Abstract
This study aimed to determine the phenolic content and antioxidant effect of grape pomace extract on silver carp fillets during refrigerated storage. Total phenolic content of grape pomace extract was quantified by colorimetric methods. Silver carp samples were treated with 0, 2, and 4% of grape pomace extract (g extract/100 g flesh) and stored 15 days in a refrigerator (4°C). Changes in pH, peroxide value, thiobarbituric acid, and heme iron at 0, 3, 6, 9, 12, and 15 days of storage were investigated. Results showed that the values of pH, peroxide value, and thiobarbituric acid increased and iron levels decreased in all treatments during storage (p < 0.05). The phenolic contents were generally much more influenced by the quality parameters treated compared to the control. The addition of grape pomace extract delayed lipid oxidation in silver carp fillet considerably during refrigerated storage. These results suggested that grape pomace extract has the potential to be used as a natural antioxidant.
Keywords:
INTRODUCTION
Fish is considered a vital component of a healthy diet and an excellent source of protein, essential minerals, trace elements, and vitamins. Furthermore, marine lipids contain high concentrations of polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA).[Citation1] Silver carp is highly susceptible to oxidation because of its relatively high content of PUFA. Lipid oxidation leads to an unpleasant odor, rancid taste, and discoloration.[Citation2] The degradation of PUFA by auto-oxidation during storage easily leads to the formation of free radicals, hydroperoxides, and volatiles associated with rancidity.[Citation3,Citation4] The use of antioxidants is a major strategy for preventing lipid oxidation and improving shelf life in food products.[Citation5–Citation7] The extracts contain bioactive phenolic compounds which have recently been recognized for their efficacy in providing significant antioxidant activity to human foods.[Citation8,Citation9]
The antioxidant activity of grapes has been positively associated with their phenolic composition such as anthocyanins, flavonols, flavan-3-ols, procyanidins, and phenolic acids.[Citation5,Citation10–Citation13] Additionally, these compounds have been shown to reduce hydroperoxide formation and inhibit both lipid and protein oxidation.[Citation14] In addition to antioxidant effects, the grape phenolics have been reported to inhibit platelet aggregation,[Citation15] exert antimicrobial,[Citation16] anti-inflammatory,[Citation17] and anti-aging activities.[Citation18] Overall, there is clear evidence of the health promoting qualities of consuming grapes and grape derived products. The objective of this work was to determine the effect of grape pomace extract on quality changes in silver carp (Hypophthalmichthys molitrix) fillets during refrigerated storage. Silver carp was selected because of its popularity and high value, demanding the best handling to preserve its sensorial quality.
MATERIAL AND METHODS
Sample Preparation
Experiments were carried out with fresh silver carp (Hypophthalmichthys molitrix) which were caught from Zabol Chahnimeh located in southeastern Iran. The carp were transported in isothermal iceboxes to the laboratory 5 h after being caught. Fresh carp of a similar weight (1 to 1.5 kg) were selected for each experiment. The fish were cleaned and filleted. Concentrations of 0, 2, and 4% total phenolic of red grape pomace extract were added to the same weighed fillets (about 100 g). The samples were placed in moisture-impermeable plastic bags, stored in a refrigerator (+4°C), and taken for analysis on days 0, 3, 6, 9, 12, and 15. The experiences were performed with three replicates.
Extraction
Fresh red grapes (Vitis vinifera) were prepared from Shahryar (Iran) and were transferred to the laboratory. They were stored at –20°C until they were made into pomace. Grape pomaces were dried at 45°C for 72 h and milled to a particle size less than 0.5 mm. Dried grape pomace (200 mg) was placed in a test tube, then 20 mL Diethyl ether containing 1% acetic acid was added to remove pigments and fat. The solution was thoroughly shaken at room temperature for 20 min and centrifuged at 3000 g and 4°C for 10 min, after which the supernatant was recovered. Ten milliliters of acetone 70% (V/V) was added to the residue, and shaking and centrifugation were repeated. Extractions were performed to calculate the total phenolic content.[Citation19]
Determination of Total Phenolic Content and pH
Total phenolic content was determined by Folin-Ciocalteu reagent[Citation19] using tannic acid as the standard. A mixture of 0.1 mL of extract, 0.5 mL of distilled water, 0.25 mL of Folin-Ciocalteau reagent, and 1.25 mL of Na2CO3 was introduced into a 25 mL volumetric flask. After reacting for 40 min, the absorbance was measured at 725 nm using an ultraviolet-visible spectrophotometer (Jenway, 6305). The results were expressed as g of tannic acid equivalents per kg of grape pomace dry matter (DM). The sample was thoroughly homogenized with 10 mL of distilled water, and the homogenate was used in determining the pH using a digital pH-meter (model Wagtech-cyber scan 510, Germany).[Citation20]
Peroxide Value (PV) and Thiobarbituric Acid Value (TBA)
The peroxide content in the total lipid extracts was determined according to the method of (AOAC, 2000).[Citation21] Results were expressed in meq O2/kg lipid. The TBA value (as malonaldehyde) was determined calorimetrically.[Citation22] A mixture of 200 mg sample and 1 mL of 1-Butanol for dissolving was added to a 25 mL volumetric flask. Five milliliters of the mixture was pipetted into a dry stoppered test tube, and 5 mL of TBA reagent (prepared by dissolving 200 mg of 2-TBA in 100 mL 1-Butanol, filtered, stored at 4°C for not more than seven days) was added. The test tubes were stoppered, vortexed, placed in a water bath at 95°C for 120 min, and then cooled. Absorbance (As) was measured at 530 nm against water blank. A reagent blank was run and absorbance (Ab) was recorded. TBA value (mg malonaldehyde/kg) was obtained using the formula:
Heme Iron Content
Heme iron was determined using the acidified acetone extraction method explained by Clark et al.[Citation23] Approximately 10 g of ground sample was weighed into 50 mL centrifuge tubes, and 20 mL of acid-acetone mixture (40 mL of acetone, 9 mL of water, and 1 mL of concentrated hydrochloric acid) was added. Each sample was homogenized for 30 s with a blender (Panasonic). Then, an additional 20 mL of acid-acetone mixture was added, and the samples were mixed thoroughly. The tubes were capped tightly and kept in the dark for 1 h. The extract was centrifuged at 2200 g for 10 min. The supernatant was filtered through glass microfiber filters (Whatman GF/A), and the absorbance was measured at 640 nm against a reagent blank. The absorbance was multiplied by the factor of 6800 and then divided by the sample weight to give the concentration of total pigments in the meat as μg hematin per gram sample. The iron content was calculated with the factor of 0.0882 μg iron per g hematin.
Statistical Analysis
Data were analyzed by two way ANOVA design using SPSS version 15.0 software. When differences were significant (p < 0.05), mean values were evaluated by the Student-Newmann-Keul (SNK) method.
RESULTS AND DISCUSSION
Total Phenolic Content of Grape Pomace Extract
Phenols are one of the most important groups of natural antioxidants. They occur only in material of plant origin and are known to protect easily-oxidizable constitutes of food from oxidation. The phenolic content and composition greatly differ with the type of grape, and the extraction yield is greatly affected by the solvent. In this study, the total phenolic content of red grape pomace was found to be 65 g/kg DM. Negro et al.[Citation24] found that the total phenolic content of Italian red grape pulp was 161 g/kg DM. Alipour and Rouzbehan[Citation25] reported that the total phenolic content of Iranian grape pomace was 22.70 g/kg DM. It can be concluded from these results that the phenolic content of grape pomace changes according to cultivar (location, species, and stage of maturity) and solvent(s) used in the extraction.[Citation19,Citation26]
Antioxidant Effects of Grape Pomace Extract on pH
shows the pH values for the silver carp fillets subjected to grape pomace extract during refrigerated storage. Significant pH changes can be seen between treatment and storage time. There was no significant difference among the treatment after three days of storage, showing that the grape pomace extract had little impact on overall pH change in fish samples. Moreover, a significant difference in the pH was noticed between the control and the grape pomace extract treatment after nine days of storage. An increase in pH was observed after storage for all treatments, but this increase was more in the control when compared with the grape pomace extract treatment.
TABLE 1 Effect of grape pomace extract on pH of silver carp fillets during refrigerated storage
TABLE 2 Effect of grape pomace extract on PV (meq O2/kg lipid) of silver carp fillets during refrigerated storage
pH is an important and effective indicator of meat quality.[Citation27] The lower pH observed in the samples treated with grape pomace extract can be attributed to its antibacterial properties.[Citation28] Phenolic compounds of grape pomace extract can increase microbial inhibition, protect fillets against internal protease, and inhibit protein breakdown and amine production.[Citation29] The pH values of the silver carp fillets increased during storage, possibly due to the production of alkaline compounds.[Citation30]
Antioxidant Effects of Grape Pomace Extract on PV
PV is one of the important indices of meat spoilage.[Citation31] It indicates the existence of concentrations of peroxides and hydroperoxides that are produced during the early stages of lipid oxidation. The PV of silver carp fillets was modified during refrigerated storage. The PV values of the samples are shown in . There were no significant differences among all treatments on the first day; however, a significant difference in PV was noticed between the control and 2 and 4% grape pomace extracts during storage (p < 0.05). The PV values of the 2 and 4% grape pomace extracts were lower than the control. Lower PV values in the samples treated with grape pomace extract might be linked with phenolic content.[Citation32] The use of antioxidants was very effective in reducing lipid oxidation in fish fillets because of its phenolic compounds which act as inhibitors for radical reactions on autoxidation.[Citation33] Ojagh et al.[Citation34] reported that the PVs increased in all treatments, but this increase was lower in the samples treated with chitosan and cinnamon due to their antioxidant activity. PV increased during the ninth day of storage. After this, a sudden decrease in all treatments was observed (), which may be related to secondary oxidation products and volatile compounds.[Citation35] The PV of fillets increased between the 12th and 15th day of storage (). These results were in agreement with the findings of Ozogul et al.[Citation36] on Anguilla anguilla.
Antioxidant Effects of Grape Pomace Extract on TBA Values
The TBA value is most widely used for measuring the extent of oxidative deterioration of lipids.[Citation37] Malondialdehyde (MDA) is one of the most abundant aldehydes generated during secondary lipid oxidation, and it is probably the one most commonly used as an oxidation marker.[Citation35] The evolution of TBA values of the silver carp fillets during refrigerated storage are shown in . On the first day, the grape pomace extract had a significant effect on the TBA values of 2 and 4% treatments in comparison with the control. The decrease of TBA in the treatments could be due to the effects of antioxidants in reducing peroxide.[Citation38,Citation39] Increases in TBA values in different samples indicated oxidation in the silver carp fillets during refrigerated storage. This was probably due to the destruction of hydroperoxides into secondary oxidation products, especially aldehydes in the later stages of lipid oxidation.[Citation40,Citation41] A significant difference in the TBA value was observed between the control and the 2 and 4% grape pomace extracts during storage (p < 0.05). Similarly, Tajik, Farhangfar, Moradi, and Razavi Rohani,[Citation42] who observed strong antioxidant effects from clove and grape seed extracts, reported that the antioxidant potential of this extract significantly restrained TBA values in silver carp fillets. The lowest increase in TBA value was observed in the 4% treatment. According to Goulas and Kontominas,[Citation43] the TBA value of 1–2 mg MDA/kg of fish flesh is usually regarded as the limit beyond which fish will normally develop an objectionable odor/taste. The TBA values at the end of storage for all samples of this study were lower than the limited value.
TABLE 3 Effect of grape pomace extract on TBA (mg malonaldehyde/kg) of silver carp fillets during refrigerated storage
Antioxidant Effects of Grape Pomace Extract on Heme Iron
Metal ions can play an important role in lipid oxidation as Proxidan.[Citation44] shows the heme iron values for the silver carp fillets subjected to grape pomace extract during refrigerated storage. There were no significant differences between all treatments in the first day; however, a significant difference in heme iron values was observed between the control and the 2 and 4% grape pomace extracts during storage (p < 0.05). Metal ions, such as iron, can catalyze the production of free radicals. Natural polyphenols are capable of removing free radicals, chelating metal catalysts, activating antioxidant enzymes, and inhibiting oxidases.[Citation45] The lowest and highest heme iron values were obtained for the control and the 4% grape pomace extract, respectively (). Low iron levels indicate an indirect increase in fat oxidation.[Citation44] The heme iron values decreased significantly in all treatments during storage (p < 0.05). Hokey et al.[Citation46] reported that the negative relationship between iron and lipid oxidation indices reveals that wherever heme iron is reduced and non-heme iron is increased, oxidation spoilage also increases.
TABLE 4 Effect of grape pomace extract on Heme iron (μg/g) of silver carp fillet during refrigerated storage
CONCLUSIONS
This study showed that grape pomace extract plays an important role in the lipid oxidation of silver carp fillets. Significant increases in PVs and TBA of fish fillets appeared during refrigerated storage. The value of heme iron decreased when lipid oxidation increased in the fish fillets. The samples treated with 4% total phenolic content were better preserved compared with other treatments. Thus, grape pomace extract has a potential use in improving the quality of fish and suggests itself as a natural antioxidant to prevent the deterioration of stored fish. Moreover, the use of a by-product such as grape pomace can supply an opportunity to evaluate it economically in the food industry.
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