Optimization of enzymatic hydrolysis conditions for yellowfin tuna rest raw materials using alcalase enzyme

MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY MUSIIGE DENIS OPTIMISATION OF ENZYMATIC HYDROLYSIS CONDITIONS FOR YELLOW FIN TUNA REST RAW MATERIALS USING ALCALASE ENZYME M

MINISTRY OF EDUCATION AND TRAINING

NHA TRANG UNIVERSITY

MUSIIGE DENIS

OPTIMISATION OF ENZYMATIC HYDROLYSIS

CONDITIONS FOR YELLOW FIN TUNA REST RAW

MATERIALS USING ALCALASE ENZYME

MASTER THESIS

KHANH HOA - 2020

MINISTRY OF EDUCATION AND TRAINING

NHA TRANG UNIVERSITY

MUSIIGE DENIS

OPTIMISATION OF ENZYMATIC HYDROLYSIS

CONDITIONS FOR YELLOW FIN TUNA REST RAW

MATERIALS USING ALCALASE ENZYME

MASTER THESIS

Decision on establishing the Committee:

Supervisors:

Assoc Prof Nguyen Van Minh

Dr Pham Duc Hung

Chairman:

Assoc Prof Trang Si Trung

Faculty of Graduate Studies:

(Full name)

KHANH HOA - 2020

UNDERTAKING

I undertake that the thesis entitled: “Optimization of enzymatic hydrolysis

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

this thesis is submitted

Khanh Hoa, Date 25 month 09 year 2020

Musiige Denis

FUNDING

This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106.05-2019.46 to Dr Pham Duc Hung

ACKNOWLEDGEMENT

I am extremely honored for the opportunity bestowed upon me to work under the versatile guidance of Assoc Professor Nguyen Van Minh, Faculty of food technology, Nha Trang University for his excellent guidance, continuous support, resourceful advice, encouragement and understanding throughout the experimental period until thesis completion His uncommon scientific knowledge, despite his busy schedule provided timely feedbacks and correction to my thesis, making it a useful library and reference material It is my privilege to record a deep sense of gratitude for the invaluable and constant inspiration, help, kind, constructive criticism, unfailing interest, meticulous planning right from suggesting the problem till the completion of this thesis

I appreciate with immense pleasure the support obtained from my second supervisor, Dr Hung, Institute for Aquaculture, Nha Trang University providing all sorts of resources to me for easy completion of my work and for his constant supervision, invaluable guidance and all the facilities extended in the course of this investigation

My sincere gratitude goes to the entire VLIR international master’s program Management Board at Nha Trang University with special regards to the vice dean, faculty of food technology, Dr Mai Thi Tuyet Nga and the Graduate Studies Department, Nha Trang University for making my stay in Vietnam a successful one I

am also indebted to the Nha Trang University’s entire teaching staff and my classmates for providing me with a good and world-class working environment I am extremely grateful for the love, care and all the support provided by the department of external cooperation, Nha Trang University which made my stay in Vietnam worthwhile with special consideration to the head, Dr Ngan for the timely assistance

as and whenever sought

I am as well grateful to my biological and spiritual family for their unending boost, patience and understanding Special thanks goes to my sister Lydia and all my friends for their moral support, and motivation during this research work

Musiige Denis

September 2020, Nha Trang, Vietnam

TABLE OF CONTENTS

UNDERTAKING iii

FUNDING iv

ACKNOWLEDGEMENT v

TABLE OF CONTENTS vi

LIST OF SYMBOLS ix

LIST OF ABBREVIATIONS x

LIST OF TABLES xi

LIST OF FIGURES xiii

ABSTRACT xiv

Chapter 1 INTRODUCTION 1

1.1 Problem statement and purpose of study 5

1.2 Objectives of the study 5

1.2.1 Main objective 5

1.2.2 Specific objectives 5

Chapter 2 LITERATURE REVIEW 6

2.1 Tuna 6

2.1.1 Tuna waste 7

2.1.2 Applications of tuna waste/by-products (rest raw materials) 8

2.1.2.1 Pet food sources from Tuna dark muscle 8

2.1.2.2 Oil from Tuna 9

2.1.2.3 Tuna collagen and gelatin 9

2.1.2.4 Tuna bone powder 10

2.1.2.5 Tuna digestive enzymes 11

2.2 Fish protein hydrolysates 11

2.3 Recovery methods of fish protein from fish rest raw materials 12

2.3.1 Chemical hydrolysis 12

2.3.1.1 Acid hydrolysis 12

2.3.1.2 Alkaline hydrolysis 12

2.3.2 Fermentation Hydrolysis 13

2.3.3 Isoelectric Solubilization and Precipitation (ISP) 13

2.3.4 Enzymatic hydrolysis 15

2.3.4.1 Enzymes 15

2.3.4.2 Application of enzymes 16

2.3.4.3 Factors that influence enzyme activity during hydrolysis 16

2.3.4.4 Alcalase 18

Chapter 3 MATERIALS AND METHODS 25

3.1 Materials 25

3.1.1 Head and viscera from Yellow fin Tuna 25

3.1.2 Enzyme and chemicals 25

3.2 Experimental design 25

3.2.1 Preparation of protein hydrolysates 25

3.2.2 Experimental design for optimization and analysis of data 26

3.3 Analysis methods 29

3.3.1 Proximate Chemical composition 29

3.3.2 Determination of the degree of hydrolysis 29

3.3.3 Determination of protein solubility 29

3.3.4 Amino acid analysis 30

3.4 Statistical analysis 30

Chapter 4 RESULTS AND DISCUSSION 31

4.1 Proximate composition of the rest raw materials 31

4.2 Optimization of hydrolysis parameters for DH and solubility of viscera 31

4.2.1 Optimal plot for DH and Solubility of viscera 43

4.3 Optimization of hydrolysis parameters for DH and solubility of head 44

4.3.1 Optimal plot for DH and solubility of head 52

4.4 Optimization and validation of the models 53

4.5 Proximate composition of the hydrolysates 55

4.6 Amino acid composition 55

Chapter 5 CONCLUSIONS AND RECOMMENDATIONS 58

5.1 Conclusions 58

5.2 Recommendations 58

REFERENCES 60

APPENDICES I

Appendix 1 I

Appendix 2 VII

Appendix 3 XX

Regression coefficient for linear effect

Regression coefficient for quadratic effect

Regression coefficient for interaction effect

Broken peptide bonds

Total number of peptide bonds

LIST OF ABBREVIATIONS

ANOVA Analysis of variance

AOAC Association of Analytical Communities

AU Anson unit

CCD central composite design

CPHA Cuttlefish protein hydrolysates Alcalase CPHP Cuttlefish protein hydrolysates protamex CPHS Cuttlefish protein hydrolysates

DH Degree of hydrolysis

DHA Docosahexaenoic acid

DNFB Dinitrofluorobenzene

DPPH 2, 2- diphenyl-1-1picryhydrazyl

EPA Eicosapentaenoic acid

FAD Fish aggregation devices

FAO Food and agricultural organization

FPH Fish protein Hydrolysate

FPI Fish protein isolate

ISP Isoelectric solubilization and precipitation KDa kilo Dalton

LAB Lactic acid bacteria

NRC Nutritional research council

pH Potential of hydrogen ions

PI Isoelectric point

PUFA Polyunsaturated fatty acids

RSM Response surface methodology

SAS Statistical analysis system

WHO World health organization

LIST OF TABLES

Table 3.1 Experimental range and values of the independent variables in the central composite design for optimization of enzymatic hydrolysis conditions for visceral and

head waste proteins of tuna from yellow fin tuna (Thunnus albacares) 27

Table 3.2 A complete composite design for the optimization of degree of hydrolysis and solubility of both viscera and head hydrolysates 28

Table 4.1 Proximate chemical composition of Yellow fin tuna rest raw materials 31

Table 4.2 Experimental design used in the experiment and the response for DH and solubility for viscera 32

Table 4.3 Parameter estimates for Degree of hydrolysis (viscera) 33

Table 4.4 Parameter estimates for Solubility (viscera) 34

Table 4.5 Results of ANOVA for degree of hydrolysis (Viscera) 35

Table 4.6 Results of ANOVA for solubility (Viscera) 35

Table 4.7 Optimum conditions as coded and un-coded data for tuna DH and solubility of visceral protein hydrolysates 36

Table 4.8 Experimental design used in the experiment and the response for degree of hydrolysis and Solubility values for viscera (observed and predicted values) 41

Table 4.9 Experimental design used in the experiment and the response for DH and solubility for head 44

Table 4.10 Parameter estimates for Degree of hydrolysis (head) 45

Table 4.11 Parameter estimates for Solubility (Head) 45

Table 4.12 Results of ANOVA for degree of hydrolysis (Head) 46

Table 4.13 Results of ANOVA for solubility (Head) 47

Table 4.14 Optimum conditions as coded and un-coded data for tuna DH and solubility of head protein hydrolysates 48

Table 4.15 Experimental design used in the experiment and the response for degree of hydrolysis and Solubility values for head (observed and predicted values) 50

Table 4.16 Optimum conditions as coded and un-coded data for tuna visceral and head protein hydrolysates for combined variables, degree of hydrolysis and solubility 54

Table 4.17 Proximate chemical composition of Yellow fin tuna FPH 55

Table 4.18 The amino acid composition of yellow fin tuna visceral and head protein hydrolysates (g/100g) and chemical score in comparison with FAO /WHO reference protein 57

Figure 4.2 Response surfaces and contour plots for the effect of variables on solubility (Viscera) as a function of different hydrolyzing conditions: A; time and temperature, B; time and enzyme concentration, C; temperature and enzyme concentration 40

Figure 4.3 Relationship between the observed/actual and predicted values of the degree of hydrolysis (Viscera) 42

Figure 4.4 Relationship between the observed/actual and predicted values of solubility (Viscera) 42

Figure 4.5 A plot showing the optimal conditions for degree of hydrolysis and solubility for viscera 43

Figure 4.6 Response surfaces and contour plots for the effect of variables on DH (Head) as a function of different hydrolyzing conditions: A; time and temperature, B; time and enzyme concentration, C; temperature and enzyme concentration 48

Figure 4.7 Response surfaces and contour plots for the effect of variables on solubility (head) as a function of different hydrolyzing conditions: A; time and temperature, B; time and enzyme concentration, C; temperature and enzyme concentration 49

Figure 4.8 Relationship between the observed/actual and predicted values of the degree of hydrolysis (Head) 51

Figure 4.9 Relationship between the observed/actual and predicted values of solubility (Head) 51

Figure 4.10 A plot showing the optimal conditions for degree of hydrolysis and solubility for head 52

ABSTRACT

Protein hydrolysates were prepared from visceral and head wastes/rest raw materials of yellow fin tuna Hydrolysis conditions (viz., temperature, time, and enzyme to substrate level) for preparing protein hydrolysates from yellow fin tuna visceral and head wastes using in situ pH of the visceral and head mass were optimized by a complete composite design (CCD) of response surface methodology (RSM) The regression coefficient observed during both experimental and validation runs was close to 1.0, showing the validity of prediction models All the hydrolysis conditions had a significant effect (P˂0.05) on both the degree of hydrolysis and solubility for both viscera and head A hydrolysis time of 6.7 h, temperature of 53.4 °Cand an enzyme to substrate level of 0.88 % (v/w), were found to be the optimum conditions to obtain a higher degree of hydrolysis of 66% and solubility 71.0% for visceral hydrolysis While optimal conditions for head hydrolysis were found to be 7 h for hydrolysis time, 55 °C

for hydrolysis temperature and 0.82% (v/w) enzyme to substrate level yielding a higher degree of hydrolysis of 28% and solubility 89.1% with Alcalase the protease enzyme employed in both cases The profile of the amino acid of both, visceral and head protein hydrolysates obtained with the optimized conditions revealed that the protein hydrolysates were similar to FAO/WHO reference protein The protein hydrolysates has the potential for application as an ingredient in balanced fish diets for fingerlings

Keywords: Alcalase protease; Yellow fin tuna waste; RSM; Protein hydrolysates; Optimization

Chapter 1 INTRODUCTION

Tuna (Thunnus spp) refers to certain members of the family Scombridae, a

group of marine fishes including tunas, bonitos, mackerels, seer fishes and the butterfly kingfish Conversely, for ichthyologists, tuna refers to any of the 14 species

of the tribe Thunnini within the family Scombridae (Jolla & Klawe 1977) They are

classified as tropical tunas, like big eye (Thunnus obesus), skipjack (Katsuwonus

pelamis), yellow fin tuna (Thunnus albacares) as well as temperate tunas for instance

albacore (Thunnus alalunga), Atlantic blue fin tuna (Thunnus thynnus), Pacific blue fin tuna (Thunnus orientalis), and Southern blue fin tuna (Thunnus maccoyii) (Herpandi et

al, 2011) The most frequently tuna species’ sizes caught vary from 30 to 200 cm with biggest size and weight stretching from 70 to 300 cm and 9 to 650 kg, respectively Atlantic blue fin tuna exhibit the largest size and weight, whereas black skipjack poses the smallest sizes (Herpandi et al, 2011)

On a global scale, tuna production has stretched close to 4.5 million tons per year Yellow fin tuna, is the 2nd major species caught following skipjack that accounts for 59.1% of total production It’s the 3rd largest species following blue fin and big eye which increases its suitability and availability for the canning industry (Herpandi et al, 2012) Raw materials used in the canning industry like fresh and frozen precooked loins, tuna for immediate consumption such as sashimi, as well as and canned tuna products like solid packs, flakes, and chunks are the leading traded forms of tuna globally They are greatly composed of omega 3 fatty acids, proteins, selenium and vitamin D (FAO, 2014) Its demand has progressively increased due to the development of canning industry (Herpandi et al, 2011) Huge amounts of rest raw materials are generated since the industry is interested in only white meat 450000 tons per year of processing discards is estimated to come from tuna canning industry (Herpandi et al, 2012) Muscle after the removal of loins, viscera, gills, dark muscle, head, bones, and skin, are some of the solid rest raw materials generated from the processing industry and can constitute close to 70% of the starting material (Wisuthiphaet et al, 2016; Guerard et al, 2002) Of the 70%, 20 to 35% solid waste and

20 to 35% liquid wastes with products only 30 to 35% (Sayana Sirajudheen , 2017)

The disposal of these wastes produce a major problem to the environment because of their odor and high moisture content when are dumped as commercial or domestic waste (Guerard et al, 2002) However, fish waste could be utilized for production of animal feeds, biodiesel, natural pigments, food products and pharmaceuticals, the recovery of protein and potential generation of bioactive peptides from proteins present in fish trimmings, skin, and other organs; the production of collagen and gelatin from skins; the recovery of enzymes from intestines; oil from fish frames, head, gut, liver, and roe; and in addition calcium, glucosamine, and chitosan are acknowledged to be valuable materials that provide significant opportunities for development of value-added products (Shavandi et al, 2018)

Tuna dark muscle can be used as a source of pet food, production of tuna oil usually from head and bone but not viscera as it’s a good source of poly unsaturated

fatty acids (PUFAs) with ω–3 (Wongsakul et al, 2000) These are good nutritionally

and for human health as they lessen the risks associated with coronary diseases on top

of boasting functionality of the immune system and averting some cancers Tuna bones and fins are good sources of tuna collagen and gelatin like skipjack tuna yields 53.6% collagen whereas collagen content in yellow fin tuna was 27.1% (Woo et al, 2008) Between 60 to 70% of minerals like calcium phosphate and hydroxyapatite are found in tuna bone powder obtained from fish bone which suits them for their application as calcium food supplement (Ae et al, 2005) Several digestive enzymes are found in the tuna inner organs for example gastric mucosa secrets pepsin, a proteolytic enzyme, pancreas secrets trypsin plus chymotrypsin with spleen producing proteinases (Klomklao et al, 2007)

Yellow fin tuna (Thunnus albacares) whose rest raw materials i.e viscera and

head is under study refers to a large epipelagic species living in vast parts of tropical and subtropical waters of the major oceans (Lee et al, 2016; Zudaire et al, 2013) It’s

an intensely exploited fish due to its high demand, and is harvested widely, by employing various types of fishing gears (Sánchez-Zapata et al, 2011) Huge quantities

of yellow fin tuna find a lot of application in canned and dry-salted products like cured tuna loin and is also widely used in raw fish dishes as sashimi, a raw fish product common in Asian countries of Korea and Japan The worldwide annual production of

On the other hand, some of these protein-rich rest raw materials are processed into low value market products, ranging from animal feeds to fertilizers in spite of the emergency of its new found application as ingredients in functional foods Fish Protein Hydrolysate (FPH), by hydrolysis of these rest raw materials, which once obtained plays an important role in food processing companies as ingredients providing functional properties ranging from gelling, whipping, as well as texturing properties (Taylor et al, 2010)

Protein hydrolysates refers to small peptides and polypeptides with several amino acids (Chalamaiah et al, 2012) They can be obtained by using chemical methods, solvents and enzymatic hydrolysis (Noman et al, 2018) However, the use of chemicals limits the products’ application in food industry, yet enzyme hydrolysis results into a product with improved nutritional value as well as other functional properties (Quaglia & Orban, 1990) Enzymatic hydrolysis requires significantly small amounts of enzyme for easy deactivation at mild conditions like temperature and pH

Enzymes are highly available from different sources, it has no effect on amino acids and the resulting peptide mixture is easy to purify (Pasupuleti & Demain, 2010) Enzymatic hydrolysis is carried out with Proteolytic enzymes to breakdown peptide bonds so as to produce fish protein hydrolysates (FPH) They can either be endogenous or exogenous i.e obtained from other sources for example plants, animals, and microbes The pre-condition for using exogenous enzymes for FPH production is

they must be of food grade and nonpathogenic in case they are of microbial origin The most common commercial proteases used for the hydrolysis of fish protein are from plant sources and animal sources, such as chymotrypsin, pepsin and trypsin (Klomklao

et al, 2007)

Nevertheless, fish protein have been hydrolyzed with enzymes from microbial sources Considering enzymes from animal and plant sources, those from microbial origin exhibit a multitude of advantages ranging from a couple of catalytic activities to greater pH and temperature stabilities (Diniz & Martin, 1997) Microbial protease

enzymes like Alcalase operating at alkaline pH, produced from Bacillus licheniformis

have proved to be the most effective with regards to fish proteins hydrolysis considering technical and economical perspective (Dufossé et al, 2001;Wasswa et al, 2007;Pacheco-Aguilar et al, 2008) A variety of enzymes including papain, pepsin, neutrase, trypsin, proteases, pancreatin, pronase, bromelain, and validase have been employed to hydrolyze the fish rest raw materials to produce FPH (Noman et al, 2018) Protamex, flavourzyme, corolase umamizyme, kojizyme, and orientase are the other enzyme formulations that have demonstrated an excellent potential for fish protein hydrolysis to produce FPH with high functional properties

Over the time, FPH were proved to be a good source of antioxidants with peptides possessing anti-cancer and anti-anemia properties as well as microbial growth media components (Herpandi et al, 2011) The current study is therefore aimed at investigating the effects of Alcalase enzyme concentration, hydrolysis temperature, and incubation time on the Degree of Hydrolysis (DH) and solubility of visceral and head waste proteins of yellow fin tuna and hence the entire optimization of the hydrolysis process leading to the highest yields for their suitability as feed for fingerlings Since the central composite design (CCD) of Response surface methodology (RSM) proved to be a relevant, effective and time efficient tool for optimizing the conditions of hydrolysis (Roslan et al, 2014) It was therefore employed

in this study whose results can be utilized for potential commercial and industrial applications

1.1 Problem statement and purpose of study

Rest raw materials of tuna canning industry amounts up to 70% of the original material and their disposal poses a big threat to the environment because of their odor and high moisture content which causes air pollution and can result in diseases (Guerard et al, 2002) All this comes at a time when aquaculture is growing at a faster rate as more people are engaging in fish farming and has therefore resulted into an increased need for high quality proteins as feed for these aquatic products, however, different growth stages require different types of proteins With juvenile and fingerlings being critical stages of growth, they poses no mechanism for digesting long chain peptides, hence the need for hydrolysis of long chain peptides to short chain peptides to make it available and suitable for easy absorption by this category of fish

Considering the value of Tuna fish rest raw materials and the way it’s being utilized, it is therefore imperative to assay a better mechanism for exploitation, Fish protein hydrolysates (FPH), by analyzing the influence of enzymatic hydrolysis conditions on the degree of hydrolysis of visceral and head waste proteins of tuna for yield optimization as well as the solubility of the Hydrolysate for their suitability as quality feed for the growth fingerlings

1.2 Objectives of the study

ii To determine the chemical properties and amino acid profile of visceral and head protein hydrolysates

Chapter 2 LITERATURE REVIEW 2.1 Tuna

Tuna (genus Thunnus), sometimes referred to as tunny, is used to describe

whichever of the seven species belonging to oceanic and or marine fishes, where the

oversized ones are associated with genus Thunnus and their commercial value as food

is tremendous (Ottolenghi, 2008) They are related to mackerels belonging to the same family, Scombridae with great variations within as well as amongst species

Tunas are extended, vigorous, and rationalized kind of fish with possession of a smooth-edged body tapering to a slim tail base with a crescent-shaped tail They are majorly dark in color on the top/above whereas the bottom part/below surface appears silvery, in most cases with an iridescent shine Both sides of the tail base possess a conspicuous keel together with a series of small finlets behind dorsal and anal fins, along with a corselet of enlarged scales in the shoulder region The other distinguished feature is a well-developed network of blood vessels underneath the skin to regulate temperatures during long-term, slow swimming (Palstra & Planas, 2013) Tunas are distinctive among fishes for their ability to maintain the temperature of their bodies above that of the surrounding water, at around 5 to 12 °C above ambient water temperature due to their vascular system with some muscles going as much as 21 °Cabove the surrounding water

The seven species of genus Thunnus, include the northern Bluefin tuna (T

thynnus), albacore (T alalunga), yellow fin tuna (T albacares), southern blue fin tuna

(T thynnus maccoyii), big eye tuna (T obesus), black fin tuna (T atlanticus), and long tail tuna (T tonggol) These species vary from medium to extra-large sizes (Herpandi et

al, 2011) The largest is the northern blue fin tuna, and can go to a maximum length and weight close to 4.3 metres and 800 kg respectively Others like the yellow fin tuna can weigh as much as 180 kg, with the least albacore growing to as much as around 36 kg

The northern blue fin tuna typically poses yellow finlets with silvery spots or bars often marked on it Over fishing has significantly contributed to the decline of northern blue fin tunas in the Atlantic Ocean since pre industrial times This has caused scientists and environmental organizations to call for a cessation of this specie capture Though, pending implementation Albacore is the other equally essential

specie, with a shining blue stripe on each side; the yellow fin, with yellow fins and a golden stripe on either side; as well as the big eye, which is vigorous fish with moderately large eyes

Tunas swim long distances throughout the world’s oceans and occupy tropical,

temperate, and even some cooler waters (National tuna management plan in vietnam,

2012) The only two species of relatively limited distribution are the black fin tuna (western Atlantic) and the long tail tuna (Indo-Pacific region) Fishes, squids, shellfish, and a variety of planktonic organisms serves as feed for tunas while spawning the open sea over very large areas

Numerous other species belonging to Scombridae family are most oftenly

referred to as tuna, such as the skipjack tuna (Katsuwonus, or Euthynnus, pelamis),

which is found worldwide and can grow up to about 90 cm and 23 kg Others include

the bonitos, of the genus Sarda, which are tuna-like fishes, also found worldwide with

both commercial and sporting value

2.1.1 Tuna waste

The rest raw materials commonly known as wastes are described with reference

to either harvesting or the processing method employed Normally, the part that has the fillets forms the major product in the tuna processing industry whereas the guts/intestines or viscera, head, backbones, trimmings and the skin forms the rest raw materials casually referred to as wastes (Wasswa et al, 2007; Kristbergsson & Arason, 2007) However, due to the growing demand of so many products and bioactive compounds that can be processed/obtained from these wastes/by-products, there description has of late changed from being referred to as “waste/by-products” to now

“rest raw materials” Total yield from the tuna processing industry is determined by the gutted fish and the head where 62% constitutes the edible fresh with skinless tuna amounting to 46% (Kristbergsson & Arason, 2007) There is always relatively little meat in fish heads which is oftenly disposed of and sometimes given to animals as feed save for a few parts of the tuna head with possibility of being consumed as sources of meat such as tongue, cheeks, and collar Owing to their distinctive taste and outstanding texture, tongues and cheeks are regarded as delicacies by a section of consumers Research shows that tuna loins constitutes of 37.1% whereas fillets 17.9%

of a headless tuna (Fisheries & Countries, 2007) With the two considered tuna industry’s extracted major and integral components, this means that utilization of only these parts from a single tuna leaves a lot of rest raw material redundant The same article reported that bones and dark muscle, that are regarded as waste, weighed close

to 18% of a headless tuna, with skin and viscera comprising 13%, belly 6.2%, whereas the remaining frame scrap amounting to 7.9% Subject to maturity and season, viscera, comprising of a combination of liver plus roe, could result into a net weight of a whole tuna in the range of 10 to 25% Pyloric caeca, which also belongs to the gut is always not consumed though poses a huge potential as a bioactive compounds source for example enzymes, with several applications

2.1.2 Applications of tuna waste/by-products (rest raw materials)

Currently, large amounts of food continues to be dumped at a commercial or domestic level Despite the need to reduce the waste worldwide, huge amounts of waste generated keeps on increasing every year Thus, of late, there is a growing interest in exploring available mechanisms for better utilization of underutilized resources and wastes from industries, including tuna rest raw materials Precisely, canning industries that deal in tuna generate waste as much as 70% of the original material (Wisuthiphaet et al, 2016; Guerard et al, 2002) Out of the 70%, 20 to 35% solid waste and 20 to 35% liquid wastes with products only 30 to 35% (Sayana Sirajudheen , 2017) Hence the need to explore more advanced, sustainable and environmentally friendly ways of utilizing these waste products

2.1.2.1 Pet food sources from Tuna dark muscle

A large percentage of canned pet food in several markets of pet food products are based on tuna The major constituent of tuna based pet food being blood meat (tuna dark muscle) with its major purpose of giving flavor to feed Before canning Tuna for human consumption, this dark meat is always cut off Whole tuna loins are used to produce Hedonistic pet feed, which is basically human-grade though in limited quantities Processing of Canned pet feed tuna is no different from that of other tuna products, with the existence of a variety of formulations, including but not limited to packaging in water with vitamin and mineral supplements, antioxidants, vegetable oils,

coloring agents, and occasionally powdered tuna frames for enhancement of calcium composition

2.1.2.2 Oil from Tuna

Tuna processing industry is generating an essential by-product in Tuna oil Waste products from the tuna canning industry are used to produce refined oil, with little odor and light yellow color Head, meat, and bones are used in the process for tuna oil production excluding the viscera and tuna livers Crude tuna oil is obtained from tuna waste by steam and later purification After which, it’s then taken to a refinery where it undergoes a 4-step process beginning with neutralization, bleaching, and winterizing to get rid of crystallized fats From there, an aromatizing process is carried out to remove odor-causing contaminants It’s from here that the oil is then either transported in bulk or sent to final consumers such as pharmaceutical industries Tuna oil poses a lot of health and nutritional benefits ranging from being a polyunsaturated fatty acids (PUFAs) source, in particular EPA (eicosapentaenoic acid,

C22:5n3) and DHA (Docosahexaenoic acid, C22:6n3), that are essentially omega-3

fatty acids with at least 5.7% EPA and 18.8% to 25.5% DHA (Chantachum et al, 2000b; Wongsakul et al, 2003) PUFAs in particular have gotten an immense role they play as far as human health and nutrition are concerned, this ranges from reducing risks associated with coronary disease risks, preventing some cancers types, as well as improving body’s immunity Shen et al (2007) reported an appropriate technique for provision of ɷ-3 fatty acids with application of oil-in-water emulsions The highly unsaturated nature of long chain PUFAs increases their oxidation susceptibility However, the encapsulation of this oil through the addition of antioxidants is a proper remedy to lipid oxidation (Klinkesorn et al, 2004)

2.1.2.3 Tuna collagen and gelatin

Gelatin, a derivative of collagen is obtained by partial hydrolysis of collagen, an abundant protein from animal sources They are diverse forms of the identical macromolecule and enjoy a wide application in pharmaceutical industry, food industry, cosmetics and cell cultures, and of late, its new found industrial application has escalated its consumption (Karim & Bhat, 2009) A lot of commercial products are made from collagen and gelatin whose major sources are cows together with pigs

However, mammalian diseases like foot and mouth disease limit their application owing to safety problems arising from the risk of transmitting to humans the disease

On the other hand, the risk of pathogen transmission in collagen and gelatin from fish

is minimal, at the same time, they don’t controvert religious sensitivities of Islamic and Hindu/Buddhist food laws as opposed to pigs and cows’ products Despite being dumped as waste, fish skin, bone, and fins are good sources of collagen and gelatin Their collagen yield can go as high as 54% (Nagai & Suzuki, 2000) Woo et al (2008) stated that; close to 30% of most organisms entire protein is collagen Fish gelatin though, to be applied in the food and pharmaceutical industries, must have these unique features with the first one being the possession of a large quantity of rest raw materials and its efficient collection to ensure continuous production in industry Secondly, is the rheological properties including gel strength, gelling, and melting points of gelatin from fish by-products shouldn’t be any different from those of mammalian origin It’s equally important to note that yellow fin tuna skin gelatin has a higher gel strength compared to that from bovine and porcine though, with lower gelling and melting points (Cho et al, 2005) The viscoelastic properties of gelatin from tuna skin is similar to those from mammals while that from dorsal skins of yellow fin tuna had better solubility and viscosity attributes (Woo et al, 2008)

Enzymatic digestion of tuna gelatin with pepsin for 3 h resulted into a degree of hydrolysis which was higher in reference to the one obtained with Alcalase On the other hand, gelatin from squids exhibited a degree of hydrolysis which is higher upon Alcalase digestion compared to the one with pepsin from which a conclusion can be drawn that different degrees of hydrolysis are obtained with different enzymes (Alemán et al, 2011)

2.1.2.4 Tuna bone powder

The fish bone has both organic and inorganic components with 30% of the former made up of collagen and 70% of the latter constituting calcium phosphate and hydroxyapatite (Nagai & Suzuki, 2000) This therefore makes it rich in valuable inorganic substances with a balance of calcium and phosphorus suitable for application

as a calcium food supplement (Yoon et al, 2005) Though, until now, it is essentially and primarily used in animal feed Fish bone’s structure necessitates softening to transform it into an edible form for it to be integrated into calcium-fortified food

2.1.2.5 Tuna digestive enzymes

Viscera from the fish are the most essential rest raw materials from the fishing industry due to their abundant source of digestive enzymes Gastric mucosa from viscera secrets pepsin, pancreas secrets trypsin and chymotrypsin A vast number of researchers have isolated numerous digestive Proteolytic enzymes from fish’s internal organs, separated and purified the enzymes from tuna internal organs, such as the spleen of skipjack tuna (Klomklao et al, 2007), the spleen of yellow fin tuna (Klomklao et al, 2007; Li et al, 2006) and the stomach of albacore tuna (Nalinanon et

al, 2009) Main proteinases in the spleen of 3 tuna species (skipjack, yellow fin, and tongol) included trypsin-like serine proteinases (Klomklao et al, 2007) Proteinases from yellow fin tuna amongst the rest exhibited the highest activity which suits them for a lot of application in protein hydrolysis processes in industries (Kikuchi, 2010)

2.2 Fish protein hydrolysates

Hydrolysates refers to a complex mixture of oligopeptides, peptides and free amino acids of various sizes that are produced by the breakdown of proteins either by partial or extensive hydrolysis On the other hand, bio peptides or bioactive peptides refers to peptides that poses beneficial pharmacological properties Chemicals (acids

or bases) or biological (enzymes) means are employed to degrade the proteins The peptide bonds of proteins are cleaved by acids and bases during chemical hydrolysis resulting into products of contrasting chemical composition and functional properties Despite the low cost and simplicity which makes chemical process preferable to biological process by industries, the products are restricted for use only as flavor enhancers due to low nutritional qualities and poor functional properties which limits their applications in food ingredients (Taylor et al, 2010) Several processes have been

2.3.1.1 Acid hydrolysis

Acid hydrolysis normally breaks down the proteins into distinct amino acid plus smaller peptides, the process is associated with the loss of some essential amino acids, like cystine, cysteine, methionine, and tryptophan (Pasupuleti & Demain, 2010) 6 M HCl at 118°C for 18 h are the optimal conditions for the total hydrolysis of fish protein (Taylor et al, 2010) Thorough hydrolysis process increases the products’ solubility with the release of large amount of salt due to the neutralization process resulting in to product unsuitable for food This limits its application for as human or pet food flavor enhancer in spite of the possibility of partial or complete removal of salt with Nano filtration and ion-exchange resins (Pasupuleti & Demain, 2010)

2.3.1.2 Alkaline hydrolysis

Protein degradation with a base is a simple and a candid process where the solubilized heated protein is mixed with calcium, sodium, and potassium The product has poor functionality and, accompanied with adverse effects on the nutritional properties of the protein (Kristinsson & Rasco, 2000) There is a potential formation of

toxic substances such as lysioalanine, ornithinoalanine, lanthionine, and β-amino

alanine as a result of disulfide bonds losing cysteine, serine, and threonine On top of that, alkaline hydrolysis reduces the hydrolysis rate through the production of products with an inhibiting effect on proteolytic enzymes Amidst a series of limitations, the method still finds a limited application food industries for recovery and solubilization

of a plethora of proteins

2.3.2 Fermentation Hydrolysis

Fermentation hydrolysis basically refers to biochemical break down of fish proteins into peptides and amino acids using microorganisms Hydrolysates from fish protein has been produced by various identified and specific microorganisms The functionality of FPHs recovered by fermentation hydrolysis vary because microorganisms used in the culture are different (Daliri et al, 2017) Examples of bacteria that has been used to produce Protein hydrolysates through fermentations

include as Enterococcus faecium NCIM5335 (Balakrishnan et al, 2011) and lactic acid bacteria (LAB) Pediococcus acidilactici NCIM5368 (Chakka, Elias, Jini, Sakhare, & Bhaskar, 2015) FPH prepared from sardinelle, using proteolytic bacterium, Bacillus

subtilis A26 had excellent solubility and interfacial properties, with a high antibacterial

and antioxidant activities (Jemil et al, 2014)

Three proteolytic Lactic acid bacteria (P acidilactici NCIM5368, E faecium NCIM5335, and P acidilactici FD3) used to ferment fish head rest raw materials at

10% (w/w) glucose, 2% (w/w) NaCl, and 10% (v/w) LAB cultures at 37 °C

were isolated from fish processing The resulting degree of hydrolysis was 38.4% without significant effect on lipid fatty acid profiles (Ruthu et al, 2014) On the other hand, Rai

et al (2011) in a related experiment, utilized 5 various Lactic acid bacteria obtained from rest raw materials after fish processing in fermentation of fish head waste under the above conditions The resulting fermentation liquor mainly contained FPH with a high antioxidant and antibacterial properties It is of paramount importance to understand the auxiliary role fermented fish protein in the removal of hyper allergic or anti-nutritional components normally in ingredients such as trypsin inhibitors, glycinin, β-conglycinin, phytate which characteristic/property/advantage can’t be realized with other methods FPH recovery (Hou et al, 2017)

2.3.3 Isoelectric Solubilization and Precipitation (ISP)

Also called pH-shift method, isoelectric solubilization and precipitation (ISP),

is a method protein recovery that depends on the isoelectric point (pI) of proteins This

pH is protein specific, and hence varies with different proteins The process is a mild, non-thermal pasteurization process, owing to extreme pH shifts involved (Lansdowne

et al, 2009) Several researchers have successfully employed this method in protein

recoveries from fish and fish rest raw materials (Tahergorabi et al, 2012; Chen et al,

2009; Choi & Kim, 2005) beef and chicken meat (Tahergorabi et al, 2012; DeWitt et

al, 2002) from plant sources including soy (Foh et al, 2012; Rickert et al, 2004) and wheat protein (Liu et al, 2013) ISP has also been effective in lowering populations of

Escherichia coli in fish protein isolate (FPI) among other applications (Lansdowne et

al, 2009)

ISP’s wide application is attributed to the fact that it allows selective and efficient recovery of protein, while at the same time separates lipids from other rest raw materials such as bones, skins, and scales unsuitable for consumption by humans (Ananey-Obiri et al, 2019; Segneanu et al, 2013) After ISP, the resulting protein from fish is termed as FPI with high stability, and functionalities (Lee et al, 2016) Various research findings acknowledged the significant role of pH to FPI functionalities like emulsification, gelation, and water absorption (Liu et al, 2013)

The structure, yield, and the level of unfolding of protein isolate can be enhanced

by the extraction method and precipitation pH (Abugoch et al, 2008) Its adoption as a method of protein recovery led to a significant yield of protein The ISP processes are essential stages for they determine quantity of protein formed (Liu et al, 2013)

The pH shift generally embroils the solubilization of the protein by altering the

pH and ensuing drop to suitable pH for protein precipitation At this pH, the protein charges assume equilibrium, with the net ionic charge on the protein becoming statistically zero Upon acid addition to the solution of proteins, it dissociates to produce hydronium (H3O+) The low pH induces protonation on glutamyl or aspartyl

of negatively charged side chains Conversely, a dissociation occurs to produce hydroxide ions (-OH) upon base addition The process causes loss of hydrogen ions by side chains on tyrosyl, tryptophanyl, cysteinyl, lysyl, argininyl, or histidine residues, hence deprotonation (Ananey-Obiri et al, 2019)

ISP processing includes first, the release of the protein by breaking the fish muscle cells and afterwards, homogenization with distilled water follows at 1:6 ratio (crushed fish: water, w-v) The pH of the resultant mixture is raised to around11 for 10 minutes for solubilization of the protein It’s then centrifuged resulting into three layers, namely: upper layer (oil), central layer (fish muscle protein solution) and lower

layer (insoluble i.e bones, proteins, and membrane lipids) The central layer is removed and the pH is adjusted isoelectrically to about 5.5 for 10 minutes to precipitate the protein It’s then centrifuged into two layers: bottom layer (process water) and the upper layer (FPI) (Ananey-Obiri et al, 2019)

2.3.4 Enzymatic hydrolysis

2.3.4.1 Enzymes

Enzymes are essentially proteins that catalyze most of the chemical reactions occurring in living organisms, ensuring stable metabolism of substances in living organisms Thus, they contribute to the body’s metabolism and the environment to maintain life They improve catalytic efficiency up to millions of times compared to other inorganic and organic catalysts They can function well under normal pressure, temperature, and ambient pH close to physiological pH Furthermore, they are specific

to the reactions and substrates involved in the catalytic process which characteristic renders them of a great significance for research and application

In industrial production, enzymes are often used as catalysts instead of chemicals to reduce toxic substances released into the environment to reduce environmental pollution in response to the current development trend of industrialization and modernization Recently, the biotechnology industry has grown strongly and brought about high economic value especially the technology producing enzyme preparations such as protease enzymes The protease enzyme is the catalyst of the hydrolysis of peptide (-CO-NH-) bonds present in the protein molecule and polypeptides to form shorter peptide chains

The protease classification is based on; appropriate pH for the activity of each enzyme which may be acid, alkaline or a neutral protease; specific properties of substrates with endo peptidase and exo-peptidase, amino peptidase, carboxyl peptidase, di peptidase and according to the origin of the enzyme, such as internal enzymes, muscle enzymes, pancreatic enzymes

Basing on the specific properties of substrates, Amino peptidase catalyzes the hydrolysis of peptide bonds at the nitrogen end of the polypeptide chain, Carboxyl peptidase which catalyzes the hydrolysis of peptide bonds at the carbon end of the

is widely used for the production of inoculants for the food industry and other industries in general In the food industry, Protease is extracted from pineapple, papaya, animal organs and microorganisms to help tenderize meat, promote the hydrolysis of fish meat to obtain fish protein hydrolysates with a more nutritional, functional and bioactive properties

2.3.4.3 Factors that influence enzyme activity during hydrolysis

Effect of enzyme concentration

The lower the enzyme concentration, the lower the hydrolysis rate When the concentration of the enzyme increases, the hydrolysis reaction speed increases, but to a limit value v=vmax, if the concentration of the enzyme increases beyond that, the rate of hydrolysis by enzyme will not increase significantly

Effects on inhibitors and activators

Activators are substances that increase enzyme activity, their chemical nature is metal ions, anions or organic substances However, their efficacy is only in defined concentration limits as the enzyme concentration When used in excess of the permitted concentration, the enzyme activity will decrease Inhibitors on the other

hand are inorganic or organic substances that causes a reduction in enzyme activity Great attention should be paid to the inhibitors that affect each enzyme since each has different inhibitors

Effect of temperature

Enzymes are basically proteins with catalytic activity, so they are less resistant

to heat, and high temperatures will make them denatured In the appropriate temperature range their activity is best As the temperature increases, the rate of hydrolysis reaction increases by a characteristic by a factor;

Where kt is the reaction rate constant at temperature t, kt + 10 is the reaction rate constant at t + 10 °C

The Q10 coefficient of most enzymes in the fish body ranges from 2-3, except the hemoglobin reaction in fish blood can be up to 7 At the appropriate temperature of the enzyme, the enzyme activity is high There is a value of temperature at which the enzyme reaction reaches its maximum called the optimum temperature The most suitable temperature range is in the range 40-50 °C Most enzymes have a critical temperature of about 90 °C that inactivates the enzyme especially thermostable enzymes such as bromelain, papain, and a higher critical temperature The appropriate temperature for an enzyme also depends on pH and substrate concentration

One of the attributes that qualifies an exogenous enzyme for the production of FPH with desired properties is the potential and ability to cleave peptide bonds at specific places Such must be food-grade and nonpathogenic if they are of microbial origin A wide range of food-grade Proteolytic enzymes is extensive and grants enzymologists a chance to prepare FPH from fish rest raw materials They include but not limited to those from plant sources for example, papain (Noman et al, 2018) and animal sources, such as pepsin, chymotrypsin and trypsin (Klomklao et al, 2007)

Microbial protease enzymes like Alcalase operating at alkaline pH, produced

from Bacillus licheniformis have proved to be most effective with regards to fish

proteins hydrolysis considering technical and economical perspective (Dufossé et al, 2001;Wasswa et al, 2007;Pacheco-Aguilar et al, 2008) Other enzyme formulations with excellent potential for hydrolyzing fish protein to produce highly functional FPHs, include protamex (Liaset & Espe, 2008), flavourzyme (Thiansilakul et al, 2007), corolase (Kristinsson & Rasco, 2000) umamizyme (Guerard et al, 2002) kojizyme (Nilsang et al, 2005), and orientase (Hsu et al, 2009)

2.3.4.4 Alcalase

Alcalase is an endo-protease of the serine type with a very broad substrate specificity meaning it can hydrolyze most peptide bonds within a protein molecule Peptides and amino acids are formed which are either dissolved or dispersed in the washing

water It is active between pH 6.5 and 8.5 and functions between 45 and 65 °Cwith maximum activity at about 60 °C, above which the activity falls rapidly It’s an

alkaline enzyme prepared from Bacillus licheniformis and industrialized by Novo

Nordisk (Bagsvaerd, Denmark) Several researchers have proved Alcalase as one of the best enzyme for preparation of functional FPH (Taylor et al, 2010; Benjakul & Morrissey, 1997; Shahidi et al, 1995) It has a wide variety of available catalytic activities with a higher proteolytic activity Greater pH and temperature stabilities Higher DH can be achieved in a short time and moderate pH conditions Produces less bitter hydrolysates with better functional and nutritional properties with superior protein recoveries Produces hydrolysates with a lower lipid content and it’s a cost effective enzyme The above mentioned factors guided our choice of Alcalase over other proteases for the hydrolysis of yellow fin tuna and viscera and head rest raw materials

In the production of interesting peptides fractions by enzymatic hydrolysis of tuna dark muscle by-product using Alcalase, optimization of hydrolysis conditions by response surface methodology (RSM) revealed that optimal conditions developed by RSM produced hydrolysates with a low rate of peptide fraction of molecular weight of 4-1 kDa (Saidi et al, 2016) Results from a complementary study at 55 °C, for 60 min, with 1% enzyme concentration, and pH 8.5 produced hydrolysates with a high rate of the peptide fraction of molecular weight of 4-1 kDa The resulting hydrolysates composed of amino acid with a potential for application as a constituent in balanced diets of fish and as a nitrogen source in microbial growth media (Saidi et al, 2016)

Optimization by response surface methodology (RSM) using a factorial design

in the preparation of hydrolysates from visceral waste proteins of catla (catla catla) with a commercial protease revealed that enzyme to substrate ratio of 1.5% (v/w), pH 8.5 at 50 °C for 135 min were the optimum conditions for a higher DH close to 50% with Alcalase (Bhaskar et al, 2008) The amino acid profile of the Hydrolysates prepared at optimized conditions were similar to FAO/WHO reference protein It further indicated that methionine is the most limiting amino acid, hence, the Hydrolysate can be replaced for juvenile common carp amino acid requirements and henceforth unveiling its potential application as a constituent in balanced fish diets (Bhaskar et al, 2008) In a comparative study, Guerard et al (2002) hydrolyzed rest raw

materials from the tuna canning industry utilizing a commercial protease

“Umamizyme” The hydrolysis was carried out in a 1-l batch reactor at pH 7 and 45

°C.The effect of enzyme to protein substrate ratio in the range of 0.1 to 1.5% (w/w) protein was analyzed in relation to the degree of the Proteolytic degradation, quantity

of nitrogen released and the molecular weight distribution of the peptides and a DH of

up to 22.5% was achieved with an enzyme/substrate ratio of 1.5%, after 4 h Protease

“Umamizyme” proved to be as effective as Alcalase 2.4 L for the tuna waste solubilization with reference to the results obtained Though, with a lower stability as compared to Alcalase 2.4 L

Optimizing the enzymatic hydrolysis of skipjack tuna (Katsuwonus pelamis)

dark flesh using Alcalase enzyme at various concentrations (1, 1.5, 2, 2.5, & 3%) at

pH from 6 to 10, temperatures from 35 to 75°C and times of 2 to 6 hours showed that optimal hydrolysis conditions were, Alcalase 2%, pH =8.86, Temperature 65.4°C and time 5.74 h with resulting optimum degree of hydrolysis as 20.74% (Mohammad &

Yusuf, 2016) In a comparative study, skipjack tuna (Katsuwonus pelamis) protein

hydrolysates from the dark flesh produced with different types of industrial proteases including Alcalase®2.4L FG, Protamex®, Neutrase®1.5MG and Flavourzyme®500MG) for 1, 2, 3 and 4 hours with level of proteases used of 0.5, 1.0, 1.5 and 2% of the original weight of raw material indicated that longer time with higher concentration of enzyme increased the degree of hydrolysis Alcalase®2.4L FG had the highest degree

of hydrolysis among all proteases followed by Protamex®, Flavourzyme®500MG and Neutrase® 1.5MG (Herpandi et al, 2012)

Enzymatic hydrolysis of stomach proteins from yellow fin tuna (Thunnus

albacares) wastes using Alcalase in a batch reactor relating the influence of the

process variables (enzyme/substrate ratio; effect of intermediate substrate and enzyme addition) was studied with regards to the extent of Proteolytic degradation and to the molecular weight distribution of the peptides (Guérard et al, 2001) A linear correlation resulted between the degree of hydrolysis DH and the enzyme concentration Upon addition of more substrate in the due process of hydrolysis, the final DH attained was proportional to the substrate added, signifying that the concentration of hydrolysable bonds was one of the main factors influencing the hydrolysis rate

Pacheco-Aguilar et al (2008) used Pacific whiting (Merluccius productus)

muscle to produce fish protein hydrolysates with 10%, 15% and 20% degree of hydrolysis (DH) utilizing the commercial protease Alcalaseand were characterized at

pH 4.0, 7.0 and 10 according to their solubility, emulsifying and foaming properties The rate of Protein recovery in soluble fractions was found to increase correspondingly with the hydrolytic process, resulting into 48.6±1.9, 58.6±4.1 and 67.8±1.4 of the total protein after 10%, 15% and 20% DH, respectively Freeze-dried hydrolysates presented almost 100% solubility (P>0.05) at the different pH

Protein hydrolysates obtained by enzymatic hydrolysis of grass carp

(Ctenopharyngodon idella) skin using Alcalase and terminating the hydrolysis reaction

by heating the mixture to 95°Cfor 15 min revealed that at 5.02%, 10.4%, and 14.9 % degree of hydrolysis (DH), the protein hydrolysates had desirable essential amino acid profiles thus, their potential application as functional food ingredients as emulsifiers and binder agents (Wasswa et al, 2007) In a comparative study, optimization of enzymatic hydrolysis conditions (temperature, time and enzyme to substrate ratio) of

tilapia (Oreochromis spp.) scale gelatin by utilizing response surface methodology

(RSM) with a commercial Alcalase 2.4 L, a protease enzyme to breakdown the peptide chains present in the gelatin Gelatin Hydrolysate obtained showed that a hydrolysis temperature of 57.6 °C together with a hydrolysis time of 80 min and enzyme to substrate ratio of 1.20% (v/w) were the optimum conditions to obtain the highest degree of hydrolysis (10.91%) (Mohammad et al, 2014)

Fish protein hydrolysates prepared from cuttlefish (Sepia pharaonis) muscle

produced by two commercial enzymes using Alcalase and protamex methods revealed that cuttlefish protein hydrolysates using Alcalase (CPHA), yields higher degrees of hydrolysis, proximate composition, yield, solubility, than cuttlefish protein hydrolysate using protamex (CPHP) Amino acids profiles of the CPHS were higher in essential amino acids compared to the recommended pattern of requirement by FAO/WHO and NRC standards (Raftani Amiri et al, 2016)

Shahidi et al (1995) effectively optimized the hydrolysis conditions with Alcalase enzyme for the production protein hydrolysates from capelin Resulting products had a greater protein recovery of about 70.6% with reference to other alkaline

protease concentration has been presented elsewhere (Rebeca, 1991)

Protein hydrolysates produced at 5, 10, and 15% DH out of pulverized salmon muscle using one of the four alkaline proteases (Alcalase, Flavourzyme, Corolase PN-

L, and Corolase 7089) or endogenous digestive proteases Conditions of reactions were fixed at pH 7.5, 40 °C, and 7.5% protein content, and enzymes in Azocoll units were added Protein content for the hydrolysates varied between 71.7 and 88.4%, with

a very low lipid content Nitrogen recovery was between 40.6 and 79.9% (Kristinsson

& Rasco, 2000)

Nilsang et al (2005) produced FPH from fish soluble concentrate (FSC), a rest raw material from fish canning industry with Flavourzyme and Kojizyme Hydrolysis conditions were optimized with RSM and the model equations were suggested with regard to the influence of temperature, time, and enzyme concentration (E) on the DH The optimal values for Flavourzyme concentration, substrate concentration, temperature, and hydrolysis time were 50 Leucine Amino peptidase Unit (LAPU/g) protein, 20% (w/w), 45 °C, and 6 h, respectively whereas for Kojizyme were 40LAPU/g protein, 20% (w/w), 50 °C, and 6 h Kojizyme led to the production of some bitter-taste amino acids like tryptophan during unlike Flavourzyme The spray-dried FPH produced with Flavourzyme contained high protein content (66%) The DH with Kojizyme and Flavourzyme was 68 and 62%, respectively

Thivel et al (2005) evaluated the effects of different Proteolytic enzymes and different reaction durations of 25, 50, 75 min on functional and nutritional attributes of

red salmon head hydrolysates DH values for the 75-min hydrolysis varied between 6.4% and 16.7% Protein hydrolysates appeared yellow with a protein content of 62.3% to 64.8% and high levels of essential amino acids A weak correlation was observed between increased DH values and increased hydrolysates solubility

Enzymatic hydrolysis of yellow fin tuna visceral protein using Neutrase with RSM using factorial design was carried out and DH was determined as a response to the hydrolysis conditions Enzyme activity of 39.61 AU/kg protein, temperature of 53

°C, and hydrolysis time of 141 min were found to be the optimal conditions to obtain a 30% DH The tuna visceral protein hydrolysates possessed a significantly high protein content of 74.56%, with a low lipid content of 1.86% (Motamedzadegan et al, 2010)

Ovissipour et al (2009) prepared Protein hydrolysates from the Persian sturgeon

viscera (Acipenser persicus) Hydrolysis was carried out at three different temperatures

i.e 35, 45 and 55 °C, pH 8.5, with a protease enzyme - Alcalase and an enzyme to substrate ratio of 0.1 AU/g viscera protein for a period of 205 min Protein and lipid content of the resulting hydrolysates were 65.82%, and 0.18%, respectively Protein recovery and DH varied between 34.97% to 61.96% and 13.32% to 46.13%, correspondingly The highest degree of hydrolysis was observed at 55 °C after 205 min

Nguyen et al (2011) investigated Long-term proteolysis of tuna by-products i.e head, viscera and tail by a protease - Protamex DH achieved at the end of 12 h of hydrolysis for head, viscera and tail were 32.3, 16.8 and 22.2 %, respectively Nitrogen recovery in the soluble fractions was 73.6 %, 82.7 % and 85.8 % for head, viscera and tail correspondingly

Benjakul & Morrissey (1997) studied further Alcalase at pH 9.5, 60 °C and Neutrase at pH 7.0, 55 °Con Pacific whiting solid waste Alcalase proved to have a higher activity compared to Neutrase with an enhanced and efficient hydrolysis Optimal conditions for Alcalase were 20 (AU)/kg, 1 h, and waste: buffer ratio of 1:1 (w/v) at 60 °C and pH 9.5 with the resulting hydrolysates rich in protein content as well as better nitrogen recovery close to 70% and an amino acid composition related

to that of fish muscle Lastly, Alcalase proved to be the most cost-effective enzyme in relation to the five enzyme preparations analyzed on hydrolyzed salmon muscle proteins

With reference to the available literature, it’s evident that a lot of research has been done about the production of fish protein hydrolysates from various kinds of marine and sea organisms or their by-products for different applications as well as optimization of hydrolysis conditions using Response surface methodology (RSM) in the production of hydrolysates However, it’s important to note that all these researches have been done using the fish as a whole or by-products mixed together This therefore creates a gap especially for the case where, by-products are utilized since you can’t determine which of the by-products whether visceral, head, bones, dark muscle or fins has a significant effect on the degree of hydrolysis, solubility, amino acid profile of the hydrolysates

This work is therefore aimed at bridging the above mentioned gap by investigating the effects of Alcalase enzyme concentration, hydrolysis temperature, and incubation time on the Degree of Hydrolysis of visceral and head waste proteins of Yellow fin tuna and consequently evaluate the Solubility of the attained product for its suitability as feed for fingerlings

Chapter 3 MATERIALS AND METHODS 3.1 Materials

3.1.1 Head and viscera from Yellow fin Tuna

Visceral waste devoid of airbladder and head waste obtained from the

processing of Yellow fin Tuna (Thunnus albacares) were collected from Hong Ngoc

Seafood Company, Hoa Hiep industrial zone, Dong Hoa district, Phu Yen province, Vietnam Samples were frozen and transported to the Labs of Nha Trang University where experiments were carried out Samples were thawed and minced separately that is head and visceral waste, vacuum packaged and then stored at -202°Cuntil use A day before hydrolysis, they were shifted to the refrigerator for thawing at 42

°C for 12 h

3.1.2 Enzyme and chemicals

The protease employed for the optimization studies was Alcalase (with a declared activity of 2.4 AU/g and a density of 1.18 g/ml) which is a bacterial endo-

proteinase from a strain of Bacillus licheniformis It was provided by the Danish

company Novozymes The enzyme was immediately stored at 41 °C

All chemicals and reagents employed in the study were of undisputed purity and analytical grade They were purchased from Asia Laboratory Instruments Company Limited, 594/23 Au Co Street, Tan Binh district, Ho Chi Minh City, Vietnam

3.2 Experimental design

3.2.1 Preparation of protein hydrolysates

To establish the effect of enzymatic hydrolysis conditions of yellow fin tuna rest raw materials i.e visceral and head rest raw materials by using Alcalase enzyme

on the degree of hydrolysis and solubility of the hydrolysates, a full factorial design with three factors, two levels and four center points was used (Table 3.1) Protein hydrolysates were prepared according to the method of Ovissipour et al (2009) with some modifications 50g of the minced substrate was mixed with 0.1M sodium phosphate buffer in the ratio 1:1 to maintain the pH constant during the course of hydrolysis The stages of hydrolysis are charted in Figure 3.1 The solutions were put

in different 250 ml glass beakers and enzyme was added at a ratio 0.5-1.0% of the total

proteins in the fish rest raw material Samples were then transferred to a water bath with constant stirring at a temperature of 50-60 °C for 4-10 h, which was maintained constant throughout the hydrolysis time The initial conditions of hydrolysis for the first run (0.5% enzyme, Temperature of 50 °C and time of 4 h) were chosen according

to the central composite design of response surface methodology generated for optimization of these rest raw materials Subsequent conditions were used for the next runs according to the run order of the same design For enzyme activity deactivation, the resulting product was heated to 90 °Cfor 15 min in a water bath, and then it was cooled to room temperature with iced water and centrifuged at 7500 rpm at 4 C for 45 min for visceral waste and 5000 rpm at 4 °Cfor 25 min for head waste The surface oil layer was removed with a plastic pipette and the supernatant (FPH) was collected and stored at −202 °Cuntil further analysis

Figure 3.1 Scheme for the Preparation of the fish protein Hydrolysate (FPH) from yellow fin tuna viscera and head rest raw materials

3.2.2 Experimental design for optimization and analysis of data

A central composite design was used for the optimization of Degree of hydrolysis and solubility of fish protein hydrolysates obtained from the enzymatic hydrolysis of yellow fin tuna rest raw materials Complete composite design (CCD) in the experimental design consisted of 23 factorial points, six axial/star points (α =1.682)

Mince the tuna

rest raw

materials (50g)

Add a mixture of water and sodium phospahte buffer to the minced materials in the ratio of 1:1 (50mls:50g)

Adjust the pH for optimum activity

60 °C, 4-10 h)

Terminate the reactions by heating the solution at 90 °C for 15 minutes, to inactivate enzymes

Cool with ice to

room temperature

at (22 - 25° C) and

then centrifuge

Collect the supernatant, (store at -20°C, untill further analysis)

Fish protein hydrolysate (FPH)