CHAPTER 1

INTRODUCTION

1.1 Background of study

Marine food products have been essential part of human intakes since people

started fishing the seas and producing food through aquaculture. Seafood products

such as fried fishes, prawns, squids and many others are popular local cuisine, loved

by many consumers. In fact, the worldwide consumer demand for such nutritious

products for aquatic animal food products are marketed internationally rather than all

meat and poultry traded together (Tacon & Metian, 2013). In response, many food

scientists and technicians are focusing on fish proteins as an effort to develop more

protein sources and apply them as functional or nutritional ingredients.

Fish protein hydrolysate (FPH) have gained so much attention for years due to

a lot of health-beneficial effects and has been used in human everyday life. It has

become a big target for researchers to identify new technologies with better added

value for FPH (Herpandi et al., 2011). Raw materials for FPH production can be both

fish and fish leftovers. Fish flesh, skin and waste such as the latter include head,

trimmings, fins, frames, viscera and roe are three major components of fish widely

used as sources of FPH (Halim et al., 2016). The underutilized and underestimated

fishing leftovers from the main production can be processed to get FPH, which can

increase the efficiency of fish processing by redirection of the by-products for human

consumption (Muzaifa et al., 2012). This will be more profitable and environmentally

friendly. In the case of FPH, the fishing industry can avoid the fishing leftovers
consumption to feed animals, which will be consumed by human that has been proven

to provide numerous health benefits to human. Hydrolysates of proteins are

degradation products of the enzymatic conversion of proteins to become small

fragments of peptides, containing 2 to 20 amino acids.

Freeze drying is a technique which utilizes the principle of moisture

sublimation under lowered pressure. Freeze drying is a highly applicable method

used for FPH drying at laboratory scale due to its simplicity and speed. A large

number of authors have used freeze drying for the recovery of FPH in a number of

researches, which studied different FPH purposes such as (Noman et al., 2018),

Jenkelunas & Li-Chan, 2018, Samsudin et al., 2018 and many others. Through a

number of research work, freeze drying showed gentle treatment of the hydrolyzed

mixture and a better protein recovery together with low moisture content in final

product. However, at large-scale production the cost and limited producing capacity

of freeze-drying plays a negative role, where freeze drying is mostly used for the

production of the highest quality FPH. Hence, other alternative which is vacuum

drying will be used in order to compare both drying methods.

Protein hydrolysates can be extracted by using chemical hydrolysis (Herpandi

et al., 2011) either acid or alkali, by heat treatment (Korczek et al., 2020) and by

enzymatic hydrolysis (Noman et al., 2018). As compared with the chemical process,

proteolysis by enzymes has several advantages. These include mild process

circumstances, specificity, high reaction velocity and a lot of choices (Robinson,

2015). By optimizing hydrolysis conditions, using different enzymes and fish species

as substrates, it is possible to create a wide variety of hydrolysates with desirable

physical, chemical and biological properties (K. Elavarasan et al., 2014) as they play a

major role in deciding the degree of hydrolysis. Proteolytic enzyme, also called
protease, proteinase, or peptidase, any of a group of enzymes that break the long chain

like molecules of proteins into shorter fragments (peptides) and eventually into their

components, amino acids (Mótyán et al., 2013). The enzymes used are digestive

proteolytic enzymes (such as pepsin, chymotrypsin, trypsin and so on) obtained from

animals, or food grade enzymes obtained from edible parts of plants and from

microorganisms with an accepted safe use in human nutrition (Fernandes, 2016).

In recent development, most of the studies on bioactive properties as well as

functional properties of FPH have been carried out with reference to type of enzymes,

nature of substrates and degree of hydrolysis. The bioactive properties of fish protein

hydrolysate (FPH) such as antioxidant (Korczek et al., 2020; Morales-Medina et al.,

2016), angiotensin–I converting enzyme (ACE) inhibitory activity (Sila et al., 2013),

anticancer (Nura Ruhaya Abdul Halim et al., 2018), immunomodulatory (Kiewiet et

al., 2018), anticoagulant (Rajapakse et al., 2005), anti-diabetic (Harnedy et al., 2018)

and mineral binding properties have been studied. The most studied properties of

FPH are on the antioxidant properties.

The effect of nature of drying on the properties of protein hydrolysates likely

to depend on nature of peptides present in the hydrolysate. Previous studied on

porcine placenta hydrolysates prepared using vacuum drying resulted in lower

antioxidant properties than the freeze drying and spray drying (Tang et al., 2013).

Besides, according to (Hau, Mohd Zin, et al., 2018), the best technique to produced

Yellowstripe scad’s powdered protein hydrolysate was subjected to freeze drying

using 2.0% of Alcalase enzyme at 2 hours of hydrolysis time. High degree of

hydrolysis (95%) successfully obtained and gives better functional properties of

protein hydrolysate. Due to their high antioxidant properties, fish protein hydolysates

(FPH) have aroused special interest (Petrova et al., 2018). A significant aspect of
industrial production of FPH is related to the concentration methods that can be used,

such as oven, freeze or spray drying. Among them, freeze drying is the preferred

operation in industry, since it causes less damage to peptides. However, high capital

and running costs have led to a search for alternate drying methods. Recently,

(Krishnamoorthy Elavarasan & Shamasundar, 2016) compared the effect of oven

drying and freeze drying on the antioxidant properties of FPH with papain from

Cirrhinus mrigala, suggesting that oven drying may be recommended. Next, the

newest recent studied had been done by Kim et al., (2020) regarding the effects of hot

air drying (HAD), low temperature vacuum drying (LVD), and freeze drying (FD) on

physicochemical characteristics and textural features of Yellow Croaker

(Larimichthys Polyactis). It summarized that LVD rather than HAD and FD provide

good qualities of dried fish in terms of physicochemical characteristics and textural

properties. However, investigation on the effect of drying process on the

physicochemical and functional properties of FPH is still imited.

1.2 Problem Statements

The yield of small pelagic such as shortfin scad (Decapterus Macrosoma)

caught from sea was high but underutilized in processing industries. The issues have

arisen when there are almost 60-70% of post-processing leftovers (flesh, head, skin,

bones, scales and viscera) from the processing of fish sausage in east coast region of

Malaysia. In 2015, total number of shortfin scad caught in Malaysia was

approximately increased from 102 644 in 2014 to 117 155 in 2015 (Department of

Fisheries, 2015). This kind of species are rarely used for specific usage such as

canning, new product development or protein characteristic. It was eaten as normal

dish either fried or cooked with sauce. Since the fish rarely process into other product,

the shelf life for storage is limited due to high amount of protein is prone to
degradation, oxidation and other undesirable processes (Papuc et al., 2017).

Production of various value-added materials such as proteins (Ghaly et al., 2013),

bioactive peptides (Ghaly et al., 2013), gelatine (Ghaly et al., 2013) and collagen (Chi

et al., 2014) are an option to fix this problem.

Furthermore, seeing the importance of these fish protein hydrolysate, the

shortfin scad waste from the processing of fish sausage in east coast region of

Malaysia has high potential to be converted into fish protein hydrolysate. There has

been a lot of development of these fish products. Applications that have been carried

out on protein hydrolysate are currently concentrated more on fish but still lack of

studying regarding the effect of vacuum drying and freeze drying on physicochemical

and functional properties for shortfin scad was reported.

1.3 Justification

The research will improve the usage of low value of fish which is less utilized

also reduce the wastage of resources in fish processing industries. This study can help

to widen the use of shortfin scad to produce fish protein hydrolysate rather than only

domestically used or processed into feeds. As these fish are low valued fish, most of

them are kept in improper conditions which can reduce the shelf life of the fish. Since

fish protein powder has better stability prior to storage in proper condition, thus it can

be commercialised for higher revenue also increase the value of fish and to promote

the healthy living. This knowledge can lead to more scientific research of functional

properties of protein which relate in processing industries.

The research also will furnish information regarding the effect of vacuum

drying and freeze drying on the physicochemical and functional properties of fish
protein hydrolysate which can serve as reference for further study. This knowledge

can lead to more scientific research which can greatly widen the use of protein

hydrolysate based on its physicochemical and functional properties in food processing

industries.

1.4 Objectives

1. To determine the effect of vacuum drying and freeze drying on the

physicochemical properties of fish protein hydrolysate

2. To determine the effect of vacuum drying and freeze drying on the functional

properties of fish protein hydrolysate

 CHAPTER 2

LITERATURE REVIEW

2.1 Protein source

Protein is a complex macromolecule, which consists of carbon, nitrogen,

oxygen, hydrogen, and sulphur. It is a fundamental component of all living cells.

Protein consists of unbranched chains of smaller units called amino acids to form a

complex organic compound which is the second largest component in human body

after water (Hoffman & Falvo, 2004). A total of 20 different kinds of amino acids

which is essential and non-essential amino acids are used to make all types of protein

(Weijs et al., 2014).

Animals and plants are sources of protein. There are two types of proteins

which are complete and incomplete. Complete proteins consist of all the essential

amino acids. Complete protein which is high quality sources can be found in animal

proteins such as meat, poultry, fish, milk, eggs and cheese. Meanwhile, proteins

extracted from plants usually can be found in nuts, seeds, vegetables, legumes (peas

and beans) and grains and most of plant-based food are incomplete proteins, low in

one or more essential amino acids but some plant proteins, such as soybeans, are also

complete proteins (Palmer, 2014).

2.1.1 Fish and Seafood

Seafood is one of the best protein sources. It usually consists of low amounts

of fats. According to Wisuthiphaet et al. (2016), seafood products reduce hunger

vulnerability by providing a complementary source of food when other food sources,

such as agriculture, are seasonally low. Fish such as salmon are highly nutritious food

items contains decent quantities of fats but these are mostly beneficial lipids, such as

essential omega-3 fatty acids, which are good for cardiovascular health (Richter et al.,

2015). In most countries, fish is one of the major sources of animal protein and

essential nutrients for maintaining health and protecting against various types of

diseases, such as blood pressure, cancer and heart attack (Abraha, 2017).

Praveen Kumar et al. (2017), stated in the findings, fish is a highly

proteinaceous food consumed by a greater percentage of population in the world due

to its abundance and palatability. According to United Nations, Department of

Economic and Social Affairs, Population Division (2017), the world population due to

reach 9.8 billion by 2050, the demand for such food products is set to increase further.

In addition to acting as potential sustainable food sources to meet future demand,

marine proteins contain a wide range of bioactive components in the form of peptides

encrypted within their primary sequences. Fish and fishery products are denote as

important and valuable source of nutrients of fundamental importance for diversified

and healthy diets since they are considered as high quality protein, consisting of

complete amino acids (K. Elavarasan et al., 2014). Recent researchers are more

focusing on fish protein that being used as functional food to promote healthy living

making the finding more acceptances because of its special biochemical and

nutritional qualities. Fish proteins can be found in all parts including flesh, head,

frames, fin, tail, skin and guts of the fish in varying quantity of proteins

(Ramakrishnan, 2013). Unutilized fish, underutilized fish and fish processing waste

can be used to produce fish proteins as large amount of fish by-product are currently

disposed or used for low–value products. There is a huge potential for reducing the
amount of by-product and to develop a larger amount of the by-product for value

added product for human consumption (Muzaifa et al., 2012).

2.1.2 Functions of protein

Proteins are required to produce, maintain, and repair all components of the

body such as bones, hair, skin, muscles, and other organs (Hermann et al., 2005).

Proteins play a crucial role in the production and regulation of hormones, enzymes,

and have a vital role in the coding and sequencing of genes (Vaidyanathan, 2016).

They also supply energy, but not as much as carbohydrates. Proteins are essential for

normal functioning of the body (Hoffman & Falvo, 2004). They also significantly

contribute towards the body’s immune system, cell signalling, cell cycle, and cellular

adhesion (Hoffman & Falvo, 2004). Proteins are required to make haemoglobin,

which is an important part of red blood cells and making antibodies to protect us from

illness and infections. (Van Goudoever et al., 2014; Hermann et al., 2005). Proteins

play a significant role in transporting materials in and via the body fluids (Buxbaum,

2007). Proteins are essential nutrients and an integral part of a balanced, healthy diet.

Eating appropriate quantities of proteins is necessary for stronger muscles and healthy

body tissues (Hoffman & Falvo, 2004). Since proteins are the building blocks of all

biological tissues, it is obvious that they handle the job of cell repair and rejuvenation

beautifully well (Artmann, 2018).

Amino acids are important biomolecules that both serve as the basic building

blocks of proteins, consisting of 20 different properties of amino acids. They consists

of carbon, hydrogen, nitrogen and oxygen with two out of 20 contain sulphur

(Brosnan, 2006). The amount of amino acid content varies depend on the various

factors such as species, size, maturity, fishing season, water salinity and food sources
(Doğan & Ertan, 2017). Fishes contain high amount of essential amino acids which

are methionine, lysine, tryptophan, cysteine and threonine as they play a crucial

function in regulating health and body nutrition (Mohanty et al., 2014). Fish provides

not only high-value protein, but also a wide range of essential micronutrients,

including many types of vitamins (D, A and B), minerals including calcium, iodine,

zinc, iron and selenium and polyunsaturated omega-3 fatty acids (docosahexaenoic

acid and eicosapentaenoic acid) (FAO 2012). Industrial research in the field of fish

processing and product development has resulted in a variety of approaches and

formulations that use fish as an ingredient for human food as such products are more

diverse and far-reaching than poultry or meat products (Thilsted et al., 2014). The

versatility resulting from the diversity of fish species contributes to the production of

high-protein foods, both with a low cost and high value product (Thilsted et al., 2014).

2.2 Shortfin scad (Decapterus Macrosoma)

Shortfin scad (Figure 2.1) or also known as selayang, basung and sardine

belongs to the small pelagic group which is categorized as low-value fishes (Rasli &

Sarbon, 2019). It is a type of fish that easily found in Malaysia. Shortfin scad is

extremely important in Terengganu, Perlis and East Johor as the main ingredient for

‘keropok lekor’, fish crackers and fish sausages processing. Other than used for

human consumption, shortfin scads are also used as bait for local and foreign tuna

fishing (Asni et al., 2019). In Malaysia, the amount of shortfin scad caught in

Malaysia around 100,000 to 110,000 tons per year (Department of Fisheries, 2015).

Marine fishes can be divided into two categories; pelagic and demersal fish. Pelagic

fishes are those found on the surface or the middle depth of body water (Hau, Amiza,

et al., 2018). Marine pelagic fishes are further categorized into coastal fish and

oceanic fish depending on the mainland shelf they inhabit. The word 'small pelagic
fishes' refers to a various group of mainly planktivorous fishes sharing the same

habitat, the surface layers of the water column, usually above the continental shelf and

in waters not exceeding 200 meters depth (Dalzell, 1993). Hau et al. (2018) reported

that these pelagic fishes commonly achieve a maximum weight of less than 500 gram

each.

Figure 2.1 Shortfin scad (Decapterus macrosoma)

2.2.1 Taxonomy and phylogeny

Decapterus spp. as a member of the Carangidae, is one of the fish that has

been used as a commodity of trade and economic resources (Kimura et al., 2013).

They are found throughout the world, widely distribution in Indo-Pacific, from East

Africa to Hawaii and eastern Pacific Ocean from Gulf of California to Peru (Matsuma

et al., 2011). In the East China Sea, there are several of Decapterus species (D.

maruadsi, D. akaadsi, D. macrosoma, D. macarellus, D. tabl, and D. russelli) with D.

maruadsi and D. macrosoma being the most abundant (Shiraishi et al., 2010). The

fishes of the family Carangidae including the genus Decapterus are economically

crucial resources in tropical and subtropical waters of the world (Shiraishi et al.,

2010).
 According to Matsuma et al., (2011) Shortfin scad is a small species fish,

commonly found to be less than 25 cm, although some have maximum length of 30

cm. The length of shortfin scad ever recorded is 30 cm while the highest weight ever

recorded was around 300 grams. This species has a typical scad body shape, with

elongate, slender and nearly rounded or cylindrical body shape. Its body colour is

blue-gray dorsally, silvery ventrally. Their operculum usually has small black spots

with 9 dorsal fin spines and 3 anal fin spines. (Matsuma et al., 2011). These fishes

also have adipose eyelid well developed and completely covering eye except for a

vertical slit centered on pupil; predorsal scaled area not reaching to level of center of

eye and straight part of lateral line with scutes (24-40) posteriorly.

It was reported that maturity age of these Shortfin scads was estimated to be 2

years old based on results of age determination and size at maturity (Shiraishi et al.,

2010). It is a schooling species habitat, at a depth of 30-70 m above the water surface

(Alatorre-Ramirez et al., 2013). According to (Lubis et al., 2019) in their findings, the

composition of shortfin scads food consists of five groups namely fish,

phytoplankton, zooplankton, shrimp, and debris. The taxonomic hierarchy of shortfin

scad is shown in Table 1 (Myers, 2020).

Table 1. The taxonomic hierarchy of shortfin scad

Taxonomic Hierarchy and Nomenclature

Kingdom Animalia 
Subkingdom Bilateria 
Infrakingdom Deuterostomia 
Phylum Chordata 
Subphylum Vertebrata 
Infraphylum Gnathostomata 
Superclass Actinopterygii 
Class Teleostei 
Superorder Acanthopterygii 
Order Perciformes 
Suborder Percoidei 
 Family Carangidae 
Genus Decapterus 
Species Decapterus macrosoma

2.2.2 Nutritional content

Pelagic fish is a good energy source and rich in micronutrients that are not

commonly present in the basic food (Huan et al., 2017). According to Ishak & Sarbon,

(2018) reported that the major constituents of Shotfin scad were protein, fat, water and

ash. Ishak & sarbon (2018) also reported that there is carbohydrates, vitamins,

nucleotides, other non-protein nitrogenous compounds etc. also present in small

quantities. The proximate composition of the raw and hydrolysate of shortfin scad

waste is shown in Table 1.

Table 1. Chemical composition of raw shortfin scad waste and hydrolysate of shortfin

scad waste.

Component Raw shortfin scad waste Shortfin scad waste

(%) hydrolysate
b
Crude protein 22.97 ±0.82 73.08 ± 1.54 a
Fat 9.80 ±0.59a 7.55 ± 0.90b
Moisture content 56.88 ±0.48a 5.06 ± 0.47 b
Ash 3.17 ±0.47 b 10.40 ± 0.13a
Different letter (ɑ-b) means significantly different ( p<0.05 ¿ between the column.
Data reported were means values ± standard deviations.

Thus, chemical composition results indicated that Shorfin scad waste hydrolysate is

an excellent source of high quality protein that provides all of the composition needed

by the human body. According to Rasli & Sarbon, (2019) stated in their findings on

proximate analysis of of shortfin scad skin gelatine hydrolysate (SSGH) and shortfin

scad skin gelatine (SSG).

Table 2: The proximate analysis of of shortfin scad skin gelatine hydrolysate (SSGH)

and shortfin scad skin gelatine (SSG).

Component SSGH SSG

(%)
Moisture content 13.82 ± 2.42 73.08 ± 1.54 a
Protein 90.05 ± 0.28 7.55 ± 0.90b
Fat 1.95 ± 0.71 5.06 ± 0.47 b
Ash 12.48 ± 1.63 10.40 ± 0.13a
Data reported were means values ± standard deviations.

Thus, the chemical composition obtained also indicated that SSGH was a good source

of high-quality protein.

2.3 Protein hydrolysate

In developing countries, the aquaculture sector has problems with the

evaluation of fish waste at various rates (Cilbiz N & Hanol Z, 2015). Despite the fact

that the world's waste needs to be reduced, the amount of waste generated continues

to increase every year. Therefore, in order to reduce this issue, interest has been

directed towards on the potentially marketable of processing fish leftovers which have

long been discarded as waste to increase the value and utilization of low value

proteinaceous fish. Herpandi et al. (2011) had reported that processes such as protein

hydrolysis via enzymatic hydrolysis can be employed to produce more marketable and

functional protein hydrolysate. The underutilized products that would otherwise be

discarded become useful food ingredients for human consumption (Kristinsson &

Rasco, 2000). Hydrolysate is defined as proteins that are broken down into peptides of

various sizes.

The wastes of the fish processing industry are raw materials with huge

potential to produce FPH with functional characteristics that can be used not only as
animal feed but also give numerous health advantages to human (Ghaly et al., 2013).

Production of FPH is affected by many factors, such as the composition of raw

material, type of enzyme used, hydrolysis conditions, and degree of hydrolysis.

Parameters such as pH, temperature, time and concentration of enzyme will affect the

enzymatic activities, thus giving possibilities to control the process (Tanuja et al.,

2012). Therefore, many researches continue to seek for improvement of the utilization

of fishery by-products. The preparation of protein hydrolysate can be carried out

either chemically (using acids or bases) or biologically (using enzymes)

(Vijaykrishnaraj & Prabhasankar, 2015). The hydrolysis of fish protein is an

appropriate strategy for economic gain under the consideration of fish processing

waste into high-value products with the improvement in both quality and quantity

(Wisuthiphaet et al., 2015).

2.3.1 Enzymatic hydrolysis

To derive the peptides from fish protein, numerous methods have been utilized

to release bioactive peptides but enzymatic hydrolysis of whole protein is vast

majority techniques (Vijaykrishnaraj & Prabhasankar, 2015). Enzymatic hydrolysis

by using proteolytic enzymes can be endogenous or from other sources, such as

animals, plants, and microbial have been commonly used as this method considered to

convert the fish into a more marketable and functional form (Bruno et al., 2019;

Herpandi et al., 2011). In comparison with animal- or plant-derived enzymes,

microbial enzymes have some advantages, including a wide variety of available

catalytic activities and greater pH and thermal stabilities (Raveendran et al., 2018).

Numerous proteases such as Alcalase, Flavourzyme, Papain, Neutrase, and Protamex

have been employed for the preparation of protein hydrolysates from the fish by-

products with different bioactivities (Neves et al., 2017). The specificity of enzymes
used for proteolysis and the degree of hydrolysis (DH) are very important to evaluate

the functional activity of protein hydrolysate (Karamać et al., 2016). Protein

hydrolysate with different DH and different functional properties could be formed by

proper control during hydrolysis (Amiza et al., 2012). Besides, physicochemical

properties of protein hydrolysate also significantly influenced by the degree of

hydrolysis, type of substrate and protease enzyme used (Amiza et al., 2012).

Enzymatic hydrolysis of proteins produces a decrease in peptide size, which

can modify functional characteristics of the proteins and improve their quality and

more gently than acids. However, the hydrolysate produced is underutilised by human

consumption due to the presence of bitterness in taste and fishy flavour, then affects

the sensory acceptability of protein hydrolysate (Huan et al., 2017). Huan et al.

(2017), stated that tryptophan is one of amino acid that usually contributes to the bitter

taste. According to Bruno et al. (2019), the advantages of using enzymatic hydrolysis

are specific, simple, safe, eco-friendly method but slightly high cost of production. It

also often favoured for not producing chlorohydrins and contains very little salt

compared to chemical hydrolysis (Robinson, 2015). It requires a relatively small

amount of enzymes that can be easily deactivated and mild conditions of hydrolysis

(Herpandi et al., 2011). For example, Alcalase (endopeptidase) is a commercially

available enzyme preparation that has been widely used in the production of protein

hydrolysates because of its thermostability (50 °C) and high optimal pH (pH 8.5),

which can minimize the growth of microorganisms (Salwanee et al., 2013). Alcalase

works at alkaline pH have been reported to be most efficient in the hydrolysis of fish

proteins (Guérard et al., 2001). Another advantage of using Alcalase is that it provides

extensive proteolysis with less bitterness of protein hydrolysates compared to others

proteases (Liceaga-Gesualdo & Li-Chan, 1999). Thus, it has been proved to be one of
the best enzymes used in the preparation of protein hydrolysate. Moreover, according

to it is critical to select a cheap source of enzyme and easily available such as papain

produced from papaya latex. Papain is also the enzyme of choice due to its optimal

activity is at neutral pH which makes the process efficient and reproducible (Hormigo

et al., 2003). Hydrolysis from other enzymes will be more expensive. Furthermore,

Hormigo et al., (2003) stated in their findings that enzymatic conversion of fish frame

waste to protein hydrolysate could be a solution to minimize the pollution issues

related to seafood processing operations, and a way to add value to processing by-

products. Hence, the availability of various enzymes from different sources enables

the manufacturer to choose the best one based on the desired final product and holds

the most promise for the future due to products with high functionality and nutritive

value (Herpandi et al., 2011).

FISH PROTEIN

HOMOGENIZATION

ENZYMATIC REACTION

TERMINATION OF HYDROLYSIS (heat or low pH)

CENTRIFUGATION
 SUPERNATANT

DEHYDRATION

FISH PROTEIN HYDROLYSATE

Figure 2: A flow sheet for the enzymatic hydrolysis of fish protein to make fish

protein hydrolysate

2.3.2 Chemical hydrolysis

Chemical hydrolysis (acid or alkaline) also considered as a conventional

method for producing protein hydrolysates where this process is more preferred in

vegetable protein hydrolysis (Wisuthiphaet et al., 2015; Ghaly et al., 2013). Acid or

alkaline are will lead to complete hydrolysis, cleaving all the peptide bonds under

high pressure and temperature in a short time followed by neutralization to pH 6.0 to

7.0, and the product was either concentrated to a paste or further dried (Herpandi et

al., 2011). This method consumes less cost and time (approximately 24 hour).

However, hydrolysis reaction by chemical is difficult to control product quality due to

its harsh reaction and unspecific peptide bonds cleaving (Wisuthiphaet et al., 2015).

Acid hydrolysis of proteins is used more commonly than hydrolysis under alkaline

conditions (Kristinsson & Rasco, 2000).

Acid hydrolysis of fish protein has usually involved reacting fish proteins with

hydrochloric acid or sulfuric acid (some cases), and the proteins are will completely

hydrolyzed at high temperature, and often high pressure. According to Kristinsson &

Rasco (2000), stated that the hydrolysate produce by acid hydrolysis contains large
amount of salt (NaCl) during neutralization of an acid hydrolysis, which can make the

product unpalatable and interferes with functionality in food systems. In addition,

Hou et al., (2017) noted that chemical hydrolysis typically breaks down the proteins

into individual amino acid and smaller peptides, but another drawback of acid

hydrolysis is some essential amino acids will be diminished during the reaction. For

example, tryptophan is usually totally lost in an acid hydrolysis while cysteine, serine

and threonine are partially broken down and asparagine and glutamine are converted

into acidic forms (Herpandi et al., 2012). Besides, vitamins also mostly destroyed by

acid hydrolysis (Alvarez et al., 2012). Moreover, FPH produced by this process

usually used as an additive in animal feed, culture media and plant fertilizer due to

low production cost and resulting extensive hydrolysis (Wisuthiphaet et al., 2015);

Kristinsson & Rasco, 2000).

For alkali hydrolysis used primarily sodium hydroxide, to hydrolyze protein

often yields poor functionality and more importantly can adversely affect the nutritive

value of the hydrolysate (Kristinsson & Rasco, 2000). Rapid cleavage to large water-

soluble polypeptides takes place, followed by further degradation at a slower rate

during the process (Chapman et al., 2018). In addition, Lysioalanine, ornithinoalanine,

lanthionine, and β-amino alanine, which are toxic substances, also may be formed.

These occur because of the elimination and addition processes that take place during

the processing which is highly undesirable in foods (Herpandi et al., 2011). Despite

all these problems, limited alkaline treatment is used in the food manufacturing to

recover and solubilize a wide range of proteins (Herpandi et al., 2011).

2.4 Applications of protein hydrolysate

 Protein hydrolysate has many functions especially to improve or modify the

physicochemical and functional properties such as solubility, emulsifying properties,

foaming properties, water holding capacity and many more without losing its

nutrition. The properties of peptides present in FPH depend on the nature of proteases

and substrate used for the hydrolysis process (Elavarasan & Shamasundar, 2016)

FPH has been recognised and used in human daily life for years. For instance,

traditional food such as fish sauce contain FPH. Fish sauce is a traditional seasoning is

broadly used in large quantities, mainly as a condiment in cooking. Fish sauces are

produced by autolysis reaction catalyzed by endogenous enzymes or the addition of

commercial enzymes during the fermentation phases. Abundant variety of raw

materials can be used for sauce production provided that the proteolytic enzyme

substance is sufficient to cause efficient tissue solubilization and protein hydrolysis

(Gildberg 2001). Fish sauce is often made from small pelagic species such as

anchovies and sardines (Park et al., 2001). The type of fish used to produce fish sauce

affects the nutritional quality of the sauce, especially its nitrogen content.

Finding new applications with better added value for FPH has become a main

target for many researchers. Previous studies have shown that protein hydrolysates

from fish sources acquire antioxidant activity, hence FPH could be a potential source

of anti-hypertensive, anti-cancer, anti-anemia peptides.

2.4.1 Antioxidants

The development of undesirable off-flavors, odors, impair the nutritional value

of foods and potentially toxic reaction products due to lipid oxidation become a great

concern to the food industry and consumers (K. Elavarasan et al., 2014). Antioxidants

are used to preserve food products by retarding discoloration and deterioration

causing from oxidation. Antioxidants are increasingly used as enhanced the shelf life

and to improve the stability of lipids and lipid-containing foods. The use of synthetic

antioxidants is under strict regulation due to its potential health hazards caused by

such compounds (Park et al., 2001). Synthetic antioxidants, such as butylated

hydroxyanisole and butylated hydroxytoluene, commonly used as food additives to

stop the deterioration. Even though these synthetic antioxidants show stronger

antioxidant activity than the natural antioxidants, there is concern about their safety

(Herpandi et al., 2011). Thus, the development of natural antioxidants as other

alternative to synthetic ones is a great attention among researchers.

In recent years, hydrolysed proteins from many animal and plant sources have

been found to possess antioxidant activity, including those from aquatic products and

fish by-products (Herpandi et al., 2011). Various hydrolysates were produced by

different proteases and their antioxidant activities were examined have proven to be

good sources of antioxidant peptides such as mackerel protein (Korczek et al., 2020),

yellowfin sole frame protein (Jun et al., 2004), yellow stripe protein (Selaroides

leptolepis)(Putalan et al., 2018), round scad protein (Decapterus maruadsi) (Jiang et

al., 2014), tuna protein (Mutamimah et al., 2018), Pacific hake protein (Samaranayaka

et al., 2010), tilapia protein (Daud et al., 2013), and silver carp protein (Wu et al.,

2013). Different methods or techniques used to evaluate antioxidant activities,

including measuring their ability to inhibit the auto-oxidation of linoleic acid,

determining their scavenging effect on the DPPH free radical, and measuring their

reducing power.

2.4.2 Anti-hypertensive peptides

 Hypertension, or high blood pressure, is one of the major independent risk

factors for cardio- vascular disease. Angiotensin-I-converting enzyme (EC 3.4.15.1;

ACE) plays an important physiological role in the regulation of blood pressure by

converting angiotensin I to angiotensin II, a potent vasoconstrictor. Therefore, the

inhibition of ACE activ- ity is a major target in the prevention of hypertension.

Synthe- sized chemical drugs, such as captopril, are the most widely used medications

to treat and prevent hypertension. Although these drugs have obvious antihypertensive

effects, the side effects (such as dry cough, angio-edema, and many other

dysfunctions of hu- man organs) are beyond tolerance (Wu and others 2008). Many

peptides with high ACE inhibitory activity have been isolated and characterized both

from natural and processed foods. These peptides are of great interest as alternatives

to synthetic drugs. Bioactive peptides that are potential ACE inhibitors have been de-

rived frommarine organisms. Although natural peptides from food proteins may be

less potent than synthetic drugs, they have not shown adverse effects. Therefore, food-

derived ACE inhibitory peptides are believed to be safer and cheaper to make than

synthetic drugs.

Bioactive peptides are all inactive in their original protein, but they can be converted

to active forms through intestinal digestion or hydrolysis by proteases. Several

peptides derived from FPH made from Alaska pollock (Byun and Kim 2001), shark

(Wu and others 2008), yellowfin sole (Jung and others 2006), bigeye tuna (Qian and

others 2007; Lee and others 2010), and skipjack tuna liver (Je and others 2009) are

known to have ACE inhibitory activities and to exhibit antihypertensive activities in

experimental animals (Table 4).

The potency of these FPH peptides to inhibit ACE activity is expressed as an IC50

value, which is the ACE inhibitor concen- tration that leads to 50% inhibition of ACE
activity (Table 4). Moreover, the inhibition modes of ACE-catalyzed hydrolysis of

these antihypertensive peptides have been determined using Lineweaver–Burk plots.

These plots suggest that ACE inhibitory peptides act as noncompetitive inhibitors to

inhibit ACE. Jung and others (2006) reported that yellowfin sole frame peptide have

an antihypertensive effect. It was tested as feed for spontaneously hypertensive rats

and showed the blood pressure significantly de- creased after peptide ingestion. These

results suggest that ACE inhibitory peptides would be a beneficial ingredient in

nutraceuticals and pharmaceuticals used to treat hypertension and its related diseases.

2.4.3 Anti-proliferative compounds

Anticancer molecules isolated from marine organisms belong to diverse

structural classes, including polyketides, terpenes, steroids, and peptides (Mayer and

Gustafson 2004). These molecules usually are obtained from sessile animals such as

corals, sponges, and ascidians, which protect them- selves from predation by

synthesizing potent cytotoxic molecules. However, fish tissues also constitute a

potential source ofanticancer molecules. For example, squalamine, an aminosterol

isolated from the liver ofthe dogfish shark (Squalus acanthias), was demonstrated to

be a potent inhibitor ofangiogenesis and tumor growth in sev- eral animal models

(Sills and others 1998; Cho and Kim 2002). Alkylglycerols, which are natural

etherlipids that are abundant in shark liver oil, were recently described as inhibitors

oftumor vas- cularization (Pedrono and others 2004). Picot and others (2006) reported

that some FPH obtained by controlled enzymatic hy- drolysis have a significant

antiproliferative effect on human cancer cell lines in vitro. These preliminary data

suggest that FPH could represent a useful source of anticancer peptides. However,

slight variations in the hydrolysis process could lead to high variations in bioactivity,

thus confirming the need to accurately control the hydrolysis process to ensure
repeatability of FPH bioactivity. Sci- entists who work with bioactive compounds

from FPH are aware of the rules and regulations required to ensure consumer safety

and efficacy of new products. In this context, demonstration of bioactive properties

via in vitro screening tests in no way proves that fish peptides (or native proteins)

exert the same beneficial effects when consumed by humans. Source of antianemia

compounds.

2.4.4 Anti-anemia

During the last decade, several studies have shown that FPH or fish peptides

possess biological properties that can regulate the immune system, gastrointestinal

functions, blood pressure, and mineral absorption (Clare and Swaisgood 2000). For

example, FPH from sardines was shown to have significant in vivo antianemia

activity on experimental anemia models induced by blood loss or cyclophos- phamide

damage to the hematogenic mechanism (Dong and oth- ers 2005). Previous animal

research revealed that hydrolyzed fish offal protein inhibited anemia in mice that was

caused by an injection with cyclophosphamide. The hydrolyzed fish protein could

significantly inhibit decreases in red blood cells, hema- tocrit, hemoglobin, and

platelets, but not white blood cells (Shang-gui and others 2004). Its antianemia action

might occur by preventing the functional groups of protein (including amido,

mercapto, hydroxyl, and carboxyl groups in the mouse body) from undergoing

alkylation with cyclophosphamide. The antianemia action of the hydrolyzed offal

protein also might be due to nutritional functions of minerals, including calcium,

phosphorus, and iron.

2.4.5 FPH as components of microbial growth media

 Growth of the biotechnological fermentation industry had led to an increas-

ing demand for microbial growth media. Usually, the most ex- pensive component of

microbial growth media is the nitrogen source, which often is termed peptone when

made from nonheat—coagulable water-soluble proteins/peptides (Green and others

1977). Peptones are defined as protein hydrolysates that are readily soluble in water

and are not precipitable by heat, alkaline conditions, or saturation with ammonium

sulfate. They are one of the most important constituents of bacterial culture media.

Each peptone has its own biological characteristics, and no peptone by itself can meet

all the requirements ofmicroorganisms during cells cultivation. Different materials

from animal and plant sources are used to produce peptones, and most of them are

relatively expensive (Parrado and others 1993; Reissbrodt and others 1995; Martone

and others 2005). Growth substrate costs often constitute the major part of the

production cost ofmicrobial cells and bio- products from the fermentation industry (de

la Broise and others 1998). Therefore, a new and reliable source of good quality pep-

tones that are less expensive compared to the currently available ones would greatly

benefit this industry.

The use of fish materials as a source of nutrients for microbes was reported as

early as 1949 (Tarr and Deas 1949). Since then, several attempts to explore the use of

fish peptones as a component of microbial growth substrates have been reported. FPH

from hake filleting waste appears to be of sufficient nutritional value to support

growth ofbacteria and archaea (Martone and others 2005). Fish peptones from tuna,

cod, salmon, and unspecified fish were compared to one made with a casein using a

new method based on Gompertz modeling of microbial growth. Cumulative results

obtained from 6 species ofbacteria, yeasts, and fungi showed that, in most cases, the

fish peptones were very effective (Dufosse and others 2001). Ghorbel and others
(2005) reported that defatted Sardinella meat protein hydrolysate may be an excellent

nitrogen source for growth ofRhizopus oryzae and the production oflipase. In

comparison with the result obtained using commercial peptone, there was a slight

improvement in lipase production with fish media. The high lipase activity obtained

with cheap fish meal clearly indicated that these substrates could be used in industrial

fermentation processes.

FPH made from cod viscera has been tested as a combined source for nitrogen,

amino acids, and vitamins in microbial growth media (Aspmo and others 2005).

Using a panel of 5 differ- ent microbes (Escherichia coli, Bacillus subtilis,

Lactobacillus sakei, Saccharomyces cerevisiae,and Aspergillus niger), the

performance of this FPH was compared to the performance of common com- mercial

peptones. The results showed that FPH is a promising alternative to currently

available commercial nitrogen sources of other origins. In the case of the food-grade

and nutritionally fas- tidious L. sakei, 2 of the fish hydrolysates were clearly superior

to all tested commercial peptones. Safari and others (2009) reported that peptones

obtained from the enzymatic hydrolysis ofyellowfin tuna head waste using alcalase or

protamex were effective in pro- moting the growth oflactic acid bacteria. Moreover,

the peptones generated using both enzymes were better at promoting lactic acid

bacteria growth than the commercial de Man, Rogosa and Sharpe (MRS) media (P <

0.05). The choice ofproteolytic enzyme used to produce the fish hydrolysate had a

considerable impact on the performance of the resulting hydrolysate, both in terms of

max- imum growth rate and biomass production. Peptones produced using alcalase,

with a higher degree of hydrolysis (34%), induced better growth and performed better

overall as a lactic acid bacteria substrate than those using protamex (19% degree

ofhydrolysis).
 Fish peptones are usually produced either through hydrolysis with endogenous

or exogenous enzymes at a low pH or by a combination of these 2 methods. Acid

hydrolysis is a lengthy process that uses either inorganic acids at very low pH (<2.5)

or short-chain organic acids at pH 3.0 to 3.8 to avoid microbial growth. Due to the

obligatory neutralization step, hydrolysis at low pH results in a high ash content for

the produced peptone. Organic acids also possess growth inhibitory properties that

may be a disadvantage for the application of the resulting hydrolysate in microbial

growth media. Enzymatic digestion at neutral or alkaline pH by exogenous enzymes

has also been described, but this strategy seems to be rarely utilized in industry.

Previous research had pointed out the nutrients and functional compounds that

essential for good health, are present in Shortfin scad. Based on research of Ishak &

Sarbon (2018), their findings demonstrate the promising potential of shortfin scad

waste hydrolysate for the application as natural bioactive sources due to high protein

content and concentration, lower molecular weight, high solubility, and high

percentage of essential amino acids fulfilling the adult human needs. Study done by

Rasli & Sarbon, (2018) reported about gelatin. Gelatin is a protein derived from

collagen, the important constituent of animal tissue (Gilsenam & Ross Murphy,

1999). The findings prooved that Shortfin scad gelatin had higher melting and gelling

temperatures than those of sin croaker gelatin. The bloom strengths of gelatins from

sin croaker and from shortfin scad were 125 and 177 g, respectively, compared to 240

g for commercial bovine gelatin.

Besides, Angiotensin Converting Enzyme (ACE) plays an important role in

the regulation of blood pressure as well as fluid and salt balance in mammals.

Regarding the cardiovascular system, ACE converts the inactive decapeptide,

angiotensin I, to the active octapeptide, angiotensin II, which is potent vasoconstrictor

that degrades the antihypertensive vasodilator, bradykinin. This process results in

increased blood pressure (Brown and Vaughan, 1998). Hence, ACE inhibitors are

believed to lower blood pressure and help prevent cardiovascular disease. Many

researchers have successfully isolated ACE-inhibitory peptides from (Syngnathus

schlegeli) (Wijeskera et al., 2011); and salmon byproducts (Ahn et al., 2012). ACE

inhibitory peptides deriving from fish protein hydrolysates are considered natural

alternative bioactive peptides that are safer than synthetically produced ACE

inhibitors

Predicted values for yield and ACE inhibition were higher while DH was lower on

validation outcomes tests. These confirmed RSM results suggest that SSGH can be

used

2.5 Drying methods

Drying of protein hydrolysate is a process that allows ensuring a stable shelf-

life of final product by reducing the water content and making the transportation of

protein hydrolysate become easier due to the reduced mass and volume of the final

product (Petrova et al., 2018). Historically, various drying techniques have been used

that available to produce protein hydrolysate in powder form such as spray drying,

freeze drying, vacuum drying and oven-drying, (Badmus et al., 2019). The most

commonly used drying systems at large scale in FPH production are spray drying and

freeze drying (Petrova et al., 2018). Although, there are different drying techniques

available, several factors such as cost, energy consumption, effectiveness and impact

on quality all need to be considered when selecting the most applicable method

(Badmus et al., 2019). Spray drying is a popular method used at different productions

over years because of the high productivity (Parvathy et al., 2018). Nevertheless, for
research purposes its use is limited by the availability at a certain research laboratory,

where freeze dryers are rather available. However, several research laboratories are

provided by spray driers and a number of research works on FPH have been published

such as (De Paris et al., 2016) used spray drying conditions to evaluate the FPH

obtained from tilapia processing by-products in an animal nutrition industry,

(Morales-Medina et al., 2016) also used spray drying for the microencapsulation of

fish oil by FPH made from sardine and horse mackerel and many others.

Freeze drying is a technique which utilizes the principle of moisture

sublimation under lowered pressure. The advantages of using freeze drying as highly

applicable method used for FPH drying at laboratory scale because of its simplicity

and speed. There are many researchers reported have been used freeze drying for the

recovery of FPH, which studied different FPH purposes (Huan et al., 2017)

(Jenkelunas & Li-Chan, 2018)(Noman et al., 2018)(Samsudin et al., 2018)(Rasli &

Sarbon, 2019)(Niu et al., 2019) and many others. For example, the work of

( Elavarasan & Shamasundar, 2016) used freeze drying in the evaluation of bioactive,

clinical and structural properties of freeze-dried FPH from freshwater fish (Cirrhinus

mrigala). Thus, freeze drying has shown gentle treatment of the hydrolyzed mixture

and a good protein recovery along with low moisture content in final dried FPH

through a number of published research. However, the cost and limited production

capacity of freeze-drying become a negative role at large-scale production, where

freeze drying is mostly used to produce FPH at the highest quality (Krittalak et al.,

2018). Considering the high capital and running cost in freeze drying process since

more energy is required, alternate drying methods, which is vacuum drying are being

explored for drying of FPH.

 In comparison to the freeze drying, vacuum drying allows the drying

temperature to be reduced and a higher quality to be obtained than the conventional

air drying process at atmospheric pressure. A major advantage of vacuum drying is

less energy is needed for drying (Parikh, 2015). Parikh (2015) stated that energy

conservation results in cutting down on the economic and environmental costs. A

studied has been reported using the vacuum drying as the method to produce FPH

such as (Tanuja et al., 2014) that observed the functional and antioxidative properties

of frame meat of striped catfish (Pangasianodon hypophthalmus). Tang et al., (2013)

stated that vacuum drying also can be carried out at higher temperatures than freeze

drying. However, there are still lack of publications regarding the effect of vacuum

drying method on the FPH properties that will fill the gap in the existing knowledge.

It is crucial that the FPH is not overheated during drying process as excessive

heating results in poor functional properties due to the aggregation of the denatured

protein molecules that occurred (Lourenço da Costa et al., 2007). The reducing of

essential amino acid also can occur which subsequently reduce the nutritional quality

of protein hydrolysate as it happen due to destruction of biological and functional

qualities of protein (Lourenço da Costa et al., 2007).

 CHAPTER 3

MATERIALS AND METHODS

3.1 Materials
Fresh Shortfin Scad by-products (Decapterus Macrosoma) is purchased from a

local factory named in Kilang Keropok SEAmEQ GONG BADAK at Terengganu,

Malaysia. The fish will be transported directly to the laboratory. The cleaning will be

done on the fish frames (head and bone). Shortfin Scad by-products are minced using

a blender, packed in polyethylene bags, frozen, and stored at -20°C until further use.

Papain enzyme (from latex of Carica papaya, activity ≥ 3 U/mg) are purchased from

Sigma Aldrich (St. Louis, MO, USA). All other chemical reagents used are analytical

grade reagent.

3.2 Methods

3.2.1 Preparation of Fish Protein Hydrolysates

Shortfin Scad by-product is minced then the mixture will undergo hydrolysis

process. The hydrolysis will be carried out following the method described by

Elavarasan et al. (2014). The enzyme used for hydrolysis is papain. Conditions needed

for hydrolysis process such as enzyme to substrate ratio (E/S), pH and duration of

hydrolysis will be 0.26 %, 6.5 and 60 min, respectively. The E/S ratio is derived from

the plot of log10enzyme to substrate ratio (2.5, 5.0, 7.5 and 10 %) and degree of

hydrolysis (data not given) to achieve 5 % degree of hydrolysis (DH). The hydrolysis

reaction will be terminated by heating the mixture in a boiling water bath for 15 min.
Mixture will be filtered using Whatman filter paper grade 4 (GE Healthcare UK Ltd.,

Amersham Place Little Chalfont, Bucking Hamsphire, UK) to produce supernatant.

The supernatant obtained will be subjected to the drying experiments.

3.2.2 Drying experiments

For drying process, the hydrolysate supernatant obtained will be divided into

two equal aliquots and each aliquot will be dried by low temperature vacuum drying

and freeze drying methods. All tests will be conducted in triplicate.

3.2.2.1 Vacuum drying

The vacuum drying experiments were carried out in a low temperature vacuum

dryer (Vacuum Oven ADP-21, Yamato, Mexico). The heated wall temperatures will

be in the range 50–60 °C. A vacuum pump is attached to the drying chamber to lower

the pressure. The vacuum levels will be set at −720 mmHg. The drying time to reduce

moisture content to 35%–50% was 20 h. The dried hydrolysate will be designated as

VD-FPH. The samples will be stored in air tight container under desiccated conditions

for further analysis.

3.2.2.2 Freeze drying

For freeze drying, the hydrolysate will be poured to the drying trays and

frozen at −45 °C for 60 min. The thickness of the solution layer in the trays is less

than 10 mm. Freeze drying will be carried out in freeze dryer (Freeze dry system,

Freezone 6, LABCONCO, Kansas City, Missouri) for 24 h at a temperature of -54°C

with 0.3 mbar. The final moisture content of dried hydrolysates was less than 50%.
This hydrolysate will be designated as FD-FPH and stored in air tight containers

under desiccated conditions for further analysis.

3.3 Degree of hydrolysis (DH)

Degree of hydrolysis (DH) will be calculated as described by Hoyle & Merritt,

(1994). After hydrolysis, 20 ml of FPH will be added to 20 ml of 20% (w/v) TCA

(trichloroacetic acid) to produce 10% TCA soluble material. The mixtures will be left

to stand for 30 min to allow precipitation, followed by centrifugation (7800 × g for 15

min). The supernatant will be analyzed for protein content by using the Kjeldahl

method. The degree of hydrolysis (DH) was computed as the formula below:

10 % TCA −soluble nitrogen∈the sample

DH (%)= [ % total nitrogen∈the sample ]
× 100 %

3.4 Determination of Physicochemical properties

3.4.1 Protein content

Protein content in the sample will be estimated using Kjeldahl method

according to standard procedure given in Association of Official Analytical Chemists

(AOAC, 1995), involves digestion, distillation and titration. FPH samples (1g) will be

taken in the digestion tube, to this anhydrous potassium sulphate will be added to

increase boiling point, and copper sulphate will be added as a catalyst or to speed up

the reaction, concentrated sulphuric acid will be added as oxidizing agent. The

material will be left over night to enhance digestion and then the material will be

digested in the digestion blocks at the temperature of 420°C, this process is called
digestion. The sample will be distilled by passing steam, ammonia liberated due to

addition of alkali and trapped in boric acid. Boric acid in NH3 will be titrating against

the standardized hydrochloric acid using methyl red indicator. The percent nitrogen is

calculated using the titer value. The protein content will be obtained by multiplying

the percent of nitrogen with the factor 6.25.

(V 1−V 2 )× normality × 14.01

Nitrogen ( % )= ×100 %
w ×1000

Where,

V1 = volume of H2SO4, ml

V2 = volume of blank, ml

W = weigh of sample, g

Protein content = Nitrogen (%) × 6.25

3.4.2 Hygroscopicity

Hygroscopicity will be determined according to the methodology described by

(Correia et al., 2017). Samples (0.5 g) will be placed in a desiccator containing a

saturated solution of NaCl (RH 75.3%). The results will be expressed as the mass of

water absorbed per 100 g of sample after 7 days of storage.

3.4.3 Colour Measurement

A chromameter (Minolta Chroma Meter CR-300, Japan) will be used for

colour measurement of the hydrolysates. The colour of VD-FPH and FD-FPH

samples will be measured by colorimeter that will be calibrated with a white tile prior

to colour measurement and reported by the CIE system. L* value (lightness), a* value

(redness/greenness), and b* value (yellowness/blueness) will be recorded.

3.5. Determination of functional properties

3.5.1 Protein Solubility

Solubility of protein from VD-FPH and FD-FPH samples will be determined

following a method described by Sukkwai et al., (2011). The dried FPH will be

dissolved in distilled water at 60°C to obtain a final concentration of 2% (w/v) and the

mixture will be stirred at room temperature until the hydrolysate was completely

solubilised. The solution will be adjusted to different pHs (4, 7 and 10) with either 6

N NaOH or 6 N HCl. The volume of solution will be made up to 10 ml with distilled

water, previously adjusted to the same pH of hydrolysate solution. The solution will

be centrifuged at 8500 rcf at room temperature for 10 mins. Protein content in the

supernatant will determined by the biuret method. Three replicates of each sample

will be tested and the solubility of dried hydrolysates will be calculated using the

following expression.

amount of protein∈supernatant
Protein solubility ( % )= ×100
total amount of protein ∈sample

3.5.2 Emulsifying properties

Emulsifying capacity and emulsifying stability of VD-FPH and FD-FPH

samples will be determined following the methods of Neto et al. (2001). The

hydrolysate of 0.05 g will be dispersed with 5 mL distilled water to produce 10 mg/ml

protein dispersion. Then, the dispersion will be homogenized with 5 mL of corn oil at

speed 2 (10,000/ min) for 1 min by using homogenizer (IKA@ T18 Basic, IKA

Works Inc., USA). The emulsion will be centrifuged at 1100 rpm for 5 min. The

height of the emulsion layer and the total content in the tube can be determined. The

emulsifying capacity was calculated as follows:

 height of emulsified layer
emulsifying capacity ( % ) = × 100 %
height of the total content

Emulsifying stability will be determined by heating the emulsion at 55°C for 30 mins

before centrifuging at 2000 rpm for 5 mins. The emulsifying stability can be

calculated according to the equation below:

height of emulsified layer after heating

emulsifying stability ( % )= ×100 %
height of thetotal content before heating

3.5.3 Foaming Properties

Foaming capacity and foam stability of VD-FPH and FD-FPH samples will be

determined according to the method of Razali et al. (2015) with some modifications.

For foaming capacity, about 20 ml of sample solution (0.2 g/ml) was adjusted to pH 2,

4, 6, 8 and 10. The mixture was homogenized (Homogenizer with generator, Eurostar

power control-visc, IKA-WERKE, Germany) at a speed of 1600 rpm in order to

incorporate the air in the sample solution for 2 min at room temperature. The whipped

sample was immediately transferred into a 25 ml cylinder and the total volume was

taken after 30 s. The difference in volume will be expressed as the volume of the foam

and foaming capacity will be expressed as percentage of volume increase upon

homogenizing. The foaming capacity was calculated as follows:

Volume of foamliquid −Initial volume of liquid

Foam Capacity(%) ×100 %
Initial volume of liquid

For foam stability, the whipped sample will be allowed to stand at room temperature

for 30 min and the volume of the whipped sample then will be recorded. All

determinations will be derived from the means of three measurements. The foaming

stability will be calculated as follows:

 Volume of foam after 30 min−Initial volume of liquid
Foam stability ( % )= ×100
Initial volume of liquid

3.5.4 Water Holding Capacity (WHC)

Water holding capacity (WHC) of the VD-FPH and FD-FPH samples will be

determined using the centrifugation method outlined by Medcalf and Gilles (1965). A

suspension of 5 g protein hydrolysate (dry weight) will be added to 75 ml of distilled

water. The mixture will be agitated at 25°C for 1hr, and centrifuged at 3000 rpm for

10 mins. Free water will be removed and drained for 10 mins. The pallet left in the

centrifuge tube will be weighed. The value of water holding capacity will be

calculated as follows:

Weight of pallet
Water holding capacity ( % )= ×100
Weight of hydrolysate
3.6 Experimental Design

FISH PROTEIN HYDROLYSATE

Drying Methods

Vacuum Drying Freeze drying

Physicochemical and Functional Properties

1. Proximate analysis
2. Hygroscopicity
3. Colour
4. Protein solubility
5. Emulsifying properties
6. Foaming properties
7. Water holding capacity

Data analysis
 Components of experimental design Description
Experimental unit Fish Protein Hydrolysate from Shortfin

Scad (Decapterus Macrosoma)

Replication 3
Independent variable & Level Types of drying method

1. Vacuum drying

2. Freeze drying
Dependent variable 1. Physicochemical properties

Assignment Rule
Arrangement Rule
Total experimental units
Statistical Analysis
2. Functional properties
Completely Randomized Design (CRD)
2 treatments × 1 factor = 2 full factorial
2 treatments × 3 replications = 6
One-way ANOVA

3.7 Statistical Analysis

All experiments will be carried out in triplicate (n=3). All data will be reported

as mean ± standard deviation and analysed statistically using One-way ANOVA with

significance level at p<0.05 by using Minitab 14 software (Minitab Inc., USA).

Significant difference between means of results will be determined by using Tukey’s

multiple comparison test at α = 0.05.

CHAPTER 4

EXPECTED RESULT
 The methods of drying which are the vacuum drying and freeze drying will

generally affected the physicochemical and functional properties of fish protein

hydrolysate obtained from the fish frames of Shortfin Scad (Decapterus macrosoma).

The effects will be assessed in terms of protein content, hygroscopicity, colour,

protein solubility, water holding capacity (WHC), emulsifying and foaming

properties. It is expected that the freeze-dried hydrolysate (FD-FPH) may very

hygroscopic and high WHC. This might be due to higher concentration of polar

groups such as NH2 and COOH which can absorb higher amount of water. Moreover,

it is assumed that FD-FPH give higher protein content compared to vacuum-dried

hydrolysate (VD-FPH) probably due to low temperature drying which reduced protein

denaturation. Drying of protein using high temperature induces few stresses that can

denature protein by modifying protein structures. Besides, the VD-FPH may have

higher browning intensity colour compared to FD-FPH as it uses higher temperature.

VD-FPH may exhibit lowest solubility as it lead to more aggregation and the tight

structure of the sample, it is hard to combine with water. VD-FPH may exhibit lowest

emulsifying capacity due to lower solubility and hygroscopicity meanwhile FD-FPH

may exhibit highest emulsifying stability due to partial unfolding of protein molecules

which improved flexibility to prevent coalescence. FD-FPH may exhibit lowest

foaming properties which could be mainly attributed by its solubility which influence

by drying process.
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