*2.6. Determination of Non-Covalent Bonds*

The chemical interactions or bonds were determined according to the method of Yan et al. [23] with slight modifications. Briefly, 1 g of surimi gels was added 10 mL 0.05 M NaCl (A), 0.6 M NaCl (B), 0.6 M NaCl + 1.5 M urea (C), and 0.6 M NaCl + 8 M urea (D) and homogenized for 2 min by homogenizer (Shanghai Fokker Equipment Co. Ltd., Shanghai, China). The mixtures were placed at 4 ◦C for 1 h and subsequently centrifuged at 15,000× *g* for 10 min by refrigerated centrifuge (CR21GШ, Hitachi High-Tech Co., Ltd., Shanghai, China).

The protein concentrations of supernatant collected were determined by using the Biuret method [24]. The contents of nonspecific associations (A), ionic bonds (B), hydrogen bonds (C), and hydrophobic interactions (D) were determined by the concentrations of soluble protein in supernatants.

#### *2.7. Determination of Total Sulfhydryl Groups*

The measurement of total SH content was described by Zhang et al. [25]. According to the following equation, the content of SH was calculated.

$$\text{SH groups }\upmu\text{mol/ g protein} = \frac{\text{A} \times \text{D}}{\text{c} \times \text{d} \times \text{c}} \times 10^6\tag{1}$$

In the equation, A and D represent absorbance at 412 nm and the dilution ratio, respectively; ε refers to molar extinction coefficient, 13,600 L/(mol·cm); d is the thickness of cuvette, 1 cm; and c is the protein concentration, 4 mg/mL.

#### *2.8. Fourier Transform Infrared (FT-IR) Spectroscopy*

The surimi gels were freeze-dried and ground into powder. An amount of 1.5 mg of powder was mixed with 150 mg KBr and then pressed into a transparent sheet. The spectrums of the matrix were observed by a Nicolet iS5 FT-IR spectrometer (Thermo Fisher Instruments Co., Ltd., Shanghai, China). Samples were then scanned 32 times from 400 to 4000 cm−<sup>1</sup> with a resolution of 4 cm−<sup>1</sup> [26]. Relative intensity was analyzed by OMNIC 9.2 software (Thermo Fisher Scientific Inc., Waltham, MA, USA) and PeakFit 4.12 software (Systat Software Inc., San Jose, CA, USA).
