Comparison of Surimi and Solubilized Surimi for Kamaboko Production from Farmed Chinook Salmon

COMPARISON OF SURIMI AND SOLUBILIZED SURIMI FOR KAMABOKO PRODUCTION FROM FARMED CHINOOK SALMON

JILL MARIE RICHARDSON

B.Sc, The University of Alberta, 1993

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF

THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

THE FACULTY OF GRADUATE STUDIES

(Department of Food Science)

We accept this thesis as conforming to the required standard

THE UNIVERSITY OF BRITISH COLUMBIA

April 1999

©Jill Marie Richardson, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.

Department of Food Science.

The University of British Columbia Vancouver, Canada

Date vTun^ /b. /Qtytf^B) -; Abstract

The thesis hypothesis of this research was that farmed chinook salmon could be made

into better quality functional kamaboko when made from solubilized frozen surimi than when made from conventional frozen surimi.

An 84 day storage study compared kamaboko gel quality made fromsolubilize d and traditional surimi. Fresh farmed chinook salmon (Oncorhynchus tshawytscha) was used to make both solubilized surimi and surimi (control). Solubilized treatments contained varying concentrations of calcium chloride, sodium chloride and water. The Random

Centroid Optimization (RCO) program randomly generated concentration values of additives. All surimi treatments (solubilized and control) contained 8.3% cryoprotectants. Treatments were taken from storage on days 3, 7, 14, 28, 56 and 84 from an -8°C freezer and made into kamaboko. Solubilized treatments were diluted after frozen storage and then centrifuged to constant moisture content.

All kamaboko gels had respective moisture, protein, crude fat and ash contents of 74.5 +

5.5%, 13.6 ± 1.7%, 4.4 + 2.4%, and 5.6 ± 3.1%. Salts added to solubilized treatments influenced ash content. No proximate analysis trends were observed between treatments during the storage study. Variation in protein and water concentrations within the range of this study did not appear to affect overall kamaboko quality.

ii Treatments had similar Hunter "L", "a", "b" values and values remained relatively

consistent over the storage study. The TA-TXT2 Texture Analyzer was used to conduct

punch tests. A 6 point fold test scale was employed to evaluate kamaboko elasticity.

ANOVA suggested that gel strengths and fold test scores did not change over time, but

treatments were significantly different (p < 0.05).

All factors (sodium chloride, calcium chloride, and dilution) proved significant (P < 0.05)

in contributing to the treatment effect using multiple regression. However, sodium

chloride and calcium chloride had a larger impact than the dilution factor on gel strength

and fold test scores. Although, treatments were not significantly different on different

days of the storage study, treatment 7 was clearly the best treatment when compared to the control and other treatment gel strengths and fold test scores.

Each kamaboko treatment (before cooking) for each storage day was examined by SDS-

PAGE. Variations in gel strength were consistent with degradation in the myosin heavy chains of the SDS-PAGE Phast gels. Lower gel strengths were observed in treatments that had more degradation of the myosin heavy chain.

iii Table of Contents

Abstract ii

Table of Contents iv

List of Tables viii

List of Figures ix

Acknowledgments x

Chapter 1: Introduction 1

1.1 Definition of surimi 1

1.2 Historical background of surimi 2

1.3 Surimi products 4

1.4 Species used for surimi production 5

1.5 The modern surimi process 6

1.5.1 Sorting and cleaning 7

1.5.2 Filleting 7

1.5.3 Separation of meat 8

1.5.4 Leaching 8

1.5.5 Intermediate dewatering 9

1.5.6 Refiner 10

1.5.7 Final dewatering and the importance of pH 11

1.5.8 Blending 1 11

1.6 Cryoprotectants 12

1.7 Solubilization 13

iv 1.8 Kamaboko making 15

1.9 Surimi quality 15

1.10 Rheological properties of surimi gels 16

1.10.1 Effect offish freshness, rigor condition, seasonality 16

1.10.2 Effect of chopping temperatures 17

1.10.3 Effect of moisture content 17

1.10.4 Effect of functional additives 18

1.10.5 Effect of storage temperature 18

1.10.6 Effect of low temperature setting 19

1.10.7 Effect of refrigerated storage on gels prior to evaluation 19

1.10. Effect of test temperatures 19

1.11 Random Centroid Optimization 20

1.12 Compositional aspects of surimi processing 20

1.12.1 Lipids in fish 20

1.12.2 Proteins 21

1.12.2.a Sarcoplasmic proteins 21

1.12.2.b Myofibrillar proteins 22

1.12.2.C Connective tissue 23

1.13 Thesis hypothesis 23

Chapter 2: Materials and Methodology 42

2.1 Optimization of surimi treatments using RCO 42

2.2 Surimi production 42

V 2.2.1 Fish source 42

2.2.2 Filleting 43

2.2.3 Grinding 43

2.2.4 Leaching 43

2.2.5 Dewatering 45

2.3 Surimi solubilization 45

2.4 Kamaboko making 47

2.5 Compositional evaluation of kamaboko 49

2.5.1 Moisture determination (oven method) 5 0

2.5.1.2 Moisture determination (microwave method) 50

2.5.2 Crude fat determination 50

2.5.3 Protein determination 51

2.5.4 Ash determination 51

2.6 Sample preparation 51

2.7 Functional evaluation of kamaboko 52

2.7.1 Color 52

2.7.2 Gel strength 52

2.7.3 Fold test , 53

2.7.4 SDS-PAGE electrophoresis 53

2.8 Statistical analysis 55

Chapter 3: Results and Discussion 58

3.1 Compositional evaluation results of kamaboko 58

vi 3.1.1 Crude protein content 5 8

3.1.2 Moisture content 59

3.1.3 Crude fat content 61

3.1.4 Ash content 62

3.1.5 pH 63

3.2 Functional evaluation results of kamaboko 64

3.2.1 Color 64

3.2.2 Gel strength 65

3.2.3 Fold test scores 66

3.2.4 SDS-PAGE electrophoresis 67

Chapter 4: Conclusion and future recommendations

4.1 Conclusion and future recommendations 79

Bibliography 81

Vll List of Tables

Table 1. Nine solubilized treatments and the control 56

Table 2. Fold test scale 57

Table 3. Crude protein fraction in kamaboko gel (wet basis, n =3) 68

Table 4. Moisture fraction in kamaboko gels (wet basis, n = 3) 69

Table 5. Fat and ash fractions in day 84 kamaboko gels (wet basis, n=3) 70

Table 6. Fat and ash fractions in treatment 7 kamaboko gels (wet basis, n =3) 71

Table 7. PH values of solubilized kamaboko throughout the storage study

(n =18 (3 X6 storage days)) 72

Table 8. Hunter "L", "a", "b" values of kamaboko gels (n = 5) 73

Table 9. Standard deviations of Hunter "L", "a", "b" values in kamaboko

gels (n = 5) 74

Table 10. Gel strengths (N X mm) of kamaboko gels (n = 5) 75

Table 11. Multiple R values for gel strengths and treatment factors 76

Table 12. Fold test scores df kamaboko gels (n=5) 77

viii List of Figures

Figure 1. Effect of the discovery of cryoprotectants and tighter catch controls on the Japanese fishing industry during 1965-1985 25

Figure 2. Comparison of traditional and modern surimi production 26

Figure 3. Examples of surimi based products 27

Figure 4. Flow diagram of the surimi process and subsequent yield 28

Figure 5. Typical design of a surimi meat separator 29

Figure 6. Effect of agitation and water-protein contact time during leaching 30

Figure 7. Effect of ionic strength of wash water during leaching 31

Figure 8. Effect of meat: water ratio and washing cycles on the removal of 32 soluble proteins during leaching

Figure 9. Effect of pH on the water holding capacity of minced meat 33

Figure 10. Effect of wash water temperature on the efficiency of dewatering 34

Figure 11. Effect of moisture content (%) on gel strength and elasticity 35

Figure 12. Effect of functional additives on kamaboko gel strength

And elasticity 36

Figure 13. Effect of frozen storage temperature on gel strength - 37

Figure 14. Effect of refrigerated storage prior to gel strength and elasticity

evaluations 38

Figure 15. Effect of test temperature on gel strength 39

Figure 16. Schematic of myosin 40

Figure 17. Heat gelation of surimi 41

Figure 18. SDS-PAGE results 78 Acknowledgment

I would like to express my immense respect and appreciation to a wonderful mentor, my supervisor, Dr. Durance. Throughout my studies at UBC, Dr.

Durance provided me with outstanding advice, support, and guidance. He also tolerated my bad jokes, my stubborn ways and he supported my non-academic pursuits (sports). For these reasons, and countless others I would like to thank him.

I would also like to thank my committee members, the Department of Food

Science, friends and family for their exceptional support.

Finally, I would like to thank a dear friend, Peter. For without his love, support and belief in me, I would never have entered the realm of graduate school. Chapter 1. Introduction

Chapter 1

Introduction

1.1 Definition of surimi

Surimi is a Japanese term meaning "minced meat" (Sonu, 1986). Although surimi

can be made from both land and sea based animals, surimi is almost always

derived from fish muscle (Lanier, 1997). In simple terms, surimi is deboned,

washed, concentrated fish muscle (Lanier, 1997). It is necessary to distinguish

"minced fish", "raw surimi" and "frozen surimi". When fish flesh is separated

from bones and skin, it is called "minced fish", the starting material for surimi

production. Once minced fish is washed to remove fat and water-soluble

components, it becomes "raw surimi". "Raw surimi" is a wet concentrate of the

myofibrillar proteins of fish and possesses enhanced gel-forming, water-holding,

fat-binding, and other functional properties relative to "minced fish". However,

the myofibrillar proteins will lose their functional properties once they are frozen

(Okada, 1992). The primary properties of surimi are its long term stability in

frozen storage with cryoprotectants and its ability to form thermo-irreversible gels

(Sonu, 1986; Lanier, 1986). Because surimi can be converted to an elastic and

chewy gel, it is used as an intermediate raw material in a variety of fabricated

seafoods and other traditional products (Ma et al, 1996; Lee, 1984). For the

purpose of this thesis, surimi refers to "raw surimi" made from farmed chinook

salmon that has been mixed with cryoprotectants and frozen.

1 Chapter 1. Introduction

1.2 Historical background of surimi

Commercial surimi production in Canada and the US is about 40 years old (Sonu,

1986; Okada, 1992; Lee, 1984). However, the Japanese have been making surimi

for approximately 1500 years (Sonu, 1986). Traditionally, surimi was made from

fresh fish and processed into kamaboko. Kamaboko is a generic term that refers

to a range of traditional Japanese surimi based products (Okada, 1992). For most

of surimi's long history, surimi production occurred daily and depended on the

availability of fresh fish and manual labor. In the early 1900's production

increased with the arrival of the western trawl method of fishing (Okada, 1992).

However, surimi production remained limited due to its poor shelf life. In 1959,

Nishiya's research group in Hokkaido Japan discovered sugars effectively

prolong and stabilize protein functionality in surimi during frozen storage

(Mackie, 1993; Okada, 1992; Lee, 1984). Initially, surimi contained 5% glucose.

Later sucrose and sorbitol replaced glucose at the 8-10% level to improve'stability

and to reduce Maillard browning products (Okada, 1992). This discovery enabled

industry to stockpile surimi. Production efforts moved from onshore to

mechanized offshore production and output dramatically increased (Lee, 1984).

Many more advancements in surimi technology occurred during the early 1970's

(Okada, 1992). Collaborative efforts from the Japanese surimi industry and

various research institutes laid the basis for modern surimi production. These

efforts resulted in the establishment of physical and chemical principles for surimi

production and preservation.

2 Chapter 1. Introduction

More specifically, scientists discovered the following important facts:

i) Solubilization of myofibrillar proteins and crosslinking of the protein gel network is key in producing high quality surimi

ii) Gel forming ability of surimi based products is species dependent and is affected by the freshness, biological state of fish, and frozen storage treatment

iii) Surimi based products are affected by the level and type of salt, pH, and heating schedule

iv) Starches have a strong gel strengthening ability and participate in a dispersed phase, whereas protein functions in a continuous phase (Okada, 1992)

Several processing factors influence the storage stability of surimi based products.

For example, moisture content, packaging material, cooking procedures and the

use of preservatives all have impact on storage stability (Okada, 1992). Figure 1

depicts the dramatic increase in surimi production during the years following

Nishiya's discovery (Sonu, 1986). The rise in surimi production was due to the

rapid growth of the Japanese economy during these times and due to the abundant

supply of frozen surimi. A decline in surimi production began in 1974 due to an

increase in the cost of raw materials and a change in consumer perceptions. From

1974 to the present, surimi prices have almost doubled due to the water pollution

act of 1970, the oil crisis of 1974, and an increase in catch regulations by nations

controlling the pollock resource. Consumers perceptions changed as they became

concerned with food additives such as hydrogen peroxide, and nitrofuran

compounds used in surimi production (Okada, 1992).

3 Chapter 1. Introduction

As the production of surimi has evolved, so has the machinery used to make it.

Equipment advancements include: fishwashers, descalers, large capacity meat

separators, washing tanks, rotary sieves, refiners, screw presses and decanters.

Fishwashers, descalers and meat separators facilitate the cleaning and isolation of

white flesh. Rotary sieves have increased leaching efficiency, while refiners have

replaced batch type dewatering processes. Decanter centrifuges recover fine

muscle particles suspended in the effluent water (Okada, 1992; Lanier et al.,

1992).

1.3 Surimi products

Surimi production peaked during the late 1960's to mid 1970's (Okada, 1992).

Production has since declined due to an increased cost in raw products due to

stricter catch controls and tighter effluent disposal regulations (Okada, 1992;

Marris, 1991). As a result, efforts to produce new products, minimize effluent,

utilize other species, attract new consumers, and find alternate ways to preserve

surimi based products are being made. Examples of surimi based product

development include: crab leg analogs, binding agents, high fiber drinks, meat

flavored analogs, and dehydrated surimi (Morris, 1988; Okada, 1992, Andres,

1987). Figure 2 depicts some of the advancements in surimi production (Okada,

1992)

4 Chapter 1. Introduction

1.4 Species used for surimi production

Presently, the most popular species used for surimi production is Alaska pollock

(Theragra Chalcogramma) (Whittle and Hardy, 1992; Okada, 1992; Mackie,

1993). The volume, availability, subtle flavor, odor characteristics and low cost

of this species makes it optimal for surimi production (Roussel and Cheftel,

1988). However, the recent decline in the Alaska pollock fishery has prompted

studies on the suitability of various other fish species for surimi production (Dora

and Hiremath, 1991; Okada, 1992). Presently, industry is actively seeking more

profitable under-utilized species. Whittle and Hardy (1992) have defined under•

utilized species as fish that are:

i) Available, but difficult to catch, process, or market

ii) Caught in quantity or as a by-catch and used for low value industrial products, but which could be upgraded for human consumption

iii) Waste of edible flesh generated because of inefficient handling, processing or distribution

iv) Simple, but technically unjustified loss of quality and value in the handling and sale of fishery products

According to the FAO, (Whittle and Hardy, 1992) future demands for fish will

come from developing countries, particularly Asia. Japan already uses under•

utilized species (mackerel and sardine surimi) for its school lunch program (Dora

and Hiremath, 1991). By the year 2000, demand will surpass supplies from

conventional fish sources. Figure 3 lists a variety of traditional and surimi based

products (Sonu, 1986). Therefore, it seems logical economically to use under•

utilized fish species as additional resources (Whittle and Hardy, 1992).

5 Chapter 1. Introduction

According to the above definition of under-utilized, farmed salmon may be

classified as under-utilized due to the loss of value incurred when salmon steak

sides are processed into canned salmon. Unfortunately, under-utilized species are

not always easy to process. Examples of processing difficulties include;

migratory patterns of species, seasonality of species, fluctuations in yield,

variability in composition, dark flesh, strong flavors, oxidation and rancidity in

frozen storage, and consumer prejudices (Whittle and Hardy, 1992). However,

there are some methods to overcome these hurdles. For instance, the following

approaches are useful in limiting lipid oxidation. Sodium bicarbonate removes fat

from high fat fish during washing. Super-decanters separate fish oil from meat.

Pressure showers mechanically remove dark muscle, skin and subcutaneous fat

(Putro, 1989). Examples of under-utilized species include; late run pacific chum

salmon, whiting, krill, hoki, hake, herring, and mackerel (Whittle and Hardy,

1992; Marris, 1991). As surimi production grows, so does the list of species used.

No longer are firm fleshed white fish the only raw material used for surimi.

Perhaps, fish protein technology and not the inherent fish characteristics will

eventually determine the usefulness of a species for surimi production (Holmes et

al., 1992).

1.5 The modern surimi process

Regardless of the species, modern production of surimi employs one of two

methods; onshore or offshore processing. Fresh fish makes the best quality

surimi. Therefore factory trawlers produce the highest quality surimi followed by

6 Chapter 1. Introduction

factory mother ships and land based factories (Toyoda et al., 1992). Factory

trawlers process surimi within 24 hours post mortem.

1.5.1 Sorting and cleaning

Target species are separated from other species in the catch before fish are

processed. Typically, this step is manual. Next, fish are sorted by size to increase

final yield. The body temperature of fish should be kept just above the freezing

point, stored in crushed ice or in refrigerated sea water prior to processing

(Ohshima et al., 1993; Lee, 1986).

1.5.2 Filleting

Once sorted, fish are descaled or skinned and filleted. If skins remain on the fish,

they are descaled to prevent clogging of the deboning machine (Toyoda et al.,,

1992). It is important to process fish after rigor, otherwise extreme muscle

contractions result causing unacceptable textural qualities (Mackie, 1993).

Filleting involves heading, deboning, and evisceration. This may be manual or

automatic. Higher grade surimi results from proper viscera removal. Viscera

contains proteolytic enzymes and spoilage microorganisms that are detrimental to

surimi quality (Ohshima et al., 1993). Enroute to the separator via a conveyor

belt, fillets are spray washed to removed excess scales, viscera and manually

inspected for quality. Yield varies depending on season, presence and or absence

of roe and the original size of the fish (Lee, 1986). Figure 4 illustrates a flow

diagram of the surimi process and the processes' typical yield (Lee, 1985).

7 Chapter 1. Introduction

1.5.3 Separation of meat

A meat separator separates fish from bone and skin. Belt drums physically

remove unwanted fish parts while mincing the fish. The relatively soft fish flesh

is pressed through a screen to the belt drums' interior while the bone, skin and

viscera remain on the drums' exterior. The diameter of the screen affects output,

quality and the effectiveness of dewatering and leaching (Ohshima et al, 1993;

Toyoda et al., 1992). Medium size screen perforations are preferable because

they maintain decent yield and quality. Figure 5 depicts a typical design of a meat

separator (Lanier, 1992).

1.5.4 Leaching

After mincing, fresh water is used to leach the fish. Leaching removes

undesirable blood pigments, digestive enzymes, inorganic salts, sarcoplasmic

proteins, and trimethylamine oxides while concentrating the protein and

improving gel strength (Ohshima et al., 1993; Babbit, 1986; Lee, 1984). The

effectiveness of leaching is dependent on water-protein contact time, hardness of

the water, meatwater ratio, and effects of pH (Fig. 6, 7, 8, 9) (Ohshima et al.,

1993; Toyoda et al, 1992). Recent developments in the leaching process utilize

vacuums to accelerate lipid flotation and high-lipid content dark flesh removal

(Nishioka, 1984). Protein extraction appears to be maximized when agitated for

nine minutes (Toyoda et al., 1992). However, dewatering is more difficult when

protein residence time in water is extended. Therefore, protein residence time in

water should be kept to a minimum. Fifteen to twenty minutes for the entire

8 Chapter 1. Introduction

leaching process is industry standard (Toyoda et al., 1992). Wash water should be

near or below the temperature of the water normally inhabited by the fish to

maintain protein functionality and to prevent microbial proliferation (Hashimoto

et al., 1982). Generally, functional properties of protein decreases with increased

leaching temperatures. However, extraction of undesired sarcoplasmic proteins

increase with increased leaching temperatures. The first two washes use fresh

water, while the last wash contains 0.3% salt solution to facilitate dewatering

through osmotic pressure (Lee, 1986). Three washes of a 3.T water to meat ratio

is considered appropriate. However, due to effluent concerns lower water: to

meat ratios have show to be effective and economical (T. Lin, 1997).

1.5.5 Intermediate dewatering

Intermediate dewatering takes place between each washing cycle to ease the final

dewatering cycle. About 8% of the starting mince is lost via dewatering screens.

However, centrifugation methods recover most mince (Toyoda et al., 1992).

Warm water facilitates water removal but may be deleterious to gel forming

abilities of the protein. High ionic strength water also facilitates dewatering.

Low ionic strength wash water results in swollen high moisture fish. Therefore,

producers often add a small amount of salt in the final washing cycle. High salt

levels solubilize the protein and cause premature setting. Calcium and

magnesium in hard water are believed to cause color deterioration in frozen

surimi. Iron and manganese in hard water are believed to cause textural

deterioration in frozen surimi (Lee, 1986). Researchers believe calcium increases

9 Chapter 1. Introduction

crosslinking between myosin heavy chains leading to syneresis and the

development of hard rubbery surimi (Lee, 1986).

1.5.6 Refiner

Refiners separate the tissue, skin, and scales by using selective screen sizes from

partially leached and dewatered surimi prior to the final dewatering step. After

two washes, moderately wet meat (87-90%) is considered appropriate for refining

(Lee, 1986). Refiners typically consist of a cylindrical screen and a screw shaped

rotor. The washed mince is fed into the machine, and the soft meat is selectively

forced through the perforations under the compressive force generated by the

rotor. White, soft meat emerges from the front part of the refiner. Towards the

rear part of the refiner, the meat becomes increasingly harder and of poorer

quality. Only hard materials such as connective tissues, skin, bones, and scales,

which cannot pass through the perforations, are discharged through the end of the

refiner as reject. Strainers were the precursors to refiners. Strainers were used

after the final wash, and resulted in elevated temperatures due to lower moisture

contents. Lower moisture contents led to friction and subsequent protein

denaturation. Thus, refiners have alleviated this problem since tissue, skin, and

scales are separated when the minced fish flesh has a moderately wet meat

content, thus minimizing friction and subsequent premature protein denaturation

(Toyoda et al., 1992).

10 Chapter 1. Introduction

1.5.7 Final dewatering and the importance of pH

A screw press completes the final dewatering step. The mince is pressed through

a screen to a desired moisture content (usually 74-84%). The isoelectric point of

fish protein is near a pH of 5.5 (Sonu, 1986). Normally the pH is just above the

isoelectric point to facilitate dewatering. Figure 10 illustrates the effect of wash

water temperature on the efficiency of dewatering (Toyoda et al., 1992). A low

pH will destabilize the protein and cause a reduction in the proteins' gelling

abilities. High pH values leads to dewatering difficulties due to the increase of

net negative charges on the proteins. Figure 9 illustrates dewatering difficulties

encountered at various mince and wash water pH (Sonu, 1986). Therefore, the

leach water maintains a pH of 7 to maximize water-holding capacity (Toyoda et

al., 1992; Lee, 1986).

1.5.8 Blending

Blending is the uniform incorporation of cryoprotectants into surimi to minimize

freeze denaturation of fish proteins during frozen storage (Lee, 1986). Generally,

cryoprotectants consist of 4% sucrose, 4-5% sorbitol and 0.2-0.3%

polyphosphates (Ohshima et al., 1993). Silent cutters and continuos blenders mix

cryoprotectants with dewatered surimi. A temperature increase often results from

increased mixing. Therefore, blending should be quick and efficient to prevent

loss of protein functionality (Lee, 1986). Chopping occurs at 15 second intervals

to avoid excessive temperature increases (Lee, 1986). Next, surimi is frozen

11 Chapter 1. Introduction

quickly in a contact freezer prior to storage in a controlled freezer (Ohshima et al,

1993).

1.6 Cryoprotectants

Fish protein is especially susceptible to protein denaturation unless mixed

immediately with cryoprotectants before freezing. Without cryoprotectants,

myofibrillar proteins in surimi will denature and aggregate during extended frozen

storage. Numerous compounds have cryoprotective effects such as amino acids

and carboxylic acids. The surimi industry tends to use sucrose and or sorbitol at

the 8% level with 0.2-0.3% polyphosphates to prolong frozen storage

(MacDonald and Lanier, 1991; Ohshima et al., 1993). These cryoprotectants are

readily available and economical. There are several theories to explain the

cryoprotective effects of sugar. Cryoprotectants, particularly low molecular

weight sugars may preferentially hydrate protein and decrease the amount of

water removed from the protein. However, some sugars that are effective

cryoprotectants do not meet stearic hindrance requirements to allow such

preferential hydration (MacDonald and Lanier, 1991). This preferential hydration

involves sugar molecules that are small enough to bind to the surface of the

protein in effect, replacing water molecules on the surface of the protein. The

glass transition concept has been postulated to explain the cryoprotective nature

of high molecular weight carbohydrates. Glass is an amorphous solid that has a

liquid like structure. With a drop in temperature a solutions' viscosity will

increase and the molecular movement will decrease. Ice crystallization

12 r 1. Introduction

practically ceases at the glass transition point. Proteins and other reactive

components trapped in a glass matrix are very stable. However, glass transition

behavior is less predictable in complicated protein systems (Ohshima et al., 1993).

The exact mechanism of cryoprotectants is unknown and more research is needed

to fully explain the action of cryoprotectants on fish proteins in frozen muscle

(Mackie, 1993).

Moisture also plays an important role in freeze thaw stability. The higher the

water activity, the more susceptible surimi is to freeze destabilization. One

approach to overcoming freeze thaw instability without reducing the water

activity is by using cryoprotective ingredients (Lee, 1986).

1.7 Solubilization

Saito et al. (1996) proposed a novel method called solubilization for prolonging

frozen storage of surimi. Solubilization dilutes surimi for frozen storage in an

attempt to minimize protein-protein and protein-fat interactions that may cause

protein denaturation. Solubilized samples consist of minced fish, cryoprotectants,

water, calcium chloride, and sodium chloride.

Cryoprotectants at the 8.30% level are added to the solubilized treatments to

retard protein denaturation. Water is added to the minced fish to physically

separate myofibrillar proteins.

13 Chapter 1. Introduction

Calcium chloride is believed to form salt linkages between negatively charged

sites on two adjacent proteins. Through such a mechanism, calcium may

contribute to gel strengthening of surimi gels, but does not cause premature

gelation of surimi. Furthermore, the unique ability of surimi to gel at low

temperatures called the "setting" reaction is mainly due to the formation of

covalent bonds. These bonds form between neighboring amino acids of

glutamine and lysine in the presence of transglutaminase. The addition of

calcium to improve surimi gel strength may be due more to crosslinking of

transglutaminase than forming ionic linkages between proteins (Lanier, 1997).

Sodium chloride is required for proper gelation of surimi. Salt ions individually

hydrated with water bind oppositely charged groups exposed on the protein

surface. Intermolecular ion linkages among myofibrillar proteins are ruptured and

the proteins are dissolved in water because of their increased affinity for water.

Salt addition must undergo sufficient grinding to enhance protein solubilization

(Lanier, 1997). However, premature addition of salt will result in premature

setting of surimi. Therefore, it is likely that salt in solubilized treatments has a gel

strengthening effect rather than causing premature surimi gelation based on

results from other researchers (Saito et al., 1996).

Optimum levels of dilution, calcium chloride, and sodium chloride may exist for

a given surimi process and a given species based on the varying treatments results

obtained from Saito et al. (1996). Saito and coworkers solubilized surimi for

14 Chapter 1. Introduction

frozen storage and then reconstituted the surimi upon thawing into kamaboko

gels. They were able to produce a surimi gel that was better than the control.

1.8 Kamaboko making

Kamaboko is a traditional Japanese surimi-based product made from surimi. For

gelation, it is imperative that myofibrillar proteins are in the native state for the

first stage of kamaboko production (Mackie, 1993). Salt (usually 3%) chopped

into frozen surimi induces dissociation of the actomyosin, allowing a sol-gel to

form (Mackie, 1993). Functional additives such as whey protein and protease

inhibitors assist with maximizing kamaboko gel strength (Lee et al., 1992; Kim

and Park, 1997). Once blended, kamaboko is extruded into lubricated casings

and sealed. Next, kamaboko cooks for 20 minutes at 90°C in a water bath. Some

believe gelation of washed mince occurs from the heat-induced dissociation of the

actin-myosin complex followed by the formation of covalent linkages between

myosin chains (An et al., 1996; Saeki, 1996). The heat process varies in time and

temperature based on the species being used. After cooking, gels are immersed in

an ice bath, and then stored in a cold room for later evaluation of color, gel

strength, fold test, fat, moisture, protein, ash and SDS-PAGE electrophoresis.

1.9 Surimi quality

Surimi quality is often classified into 2 major categories: functional and

compositional. Compositional quality can be defined as chemical constituents

measured as mass or volume percentage of a unit quantity of surimi. The surimi

15 Chapter 1. Introduction

constituents of interest to food processors are protein, moisture, fat and visual

contaminants (Anon, 1991). Functional properties can be defined as the effects an

ingredient has on either the organoleptic properties of a food (flavor, odor,

texture, appearance) or the processing properties of a food, (resistance to tear or

breakage, etc). This definition implies functional properties are affected by the

quantity of ingredient added to the food and by the manufacturing process. All

measurements of functionality should be evaluated on a cooked product model, i.e

kamaboko (Anon, 1991). Gelling potential and color are the two most important

quality attributes of surimi (Saeki, 1996; Hsu et al, 1997). Standard methods for

measuring and specifying these properties of surimi have been proposed by the

surimi committee of the National Fisheries Institute (Anon, 1991) out of a need to

standardize industry practices.

1.10 Rheological properties of surimi gels

1.10.1 Effect of fish freshness, rigor condition, seasonality

There are several factors that affect the quality of surimi such as fish freshness,

size and seasonality. Generally freshness decreases with storage time.

Consequently, surimi produces its' maximum quality when it is processed shortly

after it is caught. The earliest fish can be processed is 5 hrs after catch, when the

fish has passed the rigor state. Fish in rigor state is difficult to process because it

is too stiff for machine handling. As a rule, fish maintains surimi-making quality

for 1-2 days after being caught (Lee, 1986). Fish caught during a feeding period

produce higher grade surimi (Lee, 1986).

16 Chapter 1. Introduction

1.10.2 Effect of chopping temperatures

Frozen surimi blocks are either broken into small pieces or partially thawed prior

to chopping. Research has shown that chopping surimi close to 0°C yields the

maximum quality of gels. Since chopping at 0°C is not always feasible, chopping

at 5°C still results in good gels (Kim and Park, 1997).

1.10.3 Effect of moisture content

Moisture content contributes to the economics of surimi and it contributes to

texture. Generally as moisture content (wet basis) increases from 75-85.5%, shear

' stress and strain decrease. A sample difference of 1% is acceptable for measuring

gel strengths using the puncture test. Figure 11 depicts the effect of moisture

content on gel strength and elasticity of high and low grade Pacific whiting and

Alaska pollock surimi. High grade surimi was prepared with sugar, sorbitol, and

sodium tripolyphosphate. In addition, high grade Whiting was prepared with

beeef plasma protein as an enzyme inhibitor. Low grade surimi was prepared by

thawing (48 hr at 25°C) and refreezing the high grade surimi (Reppond and

Babbit, 1997). Results from Yoon et al., (1997) indicate shear strain is

influenced less by moisture content than shear stress. Similarily, other

researchers have found water has a significant effect on shear stress but no effect

on shear strain of pollock surimi (Lanier et al., 1985; Hamann and MacDonald,

1992). Furthermore, at constant temperatures, shear stress values are proportional

to the concentration of crosslinked polymers and shear strain values are

proportional to the mean molecular weight of protein chains adjoining crosslinks

17 Chapter 1. Introduction

(Hamann and MacDonald, 1992). Hamann and MacDonald (1992) reported that

higher strain values were associated with lower free moisture in surimi gels

depending on species. These results are suppoerted by the trends depicted in

Figure 11 (Yoon et al., 1997).

1.10.4 Effect of functional additives

There are numerous effective and economic additives used by the surimi industry

to increase gel strength and elasticity. Least cost linear programming minimizes

ingredient cost (Hsu et al., 1997). However, not all quality relationships are linear

in nature. Figure 12 illustrates typical functional additives used in surimi

production (Park, 1994).

1.10.5 Effect of storage temperature

Time and temperature of surimi in frozen storage affects gel quality. Figure 13

illustrates the effect of cold storage temperature on the ashiness of Alaska pollock

surimi (Sonu, 1986). Ashiness of surimi made from fresh fish does not change

significantly when held below -20°C. However frozen temperatures above -10°C

causes the ashiness ability to gradually decrease and the surimi becomes useless

after 3 months. "Ashi" is defined as the combined effect of "springiness" and

"cohesiveness" and is evaluated by at least a 3 person panel of experts. A 10-

point grade system is used to evaluate the ashiness of 3 mm thick slices of

kamaboko. The 10-point grade system is as follows: 1 = crumbly, 2 = extremely

18 Chapter 1. Introduction

weak, 3 = very weak, 4 = weak, 5 = somewhat weak, 6 = average, 7 = somewhat

strong, 8 = strong, 9 = very strong, and 10 = eytremely strong (Sonu, 1986).

1.10.6 Effect of low temperature setting

Low temperature setting prior to cooking affects stress values. The optimum

setting temperature often corresponds to the water temperature of fishing grounds

that the fish are caught in (Kim and Park, 1997).

1.10.7 Effect of refrigerated storage on gels prior to evaluation

During 6 days of refrigerated storage, both shear stress and shear strain values

will increase. This is likely due to the increase of hydrogen bonding at lower

temperatures (Kim and Park, 1997). Figure 14 illustrates an increase in shear

strength and shear strain of Alaska pollock and Pacific whiting during 6 days of

refrigerated storage (Park, 1997). Therefore, for reproducibility, it is important to

measure gel strength at a constant time after forming kamaboko.

1.10.8 Effect of test temperatures

Gels exhibit maximum shear stress when tested at room temperature. On the

contrary, maximum shear strain values are observed at higher temperatures (50-

60°C) (Kim and Park, 1997; Howe et al., 1991). Figure 15 illustrates the effect of

test temperature on the shear strain and stress of Alaska pollock and Pacific

whiting kamaboko with various salt concentrations (Park, 1995).

19 Chapter 1. Introduction

1.11 Random Centroid Optimization

Random Centroid Optimization (RCO) is a technique that combines random

search and centroid search (Dou et al., 1993). Advantages of this optimization

method include; simplicity in theory, high search efficiency, no need for boundary

constraints resulting in a search stall, less chance of homing in on a local optima

rather than a global optimum, and the possibility of finding an unexpected

combination of conditions that may revolutionize conventional routines. RCO

also allows one to optimize a product of a poorly understood subject with a

minimum number of experiments. It is especially useful for optimizing non-linear

relationships with interactions between factors, such as many quality functions

(Dou et al., 1993; Nakai, 1990). Research done by Nakai et al, (1998) has

shown that RCO can be applied to find the global optimum of a multimodal

function.

1.12 Compositional aspects of surimi processing

1.12.1 Lipids in fish

Most lipids in fish are found in the head, liver, dark muscle and beneath the skin.

Therefore, if fish are properly gutted, skinned and cleaned most fat is removed.

However, there is some lipid in fish muscle known as phospholipid, which is

sensitive to oxidation. Phospholipid is contained in dark muscle fibers. Therefore

removal of dark muscle prior to leaching helps eliminate this unstable lipid

fraction. Phospholipids incorporated into refined fish proteins are especially

unstable in the presence of pro-oxidants (Mackie, 1993).

20 Chapter 1. Introduction

1.12.2 Proteins

Fish muscle proteins are grouped based on their solubility's in salt solution and

water. These groups are sarcoplasmic, myofibrillar, and connective tissue

proteins (Mackie, 1993).

1.12.2.a Sarcoplasmic proteins

Twenty to twenty five percent of total protein comes from proteins that are

soluble in water and dilute salt solutions (Haard et al, 1994). They include

myoglobin, enzymes and other albumins. These proteins may interfere with

myosin crosslinking during gel matrix formation because they do not form gels

and have low water holding capacities (Sikorski and Kodakowska, 1994).

Therefore, sarcoplasmic proteins are undesirable components in surimi.

Sarcoplasmic proteins such as hemoglobin and myoglobin are water soluble.

However, these proteins are easily oxidized by air such that the whole protein or

color imparting heme portion can attach to the water insoluble proteins and

survive the leaching process. This causes discoloration of the surimi and

introduces ferric iron which is a known catalyst of lipid oxidation (Lanier, 1997).

Therefore, the removal of color pigments is dependent on keeping the pigments in

their native state. Heme is very difficult to remove since their deposits are found

within the muscle (Greene, 1926). However, most myoglobin is found in dark

muscle. Therefore, physical removal of myoglobin is accomplished by separating

dark muscle from light muscle. Besides myoglobin and hemoglobin, there are

other soluble proteins in fish muscle that contribute to poor gelation. For

21 Chapter 1. Introduction

example, most fish possess heat stable proteolytic enzymes. These enzymes are

species dependent and are usually active in certain pH and temperature ranges

(Lanier, 1997).

1.12.2.b Myofibrillar proteins

Sixty to seventy-five percent of total protein are myofibrils. The major

constituents of myofibrils are actin, myosin, and tropomyosin (Mackie, 1993).

These proteins contribute to the unique gelling properties of surimi. Myosin

makes up the thick filamento f myofibrils. It consists of 2 heavy chains coiled

around each other. At the end of myosin, a globular site associates with the

contractile mechanism between the thick and thin filaments. Attached to each

globular head are 2 light chains associated by non covalent bonds. Figure 16

depicts a schematic of actomyosin (Mackie, 1993). During frozen storage,

myosin undergoes aggregation resulting in toughening and loss of water holding

capacity. Generally, fishmyosi n is less stable than myosin of mammals or birds

(Sikorski, 1994). Addition of lower molecular weight sugars deters protein

denaturation during frozen storage (MacDonald and Lanier, 1991).

Myofibrillar proteins are highly reactive in their unfolded state. A gel results

when reactive protein] surfaces form a 3 dimensional network entrapping water.

Figure 17 illustrates the 3 dimensional protein network formed during gelation of

surimi (Morrissey et al., 1995). There are 4 types of bonds that can link proteins.

Hydrogen bonds stabilize water within a gel particularly at colder temperatures

22 Chapter 1. Introduction

(Lanier, 1997). Calcium ions form positive salt linkages between negative sites of

two adjacent proteins. Salt ions act as a bridge between protein side groups and

water. Hydrophobic bonds form with exposed sites of denatured proteins. This

usually occurs at elevated temperatures. S-S linkages form with the oxidation of

two cysteine molecules on adjacent protein chains (Lanier, 1991).

Fifteen to thirty percent of the myofibrillar protein is actin. Actin is more stable

than myosin during frozen storage. Other myofibrillar proteins include

tropomyosin and troponins (Mackie, 1993; Sikorski, 1994).

1.12.2.C Connective tissue

Connective tissue accounts for 3-10% of total protein in fish. Upon cooking,

collagen breaks down to form gelatin. Since collagen lowers gel strength in

surimi, producers remove it during processing (Mackie, 1993).

1.13 Thesis hypothesis

This thesis was motivated by two needs: to determine if under-utilized salmon has

potential to make a high quality surimi gel and to determine if solubilization can

prolong frozen storage of salmon surimi. Rather than using pink salmon that has

a limited season and is locally unavailable fresh, farmed chinook salmon

(Onchorhynchus tshawytscha) was used for this experiment. Local industry

experts have identified the need to process leftover steak sides into a value added

product from farmed chinook salmon (Oncorhynchus tshawytscha), in order to

23 ter 1. Introduction

maximize profits (Agri-marine, 1996). These leftover steak sides may have

potential as a surimi based value added product.

Thesis hypothesis: Farmed chinook salmon can make better quality functional

kamaboko after solubilized frozen storage than surimi made from farmed chinook

salmon. Quality functional kamaboko will be evaluated on the basis of gel

strength, fold test scores, and SDS-PAGE electrophoresis gels.

24 Chapter 1. Introduction

Figure 1. Effect of the discovery of cryoprotectants and tighter catch controls on the Japanese fishing industry during 1965-1985 (Sonu, 1986)

25 Chapter 1. Introduction leaching A) Traditional Surimi Production i m' filleting

stone-grinding with salt and ingredients

shaping of kamaboko steaming of kamaboko

Figure 2. Comparison of traditional and modern surimi production

26 Chapter 1. Introduction

RAW OR FROZEN SURIMI

GRINDING WITH

SALT AND

INGREDIENTS I

STEAMED FRIED BROILED OTHERS I

• KAMABOKO B TEMPURA CHIKUWA FISH

IMITATION H SATSUMA- SAUSAGE CRAB MEAT, AGE SHRIMP & FISH HAM SCALLOPS

HANPEN

NARUTO

Figure 3. Examples of surimi based products (Sonu, 1986)

27 Chapter 1. Introduction

10 0 - 1 WASTE

WASTE DEBONER 3 1.9 [—HIHCED WASHING 2 1.2 -WASHED REFINER 1 9.1 —REFINED DEHYDRATION 1 8.0 [—DEHYDRATED iY CRYOPROTECTANTS 19.5 I— SURIMI 2 3.8 AFTER RECOVERY

Figure 4. Flow diagram of the surimi process and subsequent yield (Le< 1985)

28 Chapter 1. Introduction

AdjustableCrusherRoll AdjustableBeltTension Rol Headed & Gutted Fish

Perforate_ d Drum \/\

s11 Extruded Fish Flesh ,C7 ced to Inside ofDrum

(:?^H^U-CW?vc^-Scraper

Waste Chute

Adjustable Main Pressure Roll -i

Waste,

Figure 5. Typical design of a surimi meat separator (Lanier, 1992)

29 Chapter 1. Introduction

Figure 6. Effect of agitation time on protein concentrations (g/1) with varying meat:water ratios (Toyoda et al., 1992)

30 Chapter 1. Introduction

87 600

Q o GO _ 400 T3 85 ** / s 0Q

i-1 Ao b 3.

8 83 200 o U

| MEDIUM VERY. SOFT. 1 SOFT J HARD. ' VERY HARD 1 1 1 1 1 1 I 4 6. 8 10 12 14 16

Hardness of Leaching Water

Figure 7. Effect of leach water hardness on gel strength and water content of croaker (Sonu, 1986)

3'1 Chapter 1. Introduction

J L L_ 1- 1 2 3 4 Washing Cycle

Figure 8. Effect of meat: water ratios and washing cycles on the removal of soluble proteins during leaching (Lee, 1986)

32 Chapter 1. Introduction

Figure 9. Effect of pH on the water holding capacity of minced meat (Sonu, 1986)

* The y-axis denotes the de watered volume of 3:1 water:meat mixture after it has been centrifuged, dewatered, expressed as % of initial volume

33 Chapter 1. Introduction

0 10 20 30 Time (min)

Figure 10. Effect of wash water temperature on the efficiency of dewatering (Toyoda et al., 1992)

34 Chapter 1. Introduction

Figure 11. Effect of moisture content on gel strength and elasticity of alaska pollock and pacific whiting (Yoon et al., 1997)

*high = high quality surimi (made under standard industry processing conditions)

low = low quality surimi (high quality surimi that is subjected to refreezing after 48 hrs.at25°C)

35 Chapter 1. Introduction

60 3 •Shear Stress ESlShear Strain 50 2.5

Ki 40 - 2

W (D 30 00 - 1.5

00 20 1

10 :- 0.5

0 Control BPP DEW FEW SPI WG WPC WPI 0., 1% Dried Additive

Figure 12. Effects of functional additives on kamaboko gel strength and elasticity (Park, 1994)

BPP = beef plasma protein, DEW = dried egg white, FEW = frozen egg white, SPI = soy protein isolate, WG = wheat gluten, WPC whey protein concentrate, WPI = whey protein isolate

36 Chapter 1. Introduction

STORAGE TEMPERATURE / •© © . -35°C

CO w • 60

I 3

'i 2

0 0 1 2 3 4

Storage Duration (Months)

Figure 13. Effect of frozen storage temperature on gel strength (Sonu, 1986)

37 Chapter 1. Introduction

2 3 4 5. Refrigerated Storage (Day)

Refrigerated Storage (Day)

Figure 14. Effect of refrigerated storage prior to gel strength and elasticity evaluations of alaska pollock and pacific whiting (Park, 1997)

38 Chapter 1. Introduction

60 ••0.5 -Ari -*-1.5 *2%NaCl

i i i i i i i i i r 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

Temperature of Gels (°C) at Fracture

Figure 15. Effect of test temperature on gel strength of alaska pollock (Park, 1995)

39 Chapter 1. Introduction

Figure 16. Schematic of myosin (Mackie, 1993)

40 Chapter 1. Introduction

Myofibrillar proteins Protein interaction sites

Figure 17. Heat gelation of surimi (Morrissey et al., 1995)

41 Chapter 2. Materials and Methodology

Chapter 2

Materials and Methodology

2.1 Optimization of surimi treatments using random centroid optimization (RCO)

Three factors were entered into the random centroid optimization program;

calcium chloride, sodium chloride, and dilution. The ranges for each factor were

0.0-1.0 %, 0.0-3.0%, and 200-500% in total weight of solubilized samples,

respectively. The ranges entered for each of the factors came from previous RCO

work done by Saito et al., (1996). Once these ranges were entered into the RCO

program, the program randomly generated values for 9 treatments. Table 1 lists

the composition of the 9 treatments of solubilized surimi and the control surimi.

2.2 Surimi production

Due to processing limitations of the experimental plan, treatments were processed

in 2 batches. The firstbatc h consisted of treatments 1, 2, 3 and the control. The

second batch consisted of treatments 4 through 9.

2.2.1 Fish source

In batch one, 87.76 kilograms of whole, gutted premium farmed chinook salmon

(Walcan Co., Vancouver) were transported by ferry to Campbell River and flown

by Canadian freight in 4 iced boxes to the Vancouver International Airport. Fish

arrived at the UBC Department of Food Science pilot plant for processing.

Salmon were approximately 10-15 hours post-mortem when received.

42 Chapter 2. Materials and Methodology

2.2.2 Filleting

Upon arrival, fish underwent a basic quality inspection. Fish appeared healthy

apart from a few eye lesions. Ice was removed and whole fish were weighed.

After weighing, fish were put on ice before filleting. Manual filleting commenced

in the pilot plant (25°C). Once filleted, and cut (approximately 5 X 5 cm pieces)

salmon was placed on half iced tubs in a 4°C cold room.

2.2.3 Grinding

Once filleted, grinding of fish commenced. Salmon pieces were fed through a

grinder (Beem Gigant, Germany) using a l/8th inch diameter extrusion

attachment. Ground fish were deposited into clean, dry tubs. The final weight of

minced fishwa s 36.30 kg.

2.2.4 Leaching

Fish were washed three times (batch style) in a large fish tote. The pH was

monitored through out the wash cycle to ensure a pH of 6-7. The pH was

measured by taking triplicate 50-mL samples of the minced fish in the wash water

with a pH meter (Corning, pH meter 220, Richmond Hill, Ont). Although not

used, one molar sodium bicarbonate solution was available for altering the pH, if

the minced fish pH fell below 6.

Wash 1

Minced fish (36.3 kg) was washed in 80L of precooled (4°C) tap water in a fish

43 Chapter 2. Materials and Methodology

tote. Temperatures were monitored (VWR thermometer, VWR Canlab, Edmonton

AB) through out the washings to ensure the wash water did not exceed 10°C. The

minced fish was mixed manually with a spoon at an approximate rate of 25

revolutions per minute for 5 minutes. Efforts to skim most of the fat off the

surface of the wash water were made before removing the washed fish. After

each wash cycle, consecutive 2L aliquots of minced fish were poured through

stainless steel sieves (1mm diameter) for initial dewatering. Once in the sieve,

minced fish was stirred constantly to remove excess water. Washed and partially

dewatered fish were stored in clean tubs until all of the fish was dewatered from

the fish tote. The final weight of minced fish was 46.25 kg.

Wash 2

Next, the fish tote was cleaned before adding the second wash water (4°C). Wash

two followed the same procedure as wash one. However, 46.25 kg of washed fish

was placed into 70L of tap water. The final weight of minced fish was 38.90 Kg.

Wash 3

Before the final wash, distilled water rinsed the fish tote. Next, 65L of 0.3% salt

in distilled water (4°C) was added to the fish tote. The fish was mixed manually

with a spoon for 5 minutes at an approximate rate of 25 revolutions per minute.

The solution stood for 10 minutes to solubilize the proteins. Sieves and

cheesecloth assisted in the dewatering of fish. Surimi was placed in a winepress

lined with cheesecloth (Grade 80, Vancouver Textiles Ltd., CA). Pressure (less

44 Chapter 2. Materials and Methodology

than 2 tons) was applied to dewater the minced fish using a hydraulic press. The

final weight of the fish after preliminary dewatering was 26.40 kg. The fish had a

high water content (based on experience from previous experiments) and

subsequently required centrifuging.

2.2.5 Dewatering

12-500 mL centrifuge containers held minced fish (Nalgene, Rochester NY) with

sealing caps in pre-cooled (4°C) GSA rotors (Dupont Sorvall, Missisauga, Ont).

Rotors were placed in Sorvall centrifuges (less than 10°C) (Sorvall RC 5B Plus,

Dupont Sorvall, Missisauga, Ont.) and centrifuged at 10,000 rpm. Supernatants

were discarded after each centrifugation until surimi reached a final moisture

content of 75% (w/v) and a weight of 20.65 kg.

2.3 Surimi solubilization

In batch one, minced fish was used to prepare treatments 1,2, 3 and the control.

Treatments 1, 2, and 3 were split into 5.25 kg aliquots for solubilization and the

control was split into 7-500 g aliquots. The solubilized treatments were made in a

4°C cold room. Distilled water dissolved pre-weighed sugars and salts in a

stainless steel bowl. Minced fish (5.25 kg), distilled water, dissolved sugars and

salts were combined to treatment weight. A balance determined final treatment

weights. A tabletop scale (Ishida, MTC-20, Japan) was tared and the distilled

water was added to the solubilized surimi until the appropriate weight was

reached for each treatment. Weights were calculated based on values given in

45 Chapter 2. Materials and Methodology

Table 1. For example, treatment one consisted of 5.25 kg of minced fish, 125g

CaCl2, 386g of NaCl,1.73 kg of cryoprotectants, and 7.49 kg of distilled water.

The final weight of treatment 1 (solubilized) = 3.97 X 5.25 kg = 20.84kg. Next,

treatments were put into 2L bags, sealed and frozen for about 5 hours in a -18°C

walk in freezer. This initial freezing took place because both -8°C freezers used

for the storage study (Refrigerator Incubator, Forma Scientific) had insufficient

power to freeze samples. Treatments were frozen in random order to

accommodate different chill rates within the freezer. When samples became

solid, they were assumed frozen and transported to the -8°C freezer for the storage

study. On days 3, 7, 14, 28, 56, and 84, treatments and controls were pulled from

storage and made into kamaboko. The -8°C freezer was chosen for the duration

of this study in order to speed up the progress of this experiment.

Batch two

The second batch of salmon was used to prepare treatments 4 through 9. The

methodology outlined in sections 2.21-2.3 of this thesis were followed for batch

one and batch two. However 136.2 kg of whole gutted premium chinook salmon

was received for the second batch. After filleting, and grinding there was 58.29

kg of minced fish. 116L and 102L of tap water were used for the first and second

washes, respectively. 94L of distilled water with 0.3% salt were used for the third

wash. After preliminary dewatering with the hydraulic press and final

centrifuging, 33.22 kg of surimi remained with a 75% moisture content for

kamaboko making. Four percent sucrose, 4% sorbitol, 0.15% tripolyphosphate

46 Chapter 2. Materials and Methodology

pentasodium, (Practical Grade Sigma) and 0.15% sodium pyrophosphate

(Mallinkrodt Chemical Works, St. Louis, MO) were mixed into the control using

a vacuum mixer (Stephan model UM-5, Columbus, Ohio). The solubilized

treatments in batch one and two used the same concentration of cryoprotectants

as the control. Batch one and two yielded 23.5% and 24.9% of the original whole

fish weight, respectively.

2.4 Kamaboko making

Treatments were removed from the freezer (-8°C) the day before kamaboko

making. Samples thawed overnight in a 4°C cold room. Treatments were pulled

from the cold room, diluted with varying amounts of chilled distilled water in 4L

plastic beakers and mixed (Nalgene, Rochester NY)(Fig. 23). This dilution step

was necessary to make water removal possible. Samples were initially dewatered

at room temperature. However, samples did not exceed 10°C. A pH Meter

(Model 620-Fischer Accumet, Nepean, Ont.) was used to measure triplicate

samples of diluted solubilized samples. Samples underwent preliminary

dewatering with grade #80 cheesecloth to minimize the volume of solubilized

surimi requiring centrifuging after pH determination.

Next, treatments were placed in 500 mL centrifuge containers (Nalgene,

Rochester, NY) and labeled. Containers were placed in GSA rotors (less than

10°C) (Dupont Sorvall, Missisauga Ont.). Treatments centrifuged multiple times

at a maximum speed of 10,000 rpm to a moisture content of 75% (wet basis).

47 Chapter 2. Materials and Methodology

Typically, 4-5 centrifugations were required. However, the number of

centrifugations required varied between treatments. The quick microwave method

(section 2.5.1.2) was used after each centrifugation to determine moisture content.

Centrifuged treatments were weighed and the amount of moisture needed to be

removed was calculated using the rapid microwave method. Likewise, if samples

were below the desired moisture content, the appropriate weight of distilled water

was added to the treatment based on the moisture content determined by the rapid

microwave method. Treatments were sealed in ziploc freezer bags, labeled and

placed in the cold room until all treatments were adjusted to the appropriate

moisture content.

A vacuum mixer (Stephan.model UM-5, Columbus, OH) was pre-cooled to 0°C

with a circulating ice bath made from saturated salt solution. Saturated salt

solutions were prepared by adding table salt to IL of water until salt precipitated

out of solution and then kept in the freezer at - 18°C overnight until used. Three

percent salt was added to each treatment to solubilize the mixture. Salt and

treatments were mixed at power one under vacuum (-50bar) for 5 minutes. When

the temperature exceeded 10°C due to blade friction, the mixer was stopped.

Mixing resumed at temperatures below 10°C. Immediately after mixing,

treatments were placed in a plastic bag, vacuum sealed (Model 30PI, Packaging

Aids Corp., San Francisco, CA) and placed in a cold room (4°C), until all

treatments were packaged.

48 Chapter 2. Materials and Methodology

The 5 lb. sausage stuffer (The Sausage Maker Inc., Buffalo, NY) was mounted on

plywood and fastened with "c" clamps to the tabletop in the cold room for

kamaboko extrusion. A hole was cut on each treatment bag and placed in the

stuffer with the opening facing the stuffer tube. The stuffer tube (2.2 cm

diameter) was sprayed with lubricant (Country Pure Vegetable Oil Cooking

Spray, Lucerne Foods Ltd.). One end of the casing was tied with a knot, the other

end with twine. Care was taken to extrude treatments with no air bubbles (Fig.

26). Occasionally, the stuffer tube was detached and a smaller secondary tube

assisted in pushing the surimi into the casing. This method of stuffing was only

1 used when an insufficient amount of surimi was obtained using the preferred

stuffing method. Treatments remained in the cold room (4°C) until all treatments

were stuffed. Each treatment had a constant diameter. Due to variation in

recovery for kamaboko making, sausages for each treatment varied in number and

length. This methodology produced sufficient kamaboko for both compositional

and functional analysis.

Treatments were then placed in a rectangular shaped plastic colander and cooked

at 90°C for 20 minutes. Kamaboko was then stored overnight in a 4°C cold room.

2.5 Compositional evaluation of kamaboko

2.5.1 Moisture determination (oven method)

Triplicate 2-5g (Sartorius AG, Germany) samples of treatments were dried at

100°C (Blue M Electric Co. Stabil Therm, Blue Island IL) to a constant weight in

49 • / Chapter 2. Materials and Methodology

aluminum dishes. After 18 hours, samples were weighed again on an analytical

balance to determine moisture content. This method was adopted from the

manual of standard methods for measuring and specifying properties of surimi

(Anon, 1991). Samples were pre-dried for crude fat, protein and ash

determination using this method.

2.5.1.2 Moisture determination (microwave method)

A quick microwave (120V 60Hz microwave, Matsushita Electric Inc, Japan)

method determined moisture content during centrifugation. Triplicate 5 g

samples were dried to a constant weight on filter paper (Whatman #2) using 2

^ minute intervals at power 10. This method was adopted from a rapid drying

method (Bostian et al., 1985).

2.5.2 Crude fat determination

Triplicate pre-dried samples were sealed in plastic bags. Before fat extraction,

samples were ground in a Retch ultracentrifugal mill (model ZM 100, Glenn Mills

Inc., Clifton, NJ) and placed in ziploc bags. Ground (2-3g) samples were

(Sartorius AG, Germany) placed in tared cellulose extraction thimbles (22 X 80

mm, Whatman International Ltd., Maidstone England). Pre-dried milled samples

in thimbles were placed in a goldfish apparatus (Lab Con Co., Fisher Scientific

Inc., Nepean, Ont.) and petroleum ether extracted was used to extract the fat for 6

hours (Strugnell, 1989). The following equation determined % fat: % fat =

weight of crude fat/total weight of initial pre-dried sample (corrected for moisture

50 Chapter 2. Materials and Methodology

content). Crude fat was performed on all treatments on storage day 84, and for

each storage day for treatment 7.

2.5.3 Protein determination

Pre-dried samples (less than 1/3 gram) were ground using the Retch ultra-

centrifugal mill (model ZM 100, Glen Mills Inc., Clifton, NJ) and defatted using

the crude fat determination method. These samples were analyzed by the Dumas

method (Leco FP-428, Leco Corp. St. Joseph, MI) for nitrogen determination.

This method was adopted from the AOAC method (968.06) for protein (crude) in

animal feed. A factor of 6.25 was employed to estimate crude protein content.

Protein determination was performed in triplicate per treatment, for each storage

day.

2.5.4 Ash determination

Triplicate pre-dried samples (2 g) were weighed and placed in tared pre-dried

porcelain crucibles. Dry ashing was done with a muffle furnace at 550°C (Blue

M Box type muffle furnace, Blue Island, IL) for 24 hours. Percent ash was

defined as weight of ash/initial wet weight of sample. This method was adopted

from the AOAC method (900.02) for ash determination in meat (1990) (crude).

Dry ashing was performed on all treatments of storage day 84, and treatment 7.

2.6 Sample preparation

A miter box (35.6 X 13.0 X 7.6 cm- Mastercraft, Canada) helped to cut kamaboko

51 Chapter 2. Materials and Methodology

samples. Doctor blades (20 cm X 5 cm, razor sharp) fit perfectly into the miter

box guides and subsequently assisted with the cutting of kamaboko. Widths of 3

mm, 2.5 cm, and 5.0 cm were used to cut kamaboko samples for the fold test, gel

strength, and color tests, respectively.

2.7 Functional evaluation of kamaboko

2.7.1 Color

Samples were pulled from the cold room (4°C) the morning after they were

cooked and the casings were removed. Samples were allowed to reach room

temperature. Five samples were cut per treatment per storage time. Once all

treatments were cut, the Hunter II Labscan Calorimeter (Hunter Associates

Laboratory Inc., Reston, IL) was standardized for white and black tiles. Samples

were situated on top of a 1 cm diameter measuring port. "L", "a", "b" values were

recorded for each of the treatments with a computer. "L" denotes lightness on a 0

to 100 scale from black to white: positive "a" values mean red and positive "b"

values mean yellow. This method was adapted from a manual of standard

methods for measuring and specifying the properties of surimi (Anon, 1991).

2.7.2 Gel strength

Five samples were evaluated per treatment per storage time. Samples were

centered on a Texture Analyzer base (TA-TXT2 Texture Analyzer, Texture

Technologies Corp. Stable Micro Systems, Scarsdale, NY) and punctured with a

5-mm diameter probe at the rate of 1 mm per second (Fig. 28). Each graph

52 Chapter 2. Materials and Methodology

recorded the peak height (N) and the distance on the x axis to the peak (mm).

Peak height was defined as the highest point where the slope no longer continued

to form a straight line. This method was adapted from the manual of standard

methods for measuring surimi (Anon, 1991).

2.7.3 Fold test

Five samples were evaluated per treatment per storage time. Samples were taken

and folded up to two times with a forefinger and thumb based on the 6-point scale

for fold test. Table 2 lists the fold test scale used to evaluate gel elasticity

(Bouraaoui, 1995).

2.7.4 SDS-PAGE electrophoresis

Kamaboko gels (9 treatments and control) were added to Tris buffer (pH 8.0)

containing 5 mmol Tris, 8 M urea, 10% SDS w/v in a 1:10 volume ratio and

placed in centrifuge tubes (Safelock microcentrifuge tubes, Eppendorf, Westbury,

NY). Samples shook overnight in a shaker (Waterbath shaker, Eberach Co, Ann

Arbor, MI). Tubes were then centrifuged at 10, 000 rpm for 3 minutes (model

5415C, Eppendorf, Germany). Fifty ul of supernatant were transferred into

plastic centrifuge tubes. To each tube, 12.5 ul of SDS clear and 37.5 ul of 1 mmol

EDTA Tris buffer (pH 8.0) was added in a ratio of 1:3. In a fume hood, 3 ul of

B-mercaptoethanol, and 5 ul of bromophenyl blue indicator were pipetted into a

centrifuge tube. Tubes vortexed for one minute (Fischer Vortex, Genie 2, Fisher

Scientific). Tubes were boiled for 5 minutes in a rack to accelerate denaturation

53 Chapter 2. Materials and Methodology

and cooled at room temperature. Samples centrifuged (Eppendorf 5415C,

Germany) for 3 minutes at 10, 000 rpm. Three microlitres of supernatant for each

treatment were delivered into wells and transferred onto SDS-PAGE gels (Phast

Gel Gradient 10-15, Pharmacia Biotech, Uppsala Sweden) using SDS buffer strips

and SDS sample applicators (Pharmacia Biotech, Uppsala Sweden). SDS gels

were run on the SDS Phast System (Pharmacia Biotech, Uppsala Sweden). Once

finished, gels were stained for 20 minutes with a 1:1 20% acetic acid and

Coomassie Brilliant Blue stock solution. Next, gels were de-stained twice with

30% methanol and 10% acetic acid. Gels were re-stored with 5% glycerol and

10% acetic acid. Wide range molecular weight standards determined molecular

weights (M.W. 6 500-205 000, Sigma, Aldrich Co., CA). The standards and there

molecular weights were: aprotinin, bovine lung (6, 500), a-lactalbumin, bovine

milk (14, 200), trypsin inhibitor, soybean (20, 000), trypsinogen, bovine pancreas

(24, 000), carbonic anhydrase, bovine erythrocytes (29, 000), glyceradldehyde-3-

phosphate dehydrogenase, rabbit muscle (36, 000), ovalbumin, chicken egg (45,

000), glutamic dehydrogenase, bovine liver (55, 000), albumin, bovine serum (66,

000), fructose-6- phosphate kinase, rabbit muscle (84, 000), phosphorylase b,

rabbit muscle (97, 000), galactosidase, E. coli (116, 000), myosin, rabbit muscle

(205, 000). This method was adopted by procedures followed in the Food

Science Departmernt at the University of British Columbia (Ogawa, 1997). SDS-"

PAGE electrophoresis was done on each treatment per storage time.

54 Chapter 2. Materials and Methodology

2.8 Statistical analysis

Excel spread sheets were used to conduct both one-way analysis of variance

between treatments and two-way analysis of variance for differences within a

treatment on a given day. Tukey's test determined what treatments or days were

significantly different. Multiple regression was conducted for RCO factors for

each treatment on a given storage day.

55 Chapter 2. Materials and Methodology

Table 1. Nine solubilized treaments and the control

Treatment Dilution (%) CaCI2 (%) NaCI (%) 1 397 0.60 1.85

2 421 0.28 2.6

3 271 0.35 2.56

4 202 0.30 0.49

5 487 0.44 0.70

6 452 0.48 0.86

7 258 0.05 0.38

8 240 0.29 1.82

9 372 0.31 2.70

control 0.00 0.00 0.00

56 Chapter 2. Materials and Methodology

Table 2. Fold test scale

Fold test score Kamaboko gel attribute

1 Breaks by finger pressure

2 Does not break by finger pressure

3 Cracks immediately when folded

4 Cracks gradually when folded

5 No cracks showing after first fold

6 No cracks showing after second fold

57 Chapter 3. Results and Discussion

Chapter 3

Results and Discussion

3.1 Compositional evaluation results of kamaboko

3.1.1 Crude protein content

Table three provides a summary of crude protein content (wet basis) in kamaboko

samples. Each treatment had a relatively low standard deviation for a given day,

indicating sample homogeneity. However, a substantial discrepancy between

protein values existed for treatments over time (13.59 ± 1.69%, n = 180). The

inconsistent crude protein concentration may have been due to inconsistent

sample preparation. Differences in sample density and consequently protein

concentration could have resulted from inconsistent sample extrusion (Reppond

and Babbit, 1997). Extrusion speed error and resistance error to the surimi

entering the casing likely contributed to variations observed in protein

concentrations. The variation in protein concentration was substantial when

compared to consistent values published by other researchers. For example,

Reppond et al., (1995) reported kamaboko protein concentrations of 17.7 ± 0.3%.

However, this value was for one sample at one storage time and commercial

surimi equipment produced the kamaboko. Other researchers have reported a

protein concentration mean of 11.25 ± 0.65% for surimi products. Therefore, the

crude protein average in this study appears appropriate, but the variation in

protein values does not.

58 Chapter 3. Results and Discussion

Other factors, such as the proportion of total fat, salts, sugars, and water in the

kamaboko samples could have affected crude protein content. Since there is no

trend corresponding to treatments with high non-protein content, it is reasonable

to suggest that the variation was due to poor sample preparation. Fortunately,

protein concentration within the ranges studied whether, high or low, seemed to

have little effect on overall gel quality. For example, treatments that had

relatively low protein concentrations also had high gel strengths and fold test

scores (control, day 28). Perhaps the various mean protein concentrations in this

study were not significant enough to show that increased protein concentrations

leads to higher gel strengths (Hamada, 1992; Hamann and MacDonald, 1992).

3.1.2 Moisture content

The mean moisture content of kamaboko gels were 74.5 ± 5.5% (n =180) after

cooking. Table 4 presents a summary of the moisture contents (wet basis) in

kamaboko gels. These values are consistent with prominent surimi manufacturers

company values. Anon (1993), reported a final screw press and kamaboko

moisture content of 82-84% and 74-75.5% in Alaska pollock, respectively. The

high moisture content is economical from industry's perspective since water is

cheaper than protein. However, as moisture increases, gel strength decreases

(Lanier, 1992)? Reppond et al., (1993) recognized the difficulty in producing

surimi with the same moisture content. Subsequently, a correction factor was

developed to compensate for surimi with different moisture contents.

Unfortunately, this factor is only suitable for surimi evaluated for shear stress and

59 Chapter 3. Results and Discussion

strain (not punch force). Due to the lack of uniformity of samples using a punch

test, mathematical models cannot predict gel quality based on various moisture

contents. This is not surprising, since other researchers (Hamann and

MacDonald, 1992) have found the torsion test to be far more reliable than the

punch test due to the geometry of samples. The torsion test is preferable to the

punch test for several reasons. The torsion test does not cause the gel to undergo

major geometric changes so that shear stress and strain may be calculated. On the

contraty, gross shape changes occurs during the punch test and stress and strain

cannot be easily determined quantitatively. Hamann and MacDonald (1992)

provide a detailed explanation on how to prepare surimi gels for the torsion test.

Due to equipment limitations, the punch test was used instead of the torsion test in

this thesis. Reppond et al., (1997) suggested a 1% moisture level difference was

comparable without mathematical or physical adjustments. Therefore, it was not

reasonable to compare treatments in this experiment due to the range in moisture

values. However, it was appropriate to make a few general conclusions. For

example, the control had the lowest average moisture content, and yet it did not

have the highest average gel strength. This indicates that some of the solubilized

treatments were clearly better than the control since both their gel strengths and

moisture contents were significantly higher than the control (i.e. treatment 7).

Results from one-way ANOVA and Tukey's test support this conclusion.

Surimi samples were stuffed into casing with mean moisture contents of 77 ±

2% (n =180) but lost an average of 2.5% moisture during the cooking process.

60 Chapter 3. Results and Discussion

Cavestany et al., (1994) also found a 2% expressible moisture loss of kamaboko

gels after cooking. The casings were not waterproof since casings had knots at

both ends.

3.1.3 Crude fat content

Crude fat content was analyzed for all samples on day 84 and for the best

treatment (treatment 7). Day 84 was chosen for fat analysis for two reasons.

Firstly, day 84 was the last storage day in this study, and subsequently crude fat

content values should represent the poorest quality kamaboko due to protein

degradation over the storage study. Secondly, day 84 crude fat content analysis

indicated fat values between treatments. Treatment 7 was chosen for crude fat

analysis, since it produced the best quality kamaboko gel. Furthermore, treatment

7 crude fat values might have indicated whether or not sample homogeneity

between storage days for a given treatment existed. The crude fat content of

kamaboko gels for storage day 84, and the best treatment (7) was 4.4 ± 2.4%.

Table 5 lists mean and standard deviations of crude fat contents in kamaboko gels.

Reppond et al., (1993) reported a fat content of 0.9 ± 0.0% for herring surimi.

However, they suggested this value was likely unrealistically low due to low belt

pressure used during mechanical deboning. Since herring is a fatty fish (James,

1988), one might expect similar fat values for farmed chinook salmon kamaboko.

Unfortunately, there are no values in the literature specifically cited for kamaboko

made from farmed chinook salmon to make an appropriate comparison. Industry

typically uses low fat fish such as pollock for surimi production (Anon, 1993).

61 Chapter 3. Results and Discussion

The high fat values in this experiment are likely due to the batch process used to

wash the minced fish, the initial fat content of the salmon, and the omission of the

"refiner step". A fat layer on the wash water surface contaminated the minced

fish as it was removed from the fish tote, even though researchers tried to remove

the fat prior to collecting the washed minced fish. A continuous washing system

(commercial surimi equipment) would have been a much more effective way to

remove the fat from the minced fish. The advantages of low fat kamaboko are

numerous. For example, samples will have a better shelf life and quality.

According to Kennish et al., (1990) farmed chinook salmon {Oncorhynchus

tshawytscha) generally have a muscle crude fat content of 2.6-4.0%. However,

variation is greatly dependent on the diet, and the genetic make up of the salmon.

Others have reported higher crude fat content values of salmon in the range of 1.6

-18% (Greene, 1926). The fanned chinook salmon used for this experiment had a

muscle crude fat content of 17 ± 1% for batch one and 5.5 ± 0.5% for batch two

(n=3). The variation in fat content between batch one and batch two is likely due

to sampling procedures, not seasonality of the fish. Industry experts reported that

west coast salmon fish farms have a consistent crude fat content during the time

period that farmed fish was obtained for this experiment (Mitchell, 1998).

Regardless of the crude fat content of the salmon fillets, kamaboko treatments had