Bio-based film as well as preparation method and application thereof

Abstract

The invention belongs to the field of bio-based composite materials, and particularly relates to a bio-based film as well as a preparation method and application thereof. The bio-based film is of a double-layer composite structure and comprises an outer layer, the outer layer is formed by cross-linking chitosan and cellulose nanofibers through a cross-linking agent genipin and hydrophobic modification through caprylic acid, and a high-barrier transparent protective layer is formed; and the inner layer is formed by compounding thermoplastic starch and a hydrophobic deep-eutectic solvent consisting of thymol and octanoic acid, so that the heat sealing layer with antibacterial and antioxidant functions is formed.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bio-based composite materials, and particularly relates to a bio-based film and a preparation method and application thereof. BACKGROUND

[0002] It has become a research hotspot and development trend in the field of polymer materials and food science to develop bio-based packaging materials derived from renewable resources, which are environmentally friendly and biodegradable, to replace traditional petroleum-based plastics. Natural biopolymers such as chitosan, cellulose and starch are considered to be ideal building blocks for constructing a new generation of packaging materials due to their inherent advantages such as abundant reserves, low cost, non-toxicity and safety, and complete biodegradability, and exhibit great application potential.

[0003] However, direct application of a single natural biopolymer in the field of high-performance packaging still faces many technical bottlenecks. Chitosan has excellent film-forming property and natural antibacterial property, but the mechanical strength of the chitosan film is low, and the performance will decrease significantly in a humid environment. Although cellulose nanomaterials can provide excellent mechanical support, their inherent strong hydrophilicity leads to poor water vapor barrier ability. Although physical blending of the two can improve some properties, the moisture resistance and long-term stability of the composite material are still insufficient due to the lack of stable interfacial bonding. On the other hand, starch-based materials have thermoplastic processing potential and are expected to achieve heat sealing function, but they generally have poor mechanical properties, strong moisture absorption and weak barrier properties. In order to overcome these shortcomings, various modification methods have been tried in the prior art, such as hydrophobic treatment by grafting long-chain fatty acids or silanes, but these methods often severely damage the optical properties of the material while improving its water resistance, resulting in a significant decrease in film transparency and an increase in haze, which affects the visibility of the packaging contents and limits its commercial application value.

[0004] In summary, the bio-based packaging materials provided by the prior art often have difficulty in achieving a balance of multiple key performance indicators in a single film. There is still an urgent need in the art to develop a new type of bio-based film material that not only can be completely biodegradable, but also must have excellent mechanical properties comparable to traditional plastics, high barrier properties to water vapor and oxygen, and good surface hydrophobicity; most importantly, the film must maintain high transparency to meet the appearance requirements of commercial packaging while achieving the above properties, and must also have the necessary heat sealing properties for rapid sealing on an industrial automated production line; in addition, if the film is further endowed with long-term antibacterial and antioxidant activity, it will greatly expand its application prospects in the field of active food preservation. SUMMARY

[0005] The present application aims to solve the above problems and provides a bio-based film and a preparation method and application thereof.

[0006] Firstly, a bio-based thin film employs the following technical solution: A bio-based thin film having a bilayer composite structure, comprising: The outer layer, formed by cross-linking chitosan and cellulose nanofibers with the cross-linking agent genipin and then modifying them with octanoic acid hydrophobicity, constitutes a high-barrier transparent protective layer; and The inner layer is composed of thermoplastic starch and a hydrophobic eutectic solvent consisting of thymol and caprylic acid, forming a heat-sealing layer with antibacterial and antioxidant functions.

[0007] Furthermore, the mass ratio of chitosan to cellulose nanofibers in the outer layer is 1:1 to 3:1.

[0008] Furthermore, the thermoplastic starch in the inner layer comprises starch, polyvinyl alcohol, and glycerol.

[0009] Furthermore, in the hydrophobic eutectic solvent, the molar ratio of thymol to octanoic acid is 1:1.

[0010] Secondly, a method for preparing a bio-based thin film adopts the following technical solution: A method for preparing a bio-based thin film includes the following steps: Chitosan and cellulose nanofibers were mixed in a solution, genipin was added for cross-linking, and octanoic acid was added for hydrophobic modification to obtain an outer layer material solution. Thermoplastic starch is mixed with a hydrophobic eutectic solvent composed of thymol and octanoic acid in solution to obtain an inner layer material solution; The outer layer material solution and the inner layer material solution are cast layer by layer and dried to obtain the bio-based film with the double-layer composite structure.

[0011] Furthermore, in the hydrophobic modification process, octanoic acid is covalently grafted onto the amino group of chitosan via amide bonds by using 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide as coupling agents.

[0012] Further, the step of providing the inner layer material solution includes: Starch and polyvinyl alcohol were dispersed in water and heated to gelatinize. After cooling, they were mixed with glycerol to obtain a thermoplastic starch matrix. Thymol and octanoic acid are mixed to form the hydrophobic eutectic solvent; The thermoplastic starch matrix is ​​mixed evenly with a hydrophobic eutectic solvent to obtain the inner layer material solution.

[0013] Furthermore, the step of layer-by-layer casting and drying includes: casting the outer layer material solution into a film and drying it to form a solid viscous surface; casting the inner layer material solution onto the viscous surface and drying it to obtain the bio-based film.

[0014] Thirdly, the application of a bio-based thin film employs the following technical solution: Application of a bio-based film in the preparation of food preservation packaging materials.

[0015] Furthermore, the heat-sealing properties of the inner layer of the bio-based film are used to package and seal food, and the high barrier, antibacterial and antioxidant properties of the bio-based film are used to extend the shelf life of the food inside the package.

[0016] The beneficial effects of this invention are: This invention provides a bio-based film that overcomes many technical bottlenecks in the field of existing bio-based packaging materials through its unique double-layer composite structure. This bio-based film uses a thermoplastic starch composite system as its functional inner layer, overcoming the fundamental problem of traditional chitosan / cellulose-based films lacking thermoplasticity and being unable to undergo industrial heat sealing. The outer layer utilizes the natural crosslinking agent genipin to chemically crosslink chitosan and cellulose nanofibers, constructing a stable network framework that significantly improves the film's mechanical strength and moisture resistance, ensuring its structural integrity in humid environments. The outer layer is further modified with octanoic acid for hydrophobicity, imparting excellent waterproof properties while maintaining extremely high optical transparency, perfectly resolving the inherent contradiction of traditional hydrophobic modification methods sacrificing transparency. This dense, crosslinked outer layer and the functional inner layer work synergistically to form a highly efficient barrier against water vapor and oxygen. Simultaneously, the low-eutectic solvent in the inner layer further endows the film with long-lasting antibacterial and antioxidant activity. This bio-based film integrates multiple excellent properties such as high mechanical properties, high barrier properties, high transparency, waterproof properties, heat sealability, and bioactivity, providing a comprehensive bio-based packaging solution with superior performance and practical industrial application value. Attached Figure Description

[0017] Figure 1Scanning electron microscope (SEM) images of the surface (a) and fracture cross-section (b) of C1-OA-T, C2-OA-T, and C3-OA-T provided in Example 1. Two-dimensional height maps (c) of C1-OA-T, C2-OA-T, and C3-OA-T by atomic force microscopy (AFM). Fourier transform infrared (FTIR) spectra of the films (d). X-ray diffraction (XRD) spectra of C1-OA, C2-OA, and C3-OA (C layer) in the bilayer film samples C1-OA-T, C2-OA-T, and C3-OA-T (e). X-ray diffraction (XRD) spectra of thermoplastic starch (TPS, T layer) in the bilayer film samples C1-OA-T, C2-OA-T, and C3-OA-T (f).

[0018] Figure 2 Appearance (a), light transmittance (bd) of different groups of bio-based films provided in Example 1. Thermogravimetric analysis (TGA) curves of the films and corresponding derivative thermogravimetric analysis (DTG) curves (eg). Tensile strength (h) and elongation at break (i) of the films. Heat seal strength of C3-OA-T and photographs taken during the test (j).

[0019] Figure 3 Swelling degree (a) and water solubility (b) of different groups of bio-based films provided in Example 1. Water vapor transmission rate (c) and oxygen transmission rate (d) of the films. ABTS (e) and DPPH (f) scavenging activity of the films. Photographs (g) and data (h) of the film contact angles.

[0020] Figure 4 To assess the antibacterial effects of the different groups of bio-based films provided in Example 1, the antibacterial effects of the films against *Escherichia coli* and *Staphylococcus aureus* were detected using the plate coating method (a). The antibacterial efficiency of the films against *Escherichia coli* (b) and *Staphylococcus aureus* (c) is shown. Data (d) and photographs (e) of the antibacterial inhibition zones of the films are also presented. An image of the films showing viability / deactivation staining is shown (f).

[0021] Figure 5 Test results of different groups of bio-based films provided in Example 1 for food packaging; the first column shows examples of packaged samples, and the images from days 1-5 were taken after the film was removed (a). Weight loss rate (b) and OD595 value (c) of strawberries in different treatment groups during storage. Photographs of the film's natural degradation ability (d).

[0022] Figure 6 This is a schematic diagram illustrating the synthesis method of C3-OA-T and the thermoplastic film preservation mechanism provided by the present invention.

[0023] Data were obtained from three independent experiments (n=3) and are expressed as mean ± standard deviation (SD). One-way ANOVA was used to compare groups to assess statistical significance. The significance thresholds were as follows: *p<0.05, **p<0.01, and ***p. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the present application will be further described in detail below with reference to the accompanying drawings. The described embodiments should not be regarded as limitations on the present invention. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0025] In the following description, references to "some embodiments" refer to a subset of all possible embodiments; however, it is understood that "some embodiments" may be the same or different subsets of all possible embodiments and may be combined with each other without conflict. Unless otherwise defined, all technical and scientific terms used in the embodiments of the invention have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments of the invention pertain. The terminology used in the embodiments of the invention is for the purpose of describing the embodiments of the invention only and is not intended to limit the invention.

[0026] Those skilled in the art should understand that, in the following description of the embodiments of the present invention, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention.

[0027] The terminology used in the embodiments of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The singular forms “a” and “the” as used in the embodiments of this invention and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0028] Those skilled in the art will understand that the numerical ranges in the embodiments of the present invention should be understood to specifically disclose each intermediate value between the upper and lower limits of the range. Each smaller range between any stated value and an intermediate value within the stated range, as well as any other stated value or an intermediate value within the stated range, is also included within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, the technical / 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 are described herein, any methods and materials similar or equivalent to those described herein may be used in embodiments or test cases of the invention. All references to this specification are generally incorporated herein by reference to disclose and describe methods and / or materials associated with said references. In the event of any conflict with any incorporated reference, the contents of this application shall prevail.

[0030] It should be noted that all raw materials and / or reagents in the embodiments of the present invention were purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0031] Reagents and materials: Chitosan (degree of deacetylation ≥95%, viscosity 100-200 mPa·s, Macklin), caprylic acid (OA, purity >99%, Adamas), potato starch (C6H2O) 10 O5) n Aladdin Holding Group Co., Ltd. (Shanghai, China) provides the following: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, purity >98%, Adamas), n-hydroxysuccinimide (NHS, purity >99%), sodium hydroxide (purity >99.7%), glycerol, thymol, polyvinyl alcohol, and sugarcane pulp, supplied by Shenzhen Yutong Packaging Technology Co., Ltd.; 2,2,6,6-tetramethyl-1-piperoyloxy (tempo, purity >99%, Aladdin); sodium hypochlorite solution (NaClO, purity >99.7%), sodium chlorite (NaClO2, purity bbb80%), CaCl2 (purity >96%, Macklin); reduced iron powder (purity >99%, 300 mesh, Macklin); powdered activated carbon (10-24 mesh, Aladdin); and *Escherichia coli* (ATCC 25922) and *Staphylococcus aureus* (ATCC). 25923) was donated by Southeast Asian University.

[0032] Example Example 1 Example 1 provides a bio-based thin film with a bilayer composite structure, comprising: The outer layer, formed by cross-linking chitosan and cellulose nanofibers with the cross-linking agent genipin and then modifying them with octanoic acid hydrophobicity, constitutes a high-barrier, transparent protective layer; and The inner layer is composed of thermoplastic starch and a hydrophobic eutectic solvent consisting of thymol and caprylic acid, forming a heat-sealing layer with antibacterial and antioxidant functions.

[0033] Example 1 also provides a method for preparing a bio-based thin film, comprising the following steps: 1. Preparation of cellulose nanofibers: Cellulose (1 g) was suspended in a beaker containing 90 mL of 0.05 M acetate buffer (pH 3.8 or 4.5) containing TEMPO (0.016 g, 0.1 mmol) and sodium chlorite (80%, 1.13 g, 10 mmol). A 2 M sodium hypochlorite solution (0.5 mL, 1.0 mmol) was diluted to 0.1 M with the same 0.05 M buffer as the oxidation medium and added to the beaker all at once. The beaker was immediately stoppered, and the suspension was stirred at 1000 rpm and 60 °C for the specified 12 h. After cooling the suspension to room temperature, the TEMPO-oxidized cellulose was thoroughly washed with water via filtration.

[0034] 2. Preparation of Chitosan-Cellulose Nanofiber Solution: 0.5 g of chitosan was dissolved in 50 mL of 1% (v / v) glacial acetic acid solution. Different amounts of CNF (equivalent to 100%, 50%, and 33% (w / w) of chitosan weight, respectively) were added to the dissolved chitosan solution, and these mixtures were labeled as groups C1, C2, and C3, respectively. 0.15 mmol / L genipin was added to each mixture, the solution was stirred for 30 minutes, and then sonicated for 30 minutes to prepare the CS / CNF solution. CS / CNF solutions with different weight ratios were recorded as groups C1, C2, and C3.

[0035] 3. Preparation of CS / CNF / OA membrane: 0.5 g of octanoic acid was dissolved in sodium octanoate solution. Then, 0.40 g of N-hydroxysuccinimide (NHS) and 0.67 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were added to the prepared CS / CNF solution and mixed thoroughly. The sodium octanoate solution was slowly added dropwise to the mixture, and the reaction was carried out with magnetic stirring at room temperature for 17 hours. After the reaction was completed, anhydrous ethanol was added to the solution, and then the mixture was centrifuged at 12000 rpm for 10 minutes. The resulting product was dried to obtain CS / CNF / OA. After drying, 0.5 g of the sample was dissolved in 50 mL of 1% (v / v) glacial acetic acid solution to obtain the CS / CNF / OA solution, which is the final outer layer material solution. CS / CNF / OA with different weight ratios were recorded as the C1-OA group, the C2-OA group, and the C3-OA group.

[0036] 4. Preparation of eutectic solvent (DES): Mix thymol and octanoic acid in a 1:1 molar ratio and stir in a magnetically stirred water bath at 60°C for 20 minutes until a homogeneous liquid is obtained.

[0037] 5. Preparation of Thermoplastic Starch (TPS) Solution: 1.2 g of potato starch (2.4% (w / v)) and 0.4 g of polyvinyl alcohol (PVA) were dispersed in distilled water. The resulting suspension was heated in an oil bath at 95°C with stirring at 500 rpm for 30 minutes to promote starch gelatinization. The starch solution was then cooled to approximately 40-50°C, followed by the addition of glycerol (20% w / w of the dry starch basis) and DES. The mixture was stirred for another hour to ensure uniform dispersion. Finally, the starch solution was mixed with the glycerol / DES mixture to obtain the thermoplastic starch solution, which is the final inner layer material solution.

[0038] 6. Preparation of the bilayer membrane: Solutions of chitosan (CS) / nanocellulose (CNF) / oleic acid (OA) in different proportions were cast onto plastic plates with a diameter of 90 mm and dried at room temperature until the surface became solid but still viscous. Then, a TPS solution was dropwise added to the surface of the first layer using a pipette, and the mixture was dried at room temperature for 12 hours until the solvent had completely evaporated. The solution was then completely peeled off from the plastic plate to obtain a transparent and flexible bio-based film with a bilayer composite structure. Different weight ratios of CS / CNF / OA / TPS were recorded as the C1-OA-T group, C2-OA-T group, and C3-OA-T group. The synthesis methods and thermoplastic film preservation mechanisms are detailed in [link to documentation]. Figure 6 As shown.

[0039] Example 2: Morphological Characterization of CNFs and Bio-based Thin Films The surface and cross-sectional morphology of the bio-based thin film provided in Example 1 were analyzed using scanning electron microscopy (SEM). Micrographs were taken using a Hitachi SU8010 scanning electron microscope at an operating voltage of 3.0 kV. The surface roughness of the bio-based thin film was analyzed using atomic force microscopy (AFM). The morphology of the cellulose nanofibers (CNFs) was characterized using a HT7700 (Hitachi, Japan) transmission electron microscope (TEM).

[0040] The morphology of CNF and synthetic bio-based films was characterized by transmission electron microscopy. Figure 1 ,Depend on Figure 1 As can be seen from the transmission electron microscope images, the CNF prepared in Example 1 exhibits a typical fiber network structure with a uniform diameter distribution. Clear fiber boundaries are visible, indicating successful dispersion of the nanofibers without significant agglomeration. The high aspect ratio of CNF is beneficial for improving the mechanical properties of the material. The surface and cross-sectional morphology of the different groups of sample films provided in Example 1 were characterized using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Surface scanning electron microscope images of all sample films (…) Figure 1a) It exhibits a highly uniform and continuous structure, without obvious defects such as bubbles or cracks. Cross-sectional scanning electron microscope image ( Figure 1 (b) The bilayer film structures of samples C1-OA-T, C2-OA-T, and C3-OA-T are clearly shown, with tight interfacial bonding between each layer and uniform thickness. Two-dimensional height maps obtained using atomic force microscopy (AFM) are also presented. Figure 1 c) Analysis revealed island-like protrusions on the surface of the sample membrane.

[0041] Example 3: Fourier Transform Infrared Spectroscopy (FTIR) The different groups of bio-based thin film samples provided in Example 1 were tested using a TGA8000S (PerkinElmer, USA) at 4000-400 cm⁻¹. -1 Analysis was performed within the specified wavelength range. Fourier transform infrared spectroscopy (FTIR) was used to analyze the chemical bond changes in all sample films. Figure 1 d). All membrane samples were tested at 3200 cm⁻¹. -1 Up to 3600 cm -1 Broad peaks were observed across the range. For chitosan (CS) sample membranes, these peaks corresponded to OH and NH stretching vibrations; for cellulose nanofiber (CNF) and thermoplastic starch (TPS) sample membranes, they corresponded to strong OH stretching vibrations. Furthermore, all membrane samples showed peaks at 2850 cm⁻¹. -1 and 2950 cm -1 The presence of absorption peaks between these peaks is attributed to the CH stretching vibration.

[0042] Crosslinking with genipin resulted in C3 at 1410 cm⁻¹. -1 An absorption peak appears at 1650 cm⁻¹, corresponding to the CN stretching vibration. This peak is a result of nucleophilic substitution of the olefinic carbon of genipin by the amino group of chitosan (CS), which opens the dihydropyran ring to form a heterocyclic amine. Furthermore, at 1650 cm⁻¹... -1 The absorption peak at 1540 cm⁻¹ is attributed to the substitution of the ester group on genipin by chitosan during the reaction. -1 The absorption peak at that point is due to the bending vibration of the NH group.

[0043] After activating the carboxyl group of octanoic acid (OA) with NHS / EDC and grafting it onto the C3 surface, 1650 cm -1 The absorption peak at C3 is enhanced, corresponding to the amide I band (C=O stretching vibration) in the amide bond. Furthermore, the amide II band at C3 (1550 cm⁻¹) is also enhanced. -1 The intensity of the NH bending vibration at 1375 cm⁻¹ decreased significantly, which may be due to the reduction in the number of free amino groups during the reaction, leading to a decrease in peak intensity. The sample C3-OA showed a peak intensity at 1375 cm⁻¹. -1The absorption peak at this point can be attributed to the amide III band, which is caused by the weak coupling vibrations of CN and NH in the secondary amide. These results indicate that octanoic acid is chemically bonded to C3 via an amide bond, successfully terminating the hydrophobicity of the material.

[0044] Furthermore, after introducing a thymol / menthol eutectic solvent (TPS) containing a eutectic solvent (DES) into trioctyl citrate oleate (C3-OA), the infrared spectrum was observed in the 3000–3100 cm⁻¹ range. -1 A weak vibrational peak was observed within the range, which is associated with the stretching vibration of aromatic CH and originates from the benzene ring structure of thymol. These findings confirm that C3-OA-T achieves complexation of TPS with C3-OA with the participation of DES.

[0045] Example 4 X-ray diffraction (XRD) The XRD spectra of the different groups of bio-based thin films provided in Example 1 were obtained using an Empyrean X-ray diffractometer (Marven Panaco Ltd, Marven, UK), with an angle range of 5-90° (2θ) and a scanning speed of 10° / min. Figure 1 The TPS shown in f exhibits low intensity and no sharp peaks between 15° and 25°, confirming its amorphous properties. C1-OA-T, C2-OA-T, and C3-OA-T represent bilayer structures, where the C layer consists of samples C1-OA, C2-OA, and C3-OA, and the T layer is composed of sodium alginate (TPS) mixed with a eutectic solvent (DES). Figure 1 The image shows the X-ray diffraction (XRD) spectrum of layer C, which exhibits a broad range of amorphous diffuse peaks between 15° and 25°. These peaks differ from those observed in samples C1-OA, C2-OA, and C3-OA. Notably, a novel absorption peak appears in layer C at 19.5°, attributed to the infiltration of layer T into layer C during bilayer preparation. Polyvinyl alcohol (PVA) in layer T acts as a plasticizer, contributing to the absorption peak at 19.5°.

[0046] The T layer exhibits a broad, low-intensity peak between 15° and 25°, and also shows an absorption peak at 19.5°, which is stronger than that of the C layer. This further confirms that the peak originates from PVA in the T layer. The T layer does not show any sharp diffraction peaks, indicating that the eutectic solvent (DES) is completely amorphous, which is consistent with the typical X-ray diffraction (XRD) characteristics of low-cohesion solvents.

[0047] Example 5 Physical properties of bio-based thin films (1) Color and light transmittance test The brightness (L), red-green value (a), and yellow-blue value (b) of the different groups of bio-based films provided in Example 1 were measured using a CS-5960GX spectrophotometer. A standard white plate (L* = 93.39, a* = 0.07, b* = 2.53) was used as a control. The total color difference (ΔE) was calculated according to formula (1):

[0048]

[0049] The transmittance of the thin film was measured in the wavelength range of 380-780 nm using a CS-5960GX spectrophotometer with a sampling interval of 2 nm.

[0050] like Figure 2 As shown in Figure a, all sample films exhibited high transparency, with no obvious color visible to the naked eye. Table 1 lists the color parameters of the measured films. The L* values ​​of C1-C3 films ranged from 93.57 to 94.14, indicating high brightness. After oleic acid (OA) modification (C3-OA) and the introduction of a eutectic solvent (DES) (C3-OA-T), the L* values ​​were further increased to 94.46 and 95.56, respectively.

[0051] Table 1. Color parameters of chitosan films with different film plasticization.

[0052]

[0053] The trend of the whiteness index (WI) is consistent with that of the L* value, with WI gradually increasing from 75.56 for C1 to 82.72 for C3-OA-T. These changes in L* and WI can be attributed to the chemical modification with oleic acid (OA) and the introduction of a eutectic solvent (DES), which reduces the crystallinity of the material. The decrease in crystallinity leads to a significant reduction in light scattering, thereby increasing both the L* and WI values ​​of the material.

[0054] All film samples had negative a* values ​​(-0.81 to -0.16), indicating that these films generally had a greenish tint. The b* value and yellowness index (YI), reflecting the yellowness of the material, ranged from 0 to 2.5 and 0 to 5 for all samples, indicating that these films had a yellowish tint.

[0055] All materials had ∆E values ​​less than 5. Studies show that when ∆E exceeds 2, consumers may notice a color change, while a ∆E value greater than 5 indicates a significant color difference. Among all membrane materials, only samples C1, C1-OA, and C1-OA-T had ∆E values ​​exceeding 2, while the ∆E values ​​of the remaining samples were all below 2. This suggests that consumers might not perceive a significant color change in the latter group of samples.

[0056] The color changes in C1, C1-OA, and C1-OA-T can be attributed to the cross-linking reaction between chitosan (CS) and cellulose nanofibers (CNF) via genipin. The higher CNF content in these samples may have promoted this reaction, making the color changes more pronounced.

[0057] Figure 2 bd shows the transmittance of bio-based thin films in the wavelength range of 380 nm to 780 nm. The transmittance of C1, C2, and C3 is between 70% and 90%, with lower transmittance in the near-ultraviolet region. The transmittance of C1-OA, C2-OA, and C3-OA ranges from 75% to 90%, while the transmittance of C1-OA-T, C2-OA-T, and C3-OA-T exceeds 80%.

[0058] The transmittance curves of all samples were smooth, with no obvious absorption peaks. This indicates that the materials are uniform and free of significant defects. The high transmittance of the film provides consumers with a direct sense of product quality.

[0059] (2) Thermogravimetric analysis (TG) The thermal stability of the different groups of bio-based films provided in Example 1 was evaluated using a STA6000 (PerkinElmer). Approximately 4 mg of sample was heated from 30 °C to 800 °C at a heating rate of 10 °C / min under a nitrogen atmosphere.

[0060] Thermogravimetric analysis (TGA) was used to evaluate the thermal stability of the membrane. Figure 2 The example shows the TGA and DTG curves of all sample membranes in the temperature range of 35℃ to 800℃. All sample membranes exhibit two main weight loss phases.

[0061] The first stage occurs between 35°C and 150°C, primarily corresponding to the loss of moisture and volatile substances. During this stage, the highest weight loss rate occurs at approximately 80°C. Treatment of C1, C2, and C3 with octanoic acid (OA) to prepare C1-OA, C2-OA, and C3-OA significantly reduced the weight loss rate, decreasing from 9%, 12%, and 7% to 4%, 5%, and 4%, respectively. This reduction indicates that the hydrophobic octanoic acid chain effectively inhibits water absorption by the bio-based membrane.

[0062] The weight loss rates of C1-OA-T, C2-OA-T, and C3-OA-T were 4%, 4%, and 9%, respectively, which are very close to those of the OA-modified samples. The slightly higher weight loss in some samples can be attributed to the water absorption of thermoplastic starch (TPS) and slight volatilization of the diethyl eutectic solvent (DES). The second stage of weight loss occurred between 200°C and 400°C, primarily due to the thermal degradation of the polymer backbone.

[0063] The main peak temperatures of C1, C2, and C3 were observed to be 330℃, 293℃, and 288℃, respectively. Compared with pure chitosan (CS) (differential thermogravimetric (DTG) peak around 255℃), the cross-linked structure significantly improved the thermal stability of the material.

[0064] After treatment with octanoic acid (OA), the main peak temperature shifted to approximately 253 °C, indicating a decrease in thermal stability. This decrease can be attributed to the disordering of molecular arrangement and a reduction in crystallinity, characteristic of OA-modified samples. Furthermore, the increased residual carbon content in C1-OA, C2-OA, and C3-OA is likely due to the carbonization tendency of the long carbon chains in octanoic acid. The main peak temperatures of C1-OA-T, C2-OA-T, and C3-OA-T are approximately 266 °C, consistent with the thermal stability of the OA-treated materials.

[0065] (3) Mechanical properties The tensile strength (TS) and elongation at break (EB) of the different groups of bio-based films provided in Example 1 were determined using a microcomputer-controlled electronic universal testing machine. The bio-based films were cut into rectangles (1.5 cm × 12 cm) and tested at a rate of 5 mm / min until mechanical breakage occurred. The heat-sealing operation was performed using a manual sealing machine (Deli, China) equipped with a 2 mm sealing strip and a heat-sealing adjustment button. The bio-based films were cut into rectangular strips, each 60 mm long and 15 mm wide, and then fixed to the machine. The film samples were heat-sealed for 2 seconds at setting 4. The two ends of the sample were clamped in the tensile testing machine, ensuring the heat-sealed area was equidistant between the clamps to guarantee the sealing edge was perpendicular to the direction of the applied force. The testing speed was 20 mm / min.

[0066] By tensile strength (TS) Figure 2 h), Elongation at break (EB) Figure 2 i) The mechanical properties of the films were characterized using the corresponding stress-strain curves. The highest tensile strength (25.44 MPa) was observed in the C1 sample with a low chitosan (CS) content. This is mainly because stress transmission is dominated by the rigid network of cellulose nanofibers (CNFs). However, its brittleness resulted in a low elongation at break, only 1.46%. With increasing flexible-chain chitosan content, the continuous rigid structure of the CNFs was disrupted, and the strength gradually decreased, dropping to 14.43 MPa in the C3 sample, while the elongation at break increased to 3.29%. Modification with octanoic acid (OA) significantly reduced the mechanical properties of the films, with the fracture strength decreasing by 46.0%–54.9% from C1-OA to C3-OA. This is mainly attributed to the disruption of the hydrogen bond network between CS and CNFs by the long-chain octanoic acid. Notably, the elongation at break of C3-OA increased to 5.26% due to the presence of octanoic acid.

[0067] By tensile strength (TS)Figure 2 h), Elongation at break (EB) Figure 2 i) The mechanical properties of the films were characterized. The highest tensile strength (25.44 MPa) was observed for C1, with a lower chitosan (CS) content. This is mainly because stress transmission is dominated by the rigid network of cellulose nanofibers (CNFs). However, its brittleness resulted in a low elongation at break, only 1.46%. With increasing flexible-chain chitosan content, the continuous rigid structure of the CNFs was disrupted, leading to a gradual decrease in strength, dropping to 14.43 MPa at C3, while the elongation at break increased to 3.29%. Modification with octanoic acid (OA) significantly reduced the mechanical properties of the bio-based films, with the tensile strength decreasing by 46.0%–54.9% from C1-OA to C3-OA. This is mainly attributed to the long octanoic acid chains disrupting the hydrogen bond network between CS and CNFs. Notably, the elongation at break increased to 5.26% in C3-OA due to the presence of octanoic acid. For the bilayer films C1-OA-T, C2-OA-T, and C3-OA-T, compared with C1-OA, C2-OA, and C3-OA, their mechanical properties were enhanced, with tensile strengths of 27.29 MPa, 23.81 MPa, and 11.44 MPa, respectively. Their elongation at break also increased, reaching 2.56%, 3.94%, and 5.73%, respectively. Previous studies have shown that directly incorporating octanoic acid-thymol eutectic solvent (DES) into the chitosan / gelatin system leads to a decrease in mechanical properties. However, in this study, this trend was successfully reversed by introducing polyvinyl alcohol (PVA) as a plasticizer and interfacial compatibilizer. The enhancement mechanism is mainly attributed to the formation of a hydrogen bond network between the PVA molecular chains and starch. Furthermore, the flexible segments of the PVA chains enhance surface toughness through entanglement.

[0068] Heat seal strength is a key performance indicator for packaging materials, directly affecting the integrity of the packaging, the protection of the contents, and the efficiency of automated production. Test results ( Figure 2 j) indicates that the material exhibits excellent interfacial bonding under standard heat-sealing conditions, with a peak strength of 19.9 N / 15 mm. This is comparable to the heat-sealing strength of high-density polyethylene processed at 126°C, meeting the typical requirements of traditional packaging materials.

[0069] Example 6 Waterproofing of Bio-based Films (1) Dissolution rate After weighing the different groups of bio-based films (2 cm × 2 cm) provided in Example 1, they were soaked in 30 mL of distilled water at room temperature for 24 hours. After soaking, the water on the surface of the film was removed with filter paper, and then the swollen film was weighed. The swelling rate of the film was calculated according to formula (2):

[0070]

[0071] Where W1 and W2 are the initial mass and dissolved mass of the bio-based film, respectively.

[0072] Figure 3 Example a illustrates the swelling properties of different groups of bio-based films provided in Example 1. Pure chitosan (CS) film and pure thermoplastic starch (TPS) film exhibited extremely high swelling ratios, reaching 1683% and 1377%, respectively, while the swelling ratio of cellulose nanofiber (CNF) film could not be measured due to its complete dissolution in water. When CS and CNF were crosslinked via genipin, the movement of the molecular chains was restricted, resulting in a significant decrease in the swelling ratio of the film. Oleic acid (OA) modification further modulated the swelling balance, with the swelling ratios of OA-modified CS / CNF films (C1-OA and C2-OA) approaching the lowest values ​​observed for these materials. However, the swelling ratio of the OA-modified CS / CNF film (C3-OA) was only slightly lower than that of the unmodified CS / CNF film (C3), attributed to its higher CS content. The addition of a thermoplastic starch layer slightly increased the swelling ratio of the film, attributed to the higher swelling ratio of TPS and the addition of polyvinyl alcohol (PVA).

[0073] (2) Water solubility After weighing the different groups of bio-based films (2 cm × 2 cm) provided in Example 1, they were immersed in 30 mL of distilled water at room temperature for 24 hours. After immersion, the films were removed, dried at 25°C for 24 hours, and then weighed. The water solubility of the films was calculated according to formula (3):

[0074]

[0075] In the formula, W1 and W2 are the initial mass and dried mass of the bio-based film, respectively.

[0076] Figure 3 b illustrates the water solubility of the bio-based film provided in Example 1. Due to the small size and high hydroxyl content of nanocellulose (CNF), the CNF film exhibits complete solubility. Although chitosan (CS) and thermoplastic starch (TPS) are also rich in hydroxyl groups, the entanglement of molecular chains in the CS and TPS films results in slightly lower solubility, at 63% and 28%, respectively. The solubility of the film further decreases when CS and CNF crosslink to form a network structure. Modification with oleic acid (OA) reduces the water solubility of C1-OA and C2-OA, while the trend for C3-OA is consistent with the solubility data SD. The addition of thermoplastic starch increases the water solubility of the film, which is attributed to the higher water solubility of the composite TPS, leading to an overall increase in the water solubility of the material.

[0077] (3) Water contact angle At room temperature, the water contact angle (WCA) was measured using a Theta Flex (Attension, Biolin Scientific) to evaluate the hydrophobicity of the different groups of bio-based films provided in Example 1. A 2 μL water droplet was placed on the film surface using a precision syringe, and the WCA value was recorded using a camera.

[0078] To study the interaction between water droplets and the thin film surface, the water contact angle (WCA) of the thin film was measured. Figure 3 (g, h). According to the WCA value, when WCA < 65°, the film exhibits hydrophilicity; when WCA > 65°, the film exhibits hydrophobicity. The control film has a hydrophilic surface at 0 s.

[0079] Pure TPS and CNF membranes exhibit strong hydrophilicity due to their abundant polyhydroxyl structures, with water contact angles (WCA) of 41.3° and 41.7°, respectively. In contrast, the pure CS membrane achieves a contact angle of 89°.

[0080] The contact angles (WCA) of C1, C2, and C3 films are slightly lower than those of pure chitosan (CS) films, ranging from 87° to 88°. This reduction can be attributed to the addition of cellulose nanofibers (CNFs), which, due to their hydrophilicity, lower the overall contact angle, making the films more hydrophilic.

[0081] After hydrophobication treatment with oleic acid (OA), the contact angle of the film increased significantly (P<0.001), ranging from 107° to 110°. This indicates that the long alkyl chain of octanoic acid was successfully grafted onto the chitosan amino group, forming a more hydrophobic surface. The difference in water contact angle (WCA) between substrates with different chitosan contents modified with octanoic acid was less than 3°, indicating that the hydrophobicity mainly depends on the density of interfacial amide bonds.

[0082] After incorporating TPS functional layers, the water contact angle (WCA) of C1-OA-T, C2-OA-T, and C3-OA-T films decreased by 10° to 15°. Despite this reduction, the WCA of these films is still higher than that of the C1, C2, and C3 films. The decrease in contact angle is mainly attributed to the hydrophilicity of TPS, which contains a large number of hydroxyl groups that expose more hydrophilic sites, thus leading to a decrease in the overall water contact angle.

[0083] Example 7 Barrier Properties of Bio-based Thin Films (1) Water vapor transmission rate (WVP) 30 g of anhydrous calcium chloride was placed in a beaker with a diameter of 5.0 cm, and then the beaker was covered and sealed with different groups of bio-based films provided in Example 1. The beaker was placed in an environment with a temperature of 25°C and a relative humidity of 70%, and the weight of the beaker was measured every 12 hours. The water vapor transmission rate of the bio-based film was calculated according to formula (4):

[0084]

[0085] Where Δm / Δt is the moisture absorption rate (g / s), and A is the effective area of ​​the membrane (cm²). 2 ), d is the thickness of the membrane (cm), and Δp is the water vapor pressure difference across the membrane (Pa).

[0086] Water vapor transmission rate (WVP) is an important parameter for packaging films. Lower water vapor transmission rate can reduce the likelihood of food spoilage, reduce moisture loss, and extend the shelf life of food. Figure 3 c shows the water vapor transmission rates of different groups of bio-based films. Among the pure components, cellulose nanofibers (CNF) had the highest water vapor transmission rate, at 5.45 × 10⁻⁶. -13 g·cm·cm -2 ·s -1 ·Pa -1 This can be attributed to its high hydrophilicity and porous fibrous network structure. The water vapor permeability of thermoplastic starch (TPS) is slightly lower, at 4.40 × 10⁻⁶. -13 g·cm·cm -2 ·s -1 ·Pa -1 This is likely due to the tight packing of starch molecular chains. The water vapor permeability of pure chitosan (CS) is 5.05 × 10⁻⁶. -13 g·cm·cm -2 ·s -1 ·Pa -1 Despite its high contact angle (WCA), the increased water vapor transmission rate indicates the presence of micropore defects within the film.

[0087] After crosslinking chitosan (CS) and cellulose nanofibers (CNF), the water vapor transmission rate (WVP) of the films (C1, C2, and C3) decreased compared to pure CS and pure CNF, with C2 exhibiting the lowest WVP at 3.56 × 10⁻⁶. -13 g·cm·cm -2 ·s -1 ·Pa -1 This indicates that the mCS:mCNF = 2:1 composition forms a dense three-dimensional network that effectively hinders the diffusion of water molecules.

[0088] After modification with oleic acid (OA), the water vapor transmission rate (WVP) of C1-OA increased to 5.35 × 10⁻⁶. -13 g·cm·cm -2 ·s -1 ·Pa -1 This may be due to the competition between excessive nanocellulose (CNF) and oleic acid, resulting in more voids and structural defects in the film, weakening its structural integrity. On the other hand, C2-OA (2.66 × 10⁻⁶) -13 g·cm·cm -2 ·s -1 ·Pa -1 ) and C3-OA (2.21×10⁻¹³ 10 -13 g·cm·cm -2 ·s -1 ·Pa -1 The reduced water vapor permeability of these materials can be attributed to their higher chitosan (CS) content, which allows oleic acid to graft and form a dense, continuous hydrophobic layer.

[0089] The addition of a thermoplastic starch (TPS) layer significantly reduced the water vapor transmission rate (WVP) of the materials. Among them, C3-OA-T exhibited the lowest WVP, at 0.58 × 10⁻⁶. -13 g·cm·cm -2 ·s -1 ·Pa -1 Cross-sectional scanning electron microscope (SEM) image of the double-layer film ( Figure 1 b) shows that the C-OA layer structure is loose, with some defects and voids, while the TPS layer structure is denser, with fewer defects and voids, and can effectively block the transport of water vapor. In addition, the multilayer structure may increase the diffusion path of water vapor, further reducing its transport rate.

[0090] (2) Oxygen permeability (OP) Oxygen permeability (OP) was measured using the following method. 5 g of deoxidizing agent (reduced iron powder = 1.25 g, powdered activated carbon = 2.5 g, NaCl = 1.25 g) was added to the bottom of a centrifuge tube, which was then sealed with the different groups of bio-based membrane samples provided in Example 1 and initially weighed. The centrifuge tube was then placed in an environment at 25°C and 70% relative humidity, and its weight was measured every 24 hours. The oxygen permeability of the membrane was calculated according to formula (5):

[0091]

[0092] Where Δm is the weight change of the centrifuge tube (kg), d is the membrane thickness (m), and A is the membrane area (m²). 2 ), where t is the equilibrium time (s).

[0093] Oxygen permeability (OP) is a critical factor in packaging because oxygen accelerates fat oxidation and promotes the growth of aerobic bacteria. Therefore, films with low OP can extend the shelf life of food. After crosslinking chitosan (CS) with cellulose nanofibers (CNF), C2 exhibited the lowest OP, at 4.99 × 10⁻⁶. -11 kg·m·m -2 ·s -1 This is similar to the trend of water vapor transmission rate (WVP).

[0094] After modification with octanoic acid (OA), the oxygen permeability (OP) values ​​of all bio-based membranes decreased. Among them, C3-OA had the lowest OP value, at 2.20 × 10⁻⁶. -11 kg·m·m -2 ·s -1 This indicates that the long alkyl chains of octanoic acid form a dense physical barrier, effectively hindering oxygen permeation. The addition of a thermoplastic starch (TPS) layer further reduced the oxygen permeability of the bilayer film. This reduction, consistent with the moisture permeability (WVP) results, can be attributed to the denser structure of the TPS layer, which effectively blocks oxygen transport.

[0095] Example 8 Antioxidant activity of bio-based thin films (1) Radical scavenging ability of 2,2-diphenyl-1-picrylhydrazine (DPPH) 20 mg of each of the different groups of bio-based thin film samples provided in Example 1 were weighed and placed in 4 mL of ethanol / water (50% by volume) mixture, followed by 250 μL of 1 mM DPPH ethanol solution. The mixture was incubated at room temperature with gentle stirring (80 rpm) for 2 hours in the dark. After incubation, the absorbance of the solution was measured at 517 nm, and the DPPH free radical scavenging rate was calculated according to formula (6):

[0096]

[0097] Among them, A sample A is the absorbance of the ethanol solution containing the membrane sample. DPPH This is the absorbance of the control (i.e., an ethanol / aqueous solution containing DPPH but without a membrane sample).

[0098] (2) Free radical scavenging ability of 2,2'-azobis(3-ethylbenzothiazole-6-sulfonic acid) (ABTS) ABTS solution (7 mM) was prepared by diluting with potassium persulfate (2.45 mM) and incubating overnight in the dark. 20 mg of each of the different groups of bio-based film samples provided in Example 1 was weighed and placed in 4 mL of water, then 250 μL of ABTS solution was added. The mixture was gently stirred (80 rpm) and incubated for 30 minutes in the dark at room temperature. After incubation, the absorbance of the solution was measured at 734 nm, and the ABTS radical scavenging activity was calculated according to formula (7):

[0099]

[0100] Among them, A sample A is the absorbance of the aqueous solution containing the membrane sample. ABTS It is the absorbance of the control (i.e., an aqueous solution containing ABTS but without a membrane).

[0101] Antioxidant properties are crucial for food preservation because oxidation typically occurs in foods containing lipids and pigments. Once oxidation occurs, it produces unpleasant odors and causes color changes, affecting food safety and consumer purchasing decisions. Figure 3 Figures e and 3f demonstrate the membrane's ability to scavenge ABTS and DPPH free radicals. ABTS is stable in aqueous phase, making it ideal for detecting hydrophilic antioxidants, while DPPH is better suited for lipid-soluble systems and can effectively mimic the lipid oxidation environment in food.

[0102] Chitosan (CS) membranes exhibit certain antioxidant properties, which can be attributed to the inherent antioxidant characteristics of CS itself. However, tea polysaccharide (TPS) membranes and cellulose nanofiber (CNF) membranes have almost no antioxidant capacity.

[0103] Both CS and CNF membranes exhibited certain antioxidant capabilities after crosslinking, which can be attributed to the inherent antioxidant properties of CS. Modification with OA slightly enhanced the membrane's ability to scavenge ABTS and DPPH free radicals. Eutectic solvents containing organic acids exhibited strong H2O2 scavenging capabilities, which may explain the enhanced free radical scavenging ability of C-OA compared to C-OA. Introducing a TPS functional layer significantly improved the membrane's free radical scavenging ability, exceeding 96% for ABTS and over 83% for DPPH. This superior antioxidant performance is a result of the combined effect of the catechol structure in thymol and the organic acid properties of OA.

[0104] Example 9 Antibacterial activity The antibacterial activity of different groups of bio-based films provided in Example 1 against *Escherichia coli* and *Staphylococcus aureus* was tested using the plate count method. First, the different groups of bio-based films (2 mg) provided in Example 1 were sterilized by ultraviolet irradiation. Then, the sterilized sample films were immersed in 500 μL of bacterial suspension (10... 5 In a solution of CFU / mL, gently shake at 200 rpm for 4 hours at 37°C. Take a certain volume of the bacterial suspension and dilute to 10⁻⁶. 4 50 μL of the diluted suspension was inoculated onto LB agar medium. The plates were incubated at 37°C for 12 hours to observe bacterial growth. The control group did not contain any sample membrane.

[0105] The antibacterial activity of the membranes was evaluated using the inhibition zone method. Different groups of bio-based membranes and filter paper provided in Example 1 were cut into circles with a diameter of 6 mm and sterilized under ultraviolet light for 15 minutes. The circular membrane samples were then carefully placed in a container containing Staphylococcus aureus or Escherichia coli (10 μL). 6 On the corresponding plates (CFU / mL), tetracycline at 0.15 mg / mL was used as a positive control. The membrane was incubated at 37°C for 12 hours, and the shape and diameter of the inhibition zone were recorded.

[0106] The antibacterial efficacy of the different groups of bio-based films provided in Example 1 was further evaluated using live-death staining analysis. Bacterial solutions were prepared following the same procedures as the plate sterilization test. To prepare the live-death staining working solution, 10 μL of propidium iodide (PI) and 10 μL of 1,3-dimethylamine-2-oxo-1-azacyclopropane-1-methanol (DMAO) were mixed with 80 μL of detection buffer. Then, 0.5 mL of the treated bacterial solution was mixed with 5 μL of the staining working solution and incubated at 37°C in the dark for 15 minutes. Finally, the stained bacterial solutions were photographed using a fluorescence imaging microscope (ZEISS Primostar3, Carl Zeiss Oberkohenk GmbH, Germany).

[0107] Pathogenic microbial contamination poses a significant threat to food safety. Antimicrobial food packaging can effectively prevent food spoilage, ensure food safety, and reduce waste caused by contamination. In this embodiment, a bio-based film was co-cultured with *Escherichia coli* and *Staphylococcus aureus*. The antimicrobial properties of the film were evaluated using the plate coating method, inhibition efficiency, viability staining method, and inhibition zone diameter. Figure 4 a shows the results of the antibacterial test using the plate coating method.

[0108] Compared to the blank control group, TPS and CNF films showed no antibacterial effect. In fact, these films provided nutrients for bacteria, promoting bacterial growth and resulting in a higher colony count compared to the control group. CS has inherent antibacterial properties, therefore the CS film exhibited some antibacterial activity, with a reduced colony count compared to the blank group. However, the films modified with OA (C1-OA-T, C2-OA-T, C3-OA-T) showed no colony growth, indicating that these samples possess excellent antibacterial properties.

[0109] Figure 4 Figures b and 4c demonstrate the antibacterial efficiencies of different groups of bio-based films provided in Example 1 against *Escherichia coli* and *Staphylococcus aureus*. The results showed that the TPS and CNF membranes had an inhibition rate of 0%. The CS membrane exhibited an antibacterial efficiency of 26% against *E. coli* and 56% against *S. aureus*. In contrast, the C1-OA-T, C2-OA-T, and C3-OA-T membranes all showed antibacterial efficiencies greater than 98% against both *E. coli* and *S. aureus*. This high antibacterial performance can be attributed to two factors: first, the electrostatic interaction between chitosan (CS) and the microbial outer membrane disrupts the cell membrane; second, the low-pH eutectic solvent (DES) contains a high concentration of -OH and -COOH groups, which damage the cell membrane and its proteins. Furthermore, thymol in the DES contributes to broad-spectrum antibacterial activity against a variety of foodborne pathogens.

[0110] Figure 4 Figures d and 4e demonstrate the size of the inhibition zones of the different groups of bio-based films provided in the examples against *Escherichia coli* and *Staphylococcus aureus*. Tetracycline was used as a positive control. Figure 4 It was observed that C3-OA and CS films without a functional TPS layer and without added DES did not form inhibition zones. However, since chitosan (CS) is a non-soluble antibacterial material, the CS film exhibited some antibacterial activity when using the plate coating method. The inhibition zones of the C3-OA-T film against Escherichia coli and Staphylococcus aureus were 11 mm and 22 mm, respectively, which are comparable to the inhibition zones of tetracycline against these bacteria. This further confirms that the material has strong antibacterial properties. The antibacterial effect of C3-OA-T against Staphylococcus aureus was more significant than that against Escherichia coli, which may be due to the thicker cell walls of Gram-positive bacteria, composed of multiple layers of peptidoglycan. The inhibition zone sizes of C1-OA-T, C2-OA-T, and C3-OA-T against Escherichia coli and Staphylococcus aureus were similar, indicating that different substrate materials did not significantly affect the overall antibacterial efficiency of the film.

[0111] Fluorescent staining results of live and dead bacteria ( Figure 4f) indicates that C3-OA-T exhibits excellent antibacterial properties against both Staphylococcus aureus and Escherichia coli. After 4 hours of incubation, the percentage of dead Escherichia coli in the experimental group reached 99.8%, and that of dead Staphylococcus aureus reached 92.9%, significantly higher than that in the LB blank control group.

[0112] Example 10: Experiment on the use of bio-based films in food packaging Strawberries of similar maturity and size were selected for the experiment. Three strawberries were randomly placed into three different types of film packaging: different groups of bio-based film samples provided in Example 1, PE / PET composite film, and PLA / PBAT composite film. The samples were then sealed using a heat sealer (Deli). Unpackaged strawberries served as a blank control. All prepared samples were stored at 25°C and 60% relative humidity for 5 days. The weight loss and microbial count of the samples were recorded daily. At least three parallel experiments were conducted under each condition.

[0113] (1) Weight loss rate The weight loss rate is calculated using formula (8):

[0114] Wherein, W1 and W2 are the initial mass of the strawberries and the mass after being stored for different numbers of days, respectively.

[0115] (2) Number of microorganisms A thin slice (approximately 1 gram) was cut from the surface of each strawberry from different groups. The slice was then immersed in 9 mL of 0.85% physiological saline solution, ensuring thorough mixing. The samples were incubated at 37°C for 4 hours, after which the absorbance of the samples was measured at 595 nm using a microplate reader.

[0116] Strawberries, a highly perishable fruit, were used as a model to evaluate the film's preservation capabilities. Based on previous experiments, C3-OA-T was selected as the experimental group due to its excellent water vapor and oxygen barrier properties, which play a crucial role in preservation. Since C3-OAT is a bilayer film, it was compared with two control groups: one was a petroleum-based polymethyl methacrylate and polyethylene bilayer composite film (PET + PE), and the other was a biodegradable polylactic acid and polybutylene terephthalate (PLA + PBAT) bilayer composite film.

[0117] like Figure 5As shown in Figure a, unpackaged strawberries lost a significant amount of moisture and shrank noticeably after 5 days of storage, with extensive mold growth on their surface. Strawberries packaged in PET+PE showed skin damage on day 4, mold appeared on day 5, and some areas of the skin oxidized and turned brown. Strawberries packaged in PLA+PBAT showed skin damage as early as day 3, and mold growth was observed on day 4. In contrast, strawberries treated with C3-OAT showed minimal changes in appearance throughout the storage period and showed no signs of mold. This preservation effect is attributed to the excellent antibacterial and antioxidant properties of the low co-solvents (containing thymol and caprylic acid) in C3-OA-T.

[0118] Freshness is further assessed by measuring weight loss rate and microbial count. Fruit weight loss is primarily due to water evaporation caused by transpiration and respiration, and strawberries, with their thin and delicate skin, are particularly prone to rapid water loss. Figure 5 As shown in b, after 5 days, unpackaged strawberries had the highest weight loss rate, reaching 41%. Strawberries packaged in PET+PE had the lowest weight loss rate, at 2%, thanks to the excellent barrier properties of the PET+PE composite film against water, oxygen, and carbon dioxide. Strawberries packaged in PLA+PBAT had a weight loss rate of 11%, while those packaged in C3-OA-T had a lower weight loss rate of 8%, effectively reducing moisture loss and helping to maintain freshness.

[0119] The number of microorganisms in different groups of bio-based films provided in Example 1 during storage was measured using OD595. Figure 5 c). Unpackaged strawberries, directly exposed to air, experienced a continuous increase in microbial numbers during storage, reaching a peak OD595 value of 0.844 on day 5. PLA+PBAT packaging offered poor barrier properties against water and oxygen, providing favorable conditions for microbial growth. Consequently, strawberries packaged in PLA+PBAT exhibited a higher microbial count, with an OD595 value of 0.70 on day 5. In contrast, PET+PE, due to its superior barrier properties, limited the growth of aerobic bacteria, allowing only limited growth of anaerobic bacteria. Nevertheless, the microbial count still increased over time, reaching an OD595 value of 0.21 on day 5. Although C3-OA-T offered less effective barrier properties against water and oxygen than PET+PE, its strong antimicrobial properties helped control microbial numbers within a lower range. Throughout the 5-day storage period, the microbial count did not increase significantly, and the OD595 value remained at 0.07 on day 5.

[0120] Example 11 Degradation rate of bio-based thin films The biodegradability of the film materials in the natural environment was qualitatively assessed using a disintegration method. Three types of films—PE / PET composite film, PLA / PBAT composite film, and different groups of bio-based films provided in Example 1—were buried in soil at a depth of 10 cm. Film samples were removed from the soil at specific time intervals, photographed to assess their degree of degradation, and the biodegradability of the films was determined by visually observing the integrity of the films.

[0121] Soil burial degradation experiment ( Figure 5 (d) This indicates that C3-OA-T exhibits superior biodegradability compared to traditional packaging materials. In a 30-day landfill test, C3-OA-T showed a degradation rate exceeding 85%, while PET+PE and PLA+PBAT films showed almost no significant degradation. PET+PE is difficult to degrade due to its high crystallinity and dense molecular structure, while PLA+PBAT, although theoretically degradable, suffers from degradation stagnation due to the rigidity of its molecular chains and the lack of specific microbial populations in the soil. These results confirm that C3-OA-T is an environmentally friendly packaging material with excellent biodegradability, providing a promising alternative to petroleum-based plastics.

[0122] This invention provides an asymmetric bilayer biofilm that mimics the "rigid-flexible synergy" effect of petroleum-based packaging. The poor performance of bio-based materials in high-humidity environments is addressed by covalently crosslinking the CNF / CS network with genipin. A balance between hydrophobicity (contact angle 110°) and transparency (visible light transmittance >80%) is achieved through short-chain modification with octanoic acid. Furthermore, the antibacterial properties of the thermoplastic starch (TPS) layer are enhanced using a low-eutectic solvent (thymol / octanoic acid), while simultaneously imparting heat-sealing functionality to the film. The final material exhibits excellent mechanical strength, superior heat-sealing performance (19.9 N / 15 mm), high barrier properties, and strong antioxidant / antibacterial activity (ABTS scavenging rate >96%, DPPH scavenging rate >83%, antibacterial rate >98%). Strawberry preservation experiments confirmed its effectiveness; no mold growth was observed after 5 days, with only 8% weight loss, and soil degradation rate exceeded 85% after 30 days, significantly outperforming traditional plastics. Future research will focus on further optimizing the system, improving the material's long-term water resistance, expanding its application in other perishable food preservation applications, and assessing its long-term safety.

[0123] The above description is merely a specific embodiment 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 technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A bio-based film, characterized in that, The bio-based film has a double-layer composite structure, comprising: an outer layer formed by cross-linking chitosan and cellulose nanofiber through a cross-linking agent, genipin, and hydrophobically modified by octanoic acid, constituting a high-barrier transparent protective layer; and an inner layer formed by compounding thermoplastic starch with a hydrophobic eutectic solvent composed of thymol and octanoic acid, constituting a heat-seal layer with antibacterial and antioxidant functions.

2. The bio-based film according to claim 1, wherein, The mass ratio of chitosan to cellulose nanofiber in the outer layer is 1:1 to 3:

1.

3. The bio-based film according to claim 1, wherein, The components of the thermoplastic starch in the inner layer include starch, polyvinyl alcohol, and glycerol.

4. The bio-based film of claim 1, wherein, In the hydrophobic eutectic solvent, the molar ratio of thymol to octanoic acid is 1:

1.

5. A method of preparing the bio-based film of claim 1, wherein, The steps include: mixing chitosan and cellulose nanofiber in a solution, adding genipin for cross-linking, and adding octanoic acid for hydrophobic modification to obtain an outer layer material solution; mixing thermoplastic starch with a hydrophobic eutectic solvent composed of thymol and octanoic acid in a solution to obtain an inner layer material solution; casting the outer layer material solution and the inner layer material solution layer by layer and drying to obtain the bio-based film with a double-layer composite structure.

6. The production method according to claim 5, wherein In the hydrophobic modification process, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are used as coupling agents to covalently graft octanoic acid onto the amino group of chitosan through an amide bond.

7. The preparation method according to claim 5, characterized in that, The steps of the inner layer material solution include: dispersing starch and polyvinyl alcohol in water and heating to gelatinize, then blending with glycerol after cooling to obtain a thermoplastic starch matrix; mixing thymol and octanoic acid to form the hydrophobic eutectic solvent; mixing the thermoplastic starch matrix and the hydrophobic eutectic solvent uniformly to obtain the inner layer material solution.

8. The preparation method according to claim 5, characterized in that, The step of layer-by-layer casting and drying includes casting the outer layer material solution into a film and drying to form a solid sticky surface; casting the inner layer material solution on the sticky surface and drying to obtain the bio-based film.

9. Use of the bio-based film of claim 1 in the preparation of food preservation packaging materials.

10. Use according to claim 9, characterized in that, The bio-based film is used to package and seal food by taking advantage of the heat-seal performance of the inner layer, and to extend the shelf life of the packaged food by taking advantage of the high barrier property, antibacterial, and antioxidant performance of the bio-based film.

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