Antioxidant activity of black currant (Ribes nigrum L.) extract and its inhibitory effect on lipid and protein oxidation of pork patties during chilled storage

Meat Science 91 (2012) 533–539

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Antioxidant activity of black currant (Ribes nigrum L.) extract and its inhibitory effect on lipid and protein oxidation of pork patties during chilled storage Na Jia, Baohua Kong ⁎, Qian Liu, Xinping Diao, Xiufang Xia College of Food Science, Northeast Agricultural University, Harbin, Heilongjiang 150030, China

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Article history: Received 12 October 2011 Received in revised form 24 January 2012 Accepted 13 March 2012 Keywords: Black currant extract Antioxidant activity Protein oxidation Lipid oxidation Pork patties

a b s t r a c t This experiment was conducted to assess the antioxidant efficacy of black currant (Ribes nigrum L.) extract (BCE) in raw pork patties during chilled storage. The extracting conditions of frozen BCE including ethanol concentrations (0–100%) and extracting times (0.25–12 h) were studied. BCE extracted with 40% ethanol for 2 h had the highest anthocyanin content, the strongest radical scavenging activities as well as the second strongest reducing power. BCE was condensed and added to pork patties at 5, 10 or 20 g/kg. Compared with the control, BCE treatments significantly decreased the thiobarbituric acid-reactive substance values and carbonyls formation and reduced the sulfhydryl loss of pork patties in a dose-dependent manner (P b 0.05), which showed that the BCE significantly inhibited lipid and protein oxidation. The BCE-treated patties showed significantly higher redness (P b 0.05) than the control. The findings demonstrated strong potential for BCE as a natural antioxidant in meat and meat products. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Lipid oxidation is one of the main causes of quality deterioration in meat and meat products because it leads to discolouration, offflavours, texture deterioration, and loss of nutrients, all of which are the major quality factors affecting consumers' acceptance of meat (Min & Ahn, 2005). In addition, protein oxidation affects meat quality, including tenderness, water-holding capacity, and nutritional quality, and has attracted considerable attention in recent years after having been ignored for decades (Lund, Heinonen, Baron, & Estévez, 2011). Complex mechanisms and reaction processes are involved in lipid and protein oxidation, while it is generally accepted that both types of oxidation occur mainly via a radical chain reaction including initiation, propagation, and germination stages (Gardner, 1979; Lund et al., 2011; Shahidi & Zhong, 2010); thus, one effective method for slowing oxidation is to use antioxidants to break the radical chain reaction to maintain the nutritional and sensory qualities of meat. Compared with synthetic antioxidants, natural antioxidants are of great interest because of their safety and health characteristics. Extracts from plant materials, such as fruits, vegetables, herbs, spices, and their constituents, are good sources of natural antioxidants (Duan, Jiang, Su, Zhang, & Shi, 2007; Kanatt, Chander, & Sharma, 2007). In particular, phenolic compounds, as an important part of natural antioxidants, have received much attention because of their remarkable radical scavenging activity and their reducing ability. Many

⁎ Corresponding author. Tel.: + 86 451 55191794; fax: + 86 451 55190577. E-mail address: [email protected] (B. Kong). 0309-1740/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2012.03.010

plant extracts rich in phenolic compounds have been reported to have positive effects on the inhibition of lipid and/or protein oxidation in diverse meat systems, for example, rosemary extracts (Lund, Hviid, & Skibsted, 2007), olive leaf extracts (Hayes et al., 2009) and rapeseed and pine bark phenolics (Vuorela et al., 2005). Recently, fruit extracts containing abundant phenolic compounds were also added to muscle foods to retard lipid and/or protein oxidation and discolouration; these extracts include pomegranate juice phenolics (Vaithiyanathan, Naveena, Muthukumar, Girish, & Kondaiah, 2011), white grape extract (Jongberg, Skov, Tørngren, Skibsted, & Lund, 2011) and extracts from strawberry, dog rose, hawthorn, arbutus berry, and blackberry (Ganhão, Estévez, Kylli, Heinonen, & Morcuende, 2010; Ganhão, Morcuende, & Estévez, 2010). Fruits, especially berries, have been found to possess pharmacological and biochemical properties that are caused mainly by the antioxidant activity of their diversified compositions (Szajdek & Borowska, 2008). Black currant (Ribes nigrum L.), a dark-pigmented fruit native to northern and central Europe and northern Asia and now also cultivated in several states in the U.S. (Oregon, Vermont, New York, and Connecticut), have been found to possess high anthocyanin content and superior antioxidant activity compared with that found in many other fruits. Many studies have demonstrated the excellent antioxidant activity of black currant extract (BCE) and its health benefits, including anticarcinogenic activity (Wu, Koponen, Mykkänen, & Törrönen, 2007), yet little is known about its application as an antioxidant in food systems, including muscle foods. Furthermore, many factors, such as the polarity of extracting solvents and extracting temperature and time, affect the extraction efficiency of anthocyanins from black currant (Cacace & Mazza, 2003);

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therefore, it is necessary to determine the optimal condition for obtaining BCEs with both high antioxidant activity and high anthocyanin content. The objectives of this research were to determine the optimal extracting condition of frozen crude BCE with aqueous ethanol by determining the extract's anthocyanin content, reducing and radical scavenging ability. Additionally, the inhibitory effect of BCE on lipid and protein oxidation and colour deterioration in raw pork patties during chilled storage was investigated. 2. Materials and methods 2.1. Materials and chemicals Frozen black currants (Ribes nigrumL.) were purchased from Gaotai Food Corporation (Binxian, Heilongjiang, China) and stored at −20 °C until use. Pork trims were obtained within 24 h postmortem from a local packing plant (Beidahuang Meat Processing Co., Harbin, China), kept in ice and transported to the laboratory. Testing chemicals, including 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,4dinitrophenylhydrazine (DNPH), 2,2′-azinobis (3-ethylbenzothiazoline6-sulfonic acid) diammonium salt (ABTS), DTNB [5,5′-Dithiobis (2nitrobenzoic acid)], butylated hydroxyanisole (BHA), and ethylene diamine tetraacetic acid disodium salt (EDTA), were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals and reagents were purchased from Solabio Corporation (Beijing, China). 2.2. Preparation of BCE Frozen black currants were thawed at room temperature and then homogenised for 3 min using a blender. An aliquot (10 g) of homogenised black currants was mixed into 100 mL of different concentrations of ethanol (0, 20, 40, 60, 80, or 100%) at 35 °C for 2 h or mixed into 100 mL of 40% ethanol for different times (0.25, 1, 2, 4, 8 or 12 h) in enclosed flasks with constant agitation. The homogenate was filtered through Whatman No. 2 filter paper, and the anthocyanin content and reducing and radical scavenging ability of the filtrate were determined to obtain the optimal extracting condition. BCE, which has high reducing and radical scavenging abilities (extracting condition: 40% ethanol for 2 h at 35 °C), was used for further study to evaluate its inhibitory effect on lipid and protein oxidation and colour deterioration in pork patties. Another 100 g homogenised frozen black currants was mixed into 1000 mL 40% ethanol at 35 °C for 2 h in an enclosed flask with constant agitation followed by first filtering through Whatman no. 2 filter paper and then through a 0.45 μm filter membrane. These filtrates were subsequently concentrated on a rotary vacuum evaporator (Eyela N-1000, Tokyo Rikakikai Co. Ltd., Tokyo, Japan) at 40 °C. The semidried samples were then reconstituted in a final volume of 50 mL of distilled water; this procedure showed that 1 mL of BCE is extracted from 2 g of frozen black currants. 2.3. Determination of total anthocyanin content Total anthocyanin content was determined by the pH differential method (Lee, 2005). Briefly, 0.25 mL of BCE filtrate was diluted 20 times with a pH 1.0 potassium chloride buffer (0.025 M) or a pH 4.5 sodium acetate buffer (0.4 M), respectively. The absorbance at 520 nm was compared with a distilled water blank after incubation in darkness for 15 min. Anthocyanin content, expressed as cyanidin3-glucoside equivalents, was calculated as: anthocyanin content (mg/L) = (ApH 1 − ApH 4.5) × 449.29 × DF × 1000 / (26,900 × 1) where ApH 1 and ApH 4.5 are the absorbance of the BCE filtrate in a pH 1.0 and a pH 4.5 buffer, respectively; 449.29 is the molecular weight of cyanidin-3-glucoside (g/mol); DF is the dilution factor (20); 1000 is the factor for conversion from g to mg; 26,900 is the molar extinction coefficient (M − 1·cm − 1); and 1 is the pathlength (cm).

2.4. Determination of antioxidant activity 2.4.1. DPPH radical scavenging activity The DPPH radical scavenging activity was determined according to Wu, Chen, and Shiau (2003). The mixture of 0.1 mL of BCE filtrate and 3 mL of 0.1 mM DPPH solution (dissolved in 95% ethanol) was incubated in darkness for 30 min, and the absorbance at 517 nm was read. DPPH radical scavenging activity (%) was calculated as (Ac − As) × 100/Ac, where Ac is the absorbance of the control (DPPH solution without BCE filtrate) and As is the absorbance of the sample. 2.4.2. ABTS radical scavenging activity The method described by Ozgen, Reese, Tulio, Scheerens, and Miller (2006) was used to measure the ABTS radical scavenging activity. Briefly, 20 μL of BCE filtrate was mixed with a diluted ABTS •+ solution (absorbance of 0.70 ± 0.01 at 734 nm). The mixture was incubated in darkness for 6 min, and the absorbance at 734 nm was read. ABTS radical scavenging activity (%) was calculated as (Ac − As) × 100/Ac, where Ac is the absorbance of the control (ABTS •+ solution without BCE filtrate) and As is the absorbance of the sample. 2.4.3. Reducing power The reducing power was determined using the method of Oyaizu (1986). An aliquot (0.5 mL) of BCE filtrate was diluted in 2.5 mL of a sodium phosphate buffer (0.2 M, pH 6.6), and then 2.5 mL of 1% potassium ferricyanide was added. After incubation at 50 °C for 20 min, 2.5 mL of 10% trichloroacetic acid (w/v) was added. The mixture was centrifuged at 1000 g for 10 min. An aliquot (5 mL) of the supernatant was mixed with 5 mL of distilled water and 1 mL of 0.1% ferric chloride, and the absorbance at 700 nm was read after a 10 min incubation. The absorbance increase indicates an increase in the reducing power. 2.5. Preparation of pork patties The antioxidant efficacy of BCE was assessed with pork patties. The pork trims (10% fat) were ground through 4 mm plates. A 5 × 4 (formulation × storage time) factorial design was used. Specifically, the following five-patty formulations, each with 20 g/kg NaCl, were prepared: 1 control (without BCE) and 4 treatments, namely, with 5, 10 or 20 g/kg of BCE, or with 0.2 g/kg BHA. For each product formulation, 20 patties were made from fresh pork trims (1 kg), and for each storage time, 5 patties were used for analysis. In patty preparation, fresh pork trims and ingredients were mixed by blending for 5 min with a Kitchen Aid mixer. Duplicate patties of 50 g each were shaped by hand into approximately 8 cm diameter and 1 cm thickness. Patties were placed in polypropylene trays, overwrapped with an oxygen-permeable polyvinyl chloride film (Weiguang Plastic Co., Ltd., Jiangsu, China), and stored at 4 °C for 1, 3, 6 or 9 days under fluorescent lights to simulate supermarket retail display conditions. A strict sanitation procedure was followed to avoid microbial contamination. Lipid oxidation, protein oxidation and colour were measured. 2.6. Measurement of lipid oxidation Lipid oxidation was evaluated by thiobarbituric acid-reactive substance (TBARS) according to Jongberg et al. (2011). Briefly, 5 g of meat was homogenised (11,000 rpm, 1 min) in 15 mL of 7.5% trichloroacetic acid with 0.10% propyl gallate and 0.10% EDTA and then filtered. Five millilitres of the filtrate was mixed with an equal volume of 20 mM thiobarbituric acid. The mixture was incubated at 100 °C in a water bath for 40 min, and the absorbance at 532 nm was read. The TBARS value, expressed as mg of malonaldehyde/kg of the meat sample, was calculated using a molar extinction coefficient (156,000 M − 1·cm − 1).

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2.7. Measurement of protein oxidation 2.7.1. Preparation of myofibrils protein Myofibril protein (MP) was extracted according to the method employed by Park, Xiong, Alderton, and Ooizumi (2006), stored in a tightly capped bottle at 4 °C, and utilised within 24 h. 2.7.2. Protein carbonyls Protein carbonyls were determined by the method of Levine et al. (1990). A 1 mL aliquot of 6 mg/mL MP was reacted with 1 mL of 10 mM DNPH in 2 M HCl at room temperature for 1 h; another 1 mL of sample was added with 1 mL of 2 M HCl (control). After incubation, 1 mL of 20% trichloroacetic acid was added, and the mixture was centrifuged at 10,000 rpm for 5 min. The pellet was washed three times with 1 mL of ethanol/ethyl acetate (1:1), dissolved in 3 mL of 6 M guanidine hydrochloride (pH 2.3) at 37 °C for 15 min, and then centrifuged at 10,000 rpm for 3 min. Absorbance of the DNPHtreated sample was measured at 370 nm. Absorbance of the control was read at 280 nm to calculate the protein concentration in the final pellet using a bovine serum albumin (BSA) standard. The carbonyl content was expressed as nmol carbonyl/mg protein using an absorption coefficient of 22,000 M − 1·cm − 1 for protein hydrazones. 2.7.3. Sulfhydryl content Sulfhydryl (SH) content was determined using Ellman's (1959) method. Briefly, 1 mL of 2 mg/mL MP was mixed with 8 mL buffer (0.086 M Tris, 0.09 M glycine, 4 mM EDTA, pH 8), and centrifuged at l0,000 g for 15 min. An aliquot of 4.5 mL of the supernatant was added with 0.5 mL of Ellman's reagent (10 mM DTNB). The mixture was incubated at room temperature in darkness for 30 min, and the absorbance at 412 nm was read. The SH concentration was calculated using a molar extinction coefficient of 13,600 M − 1·cm − 1. Protein concentrations were determined using a BSA standard.

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also showed the significant lower reducing power and lowest DPPH • and ABTS •+ scavenging activities compare to different concentrations of ethanol extracts except to 100% ethanol. When ethanol was included in the extracting solvent, the anthocyanin content was significantly higher than that achieved with the water extract (P b 0.05). Extraction with different concentrations of ethanol (20–100%) had no significant influence on anthocyanin content in frozen BCE (P > 0.05) but could significantly affect the antioxidant activities (P b 0.05) of BCE: the reducing power, ABTS •+ scavenging activity up to a maximum at 40% ethanol, and DPPH • scavenging activity up to a maximum at 60% ethanol (which had no significant difference compared with 40% ethanol). These antioxidant indexes decreased with further increase in ethanol concentration; thus, the 40% ethanol extract was selected as the optimal extracting solvent and used for further experiments. Cacace and Mazza (2003) also showed that the extraction of anthocyanins from black currants did not require a high ethanol concentration, and the maximum yield was at approximately 50% ethanol. The 100% ethanol BCE had the lowest (P b 0.05) reducing power and the lowest DPPH • and ABTS •+ scavenging activities among ethanol BCEs. This result indicated that pure ethanol could not extract some hydrophilic chemical compounds in frozen black currants; these compounds could also have effective antioxidant activity. The effects of extracting time on total anthocyanin content and antioxidant activity of crude BCE are shown in Fig. 2. The anthocyanin content increased with extracting time up to a maximum at 2 h and then decreased with a further increase in extracting time. Of the six extracting times, the 2 h extract showed the highest DPPH and ABTS scavenging activities and the second highest reducing power. Although the 4 h extract showed the highest reducing power, a long extracting time is not economical and not easy to control in future industry production. Although 1 h was a short extracting time, the 200

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A 2.8. Colour measurements The surface colour of pork patties was measured by a ZE-6000 colorimeter (Juki Corp, Tokyo, Japan) using the D65 light source and a 10° observer with an 8 mm diameter measuring area and a 50 mm diameter illumination area. The instrument was calibrated with a white reference tile (L* = 95.26, a* = −0.89, b* = 1.18). The values, expressed as L* (lightness) and a* (redness) units, were obtained from four different areas on the surface of each patty, and a minimum of 3 pork patties per treatment block were analysed to obtain an average value.

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3.1. Anthocyanin content and the reducing and radical scavenging abilities of BCE Anthocyanins, as the main phenolic compounds in black currants, were used as an indicator of extraction efficiency. As shown in Fig. 1, the lowest anthocyanin content (119.5 mg/L) was observed when pure water was used as the extracting solvent; the water extract

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1 h extract showed significantly lower reducing power than that of the 2 h and the 4 h. Therefore, 2 h was used for extracting BCE in the subsequent experiment. The most widely used method for extracting anthocyanins from plants is organic solvent extraction, most commonly aqueous mixtures with ethanol, methanol or acetone (Kähkönen, Hopia, & Heinonen, 2001). Nevertheless, it is difficult to develop a standard extraction procedure that could extract all plant phenolics because the solubility of phenolics is associated with both their chemical nature and the polarity of the extracting solvent used (Naczk & Shahidi, 2006). Acetone was found to be the most effective for extracting phenolics from buckwheat compared with methanol, ethanol, butanol and ethyl acetate (Sun & Ho, 2005), whereas for black sorghum, aqueous acetone was not better than acidified methanol when extracting anthocyanins (Awika, Rooney, & Waniska, 2004). Obviously, the extracting efficiency was dependent on both the phenolics source and the extracting solvent used, meaning that the extracting solvent suitable for extracting phenolics from this source may be not suitable for extracting phenolics from another source. In our tests, a mixture of ethanol and water was selected because of the polar characteristics of anthocyanins and the acceptability of ethanol in the food industry. The crude frozen BCE produced by this method also contained other non-phenolic components that may contribute to the antioxidant activity as well (Kapasakalidis, Rastall, & Gordon, 2006); thus, the BCE was not purified in further experiments. Moreover, an acidified organic solvent was reported to be efficient for extracting anthocyanins (Patil, Madhusudhan, Ravindra Babu, & Raghavarao, 2009); however, the labile acyl and sugar residues might be degraded during concentrating the acidic extracts of anthocyanins (Naczk & Shahidi, 2006). That potential degradation was also confirmed in our preliminary experiments; thus, the acidified solvent was not used to avoid

Lipid oxidation, expressed as TBARS, in control pork patties increased rapidly during refrigerated storage, reaching 2.13 mg/kg by day 9 (Fig. 3). The TBARS production was significantly inhibited (P b 0.05) in pork patties treated with 5, 10, or 20 g/kg of BCEs and decreased by 74.9, 90.6, and 91.7%, respectively, compared with the control over a 9-day storage period. The efficacy of BCE was comparable with that of BHA, which was added at the 0.2 g/kg concentration level, and 10 and 20 mg/kg BCE treatments both reduced TBARS values similar to that of 0.2 g/kg BHA. It was observed that the TBARS values of BCE-treated patties were below the acceptable sensory threshold limit for exhibiting rancid flavour (1 mg/kg) (Ockerman, 1976). Thus, frozen BCE is a good source of natural antioxidants and may be used as a substitution for BHA to protect meat against lipid oxidation. Recently, extracts from some fruits have been added to muscle foods as inhibitors of lipid oxidation. Wild fruit extracts such as strawberry, hawthorn, dog rose, and blackberry displayed remarkable antioxidant activity against lipid oxidation in raw pork patties stored at 2 °C for 12 days (Ganhão et al., 2010). Pomegranate fruit juice phenolics were found to reduce TBARS values of spent hen breast muscle at 4 °C for 28 days (Vaithiyanathan et al., 2011). White grape extract inhibited TBARS values in beef patties during 9 days of storage at 4 °C (Jongberg et al., 2011). Extracts from avocado peels and seeds protected lipids against oxidation in pork patties stored for 15 days at 5 °C (Rodríguez-Carpena, Morcuende, Andrade, Kylli, & Estévez, 2011; Rodríguez-Carpena, Morcuende, & Estévez, 2011). In oil-inwater emulsions containing MP, phenolic compounds were found to exhibit an intense effect against lipid oxidation by mainly locating to the inner layer of the interphase and being exposed to the lipid phase where lipid oxidation occurs (Estévez, Kylli, Puolanne, Kivikari, & Heinonen, 2008). Our results were consistent with these previous findings and suggested the potential application of BCE as an antioxidant for the production of meat foods with improved quality traits. The inhibitory effect of BCE on lipid oxidation may be attributed to its phenolic compounds and other biochemical compounds that mainly contribute to the antioxidant activity. Light, heat, enzymes, metals, metalloproteins, and microorganisms could induce

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Fig. 3. Influences of BHA and different concentrations of black currant extract (BCE) on thiobarbituric acid reactive substance (TBARS) values of pork patties stored for 9 days at 4 °C. Error bars refer to the standard deviations obtained from triplicate sample analysis. Means with different letters (a–d) at the same storage time differ significantly (P b 0.05).

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lipid oxidation, and most of the oxidation processes, including autoxidation, photooxidation, and thermal or enzymatic oxidation, involve free radical and/or other reactive species as the intermediate (Shahidi & Zhong, 2010). Accordingly, BCE may inhibit lipid oxidation by blocking radical chain reaction in the oxidation process because it has been proven to have remarkable radical scavenging activity and reducing power; these activities indicate that it has excellent potential to donate electrons or hydrogen to free radicals and convert them into stable diamagnetic molecules. 3.3. Inhibition of protein oxidation in raw pork patties Protein oxidation, another major cause of meat quality deterioration in addition to lipid oxidation, is an innovative research field and has drawn increasing interest in recent years (Lund et al., 2011). The extent of protein oxidation was assessed by measuring both the formation of carbonyls and the loss of sulfhydryl groups. As presented in Fig. 4A, the amount of carbonyls gradually increased in all patties with the highest values observed in control patties on all days of analysis. Patties with 5 g/kg BCE or 0.2 g/kg of added BHA showed a slight, though not significant, decrease in the carbonyl content throughout the storage period (P > 0.05) compared to that of the

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control, while 10 g/kg and 20 g/kg BCE significantly inhibited carbonyl formation at 6 and 9 days storage (P b 0.05) compared to the control. The reported effects of plant extracts on protein oxidation are inconsistent, unlike their solid inhibitory effects on lipid oxidation. Extracts from strawberry, hawthorn, dog rose and blackberry inhibit protein oxidation in cooked burger patties (Ganhão, Morcuende, & Estévez, 2010). Pomegranate fruit juice phenolics (Vaithiyanathan et al., 2011), white grape extract (Jongberg et al., 2011), and extracts from avocado peels and seeds (Rodríguez-Carpena, Morcuende, & Estévez, 2011; Rodríguez-Carpena, Morcuende et al., 2011), which have been shown to inhibit lipid oxidation as reviewed above, also have exhibited an inhibitory effect on protein oxidation. However, Lund et al. (2007) reported that the rosemary extract did not inhibit protein oxidation in beef patties and, in contrast, the ascorbate/citrate promoted protein oxidation. Apple polyphenols were also found to have no effect against protein oxidation in both pork and beef sliced cooked hams (Sun, Zhang, Zhou, Xu, & Peng, 2010). These conflicting results may be attributed to the different chemical structure of phenolic compounds and the nature and conformation of the proteins (Estévez et al., 2008). In the present study, BCE inhibited the formation of protein carbonyls to some extent, while the inhibitory effect was less intense than that in lipid oxidation. These results may be attributable to the faster occurrence of lipid oxidation than that of protein oxidation and to the likely covalent binding of phenolic compounds to protein that would mask their antioxidant activity on proteins (Arts et al., 2002; Estévez et al., 2008; Lund et al., 2007). Apart from numerous environmental factors, transition metals, myoglobin and oxidising lipids are the main initiators of protein oxidation, especially protein carbonylation (Estévez, 2011); for this reason, plant extracts may protect protein against oxidation by retarding the above initial factors. Estévez and Heinonen (2010) suggested that phenolic compounds inhibited MP oxidation by acting as metal chelators and radical scavengers. Furthermore, the effect of phenolic compounds against protein oxidation was also related to their covalent and non-covalent interactions with proteins (Viljanen, Kivikari, & Heinonen, 2004), which may protect proteins from attracting free radicals. However, the exact mechanisms of the inhibition of protein oxidation by phenolic compounds and their interactions are still not completely understood, and further investigations are needed to clarify the mechanisms. The loss of sulfhydryl groups as a consequence of disulphide formation is another well-described marker of oxidation reactions in muscle protein (Soszyński & Bartosz, 1997). The oxidation of cysteine sulfhydryl groups to form disulfide bonds, inducing protein crosslinks, is responsible for the loss of sulfhydryl groups. As shown in Fig. 4B, the effect of the BCE on the changes in the total sulfhydryl content of MP followed a different pattern than that found for the protein carbonyl content: the level of sulfhydryl groups was decreased in all patties from 1 to 9 days storage. The addition of BCE and BHA could significantly decrease the loss of sulfhydryl content. However, among the BCE-treated patties, the higher concentration of BCE resulted in a higher, though not significant, loss of sulfhydryl content (P > 0.05), which may result from the interaction between sulfhydryl groups and phenolic compounds (Jongberg et al., 2011). Sulfhydryl groups could convert into disulfides and other oxidised species, which is one of the earliest observable events during the radical-mediated oxidation of proteins (Dean, Fu, Stocker, & Davies, 1997); therefore, BCE may reduce the loss of sulfhydryl content by scavenging free radicals. In addition, sulfhydryl groups can trap free radicals (Vaithiyanathan et al., 2011); thus, BCE could compete with sulfhydryl groups for trapping free radicals to protect sulfhydryl groups themselves from oxidation. Vaithiyanathan et al. (2011) found that chicken meat dipped in pomegranate fruit juice phenolics exhibited a higher content of total sulfhydryl and protein-bound sulfhydryl groups than meat that was not treated. Rosemary and lemon

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and BHA-treated patties showed significantly higher redness as 20 g/kg BCE > 10 g/kg BCE > 5 g/kg BCE > 0.2 g/kg BHA than the control (P b 0.05). Contrary to a*-value, L*-value showed a steady increase in all the groups during the 9-day storage period, and BCE treatments could significantly decrease L*-value compared with the control on all storage days (P b 0.05) (Fig. 5B). As shown in Fig. 5C, patties with added BCE displayed a distinctive purple colour due to the presence of the major pigment in BCE, anthocyanins, which were blended with the patties and dyed the patties during manufacturing; BCE was also the main cause of the increase in redness and the reduction in lightness. It is unknown whether consumers would accept the purple colour; however, above 3 days of storage, the colour of BCEtreated patties became more acceptable than the brown colour in the control and the initial purple colour. Similarly, Ganhão, Morcuende, and Estévez (2010) found that blackberry, which is rich in anthocyanins, enhanced the colour of cooked burger patties, while the other fruits containing small amount of anthocyanins did not influence colour. Other phenolic-rich extracts have also been reported to inhibit the colour change in pork meat during chilled storage, for example, extracts from mustard leaf kimchi (Lee et al., 2010) and avocado peels and seeds (Rodríguez-Carpena, Morcuende, & Estévez, 2011; Rodríguez-Carpena, Morcuende et al., 2011). The discolouration in pork was caused by the formation of

balm extracts also inhibited a loss of sulfhydryl groups in cooked pork patties (Lara, Gutierrez, Timón, & Andrés, 2011). In contrast, rosemary extract, green tea extract, ascorbic acid, and tocopherol had little effect on the sulfhydryl groups in pork patties (Haak, Raes, & De Smet, 2009). Interestingly, Jongberg et al. (2011) found a faster rate for the loss of sulfhydryl groups in white grape extract-treated beef patties than in untreated patties, but less myosin cross-link formation, indicating the interactions between white grape extract and the sulfhydryl groups of MP that reduced cross-link formation in beef patties. Nevertheless, the present results were consistent with the carbonyl content assays and revealed that BCE possesses the ability to inhibit protein oxidation by decreasing the formation of carbonyls and increasing the content of sulfhydryl groups to some extent compared to control. 3.4. Inhibition of colour deterioration in raw pork patties Colour plays a great role in both the quality and consumer's acceptance of porcine meat. The changes of a*- and L*-value of pork patties with and without antioxidants during chilled storage are shown in Fig. 5. There was a steady decrease in red colour (a*-value) in the control and BCE- and BHA-treated raw pork patties, and the total loss reached 2–5 units over a 9-day storage period (Fig. 5A). The BCE-

A

Control 5 g/kg BCE 10 g/kg BCE 20 g/kg BCE 0.2 g/kg BHA

18 a b

14

a*-value

b

c

a

b

a

b

d

b b

c c

8

a a

b a

a

a

a

c

b

a

12

Control 5 g/kg BCE 10 g/kg BCE 20 g/kg BCE 0.2 g/kg BHA a

a

c

10

60

L*-value

16

B 65

d

55

b b c

c 50

c

d d

e

d

c

c

e

d 45

6 4

40 1

3

6

9

1

Days of Storage

C

Control

5 g/kg BCE

3

6

9

Days of Storage

10 g/kg BCE

20 g/kg BCE

0.2 g/kg BHA

1d

3d

6d

9d

Fig. 5. Influences of BHA and different concentrations of black currant extract (BCE) on a*- and L*-values of pork patties (A and B) and corresponding photographs (C) during 9 days of storage at 4 °C. Error bars refer to the standard deviations obtained from triplicate sample analysis. Means with different letters (a–e) at the same storage time differ significantly (Pb 0.05).

N. Jia et al. / Meat Science 91 (2012) 533–539

metmyoglobin and non-enzymatic browning reactions between lipid oxidation products and amine in meat (Xia, Kong, Liu, & Liu, 2009). Otherwise, primary and secondary lipid oxidation products were both associated with the accumulation of metmyoglobin (Faustman, Sun, Mancini, & Suman, 2010); therefore, BCE may retard the formation of metmyoglobin by inhibiting lipid oxidation and may protect the pork patties from discolouration in addition to dying the patties. 4. Conclusions Frozen BCE, with higher anthocyanin content, stronger reducing power and stronger DPPH • and ABTS •+ scavenging activities, could be extracted with 40% ethanol in water for 2 h. In situ testing confirmed BCE to be a highly effective antioxidant in raw pork patties because it inhibited both lipid and protein oxidation and stabilised the red colour during refrigerated storage. These data strongly suggest that frozen BCE has potential as a natural antioxidant source for meat and meat product quality preservation. Further research is needed to identify the specific and main phenolic compounds in frozen BCE that are responsible for the overall antioxidative capability in muscle foods. Acknowledgements This study was supported by the Heilongjiang Natural Science Youth Fund (Grant no. QC2011C001), the Science Foundation of National Public Beneficial Vocation in China (Grant no. 200903012-02) and the Programme for Innovative Research Team of Northeast Agricultural University (Grant no. CXZ011). References Arts, M. J. T. J., Haenen, G. R. M. M., Wilms, L. C., Beetstra, S. A. J. N., Heijnen, C. G. M., Voss, H. -P., et al. (2002). Interactions between flavonoids and proteins: Effect on the total antioxidant capacity. Journal of Agricultural and Food Chemistry, 50, 1184–1187. Awika, J. M., Rooney, L. W., & Waniska, R. D. (2004). Anthocyanins from black sorghum and their antioxidant properties. Food Chemistry, 90, 293–301. Cacace, J. E., & Mazza, G. (2003). Optimization of extraction of anthocyanins from black currants with aqueous ethanol. Journal of Food Science, 68, 240–248. Dean, R. T., Fu, S., Stocker, R., & Davies, M. J. (1997). Biochemistry and pathology of radical-mediated protein oxidation. Biochemical Journal, 324, 1–18. Duan, X., Jiang, Y., Su, X., Zhang, Z., & Shi, J. (2007). Antioxidant properties of anthocyanins extracted from litchi (Litchi chinenesis Sonn.) fruit pericarp tissues in relation to their role in the pericarp browning. Food Chemistry, 101, 1365–1371. Ellman, G. L. (1959). Tissue sulfhydryl groups. Archives of Biochemistry and Biophysics, 82, 70–77. Estévez, M. (2011). Protein carbonyls in meat systems: A review. Meat Science, 89, 259–279. Estévez, M., & Heinonen, M. (2010). Effect of phenolic compounds on the formation of α-aminoadipic and γ-glutamic semialdehydes from myofibrillar proteins oxidized by copper, iron, and myoglobin. Journal of Agricultural and Food Chemistry, 58, 4448–4455. Estévez, M., Kylli, P., Puolanne, E., Kivikari, R., & Heinonen, M. (2008). Oxidation of skeletal muscle myofibrillar proteins in oil-in-water emulsions: Interaction with lipids and effect of selected phenolic compounds. Journal of Agricultural and Food Chemistry, 56, 10933–10940. Faustman, C., Sun, Q., Mancini, R., & Suman, S. P. (2010). Myoglobin and lipid oxidation interactions: Mechanistic bases and control. Meat Science, 86, 86–94. Ganhão, R., Estévez, M., Kylli, P., Heinonen, M., & Morcuende, D. (2010). Characterization of selected wild mediterranean fruits and comparative efficacy as inhibitors of oxidative reactions in emulsified raw pork burger patties. Journal of Agricultural and Food Chemistry, 58, 8854–8861. Ganhão, R., Morcuende, D., & Estévez, M. (2010). Protein oxidation in emulsified cooked burger patties with added fruit extracts: Influence on colour and texture deterioration during chill storage. Meat Science, 85, 402–409. Gardner, H. W. (1979). Lipid hydroperoxide reactivity with proteins and aminoacids: A review. Journal of Agricultural and Food Chemistry, 27, 220–229. Haak, L., Raes, K., & De Smet, S. (2009). Effect of plant phenolics, tocopherol and ascorbic acid on oxidative stability of pork patties. Journal of the Science of Food and Agriculture, 89, 1360–1365. Hayes, J. E., Stepanyan, V., Allen, P., O'Grady, M. N., O'Brien, N. M., & Kerry, J. P. (2009). The effect of lutein, sesamol, ellagic acid and olive leaf extract on lipid oxidation and oxymyoglobin oxidation in bovine and porcine muscle model systems. Meat Science, 83, 201–208.

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