Enhancing the quality of fresh snakehead fish fillets through improved handling and storage

MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY SONKARLAY KARNUE ENHANCING THE QUALITY OF FRESH SNAKEHEAD FISH FILLETS THROUGH IMPROVED HANDLING AND STORAGE MASTER THESIS..

MINISTRY OF EDUCATION AND TRAINING

NHA TRANG UNIVERSITY

SONKARLAY KARNUE

ENHANCING THE QUALITY OF FRESH SNAKEHEAD FISH

FILLETS THROUGH IMPROVED HANDLING

AND STORAGE

MASTER THESIS

MINISTRY OF EDUCATION AND TRAINING

NHA TRANG UNIVERSITY

Topic Allocation Decision

Decision on establishing the Committee

Defense Date:

Supervisor:

Assoc Prof Nguyễn Văn Minh

Chairman:

Assoc Prof Trang Si Trung

Faculty of Graduate Studies:

KHANH HOA - 2020

UNDERTAKING

I hereby declare that the thesis in title “Enhancing the quality of fresh snakehead

fish fillets through improved handling and storage” was compiled and written

exclusively by me and it has never been submitted, in whole or in part, in any previous application for an academic degree until submission to Nha Trang university for assessment and granting of MSc degree in Food Technology

Khanh Hoa, September 7, 2020

Sonkarlay Karnue 60CH299

FUNDING

This research is funded by the Vietnam Ministry of Education and Training (MOET)

under grant number CT2020.01.TSN.02; “Nghiên cứu công nghệ sơ chế và bảo quản cá lóc

tươi (Channa striata) và phi lê cá lóc” to Assoc.Prof Nguyen Van Minh

ACKNOWLEDGEMENT

I would not have succeeded in writing this thesis without the guidance and motivation of other professional individuals with expertise in food science I wholeheartedly extend my gratitude to my supervisor, the most dynamic and energetic Assoc.Prof Nguyễn Văn Minh for his patience, enthusiasm and sincerity in guiding me through my research time I cannot express all his good deeds, encouragements and moral lesson he thought, Prof Minh has been of great help throughout my research time

I must acknowledge the Nha Trang University for according me the scholarship opportunity and a suitable learning facility in which this thesis research was done Not only, but also the peaceful environment and caring services I have received are valuable contributions to the success of the thesis writing More besides, I must appreciate Dr Mai Thi Tuyet Nga, the director for VLIR in Nha Trang University for allowing me to use her personal equipment for my research

Away from this, I am glad to acknowledge the director general and all the officials and staff of the national fishery and aquaculture authority (NaFAA) of Liberia for their support and encouragements without which this thesis could not be written Nobody has been more concerned about me in the pursuit of this project than my family I would like

to thank my parents, Mr and Mrs Teewon, and all friends and relatives who love and guide me in whatever I pursue Most importantly, I wish to thank my loving wife and daughter for their patience and support

Thanks

Khanh Hoa, September 7, 2020

Sonkarlay Karnue 60CH299

TABLE OF CONTENTS

UNDERTAKING iii

FUNDING iv

ACKNOWLEDGEMENT v

TABLE OF CONTENTS vi

LIST OF ABBREVIATIONS xi

LIST OF TABLES xii

LIST OF FIGURES xiii

ABSTRACT xv

CHAPTER 1: INTRODUCTION 1

1.1 Preamble 1

1.2 Background 2

1.3 Problem statement 3

1.4 Justification of the study 4

1.5 Research Questions 5

1.6 Hypothesis 5

1.7 Objectives 6

1.7.1 General objective 6

1.7.2 Specific objectives 6

1.8 Scope of the study 6

1.9 Limitations of the study 6

Chapter 2: LITERATURE REVIEW 7

2.1 Snakehead Fish 7

2.2 Nutrient composition of snakehead fish 8

2.3 Fish freshness 9

2.4 Fish Spoilage 9

2.4.1 Enzymatic spoilage 10

2.4.2 Glycolysis 10

2.4.3 Lipolysis 11

2.4.4 Proteolysis 11

2.4.5 Nucleotide and nucleoside degradation 12

2.5 Fish Lipid 12

2.5.1 Lipid Oxidation 13

2.5.2 Mechanism of Lipid Oxidation 13

2.5.2.1 Initiation 13

2.5.2.2 Propagation 14

2.5.2.3 Termination 14

2.5.3 Photo-Oxidation 14

2.5.4 Enzymatic Oxidation 15

2.5.5 Hematin Compounds-induced Oxidation 15

2.5.6 Protein Oxidation 16

2.5.7 Effects of Oxidation on the quality of Fish/Fishery Products 17

2.5.8 Monitoring Lipid Oxidation 18

2.6 Fish Handling 19

2.6.1 Methods of Killing Fish 19

2.6.2 Fish Bleeding 20

2.6.3 Fish Gutting 21

2.6.4 Filleting and Skinning Fish 21

2.7 Fish Preservation 22

2.7.1 Preservation by Low Temperature 22

2.7.2 Chilling and Chilling Media 23

2.7.3 Superchilling 24

2.8 Packaging methods 25

2.8.1 Modified atmosphere packaging (MAP) 25

2.8.2 Vacuum Packaging 26

2.8.3 Air packaging 26

2.8.4 Packaging Impacts and Fish Quality Changes 27

Chapter 3: MATERIALS AND METHODS 29

3.1 Materials 29

3.1.1 Snakehead fish 29

3.1.2 Chemicals 29

3.2 Experimental design 29

3.2.1 Preliminary experiment 29

3.2.1.1 Development of QIM and Torry Schemes for Snakehead fish (SHF) fillets 29

3.2.1.2 Determination of freezing point of snakehead fish (SHF) fillets 30

3.2.2 Main experimental design 30

Experiment 1: Cooling media effects on whole snakehead (SHF) quality 31

3.3 General Sample Preparation 33

3.3.1 Experiment 1: 33

3.3.1.1 Cooling media effects on whole snakehead (SHF) quality 33

3.3.1.2 Temperature Sensor Insertion into fish muscle 33

3.3.1.3 Cooling Rate of Cooling Air, Crushed Ice and Slurry Ice 33

3.3.2 Experiment-2: the effect of packaging methods and storage temperature on the quality of Snakehead fish fillets 34

3.4 Quality analysis methods 34

3.4.1 Sensory analysis using QIM Scheme 34

3.4.2 Evaluation of cooked SHF fillets with Torry Scheme 35

3.4.3 Cooking Yield Determination 35

3.4.4 Texture Profile Analysis 36

3.4.5 Proximate Composition Analysis 37

3.4.6 Determination of pH and soluble protein content 38

3.4.7 Lipid hydroperoxide and TBARS analysis 38

3.5 Statistical analysis 39

Chapter 4: RESULTS AND DISCUSSION 40

4.1 Preliminary experiment 40

4.1.1 Development of QIM and Torry Schemes for snakehead fish fillets 40

4.1.2 Determination of snakehead fish muscle freezing point 42

4.2 The effects of cooling media on the quality of whole snakehead fish 43

4.2.1 Cooling Rate 43

4.2.2 Fish appearance 45

4.2.3 Sensory attributes of fish fillets using QIM 46

4.2.4 Sensory attributes of fish fillets using Torry scores 48

4.2.5 Cooking yield 49

4.2.6 Effect of cooling media on the texture profile of Snakehead fish fillets 50

4.3 Packaging method and storage temperature effects on the quality of snakehead fish fillets 52

4.3.1 Proximate Composition Analysis 52

4.3.2 Changes in pH and soluble protein 54

4.3.3 Changes in peroxide value and TBARS contents 55

4.3.4 Sensory analysis of packaged fillets using QIM 57

4.3.5 Changes in Torry Scores of chilled and superchilled packaged fillets 62

4.3.6 Cooking yield of chilled and superchilled packaged fillets 63

4.3.7 Texture profile analysis of chilled and superchilled fillets 65

Chapter 5: CONCLUSION AND RECOMMENDATION 70

5.1 Conclusion 70

5.2 Recommendation 71

REFERENCES 72 APPENDICES I Appendix A: I Appendix B V Appendix C VII

LIST OF ABBREVIATIONS

CA : Cooling air

CAP : Chilled air packed fillet

CI : Crushed ice

CPO : Cumene peroxide

CVP : Chilled vacuum packed fillet

FSC : Flesh side color

Odo : Odor

PL : Phospholipid

PV : Peroxide value

QI : Quality index

QIM : Quality index method

SAP : Superchilled air packed fillet

LIST OF TABLES

Table 2.1: Nutrient distribution in different size of snakehead fish 8

Table 2.2: The Shelf life of fresh fishery products under vacuum or air packaging 28

Table 3.1: Definition of treatments and sampling schemes 34

Table 4.2: The QIM Scheme developed for Snakehead fish 41

Table 4.3: Torry Scores for cooked Snakehead fish fillets 42

Table 4.4: Changes in proximate composition of SHF fillets 53

Table 4.5: Changes in the Texture Profile parameters of air and vacuum packed snakehead fish fillets during chilled and superchilled storage 68

LIST OF FIGURES

Figure 2.1: A pictorial diagram of snakehead fish (Channa straitus) 8

Figure 2.2: Myoglobin and Hemoglobin oxidation 16

Figure 3.1: Snakehead fish used in this study 29

Figure 3.2: Experimental design for evaluating Cooling media effects on whole snakehead (SHF) quality 31

Figure 3.3: Experimental design for evaluating the effects packaging method and storage temperature on snakehead fish fillet quality 32

Figure 3.4: A portion of fillet cut for the evaluation of cooking yield 36

Figure 3.5: Fillet portion for TPA (A) and a SUN RHEOMETER (B) 37

Figure 4.1: The initial freezing point of Snakehead fish muscles 43

Figure 4.2: Temperature profile of cooling the central muscles of whole snakehead fish in cooling air, crushed ice and slurry ice 45

Figure 4.3: Difference in the appearance of whole snakehead fish after 3 days of storage in different cooling media 46

Figure 4.4: Principal components of QIM scores for whole snakehead fish store in cooling air (CA), crushed ice (CI), and slurry ice (SI) 48

Figure 4.5: Changes in the Quality index (QI) of whole snakehead fish (A) and attribute scores (B) determined after 3 days of storage cooling air (CA), 48

slurry ice (SI) and crushed ice (CI) 48

Figure 4.6: Changes in the Torry scores of whole snakehead fish after 3 days of cooling in different ice media 49

Figure 4.7: changes in the cooking yield of whole snakehead fish after 3 days of cooling in different ice media 50

Figure 4.8: Changes in the hardness (A) and texture profile (B) of whole snakehead fish after 3 days of cooling in different ice media 51

Figure 4.9: Changes in the pH (A) and soluble protein of content (B) of air and vacuum packed snakehead fish fillets before and after chilled and superchilled storages 55

Figure 4.10: Changes in PV (A) and TBARS values (B) 56

Figure 4.11: Principal components (A) and original bi-plot (B) of the QIM scores for air and vacuum packed snakehead fish fillets from chilled and superchilled storages 57 Figure 4.12: Changes in the QIM scores for attributes of air and vacuum packed fillets from chilled (A and B) and superchilled (C and D) storages 61 Figure 4.13: Correlation between storage time and quality index (QI) of air and vacuum packed snakehead fish fillets during chilled and superchilled storage 61 Figure 4.14: Changes in the Torry scores of air and vacuum packed snakehead fish fillets stored at chilled and superchilled storages 63 Figure 4.15: Changes in the cooking yield of air and vacuum packed snakehead fish fillets after chilled and superchilled storages 64 Figure 4.16: Changes in the hardness (A) shearing energy (B) and peak distance (c) of air and vacuum packed fillets during chilled and superchilled storage 68

ABSTRACT

Snakehead fish (Channa straita), as one of the most valuable freshwater fish, is

immensely used as food and traditional medicines mostly in Asia However, the shortcoming associated with the broader consumption of the snakehead fish is the relatively short shelf life derived from mishandling and inaccurate storage condition Therefore, the purpose of this study was to determine a suitable handling and storage method to enhance the quality and extend the shelf life of the snakehead fish fillets The specific objective of the study was to investigate the effect of cooling air, crushed ice, and slurry ice on the fresh quality of the whole snakehead fish The effects of vacuum packaging in comparison to air packaging method on the quality of the snakehead fish fillets was studied at both chilling and superchilling storage temperatures

Preliminary experiments showed that the freezing point of the snakehead fish muscle was -1.3 C It was also preliminarily observed that the skin-side color, flesh-side color, texture, stickiness and odor of the snakehead fish (SHF) fillets were parameters that changed the most along storage time Based on this, a QIM scheme with 12 demerit points and a Torry scheme spanning a score range 10 to 3 were developed for further use during the experiment one and two

During experiment one, it was also found that, slurry ice showed a faster cooling rate of whole snakehead fish and a fresh quality preservation capability over crushed ice and cooling air Fish from slurry ice fish showed a fresh appearance similar to that of the fresh fish before ice storage A lower freshness (QIM) score and a harder texture were revealed for slurry iced fish fillets than fillets of fish from crushed ice or cooling air

Proximate composition analysis showed no significant change in the moisture, total protein, total lipid, and ash contents of the air packed and vacuum packed fillet groups from both chilled and superchilled storage pH analysis at the end of chilled and superchilled storage (day 14 and 25) indicated that fillets from chilled storage have lower

pH and less soluble protein content as compared to fillets from superchilled storage Superchilled vacuum packed fillets revealed the highest percentage of soluble protein A higher degree of lipid oxidation was reflected by hydroperoxide (PV) and TBARS values

in chilled stored fillets than superchilled groups Final TBARS values in superchilled vacuum packed fillets was not significantly different (p > 0.05) from that of the fresh

fish Sensory analysis using QIM scheme and Torry scores rejected air packed fillets and vacuum packed fillets on day 10 and 14 at chilled storage The same fillet groups were acceptable up to 14 and 25 days at superchilled storage Instrumental texture analysis indicated that superchilled vacuum packed fillets were harder, more springy, cohesive, chewy, and gummy than its chilled stored counterparts

With this result in agreement with lipid oxidation study, the shelf life of the air and vacuum packaged snakehead fish (SHF) fillets was 9 and 13 days in chilled storage and

12 and 25 days in superchilled storage Based on our results, it can be reasonably concluded that slurry ice positively enhanced the fresh organoleptic properties of the whole snakehead fish, and vacuum packaging and storage at -2.5 ± 0.5 C was more efficient in preserving the fresh quality of the snakehead fish fillets

Keywords: snakehead fish, fresh quality, slurry ice, vacuum packaging, superchilling

CHAPTER 1: INTRODUCTION

1.1 Preamble

Known worldwide for its predatory effect, snakehead fish has so much to offer the hunger-striking human race In the face of increasing human population and its accompanying shortage of food, diseases resulting from imbalanced diet, and the inadequacy of terrestrial crop production as the result of global warming, snakehead fish stands to provide high-quality protein, vitamins, minerals, and omega-3 fatty acids very essential for human health This has made snakehead fish one of the most cultivated fish species in Vietnam and Asia at large (Chung, 2011; Sinh et al, 2014; Grimm-Greenblatt

et al, 2015)

However, the challenge faced by the snakehead fish industry is the rapid deterioration in quality and the shortening of its shelf life during storage The freshness of the fish is considered essential to its traditional consumers A number of factors including improper handling, and inadequate storage condition, are implicated in the snakehead fish (SHF) freshness degradation The demand for fresh snakehead fish (SHF) meat cannot be met if processing and preservation methods are inadequate

This study purposely sought to establish an optimum preservation method to

enhance the quality of the snakehead fish (Channa straitus) The study investigated the

effects of cooling in ice media, chilling, superchilling, and vacuum packaging on the

quality of snakehead fish (Channa straitus) The results obtained are organized into the

research presented in this thesis

The research presented in this thesis is the result of background investigation (chapter-1) that highlights current issues about snakehead fish and the method of preservation selected Reviews of existing literature on factors that interplay with fish quality and the significance of the preservation methods used in this study are presented

in chapter-2 The chapter-3 of this study details the general methodology, materials, and

chemicals used to assess quality changes in the snakehead fish (Channa straitus) The

core experiments of the thesis are found in chapter-4 that presents the effect of different cooling media, storage temperature, and packaging methods on the quality of snakehead fish (SHF) fillets The summary of the study that highlights key findings, future study needs, and relevant literatures cited are contained in chapter-5

1.2 Background

Fish is an important source of food worldwide that provides more than three billion people with daily animal protein (Tacon & Metian, 2013; FAO, 2018) Fish contains high-quality protein, vitamins, minerals, and omega-3 fatty acids very essential for the prevention of cardiovascular diseases, brain development in infants among others (Habibur & Molla, 2018) Beyond food needs, fish can also be utilized for the production

of pharmaceutical therapies (Jais & Manan, 2007), animal feeds, or used for recreational and cultural purposes This secures for fish a top spot among the most demanded food products in the world

Meeting such demand requires processing and preserving fish with high food value and transporting it from the point of production to places where the cost of livestock meat has risen beyond the reach of the low-income people One high-value fish

of which little is known about its processing condition is snakehead fish The need to preserve Snakehead fish is an increasing concern that cuts across several interest groups including farmers, processors, consumers, and pharmacologists Specifically, Snakehead fish contains important amino acids such as leucine, alanine, aspartic acid, and glutamic acid highly essential for health (Biosci et al, 2014) In a randomized, placebo-controlled

trial comparing the effects of oral snakehead (C striatus) extract (500 mg/day) with

placebo given for 3-month period to patients with primary knee osteoarthritis, snakehead

(C striatus) extract revealed a significant improvement of pain (Kadir et al, 2014) The

wound healing properties of snakehead fish (SHF) among post-operative patients was studied, and the results confirmed that the fish contained essential amino acid fatty acids along with 19% of arachidonic acid (C20:4) which is a precursor of prostaglandin and thromboxane noted for tissues growth (Rahman et al, 2018)

Several researchers have investigated different parameters in Snakehead fish

Study on the nutrients composition in the skin of the snakehead fish (Channa straitus)

revealed that the fish contains 77.2±2.39% moisture, 13.9±2.89% protein, 5.9±0.45 fat and 0.77±0.12% ash (Marimuthu et al, 2012) The meat yield of snakehead fish was investigated and found to be 63% which is 3 - 5% higher than tilapia and channel catfish (Qiufen et al, 2013) These among other reasons such as the ability to endure extreme environmental conditions, and high survival ratio during weaning to formulated feed (Thi

et al, 2017), make snakehead fish the best candidate for aquaculture The fish is widely cultured in Asia and some part of Africa where it is immensely used as food, traditional medicines, and pharmacological therapy owing to its anti-microbial, anti-inflammatory, cell growth, and anti-nociceptive properties (Qiufen et al, 2013; Rahman et al, 2018) Government initiative of providing trade incentives and bank loan to fish farmers in Vietnam has boosted snakehead fish production in the aquaculture rich Mekong delta region of Vietnam Such effort has increased snakeheads production from the total of 5,300 tons in 2004 to 30,000 tons in 2009 and 40,000 tons in 2010 (Chung, 2011; Sinh et

al, 2014; Grimm-Greenblatt et al, 2015) In 2014, China produced 510,000 metric tons of snakehead fish (Sagada et al, 2017) and the most recent production in China is 510,340 metric ton (Cao et al., 2020) In India, snakehead accounted for 13% of marketable freshwater fish (Haniffa, 2015) Snakehead fish production has contributed more than 70% of the total aquaculture production in Cambodia due to its popularity as food fish (So et al, 2009) In 2012, snakehead production in Malaysia accumulated to 1,284 tons

(Iliyasu et al, 2016) A total of 297 tons of snakehead fish (Parachanna obscura) was

reported from aquaculture in Nigeria (Moehl et al, 2004)

1.3 Problem statement

Snakehead fish (Channa straita), as one of the most valuable freshwater fish, it is

immensely used as food and traditional medicines mostly in Asia However, the shortcoming associated with the broader consumption of the snakehead fish is the relatively short shelf life derived from mishandling and inaccurate storage condition Marketing Snakehead is a localized business in Asian countries However, the value and market-share reducing factor for the fish seems to be the general perception that farmed seafood is inferior in quality to wild-caught stock This has led to little attention paid to the handling and processing of the farmed snakehead fish The spoilage of snakehead fish begins from the point of harvest through its marketing chain as the result of poor handling and improper storage conditions Based on high vitality, the fish can be sold whole and alive Due to change in environment, the fish endures stress, depleting limited cellular energy source (ATP) which hastens enzymatic autolysis In response to stress, escape behavior can be exhibited during which the live fish secrets mucus/slimes on its body surface The high concentration of slimes renders fish difficult in handling leading

to rough treatments that cause physical damage to the fish muscle (Shephard, 1994) After death, the mucus/slime also becomes a nutrient for surface bacterial growth, and bruises on the skin provide access to the fish muscle for bacterial contamination Other than superbranchial cavity for aerial respiration, snakehead fish also employs mechanisms such as depressed metabolic demands and increased oxygen transport which are associated with the accumulation of blood ammonia (Duan et al, 2018) Excessive level of ammonia in fish has the ability to reduce total protein in fish (Shin et al, 2016) Fish spoilage has been confirmed by many studies to be the shared function of microbial, enzymatic, and chemical reactions in the post-mortem fish muscles (Gatica et al., 2010; Lougovois et al., 2014; Mathew, 2017; Boziaris & Parlapani, 2017; Daskalova, 2019) These reactions are maximized at elevated temperatures with negative impact on the quality of the fish

Few studies have examined the relationship between storage temperature, time and quality-related variations in the snakehead fish muscle, but with focus on freezing and the frozen storage of the fish (Sahu et al, 2012; Tan et al, 2014; Hidayati et al, 2018) Freezing and frozen storage may denature and aggregate proteins, leading to an alteration

in functional properties, textural characteristics, water holding capacity, and juiciness of the fish Meeting consumer demand for fresh or minimally processed fish products without changing the natural quality parameters requires more than a single preservative technique to enhance the fresh quality of the fish

1.4 Justification of the study

The quality of fish relies immensely on the safety, nutritional value, freshness, and availability of the fish It is a fact that spoiled fish contains several pathogenic microorganisms and harmful chemical substances Not only does the spoilage of fish cause economic downtrend in the fish industry, but it also poses a serious threat to consumers’ health Therefore, it is imperative to understand the causes of spoilage and devise mechanisms that inhibit such spoilage in the snakehead fish This study plays a significant role in sufficiently assessing the intrinsic and extrinsic parameters that

interfere with the quality and shelf life of the snakehead fish (Channa striata) The

optimum storage conditions devised in the study are considered new and efficient in enhancing the keeping quality of the snakehead fish

As the outcomes of this study relied heavily on empirical evidence, relevant concepts, objective considerations, and ethical neutrality, they are therefore expected to assist the fish industry in optimizing time-temperature index that assures the fresh quality and longer shelf life of the snakehead fish The results are also expected to increase existing research based knowledge in the field of food technology at Nha Trang University

1.5 Research Questions

Several research findings have suggested that temperature control, and packaging are effective measures in inhibiting spoilage reactions and maintain freshness in post-mortem fish (Txdolw et al, 2018; Shahidi et al, 2020) The effectiveness of preservation

by temperature control depends on the media of cooling (chilling, superchilling) and the estimation of cold storage temperature largely depends on the freezing point of the fish muscle It is imperative to know

i How does chilling affect the snakehead fish (Channa straitus) quality and

which chilling method more efficiently retards deteriorative reactions and maintains the fresh quality of the snakehead fish?

ii Which packaging method is more effective and to what degree does the packaging method extend the shelf life of the snakehead fish?

1.6 Hypothesis

Based on previous publications, it can be predicted that cooling in slurry ice has the ability to maintain the fresh organoleptic properties of the whole snakehead fish Vacuum packaging and superchilling have the potential to inhibit microbial growth and retard oxidative and enzymatic reactions, thus enhancing the snakehead fish fillet quality and shelf life extension This is projected considering that temperature plays a critical role in the growth cycle of microorganisms and the viability of enzymatic activity On the other hand, vacuum packaging creates barrier to gaseous exchange and microbial infiltration that may otherwise cause the fish spoilage

1.7 Objectives

1.7.1 General objective

To determine a suitable handling and storage method to enhance the quality and

extend the shelf life of the snakehead fish (Channa straitus) fillets

1.8 Scope of the study

This research was conducted in the food laboratory facility in Nha Trang University, Nha Trang City, Vietnam Southeast Asia The stipulated time frame for this research was 10months (Nov 2019 – August 2020) The activity plan for this study included background study and proposal writing – Nov to Dec 2019; proposal defense, sample identification and collection – Jan 2020; experimentations, data collection, and thesis writing – Feb to July 2020; and thesis submission and defense – September 2020

1.9 Limitations of the study

A major challenge that confronted this study was time The full timetable designed for this study could not be implemented due to the outbreak of COVID-19 in Vietnam which resulted into the abrupt closure of the school

Chapter 2: LITERATURE REVIEW

2.1 Snakehead Fish

The snakehead fish (Channa striatus) from the channidae family, is an air-breathing

freshwater fish with a pair of suprabranchial cavities for aerial respiration Other mechanisms of aerial respiration in snakehead include depressed metabolic demands and increased oxygen transport These mechanisms can lead to accumulation of blood ammonia (Duan et al, 2018) The fish has a dark brown dorsal appearance and a white or grey ventral side Naturally snakehead lives in streams, canals, rivers, ponds, reservoir, and lakes Scientific classification placed snakehead fish into

Genus: Channidae and

Species: Channa striata

Currently there are approximately 28 species with two genera namely channa

(including Channa striata) popular in Asia and parachanna associated with African

origin Snakehead can be distinguished from other similar fish, like Bowfin fish, by a long dorsal and anal fins on its compressed and long rod-shaped body with expanded lower jaw, wider mouth and eyes position similar to that of Snake, a characteristic that

defines the common name (snakehead) of the Channidae spp (Kadir et al, 2014)

Snakehead in its area of popular use can be referred to as snakehead murrel, chevron snakehead, striped snakehead etc Snakehead consists of basic nutrients and other biomedical properties that validates its pharmacological usage

Figure 2.1: A pictorial diagram of snakehead fish (Channa straitus)

2.2 Nutrient composition of snakehead fish

The nutrient composition of fish consists of protein, lipid (fat), moisture, and mineral (ash) These are important 9±0.45 fat and 0.77±0.12% ash (Marimuthu et al, 2012) Nutrients distribution in fish can be components of fish that attract consumers The protein and fat content of fish are the most valuable nutrients on which fish quality determination is based Snakehead contains protein, fat, mineral and vitamins in amount essential for human nutrition In 2012, a group of researchers investigated the nutrient

composition of fresh Snakehead fish (Channa straitus) and revealed that the fish contains

77.2±2.39% moisture, 13.9±2.89% protein, 5.9±0.45% fat and 0.77% ash (Marimuthu et

al, 2012) Nutrients distribution in fish can be affected by the age, body size and other factors (Breck, 2014) Rosmawati et al (2018) investigated the nutrient composition in the

small size (SS), medium size (MS) and large size (BS) of snakehead fish (Channa

straitus) and report the result shown in Table 2.1

Table 2.1: Nutrient distribution in different size of snakehead fish

2.3 Fish freshness

Freshness is a relative term that attempts to define the quality state of fish/fishery products in terms of time, changes that occur in fish after catch, spoilage and so on The time the fish was caught up to the time it was delivered to the consumers, how the fish was processed (excluding canning, cooking, curing and freezing) and the inherent sensorial attributes such as appearance, odor, flavor, and texture are determining factors

Farmers view freshness as a higher price accumulating and market share negotiating factor, while processors consider the fresh state of fish/fishery products as a point of upturn in production volume On the other hand, consumers conceive freshness

as a point of maximum nutritional value The fresh state of fish may be indicated by a shining, lustrous skin covered with a thin layer of evenly spread, nearly transparent slime with bright, convex eyes consisting of a jet-black pupil and transparent cornea and with gills generally bright pink or red without any free visible slime (Lougovois & Kyrana, 2014) Fish/fishery products considered fresh are expected to maintain characteristic color, texture, flavor, and odor and all other identifiable species-specific features

2.4 Fish Spoilage

Fish spoilage is a change in the quality of fish that affects the nutritional worth (moisture content, protein, fat, and mineral), availability, convenience, integrity, physical attributes of the species While safety and freshness are undeniably the most essential quality parameters, sensory features such as odor, flavor, and palatability are the major attributes that consumers can readily use to accept or reject a fish product Fish quality degradation is relative to enzymatic, microbial, and chemical reactions in the fish muscles after death Research has confirmed that the loss of 30% of fish landed globally are

caused by microbial activity alone While 25% of primary fishery products are lost to chemical deterioration every year, about 4-5 million tons are lost to enzymatic and microbial spoilage due to improper storage (Brooks et al, 2010)

2.4.1 Enzymatic spoilage

Enzymes are found in both the intracellular and extracellular sections of fish muscle, but in varying specific concentrations In live fish, enzymes such as phosphorylase B, chymotrypsinogen, etc., are inactive precursors And they require cofactors for activation While other enzymes such as hydrolases are kept inside lysosomes and isolated from their substrates There are also some enzyme systems such

as adenylate cyclase, ATP-ase, etc., which are present in an active form in tissues, though

in low concentration and are distributed only in specialized tissues (Mukundan et al, 1986) These enzymes aid digestion of complex molecules as well as production of energy for cellular activities in live fish Deterioration in postmortem fish usually derives from enzymatic breakdown in carbohydrate, fat and protein contents of a fish

2.4.2 Glycolysis

The amount of carbohydrates in fish is relatively low but can influence rigor mortis and autolysis induced by enzymes Carbohydrates in all animal tissues are converted into glucose and other compounds to power cellular activities Excess glucose can be stored as glycogen After death, intracellular glycogen is broken down by enzymes

in a process called glycolysis Glycolysis in fish is a function of phosphorylase Its optimum activity occurs at neutral pH and ambient temperature In postmortem fish, glycolytic enzymes (phosphorylases) break down glycogen Principally, lactic acid and pyruvic acids are produced as its end-products with only two moles of ATP released through an anaerobic pathway ATP provides energy for muscle contraction by the release of Ca2+ ions in the sarcoplasm which prevents contracting interaction between actin and myosin thus keeping the fish muscle relaxed, pliable and elastic (V.P Lougovois & Kyrana, 2014) Over a period, due to lack of oxygen and nutrient supply, the level of creatine triphosphate (CTP), and ADP, ATP in the anaerobic muscle gradually decline from normal range of 7-10 moles/g to less than 1.0 mole/g tissue This causes the bridging of actin and myosin that contracts the muscles into rigor mortis Anaerobic glycolysis results in the accumulation of more protons from lactic acid

that in turn lowers the fish muscle pH from 7.0 – 7.2 to 6.2 – 6.5 (Lakshmanan, 2000) Post-mortem lowering of pH can lower the net ionic charge on muscle protein This is noticed to partially denature protein and cause change in the muscle texture, water holding capacity (Gatica et al, 2010) and lessen resistance to microbial growth (Daskalova, 2019) Loss of water from fish flesh can lead to change in light reflection at the surface known as color change (Gatica et al, 2010; Brooks et al, 2010)

et al, 1986) A higher concentration of unsaturated fatty acids released from lipolysis is quite susceptible to reaction with atmospheric oxygen reaction The result of such a reaction is oxidative rancidity

2.4.4 Proteolysis

Acidic pH can actuate many autolytic enzymes Small organelles in cells known as lysosomes contain about 36 hydrolytic enzyme systems These enzyme systems are capable of degrading protein, carbohydrate, fat, nucleic acids, etc (Mukundan et al, 1986) Cathepsins and proteases are the dominant proteolytic enzymes and confined in fish lysosomes Fluctuation in temperature as well as an increase in acidity can rupture the lysosomal membrane The breakdown of lysosomal membrane can release cathepsins, proteases, and several other hydrolases into muscle tissue Cathepsins are active at acidic

pH (3-4.4), and a temperature range from 37 up to 50 C This enzyme is capable of breaking down peptide bonds and digesting the entire fish body protein in less than 24hrs (Mukundan et al, 1986) Cathepsin activity in fish is at least 10 times greater than that of mammals (Mukundan et al, 1986) Protein degradation in postmortem fish produces an increased concentration of free fatty acid and amino acid

2.4.5 Nucleotide and nucleoside degradation

Nucleotide (containing phosphate group) and nucleoside are nitrogenous bases and sugar The degradation of these ATP related compounds in fish muscles produces several flavoring compounds During autolysis, adenosine triphosphate (ATP) rapidly degrades

to adenosine diphosphate (ADP) ADP can be broken down to adenosine monophosphate (AMP) and inosine monophosphate (IMP) IMP is responsible for the desirable sweet meaty characteristic and umami flavor in fresh fish (Lakshmanan, 2000) As autolysis continues, IMP decomposes into neutral-tasting inosine In further degradation, bitter-tasting hypoxanthine (Hx) that characterizes the spoilage of fresh fish can be released (Lakshmanan, 2000) Autolytic spoilage of fish is also characterized by belly bursting caused by leakage of proteolytic enzymes from pyloric caeca and intestine to the ventral muscle (Dong et al, 2019) An increased concentration of these enzymes may digest protein in the gut muscles of heavily feeding fish and provide a substrate for the proliferation of bacteria which produces CO2 and H2 gases that cause the belly bursting (Lakshmanan, 2000)

2.5 Fish Lipid

Lipids are a group of naturally occurring compounds, which are readily soluble in organic solvents, such as hexane, toluene, chloroform, ethers, and alcohols Physico-chemically, lipid molecules are classified into neutral and polar lipids typically found in all living organisms Neutral lipids comprise of triglycerides primarily contained in adipose tissues which are a major source of energy for living organisms In contrast, polar lipids consist of phospholipids and glycolipids found in the membrane which serve as structural compounds that form a barrier separating the living cell from the outside world

Generally, lipid contains mainly nutritional fat-soluble vitamins (A and D), polyunsaturated fatty acids combined with glycerol (glycerides and glyceryl ethers), fatty alcohols (wax esters), sterols (sterol esters), phosphoric acid, and amines Differing from other sources, fish lipids contain a wider range of fatty acids, longer-chain fatty acids, and a larger proportion of highly unsaturated fatty acids, particularly (eicosapentaenoic acid, EPA) and (docosahexaenoic acid, DHA) (Nguyen, 2018) Fish lipid content is said

to be heterogeneously distributed throughout the body of marine species, probably due to physiological and anatomical factors (age, sex, and sexual maturation) For instance, the

lipid content of lean fish is proportionally less than 2% while fatty fish contains up to 30% lipid concentration (Bao & Ohshima, 2014)

2.5.1 Lipid Oxidation

A major challenge in the preservation and storage of fish/fishery products is lipid oxidation, a complex process in which unsaturated fatty acid reacts with oxygen and metal ions (Fe3+ and Cu2+) and H2O2, and UV light in a free radical propagated chain reaction to form peroxides This process also entails a series of secondary reactions that lead to lipid degradation and rancidity Sequential removal of a hydrogen atom from the fat acyl chain decomposes lipid and forms volatile compounds such as aldehydes, ketones, alcohol, etc which contributes to unacceptable off-flavor and off-odor in fish products (Vieira et al, 2017) Free radical and peroxide can also destroy vitamin A, C, and E and carbonyl products can react with cysteine, methionine, tryptophan, and lysine

to break down protein through Maillard reaction that gives rise to undesirable brown discoloration (Vieira et al, 2017) The chemistry of polyunsaturated fatty acids and their reactivity to oxygen and carbon to carbon bond cleavage or rearrangements in foods have been immensely studied and their detrimental effects are documented

yellow-2.5.2 Mechanism of Lipid Oxidation

Lipid can be oxidized by many catalytic systems which include light, temperature, enzymes, metals, metalloproteinase, and microorganisms (St Angelo, 1996) The three mechanisms of lipid oxidation include autoxidation, Photo-Oxidation, Oxidation by Lipoxygenase, and Hematin Compounds-induced Oxidation Autoxidation is a spontaneous reaction of molecular oxygen with lipids through a free radical chain reaction that involves three phases (Secci & Parisi, 2016):

2.5.2.1 Initiation

The initiation of lipid oxidation is a complex process yet to be made clear beyond all reasonable doubts However, it entails hemolytic hydrogen atom abstraction from the methylene group of polyunsaturated fatty acid , which leads to alkyl radical (R•) formation Molecular oxygen known to initiate this reaction contains a triplet electronic state while the double bond of fatty acid on which it must react as a singlet-state of an electron (Pateiro et al, 2019) This explains the need for energy source (heat or light) Or

the presence of catalytic metal ions to activate oxygen to form singlet oxygen (O2) or reactive oxygen species such as hydrogen peroxide (H2O2), superoxide anion (O2-), and hydroxyl radical (OH-) that react with fatty acid double bonds (abstract hydrogen atoms)

to form alkyl radicals Alkyl radical formation is followed by isomerization that reduces the energy level of the alkyl radical to form a conjugated double bond (Barden 2014)

2.5.2.2 Propagation

The decomposition of alkyl radicals, which results from combining with existing oxygen, leads to the formation of peroxyl radicals (ROO•) Peroxyl radicals with sufficient energy can react with unsaturated fatty acids which usually results into the formation of hydroperoxides (ROOH) and another alkyl radical (Barden, 2014) Decomposition of hydroperoxide leads of the formation of aldehydes or a rancid flavor in fish muscle, whereas the free radicals produced can damage vitamins and protein (Nguyen et al, 2014)

co-2.5.2.3 Termination

Termination is the completion of the free radical reaction process in which radical products are formed by interaction of fatty acid radical and peroxy radical

non-2.5.3 Photo-Oxidation

Photo-oxidation can occur in two ways; which the first transpires by the migration

of electron or a hydrogen atom from an excited triplet sensitizer to a substrate (PUFAs), which produces free radicals or radical ions secondly, by the excitement of triplet oxygen to singlet oxygen by light which reacts with the double bond of unsaturated fatty acids, producing an allylic hydroperoxide (Wasowicz et al, 2004)

2.5.4 Enzymatic Oxidation

Enzyme-catalyzed lipid oxidation and free radical initiation are similar; only the formation of hydroperoxides initially in fatty acid differentiates the two (Zhang, 2018) Lipoxygenase is a group of enzymes that contains an iron atom, which is in high spin state Fe (II) and must be oxidized to Fe (III) by fatty acid hydroperoxides or hydrogen peroxide The active enzyme abstracts a hydrogen atom from the methylene group of a polyunsaturated fatty acid to form a conjugated diene system that reacts with molecular oxygen (Secci & Parisi, 2016) Lipoxygenase produces similar flavor volatiles to those produced during autoxidation

2.5.5 Hematin Compounds-induced Oxidation

Myoglobin (found in muscle tissues) and hemoglobin (found in blood tissues) are iron-containing color pigments with physiological function of distributing oxygen in a biological system Postmortem, non-enzymatic oxidation also occurs by these hematin compounds (hemoglobin, myoglobin and cytochrome) as a result of their iron content Both hemoglobin and myoglobin initially exist in deoxy form but may combine with molecular oxygen to form oxy-hemoglobin/myoglobin

During storage, ferrous oxymyoglobin (Fe2+) may oxidize to ferric metmyoglobin

Fe3+ (Baron & Andersen, 2002) This causes discoloration in fish fillets Ferric metmyoglobin (Fe3+) content can be kept in balance with ferrous oxymyoglobin (Fe2+) through enzymatic reduction In postmortem fish, pH fall can stimulate acid-catalyzed

autoxidation (Baron & Andersen, 2002) As seen in (Figure 1.2), ferric metmyoglobin

Fe3+ can be recycled to its deoxy form

Figure 2.2: Myoglobin and Hemoglobin oxidation

Adopted from (Suman & Joseph, 2013)

This occurs through reaction with superoxide and metmyoglobin reductase (Nguyen et al, 2014) Reactive ferric metmyoglobin Fe3+ can also react with hydrogen peroxide to form an unstable hyper-valent perferryl myoglobin MbFe (IV) Such reaction rapidly reduces hyper-valent perferryl myoglobin to a stable ferryl myoglobin MbFe (IV) that can again change to metmyoglobin at physiological pH (Baron & Andersen, 2002) Myoglobin structure can also be changed by aldehydes through covalent bonds, resulting

to fillet color change during storage time (Suman & Joseph, 2013) Additionally, brown pigments in fish can be generated via lipid-protein interaction In this case, lipid peroxide can interact with active types of proteins and lead to the transformation of the light-colored or colorless precursor to brown pigments (Hidalgo & Zamora, 2000)

2.5.6 Protein Oxidation

Protein oxidation is the change in the protein covalent structure (bonds) that result from reaction directly with reactive oxygen species (ROS) or indirectly with secondary lipid oxidation by-products such as malondialdehyde, hydroxynonenal and acrolein that covalently bind to protein residues (Requena et al, 1997) Oxidation of amino acid side chains and protein

Metmyoglobin/

haemoglobin MetMb(Fe3+)

Reduction

+CO

Oxidation

Oxidation -O

O 2

backbones induced by ROS simply leads to protein fragmentation or protein–protein linkages (Zhang, 2018) Among all amino acids, cysteine and methionine are more susceptible

cross-to oxidation due cross-to their sulfur group Oxidative change in protein can confer change in physical and chemical properties, including conformation, structure, solubility, vulnerability to proteolysis, and other enzyme activities (Zhang, 2018)

While non-radical pro-oxidant reaction with protein backbone might be very slow, -OH radical induced abstraction of hydrogen from the alpha-carbon site of amino acid residues is a rapid process with damaging effect on protein backbone (Stadtman, 2006) This protein backbone oxidation can lead to formation of carbon-center radicals that react with O2 to form alkylperoxyl radicals that may react with HO2 to produce hydroperoxides or may be eliminated to produce imines (Zhang, 2018) Without oxygen involvement, two carbon-center radicals can associate to create cross-linkage within and between proteins (Stadtman, 2006) Hydroperoxides reaction with alkoxyl radicals can result into peptide bonds cleavage while imines hydrolysis can cause protein backbone fragmentation (Davies, 2005)

2.5.7 Effects of Oxidation on the quality of Fish/Fishery Products

Lipid oxidation can be implicated in the loss of functional properties, nutritional values, the formation of toxic compounds and colored products (Secci & Parisi, 2016) Several research accounts have documented the negative impacts of lipid oxidation on the sensory aspect of fish (Hematyar et al, 2019; Yin et al, 2014) Decrease in sensory parameters such as color, odor, texture and muscle elasticity was reported by (Yin et al,

2014) from study on the effects of frozen storage on grass carp (Ctenopharyngodon idellus)

fillet Interaction between protein and lipid can hinder the functional properties of fish

During frozen storage, ice crystals formation and recrystallization can disrupt cell organelles This usually leads to the release of lipase that causes the formation of free fatty acid via lipolysis Free fatty acid and hydroperoxide can interact, cross-link, and fragment sarcoplasmic and myofibrillar protein Denatured protein can lose water holding capacity through drip loss as the result of damage in the food texture (Nguyen et al, 2014)

Protein oxidation by ROS does not only cause backbones and side chains modification of proteins but also changes in the structure can lead to variation in quality attribute including tenderness and other physical and chemical properties of protein

(Zhang, 2018) The effect of ROS-induced oxidation on protein was confirmed when treatment of cod muscle with free radical generating system (-OH) resulted into decrease

in protein solubility, and increase in gel-formation, and water-binding activity (Srinivasan

& Hultin, 1997) Increased gel formation is an accompanying factor of cross linking of polypeptides and protein via disulfide bonds, which decrease the mobility of gel matrix thereby stabilizing other non-covalent bonds within the gel matrix This is followed by protein aggregation and polymerization that usually resists enzymatic degradation, thus altering digestibility with negative impact of nutritional values of fish muscles

2.5.8 Monitoring Lipid Oxidation

Lipid oxidation is a complex reaction that produces several biochemical compounds; each with its own characteristic feature specific for indicating an extent of lipid oxidation Different composition of food products and the intricacy of reactions and interactions that occur during lipid oxidation, have made it difficult to develop a single technique to measure degree of oxidation Consequently, several techniques have been categorized into sensory evaluation and several chemical analysis procedures In food industry oxidative rancidity can be detected by taste or smell through the use of human panelists whose sensitivity allow the recognition of oxidative deterioration at a point where chemical method cannot detect

However, there can be dissimilarity in individual panelists’ sensitivity to off-flavor and off-odor Such dissimilarity is driven by an individual's health status and can influence performance accuracy With this, the chemical method can be applied to validate sensory data The state of oxidation can be assessed primarily from changes in fatty acids, the formation of lipid hydroperoxides and conjugated dienes The amount of free fatty acid (FFA) is a value for lipolysis and can be oxidized faster than bound fatty acid (FA) Therefore, they can be regarded as a measure of increased oxidative reactivity

of fish muscle (Chowdhury & Sciences, 2015)

In most cases, hydroperoxide value is used to indicate initiation of lipid oxidation These primary oxidation products are unstable, therefore, a reliable quantification of oxidative deterioration should include estimation of secondary products such as carbonyls and aldehydes (malondialdehyde) (Pateiro et al, 2019) The reactivity of aldehydes with thiol containing acid, has made the thiobarbituric acid (TBA) test is an extensive method

of quantifying oxidative damage in food system Malondialdehyde is the principal secondary oxidation product from PUFA that reacts with TBA to give the red pigment on which the test is based This red colored complex of malonaldehyde-TBA can be measured spectrophotometrically at 532 nm (Taylor, 2010a)

The processes listed above are major causes of fish spoilage, however their initiation is not only derived from intrinsic means but human contact, mishandling, and inadequate preprocessing operations are factors among several extrinsic means that enhance fish spoilage

2.6 Fish Handling

Fish handling is a series of practices that ensure the best quality of fish It entails but is not limited to method of catching, slaughtering, bleeding, gutting, washing, and filleting operations (Borderías & Sánchez-alonso, 2011) Hygiene and sanitation also play

a critical role in the safety and quality of fish The use of nets, hook, pots, etc in catching wild fish is considered to have caused fish a period of desperate struggle and asphyxiation on board This results into muscle glycogen depletion, more lactates and bruises that favor rapid on-set of rigor mortis, enzyme activities, bacteria penetration and thus spoilage (Borderías & Sánchez-alonso, 2011) Unlike wild fish, farmed fish are harvested under controlled condition where sometimes fish are kept at low density (usually about 5 – 10 kg/m3) inseparate holding unit without much stress Starvation is another handling method that evacuates the gut content of farmed fish and delay spoilage

by reducing digestive enzyme activity (Huidobro & Tejada, 2004)

2.6.1 Methods of Killing Fish

Since fish response to stress can lead to quality depreciation, the method of killing/slaughtering fish is of paramount concern Of recent, chilling live fish in ice slurry (asphyxia) and the exposure of fish to ammonia or salt bath are methods recorded for killing fish Other methods include freezing, bleeding (exsanguination) by cutting blood vessels through the gills, and the transfer of fish to water saturated with carbon dioxide gas (Vis & Abbink, 2014) However, these methods cannot prevent stress and discomfort

as fish remains conscious and sensitive to struggle with their effects It was been

demonstrated, in the killing of cobia (Rachycentron canadum), that percussive stunning

(a blow to the head) can induce immediate loss of consciousness and insensitivity which prevents avoidable stress and enhance fish flesh quality (Cesar et al, 2017)

Bleeding common Carp (Cyprinus carpio) by cutting the gill arches reduced the total heme content, from 9.6±1.6 in unbled to 2.34±0.8 μmol/kg of hemoglobin in bled carp (Sterniša et al, 2018) The same study also revealed a significant reduction in peroxide value from 88.9±4.2 in unbled to 62.1±2.9 μmol/kg in bled carp, as well as TBARS value that reduced from 4.2±0.5 in unbled to 2.6±0.4 μmol/kg of malondialdehyde in bled The sensory analysis also showed improved color, odor and overall acceptability of bled raw fillets

Bleeding can be affected by pre-handling stress When fish are exposed to stress factors, they exhibit escape behavior during which blood flow is directed to the locomotive white muscle to meet the increased demand for oxygen Stress also reduces the time required for blood clotting This results in poor blood drainage induced by catecholamine through intravascular coagulation and vasoconstriction (Erikson et al, 2010)

It is also predicted that temperature plays a critical role in blood coagulation At low temperatures (3 – 5 C), blood coagulation time is extended, which keeps the blood fluid up to an hour, thus improving bleeding (Roth et al, 2009) The practice of bleeding largely depends on the type of species, the size, the season of capture, and most importantly the time interval between capture/slaughter and bleeding

In 2014, a researcher demonstrated various methods of bleeding that reduced residual blood and improved fillet whiteness of cod fish However there was no clear

trends reported between bleeding methods except for a two-stage approach of direct gutting immediately after capture and exsanguination for 30min prior to gutting It was concluded that time between slaughter and bleeding was the only single factor that influenced proper blood drainage from fish muscles (Olsen et al, 2014)

2.6.3 Fish Gutting

In fish handling, gutting influences quality attributes However, at what time and how gutting is done are key factors that lend the practice its importance in enhancing fish

shelf life The quality parameters of gutted and ungutted Arctic charr (Salvelinus alpinus)

stored on ice for 17 days were studied and results revealed reduced bacteriological count that correlated well with sensory scores from the quality index (QIM) (Akankwasa, 1998) Akankwasa also concluded that fish that were gutted and iced immediately showed better quality than those that were delayed for 18 hours Similar result was

reported when the storage of gutted Meagre (Argyrosomus regius) at refrigerated

temperature (4 C) proved advantageous than it counterpart in terms of chemical, microbiological and sensory analyses (Bİlgİn et al, 2016; Uddin et al, 2017; Changeswhole et al, 2008 ) The effectiveness of gutting can be ascribed to the removal viscera and gill which contain a lot of bacteria and enzymes

2.6.4 Filleting and Skinning Fish

Following slaughter, bleeding and gutting, fish can be filleted by separating the muscle mass from the vertebrae through cutting along the upper and lower appendices on the spine, removing the ribs with a thin blade knife Filleting is followed by trimming; removing the backbone, visible fat, pin bones, and skin to satisfy consumer demand Factors affecting the fillet yield of farmed fish have been the species of fish, size, sex, anatomy (Bencze Rora, 2017) as well as farming condition (feeding, water temperature etc.) (Borderías et al, 2011) The belly flaps of fillets are liable to discoloration; its removal could enhance better appearance during storage Filleting can be done manually

or by machine in commercial setting Maximum yields obtained from manually filleting

farmed Atlantic Halibut (Hippoglossus hippoglossus) ranged from 55.2±0.3 to 58.8±3.5

for skin-on and 49.0±1.6 to 52.9±2.9 skinless fillets (Davísson, 2013)

2.7 Fish Preservation

Fish spoilage is an intricate post-mortem process that involves enzymes, bacteria, and inherent chemical interactions in fish muscles These processes occur very fast due to high moisture, less connective tissues, and highly unsaturated fatty acid contents associated with fish Spoilage induces loss of fish nutritional value and other functional properties which consequently lead to a downward trend in the economic productivity of its stakeholders Inhibiting fish spoilage requires more than a single treatment to preserve the delicate fresh quality often demanded by consumers Meeting consumer demand without changing the natural quality parameters of fish necessitates the use of temperature, packaging, and chemical additives to delay, reduce, or inhibit spoilage reactions The responses of enzymes, bacteria and biochemical components of fish to these preservative measures have duly been investigated (Co et al, 2016; Mahmud et al, 2018; Pan et al, 2019)

2.7.1 Preservation by Low Temperature

Enzymatic autolysis and microbial deterioration actions in fish largely depend on the temperature at which the fish is stored (Gandotra, 2012) Usually, fish spoilage can occur rapidly at elevated temperature that favors bacterial and enzyme activities However, the proliferation and deteriorative impacts of bacteria and enzyme are often retarded at low temperature For every ten degree reduction in temperature, the rate at which fish deteriorates decreases by a factor of 2 – 3 (Txdolw et al, 2018) The effect of low temperature on bacterial growth can be exerted by decreasing the bacteria’s affinity for substrates, reducing its cell fluidity and extending its lag phase (Nedwell, 1999) Low temperature also reduces the pH of biological buffers which often affects charges of amino acids and protein solubility (Georlette et al, 2004) Reduction of temperature also causes the unfolding of protein molecules on account of hydration of polar and non-polar groups of proteins that weakens the hydrophobic forces crucial for protein folding and stability (Ashwini Charpe et al, 2019) Low temperature effect of increasing water viscosity can also affect the transfer of materials across the cells of spoilage bacteria (Ashwini Charpe et al, 2019) Low temperature similarly induces loss of enzyme activity through cold denaturation of enzyme (Georlette et al, 2004) Low temperature is widely noted for extending the shelf life of aquatic food through its interference with normal

physiological processes in bacteria and enzymes The use of low temperature to preserve fish can be achieved through chilling, superchilling and freezing

2.7.2 Chilling and Chilling Media

Chilling is the process of cooling down fish or fish products to a temperature close

to but not below the freezing point of the fish muscle Though chilling cannot obviate spoilage, it holds advantages in prolonging the shelf life of fish, which it does by slowing down the action of enzymes and bacteria, and their resulting chemical and physical

processes that can affect the fish quality (Köse & Erdem, 2001)

Media used for chilling include cooling air or ice of different forms and varying degrees of temperature The major advantage ice has in chilling is its high latent heat of fusion During the phase transition, one kg of ice can absorb 80kcal of heat which is sufficient to cool about 3kg of fish from 30C to 0C (Graham et al, 1993; Txdolw et al, 2018) Ice appears in several forms including blocks, plates, tubes, shells, and flakes Of these, flake ice has a lasting cooling capacity and efficiency due to its large surface area for heat exchange (Txdolw et al, 2018) Ice can be manufactured in the block form, and crushed to smaller and irregularly sized pieces for icing Crushed ice has a large surface area, which ensures rapid cooling than the large blocks It has a relatively slow melting rate and minimum losses during storage and distribution One advantage of block ice extended to its crushed form is its comparative longevity in storage compared with other forms of ice (Shawyer & Pizzali, 2003)

However, the use of slurry ice in chilling fish has proven advantageous over flake

or block ice This is due to (i) the sub-zero storage temperature of slurry ice (ii) faster chilling rates due to the higher heat exchange power, (iii) little or no physical damage caused to the fish surface due to its microscopic spherical crystals (0.1 to 1 mm in diameter), and (iv) the avoidance of dehydration due to the full coverage of the fish surface (Losada et al, 2007)

An alternative cooling agent to ice is cooling (refrigerated) air Cold air passed over the surface of a fish rapidly cools it In a chill room, heat from the fish usually warms the air around it The warm air rises and is cooled by the refrigeration system This cold air then falls or is blown by fans, back to the fish surface (Hanjabam et al,

2017) Blowing air has the disadvantage of dehydrating the fish surface Although heat removal is faster through good air circulation, air requires 10,000 times less heat to warm from 0 to 0.5 C, than the same volume of ice

Generally, chilling retards spoilage for a relatively short period by inhibiting the growth of microorganisms as well as enzyme activities However, some microorganisms such as psychrophiles and psychrotrophs (with growth temperature of 0 to 20 C) are not affected by traditional chilling temperature, usually at or above 0 C (Ashwini et al, 2019) This necessitates the concept of reducing the fish temperature slightly below the freezing temperature of the fish muscle

2.7.3 Superchilling

Since the discovery of superchilling by Le Danois in 1920, several definitions and methods of superchilling have emerged, but the principle of superchilling remains unchanged Superchilling is the reduction of the fish temperature 1 – 2 degrees below the initial freezing point of the fish muscle The freezing point of seafood differs with species composition, it is generally found within a range of -0.5 to -2.8 C (Chun-hua et al, 2014) Superchilling, in more cases, is done in the temperature range from -1 to -5C, where half of the water in the fish muscle freezes (Chun-hua et al, 2014; Zakhariya, 2014) During this freezing, the formation and growth of ice crystals absorb water from the surrounding tissues leading to an increase in solute concentration and making water less available for microbial and enzymatic activities (Chun-hua et al, 2014) Superchilling combines the favorable effect of low temperature and ice crystals formation to inhibit microbial and enzymatic activities, thereby increasing the shelf life of muscle food two to four times compared to traditional chilling (Banerjee & Maheswarappa, 2018) Superchilling advantageously maintains fish freshness and suppresses microbial growth, however protein denaturation due to large ice crystals formation and lipid oxidation due

to pro-oxidant accumulation is associated with a slow rate of superchilling (Kaale et al, 2011) Since chilling cannot stop spoilage altogether, it can be inarguably more effective when used in synergism with other preservative measures