Study on the application of liquid ice for handling and preservation of yellowfin tuna

Among the studied cooling and storage media, the liquid ice of 3.5% NaCl, 48% ice content, initial temperature of 4.0°C showed the best preservation effect for yellowfin tuna sensory qua

MINISTRY OF EDUCATION AND TRAINING

NHA TRANG UNIVERSITY

OLANREWAJU AKIN YINKA

STUDY ON THE APPLICATION OF LIQUID ICE FOR HANDLING AND PRESERVATION OF YELLOWFIN TUNA

MASTER THESIS

ii

MINISTRY OF EDUCATION AND TRAINING

NHA TRANG UNIVERSITY

OLANREWAJU AKIN YINKA STUDY ON THE APPLICATION OF LIQUID ICE FOR HANDLING AND PRESERVATION OF YELLOWFIN TUNA

Department of Graduate Studies:

KHANH HOA - 2020

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UNDERTAKING

I undertake that the thesis entitled: “Study on the application of liquid ice for

handling and preservation of yellowfin tuna” is my own 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 in

Vietnam I joint the project since January 2020, when the final phase of the project

studying on slurry ice and crushed block ice was going on, and luckily allowed to use

the data previously collected by my teammates

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

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ACKNOWLEDGMENTS

First and for most, my sincere appreciation goes to God for granting me such a great opportunity to be alive and healthy to complete this program I specially honor the Lord Jesus and the Holy Spirit for the deep inspiration, supernatural strength, and great opportunity made available to be awarded a scholarship in Vietnam

Secondly, I really want to appreciate my supervisor and promoter in the person of Dr Mai Tuyet Nga for her unrelenting supports and great contributions towards the success of this research study I acknowledge the support of the HOD, secretary of the program and other staff in the department

Thirdly, I would like to express the deepest appreciation to my good friend for his immense contribution towards the success of my thesis My good colleagues, you are well appreciated for your love and kindness Especially, I appreciate my family and friends in the diaspora for your love, prayers, and support

I would like to appreciate the support of the VLIR Network Vietnam, Nha Trang University, Food Technology Department for the success of this research study Food Technology Laboratories at Nha Trang University for allowing me to carry out this research study with their up-to-date equipment

Last but not least, I would like to thank project KC.05.10/16-20 of Vietnam

“Studying, designing, and manufacturing a liquid ice production system for handling and preservation of ocean tuna” for financial support and permission to use its data for my MSc thesis

that could be used for port-harvested Yellowfin tuna Among the studied cooling and

storage media, the liquid ice of 3.5% NaCl, 48% ice content, initial temperature of 4.0°C showed the best preservation effect for yellowfin tuna sensory quality The second most effective medium was the liquid ice of 3.0% NaCl, 44% ice content, initial temperature of -3.1°C Fish chilled down either in slurry ice of initial temperature of -4.0°C or in crushed ice, and then stored in crushed ice had the second worst and the worst sensory quality, respectively, indicating the weakness of traditional icing However, no cooling or storage media effect, as well as no size influence on TVC of tuna samples has been found so far These results vividly showed that liquid/slurry ice has a large scope of preservation effect and has the potential to improve significantly the quality and as well as extends the product shelf life Further study on on-board cooling and salt uptake of fish stored for long period

-in liquid ice is needed

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TABLE OF CONTENTS

UNDERTAKING iii

ACKNOWLEDGMENTS iv

ABSTRACT v

TABLE OF CONTENTS 1

LIST OF ABBREVIATIONS 3

LIST OF FIGURES 4

LIST OF TABLES 5

CHAPTER 1 INTRODUCTION 6

1.1 INTRODUCTION 6

1.2 MAIN OBJECTIVE 9

1.3 SPECIFIC OBJECTIVES 9

1.4 PROBLEM STATEMENTS 9

CHAPTER 2 LITERAURE REVIEW 11

2.1 YELLOWFIN TUNA 11

2.1.1 Production 11

2.1.2 Habitat of Yellowfin Tuna 12

2.1.3 Size, age, and growth 12

1.3.4 Reproduction 13

2.2 POST-MORTEM QUALITY CHANGES OF FISH-RELEVANT FACTORS AND VARIABLES 14

2.2.1 Autolytic changes 14

2.2.2 Chemical spoilage 14

2.2.3 Microbiological spoilage 15

2.3 HANDLING AND PRESERVATION OF FISH 16

2.4 REFRIGERATED METHODS OF SEAFOOD PRESERVATION 18

2.4.1 Icing and iced storage of fish 21

2.4.2 Pre-cooling and cooling by slurry ice 22

2.5 EVALUATION OF FRESHNESS AND QUALITY CHANGES OF FISH 24 2.5.1 Sensory analysis 25

2.5.2 Microbiological analysis 27

CHAPTER 3 MATERIALS AND METHODS 28

3.1 MATERIALS 28

3.2 APPARATUS AND TOOLS 28

3.3 METHODS 29

3.3.1 Experimental plan 29

3.3.2 Experimental factors 30

3.3.3 Sampling 30

3.3.4 Determination of sensory quality 32

3.3.5 Determination of total viable count 33

3.4 DATA COLLECTION AND ANALYSIS 34

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CHAPTER 4 RESULTS AND DISCUSSION 35

4.1 CHANGES OF TVC IN TUNA DURING STORAGE OF FISH 35

4.1.1 Changes of TVC in tuna cooled and stored in liquid ice over time 35

4.1.2 Changes of TVC in tuna cooled in ice slurry and stored in crushed block ice 40

4.1.3 Changes of TVC in tuna cooled and stored in crushed block ice 41

4.2 SENSORY CHANGES OF TUNA DURING STORAGE 43

4.2.1 Sensory changes of tuna cooled and stored in liquid ice over time 43

4.2.2 Sensory changes of tuna cooled in ice slurry and stored in crushed block ice 47

4.2.3 Sensory changes of tuna cooled and stored in crushed block ice 48

4.2.4 Comparison of sensory quality of yellowfin tuna cooled and stored in different media 49

CONCLUSIONS AND RECOMMENDATIONS 58

CONCLUSIONS 58

RECOMMENDATIONS 58

REFERENCES 59

APPENDICES - 1 -

NaCl - Sodium Chloride

TVC - Total viable count

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LIST OF FIGURES

Figure 3 1 Experimental flowchart 29

Figure 3 1 Yellowfin tuna 31

Figure 3 2 Sampling tool 31

Figure 3 3 Procedures of TVC determination 33

Figure 4 1 Changes of TVC of Fish 2 (30 kg up) during storage in liquid ice of 3.0% of NaCl, 44% initial ice concentration, and initial temperature of -3.1°C 35

Figure 4 2 Changes of TVC in Fish 3 (20 kg up) during storage in liquid ice of 3.5% of NaCl, 48% initial ice concentration, and initial temperature of -4.0 °C 37

Figure 4 3 Changes of TVC in Fish 4 (30 kg up) during storage in liquid ice of 3.5% of NaCl, 48% initial ice concentration, and initial temperature of -4.0°C 38

Figure 4 4 Changes of TVC in Fish 5 (40 kg up) during storage in liquid ice of 3.5% NaCl, 48% initial ice concentration, and initial temperature of -4.0°C 39

Figure 4 5 Changes of TVC in Fish 8 (30 kg up) cooled in ice slurry with an initial temperature of -4.0°C and stored in crushed block ice 40

Figure 4 6 Sensory changes of Fish 2 (30 kg up) stored in liquid ice of 3.0% NaCl, 44% initial ice mass, and initial temperature of -3.1°C 43

Figure 4 7 Sensory changes of Fish 8 (30 kg up) cooled in ice slurry with an initial temperature of -4.0°C and stored in crushed block ice 47

Figure 4 8 Comparison of the sensory quality of all the fish at day 0 49

Figure 4 9 Comparison of the sensory quality of all the fish at day 3 50

Figure 4 10 Comparison of the sensory quality of all the fish at day 6 51

Figure 4 11 Comparison of the sensory quality of all the fish at day 9 51

Figure 4 12 Comparison of the sensory quality of all the fish at day 12 52

Figure 4 13 Comparison of the sensory quality of all the fish at day 15 53

Figure 4 14 Comparison of the sensory quality of all the fish at day 18 54

Figure 4 15 Comparison of the sensory quality of all the fish at day 21 54

Figure 4 16 Comparison of the sensory quality of all the fish at day 24 55

Figure 4 17 Comparison of the sensory quality of all the fish at day 27 56

Figure 4 18 Comparison of the sensory quality of all the fish at day 30 56

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LIST OF TABLES

Table 3 1 Studied parameters of liquid ice 30 Table 3 2 Cooling and storage media for tuna 30 Table 3 3 Control sensory sheet 1 32

Table 4 1 Changes of TVC in Fish 6 (40 kg up) cooled and stored in crushed block ice 41 Table 4 2 Changes of TVC in Fish 7 (30 kg up) cooled and stored in crushed block ice 42 Table 4 3 Sensory scores of Fish 3 (20 kg up) cooled and stored in liquid ice of 3.5% NaCl, 48% initial ice concentration, and initial temperature of -4.0°C 45 Table 4 4 Sensory scores of Fish 6 (40 kg up) and Fish 7 (30 kg up) cooled and stored in crushed block ice 48

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CHAPTER 1 INTRODUCTION 1.1 INTRODUCTION

Yellowfin tuna (Thunnus Albacares) part of the member of family Scombridae and

falls among the pelagic large size marine fish (Jinadasa, Galhena and Liyanage, 2015) Yellowfin tuna is a big species of tuna mostly found in Atlantic, Pacific and Indian oceans It is one of the specific focus species for the tuna fishing system and the most commonly caught marine fish on overseas fishery Yellowfin Tuna can be seen all over the environment that is warm-temperate and major occupied with tropical waters of all oceans staying in the environment with the temperatures between 15oC and 31oC (Nsw, 2000)

The principal components of Yellowfin Tuna are fat, water, vitamin, minerals and protein compounds The protein constituents of Yellowfin Tuna is within 15- 20% but there is variation as per the time period (Jinadasa, Galhena and Liyanage, 2015) The spoilage processes of fish is a difficult process which existed because of the actions of enzymes, microorganisms and chemical components (Nwaigwe, 2017) Yellowfin Tuna is a rapidly growing fish with females reaching 5 kg after one year and becomes matured after about 2 years at 25 kg Yellow fin Tuna mostly feeds on small fish, crustaceans and squids ((Nsw, 2000) According to Food Agricultural Organizations, it reported that the post-harvest losses getting to 35% In most of the developing countries, fish quality and their outcomes are the most imperative concern

in the industries where fish and fish materials are produced The losses are said to be higher in the countries where they consume lower protein Most of the fishermen have realized the benefit of high standard products in view of getting good outcome of the catch When there are physical damages to the body of the Yellowfin Tuna, it results

to spoilage, reduce the quality and shelf life (Razak and Hassan, 2002) Yellow tuna

is commonly used in the preparation of raw cuisine such as sashimi and sushi

(Nurilmala et al., 2013)

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Ice slurry has been seen to be a promising medium for preserving aquatic products Slurry ice has high benefits on local freshwater ice (block ice) and it brings down the temperature very quickly, has faster chilling rate and causes almost no physical damage to the fish Slurry ice also slows down the rate of microbial development and gives longer shelf life for many species It was recorded that the slurry ice has preventive effects over the multiplication of microbial loads when it is utilized for the preservation of some species on-board storage (Zhang, 2017) Deterioration of fish begins immediately after they die and as the degradation rate is mostly rely on the temperature, the quicker the fish can be cooled the better This is one of the reasons why the fish quality is prevented for enough time when there is sufficient ice quantity and it is distributed accordingly Slurry ice is especially efficient for cooling large pieces (Ronsivalli and Baker, 1981) The smaller the ice particles, the greater the interaction between fish and ice, it has positive effect on the rate of heat removal (Ronsivalli and Baker, 1981)

The proper handling of fish plays high role in the extension of storage life and fish quality after catch Any delay in fish cooling after its catch under the environmental temperatures of 15-20oC will bring down the seafood storage life for quite a number

of days (Razak and Hassan, 2002) Some tools are required for proper handling of fish and these include gaffs, tuna missile, fish bat or club, spike, knives, nylon brushes, meat hook, gloves, mutton cloth or plastic body bags, ice shovel and all these tools should be kept clean to prevent cross contamination to the fish One of the such good handling practices is to ensure that captured live fish are not permitted to fight and die of asphyxia oxygen starvation (Tawari and Abowei, 2011) There are so many factors responsible for the spoilage of fish and these includes microbiological, enzymatic, oxidative and hydrolytic (‘Recent Developments in Fish Processing, 1953) Microorganism contamination of fresh fish is the most causative agents of fish storage When fish is kept under low temperature, growth of bacteria and as well as spoilage is minimized (Tawari and Abowei, 2011) Inappropriate handling practices and insufficient infrastructure facilities have immediate impact on the quality of yellowfin tuna which can result in major post-harvest losses

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The two most common problems when it comes to selling and marketing of seafood are their openness to deterioration and unhealthy cleaning quality The major objective of fish preservation is to inhibit microbial, chemical and enzymatic spoilage and which is only possible by controlling the storage temperature, sustaining appropriate pH, aW or through the use of preservatives Temperature plays a main role

in fish spoilage which have major control on microbial growth and the autolytic degradation (Co, Cn and Tk, 2016)

Preservation of fish is necessary in order to get the fish to an end user in good and usable conditions The necessary steps to accomplish this starts immediately before the fishing expedition starts and do not stop until fish is eaten or processed into any other form (Nwaigwe, 2017) When fish is stored at the temperature that is low, it can extend its storage life and improves the quality as a result of reducing the chemical and microbiological processes The higher the temperature during storage, the shorter the shelf life of fish and also affects the quality of the fish (Agustini, 2002) Freshness quality of fish is said to be a great important factor on the overall quality of a particular fish product This quality included appearance, flavour, odour, skin colour and texture of each fish product, is knowingly and unknowingly determined by every consumer If the fisheries item comes across the expectation of the buyer in respect

to freshness quality, it will be more likely to be purchased

Fish that is not properly taken care of might not be noticeably bad, but it loses its quality because of off-flavours, soft texture, or discoloration that discourage a potential buyer from buying (Nwaigwe, 2017) The major causes of fish spoilage are microorganism contaminations, such as bacteria, fungi, virus etc., and their growth

It is generally stated that if fish are kept clean at low temperature, then development

of bacteria, consequently spoilage is kept at minimum (Tawari and Abowei, 2011) Therefore, the aim of this study is to show the difference and the effect of ice slurry and block ice on the sensory, microbiological and chemical parameters of yellowfin tuna possessing unique attention applied to the quality deteriorations in the fish during the chilled preservation

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1.2 MAIN OBJECTIVE

To evaluate the suitable handling and preservation method that can be used for

port-harvested Yellowfin Tuna

1.3 SPECIFIC OBJECTIVES

1 To monitor and compare the sensory changes during storage of yellowfin tuna cooled and stored by various media, namely liquid ice of different salt and initial ice concentration, mixture of ice and salted water, crushed block ice or combinations of those

2 To monitor and compare the total viable counts (TVC) changes during storage

of yellowfin tuna cooled and stored by the above mentioned media

3 To find out the suitable medium/media for post-harvested yellowfin tuna handling and preservation

1.4 PROBLEM STATEMENTS

One of the most challenging demands faced by the fish industry is to maintain the quality and improve the yield of the fish products It is generally known that tuna has been regarded as a palatable and valuable fish species and its freshness has become the concern of many researchers

Block and flake do not have direct contact with the flesh of the fish thereby limiting the quality of the yellowfin tuna Most of these forms of ices require a certain degree

of manual operation for transportation from one place to another, as there is an additional income procurement which in turn affects the outcome of the final product

The block ice possess rather sharp edges that damages a product’s surface when used for direct contact chilling, when the sharp edges pierce through to the flesh of the fish, this tends to open up the flesh of the fish muscle providing medium for quick deterioration in the quality of the fish muscle by chemical, biological and

microorganism activities

other media and this gives it good cooling system

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CHAPTER 2 LITERAURE REVIEW 2.1 YELLOWFIN TUNA

2.1.1 Production

Yellowfin tuna (Thunnus albacares) is important in tropical and subtropical season

Near-surface schooling yellowfin tuna are mainly caught by purse seines and by and-line fishing, than trolling and gillnetting (Science, 2010) The most popular fishing method for deep swimming yellowfin tuna is long lining The total catch of this species in 1999 was reported to be 1,258,386 t, of which Indonesia 176,320 t and Mexico 121,884 t (Science, 2010)

pole-Vietnam, with a long coastline of over 3,260 km, 226,000 km2 of the inland water is about 226,000 km2, and above 1 million km2 of the Exclusive Economic Zone (EEZ), has significant potential for seafood exploitation Yellowfin tuna poses a mild, meaty flavor and it can be called swordfish Yellowfin tuna is leaner compared to Bluefin

Fish has contributed around 50% of the total protein source for human beings (Vietnam, 2020) Yellowfin tuna was recorded as the second dominant component of coastal based tuna fishery and they formed about 24.5% of the overall tuna catch in coastal fishery with an average annual production of 27,277 t during the year 2006-

2010 (Ghosh, 2012) When the fish meat is in its raw form, it possesses bright red but when it is cooked, it changes from brown to grayish-tan, firm and moist skin with big flakes There are important yellowfin tuna fisheries throughout tropical and subtropical seas (FAO, 2010) A number of researchers have seen vertical movements and environmental preferences with the aid of acoustic and satellite telemetry and also archival data loggers One of the major limitation of yellowfin tuna and all fishes

is the oxygen concentration though there is a large level of differences which occurs for endurance to hypoxic conditions (Weng, Stokesbury and Boustany, 2018)

Vietnamese tuna fisheries, occurring mainly in Binh Dinh, Phu Yen and Khanh Hoa provinces, use four main fishing gears, which are longline, purse seine, hand line and

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gillnet There were 714 and 1,678 long line fishing vessels in 2011 and 2012 respectively

2.1.2 Habitat of Yellowfin Tuna

Yellowfin tuna (Thunnus albacares) are found mostly on the surface layer of all warm

seas of the world apart in the Mediterranean Sea The gear types determine the varieties of fish sizes Most of the long liners delivers majorly fish > 100 cm, while purse seiners (and pole-and-line boats) focus both small (40- 60 cm) and large fish (Lehodey and Leroy, 1999)

Thermal structure of the water column affects the vertical water column distribution

of the, as is revealed by the high correlation between the susceptibility of the fish to purse seine capture, the mixed layer depth, and the strong point of the thermocline temperature gradient Yellowfin tuna live mainly at the upper 100 m of the water column with noticeable oxyclines (Science, 2010)

2.1.3 Size, age, and growth

Yellowfin tuna can reach a dimension of 280 cm long with the highest weight of 400

kg 176.4 kg as reported by the International Game Fish Association (IGFA) This fish species is popular in warm waters all over the Pacific and the Atlantic

Yellowfin spawning seems to be Pacific-wide and confined in its northern and southern extremes by the 26°C surface isotherm While the incidence of larvae is unceasing transversely the equatorial Pacific inside a zone nearly 10° north and south

of the equator, three areas of higher larval density have been uncertainly documented: 180-160°W, east of 110°W and 130-170°E The occurrence of spawning in yellowfin tuna is seen all year round, possibly with a peak in the November–April period Some records also recommend diverse spawning times for areas east (March–September) and west (November–April) of 180° The smallest female yellowfin tuna found in the eastern pacific with mature ovaries was 84 cm, and the projected length at 50-percent development was 95 cm A few yellowfin tuna in the central equatorial Pacific mostly

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reaches maturity at around 70-80 cm According to the recent data recorded by the University of Hawaii shows that the majority of yellowfin tuna do not mature until they get to 100-110 cm Nevertheless, the food supply plays important roles in determining the time needed to reach spawning state (Lehodey and Leroy, 1999) The optimum fork size is over 200 cm, common fork size is 150 cm The smallest mature fish were found within the size group from 50 to 60 cm fork length at an age of roughly 12 to 15 months in Philippines and Central America, but within 70 and 100

cm fork length indicates that the percentage of mature individuals is much higher All fish reaches sexual maturity when they gets to the length over 120 cm (Science, 2010)

1.3.4 Reproduction

Yellowfin tuna sizes vary by regions, especially at the maturity stage, and differences could also be observed between individuals seen near- and offshore When yellowfin reaches the state of maturity by the time they become 120 cm long at the age of about 2-3 years However, some cases are seen where fish become mature at the size of 50

to 60 cm in fork length at an age of about 12 to 15 months The percentage of the sex

is nearly 1:1 in immature fish and in grownup up to 140 cm This fish shows full sexual maturity from 50 cm upward and as well as pawn at a much smaller size at an age of almost one year, especially in Indian waters (Ghosh, 2012)

Yellowfin tuna reproduces all year round, but it happens mostly during the summer

in each hemisphere In the tropical water of Mexico and Central America, it has been resolute that yellowfin spawn at minimum of two times in a year Record shows that the minimum temperature for yellowfin tuna fish spawning is 26oC and each female lays multiple million eggs per year

in the muscle of fish muscle as at when they are dead Self-digestion weakens the belly wall (Mayer and Wolf, 2015)

Rough handling coupled with autolysis can cause the bursting of the belly which is rely mostly on the storage temperature and time Nucleotide catabolites from autolytic changes could be responsible for fish spoilage The major autolytic method

in the fish muscles involves carbohydrates and nucleotides Subsequently, the process

of rigor mortis sets in, it serves as a basis for advance autolytic deterioration The belly of some fish (e.g herring, capelin, sprats and mackerel) during the period of heavy feeding is very vulnerable to tissue dilapidation and may rupture inside some hours after the catch (Mayer and Wolf, 2015)

2.2.2 Chemical spoilage

Peroxidation occurs in Herring fillets because of high polyunsaturated fatty acids, great level of speed up of haeme-proteins and made up of relatively small postmortem muscle pH and this leads to susceptibility High content of these compound in the fish

is particularly on of the major factors that brings about the quality reduction, especially all through the post-harvest handling They eventually plays a major role

to catalyze the development of rancidity, texture changes, pigmentation and that of nutritional value loss (Mayer and Wolf, 2015) Oxidation of a purely chemical nature

is the major important changes that takes place in the lipid fraction and these variations may possess serious quality depreciation challenges, which includes rancid

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odours, flavours, and discolouration Auto-oxidation and lipid autolysis are the two types of rancidity found Auto-oxidation is described as a response involving oxygen and unsaturated lipid which is enhanced by heat and light Lipid autolysis which is

an enzymatic hydrolysis consisting of free fatty acid and glycerol as most important products The breakdown of sulphur-containing ammonium acids to methyl mercaptan, hydrogen sulphide and dimethylsulphide play major role toward the resultant smell of the damaged fish When peptide is broken down to ammonia, it gives off the ammonia and sulphate odours Due to the fact that most small and medium sized fatty pelagic fish are caught in excessive number and they are not gutted directly when catch which results to a challenge due to the rate at which rancidity sets in (Mayer and Wolf, 2015)

of the genera Pseudomonas, Shewenella, and Alteromonas cause disintegration at a

speedy rate The major cause of the organoleptic spoilage in raw fish is due to the accumulations of metabolic products of bacteria thereby producing the characteristics fishery ammonia and sulphide odour, and this affects the texture by changing it to the pulpy and slimy features of damaged fish (Gormley, 1990)

Several researches reported important inhibition of histamine development in fish preserved at low temperatures (Taylor, 1986) Nevertheless, the histamine

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accumulated in mackerel stored for elongated times (14 days) at 5°C Others researchers have recounted on a group of psychrophilic halophiles which indicates as part of the ordinary surface micro-flora of marine fish and can yield big volumes of

histamine at temperatures as low as 2.5°C (Okuzumi et al., 1984) These bacteria

occur artificially in the external layer of the on the gills, skin and intestine of marine fish and several of these bacteria need proteins and amino acids for their growth (Prescott, Harley and Klein, 1996) Spoilage micro-organism grow even at low temperatures in refrigerated seawater (RSW) systems in the presence of NaCl because they are modified to the environment where the fish is gathered

2.3 HANDLING AND PRESERVATION OF FISH

Seafood spoilage is caused by a number of factors which include endogenous enzymatic processes, lipid oxidation and processing techniques, most times the spoilage that occurs above freezing temperature is as a result of the presence of bacteria on the fish flesh when it is harvested Since fish are poikilothermic, the microflora present during the period of catch is greatly influenced by the microorganisms in their habitat and temperature (Keys, 2015) To properly retard the enzymatic and microbial activity after harvest, the fish should be cooled to temperatures between 0 and -2°C as quickly as possible and even a short time of temperature abuse can promote rapid decomposition and loss of quality (Keys, 2015) Correct handling of fish through the aid of quick cooling and keeping on board play major roles in dictating the quality of the fish And also, adequate processing kinds and methods established on capacity and demand are also the key factors for real assistances (Mayer and Wolf, 2015)

Another major factors that can be responsible for seafood contamination and growth

of pathogenic micro-organisms is inappropriate handling and processing of seafoods Also, the regular incidence of aquatic bio-toxins and regular pathogenic flora of the aquatic surroundings also have contributing impact to seafood borne diseases

(Mahmud et al., 2018) In order to improve the marketing chances regionally as

quality standards are becoming as important criteria for selling fish across borders,

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there must be taken into considerations the improved quality and sanitation issues People who are involved in the fish value chain should have their capacity built because this is important of improving standards and quality (Ward and Beyens, 2012)

One of the major factors that the body support of the fish and techniques needs is safe handling and this in turn helps to protect against wound The common examples of these techniques are using wet hands and not grabbing fish by the jaws, gills or eyes All the necessary precautions must be adhered to such as time out of the water should

be reduced and all measures should be carried out low and on the water tub so that should in case a fish slides off the measuring board it will fall softly into the tub of water and not on a rigid boat outward (Hawkins, 2008) The way fish is handled onboard the vessels i.e how they are preserved, packed and moved from one place to another greatly influences the quality of fish getting to the consumer or processing factories Personnel on board the vessels play the major role of preserving the catch after hauling till it is unloaded at landing centers To ensure high quality for the end product, big tuna must be gaffed carefully, spiked, bled and then quickly placed in the slurry in order to prevent a rise in the temperature which will later make them prone to quick spoilage

Preservation means putting microorganisms in an unfavorable environment of their normal activities to delay or inhibit their growth, shorten their survival or cause their death (Tsironi, Houhoula and Taoukis, 2020) The quality of end seafood product is determined by the standard of raw materials The quality lost during handling cannot

be recovered back during processing Most of the fish produced from low quality fish does not pose any safety risk but the quality as well as the shelf life of the final product would be affected (Quang, 2005) In Vietnam, one of the major challenges is how to maintain fish raw material standard The interval frame between catching and acceptance at the processing plants can be extended while the raw materials temperature is not sufficiently low adequately to inhibit decomposition or

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deterioration The fishing sector in Vietnam has been faced with the challenges of stagnation in catching and also deterioration in the quality of raw materials

2.4 REFRIGERATED METHODS OF SEAFOOD PRESERVATION

The purpose of the preservation of foods by refrigerated system is to decrease and sustain the food temperature which in turn stops, or greatly decreases the rate at which deterioration changes take place in the food Examples of these changes can be microbiological (i.e development of microorganisms), biochemical (e.g browning reactions, lipid oxidation, and pigment degradation), and/or physical (such as moisture loss) physiological (e.g ripening, senescence, and respiration) (James and James, 2014) Some foods possess the features that enable them to be easily processed and preserved while others, such as fresh fish, poses a challenge to retailers, processors and consumer Fish is highly prone to deterioration because of the presence on inherent constituents, which are responsible for the growth of bacteria The quality of fish quality will become depreciated after some hours or days if appropriate storage condition which include refrigeration or preservation treatments (e.g salting, heating and irradiation) are not put in place When fish are caught off-shore, special procedures are used to enable low amount of microbial counts through processing and to assure consumer product’s safety (Barbut, 2015)

Some of the most dominant preservation methods used presently have been established in several years ago before the knowledge science about micro-organism/chemical decomposition and pathogen existence In the past, people extend the shelf life of food by using different preservative methods and these include cooling, drying, freezing, heating, fermenting and adding ingredients such as salt, sugar, etc Today, antimicrobial compounds, produced by selected strains of microorganisms resulted to the development of molecular biology, are used to inactivate pathogens during fermentation of fish (Barbut, 2015) The skin outward of not alive fish is a perfect growth medium for micro-organism, which results in fish spoilage When the temperature is altered, the growth of many bacteria that causes

the deterioration will be retarded but not completely eliminated (Txdolw et al., 2018)

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Physical factors can aids either chemical or bacteriological processes such as bruising cutting and tearing etc can open up fish muscles to enables quick bacterial growth and this causes blood to flow out which in turn darken the fillets and reveal greater

surface area for chemical corrosion (Txdolw et al., 2018)

To make available hygienically safe food products of high organoleptic standard (such as smell, taste texture, appearance), sensible effort must be paid to every aspect

of the cold chain from primary freezing or chilling of the raw ingredients, packing and transport to retail exhibition outlet handling A competent and appropriate cold-chain is made to enable the optimum conditions for retarding, or hindering these variations (James and James, 2014)

In view to understand the degree of perishability of foods, they are often categorized

as less delicate, moderately delicate and highly delicate Foods such as cereals, grains, and nuts are categorized as less delicate and more stable, vegetables as moderately delicate and seafoods as highly delicate food items Seafoods are prone to spoilage as

a result of their high moisture content, inherent enzymes, weak connective tissue, neutral pH and nutrients availability which enhances the growth of microorganisms

(Jiang et al., 2018) Furthermore, the quality of seafood is greatly dependent on its initial quality, catchingtime, catching location or habitat, gender, body composition, species, and methods of handling (Jiang et al., 2018) Studies shows that the decomposition of fish occurs from three major factors: autolysis, enzymatic,

oxidation and microbial growth (Ghaly et al., 2010)

Different types of preservation methods have been used both traditionally and in modernized ways which include drying, salting, smoking, freezing, chilling, brining, fermentation and canning These methods have been reported to extend the shelf life

of seafoods and meat products, although at different degrees However, the most common methods in the industry are the chemical and low temperature storage techniques for controlling water activity, enzymatic, oxidative and microbial

development (Ghaly et al., 2010)

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Principles guiding the various methods include controlling water activity, low temperature storage and controlling oxidative spoilage, while other means of preservations can be grouped into chemical, physical, and microbial controlling methods The major objective of proper handling and preservation is to control or reduce those factors that can cause spoilage in fish and fishery products so that the end product is wholesome and safe for the consumer Fish and fishery products in a well-kept situation will commonly have advanced value placed on them both at the wholesale and retail levels and thereby gives higher profits to the seafood operators

(Txdolw et al., 2018)

Chilling plays an important role in reducing decomposition in fish if it is done speedily and if the fish are chilled and kept cautiously and hygienically Immediate chilling of fish ensures high quality products For every 10oC reduction in

temperature, the rate of deterioration decreases by a factor of 2-3 (Txdolw et al.,

2018) Chilling is obtained when the top layer of the seafood is covered with ice However, chilling techniques is not encouraged for a long-term preservation, because melted ice produces excess water which may be containing washed bacteria and therefore bring about leaching of valuable flesh content responsible for flavour and desirable taste Meanwhile, well-iced seafoods are able to stay six to seven days without any difference in their taste and a freshly caught seafood Also, antibiotics are sometimes added to the ice used in the chilling process (Tawari and Abowei, 2011)

When a product has been chilled, the temperature must be preserved by refrigerated storage Cold air plays Chill stores are normally cooled by circulation of cold air produced by mechanical refrigeration units, and foods may be stored on pallets, racks,

or in the case of carcass meats, hung from hooks (Richardson, 2001) The size of the refrigeration system is different depending on the level of heat that needs to be eliminated and preferably the heat capacity will be reduced During the time of cooling, heat will be eliminated out of the products during storage, transportation and retailing the only means by which heat loads should gain access to the product should

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be across the structures, penetrating through the door opening (Evans, 2020) The increase in the deterioration of seafood is still the major problem as a result of seafood distribution and safety It is paramount to provide high quality products for the consumer consumption with high quality products by prolonging the time of freshness Research is still on going about the super-chilling storage on seafood and

there are many challenges not yet determined (Ando et al., 2004)

In chilling or freezing, the rate of heat removal from products will determine the size

of the plant At the initial stage of cooling, the maximum rate of heat release occurs when the temperature gradient between the outward of the product and the refrigerating medium is at its peak (Evans, 2020) The most suitable method for chilling fish when transporting is to stock the fish in an ice-box containing ice slurry made from sea water and ice The subzero point of seafood is referred to be around -2oC and it is neither a chilled nor a partially frozen temperature When seafood is exposed to temperatures below 0°C, it undergoes from low temperature stress, and it

is assumed that the free amino acid and sugar level of the cells multiply so has to resist from becoming frozen When seafood is kept under low temperature, the growth

of microorganism is greatly altered (Ando et al., 2004)

2.4.1 Icing and iced storage of fish

Ice storage is one of the types of preservative technique Ice serves as an ideal medium for chilling of fish because of the high heat absorption capacity The use of ice in the preservation of fish is one of the easiest means of preserving fish and food because the medium is generally available everywhere and the fish can be kept for a number

of weeks (e.g 20days to 30 days) in suitable standard if appropriate icing is done In most circumstances due to the insufficient of knowledge, icing is not accurately engaged in traditional fish handling and preservation Icing plays major role in reducing the rate of post-harvest damages as well as maintaining the quality of fish (Clucas and Ward, 1996)

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Popular forms of ice used in the seafood industry are crushed ice and flake ice Ice melt-water majorly removes the surface micro-organisms and contaminants Ice melt-water also preserves the fish surface that prevents lack of fluids and conserves the glossy presence, also because of the direct contact of ice-melt water makes a good conductor of heat that enable cooling As ice melts at 0oC, ice will not freeze the fish but automatically controls the temperature at the appropriate chill level (Clucas and Ward, 1996)

2.4.2 Pre-cooling and cooling by slurry ice

For so long now, different methods of preserving such as refrigerated sea water, traditional flake ice and chemical additives have been used The use slurry ice which

is also called liquid ice has seen to be a technique with future existence for the preservation of aquatic food products in an ice-water suspension at frozen temperature Ice slurry has proven to provide several benefits over flake ice or block ice and these advantages include lower physical damage to products because of its tiny microscopic particles and spherical geometry of the ice crystals, faster cooling

of the body temperature and better heat exchange power (Gao et al., 2010)

The use of ice for extending the food shelf life date back many years, when ice used for cooling was taken from the nature such as arctic ice or winter snow In order to bring down the temperature, the natural ice was sometimes mixed with salt Ice slurry

is a homogenous mixture of fine ice particles and the carrier liquid, which may be pure freshwater or a binary solution of water and a freezing point depressant, such as sodium chloride, ethanol, ethylene glycol and propylene glycol (Kauffeld, M., Kawaji, M., Egolf, 2005)

Slurry ice contact is gentle to the fish, and little surface damage is incurred Salinity, temperature, and ice packaging factor (IPF) can be easily controlled Based on these advantages of slurry ice, it has been used to chill fish following landing The effect

of this chilling technique on the quality of chub mackerel has been shown to preserve high quality fish flesh with high ATP and high pH 6 There are, however, problems

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of chilling fish by slurry ice If the fish is immersed for too long, cold shock rigor may set in Hence, fish should normally be stored on ice after certain time of immersion in slurry ice The eyes become dull due to the high salinity and low temperature of slurry ice Therefore, the salinity and temperature should be controlled

(Maeda et'al., 2014)

Ice slurry because of the latent heat of fusion, it has a high energy storage density The fast cooling rate of ice slurry is due to the large heat transfer surface area generated by its recurrent particles The ice slurry sustains a persistent low temperature form during the cooling procedure and make available a higher heat transfer coefficient than water as well as other single phase liquids These features of ice slurry make of great important in several applications Slurry ice submerges the entire fish surface including the abdominal cavity thereby creating direct contact of the fish with the cooling medium and this create an immediate or rapid decrease of the fish core temperature Slurry ice advantages include pumpability, less damage to the product, and it has direct contact with the product and this in return gives it the ability to absorb heat (Keys, 2015) Ice slurry also has some imperfection, and these includes the ability of the ice to suspend to the surface of the container and form tough ice layers which have to be substantially fragmented (mostly with a shovel or any other implements) by personnel and this in return damages product in most times The tiny‐sized and gel‐like in texture ice crystals in the slurry will be less abrasive The lesser the ice crystal size, nevertheless, may bring about more quick melting as well as has less operative cooling effects than traditional ice slurry (Keys, Lowm der and Mireles DeWitt, 2018)

Ice slurry has gained more and more application in the seafood industry to take full advantage of the chilling swiftness effects on the fish This ice possess a wide range covering capacity which gives fish or any other food products a better protection against oxidation and dehydration, and which can also reacts with some other agents

known as ozone and melanosis inhibitors (Losada et al., 2004)

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There has been a study of the change in use of ice slurry as initial storing system

preceding to the method of freezing and frozen preservation of fish called sardine (S pilchardus) to control rancidity development in the frozen products As a matter of

fact, the initial ice slurry cooling step resulted to elongated storage life of the frozen products, as shown by sensory analysis (flesh, external odour, colour and appearance) (Medina, Gallardo and Aubourgs, 2009)

2.5 EVALUATION OF FRESHNESS AND QUALITY CHANGES OF FISH

There is rise in the consumer’s craving to have high standard fresh seafood Freshness refers to odor, flavor, texture and appearance, and all these has greater impact on the overall quality of fish Fish spoilage and quality may be tested through microbial and sensory analysis, which indicates freshness by tracking bacterial multiplications as well as physical degradations (Keys, 2015) Physical deterioration may include changes in odor, flavor, and texture that sensory analysts may be able to distinguish (Simpson BK, 1997) Sensory changes are often the result of bacterial metabolic processes that form a series of chemical end products including amines, sulfides, aldehydes, and ketones These metabolic by-products are often associated with unfavourable off odors and off-flavors that sensory analysts can use to indicate fish (Olafsdottir and Jonsdottir, 2009) After the death of fish, sensory changes (in odor, color, texture, flavor and appearance attributes) that occur vary considerably with season, catching method, species fishing ground and storage conditions which eventually shows the chemical, autolytic and microbial deterioration processes that makes fish unfit for human consumption

The fresh flesh has a translucent appearance as an outcome of the incident light scattering reflectively as well equally When melting ice comes in contact with the fish, it resulted to penetration of water to the skin and reduce translucency As spoilage increases the flesh shows the appearance of opaque because the occurrence light is not scattered equally as a result of the gradual breaking down of myofibrils and their wider and more random intracellular distribution The peritoneum becomes dull and can be progressively more easily detached from the internal walls of the

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visceral cavity (Lougovois and Kyrana, 2005) During spoilage of intact fish, the blood contained in the kidneys, its associated vessels and main artery along the backbone gradually diffuses into the adjacent flesh causing blood discoloration Rough handling may also be responsible for rupture of blood vessels, bruises and blood moving directly inside the tissue Depending on the extent of occurrence of blemishes and defects such as bloodstains, bruising and discoloration, the fish may become unacceptable for some purposes like retail display of wet fillets (Lougovois and Kyrana, 2005)

2.5.1 Sensory analysis

When considering sensory analysis which includes the appearance, flavour, texture and odour are mostly examined with the aid of human senses Scientifically speaking, the procedure can be segmented into three steps Detection of a stimulus by the human sense organs; assessment and analysis with the aid of mental process; and thereafter, the answer of the judge to the stimuli Differences among individuals in the answer

of the same level of stimuli can be differs and can add to an incomplete answer of the test People can, for instance, different broadly in their feedback to colour (colour blindness) and also in their alertness to chemical stimuli It is very paramount to be notified about the differences when picking and training assessors for sensory test Interpretation of the stimulus and response must be trained very cautiously in order

to receive objective responses which shows features of the fish being estimated It is very convenient to give an objective answer to the asked question: such as is the fish

in rigor (perfectly stiff), but more training is required if the assessor has to choose

whether the fish is post or pre-rigor (Huss, 1988)

2.5.1.1 Quality Index Method (QIM)

The quality of fish needs to be kept as fresh as possible because fresh fish is a greatly perishable product The distribution of fishes are not constant and fresh fish can be stored only for a limited time Therefore, the need for quick analytical methods to measure the food standard and freshness is more than ever before The method called

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Quality index method which was developed by the European fisheries research institutes serves as an effective method for carrying out sensory assessment QIM is seen as a practical and objective material for estimating the fresh fish production management in the seafood monitoring and also the other parts of the chain (Bernardi, Mársico and de Freitas, 2013) A well-organized scaling method, the QIM, is given

as a practical and objectives material for examining fresh fish in production management in the official seafood inspections as well as other aspects of the chain Sensory analysis of the whole fish is completely carried out by trained assessors at fish manufacturing factories The sampling is very paramount but there is no difference between the generally practiced for quality management The sampling must be randomized, and the number rely solely on the lot The examination area must be as neutral as possible relating to noise and smell Trained assessors can examine as much as 40fish with the aid of QIM in 20minutes and the method doesn’t have any harm (Hyldig, 2004)

2.5.1.2 Torry method

The Torry schemes were created in the year 1950s at the Torry Research station in Aberdeen The method called Torry scheme was organized using a small number of species and focused on the types of fish This method majorly serves as effective objection system for sensory evaluation of fish and also gives comprehensive examination of the organoleptic qualities of fish Torry schemes are also available for raw (whole) and cooked fish Torry scheme can be used to evaluate the cooked fish and all fish fillets and Torry assessment can serve as an indicator of freshness quality over time in general (Workbook, 2015)

2.5.1.3 Quantitative descriptive analysis

This method is majorly used to determine the shelf life of a product This method may be used for fish Cook to determine the maximum storage time outside of work and also describe the detailed description of the individual's product characteristics (Hạnh, 2012)

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2.5.2 Microbiological analysis

In microbiological analysis, listing of micro-organisms is an important microbiological work for so many industrial and research applications Several tests for identification and numbering of micro-organisms are used most times in the recent age and this includes standard plate count with or without membrane filtration, Adenosine Triphosphate testing, flow cytometry detection, IR spectroscopy and polymerase chain reaction (PCR) (Bogomolny and Vanholsbeeck, 2013) Foodborne pathogens such as bacteria or toxins, viruses or parasites may result to human disease when polluted food is taken The basis of pollution may fluctuate but destructive microbes are mostly in control for triggering gastrointestinal contaminations The foundations could be the animal, the environment or contamination during food handling (Congress and Pdhi, 2018)

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CHAPTER 3 MATERIALS AND METHODS 3.1 MATERIALS

Yellowfin Tuna caught from Vietnam was used for the experiment Yellowfin Tuna

of three different sizes, namely 20, 30, and 40 kg up, was purchased from the Hon Ro Seaport (Nha Trang city, Khanh Hoa province, Vietnam) and was transported to the Food Technology Laboratories of Nha Trang University On the arrival of the fish to the laboratories, the fish temperature was checked raised immediately to 24-28oC in order to simulate the tuna body temperature right after catch and cooled down to around 0oC before actual storage began

Cooling and storage media have been used in the study:

- Liquid ice, produced from the liquid ice machine, with:

o 3.0% NaCl, 44% initial ice concentration, initial temperature -3.1°C;

o 3.5% NaCl, 48% initial ice concentration, initial temperature -4.0°C; Salinity of liquid ice was similar to that of seawater

- Slurry ice produced by mixing ice and salted water to create an initial temperature of about -4.0°C For this purpose, crushed block ice was mixed with saturated NaCl solution until the desired temperature was reached;

- Crushed block ice (control)

3.2 APPARATUS AND TOOLS

- Electrical heated incubator thermostatically controlled

- Pipette 1000 microlitre with disposable

- Petri dishes, approx 9 cm in diameter

- Pipette 100 microlitre with disposable

- Laboratory balance

- Pyrex flask with screw cap, 100ml

- Substrate tubes 10mm x 16 mm with cap

Figure 3 1 Experimental flowchart

Ice crystal ratio

Evaluation of the Parameters

Total viable count

Yellowfin Tuna

Cool and preserved with liquid ice

Salt concentration Fish size

Periodic sampling with 2 samples points

(back and abdomen)

Sensory

Statistical analysis

Conclusions

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3.3.2 Experimental factors

Table 3 1 Studied parameters of liquid ice

Detail information of the experimental variables are depicted in Table 3 2

Table 3 2 Cooling and storage media for tuna Tuna Fish size Cooling medium Storage medium Storage days

Liquid ice of 3.0%

NaCl, 44% initial ice concentration, initial temperature -3.1℃

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F3 20 kg up Liquid ice of 3.5% NaCl, 48% initial ice

concentration, initial temperature -4.0℃

(LI3.5)

Liquid ice of 3.5%

NaCl, 48% initial ice concentration, initial temperature -4.0℃

of LI3.5

Crushed block ice 30

Note: F6 and F7 are control samples (cooled and stored in crushed block ice)

3.3.3 Sampling

Samples were taken at the following time points: upon arrive to the laboratories, at the beginning of the cooling process, at the beginning of storage and every 1-3 days

Back core

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during the preservation process up to 12-39 days until the fish spoilage Day 0 is considered as start of storage, day -2 as arrival day to the laboratories, and day -1 as start of cooling

Figure 3 2 Yellowfin tuna

Samples were drawn from the back and belly flesh of the fish with the support of a sampling tool (Figure 3 3)

Figure 3 3 Sampling tool

Belly core Back core