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

Major:

MSc. In Food Technology

Topic allocation Decision

192/QD-DHNT

Decision on establishing the Committee:
Defense date:

18/09/2020

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
conditions for yellow fin tuna rest raw materials using Alcalase enzyme” is my
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

iii



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.

iv


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.

v


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

vi


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
vii


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

viii



LIST OF SYMBOLS
Time
Temperature
Enzyme concentration
Percentage
h

Hours
Degrees Celsius
Axial/ star points
Intercept/constant/offset term
Regression coefficient for linear effect
Regression coefficient for quadratic effect
Regression coefficient for interaction effect
Broken peptide bonds
Total number of peptide bonds
⁄

Volume/ weight

⁄

Volume/volume
Nanometres
Millilitres
Microlitres
Litres
Molarity


ix


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

x


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
xi


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

xii


LIST OF FIGURES
Figure 3.1. Scheme for the Preparation of the fish protein Hydrolysate (FPH) from

yellow fin tuna viscera and head rest raw materials...................................................... 26
Figure 4.1. Response surfaces and contour plots for the effect of variables on DH
(Viscera) as a function of different hydrolyzing conditions: A; time and temperature,
B; time and enzyme concentration, C; temperature and enzyme concentration. .......... 38
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

xiii


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 °C
and 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

xiv


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

1


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

2


3,400,000 MT has enhanced its application as sashimi in several other countries (Woo
et al, 2008).
The industrialization of Yellow fin tuna is increasing fishery rest raw materials
with the amount of by-products generated in the due course reaching approximately
75% of the entire fish weight (Wisuthiphaet et al, 2016; Guerard et al, 2002). Some of
these are used to produce fish sauces and food products such as dry-salted roe (1.5–
3.0% of total weight). Yellow fin tuna roe could also be used in animal feed or pet
food preparation (Chalamaiah et al, 2013;Klomklao et al, 2014). However, a big
proportion of these rest raw materials is just thrown out which is a big cause of
contamination of the environment thereby contributing further to an annual increase in
hazardous waste quantities out of fish processing.
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
3


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.

4


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
1.2.1. Main objective.
To optimize enzymatic hydrolysis conditions for the production of fish protein
hydrolysates from yellow fin tuna rest raw materials.
1.2.2. Specific objectives.
i.

To determine the effects of Alcalase enzyme concentration, temperature,

and hydrolyzing time on the degree of hydrolysis (DH) and solubility of visceral and
head rest raw materials of yellow fin tuna.
ii.

To determine the chemical properties and amino acid profile of visceral and

head protein hydrolysates.

5


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 °C
above 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
6


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%
7



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,

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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.
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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.
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Physiognomies study of calcium from bones of skipjack tuna bone recommends
pretreatment for enhanced absorption (Yoon et al, 2005).
Tuna bone is associated with anti-oxidant properties. The backbone protein
from tuna poses an antioxidant peptide which significantly inhibits lipid oxidation in
emulsion system of linoleic acid and at the same time quenches free radicals 2, 2Diphenyl- 1-picryhydrazyl (DPPH) (Je et al, 2007).
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

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