*2.2. pH Measurement*

The pH value of the beef muscles was measured at 1, 3, 7, 9, 11, and 14 days postmortem following the method described by Kim et al. [24], with minor modifications. Chopped meat (10 g) was mixed with 100 mL of distilled water for 15 s and then homogenized using an Ultra-Turrax T25 homogenizer (IKA-Werke, Gmbh & Co., Staufen in Breisgau, Germany) at 2800 g. The pH of the homogenate was measured using a pH meter equipped with an electrode (PB-10, the precision was 0.01, made by Sartorius Group, Goettingen, Germany). Each sample was determined three times.

#### *2.3. Myofibril Fragmentation Index (MFI)*

MFI was determined by modification of the method by Hou et al. [25]. Two gram samples were homogenized with a homogenizer (FJ200-S, Shanghai Specimen Model Co., Shanghai, China) at 10,000 g for 60 s (3 × 20 s with a 60 s break between bursts) at 4 ± 2 ◦C in 20 mL ice-cold buffer (100 mM KCl, 20 mM K2HPO4, 1 mM EGTA, 1 mM MgCl2, and 1 mM NaN3, pH 7.0). The homogenates were centrifuged using a LG10-24A model centrifuge (Peking Medical centrifuge Factory, Beijing, China) at 3000 g for 15 min at 4 ◦C and the supernatant was discarded. The pellets were homogenized in 20 mL of homogenizing buffer and centrifuged and the supernatant was discarded again. The resulting pellets were then resuspended in 5 mL of homogenizing buffer and filtered through a polyethylene strainer (200-mesh) to remove the fat and connective tissue. Then, 5 mL buffer was used to promote the passage of myofibrils through the strainer. The protein concentration of suspension was determined by the biuret method [26]. The protein concentration was diluted to 0.5 mg/mL and measured spectrophotometrically at 540 nm (UV 6100, Metash, Shanghai, China). MFI was calculated by multiplying A540 by 200.
