A composite film containing ergothioneine gold nanomaterials, and a preparation method and application thereof

Abstract

This invention provides a composite film containing ergothioneine and gold nanomaterials. The composite preservation film is obtained by mixing and stirring a complex prepared from ergothioneine and gold nanoclusters with a mixture prepared from chitosan and polyvinyl alcohol, followed by molding and drying. The preparation process is as follows: S1, ergothioneine solution, chloroauric acid solution, and ultrapure water are mixed, the pH is adjusted, the mixture is stirred, and then placed on a shaker overnight. After dialysis and freeze-drying, AuNSs-EGT is obtained; S2, chitosan is dissolved in an aqueous acetic acid solution to obtain a chitosan solution; polyvinyl alcohol is dissolved in deionized water to obtain a polyvinyl alcohol solution; the two solutions are mixed, a plasticizer is added, and the mixture is stirred to obtain a CS-PVA mixture; S3, AuNSs-EGT is added to the CS-PVA mixture and mixed evenly. After ultrasonic treatment, the mixture is poured into a mold and dried to obtain the composite preservation film. The composite film prepared by this invention possesses excellent physical and chemical properties and can be used in the field of food preservation.

Description

Technical Field

[0001] This invention belongs to the field of food preservation film preparation technology, specifically relating to a composite film containing ergothionein gold nanomaterials, its preparation method, and its application. Background Technology

[0002] Ergothioneine (EGT) is a natural antioxidant derived from histidine, primarily found in certain plants and microorganisms. In food, ergothioneine can be used as a dietary supplement. A dietary supplement containing ergothioneine, triterpenes, carotenoids, and vitamin C can improve various symptoms caused by oxidative stress and enhance physical fitness. Ergothioneine is non-toxic, colorless, and odorless, and has better thermal and pH stability compared to vitamin C. Numerous studies have shown that ergothioneine possesses multiple physiological functions, including free radical scavenging, antioxidant, and anti-inflammatory effects. In the human body, ergothioneine protects cells from oxidative stress damage, playing a vital role in maintaining normal cellular physiological functions and preventing chronic diseases. In the food industry, ergothioneine effectively inhibits lipid and protein oxidation in food, extending shelf life, maintaining food quality, and providing excellent color protection for meat products such as beef, tuna, and salmon. Its good biocompatibility and antioxidant properties make it an ideal choice for food additives and nutritional fortifiers. While international studies have reported that ergothioneine can be used as a reducing agent and stabilizer to prepare gold nanoparticles with good dispersibility and stability, research on its antibacterial properties is limited, with only preliminary explorations of its inhibitory effects on a few bacterial species under specific conditions. Domestic research in this field is also in its early stages. Although some studies have attempted to prepare ergothioneine gold nanoclusters, progress in exploring antibacterial mechanisms and developing practical applications lags behind, particularly in in-depth research on the systemic and comprehensive antibacterial mechanism against Gram-negative bacteria.

[0003] With the development of nanomedicine, an increasing number of nanomaterials and novel antibacterial therapies are being used for antibacterial treatment. The advancement of nanotechnology has provided new methods for combating bacterial infections. Currently, a wide variety of nanomaterials are being used for bacterial eradication, including carbon-based nanomaterials such as carbon dots, graphene quantum dots, carbon nanotubes, graphene oxide, and graphitic carbon nitride; metal-based nanomaterials such as zinc oxide, titanium dioxide, magnesium oxide, copper oxide, iron tetroxide, silver nanoparticles, and gold nanoparticles; as well as various polymer nanoparticles and antibacterial drug delivery systems. These nanomaterials exert their antibacterial effects through different mechanisms, such as generating reactive oxygen species, damaging cell membranes or cell contents, providing new pathways for antibacterial therapy. Ergothioneine is a green reducing agent, aligning with environmental principles of sustainability and low toxicity. It possesses strong reducing and coordination abilities, enabling it to induce chloroauric acid to self-assemble into gold nanoclusters with unique properties. Gold nanoclusters, due to their ultra-small size, ease of modification, low toxicity, and good biocompatibility, are being used for bacterial eradication. Gold nanomaterials typically exert their antibacterial properties by generating reactive oxygen species, damaging cell membranes, or disrupting cell contents. Furthermore, the size and surface properties of gold nanomaterials influence their antibacterial activity. Moon et al. coated Ni-Ti-0 nanotubes with gold (Au) nanoparticles, achieving highly efficient antibacterial properties in nickel-titanium alloys under 470 nm light irradiation, thus expanding the application of this material in dentistry. However, despite the antibacterial effects shown by gold nanomaterials, their antibacterial mechanisms require further investigation, which limits their further development and application.

[0004] In today's society, the development of food preservation and microbial control technologies is crucial for ensuring food safety, reducing food waste, and protecting public health. Traditional food preservation films mostly lack antibacterial properties and cannot effectively inhibit the growth and reproduction of microorganisms. This can lead to food remaining susceptible to microbial contamination after packaging, thus shortening its shelf life. Chitosan (CS), as a linear macromolecular polysaccharide, is one of the most commonly used materials for coatings, film formation, and impregnation due to its biodegradability, sustainability, and biodegradability. However, its relatively low mechanical and barrier properties limit the application of chitosan-based food packaging in the food industry.

[0005] Therefore, there is an urgent need to provide a membrane that has good safety and excellent physical and chemical properties for food preservation, in order to meet the needs of the food industry. Summary of the Invention

[0006] Based on this, the present invention provides a novel green food preservation film by inducing the self-assembly of gold nanoclusters using ergothioneine and chloroauric acid as raw materials, conducting in-depth research on their antibacterial mechanism against Gram-negative bacteria (Escherichia coli, Enterobacter aerogenes, Salmonella), and introducing the gold nanoclusters into a chitosan / polyvinyl alcohol composite film.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A composite film containing ergothioneine and gold nanomaterials is obtained by mixing and stirring a complex prepared from ergothioneine and gold nanoclusters with a mixture prepared from chitosan and polyvinyl alcohol, followed by molding and drying.

[0008] A method for preparing the above-mentioned composite food preservation film includes the following steps: S1. Mix ergothioneine solution, chloroauric acid solution and ultrapure water evenly, adjust the pH of the mixture to 8-12, stir the reaction, place it on a shaker overnight after the reaction is completed, then dialyze the solution and freeze dry to obtain gold nanoclusters-ergothioneine complex. S2. Dissolve chitosan in an aqueous acetic acid solution and stir to obtain a chitosan solution; dissolve polyvinyl alcohol in deionized water and stir to obtain a polyvinyl alcohol solution; mix the chitosan solution and polyvinyl alcohol solution evenly, add plasticizer and stir to obtain a chitosan-polyvinyl alcohol mixture; S3. Add the gold nanoclusters-ergothionein complex prepared in S1 to the chitosan-polyvinyl alcohol mixture prepared in S2, mix evenly, sonicate, pour into a mold, and dry to obtain a composite preservation film.

[0009] Furthermore, in step S1, the concentration of ergothioneine solution is 21.5~107.5 mmol / L, and the concentration of chloroauric acid solution is 15~25 mmol / L; The ratio of ergothioneine to chloroauric acid in the mixture is (1~10):1.

[0010] In some specific embodiments, preferably, the ergothioneine solution concentration is 43 mmol / L and the chloroauric acid solution concentration is 20 mmol / L; The ratio of ergothioneine to chloroauric acid in the mixture is 2:1.

[0011] Furthermore, the stirring reaction conditions in step S1 are: temperature 80~100℃, rotation speed 450~550 rpm, and time 10~14h.

[0012] In some specific embodiments, preferably, the stirring reaction conditions in step S1 are: temperature 90°C, rotation speed 500 rpm, and time 12 h.

[0013] Furthermore, in step S2, the concentration of the acetic acid aqueous solution is 0.5~1.5% (v / v), the concentration of the chitosan solution is 1.5~2.5 wt%, and the concentration of the polyvinyl alcohol solution is 2.5~3.5 wt%. Chitosan solution and polyvinyl alcohol solution were mixed at a solute mass ratio of 2:3. The plasticizer is glycerol, accounting for 10-20% of the total volume of the chitosan-polyvinyl alcohol mixture.

[0014] In some specific embodiments, preferably, in step S2, the concentration of the acetic acid aqueous solution is 1% (v / v), the concentration of the chitosan solution is 2.0 wt%, and the concentration of the polyvinyl alcohol solution is 3.0 wt%. Chitosan solution and polyvinyl alcohol solution were mixed at a solute mass ratio of 2:3. The plasticizer is glycerol, accounting for 15% of the total volume of the chitosan-polyvinyl alcohol mixture.

[0015] Furthermore, the stirring conditions for preparing the chitosan solution in step S2 are: temperature 65~75℃, rotation speed 400~600rpm, and time 3h. Stirring conditions for preparing polyvinyl alcohol solution: temperature 75~85℃, speed 200~300 rpm, time 3h.

[0016] In some specific embodiments, preferably, the stirring conditions during the preparation of the chitosan solution in step S2 are: temperature 70°C, rotation speed 500 rpm, and time 3 hours. Stirring conditions for preparing polyvinyl alcohol solution: temperature 80℃, speed 300 rpm, time 3h.

[0017] Furthermore, in step S3, the amount of gold nanoclusters-ergothionein complex added is 0.5~1.5% of the mass of the chitosan-polyvinyl alcohol mixture.

[0018] In some specific embodiments, preferably, the amount of gold nanoclusters-ergothionein complex added in step S3 is 1.5% of the mass of the chitosan-polyvinyl alcohol mixture.

[0019] Furthermore, the ultrasonic treatment conditions in step S3 are: power 80~150 W, time 40~60 min; The drying conditions are: temperature 65~75℃, time 240~360 min.

[0020] In some specific embodiments, preferably, the ultrasonic treatment conditions in step S3 are: power 100 W, time 50 min; The drying conditions are: temperature 70℃, time 360 ​​min.

[0021] The above-mentioned composite preservation film is used in food preservation.

[0022] Furthermore, the food includes fresh meat and vegetables.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention successfully prepared a CS-PVA / AuNSs-EGT composite film. First, EGT solution and chloroauric acid solution were prepared in sequence, and then AuNSs-EGT was prepared to prevent the problem of excessive reaction and easy agglomeration caused by directly adding EGT and chloroauric acid (with crystal structure and average particle size of 1.3nm). Then, CS solution and PVA solution were prepared in sequence and mixed to obtain a uniform CS-PVA mixture. Finally, AuNSs-EGT was added to form a CS-PVA / AuNSs-EGT composite film with excellent optical properties (reducing oxidation caused by food exposure), physical properties (thickness is not affected by AuNSs-EGT, and thermal degradation temperature is as high as 280℃), and mechanical properties (significantly increased tensile stress).

[0024] (2) Further, the antibacterial and toxicity tests were conducted using the prepared AuNSs-EGT, which showed that the minimum bactericidal concentrations of AuNSs-EGT against Escherichia coli, Enterobacter aerogenes, and Salmonella were 256 μg / ml, 512 μg / ml, and 256 μg / ml, respectively. It can also increase the production of reactive oxygen species (ROS). Through the study of bacterial membrane damage, staining of live and dead cells revealed that significant bacterial membrane damage occurred after treatment with high concentrations of AuNSs-EGT. The biocompatibility test was conducted to detect the cytotoxicity of AuNSs-EGT. The results showed that the cell survival rate of HeGp2 cells was ≥80% when the concentration of AuNSs-EGT was below 128 μg / mL, indicating that AuNSs-EGT has low cytotoxicity.

[0025] (3) Furthermore, the preservation test of pork using CS-PVA / AuNSs-EGT composite film showed that it can maintain better color (good color and moisture even on the 6th day), pH value (pH value rises slowly), juice loss rate (juice loss rate rises slowly), texture (elasticity and cohesiveness recovery are both high), total bacterial count (less than 6 log CFU / g before 12 days), TBARS value (still less than 0.6 mg MDA / kg after 12 days), etc., which can delay the decline in pork quality and have a good preservation effect. Attached Figure Description

[0026] Figure 1 This is a flowchart of the technical solution of Embodiment 1 of the present invention.

[0027] Figure 2 This is a TEM and particle size distribution diagram of AuNSs-EGT in Example 1 of the present invention.

[0028] Figure 3 The fluorescence spectrum of AuNSs-EGT in Example 1 of the present invention is shown; wherein, (A) is a three-dimensional contour plot and (B) is a UV-vis and fluorescence excitation-emission spectrum.

[0029] Figure 4 This is a photostability diagram of AuNSs-EGT in Embodiment 1 of the present invention.

[0030] Figure 5 The images show the FT-IR spectra of EGT and AuNSs-EGT in Embodiment 1 of the present invention.

[0031] Figure 6 The above are XPS images of AuNSs-EGT in Embodiment 1 of the present invention; wherein, (A) is the full spectrum of AuNSs-EGT; (B) is the Au4f spectrum of AuNSs-EGT; (C) is the C1s spectrum of AuNSs-EGT; (D) is the N1s spectrum of AuNSs-EGT; (E) is the O1s spectrum of AuNSs-EGT; and (F) is the S2p spectrum of AuNSs-EGT.

[0032] Figure 7 The MBC of AuNSs-EGT against three donor bacteria in Example 1 of this invention is as follows: (A1~A3): Control, 128 μg / mL, 256 μg / mL AuNSs-EGT; (B1~B3): Control, 256 μg / mL, 512 μg / mL AuNSs-EGT; (C1~C3): Control, 128 μg / mL, 256 μg / mL AuNSs-EGT.

[0033] Figure 8 The time-dependent killing dynamics curves of AuNSs-EGT against three donor bacteria in Example 1 of this invention are shown; where (A) is Escherichia coli, (B) is Enterobacter aerogenes, and (C) is Salmonella.

[0034] Figure 9 The results show the size of the inhibition zone under different conditions in Example 1 of the present invention; where (A) Escherichia coli, (B) Enterobacter aerogenes, (C) Salmonella; C, Control (PBS); TE, Tetracycline hydrochloride 50 μg / mL; EGT, EGT 5 mg / mL; 1, AuNSs-EGT 512 μg / mL; 2, AuNSs-EGT 256 μg / mL.

[0035] Figure 10 The effect of different concentrations of AuNSs-EGT on the ROS of three donor bacteria in Example 1 of this invention ( ±s, n=3); where (A) Escherichia coli, (B) Enterobacter aerogenes, (C) Salmonella; p<0.05, a compared with the control group, b compared with the 64 μg / mL AuNSs-EGT group.

[0036] Figure 11 Representative fluorescence images (A) and bar charts (B, Escherichia coli; C, Enterobacter aerogenes; D, Salmonella) of three donor bacteria treated with different concentrations of AuNSs-EGT in Example 1 of this invention after double staining with Hoechst 33342 and PI dye. (±s, n=3); where p<0.05, a compared with the control group, b compared with the 64μg / mL AuNSs-EGT group.

[0037] Figure 12 This is a schematic diagram of the membrane potential of three test bacteria after treatment with different concentrations of AuNSs-EGT in Example 1 of the present invention. (±s, n=3), where (A) Escherichia coli; (B) Enterobacter aerogenes; (C) Salmonella; p<0.05, a compared with the control group, b compared with the 64 μg / mL AuNSs-EGT group.

[0038] Figure 13 The images show the morphological changes of Escherichia coli after treatment with different concentrations of AuNSs-EGT in Example 1 of this invention; wherein, (A) 0 μg / mL AuNSs-EGT; (B) 256 μg / mL AuNSs-EGT; (C) 512 μg / mL AuNSs-EGT.

[0039] Figure 14 The cytotoxicity of AuNSs-EGT on HepG2 cells in Example 1 of this invention ( ±s, n=3); where p<0.05, a compared with the control group.

[0040] Figure 15 This describes the effect of different AuNSs-EGT addition amounts on the UV resistance properties of the composite film in Example 1 of the present invention.

[0041] Figure 16 The effect of AuNSs-EGT concentration on the mechanical properties of the composite membrane in Example 1 of this invention. Figure 17 The image shows the infrared spectrum of the CS-PVA / AuNSs-EGT composite film in Example 1 of this invention; where gray represents the CS-PVA film; red represents 0.5% of CS-PVA / AuNSs-EGT; blue represents 1.0% of CS-PVA / AuNSs-EGT; and green represents 1.5% of CS-PVA / AuNSs-EGT.

[0042] Figure 18 The thermal stability of the CS-PVA / AuNSs-EGT composite film in Example 1 of this invention (A, thermogravimetric curve; B, differential thermogravimetric curve) Figure 19 The appearance changes of pork samples treated with different methods in Example 2 of this invention during 0-12 days. Figure 20 The color changes of pork treated with different methods during storage at 4°C in Example 2 of the present invention are shown in Figure 2 (A, L* value; B, a* value; C, b* value).

[0043] Figure 21 This refers to the pH changes of pork treated with different methods during storage at 4°C in Example 2 of the present invention.

[0044] Figure 22 This illustrates the changes in juice loss rate of pork treated with different methods during storage at 4°C in Example 2 of the present invention.

[0045] Figure 23 The changes in the total bacterial count of pork under different treatment methods in Example 2 of this invention are shown.

[0046] Figure 24 The variation of TBARS values ​​in pork under different treatment methods in Example 2 of this invention. Detailed Implementation

[0047] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0048] Example 1 This embodiment provides a composite film containing ergothionein gold nanomaterials, including the following preparation process (see process flow diagram). Figure 1 ): 1. Synthesis and characterization of gold nanoclusters-ergothionein complex (AuNSs-EGT) 1.1 Synthesis of AuNSs-EGT First, ergothioneine solution and chloroauric acid solution were prepared sequentially. Then, using a one-pot method, the ergothioneine solution, chloroauric acid solution, and 40 mL of ultrapure water were added to a 100 mL round-bottom flask. After gentle stirring on a magnetic stirrer, sodium hydroxide solution was added to adjust the pH. The mixture was heated in an oil bath to 90°C and reacted at 500 rpm for 12 h. Next, the mixture was placed on a shaker overnight for 12 h to promote the formation of AuNSs-EGT. After the reaction was complete, a pale yellow transparent solution was obtained. This solution was then dialyzed in a dialysis bag containing deionized water for 12 h to further purify AuNSs-EGT and remove small molecule impurities. Finally, the dialyzed solution was collected and freeze-dried to obtain AuNSs-EGT.

[0049] 1.2 Optimization of the synthesis process of AuNSs-EGT To investigate the effects of material ratio, reaction time, and pH on the results in the preparation of AuNSs-EGT, the following investigations were also conducted: (1) AuNSs-EGT material ratio optimization First, EGT solutions of different concentrations (21.5, 43, 64.5, 86, 107.5 mmol / L) were prepared. Then, 0.5 mL of 20 mmol / L chloroauric acid was added to each flask to make the EGT:chloroauric acid (HAuCl4·3H2O) ratio 1:1, 2:1, 4:1, 6:1, 8:1, and 10:1, for a total of 6 groups, and AuNSs-EGT products with the corresponding ratios were obtained.

[0050] Then, following the CLSI 2015 guidelines, the minimum inhibitory concentration (MIC) of AuNSs-EGT was determined using the plate dilution method. 0.1 mL of LB medium was added to each well of a sterile, clear 96-well plate. The highest concentration of AuNSs-EGT was added to well 1, and serially diluted AuNSs-EGT was added to wells 2-128, with a final volume of 0.1 mL. Well 12 served as the negative control, receiving no drug. *E. coli*, *Salmonella*, and *Enterobacter aerogenes* were cultured to the logarithmic growth phase and diluted with medium to a concentration of 1 × 10⁻⁶. 5 The concentration of CFU / mL was determined. 10 μL of bacterial solution was added to each well, and the plate was incubated at 37°C for 24 hours. Each concentration had three replicates. The absorbance (optical density, OD) of each well was measured at 600 nm using a microplate reader. 600 The lowest concentration corresponding to the transparent well is considered the MIC value of AuNSs-EGT. OD 600 This is a semi-quantitative experiment that can roughly estimate the number of bacteria in a solution. All data are from independent experiments repeated three times (results are shown in Table 1).

[0051] Table 1 EGT: Minimum inhibitory concentration (μg / mL) of chloroauric acid in different materials

[0052] Table 1 shows that when the mass ratio of EGT to chloroauric acid was 1:1, 2:1, 4:1, 6:1, 8:1, and 10:1, AuNSs-EGT exhibited different antibacterial activities against the three donor bacteria. The best antibacterial effect was observed at a mass ratio of 2:1, with MICs of 64 μg / mL, 128 μg / mL, and 128 μg / mL against Escherichia coli, Enterobacter aerogenes, and Salmonella, respectively. However, the substance synthesized when the mass ratio of EGT to chloroauric acid was 1:1 showed almost no antibacterial activity against Enterobacter aerogenes and Salmonella. This indicates that the antibacterial performance of AuNSs-EGT is related to the mass ratio of EGT to chloroauric acid.

[0053] (2) Optimization of reaction time for AuNSs-EGT The optimal ratio of ergothioneine to chloroauric acid (2:1) was selected, and the mixture was synthesized according to the above method. The reaction time was adjusted to 4, 6, 8, and 10 hours in sequence. The optimal reaction time was selected by determining the minimum inhibitory concentration (MIC) (the results are shown in Table 2).

[0054] Table 2 Minimum inhibitory concentrations (μg / mL) of EGT:chloroauric acid (2:1) at different reaction times

[0055] As shown in Table 2, the antibacterial properties of products formed by different reaction times vary. The antibacterial effect is best with a reaction time of 6 hours, and its MIC concentration against Escherichia coli and Salmonella is 64 μg / mL.

[0056] (3) pH optimization of AuNSs-EGT reaction The pH of AuNSs-EGT was optimized after the material ratio (EGT:chloroauric acid = 2:1) and time (6h) were optimized. It was synthesized according to the above method, and different doses of NaOH were added to adjust the pH of the solution to 8, 10, 11 and 12 in sequence. The optimal pH was selected by the minimum inhibitory concentration determination (the results are shown in Table 3).

[0057] Table 3 Minimum inhibitory concentrations (μg / mL) of EGT:chloroauric acid (2:1, 6 h) at different pH values

[0058] As shown in Table 3, the antibacterial properties of products formed under different pH conditions vary, with the best antibacterial effect observed at pH 10.

[0059] Based on the above series of investigations, and considering the antibacterial effect and actual economic cost of the synthesized AuNSs-EGT, subsequent experiments will use AuNSs-EGT with a mass ratio of 2:1, a reaction time of 6 h, and a pH of 10 as the final reaction conditions for the synthesis of AuNSs-EGT, and then characterize it to explore its antibacterial mechanism and biocompatibility.

[0060] 1.3 Optical performance of AuNSs-EGT To understand the optical properties of AuNSs-EGT prepared under optimal process conditions, the following tests were also conducted: (1) Transmission electron microscopy of AuNSs-EGT The morphology of AuNSs-EGT was characterized using transmission electron microscopy (TEM). AuNSs-EGT was uniformly dispersed in an appropriate amount of water, and 10 μL was dropped onto a copper mesh. After the solvent evaporated, the mixture was placed in a TEM chamber, and its morphology and structure were observed under vacuum conditions (results are shown in [link to TEM]). Figure 2 ).

[0061] Depend on Figure 2 It can be seen that the size, shape, and crystal structure of gold nanoparticles can be easily observed. To determine the morphology and composition of AuNSs-EGT, the morphology and microstructure of the product were recorded using transmission electron microscopy. As shown in Figure A, the transmission electron microscope (TEM) image shows that AuNSs-EGT is uniformly distributed and spherical. Figure B clearly shows the crystal structure of AuNSs-EGT, and a lattice stripe pattern is observed in the image, confirming the crystal structure of the particles, where 0.027 nm corresponds to the (1328) crystal plane of gold. The particle size of AuNSs-EGT ranges from 0.6 nm to 2.1 nm. By measuring the particle size of 150 nanoclusters, the average particle size of AuNSs-EGT was found to be 1.3 nm.

[0062] (2) Fluorescence spectrum of AuNSs-EGT The fluorescence spectrum of AuNSs-EGT solution was scanned at room temperature using a fluorescence spectrophotometer (F-2700) to investigate its excitation and emission wavelengths (see results). Figure 3 ).

[0063] (3) UV-Vis absorption spectrum of AuNSs-EGT The UV-Vis absorption spectrum of AuNSs-EGT solution in the 800–200 nm range was scanned at room temperature using a UV-Vis spectrophotometer (UV-2550). The lyophilized sample was dissolved in ultrapure water, and measurements were performed using a quartz cuvette with an optical range of 1 cm. The solution spectrum was used as a background to eliminate interference (results are shown in [link to results]). Figure 3 ).

[0064] Depend on Figure 3 As shown in Figure A, a three-dimensional contour plot of AuNSs-EGT, its fluorescence intensity is strongest at excitation wavelength of 330 nm and emission wavelength of 481 nm. Figure B indicates that the excitation wavelength of AuNSs-EGT is 325 nm, and the emission wavelength is approximately 481 nm. The UV-vis diagram shows that the absorption spectrum of AuNSs-EGT at a 2:1 mass ratio begins at approximately 396 nm, and due to its molecular-like properties, it rises rapidly below 396 nm, with a shoulder peak appearing at approximately 250 nm. This is related to the π→π* electronic transition on the imidazole ring group.

[0065] (4) Quantum yield of AuNSs-EGT Rhodamine 6G (QY = 0.94, ethanol) was used as a reference to calculate the quantum yield of AuNSs-EGT. The relative fluorescence quantum yield of AuNSs-EGT was calculated using the following formula: Фx=Ф st ( D x / D st () A st / A x () n x 2 / n st 2 ).

[0066] Where Φ, D, and A refer to the standard quantum yield, the integral area of ​​the corresponding fluorescence spectrum, and the absorbance at the excitation wavelength of the selected reference material, respectively; n is the refractive index of the solvent; and the subscripts x and st represent the sample and the standard material, respectively. To minimize the reabsorption effect, the ultraviolet absorption at the excitation wavelength should always be kept below 0.05.

[0067] Using Rhodamine 6G as a standard, the fluorescence quantum yield of AuNSs-EGT was determined and analyzed using the reference method. The quantum yield was calculated to be 36% according to the quantum yield formula.

[0068] (5) Fluorescence stability of AuNSs-EGT The prepared AuNSs-EGT was placed under a UV lamp, and its fluorescence intensity F was measured at 0, 5, 10, 15, 20, 25, and 30 min to assess its stability (see results). Figure 4 ).

[0069] Depend on Figure 4It can be seen that when the aqueous solution of AuNSs-EGT is continuously irradiated for 1800 s, the fluorescence intensity decreases by 23%, as shown in the figure. The experiment shows that AuNSs-EGT has good resistance to photobleaching.

[0070] 1.4 Surface properties of AuNSs-EGT To understand the surface properties of AuNSs-EGT prepared under optimal process conditions, the following tests were also conducted: (1) Fourier transform infrared spectrum of AuNSs-EGT Infrared spectroscopy of AuNSs-EGT was performed using Fourier transform infrared spectroscopy (FT-IR). After lyophilizing the sample, 400 mg of lithium bromide (LiBr) was added to 0.5%–1% of the sample to prepare LiBr sample slides, which were then measured using FT-IR. The chemical bonds and functional groups in the sample were analyzed using infrared spectroscopy to verify whether the ligands were successfully modified onto the gold nanoparticles (results are shown in [link to results]). Figure 5 ).

[0071] Depend on Figure 5 It can be seen that the infrared spectrum of EGT is at 3448 cm⁻¹. -1 An absorption peak is observed at 1615 cm⁻¹, which is attributed to the stretching vibration of the amino NH bond in EGT. The stretching vibrations of C₂C₃ and C=O may contribute to the peak at 1615 cm⁻¹. -1 The appearance of characteristic absorption peaks indicates the presence of amino groups (-NH2) and carbonyl / conjugated double bond functional groups containing CC / C=O in the (EGT) molecule, and that the chemical bonds of these functional groups maintain their intrinsic vibrational characteristics, verifying the integrity of the characteristic molecular structure of EGT. EGT exhibits a stretching vibration absorption peak (vS-H) of SH (thiol group) at 2630 cm⁻¹, while the infrared spectrum of AuNSs-EGT shows a peak at 2630 cm⁻¹. -1 The SH (thiol group) disappears. This is because after EGT reacts with chloroauric acid, the SH bond is converted to Au-S (gold-sulfur bond) and fixed on AuNCs-EGT, which helps to prove the synthesis of AuNSs-EGT.

[0072] (2) X-ray photoelectron spectroscopy XPS can be used to investigate the chemical elements present on the sample surface and the chemical bond relationships between these elements. 1.0 mg of AuNSs-EGT powder, thoroughly dried and ground, was compressed into a tablet, and the C, N, O, Au, and S elemental contents on its surface were analyzed using K-Alpha X-ray photoelectron spectroscopy. Peak fitting of the data was performed using Avantage software (results are shown in [link to results]). Figure 6 ).

[0073] Depend on Figure 6It can be seen that the spectrum shows that it mainly contains five elements: Au 4f, C 1s, N 1s, O 1s, and S 2p. Figure 6 A), such as Figure 6 B shows that the Au 4f spectrum is deconvolved into 4 peaks, namely Au(0)4f. 7 / 2 (83.9eV), Au(I)4f 7 / 2 (84.4eV), Au(0)4f 5 / 2 (87.6 eV) and Au(I)4f 5 / 2 (88 eV), indicating the simultaneous presence of Au(0) and Au(I). The C 1s spectrum describes the three forms of carbon bonding ( Figure 6 The values ​​for C were 284.3 eV (CC), 285.7 eV (CO), and 287.8 eV (C=O), respectively. The N1s spectrum yielded two characteristic peaks ( Figure 6 D), 399.3 eV (CN) and 402 eV (NH), respectively. Similarly, the O1s spectrum shows three characteristic peaks ( Figure 6 The peak values ​​(E) were 530.6 eV (H2O), 531.4 eV (C-OH), and 535.2 eV (OC=O). The S2p spectrum was fitted with three peaks (E). Figure 6 The F values ​​are RS-Au (162 eV) and R-SO3 (168.4 eV), respectively. This indicates that EGT was successfully modified onto gold nanoparticles.

[0074] 1.5 Antibacterial activity of AuNSs-EGT The antibacterial mechanism of gold nanoclusters is highly complex, and its antibacterial activity is related to many factors, such as the surface ligands, size, and shape of the gold nanoclusters. These antibacterial nanomaterials exert their antibacterial effects by influencing the integrity of bacterial cell membranes and generating reactive oxygen species. Therefore, the antibacterial performance, mechanism, and biosafety of AuNSs-EGT against Gram-negative bacteria will be evaluated and discussed in the following section.

[0075] 1.5.1 Bacterial Culture LB broth medium: 1.25g dissolved in 50ml distilled water, autoclaved at 121℃ for 15 minutes, then used for bacterial culture. LB agar medium: 20g dissolved in 500ml distilled water, autoclaved at 121℃ for 15 minutes, then used for bacterial culture. After inoculating the bacteria into the medium, shake in a constant temperature shaker at 37℃ and 200rpm for approximately 8 hours, then store at 4℃ for later use.

[0076] 1.5.2 Antibacterial properties of AuNSs-EGT (1) Determination of minimum bactericidal concentration (MBC) AuNSs-EGT was diluted to 512, 256, and 128 μg / mL according to the minimum inhibitory concentration (MIC) assay. The diluted sample solutions and diluted bacterial suspensions were mixed at a 1:1 volume ratio and incubated for 12 h. This was used to determine the minimum bactericidal concentration (MBC), with a blank control group. In a sterile environment, 10 μL of bacterial suspension containing different concentrations of AuNSs-EGT was taken from each solution and diluted 10⁵ times. 100 μL of the diluted suspension was evenly spread onto LB agar and incubated at 37°C for 24 h. Colony growth on the solid culture medium was observed, and photographs were taken to record the bacterial colony growth on the plates. The concentration of AuNSs-EGT at which no bacterial colonies grew in the petri dish was considered the minimum bactericidal concentration of AuNSs-EGT (see results below). Figure 7 ).

[0077] Depend on Figure 7 It was found that when AuNSs-EGT concentrations of 256 μg / mL, 512 μg / mL, and 256 μg / mL were co-cultured with *E. coli*, *Enterobacter aerogenes*, and *Salmonella*, no colonies formed on the culture medium. This indicates that AuNSs-EGT can completely inhibit the growth and reproduction of *E. coli* and *Salmonella* at relatively low concentrations. The mean corpuscular mass index (MBC) for *E. coli* and *Salmonella* was 256 μg / mL, and for *Enterobacter aerogenes* it was 512 μg / mL.

[0078] (2) Determination of time-kill curve Escherichia coli, Salmonella, and Enterobacter aerogenes in the logarithmic growth phase were cultured to an initial OD value of 0.3, and then treated with 0, 64, and 128 μg / mL AuNSs-EGT, respectively. The OD values ​​of the bacterial cultures were measured at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 hours. Three parallel control groups were set up, and time-kill curves were plotted (results are shown in...). Figure 8 ).

[0079] Depend on Figure 8 It is known that using optical density (OD) to reflect the concentration or quantity of microorganisms is a commonly used method. Within a certain range, the concentration of a bacterial suspension is directly proportional to transmittance and inversely proportional to optical density. Generally, the wavelength range for measuring bacterial density is 580 nm to 660 nm. As shown in the figure, OD... 600 The smaller the nm value, the stronger the antibacterial effect. AuNSs-EGT has good antibacterial activity against the three donor bacteria, and the higher the concentration, the stronger the antibacterial effect.

[0080] (3) Determination of inhibition zone First, place the Oxford cup into a sterilized agar plate, pour in approximately 10 mL of agar medium, and allow it to solidify horizontally. Once solidified, remove the Oxford cup. Then, inoculate 10 μL of bacterial suspensions of *E. coli*, *Salmonella*, and *Enterobacter aerogenes*, spreading them evenly. Add equal volumes of physiological saline, tetracycline hydrochloride (50 μg / mL), EGT (5 mg / mL), AuNSs-EGT 1.024 mg / mL, and 0.512 mg / mL to the wells, respectively. Incubate at 37°C for 24 hours, observe, and measure the size of the inhibition zone with calipers (see results below). Figure 9 ).

[0081] Depend on Figure 9 It was observed that inhibition zones formed around the wells of both tetracycline hydrochloride and AuNSs-EGT on agar plates, while no inhibition zones formed around the wells of the EGT-treated group and the PBS control group. AuNSs-EGT at 512 µg / mL was highly sensitive to *Escherichia coli* and *Enterobacter aerogenes*, and moderately sensitive to *Salmonella*. The results indicate that EGT alone cannot inhibit microbial growth, while the synthetic AuNSs-EGT can inhibit the growth of all three donor bacteria. Furthermore, the inhibition diameter was 512 / 256 µg / mL for AuNSs-EGT > 50 µg / mL for tetracycline hydrochloride > EGT / PBS.

[0082] 1.5.3 Antibacterial Mechanism of AuNSs-EGT (1) Analysis of bacterial reactive oxygen species (ROS) production Intracellular ROS levels were assessed using 2',7'-dichlorofluorescein diacetate (DCFH-DA) dye. In the presence of ROS, intracellular esterases deesterify DCFH-DA, converting it into fluorescent 2',7'-dichlorofluorescein (DCF). First, three donor bacteria growing in the logarithmic growth phase were collected and washed several times with PBS buffer. Then, 10 μM DCFH-DA probe was added to the bacterial solution and incubated at 37°C, 200 rpm for 1 h. After washing several times to remove excess probe, the probe-loaded bacteria were incubated with 64 or 128 μg / mL AuNSs-EGT or control solution (PBS) at 37°C, 200 rpm for 2 h. The fluorescence intensity of each solution was then measured using a microplate reader at excitation / emission wavelengths of 488 / 525 nm. The fluorescence intensity indicated the concentration of DCF generated, reflecting the intracellular ROS level (results are shown in [link to results]). Figure 10 ).

[0083] Depend on Figure 10It was found that compared with the blank control group, the AuNSs-EGT treatment group showed increased ROS production, and the higher the concentration, the more ROS was produced. A sudden increase in ROS levels in cells is usually accompanied by a weakening of antioxidant activity systems. Microbial enzymatic antioxidant systems include glutathione reductase (GR), catalase (CAT), and superoxide dismutase (SOD). Therefore, it is speculated that the increased intracellular ROS levels in bacteria treated with AuNSs-EGT may be due to AuNSs-EGT inhibiting antioxidant enzymes, thereby increasing ROS, slowing bacterial growth, or causing oxidative damage. Intracellular reactive oxygen species (ROS) level analysis results indicate that AuNSs-EGT can induce oxidative stress in the cells of three tested bacteria—Escherichia coli, Enterobacter aerogenes, and Salmonella—leading to a reduction in viable cells.

[0084] (2) Study on bacterial membrane damage The degree of bacterial cell membrane damage was assessed using Hoechst 33342 and propidium iodide (PI) staining. Three donor bacteria growing in the logarithmic growth phase were incubated with PBS buffer or 64 or 128 μg / mL AuNSs-EGT solution in a shaker at 37°C and 200 rpm for 2 h. Then, 5 μg / mL Hoechst 33342 and 10 μg / mL PI were added to the treated bacterial solution, and the mixture was incubated in the dark for 15 min. After washing the bacterial solution several times with PBS buffer to remove excess dye, the staining was observed using an upright fluorescence microscope to assess the degree of bacterial membrane damage. Fluorescence intensity was detected at an excitation wavelength of 488 nm and an emission wavelength of 612 nm (results are shown in [see table]). Figure 11 ).

[0085] Depend on Figure 11 It was found that after treatment with AuNSs-EGT, the red fluorescence intensity of Escherichia coli, Enterobacter aerogenes, and Salmonella was significantly increased, and positively correlated with the AuNSs-EGT concentration. In contrast, the control group bacteria showed almost no red fluorescence signal, indicating that the increased fluorescence intensity in the treated group was a direct result of the action of AuNSs-EGT, rather than due to factors inherent to the bacteria themselves.

[0086] (3) Measurement of bacterial membrane potential The changes in membrane potential were measured using DiBAC4(3) dye. Three donor strains growing in the logarithmic growth phase were incubated for 3 h at 37°C and 200 rpm in a shaker with PBS buffer or 64 or 128 μg / mL AuNSs-EGT solution. After centrifugation, the cells were washed three times with PBS buffer, and 3 μM DiBAC4(3) was added to the bacterial culture. The culture was incubated at 37°C in the dark for 30 min, and then different concentrations of gold nanoparticles (0, 64, and 128 μg / mL) were added. The culture was incubated at 37°C in the dark for 1 h. The fluorescence intensity of each group and its background was measured using a fluorescence microplate reader (Ex / Em=485nm / 535nm) (results are shown in [link to results]). Figure 12 ).

[0087] Depend on Figure 12 It was found that, compared with the control group, the fluorescence intensity of the three bacterial cells significantly increased after AuNSs-EGT treatment (p<0.05), indicating an increase in cell membrane potential and bacterial cell depolarization. Based on the above results, it is speculated that the inhibition of the growth of the three donor bacteria by AuNSs-EGT may be through its action on the cell membrane, affecting bacterial membrane permeability and integrity. The cell membrane, as the outer protective barrier of bacteria, protects the integrity of its cellular structure; if damaged, it allows important ions and biomolecules to escape from the bacterial cell, thus affecting normal energy and material metabolism and leading to bacterial apoptosis.

[0088] (4) Bacterial morphology analysis (SEM) The above results were further verified by observing morphological changes in Escherichia coli using SEM. Figure 13 As shown, the blank control group of *E. coli* exhibited a typical rod-shaped structure with a smooth, intact cell membrane and uniform size. However, after treatment with different concentrations of AuNSs-EGT, varying degrees of damage were observed. After treatment with 256 μg / mL AuNSs-EGT, the cell surface of *E. coli* showed shrinkage and depression, the rod-shaped three-dimensional structure partially disappeared, and cell adhesion occurred. When the AuNSs-EGT concentration reached 512 μg / mL, the three-dimensional structure of the bacteria almost completely disappeared, and the cell morphology was damaged. This indicates that AuNSs-EGT treatment of *E. coli* can lead to bacterial cell rupture, thereby disrupting the integrity of the cell membrane.

[0089] 1.5.4 Biocompatibility of AuNSs-EGT Using hepatocellular carcinoma cells as a model, the biocompatibility of AuNSs-EGT was assessed using the 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) assay. First, cells were seeded into 100 μL per well of a 96-well cell culture plate and incubated for 24 h. Then, the original culture medium was replaced with cell culture medium containing different concentrations of 100 μL AuNSs-EGT (0, 16, 32, 64, 96, and 128 μg / mL in three parallel wells per group), and incubated for another 24 h. The culture medium was then discarded, and 100 μL of fresh DMEM medium and 10 μL of MTT solution (5 mg / mL) were added to each well, followed by incubation for another 4 h. Finally, the culture medium was removed, and 150 μL of LDMSO was added to each well. The plate was shaken for 5 minutes to completely dissolve the blue-purple MTT crystals. The absorbance (A) at 490 nm was measured using a microplate reader, and cell viability was assessed using the following formula: Cell viability (%) = A treat / A contol ×100% Among them, A treat A represents the average absorbance of the experimental group with added material after background subtraction. control The average absorbance of the control group without added materials after background subtraction (see results). Figure 14 ).

[0090] Depend on Figure 14 It can be seen that the survival rate of HepG2 cells was higher than 89% at a concentration of 96 μg / mL with AuNSs-EGT, and it was still higher than 80% at a concentration of 128 μg / mL, indicating that AuNSs-EGT has very low cytotoxicity.

[0091] 2. Preparation and characterization of CS-PVA / AuNSs-EGT composite membrane Chitosan (CS), a natural polysaccharide, has been studied for its use in food packaging due to its excellent biodegradability, biocompatibility, and film-forming properties. However, its poor mechanical and barrier properties limit its application. Polyvinyl alcohol (PVA), a water-soluble polymer containing many hydroxyl groups, possesses advantages such as excellent mechanical properties, biodegradability, strong adhesion, and low air permeability. Preparing composite films by mixing PVA and CS is an effective strategy for obtaining biodegradable food packaging films with good mechanical properties. However, the preservation effect of CS-PVA films is limited. Currently, various inorganic nanoparticles have been incorporated into CS-PVA films as additives to improve the overall performance of the composite films. Therefore, this study prepared a CS-PVA / AuNSs-EGT composite food preservation film by mixing AuNSs-EGT with CS-PVA. The thickness, color difference, mechanical properties, and thermal stability of the composite film were investigated.

[0092] 2.1 Preparation of CS-PVA / AuNSs-EGT composite membrane CS was dissolved in a 1% (v / v) aqueous acetic acid solution and stirred at 70°C for 3 hours to obtain a CS solution (2wt%). PVA was dissolved in deionized water and stirred at 80°C for 3 hours to obtain a PVA solution (3wt%). The two solutions were then mixed according to the solute mass ratio, and 15% plasticizer (glycerol) was added to improve plasticity, and stirring was continued for 30 minutes. Subsequently, different weight percentages of AuNSs-EGT (0, 0.5, 1.0, 1.5%) were added in portions and quickly mixed until homogeneous. Air bubbles in the mixture were removed by ultrasonic vibration (100 W power, 50 min time). The solution was poured into a mold by casting and dried in a 70°C oven to finally obtain a CS-PVA / AuNSs-EGT composite film.

[0093] Among them, CS-PVA / AuNSs-EGT-0 (0%), CS-PVA / AuNSs-EGT-0.5 (0.5%), CS-PVA / AuNSs-EGT-1 (1.0%) and CS-PVA / AuNSs-EGT-1.5 (1.5%) represent food preservation films with different AuNSs-EGT contents, and the composite film without AuNSs-EGT is named CS-PVA.

[0094] 2.2 Characterization of CS-PVA / AuNSs-EGT composite membrane (1) Determination of the thickness of CS-PVA / AuNSs-EGT composite film The thickness of the starch film was measured using a handheld electronic vernier caliper with an accuracy of 0.01 mm. Several points were randomly selected on the composite film, and the measurements were taken at these points using the vernier caliper. The results are recorded in Table 4.

[0095] Table 4. Thickness (mm) of composite membranes with different AuNSs-EGT concentrations

[0096] As shown in Table 4, many factors affect the final performance of edible films, such as the protein, carbohydrate, and lipid content in the raw materials. It can be seen that there is no significant difference in the thickness of the composite films (P>0.05), indicating that AuNSs-EGT has little effect on the change in composite film thickness, which may be related to the low solid content in the composite film.

[0097] (2) Measurement of color difference of CS-PVA / AuNSs-EGT composite film The color difference of CS-PVA / AuNSs-EGT composite films was measured using a portable colorimeter. The color of composite films with different concentrations of CS-PVA / AuNSs-EGT was determined before measurement. A white A4 sheet of paper was used to calibrate the instrument before measurement. The color difference data (L*, a*, b* values) were recorded by the instrument and are shown in Table 5.

[0098] Table 5 Color difference of composite films with different AuNSs-EGT concentrations

[0099] As shown in Table 5, the color of food packaging film materials affects the appearance of food and thus influences consumer choices. Compared with CS-PVA film, the addition of AuNSs-EGT leads to a significant decrease in the L* value and red / green value (a* value) of CS-PVA / AuNSs-EGT composite film, and a significant increase in the yellow / blue value (b* value). In application, the film's own pale yellow color helps to reduce the oxidative deterioration of food caused by exposure to ultraviolet and visible light.

[0100] (3) Determination of transmittance of CS-PVA / AuNSs-EGT composite film The experimental data were obtained using a UV spectrophotometer. The film was cut into strips of 1cm × 3cm and placed in a cuvette. The transparency of the starch film was determined by measuring the transmittance of light at 600nm (T600). The transmittance value was calculated using the following formula: Transmittance value = -(log T600) / H).

[0101] Where T600 is the transmittance fraction at 600 nm, and H is the film thickness (mm). A larger value indicates lower film transparency (see results). Figure 15 ).

[0102] Depend on Figure 15 It is evident that UV resistance can affect the rate of oxidation of nutrients in food. As the AuNSs-EGT content in the composite film increases, the light transmittance gradually decreases, indicating a gradual enhancement in light blocking performance. Furthermore, all CS-PVA / AuNSs-EGT composite films exhibit low transmittance values ​​in the 200nm and 400nm wavelength ranges, demonstrating their excellent UV blocking properties. For packaged foods, films with excellent UV blocking properties can slow down lipid oxidation, thereby extending the shelf life of the food.

[0103] (4) Determination of mechanical properties of CS-PVA / AuNSs-EGT composite membrane The mechanical properties of CS-PVA / AuNSs-EGT composite membranes were evaluated using an electronic tensile testing machine. CS-PVA / AuNSs-EGT composite membranes were cut into standard mold shapes and fixed on test fixtures for mechanical tensile testing. The predetermined test speed was set to 0.5 mm / s. During the tensile process, the stress-strain curves of the fiber membranes were carefully recorded to determine key mechanical parameters. Each sample underwent five repeated measurements to ensure the accuracy and reliability of the results (see [Results]). Figure 16 ).

[0104] Depend on Figure 16 It is known that the mechanical properties of a membrane are mainly related to the distribution and density of polymer chains in the membrane matrix through intermolecular and intramolecular interactions. The tensile strength of the CS-PVA membrane is 32.66 MPa, while the tensile strength of the composite membrane with added AuNSs-EGT is significantly higher than that of the CS-PVA membrane, with the CS-PVA / AuNSs-EGT 1.5% membrane reaching 89.16 MPa. This is related to the hydrogen bonding between CS-PVA and AuNSs-EGT. The same phenomenon is also observed in tensile stress; as the addition of AuNSs-EGT increases from 0% to 1.5%, the tensile stress of the CS-PVA / AuNSs-EGT composite membrane increases significantly. Therefore, the addition of AuNSs-EGT significantly improves the mechanical properties of the material.

[0105] (5) Fourier transform infrared spectroscopy determination of CS-PVA / AuNSs-EGT composite film Different membrane samples were analyzed using Fourier transform infrared spectroscopy, with the analytical parameters ranging from 4000 to 500 cm⁻¹. -1 1cm interval -1 The resolution is 4cm. -1 (See results) Figure 17 ).

[0106] Depend on Figure 17 It can be known that: in the range of 3600-3000 cm -1 The broadband frequency band at 2933 cm⁻¹ is called the stretching vibration of -OH. The CS-PVA film at 2933 cm⁻¹... -1 The vibrational bands observed at 1419 and 1031 cm are attributed to the antisymmetric stretching of CH in the alkyl group. -1 The peaks at 850 cm⁻¹ correspond to the stretching of the CH and COC groups, respectively. -1The small peak at [value] corresponds to the CC backbone. Compared to the CS-PVA film, the infrared spectrum of the CS-PVA / AuNSs-EGT composite film showed no significant change in the characteristic peaks. The CS-PVA / AuNSs-EGT composite film obtained by loading AuNSs-EGT into the CS-PVA matrix showed a slight shift in the -OH peak due to the interaction between CS-PVA and AuNSs-EGT. With the addition of AuNSs-EGT, a peak at 2933 cm⁻¹ was observed in the CS-PVA film. -1 The peak value shifts to a lower position (2929 cm⁻¹) in the CS-PVA / AuNSs-EGT composite membrane as the intensity decreases. -1 Furthermore, the FT-IR spectrum of the CS-PVA film shows that at 3273 cm⁻¹... -1 A peak corresponding to the -OH stretching vibration was observed at a higher wavelength (3281 cm⁻¹) in the CS-PVA / AuNSs-EGT composite film. -1 Slight shift. This change observed in the stretching vibrations of the -OH band may be due to the incorporation of AuNSs-EGT altering the formation or strength of intermolecular hydrogen bonds in CS-PVA, thereby affecting the stretching vibrational frequency of the hydroxyl group.

[0107] (6) Determination of thermal stability of CS-PVA / AuNSs-EGT composite film Thermogravimetric analysis (TGA) parameters of different samples were determined using a thermogravimetric analyzer. The parameters were set as follows: 10 mg of sample was heated at 5 °C. -1 The heating rate was increased to 600℃, and the results were analyzed using Origin software (see results below). Figure 18 ).

[0108] Depend on Figure 18 It is evident that the thermal stability of the plastic wrap is crucial for maintaining packaging integrity under broader conditions. The first stage of weight loss in the CS-PVA / AuNSs-EGT composite film occurred below 200°C. This is likely due to the evaporation of water in the sample at relatively low temperatures, a physical change. The second stage of weight loss occurred in the temperature range of 200 to 400°C. 0 C. In this stage, the plastic wrap decomposes into CO2 and water vapor. Simultaneously, the CS-PVA / AuNSs-EGT composite film undergoes further dehydration. After the third stage at 400℃, the structure of the plastic wrap is completely destroyed. The highest thermal degradation temperature of the composite film is around 280℃, and the addition of AuNSs-EGT has little effect on this temperature.

[0109] Example 2 In this embodiment, the composite film prepared in Example 1 is used to further study its preservation effect on pork, as detailed below: A practical performance experiment on the preservation of fresh pork was conducted using the successfully prepared CS-PVA / AuNSs-EGT composite membrane. The entire experiment lasted 12 days. During this period, the color value, pH value, juice loss rate, texture, total bacterial count, and lipid oxidation of the fresh pork were measured every 3 days. The spoilage of the fresh pork was determined using these evaluation criteria, and the preservation effects of the above-mentioned membranes on fresh pork were investigated.

[0110] 2.1 Preparation of pork samples Purchase commercially available fresh pork, remove the outer layer to reduce the bacterial content of the sample, and cut the pork into approximately equal-weight pieces. These pieces are randomly divided into 6 groups. Group 1 is not covered with plastic wrap; Group 2 is packaged with commercially available food-grade plastic wrap (PE film); Group 3 is packaged with CS-PVA film; Group 4 is packaged with CS-PVA / AuNSs-EGT 0.5 (0.5%); Group 5 is packaged with CS-PVA / AuNSs-EGT 1.0 (1.0%); and Group 6 is packaged with CS-PVA / AuNSs-EGT 1.5 (1.5%). The pork is refrigerated at 4°C, and its physicochemical properties are measured at 0, 3, 6, 9, and 12 days (see section on appearance changes). Figure 19 ), to investigate preservation status.

[0111] Depend on Figure 19 It is evident that the color of fresh meat is a crucial visual indicator for consumers to judge its freshness. Under normal circumstances, fresh meat should exhibit a vibrant red color, primarily due to the formation of oxymyoglobin from the combination of myoglobin and oxygen. This chemical reaction not only gives fresh meat its distinctive red color but also reflects its freshness and oxygen content. As storage time increases, the surface color of pork in the Control and PE groups gradually darkens, caused by the discoloration of myoglobin due to oxidation. Furthermore, the oxidation phenomenon is more pronounced in the PE group. In contrast, pork in the CS-PVA / AuNSs-EGT composite film group maintained good color and moisture even on day 6.

[0112] 2.2 Determination of pork color The surface color of the sample was measured using a colorimeter, and the parameters L* (brightness), a* (red), and b* (yellow) were recorded. Five measurement points were randomly selected on the surface of the meat sample, and the average value was taken (see results). Figure 20 ).

[0113] Depend on Figure 20It can be seen that: the larger the L* value, the greater the brightness of the sample; the larger the a* value, the more the sample's color shifts towards red; and the larger the b* value, the more the sample's color shifts towards yellow. As shown in Figure A, with the increase of chitosan concentration, the L* value shows a linear decreasing trend, with the Control group showing the most significant decrease, dropping to 27.51 on day 12, while the brightness value covered by the PE film shows an increasing trend. The L* value is highest when the AuNSs-EGT concentration reaches 1.5%. Similarly, analyzing the a* value, as shown in Figure B, it shows a continuously decreasing trend. This trend indicates that during storage, fresh meat gradually loses its original bright red color, a typical manifestation of meat spoilage. The decrease in a* value not only affects the appearance of the meat but is also an important indicator of quality decline. In particular, the Control and PE groups show the most rapid decrease in a* value. This indicates that the PE film is relatively ineffective in maintaining the color of meat. In contrast, the a* value of the CS-PVA / AuNSs-EGT composite film packaging group decreased significantly more slowly, indicating that this packaging method has an advantage in maintaining the freshness and color of meat. This difference is mainly due to the different properties of the packaging materials. As shown in Figure C, the b* value (yellowness) of each group also increased with increasing storage time. Meanwhile, the b* value of the CS-PVA / AuNSs-EGT composite film group was lower than that of the Control and PE groups. Furthermore, the CS-PVA / AuNSs-EGT composite film with a higher AuNSs-EGT content had a much lower b* value, possibly due to its significantly better antioxidant properties. In summary, the color change of the CS-PVA / AuNSs-EGT group was slower than that of the Control, PE, and CS-PVA groups, indicating that the pork wrapped in the CS-PVA / AuNSs-EGT composite film was of better quality. In addition, the CS-PVA / AuNSs-EGT composite film with a higher AuNSs-EGT content can more effectively maintain the freshness of pork.

[0114] 2.3 Determination of pH value in pork Meat samples and distilled water were mixed at a ratio of 1:9 and homogenized for 2 minutes. The homogenate was filtered, and the pH value of the filtrate was measured using a pH meter. Each group of samples was repeated three times (results are shown in [link to results]). Figure 21 ).

[0115] Depend on Figure 21It can be seen that for fresh pork, a pH value of 5.8-6.2 indicates first-grade freshness, 6.2-6.7 indicates second-grade freshness, and pH > 6.7 indicates spoiled meat. The pH changes of pork treated with six different packaging methods during storage at 4℃ showed that, overall, the pH value of pork treated with all six packaging methods gradually increased over time. The pH increase trend of the CS-PVA / AuNSs-EGT 1.5% composite membrane group was relatively slow compared to the other groups, while the PE group showed the fastest increase. This is because cellular respiration in the meat initially produces acidic substances such as lactic acid and phosphoric acid through glycogenolysis for energy, causing the pH of the meat sample to gradually decrease. Subsequently, under the action of microorganisms, the decomposition of proteins and amino acids in the meat produces amine metabolites, causing the pH of the meat sample to increase.

[0116] 2.4 Determination of pork juice loss rate Before the experiment, accurately weigh each meat piece and record its mass (W0, g). Every 3 days, gently wipe the surface of the sample to remove moisture, weigh it again, and record its mass (W1, g). Juice loss rate was calculated using the following formula (see results). Figure 22 ): Juice loss rate % = (W0-W1) / W0×100%.

[0117] Depend on Figure 22 It can be seen that the juice loss rate of the membrane-free group increased significantly with the extension of refrigeration time, far exceeding that of the other groups. The CS-PVA / AuNSs-EGT 1.5% composite membrane performed better than the PE membrane group. This may be because the CS-PVA / AuNSs-EGT 1.5% composite membrane has a higher water vapor permeability than the PE membrane, resulting in a lower water retention effect. A moderate amount of juice discharge is beneficial for maintaining a dry environment, while excessive juice will accelerate the growth of spoilage bacteria and pathogenic bacteria.

[0118] 2.5 Determination of pork texture Elasticity, resilience, and cohesion of samples were determined using a texture analyzer in TPA mode. A P / 36R probe was used, with a pre-measurement velocity of 2 mm / s, a measurement velocity of 1 mm / s, a post-measurement velocity of 1 mm / s, a compression height of 30%, a measurement interval of 5 s, and automatic triggering. The elasticity, cohesion, and resilience of pork under different storage times were obtained based on compression curves. Three parallel samples were tested in each group, and the results were averaged (results are shown in Table 6).

[0119] Table 6. Pork processed by different methods during storage at 4°C Changes in elasticity, cohesion, and resilience

[0120] Table 6 shows that on day 0, the elasticity of pork was 0.96 mm, cohesion was 0.67, and resilience was 0.28. With increasing storage time, the elasticity of pork in all treatment groups showed a decreasing trend, with significant differences (P<0.05). The decrease in elasticity is caused by the degradation of proteins into smaller molecules, leading to the disruption of the protein network structure. Compared with the Control group and PE, the elasticity of the CS-PVA / AuNSs-EGT composite membrane treatment group was greater than that of the Control and PE groups (P<0.05), and the high-concentration CS-PVA / AuNSs-EGT 1.5% composite membrane showed the slowest decrease. Resilience refers to the degree to which meat returns to its original shape under external pressure. Resilience is correlated with elasticity, showing an overall decreasing trend. With increasing storage time, the cohesion of pork gradually decreases. At day 12, the cohesion of all treatment groups was significantly greater than that of the control group and the PE group (P<0.05). The cohesion of the control group was 0.25, while that of the CS-PVA / AuNSs-EGT 1.5% treatment group was 0.48. This indicates that the composite membrane treatment effectively reduced the decline in meat quality and that the CS-PVA / AuNSs-EGT composite membrane plays a positive role in maintaining pork quality. Compared with the pork samples treated in the Control and PE groups, the samples in the composite membrane treatment group showed higher elasticity, cohesion, and resilience. This suggests that the CS-PVA / AuNSs-EGT composite membrane can better maintain pork texture, and the higher the AuNSs-EGT concentration, the better the effect on maintaining pork texture.

[0121] 2.6 Determination of total bacterial count in pork The procedure was performed according to GB4789.2-2022, "National Food Safety Standard - Microbiological Examination of Food: Determination of Total Colony Count". Under aseptic conditions, 1g of minced pork was placed in a sterile bag containing 9mL of physiological saline and homogenized. After homogenization for a certain period, a sample was taken and diluted 10-fold. Then, 0.1mL of each of the three appropriate dilution ratios was taken. The diluted solutions were added to prepared agar plates. The plates were inverted and incubated at 36℃ for 48 hours. Afterward, the colony count was performed (see results below). Figure 23 ).

[0122] Depend on Figure 23 It is known that microorganisms are one of the key factors in protein deterioration; therefore, total bacterial count is commonly used to determine the level of microbial contamination in food. Total bacterial count (TVC) is one of the important indicators for assessing the freshness of meat. National standards stipulate that the TVC value of pork should be below 61 g CFU / g. The changes in total bacterial count of pork under different treatment methods are shown below. Figure 23As shown, the total bacterial count increased continuously with increasing storage days. The total bacterial count in the CS-PVA / AuNSs-EGT composite membrane treatment group was lower than that in the control group. The total bacterial count in the control group increased rapidly after 3 days, from 3.56 log CFU / g at 0 days to 7.6 log CFU / g at 12 days. However, the CS-PVA / AuNSs-EGT composite membrane treatment group showed an inhibitory effect. The total bacterial count in the CS-PVA / AuNSs-EGT 1.5% composite membrane treatment group was less than 6 log CFU / g before 12 days and reached 6.45 log CFU / g at 12 days. The total bacterial count of the CS-PVA membrane at 12 days was 7.2 log CFU / g. Therefore, CS-PVA / AuNSs-EGT composite membrane treatment can effectively inhibit microbial growth and delay the spoilage of pork. The higher the concentration of AuNSs-EGT, the slower the colony growth. It is possible that the combination of AuNSs-EGT and CS-PVA enhances the antibacterial effect, thereby extending the shelf life of pork.

[0123] 2.7 Determination of lipid oxidation in pork Weigh 4g of meat and add 10mL of 20% trichloroacetic acid (20g in 100mL of water). Homogenize and centrifuge for 15min. Take 2mL of the supernatant and add 2mL of 0.02M thiobarbituric acid (TBA) solution (0.2883g in 100mL of water). Heat in a boiling water bath for 30min. After cooling, measure the absorbance at 532nm. Three parallel samples were tested for each group, and the average value was taken (see results). Figure 24 The TBARS value is expressed as malondialdehyde content, and the calculation formula is as follows: TBARS （mg / kg） = (A 532 +0.002) × 2.587 Depend on Figure 24 It is known that lipid oxidation is one of the main causes of meat spoilage, and the TBARS value is commonly used to assess the degree of lipid oxidation in pork. When the TBARS value of pork is higher than 0.6 mg MDA / kg, the pork usually emits an unpleasant odor. Figure 24 The study shows the trends in thiobarbituric acid (TBARS) levels in pork treated with different methods. Overall, the TBARS values ​​of pork in all treatment groups showed a continuous upward trend during storage, indicating that the degree of oxidation was increasing and freshness was gradually decreasing. The Control group showed the fastest growth rate and the highest value; while the CS-PVA / AuNSs-EGT composite membrane group showed the slowest trend and the lowest value. This is because the CS-PVA / AuNSs-EGT composite membrane contains certain antioxidants that delay pork spoilage.

[0124] In summary, this application, through a series of investigations, ultimately obtained a CS-PVA / AuNSs-EGT composite membrane suitable for preservation. The prepared composite membrane exhibits excellent performance in optical properties, mechanical properties, and thermal stability. Using the prepared composite membrane for pork preservation demonstrates significant advantages in terms of color, pH, juice loss, texture, and total bacterial count.

[0125] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0126] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. 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 composite membrane containing ergothionein gold nanomaterials, characterized in that, The composite preservation film is obtained by mixing and stirring a complex prepared from ergothioneine and gold nanoclusters with a mixture prepared from chitosan and polyvinyl alcohol, followed by molding and drying.

2. A method for preparing the composite food preservation film according to claim 1, characterized in that, Includes the following steps: S1. Mix ergothioneine solution, chloroauric acid solution and ultrapure water evenly, adjust the pH of the mixture to 8-12, stir the reaction, place it on a shaker overnight after the reaction is completed, then dialyze the solution and freeze dry to obtain gold nanoclusters-ergothioneine complex. S2. Dissolve chitosan in an aqueous acetic acid solution and stir to obtain a chitosan solution; dissolve polyvinyl alcohol in deionized water and stir to obtain a polyvinyl alcohol solution; mix the chitosan solution and polyvinyl alcohol solution evenly, add plasticizer and stir to obtain a chitosan-polyvinyl alcohol mixture; S3. Add the gold nanoclusters-ergothionein complex prepared in S1 to the chitosan-polyvinyl alcohol mixture prepared in S2, mix evenly, sonicate, pour into a mold, and dry to obtain a composite preservation film.

3. The preparation method according to claim 2, characterized in that, In step S1, the concentration of ergothioneine solution is 21.5~107.5 mmol / L, and the concentration of chloroauric acid solution is 15~25 mmol / L; The ratio of ergothioneine to chloroauric acid in the mixture is (1~10):

1.

4. The preparation method according to claim 3, characterized in that, The stirring reaction conditions in step S1 are: temperature 80~100℃, rotation speed 450~550 rpm, and time 10~14 h.

5. The preparation method according to claim 2, characterized in that, In step S2, the concentration of the acetic acid aqueous solution is 0.5~1.5% (v / v), the concentration of the chitosan solution is 1.5~2.5 wt%, and the concentration of the polyvinyl alcohol solution is 2.5~3.5 wt%. Chitosan solution and polyvinyl alcohol solution were mixed at a solute mass ratio of 2:

3. The plasticizer is glycerol, accounting for 10-20% of the total volume of the chitosan-polyvinyl alcohol mixture.

6. The preparation method according to claim 5, characterized in that, Stirring conditions during the preparation of chitosan solution in step S2: temperature 65~75℃, rotation speed 400~600rpm, time 3h; Stirring conditions for preparing polyvinyl alcohol solution: temperature 75~85℃, speed 200~300 rpm, time 3h.

7. The preparation method according to claim 2, characterized in that, In step S3, the amount of gold nanoclusters-ergothionein complex added is 0.5~1.5% of the mass of the chitosan-polyvinyl alcohol mixture.

8. The preparation method according to claim 7, characterized in that, Ultrasonic treatment conditions in step S3: power 80~150W, time 40~60min; The drying conditions are: temperature 65~75℃, time 240~360 min.

9. The application of the composite preservation film according to claim 1 in food preservation.

10. The application according to claim 9, characterized in that, Food includes fresh meat and vegetables.

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