Improved Oxidative Stability of Enhanced Pork Loins Using Red Perilla Extract

Dried leaves of red perilla were purchased in a local market. Dried leaves (50 g) of red perilla were mixed with distilled water (1.95 L) followed by the extraction in a shaking water bath for 24 h at 70℃. The extract was centrifuged for 30 min at 6,710 × g (CR 20B2, Hitachi Koki Co., Ltd. Japan), and the supernatants were recovered by gravity filtration using Whatman No. 4 filter paper (Whatman Inc., England). The filtrate was lyophilized (Ilshin Co., Korea). The resulting powdered extract of red perilla leaves (ERP) was stored in a deep freezer at −70℃ until use.

The total phenolic content of ERP was measured using the Folin-Ciocalteu method described by Subramanian et al. (1965). A 0.1 mL sample in distilled water was added to 0.2 mL of Folin-Ciocalteu reagent and kept at 23℃ for 1 min. A 5% sodium carbonate solution (3 mL) was added to the mixture and incubated in the dark at 23℃ for 2 h. The absorbance of the mixture was measured at 765 nm using a spectrophotometer (DU 530, Beckman Instruments Inc., USA). The quantification of phenolics was based on a standard curve generated with the gallic acid and expressed as gallic acid equivalents (GAE).

Boneless pork loins (approximately 2.5-2.8 kg) were obtained from a local market (Korea). Pork loins were assigned to the injection of one of three treatments (control, brine containing no ERP; ERP 0.2, brine containing 2 g of ERP per kg of pork loin; and ERP 0.4, brine containing ERP at 4 g per kg of pork loin) (Table 1). The appropriate amount of ERP (2 or 4 g/kg of pork loin) was dissolved in brine before injection. Pork loins were weighed individually, and injected by the manual multiple-needle injector (a customized equipment) with brines (15% by weight). The injection process was repeated three times on the same day to manufacture three pork loins for each treatment. Pork loins were sealed in polyethylene bags and stored in a refrigerator at 4℃ for 24 h. Each pork loin was then cut into three pieces, which were individually packaged in polyethylene bags, and stored in a refrigerator at 4℃ for 0, 3, and 7 d. On each storage interval, a piece of each pork loin was cut into 2 cm slices, three of which were randomly selected for testing.

The color (CIE L*, a*, and b* values) of raw pork loin slices was measured using a colorimeter (CM-3500d, Minolta, Japan). Measurements were taken perpendicular to the surface of the slice with an illumination diameter of 30 mm at three different locations per sample. The results were analyzed using Spectra Magic Software (Minolta, Japan).

The raw pork sample (1 g) was mixed with 9 mL of distilled water and homogenized (T25 basic, IKA GmbH & Co. KG, Germany) at 1,130 × g for 1 min. The homogenate was centrifuged (Continent 512R, Hanil Co., Ltd., Korea) at 2,265 × g for 10 min and the supernatants were recovered by gravity filtration using Whatman No. 4 filter paper (Whatman Inc., England). The pH of each filtrate was determined measured with a pH meter (SevenEasy, Mettler-Toledo, Switzerland) which was pre-calibrated using standard buffers (pH 4.01, 7.00, and 9.21).

The pork loin slices were cooked in an electric steam oven (EON-C305CSM, Tongyang Magic Co., Korea) at 180℃ for 25 min until the internal temperature of the pork loin reached 75℃, and subsequently cooled at room temperature for 30 min. Cooking loss was calculated by the weight loss (%) of each slice after cooking.

Lipid oxidation of cooked slices of pork loin was monitored by the detection of malondialdehyde (MDA). This procedure was conducted according to the method described by Jung et al. (2016). For this analysis, MDA was extracted from the samples with acetonitrile as follows. The sample (3 g) was homogenized with 6 mL of distilled deionized water and 50 µL of 7.2% 2,6-di-tert-butyl-4-methylphenol in ethanol using a homogenizer (T25 basic) at 16,000 rpm for 1 min. Next, 500 µL of the homogenate was transferred into an Eppendorf tube, and 100 µL of 6 M NaOH solution (final concentration: 1 M) was added for the alkaline hydrolysis of the protein-bound MDA. The tubes were incubated in a water bath at 60℃ for 45 min. After cooling in ice for 5 min, 1 mL of acetonitrile was added to the tube, and the mixture was vigorously vortexed. The tube was centrifuged at 13,000 × g for 10 min (HM-150IV, Hanil Co., Ltd., Korea). The upper clear phase of the supernatant contained the MDA extract. As an MDA standard, the solution of 1,1,3,3-tetraethoxypropane (3.2 mM) was diluted with distilled to a concentration of 0.1, 0.2, 0.4, 0.8, or 1.6 mM. Subsequently, 1 mL of the MDA extract, standard, or deionized water (blank) was passed through a 0.2-µm PVDF syringe filter (Whatman), and the filtrate was collected in a vial. The concentration of MDA was analyzed by HPLC (ACME 9000, Younglin Instruments Inc., Korea), using an Atlantis T3 C18 RP column (4.6 × 250 mm, 5 µm particles) with a mobile phase consisting of 30 mM K₂HPO₄ (pH adjusted to 6.2 with H₃PO₄). The isocratic flow rate of the mobile phase was 1.2 mL/min, and the injection volume was 50 µL. The column temperature was maintained at 35℃ and the UV/Vis detector was set to a wavelength of 254 nm. The concentration of MDA in each sample was expressed in MDA mg/kg of pork loin.

Sensory evaluation of cooked pork loin was conducted independently during three sessions. Each session was arranged with pork loins from each batch. Pork loin slices were cooked at 180℃ for 25 min using an electric steam oven (EON-C 305CSM). The cooked pork loin slices were reheated at 180℃ for 5 min and cut into even slices immediately before sensory analysis. Pork loins were served on white glass plates to ten panelists who evaluated the color, flavor, taste, odor, tenderness, and overall acceptability of the pork loins. Every parameter but odor was ranked on a 9-point hedonic scale (1 = extreme dislike, 9 = extreme like).

This study was conducted in triplicate. Data was analyzed using the PROC GLM procedure in a randomized complete block design (batch as a block). The experimental unit was a slice of pork loin. The major effect included in the statistical model was ERP concentration. In the analysis of the data from the sensory evaluation, the panel was included in the model as a random effect. Specific comparisons were made by Tukey’s multiple range test when the main effect was significant. Results were reported as least-square mean values and the standard error of the least-square means (SEM). Statistical significance was defined as p<0.05. SAS software (version 9.3, SAS Institute Inc., USA) was used for statistical analyses.

The L*-value of raw pork loins was influenced by brine injection containing ERP (Table 2). ERP 0.2 had a significantly lower L*-value than the control at 4 and 7 d of storage (p<0.05). In addition, the L*-value of ERP 0.4 was lower than that of the control for all storage days (p<0.05). There were no significant differences between the L*-values of ERP 0.2 and ERP 0.4 for all storage days (p>0.05). During storage periods, no variance in L*-values was found in the control group. The L*-values of ERP 0.2 and ERP 0.4 at 4 d of storage were significantly decreased compared with those in 0 d of storage (p<0.05). However, there was no significant differences of the L*-values of ERP 0.2 and ERP 0.4 between 0 and 7 d of storage (p>0.05). ERP 0.2 and ERP 0.4 had significantly higher a* and b*-values compared to that of the control for all storage days (p<0.05). There were no changes in the a* and b*-values across all treatments during storage periods except for the b*-value of the control (p>0.05).

The leaves of red perilla contains anthocyanin, which is composed of red, purple, and blue colored pigments (Asif, 2012; Gai et al., 2017; Jung and Joo, 2013). Lee et al. (2016) reported that the addition of brown soybean extracts containing a large quantity of anthocyanins into pork patties resulted in the decrease of L*-values and the increase of a*-values. Previous studies have also reported the decrease of L*-values and the increase of a*-values due to naturally sourced anthocyanins added to meat (Ganhao et al., 2010; Kim et al., 2015). Therefore, the addition of natural preservatives in meat and meat products leads to change the color due to their pigmentation. The color of meat is an important quality parameter because consumers generally judge the freshness of meat by the meat color. Generally, pork with the highest L*-values and the lowest a*-values, which appear similar to pale, soft, and exudative (PSE) meat, is considered poor-quality pork although it is not PSE (Jung et al., 2015). In the present study, ERP decreased the L*-value and increased the a*-value of the enhanced pork loin. Therefore, it may be used for the improvement of the color of pork loins.

pH is an important quality parameter in meat. At pH values near that of the isoelectric point of muscle protein (approximately 5.2-5.4), the water holding capacity of the tissue diminishes due to the decrease in net charges (Huff-Lonergan and Lonergan, 2005). The injection of brine containing ERP did not influence the pH of raw pork loins at 0 and 4 d of storage (p>0.05, Table 3). However, ERP 0.4 had significantly a lower pH than that of the control after 7 d of storage (p<0.05). There was no change in the pH of the control and ERP 0.2 during the storage periods (p>0.05). Although the pH of ERP 0.4 changed during storage, no significant difference in pH was found between 0 and 7 d of storage. The pH of the red perilla extract solution was approximately 5.6 (data not shown). Lee et al. (2015) reported that the pH of red perilla extract ranged from 5.4 to 5.6 that its subsequent addition to beef patties at a concentration of 6 g/kg decreased the pH of beef patty. ERP was added at lower concentrations than in previous works (Lee et al., 2015). In the present study, sodium pyrophosphate was also injected into the pork loins. As an alkaline additive, sodium pyrophosphate has the capacity to buffer pH (Lee et al., 2018). Therefore, the influence of ERP on the pH of pork loins is minor with the exception of the low pH of ERP 0.4 after 7 days of storage. The cooking loss of pork loins did not vary by treatment or storage period (Table 4).

The lipid oxidation of cooked pork loins was measured by detecting MDA, an abundant secondary product of lipid oxidation (Jung et al., 2016). The rate of lipid oxidation in raw meat increases with storage time, and highly oxidized raw meat samples show a substantial degree of lipid oxidation after cooking (Ferioli et al., 2008). The MDA contents of cooked pork loins did not vary by treatment before storage (Table 5). However, ERP 0.2 and ERP 0.4 contained less MDA than the control sample after 4 and 7 days of storage (p<0.05). Furthermore, the MDA contents of ERP 0.2 and ERP 0.4 at 4 and 7 d of storage did not differ significantly compared to that of the sample before storage, while the MDA contents of the control increased over time. These results indicate that ERP inhibited the lipid oxidation process in the enhanced pork loins. Lee et al. (2015) reported that the lipid oxidation of beef patties was inhibited when an aqueous extract of red perilla was used as an additive. Phenolic compounds within ERP are responsible for this antioxidant activity.

ERP contained phenolic compounds at a concentration of 71.76 mg GAE/g (data not shown). Red perilla leaves contain several phenolic and flavonoids compounds (Asif, 2012). Previous studies reported that ERP contains three phenolic acids, including coumaroyl tartaric acid, caffeic acid, and rosmarinic acid, and six flavones, including apigenin 7-O-caffeoylglucoside, scutellarein 7-O-diglucuronide, luteolin 7-O-diglucuronide, apigenin 7-O-diglucuronide, luteolin 7-O-glucuronide, and scutellarein 7-O-glucuronide (Asif, 2012). In addition, malonylshisonin is a key anthocyanin in the water extract of red perilla (Asif, 2012). Phenolic compounds can inhibit lipid oxidation by scavenging free radicals and therefore preventing the initiation of the process and by donating an electron to terminate the radical chain reaction (Rice-Evans et al., 1997). Furthermore, unlike vitamin C and E, it can act as a chelator for transition metal ions (Nimse and Pal, 2015). Transition metal ions in meat, especially free iron ions, promote lipid oxidation through Fenton's reactions (Fe²⁺ or Fe³⁺/H₂O₂), in which free iron ions generate hydroxyl radicals by reacting with hydrogen peroxides, resulting in the generation of lipid and peroxyl radicals from lipid hydroperoxides (Jung et al., 2012; Nimse and Pal, 2015).

The scores for both the color and tenderness of the cooked pork loins were not significantly different between each of the treatments at each of the storage intervals (Table 6). The taste scores of ERP 0.2 and ERP 0.4 were significantly lower than those of control at 0 and 4 d of storage (p<0.05). However, the taste score of ERP 0.2 was not significantly different than that of the control after seven days of storage (p>0.05). Similar results were attained for the flavor of the cooked pork loins. Although ERP 0.2 and ERP 0.4 had significantly lower scores for flavor compared with those of the control at 0 and 4 d of storage (p<0.05), there was no difference between the flavor scores for all treatment after 7 d of storage. For overall acceptability, significantly lower scores were found in ERP 0.2 and ERP 0.4 compared to that of the control at 0 and 4 d of storage (p<0.05), however, ERP 0.2 was no different than the control at 7 d of storage. These data show that enhancing pork loins with ERP at a concentration of 4 g/kg (ERP 0.4) consistently resulted in the decrease in their sensory desirability. Mo et al. (1999) reported that food treated in a similar fashion had a lower sensory score due to the unique flavor of red perilla leaves. However, ERP 0.2 showed similar sensory properties to the control after seven days of storage despite its lower scores after 0 and 4 d of storage. These results might be attributed to the inhibitory action of red perilla extract toward lipid oxidation. In the present study, lower levels of lipid oxidation were found in ERP 0.2 compared than in the control.