Zinc-doped egcg carbon dots and preparation method and application thereof

Abstract

The application provides a zinc-doped EGCG carbon dot, which is a nanoparticle prepared by a hydrothermal carbonization method with epigallocatechin gallate as a carbon source and zinc ions as a doping element. The application also provides a zinc-doped EGCG carbon dot, which comprises the following steps: adding EGCG and zinc salt into ultrapure water according to a mass ratio of (0.2-1):(0.05-0.5) and uniformly ultrasonic dispersing; transferring the mixed solution into a reaction kettle and performing hydrothermal reaction at 160-220 DEG C for 2-6 hours; and after the reaction is completed, performing purification treatment on the product to obtain the zinc-doped EGCG carbon dot. Based on the zinc-doped EGCG carbon dot, the application also provides an application of the zinc-doped EGCG carbon dot in preparation of an antibacterial agent or a food preservative. The zinc-doped EGCG carbon dot provided by the application has significantly enhanced broad-spectrum antibacterial and antifungal activities, excellent biocompatibility and fruit and vegetable preservation effects.

Description

Technical Field

[0001] This invention relates to the field of antibacterial nanomaterials technology, specifically to a zinc-doped EGCG carbon dot, its preparation method, and its application. Background Technology

[0002] Food spoilage is one of the major challenges facing the world today, causing not only huge economic losses but also seriously threatening public health. The growth and reproduction of microorganisms (such as bacteria and fungi) is the main cause of food spoilage. Therefore, developing efficient and safe antimicrobial materials for food preservation is of great significance.

[0003] Epigallocatechin gallate (EGCG) is the most abundant natural catechin in tea, possessing various biological activities such as antioxidant, anti-inflammatory, and antibacterial properties. However, EGCG has significant limitations in practical applications: its chemical structure is highly reactive, easily degraded by light, oxygen, and temperature, and its water solubility and stability are poor, limiting its effectiveness in food preservation.

[0004] In recent years, carbon dots (CDs), as a novel type of nanomaterial, have attracted widespread attention in the fields of antibacterial and food preservation due to their good biocompatibility, tunable fluorescence properties, and surface functionalization potential. However, the improvement in antibacterial activity of carbon dots prepared using EGCG as a single precursor is currently limited, making it difficult to meet the needs of efficient food preservation.

[0005] Therefore, developing a novel functionalized carbon dot based on EGCG to improve its antibacterial properties and stability is of great practical significance. Summary of the Invention

[0006] This invention aims to overcome the shortcomings of existing technologies and provide a zinc-doped EGCG carbon dot (EGCG-Zn²⁺-CDs), its preparation method, and its applications. This invention utilizes a one-step hydrothermal method to carbonize EGCG with zinc ions. The resulting carbon dots exhibit uniform particle size, good dispersibility, abundant surface functional groups, and excellent fluorescence properties. Furthermore, they demonstrate significantly enhanced broad-spectrum antibacterial and antifungal activity, as well as excellent biocompatibility and fruit and vegetable preservation effects.

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

[0008] The first aspect of the present invention is to provide a zinc-doped EGCG carbon dot, wherein the carbon dot is a nanoparticle prepared by hydrothermal carbonization using epigallocatechin gallate as the carbon source and zinc ions as the dopant element.

[0009] Furthermore, the zinc-doped EGCG carbon dots have a particle size of 1-3 nm and contain hydroxyl and carboxyl oxygen-containing functional groups on their surface. Zinc ions are bound to the carbon dot surface through coordination.

[0010] Furthermore, the zinc-doped EGCG carbon dots have a graphite-like structure with a lattice spacing of 0.20 nm;

[0011] The zinc-doped EGCG carbon dots exhibit visible light emission characteristics at an excitation wavelength of 322 nm and have an ultraviolet absorption peak at 268 nm.

[0012] A second aspect of the present invention is to provide a method for preparing zinc-doped EGCG carbon dots as described in the first aspect, comprising the following steps:

[0013] Step S1: Add EGCG and zinc salt to ultrapure water at a mass ratio of (0.2~1):(0.05~0.5) and disperse evenly by ultrasonication;

[0014] Step S2: Transfer the mixed solution to a reaction vessel and hydrothermally react at 160~220℃ for 2~6 hours;

[0015] Step S3: After the reaction is complete, the product is purified to obtain the zinc-doped EGCG carbon dots.

[0016] Further, in step S3, the purification process includes centrifugation, filtration, dialysis, and drying; wherein the centrifugation speed is 4000-6000 rpm, the filtration uses a 0.22 μm microporous membrane, the dialysis uses a dialysis bag with a molecular weight cutoff of 0.5 kDa, and the dialysis time is 20-28 hours.

[0017] A third aspect of the invention is to provide the use of the zinc-doped EGCG carbon dots described in the first aspect in the preparation of antibacterial agents.

[0018] Furthermore, the antibacterial agent is used to inhibit at least one of Gram-positive bacteria, Gram-negative bacteria, and fungi.

[0019] A fourth aspect of the present invention is to provide the application of the zinc-doped EGCG carbon dots described in the first aspect in the preparation of a fruit and vegetable preservative for postharvest preservation of fruits and vegetables.

[0020] Furthermore, the fruit and vegetable preservative is an aqueous solution of zinc-doped EGCG carbon dots, with a concentration of 100~200 μg / mL.

[0021] Furthermore, the fruit and vegetable preservative is applied to the surface of fruits and vegetables by spraying or soaking.

[0022] Compared with the prior art, the zinc-doped EGCG carbon dots, their preparation method, and applications provided by this invention have the following advantages:

[0023] I. The zinc-doped EGCG carbon dots provided by this invention use natural polyphenol EGCG as the carbon source and introduce Zn. 2+ EGCG-Zn with antibacterial activity was successfully prepared by a one-step hydrothermal method. 2+ -CDs. Characterization results show that EGCG-Zn 2+ -CDs have uniform particle size and good dispersibility, and their surface is rich in oxygen functional groups such as hydroxyl and carboxyl groups, which stably bind Zn through coordination. 2+ Through a synergistic strategy of zinc doping and carbonization, the prepared EGCG-Zn²⁺-CDs achieved a minimum bactericidal concentration (MBC) of 50 μg / mL against Staphylococcus aureus and Escherichia coli, which is 10-15 times higher than that of undoped EGCG carbon dots (EGCG-CDs) and 100 times higher than that of a simple mixture of EGCG and zinc acetate, demonstrating broad-spectrum and highly efficient bactericidal capabilities.

[0024] II. The zinc-doped EGCG carbon dots provided by this invention can achieve multi-target inhibition by disrupting bacterial cell structure and interfering with the expression of key functional genes such as DNA replication, biofilm formation, and energy metabolism; simultaneously, they can exert multi-target antibacterial effects by interfering with the expression of genes related to amino acid synthesis and sugar metabolism. In practical applications, using zinc-doped EGCG carbon dots for post-harvest preservation of fruits and vegetables can effectively delay the fruit spoilage process, reduce weight loss, and maintain good appearance quality, demonstrating clear prospects for industrial application. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The UV-Vis absorption spectrum of EGCG-Zn²⁺-CDs prepared in Example 1 of this invention.

[0027] Figure 2 The fluorescence spectrum of EGCG-Zn²⁺-CDs prepared in Example 1 of this invention is shown; the inset shows the appearance of the solution under natural light (left) and 365 nm ultraviolet light (right).

[0028] Figure 3 This is a comparison of the Fourier transform infrared (FTIR) spectra of EGCG-Zn²⁺-CDs prepared in Example 1 of this invention with EGCG and zinc acetate.

[0029] Figure 4High-resolution transmission electron microscopy (HRTEM) image (A), transmission electron microscopy (TEM) image (B), and particle size distribution diagram (C) of EGCG-Zn²⁺-CDs prepared in Example 1 of this invention.

[0030] Figure 5 XPS full spectrum (A) and high-resolution C1s (B), O1s (C), and Zn2p (D) spectra of EGCG-Zn²⁺-CDs prepared in Example 1 of this invention.

[0031] Figure 6 This is a comparison of the diameters of the inhibition zones of different concentrations of EGCG-Zn²⁺-CDs, EGCG-CDs, and raw materials against Staphylococcus aureus and Escherichia coli.

[0032] Figure 7 The growth curves of Staphylococcus aureus (A) and Escherichia coli (B) after treatment with different concentrations of EGCG-Zn²⁺-CDs according to the present invention are shown.

[0033] Figure 8 The following are bar charts showing the bacterial survival rates after co-culturing different concentrations of EGCG-Zn²⁺-CDs with Staphylococcus aureus (A) and Escherichia coli (B) for 24 hours: bar charts showing the bacterial survival rates after co-culturing different concentrations of EGCG and zinc acetate mixtures with S. aureus (C) and E. coli (D) for 24 hours: bar charts showing the bacterial survival rates after co-culturing different concentrations of EGCG-CDs with S. aureus (E) and E. coli (F) for 24 hours: bar charts showing the bacterial survival rates after co-culturing different concentrations of EGCG-CDs with S. aureus (E) and E. coli (F).

[0034] Figure 9 These are photographs of Staphylococcus aureus and Escherichia coli colonies on LB agar plates after treatment with different concentrations of EGCG-Zn²⁺-CDs in this invention.

[0035] Figure 10 The antibacterial mechanism of EGCG-Zn²⁺-CDs in this invention is as follows: Figure 10 A represents scanning electron microscope (SEM) images of Staphylococcus aureus before and after EGCG-Zn²⁺-CDs treatment. Figure 10 B represents scanning electron microscope (SEM) images of E. coli before and after EGCG-Zn²⁺-CDs treatment. Figure 10 C indicates the quantitative analysis of relative gene expression in Staphylococcus aureus after treatment with EGCG-Zn²⁺-CDs. Figure 10 D indicates the quantitative analysis of the relative gene expression levels in Escherichia coli after treatment with EGCG-Zn²⁺-CDs.

[0036] Figure 11The images (A) and (B) show the inhibition of colony growth of Penicillium digitatum by different concentrations of EGCG-Zn²⁺-CDs in this invention, as well as the colony diameter measurement curve.

[0037] Figure 12 The images show heatmap (A) and volcano plot (B) showing the effects of EGCG-Zn²⁺-CDs treatment on the transcriptome of Penicillium fingerling in this invention.

[0038] Figure 13 The images show the GO functional enrichment analysis (A) and KEGG pathway enrichment analysis (B) of EGCG-Zn²⁺-CDs treatment on Penicillium digitatum in this invention.

[0039] Figure 14 The results of cytotoxicity experiments on AmI-12 normal hepatocytes (A) and HepG2 liver cancer cells (B) at different concentrations of EGCG-Zn²⁺-CDs in this invention are shown.

[0040] Figure 15 The images show the appearance changes (A), weight loss rate changes (B), and rot rate changes (C) of citrus fruits treated with different concentrations of EGCG-Zn²⁺-CDs and untreated fruits during 28 days of storage. Detailed Implementation

[0041] To enable those skilled in the art to better understand the technical solutions in the embodiments of the present invention, and to make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be further described below.

[0042] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0043] Example 1: Preparation of Zinc-Doped EGCG Carbon Dots (EGCG-Zn²⁺-CDs)

[0044] Weigh 0.2 g EGCG (analytical grade, Shanghai Yuanye Biotechnology Co., Ltd.) and 0.1 g zinc acetate (analytical grade, Tianjin Zhonglian Chemical Reagent Co., Ltd.), add to 20 mL ultrapure water, and sonicate for 5 minutes to ensure thorough dispersion. Transfer the mixture to a 50 mL stainless steel reactor lined with polytetrafluoroethylene (PTFE), and react at 200 °C for 4 hours to complete carbonization. After the reaction, allow it to cool naturally to room temperature. Centrifuge the product at 5000 rpm for 10 minutes to remove large particulate impurities, and then filter through a 0.22 μm microporous membrane. Place the filtrate in a dialysis bag with a molecular weight cutoff of 0.5 kDa and dialyze with ultrapure water for 24 hours (changing the water every 8 hours) to remove unreacted precursors and small molecule impurities. Finally, freeze-dry the purified solution to obtain brown EGCG-Zn²⁺-CDs powder, and store at 4 °C protected from light.

[0045] Example 2 Characterization of EGCG-Zn²⁺-CDs

[0046] The EGCG-Zn²⁺-CDs prepared in Example 1 were characterized, and the results are as follows:

[0047] UV-Vis absorption spectra, such as Figure 1 As shown, a distinct absorption peak appears at 268 nm, corresponding to the π→π* transition in the aromatic structure. The fluorescence spectrum is as follows. Figure 2 As shown, it exhibits strong visible light emission under 322 nm excitation, and the solution shows blue fluorescence under ultraviolet light.

[0048] FTIR spectra such as Figure 3 As shown, compared with EGCG, EGCG-Zn²⁺-CDs still retain a broad OH stretching vibration peak at 3200-3500 cm⁻¹, indicating that its surface is still rich in hydroxyl groups. However, the peak shape shows a certain degree of broadening and shift compared with EGCG, indicating that hydroxyl groups are involved in hydrogen bond reconstruction or metal coordination processes. Meanwhile, the obvious aromatic ring-related characteristic peaks in EGCG are present in EGCG-Zn²⁺-CDs. 2 + The significant reduction in -CDs indicates that the aromatic structure underwent partial carbonization and reconstruction during the hydrothermal process. EGCG-Zn 2+ -CDs at approximately 1700cm -1 The C=O absorption peak at 1200-1400 cm⁻¹ and the absorption peak at 1200-1400 cm⁻¹ -1 The CO and COC vibrational peaks within the region are significantly enhanced and shifted, which is generally considered to be an important characteristic signal of carboxyl groups participating in metal coordination.

[0049] Transmission electron microscopy (TEM / HRTEM) such as Figure 4 As shown, by Figure 4It can be seen that EGCG-Zn²⁺-CDs exhibit a uniformly dispersed near-spherical morphology with a particle size of 1-3 nm. HRTEM shows a lattice spacing of 0.20 nm, indicating that it has a certain graphite-like structure.

[0050] XPS full spectrum as follows Figure 5 As shown, by Figure 5 It can be seen that EGCG-Zn 2+ -CDs are mainly composed of C and O elements; characteristic peaks such as CC / C=C, CO, C=O, and OC=O can be decomposed in the high-resolution C1s and O1s spectra, indicating that EGCG-Zn 2+ -CDs are rich in various oxygen-containing functional groups on their surface. These functional groups not only enhance EGCG-Zn 2+ The water dispersibility of CDs also provides potential active sites for their interfacial interactions with microorganisms. 2+ Mainly through hydroxyl and carboxyl groups with EGCG-Zn 2+ -CDs surface coordination achieves stable anchoring. This surface coordination method maintains EGCG-Zn 2+ -CDs possess water solubility and structural stability, and may enhance their interaction with microbial membranes, providing a basis for improved antimicrobial properties.

[0051] Example 3 Antibacterial activity and antibacterial mechanism

[0052] 1. Zone of inhibition test: E. coli and S. aureus were diluted to 1×10⁻⁶. 8 After reaching CFU / mL, the solution was evenly spread on the surface of the solid culture medium and allowed to stand for approximately 5 minutes. Then, under aseptic conditions, Oxford cups were placed, and 100 µL of EGCG-Zn at different concentrations was added to each cup. 2+ -CDs solutions (2000, 1500, 1000, 500, 200, 150, 100, 50, 0 µg / mL), with sterile water as a control. After incubation at 37℃ for 24 h, the diameter of the inhibition zone was measured to evaluate the EGCG-Zn... 2+ The antibacterial effect of CDs was quantitatively evaluated.

[0053] Please see Figure 6 EGCG-Zn 2+ -CDs exhibited a clear concentration-dependent antibacterial activity, with the inhibitory effect on *S. aureus* and *E. coli* gradually increasing with increasing treatment concentration. In contrast, undoped Zn... 2+While EGCG-CDs also exhibit a certain concentration dependence, their antibacterial effect is mainly concentrated in the higher concentration range, with little inhibitory effect at low concentrations, reflecting their relatively limited antibacterial efficiency. Neither EGCG nor zinc acetate showed significant antibacterial activity when used alone, and the antibacterial effect of their mixed solution was also limited, showing no obvious synergistic enhancement. However, Zn... 2+ EGCG-Zn constructed by doping 2+ -CDs exhibit stronger antibacterial activity under the same conditions, indicating that the introduction of metal ions can enhance the function of CDs at the structural level. This enhancement effect is more likely due to the regulation of the surface chemical properties and interfacial interactions of CDs by the doping process.

[0054] 2. EGCG-Zn 2+ Bacterial growth curve under the action of CDs

[0055] Dilute E. coli and S. aureus to 1×10⁻⁶ 8 After setting the CFU / mL concentration, 50 μL of LLB broth and 100 μL of EGCG-Zn at different concentrations were added sequentially to the 96-well plate. 2+ CDs solution (final concentrations of 100, 75, 50, 25, 10, 7.5, 5, and 0 μg / mL) was inoculated with 50 μL of bacterial suspension, using sterile water as a blank control. The mixture was incubated at 37°C for 24 hours, with OD measured every 2 hours. 600 Plot bacterial growth curves, such as Figure 7 As shown.

[0056] Depend on Figure 7 This indicates that the addition of EGCG-Zn 2+ Following CDs, the growth of both bacteria was significantly inhibited, and the inhibitory effect increased with increasing concentration. At low concentrations (5–10 µg / mL), the bacteria could still grow slowly, but the growth rate was significantly lower than the control group; when the concentration reached 25 µg / mL, OD... 600 The growth rate was significantly reduced, and bacterial growth was almost completely inhibited at high concentrations (50–100 µg / mL). In contrast, *S. aureus* showed significantly reduced growth at EGCG-Zn concentrations. 2+ -CDs are slightly more sensitive than E. coli.

[0057] 3. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC):

[0058] Determination of bacterial MIC: E. coli and S. aureus were diluted with LB broth to 1×10⁻⁶. 8CFU / mL. Subsequently, 50 µL of LB medium and 100 µL of EGCG-Zn at different concentrations were added sequentially to the 96-well plate. 2+ -CDs solution (final concentrations of 100, 75, 50, 25, 10, 7.5, 5, and 0 µg / mL), followed by 50 µL of bacterial suspension. Sterile water was used as a blank control. After incubating the culture plates at 37°C for 24 h, OD was measured. 600 Determination of EGCG-Zn 2+ -CDs' antibacterial activity.

[0059] Determination of bacterial MBC: After completing the MIC experiment, based on the results, take 1 mL of EGCG-Zn 2+ -CDs were mixed with 1 mL of E. coli LB broth to prepare mixtures with final concentrations of 50, 25, 12.5, and 0 µg / mL. 1 mL of EGCG-Zn was then added. 2+ -CDs were mixed with 1 mL of LB broth containing S. aureus to prepare final concentrations of 50, 25, 12.5, and 0 µg / mL. After standing for 2 h, 100 µL of the supernatant was inoculated onto fresh LB solid medium plates. The plates were incubated at 37 °C for 24 h, and colony formation was observed.

[0060] Please refer to the following: Figure 8 and Figure 9 ,Depend on Figure 8 It can be seen that the MBC of EGCG-Zn²⁺-CDs against S. aureus and E. coli is 50 μg / mL, which is 100 times higher than that of EGCG + zinc acetate mixture (MBC=5000 μg / mL), and 10 times and 15 times higher than that of EGCG-CDs (MBC=500 μg / mL for S. aureus and MBC=750 μg / mL for E. coli), respectively.

[0061] Depend on Figure 9 It can be seen that EGCG-Zn 2+ The results of EGCG-Zn mixed plating with *S. aureus* and *E. coli* showed that its antibacterial activity was significantly dose-dependent. Compared with the control group, colony formation of both bacteria was inhibited at 12.5 µg / mL, and the number of colonies was significantly reduced when the concentration was increased to 25 µg / mL. Further increasing the concentration to 50 µg / mL resulted in almost no visible colony formation of *S. aureus* and *E. coli*, demonstrating good bactericidal effect. The results show that EGCG-Zn... 2+-CDs showed a MBC of 50 µg / mL for both *S. aureus* and *E. coli*. Compared to *E. coli*, *S. aureus* showed a lower MBC at medium to low concentrations for EGCG-Zn. 2+ -CDs exhibit higher sensitivity, which may be related to differences in their cell wall structure. The above results directly validate the efficacy of EGCG-Zn. 2 + -CDs have broad-spectrum antibacterial potential against both Gram-positive and Gram-negative bacteria.

[0062] 4. Mechanism of antibacterial action

[0063] 2 mL of 10 µg / mL EGCG-Zn 2+ -CDs were added to 4 mL of 1×10 8 The experimental group was treated with LB broth solution containing CFU / mL of S. aureus and E. coli, while the control group was treated with 2 mL of sterile water added to 4 mL of 1×10⁻⁶ CFU / mL broth. 8 CFU / mL LB broth solutions of *S. aureus* and *E. coli* were cultured for 24 h. After centrifugation, the bacteria were washed three times with sterile water and fixed in 2.5% (*E. coli*) and 4% (*S. aureus*) glutaraldehyde, respectively, overnight at 4°C. After centrifugation to remove glutaraldehyde, the samples were washed three times (15 min each) with 0.1 M pH 7.0 PBS, followed by a gradient dehydration with 30%, 50%, 70%, 80%, 90%, and 95% ethanol, and then treated twice with 100% ethanol (20 min each). After dehydration, the samples were stored in 100% ethanol and dried using a critical point desiccator. Finally, conductive carbon adhesive was used to fix the samples on the stage for scanning electron microscopy. (See also...) Figure 10 A and Figure 10 B, via EGCG-Zn 2+ After treatment with CDs, the bacteria exhibited rough surfaces, collapse, and rupture, with their cell structure severely damaged.

[0064] RT-qPCR analysis was performed on E. coli and S. aureus. OD 600 =0.6 bacterial suspension with 7.5 μg / mL EGCG-Zn 2+CDs were collected after 24 h of co-culture for RNA extraction. RT-qPCR was performed using NovoStart® Fast SYBR qPCRSuperMix, with specific primers targeting genes related to cell wall biosynthesis (e.g., waaC, waaF), cell division (ftsA, ftsZ), and DNA replication (dnaE, polA). rpoA and WaaA were used as internal control genes for *S. aureus* and *E. coli*, respectively, and relative gene expression levels were calculated using the 2^-ΔΔCt method. Please refer to [link to relevant documentation]. Figure 10 C and Figure 10 Treatment with D, EGCG-Zn²⁺-CDs significantly downregulated genes related to DNA replication (dnaT), biofilm formation (icaA, asrA), and cell division (fisA, polA, dnaE) in *S. aureus*; and significantly downregulated genes related to outer membrane structure (waaF), cell division (ftsZ), and transcriptional regulation (rpoA, rpoB) in *E. coli*, confirming multi-target antibacterial activity.

[0065] Example 5 Antifungal activity test

[0066] In a sterile operating table, sterile PDA solid medium was used to prepare medium at concentrations of 600, 400, 200, 150, 100, and 50 µg / mL before solidification. The medium was then poured into sterile petri dishes and allowed to cool and solidify. A blank control group was also included. 10 µL of 1×10⁻⁶ PDA solid medium was used. 8 A CFU / mL spore suspension was placed in the center of the culture medium and incubated at 28°C for 6 days. Growth was observed daily, and colony diameter was measured. Figure 11 As shown.

[0067] Depend on Figure 11 It can be seen that in EGCG-Zn 2+ In the CDs-treated groups, colony growth was significantly inhibited, and the inhibitory effect increased with increasing concentration. In the low-concentration (50–100 µg / mL) treatment groups, colonies could still gradually expand, but the growth rate was significantly slower than that of the control group; in the medium-concentration (150–200 µg / mL) treatment groups, colony expansion was further restricted, and the diameter growth was slow; in the high-concentration (400–600 µg / mL) treatment groups, significant antibacterial effects were observed, with the 600 µg / mL treatment almost completely stopping colony growth.

[0068] Transcriptome sequencing of P. digitatum yielded the following results: Figure 12 , Figure 13 As shown. By Figure 12 , Figure 13It can be seen that the gene expression profiles of the treatment group and the control group are significantly different. The differentially expressed genes are enriched in DNA binding, transcriptional regulation, nucleic acid metabolism, ribosome production, amino acid synthesis, sugar metabolism and MAPK signaling pathway, which confirms that it achieves multi-target antifungal effects by interfering with genetic information processing, metabolism and stress regulation networks.

[0069] Example 6 Cytotoxicity Test

[0070] Cell viability was assessed using the CCK-8 assay. Once cells reached the logarithmic growth phase, they were digested with trypsin to disperse them into a single-cell suspension. Cell counts were performed on 100 µL suspensions under a microscope using a hemocytometer. After counting, cell suspensions were prepared using complete culture medium and diluted 2 × 10⁻⁶ cells / mL. 5 Seed 200 µL of cell culture into 96-well plates at a density of cells / mL. Ensure thorough mixing during seeding and add PBS to the edge of the wells to reduce edge effects. Add EGCG-Zn. 2+ After treatment with CDs, cells were cultured at 37°C and 5% CO2 humidification for 12 h, and then 10 µL of CCK-8 reagent (5 mg / mL) was added to each well. Finally, the absorbance was measured at 450 nm using a microplate reader, and cell viability was calculated according to the formula.

[0071] like Figure 14 As shown, EGCG-Zn 2+ -CDs showed good overall biocompatibility with Aml-12 normal hepatocytes and HepG2 hepatocellular carcinoma cells. With EGCG-Zn 2+ Increasing the CDs concentration from 100 µg / mL to 600 µg / mL maintained high cell viability for both cell types (over 90%), with no significant difference compared to the control group, indicating that low to moderate concentrations had minimal impact on cell viability. When the concentration was further increased to 800 µg / mL, both Aml-12 and HepG2 cell viability decreased to some extent, with significant differences compared to the low-concentration group, but overall remained above 85%, showing no significant cytotoxicity. These results indicate that EGCG-Zn 2+ -CDs exhibit good cellular safety over a wide concentration range, especially at concentrations ≤600 µg / mL, demonstrating good biocompatibility with both normal and tumor cells, providing experimental evidence for their safe use in food and biological applications.

[0072] Example 7 EGCG-Zn 2+ Application of CDs in Citrus Preservation

[0073] The citrus fruits used in the experiment were purchased from a local farm and used directly without any pretreatment. They were washed with distilled water and then air-dried at room temperature (approximately 25°C). Subsequently, 100 µg / mL and 200 µg / mL of EGCG-Zn were applied to the fruits, respectively. 2+ -CDs solution was evenly sprayed onto the surface of the citrus fruits, allowing them to be completely covered before air drying; the control group was treated with distilled water in the same way. All samples were stored at room temperature (25°C, 60% relative humidity) for 28 days, with three parallel groups of five citrus fruits per group. During storage, the appearance of the samples was recorded daily, and their spoilage and weight changes were assessed. The experimental results are as follows: Figure 15 As shown.

[0074] Depend on Figure 15 As can be seen from A, during the 28-day storage process, different concentrations of EGCG-Zn 2+ -CDs treatment showed significant differences in the storage quality of citrus fruits. Regarding appearance, the control group citrus fruits developed mold and rot on day 16 of storage, with the deterioration accelerating significantly after day 20; in contrast, 100 μg / mL EGCG-Zn... 2+ - The CDs treatment group showed a significantly slower rate of appearance deterioration, with noticeable rotting only appearing on the 24th day; while the 200 μg / mL treatment group maintained a relatively bright color throughout the entire storage period, with the lowest degree of mold and rotting.

[0075] Depend on Figure 15 As shown in B, the control group had the highest weight loss rate, which increased rapidly in the later stages of storage, while the EGCG-Zn at 100 μg / mL and 200 μg / mL... 2+ The weight loss rate of all CDs-treated groups was significantly reduced, and the weight loss rate gradually decreased with increasing treatment concentration. Among them, the 200 μg / mL treatment group maintained the lowest weight loss rate throughout the entire storage process, indicating that it can effectively reduce fruit moisture loss.

[0076] Depend on Figure 15 C indicates that the decay rate in the control group increased rapidly around day 16, while the decay in the 100 μg / mL treatment group was significantly delayed and the rate of increase was relatively slow. In contrast, the 200 μg / mL treatment group showed almost no decay in the first 20 days of storage, with only a small amount of decay occurring in the later stages of storage. This suggests that higher concentrations of EGCG-Zn... 2+ -CDs can effectively inhibit mold growth in citrus fruits.

[0077] Therefore, the EGCG-Zn of the present invention 2+-CDs can significantly improve the post-harvest storage quality of citrus fruits by delaying fruit appearance deterioration, reducing moisture loss, and inhibiting mold growth. Furthermore, their preservation effect increases with increasing treatment concentration, providing a new technical approach and application prospect for post-harvest preservation of citrus fruits. In addition, EGCG-Zn... 2+ -CDs can also be used for antibacterial and preservation purposes on other foods, such as strawberries, grapes, or other fruits and vegetables.

[0078] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations made to these embodiments without departing from the principles and spirit of the present invention still fall within the protection scope of the present invention.

Claims

1. A zinc-doped EGCG carbon dot, characterized in that, The carbon dots are nanoparticles prepared by hydrothermal carbonization using epigallocatechin gallate as the carbon source and zinc ions as the dopant element.

2. The zinc-doped EGCG carbon dots according to claim 1, characterized in that, The zinc-doped EGCG carbon dots have a particle size of 1-3 nm and contain hydroxyl and carboxyl oxygen-containing functional groups on their surface. Zinc ions are bound to the carbon dot surface through coordination.

3. The zinc-doped EGCG carbon dots according to claim 1, characterized in that, The zinc-doped EGCG carbon dots have a graphite-like structure with a lattice spacing of 0.20 nm. The zinc-doped EGCG carbon dots exhibit visible light emission characteristics at an excitation wavelength of 322 nm and have an ultraviolet absorption peak at 268 nm.

4. A method for preparing zinc-doped EGCG carbon dots according to any one of claims 1-3, characterized in that, Includes the following steps: Step S1: Add EGCG and zinc salt to ultrapure water at a mass ratio of (0.2~1):(0.05~0.5) and disperse evenly by ultrasonication; Step S2: Transfer the mixed solution to a reaction vessel and hydrothermally react at 160~220℃ for 2~6 hours; Step S3: After the reaction is complete, the product is purified to obtain the zinc-doped EGCG carbon dots.

5. The method for preparing zinc-doped EGCG carbon dots according to claim 4, characterized in that, In step S3, the purification process includes centrifugation, filtration, dialysis, and drying; wherein the centrifugation speed is 4000-6000 rpm, the filtration uses a 0.22 μm microporous membrane, the dialysis uses a dialysis bag with a molecular weight cutoff of 0.5 kDa, and the dialysis time is 20-28 hours.

6. The use of zinc-doped EGCG carbon dots as described in any one of claims 1-3 in the preparation of antibacterial agents.

7. The application according to claim 6, characterized in that, The antibacterial agent is used to inhibit at least one of Gram-positive bacteria, Gram-negative bacteria, and fungi.

8. The application of zinc-doped EGCG carbon dots as described in any one of claims 1-3 in the preparation of fruit and vegetable preservatives, characterized in that, The fruit and vegetable preservative is used for post-harvest preservation of fruits and vegetables.

9. The application according to claim 8, characterized in that, The fruit and vegetable preservative is an aqueous solution of zinc-doped EGCG carbon dots, and its concentration is 100~200 μg / mL.

10. The application according to claim 8, characterized in that, The fruit and vegetable preservative is applied to the surface of fruits and vegetables by spraying or soaking.

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