Chloramine-T

Proximate composition: The proximate composition of breast and thigh meat of each bird was determined as outlined by AOAC (1995) . Briefly, moisture content was measured by drying the samples (2 g) at 102°C for 15 h. Crude fat and crude protein contents were measured by the Soxhlet extraction system (TT 12/A, Gerhardt Ltd., Germany), and the Kjeldahl method (VAPO45, Gerhardt Ltd., Germany), respectively.
pH value: pH of breast and thigh meat from each bird was measured using a pH meter (750P, Istek Co., Korea) after calibration using buffers (pH 4.01, 7.00, and 9.21) at room temperature according to the method described by Jung et al. (2011) . The mean value of three repeated measurements from each sample was used.
Water-holding capacity: Water-holding capacity (WHC) was determined according to the method of Ryoichi et al. (1993) . Two grams of the mined sample was wrapped on a round filter paper (No. 4, Whatman Ltd. Kent, UK), subsequently placed into a centrifuge tube and centrifuged (CR 20B2, Hitachi Koki Co., Ltd. Fukuoka, Japan) at 6,710×g for 10 min. The released water absorbed into the filter paper was weighed and calculated as a percentage of the initial moisture of meat.
Color values: The color values of breast and thigh meat were measured on the surface of meat samples using a colorimeter (Spectrophotometer, CM-3500d, Minolta, Japan) which was calibrated against a black and a white reference tile. The values of lightness (CIE L*), redness (CIE a*), and yellowness (CIE b*) were obtained using the average value of three repeated measurements taken from different locations on the meat surface. Each color value was analyzed by Spectra Magic Software (Minolta Inc., Japan).
Total and insoluble collagen contents: The total collagen content of each sample was determined by acid hydrolysis as described by Palka (1999) (link). The sample (500 mg) was hydrolyzed with 25 ml of 6 M HCl at 110°C for 24 h. The hydrolysate was filtered, neutralized with 10 M and 1 M NaOH, and diluted with distilled water to a final volume of 250 ml. Hydrolysate (4 ml) and 2 ml of chloramine T solution (1.41 g of chloramines T, 10 ml of distilled water, 10 ml of n-propanol, and 80 ml of citric buffer at pH 6.0) were mixed in a test tube and allowed to stand for 20 min at room temperature. Then, 2 ml of 4-dimethyl-aminobenzaldehyde solution (10 g of p-DABA, 35 ml of HClO₄-60%, and 65 ml of isopropanol) was added and the solutions were shaken and heated at 60°C for 20 min. The samples were then cooled for 5 min in tap water and the absorbance was measured at 558 nm using a spectrophotometer (DU 530, Beckman Instruments Inc., USA). The amount of hydroxyproline was determined by comparison with a standard curve. The total collagen content (mg of collagen per g of meat) was calculated from the hydroxyproline content using a coefficient of 7.25.
Soluble collagen was extracted according to the method of Liu et al. (1996) (link). First meat samples (2 g) were homogenized with 8 ml of 25% Ringer’s solution. The homogenates were heated at 77°C for 70 min and centrifuged at 2,300×g at 4°C for 30 min. The extraction was repeated twice and the sediment was then hydrolyzed with 6 M HCl at 110°C for 24 h. The hydroxyproline content of the sediments was determined and the insoluble collagen content was calculated as described previously.
Nucleotide content: The meat samples (5 g each) were mixed with 25 ml of 0.7 M perchloric acid and centrifuged at 1,130×g for 1 min to extract nucleic acids. The extracted nucleic acids were centrifuged at 2,090×g for 15 min and then filtered through a filter paper (No.4, Whatman International Ltd. USA). The supernatant was then adjusted to pH 7.0 with 5 N KOH. The pH-adjusted supernatant was placed into a volumetric flask and made up to 100 ml with 0.7 M perchloric acid (pH 7.0). After cooling for 30 min, the supernatant was centrifuged at 1,130×g (0°C) and then filtered through a 0.2 μm PVDF syringe filter (Whatman International Ltd.). The filtrate (5 ml) was analyzed using HPLC (ACME 9000, Younglin Instruments Inc, Korea). For the analytical conditions of the HPLC, Waters-Atlantis dC18 RP column (4.6×250 mm, 5 μm particles, Waters Co., USA) was used. Mobile phase was 0.1 M triethylamine in 0.15 M acetonitrile (pH 7.0). The flow rate of the mobile phase was 1.0 ml/min and the injection volume was 10 μl. The column temperature was maintained at 35°C and the detection was monitored at a wavelength of 260 nm. The peaks of the individual nucleotides were identified using the retention times for standards: hypoxanthine, inosine, inosine-5’-monophosphate (IMP), adenosine-5’-monophosphate (AMP) (Sigma, USA), and the concentration was calculated using the area for each peak.
Fatty acid composition: Fatty acid composition was analyzed by the original method of Folch et al. (1957) (link) with some modifications done by Jung et al. (2010) (link). Lipids were extracted from breast and thigh meat by mixing meat samples (10 g) and 50 ml of Folch solution (chloroform: methanol = 2:1). To this solution, 0.88% KOH was added, followed by vigorous mixing while capped and incubation at room temperature for 2 h. Then, the upper layer was removed, and chloroform was evaporated using N₂ gas (99.999%). After cooling, 1 ml of methylating reagent (BF₃-methanol, Sigma Chemical Co., USA) was added to 100 μl of lipid, which was heated at 70°C for 30 min. The samples were removed from the water bath and allowed to cool, after which 2 ml of hexane (HPLC grade) and 5 ml of distilled water were added to the samples. The samples were then vortexed and the upper layer removed. The fatty acid methyl ester dissolved in hexane was transferred to a GC vial. Fatty acid composition was analyzed using a gas chromatograph (GC 6890N, Agillent, USA) equipped with a mass selective detector. A split inlet (split ratio, 50:1) was used to inject samples into a HP-5MS capillary column (30 m×0.25 mm×0.25 μm film thickness, Agillent, USA). The ramped oven temperature was 150°C for 3 min, increased to 180°C at 2.5°C/min and maintained for 5 min, and increased to 220°C at 2.5°C/min and maintained for 25 min. Inlet temperature was 210°C and the detector temperature 250°C. Helium was the carrier gas at constant flow of 0.7 ml/min. The temperature of the mass spectrometer (MS) source, MS quadrupole, and transfer line into the MS were 230, 150, and 280°C, respectively. The fatty acid composition was identified by a mass spectrum database (NIST Library, mass spectral search program, version 5.0, USA).