Water- and salt-soluble proteins were extracted from the mackerel muscle according to Yang et al. [23]. First, 5 g of mackerel muscle was mixed with 20 mL of precooled 20 mmol/L Tris-maleate solution (50 mmol/L NaCl, pH 7.0). The mixture was homogenized for 45 s on ice and centrifuged for 10 min at 4 °C with a centrifugal force of 8000× g. The supernatant was used for the measurement of water-soluble sarcoplasmic protein. Afterward, 20 mL of precooled 20 mmol/L Tris-maleate solution (0.6 mol/L NaCl, pH 7.0) was added to the precipitate, and the mixture was homogenized for 45 s on ice. After 15 min centrifugation at 8000× g at 4 °C, the supernatant was collected as the salt-soluble myofibrillar protein solution for subsequent experiments. The contents of water- and salt-soluble protein in the supernatant were measured using the method of Bradford, and bovine serum albumin (BSA) was used as the standard.

According to the previous method of Ganhao et al. [30], 1 mL of 2.4-dinitrophenol hydrazine (DNPH, 10 mmol/L in 2 N HCl) was mixed with 1 mL of the salt-soluble protein solution, which was incubated in a 30 °C water bath for 1 h in darkness. Then, the protein in the mixture was precipitated by the addition of 1 mL of trichloroacetic acid. After 5 min centrifugation at 8000× g, the precipitate was washed three times using 1 mL ethanol–ethyl acetate (1:1, v/v) solution to remove the redundant DNPH. Finally, the precipitate was suspended in 3.5 mL guanidine hydrochloride (6 mol/L) and kept at 37 °C for 15 min. After 3 min centrifugation at 10,000× g, the absorbance of the supernatant was measured at 370 nm to calculate the content of carbonyl groups expressed as μmol of per gram of protein.

The total SH content was determined according to Shi’s method [25] with some modifications. First, 9 mL tris-HCl buffer (0.05 mol/L with 8 mol/L urea, 10 mmol/L ethylenediamine tetra acetic acid (EDTA, pH 6.8) was added to 1 mL myofibrillar protein solution, which was incubated at 4 °C for 1 h. Then, 4 mL of the mixed solution was sampled and mixed with 400 μL Ellman’s reagent (DTNB). After incubating for 25 min at 40 °C, the absorbance of the solution was measured with a wavelength of 412 nm, and the total SH content was calculated as μmol per gram protein with 13,600 M−1 cm−1 as the molar extinction coefficient.

The content of the disulfide bond was calculated by the total SH and free SH content. The free SH content was measured using the same method of total SH except for the tris-HCl buffer, which was substituted by an equal volume of buffer without urea (0.05 mol/L tris-HCl with 10 mM EDTA, pH 6.8). The disulfide bond was calculated with the following equation:

Changes of surface hydrophobicity were determined according to the methodology of Kobayashi et al. [31]. For this experiment, 8-Anilinonaphthalene-1-sulfonic acid (ANS, 8 mmol/L in 0.1 mol/L PBS buffer, pH 7.0) was used as the fluorescent probe. Myofibrillar protein solutions with gradient concentrations of 0.05, 0.10, 0.15, and 0.2 mg/mL were prepared by the dilution of stock solution. Then, 4 mL of the diluted myofibrillar solution was mixed with 10 μL ANS. The fluorescence intensity of the mixture was determined by an F-2700 Fluorescence Spectrophotometer (Hitachi, Tokyo, Japan) using a 390 nm excitation wavelength and 470 nm emission wavelength. S0-ANS was calculated based on the slope of the curve between fluorescence intensity and protein concentration.

Freeze-dried myofibrillar protein was used for the FTIR analysis (Frontier, PerkinElmer, Norwalk, CT, USA). The sample was mixed with KBr (1:100), and the mixture was ground into a uniform powder and pressed into a transparent sheet. Under transmittance mode, all spectra were collected in the wavelength range of 4000 to 400 cm−1 with the resolution of 4 cm−1 at room temperature. Omnic software was used for basic spectrum analysis. The data of the amide I bands were fitted to a Gaussian curve by Peakfit 4.12 software (Seasolve Software, Inc. Framingham, LA, USA). The secondary structure contents of each component were calculated from the peak area.

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