Preparation method and application of EGCG (epigallocatechin gallate)-mediated hydrogel

Abstract

The invention relates to the technical field of hydrogel, in particular to a preparation method and application of EGCG-mediated hydrogel, a lysozyme solution is heated under the acidic condition to form lysozyme amyloid fibrils LAFs, EGCG is dissolved in a buffer solution and mixed with the LAFs solution obtained in the step 1 in proportion to form an LAFs-EGCG compound, and the EGCG-mediated hydrogel is prepared. The chitosan CS is dissolved in an acetic acid buffer solution, glutaraldehyde is added to serve as a cross-linking agent, the mixture is mixed with the LAFs-EGCG compound obtained in the step 2 and stirred, the composite hydrogel is formed, the concentration of EGCG is 0.2-1.0 wt%, the concentration of LAFs is 5 wt%, the concentration of chitosan is 2 wt%, the pH value of the lysozyme solution in the step 1 is 2.0, the heating temperature is 77 DEG C, the heating time is 10 hours, the concentration of EGCG in the step 2 is 0.4-0.8 wt%, and the pH value of the lysozyme solution in the step 2 is 1.5-2.5. In the step 3, the concentration of glutaraldehyde is 2%, the storage modulus G'of the hydrogel is 325-480 Pa, the hardness is 2176.71 g, and the bacteriostasis rate on staphylococcus aureus and salmonella exceeds 95%.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogels, and in particular to a method for preparing an EGCG-mediated hydrogel and application thereof. Background Art

[0002] As we all know, lysozyme (LYZ), as a natural antibacterial protein, has attracted much attention in the fields of food preservation and biomedicine due to its specific lysis of Gram-positive bacteria. However, its monomer form has defects such as narrow antibacterial spectrum, weak effect on Gram-negative bacteria, poor thermal stability, and easy aggregation and inactivation, which seriously limit its practical application. In recent years, regulating protein misfolding to form amyloid fibrils (AFs) has become a new strategy to enhance protein function. For example, Wei et al. (2021) found that the β-folded structure of lysozyme amyloid fibrils (LAFs) can significantly enhance the antibacterial activity against Staphylococcus aureus and Escherichia coli, but its preparation relies on electrostatic cross-linking such as cellulose nanocrystal composites, which easily leads to uneven fiber entanglement and loose network structure (De France et al., 2020), and the mechanical strength of single LAFs-based materials is still difficult to meet the needs of complex applications.

[0003] Chitosan (CS) is often used to construct hydrogel systems due to its biocompatibility, degradability and inherent antibacterial properties, but the gel formed by cross-linking alone has weak mechanical properties, the storage modulus is usually less than 200 Pa, and the water retention is poor. In the existing technology, the performance is mostly enhanced by introducing inorganic nanoparticles or chemical cross-linking agents such as glutaraldehyde, but the former may cause biological toxicity, and the latter is prone to excessive cross-linking and destroy the function of active ingredients such as proteins Xue et al., 2022.

[0004] Epigallocatechin Gallate (EGCG), as a representative of green tea polyphenols, can regulate protein conformation and enhance material function through non-covalent interactions such as hydrogen bonding and π-π stacking. Zhang et al. 2024 confirmed that EGCG can induce pea protein nanofibers to form high-strength hydrogels, but its mechanism of action in the rigid LAFs system is not yet clear. In addition, existing studies have mostly focused on the interaction between EGCG and single biomacromolecules such as proteins or polysaccharides, while there is a lack of research on the synergistic effect of polyphenol-protein-polysaccharide three-phase composite systems, especially the systematic regulation of EGCG concentration gradient on gel network reconstruction, antibacterial spectrum expansion and biocompatibility. The existing protein-polysaccharide composite gel has insufficient mechanical properties, a single antibacterial spectrum and insufficient biocompatibility verification. Therefore, it is necessary to propose a solution to this technical problem. Summary of the invention

[0005] (1) Technical problems to be solved In view of the deficiencies of the prior art, the present invention provides a method for preparing EGCG-mediated hydrogel and its applications.

[0006] (2) Technical solutions To achieve the above object, the present invention provides the following technical solutions: A method for preparing EGCG-mediated hydrogel, comprising the following steps: Step 1: Heat-treat the lysozyme solution under acidic conditions to form lysozyme amyloid fibrils LAFs; Step 2: Dissolve epigallocatechin gallate EGCG in a buffer solution, and mix it with the LAFs solution obtained in Step 1 in a certain proportion to form a LAFs-EGCG complex; Step 3: Dissolve chitosan CS in an acetic acid buffer solution, add glutaraldehyde as a crosslinking agent, mix it with the LAFs-EGCG complex in Step 2, and stir to form a composite hydrogel; Among them, the concentration of EGCG is 0.2-1.0 wt%, the concentration of LAFs is 5 wt%, and the concentration of chitosan is 2 wt%.

[0007] Further, the present invention is improved in that the pH of the lysozyme solution in Step 1 is 2.0, the heating temperature is 77 °C, and the heating time is 10 hours.

[0008] Further, the present invention is improved in that the concentration of EGCG in Step 2 is 0.4-0.8 wt%.

[0009] Further, the present invention is improved in that the concentration of glutaraldehyde in Step 3 is 2%.

[0010] Further, the present invention is improved in that the storage modulus G' of the hydrogel is 325-480 Pa, the hardness is 2176.71 g, and the antibacterial rates against Staphylococcus aureus and Salmonella exceed 95%.

[0011] Further, the present invention is improved in that the free water ratio T23 of the hydrogel is 5.467%-30.581%, and the content of β-sheet structure is 14.59%-29.41%.

[0012] Further, the present invention is improved in that the microstructure of the hydrogel is a uniformly distributed fiber network, the average particle size is 50-200 nm, and the Zeta potential is -10 to +5 mV.

[0013] Further, the present invention is improved in that the biocompatibility of the hydrogel is manifested as a cell survival rate ≥100%, and no dead cells are detected under a confocal microscope.

[0014] Furthermore, the present invention also provides a method for preparing EGCG-mediated hydrogel and its applications. The hydrogel is used for food preservation, medical dressings or antibacterial packaging materials.

[0015] Furthermore, the present invention is improved in that the antibacterial packaging material has a lethality rate of 89.4% against Gram-negative bacteria.

[0016] (III) Beneficial effects Compared with the prior art, the present invention provides a method for preparing EGCG-mediated hydrogel and its applications, which have the following beneficial effects: In the method for preparing EGCG-mediated hydrogel and its applications, by combining lysozyme amyloid fibrils (LAFs) with epigallocatechin gallate (EGCG), and further cross-linking with chitosan (CS) to form a composite hydrogel, its antibacterial performance is significantly improved. Specifically, the antibacterial rate of this hydrogel against Staphylococcus aureus and Salmonella exceeds 95%, and the inhibitory activity against Gram-negative bacteria such as Escherichia coli is also significantly enhanced. This is mainly due to the combination of the inherent antibacterial properties of EGCG and lysozyme fibrils, which not only enhances the antibacterial effect of the original lysozyme but also broadens its antibacterial spectrum, enabling it to effectively combat various types of bacteria. In addition, EGCG can also disrupt the formation of bacterial biofilms and promote the generation of high concentrations of reactive oxygen species, leading to bacterial death.

[0017] This composite hydrogel exhibits superior mechanical strength, with a storage modulus G' of 325 - 480 Pa and a hardness of 2176.71 g, far exceeding the mechanical properties of traditional single chitosan hydrogels. This enhancement is mainly attributed to the hydrogen bond network and π-π interactions formed between EGCG and LAFs. These non-covalent interactions not only enhance the intermolecular connection force but also promote the formation of a three-dimensional network structure, enabling the hydrogel to exhibit better elastic recovery ability when subjected to external forces. In particular, with the change of the EGCG concentration, the critical strain value also changes, indicating that low concentrations of EGCG weaken the LAFs-CS network, while high concentrations of EGCG further enhance the network stability through covalent cross-linking between glutaraldehyde and the amino groups of chitosan.

[0018] By finely regulating the concentration of EGCG, the microstructure of the hydrogel can be optimized to present a uniformly distributed fiber network with an average particle size of 50 - 200 nm. This microstructure helps to improve the overall stability and dispersibility of the hydrogel. At the same time, the Zeta potential varies in the range of -10 to +5 mV, indicating that the surface of the hydrogel particles carries appropriate charges, which is conducive to maintaining the electrostatic repulsion between particles, reducing the occurrence of aggregation phenomena, and ensuring the good dispersibility of the hydrogel in the solution.

[0019] The experimental results show that the hydrogel has excellent biocompatibility, with cell viability close to or exceeding 100%, and no dead cells are observed under confocal laser scanning microscopy. This means that the material is non-toxic to human cells and is suitable for use in the biomedical field. For example, when used as a medical dressing, it can not only provide effective wound protection, but also promote cell proliferation and accelerate the wound healing process. The results of both the MTT assay and the live / dead fluorescence double staining method confirm this, indicating that after adding EGCG, the composite hydrogel can not only maintain good cell viability, but also further promote cell proliferation. Description of the Drawings

[0020] Figure 1 Schematic diagram of the particle size distribution, average particle size (A), zeta potential, and turbidity (B) of the LAFs-EGCG complex in the present invention. Letters with the same index indicate significance (P<0.05). L and LE meas represent lysozyme and the LAFs-EGCG complex, and 0-1.0 represents the EGCG concentration; Figure 2 AFM topographic spectrum of the LAFs-EGCG complex in the present invention; Figure 3 UV spectrum of the LAFs-EGCG complex in the present invention. L and LE meas represent lysozyme and the LAFs-EGCG complex, and 0-1.0 represents the EGCG concentration graph; Figure 4 Intrinsic fluorescence spectrum of the LAFs-EGCG complex in the present invention. L and LE meas represent lysozyme and the LAFs-EGCG complex, and 0-1.0 represents the EGCG concentration graph; Figure 5 NMR spectrum of the LAFs-EGCG complex in the present invention. L and LE meas represent lysozyme and the LAFs-EGCG complex, and 0-1.0 represents the EGCG concentration; Figure 6 FTIR spectrum of the LAFs-EGCG complex in the present invention. L and LE meas represent lysozyme and the LAFs-EGCG complex, and 0-1.0 represents the EGCG concentration; Figure 7 Synchronous diagram of the LAFs-EGCG complex in the present invention. L and LE meas represent lysozyme and the LAFs-EGCG complex, and 0-1.0 represents the EGCG concentration; Figure 8 Asynchronous diagram of the LAFs-EGCG complex in the present invention. L and LE meas represent lysozyme and the LAFs-EGCG complex, and 0-1.0 represents the EGCG concentration; Figure 9This is the Raman spectrum of the LAFs-EGCG-CS hydrogel of the present invention. L, LE, and LEC represent lysozyme, LAFs-EGCG complex, and LAFs-EGCG-CS hydrogel, respectively. 0 - 1.0 represents the EGCG concentration; Figure 10 This is a schematic diagram of the network cross-linking part of the LAFs-EGCG-CS hydrogel of the present invention. L, LE, and LEC represent lysozyme, LAFs-EGCG complex, and LAFs-EGCG-CS hydrogel, respectively. 0 - 1.0 represents the EGCG concentration; Figure 11 This is a schematic diagram of the water molecule-gel interaction of the LAFs-EGCG-CS hydrogel of the present invention. L, LE, and LEC represent lysozyme, LAFs-EGCG complex, and LAFs-EGCG-CS hydrogel, respectively. 0 - 1.0 represents the EGCG concentration; Figure 12 This is a schematic diagram of the secondary structure of the LAFs-EGCG-CS hydrogel of the present invention. L, LE, and LEC represent lysozyme, LAFs-EGCG complex, and LAFs-EGCG-CS hydrogel, respectively. 0 - 1.0 represents the EGCG concentration; Figure 13 This is a schematic diagram of the strain scanning of the LAFs-EGCG-CS hydrogel of the present invention. LC and LEC represent LAFs-CS and LAFs-EGCG-CS hydrogels, respectively. 0 - 1.0 represents the EGCG concentration; Figure 14 This is a schematic diagram of the strain frequency of the LAFs-EGCG-CS hydrogel of the present invention. LC and LEC represent LAFs-CS and LAFs-EGCG-CS hydrogels, respectively. 0 - 1.0 represents the EGCG concentration; Figure 15 This is a schematic diagram of the LF-NMR results of the present invention. LC and LEC represent LAFs-CS and LAFs-EGCG-CS hydrogels, respectively. 0 - 1.0 represents the EGCG concentration; Figure 16 This is a schematic diagram of the water molecule type of the present invention. LC and LEC represent LAFs-CS and LAFs-EGCG-CS hydrogels, respectively. 0 - 1.0 represents the EGCG concentration; Figure 17 This is a diagram of the structural elasticity and resilience performance of the LAFs-EGCG-CS hydrogel of the present invention. LC and LEC represent LAFs-CS and LAFs-EGCG-CS hydrogels, respectively. 0 - 1.0 represents the EGCG concentration; Figure 18This is the SEM micrograph of the LAFs-EGCG-CS hydrogel of the present invention. LC and LEC measure LAFs-CS and LAFs-EGCG-CS hydrogels, and 0-1.0 represents the EGCG concentration; Figure 19 This is the schematic diagram of the MTT absorbance of the present invention at A490 nm for 1 and 3 days. LC and LEC measure LAFs-CS and LAFs-EGCG-CS hydrogels, and 0-1.0 represents the EGCG concentration; Figure 20 This is the schematic diagram of the cell viability of huvec cells treated with the LAFs-EGCG-CS hydrogel of the present invention. LC and LEC measure LAFs-CS and LAFs-EGCG-CS hydrogels, and 0-1.0 represents the EGCG concentration; Figure 21 This is the merged graph of cell survival and death stained by AM and PI of the present invention. LC and LEC measure LAFs-CS and LAFs-EGCG-CS hydrogels, and 0-1.0 represents the EGCG concentration; Figure 22 This is the schematic diagram of the inhibition rate of the hydrogel of the present invention against Staphylococcus aureus and Salmonella. LC and LEC measure LAFs-CS and LAFs-EGCG-CS hydrogels, and 0-1.0 represents the EGCG concentration; Figure 23 This is the schematic diagram of the lethality rate of the hydrogel of the present invention against Salmonella. LC and LEC measure LAFs-CS and LAFs-EGCG-CS hydrogels, and 0-1.0 represents the EGCG concentration; Figure 24 This is the schematic diagram of the staining of live and dead Salmonella by the hydrogel of the present invention. LC and LEC measure LAFs-CS and LAFs-EGCG-CS hydrogels, and 0-1.0 represents the EGCG concentration. Detailed implementation manners

[0021] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0022] Please refer to Figures 1 - 24 , the present invention is a method for preparing an EGCG-mediated hydrogel, including the following steps: Step 1: Heat-treat the lysozyme solution under acidic conditions to form lysozyme amyloid fibrils (LAFs); Step 2: Dissolve epigallocatechin gallate (EGCG) in a buffer solution and mix it with the LAFs solution obtained in Step 1 in a certain proportion to form a LAFs-EGCG complex; Step 3: Dissolve chitosan (CS) in an acetic acid buffer solution, add glutaraldehyde as a cross-linking agent, and mix it with the LAFs-EGCG complex in Step 2, and stir to form a composite hydrogel; Among them, the concentration of EGCG is 0.2-1.0 wt%, the concentration of LAFs is 5 wt%, and the concentration of chitosan is 2 wt%.

[0023] In this scheme, the pH of the lysozyme solution in Step 1 is 2.0, the heating temperature is 77 °C, and the heating time is 10 hours.

[0024] In this scheme, the concentration of EGCG in Step 2 is 0.4-0.8 wt%.

[0025] In this scheme, the concentration of glutaraldehyde in Step 3 is 2%.

[0026] In this scheme, it is characterized in that the storage modulus (G') of the hydrogel is 325-480 Pa, the hardness is 2176.71 g, and the antibacterial rate against Staphylococcus aureus and Salmonella exceeds 95%.

[0027] In this scheme, the proportion of free water T23 of the hydrogel is 5.467%-30.581%, and the content of β-sheet structure is 14.59%-29.41%.

[0028] In this scheme, the microstructure of the hydrogel is a uniformly distributed fiber network, the average particle size is 50-200 nm, and the Zeta potential is -10 to +5 mV.

[0029] In this scheme, the biocompatibility of the hydrogel is manifested as a cell survival rate ≥100% and no dead cells are detected under a confocal microscope.

[0030] Furthermore, the present invention also provides an application of EGCG-mediated hydrogel, and the hydrogel is used for food preservation, medical dressings or antibacterial packaging materials.

[0031] In this scheme, the lethality rate of the antibacterial packaging material against Gram-negative bacteria reaches 89.4%.

[0032] Preparation method and application of EGCG-mediated hydrogel. Lysozyme (LYZ) and epigallocatechin gallate (EGCG) were purchased from Sigma-Aldrich (Shanghai, China). Low molecular weight chitosan (CS, Mw: 100-300 kDa) was from Macklin Biochemical Co., Ltd. (Shanghai, China). Fluorescein 5-isothiocyanate (FITC), calcein acetoxymethyl ester (AM), rhodamine isothiocyanate (TRITC) and propidium iodide (PI) were purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). All other chemicals were analytical grade chemicals and did not require further purification when used.

[0033] Preparation of LAFs-EGCG complexes: 10 g of lysozyme was dissolved in 90 mL of deionized water to obtain a 10 wt% lysozyme solution. Undissolved components were removed, and the pH was adjusted to 2.0 with 6 M hydrochloric acid. Then, the solution was centrifuged at 4°C for 30 min. Lysozyme amyloid fibrils (LAFs) were prepared by heating and stirring the solution at 77°C for 10 h. EGCG was dissolved in 10 mM Bis-Tris buffer (pH 5.8) at a concentration of 0.2-2 wt%. Equal volumes of 5 wt% LAFs solution (diluted with Bis-Tris buffer) and 0.2-2 wt% EGCG solution were magnetically stirred for 30 min to form LAFs-EGCG complexes, and the final EGCG concentrations were 0.2, 0.4, 0.6, 0.8, and 1.0 wt%. The control group was composed of an equal volume of LAFs solution mixed with buffer without EGCG. The samples were labeled as L, LE0.2, LE 0.4, LE 0.6, LE 0.8, and LE 1.0, respectively.

[0034] Particle size and zeta potential: The particle size distribution, average particle size, and zeta potential of the LAFs-EGCG complexes were measured using a Nano ZS-90 instrument (Malvern Instruments, Worcestershire, UK). It was diluted to 0.1% (w / v) with distilled water, equilibrated in the instrument for 120 s, and measured at 25°C. At least 12 consecutive readings were recorded, and the average particle size and zeta potential were calculated from three independent measurements.

[0035] Atomic force microscopy (AFM): According to the method described by Strzdat et al., the EGCG-LAFs complexes were diluted to 10 μg / mL, and 10 μL of the solution was drop-cast on the surface of freshly cleaved mica and air-dried. The complexes were imaged and analyzed using an atomic force microscope (Dimension Icon, Bruker).

[0036] Nuclear magnetic resonance (NMR): The complexes were analyzed by obtaining the ^1H nuclear magnetic resonance spectra using a Bruker Avance 400 spectrometer operating at 400 MHz. The samples were scanned in deuterium oxide (D2O) at 25 °C.

[0037] Fourier transform infrared spectroscopy (FTIR): FTIR analysis was performed using the method described by Hu et al. [5]. 1.2 mg of the freeze-dried sample was mixed with 100 mg of dry potassium bromide in an agate mortar. Compression and scanning were carried out 16 times in the range of 400 - 4000 cm^-1 using an FTIR spectrometer (NicoletiS50, Thermo Fisher Scientific, USA).

[0038] Ultraviolet-visible spectroscopy and intrinsic fluorescence analysis: To determine the protein content of the EGCG-LAFs complex, the protein concentration was diluted to 1 mg / mL, and the spectrum was scanned in the range of 200 - 400 nm (UV2600). For intrinsic fluorescence analysis, 4 mL of the sample (1 mg / mL) was excited at 280 nm, and the fluorescence spectrum was recorded in the range of 300 - 600 nm (RF5301-PC, Shimadzu, Tokyo, Japan).

[0039] Preparation of LAFs-EGCG-CS hydrogels: Chitosan (CS) was dissolved in an acetic acid buffer solution, and its concentration was adjusted to 2% (w / v) after complete dissolution. Then, 20 mL of the CS solution was mixed with 5 mL of a 2% glutaraldehyde solution and magnetically stirred for 10 minutes. Subsequently, 5 mL of EGCG complex solutions with EGCG concentrations of 0%, 0.2%, 0.4%, 0.6%, 0.8%, and 1.0% were added, and then magnetically stirred for another 30 minutes to prepare LAFs-EGCG-CS composite hydrogels. These hydrogels were labeled LEC 0, LEC 0.2, LEC 0.4, LEC 0.6, LEC 0.8, and LEC 1.0, respectively.

[0040] Raman spectroscopy: The hydrogels were cut into thin slices, wrapped with aluminum foil, and Raman experiments were carried out. The excitation wavelength was 785 nm, and the emission power was 300 mW. Raman spectra were measured in the range of 400 - 3600 nm. Each sample was scanned at least 3 times, and the normalization process was based on the phenylalanine internal standard at 1004 cm⁻¹ as a reference for the intensity change of each Raman peak. The spectra were fitted using Peakfit 4.2 software.

[0041] Rheological properties: The rheological properties of LAFs-EGCG-CS hydrogels were measured using a rheometer (MCR301, Anton Paar, Austria). The frequency sweep test was carried out at 25 °C, with a frequency range of 1 - 100 rad / s and a strain of 2%. The storage modulus (G’) and loss modulus (G’’) were recorded. For the amplitude sweep test, the parameters were set at 25 °C, a strain range of 0.01 - 1000%, and a frequency of 1 rad / s.

[0042] Low-field nuclear magnetic resonance (LF-NMR): Gel blocks (1 cm×1 cm×1 cm) were placed in the center of the nuclear magnetic resonance tube. The transverse relaxation time (T2) was phase cycled using a Carr-Purcell-Meiboom-Gill (CPMG) sequence to eliminate artifacts. The τ value (the time between 90° and 180° pulses) was set at 200 μs, and the interval between two consecutive scans was 3000 ms.

[0043] Scanning electron microscopy (SEM): The microstructure of LAFs-EGCG-CS hydrogels was observed using a scanning electron microscope (SEM). The gel samples were cut into thin slices, quickly frozen in liquid nitrogen, and then freeze-dried. The freeze-dried gels were coated with a layer of gold (about 100 Å) and imaged under high vacuum at an acceleration voltage of 5 kV with a magnification of 500×.

[0044] Texture profile analysis (TPA): Texture profile analysis was performed using a TA-XT Plus texture analyzer (Stable Micro Systems, Surrey, England). The EGCG-LAFs composite gels were cut into 1 cm³ cubes and manually placed on the texture analyzer platform. Each sample was compressed to half of its original height using a 36-mm cylindrical compression probe. The pre-test, test, and post-test speeds were set at 1 mm / s, 1 mm / s, and 5 mm / s, respectively, with an interval of 5 s.

[0045] Biocompatibility: The biocompatibility of LAFs-EGCG-CS hydrogels was evaluated using a cytotoxicity test method. A single-cell suspension of HUVECs was prepared in a medium containing 10% fetal bovine serum and seeded at a density of 10³ - 10 5Cells were inoculated into 96-well plates at a density (200 µL per well) at a certain speed. After culturing for 3 - 5 days, 10 µL of MTT solution (5 mg / mL in PBS, pH 7.4) was added to each well and incubated for 4 h. The culture supernatant was carefully removed, and the supernatant was taken out after centrifuging the suspended cells. Then, 100 µL of DMSO was added to each well and shaken for 10 min to completely dissolve the formazan crystals. The absorbance at 490 nm was measured using a microplate reader to calculate biocompatibility. For live / dead cell staining, 100 µL of staining solution (2 µM AM and 5 µM PI) was added to 200 µL of cell suspension, mixed thoroughly, and incubated at 37 °C for 15 min. Cell viability was observed under a confocal laser scanning microscope (AM: excitation wavelength 494 nm, emission wavelength 517 nm; PI: excitation wavelength 535 nm, emission wavelength 617 nm).

[0046] Antibacterial activity: The antibacterial activities of EGCG-amyloid fibril hydrogels against Staphylococcus aureus and Escherichia coli were determined by the plate counting method. Bacterial density was measured at an OD value of 600, and the suspension was diluted with PBS to about 10 6 CFU / mL. 100 µL of the bacterial suspension was mixed with 1 g of crushed LAFs-EGCG-CS hydrogel, plated, and incubated at 37 °C for 24 h. The survival rate was calculated by comparing with the control group (without hydrogel). To expand the antibacterial spectrum of LYZ, after co-culturing the bacterial suspension with the hydrogel, it was stained with TRITC (1 mg / mL, excitation wavelength 545 nm, emission wavelength 595 nm) and FITC (0.2 mg / mL, excitation wavelength 480 nm, emission wavelength 520 nm) to analyze the number of live and dead bacteria. The lethality rate of LAFs-EGCG-CS against Gram-negative bacteria was further calculated.

[0047] Statistical data were expressed as mean ± standard deviation (X ± SD). The experimental data were analyzed for significance by one-way analysis of variance, and then Duncan's multiple comparison test was performed using SPSS Statistics 25.0 software. The significance level was P < 0.05. The results were plotted using Origin software.

[0048] Particle size and zeta potential are usually used to evaluate the stability of colloids or solutions. Figure 1 The particle size distribution, average particle size, and zeta potential of the complex are shown. Higher zeta potential values usually indicate greater stability and smaller molecular or particle sizes. As Figure 1As shown in Figure A, with the increase of EGCG concentration, the particle size, zeta potential and turbidity of the LE complex all showed significant changing trends. The particle size distribution of the complex was mostly unimodal. With the increase of EGCG concentration (0 - 0.8 wt%), the position of the main peak gradually shifted to the left, indicating that there was a spontaneous and significant interaction between EGCG and LAFs. When the EGCG concentration reached 0.6 wt% and 0.8 wt%, the particle size distribution became bimodal, reflecting the complexity of the interaction between EGCG and LAFs. With the increase of EGCG concentration, the average particle size of the LE complex decreased significantly (P < 0.05), indicating that a higher EGCG concentration promoted the self - aggregation of LAFs. At the same time, with the increase of EGCG concentration, the zeta potential of the complex decreased significantly (P < 0.05). This was mainly attributed to the multiple hydroxyl groups and aromatic ring structures of EGCG, which interacted with LAFs, resulting in the redistribution of surface charges on the complex. In addition, the hydrogen bond or electrostatic interaction between the hydroxyl groups of EGCG and the positively charged groups on the surface of LAFs neutralized or shielded the surface charges, resulting in a decrease in zeta potential. With the increase of EGCG concentration, the turbidity decreased significantly (P < 0.05). This might be due to the surface properties of the complex being conducive to dispersion rather than aggregation, and the weaker light scattering effect of the smaller particles formed. The addition of EGCG might also improve its solubility.

[0049] Atomic force microscopy (AFM) has been widely used to observe the microscopic morphological characteristics of amyloid fibrils. As Figure 2 shown, we used AFM to observe the morphology of lysozyme amyloid fibrils (LYFs) formed by heating at 95°C and the LE complex formed at different EGCG concentrations. The presence of EGCG significantly affected the morphology of lysozyme fibrils, and this effect showed an obvious dose - dependent relationship. In the absence of EGCG, the lysozyme fibrils were unevenly distributed and aggregation occurred in some areas. After adding 0.2 wt% EGCG, the fiber aggregation became more obvious, and only amorphous aggregates were observed in the AFM image. When the EGCG concentration increased to 0.4 wt% and 0.6 wt%, dispersed short fibrils and amorphous particles could be observed, indicating that EGCG inhibited the formation of long fibrils. When the EGCG concentration was 0.8 wt%, uniformly dispersed long fibrils could be observed, indicating that a high EGCG concentration promoted the formation of long fibrils. However, further increasing the EGCG concentration led to the reappearance of short fibrils and amorphous particles, which might be due to the interference of excessive EGCG with the stability of the fibrils. In summary, low EGCG concentrations promoted the formation of aggregates, medium concentrations inhibited the formation of long fibers, favored short fibers and amorphous particles, while high concentrations promoted the formation of long fibers.

[0050] Figure 5 The nuclear magnetic resonance (NMR) spectra of LAFs-EGCG complexes after treatment with different concentrations of EGCG are shown. The chemical shifts of hydroxyl groups appear in the range of 3 - 5 ppm, while the proton signals of aromatic rings are concentrated in the range of 6 - 8 ppm. With the increase in EGCG concentration, the signal intensities at 2.95 ppm (hydroxyl group), 6.00 ppm, 6.42 ppm, and 6.87 ppm (aromatic protons) gradually increase, indicating a significant interaction between EGCG and LAFs. This is attributed to the hydrogen bonds between EGCG and LAFs. The hydroxyl groups of EGCG molecules form hydrogen bonds with polar groups (such as amino or carboxyl groups) on the surface of LAFs, changing the chemical environment of the hydroxyl protons and shifting the signal to 2.95 ppm. Meanwhile, with the increase in EGCG concentration, the π-π aromatic rings of EGCG and aromatic amino acid residues (such as phenylalanine or tyrosine) on the surface of LAFs are enhanced, resulting in a significant increase in the signal intensities of aromatic protons (6.00 ppm, 6.42 ppm, and 6.87 ppm).

[0051] Currently, there are various analytical tools available for studying the conformation of proteins, among which Fourier transform infrared spectroscopy (FTIR) is a powerful technique for studying protein structure. As Figure 6 shown, there are significant differences in the FTIR spectra of LAFs treated with different EGCG concentrations at 1530, 1659, and 3305 cm⁻¹. The amide I and amide II bands are sensitive indicators of changes in the secondary structure of proteins in the FTIR spectra. The peak at approximately 1651 cm⁻¹ is attributed to the amide I band (mainly C=O stretching vibration), while the amide II band (N-H bending vibration) is located at 1539 cm⁻¹. The peak at 3305 cm⁻¹ represents -OH stretching vibration, indicating the presence of intramolecular and intermolecular hydrogen bonds in the EGCG-LAFs complex. After adding EGCG, the absorbance of the LAFs peak decreases, probably due to the formation of hydrogen bonds between multiple hydroxyl groups of EGCG and LAFs, restricting the strong vibrations of groups at different wave numbers and thus reducing the absorbance.

[0052] The contour plots of synchronous and asynchronous two-dimensional correlation spectra are as Figure 7 and Figure 8As shown, the synchronous spectrum is symmetric along the diagonal (corresponding to the spectral coordinates v1 = v2), and correlation peaks appear on or off the diagonal. The peaks on the diagonal are called auto-peaks, and their intensities reflect the sensitivities of the corresponding frequency components to external perturbations. Cross-peaks are located in the off-diagonal regions of the synchronous spectrum, indicating that two different frequency components exhibit co-variation in response to external perturbations. If two frequency components form positive cross-peaks in the synchronous spectrum, it means that they change in the same direction under external perturbations. In the synchronous spectrum, auto-peaks mainly appear at (1600, 1600) and (3200, 3200), showing strong auto-correlation peaks. In the asynchronous spectrum, two cross-peaks are observed, indicating that the spectral intensities at 1600 and 3200 cm⁻¹ change significantly with the EGCG concentration, representing the sensitive bands of the EGCG-LAFs complex, which are mainly related to the stretching vibrations of C=O, C-N, and N-H.

[0053] As Figure 3 shown, we used the ultraviolet-visible absorption spectrum of LYZ in the range of 240 - 360 nm to evaluate the interaction between EGCG and LAFs and the structural changes of LAFs. The absorption peak at 280 nm is caused by the π→π* transition of aromatic amino acid residues. Low concentrations of EGCG (0.2 - 0.4 wt%) result in a decrease in the absorption intensity of LAFs, while high concentrations of EGCG (0.6 - 1.0 wt%) result in a recovery of the absorption intensity, accompanied by an obvious blue shift. EGCG contains phenolic hydroxyl groups and is negatively charged, and it is easy to form hydrogen bonds with the positively charged amino acid residues in LAFs, leading to structural rearrangement, conformational changes, and burial of chromophores in LAFs, thus reducing the absorption intensity. However, at high concentrations, EGCG tends to self-aggregate to form larger complexes, reducing the effective contact area with LAFs and enabling LAFs to maintain their structures, thereby increasing the absorption intensity.

[0054] The LAFs molecule contains 6 tryptophan (Trp) residues, 3 of which (Trp62, Trp63, and Trp108) are located near the cleft, and the other 3 are in the core region, making lysozyme produce intrinsic fluorescence. As Figure 4As shown, with the increase in the concentration of EGCG, the intrinsic fluorescence intensity of LAFs gradually decreases. When the EGCG concentration reaches 1.0 wt%, the change in fluorescence intensity tends to stabilize. This is mainly because the fluorescent aromatic amino acid groups (such as tryptophan and tyrosine) in LAFs molecules form non-fluorescent complexes with EGCG. With the increase in the EGCG concentration, stable non-fluorescent complexes are formed between EGCG and LAFs through non-covalent interactions such as hydrophobic interactions and hydrogen bonds. These complexes are embedded in the fiber network structure, resulting in a decrease in fluorescence intensity. When the EGCG concentration reaches 1.0 wt%, the number of fluorescent chromophores and EGCG binding sites tends to saturate, leading to the stabilization of fluorescence intensity. In addition, the attachment of EGCG on the surface of LAFs causes a quenching effect, further reducing the fluorescence emission intensity. The addition of EGCG causes a red shift in the maximum fluorescence wavelength (λmax) of LAFs, indicating the interaction between EGCG and LAFs, the unfolding of the protein backbone, and the exposure of buried Trp and Tyr residues to a more hydrophilic environment. Similar phenomena were also observed by Jia et al. in their study on the interaction between EGCG and whey protein, confirming that EGCG can cause a red shift in the maximum wavelength of protein intrinsic fluorescence.

[0055] Figure 9 The Raman spectrum of LAFs-EGCG-CS is shown. The amide I band corresponds to 1626 - 1712 cm⁻¹, and the amide III band corresponds to 1220 - 1330 cm⁻¹, which reflect the backbone structure of the protein and are mainly stabilized by hydrogen bonds between N-H and C=O groups. The spectral intensities of the amide I and amide III bands first decrease and then increase, indicating a change in the backbone structure of LYZ. The peak at 587 cm⁻¹ is attributed to the s bond, 952 cm⁻¹ corresponds to carbon vibrations, and 1150 - 1160 cm⁻¹ corresponds to carbon bond peaks. 760 cm⁻¹ corresponds to tryptophan residues, and the band at 1450 cm⁻¹ corresponds to aliphatic residues (C-H, -methylene, and -methyl bending vibrations). The spectral intensities of both tryptophan and aliphatic amino acids decrease, which may be related to the hydrophobic interactions in the protein, indicating that the tryptophan residues in LAFs are buried in a hydrophobic environment, weakening the intermolecular hydrophobic interactions, while aliphatic amino acids are exposed on the outside of the molecule.

[0056] Further analyze the crosslinking between LAFs-EGCG-CS and the interaction between water molecules and the gel network Figure 10 and Figure 11 show the Raman spectrum at (C-O-C vibration), (C-H vibration), (strong hydrogen bond interaction), and (weak hydrogen bond interaction or water molecule migration). The results show that with the increase in the EGCG ratio, and The peak at showed a red shift and its intensity decreased. The peak at 1160 cm⁻¹ is the glycosidic bond in CS and the ester bond in EGCG, while the peak at 2950 cm⁻¹ mainly reflects the C-H bonds in chitosan and LAFs. These changes indicate that the bonding strength of the glycosidic bond or ester bond in the gel network increases, the intermolecular interaction enhances, and the network arrangement becomes more ordered. The analysis of the water-gel interaction peaks shows that the strong hydrogen bond interactions of each group all show a blue shift, and the peak intensity of the weak hydrogen bond interaction decreases. The introduced EGCG forms stronger hydrogen bonds or π-π stacking interactions with LAFs and CS, restricting the movement of water molecules. As a result, more water molecules bind in the gel network to form strong hydrogen bond water, leading to the peak position of the strong hydrogen bond interaction moving to a lower wavenumber, while the proportion of free water or weak hydrogen bond water decreases.

[0057] Figure 12 shows the changes in the secondary structure of LAFs. With the increase in the concentration of EGCG, the α-helix content increased from 31.06% to 34.9%, the β-sheet content decreased from 29.41% to 14.59%, and the β-turn content increased from 18.78% to 29.31%. This is because the addition of EGCG disrupted the existing β-sheets in LAFs, resulting in the unfolding of the β-sheet structure and its transformation into β-turn. At the same time, hydrogen bonds are formed between EGCG and LAFs through electrostatic interactions, contributing to the increase in the α-helix content. Acetic acid changed the pH environment of LAFs, leading to the protonation of the protein side chains, while glutaraldehyde competed with LAFs for surface hydration, destroying its structure and promoting the transformation of β-sheets to β-turns.

[0058] As Figure 13 shown, the amplitude sweep results of the LAFs-EGCG-CS hydrogel indicate that the storage modulus (G’) remains stable within the linear viscoelastic region (LVR), showing the typical three-dimensional cross-linked structure of the gel network. When the shear strain exceeds the critical value (γc), both G” and the loss modulus (G”) decrease significantly, and G” is always higher than G’, confirming the solid characteristics of the gel. It is worth noting that as the concentration of EGCG increases from 0.2 wt% to 0.8 wt%, the critical strain (the intersection strain value of G and G’) gradually decreases (from 12.5% to 6.8%), indicating that a low concentration of EGCG weakens the LAFs-chitosan network through hydrogen bonds. However, when the EGCG concentration further increases to 1.0 wt%, the critical strain rebounds to 9.2%, probably due to the covalent cross-linking of high-concentration EGCG with LAFs / chitosan and the Schiff base reaction mediated by glutaraldehyde, further enhancing the stability of the network.

[0059] The frequency sweep results are as Figure 17As shown, G′ showed frequency dependence at all EGCG concentrations and was always higher than G″, which is consistent with the typical characteristics of weak gels. When the EGCG concentration was 0.4 wt%, G′ reached a local maximum (325 Pa), while the G′ of the 1.0 wt% group increased significantly to 480 Pa. This non-linear change can be explained as follows: low concentrations of EGCG (0.2 - 0.4 wt%) enhanced the intermolecular forces through π-π stacking and hydrogen bonding, while medium to high concentrations (0.6 - 0.8%) reduced the crosslinking efficiency due to the steric hindrance effect of EGCG. When the concentration increased to 1.0 wt%, the excess EGCG was likely to form covalent crosslinks with the amino groups of chitosan through glutaraldehyde, thus forming a denser network. It should be noted that the decrease in G″ at low frequencies (<1 rad / s) may be related to the relaxation behavior of molecular chains, while the increase at high frequencies reflects the elastic response of the network structure on a short time scale.

[0060] The relaxation time (T2) and peak area measured by LF-NMR are important parameters for characterizing the water distribution in the LYZ hydrogel. T2 consists of four components: free water (T23), semi-bound water (T22), bound water (T21), and strongly bound water (T2b). Figure 15 and Figure 16 shows the effect of different EGCG concentrations on the water distribution in the LAFs-EGCG-CS hydrogel. In all samples, there were significant differences in the peaks of the LAFs-EGCG-CS hydrogel, and the contents of T22, T23, T2b, and T21 decreased in sequence, indicating that most water molecules were bound in the gel network. With the increase in EGCG concentration, the content of T23 initially increased from 8.422% to 30.581%, then decreased to 5.467%, and finally increased to 17.288%. When EGCG was initially added to LAFs, their interaction was weak, releasing the tightly bound water molecules in the protein, resulting in an increase in the content of free water and a decrease in the content of bound water. With the increase in EGCG concentration, the non-covalent interaction with LAFs led to structural folding, capturing the free water in the protein. Further increase in EGCG concentration led to the competition of glutaraldehyde for hydration on the surface of LYZ, making its structure unstable and promoting the transformation from β-sheets to β-sheets, releasing the captured free water. Therefore, the distribution and content of water molecules are affected by the interaction force between EGCG and LAFs, with glutaraldehyde playing an auxiliary role.

[0061] The texture properties of the LAFs-EGCG-CS hydrogel are as Figure 17As shown, the hardness and elasticity of the hydrogel first increased and then decreased, reaching a maximum value, while there was no significant difference in elasticity (P > 0.05). Low concentrations of EGCG (0.2 - 0.8 wt%) formed hydrogen bond networks with LAFs and CS through phenolic hydroxyl groups, and synergistically increased the density of the three-dimensional network through glutaraldehyde-mediated covalent cross-linking, thereby improving the mechanical strength. However, excessive EGCG (1.0 wt%) hindered the contact of cross-linking sites due to steric hindrance effects, and its tendency to self-aggregate led to local phase separation, resulting in a decrease in structural uniformity. The stability of elasticity may be related to the rigid benzene ring structure of EGCG, which limits the extension of molecular chains and offsets the influence of changes in cross-linking density on flexibility. In addition, the free water content of the LEC 0.8 group was the lowest (T23 = 5.467%), indicating that tightly bound water molecules improved the energy storage efficiency, thereby enhancing the elasticity.

[0062] As Figure 17 shown, the addition of EGCG significantly changed the macroscopic appearance and microscopic morphology of the composite hydrogel. The hydrogel without EGCG was uniform, semi-transparent, and orange in color, while as the EGCG concentration (0.2 - 1.0 wt%) increased, the color of the gel gradually changed from dark brown to yellowish brown, and the transparency decreased until the gel became completely opaque ( Figure 17 ). This is attributed to the π-π conjugation effect between the pyrrolidine triol group of EGCG and tyrosine residues (characteristic absorption of dark brown) and the Schiff base reaction between the oxidized quinone product of EGCG and the amino group of chitosan (yellow). The microscopic structure of the hydrogel without EGCG showed a smooth surface, while increasing the proportion of EGCG led to the formation of more cross-linked gel surfaces, indicating that low concentrations of EGCG promoted the cross-linking of LAFs-CS molecular chains through hydrophobic interactions and hydrogen bonds, forming a three-dimensional network. When the EGCG concentration increased to 0.6 - 0.8 wt%, a granular structure formed on the gel surface, possibly due to the local crystallization of molecular chains caused by the rigid benzene ring structure of EGCG, resulting in the formation of phase separation microdomains. It should be noted that the 1.0 wt% EGCG group showed more heterogeneous aggregates because high concentrations of EGCG preferentially reacted with glutaraldehyde to form oligomers, disrupting the continuous network of LAFs-CS.

[0063] The biocompatibility of the composite hydrogel was evaluated by the MTT method and live / dead fluorescence double staining method. The MTT results showed that the OD value at 490 nm reflected cell viability, and the higher the OD value, the more viable cells were present on the hydrogel sample. As Figure 19As shown, the proliferative activities of HUVEC cells in the composite hydrogel experimental group and the control group gradually increased over time. Notably, on the 3rd day, the OD490 value of the composite hydrogel experimental group was significantly higher than that of the control group (P < 0.05), indicating that the addition of EGCG promoted cell proliferation. According to the literature, if the cell viability is lower than 70% after co-culture for a certain period, the biomaterial is considered to have cytotoxicity. However, as Figure 20 shown, after 3 days of culture, the cell viability of all samples was close to or exceeded 100%, further confirming the non-cytotoxicity of the hydrogel. To more intuitively evaluate the cell state, we used confocal laser scanning microscopy (CLSM) to observe the live / dead morphology of the cells. As Figure 21 shown, the number of cells in both the experimental group and the control group increased significantly over time, and almost no dead cells were observed, which was consistent with the trend of the OD values in the MTT assay. After 3 days of culture, there was a significant difference in the number of cells between the experimental group and the blank control group, indicating that the composite hydrogel not only exhibited good cell compatibility but also further promoted cell proliferation. In summary, the LAFs-EGCG-CS composite hydrogel has good biocompatibility and the ability to promote cell proliferation, providing important evidence for its potential application in the biomedical field.

[0064] To study the effect of the EGCG-conjugated lysozyme amyloid fibril hydrogel on bacteria, Escherichia coli and Staphylococcus aureus were used as representative microorganisms, and the plate counting method was used to evaluate its antibacterial effect. The calculated antibacterial rates are as Figure 22 shown. Lysozyme itself has strong antibacterial activity against Gram-positive bacteria, and both LAFs and LAFs-EGCG-CS have significant antibacterial effects on Staphylococcus aureus, with an antibacterial rate exceeding 95%. However, lysozyme has weak inhibitory activity against Gram-negative bacteria, so the LC group had no inhibitory effect on Escherichia coli. After the incorporation of EGCG, with the increase in the EGCG concentration, the inhibition rate against Escherichia coli increased significantly (P < 0.05), indicating that EGCG broadened the antibacterial spectrum of LYZ and provided a promising material for a wider range of applications. Fluorescence images showed that with the increase in the EGCG content, the number of live cells decreased while the number of dead cells increased. In addition, as Figure 24As shown, with the increase in the concentration of EGCG, the number of dead cells increased significantly (P < 0.05). When the EGCG concentration was 1.0 wt%, the inhibition rate of cell proliferation reached 89.4%. The significant antibacterial effect of the lysozyme amyloid fibril hydrogel combined with EGCG can be attributed to the following reasons. On the one hand, the inherent antibacterial properties of EGCG and lysozyme fibrils partially inhibited the growth of bacteria. On the other hand, the addition of EGCG disrupted the formation of bacterial biofilms and promoted the production of high-concentration reactive oxygen species, resulting in bacterial death. Therefore, the EGCG-bound LAFs hydrogel exhibited good multi-channel synergistic antibacterial ability. These results were consistent with previous studies on the synergistic antibacterial effect of EGCG and fibrils.

[0065] Irreversible rigid LAFs were prepared, and by regulating the interaction between the EGCG concentration and LAFs, stable complexes were formed, and the relationship between the phenotype and structure of the complexes was established. It was found that EGCG enhanced the structural stability of LAFs through hydrogen bond networks and π-π interactions, forming a mesoscopic structure with a fiber network of small particles and neutralized surface charges. Spectral analysis showed that EGCG enhanced the vibration of active groups such as C-O and N-H in LAFs. By loading the LAFs-EGCG complex into a glutaraldehyde-mediated chitosan hydrogel, the complex enhanced the regularization of the gel network, strengthened the water-gel network structure, thereby improving the texture properties, rheological properties, and water retention. However, an excessive amount of the EGCG-glutaraldehyde complex increased the activity of free water in the system. All hydrogels had good biocompatibility and could be used for the preservation of food-medicine homologous materials. In addition, the hydrogels significantly improved the antibacterial activity against Staphylococcus aureus and Salmonella, with a stronger lethality against Salmonella.

[0066] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

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Claims

1. A method for preparing EGCG-mediated hydrogel, characterized in that: The following steps are involved: Step 1: Heat the lysozyme solution under acidic conditions to form lysozyme amyloid fibrils LAFs; Step 2: dissolving epigallocatechin gallate EGCG in a buffer solution, and mixing it with the LAFs solution obtained in step 1 in a certain proportion to form a LAFs-EGCG complex; Step 3: dissolving chitosan CS in acetic acid buffer, adding glutaraldehyde as a cross-linking agent, mixing with the LAFs-EGCG complex of step 2, and stirring to form a composite hydrogel; Among them, the concentration of EGCG was 0.2-1.0 wt%, the concentration of LAFs was 5 wt%, and the concentration of chitosan was 2 wt%.

2. The method for preparing EGCG-mediated hydrogel according to claim 1, characterized in that: The pH of the lysozyme solution in step 1 is 2.0, the heating temperature is 77° C., and the heating time is 10 hours.

3. The method for preparing EGCG-mediated hydrogel according to claim 2, characterized in that: The concentration of EGCG in step 2 is 0.4-0.8 wt %.

4. The method for preparing EGCG-mediated hydrogel according to claim 3, characterized in that: The concentration of glutaraldehyde in step 3 is 2%.

5. The method for preparing EGCG-mediated hydrogel according to any one of claims 1 to 4, characterized in that: The hydrogel has a storage modulus G' of 325-480 Pa, a hardness of 2176.71 g, and an antibacterial rate of more than 95% against Staphylococcus aureus and Salmonella.

6. The method for preparing EGCG-mediated hydrogel according to claim 5, characterized in that: The free water ratio T23 of the hydrogel is 5.467%-30.581%, and the β-folded structure content is 14.59%-29.41%.

7. The method for preparing EGCG-mediated hydrogel according to claim 5, characterized in that: The microstructure of the hydrogel is a uniformly distributed fiber network, the average particle size is 50-200 nm, and the Zeta potential is -10 to +5 mV.

8. The method for preparing EGCG-mediated hydrogel according to claim 5, characterized in that: The biocompatibility of the hydrogel is manifested in that the cell survival rate is ≥100%, and no dead cells are detected under a confocal microscope.

9. The use of EGCG-mediated hydrogel according to any one of claims 5 to 8, characterized in that: The hydrogel is used for food preservation, medical dressing or antibacterial packaging material.

10. The use of EGCG-mediated hydrogel according to claim 9, characterized in that: The lethality rate of the antibacterial packaging material to Gram-negative bacteria reaches 89.4%.

Images