*3.1. Bacteriostatic Properties*

The broad-spectrum bacterial inhibitory properties of chitosan have been confirmed by in vitro experiments and complex food antimicrobial assays [29–32]. In the process of food preservation, the use of chitosan coating or film covering the surface of food can, to a certain extent, resist microbial attack, slow down food spoilage, and thus extend the shelf life of food. For example, Wei et al. [33] studied the effects of different concentrations of chitosan on the color and surface microbial populations of frozen duck skin. The results showed that chitosan could form oxygenation competition with color-presenting substances, and 3% chitosan could effectively reduce the total number of surface colonies of duck skin during storage. In addition to the effect of chitosan concentration on the inhibition effect, molecular weight, degree of deacetylation, and bacterial species have also been shown to have an effect. Benhabiles et al. [6] studied the effect of molecular weight and degree of

deacetylation on the inhibition effect, comparing the inhibition performance of chitosan on four Gram-positive and seven Gram-negative bacteria. The results showed that chitosan with low molecular weight and a high degree of deacetylation had a better inhibition effect, and among the tested species, chitosan had an inhibition effect on all bacteria except *Salmonella typhimurium*. The pH of the solution and the presence of metal ions also have an effect on the bacterial inhibitory effect of chitosan. Jing et al. [34] used ANOVA to test the data and concluded that the inhibitory activity of chitosan increased with decreasing pH in the range of >4 and <6. Comparing the effects of metal ions on the inhibitory activity of chitosan at a pH of 4, the results showed that Ca2+ and Mg2+ significantly reduced the inhibitory ability against Gram-negative bacteria. It can be seen that the inhibitory activity of chitosan is related to a variety of factors, with interlocking effects among them, and the best inhibition results can be obtained only by comprehensively analyzing all of the factors.

Although the information on the antimicrobial activity of chitosan is controversial, it is generally accepted that yeasts and mycobacteria are the most sensitive organisms to chitosan, followed by Gram-positive and Gram-negative bacteria [35]. The antifungal activity of chitosan is mainly attributed to the inhibition of mycelial growth and spore germination [36]. Early studies showed that chitosan inhibits spore germination, budding tube elongation, and radial growth by sequestering metals, minerals, trace elements, or nutrients required for fungal growth [37]. The inhibition of pathogenic fungi increases with increased chitosan concentration [38], but its application is hindered by low solubility at physiological pH due to its low charge density. Therefore, increasing the positive charge density of chitosan may be the most effective way to improve its solubility and antifungal activity [39]. The antifungal activity of chitosan also depends on its molecular weight [40], which affects the morphogenesis of the fungal cell wall [41]. It has also been shown that chitosan's inhibition of pathogenic fungi in fruits may be related to its ability to induce increased production of, e.g., polyphenol oxidase and peroxidase [42].

As research progressed, hypotheses on the mechanism of chitosan inhibition were put forward, but the exact mechanism of inhibition has still not been determined. Feng et al. [43] measured the OD of cell contents under UV light at a wavelength of 260 nm and showed an increase in OD. Using transmission electron microscopy to observe changes in the ultrastructure of *E. coli* and *S. aureus* before and after the action of chitosan, it was observed that the cell membrane was broken, accompanied by a large amount of content spillage (DNA and mRNA) at the periphery and bacterial apoptosis. Similarly, Hui et al. [44] evaluated the antibacterial activity of chitosan acetate solution against *E. coli* and *S. aureus*. The morphology of bacteria after chitosan treatment was observed by transmission electron microscopy. The integrity of the cell membranes of both bacteria and the permeability of the inner and outer membranes of *E. coli* were investigated by measuring the absorption values at 260 nm UV, fluorescence changes in cells treated with the fluorescent probe 1-N-phenylnaphthylamine, and the release of cytoplasmic h-galactosidase activity. The results showed that chitosan ultimately damaged the bacteria by increasing the permeability of the inner and outer cell membranes, releasing the cell contents. This damage is most likely caused by electrostatic interactions between the NH3+ group in the chitosan acetate solution and the phosphoryl group of the phospholipid fraction of the cell membrane. Imelda et al. [29] confirmed the disruptive effect of chitosan on protein synthesis by β-galactosidase expression assay.

The above studies showed that chitosan can inhibit bacteria by disrupting bacterial cell membranes and protein synthesis. Ming et al. [45] pointed out that chitosan has high chelating ability for various metal ions (including Ni2+, Zn2+, Co2+, Fe2+, Mg2+, and Cu2+) under acidic conditions. As a chelating agent, chitosan selectively chelates metal ions, thus playing a key role in microbial growth by inhibiting the growth and reproduction of microorganisms. Gooday et al. [46] pointed out that when chitosan crosses the cell wall of fungal pathogens with plant hydrolases as host, it penetrates the nucleus of the fungus, and the positively charged chitosan interacts with the negatively charged DNA, affecting RNA transcription and protein synthesis, thus achieving fungal inhibition.

In summary, there are three generally accepted theories about the mechanism of chitosan inhibition, the first of which is inter-charge interactions. As shown in Figure 3 [17], the electrostatic interaction between R-NH3+ of chitosan and negatively charged groups on the bacterial surface destabilizes the structure of the cell membrane, and the leakage of intracellular material leads to microbial death. The second theory concerns the chelating property of chitosan with metal ions. Chitosan chelates metal ions on the surface of bacteria, forming a microbially unavailable chelate, thus inhibiting microbial growth. The third theory holds that chitosan enters the cell and binds to DNA, and affects RNA transcription and protein expression, thus achieving the effect of bacterial inhibition. Although the above views are generally accepted, there are still many scholars who believe that the molecular weight and degree of deacetylation, and the differences between bacterial species, should also be considered when investigating the inhibition mechanism of chitosan.

**Figure 3.** Mechanisms used to explain antibacterial activity of chitosan.
