*2.2. Extraction of Myofibrillar Protein*

The extraction of myofibrillar protein was carried out according to the method of Ogawa et al. [17]. Briefly, 2 g fillets were weighed and washed by a fivefold volume of Tris-HCl buffer (pH 7.0, 10 mmol/L). Then, a fivefold volume of KCl-Tris buffer was added to the abovementioned solutions, followed by homogenization in an ice bath for 90 s (12,000 rpm), with a brief pause in the middle of the homogenization process to prevent overheating. The homogenate was centrifuged three times at 5000× *g* for 10 min. Subsequently, a fivefold volume of 10 mmol/L Tris-HCl buffer (0.6 mol/L NaCl, pH 7.0) was added and centrifuged repeatedly for 10 min at 5000× *g*. The supernatant was a myofibrillar protein extract, which was stored at −80 ◦C in a refrigerator for further use.

Protein concentration was determined by the method of Abbey et al. [18]. Standard curves were prepared by BSA (Bull Serum Albumin). Protein solution (0.05 mL) was added to 3 mL of Bradford reagent, mixed, and kept still for 10 min. The OD (optical density) value was measured by a spectrophotometer at 595 nm. At the same time, the following protein concentrations were determined by the same method.
