quality changes during frozen storage and cooking of milk …

JOURNAL OF INTERNATIONAL ACADEMIC RESEARCH FOR MULTIDISCIPLINARY Impact Factor 1.393, ISSN: 2320-5083, Volume 2, Issue 4, May 2014

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QUALITY CHANGES DURING FROZEN STORAGE AND COOKING OF MILK SHARK (SCOLIODON SORRAKAWAH) MUSCLE TISSUE: EFFECT ON

STRUCTURAL PROTEINS AND TEXTURAL CHARACTERISTICS

MAYA RAMAN* SALEENA MATHEW**

*Dept. of Biotechnology, Indian Institute of Technology Madras, Chennai, India

**School of Industrial Fisheries, Cochin University of Science and Technology, Fine Arts Avenue, Cochin, India

ABSTRACT

Structural protein characteristics and textural quality of shark muscle tissue during

frozen storage and subsequent cooking were evaluated. Myofibrillar protein (45.62 %) and

pepsin soluble collagen (13.69 %) contributed to major proportion of total protein (22.01 %)

and significantly affect textural characteristics. Urea, contribute to the off-odour in fresh

shark muscle and add to the non-protein nitrogen (NPN) fraction that are hydrolyzed during

processing conditions. Freezing and frozen storage effect protein extractability through

protein aggregation and cooking caused gelatinization of protein molecules. Conformational

changes in protein molecules were reflected in SDS-PAGE, TPA and histochemical analysis.

Fresh samples showed optimum texture at 70 °C (H1-1.93 kgf) that modified to 80 °C (H1-2

kgf) with frozen storage. Histochemical studies of fresh tissue indicated parallel myotome

bundles enveloped by collagenous sheath that undergo gelatinization during cooking.

Quantity and distribution of structural proteins contribute considerably to the muscle texture.

Myofibrillar protein and collagen significantly affect the textural properties during frozen

storage and cooking. Collagenous sheath that envelops the myotomes (myofibrillar protein)

lose its integrity during frozen storage and consequent cooking, thereby, allowing muscle

bundles to slide over at high cooking temperatures. Due to denaturation, water holding

capacity of protein is reduced during frozen storage and cooking.

KEYWORDS: Myofibrillar Protein; Collagen; SDS-PAGE; Texture Profile Analysis;

Scoliodon sorrakawah.

1. INTRODUCTION

Fish quality is a multifaceted concept, with key aspects being safety, nutritional quality and

availability, freshness, eating quality, species and size, and product type. Variations in

flavour, odour, texture and colour directly reflect meat freshness. Nutritional and textural

characteristics of fish are the major features that verify the consumer preferences’. Fish


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muscle protein and its distribution are the primary determinants of the nutritional significance

and commercialization [1]. Structural proteins, comprising of myofibrillar proteins and

collagen, account for 70-80 % of the total protein; and organize the structure and the specific

textural properties of muscle tissue. Arrangement of structural components and the changes

occurring in these components during processing affect texture in a significant manner.

Hence, textural characteristics are influenced equally by both intrinsic and extrinsic factors.

Collagen, though contribute a minor fraction, is vital in determining chemical and

organoleptic properties [2]. In raw fish meat, the firmness is closely associated with collagen.

The systematic arrangement of collagen fibre plays an important role in determining the

tissue properties [3]. Protein functionality, thus, is determined to a large extend by its

physiochemical and structural properties [4]. Consequently, these basic functional

components considerably determine the nutritional and textural properties of fish tissue.

Understanding shark proteins and the effect of protein components on textural quality is

needed that could be realized by analyzing protein characteristics and variations caused in the

components during frozen storage and cooking.

Sharks are commercially important and valuable source of protein [5]. Shark proteins are

highly popular and are appreciated dietary supplement and an alternate protein source.

Typically, cartilaginous tissue is used to extract collagen and glycoproteins [6]. Dewi et al.

reported that smaller sharks are used as fresh, chilled or as frozen meat, whilst the larger

sharks are used to provide fins and hides [7]. Shark by-products, such as, fin, liver, meat, fat

and skin find various uses. Shark liver oil and shark fins are reasonably high in demand and

are exported in considerable quantity to the Eastern Countries [8]. In addition, shark collagen

is of health significance and contains antiangiogenic and antitumor compounds [9]. Shark

cartilage is said to be of significant considerable health benefits, which needs scientific

confirmation. From economic and nutritional perspective, an appropriate utilization of the

shark meat is necessary.

Though, freezing is a widely accepted preservation technique, long-term frozen storage of

muscle tissue might lead to marked increase in toughness and dryness of the tissue [10].

Formation of cell compartments, protein coagulation, myofibrillar protein aggregation,

dehydration, loss of water-holding capacity after thawing and changes in flavour are the

common variations reported to occur during frozen storage. These alterations depend on

species, freezing method and time-temperature profile during frozen storage [10]. Cooking

also produces a notable change in the muscle components and texture that could be correlated


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to flesh quality. Texture of cooked flesh depends on the size of muscle fibres after cooking,

the quantity of coagulated proteins in the interstices and the gel formed by collagen and lipids

that allow sliding of the muscle fibres and myomeres. Baylan et al. reported that during

cooking, secondary and tertiary structures of proteins were lost due to the split in hydrogen

bonds, resulting in unfolding of native conformation [11].

The present study investigates the protein characteristics and textural variations in shark

muscle tissue (Scoliodon sorrakawah) that are subjected to different cooking temperatures.

The study attempts to understand the combined effects of the frozen storage and cooking on

the physicochemical properties, protein fractions, textural and histochemical properties.

2. Materials and method

2.1. Raw material

Milk shark (Scoliodon sorrakawah), an elasmobranch commonly found in the inshore waters

of Kerala coast along the South-West coast of India, were obtained from commercial fishing

boats. Samples measuring 70-100 cm in length and weighing 1-1.5 kg were individually

wrapped in polythene sheets and were brought to the laboratory within 8 h in iced-stored

conditions. They were then headed, cleaned and eviscerated.

2.2. Sample preparation

Unfrozen samples (F, 5 kg) were packed in polyethylene bags and kept in freezer for analysis

while for frozen-storage study, cleaned-eviscerated samples were individually quick-frozen

using air blast tunnel freezer at -40 C, wrapped separately in polythene bags and stored in 5-

ply cartons at -18 C. After six months (Fr), the samples (5 kg) were randomly selected and

enclosed in polythene bags and thawed at room temperature for 30 min, according to

CODEX alimentarius, before analysis [12].

During cooking experiments, both unfrozen and frozen samples were cut in small cubical

pieces (2 cm3), wrapped in aluminium foils and subjected to different cook temperatures

(45-90 C) for one minute [13]. The abdominal muscular region was considered for the

analysis.

2.3. Chemicals

All chemicals used in the study were of analytical grade. Chemicals for electrophoresis were

purchased from Genei, Bangalore, India.


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2.4. Physicochemical Analysis

Homogenate was prepared by blending 10 g of sample with 90 ml of distilled water for 30 s

and pH was measured using a calibrated Cyberscan pH 500 (digital pH meter, MERCK).

Expressible moisture (EM) was measured according to Ng [14]. For determining expressible

moisture, pressure of 10 kg/cm2 was applied on tissue (1 g) placed between two pre-weighed

filter papers for 10 s and the weight differences was expressed as the percentage of sample

weight. For water binding potential, the tissue was homogenized (Yorco Micro Tissue

Homogenizer, India) with water (1: 30) in pre-weighed polycarbonate tubes, heated at 60 ºC

for 30 mi, cooled and centrifuged (4000 rpm for 10 min) at room temperature. Water binding

potential (WBP) was calculated as the weight difference and accounted for the amount of

water held per gram sample [15]. Proximate composition comprising of moisture 934.01

(4.1.03), protein 955.04 (2.4.03), fat 920.39 (4.5.01) and ash 942.05 (4.1.10) were evaluated

according to Official Methods of Analysis of the Association of Official Analytical Chemists

[16]. All measurements were performed in triplicate and were expressed on wet weight basis.

2.5. Protein fractionation

Muscle proteins were fractionated according to the methodology of Hashimoto et al. [17] and

Mizuta et al. [18], which was standardized in our laboratory [19]. 10 g of tissue was

homogenized with 10 v (100 ml) of 0.05 M phosphate buffer (pH 7.5) and supernatant was

collected after centrifugation (4000 rpm for 10 min, MB-20 Superspeed Refrigerated

Centrifuge). Sarcoplasmic fraction (SP) was precipitated from the supernatant using 10 %

trichloroacetic acid. Residual tissue was further homogenized with 10 v (100 ml) 0.6 M

NaCl-phosphate buffer (pH 7.5) and supernatant containing myofibrillar protein fraction

(MY) was collected after centrifugation (4000 rpm for 10 min, MB - 20 Superspeed

Refrigerated Centrifuge). SP and MY were fractionationed at 4 C. The residual tissue were

rigorously extracted overnight with alkali and washed in distilled water, and were suspended

in 10 v (100 ml) of 0.5 M acetic acid, digested at room temperature for 48 h using enzyme

pepsin (1: 20 w/w, SIGMA Co. Ltd., USA). Supernatant collected were pepsin soluble

collagen fraction (PSC). The fractions were evaluated for nitrogen content by micro-kjeldahl

and converted to protein using the conversion factor 6.25 (AOAC, 2000). All analyses were

done in triplicates.

2.6. SDS-PAG Electrophoresis

Samples with equal protein content of 30 µg were subjected to discontinuous polyacrylamide

gel electrophoresis (Large Vertical Model Electrophoreses unit, Genei, Bangalore, India), as


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described by Laemmli [20], with slight modifications. Electrophoretic separation was

performed at 20 °C for 6 h, using 10 % polyacrylamide gel concentration for sarcoplasmic

and myofibrillar fraction and 7% gel concentration for pepsin soluble collagen using

discontinuous Tris-HCl/ glycine buffer system. Stacking gel strength was 4%. The

electrophoresis was initially adjusted to 3 mA/ well that was increased to 10 mA/well towards

the end of the gel run. Protein bands were stained with Coomassie Brilliant Blue R-250 (0.05

% w/v) in 15 % methanol (v/v) and 5 % acetic acid (v/v). De-staining was performed in an

aqueous solution of 5 % methanol and 7.5 % acetic acid, and the samples were stored in a

solution of 10 % glycerol and 7.5 % acetic acid.

Molecular mass calibration kits of broad range and high range (Genei, Bangalore, India)

consisting of myosin (205 kD), phosphorylase b (97 kD), bovine serum albumin (66 kD),

ovalalbumen (43 kD), carbonic anhydrase (30 kD), soyabean trypsin inhibitor (20.1 kD),

lysozyme (14.3 kD), aprotinin (6.5 kD) and insulin (3 kD), were used as standards for

molecular mass determination.

2.7. Texture Profile Analysis (TPA) and Sensory Evaluation

Texture Analyzer (Lloyd Instruments, UK, model LRX PLUS) and Nexygen software

(Lloyds Instruments) was used for instrumental texture analysis of unfrozen and frozen shark

muscle tissue, cooked at various temperatures. Flat-faced cylindrical probe of 50 mm

diameter compressed the bite size piece (2 cm3) twice in a reciprocating motion, imitating

the mouth action. Samples were subjected to a double compression of 40 %; and probe test

speed and trigger force were maintained at 12 mm/min and 0.5 kgf, respectively. From the

force-time plot, hardness 1- after first compression (H1, kgf), hardness 2- after second

compression (H2, kgf), cohesiveness (C), springiness (S, mm) and stiffness (SF, kgf/ mm)

were evaluated [21].

A six-member panel performed the sensory evaluation using 7-point scale with scores being

7-like extremely or much more, 4-like or dislike or neither more or less and 1-dislike

extremely or much less. The selected characteristics were tested as defined by Jowitt [22].

Wateriness, firmness, elasticity, cohesiveness, juiciness and hardness were the attributes

evaluated. The sensory panel also recorded appearance, colour, odour, texture and flavour

and finally giving overall organoleptic scores. Replicates of five were measured for each

sample.


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2.8. Histochemical Analysis

Samples (1 cm thick) were fixed overnight in Bouins’ fixative containing picric acid, 40 %

formalin and glacial acetic acid (15: 5: 1) and were subjected to serial dehydration using

ethanol of various dilutions (70 % for 24 h and 80-96% for 30 min each and absolute alcohol

for 20 min). Finally, the dehydration was completed with the samples being dipped in

acetone. The dehydrated samples were embedded in paraffin wax with ceresin (congealing

point-60 C) and sectioned (8 m, thickness) using rotary microtome (SIPCON SP 1120

Rotary Microtome, India) and fixed on slides. Double staining technique using Weigert’s

Haematoxylin stain for 5 min followed by acid: alcohol for 30 s and Van Geison’s stain for 5

min were used to stain collagen fibrils (red) and myofibrillar proteins (yellow). The stained

slides were passed through alcohol series for 10 min, followed by xylene for another 10 min

and were mounted using DPX mountant. The sections were photographed using Nikon

Eclipse E-200 compound light microscope fitted with Nikon DN 100 Digital Net Camera.

2.9. Statistical Evaluation

Results are expressed as means ± standard error of means. Statistical difference among the

results were analyzed using one way analysis of variance (ANOVA) using a Minitab software

(version 14.0, Minitab Inc, State College, PA, USA). P values of <0.05 were considered

significant. Automated surface fitting analysis was performed using TableCurve 3D (Systat

Software Inc., Bangalore, India) to analyse the affects of temperature and protein fractions

(sarcoplasmic protein, myofibrillar protein and collagen) on muscle hardness.

3. Results and discussion

3.1. Physicochemical properties

Variations in expressible moisture (EM) and water binding potential (WBP) of shark muscle

tissue with frozen storage and subsequent cooking are shown in Table 1. Fish muscle pH is

considered as a shelf-life and spoilage index. Fresh samples upon cooking showed pH

ranging between 6.3 and 6.4. Ali reported that pH in seafood varied from 5.8-7.2 depending

on the struggle at the time of harvest although normal variation was between 5.8 and 6.5 [23].

In frozen stored-cooked samples, pH showed a significant reduction and varied between 5.7

and 5.8. This could probably be due to ante-mortem stress, protein degradation and loss of

hydration property during freezing and subsequent cooking. The reduced pH with frozen

storage makes tissue soft, thereby, the tissue losing its inherent water binding property, which

is evident from increased EM. The pH affects water binding properties and subsequent, gel-


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forming ability. There was no significant difference in the pH with frozen storage and

subsequent cooking. Ante-mortem stress and post-mortal glycolysis lead to the accumulation

of lactic acid in the muscle tissue possibly contributing to the lower pH in frozen samples [24].

Expressible moisture (EM) is lowest at 55 C, for fresh (67.1 %) and frozen stored sample

(73.28 %), whilst water binding potential (WBP) is observed to be high at 65 C (2.07 g) and

50 C (2.69 g) for fresh and frozen samples, respectively. EM and WBP showed significant

difference between fresh and frozen-stored samples. Hydration property is a function of

protein functionality and vice-versa; and protein and water binding potential possibly have

positive correlation; and varies with temperature and frozen storage period [25]. It could be

possible that irreversible bond formation in actomyosin, presence of charged groups on

proteins and physical configuration of myofibrillar proteins and collagen, together with

sarcolemma, affect the water retaining capacity of the muscle tissue. Foh et al. reported that

amino acids, protein conformation and surface hydrophobicity/ polarity influence the

hydration properties of muscle protein [4]. Interaction of muscle proteins together with water

and lipid are also vital determinants in food texture.

Proximate composition of shark muscle tissue show moisture 72.46 %, crude protein 22.04

%, ash 2.36 % and lipid 0.90 %. Non-protein nitrogen fraction (NPN) could be a contributing

factor towards the total nitrogen content, thus adding to the crude protein content. Protein

content showed significant decrease with frozen storage (P<0.05). The variations showed in

protein content in fresh and frozen-stored samples with cooking temperature could be

attributed to protein denaturation associated with temperature variations [26]. Lipid content is

higher in fresh samples that reduced with frozen storage, which could be attributed to fat

oxidation [26]. Increase in ash content with frozen storage could be credited to the drip loss

and dehydration associated with frozen-storage and cooking that lead to moisture loss [27].

The effects of cooking on proximate composition has been previously reported and were

comparable with results obtained in atlantic sharpnose, lemon shark, scalloped hammerhead

shark and tiger shark [28].

3.2. Quantitative and qualitative fractionation of protein fractions

Fractionation of sarcoplasmic protein fraction (SP), myofibrillar protein fraction (MY), total

collagen (TC) and pepsin soluble collagen (PSC) during frozen storage and cooking are

shown in Fig. 1. In fresh uncooked samples, sarcoplasmic protein content was 16.07 % that

reduced during frozen storage (14.31 %). Reduced pH during frozen storage may affect the

extractability of sarcoplasmic protein by protonizing ionisable carboxylic acid side chain


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leading to hydrophobicity and consequently, the aggregation and accumulation of these

proteins in the interfibrillar spaces [29]. Non-protein nitrogen including urea also contributes

to SP in fresh samples. High concentration of urea and trimethylamine-oxide (TMAO) in the

tissue offers an off-odour to fresh samples that are lost during frozen-storage. This may also

counteract as protein-destabilizing force and affect the texture during frozen-storage [30].

The ammonical odours of fresh samples are lost during frozen-storage possibly due to its

hydrolysis. Myofibrillar protein contributes the major fraction of total protein (45.62 %).

With frozen storage and cooking, the extractability of myofibrillar proteins showed

significant decrease possibly due to aggregation and gelatinization, respectively. Collagen

content was higher (13.69 %) that was in accordance with the earlier findings [31]. More than

80 % of the total collagen were solubilised by enzyme pepsin and collected as pepsin soluble

collagen. PSC showed similar trend in both fresh and frozen-stored samples. Cooking result

in the shrinkage of collagen due to the breakdown of hydrogen bonds in the protein and could

be the reason.

Frozen storage reduced protein extractability possibly modifying the proteins by oxidation.

The aggregation/ denaturation of proteins were more severe during frozen storage. Protein

denaturation induced during cooking lead to the loss of hydration property and possibly

contributing to the cooking loss. Association-dissociation-denaturation of proteins during

frozen storage and subsequent cooking is the main contributing factor for the reduced

extractability of protein fractions in the shark muscle tissue. Weakening of fibrous linkages in

muscle structure during initial stages of processing could also contribute to the reduced

extractability of protein fractions [32]. Gelation, emulsification, water binding and lipid

binding specify the conformational status of protein system. SDS-PAGE indicates the

disintegration and conformational changes in protein molecules during frozen storage and

cooking (Fig. 2). SP included protein bands ranging between 40 to 60 kD and might include

myoalbumin, globulins and enzymes. The band between 29 and 43 kD could be

glyceraldehyde-3-phosphate dehydrogenase. Presence of additional bands with frozen-storage

(65 C) and cooking could be due to the disintegration and conformational changes in this

fraction. Low molecular weight enzymes are probably lost with frozen storage. Sarcoplasmic

proteins contribute to thermal gelation of structural proteins, thereby, affecting the textural

characteristics of the muscle tissue [33]. MY showed myosin (200 kD), -actinin (105 kD)

and thick fused band at 29 kD that could be overlapped actin, tropomyosin and troponin [34].

With frozen storage, the myosin heavy chain molecules (MHC) became intense and new band


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was observed at the top of the gel possibly due to aggregation that were too large to enter the

gel. It could be due to covalent bond formation. Cooking cleaved MHC and actin into smaller

polypeptide chains. Protein bands with low molecular weight (<6.5 kD) were not separated

distinctly in PAGE. Collagen bands in the lane 1 (F-c) indicate type I collagen consisting of

two -chains (1 and 2) and one -chain. The estimated molecular weights for the 1 and

2 chains were approximately 120 and 112 kD, respectively. The -chain is could be a dimer

with high molecular weight (205 kD). The SDS-PAGE patterns of collagen were similar to

other fish species [35]. With frozen-storage, low molecular weight bands fade while

aggregation cause accumulation cause formation of protein band at the top of the gel and with

subsequent cooking, high molecular weight components disappeared due to hydrolysis into

small molecular weight proteins [6]. Functional properties of the proteins are highly

influenced by molecular weight distribution. Aggregation and gelatinization of protein

molecules were observed to hinder the electrolytic mobility of proteins. These conformational

changes in the protein fractions significantly affected the muscle texture. Cooking cause

solubilisation of proteins and hence, leads to loss of proteins.

3.3. Texture Profile Analysis

Frozen storage and cooking contribute to denaturation and conformational changes in protein

molecules that affect the fish muscle texture as evident from TPA results (Fig. 3). Fresh

samples indicated first phase of hardening (H1) at 50 °C (4.33 kgf) and the second phase of

hardening (H2) at 70 C (1.93 kgf) with maximum juiciness. The variations in hardening

observed during frozen storage and subsequent cooking may possibly be due to the

differential freeze denaturation of the structural proteins. In frozen-stored sample, optimum

texture was observed at 80 °C (H1, 2 kgf). Three dimensional surface plot analyses were

done to analyse the role of temperature on protein fraction and textural parameter together.

The data for protein fractions, hardness (H1) and cooking temperature for fresh samples were

fitted into surface plot to simulate the response surfaces to optimize and indicate the

interrelationship between these three parameters. Surface plot analysis also emphasized that

sarcoplasmic protein (P<0.05), myofibrillar protein (P<0.001) and pepsin soluble collagen

(P<0.05) in fresh samples showed significant variation with cook temperature with optimum

texture being obtained at 70 C. It is observed that the affect of MY on texture with respect to

temperature is significantly higher (R2= 0.89) compared to SP (R2= 0.58) and PSC (R2=0.65)

(Fig. 4). Hardness and springiness were low at 60 C possibly because collagen fibres

become irreversibly solubilised and encourage textural changes due to sliding of myotomes


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that are related to heat denaturation. Hardness 1, hardness 2, springiness and stiffness showed

highly significant variation (p<0.01) between temperatures. High cohesiveness at 50 C could

also be due to denaturation of collagen. At 70 C, myofibrillar proteins affected cohesiveness

and are associated with the loss water molecules and hydration property of the tissue. An

increased opacity of fish flesh during cooking is observed probably due to the precipitation of

thermally denatured sarcoplasmic proteins that commence at 45 C [33]. In raw condition and

at 45 C, the textural parameters were high and decreased considerably with increase in

temperatures probably due to dissociation of actin-myosin complex and denaturation of

myosin tail. Heat induced gelation of myofibrillar proteins is an important functional property

and gives desirable texture and stabilization of lipids and water during processing [36].

Sensory analyses determine consumer preferences’. Cooked muscle tissue showed second

phase of hardening during later stage of cooking that concur with the second phase of

hardening as observed in TPA results (Fig. 3). Overall scoring indicated first phase of

hardening for unfrozen samples at 50 C and second phase of hardening at 70 C with

maximum juiciness. These conditions varied in frozen stored mantle probably due to

differential freeze denaturation.

3.4. Histological analysis

The arrangement of structural proteins and the changes brought in these with frozen storage

and subsequent cooking effect texture and it’s evident in histological analysis (Fig.5). The

myotomes in shark muscle tissue are arranged parallelly and enveloped by collagenous sheath

that are fibrous in nature. During frozen storage and subsequent cooking, sarcolemma

disintegrates and plasmolemma splits-off from fibre and large spaces containing fragmentary

and precipitated material are formed. Apart from contraction, the predominant change

observed is the gradual increase in permeability of membranous structures in frozen storage.

On freezing, the ammoniacal odour in the tissue is lost considerably making it more

acceptable. The cell detachment during cooking could be due to the loss of intercellular

integrity through loosening of collagen fibres. The firmness of cooked muscle is observed to

be weak and constant. The specific influences of connective tissue on texture depend on their

thickness, amount of collagen, the density and type of cross-linkages between fibrils. Shark

muscle with its higher collagen content would contribute significantly to the muscle texture.

Gelation of shark collagen is also a contributing factor in effecting the texture during frozen

storage and cooking. Nomura et al. observed that shark collagen denature at 15 C or lower

temperatures [37]. With progressive cooking in both fresh and frozen stored samples, the


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collagen undergoes gelatinization and myotomes disperse in the gel. Due to low denaturation

temperature of shark collagen, this gelation could be more prominent in frozen samples and

myotomes fragment and feebly scatter in the gel. This is due to the structural deterioration of

myofibrillar proteins and collagen, and loss of inherent water holding capacity by the protein

fractions [38]. In fresh samples, myotomes are compact and restrained within the collagen

gel. Additionally, freezing and frozen storage cause formation and development of ice

crystals in the tissue that causes lyses and disintegrate the tissue integrity. However, this

phenomenon is not distinguished in shark probably because of low temperature denaturation

and gelatinization of protein molecules.

4. Conclusions

Shark has high protein content (22.01 %) of which myofibrillar protein (45.62 %) and pepsin

soluble collagen (13.69 %) contribute significantly to the textural variations. Non-protein

nitrogen including urea provides off-odour to shark meat and contributed to the sarcoplasmic

protein fraction, which was hydrolysed during frozen storage and cooking giving no odour in

the fish tissue. Water binding potential and pH influence protein functionality during frozen

storage and cooking. Frozen storage and subsequent cooking affect myofibrillar protein and

collagen and affect muscle texture of shark muscle tissue. Frozen storage affect extractability

through aggregation while cooking caused gelatinization of protein molecules. Histochemical

studies indicated parallel arrangement of myotomes enveloped by collagenous sheath.

Optimum cook temperature for fresh shark tissue samples was 70 C while it shifted to 80 C

with frozen storage.

Acknowledgement

Sincere acknowledgements are due to Mr. Baiju, School of Industrial Fisheries, Cochin

University of Science and Technology, for helping us with the statistical analysis.

List of figure captions Figure 1. Variation in protein fractions: (a) Sarcoplasmic (SP) and myofibrillar fractions

(MY), (b) Total collagen (TC) and pepsin soluble collagen (PSC) in fresh (F) and frozen-

stored samples (Fr), (Mean±SE, n=3)

Figure 2. SDS-PAGE of protein fractions of shark muscle tissue, F-Fresh, Fr-Frozen-stored,

a-sarcoplasmic protein, b-myofibrillar protein, c-pepsin soluble collagen.


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Figure 3 Texture profile analysis: (a) Hardness 1 and Hardness 2, (b) Cohessiveness, (c)

Springiness, (d) Stiffness and (e) Organoleptic score

Figure 4 Automated surface fitting analysis using TableCurve 3D. 5 (a) three dimensional

plot of sarcoplasmic protein and hardness, 5 (b) myofibrillar protein and hardness, and 5 (c)

collagen and hardness, at varying cook temperatures

Figure 5 Histological sections of fresh and frozen samples of shark muscle tissue, Scoliodon

sorrokawah, Fresh sample: (a) uncooked, (b)-50C, (c)-70C and (d)-90C, Frozen stored

samples: (a) uncooked, (b)-50C, (c)-80C and (d)-90C

Table 1. Physical characteristics: expressible moisture and water binding potential of fresh and

frozen-stored shark muscle tissue cooked at various temperatures (Meanse; n=3)*

F- Fresh, Fr- Frozen stored six months

UC-Uncooked, C1-45C, C2-50C, C3-55C, C4-60C, C5-65C, C6-70C,C7-75C, C8-80C, C9-

90C

Means with different uppercase indicate significant differences within the condition (A-P<0.001, B-

P<0.01, C- P<0.05); Means with different lowercase indicate significant differences between the

conditions (a-P<0.05)

Treatments EM (%) WBP(g)

F Fr F Fr

UC 74.20.2C 87.50.0Ba 1.70.0 1.60.0Aa

C1 71.70.0B 88.20.0Ba 1.50.0 1.80.0Aa

C2 75.40.0C 77.80.0Aa 1.80.0 2.70.0Ba

C3 67.10.1A 73.30.1Aa 1.90.1 2.30.0Ba

C4 77.50.0C 85.80.0Ba 2.00.0A 2.00.0Ba

C5 74.20.1C 85.80.0Ba 2.10.0A 2.30.0Ba

C6 73.90.1C 86.90.1Ba 1.70.0 1.90.0Ba

C7 76.40.0C 80.20.0Aa 1.50.0 1.90.0Ba

C8 79.00.1C 81.20.0Aa 1.30.0A 1.90.0Ba

C9 75.20.0C 75.60.0Aa 1.20.0A 1.40.0Aa


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Fig. 1

Fig. 2

Fig. 3


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Fig. 4. Fig. 5.


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