*2.5. Total Sulfhydryl and Disulfide Bond Content*

Total sulfhydryl and disulfide bond content was evaluated according to the method of Benjakul et al. [21]. One milliliter of myofibrillar protein solution (4 mg/mL) was added to 9 mL 0.2 mol/L Tris-HCl buffer (pH 6.8, 8 mol/L urea, 2% SDS, and 10 mmol/L EDTA (Ethylene Diamine Tetraacetic Acid)). Then, 0.4 mL 5,5-dithio-bis (2-nitrobenzoic acid) (0.1%) was added to the resulting mixture and incubated for 25 min at 40 ◦C. The absorbance was measured at 412 nm. At the same time, 0.6 mol/L KCl as a blank was also subjected to this step. The extinction coefficient of 13,600 mol−<sup>1</sup> cm−<sup>1</sup> was used to calculate the total -SH group content. The content of the disulfide bond was calculated according to the method described by Thannhauser et al. [22].
