Application of UV light regulated Fe / Lyz@CDs nanoszyme in fruit preservation

Abstract

This invention discloses the application of UV-regulated Fe / Lyz@CDs nanozymes in fruit preservation. Using lysozyme and diethylenetriaminepentaacetic acid as precursors, Fe-doped lysozyme-based carbon dot (Fe / Lyz@CDs) nanozymes were prepared via a simple hydrothermal method. The Fe / Lyz@CDs nanozymes exhibit UV-responsive peroxidase (POD) and superoxide dismutase (SOD)-like catalytic properties. Fe / Lyz@CDs catalyze the generation of reactive oxygen species (ROS), which are beneficial for *Escherichia coli* (E. coli). E.coli Staphylococcus aureus ( S.aureus Fe / Lyz@CDs exhibits significant bactericidal effects against four typical foodborne pathogens: multidrug-resistant Escherichia coli (AREC) and methicillin-resistant Staphylococcus aureus (MRSA). Fe / Lyz@CDs is dispersed in a polyvinyl alcohol (PVA) matrix to prepare Fe / Lyz@CDs-PVA film. Using this film for mango preservation can maintain the quality of mangoes and extend their storage time.

Description

Technical Field

[0001] This invention belongs to the field of agricultural product preservation technology, specifically, it relates to the application of a UV-regulated Fe / Lyz@CDs nanoenzyme in fruit preservation. Background Technology

[0002] Microbial contamination is a significant factor in food spoilage and a major cause of shortened shelf life. Existing methods for extending shelf life, including low-temperature storage, controlled atmosphere storage, wax coating, irradiation, and chemical treatment, are often limited by technical complexity, high cost, and insufficient effectiveness. Packaging films containing active substances can impart antibacterial and antioxidant properties, effectively inhibiting microbial growth and preventing oxidation reactions, thereby protecting food quality. Polyvinyl alcohol (PVA), due to its excellent hydrophilicity and gas barrier properties, is an ideal choice for fruit preservation packaging films. Its hydrophilicity creates a moisturizing film on the fruit surface, reducing moisture evaporation and maintaining fruit hydration. Furthermore, its excellent gas barrier properties help regulate the concentration of oxygen and carbon dioxide around the fruit, thereby reducing respiration and extending freshness. However, pure PVA films lack antibacterial activity and mechanical strength. The resulting films are prone to stretching, friction, and breakage during packaging and handling. Once the film is damaged, it cannot maintain a stable internal environment, adversely affecting food quality and freshness.

[0003] Carbon dots (CDs) are a class of materials that have... sp 2 Zero-dimensional carbon-based nanoparticles (<10 nm) with carbon structure, unique optical properties, biocompatibility, antioxidant and antibacterial properties have recently become a novel strategy for utilizing light to reversibly regulate enzyme activity due to their absorption over a broad spectral region. Summary of the Invention

[0004] This invention provides a novel application of UV-regulated Fe / Lyz@CDs nanozymes, specifically their use in fruit preservation. The UV-regulated Fe / Lyz@CDs nanozymes are prepared via a simple hydrothermal method using lysozyme and diethylenetriaminepentaacetic acid (DTPA) as precursors. The prepared Fe / Lyz@CDs nanozymes exhibit UV-responsive peroxidase (POD) and superoxide dismutase (SOD)-like catalytic properties. The Fe / Lyz@CDs nanozymes effectively generate reactive oxygen species (ROS), which are effective against four types of bacteria, including *Escherichia coli*. E. coli Staphylococcus aureus ( S.aureusIt exhibits significant bactericidal effects against typical foodborne pathogens such as multidrug-resistant Escherichia coli (AREC) and methicillin-resistant Staphylococcus aureus (MRSA). Based on POD activity, glutathione (GSH) oxidase activity, and surface positive charge, the minimum bactericidal concentration (MBC) is 60 μg / mL. Fe / Lyz@CDs nanozymes were dispersed in a polyvinyl alcohol (PVA) matrix to prepare Fe / Lyz@CDs-PVA films with good water vapor permeability, antioxidant activity, and excellent UV-Vis barrier properties.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution: 1. Place 0.7-0.9g of diethylenetriaminepentaacetic acid, 0.3-0.4g of FeCl3·6H2O, and 100-150μL of lysozyme in 30-40mL of deionized water and sonicate for 10-20min. Transfer the mixture to a polytetrafluoroethylene container and react at 180℃ for 5-6h. After cooling the reaction product, centrifuge, filter the supernatant through a 0.22μm filter membrane, and vacuum dry to obtain Fe-doped lysozyme-based carbon dot Fe / Lyz@CDs nanozyme. The concentration of the lysozyme is 35-55 mg / mL; 2. Dissolve 5-6g of polyvinyl alcohol in 100-120mL of ultrapure water and stir at 90-100℃ for 25-35min. Then add 1-1.2g of glycerol and 4-5mL of 30-35mg / mL Fe / Lyz@CDs solution. Mix well and coat the mixture onto the fruit. Dry to obtain Fe / Lyz@CDs-PVA film. 3. Application of Fe / Lyz@CDs-PVA film in fruit preservation The Fe / Lyz@CDs-PVA film exhibits ultraviolet (UV) light-responsive peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) catalytic activities, and possesses broad-spectrum antibacterial and antioxidant properties. The ultraviolet light has a wavelength of 365nm and a power of 6.5-7.0mW / cm². 2 .

[0006] The beneficial effects of this invention are: 1. This invention utilizes positively charged lysozyme and diethylenetriaminepentaacetic acid (DTPA) with five -COOH groups as precursors to prepare Fe-doped lysozyme-based carbon dot (Fe / Lyz@CDs) nanozymes via a simple hydrothermal method. DTPA with five -COOH groups can bind to PVA through hydrogen bonding and fibrous entanglement networks. This reinforcement improves the mechanical strength of the hydrogel membrane, making the freshness packaging more durable during storage and transportation. Lysozyme (Lyz) is a recognized enzyme with strong antibacterial activity, its antibacterial activity mainly derived from a unique mechanism: disrupting the structural integrity of bacterial cell walls. Furthermore, Lyz can serve as an effective carrier for binding certain metal ions, demonstrating its significant carrying and protective effects. The interaction between metal ions and Lyz not only improves the stability of Lyz itself but also endows Lyz with excellent antibacterial properties. The Fe / Lyz@CDs nanozyme prepared in this invention exhibits UV-responsive peroxidase (POD) and superoxide dismutase (SOD) catalytic properties. Under UV activation, the Fe / Lyz@CDs nanozyme effectively generates reactive oxygen species (ROS), demonstrating broad-spectrum antibacterial effects against Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, multidrug-resistant Escherichia coli, and Penicillium expansum, with a minimum bactericidal concentration (MBC) of 60 μg / mL. Furthermore, the inhibition rate of biofilms treated with Fe / Lyz@CDs exceeds 80%. 2. Loading Fe / Lyz@CDs nanozymes onto PVA to prepare Fe / Lyz@CDs films improved the mechanical properties, UV-Vis light blocking properties, water vapor transmission rate, and antioxidant activity of the PVA films. These films exhibited low toxicity and good biocompatibility. When applied to mango preservation, they effectively extended the fruit's shelf life and significantly reduced the incidence of mold and browning, as well as the degree of quality deterioration. The results indicate that Fe / Lyz@CDs-PVA films have the potential to inhibit browning and maintain the quality of mangoes during storage. Attached Figure Description

[0007] Figure 1 These are TEM images of the Fe / Lyz@CDs nanozyme from Example 1 of the present invention, where image a shows the particle size distribution and image b shows the lattice fringes. Figure 2 The image shows the XRD pattern of the Fe / Lyz@CDs nanozyme of Example 1 of this invention. Figure 3 The image shown is the FT-IR spectrum of the Fe / Lyz@CDs nanozyme of Example 1 of this invention. Figure 4 The high-resolution XPS spectrum of Fe 2p of the Fe / Lyz@CDs nanozyme in Example 1 of this invention; Figure 5 The UV, fluorescence excitation and emission spectra of the Fe / Lyz@CDs nanozyme of Example 1 of this invention are shown below. Figure 6 The figures show the performance results of the Fe / Lyz@CDs-PVA film in Example 1, where figure a is the SEM image and figure b is the Zeta potential diagram. Figure 7 The figures show the light transmittance results of the Fe / Lyz@CDs-PVA films in Example 1. In the figures, Fe / Lyz@CDs-PVA0.038 represents a Fe / Lyz@CDs doping amount of 0.038 wt%; Fe / Lyz@CDs-PVA0.076 represents a Fe / Lyz@CDs doping amount of 0.076 wt%; and Fe / Lyz@CDs-PVA0.151 represents a Fe / Lyz@CDs doping amount of 0.151 wt%. Figure 8 The ultraviolet shielding properties of Fe / Lyz@CDs-PVA thin films are shown in (a) and WCA diagram (b). Figure 9 The stress-strain curves (a) and Young's modulus and toughness (b) of the Fe / Lyz@CDs-PVA film in Example 1 are shown. Figure 10 TGA (a) and DTG curves (b) for PVA and Fe / Lyz@CDs-PVA; Figure 11 The POD-like (a) and SOD-like activities (b) of Fe / Lyz@CDs-PVA prepared in Example 1 of this invention are shown. Figure 12 In Example 1, different concentrations of Fe / Lyz@CDs-PVA were compared with those under UV irradiation and without irradiation. E. coli and AREC (Figure a), S.aureus The effect of UV irradiation on the antibacterial properties of MRSA (Figure b); the effect of different materials on the antibacterial properties under UV irradiation and no irradiation. P. expansum The effect of antibacterial properties (Figure c); Figure 13 In this embodiment 1, different materials suppress... E. coli and S. aureus The result of biofilms; Figure 14 In Example 1, ROS and SEM images of different bacteria were obtained by Fe / Lyz@CDs-PVA and Fe / Lyz@CDs-PVA+UV treatment. Figure 15 The results of Fe / Lyz@CDs-PVA scavenging DPPH free radicals (Figure a) and ABTS free radicals (Figure b) in Example 1; Figure 16 The effect of different concentrations of Fe / Lyz@CDs-PVA on the survival rate of Hep G2 cells and HUVECs cells after co-incubation for 24 h (Figure a) and the results of the hemolysis experiment (Figure b). Figure 17 This example 1 shows the different storage times of mangoes treated with different groups of materials. Detailed Implementation

[0008] The technical solution of the present invention will be described in further detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto; In the following examples, the PVA film was prepared by dissolving 5g of polyvinyl alcohol (PVA) in 100mL of ultrapure water, stirring at 95°C for 30min, then adding 1g of glycerol to obtain a PVA matrix solution, coating it on a glass substrate, and drying it. Fe / Lyz@CDs nanozymes were added to the PVA matrix solution to prepare Fe / Lyz@CDs-PVA solutions of different concentrations; the film formation method was the same as above. Example 1: Preparation, characterization and properties of Fe / Lyz@CDs nanozymes and thin films 1. Material preparation (1) Preparation of Fe / Lyz@CDs: 0.75g diethylenetriaminepentaacetic acid, 0.30g FeCl3·6H2O and 120μL lysozyme (50mg / mL) were placed in 35mL deionized water and sonicated for 15min. The mixture was then transferred to a polytetrafluoroethylene container and reacted at 180℃ for 5h. After the reaction product was cooled, it was centrifuged, and the supernatant was filtered through a 0.22μm filter membrane and vacuum dried to obtain Fe-doped lysozyme-based carbon dot Fe / Lyz@CDs nanozyme. (2) Preparation of Fe / Lyz@CDs-PVA film: 5g of polyvinyl alcohol (PVA) was dissolved in 100mL of ultrapure water and stirred at 95℃ for 30min. Then, 1.0g of glycerol and 4mL of 32mg / mL Fe / Lyz@CDs nanozyme solution were added and mixed. After stirring evenly, Fe / Lyz@CDs-PVA solution was formed. The Fe / Lyz@CDs-PVA solution was coated onto a glass substrate and dried to obtain Fe / Lyz@CDs-PVA film. 2. Characterization of Fe / Lyz@CDs nanozymes Transmission electron microscopy (TEM) confirmed that the Fe / Lyz@CDs nanozyme was spherical with good dispersibility and an average size of 2.81 ± 0.62 nm (n = 220). Figure 1 a) High-resolution TEM (HRTEM) revealed that the lattice spacing of Fe / Lyz@CDs was 0.21 nm, corresponding to sp 2- Graphite carbon (100) crystal plane ( Figure 1 b); X-ray diffraction (XRD) patterns show that at approximately 2 θ There is a broad diffraction peak at 26°, which is attributed to graphite-2H ( Figure 2 The crystal plane (02) of the sample indicates the presence of high-purity iron-based nanoparticles (Fe / Lyz@CDs); then, Fourier transform infrared spectroscopy (FT-IR) was used to analyze the distribution of surface functional groups, such as... Figure 3 As shown, at 3414cm -1 There is a broad peak at 2924 cm⁻¹, attributed to the OH / NH stretching vibration, and CH (2924 cm⁻¹). -1 ), OC=O(1739cm) -1 C=N stretching vibration (1632cm) -1 ) and CO (1396cm -1 The signal is 614cm. -1 The characteristic peak at that location is attributed to the Fe-O stretching vibration; Figure 4 In the high-resolution X-ray photoelectron spectroscopy (XPS) spectrum of Fe 2p, the Fe 2p spectrum shows that Fe 2+ (2p 3 / 2 709.9 eV, 2p 1 / 2 (724.1 eV) and Fe 3+ (715.0 eV), Fe 2+ The large spectral area of ​​the component indicates its dominance, and also suggests that CDs contain a reducing portion of Fe. 3+ The optical properties of Fe / Lyz@CDs nanozymes in deionized water were investigated using ultraviolet-visible (UV-Vis) absorption spectroscopy and fluorescence spectrophotometry. An absorption peak appeared at 260 nm in the UV-Vis spectrum, which was attributed to the C=O and C=C bonds. sp 2 n-π* transitions are associated with the carbon nucleus and carbonyl functional group, respectively; fluorescence emission spectra show that at an excitation wavelength of 370 nm, the emission wavelength is 450 nm. Figure 5 The above results demonstrate the successful synthesis of Fe / Lyz@CDs nanozymes.

[0009] 3. Characterization of Fe / Lyz@CDs-PVA The morphological characteristics of the thin film were studied using SEM images, such as... Figure 6As shown in Figure a, the PVA film surface is uniform, smooth, and dense, with a smooth surface morphology but some impurities or phase separation. This is likely due to the excellent film-forming properties of PVA. The embedding of Fe / Lyz@CDs nanozymes maintains the overall density of the film surface, free of cracks or large pores. Due to the incorporation of Fe / Lyz@CDs nanozymes, only a slight increase in surface texture was observed, with no obvious filler agglomeration, indicating good dispersion in the PVA matrix and maintaining the macroscopic dimensional stability of the composite film. At the 100 μm scale, both films exhibit continuous, non-layered, and dense cross-sections without interfacial gaps or phase separation, confirming excellent interfacial compatibility. The thickness of the pure PVA film is 208 μm, while the thickness of the Fe / Lyz@CDs-PVA film is 217 μm, a difference of only 9 μm. These results indicate that the incorporation of Fe / Lyz@CDs has no significant effect on the film thickness. Surface charge interactions were assessed by measuring the potential to evaluate the potential mechanism of antibacterial activity. Figure 6 The results showed that the Fe / Lyz@CDs-PVA membrane exhibited a positive Zeta potential (+1.1 mV), while all tested strains ( E. coli (-9.3mV), AREC (-12.3mV), S. aureus Both Fe / Lyz@CDs-PVA (-8.6 mV) and MRSA (-10.7 mV) exhibit negative surface charges. After exposure to Fe / Lyz@CDs-PVA membrane, the zeta potential of bacteria shifts towards smaller negative or positive values. This suggests that positively charged Fe / Lyz@CDs-PVA particles are adsorbed onto the negatively charged bacterial cell walls through electrostatic attraction, which may lead to membrane instability and contribute to the bactericidal effect.

[0010] References (Li Fan, Preparation of Functionalized Chitosan-Based Composite Film and Study on its Fruit Preservation Performance, Master's Thesis, Guangxi University). The optical, water contact angle (WCA), mechanical, and thermal stability properties of the film were measured. The results showed that the Fe / Lyz@CDs-PVA film exhibited a narrow color change range, showing only a slight darkening effect, with no obvious color difference or turbidity. Figure 7 );exist Figure 8In study a, pure PVA films exhibited minimal UV absorption in the 200-450 nm range, demonstrating poor UV blocking ability. However, the addition of different concentrations of Fe / Lyz@CDs nanozymes to the PVA matrix significantly enhanced UV absorption. Furthermore, the UV blocking performance of the film improved with increasing Fe / Lyz@CDs concentration. When the mass ratio of Fe / Lyz@CDs in the film was 0.076 wt%, the average transmittance in the UVB band (280-315 nm) was less than 1%, and the average transmittance in the UVA band (315-400 nm) was 3%, with a UV protection factor (UPF) greater than 50 and a UVB / UVA ratio of 0.3. This indicates that the sample possesses excellent broad-spectrum UV protection performance, especially with a highly significant blocking effect against high-energy UVB. This result confirms that CDs enhance UV protection through their nanoscale structure, promoting light reflection and scattering. Furthermore, the synergistic effect between Fe / Lyz@CDs nanozymes and PVA helps to form a denser, more opaque film structure, further limiting the transmission of ultraviolet light.

[0011] The surface wettability of the thin film was evaluated using a contact angle measuring instrument (WCA), such as... Figure 8 As shown in b, the WCA of pure PVA film is 47.99°, reflecting its inherent hydrophilicity. This indicates that it easily adsorbs water onto the fruit surface, causing adhesion and swelling, which is detrimental to preservation. Adding different concentrations of Fe / Lyz@CDs to PVA films resulted in an increase in WCA value from 54.62° to 64.30°, with a slight decrease in hydrophilicity, but not reaching the critical hydrophobic value. This avoids adhesion and reduces swelling, making it more suitable for fruit preservation films. This is attributed to the hydrophobic groups of Fe / Lyz@CDs covering the hydrophilic hydroxyl groups of PVA, altering the film surface morphology, forming hydrophobic microregions, and achieving wettability regulation, thus supporting its application in fruit preservation.

[0012] The mechanical properties of the film are evaluated by measuring tensile strength (TS) and elongation at break (EB). Figure 9 a, Figure 9 The results showed that the pure PVA membrane exhibited the lowest TS (21.46 MPa) but a high EB (653.72%). After incorporation of Fe / Lyz@CDs nanozymes (0.151 wt%), the PVA TS increased to 39.88 MPa (1.85 times that of the pure PVA membrane), and the EB increased to 673.33% (P<0.05). This behavior can be attributed to the Fe / Lyz@CDs nanozymes possessing abundant functional groups and a small particle size through chemical or physical cross-linking mechanisms.

[0013] The thermogravimetric (TG) and differential thermogravimetric (DTG) curves of the thin film show two distinct weight loss phases. Figure 10 a, Figure 10 (b) Within the temperature range of 40–200 °C, the initial weight loss is mainly attributed to the evaporation of moisture in the film. The second stage of weight loss occurs in the temperature range of 200–400 °C. This process involves the thermal degradation of PVA molecular chains, leading to the generation and escape of small molecules, thus causing a significant increase in mass loss. When the temperature is >400 °C, PVA further decomposes, and the residual organic matter gradually transforms into carbon, slowing down the weight loss. Compared with pure PVA, Fe / Lyz@CDs-PVA… 0.151 Composite membranes exhibit higher thermal decomposition temperatures, slower weight loss processes, and higher char residues, demonstrating superior thermal stability.

[0014] 4. Evaluation of POD-like and SOD-like Activities of Materials (1) The peroxidase-like (POD) activity of TMB was evaluated using TMB as a catalytic reaction substrate. 50 μL of 5 mmol / L TMB, 50 μL of 10 mmol / L H2O2, and Fe / Lyz@CDs-PVA (1×1 cm) were added. 2 Add the sample to 2 mL of 0.1 mol / L HAc-NaAc buffer solution (pH 4.0), mix thoroughly, and measure the absorbance after reacting for 10 min. A UV light control group was also set up at 6.5 mW / cm². 2 Irradiated the enzymes at 365nm UV for 10 min, keeping all other reaction conditions constant. The enzyme catalytic activity of the two groups was measured and compared. Absorbance was measured at 654nm wavelength. Results are as follows: Figure 11 a) In the presence of Fe / Lyz@CDs-PVA and H2O2, TMB underwent a significant oxidation reaction. This experimental phenomenon fully demonstrates that Fe / Lyz@CDs-PVA possesses POD-like catalytic activity, capable of mimicking the function of natural peroxidase to catalyze the oxidation of TMB by H2O2 to generate a blue product. Furthermore, after UV irradiation, the POD-like activity of Fe / Lyz@CDs-PVA was significantly enhanced, with an increase of nearly 2.0 times, indicating that Fe / Lyz@CDs-PVA exhibits UV-enhanced POD-like activity.

[0015] (2) By measuring the UV inhibition rate of nitroblue tetrazolium (NBT) and the effect of Fe / Lyz@CDs-PVA on superoxide radicals (·O2), the effect of nitroblue tetrazolium (NBT) on superoxide radicals (·O2) was determined. - The scavenging rate of Fe / Lyz@CDs-PVA was investigated to assess its SOD-like activity. The specific procedure was as follows: 50 μL of 25 μmol / L riboflavin, 50 μL of 12 mmol / L methionine, and Fe / Lyz@CDs-PVA membrane (1×1 cm²) were added. 2) and 75 μmol / L NBT 50 μL were mixed in 2 mL of 25 mmol / L pH 7.4 phosphate buffer. The mixture was reacted at room temperature for 10 min. The absorbance value at 560 nm was recorded. Superoxide radical (·O2) - The clearance rate is calculated using the following equation: ·O2 - Clearance rate (%) = (A0 - A) / A0 × 100% Where A0 and A are the absorbance values ​​without Fe / Lyz@CDs-PVA and with Fe / Lyz@CDs-PVA, respectively.

[0016] Figure 11 The results show that the Fe / Lyz@CDs-PVA membrane significantly removes O2. - The clearance rate was 65.1%, which is much stronger than that of PVA (11.9%), indicating that Fe / Lyz@CDs-PVA has strong SOD-like activity.

[0017] 5. Fe / Lyz@CDs-PVA antibacterial test The following bacterial strains were obtained from Beina Chuanglian Biotechnology Co., Ltd., using Escherichia coli (E. coli) E. coli ATCC-8099), Staphylococcus aureus ( S. aureus ATCC 6538), methicillin-resistant Staphylococcus aureus (MRSA, ATCC43300), multidrug-resistant Escherichia coli (AREC, SHBCC D25148), and Penicillium expansum (ATCC 6538). P. expansum (BNCC117714) is the experimental bacteria.

[0018] In the in vitro antibacterial experiment, the prepared concentration was 1×10 8 CFU / mL E. coli , S. aureus , P. expansum AREA and MRSA bacterial suspensions were diluted to 1×10⁻⁶ with sterile phosphate-buffered saline (PBS). 5 The antibacterial performance was evaluated using the plate count method with CFU / mL.

[0019] In the plate count method, the bacterial suspension (1×10⁻⁶) is used. 5 Different concentrations of Fe / Lyz@CDs-PVA (CFU / mL) were mixed with 5 mL of physiological saline and administered at 6.5 mW / cm². 2 Irradiate with 365nm UV light for 20 min or without UV irradiation, then spread 100μL of the suspension evenly onto an agar plate. E. coli LB medium was used for AREC; S. aureusThe plates were then incubated at 37°C for 24 hours on MRSA (using 7.5% NaCl broth medium). Figure 12 Results a and 12b show that antibacterial tests of different concentrations of Fe / Lyz@CDs-PVA determined its effectiveness under UV irradiation. E. coli ,AREC S. aureus The MBC of MRSA was 60 μg / mL. Furthermore, in Figure 12 In c, for P. expansum Antibacterial tests were conducted, with 1×10 5 CFU / mL P. expansum 200 μg / mL of Fe / Lyz@CDs-PVA was added to physiological saline and incubated under UV irradiation for 20 min, followed by 24 h. The results showed that Fe / Lyz@CDs-PVA could effectively inhibit... P. expansum The bacteria grew well and showed no turbidity in physiological saline. They were inoculated onto *Czapekella asiatica* extract agar (CYA) plates. Fe / Lyz@CDs-PVA was used to inhibit the growth of the bacteria. P. expansum Its antibacterial efficiency reaches 99%.

[0020] 6. Inhibition of biomembranes To evaluate the anti-biofilm properties of Fe / Lyz@CDs-PVA, we investigated its anti-biofilm ability using crystal violet (CV) staining. Bacterial biofilms were added to containers containing different films (PVA, Fe / Lyz@CDs-PVA, etc.). 0.038 Fe / Lyz@CDs-PVA 0.076 Fe / Lyz@CDs-PVA 0.151 In a 24-well plate containing the biofilm, 2 mL of culture medium was added to each well, and the plate was incubated at 37°C for 24 h. Excess culture medium was aspirated, and the biofilm was washed three times with PBS. Then, 200 μL of methanol was added to each well for fixation. After 3 min, excess methanol was aspirated, and the biofilm was air-dried. Then, 200 μL of 1% CV staining agent was added to stain the biofilm. After 30 min of staining, excess staining agent was rinsed off with sterile PBS buffer. 200 μL of 33% glacial acetic acid solution was added to the rinsed biofilm, and incubation continued for 30 min to dissolve the fixed biofilm. Finally, the OD value at 590 nm was measured, and the absorbance was used to assess the biofilm damage intensity of Fe / Lyz@CDs-PVA.

[0021] The results are as follows Figure 13 As shown, biofilm formation was clearly observed in the control group, and Fe / Lyz@CDs-PVA+UV significantly inhibited it after 24 h of incubation. E. coli and S.aureusBiofilm formation was observed when the Fe / Lyz@CDs-PVA addition amount was 0.151 wt%. 0.151 It exhibits the lowest biofilm survival rate.

[0022] 7. Monitoring of SEM and intracellular ROS in bacteria 1×10 5 CFU / mL E. coli or S.aureus 200 μg / mL Fe / Lyz@CDs-PVA (0.0151 wt%) was mixed in a 24-well plate with a climbing biofilm, irradiated with UV for 20 min, and then incubated for 24 h. The supernatant was then aspirated, and the climbing biofilm was characterized by SEM. The results are shown in [Figure number missing]. Figure 14 The control group and PVA group retained complete cell morphology; however, in the presence of Fe / Lyz@CDs-PVA (0.151wt%), E. coli and S.aureus The cell membranes showed wrinkling and shrinkage, while after Fe / Lyz@CDs-PVA+UV treatment, E. coli and S. aureus The cells were completely destroyed.

[0023] Intracellular ROS of bacteria were measured using laser confocal scanning microscopy. Fe / Lyz@CDs-PVA and 1 mL of bacterial suspension (1×10⁻⁶) were used. 5 The fluorescence was incubated together with CFU / mL and DCFH-DA (1 mM, 50 μL) to verify ROS generation; the more ROS generated, the stronger the observed fluorescence intensity. Figure 14 As shown, the Fe / Lyz@CDs-PVA+UV group in E. coli and S.aureus The presence of a significant green fluorescent signal indicates that the two groups produced the most ROS. This result further verifies that Fe / Lyz@CDs-PVA can effectively generate more toxic ROS, thereby achieving antibacterial effects.

[0024] 8. Material's antioxidant properties The antioxidant properties of the material were assessed using DPPH and ABTS scavenging tests. For the DPPH scavenging test, Fe / Lyz@CDs-PVA solutions of different concentrations (0.2, 0.39, 0.59, 0.79, 0.98, 1.18 mg / mL) were added to 2 mL of 0.2 mmol / L DPPH solution. The mixture was incubated in the dark for 30 min, and the DPPH concentrations of the solution (A0) and the sample mixture (A) were measured at 517 nm. i The absorbance of the sample was used to evaluate the percentage of DPPH radical scavenging activity using the following equation: DPPH free radical scavenging rate (%) = (1-A) i / A0) × 100% For the ABTS radical scavenging assay, 2.45 mmol / L potassium sulfate was mixed with 7 mmol / L ABTS solution and incubated overnight in the dark to prepare the ABTS assay solution. Then, Fe / Lyz@CDs-PVA solutions of different concentrations (0.16, 0.36, 0.64, 0.92, 1.62, 1.89 mg / mL) were added to 3.8 mL of ABTS solution. The mixture was incubated in the dark for 30 min. The ABTS assay solution (A0) and the ABTS solution containing the film sample (A2) were then recorded at 734 nm. i The absorbance of ABTS was used to assess the percentage of its free radical scavenging activity using an equation: ABTS radical scavenging rate (%) = (1-A i / A0) × 100% The results showed that the DPPH and ABTS radical scavenging abilities of Fe / Lyz@CDs-PVA increased in a concentration-dependent manner, such as... Figure 15 As shown in Figure a, compared with the control group, the DPPH radical scavenging rate was significantly increased to 78.5% after the addition of Fe / Lyz@CDs-PVA solution; a similar trend was observed for the scavenging of ABTS radicals. Figure 15 (b) The film exhibits 85% scavenging capacity, and this excellent free radical scavenging performance is likely attributed to the superior SOD-like activity of Fe / Lyz@CDs-PVA.

[0025] 9. Biosafety assessment The cytotoxicity of nanozymes was detected using a CCK-8 cell viability assay kit. Specifically, human umbilical vein endothelial cells (HUVECs) and hepatocellular carcinoma cells (Hep G2, Beina Chuanglian Biotechnology Co., Ltd.) were seeded in 96-well plates and cultured for 24 h. Cells were then incubated with different concentrations of Fe / Lyz@CDs-PVA for 24 h and 48 h, respectively. After washing with PBS, CCK-8 solution was added to each well to a concentration of 10%, and the cells were incubated at 37°C. Absorbance was measured at 450 nm. The CCK-8 analysis results (…) Figure 16 a) The results showed that Fe / Lyz@CDs-PVA was not toxic to cells; The safety of Fe / Lyz@CDs-PVA was evaluated using a hemolysis test; the results are as follows: Figure 16As shown in b, the hemolysis rate in the positive control group (0.1% Triton X-100, Control) was 100.00%, indicating complete hemolysis; the hemolysis rate in the negative control group (PBS) was 3.67%, with no significant hemolysis. The hemolysis rate in all Fe / Lyz@CDs-PVA concentration groups was <3.0%, significantly lower than the 5% safety threshold specified in ISO 10993-4. Actual photographs show that the red blood cells in the material group were intact precipitates, the supernatant was clear, and no visible hemolysis was observed. The results indicate that Fe / Lyz@CDs-PVA has excellent blood compatibility, no hemolytic toxicity, and good biosafety.

[0026] 10. Fruit preservation performance experiment of Fe / Lyz@CDs-PVA film To evaluate the application of Fe / Lyz@CDs-PVA in fruit preservation, a fruit storage experiment was conducted. Fresh mangoes were immersed in a prepared Fe / Lyz@CDs-PVA solution (the solution from step (2) of Example 1) for 3 minutes, and then irradiated with 365 nm for 5 minutes to ensure the formation of a uniform and complete coating. The preservation film adhered evenly to the surface of the mango fruit, with a tight bond and no wrinkles. The treated fruits were randomly divided into 4 groups: uncoated control group (Control), PVA film group, Fe / Lyz@CDs-PVA group, and Fe / Lyz@CDs-PVA+UV group. From the appearance ( Figure 17 After 3 days of storage, mangoes without coating and those coated only with PVA showed signs of ripening and yellowing with irregular spots. In contrast, both the Fe / Lyz@CDs-PVA group and the Fe / Lyz@CDs-PVA+UV group significantly delayed these phenomena; the Fe / Lyz@CDs-PVA+UV group showed the best results, with the mangoes remaining fresh and green after 12 days of storage. The extended preservation effect of Fe / Lyz@CDs-PVA coating on the fruit is attributed to the enzyme-like bactericidal and antioxidant activities occurring on the coated peel. More importantly, these results indicate that the Fe / Lyz@CDs-PVA film exhibits stronger antibacterial activity under UV assistance and effectively delays fruit spoilage.

[0027] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. The application of a UV-regulated Fe / Lyz@CDs nanozyme in fruit preservation, characterized by: The Fe / Lyz@CDs nanozyme is prepared by placing 0.7-0.9 g of diethylenetriaminepentaacetic acid, 0.3-0.4 g of FeCl3·6H2O, and 100-150 μL of lysozyme in 30-40 mL of deionized water, sonicating for 10-20 min, transferring the mixture to a polytetrafluoroethylene container, reacting at 180 °C for 5-6 h, cooling the reaction product, centrifuging, filtering the supernatant through a 0.22 μm filter membrane, and vacuum drying.

2. The application according to claim 1, characterized in that: Dissolve 5-6g of polyvinyl alcohol in 100-120mL of ultrapure water and stir at 90-100℃ for 25-35min. Then add 1-1.2g of glycerol and 4-5mL of 30-35mg / mL Fe / Lyz@CDs solution. After mixing, coat the mixture onto the fruit to obtain Fe / Lyz@CDs-PVA film, which is used for fruit preservation and has an enhancing effect under ultraviolet light.

3. The application according to claim 2, characterized in that: The concentration of lysozyme is 35-55 mg / mL.

4. The application according to claim 2, characterized in that: The ultraviolet light wavelength is 365nm, and the power is 6.5-7.0mW / cm². 2 .

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