storage time 50

Figure 3.6 Changes in TVB-N content of yellowfin tuna of various sizes over storage time 51

Figure 3.7 Changes in TVB-N content of 30 kg up yellowfin tuna in various media over storage time 53

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ABSTRACT

Yellowfin tuna (Thunnus albacares) is one of the most valuable marine fish

species of Vietnam Improper handling and storage of this highly perishable fish may lead to the product rejection due to unacceptable quality or/and safety problems Liquid ice has been gaining more and more application for on-board and on-shore chilling and storing aquatic materials However, its practical use on Vietnamese fishing vessels and relevant places is still very limited This study aimed to determine the proximate composition and some physical parameters of yellowfin tuna caught from Vietnam offshore, as well as to know the effect of handling and storage conditions, such as cooling and preservation media, particularly liquid ice produced from salty/sea-water,

on the sensory quality and total volatile basic nitrogen (TVB-N) content of tuna over storage time Eight gutted yellowfin tuna of 20, 30, and 40 kg up and several fish frozen steaks were used in the study Media for cooling and/or preserving fish were liquid ice

of 3.0 and 3.5% NaCl, 44 and 48% initial ice mass; slurry ice of 3.5% NaCl and initial temperature of -4.0°C; and control/crushed block ice It was found that yellowfin tuna flesh had the following average proximate compositions (%): moisture 73.95-81.02%, protein 21.60-22.26%, lipid 0.64-3.15%, total carbohydrates 0.13-0.30%, and ash 0.53- 0.89% Density of gutted tuna at 0-4°C was 1045 ± 44 kg/m3 Some thermophysical parameters of yellowfin tuna meat were as follows: freezing point: -1.88 ± 0.03°C; specific heat above and below freezing point: 0.826-0.874 and 0.457-0.468 kcal/kg.°C, respectively; Enthalpy: 20.662-21.840 kcal/kg to reduce the temperature of fish meat from 25°C to 0°C; and 21.914-22.466 kcal/kg to reduce the temperature of fish meat from 25°C to the freezing point Both TVB-N and sensory indicators of yellowfin tuna chilled down and preserved in liquid ice of 3.5% NaCl and 48% initial ice mass changed slowest compared to fish stored in liquid ice of 3.0% and 44% initial ice fraction or in crushed ice, which confirmed the advantage of the liquid ice over other media However, more study on the salt uptake phenomena of fish flesh during storage ice, on application

of proximate chemical and mass composition parameters of tuna, as well as their physical parameters in the cooling and preserving process of fish and in calculating/designing post-harvest equipment

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INTRODUCTION

1 Problem statement and purpose of the study

Tuna is a marine fish that lives near the surface of the oceans and tropical, subtropical, and temperate waters, but can also dive several hundred meters Most catches come from the Pacific Ocean (70.5% in 2008), Indian (19.5% in 2010), and Atlantic and Mediterranean Seas (10.0% in 2010) (FAO, 2016) Vietnam with a 3,200

km coastline and over 1 million km2 exclusive waters, favorable geographical location and natural conditions has many outstanding advantages for the development of the fisheries sector Tuna is concentrated in central Vietnam and the center of the East Sea Tuna resources of Vietnam are estimated to be above 600 thousand tons (FAO, 2016)

Vietnam has long been a leading seafood producer and exporter in the world Seafood export has become one of the most important sectors of the economy and plays

a vital role in the total export turnover of Vietnam Tuna is a main export item after pangasius and shrimp The annual catches (including yellowfin tuna, bigeye, skipjack, and others) are more than 200,000 tons Yellowfin and bigeye tuna have an average stock of over 45,000 tons and annual catches of 17,000-21,000 tons Tuna production in the exclusive economic zone of Vietnam is estimated at 27,000 tons Binh Dinh is the province with the largest tuna catch of 9,400 tons, followed by Khanh Hoa with 5,000 tons and Phu Yen with 4,000 tons In 2018, tuna contributed about 7.4% to the total seafood export of the country Vietnam has actively adopted international sustainability rules such as the EU IUU or EII's “Dolphin Safe” label.(VASEP, 2020)

Although our country's tuna exploitation and export volumes are large, the price is low compared to that of other countries Many different factors are affecting the competitiveness of Vietnamese tuna products in the international market One of the main reasons is inappropriate post-harvest handling and storage, especially in term of cooling temperature and rate This has led to a significant decrease in the quality of fish after catching, particularly for red meat fish such as tuna, which contains histidine that

is easily converted into histamine toxin To improve the quality of tuna, which is

That is why the thesis study entitled “Study on initial proximate compositions and

some physical parameters of yellowfin tuna; changes of total volatile basic nitrogen and sensory attributes of (super)chilled fish treated by liquid ice” is necessary

2 The objective of the study

To determine the proximate composition, specific density, and freezing point of the yellowfin tuna caught in Vietnam

To know the effect of handling and storage conditions on changes in the sensory quality index (QI) and total volatile base nitrogen (TVB-N) content of yellowfin tuna caught in Vietnam over storage time

3 Scientific and applications aspects of the topic

The study provides scientific data on changes in sensory quality and TVB-N content of yellowfin tuna over storage time under different conditions of cooling and storage The knowledge is useful for better control of the quality of fish raw material, helping the fishermen to know the quality of yellowfin tuna through sensory evaluation, thereby estimating the appropriate storage temperature and time for the fish Data on the

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proximate composition of fish is to calculate the fish thermophysical characteristics, which are the basis of the design of heat transfer equipment

Hypothesis for the research question

Using liquid ice and slurry ice for cooling and/or preserving yellowfin tuna can better retain the fish quality and extend the fish shelf life compared to tradition icing

with crushed ice produced from fresh water

4 Research question

Could using liquid ice for cooling and preserving yellowfin tuna help to improve the quality of fish?

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CHAPTER 1 LITERATURE REVIEW

1.1 Introduction a yellowfin tuna

English name: Yellowfin tuna

Science name: Thunnus albacares

Figure 1 1 Yellowfin tuna (Wiki pedia)

Tuna is classified into 4 genera (Thunnus, Euthynnus, Katsuwonus and Auxis) with a total of 15 species From the genus Thunnus, the main market tuna is albacore (T

alalunga), bigeye (T obesus), Atlantic bluefin (T thynnus), and Pacific bluefin (T orientalis), Southern bluefin tuna (T maccoyii), and yellowfin tuna (T albacares)

Skipjack (Katsuwonus pelamis) is the seventh major tuna species (Jacek Majkowski,

2007)

Yellowfin tuna (Thunnus albacares) has an elongated, round body, small head,

and long tail, and occurs in all warm waters of the world, except the Mediterranean This species has been recorded in the eastern part of the Pacific Ocean, Chile, Japan, Indonesia, Australia, Northland, and New Zealand (Science, 2010), (Schaefer et al., 1963) Due to their different distributions, specific heat resistance, and exploitation by different fisheries, there is a distinction between tropical and temperate tuna Tropical tuna is found in water with temperatures above 18°C (although they can dive in colder water) while temperate tuna is found in water as cold as 10°C but can also be found in tropical seas

Tropical tunas: skipjack and yellowfin

Temperate tunas: albacore, Pacific bluefin, Atlantic bluefin, and southern Bluefin

Figure 1.2 Global capture production for species (tons)

(Source: FAO- fish statistics)

Yellowfin tuna trend to swim in groups with other fish of the same size, including other tuna as well as other larger fish such as dolphins, whales, whale sharks They

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usually live in the continental mounds, shoals, around the scrub Usually, live in the open sea, but sometimes they will move closer to the shore, where the temperature is right and where there is a lot of food Yellowfin tuna eat other small fish, crustaceans,

or squid They often breed in the summer, in warm water, with lots of rubs and floating objects The ability to recover the population is very high On average, the minimum time to double a population is 2 - 3 years (FAO, 2016)

1.2 Present status of the tuna fisheries in Viet Nam

Vietnam tuna stock is estimated at over 600,000,000 MT, among which skipjack

is the main species (> 50%) The major fishing ground is Truong Sa/Spratly Islands and DK1 rigs Tuna fishing trips normally take 10-15 days The main catches of ocean tuna are yellowfin tuna, bigeye tuna mainly caught by longline and longline fishing, and high-yield skipjack is mostly caught by net fishing, fins and gill nets Tuna fishing grounds

in Vietnam's exclusive economic zone are mainly concentrated from the northeast, east, and southeast of the Paracel archipelago stretching to the north, in, and south of the Spratly Islands (Hiệp, 2017b)

Annual catching volume of are above 200,00 MT Yellowfin tuna and big-eye tuna have average stock of more than 45,000 MT, with a yearly yield of 17,000-21,000 MT Binh Dinh, Khanh Hoa, and Phu Yen are the most tuna catching provinces with 9,400, 5,000, ND 4,000 MT/year, respectively Currently, Quang Nam province has 750 fishing vessels over 15 m long, 790 fishing vessels fishes in the middle areas, and 1500 fishing vessels capture in coastal areas, Khanh Hoa province had 71 fishing vessels over 15 m long (15-24 m) A fishing fleet with a large capacity fisherman has been continuously fishing on Hoang Sa and Truong Sa fishing grounds of Vietnam The fishing output in the first 3 months of 2020 reached 22,000 MT, an increase of more than 4% over the

same period in 2019 (Việt, 2017), (Thang, 2015), (VASEP, 2020)

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Table 1.1 Vietnam tuna products for exports in 2018 (US$)

- Live/fresh/frozen/dried tuna (HS code 03)

(Source: Vietnam Customs)

In 2018, Vietnam exported tuna to 105 markets, earning US$ 652.9 million, up 10% over 2017 The top 8 markets are: USA, EU, Israel, ASEAN, Japan, Canada, China, and Mexico, accounted for 87 % of the total export value

In 2019, there was a decline in tuna exports to the EU market, partly due to the impact of the EU's “yellow card” on insufficient efforts in fighting illegal, undeclared, and unregulated exploitation; and because of high exporting taxes Meanwhile, Vietnam's tuna export value to ASEAN increased by nearly 7% compared to 2018, reaching about 54 million USD Tuna export value in 2020 is expected to increase by about 15% compared to 2019 (Fisheries, n.d.)(VASEP april, 2020)

Due to the geographical distance, tuna fishing boats need 3 days to reach the tuna belt on Truong Sa fishing ground, so it usually takes 10-20 days for a sea trip With such

a long time, fishermen, most of whom have limited qualifications, face a big challenge

in fish preservation, which is outdated with traditional icing by crushed ice to cool fish

in the cold compartments With this method of preservation, tuna is not cooled properly before being chill stored In addition, the low quality of water used to make ice may lead

1.3 Chemical composition of tuna

The chemical composition of fish includes water, protein, lipid, glycogen, minerals, vitamins These components vary greatly, depending on the variety, species, gender, physiological state, habitat conditions, seasons, food composition, fish size, and genetic characteristics (Mahaliyana et al., 2015), (Can Nguyen Trong, Phung Do Minh, 2006) In the fish body, there are two types of red and white flesh, they differ in chemical composition Dark meat has about 1/10th the thread of white meat but has more blood vessels Dark meat contains more glycogen, fat, cholesterol, B vitamins, vitamin C, carotene, and histidine than white meat, but contains less creatine acid than white meat

So, preserving fish with red meat such as tuna is more difficult than preserving fish with white flesh

Tuna is rich in protein, high in vitamins (especially vitamin D), high in phosphorus, low in fat, which is high in unsaturated fatty acids such as omega-3 Albacore tuna proximate composition depends on the size, habitat and seasons Albacore has a longer shelf life, which is 20 days as maximum, than other fish in the Scombridae family (Pérez‐villarreal & Pozo, 1990)

Research results of (Rani et al., 2016) on the seasonal chemical composition changes of tuna from Visakhapatnam fishing port, the east coast of India, showed that two tuna species of the same family contained high protein level The skipjack tuna

(Katsuwonus pelamis), caught from the ocean waters around Sri Lanka, was relatively

high in protein (24.13 ± 2.01%), low in fat, and rich in ω-3 fatty acids, Fe, Cu, and Zn

(Mahaliyana et al., 2015) Yellowfin tuna (Thunnus albacares) eyes are also a rich

source of ω-3 fatty acids and essential amino acids (Peng et al., 2013), among which glycine, glutamic acid concentration, and aspartic acid were found with high

1.4 Freezing point of tuna meat

The freezing point of the fish indicates the temperature that begins to freeze the muscle water when cooling/freezing the fish Water in the fish body exists as a solution,

so the freezing point follows Raoul’s ice-lowering rule and the freezing point of the fish

is lightly below 0°C When frozen, the thinnest part of the solution will frost first The freezing point of marine fish ranges from -0.60C to -2.60C, in which the freezing point

of most bony fish species is -0.80C to -1.00C (Can Nguyen Trong, Phung, Do Minh, 2006) The freezing point of the fish is one of the parameters that can be used to estimate the fish enthalpy

1.5 Enthalpy

Enthalpy is defined as the amount of heat in a system/object per unit of mass (kcal/kg or kJ/kg) The change in enthalpy used to estimate the energy/amount of heat must provide or remove from the object to change the thermal state of the object (Van Der Sman & Boer, 2005) It is necessary to determine the enthalpy of Vietnamese tuna

to calculate cooling and freezing of fish

Knowing the fish proximate composition will help to calculate its thermophysical parameters, such as specific heat, which can be used for modelling to find the optimal time and temperature for freezing, thawing, and cooking They can also be used to design, develop, and optimize equipment related to yellowfin tuna catching, post-harvest handling, and processing (Pornchaloempong et al., 2012) That is the reason why this study focused on determining the basic chemical composition and some physical properties of tuna caught in Vietnam to serve the next research on cooling and chilled storage of tuna

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1.6 Effect of temperature on the cooling process

Refrigeration is a relativistic term that denotes the decrease in thermostatic heat below its natural normal range Cold and heat have the same nature as representations

of the movements of molecules and atoms The lower the temperature, the slower atoms and molecules move

Chilling is the process of removing heat from food to lower its temperature near the freezing point but not below the freezing point of the food, i.e bring the temperature

of the free-structural water in the product down without solidifying them

Since the concentration of mineral salts and cytoplasmic solutes of a product varies depending on the type of product, each product has its own freezing point and therefore a different cooling regime The freezing point of saltwater fish is lower than the freezing point of freshwater seafood because marine species contains more mineral salts

The process of refrigeration plays an important role in preserving seafood materials, extending the storage time Different seafood products have different shelf life, but the effect of temperature on the rate of spoilage of the fish is relatively similar (Can Nguyen Trong, Phung Do Minh, 2006)

The rate of cooling represents the degree of change in the temperature of any point within the material per time unit (°C/hour)

The cooling rate mainly depends on the area per unit volume of fish in contact with the cooling and/or storage medium (e.g ice, cold air, etc.) The cooling time depends on the coolant, enthalpy, specific weight, the temperature of the chilling

medium, and the heat transfer coefficient between the fish and the environment The

larger the area per unit mass, the faster the cooling rate, the shorter the time to reach a fish's core temperature of 00C Therefore, the thicker the fish body the slower the cooling rate will be (Can Nguyen Trong, Phung Do Minh, 2006)

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Latency of cooling must be avoided It was observed that if the rigor mortis of white flesh, lean fish begins at temperatures above 170C, muscle tissue may be broken due to severe muscle contractions and connective tissue weakening This defect called

“gaping”, i.e the separation of fillet flakes from each other, spoils the seafood appearance This type of improper handling would result in more difficulty of the filleting process, create lower filleting and trimming yields, and cause a decrease of the water holding capacity of the tissue Gutted cod with delayed icing (6.5 h after catch) found to have lower fillet and trimmed yields than fish iced 1 h post-harvest (Border´ıas

& S´anchez-Alonso, 2011)

1.7 Introduction of liquid ice and application on foods

Seafood starts to deteriorate rapidly soon after its death Improper preservation may lead to bacterial, enzymatic, and chemical activity increase, which reduces the shelf life, causes drip loss, and ultimately results in product rejection The quality degradation rate depends on fish species, handling and storage conditions The deterioration process

is speeded up by elevated temperatures, physical damage, and contamination The key for seafood preservation is immediate chilling post-harvest to a temperature slightly above the freezing point and maintaining this temperature throughout the cold chain Ice slurry/liquid ice can increase the chilling speed of seafood (Kauffeld, 2005), (Kauffeld,

M Wang, M J Cabello et al., 2012)

A slurry ice system is an ice suspension/binary system, which is produced from seawater and cooled to subzero temperatures, normally used for the preservation of aquatic food products

Slurry ice has been described in scientific literature as flowing ice, iceberg or liquid ice For aquatic food products, the technological benefits from slurry ice systems can be summarized as follows: (Carmen Pin˜eiroa, Aubourga, Jorge Barros-Vela´zquez, 2004)

- Higher surface heat exchange rates (almost 4 times higher than flake-ice) allows faster cooling of seafood

Slurry ice/liquid ice has successfully been applied for pre-cooling, refrigerating, transportation and storage of various fish species such as tuna, yellowtail, salmon, cod, hake, herring, mackerel, sardines, shrimp, clams, and lobsters in Iceland, Norway, Japan and elsewhere (Carmen Pin˜eiroa, Aubourga, Jorge Barros-Vela´zquez, 2004), (Kauffeld, M Wang, M J Cabello et al., 2012)

The influence of slurry ice on the quality of skipjack tuna (Katsuwonus pelamis)

during cold storage was studied and compared with flake ice Samples treated with slurry ice showed significantly higher (p < 0.05) elasticity and toughness than those treated with flake ice This may be due to faster cooling, lower final temperatures, and greater heat exchange derived from a slurry The study also showed that slurry ice effectively slowed down the degradation of myofibrillar proteins and stabilized the tissue structure (Zhang et al., 2015)

European Hake (Merluccius) was reported to have an extended shelf life in slurry

ice than in flake ice (12 vs 5 days) (Losada et al., 2004)

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Similarly good results were obtained with slurry ice used for the onboard storage

of shrimp (Rodríguez et al., 2004) Meanwhile, (Medina et al., 2009) found that there was no significant differences (p > 0.05) in the spoilage rate of seabass, a warm-water fish species, stored in flake ice and slurry ice In the other side, cloudy eyes have been reported as a backward effect of slurry ice causing to farmed seabream

1.8 Quality changes and shelf life of chilled fish

1.8.1 Autolytic changes

Autolysis also knows as self-digestion In fish, there are different enzyme systems When the fish is alive, these enzymes are necessary for the build-up of the tissue and organs and metabolism (help to digest food) After the fish is dead, the enzymes systems, especially digestive and muscular ones, still operate and take part in the fish degradation, which lead to soft muscle and reduce quality of seafood Furthermore, autolysis products are nutrition sources for microorganisms Therefore, the activation of intracellular enzymes will enhance the spoilage of fish

Trimethylamine oxide (TMAO), an osmoregulatory substance in marine bony fish, is reduced into TMA due to bacterial activity

In some species, most often the cod family, TMAO-ase or TMAO demethylase enzyme in the muscle tissue can break down TMAO into DMA and formaldehyde (FA):

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Several studies have shown a potential relationship between an increased level

of TMA and the chilled seafood quality loss, however, TMA content does not grow much during the early stages of spoilage, e.g TMA in black tiger shrimp remained at low level at the first 4 days in ice (Le et al., 2017) The compound is not considered suitable for discriminating fish stored less than 6 days in ice (Masette, 1999) Determination of total volatile base/basic nitrogen TVB-N, including ammonia, DMA and TMA, which are products of nitrogen-containing substances’ break down, is an alternative to measuring TMA alone The contents of TVB-N and TMA in chilled cod loins maintained low till day 10, after which a sharp increases in their concentrations were observed (N T T Mai et al., 2011)

1.8.2 Bacterial changes

Microflora on newly-caught fish depends on the habitat environment rather than

on the fish species Fish caught in very cold waters has lower bacterial counts than those harvested from warm waters (Huss, 1995)

Microorganisms are found on the skin and gills, as well as in the intestines of live and newly caught fish The total number of organisms vary greatly in a range of 102-107 cfu/cm2 on the fish skin The gills and the intestines consist of 103-109 cfu/g (Huss, 1995)

When the fish gets into the processing area the bacterial count on the skin is often high If the fish is not washed well with clean water, a lot of bacteria can get in the processing area and contaminate the fish flesh during filleting The flesh can also get contaminated with mesophilic bacteria from the people in the working area So personal hygiene is also very important

Temperature plays a very important role in controlling microbial growth Higher temperatures (around 37°C) can increase microbial growth

Spoilage bacteria: Pseudomonas spp., Shewanella putrefaciens, Photobacterium

phosphoreum, Enterobacteriaceae (Gram & Dalgaard, 2002) Changes of total viable

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counts (TVC) in chill-stored seafood, such as Pangasius fillets (N Mai & Huynh, 2017),

have been found to follow lag, log, and stationary phases, which were described by Baranyi & Roberts and other models

Pathogens found in natural environment of fish: Clostridium botulinum, Listeria

monocytogenes, Vibrio parahaemolyticus, V vulnificus, Aeromonas hydrophila and Plesiomonas shigelloides Other pathogens that can contaminate from the environment: Staplylococcus aureus, Salmonella spp., Escherichia coli

1.8.3 Lipid oxidation and hydrolysis

Reactions that give rise to a variety of chemical and physical changes in lipids Hydrolysis formed of free fatty acids, normally this does not cause problems in fish but causes an off-flavour in oils (soapy)

Enzymatic or non-enzymatic oxidation: Double bonds of unsaturated fatty acids are attacked and such products/substances as aldehydes, ketones, etc are accumulated This affects nutritional value, taste, odour, and colour Some of these products may also bind covalently to fish muscle proteins, causing texture changes The relative importance of these reactions is mainly attributed by the fish species and storage temperature

Highly unsaturated lipids of fish are easily oxidized This process begins immediately post-harvest but becomes particularly impactful for the fish shelf life only

at sub-zero temperatures, when oxidation rather than microbial activity becomes a main spoilage reason

The initiation of lipid oxidation starts from several early post-mortem tissue changes, which destroy the natural balance between anti- and pro-oxidants, include the collection of active oxygen species, the hemo-protein activation, the free iron increase, and the antioxidant consumption (Hultin, 1994)

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1.9 Methods of sensory evaluation

Sensory evaluation composes of a set of techniques to measure accurately the human responses to foods through the senses of sight, smell, touch, taste, and hearing and diminishes the potentially biasing effects such as brand and other information, which affect consumer perception (Mason & Nottingham, 2002), (Francisco, 2013b)

1.9.1 Quantitative Descriptive Analysis

Quantitative Descriptive Analysis (QDA) was developed during the 1970s (Stone & Sidel, 1985) Unstructured line scales are used to describe the intensity of rated attributes

During QDA training sessions, 10–12 judges, under the guidance of a panel leader, are exposed to many possible product variations to generate a set of sensory terms or to list the descriptors on the ballot in appearance sequence Then through discussion and consensus, the whole panel ends up with a standardized QDA sheet which can use to illustrate the organoleptic differences between the samples

The judges also decide on the reference standards and/or verbal definitions to affix the terms In plus, during the training the panel decides the order of attribute assessing Moreover, the obtained results are relative rather than absolute, because assessors normally rate an attribute differently on the given scale

Advantages of QDA are the independent judgments of assessors and results are not consensus derived, easily analyzed statistically and presented graphically Panel vocabulary development is without the leader effect and based on consumer language Weakness of QDA is the requirement for panel training for each product category (Lawless & Hildegarde, 2019)

QDA, along with Torry scheme, was used to determine the maximal shelf life of Atlantic herring in ice This resulted in a about 8 days of storage (Nga, Mai Thi Tuyet;

A Martinsdóttir, 2007)

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QDA and Torry scheme were used to assess the sensory quality of Tra catfish

(Pangasius hypophthalmus) fillet stored at 1 ± 1ºC and 4 ± 1ºC The results confirmed

that fresh fish was characterized by sweet flavor, whereas long stored Tra catfish was characterized by mushy, rancid odor, spoilage flavor, and dark appearance of QDA attributes QDA and Torry scores were still within the acceptable limit for human consumption after 15 days of storage at 1 ± 1ºC, and 7 days at 4 ± 1ºC (Nga & Van, 2018)

1.9.2 Sensory Evaluation by Quality Index Method (QIM)

Quality Index method (QIM) was originally developed by the Tasmanian Food Research Unit in Australia (Martinsdottir et al., 2001)

The method is based on the characteristic changes that occur in raw aquatic materials They are related to the appearance of the eyes, skin, gills, and smell properties and the scoring system ranges from 0 to 3 points Scores for all the characteristics are aggregated to give the overall organoleptic score, known as the quality index (QI) (M.Nga et al., 2007) The QIM must be tailored to the individual fish species and product The scientific development of QIM for different species is aimed at making the

QI increase linearly with the storage time in ice QIM has several advantages, including historical and remaining shelf life estimation (Francisco, 2013a) The method is suitable for guiding inexperienced people to evaluate fish, as well as for training and monitoring the performance of workshop participants

The QIM has been developed and applied for many fish species and products, such

as iced tub gurnard (Chelidonichthys lucernus) (Bekaert, 2006) and blackspot seabream (Pagellus bogaraveo) (Sant’Ana et al., 2011)

Quality indices including TVC, quality index (QI), TVB-N, and TMA-N were determined during the ice storage of black tiger shrimp, previously treated either by immersing in polyphenol solution, vacuum packing, or a combination of polyphenol solution and vacuum packing Results showed that TVC grew dramatically at the end of the storage period TVB-N and TMA-N increased over storage time, but in two different

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stages The QI was linearly correlated with the storage time, and used for estimating the remaining shelf life (Tam et al., 2019)

For albacore tuna, the sensory score, especially the taste of ripe fish, appeared to

be better indicators of the freshness of the fish than chemical indicators, such as ATP breakdown products and amines (Pérez‐villarreal & Pozo, 1990)

In this study, QIM together with control sensory sheet and TVB-N indicator were used for quality assessment of yellowfin tuna chilled and stored in liquid/slurry ice or crushed ice

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CHAPTER 2 MATERIALS AND METHODS

2.1 Materials

2.1.1 Materials and their handling

Gutted yellowfin tuna (Thunnus albacares) caught in Vietnam, Truong Sa Island

of size of 20 kg to 45 kg/fish, were landed, purchased from Hon Ro port and transported

to the Food Technology Laboratories of Nha Trang University in insulated expanded polystyrene boxes within 20 minutes Crushed ice was filled into gill and abdominal cavities, as well as put around the fish to maintain the temperature below 4°C during the journey

Only those tuna with intact, bright and shiny skin, natural color, characteristic fresh smell of marine fish, firm muscle, and transparent eyes, without any hint of off-odor or spoilage were selected for procurement

Figure 2.1 Raw materials at the port

For simulating the on-board cooling conditions, upon arrival at the laboratories, the fish temperature was raised to its initial temperature after fishing (24-28°C) Then the fish were cooled by studied media, which were either liquid ice with salt concentrations of 3.0% and 3.5% and initial ice fractions of 44% and 48%, ice slurry prepared from NaCl solution and crushed ice to create a binary system of 3.5% NaCl

27

and initial temperature of -4.0°C, or control coolant of crushed block ice, to around 0°C or freezing points After that, the cooled fish were stored in liquid ice or crushed ice to conduct an evaluation of the quality criteria of yellowfin tuna over storage time, which were 12-39 days until clear evident of spoilage

The liquid ice used for refrigeration and storage was manufactured by a liquid ice machine, developed by the Key National Funded Project KC.05.10/16-20 Liquid ice was made from brine with a salinity of 3.0% and 3.5% (salinity is similar to that of seawater)

Temperature of the storage environment and the fish body were checked daily, and ice was added or replaced as needed to maintain the fish temperature at -1 ± 1°C

Furthermore, the study also used frozen tuna steaks, produced by Hai Vuong Group, as control samples for proximate analysis

2.1.2 Appliance and chemicals

The following types of equipment and instruments were used:

Equipment: Refrigerators, freezers, protein distillation, burettes, scales, sample homogenizer, UV Spectrophotometer, water bath

Tools: stickers, PA, PE bags for sampling, alcohol lamps, glass cup, beakers, pipettes, volumetric flasks, measuring cylinders, glass rods, funnel, glass jars, petri plates, etc

Chemicals of analytical grades:

- Trichloroacetic acid (TCA) 7.5% (Merck, Germany)

- Sodium hydroxide 20% (China)

- Clohydric acid 0.05 M (China)

- Boric acid 3% (Merck, Germany)

- Sulphuric acid 75%, 98% (Merck, Germany)

- Glucose (Merck, Germany)

- Anthrone reagent (Merck, Germany)

- Phenolphthalein 1% (China)

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- Red methyl 0.2% and green methylene 0,1% (1/1) (Merck, Germany), etc

2.2 Methods

2.2.1 General experimental design

Figure 2 2 Flow chart of the study

From the experimental study of yellowfin tuna chilled and stored in different media, such as liquid ice of 3.0% and 3.5% NaCl, 44% and 48% initial ice content;

Yellowfin tuna

Statistical analysis

Conclusion

29

slurry ice, and control/crushed block ice, drawed out the trend of quality changes in sensory attributes and TVB-N level over time In plus, proximate analysis and thermophysical parameters of received tuna were determined to provide data for a liquid ice machine research team

Yellowfin tuna was purchased from Hon Ro port and transported to the Food Technology Laboratories of Nha Trang University Eight tuna were used in this study

as follows:

- Fish 1: 30 kg (30 kg up) for the research on refrigerating, describing sensory attributes, training panelists, and preliminary estimation of fish preservation time;

- Fish 2: 35 kg (30 kg up) stored in liquid ice of 3.0% salt and 44%initial ice crystal;

- Fish 3: 20 kg up refrigerated and stored in liquid ice of 3.5% salt and 48% initial ice crystal;

- Fish 4: 33 kg (30 kg up) refrigerated and stored in liquid ice of 3.5% salt and 48% initial ice crystal;

- Fish 5: 44 kg (40kg up) refrigerated and stored in liquid ice of 3.5% salt and 48% initial ice crystal;

- Fish 6: 42kg (40 kg up) refrigerated and stored in crushed block ice;

- Fish 7: 37kg (30 kg up) refrigerated and stored in crushed block ice;

- Fish 8: 35kg (30 kg up) cooled in slurry ice and stored in crushed block ice

Cooling was conducted until fish body temperature reaches -1 ± 1°C, then chilled storage started and considered as day 0

During storage time, melted ice was removed, and an equal amount of new ice was added to keep the body’s fish temperature at -1 ± 1°C

Received fish samples were used for proximate composition analysis, as well as for the measurement and/or calculation of some physical parameters, such as density of the gutted fish, freezing point, specific heat, and enthalpy of the fish flesh

30

Periodic sampling: Samples were taken from the back and abdomen flesh every 1-3 days from the point of material receiving until the fish clearly spoilt Samples were used for the determination of total volatile base nitrogen (TVB-N), and sensory evaluation by quality index method (QIM)

2.2.2 Methods of determining the proximate composition of tuna flesh

v Determination of protein content

Kjeldahl method was used for protein determination The method is based on the wet combustion of the sample by heating with concentrated sulphuric acid in the presence of metallic and other catalysts to convert organic nitrogen in the sample to ammonia, which is retained in solution as ammonium sulfate The digest, having been made alkaline, is steam distilled to release the ammonia which is trapped in a boric acid solution and then titrated with a standard solution of hydrochloride acid

The protein content can be calculated according to the following equation:

%𝑃 = %.' () * (++

, (+++ (1) Where:

% P: Percentage of protein by weight;

V: Volume (mL) of HCl used for titration of the sample;

T: Normality (mol/L) of the HCl;

C: Conversion factor, C = 6.25;

G: Weight (g) of the sample

v Determination of the moisture and dry matter contents

The moisture was determined in accordance to TCVN 3700 – 90 (MoST, 1990),

31

similar to AOAC – 925.10 (AOAC, 2007), which use oven drying at 105°C for at least

5 h until an unchanged weight of the sample The loss in weight was calculated as the moisture content Dry matter and moisture contents of foods can be calculated as follows:

X: lipid content in fish (%)

a: Mass of flask (g);

b: Mass of flask and lipid (g);

m: Mass of the sample (g);

w: Moisture content of sample (%)

v Determination of the ash content

The ash content was conducted as described by Vietnam National standard TCVN 5105:2009 (MoST, 2009), which referred to AOAC 938.08 (AOAC, 2007) by dry/oven ashing at 500°C

The ash content in weight percentages as calculated as follows:

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% Ash (wt/wt) = 56789: ;< =>?@A6 (8)56789: ;< >=9 ([) 𝑋 100 (5)

v Determination of total carbohydrates

Content of total saccharides in a sample can be estimated by the anthrone method, which is a simple colorimetric technique with relative insensitivity to interferences from the other cellular components The first step is to hydrolyze the polysaccharide and to dehydrate the monomers (digestion with sulfuric acid addition and heat treatment) The 5-carbon and 6-carbon sugars are converted to furfural and hydroxymethylfurfural, respectively Anthrone (an aromatic compound) reacts with these digestion products to give colored compounds The amount of total carbohydrates in the sample is then estimated via reading the absorbance (at 620 nm) of the resulting solution against a glucose standard curve (Plummer, 1990)

Prepare the calibration curve of absorbance (at 620 nm) vs μg glucose Readings

of the standards and calculate the total carbohydrates of the samples in mg glucose/L from this curve

2.2.3 Methods for determining physical parameters of tuna

v Determination of density of gutted tuna

The density of gutted yellowfin tuna was determined by the volume substitution method (Michailidis et al., 2009) Briefly, weighted fish was immersed in a plastic tank full of water with known volumn This caused the water to flow out from the container The fish was then removed, the tank was refilled with water to full level Volume of the fish was that of water added The density was then calculated by dividing the weight of the fish to its volume The experiment was repeated 6 times on 3 fishes Tuna temperature during the experiment was 0-4°C

v Determination of freezing point of tuna flesh

The freezing point (Tf) of yellowfin tuna flesh was determined by the cooling curve

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method, which is one of the simplest, most accurate, and widely used procedures for determining the freezing point of a food (M.S Rahman et al., 2009) In this method, the sample temperature was recorded with a thermometer such as a regular one, thermocouple, etc (Van Der Sman & Boer, 2005)

Thermocouples of the Ellab CTF9004 device (Denmark) with measuring range from -100 to 350°C, 0.1°C resolution, and 0.1°C accuracy was used Samples were taken from the flesh of the fish, each 100 g The experiment was triplicated

v Determination of specific heat of tuna flesh

Based on the tuna proximate composition, particularly the water and dry matter contents, obtained from the above experiment, the fish specific heat above and below freezing point was calculated by the following simple formulas (Can Nguyen Trong, Phung Do Minh, 2006)

- Specific heat of fish above its initial freezing point:

𝐶] =^_+.``)a(++ (b[°*bcde) (6)

- Specific heat of fish below its initial freezing point:

𝐶] =+.g^_+.``)a(++ (b[°*bcde) (7)

Where:

W (%): water content in fish;

N (%): content of dry matters in fish;

0.334 (kcal/kg°C): specific heat of dry matters in fish;

1 (kcal/kg°C): specific heat of water in fish;

0.5 (kcal/kg°C): specific heat of water below the freezing point

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v Determination of enthalpy of tuna flesh

Enthalpy of Vietnamese yellowfin tuna flesh above the freezing point was determined by the following formula (Can Nguyen Trong, Phung Do Minh, 2006):

i = Cp*(T2 - T1) (8)

Where:

(T2 - T1) (°C) is the temperature range (above the freezing point of fish);

T1 - initial temperature of fish Assuming T1 = 25°C;

T2 - temperature to cool the fish to, T2 = 0°C (case 1) and T2 = Tf °C (case 2, cool

to initial freezing point)

“from very fat to no fat”)

0

Dorsal: loss of brightness The sides: loss of brightness Abdomen: loss of silver/ chalk look, pale

Eyes The eyes are convex and bright, the cornea is transparent,

the pupils are bright black

0

The eyes are slightly convex and bright, the cornea is

The eyes are flat and white, the cornea is opaque, the pupil is

(whole fish) The skin is intact Little scratched skin surface 0 1

Texture

(flesh) Firm, elastic Slightly firm, slightly elastic 0 1

Color

(flesh/meat) Bright red, translucent, glossy Dark red or pinkish; slightly blurry, slightly glossy 0 1

Brown or discolored (gray, blue, yellow ); fuzzy; opaque 3

QI (Quality index) (0-15)

(Source: Mai Thi Tuyet Nga, unpublished)

a: Amount of nitrogen (mg) corresponding to one mL of standard solution of HCl, for 0.05 M HCl solution a = 0.07

m: Sample weight (g);

V1: Titration volume (mL) of HCl used for the tested sample (mL);

V2: Volume of the filter after the norm (mL), V2 = 100 mL;

V3: Volume of the filtrate taken for distillation (mL), V1 = 50 mL

2.2.6 Statistical analysis

SPSS 20.0 software was used for all statistical evaluations Data were subjected to analysis of variance (ANOVA) and post-hoc Tukey for mean comparison at a significant level of 0.05 Microsoft Excel 2013 was used to calculate means and standard

deviations, as well as to build graphs