*4.1. Enhanced Freshness Preservation Performance*

Despite the many biological activities of chitosan described above, its antimicrobial and antioxidant capacities are still lacking when it is used for food preservation [73,74]. To address these issues and expand its application, many methods have been investigated to enhance its preservation performance, mainly including derivatization, chitosan nanoparticles, and combinations with natural extracts, essential oils, natural polymers, synthetic polymers, etc.

#### 4.1.1. Chitosan Derivatization

The functional groups of chitosan contain an amino group and two hydroxyl groups, which can easily react with a variety of chemical groups. Introducing new groups by modifying chitosan molecules to produce derivatives with excellent physical and chemical properties is important to expand the applications of chitosan and its derivatives [75]. Previous and current studies have shown that chitosan derivatives have positive effects in terms of antioxidant activity. Xiao et al. [76] studied the use of gallic acid–chitosan derivatives to preserve cherry tomatoes and evaluated the antioxidant and endogenous enzyme activities. The results showed that the enzyme-grafted gallic acid–chitosan derivatives had excellent antioxidant capacity in scavenging DPPH, hydroxyl, and superoxide anion radicals. This resulted in prolonged fruit ripening, reduced weight loss, high hardness, and little change in epidermal color in the treated group, and aromatic compounds remained relatively constant throughout storage due to delayed postharvest senescence.

Similarly, Neslihan et al. [77] synthesized two novel chitosan–Schiff base derivatives by condensation reactions of high- and low-molecular-weight chitosan with cotton phenol and studied their scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. They concluded that both chitosan–cotton phenol derivatives had better scavenging ability against DPPH radicals than unmodified chitosan. The modified chitosan had significantly improved antibacterial activity against foodborne bacterial spoilage. Similarly, chitosan derivatives have good film-forming and antimicrobial properties, and the use of film coating treatment has significant effects on inhibiting browning, reducing enzyme activity, maintaining fruit and vegetable quality, and prolonging freshness. Meriem et al. [78] prepared active nanocomposite films of cellulose nanocrystals reinforced with styrene-based quinoxaline-grafted chitosan by the solvent casting method and studied their antibacterial activity against five common bacteria. The films demonstrated good antibacterial activity against *Pseudomonas aeruginosa*.

Furthermore, films prepared from chitosan derivatives have different physical properties depending on the type and degree of substitution of the grafting groups. Bingnan et al. [79] found that the mechanical properties of chitosan products could be improved with a small amount of differently structured formyl saccharides by establishing intermolecular formyl–sugar bonds. When the amount of sucralfate and cottonseed aldehyde was 0.5%, the breaking strength and strain of chitosan films increased from 28 to 38 MPa and 19% to 48%, respectively, and the wet stability and toughness were improved at the same time. Currently, various chitosan derivatives have been successfully designed and applied to food preservation, as shown in Table 1. There are not many research cases of chitosan derivatives for food preservation due to the concern for their potential toxicity. Therefore, future research should not only explore more chitosan derivatives with specific and enhanced functions, but also develop green chemical processes to avoid toxic residues.

**Table 1.** Related studies on chitosan derivatives for food preservation.


#### 4.1.2. Chitosan Nanoparticles

Due to its large particle size, chitosan does not come into contact with food spoilage factors when used for food preservation, thus limiting the biological activity of chitosan alone when used for this purpose [88]. Studying chitosan nanoparticles is another efficient option to enhance the utilization of chitosan. Melo et al. [89] developed composite pectin films supplemented with copper algae mud chitosan nanoparticles and evaluated their physical and mechanical properties. The mechanical analysis of maximum stress and elongation showed that nanoparticles as fillers increased the toughness of the pectin films. Water vapor permeability tests showed that nanostructured films containing copper sulfur had better barrier properties. More studies on using chitosan nanoparticles to enhance the physical and mechanical properties of chitosan films are presented in Table 2.

Ran et al. [90] studied the use of chitosan nanoparticles for fish preservation and found that the storage life of fish fillets wrapped in composite nanofilms could be extended by 6–8 days through sensory evaluation, microbiological analysis, pH, total volatile alkaline nitrogen value, thiobarbituric acid value, color, texture, and other storage quality indicators. The plant essential oil anthocyanin composite film had the best effect on fish fillet preservation, and the anthocyanin chitosan composite nanoparticle film had the best effect on protecting fish fillet appearance. In another study, modified magnetic chitosan nanomaterials obtained by combining modified chitosan with magnetic materials retained the properties of chitosan with the strong chelating ability of metal ions and had good regenerative properties, greatly expanding the application prospects of chitosan [91].

There are several methods to prepare chitosan nanoparticles, including the ionic gel [92], microemulsion [93], emulsification solvent diffusion [94], polyelectrolyte complex [95], and reversed-phase micellar [96] methods. Chitosan nanoparticles have both the biological properties of chitosan and the small size of nanoparticles with a large contact area. The final performance of nanoparticles also depends on several other parameters, such as the composition of secondary materials in the matrix, the material concentration/ratio, and the reaction time [97]. In addition, studies have reported using different types of chitosan (varying by molecular weight, degree of deacetylation, etc.) [98,99]. The applications of chitosan-based nanoparticles are gradually increasing, and it is expected that chitosan-based nanoparticle formulations will enter the market soon.


**Table 2.** Correlation of mechanical properties of chitosan nanoparticles after film formation.

#### 4.1.3. Chitosan Plant Extract Compound

Plant extracts are often rich in low-molecular-weight bioactive ingredients, such as polyphenols, terpenoids, and terpenoids, which are considered to be powerful antibacterial and antioxidant agents [106]. Plant extracts are known not only to act as free radical scavengers in vitro but also to protect the body from free radical activity. The respiration and transpiration of fruits and vegetables during storage can cause water loss, and when water loss reaches 5%, wilting and withering can occur, which can seriously affect the edible value [107]. Table 3 summarizes some recent literature on the use of plant extracts in combination with chitosan-based coatings/films for different food products.

The use of chitosan-based composite coating and preservation solution can reduce water loss, inhibit cellular action, and prolong the storage time of fruits and vegetables. Rambabu et al. [108] investigated the application of mango leaf extract incorporated into

chitosan film to enhance the antioxidant activity and characterized the tensile strength. They found that the mango leaf extract–chitosan composite film had better tensile strength (maximum tensile strength of 23.06 ± 0.19 MPa) and less elongation compared to the pure chitosan film (18.14 ± 0.72 MPa). They also evaluated the antioxidant activity of chitosan films in terms of total phenolic content, DPPH radical scavenging ability, iron ion reducing ability, and ABTS radical scavenging ability, and the results showed that the antioxidant activity of increased with the addition of mango leaf extract. Yang et al. [109] conducted similar experiments, in which blueberry leaf extract was added to chitosan coating to maintain the postharvest quality of fresh blueberries. The composite film synthesized with chitosan and plant extracts also had UV-blocking and antioxidant abilities [110]. Wanli et al. [111] studied the process of preparing chitosan–banana peel extract composite film. Different contents of banana peel extract (4%, 8%, and 12%) were added to chitosan membranes using various characterization methods, and the experimental results showed that the chitosan–banana peel extract composite membranes had good antioxidant activity in different food samples.

Plant extracts, when synergized with chitosan, usually have an inhibitory effect on bacterial growth, which is related to metabolic disorders caused in bacteria through disruption of cell membranes, enzyme systems, or genetic material. Ali et al. [112] investigated chicken meat coated with chitosan and containing 1% essential oil of oregano and 1% or 2% grape seed extract stored in the refrigerator. On the 20th day of refrigeration, the minimum viable count of each treatment organism (Enterobacteriaceae, *Pseudomonas* spp., *Lactobacillus*, and *Saccharomyces* (yeast-mold)) was 3.54–4.51 log CFU/mL. Compared with the untreated group, the results indicated that the compound coating was effective in retarding microbial growth and oxidative activity. At present, most of the plant extracts compounded with chitosan are from easily available and nontoxic substances, such as herbs and spices, and they are used in food preservation by obtaining their essential oils, which have active antioxidant and antibacterial active properties when compounded with chitosan. However, there are still problems, such as the complex extraction process, low efficiency, and the effect on the sensory quality of food. Therefore, the extraction and bio-preservation ability of plant extracts still need to be explored in the future in order to provide better technical support for the development of food preservation.


**Table 3.** Studies on chitosan–plant extract complexes for food coatings.

#### *4.2. Freshness Preservation Technology*

In recent years, the preparation of chitosan-based preservation films has become increasingly standardized, and the growing demand for food preservation has greatly contributed to the development of chitosan biofilm preservation technology. In terms of operation, traditional coating and film-making methods for food preservation have been widely used and have shown reliable results. The thickness of chitosan films is usually in the range of 6–80 μm, with an average of 25 μm, as reported by Elena et al. [120]. In addition, emerging technologies, such as film production from plant extracts, by lamination with plastic films, and by laminated layer self-assembly (LbL), are emerging. Both direct coating and bioactive packaging film preservation methods are systematically discussed at a technical level below.

#### 4.2.1. Coating Preservation

Coating preservation refers to covering the food surface with a layer of chitosan solution, which can usually be applied by dip coating, electrostatic spraying, or brushing. Due to its outstanding advantages of convenience and operability, it is often used in food preservation research. For example, Yong et al. [121,122] used electrostatic spraying of chitosan solution to preserve strawberries, which prolonged their shelf life and slowed down the degradation of quality. Rui et al. [123] investigated the potential application of chitosan as a natural growth regulator for bean sprouts by dipping bean sprouts in different concentrations of chitosan solution. The results showed a significant increase in the hypocotyl length and fresh weight of bean sprouts compared to the blank control. In addition, chitosan solution combined with plant extracts have been used as dip coatings to retard the quality deterioration of fruits, vegetables, meat, etc. Mehran et al. [124] applied chitosan solution mixed with Berberis extract and peppermint essential oil to turkey breast meat, and microbial counts and oxidation levels were significantly reduced under refrigerated conditions.

Usually, chitosan-based dip-coating treatment consists of the following steps: First, a pre-determined concentration of chitosan solution is prepared, and then the food is immersed in the solution for a pre-determined time, depending on the characteristics of the food. Finally, the food is removed, the excess solution adhering to the surface is drained, and the product is dried in a specific airflow environment. Regarding other coating methods, all steps are similar to dip coating except for the intermediate steps. Sometimes, repeated coating is applied to enhance preservation during storage [125].

Compared to other coating methods, it is obvious that dip coating is easy to operate under simple conditions. However, it is also prone to problems with dilution and contamination in practical applications. In addition, gravity tends to cause uneven film thickness on the food surface and reduces productivity to some extent [126]. The preservation substrate determines the thickness of the coating, the equipment requirements, and the drying technology used. In addition, self-healing coatings are intelligent materials that can repair coating damage and restore its properties, reducing or eliminating the adverse effects of damage. As a method of fabricating multilayer coatings, laminated layer self-assembly (LbL) is often used to construct self-healing coatings. Yu et al. [127] investigated a self-healing coating by assembling chitosan (CS) with sodium alginate (SA) layer-by-layer, and the damaged area could be completely repaired after three assembly cycles. The self-healing schematic is shown in Figure 4. The mechanical properties, water barrier, and oxygen barrier of the repaired coating were 97%, 95%, and 63% of those of the intact coating, respectively, which basically restored the barrier properties of the intact coating, although the permeability increased. In addition, the coating reduced the effect of coating damage by restoring the barrier properties of the coating and extending the shelf life of the strawberries. Thus, it can be seen that chitosan coating not only slows down food quality degradation, but also demonstrates efficient antioxidant and antibacterial effects. However, the uniformity of covering food and rough sensory effects need to be further improved. Therefore, the application of chitosan coating for freshness is worth further study, as it has the potential to become a low-cost alternative technology for fresh food preservation.

**Figure 4.** Diagram of self-healing behavior of chitosan (CS)/sodium alginate (SA) coating.

#### 4.2.2. Film-Making and Preservation

Bioactive chitosan packaging films are made by co-blending chitosan solution with bioactive materials to make cling film in which to wrap food and provide freshness. Cling films can be produced in advance in large quantities without the condition of preserved products. They can be broadly divided into two types according to the composite materials: packaging film based on chitosan solution and hybrid film prepared in combination with food-grade plastic film.

Chitosan-based biofilms are often prepared by co-blending with extracts derived from natural plants, which are of increasing interest to the food industry because of their ecosafety and rich nutritional value. The addition of active ingredients not only enhanced the inherent bioactivity of the films, but also improved the final mechanical properties and applicability of the films. For example, Zhang et al. [128] improved the mechanical properties of chitosan films by vanillin modification and showed that the film stretch of vanillin/chitosan composite films increased by 53.3% and moisture permeability decreased by 36.5% compared to pure chitosan films. Ting et al. [129] added spice extracts to chitosan films to extend the shelf life of frozen pork. The data showed that chitosan could interact intermolecularly with the polyphenols in the spices and also caused the formation of covalent bonds between the hydrogen and water molecules of the polyphenols, increasing the hydrophilicity of the composite films. In addition, plant essential oil, an aromatic oily liquid extracted from plant tissues and organs, can not only scavenge free radicals in the body and perform antioxidant functions, but also has the functions of regulating the balance of intestinal flora, killing pathogenic bacteria, and promoting the secretion of digestive juices, which is a plant extract with more applications at present. When the plant essential oil is compounded with a chitosan solution in a certain ratio, the prepared chitosan film will also have the relevant nutritional properties of plant extracts, and the plant extracts can function at low concentrations. The selective gas (CO2 and O2) permeability and good mechanical properties of chitosan make it an excellent film-forming material. However, the low water barrier properties of chitosan films limit their application, as effective control of water transfer is a desirable property for most food packaging, especially in humid environments [130]. Several studies have focused on the moisture permeability of chitosan films containing essential oils or other plant extracts. López et al. [131] found that the addition of carvacrol (0.5, 1.0, and 1.5% *v*/*v*) significantly reduced the moisture permeability of chitosan films. When essential oils or other plant extracts (tea tree, carvacrol, cinnamon, turmeric, etc.) were added to chitosan films, the moisture permeability was significantly reduced, which may be due to the hydrophobicity of the essential oil particles. It can be seen that various properties of the film or coating can be modified by adding essential oils, and chitosan with essential oils has been shown to increase hydrophobicity, reduce water vapor permeability, and improve the antimicrobial activity of the film.

For the preparation of multilayer or hybrid structured packaging films in combination with food-grade plastic films, synthetic plastics and biodegradable polymers provide sufficient mechanical strength to meet the packaging requirements and are widely used matrix materials [132]. With regard to structured packaging films, the physical and mechanical properties depend heavily on the mixing state and compatibility between the constituent components. Shun et al. [133] prepared chitosan–polylactic acid plastic films by an extrusion method and used them to preserve grouper fillets, then analyzed their physical properties and antibacterial activity. The results showed that the water vapor transmission rate and water content of the plastic films was increased, and inhibition of *Escherichia coli*, *Pseudomonas fluorescens*, and *Staphylococcus aureus* reached over 95%. For hybrid structured packaging films, the thermal stability of the active component is a key factor to consider. Sudharsan et al. [133] prepared a compatible PLA/chitosan composite film with a thickness of 0.25 mm by the solvent casting method, and it demonstrated good thermal stability, low oxygen permeability, and high mechanical properties. Although all the above studies are still at the experimental stage and still some distance from commercialization, it is

believed that there will be a positive development trend of bioactive chitosan packaging films through the continuous development of film making technology.
