Modified polyvinyl alcohol-chitosan composite dip-coating liquid, composite antibacterial film as well as preparation method and application of modified polyvinyl alcohol-chitosan composite dip-coating liquid and composite antibacterial film

Abstract

The invention relates to the technical field of packaging and antibacterial materials, and particularly discloses a modified polyvinyl alcohol-chitosan composite dip-coating liquid, a composite antibacterial film as well as a preparation method and application of the modified polyvinyl alcohol-chitosan composite dip-coating liquid and the composite antibacterial film. According to the composite antibacterial film, lactic acid esterification modified polyvinyl alcohol and chitosan are used as main components, solution blending is performed, glycerin is added for plastification, and then drying is performed to form the film; the optimal addition amount of lactic acid in the modified polyvinyl alcohol is 20%, and the modified polyvinyl alcohol is mixed with the chitosan solution according to the mass ratio of 3: 1. The composite antibacterial film provided by the invention utilizes the hydrophobicity and interfacial compatibility enhanced by modified polyvinyl alcohol to generate a synergistic antibacterial effect with chitosan, can effectively destroy the cell membrane integrity of penicillium italicum and inhibit mycelial growth and spore germination, has excellent moisture resistance, mechanical properties and biodegradability, and can be widely applied to the field of biomedical materials. The coating is suitable for green fresh-keeping coatings of perishable foods such as citrus fruits and the like.

Description

Technical Field

[0001] This invention relates to the field of packaging and antibacterial materials technology, specifically to a modified polyvinyl alcohol-chitosan composite coating solution, a composite antibacterial film, its preparation method, and its application. Background Technology

[0002] Food is a vital material foundation for human survival, and its quality and safety are increasingly valued by consumers. However, food is naturally perishable and highly susceptible to contamination and spoilage by fungi or bacteria in the environment during post-harvest storage and transportation. Among these, Penicillium italicum (…) is particularly prone to contamination. Penicillium italicum Penicillium mold, caused by mold, is the most prevalent postharvest disease of citrus fruits, often resulting in significant economic losses. Chemical fungicides were once the primary control method, but they pose risks such as drug residues, pathogen resistance, and environmental pollution. Against this backdrop, green and environmentally friendly coating technologies have gained widespread attention due to their environmentally friendly characteristics. These coatings, based on polymers (such as polysaccharides and proteins), are applied to food surfaces through soaking, brushing, or spraying to form a protective film. This film not only provides excellent gas barrier properties, delaying spoilage, but also acts as a physical barrier against microbial invasion.

[0003] Among numerous natural film-forming materials, chitosan is considered a promising edible coating matrix due to its excellent film-forming properties, biodegradability, and broad-spectrum antibacterial activity. However, pure chitosan materials have inherent defects such as poor mechanical properties and insufficient hydrophobicity, which make them prone to structural damage in high-humidity environments, severely limiting their practical applications. Summary of the Invention

[0004] This invention provides a modified polyvinyl alcohol-chitosan composite coating solution, a composite antibacterial film, its preparation method, and its application. The composite antibacterial film provided by this invention utilizes the enhanced hydrophobicity and interfacial compatibility of modified polyvinyl alcohol to produce a synergistic antibacterial effect with chitosan. It can effectively disrupt the cell membrane integrity of *Penicillium italicum*, inhibiting hyphal growth and spore germination. Simultaneously, it possesses excellent moisture resistance, mechanical properties, and biodegradability, making it suitable for a green preservation coating for perishable foods such as citrus fruits.

[0005] This invention provides a modified polyvinyl alcohol-chitosan composite coating solution, characterized in that it is prepared by blending modified polyvinyl alcohol and chitosan solution; the modified polyvinyl alcohol is obtained by esterification reaction of polyvinyl alcohol and lactic acid, and the dosage is specified.

[0006] The composite antibacterial film provided by this invention utilizes the enhanced hydrophobicity and interfacial compatibility of modified PVA to produce a synergistic antibacterial effect with chitosan. It can effectively disrupt the cell membrane integrity of Penicillium italicum, inhibit hyphal growth and spore germination, and at the same time has excellent moisture resistance, mechanical properties and biodegradability. It is suitable for green preservation coatings for perishable foods such as citrus fruits.

[0007] Furthermore, the mass ratio of lactic acid to polyvinyl alcohol is 0.15 to 0.9:3.

[0008] Furthermore, glycerol is added as a plasticizer during the preparation of the modified polyvinyl alcohol-chitosan composite coating solution.

[0009] The present invention also provides a composite antibacterial film, which is obtained by drying the modified polyvinyl alcohol-chitosan composite coating solution.

[0010] Furthermore, it includes the following steps: Polyvinyl alcohol was dissolved in deionized water, and lactic acid was added dropwise at 34℃~36℃. The mixture was stirred at a constant temperature for 14 h~18 h to obtain a modified polyvinyl alcohol solution. Chitosan powder was dissolved in a 1% (w / w) aqueous solution of glacial acetic acid and stirred at 58℃~62℃ until completely dissolved to obtain a chitosan solution. A modified polyvinyl alcohol solution and a chitosan solution were mixed, glycerol was added, and the mixture was stirred until homogeneous to obtain a modified polyvinyl alcohol-chitosan composite coating solution; subsequently, the solution was cast and dried to form a film, thus obtaining the composite antibacterial film.

[0011] Furthermore, the volume ratio of the modified polyvinyl alcohol solution to the chitosan solution is 1~3:1~3.

[0012] Furthermore, the chitosan solution contains 1% to 5% by mass.

[0013] Furthermore, the mass-to-volume ratio of the glycerol to the modified polyvinyl alcohol-chitosan solution is 0.0525 g to 0.0675 g: 60 mL.

[0014] This invention also provides a modified polyvinyl alcohol-chitosan composite coating solution or a composite antibacterial film for inhibiting Penicillium italicum (…). Penicillium italicum Applications during growth.

[0015] Furthermore, the modified polyvinyl alcohol-chitosan composite coating solution or composite antibacterial film is used to inhibit the growth of Penicillium italicum colonies, inhibit spore germination, and exert antibacterial effects by disrupting the integrity of mycelial cell membranes.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Significantly improved physical barrier properties: The composite antibacterial film prepared by this invention introduces hydrophobic groups into the molecular chain through the esterification reaction of lactic acid and PVA, resulting in a denser microstructure and a higher water contact angle. It exhibits excellent water vapor barrier properties and moisture resistance, and can maintain structural integrity and functional stability in high humidity environments. It is not prone to moisture absorption and swelling, thus ensuring the durability and reliability of its function as a barrier for food preservation packaging, overcoming the defect of poor hydrophobicity of pure PVA materials.

[0017] 2. Synergistic Enhancement of Antibacterial Properties: Due to the introduction of lactic acid-modified PVA, the composite antibacterial film prepared in this invention has a much better inhibitory effect on Penicillium italicum than the simple unmodified PVA and chitosan blend film (the volume ratio of unmodified PVA solution to chitosan solution is 3:1). The mechanism of action is that the modified PVA enhances the adsorption capacity of the composite antibacterial film surface to bacterial cells, and synergizes with the cationic antibacterial effect of chitosan to jointly destroy the integrity of the mycelial cell membrane, leading to leakage of cell contents and effectively inhibiting spore germination.

[0018] 3. Optimized material mechanical properties and compatibility: The lactic acid modification in this invention not only improves the hydrophobicity of PVA, but also enhances its compatibility with chitosan molecules, enabling the two to form a uniform and stable three-dimensional network structure through interactions such as hydrogen bonds. This structure gives the composite antibacterial film both good flexibility and mechanical strength, overcoming the shortcomings of pure chitosan film being brittle and simple blend film having poor compatibility, making it more suitable for actual coating operations.

[0019] 4. Green, Safe, and Multifunctional: All components of the composite antibacterial film of this invention (chitosan, PVA, lactic acid, and glycerol) are edible or biodegradable materials, making it environmentally friendly and in line with the development trend of green packaging. This composite film combines excellent antibacterial, hydrophobic, and mechanical properties, effectively targeting Penicillium italicum and providing a reference material solution for the preservation of other perishable fruits and vegetables, with broad application prospects. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The effect of different amounts of lactic acid added on the hydrophobicity of modified PVA films.

[0022] Figure 2The effect of different ratios of modified PVA solution and chitosan solution on the hydrophobicity and mechanical properties of the composite antibacterial membrane is shown in the figure. In the figure, a is the water contact angle of the composite antibacterial membrane, b is the tensile stress-strain curve of the composite antibacterial membrane, and c is the elongation at break-tensile strength of the composite antibacterial membrane.

[0023] Figure 3 The inhibitory effect of different addition amounts of modified PVA-chitosan composite coating solution on Penicillium italicum.

[0024] Figure 4 The effect of different amounts of glacial acetic acid added on the diameter of Penicillium italicum colonies.

[0025] Figure 5 To compare the antibacterial effects of modified PVA-chitosan composite impregnation solution and PVA-chitosan composite impregnation solution.

[0026] Figure 6 The effect of modified PVA-chitosan composite coating solution on the cell membrane of Penicillium italicum.

[0027] Figure 7 The effect of modified PVA-chitosan composite coating solution on the germination of Penicillium italicum spores. Detailed Implementation

[0028] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0029] Example 1: A modified polyvinyl alcohol-chitosan composite impregnation solution, a composite antibacterial film, its preparation method and application.

[0030] I. Experimental Methods 1. Preparation of modified polyvinyl alcohol (PVA) Accurately weigh 3 g of PVA (PVA1788 type, Maclean, Shandong Keyuan Biochemical Co., Ltd., Lot C17312034) and dissolve it in 60 mL of deionized water. Add the PVA powder in steps, stirring briefly at 300 rpm for 30 s after each addition of 0.3 g of PVA powder. Continue adding the powder only after the PVA powder has been completely soaked. After the addition is complete, mechanically stir at 300 rpm in a 90℃ constant temperature water bath until the PVA is completely dissolved to obtain a 5% (w / v) transparent homogeneous solution, which is the PVA solution. The system temperature was stabilized at 35℃±1℃. Lactic acid (MW=90.08, ≥85%, with added amounts of 0.15 g, 0.3 g, 0.6 g, and 0.9 g, corresponding to 5%, 10%, 20%, and 30% lactic acid additions, respectively) was slowly added dropwise to the PVA solution using a constant-pressure dropping funnel. The dropping rate was controlled at approximately 0.5 mL / min to ensure uniform dispersion of the lactic acid. After the addition was complete, the reaction was mechanically stirred continuously at 35℃ for 16 h to allow the lactic acid molecules to fully esterify with the hydroxyl groups on the PVA molecular chains, obtaining a modified PVA solution. 30 mL of each of the obtained PVA solution and the modified PVA solution were transferred to 9 cm diameter plastic petri dish molds and placed in a 40℃ forced-air drying oven for 15 h to form a transparent film with a thickness of approximately 0.25 mm, obtaining the PVA film and the modified PVA film. The dried PVA film and the modified PVA film were sealed and stored at 4℃ for later use.

[0031] The role of this hydrophobic modification is to fundamentally reduce the hydrophilicity of PVA, laying the material foundation for the subsequent preparation of hydrophobic composite films. At the same time, the introduced hydrophobic segments help improve the compatibility with chitosan and promote the formation of a denser and more stable network structure.

[0032] 2. Preparation of chitosan solution Accurately weigh 3 g of chitosan powder (Shanghai Lanji Technology Development Co., Ltd., Lot 240731) and slowly add it to 100 mL of 1% glacial acetic acid (analytical grade, Tianjin Aopusheng Chemical Co., Ltd., Lot 20230308) aqueous solution (acetic acid volume / deionized water volume). After all the material has been added, in a 60℃ constant temperature water bath, adjust the magnetic stirring speed to 400 rpm and stir continuously for 2 h. The solution gradually changes from an initial turbid suspension to a transparent, viscous, homogeneous solution, thus obtaining a chitosan solution. Transfer 30 mL of the chitosan solution to a 9 cm diameter plastic petri dish mold and place it in a 40℃ forced-air drying oven for 15 h to form a film, resulting in a transparent film with a thickness of approximately 0.25 mm. Obtain the chitosan film, seal and store at 4℃ for later use.

[0033] 3. Solution Complexation and Film Formation Modified PVA solution and chitosan solution were mixed at volume ratios of 1:3, 1:2, 1:1, 2:1, and 3:1, respectively, resulting in a total volume of 60 mL. The mixture was stirred at 400 rpm for 4 h using a magnetic stirrer. Subsequently, 0.0525 g, 0.055 g, 0.06 g, 0.065 g, and 0.0675 g of glycerol were added as plasticizers, and the mixture was stirred for 12 h until homogeneous, yielding a modified PVA-chitosan composite coating solution. Finally, 30 mL of the modified PVA-chitosan composite coating solution was transferred to a 9 cm diameter plastic petri dish mold and placed in a 40°C forced-air drying oven for 15 h to form a film, thus obtaining the composite antibacterial film.

[0034] The purpose of different mixing ratios is to optimize the ratio of the two components so that, while ensuring the antibacterial function of chitosan, the modified PVA can maximize its role in enhancing mechanical properties and hydrophobicity, thereby achieving the best balance and synergistic effect in performance.

[0035] 4. Water contact angle and tensile test Water contact angle tests were conducted on PVA films with different lactic acid additions (using an optical contact angle meter, CA200, Guangdong Beidou Precision Instruments Co., Ltd.). The water contact angle was determined by measuring the static contact angle of a 2 μL water droplet. The optimal amount of lactic acid was then determined based on the water contact angle (with an unmodified PVA film at 0% lactic acid addition serving as a control group). Based on these optimized conditions, water contact angle and tensile tests were performed on composite antibacterial films prepared with different ratios of modified PVA solution to chitosan solution. The tensile strength and elongation at break of each film were tested using a tensile testing machine (CMT6502, Shenzhen Shengsiyiwei Technology Co., Ltd.). The films were cut into strips (7 mm × 40 mm), with an initial clamping distance of 20 mm and a testing speed of 10 mm / min. The optimal ratio of modified PVA solution to chitosan solution was determined based on the test results.

[0036] 5. Antibacterial performance test Italian Penicillium strain ( P.italicumThe lyophilized powder (strain number AS3.4040) was purchased from the Shanghai Microbiological Culture Collection Center (accession number D10954, this invention does not involve strain preservation). The lyophilized powder was dissolved and added to a test tube containing potato dextrose agar (PDA, Beijing Luqiao Technology Co., Ltd.). After culturing at 28°C for 7 days, one test tube was randomly selected, and mycelia on the surface of the culture medium were scraped off using a sterile inoculation loop. The mycelia were inoculated onto PDA medium and cultured in a constant temperature incubator at 28°C for 7 days. The *Penicillium italicum* cultured on PDA for 7 days was gently scraped off using a sterile inoculation loop and rinsed 5 times with sterile distilled water containing 0.05% (V / V) Tween-80. The rinse solution was collected, filtered through 4 layers of sterile gauze to remove mycelia, and then shaken for 1 min using a mini vortex mixer (L-VM-MINI, Beijing Lanjieke Technology Co., Ltd.). The spore suspension concentration was adjusted to 1×10⁻⁶ using a cell counter. 9 CFU / mL was used to obtain Penicillium italicum culture.

[0037] A co-culture method was used. 0.75 mL, 1.125 mL, and 1.50 mL of the prepared modified PVA-chitosan composite coating solution were weighed and added to 60 mL of PDA medium, then shaken well. Medium without the coating solution was used as a control group. The PDA medium was then poured evenly into petri dishes and allowed to stand for 1 h to obtain PDA plates. 100 μl of *Penicillium italicum* was added to the center of each PDA plate, and the plates were incubated at 28℃ for 7 days. The growth of *Penicillium italicum* was observed periodically, and the colony diameter was measured and recorded. To eliminate the influence of glacial acetic acid, different amounts of glacial acetic acid (0 μL, 5 μL, 10 μL, 15 μL, 20 μL) were used as controls for antibacterial testing. An unmodified PVA-chitosan composite coating solution (PVA solution to chitosan solution volume ratio of 3:1, added at 1.125 mL) was used as a control to verify the function of the modified PVA solution.

[0038] 6. Cell membrane injury research Morphological changes in *Penicillium italicum* cells treated with a modified PVA-chitosan composite coating solution (modified PVA solution: chitosan solution = 3:1) and the control group without coating solution were observed using field emission scanning electron microscopy (FE-SEM, JSM-7610FPlus, Hitachi High Technology Co., Ltd., Japan). *Penicillium italicum* mycelia (0.1 g) were collected on day 5 of culture, washed three times with PBS (0.1 M, pH=7.2), and fixed overnight at 4°C with glutaraldehyde (2.5%, v / v). Then, the mycelia were dehydrated sequentially with different concentrations of ethanol (50%, 60%, 70%, 80%, 90%, and 100%), with each concentration dehydration treatment lasting 10 min. After washing with sterile deionized water, the next concentration of dehydration treatment was performed to obtain the test samples. The sample to be tested was freeze-dried at -96°C for 12 h using a freeze dryer (VP-60X, Ipswich, UK). The dried sample was then fixed on an FE-SEM support under a vacuum of 9.6 × 10⁻⁶. -5 Platinum was sputtered at Pa for 2 min. The differences in microstructure between the treatment group and the control group were observed using field emission scanning electron microscopy (observing changes in cell surface structure to determine whether the cell membrane was damaged).

[0039] 7. Spore germination research A co-culture method was used. 0.75 mL, 1.125 mL, and 1.50 mL of the prepared modified PVA-chitosan composite coating solution were weighed and added to 60 mL of potato dextrose broth (PDB, Beijing Luqiao Technology Co., Ltd., China), respectively, and shaken well. The medium without the coating solution was used as a control group. Then, 500 μL of *Penicillium italicum* was added dropwise to the PDB medium and incubated at 28°C for 12 h, 24 h, 48 h, and 96 h, respectively. Samples were taken after incubation, and fungal spore germination was observed using a fluorescence upright microscope (DM3000-LED, Leica GmbH, Germany). The entire experiment was repeated three times.

[0040] III. Test Results 1. Effect of different lactic acid addition amounts on the hydrophobicity of modified PVA films Water contact angle test results ( Figure 1The results show that with increasing lactic acid content, the hydrophobic angle of the modified PVA film gradually increases, indicating an effective improvement in hydrophobic properties. Lactic acid, as a polycarboxylic acid chemical crosslinking agent, can undergo esterification with the hydroxyl groups on the polyvinyl alcohol molecular chain to form multiple ester bonds, thereby constructing a stable three-dimensional PVA crosslinked network. With increasing lactic acid content, the number of active sites participating in the reaction on the PVA molecular chain increases accordingly, promoting an increase in crosslinking density. This crosslinking structure introduces hydrophobic alkyl segments through ester bonds and reorganizes the arrangement of hydrophilic groups through hydrogen bonding, reducing the interaction between hydrophilic groups and water molecules, thus exposing more hydrophobic groups on the material surface, ultimately resulting in enhanced overall hydrophobicity of the material.

[0041] The results above demonstrate that the hydrophobic properties of modified PVA films can be precisely controlled by adjusting the amount of lactic acid added. When the amount of lactic acid added is 20%, the water contact angle of the modified PVA film is increased from 57° in pure PVA to 78°. Therefore, 20% is selected as the optimal amount of lactic acid added and will be used as the default amount of lactic acid added to the modified PVA solution in subsequent experiments.

[0042] 2. Effects of different formulation ratios on the hydrophobicity and mechanical properties of composite antibacterial membranes Water contact angle test results ( Figure 2 As shown in a), when the modified PVA solution and chitosan are combined at a mass ratio of 1:2, the water contact angle of the composite antibacterial membrane reaches a maximum of 100.7°, exhibiting optimal hydrophobic properties. This is attributed to the hydrogen bond network formed between chitosan molecules and modified PVA molecules, which effectively regulates the hydrophilic / hydrophobic balance of the material surface, reduces the surface free energy of the composite membrane, and thus significantly improves the hydrophobic properties.

[0043] In terms of mechanical properties ( Figure 2 (b) As the content of modified PVA solution in the composite system increases, the tensile strength of the composite antibacterial film shows a significant upward trend. This is because the hydrogen bonding between chitosan and modified PVA molecules promotes the tight bonding of the two-phase interface, constructs a uniform and dense three-dimensional network structure, and effectively enhances the material's ability to resist external forces.

[0044] Elongation at break of composite antibacterial film ( Figure 2 c) also shows a similar variation pattern to tensile strength, indicating that the introduction of modified PVA can improve the strength of the material while maintaining good flexibility, thus achieving synergistic enhancement of strength and toughness.

[0045] Based on the results of water contact angle and tensile tests, a ratio of 3:1 for modified PVA and chitosan was selected as the optimal combination, which can effectively optimize the hydrophobic and mechanical properties of the composite antibacterial membrane.

[0046] 3. Inhibitory effect of different addition amounts of modified PVA-chitosan composite coating solution on Penicillium italicum. Antibacterial tests were conducted on a modified PVA-chitosan composite coating solution prepared at a modified PVA to chitosan ratio of 3:1. Different amounts of the modified PVA-chitosan composite coating solution (0.75 mL, 1.125 mL, and 1.50 mL) were added to 60 mL of PDA solid culture medium, with the medium without the coating solution serving as the control group. The antibacterial performance was evaluated by measuring the colony diameter. Results ( Figure 3 The results showed that, compared with the control group, the colony diameter of bacteria treated with the modified PVA-chitosan composite coating solution was significantly reduced, and the antibacterial effect increased with the increase of the coating solution dosage. When the dosage reached 1.5 mL, it completely inhibited the growth of *Penicillium italicum*, demonstrating excellent antibacterial properties. This is attributed to the fact that the carboxyl groups and other functional groups in lactic acid molecules actively participate in hydrogen bond formation, and the modified PVA enhances the adsorption capacity of the membrane surface for bacterial cells. This, combined with the cationic antibacterial effect of chitosan, produces a synergistic effect, jointly disrupting the integrity of the mycelial cell membrane, leading to leakage of cell contents, thereby effectively inhibiting bacterial growth.

[0047] To verify that the antibacterial effect of the modified PVA-chitosan composite coating solution originates from its active ingredients rather than the solvent glacial acetic acid, a comparative experiment was conducted with different amounts of glacial acetic acid added (0 μL, 5 μL, 10 μL, 15 μL, 20 μL, with 0 μL as the control). Results ( Figure 4 The results showed that, among all the different amounts of glacial acetic acid added, the colony diameter of *Penicillium italicum* was not significantly different from that of the control, and the colony morphology and growth rate were basically the same. This indicates that the range of glacial acetic acid added did not have a significant inhibitory effect on the growth of *Penicillium italicum*, proving that the antibacterial effect of the modified PVA-chitosan composite coating solution comes from the active ingredient formed by the combination of modified PVA solution and chitosan solution.

[0048] To verify the effect of modified PVA solution on the antibacterial effect of modified PVA-chitosan composite coating solution, a PVA-chitosan composite coating solution with an addition volume of 1.125 mL (PVA solution to chitosan solution ratio of 3:1) was used as a comparison. Results ( Figure 5 The results showed that, at the same addition amount, the modified PVA-chitosan composite coating solution had a better antibacterial effect than the PVA solution-chitosan composite coating solution (a simple blend of unmodified PVA solution and chitosan solution), indicating that the PVA solution modified with lactic acid used in this invention has better antibacterial properties.

[0049] 4. Effects of modified PVA-chitosan composite coating solution on the cell membrane of Penicillium italicum To further investigate the inhibitory mechanism of modified PVA-chitosan composite coating solution on *Penicillium italicum*, scanning electron microscopy was used to observe the microscopic morphology of mycelial samples treated with the modified PVA-chitosan composite coating solution. Observation results ( Figure 6 The results showed that the mycelium surface structure of *Penicillium italicum* in the blank control group was intact, with smooth and plump cell membranes and a relaxed morphology, exhibiting normal fungal mycelial morphology. However, the mycelium treated with 1.125 mL of modified PVA-chitosan composite coating solution showed significant morphological changes, including obvious wrinkling and local depressions on the cell surface, and cell membrane rupture in some areas, resulting in leakage of contents (no mycelium grew in the culture medium when 1.5 mL of modified PVA-chitosan composite coating solution was added, making mycelial morphology impossible to measure; the antibacterial effect was not obvious below 1.125 mL, therefore, mycelium treated with 1.125 mL of modified PVA-chitosan composite coating solution was selected for microscopic morphological observation). This morphological change confirms that the active ingredient in the modified PVA-chitosan composite coating solution can disrupt the integrity of the *Penicillium italicum* cell membrane structure. This membrane damage leads to leakage of cell contents, affecting normal metabolic activities; on the other hand, it disrupts the osmotic pressure balance of the cell, ultimately leading to inhibited fungal growth and even death. These microscopic observations corroborate previous colony diameter measurements, jointly revealing the intrinsic mechanism by which the modified PVA-chitosan composite coating exerts its antibacterial effect by disrupting the cell membrane structure of pathogenic fungi.

[0050] 5. Effects of modified PVA-chitosan composite coating solution on the germination of Penicillium italicum spores To systematically evaluate the inhibitory effect of modified PVA-chitosan composite coating solution on the germination of Penicillium italicum spores, this study set up experimental groups with different addition amounts of modified PVA-chitosan composite coating solution (0.75 mL, 1.125 mL, and 1.5 mL). The spore germination process was dynamically observed using a fluorescence upright microscope. Samples were taken for observation after 12 h, 24 h, 48 h, and 96 h of incubation. Results ( Figure 7The results showed that the spores in the control group exhibited a normal germination process with prolonged incubation time, with most spores germinating by 48 h and forming a complete hyphal network by 96 h. However, spore germination in all experimental groups was significantly inhibited, manifested as delayed germination and reduced germination rate. This inhibitory effect showed a clear dose-response relationship, with the experimental group receiving 1.5 mL of the modified PVA-chitosan composite coating solution maintaining the lowest spore germination rate at 96 h. These results indicate that the active ingredients in the modified PVA-chitosan composite coating solution can effectively interfere with the normal germination process of *Penicillium italicum* spores. Its mechanism of action may involve disrupting the integrity of the spore cell membrane, affecting its osmotic pressure regulation and energy metabolism required for germination, thereby blocking mycelial formation. This finding further confirms the multiple inhibitory effects of the modified PVA-chitosan composite coating solution on the life cycle of *Penicillium italicum* at the spore germination level, providing important experimental evidence for the study of its antibacterial mechanism.

[0051] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0052] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A modified polyvinyl alcohol-chitosan composite dip coating solution, characterized in that, The modified polyvinyl alcohol-chitosan composite dip-coating solution is prepared by blending a modified polyvinyl alcohol and a chitosan solution.

2. The modified polyvinyl alcohol-chitosan composite dip-coating solution according to claim 1, characterized in that, The modified polyvinyl alcohol is prepared by esterification of polyvinyl alcohol and lactic acid.

3. The modified polyvinyl alcohol-chitosan composite dip-coating solution according to claim 1, characterized in that, The mass ratio of lactic acid to polyvinyl alcohol is 0.15-0.9:

3.

4. A composite antimicrobial film, characterized by, Glycerol is added as a plasticizer in the preparation process of the modified polyvinyl alcohol-chitosan composite dip-coating solution.

5. A method of preparing the composite antimicrobial film according to claim 4, characterized by, The modified polyvinyl alcohol-chitosan composite dip-coating solution of claim 1 is obtained by drying into a film. The method comprises the following steps: Dissolve polyvinyl alcohol in deionized water, add lactic acid dropwise at 34-36°C, and stir at constant temperature for 14-18 hours to obtain a modified polyvinyl alcohol solution. Dissolve chitosan powder in a 1% glacial acetic acid aqueous solution, stir at 58-62°C until completely dissolved, and obtain a chitosan solution.

6. The method of claim 5, wherein the composite antimicrobial film is prepared by a process comprising: Mix the modified polyvinyl alcohol solution and the chitosan solution, add glycerol, and stir uniformly to obtain a modified polyvinyl alcohol-chitosan composite dip-coating solution; then cast and dry into a film to obtain the composite antibacterial film.

7. The method for preparing the composite antibacterial membrane according to claim 5, characterized in that, The volume ratio of the modified polyvinyl alcohol solution to the chitosan solution is 1-3:1-3.

8. The method for preparing the composite antibacterial membrane according to claim 5, characterized in that, The mass concentration of chitosan in the chitosan solution is 1%-5%.

9. A modified polyvinyl alcohol-chitosan composite coating solution according to any one of claims 1 to 3 or a composite antibacterial film according to claim 4, in inhibiting Penicillium italicum (… The mass-volume ratio of glycerol to the modified polyvinyl alcohol-chitosan solution is 0.0525 g-0.0675 g:60 mL. Applications in ).

10. Use according to claim 9, characterized in that, Penicillium italicum The modified polyvinyl alcohol-chitosan composite dip-coating solution or the composite antibacterial film is used to inhibit the growth of Penicillium italicum colonies, inhibit spore germination, and exert antibacterial effect by damaging the cell membrane integrity of mycelium.

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