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Jasour et al., J Food Process Technol 2011, 2:5 http://dx.doi.org/10.4172/2157-7110.1000124

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Effects of Refrigerated Storage on Fillet Lipid Quality of Rainbow Trout (Oncorhynchus Mykiss) Supplemented by a-Tocopheryl Acetate Through Diet and Direct Addition after Slaughtering
Mohammad Seddigh Jasour 1, Eshagh Zakipour Rahimabadi1*, Ali Ehsani2, Mohammad Rahnama3 and Ali Arshadi1
1 2 3

Department of Fisheries, Faculty of Natural Resources, University of Zabol, 98615-538 Zabol, Iran Department of Biotechnology and Pathobiology and Quality Control, Artemia and Aquatic Animals Research Institute, Urmia University, Urmia, Iran Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, University of Zabol, 98615-538 Zabol, Iran

Abstract
This study was designed to compare the effects of -tocopherol acetate through diet (0, 300 and 500 mg/ kg) and direct addition (200 mg/kg flesh) after slaughtering on oxidative stability of rainbow trout (Oncorhynchus mykiss) fillets during 12 days of refrigerated storage. For this, fish were fed experimental diets for 58 days followed by processing to obtain boneless, skinless fillets. A solution of -tocopherol (vitamin E-ethanol-distilled water) was used for the superficial application on fillets. The fillets were packed and stored at 4 C. The samples were analyzed periodically for chemical characteristics (PV, TBA, FFA and pH). According to the results, the concentration of -tocopherol in fillets increased linearly in response to feed concentration (P<0.05). Dietary and surface application of -tocopherol improved (P<0.05) the oxidative stability of fish lipid during storage. Lower PV and TBA values were observed in the fish fillets that received dietary -tocopherol acetate in comparison with the other groups (P<0.05). In addition, there were no significant (P>0.05) differences in FFA and pH between samples held in the refrigerator during 12 days.

Keywords: Fish fillet; Lipid oxidation; Refrigerated storage; Oncorhynchus mykiss; -tocopheryl acetate Introduction
It is recommended that fish must be consumed two or three times each week. Because fish have beneficial effects in coronary heart disease, inflammatory and auto-immune disorders, aggression for human due to presence of poly unsaturated fatty acids (n-3 PUFA), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [1,2]. On the other hand, PUFA in fish lipid are very susceptible to rancidity caused by oxygen free radicals. Lipid oxidation is a major cause of quality deterioration in fish muscle. The off-orders and off-flavor that result from lipid oxidation lower the quality and thus shorten the shelflife of the muscle during storage time [3,4]. Rainbow trout is a fatty farmed fish [5], and is high demanded among the consumers. Quality deterioration of fatty fish species is primarily caused by microorganisms and lipid oxidation during the storage period [6]. Using of antioxidants in an effective way to minimize or delay the oxidation process, retarding the formation of toxic oxidation products, maintaining nutritional quality and prolonging the shelf-life of food [7]. DL--tocopherol acetate, the form of synthetic vitamin E commonly added to fish feed, functions as a lipid soluble antioxidant that protects biological membranes, lipoproteins and lipid store [7]. It has been suggested by many groups of workers that increasing dietary level of -tocopherol has increased muscle -tocopherol in tissue of fish [8-13]. Tissue -tocopherol by scavenging free radicals which are involved in the initiation and propagation of lipid oxidation has protective role against lipid oxidation [9,10,14]. Flesh quality parameters (mainly lipid oxidation) of fish fed diets containing -tocopherol has been studied by many workers, and it has been demonstrated for rohu [7], pacu [15], turbot [16], sea bass [10]
J Food Process Technol ISSN:2157-7110 JFPT, an open access journal

and beluga sturgeon [11]. In addition, the effect of dietary -tocopherol on tissue lipid quality of rainbow trout during refrigeration has studied by Yeildiz et al. [9] and Chaiyapechara et al. [13]. Comparison studies on the effects of -tocopheryl acetate through diet and direct addition after slaughtering on lipid stability of fish during storage period, have demonstrated that postmortem addition of -tocopherol improved lipid stability of tilapia hamburgers during frozen storage [17]. This study was designed to compare the effects of -tocopherol through diet and direct addition after slaughtering on lipid characteristics of rainbow trout fillets during refrigerated storage.

Material and Methods

Determine the treatment diets, sampling procedure and storage conditions
360 rainbow trout with mean initial weight of 955 g were purchased from a local aquaculture farm located at Urmia (Azarbijan Gharbi, Iran). Commercial diet was purchased from Faradaneh co. (4 mm diameter, pellet, Esfahan-Iran). The proximate compositions of the main dietary ingredients are given in Table 1. -tocopherol acetate was supplied as all-rac--tocopheryl acetate (Merck, Darmstadt,

*Corresponding author: Eshagh Zakipour Rahimabadi, Department of Fisheries, Faculty of Natural Resources, University of Zabol, 98615-538 Zabol, Iran, Tel: +98 542 2232600; E-mail: e_zakipour@yahoo.com Received September 18, 2011; Accepted October 31, 2011; Published November 02, 2011 Citation: Jasour MS, Rahimabadi EZ, Ehsani A, Rahnama M, Arshadi A (2011) Effects of Refrigerated Storage on Fillet Lipid Quality of Rainbow Trout (Oncorhynchus Mykiss) Supplemented by a-Tocopheryl Acetate Through Diet and Direct Addition after Slaughtering. J Food Process Technol 2:124. doi:10.4172/2157-7110.1000124 Copyright: 2011 Jasour MS, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Volume 2 Issue 5 1000124

Citation: Jasour MS, Rahimabadi EZ, Ehsani A, Rahnama M, Arshadi A (2011) Effects of Refrigerated Storage on Fillet Lipid Quality of Rainbow Trout (Oncorhynchus Mykiss) Supplemented by a-Tocopheryl Acetate Through Diet and Direct Addition after Slaughtering. J Food Process Technol 2:124. doi:10.4172/2157-7110.1000124

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Chemical Moisture Crude protein Crude fat Ash content Crude cellulose Phosphor Content (%) 10 42 12 13 3.7 0.85

Determination of free fatty acid (FFA): FFA analysis expressed as % oleic acid was done by the Egan et al. [20]. Determination of thiobarbiutoric acid (TBA): The content of lipid hydroperoxides and secondary oxidation compounds was determined according to the method of Egan et al. [20]. TBA had absorbance at 532 nm and expressed as mg malondialdehyde/kg lipid. pH: The pH was determined on homogenous mixtures of fish flesh and distilled water (1:10, w: v) using a digital pH -meter (crison GLP 22+, Germany) [21].

Table 1: Major basal ingredients content (%) of the commercial diets.

Germany). After acclimatization for two weeks, the fish were randomly allocated to nine tanks (30 per each tank), raised at a temperature of 151C in 4-5 m3 (3045540 cm) at the aquaculture facilities of Artemia institute (Urmia University, Iran). Supplemental aeration was provided to maintain dissolved oxygen near saturation in each tank at water flow rate 141 L/min. Temperature and dissolved oxygen were measured daily (151 C and 100.5 mg/L, respectively). The experimental diets differed only in the relative amounts of -TA (0, 300 and 500 mg/Kg feed). Soybean oil (3 %) was used for adding -TA in diets. To avoid lipid peroxidation, the diets were stored at -18 C until fed to the fish. After 2-week conditioning period, the fish were fed experimental diets to apparent satiation, twice daily at 8:00 and 17:00 h, 7 days a week. At the end of experiment (58 days), fish were killed by ice-shocking. Sample were washed with tap water, descaled, deheaded and filleted. This experiment had four treatments included: E300 (300 mg/kg -TA in diet), E 500 (500 mg/kg -TA in diet), E0 (control group, samples with commercial diet) and E200 (samples with commercial diet and 200 mg vitamin E /kg of fillets). Two fillets were packed separately in polythene bags, and stored in a refrigerated at 4 C for 12 days. The fish fillets fed commercial diet (base level of -TA) were divided into two groups. The first group defined as control group, that it is not superficially treated by spraying with -TA solation. Another group at this time treated with -tocopherol solation (-tocopherol-thanol 70%-distilled water) with concentration 200 mg -tocopherol/kg flesh. All samples were taken for chemical analysis (pH, PV, TBA and FFA) in days 0, 3, 6, 9 and 12 of refrigerated storage.

Statistical analysis
A complete randomized design (CRD) was used in this experiment. All data were presented as mean standard division (SD) and were subjected to one way analysis of variance (ANOVA), and analyzed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). The Duncan test was used for mean comparison when a significant variation was found by the ANOVA test. Regression analysis was used to determine the relationship between dietary -tocopheryl acetate and its fillet deposition. The significant means were compared at = 0.05 level (n= 3).

Results and Discussion

Diets and tissue -tocopherol: The result of the -tocopherol levels in experimental diets and fillets of fish fed the experimental diets are shown in Table 2. In the present study, it was observed that fillets -tocopherol supplementation significantly influenced concentration of dietary -tocopherol (Table 2; P<0.05). The -tocopherol concentration of fillets increased significantly (P<0.05) with increasing dietary vitamin E concentration. The dietary concentrations of -TA were 51.2, 336.1 and 566.2 mg/kg for 0, 300 and 500 mg/kg diet, respectively. At the end of study period, the fillets -tocopherol concentration for experimental diets (0, 300 and 500 mg -tocopherol/ kg feed) were 16.1, 34.2 and 47.3 mg/kg fillet, respectively (P<0.05). In addition, the correlation between -tocopherol levels in diets and fillets are shown in Figure 1. The results showed a good correlation (r2= 0.99) between -tocopherol levels in diets and fillets (Figure 1). Result of present work is in agreement with the result get for rainbow trout [9,12,13], sea bass [10], rohu [7] and beluga sturgeon [11] that have demonstrated the effects of dietary -tocopherol on tissue -tocopherol content.

Chemical analysis
Diets and tissue -tocopherol: Vitamin-E (-tocopherol) was analyzed as described by Liu and Lee [18]. 1g of homogenized sample was saponified in the presence of antioxidant (L-ascorbic acid and tert-butylhydroquinone). Exact weight of the samples were recorded and used for calculations. Freshly digestion solution (11% potassium hydroxide, 55% ethanol, 45% distilled and deionized water) was prepared to dissolve the ascorbic acid. The unsaponifiable material was extracted with iso-octane. The vitamin E was determined using normal phase HPLC and fluorescence detection. -tocopherol content in the trout fillets, reported as mg of -tocopherol per of fillets or diets. Lipid extraction: The total lipid was extracted from the fish muscle by the Bligh and Dyer [19] method. A chloroform and methanol mixture (2:1, vol/vol) was used as a solvent. Samples containing muscle lipid were transesterified with acidified methanol. Determination of peroxide value (PV): The peroxide content was determined in the total lipid extracts according to the method of Person [20]. Results were expressed in meq oxygen/kg lipid.
J Food Process Technol ISSN:2157-7110 JFPT, an open access journal

Lipid oxidation
Lipid oxidation and hydrolysis products, as main cause of a shortend shelf-life of fish and fish products were evaluated by means of the PV [22], TBA and FFA [10,11,23]. Numerous factors such as species, storage temperature and fat composition are affecting in lipid oxidation [24].
-tocopherol concentration (mg/kg samples) in treatments of E0 Trial diets Fish Fillets 51.2 16.1 E200 336.1 34.2 E500 566.2 47.3

Table 2: Trial diets and fillets (end of feeding period) -tocopherol concentration (mg/kg samples).

Volume 2 Issue 5 1000124

Citation: Jasour MS, Rahimabadi EZ, Ehsani A, Rahnama M, Arshadi A (2011) Effects of Refrigerated Storage on Fillet Lipid Quality of Rainbow Trout (Oncorhynchus Mykiss) Supplemented by a-Tocopheryl Acetate Through Diet and Direct Addition after Slaughtering. J Food Process Technol 2:124. doi:10.4172/2157-7110.1000124

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Peroxide value (PV)

The changes of peroxide value as primary products of lipid oxidation are shown in Figure 2. The PV values of 2.5, 2.05, 1.02 and 2.5 at day 0 and 27, 18.5, 15.16 and 24 mg oxygen/kg lipid at the refrigerated storage were found for dietary (0, 300 and 500 mg/kg feed) and spraying -tocopherol acetate (200 mg/kg fillet), respectively. PV content significantly increased (P<0.05) in all treatments throughout the refrigerator storage, indicating that lipid deterioration continued under the storage temperature conditions (Figure 2). There were significant differences in PV content (P<0.05) between the fillets of fish fed different levels of dietary -tocopherol, postmortem addition and control group (Figure 2). The result also showed that the highest level of dietary -tocopherol (500 mg/kg feed) was the most effective in slow down primary peroxidation, when compared with other groups (P<0.05). The postmortem addition of -tocopherol was also effective on control group (P<0.05). -tocopherol is synthetic chinone, inhibiting the lipid peroxidation by attaching free radicals to its double conjugated linkages [25]. The results of the present study indicate that -TA through diet and direct addition after slaughtering is effective in retarding the production of PV in trout fillets stored in refrigerator (4C). These results are agreement with the results of Hosseini et al. [11], who suggested that dietary -tocopherol can slow down the change of PV in beluga sturgeon fillets during storage at -18C. Also, progressive production of PV during storage of fish has reported by Ojagh et al. [26] and Rezaei et al. [27].

Figure 3: The results of ANOVA test on thiobarbituric acid (TBA) levels of rainbow trout during refrigerated storage.

was observed in control group and other treatments throughout of 12 days storage (P<0.05). When comparing the TBA of treatments, it was found that the TBA of groups that received dietary -tocopherol was lower than that received postmortem -tocopherol and control group (P<0.05). At the end of 12 days refrigerated storage, it was observed that, TBA contents of fillets were 0.4055, 03395, 0.2207 and 0.1875 mg malondialdehyde/kg lipid for dietary (0, 300 and 500 mg/kg) and spraying -tocopherol acetate, respectively. It has been reported that the maximum level of TBA value indicating good quality of the fish during storage period is 1-2 mg malondialdehyde/kg lipid [29]. In the current study, TBA values control and other treatments analyzed were much lower than such proposed limits throughout the 12 days storage period. However, Aubourg [30] has reported that TBA values may not give the actual rate of lipid oxidation, since malondialdehyde can interact with other components of fish such as nucleosides, nucleic acids, proteins, amino acids, phospholipids and other aldehydes that are end-products of lipid oxidation. In addition, in present study the fillets were stored in a dark refrigerator and protected from light. These conditions most likely contributed to the inhibition of lipid oxidation even at low -tocopherol concentration. Jitinandana et al. [12], Yeildiz et al. [9], Pirini et al. [10], Gatta et al. [31], Huang and Huang [32] and Huang et al. [33] had found lower contents of TBA in lipids of fish fed different levels of -tocopherol during storage period. It has also reported that postmortem addition of antioxidants did not bright any significant advantage over dietary supplementation [17,34-37].

Thiobarbituric acid
TBA is widely used indicator for the assessment of degree of secondary lipid oxidation [28]. In Figure 3, the TBA values for the different treatments during storage are presented. The TBA values of 0.00175, 0.0023, 0.00085 and 0.00175 mg malondialdehyde/kg of lipid at initial of trial in analyses were found for dietary (0, 300 and 500 mg/kg feed) and spraying -tocopherol acetate (200 mg/kg fillet), respectively (Figure 3; P<0.05). A continues increase in TBA value

Figure 1: Correlation between Diets and Fillets -tocopherol (mg/kg samples) concentration during the feeding period.

Lipid hydrolysis: Free Fatty Acids (FFA)

The results of the free fatty acids value of samples are given in Figure 4. The FFA contents of fillets were increased from 3.42, 3.49, 3.46 and 3.78 to 9.91, 9.92, 9.94 and 10.01 at the end of storage, for dietary (0, 300 and 500 mg/kg) and spraying -tocopherol acetate, respectively. Although, there was no significant differences in FFA content between treatments received -TA through diet and direct addition after slaughtering (P>0.05), but the release of FFA in samples stored at refrigerator for 12 days showed significant increase (P<0.05). These finding are in agreement with Hosseini et al. [11] who founds dietary -tocopherol cannot slow down the change of FFA in beluga sturgeon fillets during storage at -18 C. The progressive development in lipid hydrolysis during storage have reported for Anchovy kilka [27], rainbow trout [26,38]. The release of FFA content increased with time, as found in this

Figure 2: The results of ANOVA test on peroxide value (PV) levels of rainbow trout fillets during refrigerated storage.

J Food Process Technol ISSN:2157-7110 JFPT, an open access journal

Volume 2 Issue 5 1000124

Citation: Jasour MS, Rahimabadi EZ, Ehsani A, Rahnama M, Arshadi A (2011) Effects of Refrigerated Storage on Fillet Lipid Quality of Rainbow Trout (Oncorhynchus Mykiss) Supplemented by a-Tocopheryl Acetate Through Diet and Direct Addition after Slaughtering. J Food Process Technol 2:124. doi:10.4172/2157-7110.1000124

Page 4 of 5

Conclusions
Present work has demonstrated relationship between dietary -tocopherol levels and levels of fillet -tocopherol concentration. Also, successful inhibition of lipid oxidation in refrigerated rainbow trout fillets was possible with -tocopherol (300 & 500 mg/kg feed) and surface application after harvested (200 mg/kg flesh). But fish fed diet containing 500 mg -tocopherol/kg feed have a higher positive benefit on improved shelf life of rainbow trout fillets during refrigerated storage. These observations demonstrate the potential benefit of the fish industry of the dietary -tocopherol acetate.
Figure 4: The results of ANOVA test on free fatty acid (FFA) levels of Rainbow trout fillets during refrigerated storage.

Acknowledgments
Authors thank to Artemia and Aquatic Animals Research Institute of Urmia University for providing of facilities for this study.

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J Food Process Technol ISSN:2157-7110 JFPT, an open access journal

Volume 2 Issue 5 1000124

Citation: Jasour MS, Rahimabadi EZ, Ehsani A, Rahnama M, Arshadi A (2011) Effects of Refrigerated Storage on Fillet Lipid Quality of Rainbow Trout (Oncorhynchus Mykiss) Supplemented by a-Tocopheryl Acetate Through Diet and Direct Addition after Slaughtering. J Food Process Technol 2:124. doi:10.4172/2157-7110.1000124

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Volume 2 Issue 5 1000124