*2.3. Preparation of Frozen Surimi*

The frozen surimi was prepared according to Zhangt et al. [31]. The fish was killed and its head, scales and internal organs were removed. Fresh fish meat was cut and washed three times by five volumes of distilled ice water. Then, the surimi was dispersed in 0.5% NaCl solution at the ratio of 1:2 (*w*/*v*) and dehydrated with nylon net to reach a final moisture content at 78%. The protein content in surimi was determined to be 15.2% through the Kjeldahl method. The obtained surimi was added with CMCO-A (0.6%), CMCO-B (0.6%) or commercial cryoprotectant (4% sucrose and 4% sorbitol), respectively, and packaged in polyethylene bags with the size of 20 × <sup>15</sup> × 2 cm3. Surimi without any cryoprotectant added was set as a blank control. All samples were frozen at −18 ◦C in air (with a freezing rate of 1.2 ◦C/min) and conditioned overnight to achieve uniform initial temperatures. Then, the surimi was transferred to a freezer and stored at −18 ◦C for 0, 10, 20, 30, 45 and 60 days. The frozen surimi was thawed at 4 ◦C for 20 h before further analysis.

#### *2.4. Characterizations of MP in Frozen Surimi*

#### 2.4.1. Content of Salt-Soluble Protein

MP of the surimi was prepared by following the processes of Lin et al. with modifications [7]. Frozen surimi (5 g) was mixed with 45 mL of 0.6 M KCl solution (pH 7.0) and homogenized at 9000 rpm for 100 s (an operation of 20 s with an interval of 15 s). The homogenate was conditioned in an ice water bath for 30 s and centrifuged at 9000 rpm, 4 ◦C for 20 min. The supernatant was collected and mixed with three volumes of cold deionized water followed by centrifugation at 6000 rpm, 4 ◦C for 20 min. The precipitate was re-suspended in 20 mL of 0.6 M KCl solution and centrifuged again at 6000 rpm, 4 ◦C for 20 min. Finally, content of salt-soluble protein in the supernatant was determined by the Biuret method.
