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Available Online at http:/ / www.recentscientific.com International Journal of Recent Scientific Research Vol. 4, Issue, 7, pp.1103– 1108, July, 2013 International Journal of Recent Scientific Research ISSN: 0976-3031 RESEARCH ARTICLE IMPACT OF ASCORBIC ACID AND CITRIC ACID TREATMENT ON THE SHELF LIFE OF SILVER CARP HYPOPHTHALMICHTHYS MOLITRIX (SILVER CARP) FILLETS IN FROZEN STORAGE Roopma Gandotra, Vaini Gupta*, Meenakshi Koul and Sweta Gupta Department of zoology, University of Jammu, Jammu, 180006 ARTICLE INFO ABSTRACT Article History: The present study was aimed to investigate the effect of aqueous solutions of citric acid and ascorbic acid (0.5℅) on the nutritional quality, rancidity development and microbial quality in the muscle of Silver carp under frozen storage (-12±2⁰C) for a period of 30 days. Muscle samples of fish were divided into two groups; group A (Gp.A) treated with 0.5℅ Ascorbic acid and Citric acid (A.A & C.A) and the other group B (Gp.B) without any treatment i.e control group. After 30 days of storage, both the samples showed a significant decreasing trend in protein, lipid, ash and moisture content. The total percental decrease in Gp.A and Gp.B was 20.11% and28.24% for protein, 18.53% and 56.47% for lipid, 4.22% and 8.17% for moisture and 11.56% and 34.4% for ash respectively at the end of storage period. However, the free fatty acid (FFA), extract release volume (ERV) and thiobarbituric acid (TBA) showed a significant increasing trend in both the groups but the increase was comparatively lesser in treated Group i.e. Gp.A than Gp.B. Similarly, the bacteriological studies revealed that Total plate count(TPC), Coliform count (CC) and Psychrophillic count (PC) in both the groups increased with increase in the period of frozen storage. However, these values were found to be within permissible limits in Gp. A up to the 30th day of storage and only up to 20th day of storage in untreated Gp. B. Received 11th, June, 2013 Received in revised form 24th, June, 2013 Accepted 18th, July, 2013 Published online 30th July, 2013 Key words: Silver carp, ascorbic and citric acid, frozen period, rancidity, microbial. © Copy Right, IJRSR, 2013, Academic Journals. All rights reserved. INTRODUCTION Fish is gaining an increased importance in the diets of people due to its role in providing significant amount of important dietary factors viz. nutritional and digestive proteins including high levels of essential amino acids (lysine, methionine etc.), lipid soluble vitamins (eg. A and D), microelements like I,Fe,Ca,Cu Zn,F etc and highly unsaturated fatty acids. Also, fish is a cheap source of animal protein with little or no religious rejection of it, which gives it advantage over pork or beef. However, fish is an extremely perishable commodity and its quality and freshness declines rapidly post mortem. Deterioration or spoilage of fish begins immediately upon catching or slaughtering, and the rate at which it continues depends directly upon storage and preservation methods after death. Different spoilage mechanisms reported to be involved in this quality loss include microbial development, endogenous enzyme activity, non enzymatic lipid oxidation and enzymatic browning (Ozogul etal, 2006, Auborg, 2008) thus, decreasing the shelf life of fish. Hence, there is a necessity for developing alternate preservation methods to extend the shelf life of fish. Different methods used to extend the shelf life include low temperature storage, icing, salting, brining, smoking, frying and use of anti-oxidants. The use of anti-oxidants is emerging as an effective methodology for controlling rancidity in oils and food (Frankel, 1998, Pazos et al 2005). Ascorbic acid and Citric acid remove oxygen and reduce the first step in chain reaction of lipid oxidation (Mielnik et al, 2002). Ascorbic acid and citric acid and their salts are widely known for their role as * Corresponding author: Vaini Gupta  Department of zoology, University of Jammu, Jammu, 180006 chelators, acidulates in biological system and synergists of primary antioxidants, so that a profitable effect on fish oil and emulsions (Kelleher et al, 1992; Osborn-Barnes and Akoh, 2003), minced fish (Hwang and Regenstein, 1988; Stodolnik et al, 1992) and fish fillets (Badii and Howell, 2002; Auborg et al, 2004) have been observed. Hence, this work is aimed to study the effect of Ascorbic acid and Citric acid on extension of shelf life of Silver carp (Hypophthalmichthys molitrix). MATERIALS AND METHODS Collection of fish samples Fresh samples of Hypophthalmichthys molitrix 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 were removed and the fish was washed with large amount of water. Analytical procedures for biochemical and microbiological changes were done on 0, 10th, 20th and 30th day of storage. Fish Treatment The fish was cut in to pieces and these pieces were divided into two groups viz. Gp.A and Gp.B. Gp.A samples were dipped in the solution of 0.5% Ascorbic acid and Citric acid for 15 minutes, taken out and immediately wrapped in aluminum foil, kept in air tight plastic container and stored at12±2°C (frozen storage) while the second group, Gp.B was International Journal of Recent Scientific Research, Vol. 4, Issue, 7, pp. 1103 - 1108, July, 2013 considered as fresh (Control), freeze without pre treatment of 0.5% Ascorbic acid and Citric acid solution. Analyses The proximate composition (ash and moisture) of the fish samples were evaluated using the standard AOAC procedure (AOAC, 1995). The protein content was determined using the Lowry et al. (1951). Fat content was determined using Folch et al (1957). Thiobarbituric acid value of fish muscle during storage was determined using the method of Witte et al (1970). Free Fatty Acid (FFA) was determined by method of US Army laboratories (Natick) described by Koniecko (1979). Extract Release Volume (ERV) was determined as per the method of Strange et al. (1977). The pH of fish muscles was determined by the method of Keller et al. (1974) the microbiological profile was determined according to APHA method (1984). Data were expressed as mean ± SD and were analyzed by oneway ANOVA test using SPSS statistical programme. 5.73%, 11.40% and 20.11% on 10th, 20th and 30th day respectively. These results are in accordance with the studies of Omojowo et al (2005) in Cat fish (Clarius garipinus) and Arekemase et al (2012) in Tilapia and Mackerel who opined that the antioxidants have the ability to slow down the protein autolytic process in muscle which resulted in delayed muscle break down. Lipid content The lipid content of the untreated frozen samples of Gp. B shows a decrease from 2.27% on day 0 to 0.98% on day 30th. Total percental decrease was 22.46%, 43.61% and 56.47% on 10th, 20th and 30th day respectively during frozen storage. Similar observations were made earlier by Zoldos et al (2010) in Allaska Pollack, Siddique et al (2011) in Puntius sps. and Gandotra et al (2012) in Labeo rohita. They attributed this loss in lipid to the oxidation of lipids. 100 Statistical Analysis PROTEIN LIPID 80 Means 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. values <0.05 were considered as significant and p values <0.001were considered as highly significant p. 60 40 20 0 0 DAY Figure 1 Change in proximate composition (wet weight basis) of raw fish muscle of Silver carp (Hypophthalmichthys molitrix) stored in freezer at 12±2ºC for a period of 30 days.(Gp.B) RESULTS AND DISCUSSIONS Proximate composition Triplicate flesh samples of Silver carp i.e. with and without the treatment of (A.A& C.A) were analyzed for determining its proximate composition viz. protein, lipid, moisture and ash content during 30 days of frozen storage period. 10TH DAY 20TH DAY 30TH DAY However, the treated samples (Gp. A) also revealed a decreasing trend in lipid values but the decrease was low when compared to untreated Gp. B. The total percental decrease in Gp.A (treated) was 5.60%, 9.91% and 18.53% on 10th,20th and 30th day of storage respectively. Table1 Proximate composition (wet weight basis) of raw fish muscle of Silver carp (Hypophthalmichthys molitrix) stored in freezer at -12±2ºC for a period of 30 days. (Gp.B) Days Total protein(%) Total lipid(%) Moisture(%) Ash(%) 0 16.5a±0.01 2.27a±0.02 81.6a ±0.04 1.25 a ±0.02 10th 15.01b±0.12 1.76 b ±0.35 79.46 b±0.15 1.17 b ±0.15 Protein content During the present study the protein content of frozen muscle sample of untreated control Gp. B was 16.5% on day 0 and 11.84% on day 30th. Further, a significant (P≤0.05) percental decrease was found in total protein content i.e 9.03%, 15.27% and 28.24% on 10th, 20th and 30th day respectively. These findings are supported by the studies of Bekelvik et al (2005) in sea bass (Dicentrarchus tabreri), Siddique et al (2011) in Puntius and Gandotra et al (2012) in Labeo rohita, who suggested that loss of protein might be due to leaching effect of amino acids with melting ice. Further, muscle samples of treated Gp. A i.e A.A and C.A treated samples showed a comparatively less percental decrease in protein content i.e. 20th 13.98c±0.02 1.28 c ±0.01 76.34 c±0.01 1.11 c ±0.04 30th 11.84d±0.25 0.98 d ±0.25 74.93 d±0.02 0.82 d ±0.35 These results are favored by the findings of Arekemase et al (2012), Ehsani and Jasour (2012) and Rahimabadi and Divband (2012) who proposed that highest fat content and low free fatty acids in fish muscle samples treated with antioxidants after a prolonged storage may be due to the prevention of oxidation and hydrolysis of lipids in fish by anti-oxidants during frozen storage. Moisture content The total moisture content decreased with the increase in storage time in both Gp. A (treated) and Gp.B (untreated) muscle samples respectively. There was a 2.62%, 6.44% and 8.17% decrease in Gp. B and 1.02%, 2.43% and 4.22% decrease in Gp. A on 10th, 20th and 30th day of storage respectively. Table 1(A) Percent decrease in proximate composition of raw fish muscle of Silver carp (Hypophthalmichthys molitrix) stored in freezer at -12±2ºC for a period of 30 days.(Gp.B Days 0-10 0-20 0-30 Protein (%) 9.03 15.27 28.24 Lipid (%) 22.46 43.61 56.47 1104 Moisture (%) 2.62 6.44 8.17 Ash (%) 6.40 11.20 34.40 International Journal of Recent Scientific Research, Vol. 4, Issue, 7, pp. 1103 - 1108, July, 2013 These results get support by the findings of Le Blanc and Le Blanc (1992), Bekelvik et al (2005) in Sea Bass and Emire et al (2009) in Nile Tilapia during frozen storage. They attributed this moisture loss to the condensation of water during chilling. PROTEIN 90 80 70 60 50 40 30 20 10 0 LIPID MOISTURE ASH The total percental increase in ERV was very low in Gp.A(treated) i.e. 48% as compared to the Gp.B i.e.69.23% throughout the storage period. These results are corresponding with the results of Rostamzad et al (2011) in Persian Sturgeon, Pourashouri et al (2011) in Wels Catfish and Taheri et al(2012) in Cobia, who attributed this less increase in ERV to the increased water holding capacity in anti-oxidant (ascorbic acid and citric acid) treated samples. As water holding capacity in meat tissue is strongly related to myofibril protein structure, therefore, this less increase in ERV in present studies may be due to the effect of antioxidants in delaying the protein denaturation. 50 FFA TBA ERV 40 0 DAY 10TH DAY 20TH DAY 30 30TH DAY 20 Figure 2 Change in proximate composition (wet weight basis) of fish muscle of Silver carp (Hypophthalmichthys molitrix) treated with ascorbic and citric acid, stored in freezer at -12±2ºC for a period of 30 days.(Gp.A) 10 0 Ash content Both Gp. A and Gp. B showed a decrease in ash content during the frozen storage conditions. The total percental decrease was 3.36% and 6.4% on 10th day, 7.56% and 11.2% on 20th day and 11.76% and 34.4% in Gp. A and Gp. B respectively. Similar results were obtained by Bekelvik et al (2005) in Sea Bass and Emire et al (2009) in Nile tilapia and Okeyo et al (2009) in frozen Nile Perch. This decrease in ash may be attributed to the drip loss, resulting in loss of bulk and trace elements. 0 DAY 10TH DAY 20TH DAY 30TH DAY Figure 3 Change in bio-chemical composition of raw fishmuscle of Silver carp (Hypophthalmichthys molitrix) stored in freezer at -12±2ºC for a period of 30 days.(Gp.B) Free fatty acids (FFA) Free fatty acids are known to form off-flavour and undesirable taste producing low molecular weight compounds after oxidation. In the present study, Table 2 Proximate composition (wet weight basis) of fish muscle of Silver carp (Hypophthalmichthys molitrix) treated with ascorbic and citric acid Days Total protein(%) Total lipid(%) Moisture(%) Ash(%) 0 16.75 a ±0.02 2.32 a ±0.03 81.22 a ±0.05 1.19 a ±0.14 10th 15.79 b ±0.11 2.19 b ±0.21 80.39 b ±0.2 1.15 b ±0.3 20th 14.84 c ±0.31 2.09 c ±0.1 79.24 c ±0.01 1.10 c ±0.04 30th 13.38 d ±0.02 1.88 d ±0.02 77.79 d ±0.12 1.05 d ±0.5 Table 2(A) Percent decrease in proximate composition of fish muscle of Silver carp (Hypophthalmichthys molitrix) treated with ascorbic and citric acid, stored in freezer at -12±2ºC for a period of 30 days.(Gp.A) Days 0-10 0-20 0-30 Protein (%) 5.73 11.40 20.11 Lipid (%) 5.60 9.91 18.53 CHEMICAL ANALYSIS Extract Release Volume (ERV) The values for extract release volume in both Gp. A and Gp. B showed a progressive increase from 0th to 30th day. In Gp. B, the values were 26±0.4 ml on 0 day and 31±0.03 ml, 37.3±0.05 ml and 44.45±0.02 ml on 10th, 20th and 30th day respectively. However, in Gp. A, the initial value was 25±0.03 and after 30 days, it increased upto 37±0.04. Moisture (%) 1.02 2.43 4.22 Ash (%) 3.36 7.56 11.76 FFA was determined to investigate deterioration of fats due to their hydrolysis. Results for the untreated muscle samples of Gp. B depicted an increase in FFA under frozen storage. FFA values on day 0, 10th, 20th and 30th day were 0.54%, 4.82%, 9.02% and 14.55% respectively. Further, a gradual increase in FFA formation was observed in treated Gp. A samples, as shown in Table 4. But the increase in FFA was low in Gp. A samples as compared to Gp.B samples. Table 3 Change in bio-chemical composition of raw fishmuscle of Silver carp (Hypophthalmichthys molitrix) stored in freezer at -12±2ºC for a period of 30 days.(Gp.B) Days ERV(ml) FFA (%) TBA(mgMA/kg) 0 26 a ±0.4 0.54 a ±0.12 0 a ±0.01 10th 31 b ±0.03 4.82 b ±0.02 2.95 b ±0.15 1105 20th 37 c ±0.05 9.02 c ±0.04 3.75 c ±0.02 30th 44.45 d ± 0.02 14.55 d ±0.15 5.45 d ±0.03 International Journal of Recent Scientific Research, Vol. 4, Issue, 7, pp. 1103 - 1108, July, 2013 cfu/g on day 30th, thus crossing the permissible limit of 6 log Similar increasing trend in FFA was found by Ozogul et al (2011) cfu/g (ICMSF, 1986) on 20th day of storage. However, in Gp.A in Common sole (Solea solea) and Jezek and Buchtova (2012) in (treated samples), the values were found to be within freeze thawed fillets of Common carp and Silver carp. Aubourg et permissible limits even after final day i.e. 30th day of frozen al (2004), Pourashouri et al (2011) in Wels catfish (Silurus storage. Similar results were obtained by Sallam (2007) in glanis), Rostamzad et al(2011) in Persian sturgeon, Taheri et al refrigerated sliced salmon treated with anti-oxidants (Sodium (2011) and Pieretti et al (2012) in Rainbow trout. They associated acetate, sodium lactate and sodium citrate), Yasin and Abouthis increase in FFA to the hydrolysis of fat and oil present in fish muscle. Table 4 Change in bio-chemical composition of fish muscle of Silver carp (Hypophthalmichthys molitrix) treated with ascorbic and citric acid, stored in freezer at -12±2ºC for a period of 30 days.(Gp.A) Days ERV(ml) FFA (%) TBA(mgMA/kg) 10th 29 b ±0.01 1.08 b ±0.03 0.99 b ±0.15 0 25 a ±0.03 a 0.55 ±0.04 0 a ±0.01 This slow increase in FFA formation in Gp. A was due to the fact that Ascorbic and Citric acid act as oxygen scavengers and metal chelators, thus causing delay in lipid oxidation. (Rostamzad, 2011) 40 35 FFA TBA ERV 30 25 20 15 10 5 0 0 DAY 10TH DAY 20TH DAY 30TH DAY Figure 4 Change in bio-chemical composition of fish muscle of Silver carp (Hypophthalmichthys molitrix) treated with ascorbic and citric acid, stored in freezer at -12±2ºC for a period of 30 days.(Gp.A) Thiobarbituric acid TBA 20th 32 c ±0.03 2.21 c ±0.12 1.45 c ±0.013 30th 37 d ±0.04 3.42 d ±0.02 3.95 d ±0.04 Taleb (2007) in Mullet fish fillets treated with Marjoram and Thyme, Pezeschk et al (2012) in live and gutted fish treated with Shallot extract and Rakshit and Ramalingam (2013) in meat and fish treated with natural preservatives. A considerable increase was observed in psychrophillic count of both the samples. In Gp. A and Gp. B, the values rose from 2.1±0.05 log cfu /g and 2.03±0.2 log cfu /g on day 0 to 3.56±0.4 log cfu /g and 7.96±0.03 log cfu/g on 30th day of storage, thus Gp.B crossing the permissible limit i.e.4.6 log cfu/g on 20th day of storage while the values were within the permissible limits in Gp. A at the end of storage. Likewise, Sallam (2007) reported the reduction in psychrotrophic population in refrigerated sliced salmon treated with antioxidants (sodium acetate, sodium lactate and sodium citrate). Similarly, Yasin and Abou-Taleb (2007) observed the lowest incremental pattern in Mullet fish fillets treated with antioxidants like Marjoram and Thyme. Pezeshak et al. (2012) also reported the inhibitory effect of shallot extract on the growth of psychrotrops. The TBA value is an index of lipid oxidation measuring malondialdehyde (MDA) content and widely used for assessment of degree of lipid oxidation. MDA is formed through hydroperoxides, which are the initial reaction product of polyunsaturated fatty acids with oxygen.(Sallam,2007). Persuals of table 3 and 4 show an increase in TBA (mg malonaldehyde/ kg) values in both Gp.A and Gp.B samples with increase in storage period. However, the increase was lower in Gp.A (Treated) samples when compared to Gp. B (Control) samples. Similar results were found by Rostamzad et al(2011) in Persian sturgeon, Taheri et al(2012) in cobia (Rechycentron canadum), Zakipour and Dirband et al(2012) in Silver carp. They suggested that lower TBA values in treated samples was due to the positive effect of anti-oxidants in delaying lipid oxidation 10 TPC CC PC 8 6 4 2 0 0 DAY 10TH DAY 20TH DAY 30TH DAY Figure 5 Changes in Total Plate Count (TPC), Coliform Count (CC) and Psychrophillic Count (PC) of raw fish muscle of Silver carp (Hypophthalmichthys molitrix) stored in freezer at -12±2ºC for a period of 30 days.(Gp.B) Table 5 Changes in Total Plate Count (TPC), Coliform Count (CC) and Psychrophillic Count(PC) of raw fish muscle of Silver carp (Hypophthalmichthys molitrix) stored in freezer at -12±2ºC for a period of 30 days.(Gp.B) Days of storage TPC log cfu/g CC log cfu/g PC log cfu/g 0 day 2.15 a ±0.2 1.05 a ±0.2 2.03 a ±0.2 10th day 4.65 b ±0.12 2.23 b ±0.04 3.95 b ±0.04 Microbial Quality For the determination of freshness quality of fish before and after treatment, Total plate count (TPC), Coliform count (CC) and Psychrophillic count (PC) were analyzed during frozen storage of one month. The results presented in Table depicted that TPC in untreated muscle samples of Gp.B increased from an initial load of 2.15±0.2 log cfu/g on day 0 to 9.35±0.15log 20th day 6.86 c ±0.07 3.96 c ±0.08 5.75 c ±0.1 30th day 9.35 d ±0.15 5.99 d ±0.05 7.96 d ±0.03 The coliform count (CC) increase exponentially with increase in storage time both the samples. Gp. B (untreated) samples , the values increase from1.05±0.2 log cfu /g on day 0 to 5.99±0.05 log cfu /g on final day of storage as shown in table, thus crossing the permissible limit i.e. 2.69 log cfu /g after 20th day of storage. However the values in Gp.A (treated) samples were within the permissible limits at the end of storage. 1106 International Journal of Recent Scientific Research, Vol. 4, Issue, 7, pp. 1103 - 1108, July, 2013 Table 6 Changes in Total Plate Count (TPC), Coliform Count (CC) and Psychrophillic Count (PC) of fish muscle of Silver carp (Hypophthalmichthys molitrix) treated with ascorbic and citric acid, stored in freezer at -12±2ºC for a period of 30 days.(Gp.A) Days of storage TPC log cfu/g CC log cfu/g PC log cfu/g 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 TPC 0 DAY CC 10TH DAY 0 day 2.11 a ±0.03 1.02 a ±0.3 2.1 a ±0.05 10th day 2.89 b ±0.01 1.42 b ±0.02 2.72 b ±0.4 PC 20TH DAY 30TH DAY Figure 6 Changes in Total Plate Count (TPC), Coliform Count (CC) and Psychrophillic Count (PC) of fish muscle of Silver carp (Hypophthalmichthys molitrix) treated with ascorbic and citric acid, stored in freezer at -12±2ºC for a period of 30 days.(Gp.A) Similar results were reported by Rakshit and Ramalingam (2013) in meat and fish treated with natural preservative. 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