*3.2. Antioxidant Properties*

The antioxidant properties of chitosan have been demonstrated [10,47,48]. It is well known that oxidation is one of the most important issues affecting the quality of meat, and heterocyclic amines, which are generated during the high-temperature processing of foods containing proteins, are carcinogenic substances. Many factors influence the formation of heterocyclic amines, mainly cooking methods, processing conditions (time, temperature), and the presence of antioxidant substances. Therefore, reducing heterocyclic amines by natural compounds and reducing or inhibiting the formation of these carcinogens in cooked meat by plant extracts containing antioxidants have become hot research topics [49].

When chitosan is added to meat products as a food additive, chitosan concentration and temperature are important factors that affect the experimental results. Fatih et al. [50] investigated the effect of adding different concentrations of chitosan (0.25, 0.50, 0.75, 1% *w*/*w*)

to meatballs cooked at different temperatures on the formation of heterocyclic aromatic amines and the quality of the meatballs. The results showed that the heterocyclic amine content of the meatballs increased with increasing temperature (150, 200, and 250 ◦C), and the heterocyclic amine content showed a decreasing trend with increased chitosan concentration at the same temperature. Similarly, a study by Hojat et al. [51] showed that the addition of chitosan to huso fillets for cooking was effective in reducing heterocyclic amine production. Given that the production of heterocyclic amines usually requires hightemperature conditions, chitosan coatings or films have been less studied for reducing the production of heterocyclic amines. Kader et al. [52] studied four types of chitosan coating with different degrees of deacetylation and molecular weights for cherry preservation and evaluated the changes in total phenolic content, antioxidant capacity, total anthocyanin content, ascorbic acid, total pectin content, hardness, and color of cherries. The results showed that the antioxidant capacity of chitosan and the ascorbic acid content in cherries had a trend of increasing and then decreasing with the degree of deacetylation and molecular weight, and the chitosan with 81.22% deacetylation and 273 kDa molecular weight at 4 ◦C showed the best antioxidant capacity.

Relatively stable DPPH radicals have been widely used to test the ability of compounds to act as free radical scavengers or hydrogen donors [53]. The IC50 value is the concentration at which 50% free radical scavenging is obtained, and the lower the IC50, the more active the sample is as an antioxidant compound and the greater its ability to absorb free radicals. Rainy et al. [54] investigated the antioxidant properties of edible chitosan–galactose complex coating by compounding chitosan and galactose (0, 0.5, 1, and 1.5 g), and performed an in vitro test to evaluate the coating and analyze the parameters of antioxidant activity (DPPH method). The results showed a decreasing trend in the IC50 values, followed by an increasing trend. The lowest IC50 value of 43.20 ppm was recorded for the combination of chitosan and 1 g of galactose, which was the best antioxidant in the tested treatments. Similarly, Zhang et al. [55] reported the results of free radical scavenging experiments with different molecular weight chitosan, showing that high-molecular-weight chitosan had a high scavenging effect on hydroxyl radicals, and low-molecular-weight chitosan had a better scavenging effect on superoxide anion radicals and DPPH.

Giuseppina et al. [10] studied the effect of chitosan-based coatings on the freshness of figs by assessing the activities of enzymes, such as catalase, ascorbate peroxidase, polyphenol oxidase, and peroxidase. The results showed that the addition of chitosan coating significantly increased the total polyphenol, anthocyanin, and flavonoid contents and the antioxidant activity of stored figs, reduced oxidative stress, and prevented browning reactions compared with the untreated group. Miriam et al. [56] studied the effect of nano-chitosan/propolis coating on the shelf life and antioxidant capacity of strawberries, and the results showed higher total phenol and flavonoid content and antioxidant capacity of strawberries in the nano-chitosan/propolis coated group than in the untreated group at the end of the storage period. Chitosan delays the ripening and aging process of food, which extends the shelf life; largely maintains all sensory qualities of food; reduces enzymatic browning; decreases water loss; maintains the bright color, taste, and texture of food; and makes the aroma more durable, thus effectively maintaining the commercial value of food. There are many factors involved in the antioxidant performance of chitosan, and the main research concerns are concentration, degree of deacetylation, molecular weight, and antioxidant enzymes.

As the antioxidant mechanism of chitosan has been investigated, hypotheses have been proposed. Chitosan films in combination with other plant-based flavonoids have antioxidant properties as coatings on food surfaces [57]. Yuntao et al. [13] showed that high-molecular-weight chitosan films have a denser structure and better antibacterial properties. Braber et al. [58] concluded that chitosan biopolymers scavenge free radicals or chelate metal ions through a hydrogen or lone pair electron donor mechanism. The -OH and -NH2 groups in chitosan are the key functional groups for its antioxidant activity [59]. Zhang et al. [60] analyzed the barrier properties of chitosan–cyanidin films and

concluded that the increased hydrogen bonding between cyanidin and chitosan molecules leads to a tighter arrangement between molecules inside the film, which improves its gas barrier properties.

The comprehensive analysis of the above studies led to three conjectures regarding chitosan's antioxidant properties. The first is the barrier effect: chitosan coating can act as a barrier on the fruit surface, changing the internal gas atmosphere, reducing water loss, and delaying ripening. Second, many oxidation processes are carried out with the participation of metal ions, which play a role in transferring electrons during valence changes, and chitosan removes metal ions through chelation, thereby inhibiting oxidation reactions. Third, high-molecular-weight chitosan has denser intramolecular hydrogen bonds, which further prevents oxidation reactions from occurring when food comes into contact with air.

#### *3.3. Enzyme Activity Inhibition*

Enzymatic reactions play a non-negligible role in the softening and browning of foods. Tissue browning is inevitable in some fruits and vegetables with damaged surfaces due to the action of polyphenol oxidase. He et al. [18] studied the effect of clove oil–chitosan coating on the quality of fresh-cut lemons at four temperatures of 0, 4, 7, and 10 ◦C. By analyzing the changes in peroxidase, polyphenol oxidase, and phenolic acids, it was concluded that the chitosan coating had a stronger inhibitory effect on enzyme activity as the temperature decreased. Seafood is also highly susceptible to deterioration due to enzymatic reactions, which is the main reason for its quality decline [17].

Chitosan concentration is another important factor influencing enzyme activity. A-Dan et al. [61] investigated the effect of chitosan on papain and showed that with increased chitosan concentration, there was an inhibitory effect on papain when the concentration was greater than about 8.0288 g/L. Arisa et al. [62] investigated the effect of chitosan coating with different molecular weights at a concentration of 1% on the physicochemical properties of red bananas. The results showed that chitosan coating was able to reduce fruit respiration rate, ethylene production, and pectin hardness by inhibiting the activity of cell wall degrading enzymes (polygalacturonase and pectin lyase) that are important in pectin degradation, and with increasing molecular weight, chitosan coating showed better inhibition of enzymatic activity, thus delaying banana spoilage and deterioration. Although the mechanism by which chitosan inhibits the browning of fruits and vegetables and softening of meat tissues has not been elaborated, it must be related to the inhibition of enzymes associated with the occurrence of enzymatic reactions in foods.

With the study of the mechanism of chitosan's enzymatic activity inhibition, related hypotheses have been proposed. Kurita et al. [63] indicated that polycations may compete with divalent metals, such as Mg2+ and Ca2+, present in the cell wall, thus disrupting the integrity of the cell wall or affecting the activity of degradative enzymes. To resist local browning in fruits and vegetables, chitosan binds suspended polyphenol oxidase molecules through electrostatic interactions to inactivate the enzyme, and the inactivated polyphenol oxidase is unable to transfer electrons directly to molecular oxygen, thus acting as an anti-browning agent [56]. Riaz et al. [64] studied the effect of chitosan-based apple peel polyphenol composite coating on improving the storage quality of strawberries. The antioxidant content of the coated fruits was relatively stable compared to uncoated fruits. The reduced antioxidant activity during storage may lead to decay and senescence. The ability of chitosan-based apple peel polyphenol composite coating to reduce decay, decrease enzymatic activity, and retain the quality attributes of fruits, leading to the degradation of antioxidant compounds, is closely related to the presence of efficient oxygen radical scavengers [65]. Yage et al. [66] used chitosan/nano-titanium dioxide coating to induce film formation on the surface of mangoes, creating a microenvironment that reduced decay and water loss and delayed respiratory peaks to preserve fruit flavor.

In summary, the hypotheses on the mechanism of action of chitosan's enzyme activity inhibition include the following: First, the chelation of metal ions disrupts the enzyme activation pathway, and calpain and matrix metalloproteinases are Ca2+ dependent endogenous enzymes. Second, chitosan molecules penetrate the sarcoplasm and bind directly to the enzyme, thereby affecting enzyme activity. Third, chitosan may bind to structural proteins, which can affect the degradation and dissociation of enzyme proteins. Fourth, the chitosan coating alters the microenvironment of the sample, thus synthetically modulating the biochemical characteristics of the preserved food. Unfortunately, there are not enough relevant studies, and more research is needed to develop systematic interpretations of these hypotheses.

#### *3.4. Biodegradability*

Biodegradable polymer materials can be degraded by microorganisms or their secretions under the action of enzymes or chemical decomposition with a certain time and under certain conditions [67]. The degradation of biodegradable materials is generally accompanied by changes in their chemical and physical properties, reduced molecular weight, and the production of low-molecular-weight substances (CO2, H2O, CH4). Ruchir et al. [20] reported that chitosan coatings are environmentally friendly, biodegradable, and in most cases edible. Aris et al. [19] pointed out that although polymers synthesized with chitosan cannot be completely degraded, mixing chitosan with synthetic polymers can improve the decomposition rate of plastics that are more difficult to decompose. Polyvinyl alcohol (PVA), which is nontoxic and water-soluble, is one of the most commonly used synthetic polymers for chitosan. Yueming Li et al. [68] studied the effect of biodegradable, antibacterial chitosan starch composite film on the freshness of red grapes. The results showed that the biodegradable film had good water retention and antimicrobial efficacy and could be applied to grape preservation.

Zhang et al. [69] observed a link between degradation rate and molecular weight, degree of deacetylation, and distribution of N-acetyl D-glucosamine residues. Chitosan is a semi-crystalline polymer, and the relationship between chitosan biodegradability and the degree of deacetylation also depends on the degree of crystallinity, which reaches a maximum at a deacetylation degree of 0% (in the form of titin) or 100% (fully deacetylated chitosan) and decreases at intermediate values, with the rate of biodegradation increasing as the degree of crystallinity decreases. Similarly, Croisier et al. [70] found that acetyl residues distributed along the chitosan chain also affect the crystallinity of chitosan and, thus, the biodegradation rate. It can be concluded that smaller chitosan chains biodegrade more efficiently than higher-molecular-weight chitosan. In vivo, chitosan can be degraded by several enzymes, including lysozyme, a nonspecific enzyme present in all mammalian tissues. The degradation products are nontoxic oligosaccharides, and in vitro degradation of chitosan occurs by oxidative, chemical, or enzymatic hydrolysis. These methods are commonly used for low-molecular-weight chitosan prepared under controlled conditions.

In addition to being a polymer with an amino group, chitosan is also a polysaccharide and therefore contains readily breakable glycosidic bonds. Chitosan appears to be degraded in vivo by nonspecific enzymes, but mainly lysozyme has been reported to have this property. The biodegradation of chitosan leads to the formation of nontoxic oligosaccharides. These oligosaccharides, with variable lengths, can potentially be incorporated into metabolic pathways or excreted from the body [71,72]. Therefore, it is clear that chitosan is a strong natural candidate to replace nondegradable plastics in the future and its position as a natural alternative is established. Based on the current research, the relative application of chitosan in food packaging will be developed and commercialized shortly.
