*2.3. Carbonyl Content*

The carbonyl content was determined by following the procedures mentioned by Oliver et al. [19]. The myofibrillar protein extract was adjusted to a concentration of 5 mg/mL with phosphate buffer solution (pH 7.0), and incubated in 1 mL 0.01 mol/L 2, 4-dinitrophenylhydrazine solution at 37 ◦C for 30 min. Then, 3 mL of 20% trichloroacetic acid was added and centrifuged at 8500× *g* for 5 min. The supernatant was removed and the precipitate was washed six times with an ethyl acetate and ethanol mixture solution (1:1, *v*/*v*). Finally, the precipitate was dissolved in 5 mL of guanidine hydrochloride solution (6 mol/L) and incubated for 15 min under a 37 ◦C water bath, which was centrifuged for 10 min at 8500× *g*. Finally, the absorbance of the supernatant was measured at 370 nm. The carbonyl content was expressed as nmol carbonyl/mg protein.
