Chitosan-based active preservative film and preparation method and application thereof

Abstract

The application discloses a chitosan-based active preservative film and a preparation method and application thereof, and relates to the technical field of high polymer materials and food preservation packaging. The preservative film takes chitosan as a matrix, in-situ assembles polylysine grafted with gallic acid and cellulose nanocrystals, and introduces the chitosan film-forming system, so as to construct an active preservative film with humidity adaptability, mechanical stability, ultraviolet absorption, antioxidation, bacteriostasis and responsive active release characteristics. The core feature is that in the high-humidity packaging environment formed by fruit respiration and transpiration during blueberry storage, the preservative film has good structural integrity and mechanical reliability, and realizes the self-balancing of water migration by means of the composite network structure. When the film is used for blueberry covering packaging, the film can reduce the dew condensation and fruit shrinkage of the packaging, inhibit corruption, and improve the total phenol retention level. The film shows a higher active component release tendency under acidic conditions, which is helpful to enhance the active expression in the preservation process, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to a chitosan-based active preservation film, its preparation method and application, belonging to the field of polymer materials and food preservation packaging technology. Background Technology

[0002] Blueberries are berries with a pronounced respiratory climacteric, delicate skin, and rich anthocyanins and polyphenols. During post-harvest storage and transportation, they are prone to dehydration and shrinkage, condensation on the fruit surface, microbial infection, and nutrient degradation due to oxidation. While traditional polyethylene packaging films offer some water resistance, they easily cause moisture buildup and condensation inside the packaging, exacerbating mold growth. Ordinary bio-based films generally suffer from insufficient barrier properties, limited active functions, and poor preservation effects, making it difficult to simultaneously meet the comprehensive needs of blueberries for antibacterial, antioxidant, UV protection, and moisture regulation.

[0003] While gallic acid (GA), ε-polylysine (PL), chitosan (CS), and aldehyde-containing polysaccharide materials have been reported, the related technologies are mainly focused on medical materials, hemostatic materials, or general antibacterial materials. There is a lack of specific designs for postharvest preservation of blueberries, especially a chitosan-based active preservation film that can simultaneously inhibit condensation, maintain quality, and protect nutrients during blueberry storage.

[0004] Furthermore, methods that directly incorporate small-molecule polyphenols into film-forming systems often suffer from problems such as easy migration and oxidative inactivation of active components, limited compatibility with the matrix, and insufficient preservation efficacy. More importantly, for blueberries, which are high-respiration and high-transpiration berries, a high-humidity microenvironment is often created during packaging. Ordinary polysaccharide membranes are prone to mechanical property degradation, structural loosening, and preservation function decline due to moisture absorption, making it difficult to balance structural stability and activity expression. Therefore, it is necessary to develop an active membrane system for blueberry preservation applications that maintains good mechanical reliability and structural integrity under high humidity conditions, and combines responsive active release behavior to achieve comprehensive control over condensation, water loss, and spoilage. Summary of the Invention

[0005] To address the shortcomings of related technologies, this invention provides a chitosan-based active preservation film, its preparation method, and its application. The film system constructed using the method of this invention not only possesses ultraviolet absorption, antioxidant, and antibacterial functions, but more importantly, it maintains good mechanical reliability and structural integrity under high humidity conditions. Furthermore, it reduces condensation during packaging and fruit shrinkage through reasonable regulation of water migration. Simultaneously, the film system exhibits a higher tendency to release active components under acidic conditions, which helps enhance functional expression during the preservation process. This solves the problems of blueberry preservation materials being prone to structural instability, decreased mechanical properties, and unstable activity expression under high humidity packaging environments, and the difficulty in simultaneously achieving condensation inhibition, water loss control, and nutrient retention.

[0006] One objective of this invention is to provide a method for preparing a chitosan-based active food preservation film, specifically comprising the following steps: (1) Preparation of functionalized flexible network precursor reaction solution: Gallic acid (GA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added to buffer solution for activation to obtain activation solution. The activation solution was added dropwise to ε-polylysine (PL) solution for grafting reaction to obtain precursor reaction solution, namely PLGA reaction solution.

[0007] (2) Preparation of nanocellulose platform substrate: An oxidant was added to the cellulose nanocrystal (CNC) suspension to carry out an oxidation reaction, and then a terminator was added to terminate the reaction. After purification and drying, the nanocellulose platform substrate, namely DACNC, was obtained.

[0008] (3) Preparation of particle dispersion: Prepare a suspension of nanocellulose platform substrate (DACNC), then add the nanocellulose platform substrate suspension dropwise to the precursor reaction solution under stirring. After the addition is complete, stir the reaction to obtain a mixture. Purify the mixture to obtain a purified particle dispersion.

[0009] (4) Preparation of composite film-forming solution: Add plasticizer to acetic acid solution of chitosan (CS) to obtain CS matrix solution, add purified particle dispersion obtained in step (3) to CS matrix solution, and then perform ultrasonic degassing treatment to obtain composite film-forming solution.

[0010] (5) Preparation of chitosan-based active food preservation film: The composite film-forming liquid is cast into a mold and then dried to obtain chitosan-based active food preservation film.

[0011] Preferably, in step (1), the buffer solution is a MES buffer solution with a pH of 5.5 and a concentration of 0.1 mol / L; the GA, EDC, and NHS are added to the buffer solution in a molar ratio of GA, EDC, and NHS of 1:1:1; the amount of GA added to the activation solution is 0.29 mol / L; the activation conditions are: activation by stirring at a speed of 300-500 rpm for 0.5-1 h under ice-water bath conditions.

[0012] Preferably, in step (1), the ε-polylysine solution is prepared by ε-polylysine and MES buffer solution with pH 5.5 and a concentration of 0.1 mol / L; the concentration of ε-polylysine in the ε-polylysine solution is 0.13~0.39 mol / L; the dropping rate of the activation solution added to the precursor reaction solution is 10~15 mL / min; the activation solution is added to the ε-polylysine solution at a volume ratio of 1:1; the grafting reaction conditions are: reaction in the dark for 12~24 h.

[0013] More preferably, the grafting reaction in step (1) is carried out at room temperature.

[0014] Preferably, in step (2), the amount of cellulose nanocrystals added to the cellulose nanocrystal suspension is 0.01 g / mL; the amount of oxidant added to the cellulose nanocrystal suspension is 0.036 g / mL; and the conditions for the oxidation reaction are: reaction at 40°C in the dark for 4 hours.

[0015] More preferably, the cellulose nanocrystal suspension in step (2) is prepared from cellulose nanocrystals and deionized water.

[0016] Preferably, the oxidant in step (2) is sodium periodate; and the terminating agent is ethylene glycol.

[0017] More preferably, in step (2), ethylene glycol is added to terminate the reaction at a volume ratio of 5:100 for ethylene glycol and CNC suspension.

[0018] More preferably, the purification process in step (2) is dialysis.

[0019] Preferably, in step (3), the concentration of the nanocellulose platform substrate in the suspension is 0.01 g / mL; the nanocellulose platform substrate suspension is added dropwise to the precursor reaction solution at a volume ratio of 1:(2.5~8); the dropwise acceleration rate of the nanocellulose platform substrate suspension added to the precursor reaction solution is 10~15 mL / min; the stirring reaction conditions are: reaction in the dark at 300~500 rpm and 25~45℃ for 4~6 h.

[0020] More preferably, the suspension of the nanocellulose platform substrate in step (3) is prepared by the nanocellulose platform substrate and deionized water.

[0021] More preferably, the purification process in step (3) is performed by dialysis in deionized water.

[0022] Preferably, the plasticizer in step (4) is glycerol; the content of chitosan in the acetic acid solution of chitosan is 1.5%~2.5% by mass, and the content of acetic acid is 1% by mass; the content of the plasticizer in the CS matrix solution is 0.4%~0.8% by mass; the purified particle dispersion is added to the chitosan matrix solution at a mass ratio of (0.8~1.2):1.

[0023] More preferably, the acetic acid solution solvent for chitosan in step (4) is water.

[0024] More preferably, the conditions for ultrasonic degassing in step (4) are: ultrasonic treatment at an ultrasonic frequency of 40 kHz for 10 min.

[0025] Preferably, the thickness of the chitosan-based active preservative film in step (5) is 0.083~0.085 mm.

[0026] More preferably, the drying conditions in step (5) are: drying at 45~50℃ and relative humidity of 50%~60% for 24~36 hours.

[0027] The second objective of this invention is to provide a chitosan-based active food preservation film prepared using the method of this invention.

[0028] The third objective of this invention is to provide an application of the chitosan-based active preservation film prepared by the method of this invention in the preservation of blueberries.

[0029] Mechanism of the invention: This invention introduces gallic acid-grafted active components into a chitosan matrix, forming a composite covalent network with aldehyde-modified cellulose nanocrystals (DACNC) and the chitosan matrix. This constructs a preservation film system that combines humidity-adaptive mechanical stability with responsive active release. Specifically, ε-polylysine grafted with gallic acid is in situ assembled with oxidized cellulose nanocrystals via a Schiff base reaction, forming a composite network with rigid nanocrystals as the core and flexible grafted chains as arms. This network effectively restricts the excessive movement of chitosan molecular chains under high humidity conditions through covalent cross-linking, ensuring the preservation film maintains high mechanical reliability even under high humidity. Simultaneously, the covalently anchored gallic acid segments endow the film with UV absorption and long-lasting antioxidant capabilities, and the accelerated hydrolysis rate of Schiff base bonds under acidic conditions causes the active components to exhibit acid-responsive release characteristics, thereby enhancing the preservation function when the microenvironment changes during blueberry storage.

[0030] The beneficial effects of this invention are: (1) This invention does not simply introduce known active ingredients to give the plastic wrap antioxidant or antibacterial properties, but rather maintains good mechanical stability and structural integrity in the high humidity packaging environment of blueberries, thereby improving the applicability of the plastic wrap in actual storage conditions.

[0031] (2) Through the synergistic effect between the rigid skeleton of DACNC, the PL active component of grafted GA and the CS matrix, the preservation film exhibits a better balance between water migration regulation, condensation reduction and fruit water loss control, which is more in line with the requirements of blueberry preservation for packaging microenvironment.

[0032] (3) The preservation film obtained by the present invention not only has ultraviolet absorption, antioxidant and antibacterial functions, but also exhibits a higher tendency to release active components under acidic conditions, which helps to enhance the functional expression during the preservation process.

[0033] (4) When the preservation film obtained by the present invention is used for covering or close-fitting packaging of blueberries, it can reduce condensation and fruit shrinkage, delay weight loss, inhibit spoilage, and improve the total phenol retention level. It is suitable for high humidity packaging environment caused by fruit respiration and transpiration during blueberry storage and has clear food packaging application value.

[0034] (5) Compared with ordinary physical blended CS films or single reinforced network composite films, this invention not only endows the preservation film with preservation-related functions such as ultraviolet absorption, anti-oxidation and antibacterial properties, but more importantly, it enables the preservation film to maintain good mechanical stability and structural integrity in the high humidity packaging environment of blueberries. This system does not simply rely on the known function of a single component, but through the synergistic effect between the DACNC rigid framework, the PL active component grafted with GA and the CS matrix, a better synergistic match is formed between humidity-adaptive mechanical behavior, water migration regulation and responsive activity expression, making it more suitable for blueberry storage and preservation scenarios. Attached Figure Description

[0035] Figure 1 The images show the changes in the appearance of blueberries after they were stored in the plastic wrap of Examples 1 and 2 of this invention.

[0036] Figure 2 The graph shows the change in weight loss rate of blueberries after storing them in the plastic wrap of Example 1 and Comparative Examples 1-2 of this invention.

[0037] Figure 3 The graph shows the change in total phenolic content of blueberries after storing them in the plastic wrap of Examples 1 and 2 of the present invention.

[0038] Figure 4 The images show the transmission spectra of the plastic wrap used in the various embodiments and comparative examples 2-3 of this invention.

[0039] Figure 5The graph shows the ABTS free radical scavenging rate analysis of the plastic wrap in various embodiments and comparative examples 2-3 of the present invention.

[0040] Figure 6 This is a comparison chart of the antibacterial effects of plastic wrap in Example 1, the blank control group, Comparative Example 2, and Comparative Example 3 of the present invention.

[0041] Figure 7 This is a cytotoxicity analysis diagram of the plastic wrap extract from Example 1 of the present invention.

[0042] Figure 8 This is a diagram showing the soil degradation morphology changes of the plastic wrap in Example 1 and Comparative Example 1 of the present invention.

[0043] Figure 9 This is a comparison chart of the mechanical properties of the plastic wrap in Example 1 and Comparative Examples 2-3 of the present invention under 50% relative humidity.

[0044] Figure 10 The figures show representative stress-strain curves of the plastic wrap in Embodiment 1 and Comparative Examples 2-3 of the present invention under 70% relative humidity conditions. Detailed Implementation

[0045] To better illustrate the purpose, technical solution, and advantages of this invention, the following detailed description will be provided in conjunction with specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments and comparative examples of this invention are of commercially available analytical grade. The degree of deacetylation of CS used in the embodiments and comparative examples of this invention is ≥95%. The aldehyde content on the surface of DACNC in the embodiments and comparative examples of this invention was determined using the hydroxylamine hydrochloride-potentiometric titration method. This involves reacting the aldehyde groups in DACNC with hydroxylamine hydrochloride to generate oxime and release hydrochloric acid. The amount of acid released is then titrated with a standard NaOH solution (0.1 mol / L NaOH aqueous solution, standardized with potassium hydrogen phthalate), and the aldehyde content is calculated based on the amount of NaOH consumed.

[0046] Example 1 A method for preparing a chitosan-based active food preservation film specifically includes the following steps: (1) Preparation of functionalized flexible network precursor reaction solution: GA, EDC, and NHS were dissolved in MES buffer solution with pH 5.5 and a concentration of 0.1 mol / L at a molar ratio of 1:1:1, with GA added at 0.29 mol / L. The solution was activated by stirring at 500 rpm for 1 h under ice-water bath conditions to obtain the activated solution. The activated solution was added dropwise to the PL solution at a drop rate of 12 mL / min at a volume ratio of 1:1 (the PL solution was prepared by ε-polylysine and MES buffer solution with pH 5.5 and a concentration of 0.1 mol / L, with PL concentration of 0.39 mol / L). The grafting reaction was carried out at room temperature in the dark for 24 h. After the reaction was completed, the product solution was obtained. Without dialysis and lyophilization, MES buffer solution (pH 5.5) was added. The volume of the product solution was increased to twice that of the original product solution by adding 5.5 mol / L (0.1 mol / L) to reduce the salt content. Then the pH was adjusted to 7.0 and held for 60 min to hydrolyze and quench the residual activation intermediate. Finally, the pH was adjusted back to 5.0 to obtain the PLGA reaction solution.

[0047] (2) Preparation of nanocellulose platform substrate: Sodium periodate was added to CNC suspension (the suspension was made with deionized water as solvent, the amount of CNC added was 0.01 g / mL, and the pH was 3), and a light-protected oxidation reaction was carried out at 40℃ for 4 h. The amount of sodium periodate added to the CNC suspension was 0.036 g / mL. Then, ethylene glycol was added at a volume ratio of 5:100 to terminate the reaction. The solution was then dialyzed to neutral and freeze-dried to obtain DACNC with a surface aldehyde content of 1.2 mmol / g.

[0048] (3) Preparation of particle dispersion: A suspension of DACNC was prepared using deionized water and DACNC. The concentration of DACNC in the suspension was 0.01 g / mL. The DACNC suspension was added dropwise to the stirred PLGA reaction solution at a rate of 12 mL / min at a volume ratio of 1:2.5 between the DACNC suspension and the PLGA reaction solution. After the addition was completed, the mixture was reacted in the dark at 500 rpm and 45 °C for 6 h to allow the aldehyde group of DACNC to undergo a Schiff base reaction with the amino group of PLGA and to assemble in situ, resulting in a mixed solution. The mixed solution was then purified by dialysis in deionized water to remove unreacted small molecules and residual EDC / NHS. After dialysis, the purified particle dispersion was obtained.

[0049] (4) Preparation of composite film-forming solution: Glycerol is added to the acetic acid solution of CS and stirred. The content of CS in the acetic acid solution of CS is 2% by mass percentage and the content of acetic acid is 1% by mass percentage. The solvent is deionized water to obtain CS matrix solution. The content of glycerol in the CS matrix solution is 0.6% by mass percentage. The purified particle dispersion obtained in step (3) is added to the CS matrix solution and mixed at a mass ratio of 1:1 between the purified particle dispersion and the CS matrix solution. Then, ultrasonic degassing is performed for 10 min at an ultrasonic frequency of 40 kHz to obtain composite film-forming solution.

[0050] (5) Preparation of chitosan-based active food preservation film: The composite film-forming liquid is cast into a mold and then dried at 50°C and 50% relative humidity for 36 hours to obtain a chitosan-based active food preservation film with a thickness of 0.085 mm, namely CDPG3.

[0051] Table 1 shows the appearance score, 8-day weight loss rate, and 8-day total phenolic content of blueberries stored with plastic wrap according to this embodiment. The morphological changes of blueberries after plastic wrap storage are illustrated in the following diagram. Figure 1 As shown in the figure; the change in weight loss rate of blueberries after storage in plastic wrap in this embodiment is shown in the figure. Figure 2 As shown in the figure; the change in total phenolic content of blueberries after storage in plastic wrap in this embodiment is shown in the figure. Figure 3 As shown; the transmission spectrum analysis diagram of the plastic wrap in this embodiment is as follows. Figure 4 As shown in the figure; the ABTS free radical scavenging rate analysis diagram of the plastic wrap in this embodiment is as follows. Figure 5 As shown in the figure; a comparison of the antibacterial effect of the plastic wrap in this embodiment is shown in the figure. Figure 6 As shown; the cytotoxicity analysis diagram of the plastic wrap extract in this embodiment is shown in the figure. Figure 7 As shown in the figure; the soil degradation morphology changes of the plastic wrap in this embodiment are shown in the figure. Figure 8 As shown; the mechanical property test results of the cling film in this embodiment under 50% relative humidity conditions are as follows. Figure 9 As shown; the representative stress-strain curve of the cling film in this embodiment under 70% relative humidity is shown below. Figure 10 As shown.

[0052] Example 2 A method for preparing a chitosan-based active food preservation film specifically includes the following steps: (1) Preparation of functionalized flexible network precursor reaction solution: GA, EDC, and NHS were dissolved in MES buffer solution with pH 5.5 and a concentration of 0.1 mol / L at a molar ratio of 1:1:1, with GA added at 0.29 mol / L. The solution was activated by stirring at 300 rpm for 0.5 h under ice-water bath conditions to obtain the activated solution. The activated solution was added dropwise to the PL solution at a dropping rate of 10 mL / min at a volume ratio of 1:1 (the PL solution...). The solution was prepared by combining ε-polylysine and MES buffer (pH 5.5, 0.1 mol / L, PL concentration 0.13 mol / L). The grafting reaction was carried out at room temperature in the dark for 12 h. After the reaction, the product solution was obtained. Without dialysis and lyophilization, MES buffer (pH 5.5, 0.1 mol / L) was added to increase the volume to 10 times that of the original product solution for dilution and desalting. Then the pH was adjusted to 9.5 and held for 10 min to hydrolyze and quench residual activation intermediates. Finally, the pH was adjusted back to 6.0 to obtain the PLGA reaction solution.

[0053] (2) Preparation of nanocellulose platform substrate: Sodium periodate was added to CNC suspension (the suspension was made with deionized water as solvent, the amount of CNC added was 0.01 g / mL, and the pH was 3), and a light-protected oxidation reaction was carried out at 40℃ for 4 h. The amount of sodium periodate added to the CNC suspension was 0.036 g / mL. Then, ethylene glycol was added at a volume ratio of 5:100 to terminate the reaction. The solution was then dialyzed to neutral and freeze-dried to obtain DACNC with a surface aldehyde content of 1.2 mmol / g.

[0054] (3) Preparation of particle dispersion: A suspension of DACNC was prepared using deionized water and DACNC. The concentration of DACNC in the suspension was 0.01 g / mL. The DACNC suspension was added dropwise to the stirred PLGA reaction solution at a rate of 10 mL / min at a volume ratio of 1:4 between the DACNC suspension and the PLGA reaction solution. After the addition was completed, the mixture was reacted in the dark at 300 rpm and 25 °C for 4 h to allow the aldehyde group of DACNC to undergo a Schiff base reaction with the amino group of PLGA and to assemble in situ, resulting in a mixed solution. The mixed solution was then purified by dialysis in deionized water to remove unreacted small molecules and residual EDC / NHS. After dialysis, the purified particle dispersion was obtained.

[0055] (4) Preparation of composite film-forming solution: Glycerol is added to the acetic acid solution of CS and stirred. The content of CS in the acetic acid solution of CS is 1.5% by mass and the content of acetic acid is 1% by mass. The solvent is deionized water to obtain CS matrix solution. The content of glycerol in the CS matrix solution is 0.4% by mass. The purified particle dispersion obtained in step (3) is added to the CS matrix solution and mixed at a mass ratio of 0.8:1. Then, ultrasonic degassing is performed for 10 min at an ultrasonic frequency of 40 kHz to obtain composite film-forming solution.

[0056] (5) Preparation of chitosan-based active food preservation film: The composite film-forming liquid is cast into a mold and then dried for 24 hours at 45°C and 50% relative humidity to obtain a chitosan-based active food preservation film with a thickness of 0.083 mm, namely CDPG1.

[0057] The appearance score, 8-day weight loss rate, and 8-day total phenolic content of blueberries stored using the preservation film in this embodiment are shown in Table 1; the transmission spectrum analysis of the preservation film in this embodiment is shown in the figure. Figure 4 As shown in the figure; the ABTS free radical scavenging rate analysis diagram of the plastic wrap in this embodiment is as follows. Figure 5 As shown.

[0058] Example 3 A method for preparing a chitosan-based active food preservation film specifically includes the following steps: (1) Preparation of functionalized flexible network precursor reaction solution: GA, EDC, and NHS were dissolved in MES buffer solution with pH 5.5 and a concentration of 0.1 mol / L at a molar ratio of 1:1:1, with GA added at 0.29 mol / L. The solution was activated by stirring at 400 rpm for 0.75 h under ice-water bath conditions to obtain the activated solution. The activated solution was added dropwise to the PL solution at a dropping rate of 15 mL / min at a volume ratio of 1:1 to the PL solution. The solution was prepared by combining ε-polylysine and MES buffer (pH 5.5, 0.1 mol / L, PL concentration 0.26 mol / L). The grafting reaction was carried out at room temperature in the dark for 18 h. After the reaction, the product solution was obtained. Without dialysis and lyophilization, MES buffer (pH 5.5, 0.1 mol / L) was added to increase the volume to 5 times that of the original product solution for dilution and desalting. Then the pH was adjusted to 8.0 and held for 30 min to hydrolyze and quench residual activation intermediates. Finally, the pH was adjusted back to 5.5 to obtain the PLGA reaction solution.

[0059] (2) Preparation of nanocellulose platform substrate: Sodium periodate was added to CNC suspension (the suspension was made with deionized water as solvent, the amount of CNC added was 0.01 g / mL, and the pH was 3), and a light-protected oxidation reaction was carried out at 40℃ for 4 h. The amount of sodium periodate added to the CNC suspension was 0.036 g / mL. Then, ethylene glycol was added at a volume ratio of 5:100 to terminate the reaction. The solution was then dialyzed to neutral and freeze-dried to obtain DACNC with a surface aldehyde content of 1.2 mmol / g.

[0060] (3) Preparation of particle dispersion: A suspension of DACNC was prepared using deionized water and DACNC. The concentration of DACNC in the suspension was 0.01 g / mL. The DACNC suspension was added dropwise to the stirred PLGA reaction solution at a rate of 15 mL / min at a volume ratio of 1:4 between the DACNC suspension and the PLGA reaction solution. After the addition was completed, the mixture was reacted in the dark at 400 rpm and 35 °C for 5 h to allow the aldehyde group of DACNC to undergo a Schiff base reaction with the amino group of PLGA and to assemble in situ, resulting in a mixed solution. The mixed solution was then purified by dialysis in deionized water to remove unreacted small molecules and residual EDC / NHS. After dialysis, the purified particle dispersion was obtained.

[0061] (4) Preparation of composite film-forming solution: Glycerol is added to the acetic acid solution of CS and stirred. The content of CS in the acetic acid solution of CS is 2% by mass percentage and the content of acetic acid is 1% by mass percentage. The solvent is deionized water to obtain CS matrix solution. The content of glycerol in the CS matrix solution is 0.6% by mass percentage. The purified particle dispersion obtained in step (3) is added to the CS matrix solution and mixed at a mass ratio of 1:1 between the purified particle dispersion and the CS matrix solution. Then, ultrasonic degassing is performed for 10 min at an ultrasonic frequency of 40 kHz to obtain composite film-forming solution.

[0062] (5) Preparation of chitosan-based active food preservation film: The composite film-forming liquid is cast into a mold and then dried at 50°C and 55% relative humidity for 30 hours to obtain a chitosan-based active food preservation film with a thickness of 0.084 mm, namely CDPG2.

[0063] The appearance score, 8-day weight loss rate, and 8-day total phenolic content of blueberries stored using the preservation film in this embodiment are shown in Table 1; the transmission spectrum analysis of the preservation film in this embodiment is shown in the figure. Figure 4 As shown in the figure; the ABTS free radical scavenging rate analysis diagram of the plastic wrap in this embodiment is as follows. Figure 5 As shown.

[0064] Example 4 A method for preparing a chitosan-based active food preservation film specifically includes the following steps: (1) Preparation of functionalized flexible network precursor reaction solution: GA, EDC, and NHS were dissolved in MES buffer solution with pH 5.5 and a concentration of 0.1 mol / L at a molar ratio of 1:1:1, with GA added at 0.29 mol / L. The solution was activated by stirring at 500 rpm for 1 h in an ice-water bath to obtain the activated solution. The activated solution was added dropwise to the PL solution at a drop rate of 12 mL / min at a volume ratio of 1:1 (the PL solution was prepared by ε-polylysine and MES buffer solution with pH 5.5 and a concentration of 0.1 mol / L, with PL concentration of 0.39 mol / L). The grafting reaction was carried out at room temperature in the dark for 24 h. After the reaction was completed, the product solution was obtained. Without dialysis and lyophilization, MES buffer solution (pH 5.5) was added. The volume of the product solution was increased to 5 times that of the original product solution by adding 5.5 (0.1 mol / L) to reduce the salt content. Then the pH was adjusted to 8.5 and held for 30 min to hydrolyze and quench the residual activation intermediate. Finally, the pH was adjusted back to 5.5 to obtain the PLGA reaction solution.

[0065] (2) Preparation of nanocellulose platform substrate: Sodium periodate was added to CNC suspension (the suspension was made with deionized water as solvent, the amount of CNC added was 0.01 g / mL, and the pH was 3), and a light-protected oxidation reaction was carried out at 40℃ for 4 h. The amount of sodium periodate added to the CNC suspension was 0.036 g / mL. Then, ethylene glycol was added at a volume ratio of 5:100 to terminate the reaction. The solution was then dialyzed to neutral and freeze-dried to obtain DACNC with a surface aldehyde content of 1.2 mmol / g.

[0066] (3) Preparation of particle dispersion: A suspension of DACNC was prepared using deionized water and DACNC. The concentration of DACNC in the suspension was 0.01 g / mL. The DACNC suspension was added dropwise to the stirred PLGA reaction solution at a rate of 12 mL / min at a volume ratio of 1:8 between the DACNC suspension and the PLGA reaction solution. After the addition was completed, the mixture was reacted in the dark at 500 rpm and 45 °C for 6 h to allow the aldehyde group of DACNC to undergo a Schiff base reaction with the amino group of PLGA and to assemble in situ, resulting in a mixed solution. The mixed solution was then purified by dialysis in deionized water to remove unreacted small molecules and residual EDC / NHS. After dialysis, the purified particle dispersion was obtained.

[0067] (4) Preparation of composite film-forming solution: Glycerol is added to the acetic acid solution of CS and stirred. The content of CS in the acetic acid solution of CS is 2.5% by mass and the content of acetic acid is 1% by mass. The solvent is deionized water to obtain CS matrix solution. The content of glycerol in the CS matrix solution is 0.8% by mass. The purified particle dispersion obtained in step (3) is added to the CS matrix solution and mixed at a mass ratio of 1.2:1. Then, ultrasonic degassing is performed for 10 min at an ultrasonic frequency of 40 kHz to obtain composite film-forming solution.

[0068] (5) Preparation of chitosan-based active food preservation film: The composite film-forming liquid is cast into a mold and then dried at 48°C and 60% relative humidity for 36 hours to obtain a chitosan-based active food preservation film with a thickness of 0.085 mm, namely CDPG4.

[0069] The appearance score, 8-day weight loss rate, and 8-day total phenolic content of blueberries stored using the preservation film in this embodiment are shown in Table 1; the transmission spectrum analysis of the preservation film in this embodiment is shown in the figure. Figure 4 As shown in the figure; the ABTS free radical scavenging rate analysis diagram of the plastic wrap in this embodiment is as follows. Figure 5 As shown.

[0070] Comparative Example 1 This comparative example uses commercially available food-grade polyethylene (PE) cling film with a thickness of 0.08 mm as a control.

[0071] The PE cling film in this comparative example does not have UV shielding capabilities.

[0072] The appearance score, 8-day weight loss, and 8-day total phenols of blueberries stored using the comparative example plastic wrap are shown in Table 1; the changes in the appearance morphology of blueberries after storage using the comparative example plastic wrap are shown in the figure below. Figure 1 As shown in the figure; the change in weight loss rate of blueberries after storage in plastic wrap in this comparative example is shown in the figure. Figure 2 As shown in the figure; the change in total phenolic content of blueberries after storage in plastic wrap in this comparative example is shown in the figure. Figure 3 As shown in the figure; the soil degradation morphology changes of the plastic wrap in this comparative example are shown in the figure. Figure 8 As shown.

[0073] Comparative Example 2 A method for preparing a pure CS thin film specifically includes the following steps: A CS acetic acid solution was prepared, in which the content of CS was 2% by mass and the content of acetic acid was 1% by mass. Glycerol was added to the CS acetic acid solution to form a CS matrix solution, in which the content of glycerol was 0.6% by mass. The CS acetic acid solution was subjected to ultrasonic degassing at a frequency of 40 kHz for 10 min, and then poured into a mold. It was then dried at 50 °C and 50% relative humidity for 36 h to form a pure CS film with a thickness of 0.085 mm.

[0074] Although this comparative ratio (pure CS system) has good film-forming properties and transparency, it lacks UV shielding ability and high-efficiency antioxidant and antibacterial activity. It cannot effectively inhibit the degradation of nutrients and the growth of microorganisms during the preservation of blueberries, and its ability to regulate moisture loss is weak, resulting in obvious shrinkage and weight loss of the fruit in the later stage of storage.

[0075] The appearance score, 8-day weight loss, and 8-day total phenols of blueberries stored using the comparative example plastic wrap are shown in Table 1; the changes in the appearance morphology of blueberries after storage using the comparative example plastic wrap are shown in the figure below. Figure 1 As shown in the figure; the change in weight loss rate of blueberries after storage in plastic wrap in this comparative example is shown in the figure. Figure 2 As shown in the figure; the change in total phenolic content of blueberries after storage in plastic wrap in this comparative example is shown in the figure. Figure 3 As shown; the transmission spectrum analysis diagram of the comparative example plastic wrap is shown below. Figure 4 As shown in the figure; the ABTS free radical scavenging rate analysis of the comparative example plastic wrap is shown in the figure. Figure 5 As shown in the figure; the comparison diagram of the antibacterial effect of the plastic wrap in this example is as follows. Figure 6 As shown; the mechanical property test results of this comparative example plastic wrap under 50% relative humidity conditions are as follows. Figure 9 As shown; the representative stress-strain curve of this comparative example plastic wrap under 70% relative humidity is shown in the figure. Figure 10 As shown.

[0076] Comparative Example 3 A method for preparing a single-network composite membrane specifically includes the following steps: (1) Preparation of nanocellulose platform substrate: Sodium periodate was added to CNC suspension (the suspension was made with deionized water as solvent, the amount of CNC added was 0.01 g / mL, and the pH was 3), and a light-protected oxidation reaction was carried out at 40℃ for 4 h. The amount of sodium periodate added to the CNC suspension was 0.036 g / mL. Then, ethylene glycol was added to terminate the reaction at a volume ratio of 5:100 between ethylene glycol and CNC suspension. The reaction was then dialyzed to neutral and lyophilized to obtain DACNC with a surface aldehyde content of 1.2 mmol / g.

[0077] (2) Preparation of single-network composite membrane: Prepare an acetic acid solution of CS, in which the content of CS is 2% by mass and the content of acetic acid is 1% by mass. Add glycerol to the acetic acid solution of CS to form a CS matrix solution, in which the content of glycerol is 0.6% by mass. Prepare a suspension of DACNC using deionized water and DACNC and mix it by ultrasonication. The concentration of DACNC in the suspension is 0.01 g / mL. Add the acetic acid solution of CS to the DACNC suspension at a mass ratio of 17:1. Perform ultrasonic degassing treatment for 10 min at an ultrasonic frequency of 40 kHz. Then pour it into a mold and dry it for 36 h at 50 ℃ and 50% relative humidity to form a single-network composite membrane with a thickness of 0.079 mm, i.e., CD.

[0078] The appearance score, 8-day weight loss, and 8-day total phenols of blueberries stored using this comparative example's preservation film are shown in Table 1; the transmission spectrum analysis of this comparative example's preservation film is shown in... Figure 4 As shown in the figure; the ABTS free radical scavenging rate analysis of the comparative example plastic wrap is shown in the figure. Figure 5 As shown in the figure; the comparison diagram of the antibacterial effect of the plastic wrap in this example is as follows. Figure 6 As shown; the mechanical property test results of this comparative example plastic wrap under 50% relative humidity conditions are as follows. Figure 9 As shown; the representative stress-strain curve of this comparative example plastic wrap under 70% relative humidity is shown in the figure. Figure 10 As shown.

[0079] Application example: The application of a chitosan-based active preservation film in blueberry preservation includes the following steps: Undamaged commercially available blueberries were randomly selected, washed, and air-dried. Eight blueberries were placed in a sterile petri dish as one group. They were then fully covered with plastic wrap as described in Examples 1-4 and Comparative Examples 1-3, and the edges were sealed with tape. The treated blueberries were stored at 25°C and approximately 50% RH for 8 days. The weight changes of the blueberries were recorded regularly, and the degree of spoilage was observed. The preservation effect of the plastic wrap was evaluated by the changes in the appearance of the blueberries after storage, the weight loss rate, and the total phenol content. The specific evaluation method was as follows: Three parallel samples were taken from each of the different treatment groups of blueberries, and the initial mass W0 (g) of each sample was measured. During storage, samples were taken at set time intervals, and the mass W0 of each period was measured. i (g), and calculate the weight loss rate using the following formula: .

[0080] After storage, blueberry pulp was homogenized, and the total phenol (TP) content was determined using the Folin-Ciocalteu method to assess the retention of nutrients within the fruit.

[0081] Blueberry appearance is evaluated using a five-point scale, with the main criteria being berry plumpness, skin color, degree of wrinkling, and rot: 5 points for bright, uniformly colored, plump berries without rot; 4 points for slightly dull berries with mild wrinkling; 3 points for berries showing early rot; 2 points for berries that are noticeably wrinkled or have localized rot; and 1 point for berries that are shriveled, moldy, or severely rotten.

[0082] The specific evaluation results of blueberry preservation are shown in Table 1.

[0083] Table 1 Note: Comparative Example 3 is the control group of the single-network composite membrane. Its test data is used to illustrate the performance of the film in water regulation, nutrient retention and appearance maintenance in blueberry storage and preservation when the PLGA active network is missing.

[0084] Comparative Example 1 (commercially available polyethylene (PE) food preservation film) showed a low weight loss rate of approximately 9.05% on day 8. However, due to the extremely low moisture permeability of the PE material, the water vapor produced by the blueberries' respiration and metabolism quickly reached saturation on the inner surface of the film and underwent a phase change. The blueberry appearance morphology change graph clearly shows that Comparative Example 1 exhibited significant condensation (condensation) inside the packaging from day 2 of storage. This precipitation of liquid water provided an ideal microenvironment for the growth of pathogens. Because the PE film lacked an active inhibitory mechanism, large-scale mold infection began to appear on the blueberries from day 6 of storage. By day 8, the fruit surface was covered with a thick layer of white mycelium, corresponding to the area marked by the red dotted circle in the blueberry appearance morphology change graph. Its appearance score was only 1 point, completely losing its commercial value. The blueberry total phenol content change graph showed that the total phenol content was approximately 2.95 mg / g on day 4, decreasing to approximately 1.91 mg / g on day 8, indicating that the high-humidity spoilage environment severely damaged the internal quality of the fruit.

[0085] For Comparative Example 2 (pure CS film), its moisture regulation capability was relatively weak. The results of tests on appearance changes, weight loss rate, total phenol content, and the blueberry weight loss rate change graph showed that the weight loss rate increased sharply over time, reaching approximately 21.5% on day 8. Visually, the blueberries in Comparative Example 2 underwent severe dehydration during storage; the fruit surface showed obvious wrinkling starting on day 4, and the fruit was completely shriveled by day 8. This indicates that the single polysaccharide film-forming matrix, due to its insufficiently tight molecular chain packing and susceptibility to interference from polar water molecules, failed to form an efficient moisture barrier. The blueberry total phenol content change graph showed that the total phenol content was approximately 3.45 mg / g on day 4, decreasing to approximately 2.73 mg / g on day 8. Consequently, its mechanical integrity and moisture barrier properties were insufficient to meet the long-term storage and transportation requirements of perishable fruits, resulting in an appearance score of only 2 points.

[0086] For Comparative Example 3 (single-network composite membrane CD), the weight loss rate on day 8 was approximately 15.7%, indicating that while the introduction of DACNC alone could improve the water barrier and structural support of the pure CS membrane to some extent, it was still insufficient to inhibit water loss from blueberries. Meanwhile, the total phenol content of Comparative Example 3 on day 8 was only approximately 2.44 mg / g, and the appearance score was 2 points, indicating that the blueberries still showed significant shrinkage or localized spoilage in the later stages of storage, resulting in poor overall preservation performance.

[0087] The poor performance of Comparative Example 3 is due to the fact that this system consists only of a single-network composite structure of CS and DACNC, lacking a PLGA flexible active network grafted with GA. This prevents it from simultaneously providing high-density phenolic hydroxyl groups, ε-polylysine antibacterial activity, and the energy dissipation and interfacial synergistic effects of the flexible network, resulting in weak synergistic regulation of antioxidant, antibacterial, and moisture migration capabilities. In the high-humidity packaging environment of blueberries, the hygroscopic softening of the CS matrix and the insufficient interfacial synergy of the single rigid filler further limit the humidity-adaptive structural stability of the film, ultimately leading to poor weight loss control, total phenol retention, and appearance maintenance.

[0088] In comparison, Example 1 of this invention (chitosan-based active preservation film CDPG3) exhibits superior overall performance in blueberry preservation. As shown by the results of tests on appearance changes, weight loss rate, total phenol content, and the blueberry weight loss rate change graph, Example 1 achieved a weight loss rate of approximately 11.4% for blueberries on day 8, demonstrating a balance between weight loss inhibition and water metabolism regulation. The total phenol content change graph shows that the total phenol content was approximately 3.76 mg / g on day 4 and approximately 3.39 mg / g on day 8. Regarding the water regulation mechanism, this invention utilizes the synergistic effect of a rigid DACNC framework and a flexible PLGA network forming a dense covalent network in a CS matrix via Schiff base reaction and amide bonds. This enhances the hydrophobicity of the film, effectively inhibiting excessive water vapor penetration and helping to improve water migration during packaging, thereby mitigating the condensation problem that easily occurs in PE film systems. This relatively balanced water regulation capability, combined with the film's UV absorption characteristics, allows blueberries to maintain their firm and plump sensory characteristics after 8 days of storage, achieving an appearance score of 5.

[0089] Tests on the preservation films of Examples 2-4 showed that all examples could form chitosan-based active preservation films, and all exhibited good preservation effects during blueberry storage. Compared with the mold growth induced by condensation in Comparative Example 1 and the severe shrinkage caused by moisture loss in Comparative Example 2, the treatment groups of Examples 2-4 did not show large-area mold spots or obvious rot after 8 days of storage, and the fruit shape was well maintained. This indicates that the composite film can form a good synergistic effect between moisture migration regulation, active antioxidant and antibacterial effects.

[0090] Specifically, blueberries treated with the CDPG1 preservation film obtained in Example 2 maintained a relatively intact fruit shape in the early stage of storage, and no obvious white mycelium or large-area soft rot was observed, with an appearance score of 4 points; the weight loss rate on the 8th day was about 13.8%, indicating that the film can effectively slow down the loss of fruit moisture; at the same time, the total phenolic content of blueberries remained at about 2.76 mg / g on the 8th day, indicating that the preservation film of Example 2 can delay the degradation of phenolic nutrients in blueberries to a certain extent. The preservation film of this example has excellent comprehensive preservation performance.

[0091] Blueberries treated with the CDPG2 preservation film obtained in Example 3 exhibited excellent preservation effects. After 8 days of storage, the berries remained relatively plump, with a uniform skin color and no obvious mold growth, resulting in an appearance score of 4. The weight loss rate on the 8th day decreased to approximately 12.6%, indicating that as the matching degree between the PLGA active network and the CS matrix improved, the film's ability to regulate moisture migration in the blueberry packaging microenvironment was enhanced. Correspondingly, the total phenol content of the treatment group in Example 3 was approximately 2.78 mg / g on the 8th day, demonstrating its excellent antioxidant protection and nutrient retention capabilities.

[0092] Blueberries treated with the CDPG4 preservation film obtained in Example 4 maintained good roundness and surface integrity after 8 days of storage. The overall color of the fruit remained stable, and no obvious condensation-induced mold coverage was observed, with an appearance score of 5 points. The weight loss rate on the 8th day was approximately 11.9%, indicating that the film has strong moisture barrier and regulation capabilities. The total phenol content on the 8th day was approximately 2.94 mg / g, showing a good retention effect of phenolic substances.

[0093] Furthermore, based on the changes in appearance, weight loss rate, total phenol content test results, and the total phenol content determination results of the blueberry total phenol content change graph, the treatment group in the example can achieve a good preservation effect on the nutritional quality of blueberries after 8 days of storage. This indicates that introducing the PL component grafted with GA and DACNC into the CS system helps to improve the overall performance of the film in the blueberry preservation process, further proving that the chitosan-based active preservation film system of the present invention has good process adaptability and preservation stability.

[0094] The blueberry preservation results of this invention are not only due to the antioxidant or antibacterial functions of the film, but also closely related to its ability to maintain good structural integrity, achieve reasonable water migration regulation, and maintain active expression in a high-humidity packaging environment.

[0095] In this invention, GA is not directly added to the CS film-forming system in the form of free small molecules, but is first grafted onto the PL chain segment and then participates in subsequent composite assembly. This design is not only used to endow the film with antioxidant and antibacterial functions, but more importantly, it is used to construct a composite network with good stability under high humidity conditions together with DACNC and CS matrix, so that the film can simultaneously provide structural support, activity expression and water migration regulation during blueberry storage.

[0096] When chitosan-based active preservation films with the aforementioned composite structure are used for blueberry packaging, they can, to a certain extent, address the needs for water loss inhibition, condensation reduction, nutrient protection, and spoilage delay. Specifically, the synergistic effect of DACNC and grafted GA helps maintain the film's structural support, providing UV absorption, antioxidant, and antibacterial functions. The resulting composite network helps the film maintain good mechanical reliability and structural integrity under high-humidity packaging conditions, while also maintaining reasonable activity expression and water migration regulation behavior.

[0097] To evaluate the mechanical suitability of the film obtained in this invention under high humidity packaging environments, the films were subjected to equilibration treatment under different relative humidity conditions, and their tensile strength and elongation at break were tested. Before testing, all films were pre-dried at 50°C and 50% relative humidity for 36 hours, and then placed in environments with 30%, 50%, and 70% relative humidity for 12 hours respectively to ensure that each film sample reached a stable moisture-absorbing state before testing.

[0098] Tests showed that the food preservation film obtained in Example 1 of this invention maintained high structural integrity and mechanical reliability even under high humidity conditions. Specifically, at 50% relative humidity, the tensile strength of the pure CS film prepared in Comparative Example 2 was 34.65 MPa and the elongation at break was 49.65%, the tensile strength of the single-network composite film CD prepared in Comparative Example 3 was 47.20 MPa and the elongation at break was 31.75%, while the tensile strength of the food preservation film prepared in Example 1 reached 59.13 MPa and the elongation at break reached 81.31%.

[0099] At a relative humidity of 70% (70RH%), which is closer to the actual working conditions of fruit packaging, the tensile strength of the pure CS film prepared in Comparative Example 2 decreased to 23.05 MPa. Although the elongation at break increased to 91.71%, it showed obvious hygroscopic softening characteristics. The tensile strength of the single-network composite film CD prepared in Comparative Example 3 was 33.09 MPa and the elongation at break was 48.29%. In contrast, the preservation film prepared in Example 1 of this invention still maintained a tensile strength of 45.89 MPa and an elongation at break of 79.22% at 70% relative humidity.

[0100] The above results indicate that the chitosan-based active preservative film obtained in Example 1 not only exhibits good mechanical properties under dry conditions, but also maintains high strength and good deformability under high humidity conditions, demonstrating its excellent humidity-adaptive mechanical stability. This result is significant for the packaging and preservation of blueberries and other fruits with high respiration and transpiration rates, as these fruits easily form a high-humidity microenvironment inside the packaging during storage. Ordinary polysaccharide films often experience structural relaxation, mechanical instability, or even preservation failure due to moisture absorption. However, the film obtained in Example 1 can maintain good structural integrity in this environment, thus providing a structural basis for subsequent water migration and activity expression.

[0101] To evaluate the responsive release behavior of the chitosan-based active preservative film obtained in this invention, the preservative film obtained in Example 1 was placed at 25°C in a pH 3.0 citrate buffer solution with a concentration of 0.1 mol / L, a pH 5.0 acetic acid buffer solution with a concentration of 0.1 mol / L, and deionized water (pH approximately 7.0) for release experiments. The concentration change of GA in the release medium was measured at predetermined time points to analyze the release characteristics of the active component under different acid and alkaline conditions.

[0102] The results show that the film obtained in Example 1 of this invention exhibits a significant pH-responsive release behavior, with a higher release rate and greater release extent under acidic conditions, while the release is relatively slower under neutral conditions. The overall release process from 0 to 48 hours can be fitted using the Korsmeyer-Peppas model, where the release indices n under pH 3.0, pH 5.0, and pH 7.0 conditions are 0.1260, 0.2079, and 0.2917, respectively, all less than 0.45, indicating that the overall release behavior of this system is mainly based on the Fick diffusion mechanism; the corresponding fitting correlation coefficient R... 2 The values ​​are 0.9231, 0.9856, and 0.9754, respectively, indicating that the model can describe the overall release trend of the membrane well.

[0103] Further analysis reveals that the release process of the preservation film in Example 1 of this invention exhibits a distinct multi-stage characteristic: rapid release in the initial stage, followed by a more gradual and sustained release phase. This indicates that the membrane system does not simply experience a rapid loss of free active substances, but rather achieves staged regulation of the release of active components while maintaining membrane structural stability. Particularly under acidic conditions, the membrane system exhibits a higher release tendency, which is beneficial for enhancing activity expression when the fruit storage microenvironment changes, thereby improving the efficiency of antibacterial and antioxidant functions.

[0104] The UV absorption performance and free radical scavenging activity of the preservation films prepared in Examples 1-4, Comparative Example 2 (pure CS film), and Comparative Example 3 (single-network composite film CD) were evaluated. The antibacterial properties were further evaluated using Examples 1, Comparative Example 2, Comparative Example 3, and the blank control group as representatives.

[0105] Regarding the evaluation of UV resistance performance, the specific testing procedure is as follows: A UV-Vis spectrophotometer was used to scan the transmission spectra of each group of films within the wavelength range of 200-800 nm. UV blocking rate (SR) 紫外 The percentage calculation formula is as follows: .

[0106] Where T 平均The average transmittance of the food preservation film in the 200nm-400nm wavelength range is shown. The spectral trend of the transmission spectrum analysis reveals that the transmittance of Comparative Examples 2 and 3 increases rapidly in the transition from near-ultraviolet to visible light, reaching approximately 60% near 400nm, indicating weak interception of high-energy radiation. In contrast, the films of Examples 1-4 of this invention show a significant decrease in overall transmittance in the 200nm-400nm ultraviolet region, with almost no transmittance in the short-wave ultraviolet region, and remain significantly lower than Comparative Examples 2 and 3 near 400nm. These results demonstrate that the introduction of the PLGA active network containing GA units significantly enhances the ultraviolet shielding ability of the film, helping to mitigate the adverse effects of photo-induced oxidation on the quality of packaged food.

[0107] For evaluating antioxidant activity, the in vitro antioxidant capacity of the membrane was determined using the ABTS free radical scavenging method. The specific experimental procedure is as follows: 7.4 mmol / L ABTS ethanol solution and 2.6 mmol / L potassium persulfate solution were mixed at a volume ratio of 1:1 and incubated in the dark for 12 h to generate ABTS free radicals. The solution was then diluted with anhydrous ethanol to an absorbance of 0.70 ± 0.02 at 734 nm to obtain the ABTS working solution. Subsequently, 10 mg of the membrane sample was extracted in 10 mL of anhydrous ethanol for 24 h to obtain the membrane extract. 1 mL of the membrane extract was mixed with 4 mL of the ABTS working solution, reacted in the dark for 30 min, and then the absorbance at 734 nm was measured. The percentage of free radical scavenging rate was calculated using the following formula: .

[0108] Where A 样品 A represents the absorbance after the reaction with the added thin-film extraction solution. 对照 The absorbance was used as a control group to replace the extract with an equal amount of solvent. Experimental data from the ABTS radical scavenging rate analysis showed that the ABTS radical scavenging rate of Comparative Example 2 was approximately 45%, and that of Comparative Example 3 was approximately 70%. The scavenging rate of the treatment group in Example 1 was approximately 96%, and the ABTS radical scavenging rates of Examples 2, 3, and 4 were all approximately 95%–97%. The differences between the examples were small, but all were significantly higher than those of Comparative Examples 2 and 3, indicating that the films of each example possessed excellent antioxidant properties. This demonstrates that the high-density phenolic hydroxyl groups anchored by covalent bonds in the PLGA flexible network endow the film with good free radical scavenging ability, effectively removing free radicals and thus helping to reduce oxidative damage to packaged foods during storage.

[0109] In evaluating the broad-spectrum antibacterial properties, the film contact method was used to investigate the inhibitory effects of the film on Staphylococcus aureus and Escherichia coli. The specific experimental procedure is as follows: After sterilizing the film sample with ultraviolet light, 0.2g of the sample was added to a solution containing approximately 1×10⁻⁶... 6The film was incubated in 4 mL of sterile LB broth with a CFU / mL bacterial suspension at 37°C and 180 rpm for 24 h with shaking to ensure full contact between the film and the bacterial cells. The blank control group received no film sample, only a mixture containing approximately 1×10⁻⁶ CFU / mL bacterial suspension. 6 Four mL of sterile LB broth was used to prepare a CFU / mL bacterial suspension, and the mixture was incubated with shaking for 24 h under the same conditions. After incubation, 100 µL of the bacterial suspension from each culture tube was evenly spread onto Mueller-Hinton agar plates and incubated at 37°C for another 24 h. The antibacterial effect was then observed and colony counts were performed. The percentage of inhibition rate was calculated using the following formula: .

[0110] Wherein B represents the average total colony count in the culture dishes of the blank control group, and C represents the average total colony count in the culture dishes of the experimental group. The blank control group was a control group that did not add the film sample, but only contained bacterial suspension and LB broth, and was treated under the same shaking culture, plating, and agar plate culture conditions. The comparison of antibacterial effects shows that the blank control group had the densest colonies. Comparative Examples 2 and 3 still showed a large number of colonies growing after treatment. Although Comparative Example 2 showed a reduction compared to the blank control group, its antibacterial effect was limited, with an inhibition rate of approximately 17.1% against Staphylococcus aureus and approximately 35.0% against Escherichia coli. Comparative Example 3, due to the formation of only a CS / DACNC single-network complex structure and the lack of PLGA active components, did not show sufficient antibacterial activity, with an inhibition rate of approximately 13.2% against Staphylococcus aureus and approximately 28.0% against Escherichia coli. In stark contrast, the plate colonies in the treatment group of Example 1 of the present invention were significantly reduced, and it showed a strong inhibitory effect on both representative pathogenic bacteria, with an inhibition rate of about 95.3% against Staphylococcus aureus and about 87.5% against Escherichia coli.

[0111] The above results show that the film prepared in Example 1 has excellent performance in terms of UV shielding, anti-oxidation and antibacterial properties, indicating that introducing the PL component grafted with GA and DACNC into the CS system helps to improve the overall preservation-related performance of the film.

[0112] The above results demonstrate that the chitosan-based active preservative film obtained in this invention possesses both structural stability under high humidity conditions and responsive active release behavior under acidic conditions. This characteristic distinguishes it from ordinary physical blend films of active components, enabling it to maintain good mechanical reliability in the high humidity packaging environment of blueberries and to achieve more targeted functional expression under changes in the storage microenvironment, thus making it more suitable for blueberry preservation applications.

[0113] The biocompatibility and environmental friendliness of the chitosan-based active food preservation film described in this invention were systematically evaluated. Biocompatibility testing employed a cytotoxicity assay, specifically the MTT assay. The specific experimental procedure was as follows: Mouse fibroblast L929 cells were selected as model cells. First, the food preservation film sample was immersed in serum-free culture medium for 24 hours to prepare an extract. Then, cells were cultured at a density of 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells / well in 96-well plates and pre-cultured overnight at 37°C and 5% CO2. Then, cells were co-cultured with different concentrations (0.1 μL / mL, 1 μL / mL, 10 μL / mL, 50 μL / mL) of plastic wrap extract for 48 h. A control group containing only cells and culture medium (without plastic wrap extract) and a blank group containing only culture medium (without cell seeding) were also set up. After culture, the culture medium was discarded, and the cells were washed with 1×PBS buffer (0.01 mol / L, pH 7.4). 100 μL of 0.5 mg / mL MTT solution was added to each well, and the cells were incubated at 37°C and 5% CO2 for 4 h. The supernatant was then discarded, and 100 μL of dimethyl sulfoxide (DMSO) was added to dissolve the resulting purple formazan crystals. After gentle shaking for 10 min, the absorbance (OD) of each well was measured at 570 nm using a microplate reader. The percentage of relative cell viability was calculated using the following formula: .

[0114] Among them, OD 实验组 The absorbance of the culture group containing the plastic wrap extract, OD 对照组 The absorbance and OD values ​​are for the control group containing only cells and culture medium, without the addition of plastic wrap extract. 空白组 The absorbance is for the blank group containing only culture medium and no cells inoculated.

[0115] The experimental results shown in the cytotoxicity analysis diagram indicate that when the concentration of the preservative film extract from Example 1 is below 10 μL / mL, the relative cell viability remains above 90%, demonstrating good biocompatibility. This result demonstrates that the preservative film extract obtained in this invention exhibits good cell compatibility within the measured concentration range, indicating a sound safety basis for its packaging applications.

[0116] Soil degradation performance was evaluated using the burial method. The specific experimental procedure was as follows: Plastic wrap samples were cut to uniform sizes and buried in naturally moist soil with a humidity level maintained at approximately 40% at a depth of 10 cm. Samples were removed every 30 days and washed with deionized water, and their macroscopic morphological changes were observed. The soil degradation morphological change diagram shows that the plastic wrap of Example 1 of this invention gradually broke down and degraded over time during the burial process, indicating that the film still retained the degradable characteristics of the CS and CNC-based materials.

[0117] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for preparing a chitosan-based active food preservation film, characterized in that, Specifically, the steps include: (1) Preparation of functionalized flexible network precursor reaction solution: Gallic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide were added to buffer solution in a molar ratio of 1:1:1 to activate the solution and obtain an activation solution. The activation solution was then added dropwise to an ε-polylysine solution with a concentration of 0.13~0.39mol / L in a volume ratio of 1:1 to ε-polylysine solution to carry out the grafting reaction and obtain the precursor reaction solution. (2) Preparation of aldehyde-modified nanocellulose platform substrate: An oxidant was added to the cellulose nanocrystal suspension to carry out an oxidation reaction, and then a terminator was added to terminate the reaction. The substrate was then purified and dried to obtain the aldehyde-modified nanocellulose platform substrate. (3) Preparation of particle dispersion: Prepare a suspension of aldehyde-modified nanocellulose platform substrate with a concentration of 0.01 g / mL. Then, add the suspension of aldehyde-modified nanocellulose platform substrate to the precursor reaction liquid in a stirring state at a volume ratio of 1:(2.5~8). After the addition is completed, stir the reaction to obtain a mixture. Purify the mixture to obtain a purified particle dispersion. (4) Preparation of composite film-forming solution: Add plasticizer to the acetic acid solution of chitosan. The content of chitosan in the acetic acid solution of chitosan is 1.5%~2.5% by mass percentage, and the content of acetic acid is 1% by mass percentage to obtain chitosan matrix solution. The content of plasticizer in chitosan matrix solution is 0.4%~0.8% by mass percentage. Add the purified particle dispersion obtained in step (3) to the chitosan matrix solution in a mass ratio of (0.8~1.2):

1. Then perform ultrasonic degassing treatment to obtain composite film-forming solution. (5) Preparation of chitosan-based active food preservation film: The composite film-forming liquid is cast into a mold and then dried to obtain chitosan-based active food preservation film.

2. The method for preparing the chitosan-based active food preservation film according to claim 1, characterized in that, In step (1), the buffer solution is a MES buffer with a pH of 5.5 and a concentration of 0.1 mol / L; the amount of gallic acid added in the activation solution is 0.29 mol / L; the activation conditions are: stirring at 300-500 rpm for 0.5-1 h under ice-water bath conditions.

3. The method for preparing the chitosan-based active food preservation film according to claim 1, characterized in that, In step (1), the ε-polylysine solution is prepared by ε-polylysine and MES buffer solution with pH 5.5 and a concentration of 0.1 mol / L; the dropping rate of the activation solution added to the precursor reaction solution is 10~15 mL / min; the grafting reaction conditions are: reaction in the dark for 12~24 h.

4. The method for preparing the chitosan-based active food preservation film according to claim 1, characterized in that, In step (2), the amount of cellulose nanocrystals added to the cellulose nanocrystal suspension is 0.01 g / mL; the amount of oxidant added to the cellulose nanocrystal suspension is 0.036 g / mL; and the conditions for the oxidation reaction are: reaction at 40°C in the dark for 4 hours.

5. The method for preparing the chitosan-based active food preservation film according to claim 1, characterized in that, In step (2), the oxidant is sodium periodate; the terminator is ethylene glycol.

6. The method for preparing the chitosan-based active food preservation film according to claim 1, characterized in that, In step (3), the dropping rate of the aldehyde-modified nanocellulose platform substrate suspension added to the precursor reaction solution is 10~15 mL / min; the stirring reaction conditions are: reaction in the dark at 300~500 rpm and 25~45℃ for 4~6 h.

7. The method for preparing the chitosan-based active food preservation film according to claim 1, characterized in that, The plasticizer in step (4) is glycerin.

8. The method for preparing the chitosan-based active food preservation film according to claim 1, characterized in that, The thickness of the chitosan-based active preservative film in step (5) is 0.083~0.085 mm.

9. The chitosan-based active food preservation film prepared by the method according to any one of claims 1 to 8.

10. The application of the chitosan-based active preservation film according to claim 9 in the preservation of blueberries.

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