Gelatin-bacteriocin amino acid-dialdehyde starch composite film and preparation method and application thereof

Abstract

The application discloses a gelatin-bacteriocin amino acid-dialdehyde starch composite film and a preparation method and application thereof. The preparation method of the composite film comprises the following steps: adding a crosslinking agent into a gelatin solution, adding N-hydroxysuccinimide into the gelatin solution after activation for a set time, then adding bacteriocin amino acid into the gelatin solution, coupling for a set time under weak acid condition and in the dark, and obtaining Gel-MAAs; dissolving the Gel-MAAs in water to obtain a composite solution; dissolving dialdehyde starch in water, gelatinizing, adjusting to weak alkalinity, and obtaining a dialdehyde starch solution; adding the dialdehyde starch solution into the composite solution, mixing uniformly, reacting at 35-45 DEG C for 1-3 h, adding glycerol with a mass fraction of 1-5%, ultrasonic degassing, pouring the film solution into a mold, and drying, and thus the composite film is obtained. The composite film has excellent mechanical properties, physicochemical properties, ultraviolet resistance, good biocompatibility, and great potential in the field of active composite films.

Description

A gelatin-cytosine amino acid-dialdehyde starch composite film, its preparation method and application Technical Field

[0001] This invention belongs to the field of UV-resistant materials technology, specifically relating to a gelatin-cytosine amino acid-dialdehyde starch composite film, its preparation method, and its application. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Ultraviolet (UV) radiation exposes the environment to high levels of UV-B (280-315 nm) and UV-A (315-400 nm) radiation. UV radiation can have various harmful effects on biologically important molecules such as lipids, proteins, and DNA. It can also alter the properties and durability of non-living materials (such as rubber and textiles). Furthermore, many pharmaceuticals require light-protected storage, primarily because UV radiation accelerates drug oxidation. Some drugs can undergo photochemical degradation, which not only reduces their potency but also produces color and precipitation, severely affecting drug quality and even increasing toxicity.

[0004] UV-resistant packaging is generally made of dark-colored glass or plastic. Glass has poor mechanical properties and impact resistance, making it difficult to store. Most plastic films are petroleum-based, lacking renewability and biodegradability. This has driven innovation and application of bio-based active packaging materials. Existing edible films are often prepared using Maillard reactions, enzymatic reactions, and glutaraldehyde crosslinking. However, Maillard reactions suffer from high heating temperatures and uncontrollable reaction products; enzymatic reactions are costly; and glutaraldehyde crosslinking presents significant toxicity issues. These limitations restrict the application of edible films in pharmaceutical and food packaging. Furthermore, existing edible films have weak UV resistance, making them unsuitable for food or pharmaceutical packaging requiring light-proof or dark-protected storage. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a gelatin-cytosine amino acid-dialdehyde starch composite film, its preparation method, and its application.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] In a first aspect, the present invention provides a method for extracting cytosine-like amino acids from *Porphyra tenera*, comprising the following steps:

[0008] After pulverizing the Porphyra yezoensis, add cellulase to it, then add an ethanol solution with a mass concentration of 20-30%, extract at 40-50℃ for 1-3 hours, extract by ultrasonication for 20-40 minutes, centrifuge and collect the supernatant.

[0009] Add 2-6 times the volume of 90-97% ethanol to the supernatant, freeze to precipitate, freeze and centrifuge, freeze-dry the supernatant, and the freeze-dried product is cytosine amino acid.

[0010] Mycosporine-like amino acids (MAAs) are a class of low molecular weight (<400 Da) intracellular compounds composed of cyclohexenone or cyclohexenamine chromophores coupled with one or two amino acids responsible for ultraviolet absorption. MAAs are widely distributed in various marine organisms, especially in red algae, and have high molar extinction coefficients (ε = 28100-50000 L·mol⁻¹). -1 ·cm -1 It is hailed as the strongest ultraviolet-absorbing molecule in nature. It has the ability to absorb light in the UV-A and UV-B range without generating free radicals.

[0011] In some embodiments, the mass ratio of Porphyra tenuifolia to cellulase is 1000:1-2.

[0012] In some embodiments, the method further includes a step of purifying the prepared crude cytosine amino acid. The purification method is as follows: the crude cytosine amino acid solution is loaded into a Qapto Q column, and the ion exchange column is equilibrated with ultrapure water at a flow rate of 0.5-1.5 mL / min; then, linear elution is performed with 0.5-1.5 mol / L NaCl elution buffer at a flow rate of 0.5-1.5 mL / min, and the elution peak solution with UV absorption at 334 nm is collected.

[0013] Secondly, the present invention provides a method for preparing a gelatin-cytosine amino acid-dialdehyde starch composite film, comprising the following steps:

[0014] A cross-linking agent is added to a gelatin solution, and after activation for a set time, N-hydroxysuccinimide is added, followed by the addition of cytosine amino acids. Under weak acid conditions and in the dark, the mixture is coupled for a set time to obtain Gel-MAAs.

[0015] Gel-MAAs were dissolved in water to obtain a complex solution;

[0016] Dissolve dialdehyde starch in water and gelatinize it. Adjust the solution to a weakly alkaline state to obtain a dialdehyde starch solution.

[0017] Add the dialdehyde starch solution dropwise to the complex solution, mix well, react at 35-45℃ for 1-3 hours, add 1-5% glycerol by mass, degas by ultrasonication, pour the membrane solution into a mold, and dry to obtain the final product.

[0018] In some embodiments, the mass ratio of the Gel-MAAs complex to dialdehyde starch is 2-5:3;

[0019] Preferably, the membrane solution contains 3% glycerol, 4% Gel-MAAs, and 3% dialdehyde starch, with % being w / v.

[0020] In some embodiments, the pH value of the dialdehyde starch solution is 9-11, preferably 10.

[0021] In some embodiments, the crosslinking agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC.

[0022] Preferably, the mass ratio of gelatin, EDC, N-hydroxysuccinimide and cytosine amino acids is 0.8-1.2:0.2-0.3:0.1-0.2:0.8-1.2.

[0023] In some embodiments, the activation time is 20-40 min.

[0024] In some embodiments, the weakly acidic pH value is 4-6, preferably 5.

[0025] Thirdly, the present invention provides a gelatin-cytosine amino acid-dialdehyde starch composite film, which is prepared by the aforementioned preparation method.

[0026] Fourthly, the present invention provides the application of the gelatin-cytosine amino acid-dialdehyde starch composite film in the preparation of UV-resistant devices.

[0027] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows:

[0028] The composite membrane prepared by this invention has a compact, uniform, smooth, and flat microstructure. The composite membrane's UV blocking rate is almost 100%. Under high-intensity UV radiation, the survival rate of probiotics encapsulated in the composite membrane reaches over 70%. Cell survival rates of L929 cells co-incubated with the composite membrane within 24 hours were determined using a CCK8 cytotoxicity assay, showing that the cell survival rate of all composite membrane groups was above 80%, demonstrating the excellent biocompatibility of the composite membrane. Simultaneously, fruit storage experiments demonstrated that the composite membrane effectively preserves the appearance and color of strawberries, slows down the loss of pigments and other substances easily damaged by UV radiation, and significantly extends the shelf life of strawberries.

[0029] A gelatin-grafted spore-forming amino acid / dialdehyde starch UV-resistant composite film was prepared by casting. It has excellent mechanical properties, physicochemical properties, UV resistance, and good biocompatibility, and has great potential in the field of active composite films.

[0030] This invention uses dialdehyde starch to induce cross-linking of gelatin molecules via the Schiff base reaction. The reaction conditions are mild, which is more advantageous than commonly used methods in the preparation of edible films, such as the Maillard reaction (high reaction temperature), enzymatic reaction (high cost), and glutaraldehyde cross-linking (high cross-linking toxicity). Moreover, the reaction is easy to recycle. Attached Figure Description

[0031] 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 improper limitation of the invention.

[0032] Figure 1 is a technical roadmap of an embodiment of the present invention;

[0033] Figure 2 is a comparison chart of the effect of cellulase amount on extraction rate;

[0034] Figure 3 is a comparison chart of the effects of ethanol concentration on extraction rate;

[0035] Figure 4 is a comparison chart of the effects of the material-liquid ratio on the extraction rate;

[0036] Figure 5 is a comparison chart of the effects of ultrasonic time on extraction rate;

[0037] Figure 6 shows the response surface of the interaction of various factors on the extraction rate;

[0038] Figure 7 shows the UV-Vis spectrum analysis of MAAs;

[0039] Figure 8 is a liquid chromatogram of MAAs in Porphyra stripes;

[0040] Figure 9 is the liquid chromatogram of purified MAAs;

[0041] Figure 10 shows the mass spectrum of porphyra-334 in *Porphyra yezoensis*.

[0042] Figure 11 is the mass spectrum of shinorine in Porphyra yezoensis;

[0043] Figure 12 is a comparison of the cytotoxicity results of MAAs on L-929 cells;

[0044] Figure 13 shows the live / dead staining of cells cultured in different concentrations of MAAs for 1 day.

[0045] Figure 14 is a schematic diagram of the synthetic route of Gel-Dopa;

[0046] Figure 15 shows the infrared spectra of Gel and Gel-MAAs;

[0047] Figure 16 shows the standard curve for MAAs;

[0048] Figure 17 shows the UV scan spectra of Gel, MAAs, and Gel-MAAs complex;

[0049] Figure 18 shows the circular dichroism spectra of Gel and Gel-MAAs;

[0050] Figure 19 shows the XRD energy spectrum of the Gel-MAAs complex;

[0051] Figure 20 shows the docking results of Porphyra-334 and collagen fragments;

[0052] Figure 21 shows the docking results of Shinorine and collagen fragments;

[0053] Figure 22 shows the microstructure of the Gel-MAAs / DS composite membrane;

[0054] Figure 23 is a thermogravimetric analysis diagram of the Gel-MAAs / DS composite membrane;

[0055] Figure 24 is a comparison of the tensile strength of Gel-MAAs / DS composite films;

[0056] Figure 25 is a comparison of the elongation at break of Gel-MAAs / DS composite membranes;

[0057] Figure 26 is a comparison of the water absorption properties of Gel-MAAs / DS composite membranes;

[0058] Figure 27 is a comparison of the water solubility of Gel-MAAs / DS composite membranes;

[0059] Figure 28 is a comparison of the water vapor transmission rate of the Gel-MAAs / DS composite membrane;

[0060] Figure 29 is a comparison of the water contact angles of the Gel-MAAs / DS composite membrane;

[0061] Figure 30 shows the transmittance of the Gel-MAAs / DS composite film;

[0062] Figure 31 is a comparison of the UV protection of probiotics by the Gel-MAAs / DS composite membrane;

[0063] Figure 32 is a comparison of the survival ability of probiotics under ultraviolet light protection by Gel-MAAs / DS composite membrane;

[0064] Figure 33 shows photos of strawberries stored at 4℃ under different conditions;

[0065] Figure 34 is a comparison of the weight loss rate of strawberries under different conditions at 4℃;

[0066] Figure 35 is a comparison of the cytotoxicity results of the composite membrane on L-929 cells;

[0067] Figure 36 shows the live / dead staining of cells cultured in extract for 1 day. Detailed Implementation

[0068] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0069] Example

[0070] 1) Optimization of conditions for the extraction of MAAs from Porphyra yezoensis by cellulase method

[0071] Mouse connective tissue L-cell line 929 was obtained from the cell bank of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. It was stably cultured, passaged, and cryopreserved in Room 206, Science Laboratory Building, College of Oceanography, Shandong University.

[0072] Table 1 Experimental Materials and Reagents

[0073]

[0074] Table 2 Experimental Instruments and Equipment

[0075]

[0076]

[0077] The method for extracting MAAs from Porphyra yezoensis using cellulase is as follows:

[0078] Pulverize the seaweed to 60 mesh and collect the seaweed powder → Add 0.002g of cellulase to 2g of seaweed powder and then add 60mL of 25% ethanol solution → Extract in a water bath at 45℃ for 2h → Extract by ultrasound for 30min → Centrifuge at 5000r / min for 30min → Collect the supernatant → Add 4 times the volume of 95% ethanol to the supernatant → Freeze and precipitate the alcohol overnight at -20℃ → Freeze and centrifuge at 5000r / min for 30min → Evaporate the supernatant to one bottle bottom at 45℃ → Freeze-dry and collect the sample.

[0079] The main factors affecting the extraction rate of MAAs from *Porphyra tenuifolia* are ultrasonic time, ethanol concentration, liquid-to-solid ratio, and cellulase quality. Extraction experiments were conducted on each of these four single-factor variables. The extraction conditions were designed as follows:

[0080] Ultrasound time (min): 0, 10, 20, 30, 40;

[0081] Ethanol concentration (%): 10, 15, 20, 25, 30;

[0082] Material-to-liquid ratio (g / mL): 1:20, 1:25, 1:30, 1:35, 1:40;

[0083] Cellulase mass (g / g): 0, 0.001, 0.002, 0.003, 0.004.

[0084] When one of the single-factor parameters changes, the remaining parameters are set as follows: ultrasonic time 30 min, ethanol concentration 15%, material-to-liquid ratio 1:30 g / mL, and cellulase mass 0.002 g.

[0085] Response surface optimization experiment

[0086] By combining single-factor experiments, the appropriate range of each factor was determined. Since there was no significant difference in the effect of ultrasonic time on the extraction efficiency of MAAs, the three factors of cellulase amount, ethanol concentration and liquid-to-solid ratio were finally investigated. The Box-Behnken Designs experimental design was adopted, with the MAAs extraction rate from Porphyra tenuifolia as the response value, to further optimize the optimal conditions of MAAs extraction process. The factor levels are shown in Table 3.

[0087] Table 3. Experimental Factor Levels

[0088]

[0089] Purification steps of MAAs from Porphyra yezoensis:

[0090] The crude MAAs sample was dissolved in ultrapure water to obtain a crude MAAs extract (1 g / mL), which was then loaded into a Qapto Q column at a flow rate of 1.0 mL / min. The ion exchange column was equilibrated and stabilized using equilibration buffer (ultrapure water, pH 7.5) at a flow rate of 1.0 mL / min. Linear elution was performed using 1 mol / L NaCl elution buffer at a flow rate of 1.0 mL / min, and the elution solution with the UV absorbance peak at 334 nm was collected.

[0091] UV-Vis absorption spectroscopy scan

[0092] Take a small amount of lyophilized MAAs, dissolve it in ultrapure water and bring the volume to a final volume (0.002 g / mL). Use a UV-Vis spectrophotometer to scan the absorption spectrum and determine the spectrum of the sample in the range of 200–500 nm.

[0093] High performance liquid chromatography

[0094] The lyophilized MAAs powder was dissolved in ultrapure water and brought to a final volume (0.005 g / mL). The solution was then filtered through a 0.22 μm inorganic filter membrane, and the membrane solution was transferred to a liquid chromatography vial. A liquid chromatography detection method was established using a Bischoff-ProntoSIL-120-5-C column. 18 AQ plus (4.6×250mm, 5μm), A phase: 0.2% formic acid, B phase: chromatographic grade methanol (A:B=1:9), flow rate 1ml / min, column temperature 25℃, injection volume 10μL, analysis time 10min.

[0095] Liquid chromatography-mass spectrometry (LC-MS) analysis

[0096] Lyophilized MAAs powder was dissolved in ultrapure water and brought to a final volume (0.005 g / mL). After passing through a membrane, the solution was transferred to a liquid chromatography vial. Liquid chromatography-mass spectrometry (LC-MS) was used to analyze the molecular weight of the MAAs in the sample solution. LC conditions: Aglient poroshell 120SB-C column. 18 (3.0×150mm) Phase A: 0.2% formic acid aqueous solution; Phase B: mass spectrometry grade methanol; elution time: 15 min; injection volume: 10 μL; column temperature: 25℃; flow rate: 1 mL / min. Mass spectrometry conditions: negative ionization mode; spray pressure: 45 psi; drying gas flow rate (N2): 10 L / min; drying gas temperature: 350℃; capillary voltage: 4500 V.

[0097] Biocompatibility verification

[0098] The cytotoxicity of MAAs was detected by the CCK8 assay and live / dead cell staining.

[0099] The first step is to dissolve MAAs in culture medium and dilute them to concentrations of 5, 2.5, 1.25, 0.625, 0.32, 0.16, and 0.08 mg / mL, then sterilize them through a membrane before use.

[0100] The second step involved resuscitating mouse fibroblasts (L929) and transferring them into DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody. L929 cells in the logarithmic growth phase were then digested with trypsin containing EDTA and diluted with fresh medium to obtain 5 × 10⁶ cells / mL. 5 Cell suspension at 100 μL / mL. Add 100 μL of cell suspension to each well of a 96-well cell culture plate to achieve a cell seeding density of 1 × 10⁶ cells / mL. 4 One cell per well was incubated at 37°C for 24 hours.

[0101] Third, after observing confluence of cells under a microscope, the culture medium in the 96-well plate was removed using a pipette. 100 μL of MAAs culture medium and 100 μL of culture medium were added to both the experimental and control groups, and the cells were incubated at 37°C for 24 hours. After 24 hours, the culture medium was removed, and the suspended cells were washed with PBS. 100 μL of CCK-8 solution (diluted 10-fold with the culture medium) was added to each well, and the cells were incubated at 37°C for another hour. The absorbance was then measured at 570 nm to calculate cell viability.

[0102]

[0103] Among them, A t A0 is the absorbance of the cell culture wells containing MAAs culture medium, and A0 is the absorbance of the cell culture wells containing DMEM culture medium. e The absorbance is for the untreated cell group.

[0104] Live / dead cell staining assay: Take 1 mL of cells with a density of 6 × 10⁻⁶ 4 Cells were cultured at a density of 100 cells / mL in 24-well plates and incubated for 1 day. The supernatant culture medium was then aspirated. 1 mL of MAAs culture medium and 1 mL of fresh culture medium were added to the experimental and control groups, respectively, and the plates were incubated at 37°C for another day. The culture medium was then removed, and any remaining medium was washed with PBS. 250 μL of AO / EB reagent was added for staining for 15 min, and the results were observed using an inverted fluorescence microscope.

[0105] All experimental results were analyzed, statistically analyzed, and images were processed using GraphPad Prism 9.0 and Origin 2022 software. Quantitative data are expressed as mean ± standard deviation (Mean ± SD). ANOVA was used to analyze differences between groups, where *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 were considered statistically significant, and ns indicated no statistically significant difference.

[0106] Analysis of Experimental Results

[0107] Effect of cellulase amount on extraction rate

[0108] Under the conditions of 15% ethanol concentration, 30 min ultrasonic extraction time, and a liquid-to-solid ratio of 1:30 (g / mL), the extraction rate generally showed an increasing and then decreasing trend when the mass of cellulase varied from 0 to 0.004 g / g. The extraction rate was the highest when the amount of cellulase added was 0.001 (g / g), as shown in Figure 2. It can be seen that when the amount of cellulase exceeds a certain range, the cell viability will decrease. Therefore, the cellulase concentration range selected in the subsequent optimization experiment should be 0.001 to 0.003 g / g.

[0109] Effect of ethanol concentration on extraction rate

[0110] Under the conditions of ultrasonic extraction time of 30 min, liquid-to-solid ratio of 1:30 (g / mL), and cellulase mass of 0.002 (g / g), the extraction rate generally showed an increasing and then decreasing trend when the ethanol concentration varied from 10% to 30%. However, as the ethanol concentration increased from 15% to 30%, the extraction rate showed a decreasing trend, as shown in Figure 3. This may be because the reduction of polar water molecules affected the state of agar in laver and hindered the dissolution of MAAs. Therefore, the ethanol concentration range of 15% to 25% was selected for subsequent optimization experiments.

[0111] Effect of material-liquid ratio on extraction rate

[0112] Under the conditions of 15% ethanol concentration, 30 min ultrasonic extraction time, and 0.002 g / g cellulase, the experimental data show that the extraction rate increases and then decreases with the continuous increase of the liquid-to-solid ratio, reaching its highest value at a liquid-to-solid ratio of 1:30 (g / mL). The reason for this may be that when the liquid-to-solid ratio is low, the solvent and seaweed powder cannot fully wet and contact each other. Additionally, seaweed is rich in polysaccharides, and seaweed powder has strong water absorption, thus affecting the extraction of MAAs. When the liquid-to-solid ratio reaches approximately 1:30 (g / mL), the seaweed powder reaches water saturation, which is beneficial for the dissolution of MAAs. As the liquid-to-solid ratio increases further, the amount of ethanol in the system also increases. Under the effect of ultrasonication and heating, polar MAAs molecules may decompose or transform. Furthermore, excessively high solvent concentrations will increase losses in subsequent extraction processes. Therefore, a liquid-to-solid ratio of 1:25–1:35 (g / mL) is preferable, as shown in Figure 4.

[0113] Effect of ultrasonic time on extraction rate

[0114] Under the conditions of 15% ethanol concentration, a liquid-to-solid ratio of 1:30 (g / mL), and 0.002 (g / g) cellulase, the extraction rate reached its highest value at 20 min when the ultrasonic time varied from 0 to 40 min, as shown in Figure 5. However, there was no significant difference between the groups, indicating that the ultrasonic time had little effect on the extraction efficiency of MAAs. Therefore, 20 min, which had a relatively higher extraction rate, was ultimately selected as the reaction condition.

[0115] Experimental results of response surface optimization of MAAs extraction rate

[0116] Taking into account the results of single-factor experiments, cost, and extraction efficiency, the optimal level range for each factor was determined. Based on the Box-Behnken Designs principle, cellulase amount (A), ethanol concentration (B), and solid-liquid ratio (C) were determined as independent variables, with MAAs extraction efficiency as the response value. A response surface methodology experiment was designed, and the experimental scheme and results are shown in Table 4.

[0117] Table 4. Response Surface Experiment Scheme and Results

[0118]

[0119] The experimental data in Table 2-4 were subjected to fitting regression analysis, and the model was also subjected to analysis of variance and significance testing. The final response surface regression model is as follows:

[0120] Y=11.98+0.41A+0.36B+0.28C-0.24AB-0.44AC+0.012BC-1.57A 2 -1.60B 2 -1.02C 2 .

[0121] The response surface regression model was significant (p < 0.01), while the lack-of-fit term was not significant (p > 0.05). The lack-of-fit term means that the regression equation can well reflect the relationship between each factor and the response value, indicating that this response surface regression model can be used to analyze and predict the extraction rate of MAAs from Porphyra tenuifolia by ultrasonic cellulase extraction (Table 5).

[0122] Table 5. Results of Regression and Analysis of Variance

[0123]

[0124] Note: ** indicates highly significant (p<0.01), * indicates significant (p<0.05).

[0125] The influence of each variable in the regression equation on the response value is determined by the F-test. The smaller the p-value and the larger the F-value, the higher the significance of the relative variable. A, B, A 2 B 2 C 2 Both AC and the reaction rate significantly affected the response value (p < 0.05). Overall, the linear and quadratic terms had the most significant impact, followed by the interaction term. Based on the magnitude of F, the order of influence of each factor on extraction efficiency was: cellulase concentration > ethanol concentration > liquid-to-solid ratio.

[0126] The experimental results were processed and plotted using Design-Expert software, as shown in Figure 6. The interaction between the two factors on the response value can be reflected in the three-dimensional surface plot. The presence of a maximum point on the response surface indicates the existence of extreme values ​​within the selected experimental range.

[0127] The strength of the interaction effect can be seen from the shape of the fitted response surface and the contour lines. The curvature of the surface reflects the magnitude of the influence of a single factor on the response value. It can be observed from the response surface plot that the cellulase quality has a greater impact on the response value than the liquid-to-solid ratio and ethanol concentration, specifically reflected in the steeper curve of the surface graph, which is consistent with the results of linear regression analysis. An elliptical center on the contour lines indicates a significant interaction between the two factors, while a circular center indicates a less significant interaction. The contour lines in Figure 6 show that the interaction between ethanol concentration and cellulase quality is the most significant, while the interaction between ethanol concentration and liquid-to-solid ratio is the least significant. This also aligns with the results of the regression analysis.

[0128] The Design-Expert software fitting results showed that the extraction conditions with the highest extraction rate were as follows: when the amount of cellulase was 1.11 mg / g... -1 The extraction rate was highest at 12.0368% when the ethanol concentration was 15.53% and the material-to-liquid ratio was 1:31.

[0129] Ultraviolet scanning spectrum

[0130] As shown in Figure 7, the UV-Vis spectrum analysis of the extract from *Porphyra yezoensis* reveals a peak absorption at 334 nm. Based on a review of the literature, it is preliminarily inferred that *Porphyra yezoensis* contains MAAs.

[0131] High performance liquid chromatography

[0132] As shown in HPLC chromatogram 8, there are 6 peaks, including 2 main peaks and 4 smaller peaks, indicating that at least 6 MAAs substances exist in *Porphyra yezoensis*, but 2 are major components, with normalized peak areas of 9.11% and 84.28%. Figure 9 shows that the purified MAAs have two main peaks, proving that 2 more MAAs components remain after purification. The normalized peak area of ​​the peak with a retention time of 4.6 min is 33.89%, while the normalized peak area of ​​the peak with a retention time of 8.9 min is 66.11%, and its maximum UV absorption wavelength λmax is 334 nm.

[0133] Results of liquid chromatography-mass spectrometry analysis

[0134] The mass spectrometric structures of the main MAAs components in *Porphyra tenuifolia* were obtained by liquid chromatography-mass spectrometry (LC-MS), as shown in Figure 10. The ion peaks of the main components are [M+H]. -With m / z = 345, the estimated molecular weight is 346. Another major mass spectrum of MAAs in *Porphyra yezoensis* is shown in Figure 11, with the main component's ion peak [M+H]. - Given m / z = 331, the estimated molecular weight is 332.

[0135] Based on HPLC, UV and LC-MS spectra, and in conjunction with references, the main components of the MAAs extract from *Porphyra stylosa* were identified as porphyra-334 and shinorine.

[0136] Biocompatibility validation results of MAAs

[0137] Cytotoxicity of MAAs is also an important indicator for evaluating their use as active additives. The CCK8 assay was used to detect the cytotoxicity of MAAs solutions on L-929 cells, with MAAs concentrations ranging from 5 mg / mL to 0.08 mg / mL and a reaction time of 24 h. As shown in Figure 12, MAAs significantly promoted cell growth at lower concentrations, but at higher concentrations, they had a slight inhibitory effect, exhibiting a dose-dependent cytotoxicity. Even at MAAs concentrations as high as 5 mg / mL, cell viability remained above 90%. These results indicate that MAAs possess good biocompatibility.

[0138] The results of the live / dead cell staining assay in Figure 13 are consistent with those of CCK8. After 24 hours of co-incubation of L929 cells in MAAs culture medium, the vast majority of L929 cells maintained a spindle shape and were uniformly distributed as green live cells, with virtually no red blood cells or dead cells, indicating that MAAs have no cytotoxicity and good biocompatibility.

[0139] 2) Preparation of Gel-MAAs

[0140] Table 6 Experimental Materials and Reagents

[0141]

[0142] Table 7 Experimental Instruments and Equipment

[0143]

[0144] The preparation method of the Gel-MAAs complex is as follows: 1 g of gelatin (Gel) was weighed and dissolved in 100 mL of deionized water and stirred at 60 °C for 30 min until completely dissolved. After cooling to room temperature, 0.25 g of EDC was added and stirred until completely dissolved. After activation for half an hour, 0.15 g of NHS was added. Subsequently, 1 g of MAAs was added to the activated solution, and coupling was carried out for 3 h under dark conditions at pH 5 and room temperature with continuous stirring. After the reaction was completed, the solution was dialyzed using a dialysis bag with a capacity cutoff of 12000–14000 Da in low-temperature deionized water acidified with hydrochloric acid at pH 5 (with frequent changes) for 72 h. After dialysis, the dialyzed solution was freeze-dried to finally obtain the product Gel-MAAs, as shown in Figure 14.

[0145] Fourier transform infrared spectroscopy

[0146] Take appropriate amounts of gel, gel-MAAs, and dry potassium bromide powder (1:100), mix and grind them evenly, press them into thin sheets, and perform full-band infrared scanning with a spectral range of 4000-400 cm⁻¹. -1 The resolution is 4cm. -1 The plotting was performed using Origin 2022 software.

[0147] Construction of the standard curve for MAAs:

[0148] Construction of the MAAs standard curve: Prepare a 10 mg / mL stock solution of MAAs, dilute it sequentially, and perform UV spectrophotometry on each solution to determine the position of the maximum absorption peak of MAAs. Record the absorbance of the MAAs solution at the maximum absorption peak, and finally plot the standard curve with the concentration of MAAs on the x-axis and the absorbance on the y-axis.

[0149] Gel-MAAs grafting rate determination:

[0150] Gel-MAAs grafting rate determination: A standard solution of Gel and Gel-MAAs complex (0.002 mg / mL) was accurately prepared, and then a UV spectrophotometer was used to scan the UV spectrum from 200 to 500 nm to obtain the UV spectrum of the Gel and Gel-MAAs complex. Simultaneously, the maximum absorption wavelength of Gel-MAAs was determined, and the absorbance of Gel-MAAs was measured at the highest absorption peak. The grafting rate of MAAs was calculated based on the obtained MAAs standard curve.

[0151] Circular dichroism spectroscopy

[0152] Accurately weigh Gel and Gel-MAAs to prepare dilute solutions (0.125 mg / mL), and use a circular dichroism chromatograph (Chirascan, Applied Photophysics, UK) to perform spectral scanning of the prepared samples in the range of 190-280 nm.

[0153] X-ray diffraction analysis

[0154] The crystal structures of gel and the prepared gel-MAAs composites were investigated using an X-ray diffractometer (D8, Bruker, Germany). The sample measurement conditions were: diffraction width DS = SS = 1°, step size 0.02°, step speed 6° / min, RS = 0.3 mm, 20℃, 40 kV, 40 mA, and scanning angle (2θ) ranging from 5 to 85°. The final sample spectra were analyzed using MDI Jade software.

[0155] Molecular docking - data and simulation software

[0156] The structure of gelatin was obtained and downloaded from the RCSB PDB database (http: / / www.rcsb.org / pdb / home / home.do) (PDBID: 8h0e); the spatial structures of MAAs were obtained from the protein database (https: / / pubchem.ncbi.nlm.nih.gov / ), using Porphyra-334 (PubChem CID6857486) and Shinorine (PubChem CID10471931) as representatives. Molecular covalent docking was performed using the Schrodinger software package. Unless otherwise specified, default parameter settings were used.

[0157] Molecular docking process: The gelatin structure was prepared using the Protein Preparation Wizard module in the Schrodinger software package: protein hydrogenation, removal of water molecules, application of OPLS-2005 force field and energy optimization of the protein structure, and removal of all docked small molecules in the gelatin structure.

[0158] Ligand preparation was performed using the LigPrep module: OPLS-2005 was given a force field to generate tautomers of each small molecule at pH 7.0 ± 2.0, and the corresponding rational 3D structure of each small molecule was generated.

[0159] Covalent docking calculations were performed using the CovDock module in the Schrodinger software package. First, an active pocket was defined centered on the ligand for each crystal structure, with initial X, Y, and Z dimensions of 126×126×126 points. Once the most favorable interaction sites for each formed complex were determined, a small grid of 40×40×40 points was used to specify their respective binding energies. Then, Lys12 was selected as the reaction site for covalent docking, and the docking reaction was chosen as Iminecondensation, with other parameters left as default.

[0160] The docking results are ranked according to the docking score, and the superior docking configurations are selected, exported, and analyzed.

[0161] All experimental results were analyzed, statistically analyzed, and images were processed using GraphPad Prism 9.0 and Origin 2022 software. Quantitative data are expressed as mean ± standard deviation (Mean ± SD). ANOVA was used to analyze differences between groups, where *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 were considered statistically significant, and ns indicated no statistically significant difference.

[0162] Results Discussion

[0163] Fourier transform infrared spectroscopy

[0164] The carboxylate groups of MAAs and the amino groups of gelatin form amide bonds via EDC / NHS coupling. As shown in Figure 15, comparing the infrared spectra of gel and the gel-MAAs complex, it can be seen that the main characteristic peaks of the gel-MAAs complex are consistent with those of gel, indicating that the grafting of MAAs did not affect the overall structure of gel. Furthermore, in the spectrum of the gel-MAAs complex, at 1451 cm⁻¹... -1 and 2921cm -1 A characteristic absorption peak belonging to MAAs molecules appeared; 1636 cm⁻¹ -1 The appearance of an amide peak indicates the stretching vibration of the NC=O bond in the amide group, and this peak exhibits a higher intensity. This confirms the amidation reaction has occurred, and the Gel-MAAs grafting has been successful.

[0165] MAAs Standard Curve and UV Spectrum

[0166] A standard curve for MAAs was successfully obtained by plotting MAAs concentration on the x-axis and absorbance on the y-axis, as shown in Figure 16. As shown in Figure 17, the highest absorption peak of MAAs appears at 334 nm. While Gel does not show a significant UV absorption peak at 334 nm, the highest peak of the Gel-MAAs complex also appears at 334 nm. This result indicates that MAAs were successfully grafted onto the Gel molecule. Substituting the absorbance of the Gel-MAAs complex into the standard curve, the MAAs content in the Gel-MAAs complex can be calculated to be (148.06 ± 3.79) μmol / g.

[0167] Circular dichroism spectrum

[0168] Changes in protein secondary structure can be determined using circular dichroism spectroscopy, a widely used method. This embodiment investigates the effect of MAAs modification grafting on the secondary structure of gel molecules using circular dichroism spectroscopy. As shown in Figure 18, a significant negative peak is observed at 198 nm in the gel spectrum, due to the typical random coil conformation of gel. Simultaneously, the ellipticity of the gel-MAAs complex bands near 200 nm increases significantly, primarily due to the increased random coil conformation.

[0169] X-ray diffraction

[0170] The crystallinity of polymers can be detected using X-ray diffraction. Sharp and narrow diffraction peaks are characteristic of crystals, while broad and diffuse diffraction peaks are exhibited by the amorphous state. As shown in Figure 19, Gel exhibits a broad peak near 2θ = 20.7°, indicating its amorphous structure. The diffraction peak intensity of the Gel-MAAs composite is significantly reduced, indicating a more pronounced amorphous state; the amidation reaction decreases the crystallinity of Gel.

[0171] Molecular docking results

[0172] Covalent docking mechanisms were analyzed using Schrodinger Maestro 12.8 software, and docking was performed between Porphyra-334, Shinorine, and collagen fragments. The results demonstrated the successful covalent docking of MAAs in the selected region with Lys-12 of the collagen fragment. The covalent docking mechanism was scored using Schrodinger Maestro 12.8 software, with lower values ​​indicating easier binding to the collagen fragment. Prediction results showed that the docking score for Porphyra-334 with the collagen fragment was -3.742, and the docking score for Shinorine with the collagen fragment was -3.811, indicating strong binding affinity between MAAs and collagen fragments, with the Shinorine configuration exhibiting even stronger binding affinity.

[0173] Table 8. Results exported after docking screening of Porphyra-334, Shinorine, and collagen fragments.

[0174]

[0175] 3) Preparation of Gel-MAAs / DS composite membrane

[0176] Table 9 Experimental Materials and Reagents

[0177]

[0178] Table 10 Experimental Instruments and Equipment

[0179]

[0180]

[0181] Preparation of Gel-MAAs / DS composite membrane:

[0182] Weigh out 2, 3, 4, and 5 g of Gel-MAAs complex respectively and dissolve them in 50 ml of deionized water. Stir at 60°C for 0.5 h to ensure complete dissolution. Separately weigh out 3 g of dialdehyde starch and dissolve it in 50 ml of deionized water, then gelatinize it thoroughly (3%) and adjust the pH to approximately 10. Slowly add the dialdehyde starch solution dropwise to the gelatin solution, stirring until homogeneous. React at 40°C for 1.5 h, and add 3% glycerol as a plasticizer. After ultrasonic degassing, pour 20 g of the membrane solution into a 90 mm diameter plastic petri dish and dry it in a 30°C constant temperature drying oven. Rehydrate the membrane by placing it in a desiccator containing saturated KBr solution (RH 60%, room temperature) for 24 h, then observe it.

[0183] SEM morphology analysis of Gel-MAAs / DS composite membrane

[0184] The thin film sample was broken up in liquid nitrogen and mounted on a sample holder using double-sided tape. Then, gold was sputtered onto the thin film sample using a sputtering coating machine. The sample was observed using a field emission scanning electron microscope with an accelerating beam voltage of 20 kV.

[0185] Thermogravimetric analysis of Gel-MAAs / DS composite membrane

[0186] The sample was weighed using an electronic analytical balance, placed in a crucible, and its thermogravimetric curve was recorded within a temperature range under N2 protection. The mass change of the sample was determined by thermogravimetric analysis (TGA).

[0187] Mechanical properties of Gel-MAAs / DS composite membranes

[0188] The composite film was cut into rectangular strips of 40×10mm, and both ends were clamped flat onto the tensile probe of the texture analyzer. The initial spacing was set to 10mm, and the tensile speed was set to 60mm / min. Three parallel experiments were performed on each composite film layer, and the maximum tensile force and elongation at fracture were recorded. The average value of the results was taken.

[0189] Tensile strength(MPa)=F÷(d×W)#(4-1)

[0190] Elongationat break(%)=ΔL÷L0#(4-2);

[0191] Where F is the film fracture stress (N), d is the film thickness (mm), and W is the film width (mm); ΔL and L0 are the tensile length and original length (mm) of the film sample, respectively.

[0192] Water absorption and water solubility of Gel-MAAs / DS composite membranes

[0193] The long-term water absorption and water solubility of the film are determined by its water absorption (W). a ) and water-soluble (W s Characterized by ) The composite film was cut into square samples of 2×2cm and dried in an oven at 105℃ for 24h. The initial dry weight (W) was recorded. o Then, transfer it to 50 mL of distilled water at room temperature and soak for 24 hours. Afterward, remove it from the water, wipe it dry with filter paper, and record it as wet weight (W). t Finally, the sample was placed back into a 105℃ oven for 24 hours to dry, then transferred to a desiccator to cool and record the dry weight of the film (W). d ).

[0194] W a (%) = (W) t -W0)÷W0×100%#(4-3);

[0195] W s (%) = (W0 - W) d )÷W0×100%#(4-4).

[0196] Water vapor permeability of Gel-MAAs / DS composite membrane

[0197] Weigh 3g of anhydrous CaCl2 into a 50mm × 10mm weighing bottle. Cover the bottle opening with an intact membrane (30mm in diameter) and seal with Vaseline. Record the initial mass of the weighing bottle. Place it in a desiccator containing a saturated NaCl solution (60% RH) at room temperature. Measure the mass of the weighing bottle every 24 hours for 3 consecutive days.

[0198] WVP=(24×Δm)÷At#(4-5).

[0199] In the formula, WVP is the water vapor transmission rate (g / (m³)). 2 •24h); A is the area of ​​the thin film through which water vapor permeates (m²) 2 ); t is the time interval (h) between two stabilizations of the mass increment; Δm is the mass increase (g) after moisture absorption.

[0200] Water contact angle of Gel-MAAs / DS composite membrane

[0201] An optical contact angle meter was used to detect the hydrophilicity of the composite membrane: 1 μL of ultrapure water was placed on the sample, and the state of the water droplet on the membrane surface was photographed 3 seconds after the water droplet was dropped at ambient temperature, and the water contact angle was measured. The initial contact between the water droplet and the surface was calculated by software to obtain the water contact angle value of the membrane surface. All experiments were performed three times for each sample.

[0202] Light transmittance of Gel-MAAs / DS composite film

[0203] The composite film was cut into 1×4cm pieces of a certain thickness. 2 The rectangular shape was scanned in the 200-800 nm wavelength range using a UV-Vis spectrophotometer. The transmittance of the membrane material in the UV and visible light regions was measured using UV-Vis spectroscopy.

[0204] Study on the UV protection properties of Gel-MAAs / DS composite membranes - Verification of their protective effect on probiotics

[0205] The protective effect of the composite membrane on probiotics under ultraviolet light was verified using Lactobacillus rhamnosus.

[0206] First, prepare MRS solid and liquid culture media. For the solid culture medium: weigh 33.6g of MRS agar powder and dissolve it in 500mL of deionized water. After sterilization, transfer it to a super bench and wait until the culture medium cools to a slightly hot temperature. Then pour it into a 9cm diameter sterile petri dish for later use. After solidification, invert it for storage. For the MRS liquid culture medium: weigh 26.1g of MRS broth powder and dissolve it in 500mL of deionized water. After sterilization for 20 minutes, cool it for later use.

[0207] The preparation concentration is 1×10 6 A bacterial suspension of CFU / mL was serially diluted with sterile physiological saline and transferred to a sterile culture dish. The dish was then covered with a composite membrane and irradiated under a UV lamp for 15 min. 200 μL of the irradiated bacterial solution was evenly spread onto MRS solid medium and incubated at 37℃ for 48 h. Uncovered and PE-covered bacterial solutions were used as controls. The number of probiotic colonies was observed and recorded.

[0208] C = B ÷ A × 100% (4-6)

[0209] C represents the survival rate of probiotics, A represents the number of colonies in the group without ultraviolet irradiation, and B represents the number of colonies in the experimental group.

[0210] Verification of strawberry preservation using Gel-MAAs / DS composite film

[0211] Strawberries (purchased from the local market) were divided into three groups: a control group (uncovered), a group covered with PE plastic wrap, and experimental groups sealed with a prepared Gel-MAAs / DS composite film. They were placed in a refrigerator at 4°C, and photographed every 24 hours to observe spoilage. The strawberries were also weighed to calculate the weight loss rate.

[0212] Biocompatibility verification of Gel-MAAs / DS composite membrane

[0213] Disinfect 1g of the composite membrane in 70% ethanol for 2 hours, and wash it three times in sterile phosphate-buffered saline (PBS) for 10 minutes each time. Incubate each sterilized membrane in 10mL of complete culture medium at 37℃ for 24 hours, and collect the extract by centrifugation; the specific experimental method is as above.

[0214] All experimental results were analyzed, statistically analyzed, and image processed using GraphPad Prism 9.0 and Origin 2022 software. Quantitative data are expressed as mean ± standard deviation (Mean ± SD). ANOVA was used to analyze differences between groups, where *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, asterisks were considered statistically significant, and ns indicated no statistically significant difference.

[0215] Results Discussion

[0216] SEM results of Gel-MAAs / DS composite membrane

[0217] The surface and cross-sectional micromorphology of the freeze-dried films were observed using liquid nitrogen brittle fracture. All films exhibited dense and smooth surfaces, indicating that the polymers (Gel-MAAs and DS) were well mixed to form a compatible film. With increasing Gel-MAAs content, the crosslinked film surface still exhibited a flat and dense microstructure, and no phase separation or polymer aggregation was observed, as shown in Figure 22. These results indicate that the introduction of the Schiff-base network and the establishment of hydrogen bonding interactions improve the compatibility between components, thereby increasing the crosslinking density and uniformity. The formed covalent and non-covalent interactions significantly altered the microstructure of the film, which also provides evidence for the influence of Gel-MAAs content on the mechanical and barrier properties of the film.

[0218] Thermogravimetric analysis results of Gel-MAAs / DS composite membrane

[0219] As can be observed from Figure 23, the composite membrane exhibits three stages of heat loss. The first stage mainly occurs from room temperature to 200℃, where the mass loss is primarily due to the loss of low molecular weight substances such as water and volatile materials in the sample. The second stage mainly occurs from 250 to 500℃, primarily due to the volatilization of glycerol in the film and the thermal degradation of gelatin molecular chains and DAS macromolecular chains, which constitutes the main weight loss stage. At 600℃, the remaining mass is 28.7%.

[0220] Mechanical properties of Gel-MAAs / DS composite membranes

[0221] Mechanical properties are crucial for films in protecting product integrity. Figures 24 and 25 show the mechanical properties of the Gel-MAAs / DS composite film. As observed in Figure 24, the tensile strength of the composite film initially increases and then decreases with increasing Gel-MAAs composite concentration. This change may be because when the Gel-MAAs composite content is low, insufficient crosslinking with DS leads to stress concentration and reduced tensile strength. Furthermore, DS, as a hydrophilic natural crosslinking agent, also contributes to this phenomenon due to its plasticizing effect and hydrophilicity; when the Gel-MAAs composite content is high, DS is insufficient to achieve sufficient crosslinking with Gel-MAAs, resulting in reduced film strength. Adding 4% Gel-MAAs to DS results in a more sufficient crosslinking between DS and Gel-MAAs, achieving a tensile strength of 5.8 MPa, the highest possible tensile strength. These results are closely related to stronger crosslinking within the polymer components. The elongation at break gradually decreases with increasing Gel-MAAs composite concentration. The introduction of Schiff base and hydrogen bond interactions enhances the mechanical properties of the film. The cross-linked structure of Schiff bases enhances intermolecular forces, resulting in a denser and more compact network structure. This change restricts the movement of molecular chains and reduces their elongation ability.

[0222] Water absorption and water solubility of Gel-MAAs / DS composite membranes

[0223] The water absorption and water solubility of the composite membrane are shown in Figures 26 and 27. With increasing concentration of the Gel-MAAs composite, the water absorption and water solubility of the composite membrane increased, decreased, and then increased again. This may be because the degree of crosslinking between Gel-MAAs and DA first increases and then decreases with increasing Gel-MAAs concentration. 2% and 3% Gel-MAAs are insufficient for sufficient crosslinking with DS, and the hydrophilicity of DS leads to increased water absorption and water solubility of the composite membrane. 4% Gel-MAAs achieves the optimal crosslinking degree with DS, forming a denser and more compact network structure, thus reducing the water absorption and water solubility of the composite membrane. 5% Gel-MAAs is excessive; the hydrophilic amino and carboxyl groups in gelatin also contribute to increased water absorption and water solubility of the composite membrane. This result is consistent with the inferences regarding the mechanical properties.

[0224] Water vapor transmission rate and water contact angle of Gel-MAAs / DS composite membrane

[0225] Water vapor in the environment can promote microbial growth, directly affecting the freshness of food during preservation. Water vapor barrier properties (WVP) are an important indicator for evaluating the suitability of food preservation films, reflecting the composite film's ability to block water vapor. Hydrophobicity is the most important factor determining the suitability of polymer film materials. The hydrophobicity of the film is evaluated using the static water contact angle shown in Figure 29. The barrier capacity of a material is mainly affected by its chemical structure and microstructure.

[0226] Compared to other films, the 4% Gel-MAAs / DS composite film exhibits a denser and smoother structure, improving its hydrophobicity and enhancing its resistance to water vapor, as shown in Figure 28. These results demonstrate the density and integrity of the Gel-MAAs / DS composite film. The Gel-MAAs / DS composite film forms a tight structure between DS and Gel-MAAs through a Schiff base, generating strong intermolecular forces and exhibiting excellent barrier properties, making it a promising candidate for application in active composite membrane materials.

[0227] Light transmittance of Gel-MAAs / DS composite film

[0228] As shown in Figure 30, the control group film exhibits high transmittance (37-42% at 280-400 nm), and the commercially available PE film also shows high transmittance, indicating that they cannot shield food from ultraviolet radiation and are ineffective in preventing UV-induced oxidation. Notably, the Gel-MAAs / DS composite film exhibits almost zero transmittance in the ultraviolet spectrum (200–400 nm), demonstrating excellent UV resistance.

[0229] Protective effect of Gel-MAAs / DS composite membrane on probiotics under ultraviolet light

[0230] In modern life, the demand for probiotics is increasingly strong, and many foods, medicines, and health products contain probiotics. Ultraviolet radiation has high sterilization efficiency and is a simple, economical, and safe sterilization method. Figures 31 and 32 show the protective effect of the composite film on probiotics under strong ultraviolet radiation. The commercially available PE film in the control group has high transmittance and cannot shield probiotics from ultraviolet radiation; less than 10% of the probiotics survived. Notably, under the 4% Gel-MAAs / DS composite film coverage, more than 75% of the probiotics still survived, demonstrating excellent UV resistance.

[0231] Strawberry preservation with Gel-MAAs / DS composite film

[0232] A real-time study was conducted using Gel-MAAs / DS composite film as a packaging material to investigate its effects on fruit preservation. Strawberries were chosen as the food model in this study because they are perishable fruits with a short shelf life.

[0233] To simulate real-world conditions, strawberries stored at 4°C were compared between unwrapped, commercially available PE cling film, and Gel-MAAs / DS composite film-covered strawberries. The spoilage process of unwrapped, commercially available PE cling film-covered, and Gel-MAAs / DS composite film-covered strawberries stored at 4°C for 16 days is shown in Figures 33 and 34. Initially, the strawberries were plump and bright red. Unwrapped strawberries showed slight collapse on day 4 and mold growth on day 14, indicating spoilage; strawberries preserved with commercially available PE cling film showed signs of rotting on day 12. After day 16, more and more mold spots appeared, and the rotting and darkening of the strawberries became increasingly obvious; strawberries covered with the Gel-MAAs / DS composite film did not rot, and their appearance, color, and plumpness remained excellent. Even after day 16, no white mold was observed in the strawberries covered with the Gel-MAAs / DS composite film. Therefore, the prepared Gel-MAAs / DS composite film significantly extended the shelf life of strawberries. The Gel-MAAs / DS composite film exhibits excellent performance due to its low water vapor permeability (WVP), low hydrophilicity, and UV resistance. All strawberries showed an increasing weight loss rate within 16 days of storage, attributed to their high water content, which led to increased water loss through transpiration and respiration. The low WVP and low hydrophilicity of the Gel-MAAs / DS composite film, by limiting water evaporation, reduced respiration and oxidation in the strawberries to some extent. The weight loss rate of strawberries in the blank control group was significantly higher than that in the PE preservation film group and the experimental group. This was mainly because the strawberries in the control group were not covered by the film, allowing moisture to escape more easily into the air. While the PE preservation film had the lowest water vapor permeability, the excessively high moisture content in the environment actually promoted mold growth, which was detrimental to strawberry preservation.

[0234] Biocompatibility of Gel-MAAs / DS Composite Membranes

[0235] Cytotoxicity of Gel-MAAs / DS composite membranes is also an important indicator for evaluating their use as bio-based composite membranes. The cytotoxicity of the Gel-MAAs / DS composite membrane extract on L-929 cells was detected using the CCK8 assay. The extract was co-cultured with L-929 cells for 24 h. As shown in Figure 35, the cell viability of these composite membrane groups exceeded 100%, indicating that Sal and HA, as part of the material itself, can promote the proliferation of L929 cells. This is because these two polysaccharides provide nutrients for cell proliferation and differentiation, and the cell viability remained above 90%. The experimental results demonstrate that the Gel-MAAs / DS composite membrane has good biocompatibility.

[0236] The results of the live / dead cell staining assay in Figure 36 are consistent with those of CCK8. After 24 hours of co-incubation with the Gel-MAAs / DS composite membrane extract of L929 cells, the vast majority of L929 cells maintained their spindle shape, were uniformly distributed, and were green live cells, with virtually no red blood cells or dead cells, indicating that the Gel-MAAs / DS composite membrane extract has no cytotoxicity and good biocompatibility.

[0237] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a gelatin-cytosine amino acid-dialdehyde starch composite film, characterized in that: The process includes the following steps: Adding a cross-linking agent to a gelatin solution, activating it for a set time, then adding N-hydroxysuccinimide, followed by the addition of cytosine amino acids. Under weakly acidic conditions and in the dark, coupling is performed for a set time to obtain Gel-MAAs. Gel-MAAs are dissolved in water to obtain a complex solution. Dialdehyde starch is dissolved in water and gelatinized, then adjusted to a weakly alkaline state to obtain a dialdehyde starch solution. The dialdehyde starch solution is added dropwise to the complex solution, mixed, and reacted at 35-45℃ for 1-3 hours. 1-5% w / v glycerol is added, and after ultrasonic degassing, the membrane solution is poured into a mold and dried to obtain the final product. The cytosine amino acids are then added to the solution. The extraction method for acids includes the following steps: Pulverized *Porphyra yezoensis* is pulverized, cellulase is added, followed by the addition of a 20-30% (w / w) ethanol solution. Extraction is performed at 40-50°C for 1-3 hours, followed by ultrasonic extraction for 20-40 minutes. The mixture is then centrifuged, and the supernatant is collected. 2-6 times the volume of 90-97% ethanol is added to the supernatant, followed by freeze-precipitation, freeze-centrifugation, and freeze-drying. The freeze-dried product is the mycotoxin amino acid. The mass ratio of *Porphyra yezoensis* to cellulase is 1000:1-2. The main components of the *Porphyra yezoensis* MAAs extract are porphyra-334 and shinorine.

2. The method for preparing the gelatin-cytosine amino acid-dialdehyde starch composite membrane according to claim 1, characterized in that: The extraction method also includes a purification step for the prepared crude cytosine amino acid. The purification method is as follows: the crude cytosine amino acid solution is loaded into a Qapto Q column, and the ion exchange column is equilibrated with ultrapure water at a flow rate of 0.5-1.5 mL / min; then, linear elution is performed with 0.5-1.5 mol / L NaCl elution buffer at a flow rate of 0.5-1.5 mL / min, and the elution solution with the UV absorption at 334 nm is collected.

3. The method for preparing the gelatin-cytosine amino acid-dialdehyde starch composite membrane according to claim 1, characterized in that: The mass ratio of Gel-MAAs complex to dialdehyde starch is 2-5:

3.

4. The method for preparing the gelatin-cytosine amino acid-dialdehyde starch composite film according to claim 1, characterized in that: The membrane solution contains 3% glycerol, 4% Gel-MAAs, and 3% dialdehyde starch, with % representing w / v.

5. The method for preparing the gelatin-cytosine amino acid-dialdehyde starch composite film according to claim 1, characterized in that: The pH value of the dialdehyde starch solution is 9-11.

6. The method for preparing the gelatin-cytosine amino acid-dialdehyde starch composite film according to claim 5, characterized in that: The pH value of the dialdehyde starch solution is 10.

7. The method for preparing the gelatin-cytosine amino acid-dialdehyde starch composite film according to claim 1, characterized in that: The crosslinking agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the mass ratio of gelatin, EDC, N-hydroxysuccinimide and cytosine amino acids is 0.8-1.2:0.2-0.3:0.1-0.2:0.8-1.

2.

8. The method for preparing the gelatin-cytosine amino acid-dialdehyde starch composite film according to claim 1, characterized in that: The activation time is 20-40 minutes.

9. The method for preparing the gelatin-cytosine amino acid-dialdehyde starch composite film according to claim 1, characterized in that: The pH value of the weakly acidic solution is 4-6.

10. The method for preparing the gelatin-cytosine amino acid-dialdehyde starch composite membrane according to claim 9, characterized in that: The pH value of the weakly acidic solution is 5.

11. A gelatin-cytosine amino acid-dialdehyde starch composite film, characterized in that: Prepared by any of the preparation methods described in claims 1-10.

12. The application of the gelatin-cytosine amino acid-dialdehyde starch composite film according to claim 11 in the preparation of UV-resistant devices.

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