**1. Introduction**

Surimi is a reconstituted food used for the production of various seafood dishes, such as fish balls, cakes, and croquettes [1,2]. Among all ingredients of the fish muscle, myofibrillar protein (MP), a salt-soluble protein, is mainly responsible for surimi functionalities [3]. Like other uncured meats, the shelf life of surimi is largely depending on frozen storage [3–5], which restrains intrinsic enzyme activities, microbial growth, and lipid oxidation of surimi during long-term preservation [6,7]. However, undesirable quality deteriorations still occur (e.g., water loss, structural weakening, and nutrition decay), and most of them are related to MP denaturation [8], which owes to the effect of ice crystallization, protein concentration and oxidation [9].

**Citation:** Zhu, X.; Zhu, M.; He, D.; Li, X.; Shi, L.; Wang, L.; Xu, J.; Zheng, Y.; Yin, T. Cryoprotective Roles of Carboxymethyl Chitosan during the Frozen Storage of Surimi: Protein Structures, Gel Behaviors and Edible Qualities. *Foods* **2022**, *11*, 356. https://doi.org/10.3390/ foods11030356

Academic Editors: Jianhua Xie, Yanjun Zhang and Hansong Yu

Received: 24 December 2021 Accepted: 24 January 2022 Published: 26 January 2022

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To address those problems, appropriate uses of food-grade cryoprotectants are necessary [10]. Given the unexpected taste or high caloric values of commercial cryoprotectants, such as sugars, polyols, and phosphates [11], research interest shifted to other natural additives derived from food proteins and saccharides. Among which, oligosaccharides, i.e., carrageenan oligomer [12], cellobiose [13], and konjac oligo-glucomannan [14] etc., have drawn particular attentions owing to their pronounced health benefits and consumer acceptability [15].

Oligosaccharides function as cryoprotectants through: (1) inhibiting ice recrystallization in frozen surimi and reducing physical damages to MP structures [9], (2) forming saccharide-MP binary complex to prevent MP from freezing-induced aggregation [16]. It is noteworthy that both ice-inhibition and complexation effects of saccharides were dependent on their strong polarities [9,17]. Therefore, some intrinsic structural characteristics, such as the charge properties, of exogenous oligosaccharides are believed to govern their cryoprotective effects. For example, Zhang et al. [18,19] reported that alginate oligosaccharide and trehalose could prevent the freezing denaturation of myosin. Though both saccharides exhibited the same degree of polymerization (DP = 2n), the charged alginate oligomer had stronger protecting effect than the noncharged trehalose.

Chitosan is an acid-soluble cationic polysaccharide that has been widely used as food additives [20,21]. To improve its solubility in water, carboxymethylation is mostly carried out [22]. The derived carboxymethyl chitosan (CMCh, Figure S1) is a typical ampholytic polysaccharide [23,24]. It demonstrates outstanding moisture-retention capacity [22], which is the key to regulate the water state and ice crystallization at subzero temperatures. Furthermore, the ampholytic structure of saccharide was also reported to be effective in modulating the protein aggregation [25]. On the basis of these observations, the ampholytic CMCh was a potential cryoprotectant to proteins. However, the development of CMChbased additives remained less in frozen food when compared with other charged or neutral saccharides, such as chitosan (+), carrageenan (−), and konjac glucomannan (nonionic) [26,27]. Thus, the objective of this study is to: (1) investigate how the ampholytic structure of CMCh influences its cryoprotective effect to frozen surimi proteins, and (2) evaluate the edible properties of obtained surimi gels.

Since the functional properties of charged saccharides are highly related to their charge densities [25,26], CMCh with different degrees of carboxymethyl substitution (DS) were selected as cryoprotectants in this study, which were hydrolyzed to oligosaccharides and added to surimi before freezing. The storage stability of MP was thoroughly characterized, while the gelling behaviors and edible qualities of frozen surimi were also evaluated, including the rheological properties, microstructures, mechanical strength, water-holding capacities, sensory scores, and whiteness. This work aims at a paradigm shift for the development of ampholytic oligosaccharides as high-performance cryoprotectants for aquatic food preservations.

#### **2. Materials and Methods**

#### *2.1. Materials*

Alive farm raised-silver carp (Hypophthalmichthys molitrix, weighing 2.0 ± 0.1 kg) were purchased from a local market (Wuhan, China), transported to laboratory within 15 min, and subjected to percussive stunning. Carboxymethyl chitosan with two different DS, CMCh-A and -B, were kindly donated by Haobo biotech Corp (Henan, China). The DS values of CMCh-A and -B were characterized to be 0.8 and 1.2 by using the potentiometric titration [28]. Both CMCh exhibited similar molecular weight (Mw) at about 6.02 × 104 Da. Chitosanase, with an activity unit at 200,000 U/g, was purchased from Shengda biotech Corp (Henan, China). All other chemicals were purchased from Aladdin Bio-chem Technology Co., LTD (Shanghai, China). Ultrapure water was used in all experiments unless specified otherwise.

#### *2.2. Preparation of CMCh Oligosaccharide*

The CMCh oligosaccharide (CMCO) was prepared according to previous studies with some modifications [29,30]. Briefly, 500 mL of CMCh solution (10 mg/mL) was prepared at pH 5, and an aliquot of 60 mg chitosanase was added into the solution. The hydrolysis reaction was carried out at 50 ◦C for 5 h and terminated by heating the mixture at 100 ◦C for 10 min. The obtained solution was neutralized, filtered, and concentrated with a vacuum rotary evaporator (IkA-Works Inc., Staufen, Germany). Then, the hydrolysate was precipitated by mixing with 10 volumes of ethanol. After being redissolved in the water, the solution was filtered through a polymer membrane (Mw cut-off: 10 KDa) and the filtrates were lyophilized. The average DP for both CMCO-A and CMCO-B was characterized to be approximately 6.8, as reflected by the contents of reducing sugars.
