QUALITY CHANGES DURING FROZEN STORAGE AND COOKING OF MILK …
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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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References [1] Abbas, K.A., Mohamed, A., Jamilah, B. and Ebrahimian, M. (2008) A review on correlations between
fish freshness and pH during cold storage. American Journal of Biochemistry and Biotechnology, 4, 416-421.
[2] Johnston, I.A., Alderson, R., Sandham, C., Dingwall, A., Mitchell, D., Selkirk, C., Nickell, D., Baker, R., Robertson, B., Whyte, D. and Springate, J. (2000) Muscle fibre density in relation to the colour and texture of smoked atlantic salmon (Salmo salar L.). Aquaculture, 189, 335-349.
[3] Pati, F., Adhikari, B. and Dhara, S. (2010) Isolation and characterization of fish scale collagen of higher thermal stability. Bioresource Technology, 101, 3737-3742.
[4] Foh, M.B.K., Amadou, I., Kamara, M.T., Foh, B.M. and Xia, W. (2011) Effect of enzymatic hydrolysis on the nutritional and functional properties of nile tilapia (Oreochromis niloticus) proteins. American Journal of Biochemistry and Molecular Biology, 1, 54-67.
[5] Tidwell, J.H. and Allan, G.L. (2001) Fish as food: aquaculture’s contribution. EMBO Reports, 2, 958-963.
[6] Kittiphattanabawon, P., Benjakul, S., Visessanguan, W. and Shahidi, F. (2010) Effect of extraction temperature on functional properties and antioxidative activities of gelatin from shark skin. Food and Bioprocess Technology, 5, 2646-2654.
[7] Dewi, R.S., Huda, N. and Ahmad, R. (2011) Changes in the physiochemical properties, microstructure and sensory characteristics of shark dendeng using different drying methods. American Journal of Food Technology, 6, 149-157.
[8] Huang, Z.-R., Lin, Y.-K. and Fang, J.-Y. (2009) Biological and pharmacological activities of squalene and related compounds: potential uses in cosmetic dermatology. Molecules, 14,. 540-554.
[9] Bargahi, A. and Rabbani-Chadegani, A. (2008) Angiogenic inhibitor protein fractions derived from shark cartilage. Bioscience Reports, 28, 15-21.
[10] Hallier, A., Chevallier, S., Serot, T. and Prost, C. (2008) Freezing-thawing effects on the colour and texture of European catfish flesh. International Journal of Food Science and Technology, 43, 1253-1262.
[11] Baylan, M., Ozcan, B.D., Kucukgulmez, A. and Yanar, Y. (2011) Effects of cooking methods on electrophoretic patterns of rainbow trout. Italian Journal of Animal Science, 10, e33.
[12] Codex Alimentarius Commission. (1994) Thawing procedures for quick frozen fish blocks: air thaw and water immersion methods. Jane E. Fox-Dobson, National Marine Fisheries Service, Silver Spring MD (USA).
[13] Paul, C.P., Buchter, L. and Wlerery, A. (1966) Solubility of rabbit muscle protein after various time-temperature treatments. Journal of Agriculture and Food Chemistry, 14, 490-492.
[14] Ng, C.S. (1978) Measurement of free and expressible drips. In: Manual on analytical methods and procedure for fish and fish products laboratory. Hasegawa, H. (Ed.) Southeast Asian Fisheries Development Centre, Singapore.
[15] Borresen, T. (1980) Developments of methods to determine the water binding properties in fish minces (Norwegian). FTFI Nr. 663. 1/7/2, Tromso. 26.
[16] Association of Official Analytical Chemists (AOAC). (2000) Official Methods of Analysis of Association of Analytical chemists international. 17th Ed. Horwitz, W. (Ed). AOAC International Publishers, Maryland, USA.
[17] Hashimoto, K., Watabe, S., Konu, M. and Shiro, K. (1979) Muscle protein composition of sardine and mackerel. Bulletin of Japanese Society of Scientific Fisheries, 45, 1435-1441.
[18] Mizuta, S., Yoshinaka, R., Sato, M. and Sakaguchi, M. (1994) Isolation and partial characterization of two distinct types of collagen in the squid Todarodes pacificus. Fisheries Science, 60, 467-471.
[19] Raman, M. and Mathew, S. (2005) Effect of structural proteins on the textural characteristics of rohu and squid at various cook temperatures. Journal of Food Science and Technology, 42, 430-434.
[20] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685.
[21] Bourne, M.C. (1978) Texture profile analysis. Food Technology, 32, 62-65. [22] Jowitt, R. (1974) The terminology of food texture. Journal of Texture Studies, 5, 351-358. [23] Ali, F.H.M. (2011) Quality evaluation of some fresh and imported frozen seafood. Advanced Journal of
Food Science and Technology, 3, 83-88. [24] Tomlinson, N., Jonas, R.E.E. and Geiger, S.E. (1963) Glycolysis in ligocod muscle during frozen
storage. Journal of the Fisheries Research Board of Canada, 20, 1145-1152. [25] Kristensen, L. and Purslow, P.P. (2001) The effect of ageing on the water holding capacity of pork:
role of cytoskeletal proteins. Meat Science, 58, 17-23.
JOURNAL OF INTERNATIONAL ACADEMIC RESEARCH FOR MULTIDISCIPLINARY Impact Factor 1.393, ISSN: 2320-5083, Volume 2, Issue 4, May 2014
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[26] Arannilewa, S.T., Salawu, S.O., Sorungbe, A.A. and Ola-salawu, B.B. (2005) Effect of frozen period on the chemical, microbiological and sensory quality of frozen tilapia fish (Sarotherodun galiaenus). African Journal of Biotechnology, 4, 852-855.
[27] Sikorski, Z.F. and Sunpan, B. (1992) Preservation of seafood quality. In: Seafood chemistry, processing technology and quality. (Eds. Shahidi and Botter). London, 168.
[28] Gooch, J.A., Hale, M.B., Brown, T. Jr., Bonnet, J.C., Brand, C.G. and Regier, L.W. (1987) Proximate and fatty acid composition of 40 Southeastern U.S. Finfish species. NOAA Technical Report, NMFS. 54, 1-28.
[29] Yongsawatdigul, J. and Park, J.W. (2004) Effects of acid and alkali solubilization on gelation characteristics of rockfish muscle proteins. Journal of Food Science, 69, 499-505.
[30] Seibel, B.A. and Walsh, J.P. (2002) Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage. Journal of Experimental Biology, 205, 297-306.
[31] Hassan, F. and Mathew, S. (1999) Physicochemical, microbiological and sensory characteristics of washed mince prepared from some selected species of fish”, Journal of Food Science and Technology, 36, 459-462.
[32] Ghelichpour, M. and Shabanpour, B. (2011) The investigation of proximate composition and protein solubility in processed mullet fillets. International Food Research Journal, 18, 1343-1347.
[33] Ng, X.Y. and Huda, N. (2011) Thermal gelation properties and quality characteristics of duck surimi-like material (duckrimi) as affected by the selected washing processes. International Food Research Journal, 18, 731-740
[34] Chéret, R., Hernández-Andrés, A., Delbarre-Ladrat, C., De Lamballerie, M., & Verrez-Bagnis, V. (2006) Proteins and proteolytic activity changes during refrigerated storage in sea bass (Dicentrarchus labrax L.) muscle after high-pressure treatment. European Food Research and Technology, 222, 527-535.
[35] Tabarestani, S.H., Maghsoudlou, Y., Motamedzadegan, A., Mahoonak, S.A.R. and Rostamzad, H. (2012) Study on Some Properties of Acid-soluble Collagens Isolated from Fish Skin and Bones of Rainbow Trout (Onchorhynchus mykiss). International Food Research Journal, 19, 251-257.
[36] Hassan, F., Mathew, S. and Mukundan, M.K. (2003) Gelling and Storage Performance of Surimi Prepared from Some Tropical Fish. Indian Journal of Fisheries, 50, 431-437.
[37] Nomura, Y., Toki, S., Ishii, Y. and Shirai, K. (2000) Improvement of the Material Property of Shark Type I Collagen by Composing With Pig Type I Collagen. Journal of Agriculture and Food Chemistry, 48, 2028-2032.
[38] Makri, M. (2009) Biochemical and Textural Properties of Frozen Stored (-22°C) Gilthead Seabream (Sparus aurata) Fillets. African J. of Biotechnology, 8, 1287-1299