PHYSICO-CHEMICAL AND FUNCTIONAL PROPERTIES OF PROTEINS OF TILAPIA (OREOCHROMIS MOSSAMBICUS)

L.N. MURTHY1,3, S.K. PANDA1 and B.A. SHAMASUNDAR2

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Veraval Research Centre of Central Institute of Fisheries Technology Matsya Bhavan, Bhidia Veraval 362 269, Gujarat, India College of Fisheries, Karnataka Veterinary Animal and Fisheries Sciences University, Hoige Bazar Mangalore 575001, Karnataka, India
Accepted for Publication August 26, 2008
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ABSTRACT The properties of total proteins from fresh tilapia (Oreochromis mossambicus) have been assessed. The meat had high moisture (81.34%) and low lipid (<1%) content. The nitrogen solubility index, with water as solvent, showed minimum solubility of 25.52% of total nitrogen at pH 6.0. The solubility prole of total proteins as a function of molar concentration of sodium chloride indicated maximum solubility at 0.8 M and thereafter it decreased. The total proteins comprised three different fractions as revealed by gel ltration prole. The high molecular weight component was more predominant. The sodium dodecyl sulphate polyacrylamide gel electrophoresis pattern revealed multiple bands in the molecular weight range of 20518 kDa. Higher value of adenosine triphosphatase (ATPase) activity (4.1 mg Pi/mg of protein/ min) obtained in the present study may be due to combination of sarcoplasmic and myobrillar ATPase activity. High modori-inducing proteases (MIPase) activity at 55C was observed in the muscle extract that might interfere in the gelling ability. The gel-forming ability of tilapia meat was found to be moderate as indicated by large strain and small strain test.
jfpe_338 83..107

PRACTICAL APPLICATIONS Tilapia is one of the growing aquacultured species all over the world. In India, the mossambique Tilapia (Oreochromis mossambicus) was introduced
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Corresponding author. TEL: +91-2876-231297; FAX: +91-2876-231576; EMAIL: murthycift@ gmail.com

Journal of Food Process Engineering 34 (2011) 83107. All Rights Reserved. Copyright the Authors Journal Compilation 2009 Wiley Periodicals, Inc. DOI: 10.1111/j.1745-4530.2008.00338.x

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in inland water bodies like reservoirs to boost the production. Because of its prolic breeding habit and occupation of similar trophic niches, this species has overpopulated many bodies of water. Hence, there is growing need to utilize this species for surimi production and, consequently, generate more revenues for the inland sheries sector. Knowledge of the physicochemical and functional properties of this species would help in understanding its suitability in surimi production. The rheological properties like dynamic viscoelastic behavior, ow prole, Ca-ATPase activity, and MIPase activity would shed light on the nal gel strength of the surimi prepared from this species, and also indicate the guidelines for ameliorating the quality of the gel by addition of different concentrations of cryoprotectants or change of washing cycles.

INTRODUCTION The aquaculture sector in India has shown signicantly higher growth rate than the capture sheries during the last decade (Ayyappan and Jena 2001). In order to boost the freshwater aquaculture, many other alternate species like catshes and scampi have been attempted. The only nsh, which is aquacultured all over the world with good export potential for international trade, is tilapia. Farmed sh tilapia is becoming an increasing substitute for traditional white sh species. The global production has been greatly inuenced by rapid expansion of Nile tilapia (Oreochromis niloticus) and mossambique tilapia (O. mossambicus) cultured in China, the Philippines and Egypt (Hempel 2002). Nile tilapia now dominates global tilapia culture and its share was about 83% of total tilapia production in 2000 (FAO and FISHSTAT 2002). Asias production of tilapia from aquaculture is now more than a third of the worlds output of more than 2 million metric ton. Mossambique tilapia introduced prior to the end of the 1960s in many of the reservoirs of Southern states of India is contributing signicantly to the commercial catches in many reservoirs. Gradual dominance of this specie in the catches in many reservoirs has been documented. Sugunan (1995) reported that tropical reservoirs in India provided suitable habitats for O. mossambicus, and it has established self-sustaining populations in a number of south Indian reservoirs. In this context, suitability of this species for preparation of surimi is of utmost importance to development and diversication of shery enterprise in India. In India, mainly species of marine origin such as threadn bream (Nemipterus japonicus), ribbon sh (Trichiurus lepturus) and lizard sh (Saurida tumbil) are being used for surimi production (Muraleedharan et al. 1997). Physicochemical and functional properties of the protein of sh species intended for surimi production play a pivotal role in the end product quality. Important studies on physicochemical and functional properties of proteins

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extracted from sh species include that of Indian mackerel (Rastrelliger kanagurta) (Mohan et al. 2008), inuence of pH on the solubility and conformational characteristics of muscle proteins from mullet (Mugil cephalus) (Mohan et al. 2007), Bigeye snapper (Priacanthus hamrur) (Binsi et al. 2007), rheological properties of silver carp actomyosin (AM; Liu et al. 2008), changes in textural and rheological properties of gels from Nile tilapia muscle proteins induced by high pressure and setting (Hwang et al. 2007) and dynamic viscoelastic properties of grass carp myosin (Tao et al. 2007). Condition of the sh species and seasonal variation also inuences the functional properties of muscle proteins including gel-forming ability (Osako et al. 2003; Chopin et al. 2007). Information on characteristics of washed mince from O. mossambicus is very limited. Studies undertaken by Gopakumar et al. (1992), Hasan and Mathew (1999) and Ninan et al. (2004) have highlighted some aspects of washed mince characteristics of tilapia meat. As alterations in functional properties of sh proteins are areas of interest for commercial application (Mohan et al. 2007), in this study an attempt is made to investigate the important physicochemical and functional properties of proteins from freshly harvested mossambique tilapia (O. mossambicus) and its suitability for surimi preparation.

MATERIALS AND METHODS Fresh tilapia (O. mossambicus) caught from a natural freshwater body near Mysore, South India was used for the study. The length of sh used was 2128.5 cm, weighing 150340 g. Immediately after harvest, the sh samples were washed and iced in the ratio of 1:1 (sh : ice) and transported to the laboratory. Head and entrails were removed manually and washed with chilled water (3C) and subjected to further analysis. Proximate Composition of Tilapia Meat Meat was separated manually and macerated well using a pestle and mortar. The macerated meat was used for proximate composition. Moisture, crude protein, fat and ash content in the meat were determined as per method in AOAC (2000). pH The pH of tilapia meat samples was measured using a pH meter (Systronic 324 pH meter, Ahemdabad, India). Five grams of meat was macerated with 45 mL of distilled water, and the pH was monitored.

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Expressible Water Content Expressible water content of fresh sh meat was determined by the method of Okada (1963). A pressure of 10 kg/cm2 was applied for 10 s over the samples using an Okada expressible water-measuring instrument (Saitama Keiki Seisa Kujo Co Ltd., Japan). Expressible water content was expressed as percent of meat based on the quantity of water absorbed by lter paper. Nonprotein Nitrogen Content Nonprotein nitrogen (NPN) content of tilapia meat was determined by the method described by Velankar and Govindan (1958), using trichloro acetic acid (TCA) extract and was expressed as mg/100 g of meat. About 3.0 g of meat was macerated with 15 mL of 15% TCA for 5 min using a dried pestle and mortar. The homogenate was allowed to stand at 4C for 30 min. The slurry was ltered and made up to 50 mL with distilled water and 5 mL of aliquot was taken for nitrogen estimation using the Kjeldahl method. Solubility of Protein in High Ionic Strength Buffer Total proteins were extracted using the extraction buffer (EB; 50 mM Phosphate buffer; pH 7.5, containing 1 M NaCl). The meat was homogenized with 10 volumes of buffer using an Ultra-Turrax homogenizer (Ultra-Turrax, T25, Janke & Kunkel GMBH & Co., Staufen, Germany) at 9,000 rpm for 2 min. The homogenate was centrifuged at 9,000 g for 15 min using a refrigerated centrifuge (Intl. equipment Co., IEC, B22, Needham Heights, MA) maintained at 4C. The total nitrogen content of the clear supernatant was determined by the Kjeldahl method. The nitrogen value obtained was multiplied by a factor of 6.25 to obtain the protein content and expressed as a percentage of total protein. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) was performed for the whole protein extract as described by Laemmli (1970).The concentration of acrylamide was 10%. The thickness of the gel was 0.75 mm. Electrophoresis was carried out at a constant current mode in a vertical slab gel electrophoresis apparatus (Hoefer-Pharmacia Biotech Inc., SE-50, San Francisco, CA). A standard marker protein mixture of high range of molecular weight obtained from Sigma Chemicals (St. Louis, MO) was loaded into a separate well. Apparent Reduced Viscosity Apparent reduced viscosity of total proteins extracted in EB was measured at 25C 1C using an Ostwalds viscometer. Proteins were extracted

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with 10 volumes of EB. The slurry was homogenized at 9,000 rpm for 2 min and centrifuged at 9,000 g for 15 min at 4C. The clear supernatant obtained was used for viscosity measurements after determining protein concentration by the Lowry method (Lowry et al. 1951). The reduced viscosity at different protein concentration was calculated by the method as described by Yang (1961) and expressed as mL/mg of protein in the solution. Apparent reduced viscosity was plotted against different protein concentration (mg/mL).

red = (t1 t0 ) (t0 C )

where hred = reduced viscosity (mL/mg) t1 = ow time for protein solution (sec) t0 = ow time for solvent, EB (sec) C = concentration of protein in the solution (mg/mL) A plot of apparent reduced viscosity at a single protein concentration of 3 mg/mL (derivative value) during the study period was obtained. Gel Filtration Gel ltration prole of total proteins extracted using EB was carried out on a Sepharose 6B gel packed in a column of 1.5 80 cm (diameter height) at ambient temperature (27C). The eluant used was EB. A protein concentration of 4 mg/mL was loaded onto the column, and elution was carried out at a ow rate of 30 mL/h. Fractions (3 mL) were collected manually, and the concentration of the eluant was determined by measuring the absorbance at 280 nm using a spectrophotometer (Bausch and Lomb, Model 21-UVD, Austin, TX). Calcium-Activated ATPase Activity Calcium-activated ATPase activity was determined according to the method of Noguchi and Matsumoto (1970) and expressed as microgram of inorganic phosphorus (Pi/mg protein/min at 27C). About 1 g of meat was macerated with 10 mL of 50 mM glycine-NaOH buffer, pH 9.2. The slurry was ltered through Whatman No. 1 lter paper, and the ltrate was used as enzyme solution. The reaction mixture containing 0.06 mL of ATP solution (0.05 M), 0.4 mL CaCl2 (0.1 M), 2 mL buffer (0.05 M glycine-NaOH, pH 9.2) and 0.4 mL of meat extract was made up to 4.0 mL with buffer and incubated at 27C for 5 min. The reaction was stopped by addition of 2 mL of 15% TCA. The blank was carried out by adding 15% TCA before the enzyme was added.

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The mixture was ltered through Whatman No. 1 lter paper and the inorganic phosphorus released was estimated according to the procedure described by Taussky and Shorr (1952). Dynamic Viscoelastic Behavior Dynamic viscoelastic behavior (DVB) of tilapia meat in the temperature range of 3090C was measured using a Carri-Med Controlled Stress Rheometer (CSR-500, Carri Med, Surrey, U.K.) under oscillatory mode, using a 4-cm parallel plate measuring geometry. Fish meat devoid of connective tissue, ns, and scales was macerated well using pestle and mortar. About 4 g of macerated tilapia meat was mixed with 2.5% sodium chloride (w/w) and mixed thoroughly to get a ne ground paste. The sh paste obtained was used for DVB measurement. The gap between measuring geometry and peltier plate was adjusted to 2,000 mm. The gap was set manually at 80 using the micrometer. The applied stress of 500 Pa was within the viscoelastic region. The linear viscoelastic region was determined by a torque sweep with a frequency of 1 Hz. Measurements were made by applying a small amplitude oscillation (0.0005 rad) with a frequency of 1 Hz. A heating rate of 1C per min was achieved through peltier plate of rheometer. Applied stress was compared with the resultant strain. The results of such measurement were expressed as the storage modulus (G) and loss modulus (G). The DVB of tilapia fresh sh meat was assessed. An average of three replicates was used for plotting the results. Nitrogen Solubility Index The nitrogen solubility index (NSI) of fresh tilapia meat as a function of pH with distilled water as solvent was determined. About 2 g of fresh meat was homogenized with 28 mL of distilled water using homogenizer of Nihon Seiki Kogyo Co., Japan. The pH of the slurry was adjusted to 7.0 using either 0.1 N HCl or 0.1 N NaOH. The slurry was centrifuged at 9,000 g for 15 min at 4C using IEC B22 centrifuge. An aliquot of clear supernatant was taken for nitrogen estimation by Kjeldahl method. Nitrogen content in the supernatant was expressed as percentage of total nitrogen in the meat after due consideration of volume of acid or alkali consumed at each pH level. NSI was obtained by plotting pH versus percentage of total nitrogen solubilized. Effect of NaCl Concentration on Solubility of Total Proteins from Tilapia Meat Two grams of meat from fresh sh was homogenized with 18 mL phosphate buffer (5.0 mM, pH 7.5) containing 0, 0.3, 0.6, 0.8, 1.0, 1.5, and 2 M of sodium chloride separately at 3,000 rpm for 2 min using a homogenizer

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(Nihon Seiki Kogyo Co. Japan). Homogenate was centrifuged at 9,000 g for 15 min at 4C using IEC refrigerated centrifuge. An aliquot of supernatant was used for nitrogen estimation by Kjeldahl method. From the nitrogen value, crude protein content was determined. Solubility curve was obtained by plotting the percentage of soluble proteins of total protein against molar concentration of sodium chloride in phosphate buffer. Sulphydral Group Sulphydral group in the tilapia meat was estimated by the Ellman method (Ellman 1959). Two grams of meat sample was homogenized with 30 mL of EB for 1 min, homogenized sample was ltered, 3 mL of ltrate was taken and with phosphate buffer (50 mM, pH 8.0) and the volume was made up to 10 mL. From this, 3 mL was pipetted out and 1 mL of Ellmans reagent was added. The intensity of the color was read at 412 nm using in Philips UV/VIS Spectrophotometer using phosphate buffer as blank. Gel Strength Measurement Fish meat obtained from tilapia was ground with 2.5% sodium chloride using mortar and pestle and then maintained at 30C for 1 h. It was stuffed into synthetic Krehlon casings (3 cm 15 cm length). The casings were sealed using aluminum wire and processed at 90C for 45 min in a thermostatically controlled water bath. The gel obtained was kept at 4C overnight and same was used for the measuring of gel strength using Okada gellometer by the method described by Okada and Yamazaki (1957).The movement of stylus on the kymograph was recorded and the gel strength was measured by calculating the area under the graph. Fold test was carried out by cutting a prepared gel into 25 mm thickness, and rst, second and third folding was done manually. If it broke in the rst fold, it was graded as A, second as B and third as C. Again it was conrmed by gel strength measurements. Flow Prole Measurements (Shear Stress Sweep) The total proteins for ow prole measurements were extracted from meat using EB. Two grams of meat were homogenized with 18 mL of EB and centrifuged at 9,000 g for 15 min. The supernatant was used for measuring the ow properties. The ow properties of the protein solutions were measured at the different temperatures viz., 28C and 40C using a Carri-med Controlled Stress Rheometer by applying varying stress. The sample was equilibrated for 5 min before the shearing experiment was started. The measuring geometry

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used was 4 cm cone and plate with a truncation of 59 m. The range of stress applied varied between 26 Pa, depending on the angular velocity in the preshear experiment. The ascend and descend time were 2 min each, with a peak hold time of 1 min. Shear stress sweep of the protein solutions at different temperature was performed in triplicate and average value was taken for plotting. A ow curve was obtained by plotting log viscosity and log shear rate values. Assay of Modori-Inducing Proteases Activity Modori-inducing protease (MIPase) in tilapia meat was determined by the method as described by An et al. (1994). The crude enzyme extract was prepared by homogenizing 5 g of fresh tilapia meat with 15 mL phosphate buffer (50 mM pH 7.5) at 3,000 rpm for 2 min in a homogenizer (Nihon Seiki Kogyo Co., Tokyo, Japan). The homogenate was centrifuged at 8,000 g for 15 min. The supernatant was diluted with phosphate buffer in the ratio of 1:1 and used as the crude enzyme extract. The substrate solution was prepared by dissolving 4 mg casein in 1.25 mL phosphate buffer (50 mM, pH 7.5) and made up to 2 mL with distilled water. To determine the MIPase activity, 2 mL of the substrate solution was pre-incubated at 55 1C for 5 min in water bath. To this, 0.5 mL of crude enzyme was added and again incubated at 55 1C for 1 h for the enzymatic reaction to occur. The reaction was terminated by adding 1 mL cold 20% TCA. The slurry was centrifuged at 8,000 g for 15 min at 4C. The supernatant containing the hydrolyzed oligopeptide was collected without disturbing the pellet. The oligopeptide content in the supernatant was determined by measuring the absorbance at 280 nm in a UV-VIS Spectrophotometer (21-UVD, Austin, TX). The blank was prepared in the same manner except that the crude enzyme extract was added immediately after TCA precipitation. The enzyme activity was expressed as the difference of absorbance at 280 nm (D280 nm) between the sample and the blank.

RESULTS Physical Characteristics The tilapia (O. mossambicus) sh used in the present study had a size range of 2128.5 cm (average of 23.76 cm) and weight of individual shes varied between 150 and 340 g (average of 252.6 g). The dressed yield of whole tilapia was 64.3% and separated meat yield was 44.44% (Table 1). The proximate composition of fresh tilapia meat is shown in Table 2.

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TABLE 1. PHYSICAL CHARACTERISTICS OF FRESH TILAPIA (OREOCHROMIS MOSSAMBICUS) Average length (cm) Standard length (cm) Average weight (g) Dressed yield (%)* Separated meat yield (%) Values in parenthesis are SD. n = 25. * After removal of head, entrails and scales. From whole sh to separated meat. 23.76 (2.23) 18.9 (1.9) 252.6 (56.9) 64.3 44.44

TABLE 2. COMPOSITION AND PROPERTIES OF TOTAL PROTEINS FROM FRESH TILAPIA (OREOCHROMIS MOSSAMBICUS) MEAT Parameters Moisture (%) Total protein (%) Total fat (%) Total ash (%) Nonprotein nitrogen (mg/100 g of meat) pH Gel strength (g-cm) Expressible water content (%) of the gel Ca++ ATPase activity mg pi/mg of protein/min Sulphydral content (mM of -SH/g of meat) Extractability in extraction buffer (% total protein) High modori-inducing proteases activity DA280 nm Fresh sh 81.34 (1.7) 16.72 (0.55) 0.88 (0.02) 0.99 (0.07) 365.00 (0.52) 7.00 (0.12) 569.39 (7.93) 22.36 (1.23) 4.10 (0.07) 0.0029 (1.1 10-2) 86.51 (3.31) 0.271 (0.06)

n = 3, values in parenthesis indicate standard deviation.

Properties of Total Proteins The NSI of total proteins from fresh tilapia meat with water as a solvent is presented in Fig. 1. Maximum solubility of nitrogen was at pH 10.2 and minimum solubility of 25.52% of total protein was recorded at pH 5.8. The protein extractability from tilapia meat as a function of molar concentration of sodium chloride is given in Fig. 2. The solubility increased with increase in sodium chloride concentration and beyond 1.0 M, the solubility of proteins decreased. Nearly 8589% of proteins are soluble at the sodium chloride concentration of 0.81.0 M.

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FIG. 1. NITROGEN SOLUBILITY INDEX OF TOTAL PROTEINS FROM FRESH TILAPIA MEAT WITH WATER AS A SOLVENT

FIG. 2. SOLUBILITY PROFILE OF TOTAL PROTEINS FROM FRESH TILAPIA MEAT AS A FUNCTION OF MOLAR CONCENTRATION OF SODIUM CHLORIDE

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FIG. 3. GEL FILTRATION PROFILE OF TOTAL PROTEINS FROM FRESH TILAPIA WITH EB AS ELUANT

The apparent reduced viscosity of total proteins from fresh tilapia meat was plotted against protein concentration. With increase in protein concentration, the apparent reduced viscosity increased and gave rise to a positive slope. At single protein concentration of 3 mg/mL, an apparent reduced viscosity value of 0.09 mL/mg was obtained. The gel ltration prole of total proteins from fresh tilapia meat is depicted in Fig. 3. The prole indicated one high molecular weight component eluting at an elution volume of 67.89 mL and two low molecular weight components eluting at elution volume of 127 and 129.86 mL, respectively. The calcium ATPase enzyme activity of total proteins from fresh tilapia meat was found to be 4.10 mg Pi/mg of protein/min (Table 2). The catheptic activity of fresh tilapia muscle showed the activity at 55C (Table 2). The DA280 nm is taken as index of catheptic activity. The free sulphydral content of fresh tilapia meat was found to be 2.9 10-4 mM of -SH/g of meat (Table 2). The electrophoretic mobility of total proteins from fresh tilapia meat under reduced condition is given in Fig. 4. In the gure, lane s represents standard marker and lane a total proteins from fresh tilapia. The pattern revealed multiple bands with the molecular weight range of 20514 kD. The intensity of 205 kD bands (myosin heavy chain) was higher. The gel strength of the gel prepared from fresh tilapia meat was found to be 569.39 g-cm. The expressible water content of gel was 22.3% (Table 2). The DVB behavior of fresh tilapia meat in the temperature range of 3090C is

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FIG. 4. SDS-PAGE PATTERN OF TOTAL PROTEINS FROM FRESH TILAPIA MEAT

given in Fig. 5. The storage modulus value (G) increased with increase in temperature but rate of increase was found to be maximum between 50 and 56.7C. The loss modulus value (G) also increased with the increase in temperature, but the absolute values were considerably lower than those of G values. The temperature at which solgel transition occurred as indicated by tan d values was found to be at 50.1 and 67.6C. The shear stress sweep of total proteins from the fresh tilapia meat in EB as a solvent was carried out using ow prole software. The shear stress sweep was carried out at 28 and 40C separately. The shear stress is represented both by up and down curve. Figure 6A depicts the shear stress sweep of fresh tilapia at 28C. With increase in shear stress, there was a gradual decrease in viscosity value. With a shear stress of log -2.15 kPa, the corresponding viscosity value was found to be log -3.006 mPas. The down curve data showed, with incremental decrease in shear stress value, that there was marginal change in viscosity value, and upon reaching the original corresponding shear stress value of up curve, the viscosity value were lower (log -3.02 mPas). The shear stress sweep at 40C is depicted in Fig. 6B. At low shear stress value of log -4.31 kPa, the viscosity values obtained were log -0.46 mPas, with further increase in shear stress there was gradual decrease in viscosity. The down curve data indicated there was marginal increase in viscosity with decrease in shear stress value up to log -4.04 kPa, and with further decrease in

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FIG. 5. DYNAMIC VISCO-ELASTIC BEHAVIOR OF FRESH TILAPIA MEAT

shear stress there was a sharp increase in viscosity, reaching a value of log -3.10 mPas. The shear stress sweep carried out at 28 and 40C gave rise to a thixotropic area, and was higher at 40C in Fig. 6B.

DISCUSSION Physical Characteristics and Composition of Proteins from Fresh Tilapia Meat The shes used in the present study were relatively big, both in size and weight. It is not uncommon to nd tilapia less than 10 cm length and 50 g weight, which is an indication of stunted growth. The shes used for the study were obtained from a natural water body near Mysore and it is reasonable to expect the age to be above 1 year. The dressing yield (after removal of head, entrails) was 64.3% and the meat yield from the whole sh was 44% (Table 1). Normally bigger size sh will give higher yield compared to small size sh. The yield of separated meat of the freshwater shes like common carp and silver carp varies from 4047% (Arekere 1993; Siddaiah et al. 2001).

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FIG. 6. FLOW PROFILE OF TOTAL PROTEINS FROM TILAPIA MEAT USING EB AS SOLVENT (A) At 28C and (B) at 40C.

The composition of the meat revealed higher quantity of moisture and less of fat content compared to other freshwater shes. Many researchers have indicated that tilapia sh is a lean variety, where fat content is less than 3% (Akande 1989; Asiedu et al. 1991). The moisture content in the present study (81.34%) is slightly higher than reported value (80.33%) of the same species (Ninan et al. 2008). The total crude protein is comparable to other tilapia species and freshwater shes (Akande 1989; Arekere 1993; Siddaiah et al. 2001). The NPN content accounted for 16% of total nitrogen. This value is higher than other freshwater shes like common carps and other Indian major carps (Chakrabarti 1984). Shenoy and James (1972) found that NPN content of tilapia accounted for 10% of total nitrogen. They also conrmed that the free amino acids constituted for bulk of NPN content. The NPN constituents of marine shes are characterized by presence of trimethyl amine oxide (TMAO), imidazole derivatives, guanidino derivatives and low molecular weight peptides (Mathew et al. 1999).

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Properties of Proteins from Fresh Tilapia Fish Meat The NSI with water as a solvent has shown minimum solubility at pH 5.8 and solubility increased at either side of this pH (Fig. 1). The pH at which minimum solubility occurred is due to net charge being equal and favoring proteinprotein interaction (Hall 1996). The choice of pH and solvent for protein extraction is very critical as the amount of protein solubilized in the solvent will have a bearing on various functional properties (Kinsella 1982). The pH at which the minimum solubility occurred can be considered as isoelectric point and varies for different protein sources. For nsh and shellsh, the isoelectric point is in the range pH 56.2 (Shamasundar and Prakash 1994; Mathew and Shamasundar 2002). From cod head, major part of muscle proteins was recovered by successive extraction at room temperature in dilute NaOH (pH 11) and HCl (pH 22.6) (Arnesen and Gildberg 2006). High solubility of proteins from tilapia at acidic and alkaline pH can be attributed to hydration mediated by additional charge on the surface of proteins. Similar observations were noted by Mohan et al. (2007) in mullet (M. cephalus), where altering the pH of muscle homogenate to acidic or alkaline increased protein solubility. The solubility of total protein from tilapia as a function of molar concentration of NaCl revealed maximum solubilization between 0.81.0 M concentration. The initial increase in solubility is due to salting-in phenomenon (up to 1.0 M concentration), after which decrease in solubility was recorded due to salting-out phenomenon (Lin and Park 1998). Nakai and Li-Chan (1998) indicated the salt concentration was important in determining the functional properties by changing the protein conformation through electrostatic and hydrophobic forces. Lin and Park (1998) studied the changes in solubility prole of salmon myosin as affected by conformational changes at various ionic strength and pH. The increase in solubility correlated with increase in surface hydrophobicity and relative sulphydral content and decreased a-helicicity. In the present study, the maximum solubility occurred at a concentration of 0.81.0 M NaCl. It was decided to use 1.0 M NaCl for extraction and characterization of proteins. In hake (Merluccius merluccius), it was found that protein solubility, apparent viscosity and water-binding capacity presented maximum values at pH levels between 2 and 4, and at concentrations of less than 0.25 M NaCl (Montero et al. 1999). The protein that could be solubilized in EB from tilapia meat was found to be 86% of total proteins. Low amount of lipid in tilapia meat might be one of the reasons for high protein solubility. liyte et al. (2005) had observed raw material containing the highest amount of lipids gave the lowest percentage of solubilized proteins. Solubility in high ionic strength buffer (>0.5 M) is taken as an index of conformational status of the given protein molecule (Colmenero et al. 1988). The amount of protein

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that could be solubilized at high ionic strength or EB depends on initial freshness of sh, preprocessing condition and protein conformation. A technique developed by Kelleher et al. (2004) revealed proteins from animal muscle tissue solubilized under conditions of low ionic strength and low pH retained high yields of proteins. Once precipitated and neutralized, these proteins were capable of producing gels with good functional properties. The measurement of viscosity of protein solutions provides information on size, shape and gross conformation. The reduced viscosity as a function of protein concentration showed an increase in value with increase in protein concentration, which gave rise to positive slope. The proteins from fresh tilapia meat at a single protein concentration of 3 mg/mL yielded a hred value of 0.09 mL/mg. The value of intrinsic viscosity (h) for myosin from carp, cod and rabbit muscle have been reported to be in the range of 1.22.2 mL/mg (Webber and Portzhel 1952; Connell 1954). The variation in the reduced viscosity value at any given concentration of protein solution is inuenced by various factors such as nature of proteins and other composite substances. The intrinsic ) of solutions of randomly coiled macromolecules is often found viscosity ( h to be vastly larger than compact globular structures. Such molecules occupy a very large effective volume in solution, and intrinsic viscosity is primarily a measure of particle volume (Van Holde 1985). When a globular protein molecule is denatured, its conformation is altered to something like that of a random coil. This is accompanied with an increase in the effective volume in ) generally increases. solution so that ( h The total proteins from fresh tilapia extracted in EB (EBPhosphate buffer 50 mM, pH containing 1.0 M NaCl concentration) revealed three different peaks as indicated by gel ltration prole. The rst peak eluting at an elution volume of 67 mL is a high molecular component, which may be AM complex and other two correspond to low molecular weight fractions. In the gel ltration prole of total proteins from fresh ribbonsh, the high molecular weight component was suggested to be AM (Dileep et al. 2005). The low molecular weight components may include peptides, free amino acids and other nonprotein nitrogen constituents. The reduction of low molecular weight components was conrmed by water washing of the meat (Fig. 3). The SDS-PAGE pattern of total protein from fresh tilapia meat (Fig. 4) revealed multiple bands with wide range of molecular weight. The intensity and concentration of these bands varied. The high intensity of 205 kD band in fresh sh meat indicates high concentration of myobrillar proteins. The other bands observed are between 84 and 14 kD, which may be myosin light chain and other myobrillar protein fractions. Studies undertaken by Kim et al. (2003) revealed that sh proteins were highly degraded by acid or alkali treatment, and high activity of cathepsin B-like enzyme was detected from

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acid-aided sh proteins by sodium dodecylsulfate-polyacrylamide gel electrophoresis of Pacic whiting protein. The free sulphydral group present in the fresh tilapia sh meat was found to be 2.9 10-3 mM SH/g of meat. The data indicate the free sulphydral (SH) group present in tilapia sh meat is relatively lower as compared to common carp, milksh and tilapia hybrid, which is in the range of 8 10-2 5.13 10-2 mM/g of meat (Jiang et al. 1988, 1989; Sompongse et al. 1996). The localization of sulphydral groups are limited to myosin heavy chain and few in the light meromyosin (Mackie 1984). The number of free SH group determines the formation of disulphide bond and it is considered as one of the important linkages in improving the textural properties of heat processed products (Niwa 1992). From the present study, it is clear that free SH group in the tilapia sh is relatively lower than compared to other freshwater and marine shes. In recent studies by Mohan et al. (2007), in mullet (M. cephalus), the reactive sulphydryl groups were observed to decrease at acidic and alkaline pH with the lowest at pH 4. The Ca++ ATPase activity of total proteins extracted from TrisHCl buffer was found to be 4.1 mg Pi/mg of protein/min. This value is higher than reported ATPase activity for pink perch, common carp and milksh (Jiang et al. 1989; Arekere 1993; Ratnakumar 1999). The higher value of ATPase activity obtained in the present study may be due to combination of sarcoplasmic and myobrillar ATPase activity. Higher Ca2+ ATPase activity was also noted in Indian mackerel that contributed to the higher emulsion activity index and emulsion stability of muscle protein (Mohan et al. 2008). Measurement of myobrillar ATPase activity gives information of conformational status, and hence, can be used as a tool to monitor the process of denaturation. The proteolytic activity of tilapia meat at 55C (MIPase) has shown appreciable activity (Table 2). The MIPase are gel-weakening agents during heating. The action of these proteases vary among sh species (Morrissey et al. 1995). Yongsawatdigul et al. (2000) have revealed the proteolysis of tilapia surimi occurred as the temperature increased and attains highest activity at 65C. They have demonstrated disappearance of myosin heavy chain due to the enzyme activity at 65C for 4 h. It is important that the heating process regime should pass the optimum temperature of the enzyme as quickly as possible. It is clear that the tilapia sh does have MIPase activity and may interfere in gelling ability. Enzymatic modication responsible for the changes in protein functionality in grass carp (Ctenopharyngodon idella) skin was reported by Wasswa et al. (2007). Gelation of muscle protein results from the ordered aggregation of modied native proteins into a three dimensional elastic network. The changes in stress strain relationship during gelation process could be monitored by rheo-

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logical parameters (Egelandsal et al. 1995). The dynamic rheological test and small strain gel test have been widely used to study the heat-induced gelation of the myobrillar proteins (Visessanguan et al. 2000). The storage modulus (G) is a good index for gel-forming ability of food proteins. The higher the G value, the greater is the gel-forming ability of protein system assuming that the protein gel is made up of highly viscoelastic network. Gelation of food protein is dependent on nature of protein, pH, ionic strength, binding agents and rate of heating (Wang and Xiong 1998). The gelation behavior of a given protein can be understood by monitoring continuously the changes occurring during heating. The type of monitoring can be carried out by small strain test using controlled stress rheometer under oscillation mode. The DVB in the temperature range of 3090C of fresh tilapia sh is given in Fig. 5. The increase in elastic component (storage modulus G) in the temperature range of 3090C was found to be maximum between 6070C. The increase in storage modulus values is an indication of structure builds up reaction because of ordered aggregation on the application of thermal energy (Ziegler and Foegeding 1990). The maximum rate of increase in G values during heating below 60C is likely to involve protein unfolding and formation of disulphide bond and hydrophobic interaction (Niwa et al. 1986). Studies carried by Fukushima et al. (2007) investigated the rheological properties of sh meat pastes by dynamic rheological measurement in a range of 530C, in order to establish the optimum temperatures for shaping. Storage moduli (G) on temperature sweep analysis for walleye pollack, white croaker and threadn bream meat pastes were considerably higher than loss moduli (G) and showed maximum at 20, 27 and 28C, respectively. The G values of fresh tilapia sh meat include myobrillar and sarcoplasmic proteins and sarcoplasmic proteins may inhibit gelation process (An et al. 1994). In another study, the presence of sarcoplasmatic proteins not affecting the quality of functional properties of lms based on muscle proteins of Nile tilapia was observed by Paschoalick et al. (2003). The small strain test being more sensitive could record structure build up reaction more effectively than large strain test, which measures the resistance to application of force to the end product. The viscous element (loss modulus G) followed the same trend as that of G but the intensity of ordered aggregation was much higher, paving way for lower G values. As gel is an end product the phase transition from sol, it occurs at a particular temperature. In the present study, the temperature at which solgel transition occurred is indicated by tan d value and found to be 50.1 and 67.6C. The multiple transitions that occur in myobrillar proteins have been attributed to the oligomeric nature in the major component myosin. Sano et al. (1988) recorded three transitions for carp myosin at 36.7, 43.3 and 52C. They have attributed the rst transition due to myosin tail of the molecule, and second and third transition is due to the head portion of myosin molecule.

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Another important rheological property that helps in assessing gelforming ability is the shear stress sweep (ow prole) of total proteins at a particular temperature. Gelation prole using oscillation software provides useful data on structure build up reaction and solgel transition. The shear stress sweep provides information on resistance to shearing, thereby indicating structural impairment. The shear stress sweep of total protein from tilapia meat has been carried out using controlled stress rheometer with ow prole software. Figure 6A depicts the ow prole of total proteins from fresh tilapia meat at 28C. The prole indicated non-newtonian behavior with clear indication of pseudo plasticity. The prole indicated inability of the protein molecule to attain the original viscosity values in the down curve to that of the up curve. This is evidence of alteration in the structure due to shearing. Though the range of shear stress values applied were very small, the protein molecules gave a different up curve and down prole. The ow behavior of total protein at 40C (Fig. 6B) indicated relatively a small thixotropic area compared to prole at 28C. The down curve indicated a sharp increase in viscosity when the shear stress values were reduced incrementally. This increase in viscosity could be due to aggregation induced at the temperature studied in the experiment. The data on the ow prole of other myobrillar proteins are scanty. The myobrillar proteins from pink perch at 28C revealed a minimum thixotropic area (Karthikeyan et al. 2006). In the present study, it has not been attempted to t the mathematical model to measure the yield stress. It is evident that the total proteins from fresh tilapia at 28 and 40C exhibit non-newtonian pseudoplastic behavior. Liu et al. (2008) studied the rheological properties of AM solutions from silver carp, where AM solution behaved as pseudoplastic uid under all test conditions. Systems with higher protein concentration exhibited bigger consistency coefcients and smaller ow indices than those with lower protein concentration. Gel-forming ability is the key functional property of wide interest in sh processing technology, as it is responsible for the formation of rm texture in the end products. The gel strength indicates the ability of protein molecule to undergo ordered aggregation with entrapment of water. The gel strength of tilapia sh meat is found to be 569.39 g-cm (Table 2), which is moderately good. The gel strength of gel prepared from common carp, silver carp, pink perch, croaker and shark meat were found to be in the range of 400 1,300 g-cm (Arekere 1993; Basavanagouda 2001; Siddaiah et al. 2001; Mathew and Shamasundar 2002). The measurement of gel strength, a large strain test, is routinely used in the quality of analysis of the nal product. The choice of instrument in measuring the gel strength is very critical and the use of Okado gellometer and its modied versions are accepted as standard methods. The fresh tilapia meat has the ability to gel and give moderate gel

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strength above 500 g-cm. The corresponding folding test of the gel revealed the grades B and C. The expressible water content of prepared gels from fresh tilapia sh meat was found to be 22.36% (Table 2). A close relationship between gel texture and water holding capacity of cooked gels has been reported (Lee 1984). However, expressible water is a reection of the physical properties of protein that is related to their charge and structure (Pacheo-Aguilar et al. 1989).

CONCLUSION The present study reects the usefulness of mossambique tilapia (O. mossmbicus) for surimi production and other related value added products. As the physicochemical and other functional properties of proteins of this specie exhibit strikingly similar characteristics with that of other inland sh species cultured and captured in Indian waters, a denite possibility exists in utilization of this species in surimi production, and hence, more economic returns from inland shery sector.

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