Chitosan composite film and preparation method and application thereof

Abstract

The application discloses a chitosan composite film and a preparation method and application thereof, and belongs to the technical field of food preservation materials. The chitosan composite film comprises the following raw materials: chitosan, zein, Span 80, a natural eutectic solvent and aesculin. The preparation method comprises the following steps: mixing chitosan solution, zein solution, Span 80, the natural eutectic solvent and aesculin, stirring and ultrasonic treatment, and preparing a film-forming solution; and the film-forming solution is dried through a flow casting method to prepare the chitosan composite film. That is, the chitosan is blended with the zein, the natural eutectic solvent is used for plasticizing, and the aesculin is used for functionalization, so that the mechanical properties, the antibacterial property and the antioxidant property of the film are synergistically improved, and the fish preservation period is significantly prolonged.

Description

Technical Field

[0001] This invention belongs to the field of food preservation materials technology, and particularly relates to a chitosan composite film, its preparation method and application. Background Technology

[0002] Fish preservation plays a crucial role in maintaining its nutritional quality and economic value. Currently, traditional preservation packaging materials generally suffer from drawbacks such as difficulty in degradation, environmental pollution, and susceptibility to bacterial growth. To overcome the technological bottlenecks of traditional packaging materials, sustainable packaging technology is gradually becoming an important direction for industry development. Packaging materials with biosafety, biodegradability, and environmental friendliness are gradually replacing traditional petroleum-based materials in the food packaging field.

[0003] Natural biopolymers, with their biodegradability and environmental friendliness, have demonstrated strategic application value in the food packaging materials field. Among them, chitosan, due to its excellent biocompatibility, superior film-forming properties, and significant antioxidant and antibacterial potential, is widely recognized as one of the most promising green packaging substrates. However, pure chitosan films suffer from drawbacks such as poor mechanical strength and limited functionality, which restricts their practical application.

[0004] To address this issue, existing research often employs inorganic nanomaterials or synthetic plasticizers for modification. However, such methods may lead to risks such as reduced biocompatibility and toxic residues, posing potential threats to food safety and the ecological environment.

[0005] Therefore, developing chitosan composite films that combine excellent bioactivity and suitable mechanical strength is of great application value and practical significance for advancing fish preservation technology. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a chitosan composite film, its preparation method, and its applications.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A chitosan composite film comprising the following raw materials: chitosan, zein, Span 80, natural eutectic solvent and fraxin.

[0009] Beneficial effects: The reasons for limiting fraxin in the chitosan composite film disclosed in this invention are as follows:

[0010] Fraxinus quinone possesses both antibacterial and antioxidant activities, distinguishing it from single-function antibacterial agents and making it more suitable for the preservation needs of aquatic products. Fraxinus quinone achieves its antibacterial effect by disrupting the integrity of bacterial cell membranes and inhibiting the activity of key metabolic enzymes. It also exerts its antioxidant effect by scavenging free radicals through hydrogen donation and electron transfer via its phenolic hydroxyl groups.

[0011] Secondly, fraxin is a coumarin compound extracted from Fraxinus chinensis (a natural plant), possessing advantages such as non-toxicity and high biocompatibility. It does not release harmful metabolites into food, avoiding the risk of chemical antibacterial agent residues while preserving the protein, lipids, and other nutrients in food. Choosing fraxin meets the core requirement of "non-toxic and residue-free food contact materials."

[0012] Furthermore, fraxetine's molecular structure contains a coumarin core and glycoside substituents, exhibiting excellent chemical stability. It is not easily volatile, possesses good thermal stability, and will not be deactivated during processing. Through a hydrogen-bonded network, it can be uniformly dispersed in the membrane matrix, avoiding insufficient exposure of active sites due to aggregation, thus achieving a slow release of antibacterial and antioxidant activities and extending the shelf life.

[0013] Finally, fraxetin can compensate for the shortcomings of chitosan membranes, broadening the antibacterial spectrum, enhancing antioxidant capacity, and improving the membrane's moisture resistance. Fraxetin has inhibitory effects on both Gram-positive and Gram-negative bacteria. The phenolic hydroxyl groups of fraxetin can synergize with the antioxidant effects of chitosan, significantly improving the membrane's free radical scavenging rate. The coumarin core of fraxetin is a hydrophobic group, which can reduce the surface hydrophilicity of the membrane, decrease water adsorption, and indirectly enhance the antibacterial durability.

[0014] Furthermore, the synergistic mechanism of fraxin and other raw materials in the chitosan composite film is as follows:

[0015] 1. Synergistic effect with chitosan: The amino groups on the chitosan molecular chain form a hydrogen bond network with the phenolic hydroxyl groups of fraxin, which enhances the dispersibility of fraxin in the membrane matrix and prevents precipitation; Chitosan disrupts the bacterial cell membrane through electrostatic interaction, increasing the permeability of the cell membrane, allowing fraxin to enter the bacteria and inhibit the activity of key metabolic enzymes, thereby improving the antibacterial efficiency.

[0016] 2. Synergistic effect with natural eutectic solvents: Fraxinol is slightly hydrophobic and has low solubility in pure chitosan solution. Natural eutectic solvents can improve its solubility and dispersion uniformity in the membrane, avoiding membrane structure defects caused by excessively high local concentrations. In addition, the hydrogen bond network of natural eutectic solvents can encapsulate fraxinol molecules, reducing their contact with the external environment, delaying oxidative degradation, and thus prolonging the duration of antibacterial and antioxidant activities. Furthermore, natural eutectic solvents, as plasticizers, can enhance the mechanical strength of the membrane, compensating for the increased membrane brittleness that may be caused by the addition of fraxinol.

[0017] 3. Synergistic effect with Span 80: Span 80 can reduce the interfacial tension between different components and prevent fraxin and zein from agglomerating, thereby ensuring the uniform distribution of active ingredients.

[0018] 4. Synergistic effect with zein: The hydrophobic groups of zein form a hydrophobic interaction with the coumarin core of fraxin, while the amino groups of zein form hydrogen bonds with the phenolic hydroxyl groups of fraxin. Together, they enhance the membrane's density, improve its barrier properties against oxygen and water vapor, and reduce the oxygen supply required for bacterial growth. In addition, the dense structure formed by the two can act as a physical barrier to slow down the release rate of fraxin from the membrane to the food surface, preventing the rapid loss of active ingredients. At the same time, the antioxidant properties of fraxin can protect zein from oxidative degradation and extend the membrane's lifespan.

[0019] Optionally, the chitosan composite film comprises the following raw materials in parts by weight:

[0020] 25 parts chitosan, 25 parts zein, 0.05 parts Span 80, 1-5 parts natural eutectic solvent and 0.1-0.3 parts fraxin.

[0021] Optionally, the chitosan has a molecular weight of 150,000 Da and a degree of deacetylation ≥95%;

[0022] The molecular weight of the zein is 20 kDa.

[0023] Optionally, the natural eutectic solvent includes hydrogen bond acceptors and hydrogen bond donors;

[0024] The hydrogen bond acceptor is betaine, and the hydrogen bond donor is D,L-lactic acid.

[0025] Beneficial effects: This invention specifies betaine and D,L-lactic acid as hydrogen bond acceptor and hydrogen bond donor, respectively, because:

[0026] 1. Betaine is mainly found in sugar beets, while lactic acid is mainly produced by microbial fermentation. Both are commonly used in the food industry and have excellent biocompatibility, avoiding the toxicity risks of traditional chemical solvents.

[0027] 2. Both betaine and lactic acid are natural small-molecule organic compounds that can be degraded by microorganisms in the natural environment without causing environmental pollution. Composite films prepared with bio-based raw materials such as chitosan and fraxin are degradable, meet the requirements of sustainable packaging, and are more green and environmentally friendly.

[0028] 3. Betaine contains quaternary ammonium salt groups and carboxyl groups, while lactic acid contains hydroxyl and carboxyl groups; the two readily form a multi-hydrogen bond network. The resulting eutectic solvent has the advantages of a low melting point and stable physicochemical properties. It can disperse other raw materials without high-temperature heating, effectively preventing the deactivation of other components due to high temperatures. It does not chemically react with other raw materials, nor does it interact with proteins or lipids in fish to produce off-flavors or harmful substances.

[0029] 4. The zwitterionic properties of betaine can reduce the interfacial tension between hydrophilic and hydrophobic components, prevent aggregation, and ensure that fraxin is evenly distributed in the membrane matrix, maximizing the exposure of antibacterial and antioxidant active sites.

[0030] 5. Lactic acid can synergistically enhance the preservation effect. The weak acidity of lactic acid can moderately lower the pH value of the membrane surface, inhibiting the growth of Gram-negative bacteria, and forming a synergistic effect with the antibacterial effect of fraxin. In addition, the hydroxyl groups of lactic acid can participate in free radical scavenging, and synergistically enhance the antioxidant activity of the membrane with the phenolic hydroxyl groups of fraxin, thus more effectively inhibiting lipid oxidation in fish meat.

[0031] 6. Both betaine and lactic acid are raw materials for large-scale industrial production. They are inexpensive and have a stable supply, which reduces production costs and improves the feasibility of practical applications.

[0032] Furthermore, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:2.

[0033] Furthermore, the preparation process of the natural eutectic solvent is as follows:

[0034] The hydrogen bond donor and hydrogen bond acceptor were heated and stirred at 70°C for 20 min, and then cooled to obtain the natural eutectic solvent.

[0035] The method for preparing the above-mentioned chitosan composite film includes the following steps:

[0036] Chitosan solution, zein solution, Span 80, natural eutectic solvent and fraxin are mixed, stirred and ultrasonically treated to prepare a film-forming solution;

[0037] The chitosan composite film is obtained by casting the film-forming solution and then drying it.

[0038] Optionally, the chitosan solution is prepared by dissolving chitosan in an acetic acid solution.

[0039] Furthermore, the ratio of chitosan to acetic acid solution is 2 g: 100 mL;

[0040] The concentration of the acetic acid solution is 1 vol.

[0041] Optionally, the zein solution is obtained by dissolving zein in an ethanol solution.

[0042] Furthermore, the ratio of the amount of zein to ethanol solution used is 2 g: 100 mL;

[0043] The concentration of the ethanol solution is 90 vol.

[0044] Optionally, the stirring and ultrasonic treatment conditions are as follows: stirring at 800 rpm for 30 min, followed by ultrasonication at 80W power for 10 min.

[0045] The above-mentioned chitosan composite film is used in fish preservation.

[0046] Optionally, the fish is the pearl giant grouper.

[0047] Compared with the prior art, the present invention has the following advantages and technical effects:

[0048] The chitosan composite film disclosed in this invention is prepared by using chitosan, zein, Span 80, natural eutectic solvent and fraxin as raw materials, and can be used as a packaging material for fish preservation.

[0049] Among them, natural eutectic solvents, as plasticizers, can effectively improve the mechanical properties of chitosan composite films, exhibiting excellent tensile strength and elongation at break, achieving a good balance between strength and ductility.

[0050] Fraxin, as a bioactive ingredient, can significantly enhance antioxidant capacity and antibacterial (Staphylococcus aureus, Escherichia coli) effects, thus extending the shelf life of fish.

[0051] The chitosan composite film disclosed in this invention provides a new green alternative material for the food packaging field, effectively reducing the environmental pollution caused by plastic packaging. It also has good application prospects in the field of fish preservation, which can better maintain the quality of fish and reduce food waste, thus providing strong support for the sustainable development of the food industry. Attached Figure Description

[0052] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0053] Figure 1 Fourier transform infrared spectra of betaine, lactic acid, and the natural eutectic solvent prepared in Example 1.

[0054] Figure 2 Fourier transform infrared spectra of chitosan, zein, Span 80, the natural eutectic solvent prepared in Example 1, fraxin, and the films prepared in Examples 2 and 7.

[0055] Figure 3 X-ray diffraction spectra of the natural eutectic solvent prepared in Example 1, and the thin films prepared in Examples 2, 3, 5 and 7.

[0056] Figure 4Scanning electron microscope images of the films prepared in Examples 2-5 and Example 7.

[0057] Figure 5 Atomic force microscopy images of the thin films prepared in Examples 2, 3, 5 and 7.

[0058] Figure 6 Tensile strength test results for chitosan composite films with different raw materials and different amounts of natural eutectic solvents.

[0059] Figure 7 The results of elongation at break tests on chitosan composite films with different raw materials and different amounts of natural eutectic solvents.

[0060] Figure 8 The water vapor transmission rate test results of chitosan composite films with different raw materials and different amounts of natural eutectic solvents.

[0061] Figure 9 Thermogravimetric analysis results of chitosan composite films with different raw materials and different amounts of natural eutectic solvents.

[0062] Figure 10 The results of the antibacterial zone test for chitosan composite films with different raw materials and different amounts of fraxin.

[0063] Figure 11 The antioxidant test results are for chitosan composite films with different raw materials and different amounts of fraxin.

[0064] Figure 12 The results show the application of the chitosan composite film prepared in Example 7 in the preservation of pearl grouper. Detailed Implementation

[0065] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0066] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0067] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0068] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0069] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0070] The chitosan composite film disclosed in this invention achieves a good balance between strength and ductility by adding a natural eutectic solvent as a plasticizer, and the interfacial compatibility between the components is enhanced through hydrogen bonding. The addition of fraxin significantly improves its antibacterial and antioxidant capabilities, effectively extending the shelf life of fish and enhancing its freshness preservation. As a chitosan composite film possessing both excellent bioactivity and suitable mechanical strength, this invention provides strong support for advancing fish preservation technology.

[0071] The first aspect of the present invention provides a chitosan composite film comprising the following raw materials: chitosan, zein, Span 80, natural eutectic solvent and fraxin.

[0072] In some optional embodiments, the chitosan has a molecular weight of 150,000 Da and a degree of deacetylation ≥95%. Due to its good biocompatibility, film-forming ability, and antibacterial properties, it can be applied in the field of green packaging materials.

[0073] The molecular weight of zein is 20 kDa, and its combination with chitosan can effectively improve the barrier properties and stability of chitosan-based membranes.

[0074] Span 80, also known as dehydrated sorbitol fatty acid ester, can stabilize the system as an emulsifier and is a promising carrier for encapsulating hydrophilic components.

[0075] Fraxin is a coumarin compound with antibacterial and antioxidant properties.

[0076] The natural eutectic solvent includes a hydrogen bond acceptor and a hydrogen bond donor; the hydrogen bond acceptor is betaine, and the hydrogen bond donor is D,L-lactic acid (CAS No.: 50-21-5); the molar ratio of hydrogen bond acceptor to hydrogen bond donor is 1:2; the above-mentioned natural eutectic solvent as a plasticizer can effectively improve the mechanical properties of chitosan-based membranes, and compared with traditional organic solvents as hydrogen bond donors and acceptors, it has the significance of being green and environmentally friendly.

[0077] In some alternative embodiments, the preparation method of the natural eutectic solvent includes the following steps:

[0078] The hydrogen bond donor and hydrogen bond acceptor are heated at 70°C and stirred for 20 min until a clear, transparent and homogeneous liquid is formed. After cooling, a natural eutectic solvent is obtained.

[0079] The second aspect of this invention provides a method for preparing the chitosan composite film of the first aspect, comprising the following steps:

[0080] Chitosan solution, zein solution, Span 80, natural eutectic solvent and fraxin were mixed to prepare a film-forming solution; the film-forming solution was then dried by casting to obtain a chitosan composite film.

[0081] In some alternative embodiments, the chitosan solution is a 1 vol% acetic acid solution containing chitosan;

[0082] In some alternative embodiments, the ratio of chitosan to solvent in the chitosan solution is 2 g: 100 mL.

[0083] In some alternative embodiments, the zein solution is a 90 vol% ethanol solution containing zein;

[0084] In some alternative embodiments, the ratio of zein to solvent in the zein solution is 2 g: 100 mL.

[0085] In some optional embodiments, the method for preparing the chitosan composite film specifically includes the following steps:

[0086] Chitosan solution and zein solution were magnetically stirred and mixed, and then Span 80, natural eutectic solvent and fraxin were added sequentially and magnetically stirred and mixed to obtain film-forming solution;

[0087] Chitosan composite films were prepared by casting the film-forming solution and then drying it.

[0088] In some alternative embodiments, the magnetic stirring time is 30 minutes.

[0089] In some alternative embodiments, the drying time is 24 hours.

[0090] The third aspect of this invention provides the application of the chitosan-based composite film from the first aspect in the field of fish preservation. The fish in this application is the pearl grouper.

[0091] Unless otherwise specified, "room temperature" in this invention refers to 20-30℃.

[0092] Unless otherwise specified, the term "parts" in this invention refers to parts by weight.

[0093] All raw materials used in this invention were purchased from the market.

[0094] The technical solution of the present invention will be further illustrated by the following embodiments.

[0095] Example 1

[0096] The specific steps for preparing chitosan solution, zein solution, and natural eutectic solvent are as follows:

[0097] Preparation of chitosan solution: Dissolve 2 g of chitosan in 100 mL of 1 vol% acetic acid solution until completely dissolved to form a chitosan solution with a mass concentration of 2 wt%, which will be used for the subsequent preparation of film-forming solution.

[0098] Preparation of zein solution: Dissolve 2 g of zein in 100 mL of 90 vol% ethanol solution until completely dissolved to form a 2 wt% zein solution, which will be used for the subsequent preparation of film-forming solution.

[0099] Preparation of natural eutectic solvent: Betaine and D,L-lactic acid are mixed in a molar ratio of 1:2 and magnetically stirred in a water bath at 70°C for 20 min until a clear, transparent and homogeneous liquid is formed; the mixture is cooled to room temperature to obtain the natural eutectic solvent, which is stored in a dry container for later use; the obtained natural eutectic solvent is transparent and viscous, with no impurities precipitated, and is used for the preparation of subsequent film-forming solutions.

[0100] Example 2

[0101] The specific steps for preparing chitosan and zein (CZ) films are as follows:

[0102] Add 25 g of zein solution to 25 g of chitosan solution, stir at 800 rpm for 30 min, and then sonicate at 80 W for 10 min to obtain a homogeneous, bubble-free CZ membrane solution.

[0103] The film-forming solution was uniformly cast onto the surface of the glass substrate and dried in an oven at 40°C for 24 hours. After peeling, the CZ film was obtained.

[0104] Example 3

[0105] The specific steps for preparing chitosan, zein, and Span 80 (CZS) films are as follows:

[0106] Add 25 g of zein solution and 0.05 g of Span 80 to 25 g of chitosan solution, stir at 800 rpm for 30 min, and then sonicate at 80 W for 10 min to obtain a homogeneous, bubble-free CZS membrane solution.

[0107] The film-forming solution was uniformly cast onto the surface of the glass substrate, dried in an oven at 40°C for 24 hours, and then peeled off to obtain a CZS film.

[0108] Example 4

[0109] The specific steps for preparing thin films of chitosan, zein, and natural eutectic solvent (CZD) are as follows:

[0110] Add 25 g of zein solution and 1 g of natural eutectic solvent to 25 g of chitosan solution, stir at 800 rpm for 30 min, and then sonicate at 80 W for 10 min to obtain a homogeneous, bubble-free CZD membrane solution.

[0111] The film-forming solution was uniformly cast onto the surface of the glass substrate and dried in an oven at 40°C for 24 hours. After peeling, the CZD film was obtained.

[0112] Example 5

[0113] The specific steps for preparing films of chitosan, zein, Span 80, and natural eutectic solvent (CZSD) are as follows:

[0114] Add 25 g of zein solution, 0.05 g of Span 80 and 1 g of natural eutectic solvent to 25 g of chitosan solution, stir at 800 rpm for 30 min, and then sonicate at 80 W for 10 min to obtain a homogeneous, bubble-free CZSD membrane solution.

[0115] The film-forming solution was uniformly cast onto the surface of the glass substrate and dried in an oven at 40°C for 24 hours. After peeling, the CZSD film was obtained.

[0116] Example 6

[0117] The specific steps for preparing chitosan, zein, Span 80, and fraxin (CZSE) films are as follows:

[0118] Add 25 g of zein solution, 0.05 g of Span 80 and 0.05 g of fraxin to 25 g of chitosan solution, stir at 800 rpm for 30 min, and then sonicate at 80 W for 10 min to obtain a homogeneous, bubble-free CZSE membrane solution.

[0119] The film-forming solution was uniformly cast onto the surface of the glass substrate and dried in an oven at 40°C for 24 hours. After peeling, the CZSE film was obtained.

[0120] Example 7

[0121] The specific steps for preparing chitosan composite (CZ-DES2) films are as follows:

[0122] Add 25 g of zein solution, 0.05 g of Span 80, 1 g of natural eutectic solvent and 0.05 g of fraxin to 25 g of chitosan solution, stir at 800 rpm for 30 min, and then sonicate at 80 W for 10 min to obtain a homogeneous, bubble-free CZ-DES2 membrane solution.

[0123] The film-forming solution was uniformly cast onto the surface of the glass substrate and dried in an oven at 40°C for 24 hours. After peeling, the CZ-DES1 film was obtained.

[0124] Example 8

[0125] This embodiment provides a method for preparing a chitosan composite (CZ-DES1) film, which differs from Example 7 only in that the amount of natural eutectic solvent added in the preparation of the film-forming solution is 0.5 g.

[0126] Example 9

[0127] This embodiment provides a method for preparing a chitosan composite (CZ-DES3) film, which differs from Example 7 only in that the amount of natural eutectic solvent added in the preparation of the film-forming solution is 1.5 g.

[0128] Example 10

[0129] This embodiment provides a method for preparing a chitosan composite (CZ-DES4) film, which differs from Example 7 only in that the amount of natural eutectic solvent added in the preparation of the film-forming solution is 2 g.

[0130] Example 11

[0131] This embodiment provides a method for preparing a chitosan composite (CZ-DES5) film, which differs from Example 7 only in that the amount of natural eutectic solvent added in the preparation of the film-forming solution is 2.5 g.

[0132] Example 12

[0133] This embodiment provides a method for preparing a chitosan composite (CZ-E1) film, which differs from Example 7 only in that the amount of fraxin added in the preparation of the film-forming solution is 0.1 g.

[0134] Example 13

[0135] This embodiment provides a method for preparing a chitosan composite (CZ-E2) film, which differs from Example 7 only in that the amount of fraxin added in the preparation of the film-forming solution is 0.15 g.

[0136] Performance verification of composite membrane

[0137] I. The structure, composition and microstructure of the natural eutectic solvents and composite films prepared in Examples 1-7 were tested, as detailed below.

[0138] 1. Fourier transform infrared spectroscopy test

[0139] The natural eutectic solvent of Example 1 and the thin film sample of Example 7 were tested by FTIR with an ATR sensor, with 32 scans and a spectral resolution of 4 cm⁻¹. -1 The scanning range is 4000-500 cm. -1 The infrared spectrum of the natural eutectic solvent in Example 1 is shown below. Figure 1 As shown. The infrared spectrum of the composite membrane is as follows. Figure 2 As shown.

[0140] Fourier transform infrared spectra of lactic acid, betaine, and the natural eutectic solvent prepared in Example 1 are as follows: Figure 1 As shown. 3500-3300 cm -1 The infrared absorption bands in the region correspond to the stretching vibrations of hydroxyl groups. Lactic acid and natural eutectic solvents exhibit absorption bands at 2990 cm⁻¹. -1 and 2940 cm -1 Typical bimodalities are present at all locations, corresponding to the stretching vibrations of CH. (1300-1000 cm⁻¹) -1 The interval represents the stretching vibration of COC. Additionally, 1210 cm... -1 and 1120 cm -1 The bimodal pattern at 1720 cm⁻¹ is caused by the combined effect of the asymmetric vibration of COC and the asymmetric rocking vibration of -CH₃. Meanwhile, at 1720 cm⁻¹... -1 (C=O) and 1210 cm -1 At (CO), lactic acid exhibits the same characteristic absorption peak as the natural eutectic solvent. Due to hydrogen bonding, the stretching vibration peak of -COO in betaine increases from 1625 cm⁻¹. -1 Displaced to 1610 cm-1 These results collectively indicate that the natural eutectic solvent is formed through hydrogen bonding between betaine and lactic acid.

[0141] Figure 2 Infrared spectra of the natural eutectic solvent prepared in Example 1, chitosan, zein, Span 80, fraxin, and the films prepared in Examples 2 and 7 are shown. As can be seen from the figures, the chitosan spectrum is at 1655 cm⁻¹. -1 1597 cm -1 and 1379 cm -1 Characteristic absorption peaks are observed at 1655 cm⁻¹, corresponding to the stretching vibration of C=O in amide I, the bending vibration of NH in amide II, and the asymmetric vibration of the carboxylate ion, respectively. Zeadrin exhibits a characteristic absorption peak at 1655 cm⁻¹. -1 1538 cm -1 and 1446 cm -1 The characteristic peaks belong to the C=O stretching vibration, NH bending vibration, and CH asymmetric vibration, respectively. Span 80 has peaks in the 2800-3000 cm⁻¹ range. -1 1730-1750 cm -1 and 1100-1200 cm -1 The characteristic peaks are associated with saturated CH stretching vibrations, C=O stretching vibrations, and COC stretching vibrations, respectively. The spectrum of fraxin is in the 3300-3400 cm⁻¹ range. -1 1700-1720 cm -1 and 700-800 cm -1 The absorption peaks appearing at 3438 cm⁻¹ are due to the stretching vibration of -OH, the stretching vibration of C=O in the ester group (-COO-), and the out-of-plane bending vibration of CH in the benzene ring, respectively. In different composite membranes, the peak at 3438 cm⁻¹ is... -1 The characteristic peak intensity at this location is significantly enhanced, indicating that the hydrogen bonding between each component and chitosan is strengthened. The C=O stretching vibration peak of the amide I band in the spectrum increases from 1655 cm⁻¹. -1 Displaced to 1623-1646 cm -1 These results indicate that hydrogen bonding exists between chitosan, zein, Span 80, the natural eutectic solvent, and fraxin. The natural eutectic solvent acts as a bridge connecting the components, thereby effectively improving the interfacial compatibility between chitosan, zein, and fraxin.

[0142] 2. X-ray diffraction analysis

[0143] The structural analysis of the thin film samples from Examples 1, 2, 3, 5, and 7 was performed using X-ray diffraction. The instrument was equipped with a nickel-filtered copper target Kα radiation source, operating at 40 kV and 40 mA. The scanning range was set to 10° to 80° at the 2θ angle, and the scanning rate was 10° per minute. The XRD spectra of the composite films are shown below. Figure 3 As shown.

[0144] from Figure 3 As can be seen, the CZ film prepared in Example 2 and the CZS film prepared in Example 3 exhibit three distinct diffraction peaks, located near 2θ = 11.4°, 18.1°, and 23.1°, respectively. These results indicate that the chitosan in the film possesses highly crystalline regions and a dense internal structure. When a natural eutectic solvent was introduced as a plasticizer, all diffraction peaks in the CZSD film prepared in Example 5 and the CZ-DES2 film prepared in Example 7 changed to varying degrees. In both films, the typical diffraction peak of chitosan shifted from 23.1° to 20.8°, while the two diffraction peaks at 11.4° and 18.1° completely disappeared, indicating that the chitosan in the film gradually transformed into an amorphous state. The hydrogen bonding between the natural eutectic solvent and chitosan disrupted the inherent hydrogen bond network between hydroxyl and amino groups in the chitosan crystal, thereby leading to a decrease in crystallinity. The decrease in film crystallinity reflects an alteration in the structural integrity of chitosan and an increase in its flexibility, ultimately leading to changes in mechanical properties. Furthermore, fraxinus molecules are rich in hydroxyl groups, enabling them to form hydrogen bonds with natural eutectic solvents and interact with chitosan. Therefore, the XRD pattern of the CZ-DES2 film prepared in Example 7 is influenced by both fraxinus and the natural eutectic solvent.

[0145] 3. Scanning electron microscopy analysis

[0146] The surface and cross-sectional morphology of the thin film samples prepared in Examples 2-5 and Example 7 were observed using a scanning electron microscope. After drying, the samples were fixed on the sample stage and subjected to gold sputtering. The SEM images of the composite films are shown below. Figure 4 As shown.

[0147] Several heterogeneous regions were observed on the film surface, which was caused by the drying and demolding stages during film preparation. Compared to the CZ film prepared in Example 2, the CZS film sample with added Span 80 (Example 3) showed no significant difference. When a natural eutectic solvent was added to the chitosan film, the surface of the CZD film (Example 4) was observed to be slightly smoother, but the uniformity decreased. This is because the natural eutectic solvent functions as a crosslinking agent in the film. The hydrogen bonding between the natural eutectic solvent and chitosan molecules helps to form a more dense and smooth film structure. Compared to the relatively smooth surface and uniform texture of the CZ film prepared in Example 2, the composite films CZSD (Example 5) and CZ-DES2 (Example 7) exhibited significantly rougher surface characteristics due to the addition of more other raw materials.

[0148] The CZ films prepared in Example 2 and the CZS films prepared in Example 3 exhibit a small number of pores in their cross-sections. This may be due to residual gas inside the films or pores formed by the difference in evaporation rates between ethanol and water, thus affecting the permeability and uniformity of the films. The CZD, CZSD, and CZ-DES2 films, after introducing a natural eutectic solvent, exhibit smooth, homogeneous structures without pores in their cross-sections. This is because the natural eutectic solvent is uniformly dispersed in the chitosan network structure through hydrogen bonding. In the CZ-DES2 film, the addition of fraxin and the natural eutectic solvent may have promoted structural recombination and bonding, ultimately forming a smoother and denser cross-sectional structure.

[0149] 4. Atomic force microscopy analysis

[0150] The two-dimensional and three-dimensional morphologies of the thin film samples in Examples 2, 3, 5, and 7 were observed using atomic force microscopy (AFM). Surface roughness was calculated using Nanoscope Analysis software, and the evaluation parameters included root mean square roughness and arithmetic mean roughness. The AFM images of each composite film are shown below. Figure 5 As shown in Table 1, the roughness data for Ra and Rq are as follows.

[0151] Table 1. Ra roughness and Rq roughness of each composite film

[0152]

[0153] The film surface exhibits numerous bright annular protrusions and dark circular pores, structural features that influence surface roughness. The CZ film, serving as a control group, showed the lowest Ra roughness (11.5 nm) without any added components. Upon addition of Span 80 and a natural eutectic solvent, the Ra roughness significantly increased to 20.9 nm and 34.6 nm, respectively. This is because Span 80 and the natural eutectic solvent molecules contain numerous hydrophilic groups. These hydrophilic components disrupt the hydrogen bond network between chitosan molecules, inducing polymer chain swelling and forming a phase-separated structure composed of microregions rich in the natural eutectic solvent (liquid phase) and microregions rich in chitosan (solid phase). This structural feature leads to increased surface height variation. However, upon introduction of fraxin, the Ra roughness slightly decreased to 31.5 nm, indicating that fraxin can be uniformly dispersed in the chitosan-zein network system to form a stable structure. This result demonstrates that introducing polyphenols into chitosan-zein-based films can yield a smoother and more uniform surface morphology.

[0154] II. The performance of each composite membrane prepared in Examples 2-13 was verified.

[0155] 1. Mechanical performance testing

[0156] The films of Examples 2-5 and 7-11 were tested using a texture analyzer. The films were cut into rectangles (1.5 × 6 cm) and fixed to the handle of the instrument. The mechanical properties of the films were evaluated by tensile strength and elongation at break. Figure 6 Tensile strength test results for chitosan composite films with different raw materials and different amounts of natural eutectic solvents; Figure 7 The results of elongation at break tests on chitosan composite films with different raw materials and different amounts of natural eutectic solvents.

[0157] The pure chitosan composite film (CZ) exhibited the highest tensile strength (28.09 MPa), while the CZ-DES2 film showed the lowest (2.86 MPa). This result is mainly attributed to the interfacial incompatibility between chitosan and zein, leading to a significant decrease in the mechanical properties of the CZ film. The presence of the natural eutectic solvent significantly affected the tensile strength of the film, with films based on the natural eutectic solvent exhibiting even lower tensile strength. This is because it acts as a bridge, improving the interfacial compatibility between the hydrophobic zein and the hydrophilic chitosan. Therefore, the tensile strength of the film containing the natural eutectic solvent was lower than that of the CZ and CZS films. When the concentration of the natural eutectic solvent increased from 1% to 5%, the tensile strength of the chitosan composite film decreased significantly from 4.78 MPa to 0.78 MPa. Due to the interaction between chitosan and the natural eutectic solvent, the tensile strength of the composite film decreased with increasing natural eutectic solvent content.

[0158] Contrary to the trend in tensile strength, the elongation at break of the composite film increased significantly with the addition of a natural eutectic solvent. The highest elongation at break was 127.98% for CZ-DES2, while the lowest was only 4.74% for the CZ sample. This is due to the hydrogen bond network formed between the hydroxyl groups of the natural eutectic solvent, the amino groups of chitosan, and the carboxyl groups of zein. These hydrogen bonds are the main reason for improving the interfacial compatibility between chitosan and zein. Therefore, the CZ-DES2 film becomes a material that combines flexibility and mechanical strength. The natural eutectic solvent, acting as a plasticizer, effectively improved the elongation at break of the chitosan composite film. With increasing amounts of natural eutectic solvent, the elongation at break increased from 45.65% to 231.29%. This result indicates that the natural eutectic solvent has excellent plasticizing effects, mainly due to its enhanced mobility of chitosan molecular chains within the film. Furthermore, at higher concentrations of the natural eutectic solvent, the insoluble zein and the soluble components in the natural eutectic solvent hindered the interactions between molecular chains in the chitosan composite film through hydrogen bonding, thus disrupting the film's dense structure. These structural changes were also confirmed by cross-sectional SEM results.

[0159] 2. Water vapor transmission rate test

[0160] The films from Examples 2-5 and 7-11 were tightly mounted on test cups containing dry, anhydrous CaCl2. The test cups were placed in a storage tank containing a saturated solution (relative humidity 75%) at a temperature of 25 ± 1 °C, and the mass of the cups was measured after 24 hours. Figure 8 The water vapor transmission rate test results of chitosan composite films with different raw materials and different amounts of natural eutectic solvents.

[0161] The WVP of the thin film is calculated using the following formula (Ⅰ):

[0162] Equation (Ⅰ);

[0163] W represents the mass of the test cup (g), D represents the thickness of the film (cm), t represents the storage time (min), and A represents the area of ​​the film (cm²). 2 ), ΔP is 2339 Pa.

[0164] The water vapor transmission rates of the CZ film and the CZS film were 3.02 × 10⁻⁶ and 3.02 × 10⁻⁶, respectively. -9 and 3.35×10 -9 The addition of Span 80 and natural eutectic solvent both increased water vapor transmission rate (g / min·cm·Pa), but the water vapor transmission rate of the CZS film was lower than that of the CZD film. The increase in transmission rate caused by Span 80 may be due to its water-in-oil stable emulsion structure. The transmission rates of the CZD and CZSD films reached 6.14 × 10⁻⁶ g / min·cm·Pa.-9 and 6.69×10 -9 g / min·cm·Pa. Using a natural eutectic solvent as a hydrophilic plasticizer effectively promotes water vapor diffusion. Furthermore, the natural eutectic solvent prepared from betaine and lactic acid can increase the interchain spacing of chitosan molecules, thereby improving the water vapor permeability of the film. Therefore, Span 80 and fraxin have relatively little effect on water vapor permeability, while the introduction of the natural eutectic solvent has a significant impact. This indicates that the effect of the natural eutectic solvent on the water vapor permeability of the chitosan composite film is concentration-dependent. With increasing addition of the natural eutectic solvent, the water vapor permeability increases from 6.35 × 10⁻⁶ g / min·cm·Pa. -9 g / min·cm·Pa increased to 7.93×10 -9 g / min·cm·Pa. These data indicate that excessive natural eutectic solvents disrupt the continuous internal structure of the film, leading to a decrease in the density of the CZ-DES5 film and thus providing channels for water vapor permeation.

[0165] 3. Color Test

[0166] The color changes of the films in Examples 2-5 and 7-11 were evaluated using a colorimeter to obtain color parameters L* (brightness value), a* (red / green value), and b* (yellow / blue value). The total color difference (ΔE) was calculated using Equation (II). The color parameters are shown in Table 2.

[0167] Formula (II)

[0168] In the formula, L0, a0, and b0 are the initial color parameter values ​​of the thin film.

[0169] Table 2 Color parameters of each composite film

[0170]

[0171] With the addition of a natural eutectic solvent, both the lightness (L* value) and yellowness (b* value) of the film increased. This color change may be due to the fact that the addition of the natural eutectic solvent promoted the thermally induced Maillard reaction between the carbonyl and amino groups in the chitosan molecules. Therefore, all films containing the natural eutectic solvent exhibited a more pronounced yellow characteristic than CZ films.

[0172] 4. Thermal stability test

[0173] The thermal stability of the thin film samples from Examples 2-5 and 7-11 was tested using a thermogravimetric analyzer. Thermogravimetric analysis was performed under a nitrogen atmosphere, with approximately 10 mg of sample being heated from 30°C to 600°C at a rate of 10°C per minute. Figure 9 Thermogravimetric analysis results of chitosan composite films with different raw materials and different amounts of natural eutectic solvents.

[0174] CZSD and CZ-DES2 films exhibit a four-stage weight loss process, while CZS and CZD films show only three stages, and CZ films show only two stages. The first stage of weight loss (30-125℃) originates from the evaporation of bound water and residual acetic acid in the film matrix. The minimum mass loss in the second stage (125-220℃) is due to the degradation of Span 80, a phenomenon observed only in films containing Span 80 (CZS, CZSD, and CZ-DES2). The maximum mass loss in the third stage (220-355℃) is due to the decomposition of chitosan. Furthermore, esterification products formed from chitosan and acetic acid can also contribute to weight loss in this stage. The fourth stage (355-450℃) is related to the natural eutectic solvent, which occurs in CZD, CZSD, and CZ-DES2 films. The residual mass of films with added natural eutectic solvents after heating is lower than that of films without added natural eutectic solvents because the natural eutectic solvents act as plasticizers, reducing the energy required for the degradation of chitosan composite films.

[0175] The weight loss of chitosan composite films with different concentrations of natural eutectic solvents was most significant in the third and fourth stages. The weight loss rate decreased with increasing natural eutectic solvent concentration. Furthermore, the maximum decomposition temperature of the composite film in the third and fourth stages decreased with increasing natural eutectic solvent content. The natural eutectic solvent occupies the active adsorption sites in chitosan molecules through hydrogen bonding, thus causing the decomposition temperature of the chitosan film to decrease with increasing hydrophilic natural eutectic solvent content.

[0176] 5. Functional testing

[0177] The antibacterial properties of the films in Examples 3, 5, 6, 7, 12, and 13 were tested using *Escherichia coli* (ATCC 25922) and *Staphylococcus aureus* (ATCC 6538). 50 μL of undried film solution was added to the wells (diameter = 6 mm) of LB agar plates inoculated with bacterial suspension, and the inhibition zone diameter was measured after incubation at 37°C for 24 hours. Figure 10 The results of the inhibition zone test for chitosan composite films with different raw materials and different amounts of fraxin are shown in Table 3. The diameters of the inhibition zones are shown in Table 3.

[0178] Table 3 Diameter of the antibacterial zone of each composite film

[0179]

[0180] The antioxidant capacity of the films in Examples 2-5, 7, 12, and 13 was evaluated by DPPH radical scavenging capacity and ABTS radical scavenging capacity. Figure 11 The antioxidant test results are for chitosan composite films with different raw materials and different amounts of fraxin.

[0181] DPPH radical scavenging ability: 50 mg of the film sample was immersed in 10 mL of DPPH solution and reacted in the dark for 30 minutes. The absorbance of the solution was measured at a wavelength of 517 nm, and the DPPH radical scavenging rate was calculated according to formula (III):

[0182] Formula (III)

[0183] In the formula, A0 is the initial absorbance of the DPPH solution mixed with deionized water, and A l The absorbance of the DPPH solution after reaction with the thin film.

[0184] ABTS radical scavenging ability: A 7 mM ABTS solution was diluted with potassium persulfate and stored overnight in the dark. A 50 mg film sample was immersed in 10 mL of 2.45 mM ABTS working solution and reacted in the dark for 30 minutes. The absorbance of the solution was measured at 734 nm, and the ABTS radical scavenging rate was calculated according to formula (IV).

[0185] Formula (Ⅳ)

[0186] In the formula, A0 is the initial absorbance of the ABTS working solution mixed with deionized water, and A l The absorbance of the ABTS solution after reaction with the thin film is given.

[0187] Compared to the CZSE film, the CZS and CZSD films did not produce inhibition zones, indicating that films without fraxin lack antibacterial ability. The introduction of fraxin significantly enhanced the antibacterial activity of the chitosan composite film by increasing the number of free hydroxyl and carboxyl groups, thereby inhibiting bacterial growth. When the amount of fraxin added to the CZSD film matrix increased from 0.1% to 0.3%, the diameter of the inhibition zone against both bacteria significantly increased. This may be because the higher content of fraxin in CZ-E2 promotes the release of stronger antibacterial activity from the film. Fraxin showed broad-spectrum antibacterial activity against a variety of foodborne pathogens in the antibacterial experiment. In addition, the film showed stronger inhibitory effects against Staphylococcus aureus than against Escherichia coli, which may be related to the thick cell wall structure of Gram-positive bacteria composed of multilayered peptidoglycans, making it easier for antibacterial components to penetrate their cell walls.

[0188] Because chitosan possesses certain antioxidant capabilities, CZ films exhibit only weak free radical inhibition. However, due to the stronger free radical scavenging activity of natural eutectic solvents containing organic acids, chitosan composite films with added natural eutectic solvents demonstrate stronger antioxidant capabilities. Compared to CZ films, chitosan composite films containing natural eutectic solvents (CZD and CZSD films) show significantly improved DPPH and ABTS free radical scavenging capabilities: DPPH free radical scavenging rates increased from 34.41% to 64.26% and 64.55%, respectively; ABTS free radical scavenging rates increased from 44.46% to 66.30% and 66.95%, respectively. Furthermore, with increasing fraxin addition, the free radical scavenging capability of chitosan composite films significantly enhances: the DPPH free radical scavenging rate of films containing fraxin reaches 78.35%-83.27%, and the ABTS free radical scavenging rate reaches 83.30%-91.55%.

[0189] Example 2: Application of chitosan composite film in fish preservation

[0190] The chitosan composite film prepared in Example 7 was used for the preservation of pearl grouper. Specifically, the grouper was scaled and skinned, leaving only the flesh. The flesh was cut into regular pieces and covered with the prepared chitosan composite film. Uncovered flesh served as a control group. All samples were placed in sterile petri dishes and stored at 4°C until analysis. Changes in various indicators were measured on days 0, 1, 4, 7, and 10 of storage. Figure 12 The results are from the experiments conducted on the application.

[0191] The water-holding capacity of fish meat was characterized by centrifugal weight loss: samples were taken and weighed on days 0, 1, 4, 7, and 10, and the weight loss rate was calculated according to formula (V). pH was measured using a pH meter: the sample was mixed with potassium chloride solution at a ratio of 1:10 (w / w), homogenized, and the pH of the mixture was measured. Total volatile basic nitrogen (TVB-N) was determined using an automated Kjeldahl nitrogen analyzer. Total bacterial count (TVC) was determined by homogenizing 10 g of sample with 90 mL of sterile physiological saline for 2 minutes, then inoculating the sample solution onto plate counting agar medium for incubation and counting.

[0192] Formula (V)

[0193] In the formula, W0 is the initial weight of the fish meat, W l This represents the weight of the fish meat after centrifugation.

[0194] During the 10-day storage period, the treated group showed a smaller weight loss rate compared to the control group. Decomposition products such as organic sulfides, aldehydes, and amines generated during storage directly affect the pH value of the fish meat. The initial pH value of the fish meat was approximately 6.6, within the normal pH range for fresh fish (6.2-6.8). The pH values ​​of all samples showed a trend of first decreasing and then increasing. The pH value of the control group decreased to 6.5 on the first day of storage, increased to 7.1 on the fourth day, and further increased to 7.8 on the tenth day with increasing storage time. The pH value of the treated group decreased to 6.2 on the first day of storage, increased to 6.4 on the fourth day, and stabilized at around 6.6 after the seventh day. This is because the pH decreased in the early stages of refrigeration due to glycogen degradation into lactic acid, followed by an increase in pH due to the oxidation of fish meat producing ammonia and the decomposition of proteins by microorganisms. The pH value of the treated group was lower than that of the control group after 10 days of storage, and the overall change was slower. This is because the phenolic hydroxyl functional groups in the chitosan composite film have antibacterial and antioxidant functions. During fish storage, proteins undergo oxidative degradation under the action of putrefactive bacteria and endogenous enzymes, producing various substances that lead to an increase in total volatile nitrogen (TVR). The initial TVR content of the fish was 8.03 mg / 100 g, below the safety limit (<30 mg / 100 g). With prolonged storage, the TVR level continued to rise, reaching the limit in the control group on day 4. At the end of storage, the TVR content in the treatment group and the control group were 18.32 mg / 100 g and 143.76 mg / 100 g, respectively. The significantly lower TVR in the treatment group confirms that the chitosan composite film effectively inhibits microbial growth and prevents the accumulation of TVR. When the total bacterial count in the fish exceeds 5.70 log CFU / g (5 × 10⁻⁶), the TVR level decreases. 5 CFU / g is considered spoilage. During storage at 4℃, the control group (unpackaged) exceeded the limit on day 4, while the total bacterial count in the treated group initially decreased, then increased, and remained stable in the later stage. This is attributed to the synergistic effect of the natural eutectic solvent and fraxin in the chitosan composite film, which enhances both mechanical properties and antibacterial effects.

[0195] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A chitosan composite film for fish preservation, characterized in that, By weight, it includes the following ingredients: 25 parts chitosan solution, 25 parts zein solution, 0.05 parts Span 80, 1-5 parts natural eutectic solvent, 0.1-0.3 parts fraxin; The natural eutectic solvent includes hydrogen bond acceptors and hydrogen bond donors; The hydrogen bond acceptor is betaine, and the hydrogen bond donor is D,L-lactic acid; The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:

2.

2. The chitosan composite film for fish preservation according to claim 1, characterized in that, The chitosan has a molecular weight of 150,000 Da and a degree of deacetylation ≥95%. The molecular weight of the zein is 20 kDa.

3. A method for preparing a chitosan composite film for fish preservation as described in any one of claims 1-2, characterized in that, Includes the following steps: Chitosan solution, zein solution, Span 80, natural eutectic solvent and fraxin are mixed, stirred and ultrasonically treated to prepare film-forming solution; The chitosan composite film is obtained by casting the film-forming solution and then drying it.

4. The method for preparing the chitosan composite film for fish preservation according to claim 3, characterized in that, The chitosan solution was prepared by dissolving chitosan in an acetic acid solution; The ratio of chitosan to acetic acid solution is 2 g: 100 mL.

5. The method for preparing the chitosan composite film for fish preservation according to claim 3, characterized in that, The zein solution is obtained by dissolving zein in an ethanol solution; The ratio of zein to ethanol solution is 2 g: 100 mL.

6. The method for preparing the chitosan composite film for fish preservation according to claim 3, characterized in that, The stirring and ultrasonic treatment conditions are as follows: stirring at 800 rpm for 30 min, followed by ultrasonication at 80 W power for 10 min.

7. The application of a chitosan composite film as described in any one of claims 1-2 in fish preservation.

8. A chitosan composite film for fish preservation, characterized in that, Its preparation method includes the following steps: Add 25 g of zein solution, 0.05 g of Span 80, a natural eutectic solvent, and 0.05 g of fraxin to a 25 g chitosan solution. Stir at 800 rpm for 30 min, then sonicate at 80 W for 10 min to obtain a film-forming solution. The amount of the natural eutectic solvent used is 0.5 g, 1 g, 1.5 g, 2 g, or 2.5 g. The film-forming liquid is uniformly cast onto the surface of a glass substrate, dried in a 40°C oven for 24 hours, and then peeled off to obtain the chitosan composite film used for fish preservation. Preparation of the chitosan solution: Dissolve 2 g of chitosan in 100 mL of 1 vol% acetic acid solution until completely dissolved to form a chitosan solution with a mass concentration of 2 wt%. Preparation of the zein solution: Dissolve 2 g of zein in 100 mL of 90 vol% ethanol solution until completely dissolved to form a zein solution with a mass concentration of 2 wt%. Preparation of the natural eutectic solvent: Betaine and D,L-lactic acid are mixed in a molar ratio of 1:2 and magnetically stirred in a water bath at 70°C for 20 min until a clear, transparent and homogeneous liquid is formed; the mixture is then cooled to room temperature to obtain the natural eutectic solvent.

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