*2.8. Myofibril Fragmentation Index*

The method of Culler et al. was used to evaluate the MFI [26]. Briefly, the concentration of protein solution was adjusted to 0.5 mg/mL; then, the absorbance was measured at 540 nm, and the MFI value was equal to the OD value multiplied by 200.

#### *2.9. Free Amino Acid Content*

Samples of free amino acid extracts were prepared according to the method of Yu et al. [27]. Mobile phase 1 of an automatic amino acid analyzer (L-8800, Hitachi Co. Ltd., Tokyo, Japan) consisted of the buffer of sodium citrate and citric acid; the pH of the mixed buffers were 3.2, 3.3, 4.0, and 4.9, respectively. Mobile phase 2 was prepared by 4% ninhydrin (*v*/*v*). The test parameters were as follows: column (4.6 × 150 mm, 7 μm); column temperature (50 ◦C); channels 1 and 2 flow rates (0.4 mL/min and 0.35 mL/min, respectively).

#### *2.10. FTIR Measure*

The grouper fillets were powdered with KBr after being freeze-dried (MINFAST04, TIANLI Executive and Administration Management, Beijing, China); then, the mixed sample was pressed into flakes. An FTIR (Nicolet iS5, Thermo Scientific Inc, Waltham, MA, USA) spectrometer was used for the measurements. Infrared spectra were recorded with 32 scans in the 400–4000 cm−<sup>1</sup> range with a resolution of 4 cm−1; also, the operating environment was set at 25 ◦C. The recorded spectra were analyzed by Omnic professional software (Omnic professional, v 9.2, Thermo Nicole Inc., Waltham, MA, USA), and Gaussian fitting was used to analyze the second-derivative spectrum in the range of 1600–1700 cm−<sup>1</sup> by PeakFit software (v 412, Systat Software Inc., San Jose, CA, USA).
