Effects of Methanolic Pomegranate Peel Extract on the Chemical ...

1700 Journal of Food Protection, Vol. 79, No. 10, 2016, Pages 1700–1706 doi:10.4315/0362-028X.JFP-16-047 Copyright Q, International Association for Food Protection

Effects of Methanolic Pomegranate Peel Extract on the Chemical, Sensory, Textural, and Microbiological Properties of Gutted Rainbow Trout (Oncorhynchus mykiss) during Frozen Storage ENAYAT BERIZI, SEYED SHAHRAM SHEKARFOROUSH,*

AND

SAEID HOSSEINZADEH

Department of Food Hygiene and Public Health, School of Veterinary Medicine, Shiraz University, Shiraz 71345-1731, Iran MS 16-047: Received 29 January 2016/Accepted 4 June 2016

ABSTRACT The effects of methanolic pomegranate peel extract (MPPE) on the quality of gutted rainbow trout (Oncorhynchus mykiss) were examined periodically during 6 months of storage at 188C. Fresh samples were dipped (ratio of fish to liquid, 1:2 [wt/vol]) in sterile water or in sterile water containing 1, 2, or 4% (wt/vol) MPPE and packed in low-density polyethylene pouches. The control and treated samples were analyzed monthly for microbiological, chemical, sensory, and textural characteristics. Microbial growth in samples was significantly reduced by MPPE treatment (P , 0.05). Smaller increases in the values for total volatile base nitrogen, peroxide, and thiobarbituric acid–reactive substances and better oxidative stability were also obtained. Moreover, higher organoleptic scores were recorded. However, regarding the general acceptability, the highest score was achieved in the 1% MPPE group. Greater hardness and chewiness were obtained with 4% MPPE (P , 0.05). Therefore, dipping fish in MPPE can be considered an effective method to extend the shelf life and the overall quality of the product. Key words: Oncorhynchus mykiss; Pomegranate peel extract; Rainbow trout

The rainbow trout (Oncorhynchus mykiss) belongs to the family Salmonidae and is one of the main fish species farmed in Iran. The demand for rainbow trout in Iran and other countries’ markets has increased significantly over the past decade, and this could be due to its desirable characteristics (aroma, taste, and white flesh) resulting in a high-quality product (29). However, fish are perishable foods that generally spoil faster than do other muscle foods. Higher protein and moisture levels support microbial spoilage, whereas the presence of polyunsaturated fatty acids induces oxidation of lipids and protein. Frozen storage is a general preservation method used to control or decrease biochemical changes in fish that occur during storage. Nevertheless, frozen storage does not completely inhibit microbial and chemical reactions that lead to deterioration in the quality of fish, as fish muscle is rich in proteins and unsaturated fatty acids (38). To retain the good quality characteristics for longer and extend shelf life during frozen storage of fish, preservatives like butylated hydroxyanisole and butylated hydroxytoluene have been used widely (16). Numerous studies are currently focused on using natural ingredients to enhance food quality and shelf life and to meet consumer demands for safer foods in order to avoid the use of synthetic preservatives. The antioxidant effect of green tea leaf extracts in turkey sausages was reported by Bozkurt (8). The sensory attributes and phenolic content of * Author for correspondence. Tel: þ98 71 32286950; Fax: þ98 71 32286940; E-mail: [email protected].

precooked pork breakfast sausage with fruit purees were investigated by Leheska et al. (23). The antioxidant effects of grape seed extracts in chicken thigh meat were investigated by Brannan (9). The use of grape seed, oregano, and rosemary extracts in frozen vacuum-packaged beef and pork as an antioxidant in sausages has been reported by Rojas and Brewer (32). The addition of kinnow rind and seed powders in cooked goat meat patties improved oxidation stability (35). Similar antioxidant effects of aqueous and methanol soy extracts in minced trout muscle were investigated by D’Souza and Skonberg (12). Punica granatum (Punicaceae), commonly known as pomegranate, is a shrub or a small tree native to the Mediterranean region. The plant possesses numerous therapeutic benefits. A number of biological activities, such as antioxidant (19), antibacterial (3), antifungal (11), antitumor (31), antidiarrheal (30), and antiulcer (26) activities, have been reported with various extracts or constituents of different parts of this plant. Pomegranate peel is an inedible part, or by-product, obtained during the processing of pomegranate juice. Among the various constituents, the peel extract shows higher antioxidant activity in vitro, in good correlation with its high content of polyphenols. Pomegranate peel extract had scavenging activity against superoxide anions and inhibitory action on low-density lipoprotein oxidation (24). The antimicrobial activities of phenolic compounds may involve multiple modes of action. The type of microorganism and its cell wall structure are thought to play important roles, but phenolic

J. Food Prot., Vol. 79, No. 10

EFFECTS OF POMEGRANATE PEEL EXTRACT ON QUALITY OF RAINBOW TROUT

compounds can also bind to substrates such as minerals, vitamins, and carbohydrates, making them unavailable for microorganisms. Furthermore, phenols can be absorbed to the cell wall, resulting in a disruption of the membrane structure and function (33). However, no information regarding the use of pomegranate peel extract as a natural antioxidant and antimicrobial to maintain the quality of frozen rainbow trout has been reported. Therefore the objective of this work was to study the effect of methanolic pomegranate peel extract (MPPE) on the quality of rainbow trout under frozen storage.

MATERIALS AND METHODS Preparation of MPPE. The Rabab variety of pomegranate was obtained from a garden in Shiraz, Iran. The fruits were washed, cut manually, and peeled. The peel thus obtained was cut into small pieces and dried in an oven at 508C until reaching a constant weight and then ground to powder. Methanolic extraction was performed as described by Basiri et al. (6). The total phenolic content and DPPH radical scavenging activity were measured and reported as described in Basiri et al. (6). Fish sample preparation. A total of 72 freshly slaughtered rainbow trout with an average weight of 500 6 50 g were obtained from a local fish farm and transferred to the laboratory on crushed ice within 2 h. They were then gutted, washed carefully using tap water, and dipped for 60 s either in sterile distilled water or in sterile distilled water with 1, 2, or 4% (wt/vol) MPPE (ratio of fish to liquid, 1:2 [wt/vol]). The dipping solutions were maintained at room temperature (228C). After dipping, the samples were packed individually in low-density polyethylene pouches. All samples were stored at 188C and subjected to microbial, chemical, textural, and sensorial analysis monthly for 6 months. The fish was divided into three portions: anterior third (sensory analysis), middle third (for chemical and microbial analysis), and posterior third (for textural analysis). Microbial quality evaluation. The whole middle third of each sample was ground aseptically (Moulinex, Paris, France). Twenty-five grams of each ground sample was transferred aseptically into individual stomacher bags containing 225 ml of sterile normal saline and homogenized for 120 s in a stomacher (Stomacher BA 6021 lab blender, Seward-Medical, Worthing, England). A 10-fold serial dilution was subsequently prepared. The pour plate technique was used as described by AOAC (4). The bacteriological evaluations performed included a psychrophilic bacterial count (plate count agar at 78C for 7 days), an Enterobacteriaceae count (MacConkey agar at 378C for 48 h), and a lactic acid bacterial (LAB) count (MRS agar at 378C for 48 h) under aerobic conditions. All media were purchased from Merck (Darmstadt, Germany). After appropriate incubation, the numbers of colonies that developed on the plates were counted and expressed as CFU per gram. pH measurement. The pH of the samples was determined by blending 10 g of ground whole fish with 10 ml of distilled water for 60 s in a homogenizer (DI18B, IKA, Staufen, Germany). The pH values were measured using a standardized electrode attached to a digital pH meter (CG824, Mettler-Toledo, Giessen, Germany). Determination of TVB-N content. Total volatile base nitrogen (TVB-N) in the ground fish tissue was calculated using

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steam distillation, followed by titration (4). The TVB-N content was expressed in milligrams of N per 100 g of fish tissue. Determination of peroxide value. The peroxide content in the total lipid extracts was determined according to the method in Pearson’s Chemical Analysis of Food (14). The results were expressed in milliequivalents per kilogram. Determination of TBARS. The thiobarbituric acid reactive substance (TBARS) values of samples were determined by using the extraction method described by Benjakul and Bauer (7), with slight modifications. One gram of ground fish sample was mixed with 9 ml of 0.25 N HCl solution containing 0.375% TBA (SigmaAldrich, St. Louis, MO) and 15% trichloroacetic acid (Merck). The mixture was heated in boiling water for 10 min, followed by cooling with running water. The mixture was centrifuged at 3,500 3 g for 15 min. The supernatant was collected, and the absorbance was read at 532 nm using a spectrophotometer. The TBARS value was calculated from the standard curve of malonaldehyde (0 to 2 mg/kg; Merck) and expressed as milligrams of malonaldehyde per kilogram of sample. Sensory evaluation. The anterior third of each fish sample was evaluated by 17 trained panelists, using the following 4-point scale: 1 ¼ like extremely, 2 ¼ like moderately, 3 ¼ neither like nor dislike, and 4 ¼ dislike. The panelists were regular consumers of fish and had not had any symptoms of allergies. All the panelists were asked to evaluate color, odor, flavor, and overall likeability. Textural analysis. The area around the juncture of the anterior edge of the adipose fin and the lateral line of the fish was used for textural analysis, using a texture analyzer (CT3 Texture Analyzer, Brookfield, Middleboro, MA) with a load cell of 5 kg to perform textural profile analysis. Fish samples were removed from the pouches and compressed to 20% of their original height by two consecutive compressions using a cylindrical probe with a diameter of 10 mm. The pretest speed, test speed, and posttest speed were 5, 2, and 10 mm/s, respectively. The waiting time between the two cycles of the textural profile analysis was 10 s, and the trigger force was 5 g. Data collection and calculations (hardness and chewiness) were carried out using the Texture Expert program (TexturePro CT, Brookfield). Hardness was defined as the maximum force detected during first compression, expressed in grams. Chewiness (N per centimeter) is defined as the work needed to chew a solid sample to a steady state of swallowing (2). Statistical analysis. All experiments were performed in triplicate. Analysis of variance was performed, and mean comparisons were done with Duncan’s multiple range tests. To make comparisons between treatments, the Pearson correlation test was applied. Nonparametric data were analyzed using the KruskalWallis test. P values of less than 0.05 were considered statistically significant. Analysis was performed using an SPSS package (SPSS 16 for Windows, SPSS, Inc., Chicago, IL).

RESULTS AND DISCUSSION Effect of MPPE on microbiological changes in fish during storage. The changes in the psychrophilic bacterial counts of samples are shown in Figure 1A. In all treatments, the number of psychrophilic bacteria decreased during the first 2 months of storage (P , 0.05). At the end of the storage period, the lowest psychrophilic bacterial counts, compared

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FIGURE 1. Psychrophilic bacterial (A), LAB (B), and Enterobacteriaceae (C) counts of gutted rainbow trout samples treated with methanolic pomegranate peel extract (MPPE) at three concentrations during 6 months of frozen storage at 188 C (means 6 standard errors). *, Control; *, 1% MPPE; ~, 2% MPPE; &, 4% MPPE.

with those in the control, were obtained in the samples treated with 4% MPPE (P , 0.05). The psychrophilic bacterial counts (means 6 standard errors) of the controls and of samples treated with 1, 2, and 4% MPPE were 5.14 6 0.03, 4.87 6 0.09, 4.86 6 0.09, and 4.27 6 0.08 log CFU/g, respectively, at the end of the storage period. The changes in LAB counts during the storage period for the treated and untreated fish are given in Figure 1B. The LAB counts (log CFU per gram) in fish samples ranged from 2.20 6 0.06 log CFU/g in the samples treated with 4% MPPE to 2.47 6 0.23 log CFU/g in the controls after 2 months of storage (P . 0.05). The LAB counts of the controls and the samples treated with 1, 2, and 4% MPPE reached 3.11 6 0.03, 2.91 6 0.07, 2.89 6 0.06, and 2.45 6 0.22 log CFU/g, respectively, in month 6 of storage. An


increase in total viable counts in fish flesh during storage has been demonstrated by Fan et al. (15). The results here showed that after month 2 of storage, the LAB counts tended to increase. The increase was significantly higher (P , 0.05) for untreated samples than for treated samples. Variations in the values of Enterobacteriaceae counts during the frozen storage are presented in Figure 1C. Treatment with 1, 2, and 4% MPPE lowered Enterobacteriaceae counts compared with the values in the control group (P , 0.05). Increased concentrations of MPPE significantly improved the antimicrobial properties of MPPE against Enterobacteriaceae (P , 0.05). The highest rate in the reduction of bacterial growth was seen in the samples treated with 4% MPPE, followed by the 2 and 1% concentrations. Our results revealed that 4% MPPE significantly reduced the growth of bacteria compared with the growth in the other experimental groups (P , 0.05). The antimicrobial effects of pomegranate were studied previously. Duman et al. (13) showed that pomegranate aril extracts had antimicrobial effects against all microorganisms tested in their study. Melendez and Capriles (25) also reported that extracts from pomegranate fruits possessed in vitro antibacterial activity against many bacteria tested. Choi et al. (10) reported that P. granatum peel ethanol extract exhibited antibacterial activity against 16 strains of eight different salmonella serotypes tested. Alam Khan and Hanee (1) reported that bioactive compounds of P. granatum peel extract (polyphenols, tannins, flavonoids, and anthocyanins) have antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus strains. Shoko et al. (34) confirmed that phenolics were the most important compounds against bacteria, among which gallic acid was identified as the most active compound for inhibition of bacteria of those tested. Therefore, the phenolics inhibited microbial growth in samples treated with pomegranate extract, which may be through protein binding or enzyme inhibition (21, 22). Similar reports have also been presented regarding meat pate treated with pomegranate peel extract (17). Fan et al. (15) indicated that dipping in tea polyphenols (0.2% total phenolics) accompanied by freezing temperature delayed the spoilage of silver carp. Naveena et al. (28) have also reported inhibition of microbial growth in buffalo meat steaks dipped in clove oil which contained phenolic derivatives.

Effect of MPPE on pH of fish samples. The pH results for samples in the different treatment groups are shown in Figure 2. The pH values of fish samples in the control and MPPE treatments on day 0 were 6.30 6 0.11 and had no significant differences (P . 0.05). The pH values of the samples were significantly increased owing to storage for 6 months (P , 0.05). At the end of the storage period, the lowest pH was obtained in the samples treated with 4% MPPE, and the highest was obtained in the control group. The storage of meat at freezing temperature (188C) causes many chemical reactions, including enzymatic reactions (36). The gradual increase in pH in the samples without treatment may be due to microbial action during prolonged



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FIGURE 2. pH values of gutted rainbow trout samples treated with methanolic pomegranate peel extract (MPPE) at three concentrations during 6 months of frozen storage at 188 C (means 6 standard errors). *, Control; *, 1% MPPE; ~, 2% MPPE; &, 4% MPPE.

storage. Similar results have been reported in goat and chicken meat during post mortem aging (27, 37).

Effect of MPPE on TVB-N. There were significant (P , 0.05) differences in the TVB-N values of treated groups and controls from the fourth month of storage to the end of storage (Fig. 3). The initial value of TVB-N was 7.66 6 2.00 mg/100 g in fresh fish. In the control group, the TVB-N values reached 26.80 6 3.77 mg/100 g in the sixth month of storage. The TVB-N values of samples treated with 1, 2, and 4% MPPE reached 17.03 6 3.06, 16.16 6 3.06, and 13.50 6 2.91 mg/100 g, respectively, in the sixth month of storage. The results showed that the TVB-N values in samples treated with 4% MPPE were significantly lower than the values in other groups from the fourth month of storage to the end of storage (P , 0.05). The correlation coefficients between TVB-N content and psychrophilic bacterial and Enterobacteriaceae counts of the fish stored at 188C were 0.51 and 0.62, respectively, both of which are

FIGURE 3. Total volatile basic nitrogen values of gutted rainbow trout samples treated with methanolic pomegranate peel extract (MPPE) at three concentrations during 6 months of frozen storage at 188 C (means 6 standard errors). *, Control; *, 1% MPPE; ~, 2% MPPE; &, 4% MPPE.

FIGURE 4. Thiobarbituric acid contents (A) and peroxide values (B) of gutted rainbow trout samples treated with methanolic pomegranate peel extract (MPPE) at three concentrations during 6 months of frozen storage at 188 C (means 6 standard errors). *, Control; *, 1% MPPE; ~, 2% MPPE; &, 4% MPPE.

significant (P , 0.001). The lower TVB-N contents of the fish treated with 1, 2, and 4% MPPE in comparison with the MPPE content in the controls might be owing to the inhibitory effect of MPPE against microbes, especially spoilage bacteria (20).

Effect of MPPE on lipid oxidation. The TBA contents and peroxide values of fish with and without different treatments during storage are depicted in Figure 4A and 4B, respectively. The TBARS and peroxide values of all samples increased as the storage time increased (P , 0.05). The increases in TBARS and peroxide values suggested that, during storage, fatty acids in fish muscle underwent oxidation processes in which malonaldehyde and lowmolecular-weight compounds like aldehydes and ketones were formed. Samples treated with 4% MPPE had lower TBARS and peroxide values than the controls from months 4 and 3, respectively, to the end of the storage time (P , 0.05). These results revealed that fish treated with 4% MPPE had greater stability towards lipid oxidation than the other samples. Rainbow trout muscle contains highly polyunsaturated fatty acids, and the damage to tissues during freezing and frozen storage can induce lipid oxidation. Li et al. (24) showed that pomegranate peel extract had markedly higher antioxidant capacity than the pulp extract in scavenging or preventive capacity against superoxide anions and hydroxyl and peroxyl radicals. The contents of total phenolics,

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TABLE 1. Effect of methanolic pomegranate peel extract treatment on the likeability scores of gutted rainbow trout (Oncorhynchus mykiss) during 6 months of storage at 188 C Mean score 6 SD (n ¼ 12)a Storage time (mo)

Treatment

1

Control 1% MPPE 2% MPPE 4% MPPE Control 1% MPPE 2% MPPE 4% MPPE Control 1% MPPE 2% MPPE 4% MPPE Control 1% MPPE 2% MPPE 4% MPPE Control 1% MPPE 2% MPPE 4% MPPE Control 1% MPPE 2% MPPE 4% MPPE

2

3

4

5

6

a

Color

1.62 1.49 2.03 1.96 1.62 1.54 2.01 2.19 1.68 1.56 2.21 2.17 1.84 1.68 2.17 2.33 1.96 1.80 2.15 2.43 2.00 1.92 2.47 2.49

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

0.12 0.61 0.82 0.74 0.84 0.67 0.86 0.89 0.98 0.75 0.85 0.93 1.06 0.81 0.95 0.95 1.09 0.89 0.98 0.96 1.09 0.99 0.96 1.00

Odor A A B B A A B BC A A B B A A B B A A B C A A B B

1.52 1.52 1.62 1.68 1.50 1.50 1.62 1.68 1.60 1.49 1.58 1.66 1.82 1.50 1.52 1.52 2.11 1.50 1.41 1.49 2.41 1.39 1.37 1.37

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

0.67 0.61 0.72 0.73 0.67 0.64 0.72 0.81 0.77 0.57 0.66 0.76 0.91 0.67 0.61 0.67 0.97 0.70 0.53 0.57 1.51 0.53 0.48 0.48

Texture A A A A A A A A A A A A A A A A A B B B A B B B

1.70 1.70 1.98 1.80 1.68 1.72 1.90 1.76 1.76 1.60 1.80 1.64 2.00 1.78 1.88 1.78 2.11 1.80 1.78 1.70 2.23 1.74 1.78 1.64

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

0.72 0.70 0.81 0.80 0.73 0.77 0.80 0.73 0.76 0.66 0.72 0.65 0.91 0.87 0.90 0.83 0.99 0.87 0.80 0.80 1.03 0.82 0.87 0.77

Appearance A A A A A A A A A A A A A A A A A A A A A B B B

2.21 2.17 2.54 2.27 2.27 2.13 2.23 2.19 2.17 2.11 2.39 2.39 2.19 2.07 2.45 2.50 2.39 2.23 2.62 2.60 2.62 2.37 2.58 2.72

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

0.83 0.84 0.70 0.80 1.00 0.87 0.83 0.87 0.81 0.86 0.77 0.85 0.87 0.82 0.85 0.92 0.98 0.86 0.95 0.91 0.99 1.01 1.08 1.02

AB A B AB A A A A A A A A ABC A BC C ABC A BC C A A A A

Total acceptability

2.09 1.98 2.41 2.45 2.11 1.98 2.49 2.52 2.05 2.17 2.49 2.49 2.31 2.15 2.62 2.58 2.41 2.23 2.56 2.66 2.56 2.35 2.67 2.68

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

0.92 0.73 0.72 0.72 0.86 0.76 0.80 0.78 0.90 0.91 0.85 0.70 1.02 0.92 0.79 0.72 1.04 0.88 0.83 0.71 1.04 0.93 0.82 0.70

A AB BC C A AB B B A AB B B ABC A B BC ABC A B BC A AB B B

Likability scores: 1 ¼ like extremely, 2 ¼ like moderately, 3 ¼ neither like nor dislike, 4 ¼ dislike. Different letters in the same column within the same storage time indicate significant differences (P , 0.05).

flavonoids, and proanthocyanidins were also higher in pomegranate peel extract than in pulp extract. The large amount of phenolics contained in pomegranate peel extract may cause its strong antioxidant ability (6, 18). MPPE exhibited radical scavenging activity, reducing power, and metal-chelating activity, and thus, this compound could retard the lipid oxidation in fish.

Effect of MPPE on sensory properties of fish during storage. The results of the assessments of sensory properties for both control and experimental groups during frozen storage are summarized in Table 1. During the 6 months of storage, all attributes in all samples showed noticeably decreased likeability. Over the 6 months of storage, the highest and lowest scores for pleasant color were obtained by the 1% and 4% MPPE groups, respectively; the color quality was improved in the 1% MPPE-treated samples compared with that in the 2% and 4% MPPE experimental groups. From the fifth month of preservation, significant changes in odor were noted in the treated samples, with considerable decreases in the unfavorable odors being shown in the three MPPE-treated groups (P , 0.05). In addition, no changes in texture were shown until month 5, while during the 6 months of storage, the best score belonged to the 4% MPPE-treated group. Our results showed that various concentrations of MPPE were capable

of preserving the textural quality of samples compared with that of the controls (P , 0.05). During the storage, the overall likeability grade in the 1% MPPE-treated samples was higher than those of the other experimental groups. From the fourth month of storage, the grades in the 2 and 4% MPPE-treated groups were significantly enhanced compared with the scores for the control group. However, the general likeability was reduced concomitantly with increases in the MPPE concentration. Considering the fact that pomegranate peel extract is an aromatic and pigmented component, the increases in undesirable color and odor of the samples treated with higher concentrations of MPPE were not surprising. The best scores for undesirable changes in color, odor, and texture were seen in the 1% MPPE samples. Concomitant with increasing concentrations of MPPE, the sensory evaluation scores were reduced, which was probably because of the undesirable changes in the presence of the high concentrations of MPPE used to protect the fish from putrefactive microorganisms and, also, to reduce the fat oxidation, which resulted in overall improvement of the samples. In general, the results of the sensory evaluation indicate that the 1% concentration of MPPE produced the best sensory ranking. While the aromatic properties and texture of the product were improved at the higher concentrations, the general acceptability was simultaneously decreased.



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MPPE could potentially be used as a natural antioxidant and antibacterial agent in food products. Lipid oxidation and protein degradation cause unpleasant physical and sensory alterations even during the freezing process. In this study, an improvement in the quality parameters during frozen storage of fish was demonstrated. As a final conclusion, MPPE is recommended as a natural agent to improve the microbial, chemical, and textural properties of frozen fish during 6 months of storage.

ACKNOWLEDGMENTS This research was financially supported by ‘‘Natural Antimicrobials Centre of Excellence (NACE)’’ which is gratefully acknowledged. We would like to thank Miss M. Aghazi and Mr. G. Niknia for their technical assistance. We also thank the Liosa fish packaging company for providing fish samples.

REFERENCES

FIGURE 5. Hardness (A) and chewiness (B) scores of gutted rainbow trout samples treated with methanolic pomegranate peel extract (MPPE) at three concentrations during 6 months of frozen storage at 188 C (means 6 standard errors). *, Control; *, 1% MPPE; ~, 2% MPPE; &, 4% MPPE.

Effect of MPPE on textural properties. The textural profile analysis parameters are presented in Figure 5. The hardness and chewiness of samples were evaluated after 6 months of frozen storage. There were no significant differences for all attribute values among the samples at month 0 (P . 0.05). After 6 months of storage, all attributes showed noticeably decreased likeability (P , 0.05). At month 6, the highest scores for hardness and chewiness were found in the samples treated with 4% MPPE, compared with the control group (P , 0.05). A linear decrease was observed in the hardness until the end of the storage time (P , 0.05), indicating that the muscle became softer. Frozen storage caused changes in the muscle integrity and denaturation and aggregation of myofibrillar proteins of rainbow trout muscles that provide firmness in meat products, which is supported by the results of Bahuaud et al. (5). A downward trend was also observed for chewiness, demonstrating a significant difference (P , 0.05); this result occurred because chewiness is a derived parameter of hardness. The phenolic compounds of the pomegranate peel extract can also exhibit antioxidant activity by reducing the oxidation of protein. The results also demonstrated the antioxidant activity of 4% MPPE to inhibit protein oxidation, and thus, the hardness and chewiness of the samples were not considerably changed at the end of storage.

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