Shelf‐life enhancement of whole rainbow trout (Oncorhynchus mykiss) treated with Reshgak ice coverage

Abstract The effect of ice coverage comprised of Reshgak extract and Reshgak essential oil on shelf‐life extension of chilled whole rainbow trout (Oncorhynchus mykiss) was evaluated. Chemical (peroxide value (PV), thiobarbituric acids (TBA), total volatile nitrogen base (TVB‐N), and free fatty acids (FFA)), microbiological (total viable count (TVC) and psychrotrophic viable count (PVC)), and sensory evaluations (texture, color, flavor, and general acceptance) were investigated every 4 days during a 20‐day storage period. Results revealed that the effect of both icing systems led to considerably lower bacterial counts and chemical indices in comparison with the traditional ice coverage without such phytogenic. According to sensory analyses, fish stored in ice containing Reshgak essential oil had a longer shelf‐life (>16 days) and those stored in ice medium included with Reshgak extract possessed a shelf‐life of 16 days, whereas lot stored in traditional ice showed a shorter shelf‐life of 12 days.

attention in seafood processing as alternatives for synthetic preservatives (Abedi et al., 2016;Álvarez, García García, Jordán, Martínez-Conesa, & Hernández, 2012;Bensid et al., 2014;Li et al., 2012;Özyurt et al., 2012;Quitral et al., 2009). For instance, the inhibitory effects of essential oils and plant extracts on the microbiological and biochemical mechanisms involved in fish deterioration have been recently reported (Abedi et al., 2016;Álvarez et al., 2012;Li et al., 2012). It is conceivable that using plant bioactive compounds in cold and sanitary conditions such as ice coverage might be a promising method for increasing the seafood shelf-life.
Recently, the efficacy of icing systems containing aqueous extracts of medicinal plants has been investigated in comparison with common ice coverage in few studies. The effect of ice contains thyme, oregano, and clove extracts on chemical, sensory, and microbiological indices of gutted and beheaded anchovy (Engraulis encrasicholus) was studied (Bensid et al., 2014). The results revealed that application of ice with plant extracts could improve the maintenance quality and shelf-life of the fish. To the best of our knowledge, the efficacy of icing system containing plant essential oils to maintain fish quality during cold storage of whole fish has not been investigated.
There has been no scientific report concerning the effect of Reshgak extract (RE) and Reshgak essential oil (RO) on the shelflife of seafood. Modifying traditional preservation treatments such as ice coverage with reputable medicinal plants and spices including Reshgak seems a promising approach to extend the shelf-life of chilled fish products. Therefore, the aim of this study was to investigate the possibility of extending seafood shelf-life via RE and RO in icing system for transportation and chilled storage of rainbow trout.
In this investigation, chemical, microbiological, and sensory changes of treated rainbow trout during chilled storage were assessed.

| Plant material (preparation extract and essential oil)
Dried Reshgak was acquired from a local market in Jahrom, Fars province, Iran, and botanically identified by Department of Horticulture Science, Shiraz University, Iran. The plant was grounded to a fine powder (Pars khazar, Iran). According to the method described by Makkar and Becker (1996), the plant powder macerated in ethanol 70% at a ratio of 1:5 for 48 hr. Finally, the filtered extract (Whatman filter paper no. 2) was concentrated using a rotary evaporator (Vacuum rotary, Fara azma, Iran) at 60°C.
The essential oil of the Reshgak areal parts was isolated via hydro-distillation at Clevenger-type apparatus for 4 hr. Using anhydrous sodium sulfate, Reshgak essential oil was dehydrated, and the essential oil was kept in refrigerator at 3 ± 1°C until subsequent usage (Mexis, Chouliara, & Kontominas, 2009).

| Ice preparation
Preliminarily, various concentrations of RE (100, 200, 300, and 400 mg/L) and RO (500, 1,000, 1,500, and 2,000 mg/L) were examined. The effect of ice contains these concentrations on sensory and microbial changes of short-term cold-stored rainbow trout was studied. Concentrations with the best appearance lacking any strange odor, color, and having low microbial load were chosen (i.e., 300 mg/L RE and 1,500 mg/L RO). It is noteworthy that ice was prepared using distilled water. Also, traditional ice coverage was included in the experiment as a control group.

| Raw fish, preparation, and processing
Rainbow trout (63 individuals; weight range: 350-370 g) were acquired from a local fish farm in Sepidan, Fars province, Iran. Using a traditional icing system, they were transported to the seafood laboratory, Department of Natural Resources and Environment, Shiraz University (2.5 hr). Immediately after arrival, samples were divided into three parts. Fish were placed in covered plastic box (20 kg capacity, white colored) and covered with a layer of corresponding ice treatments (RE, RO, or control) with a fish to ice proportion of 2:1 (w/w). Ice was renewed twice a day during storage period. All boxes were refrigerated in the same holding condition during a 20-day experimental period. To conduct chemical, microbiological, and sensory assessments, samples were randomly taken every 4 days (i.e., days 0, 4, 8, 12, 16, and 20).

| Lipid extraction
Samples were subjected to chloroform/methanol/water (4:4:2.6 ratio) mixture to extract their lipid moiety according to the method described by Bligh and Dyer (1959). The wet tissue was homogenized with a mixture of chloroform and methanol in such proportions that a miscible system was formed with the water in the tissue. Dilution with chloroform and water separated the homogenate into two layers, the chloroform layer containing all the lipids and the methanolic layer containing all the nonlipids. A purified lipid extract was obtained merely by isolating the chloroform layer (Bligh & Dyer, 1959) using a rotary evaporator (Vacuum rotary, Fara azma, Iran) at 50-60°C.

| Peroxide value (PV)
The peroxide content of samples was determined according to Bensid et al. (2014). Fish oil was dissolved into chloroform-acetic acid mixture and subjected to an excess of iodide via a saturated solution of potassium iodide. The peroxides present oxidize the iodide to iodine and the iodine is then titrated to a colorimetric endpoint using sodium thiosulfate with starch as an indicator. Values were expressed as mEqO 2 kg lipid −1 .
The absorbance is measured at 530 nm after heating to 95°C for 120 min and cooling. The TBA content of samples was expressed as mg MDA kg fish −1 .

| Total volatile base nitrogen (TVB-N)
Total volatile basic nitrogen (TVB-N) of sample was determined according to Ojagh, Rezaei, Razavi, and Hosseini (2010). In brief, 2 g MgO was added to 10 g of homogenized fish samples in 400 ml distilled water. The distillate was collected using the 2% aqueous solution of boric acid and titrated using 0.05 mol/L sulfuric acid solution in the presence of the methyl red-methylene blue indicator as the endpoint detection sign. TVB-N content of samples was expressed as mg N.100 g/fish.

| Free fatty acids (FFA)
Free fatty acids contents of samples were quantified via acidimetric titration of the Bligh and Dyer (1959)

| Sensory evaluation
At least six-member trained panelists (three females and three males with age of 25 to 50 years old) performed sensory evaluations.
Samples for organoleptic evaluation were prepared by steaming for 30 min at 60°C. The texture, taste, color, and overall acceptance were rated according to 5-point hedonic scale (1, very poor to 5, excellent).

| Statistical analysis
SAS software (version 9.0) was used for statistical analysis. Data, except for sensory analysis, were subjected to one-way analysis F I G U R E 1 Changes in peroxide value of rainbow trout stored for 20 days in different icing media. Capital letters refer to comparison made among different treatments at each sampling time. Comparing the effect of storage time on each treatment was shown using lowercases. Different superscripts mean statistically significant differences (p < .05). A > B > C > D. (Extract, essential oil, and control depict samples stored in ice containing Reshgak extract, Reshgak essential oil, and those stored in icing medium without any additives, respectively) of variance (ANOVA) followed by Duncan's multiple range test for getting the conservative differences between two means. The sensory data were subjected to nonparametric Kruskal-Wallis test, followed by Mann-Whitney U test for the identification of differences.
All statistical analyses were interpreted at the significance level of p < .05. Data were presented at Mean ± SD.

| Peroxide value
The initial PV of fish samples lipid was 0.1 (mEq of O 2 kg/lipid).
The PV value of all samples significantly increased during storage (p < .05). As depicted in Figure 1, icing with RE and RO significantly hindered (p < .05) peroxide formation on days 8, 16, and 20. However, there were no significant differences (p > .05) among experimental groups on days 4 and 12 of the experimental storage of rainbow trout. The results revealed that the RE and RO were effectively slowed down lipid peroxidation of whole rainbow trout kept in bioactive icing system. According to the results, the inclusion of RE and RO in the icing system could reduce the formation of primary oxidation products. The previous research showed that major constituents of RO were α-pinene, β-myrcene, β-pinene,

| Thiobarbituric acid
Thiobarbituric acid (TBA) is a common index of the extent of lipid oxidation at the second stage of auto-oxidation, the stage of generation of aldehydes and ketones from peroxides (Fan, Chi, & Zhang, 2008;Jeon, Kamil, & Shahidi, 2002). Changes in TBA value of fish stored in different icing treatments were given in Figure 2. The initial TBA value of samples was low (0.08 mg MDA kg fish −1 ). However, it showed an increasing trend with storage time (p < .05). Meanwhile, TBA content of fish stored under icing system without any supplementary phytogenic was higher than samples stored in RE-or ROcontaining ice. The lipid oxidation products of all samples varied from 0.08 to 0.18 (mg MDA kg fish −1 ) with the highest values recorded in samples stored in common icing system at the end of the trial. It has been reported that plant extracts and essential oils may act as a high scavenger of radicals involved in lipid peroxidation protecting lipids from oxidation during storage (Kulisic, Radonic, & Milos, 2005). This antioxidant activity mainly has been related to flavonoids and polyphenolic compounds. All of these phenolic compounds have the ability to act as antioxidants by a free radical scavenging mechanism as well as ability to chelate transition metals such as iron to augmented oxidation of unsaturated fatty acids (Fan et al., 2008).
The increase in the TBA value of the present samples in the course of chilled storage was well in accordance to previous studies conducted by Abedi et al. (2016) and Gelman and Benjamin (1989) on rainbow trout and silver carp, respectively.

| Total volatile base nitrogen
TVB-N index of various experimental groups during storage is given Figure 3. TVB-N of fish at the start of the experiment was 2.8 mg N.100 g/fish. There was significant increase in TVB-N contents of whole rainbow trout during ice storage (Figure 3, p < .05).
TVB-N value reached 21.5 mg N.100 g/flesh in control group, while those of RE and RO groups was 8.9 and 5.1 mg N.100 g/flesh by the end of storage. TVB-N values increased with different rates depending on the nature of treatments. This may be attributed to the breakdown of proteins as a result of activity of microbial strains and proteolytic enzymes. The highest TVB-N contents of all experimental groups were observed on day 12 of storage, corresponding to sensory rejection of those fish stored in traditional icing system.
Icing system containing RE and RO had significantly lower TVB-N values of 13.5 and 14 mg N.100 g/flesh, respectively, in comparison with fish kept under common icing system (26.7 mg N.100 g/flesh on day 12 of storage). The TVB-N content of control samples on day 12 of storage was over the limit of acceptability of 25 mg N.100 g/ flesh (Gimenez, Roncales, & Beltran, 2002;Ojagh et al., 2010).
The statistical analysis revealed a considerable difference on days 12, 16, and 20 of storage with regard to TVB-N (p < .05) among various experimental groups, while no significant differences were observed on days 0, 4, and 8 days in this respect. There were no significant differences between RE-and RO-supplemented ice regarding the criterion through storage time (p < .05). This may be attributed to the role of such compound on microbial population and bacterial growth as antimicrobial agents.

| Free fatty acids
Free fatty acids (FFA) are products of enzymatic disintegration of lipids (Uçak, Özogul, & Durmuş, 2011). The FFA contents of chilled stored rainbow trout were depicted in

| Microbial assessment
Total viable counts (TVCs) of rainbow trout during chilling storage under different icing conditions are shown in Figure 5. The initial total viable bacterial counts indicated an acceptable fish quality was 5.97 at the end of the storage period, still well below 7, the maximal recommended limit of 7 for TVC of raw fish (Ojagh et al., 2010).
Psychrophilic bacteria are the major group of microorganisms responsible for aerobic spoilage of chilled stored fish (Ojagh et al., 2010). In the present study, the initial PVC was 2.52 ( Figure 6).
Furthermore, the variation of PVC of samples was similar to that of TVC during the chilled storage; control group showed the highest value on day 20 (7.31), followed by samples stored in ice prepared with Reshgak extract (6.62), and the lowest count (5.81) has been observed in samples stored in ice prepared with RO.
Results of microbial assessment indicated the microbiological shelf-life up to 12 days for the control samples, 16 days for samples stored in ice prepared with RE, and more than 20 days for samples stored in ice containing RO.
The subsequent sensory shelf-life determination indicated that in common icing system, the fish were acceptable up to 12 days, while F I G U R E 5 Changes in total viable counts (TVCs) of fish samples during chilling storage  (Hernández, García García, Jordán, & Hernández, 2014;Li et al., 2012;Sant'Ana & Mancini-Filho, 2000).
One might infer that antimicrobial potency of RE was less than RO in preserving rainbow trout during chilled storage. According to Figures 5 and 6, higher bacterial counts were enumerated in samples stored under traditional ice coverage followed by samples stored in ice containing RE and RO. Possibly these results are related to the bioactive compound such as polyphenolic components of the essential oil, namely flavonoids (Negi, 2012 (Janssen et al., 1984).

| Sensory evaluation
Sensory assessment has been an efficient method of evaluating the quality change of a product over storage time (Abedi et al., 2016).
Sensory characteristics including texture, color, flavor, and general acceptability of steam-cooked samples are shown in Table 1.
At the beginning of storage period, the texture was firm and flexible; however, fish kept in ice without any phytogenic supplementation showed a soft and undesirable texture and determined as spoiled sample (SD) by the experiment panelists on day 12 of storage (  (Figures 1-6) as well as sensory test (Table 1) during storage, quality attributes of the products were deteriorated due to lipid oxidation and bacterial growth which have been the main factors that determining fish quality loss and shelf-life reduction. Lipid oxidation leads deterioration of lipids and proteins decay which, in turn, contribute to the reduction in nutritional quality as well as deterioration in flavor, color, and texture of final products.

| CON CLUS ION
The proposed bio-preservatives-icing system with Reshgak extract and essential oil proved to be effective for fish preservation mainly due to their antioxidant and antimicrobial properties. Sensory evaluation also revealed that such icing system might significantly extend the seafood shelf-life. The ice supplemented with Reshgak essential oil would extend the shelf-life of whole rainbow trout for nearly further 8 days, and inclusion of extract of the plant would extend the shelflife of rainbow trout for another 4 days in comparison with traditional icing medium without any phytogenic material. Reshgak extract and essential oil combined with ice seem to be a promising solution for extending fish shelf. These results may also be of practical interest for on-board goals.

ACK N OWLED G M ENTS
The study was financially supported by Shiraz University Students