Correlation between Antioxidant Enzyme Activity, Free Iron Content and Lipid Oxidation in Four Lines of Korean Native Chicken Meat

HPLC-grade chloroform was purchased from Daejung Chemical and Metals Co. Ltd. (Korea). Distilled water (DW) was purified with a glass still (Mega-Pure^TM System MP-11A, Corning Inc., USA). All other reagents were purchased from Sigma-Aldrich Co., LLC. (USA).

Four lines of KNC (Yeonsan ogye, Hyunin black, Hoengseong yakdak and Hwangbong) were used as samples in this study and raised at Seoul National University animal farm (Korea) under similar condition. Animals were allowed to free access to feed and water. All the experimental procedures were approved by the Institute of Laboratory Animal Resources, Seoul National University (SNU-070823-5). A total of 16 roosters were randomly sampled and transferred to a chicken slaughter house (Well-cock Hyunjin Co. Ltd, Korea) within group cages for 2 h. After 2 h resting with free access to water, animals were mechanically slaughtered at the age of 12 mon (n=4 birds) using a standard procedure. The breast and thigh meat of each animal were then obtained and separately packed within oxygen-permeable bags (low-density polyethylene, 25 × 30 cm; Clean-wrap, Korea). The samples were kept chilled in an icebox and were transported to the laboratory. The samples were then stored at 4±1℃ for 10 d for oxidation studies. Analyses were performed in triplicate using samples on the initial, middle, and final days.

Lipid oxidation was measured using the 2-thiobarbituric acid reactive substances (TBARS) method by Sinnhuber and Yu (1977) with slight modification. A total of 0.5 g of sample was vortex-mixed with 0.1 g of antioxidant mixture (consisting of propylene glycol, Tween 20, butylated hydroxytoluene, and butylated hydroxyanisole), 3 mL of 1% TBA in 0.3% NaOH, and 17 mL of 2.5% trichloroacetic acid in 36 mM HCl. The sample was heated in a water bath (BW-20G, Biotechnical Services, Inc., US) at 100℃ for 30 min and then immersed in ice water for 15 min. Five milliliters of aqueous sample was then mixed with 3 mL of chloroform. The optical density (OD) of the upper layer was recorded at 532 nm against blank after centrifugation at 2,400 g for 30 min at 4℃ (1248R, Labogene, Denmark). The results were expressed in mg of malondialdehyde (MDA) per kg of meat. Each sample was analyzed in triplicate.

SOD, catalase and GSH-Px were measured in aqueous meat extract dissolved in 50 mM phosphate buffer (pH 7.0 at 25℃) according to a previous study by Renerre et al. (1996). Catalase activity was measured using a method described by Aebi (1984). First, 100 μL of filtered supernatant was mixed with 2.9 mL of 30 mM H₂O₂ within a crystal cuvette (light path: 1 cm). The decrease in absorbance at 240 nm was recorded every 10 s for 2 min. Catalase activity was expressed as units/g sample. SOD activity was determined according to the pyrogallol autoxidation method described by Marklund and Marklund (1974) with modification. The optical density of a mixture consisting of 50 μL of filtered supernatant, 3.025 mL of 50 mM Tris-cacodylate-DTPA buffer (pH 8.2 at 25℃) and 50 μL of 24.8 mM pyrogallol was recorded at 420 nm every 15 s for 2 min against a blank. One unit of SOD represents the amount of SOD required for the auto oxidation of 50% of the pyrogallol in the meat extract at pH 8.2 and 25℃. The GSH-Px activity measurement was performed according to the enzymatic protocol by Flohé and Günzler (1984) with slight modification. A total of 100 μL of filtered supernatant was mixed with 0.5 mL of phosphate buffer-0.001 M EDTA-0.1 M NaN₃, 100 μL of the assay mixture containing 5 units mL⁻¹ glutathione reductase in 50 mM phosphate buffer, 100 μL of 10 mM L-glutathione reduced, 100 μL of 1.5 mM NADPH in NaHCO₃, and 100 μL of 1.5 mM H₂O₂ within a 10-mm precision cell (104-QS, Hellma Analytics, Germany) and incubated for 5 min. GSH-Px activity was measured by recording the decrease in absorbance of the incubated mixture at 340 nm over 2 min. GSH-Px activity was expressed as units g⁻¹ sample. One unit represents the amount of meat extract needed to alter 1 μmol of NADPH with a mM extinction coefficient of 6.3 mM⁻¹ cm⁻¹ at 340 nm.

The amount of free iron in the meat was measured based on a modified version of the ferrozine method (Ahn et al., 1993; Carter, 1971). A 5 g sample was homogenized with 45 mL of citrate phosphate buffer (pH 5.5) at 13,500 rpm for 30 s using a homogenizer (Ultra-Turrax T25 basic, Ika Werke GmbH and Co., Germany). The homogenate was centrifuged at 3,200 g for 30 min at 4℃ (1248R, Labogene, Denmark). Next, 1 mL of the supernatant was mixed with 0.5 mL of freshly prepared 2% ascorbic acid in 0.2 N HCl and incubated at room temperature for 5 min. Then, the mixture was mixed thoroughly with 1.5 mL of 11.3% trichloroacetic acid followed by centrifugation at 10,000 g in an Eppendorf Micro 17R+ centrifuge (Hanil Science Industrial, Korea) for 10 min. Finally, 1 mL of supernatant was mixed with 0.4 mL of 10% ammonium acetate and 0.1 mL of ferroin indicator followed by 5 min incubation. The absorbance was read at 562 nm against a blank. The free iron value was expressed as μg/g meat.

The one-way analysis of variance (ANOVA) was used for the effect of different meat parts. Thigh meat was observed to have higher TBARS value, antioxidant enzyme activity and iron content than breast meat. A 4 × 3 factorial design with three replicates each meat part was employed with chicken lines and storage times as the main effects, using two-way ANOVA. No significant interactions were found. The statistical significance of the differences between the means from different lines and storage times was determined by Duncan’s multiple range test. P values of less than 0.05 were regarded as significant. The Pearson’s correlation coefficients and linear regression between TBARS values and other parameters were measured. Data used for regression analysis were pooled according to chicken lines, meat parts and storage times (N=24). Analyses were performed using R-version 3.2.0 with the “Agricolae” library (The R-foundation for Statistical Computing, Austria).

The degree of lipid oxidation is represented by the increasing amount of malondialdehyde content during storage. Refrigerated storage significantly increased the production of malondialdehyde for both the breast and thigh meat of each KNC (p<0.001) line. The highest malondialdehyde contents were observed on the final day of storage, ranging from 0.40 to 0.45 mg/kg meat for breast meat and 0.53 to 0.79 mg/kg meat for thigh meat (Table 1). Among KNC, the highest TBARS values were found in the thigh meat of Yeonsan ogye and Hoengseong yakdak (p<0.05). However, no significant chicken lines effects were found in breast meat.

The decline of antioxidant enzyme activity in KNC meat as affected by refrigerated storage is shown in Table 2. The SOD activity on the initial day of storage ranged from 124 to 148 units/g meat for breast and 144 to 172 units/g meat for thigh. This value then decreased on the final day of storage, ranging from 61.71 to 73.14 units/g meat and 68.57 to 98.29 units/g meat for breast and thigh, respectively. At day 0, the activity of catalase ranged from 69.14 to 73.77 units/g meat for breast and 118.07 to 133.07 units/g meat for thigh. At day 10, this value decreased to 36.29-48.55 units/g meat and 50.97-81.97 units/g meat for breast and thigh, respectively. The activity of GSH-Px decreased as well, ranging from 0.27 to 0.37 units/g meat for breast and from 0.54 to 0.62 units/g meat for thigh at day 0, dropping to 0.11-0.13 units/g meat and 0.12-0.14 units/g meat at day 10 for breast and thigh, respectively. Chicken lines affected the activity of SOD and GSH-Px in thigh meat (p<0.05), whereas no effects were observed on antioxidant enzyme activity in breast meat. The highest activity of SOD was found in thigh meat of Hwangbong and Hyunin black had the highest GSH-Px activity in its thigh meat.

The amount of iron released during refrigerated storage from KNC meat is shown in Table 3. At day 0, the amount of released iron ranged from 0.30 to 0.39 μg/g meat for breast and from 0.76 to 1.15 μg/g meat for thigh. After 10 d of storage, this value elevated to 0.41-0.49 μg/g meat and 1.21-1.25 μg/g meat for breast and thigh, respectively. No significant differences were found among chicken lines.

The correlation between TBARS values and the activity of antioxidant enzyme is shown in Fig. 1A, 1B and 1C for SOD, catalase and GSH-Px, respectively. The SOD and GSH-Px activities were negatively correlated with TBARS values in the breast and thigh meat of KNC during refrigerated storage, whereas catalase activity was not significantly associated with lipid oxidation. The correlation coefficients for SOD and GSH-Px were −0.655 and −0.493, respectively.

Fig. 1. Correlation between TBARS and (A) SOD activity (r=-0.655, p<0.001, y=144.90-114.60x), (B) catalase activity (r=-0.215, p=0.3125, y=88.79-33.86x), (C) GSH-Px activity (r=-0.493, p=0.014, y=0.44-0.47x), and (D) free iron content (r=0.552, p=0.005, y=0.36+1.08x) in four lines of KNC meat.

Download Original Figure

The association of TBARS values with the amount of released-iron of KNC meat during refrigerated storage is shown in Fig. 1D. A significant correlation was found between free iron content and lipid oxidation (p<0.01). The free iron content was positively correlated with the increasing amount of malondialdehyde content in KNC meat, with a correlation coefficient of 0.552.