JIA-2018-09

2120 ZHANG Yi-min et al. Journal of Integrative Agriculture 2018, 17(9): 2118–2125 Munk K/S ratio of MetMb was transformed to [2–(K/S 572 )/(K/ S 525 )] according Li X et al . (2012). 2.4. MetMb reducing activity MetMb reducing activity (MRA) was determined based on the procedure of Sammel et al . (2002) with some modifications as described by Liu et al . (2014). The Kubelka-Munk K/S ratios and the content of MetMb were estimated sequentially, according to the equation of AMSA (2012). The MRA was then calculated following the equation: [(Initial %MetMb– Final %MetMb)/Initial %MetMb]×100. 2.5. NADH concentration The NADH extraction followed the protocol described by Klingenberg (1974). The NADH concentration was determined based on the spectrophotometric recycling method of McCormick and Lemuel (1971). In this study, the absorbance at 600 nm was recorded (T6; Purkinje General Instrument Co., Ltd., Beijing) and the NADH concentration (nmol g –1 ) was obtained based on the standard curve by using known amounts of NADH. 2.6. Proteomics analysis Sarcoplasmic proteome extraction Beef samples were homogenized in the extraction buffer (pH 8.0) with 40 mmol L –1 Tris (pH 7.0) and 2 mmol L –1 EDTA, and 120 µL 2 mmol L –1 dithiothreitol (DTT) at the ratio of 1:5 (w/v) at 4°C. The homogenate was then centrifuged (10 000×g) for 15 min at 4°C. And the supernatant (sarcoplasmic extract) was collected and then stored at –80°C. Two dimensional gel electrophoresis The protein concentration was determined by the BCAProteinAssay Kit (Sangon Biotech, Shanghai, China). Sarcoplamic proteins (800 mg) were mixed with rehydration buffer, containing 7 mol L –1 urea, 2 mol L –1 thiourea, 4% CHAPS, 1% DTT, 0.5% IPG buffer and 0.05% bromophenol blue. After loading the protein samples onto immobilized pH gradient strips (IPG, 24 cm, pH 3–10), gels were subjected to passive rehydration for 24 h. The isoelectric focusing (IEF) was performed on IPGphor ® (GE Healthcare, USA) by applying a linear increase in voltage initially and a final rapid voltage ramping to attain a total of 80 kV. Then the IPG strips were equilibrated first with buffer I (1.5 mol L –1 Tris-HCl, pH 8.8, 6mol L –1 urea, 2%SDS, 30%glycerol, 1% (w/v) DTT) followed by buffer II (1.5 mol L –1 Tris-HCl, pH 8.8, 6 mol L –1 urea, 2% SDS, 30% glycerol, 4.5% (w/v) iodoacetamide), each for 15min. The proteins were separated in the second dimension on 12% SDS-PAGE (acrylamide:bis-acrylamide=37.5:1) using a Ettan DALT System (GE Healthcare, USA). The gels were stained with colloidal Coomassie blue for 48 h and de- stained until sufficient background clearance was obtained. Image analysis PDQuest software was adopted to analyze the gel images (Bio-Rad, USA). Each gel of SC samples on d 1 was compared with the relevant gel in the RC group. Proteins were considered differentially abundant between SC and RC treatments, if the spot density ratio was greater than 1.5, and the P -value was below 0.05. Protein identification The protein identification was based on the procedure described by Wu et al . (2015) with minor modification. The protein spots of interest, based on image analysis, were carefully excised from the gels using pipette tips, and washed by using 25 mmol L –1 NH 4 HCO 3 /50% CH 3 CN. The gel pieces were dried in vacuum centrifuge and then swollen in the following buffer for 40min at 4°C (25mmol L –1 NH 4 HCO 3 , 10 ng μL –1 trypsin). After discarding the extra buffer and replenishing the 25 mmol L –1 NH 4 HCO 3 solution, the gel pieces were incubated at 37°C for 16 h. Then the peptides were extracted and proteins were identified using a 4800 Plus MALDI-TOF-TOFMass Spectrometer (AB SCIEX, USA). The mass and mass/mass spectra were combined to search using Mascot and matched in the National Center for Biotechnology Information database. 2.7. Statistical analysis Temperature and pH values measured over the 24 h post- mortem period were analyzed by one-way ANOVA, and the differences among means were detected by the Duncan’s multiple range test at P <0.05. Meat color attributes were analyzed by the MIXED procedure (SAS, ver. 9.0), with storage time, chilling method and their interaction fitted as fixed effects, while animal (carcass) was included as a random effect. The PDIFF statement was applied to test the differences between predicted means, and significant differences were set at P <0.05. Proteins with a minimum fold change of 1.5 were detected ( P <0.05) by comparing the averaged spots intensities of the samples between step-chilling and routine chilling at d 1 of storage. The relationship between color traits and each protein marker were evaluated in Genstat (19th edition, VSN International Ltd., https://www.vsni.co.uk ) using general linear regression analyses which accounted for the effect of treatment. Level of significance was set to P <0.05. Principal component analysis was applied to L*, a* and MetMb data against the main proteins identified by regression. 3. Results and discussion 3.1. pH/Temperature It was shown that step-chilling (SC) resulted in a lower