Research Journal of Animal, Veterinary and Fishery Sciences ___________________________ ISSN 2320 – 6535 Vol. 1(5), 16-20, June (2013) Res. J. Animal, Veterinary and Fishery Sci. International Science Congress Association 16 Quality changes in the muscles of Wallago attu during frozen storage (-12±2ºC) conditionsGandotra Roopma, Vaini Gupta*, Koul Meenakshi and Gupta Sweta Department of Zoology, University of Jammu, Jammu, 180006, INDIAAvailable online at: www.isca.in Received 31st May 2013, revised 6th June 2013, accepted 20th June 2013Abstract The present study was aimed to investigate the effect of frozen storage on the proximate, biochemical and microbial profile of the muscle of a silurid cat fish (Wallago attu). The fish muscle was subjected to the frozen storage for a period of one month and the analysis was carried out at an interval of 10 days. It was observed that proximate composition viz. protein, lipid, moisture and ash content decreased significantly (P0.05) with increase in the duration of frozen period. The fresh (unfrozen) samples revealed the highest values for all i.e.15.45±0.2% for protein, 4.02±0.04% for lipid, 81.66±0.03 for moisture and 1.48±0.1% for ash while the least values were observed at the end of one month frozen storage period i.e.10.14±0.015%, 2.36±0.03%, 74±0.05% and1.33±0.02% for protein, lipid, moisture and ash respectively. pH of fish ranged between 5.9 to 7.1 while the biochemical parameters viz.,Free Fatty Acids (FFA) and Thiobarbituric acid (TBA) showed an increasing trend with increase in frozen storage period. Similarly, the microbial count for Total Plate Count (TPC), Coliform Count (CC) and Psychrotrophic Count (PC) increased gradually from 2.18±0.02 log cfu/g, 2.02±0.04 log cfu/g and 2.43±0.03 log cfu/g on day 0 to 6.87±0.1 log cfu/g, 5.25±0.2 log cfu/g and 5.99±0.02 log cfu/g on day 30threspectively. Thus, considering the importance from consumer point of view, these studies reveal that a significant loss is observed in fish during frozen storage. However, it could be implied that fish could be kept under frozen conditions when preservation is of utmost importance, so as to retain its taste and nutrition. Keywords: Frozen period, proximate, biochemical, microbial, Wallago attu. Introduction Fish as a whole is considered to have a great potential for food and thus can be expected to provide relief from malnutrition. Also, it is a very good source of protein, has a high content of water and lipid soluble vitamins, minerals and polyunsaturated fatty acids (PUFAs) of n-3 family. It is believed that Omega-3 fatty acids from fish can lower body triglycerides thus reducing the chances of heart diseases. Fish also reduces blood pressure by small but significant amounts and improve blood clotting regulation. Fish, however, is very susceptible to microbial and chemical deterioration like lipid oxidation, hydrolysis and protein denaturation etc. Moreover, the high ambient temperature of our country favours the rapid microbial growth, hence there is a need to properly handle and preserve the fish, so as to increase its shelf life. Shelf life extension can be achieved by various preservation methods, viz. salting, brining, smoking, icing, glazing, refrigeration and freezing. Since, in our country, fish in a fresh state is not always available due to seasonal fishing and the far location of major fishing grounds from cities and consuming centres, the freezing of fish becomes an update method of long term preservation. Refrigeration and freezing provide low temperature thus, inactivating the microbial growth and there by reducing enzymatic and chemical deterioration. Although many damaging processes are inhibited by such low temperature storage methods, but the undesirable reactions associated with lipids and proteins are shown to occur, leading to the detrimental changes in nutritional and sensory properties. Some disadvantages of frozen storage include freezer burn, product dehydration, rancidity and drip loss and this deterioration increases as duration of storage increases. Hence, the objective of this study is to determine the changes in nutritional, biochemical and microbial quality in the muscles of Silver carp, stored in frozen storage conditions (12±2ºC). Material and Methods Collection of fish samples: Fresh samples of Wallago attu were purchased from local market of Jammu city. They were immediately brought to the lab in polythene bags along with crushed ice. The viscera of fish was removed and the fish was washed with large amount of water. The fish was cut into pieces and these pieces were immediately wrapped in aluminum foil, kept in air tight plastic container and stored at -12±2°C (frozen storage). Analytical procedures for biochemical and microbiological changes were done on 0, 10th, 20th and 30th day of storage. Analysis: The proximate composition (protein, lipid, ash and moisture) of the fish samples were evaluated using the standard AOAC procedure. The protein content was determined using the Lowry et al method. Fat content was determined using Folch et al method. Thiobarbituric acid value of fish muscle during frozen storage was determined by using the method of Research Journal of Animal, Veterinary and Fishery Sciences ________________________________________ ISSN 2320 – 6535 Vol. 1(5), 16-20, June (2013) Res. J. Animal, Veterinary and Fishery Sci. International Science Congress Association 17 Witte et al (1970). Free Fatty Acid (FFA) was determined by method of US Army laboratories (Natick). Extract Release Volume (ERV) was determined as per the method of Strange et al. (1977). The pH of fish muscle was determined by the method of Keller et al. (1974). The microbiological profile was determined according to APHA method9. Data was expressed as mean ± SD and were analyzed by one-way ANOVA test using SPSS statistical programme. Statistical Analysis: Mean and standard errors were calculated for different parameters. The data analyses were performed using SPSS software (12.0 for Windows). Differences between treatments were analyzed using independent-measures one-way ANOVA. Post-hoc comparisons were conducted using Duncan’s test. The values were expressed as mean ± SE. p values 0.05 were considered as significant and p values 0.001were considered as highly significant. Results and Discussion Proximate Composition: Protein content: Perusals of table-1 revealed that the highest protein content (15.45±0.2%) was recorded for fresh (unfrozen) fish samples and the least protein content (10.14±0.015%) was recorded for fish samples stored for 30 days under frozen conditions. A significant percental decrease (p0.05) was found in total protein content i.e.8.67%, 20.71% and 34.3% on 10th, 20th and 30th day of frozen storage respectively table-2. In support with the present studies, Keyvan et al (2008)10 in Caspian white fish (Rutilus frisi kutum), El-deen and El-shamrey (2010)11in Gahsh (Lethrinus elongates) and Aberoumand(2013)12 in various Iranian fishes also found a protein loss during frozen storage. They related this protein loss to the denaturation of proteins and loss of nitrogen as volatile bases and nitrogenous substances formed by bacterial decomposition that escaped from tissue during frozen storage. Lipid content: The results shown in table-1 show that the lipid content decreased significantly (p0.05) from day 0 i.e. 4.02±0.04% to 2.36±0.03% on day 30th. There was 14.42%, 25.37% and 41.29% decrease on 10th, 20th and 30th day of frozen storage, table-2. Arannilewa et al13 in Tilapia, Keyvan et al10 in Caspian sea white (Rutilus frisi kutum), Siddique et al14 in Puntius sps. and Aberoumand12in various Iranian fishes also found a decrease in total lipid content during frozen storage. They opined that this protein loss must be due to oxidation and hydrolysis of lipids during frozen storage. Table-1 Change in Proximate composition of Wallago attu during frozen storage. Days of storage 0 day 10 th day 20 th day 30 th day Protein (%) 15.45 a ±0.2 14.11 b ±0.05 12.25 c ±0.03 10.14 d ±0.015 Lipids (%) 4.02 a ±0.04 3.44 b ±0.02 3.00 c ±0.07 2.36 d ±0.03 Moisture (%) 81.66 a ±0.03 79.2 b ±0.4 76.94 c ±0.1 74 d ±0.05 Ash (%) 1.48 a ±0.1 1.41 b ±0.5 1.38 c ±0.02 1.33 d ±0.02 Table-2 Percent decrease in proximate composition of fish muscle of Wallago attu under frozen storage Days Protein (%) Lipid (%) Moisture (%) Ash (%) 0-10 8.67 14.42 3.01 4.72 0-20 20.71 25.37 5.78 6.75 0-30 34.3 41.29 9.3 10.13 1020304050607080900 day10th day20th day30th day protein lipid moisture ashFigure-1 Change in proximate composition of Wallago attu during frozen storage Research Journal of Animal, Veterinary and Fishery Sciences ________________________________________ ISSN 2320 – 6535 Vol. 1(5), 16-20, June (2013) Res. J. Animal, Veterinary and Fishery Sci. International Science Congress Association 18 Moisture: The total moisture content of the fish sample decreased from 81.66±0.03% on day 0 to 74±0.05 % on day 30th. Total percent decrease was 3.01%, 5.78% and 9.3% on 10th, 20th and 30th day of frozen storage table-2. These results are favoured by the findings of Le-blanc and Le-blanc15, Arannilewa et al13, El-deen and El-shamrey11 and Aberoumand12 who proposed that decrease in moisture is due to condensation of water during frozen storage. Ash: In the present studies table-1, ash content showed a decrease with an increase in frozen storage period. Total percental decrease was 4.72% on day 10th and it increased upto 10.13% on day 30th of frozen storage. These results are in agreement with Emire et al16 in Tilapia (Oreochromis niloticus) and Aberoumand12 in Iranian fishes and Gandotra etal17 in Labeo rohita. Biochemical Composition: Thiobarbituric acid (TBA): The TBA value is an index which measures the malondialdehyde (MDA) content and is a widely used method for assessment of degree of lipid oxidation. MDA is formed through hydroperoxides, which are the initial reaction products of polyunsaturated fatty acids with oxygen18The present study showed a progressive increase in TBA value (secondary oxidation product) with increase in storage period under frozen conditions. The values rose from 0.16±0.2 on day 0 to 4.48±0.05 on 30th day of frozen storage period. These results are in consistence with the studies of] Bao et al19 in Arctic charr Salvelinus alpinus), Orak and Kayisoglu20 various fish species and Jezek and Buchtova21 in Common carp (Cyprinus carpio L.) and Silver carp (Hypophthalmichthysmolitrix V.). The increase in TBA is attributed to the fact that freezing and thawing accelerates the accumulation of secondary oxidative products released due to the destruction of cell membrane by crystals of ice and release of pro-oxidants, especially haem-iron22 Free fatty acids (FFA) : The values for Free Fatty Acids (FFA) were 0.88±0.02 on day 0 and it rose to 2.86±0.2, 4.88±0.33 and 6.93±0.05 on 10th, 20th and 30th day of frozen storage respectively. These results are in accordance with Keyvan et al10 in Caspian Sea white fish (Rutilus frisi kutum), Jezek and Buchtova23 in Silver carp and Gandotra et al24 in Mystus. These findings are devoted to the release of lipase from liposome during frozen storage which degrades the fats, thus increasing the free fatty acids25. pH: The pH values also showed an increasing trend with increase in frozen period. The pH values ranged from 5.9 on day 0 to 7.11 on 30th day. These results are in line with Arannilewa et al (2005)13 in frozen Tilapia (Sarotherodun galiaenus), Jezek and Buchtova (2011)23 in Common carp (Cyprinus carpio L.) and Silver carp (Hypophthalmichthys molitrix V.) and Erkan and Ozden (2007)26 in Gutted sardines (Sardina pilchardus)who attributed this increase to the production of basic components induced by bacterial growth. Microbial quality+: The quality of fish meat is largely dependent on its microbial contamination. Inquistive study of table- 4 shows an increasing trend for TPC, CC and PC during the frozen storage period. Initially the values for TPC were 2.18±0.02 log cfu/g and increased to 6.87±0.1 lof cfu/g at the end of storage, thus crossing the permissible limits of 6 lof cfu/g on 20th day of storage. Table-3 Change in bio-chemical composition of Wallago attu during frozen storage Days 0 day 10 th day 20 th day 30 th day TBA(mg MA/kg) 0.16 a ±0.2 1.89 b ±0.01 3.39 c ±0.4 4.48 d ±0.05 FFA (%) 0.88 a ±0.02 2.86 b ±0.2 4.88 c ±0.33 6.93 d ±0.05 pH 5.9 a 6.4 b 6.9 c 7.11 d Mean ±SD with different superscripts in a row varies significantly (P0-05) Figure-2 Change in bio-chemical composition of Wallago attu during frozen storage 0 DAY10TH DAY20TH DAY30TH DAY TBA FFA p H Research Journal of Animal, Veterinary and Fishery Sciences ________________________________________ ISSN 2320 – 6535 Vol. 1(5), 16-20, June (2013) Res. J. Animal, Veterinary and Fishery Sci. International Science Congress Association 19 Table-4 Changes in Total Plate Count (TPC), Coliform Count (CC) and Psychrophillic Count(PC) of fish muscle of Wallagoattuunder frozen storageDays of storage 0 day 10 th day 20 th day 30 th day TPC log cfu/g 2.18 a ±0.02 4.4 b ±0.12 6.1 c ±0.04 6.87 d ±0.1 CC log cfu/g 2.02 a ±0.04 3.1 b ±0.05 3.82 c ±0.06 5.25 d ±0.2 PC log cfu/g 2.43 a ±0.03 3.48 b ±0.11 4.7 c ±0.05 5.99 d ±0.02 Similarly, CC and PC also showed an increasing trend with 5.25±0.2 lof cfu/g and 5.99±0.02 log cfu/g values for CC and PC respectively at the end of frozen storage period. Likewise, Arannilewa et al13 found an increase in Coliform count with the increasing storage period in frozen Tilapia. Ozogul et al27 also reported a significant statistical increase in total viable counts of whole gutted common sole (Solea solea) over the storage period of 24 days. Simiilarly, Ola and Oladipo28 and Liu29 found an increasing trend for psychrotrophs during storage period. This increase in microbial count is attributed to growth promoting effect of moisture during frozen storage30. Conclusion The main objective of this study was to observe nutritional, biochemical and microbial changes in Wallago attu during frozen conditions. The freezing of fish at low temperature makes it less prone to spoilage by decreasing the bacterial activity. However, it was observed that there was a decrease in the nutritional parameters while an increase was observed in biochemical composition and microbial count during frozen storage. Therefore, it could be concluded that we should try to consume fish while it is fresh only. Since, all fishes are not available throughout the year; hence, freezing is best preferred when preservation of such fish species is of priority. References 1.Nettleton J. A. 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