Antioxidative and Sensory Properties of Allium hookeri Fermented by Leuconostoc mesenteroides in Pork Patties

The Allium hookeri was bought at Samchaenara Co. located Gyeongsangnamdo, Korea. The Allium hookeri was freeze dried to use as the sample at Dongil Cold Storage & Foods Co. (Icheon, Korea). Chemicals together with 1,1,3,3-tetraethoxypropane (TEP), ABTS, aluminum chloride, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu’s phenol reagent, TBA, quercetin, sodium carbonate and trichloroacetic acid (TCA) were bought at Sigma (Sigma-Aldrich, St. Louis, MO, USA). Supplementary chemicals including gallic acid and potassium persulfate were bought at Samchun (Samchun pure Chemical Co., Ltd., Pyeongtaek, Korea) and Kanto (Kanto Chemical Co., Inc., Tokyo, Japan).

The Leuconostoc mesenteroides SK 1962 obtained from the Laboratory of Feed Biotechnology and Applied Molecular Microbiology of Konkuk University, Seoul. L. mesenteroides SK 1962 was cultivated in MRS broth (Oxoid Ltd., Basingstoke, UK) at 37°C for a day, and then used as an inoculum that was incubated to get a population of 10⁷ CFU/mL on regular. Allium hookeri powder was mixed with distilled water at a 1:10 (w/v) ratio and autoclaved for 15 min at 121°C. After chilling, it was inoculated (1%, v/v) with an activated strain of 10⁷ CFU/mL on average. The mixed culture was cultivated in MRS broth at 37°C and then incubated. The samples were collected after 16 h.

Total phenol contents were measured by Folin-Ciocalteu’s methods (Singleton et al., 1999) with a few modifications. The 0.5 mL of samples and 2.5 mL Folin-Ciocalteu’s phenol reagent were assorted and then reacted for 5 min at the room temperature. Then, adding 2 mL of 75 g/L sodium carbonate to the sample and reacted at the room temperature for 2 h. The absorbance value was determined at 760 nm and for the blank the ethanol was used. To construct the standard curve garlic acid equivalents were used. Total flavonoid contents were measured by the Dowd method (Arvouet-Grand et al., 1994) with minor modifications. Temporarily, 1.5 mL of 2% aluminum chloride and 0.5 mL of diluted 10 times sample were mixed and reacted at the room temperature for 10 min. The samples were measured absorbance at 415 nm. For the standard curve the quercetin was used.

The free radical scavenging activity of each group was determined by method of Blois (Blois et al., 1958), with a few modifications. The 5 mL of 0.1 mM DPPH in 95% ethanol was mixed to a 1 mL 10-fold-diluted aliquot of each sample. The mixture of the samples was vortexing and then reacted at the room temperature for 30 min in the darkroom. Later then, the sample was purified with the 0.45 μm nylon syringe filter. The sample was determined at 517 nm of absorbance at spectrophotometer. The ABTS free radical scavenging activity was measured by Re et al method. (Re et al., 1999). The 2 mM ABTS reagent was diluted in distilled water including 2.45 mM potassium persulfate and reserved at the room temperature for 12 h in the darkroom. The ABTS solution was accustomed with a sodium phosphate buffer (0.1 M, pH 7.4) to an original absorbance at 734 nm. Then, a 0.1 mL 10-fold-diluted aliquot of each sample was mixed with 3 mL of the ABTS solution. After incubating at the room temperature for 10 min, the samples were measured at 734 nm of absorbance.

The ABTS and DPPH scavenging activity values were calculated as follows:

Pork loins and pork backfat were purchased from a local market (Seoul, Korea). Excluded the pork loin such as fat and connective tissue were removed. Lean pork (80%) and fat (20%) were ground through a 5-mm plate. The grounded mixture was mixed with 1.5 g/kg sodium nitrite. The patties were divided into three groups: CON, with no Allium hookeri powder; AH, containing 1% Allium hookeri powder; and FAH, containing 1% fermented Allium hookeri powder. The mixtures with different compositions were then formed into 80-g patties using a patty-maker (small round burger press, Spikomat Ltd., Nottingham, UK).

Crude protein and crude fat contents of the patties were measured following to the AOAC methods (2012). The moisture content was measured by the drying method at 105°C and the crude ash content was determined by the direct ash method at 550°C.

The cooking loss (%) was measured by weight changes in pork patties before and after cooking at 75°C for 45 min in a water bath, as revealed following equation (Choi et al., 2009).

The expressible drip was determined by modification of NGCS method (1987). The samples were set sandwiched between the Whatman No. 1 filter papers and forced with a force of 9.9 kg/cm² (IF 32B-S50; Ilshin Tech. Co. Ltd., Hwaseong, Korea) for 1 min and calculated with the formula as shown below.

For the pH measurement, the sample (2 g) were homogenized with 18 mL of distilled water for 1 min using a pH meter (pH 900, Precisa Co., Dietikon, Switzerland).

The TBARS values of the samples were measured by the method reported by Witte et al. (1970). The 10 g of sample was homogenized with 50 mL of 10% (w/v) TCA for 1 min. After homogenized the samples were filled up to 50 mL with distilled water. The samples were purified with Whatman No.1 paper and then 5 mL of the filtrated sample was mixed with 5 mL of TBA (2.88 g/L H₂O), which was boiled in a boiling water bath at 80°C for 10 min to develop a pink color. After cooling the samples at the room temperature, the samples were determined at 532 nm absorbance. The standard curve was measured with the malondialdehyde (MDA) prepared by acidification of TEP. The TBARS value was measured by the standard curve and presented by mg of MDA per kg of sample.

TPA of the pork patties was evaluated at the room temperature with a texture analyzer (CT31000, Brookfield Engineering Laboratories, Inc., Middleborough, MA, USA) and evaluate the hardness, springiness, cohesiveness, gumminess, and chewiness of the samples. TPA was measured with a few modifications of the Claus method (1995). The sample was cut into a 1 cm cube shape from the pork patties. The samples were crushed to 70% of its original height using a cylindrical probe with a diameter of 10 cm under a compression load of 15 kg and cross-head speed of 20 cm/min. The TPA values for hardness, springiness, cohesiveness, gumminess, and chewiness were measured as defined by Bourne (1978).

The 10 random trained panelists were joined the sensory evaluation. Each sample was graded 1 (not acceptable) to 10 (very acceptable) by the panelists. The sensory test was approved (approval number: 7001355-201910-E-102) by Konkuk Institutional Review Board (IRB). The sample patties were cut into a block with a thickness, length, and width of 1 cm each and were placed on a white plate at room temperature (Kim et al., 2015). After testing and graded one sample, the panelists were requested to wash their mouths with the pure water and wait a min before the grading the next sample for the evaluation. Grades for meat color, flavor, tenderness, juiciness, and overall acceptability were provided by the panelists. The sensory description used by the panelists was as described by Li et al. (2014).

All experiments data were evaluated by one-way analysis of variance using by SPSS software called PASW Statistics 18 (PASW 18, SPSS Inc., USA). Means were equated using the Tukey’s multiple rage test at the significance level of p<0.05.

Efficacies of the fermented Allium hookeri measured by the method used for DPPH and ABTS are presented in Table 1. According to the DPPH method, the radical scavenging activity of fermented Allium hookeri increased significantly compared to that of normal Allium hookeri (p<0.05). The ABTS radical scavenging activity of fermented Allium hookeri was as well significantly higher than that of normal Allium hookeri (p<0.05). Therefore, the addition of fermented Allium hookeri improved the DPPH and ABTS scavenging activities.

Total phenolic and total flavonoid contents of fermented Allium hookeri are shown in Table 2. These contents were conducted to measure the relationship between different antioxidant activities of fermented Allium hookeri. Enrich phenols and flavonoid foods such as vegetables are famous for high antioxidant activities cause to many bioactive substances (Park et al., 2007).

Effects of Allium hookeri and fermented Allium hookeri on the proximate composition, cooking loss, and expressible drip of pork patties are presented in Table 3.

In the proximate composition result, no significant difference was shown between the all groups. However, the cooking loss and expressible drip of FAH was significantly lower than those of other groups (p<0.05). Similar trends in cooking loss and expressible drip have been previously noted after the addition of Allium spp. in meat products (Kim et al., 2015).

Effects of Allium hookeri and fermented Allium hookeri on pH values of pork patties are summarized in Table 4. The pH values of all treated groups were significantly lower than in CON on days 1, 3, and 7 (p<0.05). On day 14, the pH value of FAH was significantly lower than that of other groups (p<0.05). The pH influences an effect on different parameters of the food product such as the shelf life, texture, and other quality characteristics. Low pH values of meat products prolong the shelf life by inhibiting the growth of microorganisms. Previous studies have indicated that adding high-antioxidant-activity additives result in a low pH value for products, as was the case in this study (Cho et al., 2015).

Effects of Allium hookeri and fermented Allium hookeri on TBARS values of pork patties are shown in Table 5. The TBARS values of FAH were significantly lower than those of CON on days 3 and 7 (p<0.05). The TBA values denote lipid oxidation of the meat products. High TBA values in meat products suggest sensory quality degradation such as rancidity and odor. A previous study reported that supplementing Allium spp. improves the TBA value by enhancing its phenolic content (Cho et al., 2015). In this study, FAH maintained the lowest TBA values among all groups. These results indicate that fermented Allium hookeri can be used as a natural antioxidant additive to enhance the sensory properties.

Effects of Allium hookeri and fermented Allium hookeri on textural properties of pork patties are shown in Table 6. FAH showed significantly decreased hardness and chewiness values (p<0.05). The moisture content affects the hardness and chewiness. However, in this study, even though the moisture content was not significantly different for the samples, the hardness and chewiness of FAH were significantly lower than those of other groups (p<0.05). A previous study indicated that addition of some additives improved the textural properties of a meat product by lowering its hardness (Yang et al., 2007). Furthermore, supplementing Allium spp. ingredients can soften the texture (Kotwaliwale et al., 2007). Therefore, this study suggests that supplanting fermented Allium hookeri can improve the texture properties by decreasing the hardness of meat products.

Changes in sensory characteristics of cooked pork patties with Allium hookeri and fermented Allium hookeri are shown in Table 7. FAH had significantly higher color, flavor, tenderness, juiciness, and overall acceptability scores compared to the other groups (p<0.05). Previous reports indicated that adding Allium spp. in the meat product improves the flavor by masking off-flavors due to its antioxidant properties (Tang et al., 2007).