Influencia del lavado y almacenamiento congelado en la fraccion de las proteinas miofibrilares de la pulpa de sardina.
El almacenamiento congelado de especies pesqueras, como la sardina, resulta en cambios significativos en sus propiedades funcionales las cuales determinan su tiempo de vida en almacenamiento. La pulpa de sardina se caracteriza por un alto contenido de grasa, musculo oscuro, y proteinas sarcoplasmaticas que inhiben la formacion de geles a base de esta pulpa. Aplicando tratamiento de lavado a la pulpa de sardina se remueven compuestos indeseables para la preparacion de productos a base de esta pulpa y a la vez aumentando su tiempo de vida en anaquel. El objetivo del presente estudio fue evaluar el efecto del almacenamiento en congelacion a -30[grados]C sobre la fraccion de las proteinas miofibrilares de la pulpa de sardina tratada con soluciones al 0,5% de bicarbonato de sodio. Lotes de pulpa de sardina se le aplico tratamiento de lavado con una solucion de bicarbonato de sodio al 0,5% y luego centrifugadas a 300 rpm por 15 min. para la eliminacion del agua remanente. Lotes de 100 gr. fueron empacados en bolsas de polipropileno y almacenadas a -30[grados]C y analizadas cada 30 dias durante150 dias. Las proteinas nmiofibrilares fueron extraidas con buffer fosfato (tris HCI, KCI, EDTA, pH 7,6), y evaluadas por la tecnica de electroforesis, SDS-PAGE. Las bandas de las diferentes proteinas y sus productos de degradacion fueron analizadas y digitalizadas utilizando un Gel Doc 2000 y un programa Quality One 4.1.1 de Bio-Rad. Las principales bandas y sus productos de degradacion fueron identificados por comparacion de estos contra un estandar de peso molecular. A los 60 dias se observo el comienzo del deterioro de las proteinas miofibrilares con pesos moleculares aparentes entre 220 y 65KD, y la formacion de agregados moleculares de alto peso molecular. A los 120 dias este deterioro se hace mas pronunciado apareciendo gran cantidad de bandas de bajo peso molecular, peptidos, los cuales incrementan a medida que transcurre el tiempo de almacenamiento congelado. Sin embargo, estos cambios son menos severos que los observados en la pulpa de sardina sin tratamiento (control). La evaluacion de los cambios que envuelven el deterioro de la pulpa de sardina en congelacion podria ayudar a establecer parametros de calidad y permitir predecir el tiempo de vida util de los productos a base de estas pulpas.
Palabras clave: Sardina, almacenamiento congelado, proteinas miofibrilares, tratamiento con bicarbonato de sodio.
Frozen storage of fish species, such as sardine, result in detrimental changes in functional properties that determine storage life. Sardine meat is characterized by high fat content, dark meat, and sarcoplasmic proteins that inhibit gel formation. Washing mince flesh with solutions such as sodium bicarbonate is very effective for removing undesirable components. The objective of this research was to study the effects of frozen storage at -30[degrees]C in the myofibrillar protein fraction of sardine mince flesh washed with 0.5% sodium bicarbonate solution. Samples of sardine-minced flesh were washed three times with a 0.5% of sodium bicarbonate solution and centrifuged at 3000 rpm for 15 minutes. These samples were divided in lots of 100 g. packed in plastic bags and stored at -30[degrees]C, and analyzed every 30 days for 150 days. The myofibrillar proteins were extracted using a phosphate buffer (tris HCl, KCI, EDTA, pH 7.6), and evaluated by SDS-PAGE. The bands were analyzed and digitalized with a Gel Doc 2000 and Quality One 4.1.1 by Bio-Rad. The main bands of myofibrillar protein were identified by comparison of these against a prestained molecular weight standard. After 60 days there was deterioration of the myofibrillar protein fraction with apparent molecular weight between 220 and 65KD, and the formation of molecular aggregates at high molecular weight occurred. After 120 days due to myofibrillar protein deterioration, protein and peptides with low molecular weight were formed and increasing throughout frozen storage. Understanding the mechanism involved in the deterioration of the mince flesh during frozen storage we would enable to help the establishment of quality parameters and ability to predict storage life for that product.
Key words: Sardine, frozen storage, myofibrillar protein, sodium bicarbonate, washing.
The sardine (Sardinella aurita) is a very important low cost fish resource in Venezuela. The annual catch is about 110,000 metric tons  with large amount of this catch used for food and canned products and a large amount of this catch is used for animal food and canned products. The consumption of fresh sardine or its frozen sardine products is not well accepted by the consumer because of the high fat content, large percentage of dark muscle, and high concentration of sarcoplasmic proteins. One alternative to increase sardine consumption could be the production of sardine mince flesh. Sardine mince flesh production is a relatively simple process in which muscle is separated from bones yielding a dark flesh meat. The process of producing mince flesh combines muscle components such as lipids, sarcoplasmic proteins, and digestive enzymes, inorganic salts, and low molecular weight organic substances that induce myofibrillar protein denaturation. Myofibrillar proteins are the most important muscle component since they are responsible for the texture attributes and functional properties of muscle in foods [4, 5, 15].
Washing treatment on fish mince flesh helps remove those components that produce denaturation of myofibrillar protein and help to increase gel formation and myofibrillar protein concentration for further mince flesh based product production. The importance of washing treatment is to removes pro-oxidants and components susceptible to lipid oxidation. Several studies have been conducted using washing treatment solutions such as sodium chloride, sodium bicarbonate; sodium phosphate and water to enhance the quality of fish mince flesh [4, 5, 10, 12, 16, 19, 20]. These studies stated that treatment of washing on the fish mince flesh significantly reduces soluble proteins, pro-oxidative enzymes, lipids, and increased gel-forming ability and improved color properties of the final product.
Frozen storage of fish mince flesh has been largely used for preservation of food by decreasing microbial. Conversely, during frozen storage fish mince flesh become unstable and undergoes a number of alterations that determine the end of its storage life. Frozen storage induces protein aggregation, causing hardening of the muscle. Myofibrillar proteins undergo denaturation and aggregation when the water and associated solutes in the tissue are lost due to dehydration by freezing. This process produces an undesirable texture for the products elaborated from this raw material. Hydrophobic interactions have been identified as a cause of lower extractability and reduction of the functionality of the myofibrillar protein. Similarly, during frozen storage formaldehyde increases its interaction with myofibrillar proteins accelerating their denaturation and aggregation [1, 3, 7, 8, 9, 13, 17]. Moreover, several researchers have concluded that washing, fish mince, will decrease the stability of its products when frozen due to the removal of oxidative compounds and the increased polarity of the residual lipids [10, 12, 20].
For a better understanding of the effects of storage and the subsequent deterioration of the mince flesh during frozen storage, and to help establish quality parameters that would be used to predict storage life for products made with sardine mince flesh the present study evaluated the effects of frozen storage at -30[degrees]C for 150 days on the myofibrillar protein fraction of sardine flesh washed with 0.5% sodium bicarbonate solution.
MATERIALS AND METHODS
Sardines were caught from Sucre State, Venezuela, and transported in insulated boxes with ice to the Food Science Technology Institute in Caracas. After reaching the laboratory, the fish were deheaded, gutted and treated with 0.5% sodium bicarbonate (NaHC[O.sub.3]) solution (1:5 mince flesh:water) (FIG. 1) following the procedure stated by Barrero and Bello . Mince flesh from sardines treated with NaHC[O.sub.3] 0.5% solution and a control (sardines mince flesh that were not washed) were divided into 100 g lots, packed, frozen at -30[degrees]C and stored at -30[degrees]C for 150 days until analysis.
[FIGURE 1 OMITTED]
Total protein extractable: Total protein content was determined by micro-Kjeldhal method A.O.A.C. . Total extractable protein in saline solution was determined according to the method of Arai , with the following modifications: 10g of mince flesh was homogenized with buffer saline (0.45M KCI, 3.38 mM [K.sub.2]P[O.sub.4] and 15.5 [Na.sub.2]HP[O.sub.4]; 1= 0.5, pH 7.5). After 24 hr the supernatant was collected and protein content was determined by micro-Kjeldhal method A.O.A.C. .
SIDS-PAGE: Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SIDS-PAGE) was performed according to Hashimoto et al. . Protein extraction was performed following the procedure stated by Ashie et al. ; 5 g of mince flesh was homogenized with buffer (tris HCI, KCI, EDTA, pH 7.6). Extracted protein was adjusted to 80 ug/ul following method stated by Lowry et al.  and subjected to electrophoresis in 12% polyacrylamide. Protein molecular weight standard markers ranging from 14.300 to 200.000 DA were purchased from Gibco BRL MA7405. After electrophoresis, the gels were stained with Coomassie Blue R-250 for 20 minutes and distained with 10% Acetic acid, 10% methanol and 80% distilled water solution for 24 hr. The bands of proteins were digitized and their optic density obtained using Gel Doc 2000 Bio-Rad and analyzed by Quantity One 4.1.1 Bio-Rad software.
Statistic analysis: All data were analyzed using Staf Grafic 6.0 (Manugistics, Inc., Rockville MD, USA). The total extractable protein was evaluated using ANOVA al a significant level of 95%. The dependent variable was the concentration for each days evaluated.
RESULTS AND DISCUSSION
Total extractable protein
The initial amount of protein extractable in saline solution was significantly (P < 0.05) higher for sardine mince flesh control (7.79%) compared to sardine mince flesh treated with 0.5% NaHC[O.sub.3] solution (6.83%) (TABLE 1). Total extractable protein decreased from 6.22% to 3.67% for the control after 150 days of frozen storage, representing 41% of the total extractable protein. The most drastic change from 7.91% to 3.67% (53%) was between 120 and 150 days of storage at -30[degrees]C. Conversely, sardine mince flesh treated with 0.5% NaHC[O.sub.3] solution decreased drastically from 6.83% to 2.81% representing 58% of the total extractable protein after 30 days of storage at -30[degrees]C; thereafter the protein extractable decreased 46% after 150 days of the frozen storage. Lack of protein solubility during frozen storage was due to interactions responsible for aggregation of the myofibrillar proteins Benjakul et al.,  stated that these interactions included disulfide bridges as well as formaldehyde formation. They evaluated physicochemical changes of some tropical fish muscle proteins during frozen storage and found that formaldehyde is an effective cross-linker that induces aggregation of protein thereby decreasing protein solubilization. They also noted a decrease in saline protein solubilization due to the exposure of reactive sulfydryl groups that induce oxidation or disulfide exchange. Moreover, formaldehyde is responsible for oxidation of sulfydryl groups inducing protein aggregation. Similarly, Careche et al.  stated that the myosin heavy chain is the most involved protein in aggregate formation. They evaluated the influence of frozen storage temperature to the type of aggregation of miofibrillar proteins in cod (Gadus morhua) fillets concluding myosin was more involved than actin in the aggregates at -30[degrees]C.
The difference in protein extractability between sardine mince flesh treated with 0.5% NaHC[O.sub.3] solution and the control could be due the lack of protection effect of the myofibrillar protein by components such as lipids that are removed during washing treatment resulting in protein aggregation at the beginning of frozen storage. Montero et al. and Tejada et al. [18, 19] stated the protective effect during frozen storage is due to the lipids contents. They reported that the washing process and cryoprotectants could modify the organization of the myofibrillar protein favoring aggregation during frozen storage.
The electrophoresis pattern of myofibrilar protein for sardine mince flesh control and mince flesh treated with 0.5% NaHC[O.sub.3] solution during frozen storage at -30[degrees]C varied between treatments (FIGS. 2 and 3, TABLES II and III). The most important myofibrillar protein bands, 200 KDa, 41 KDa, 35 KDa and 31 KDa, were correlated to each standard molecular weight marker. Optic density (OD) of myofibrillar proteins increased intensity of the band and band number during frozen storage. As frozen storage advanced, the protein extracted with saline solution increased for low molecular weight products (LMWP) 30 days and after 60 days after initiation of frozen storage for the control and sardine mince flesh treated with 0.5% NaHC[O.sub.3] solution respectively. This could be attributed to the higher proteolytic enzyme activity, high lipid content being oxidized, and trimethylamine (TMA) content that produced LMWP in the control samples. Low molecular weight proteins were responsible for the high protein extractable values obtained for the control during frozen storage. Conversely, sardine mince flesh treated with 0.5% NaHC[O.sub.3] solution had lower of LMWP at the beginning of frozen storage due to the washing treatment, which eliminated low molecular weight proteins, sarcoplasmic proteins and low molecular weight compounds that can affect degradation during frozen storage. However, sardine mince flesh treated with 0.5% NaHC[O.sub.3] solution contained a higher proportion of high molecular weight products (HMWP) throughout frozen storage. These high proportions of HMWP could be due to the production of proteins aggregations which increased the intensity of the bands between 200 to 45 KDa regions. Since myosin and HMWP are responsible for hydrophobicity, extractable protein from sardine mince flesh treated with 0.5% NaHC[O.sub.3] solution decreased during frozen storage. The decrease in protein extraction during frozen storage has been reported elsewhere. Tejada et al.  concluded that myofibrillar proteins decreased significantly as frozen storage advanced due to the gradual change of salt-extracted proteins in the protein composition. They also stated that during frozen storage there was an increase in high molecular weight band, which did not enter the gel. Futher, Montero et al.  evaluated chemical and functional properties of sardine (Sardina pilchardus W.) dark and light muscle proteins during frozen storage and the effect of washing on mince quality. They stated that the decrease in soluble protein in the treated mince was due in part to the production of high molecular weight polymers through the increase of disulfide bonds. Also, the loss of Ca-ATPase activity due to oxidation of SH-groups on the actomyosin indicated aggregation or denaturation and this loss of activity increased considerably in the first month of storage. After 60 days of frozen storage in 0.5% NaHC[O.sub.3] solution, the LMWP increased in number of eletrophoretic bands until the end of storage.
[FIGURES 2-3 OMITTED]
A remarkable difference between the control and washing treatment with 0.5% NaHC[O.sub.3] solution during frozen storage is that the washing treatment with 0.5% NaHC[O.sub.3] solution decreased protein denaturation (decrease solubilization of protein) keeping protein on stable conditions for further utilization. Washing treatment with 0.5% NaHC[O.sub.3] resulted enhance sardine mince flesh and could be recommended to decreased protein denaturation, increase storage life of further products made from sardine mince flesh. Further investigations are needed using other washing treatments such as sodium chloride, water or their combination, all of what could increase storage life of sardine mince flesh.
This study was supported by Consejo de Desarrollo Cientifico y Humanistico, Universidad Central de Venezuela, project PI 03-32-3843-2000.
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Marinela Barrero (1), Ynes Castillo y Makie Kodaira (2)
(1,2) Instituto de Ciencia y Tecnologia de Alimentos. Facultad de Ciencias. Universidad Central de Venezuela. Caracas 1042-A. Venezuela. E-mail: email@example.com
Recibido: 14/11/2005. Aceptado: 23/01/2007
TABLE I TOTAL EXTRACTABLE PROTEIN (g/100g [+ or -] SE) FOR SARDINE (Sardinella aurita) TREATED WITH 0.5% NaHC[O.sub.3] SOLUTION AND CONTROL, STORED AT -30[degrees]C FOR 150 DAYS/ PROTEINA TOTAL EXTRAIBLE (g/100g [+ or -] SE) PARA SARDINA (Sardinella aurita) ACONDICIONADA CON UNA SOLUCION DE NaHC[O.sub.3] AL 0,5% Y ALMACENADA A -30[degrees]C POR 150 DIAS Treatment Storage time Control NaHC[O.sub.3] 0.5% (days) 0 7.79 (a) [+ or -] 1.13 6.83 (b) [+ or -] 1.95 30 6.22 (a) [+ or -] 0.81 2.81 (b) [+ or -] 0.89 60 6.94 (a) [+ or -] 0.60 3.19 (b) [+ or -] 1.09 90 7.91 (a) [+ or -] 1.25 4.34 (b) [+ or -] 1.07 120 4.53 (a) [+ or -] 0.25 2.16 (b) [+ or -] 0.64 150 3.67 (a) [+ or -] 0.08 1.52 (b) [+ or -] 0.20 Result from 4 replications. ANOVA statistical analysis. Assay performed in three replications. (a,b) - means not followed by the same letter within row differ (P<0.05). TABLE II PROTEIN MOLECULAR WEIGHT (KDA) OBTAINED BY SDS-PAGE AND ANALYZED BY OPTIC DENSITY OF SARDINE MINCE FLESH (CONTROL) DURING FROZEN CONDITIONS AT -30[degrees]C. / PESO MOLECULAR DE LAS PROTEINAS OBTENIDAS POR SDS-PAGE Y ANALIDAS POR DENSIDAD OPTICA DE LA PULPA DE SARDINA (CONTROL) DURANTE EL ALMACENAMIENTO CONGELADO A- 30[grados]C Line # Standard 0 days 30 days 60 days 1 220.95 166.729 166.032 152.806 2 96.740 128.481 127.745 117.477 3 71.770 93.622 94.344 91.787 4 45.470 81.660 82.223 80.970 5 28.680 73.932 76.281 74.167 6 19.740 59.257 59.998 60.376 7 14.530 53.182 54.798 0.000 8 41.491 41.287 41.650 9 35.505 35.496 34.606 10 31.766 31.737 31.633 11 0.000 27.916 0.000 12 21.909 21.695 0.000 13 18.777 18.577 0.000 14 0.000 16.919 22.444 15 14.558 18.718 16 17.327 14.393 Line # 90 days 120 days 150 days 1 171.829 171.480 0.000 2 124.082 123.709 0.000 3 0.000 123.709 89.725 4 0.000 92.261 83.147 5 75.586 82.331 74.172 6 59.489 75.353 61.933 7 54.787 59.865 55.969 8 41.680 55.120 41.238 9 34.985 41.813 35.373 10 0.000 35.478 31.934 11 0.000 31.678 27.873 12 28.109 27.991 0.000 13 0.000 22.117 0.000 14 22.226 18.779 18.434 15 18.637 17.265 17.278 16 17.252 14.486 14.153 14.653 TABLE III PROTEIN MOLECULAR WEIGHT (KDA) OBTAINED BY SDS-PAGE AND ANALYZED BY OPTIC DENSITY OF SARDINE MINCE FLESH (CONTROL) DURING FROZEN CONDITIONS AT - 30[degrees]C/ PESO MOLECULAR DE LAS PROTEINAS OBTENIDAS POR SDS-PAGE Y ANALIZADAS POR DENSIDAD OPTICA DE LA PULPA SE SARDINA (CONTROL) DURANTE EL ALMACENAMIENTO CONGELADO A-30[grados]C Line # Standard 0 days 30 days 60 days 1 220.950 189.347 189.767 198.198 2 96.740 145.767 143.058 139.982 3 71.770 100.630 98.866 103.258 4 45.470 88.031 86.245 89.268 5 28.680 81.213 82.373 84.287 6 19.740 43.896 43.733 65.509 7 14.530 36.344 35.995 58.712 8 31.701 31.613 44.018 9 22.857 22.708 36.703 10 19.631 19.479 32.656 11 28.384 12 26.259 13 23.426 14 20.154 15 16 Line # 90 days 120 days 150 days 1 207.003 202.960 202.960 2 149.413 146.633 131.575 3 105.527 101.445 108.259 4 90.299 89.485 95.848 5 83.325 83.544 88.466 6 65.509 64.500 83.544 7 59.794 59.971 87.459 8 44.305 44.715 67.504 9 37.182 37.789 61.631 10 33.082 33.201 44.426 11 28.532 28.795 37.303 12 26.533 27.321 32.774 13 23.548 23.637 28.329 14 20.259 20.450 26.486 15 23.273 16 20.239