2.4.2. Ca2+-ATPase Activity

Ca2+-ATPase activity was determined according to Liu et al. [14]. Briefly, the Ca2+- ATPase assay was performed at 37 ◦C for 10 min in a reaction medium that consisted of 0.5 M KCl, 5 mM CaCl2, 25 mM Tris-maleate (pH 7.0), 6 mg/mL MP, and 1 mM ATP. The reaction was terminated by adding HClO4 at 5% final concentration. The mixture was centrifuged at 9000 rpm for 10 min, and the released inorganic phosphate in the supernatant was measured by using phosphomolybdate method. The activity of Ca2+- ATPase of MP was expressed as Pi amount per mg of MP per reaction time liberation min−<sup>1</sup> (μmol/mg/min)<sup>−</sup>1.

#### 2.4.3. Surface Hydrophobicity

Surface hydrophobicity of MP from the frozen surimi was characterized by using 8-anilino-1-naphthalenesulfonic acid (ANS) as the fluorescence probe according to Lin et al. with modifications [7]. The MP solution was diluted with 0.6 M KCl-10 mM phosphate buffer (pH 6.0) to achieve a series of protein concentrations of 0.2, 0.3, 0.5 and 1.0 mg/mL, and 20 μL of 8 mM ANS-0.1 M phosphate buffer (pH 7.0) was added to 1 mL of the prepared protein solution. Fluorescence intensity (FI) of the mixture was measured immediately by using a fluorophotometer (F-4600, Hitiachi High Technologies Co., Tokyo, Japan) with excitation wavelength at 390 nm and emission wavelength at 420 nm. The recorded FI vs. the concentration of MP (mg/mL) was plotted, and the initial slope was used as the index of protein surface hydrophobicity.

#### 2.4.4. Concentration of Sulfhydryl Group

The concentration of sulfhydryl (–SH) group was determined via DTNB (5,5- -Dithiobis-2-nitrobenzoic acid) assay as described by Jiang [32]. In brief, 1 mL of MP solution (4 mg/mL) was mixed with 9 mL buffer A (8 mol/L urea, 2% SDS and 10 mmol/L EDTA at pH 8.0), and 4 mL of the mixture was mixed with 0.5 mL buffer B (10 mM DTNB and 0.2 M Tris-HCl, pH 8.0). After incubation at 40 ◦C for 25 min, absorbance of the solution was determined at 412 nm. The concentration of –SH group was calculated as Equation (1):

$$\mathbf{x} = \frac{\mathbf{A} \times \mathbf{D}}{\mathbf{C} \times \mathbf{B}} \tag{1}$$

where *x* represents the SH content (mol/g); D is the dilution factor; A is the absorbance of the mixture solution; C is the molar extinction (13,600 mol−1·cm−1·L); B is the protein concentration (mg/mL).

#### 2.4.5. Intrinsic Fluorescence Intensity

Fluorescence intensity (FI) of MPs was measured with a fluorescence spectrophotometer (F-4600, Hitiachi High Technologies Co.) according to the method described by Walayat et al. [33]. Previously prepared MP solution (Section 2.3) was diluted to 0.05 mg/mL by using 0.6 M NaCl solution. The scan wavelength was in the range of 300 to 450 nm, with the scanning speed at 1000 nm/min, and the excitation wavelength was 295 nm with both excitation and emission slits at 10 nm.

#### *2.5. Preparation of Surimi Gels*

Surimi gel was prepared according to the method described by Tao et al. [8]. Frozen surimi was thawed at 4 ◦C, chopped for 5 min, mixed with 2.5% NaCl addition followed by chopping for another 5 min. The obtained surimi sol was then stuffed into a plastic polyvinylidene case and conditioned at 40 ◦C for 60 min followed by heating at 90 ◦C for 30 min (Figure S2). The obtained heat-set surimi gels were cooled in ice water for 30 min and stored at 4 ◦C overnight before further analysis.

#### *2.6. Characterizations of Surimi Gels*

#### 2.6.1. Scanning Electron Microscopy

Scanning electron microscopy (SEM) was used to evaluate the microstructures of surimi gels. A cubic Surimi gel (3 × <sup>3</sup> × 3 mm3) was conditioned in 2% glutaraldehyde at 4 ◦C for 18 h and dehydrated with a series of concentrations of ethanol (50, 70, 80, 90, and 100%), followed by lyophilization. Then, gel samples were mounted on an aluminum stub, sputter coated with gold, and observed by SEM (Hitachi S–3500 N, Hitachi, Japan) at an accelerating voltage of 10 kV.

#### 2.6.2. Rheology Test

Small amplitude oscillatory strain (SAOS) tests were performed with a Physica controlled stress rheometer (MCR-301, Anton Paar, Graz, Austria). A small piece of surimi gel was loaded between two parallel plates (20 mm diameter and 1 mm gap). Disk-shaped samples were mounted on the lower plate of the rheometer at 20.0 ± 0.1 ◦C and allowed to rest for 15 min before analyses. Then, a frequency sweep test (0.1–100 Hz) was carried out to evaluate the rheological variations of the gels prepared from surimi stored for different periods of time. The strain was fixed at 0.5% which was within the viscoelastic region

(LVR), and the storage modulus (G- ) and loss modulus (G--) were recorded. Values of tan δ were expressed as G--/Gat the testing frequency of 1 Hz and the temperature of 20 ◦C.

#### 2.6.3. Gel Strength

Gel strength was characterized on a TA. XT. plus Texture analyzer (SMS, Surrey, UK) according to the method of Liu et al. [14]. Surimi gels were cut into 25 mm high cylindrical shape slices. A slice was horizontally placed on the platform and penetrated by a spherical probe (type P/0.25). The testing speed of probe was 1 mm/s, the trigger point load was 0.1 N, and the depth of probe penetration was 10 mm. Gel strength (g/cm) was calculated by multiplying the breaking force (g) by penetration distance (mm).

#### 2.6.4. Water-Holding Capacity (WHC)

WHC of surimi gels was measured according to the method described by Li et al. with minor modifications [4]. Surimi gels were cut into the thickness of 2.5 mm and centrifuged for 10 min at 1000 g and 4 ◦C, and the released moisture was absorbed by filter paper. The weight of the gels was recorded before (W1) and after centrifugation (W2). WHC was calculated as Equation (2):

$$\text{WHC} \left( \% \right) = \left( \text{W1} - \text{W2} \right) / \text{W1} \times 100\% \tag{2}$$

#### 2.6.5. Whiteness

The whiteness of surimi gel was determined with a colorimeter (CR-400, Konica Minolta, Osaka, Japan) by measuring L\* (lightness), a\* (redness/greenness) and b\* (yellowness/blueness) values. Whiteness was calculated as Equation (3):

$$\text{Whiteness} = 100 - 100 - \text{L}^\* \text{}^2 + \text{a}^{\*2} + \text{b}^{\*2} \text{l}^{1/2} \tag{3}$$

## 2.6.6. Sensory Assessment of Surimi Gels

Surimi gels (prepared in Section 2.5) were cut into square pieces (1 × 1 × 2 cm) for sensory assessment. The evaluation panelist was composed of 20 trained individuals, and the whole evaluation process was performed in a professional sensory evaluation laboratory at room temperature. A five-point hedonic score was recorded for each surimi gel, ranging from 5 (extremely like) to 1 (extremely dislike) in terms of the taste, smell, juiciness, color, texture and overall acceptability of the cooked surimi gel.

#### *2.7. Statistical Analysis*

All characterizations were repeated three times, and the reported results were shown as the mean ± standard deviation. Significant difference among treatments was analyzed by using SPSS (Version 13.0 SPSS, Chicago, IL, USA) through analysis of variance (ANOVA) with Duncan's test. Statistical significance was accepted at *p* < 0.05.

#### **3. Results and Discussions**
