3.1.2. Ca2+-ATPase Activity

Ca2+-ATPase activity is an important indicator to measure the structural integrity of MP in fish muscle [8]. The protein conformations can be changed along with the breakdown of intermolecular noncovalent bonding during storage, resulting in the decrease of Ca2+- ATPase activity [35]. Figure 1b showed that the initial Ca2+-ATPase activity of MP of all frozen surimi samples were about at 0.45 μmol pi/mg.min before freezing and decreased to different degrees as storage time extended. The Ca2+-ATPase activity of the control group experienced the most significant decrease (~60%) to 0.17 μmol pi/mg.min at day 60. In consistent with the results of MP solubility (Section 3.1), the use of cryoprotectants (i.e., CMCO-A/-B and the commercial additive) inhibited the protein denaturation. Specifically, the values of Ca2+-ATPase activity of the CMCO-A (0.23 μmol pi/mg.min) and -B (0.28 μmol pi/mg.min) were significantly higher than that of the control group (0.17 μmol pi/mg.min) at day 60, even though they also decreased by 44.4% and 33.3%, respectively, during storage. Meanwhile, the CMCO-B group achieved comparable 60-day Ca2+-ATPase activity (i.e., 0.28 vs. 0.30 μmol pi/mg.min) to the commercial group. A similar cryoprotective effect of pullulan was also reported by Jiang et al. [37].

#### 3.1.3. Surface Hydrophobicity and Sulfhydryl Content

Variations of surface hydrophobicity reflect conformational changes of MP at different physical and chemical states. A reduced surface hydrophobicity suggests that the protein was less denatured (unfolded), and less hydrophobic groups were exposed, thus limiting the protein binding with the fluorescence probe [7]. As depicted in Figure 2a, surface hydrophobicity of the control group increased significantly from 3000 to 8800 after 60 days of storage. This phenomenon was reasonably due to the freezing-induced MP denaturation. Ice crystals that formed in surimi destroyed the hydration layer around the peptides, which resulted in more exposed hydrophobic amino acid residues and higher protein surface hydrophobicity [7]. In the presence of CMCO-A, -B and commercial additive, the surface hydrophobicity of MP was lower than that of the control group, suggesting their

cryoprotective effects on protein structures. CMCO, an ampholytic saccharide with strong polarity, may interact with proteins and increase their hydrophilicities. Consequently, undesirable protein aggregation was inhibited as evidenced by the reduced hydrophobic interaction [38].

**Figure 2.** Effects of CMCO on surface hydrophobicity (**a**) and sulfhydryl content (**b**) of MP at different storage time.

To further elaborate the storage stability of MPs, the contents of their sulfhydryl groups were measured in the presence of different cryoprotectants (Figure 2b). During the 60 days of storage, the sulfhydryl content of the control group decreased significantly from 6.22 to 4.08 mol/10<sup>5</sup> g, while the addition of CMCO-A and B mitigated such decrease with the final sulfhydryl contents of 4.31 mol/10<sup>5</sup> g and 4.73 mol/10<sup>5</sup> g, respectively. Moreover, the CMCO-B group can achieve similar sulfhydryl contents of MP to the commercial group (4.69 mol/105 g). The sulfhydryl group is chemically reactive. It could be easily oxidized to form disulfide bonds under long-term frozen storage [33], leading to serious protein aggregation, denaturation, and deterioration of food quality [39]. Owing to the strong reducing power of oligosaccharide, CMCO could inhibit the oxidation and conversion of SH to disulfide bond, thus maintaining the storage stability of MP [14].

#### 3.1.4. Intrinsic Fluorescence Intensity

The intrinsic fluorescence intensity (FI) of MP in frozen surimi was characterized by using fluorescence spectroscopy. It reflected the changes of chemical environment of tryptophan (Trp) residues, which suggested the variation of protein tertiary structures [40]. As shown in Figure 3, the FI of all MP samples was similar at Day 0, and decreased as far as the storage period elapsed, indicating the exposure of Trp residue towards solvent [41]. These results agreed with findings about the structural integrity of MP molecules (Ca2+- ATPase activity in Figure 1b), which also experienced significant decreases as the tertiary structure of MP became collapsed during long-term frozen storage [41]. In the presence of different cryoprotectants, reduction of FI became less obvious compared to the control group, and relatively higher FI was observed for the CMCO-B group than the CMCO-A during the entire storage period. A similar charge-dependent cryoprotective behavior of ampholetic saccharides was also observed in our recent study [20], in which CMCh could stabilize the microstructures of wheat gluten in frozen dough, and the protective effect was more obvious as DS of CMCh increased.

**Figure 3.** Effects of CMCO on fluorescence intensity of MP at different storage time: (**a**) day 0, (**b**) day 15, (**c**) day 30, and (**d**) day 60.

#### *3.2. Effect of CMCO on Gel Behaviors of Frozen Surimi*

3.2.1. Effect of CMCO on the Microstructure of Gels Prepared from Frozen Surimi

A porous protein network is formed during the two-stage thermal processing of surimi, which determines many important quality attributes of the gel products, such as texture, shelf life and digestion etc. [42,43]. Therefore, SEM was applied to observe the microstructure of gels prepared from frozen surimi. As shown in Figure 4, all gel samples of fresh surimi exhibited reticular and continuous protein network. After frozen storage, the gel matrix became loose for the control group, seen from the increase of heterogenic pore sizes. In contrast, the addition of cryoprotectants alleviated the breakdown of honeycomblike network of surimi gels, which were beneficial to the mechanical strength of gel matrix. Besides, a more continuous and ordered protein architecture was observed for surimi gel of the CMCO-B group than that of the CMCO-A. A similar phenomenon was also reported by Tan et al. [13], who investigated the gel morphology of frozen surimi treated by cellulosic oligosaccharide.

**Figure 4.** Microstructure of the gels prepared from frozen surimi with different cryoprotectants before and after storage for 60 days.

#### 3.2.2. Effect of CMCO on the Rheological Properties of Surimi Gels

The rheological properties of surimi gels also changed significantly as the MP became denatured during frozen storage. As shown in Figure 5a,b, the elastic moduli (G') of all surimi gels were higher than those of the viscous moduli (G") at both day 0 and day 60, indicating their solidlike behaviors. To better illustrate the gel characteristics, loss tangents (tan δ) of surimi gels during frequency sweep were recorded (Figure 5c). The reduced elasticities were observed for all groups of surimi gels, as evidenced by their ever-increased tan δ (i.e., less elastic) after freezing storage. These results were correlated with the formed heterogeneous gel networks prepared from the stored surimi (Figure 4). In the cases of cryoprotected surimi, their gel structures were maintained, thus exhibiting lower tan δ compared to the control. After 60 days, the lowest tan δ was obtained for the CMCO-B group at 0.16. These observations were in consistent with the effect of antifreeze proteins on rheological properties of the frozen surimi [33,42].

**Figure 5.** Dynamic rheological properties of the gels prepared from frozen surimi before (**a**) and after (**b**) frozen storage, and (**c**) tan δ of surimi gel within the linear viscoelastic region. Values with different uppercase letters indicate statistically significant difference among samples with different cryoprotectants at the same storage time (*p* < 0.05). Values with different lowercase letters indicate significant difference among samples with the same cryoprotectants at different storage time (*p* < 0.05).

#### 3.2.3. Effect of CMCO on the Gel Strength and Water-Holding Capacity

Gel weakening is one of the most typical quality deteriorations for surimi products, which occurs during the whole process of cold chain transportation, storage and communications [44,45]. Thus, the gel strengths of frozen surimi with different cryoprotectants were characterized (Figure 6a). Few significant differences were observed among the samples at

day 0, and the weakening effect was observed as storage time prolonged. After 60 days, gel strength of the control group exhibited the most significant decrease from 288 to 158 g·cm. The weakening effect was suppressed in the presence of cryoprotectants, and the highest gel strength of 219 g·cm was obtained for the CMCO-B group. As previously discussed (Section 3.1), introduction of CMCO to frozen surimi may preserve the MP integrity (solubility, Ca2+-ATPase, sulphydryl, and intrinsic FI), which stabilized protein networks and enhanced mechanical strengths of surimi gels.

In addition to the gel strength, WHC is another important parameter to evaluate edible qualities of gel-type foods. As shown in Figure 6b, WHC of all surimi gels kept decreasing during storage, which could be owing to the muscle filament contraction and protein denaturation/ tertiary structural changes [5]. In the presence of different cryoprotectants, such reduction of WHC became less obvious. After 60 days, the CMCO-A, CMCO-B, and commercial groups exhibited significantly higher WHC (79.20%, 85.70%, and 86.50%, respectively) than the control group (68.50%). The improved WHC may be due to the cryoprotective effect of CMCO to the gel structures. The intrinsic ampholytic characteristics of CMCO may endow the saccharide superior hydrophilicity, which helped reduce the ice crystallization, improve the gelling property and finally enhance the WHC [12]. In consistent with the results of mechanical strength, as the DS of CMCO increased, the WHC of surimi gels were enhanced (CMCO-B > CMCO-A).

**Figure 6.** Effects of CMCO on the mechanical strength (**a**) and water-holding capacity (**b**) of the gels prepared from frozen surimi at different storage time. Values with different uppercase letters indicate statistically significant difference among samples with different cryoprotectants at the same storage time (*p* < 0.05). Values with different lowercase letters indicate significant difference among samples with the same cryoprotectants at different storage time (*p* < 0.05).

Collectively, the cryoprotective mechanism of CMCO can be described from molecular perspective (Figure 7). CMCO could interact with MP through both hydrogen bonding and electrostatic complexation, which replaces the water molecules at MP surface, and prevents the freezing-induced protein aggregation [41,42]. In addition, the ampholytic structure of oligosaccharide could entrap the water, modulate the growth of ice crystals, and stabilize MP structures [46]. Thus, after thermal processing, the gel of cryoprotected surimi exhibited more organized microstructures and improved mechanical properties. It is also noteworthy that the inhibitory effect of saccharide to ice crystallization may also account for the cryoprotective behaviors of CMCO to frozen surimi [47–49], and in most cases, both the protein-stabilization and ice-inhibition effects worked concurrently.

**Figure 7.** Schematic representation of the cryoprotective mechanism of CMCO for MP in frozen surimi.

#### *3.3. Effect of CMCO on the Whiteness and Sensory Quality of Frozen Surimi*

Decreased whiteness of gel products made from frozen surimi is a common problem impairing their appearance and edible qualities. As depicted in Figure 8, the whiteness value of the control group kept decreasing from 0 to 60 days. As expected, the addition of cryoprotectants could limit the loss of whiteness of surimi gels, whose values of CMCO-A, -B, and commercial group experienced no significant difference during the entire storage period. Similar phenomenon was also reported by Tao and Walayat [5,8] who attributed the decline of gel whiteness to the combined effects of nonenzymatic protein oxidation and crystallization-induced protein denaturation. The addition of oligosaccharides could prevent those protein deteriorations and thus maintain the whiteness of gels prepared from frozen surimi [50].

**Figure 8.** Effects of the CMCO on whiteness of the gels prepared from frozen surimi at different storage time.

Sensory evaluations of all surimi gels were performed by panelists (Table 1). The average sensory scores of surimi gels enhanced in terms of flavor, taste, juiciness, texture, and color through the addition of cryoprotectants. Moreover, gels of the CMCO-B group exhibited slightly higher overall acceptability than that of the CMCO-A and commercial group. In agreement with the results of gel strength, and whiteness (Figure 6), the CMCO-B could act as a high-performance cryoprotectant to improve the edible quality of the frozen stored surimi.

**Table 1.** Effect of CMCO on sensory attributes of the gels prepared from frozen surimi after 60 days of storage.


Different letters indicate statistically significant differences among samples with different cryoprotectants (*p* < 0.05).

#### **4. Conclusions**

The present study designated the cryoprotective effects of ampholetic oligosaccharide (CMCO) on the storage stability of frozen surimi. An addition of 0.6% (*w*/*w*) CMCO can alleviate the denaturation of MP in the frozen surimi during 60 days of storage at −18 ◦C as indicated by their increased salt-protein solubility, Ca2+-ATPase activity, and sulfhydryl content. The CMCO-protected MP experienced less conformation changes (reflected by the surface hydrophobicity and FI) than the nonprotected control. Accordingly, the obtained surimi gels demonstrated significantly improved elasticity, mechanical strength, and WHC, and had more uniform microstructure upon thermal processing. Moreover, the cryoprotective effect of CMCO-B (DS: 1.2) was more pronounced than that of CMCO-A (DS of 0.8). The gel strength and WHC of the CMCO-B group were comparable to those of the commercial counterpart (4% sucrose and 4% sorbitol). Results of sensory evaluation showed that CMCO addition also improved the taste, smell, texture, juiciness, whiteness, and overall acceptability of gels prepared from frozen surimi. Findings from this study deepen the scientific insights of ampholytic saccharides as high-performance cryoprotectants in food industry.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/foods11030356/s1, Figure S1: The chemical structure of carboxymethyl chitosan; Figure S2: A schematic demonstration to the processing of surimi.

**Author Contributions:** Conceptualization, X.Z. and M.Z.; methodology, M.Z., X.L. and D.H.; validation, L.W., T.Y. and Y.Z.; investigation, X.Z., X.L., L.S. and M.Z.; resources, T.Y. and L.S.; data curation, D.H., X.Z. and M.Z.; writing—original draft preparation, X.Z., D.H. and X.L. writing review and editing, X.Z., T.Y., Y.Z. and J.X.; visualization, X.L. and M.Z.; project administration, X.Z. and T.Y.; funding acquisition: X.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by National Natural Science Foundation of China (31871870), Hubei Provincial Natural Science Foundation of China (Grant No. 2021CFB283) and Hubei University of Technology (BSQD-2020037, XBTK-2020001).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** This work is financially supported by National Natural Science Foundation of China (31871870), Hubei Provincial Natural Science Foundation of China (Grant No. 2021CFB283), Doctoral start-up fund (BSQD-2020037) and Collaborative Grant-in-Aid of HBUT National "111" center for cellular Regulation and Molecular Pharmaceutics (XBTK-2020001) of Hubei University of Technology. The authors would like to thank Yunyun Zou from the shiyanjia lab (www.shiyanjia.com) for the fluorescence test.

**Conflicts of Interest:** The authors declare no conflict of interest.
