A method for preparing a lysozyme amyloid fibril / tannic acid / zinc oxide antibacterial coating

Abstract

This invention discloses a method for preparing a lysozyme-amyloid fiber / tannic acid / zinc oxide antibacterial coating. Using a composite obtained from lysozyme-amyloid fiber and tannic acid under mild heating and stirring conditions as a substrate, zinc oxide and a plasticizer are added to prepare a lysozyme-amyloid fiber / tannic acid / zinc oxide antibacterial coating solution. This coating solution can be impregnated and dried onto the surface of cherry tomatoes to form a film, thus forming the lysozyme-amyloid fiber / tannic acid / zinc oxide antibacterial coating. This antibacterial coating exhibits good mechanical properties, adhesion, and biocompatibility, and also provides excellent preservation effects.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology and relates to the preparation of lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating and its application testing in food preservation. Background Technology

[0002] Fruits are rich in vitamins, minerals, and other nutrients. However, compared to other plant-based foods, harvested fruits are more susceptible to microbial infection and water loss through transpiration (or anaerobic respiration in the absence of air), leading to spoilage and seriously impacting food safety, orchard profits, and the economic benefits of fruit and vegetable processing enterprises. Currently, fruit preservation technologies mainly include low-temperature refrigeration, the application of chemical preservatives, and plastic wrap packaging. Low-temperature refrigeration is costly and prone to chilling injury, while chemical preservatives pose a risk of residue. Traditional plastic wrap has poor air permeability; although it can prevent water loss during storage and transportation, it simultaneously exacerbates the fruit's anaerobic respiration, hindering shelf life.

[0003] Lysozyme (LYS), a natural alkaline protease, can specifically disrupt bacterial cell walls, offering advantages such as safety and no drug resistance. However, its antibacterial efficiency is relatively low, and its material properties are poor. Lysozyme fiber, obtained through hydrothermal fiberization, possesses a higher aspect ratio and an ordered structure, known as lysozyme amyloid fibers (LAFs), exhibiting good biocompatibility and antibacterial properties (see CN120157916A). Tannic acid (TA), a natural polyphenol, possesses broad-spectrum antibacterial activity. It can also cross-link with proteins through hydrogen bonds and hydrophobic interactions, thereby enhancing material structural stability; for example, it can be used in combination with lysozyme amyloid fibers to prepare hydrogels (see CN118356396A). Zinc oxide, as an inorganic antibacterial agent, exhibits long-lasting antibacterial properties and high-temperature resistance. It can also form a metal-phenolic network structure with polyphenols through coordination bonds, improving the material's mechanical properties and antibacterial activity. Currently, there are no reports of using a combination of lysozyme, amyloid fiber, tannic acid, and zinc oxide to prepare a special antibacterial coating for fruit preservation.

[0004] Hydrogels, with their three-dimensional network structure, excellent hydrophilicity, and biocompatibility, show great promise for food preservation. Antibacterial coatings prepared using hydrogels or their synthetic raw materials as substrates primarily achieve preservation by forming a protective film directly on the fruit surface. However, these coatings often employ single antibacterial components, such as chitosan or plant essential oils, which suffer from short antibacterial duration, poor effectiveness against highly pathogenic bacteria, and susceptibility to cracking and peeling. More importantly, while combining multiple antibacterial components can enhance the antibacterial effect and has become a research hotspot in fruit preservation, the properties of the resulting coatings, especially the application method, can change significantly. For example, the composite coating obtained by the casting and drying method in CN114957751A is not actually formed directly on the food surface, and its application testing for food preservation lacks validation. Therefore, how to prepare antibacterial coatings that combine antibacterial, moisturizing, and breathable (oxygen-permeable) advantages, and ensure that such coatings meet the requirements for direct application in fruit preservation, remains a technical challenge. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating. This method is adapted to the surface characteristics of food (e.g., fruit) and can directly prepare an antibacterial coating with high antibacterial efficiency, good moisturizing properties, and good breathability on the food surface.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, a method for preparing a lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating liquid is provided, comprising the following steps: (1) Lysozyme amyloid cellulose solution was prepared by hydrothermal method using lysozyme as raw material; (2) Add tannic acid to the lysozyme amyloid fiber solution to a final concentration of 20-80 mM, and then gently heat and stir at 30-50℃ (300-500 rpm, 30-60 min) until the mixture is homogeneous. After stirring until homogeneous, stop heating and let the system stand until it cools down to obtain the lysozyme amyloid fiber / tannic acid complex. (3) Add zinc oxide to an acidic aqueous solution to a final concentration of 10-60 mM, and adjust the pH to 3.5-5 with sodium hydroxide and / or hydrochloric acid solution to obtain a zinc oxide solution. Mix the zinc oxide solution (pH=3.5-5) with the lysozyme amyloid fiber / tannic acid complex and plasticizer to obtain a lysozyme amyloid fiber / tannic acid / zinc oxide coating solution.

[0007] Preferably, in step 1, the lysozyme amyloid cellulose is obtained by heating and hydrolyzing lysozyme, and has a rich β-sheet structure. The conditions for heating and hydrolyzing include: the mass fraction of the lysozyme aqueous solution is 6%~10%, the reaction temperature is 60~90℃, and the reaction time is 12~16 h.

[0008] Preferably, step 1 specifically includes the following steps: adding lysozyme to ultrapure water to prepare a lysozyme mass fraction of 8%, then adding 0.1~0.2 M hydrochloric acid solution to adjust the pH to 1.6~2, and then reacting for 12~14 h under water bath heating at 90℃ and stirring at 300~500 rpm. After the reaction is completed, cooling to 20~30℃ (e.g., naturally cooling to room temperature) to obtain a lysozyme amyloid cellulose solution.

[0009] Preferably, in step 2, tannic acid is used as a multifunctional crosslinking agent. On the one hand, it binds to lysozyme amyloid fibers through hydrogen bonding and hydrophobic interactions. On the other hand, it can coordinate with zinc oxide through phenolic hydroxyl groups to form a metal-polyphenol network. The final concentration of tannic acid is 40~60 mM.

[0010] Preferably, in step 3, the acidic aqueous solution is selected from 0.1~0.2 M hydrochloric acid solution.

[0011] Preferably, in step 3, the plasticizer is selected from glycerin.

[0012] Preferably, in step 3, glycerol is added separately to the zinc oxide solution (pH=3.5~5) and the lysozyme amyloid cellulose / tannic acid complex at a final concentration of 30%~35% (mass fraction) before mixing, and the glycerol is uniformly dispersed. The zinc oxide solution containing glycerol is added to the lysozyme amyloid cellulose / tannic acid complex containing glycerol (the volume ratio of the zinc oxide solution containing glycerol to the lysozyme amyloid cellulose / tannic acid complex containing glycerol is 1:1~2), and then stirred for 10~30 min (100~150 rpm).

[0013] Secondly, a method for preparing a lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating is provided, comprising the following steps: The lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating liquid prepared by the method in the first aspect above is subjected to ultrasonic treatment to remove air bubbles, and then the food is impregnated and dried to form a lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating on the surface of the food.

[0014] Preferably, the ultrasonic treatment time is 5 to 15 minutes.

[0015] Preferably, the immersion conditions include: immersing the food in the de-bubbling coating liquid for 5-10 seconds and then removing it.

[0016] Preferably, the drying conditions are: air drying at 20~30°C (e.g., room temperature).

[0017] Thirdly, the method for preparing the above-mentioned lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating is provided for its application in extending the shelf life of food (such as cherry tomatoes and other fruits) or in the preservation of food (such as cherry tomatoes and other fruits).

[0018] The beneficial effects of this invention are reflected in: This invention relates to a composite antibacterial coating prepared based on lysozyme, amyloid fibers, tannic acid, and zinc oxide. This coating can effectively inhibit pathogenic bacteria on food surfaces, reduce moisture loss, and exhibit good biocompatibility. Furthermore, the preparation method of this antibacterial coating is simple and is of great significance for solving food preservation problems such as post-harvest preservation of fruits.

[0019] This invention involves immersing food, especially fruit, in a coating liquid and then drying it to form a thin antibacterial coating on the food surface. This antibacterial coating inhibits microbial growth, reduces moisture evaporation and anaerobic respiration, thereby delaying food spoilage and extending its shelf life.

[0020] Furthermore, this invention optimizes the preparation process of lysozyme amyloid fibers, thereby developing an antibacterial coating that can be combined with tannic acid and combined with zinc oxide addition ratio control (e.g., controlling the coating microstructure and water vapor and oxygen permeability), thus obtaining an antibacterial coating that can improve the room temperature storage time of cherry tomatoes. Attached Figure Description

[0021] Figure 1 The infrared absorption spectra of the lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating samples prepared using coating solutions from different treatment groups in the examples are shown.

[0022] Figure 2 Microscopic morphology images of lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating samples prepared using coating solutions of different treatment groups in the examples: A. LAFs coating solution treatment group; B. LAFs / TA coating solution treatment group; C. LAFs / TA / ZnO-1 coating solution treatment group; D. LAFs / TA / ZnO-2 coating solution treatment group; E. LAFs / TA / ZnO-3 coating solution treatment group; F. LAFs / TA / ZnO-4 coating solution treatment group.

[0023] Figure 3 The graph shows the water vapor transmission rate and oxygen transmission rate of the lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating samples prepared using coating solutions from different treatment groups in the examples.

[0024] Figure 4The adhesion test results are for lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating samples prepared using coating solutions from different treatment groups in the examples.

[0025] Figure 5 The results of cytotoxicity tests are shown for lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating samples (extracts) prepared using coating solutions from different treatment groups in the examples.

[0026] Figure 6 The lysozyme amyloid cellulose / tannic acid / zinc oxide coating solutions of different treatment groups in the examples were used to treat Escherichia coli (E. coli). E. coli ) and Staphylococcus aureus ( S.aureus The antibacterial performance test results of ).

[0027] Figure 7 The results of the preservation test of cherry tomatoes by the lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coatings prepared in different treatment groups in the examples are shown. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments described are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0029] (a) Preparation of lysozyme amyloid fibers Weigh a certain amount of lysozyme powder (animal source: egg white; manufacturer: Beijing Boao Tuoda Technology Co., Ltd.) and add it to a certain volume of ultrapure water to prepare a lysozyme solution with a mass percentage concentration of 6%~10%. Then add 0.1 M hydrochloric acid solution to adjust the pH of the 6%~10% lysozyme solution to 2. Then react for 12~16 h under water bath heating at 60~90℃ and magnetic stirring. After the reaction is completed, cool naturally to room temperature to obtain a solution of lysozyme amyloid fibers, i.e., LAFs.

[0030] For the preparation of antibacterial coatings, the three process parameters in the above LAF preparation process are the concentration of lysozyme solution (6%~10%), reaction time (12~16 h), and reaction temperature (60~90℃). Too low a concentration of lysozyme leads to insufficient coating adhesion; too short a reaction time prevents the formation of abundant β-sheet structures, while too long a reaction time causes self-aggregation and precipitation, resulting in a rough coating structure; too low a reaction temperature slows the nucleation of amyloid fibers, affecting the density of the coating structure and reducing antibacterial and other functional activities; too high a reaction temperature disrupts the hydrogen bonds, hydrophobic interactions, and disulfide bonds between amyloid fiber molecules, causing the disintegration of the ordered β-sheet structure, also affecting the density of the coating structure.

[0031] (II) Fruit preservation treatment Example 1: LAFs coating liquid treatment group (1) Preparation of lysozyme amyloid fiber coating solution A certain amount of lysozyme powder (animal source: egg white; manufacturer: Beijing Boao Tuoda Technology Co., Ltd.) was weighed and added to a certain volume of ultrapure water to prepare a lysozyme solution with a mass percentage concentration of 8%. Then, 0.1 M hydrochloric acid solution was added to adjust the pH of the 8% lysozyme solution to 2. The solution was then heated in a 90℃ water bath and magnetically stirred at 350 rpm for 12 hours. After the reaction was completed, it was naturally cooled to room temperature to obtain a solution of lysozyme amyloid fibers, i.e., LAFs. Glycerol was added to the obtained lysozyme amyloid fiber solution at a plasticizer concentration of 35% (w / w). The solution was then magnetically stirred at room temperature for 30 minutes (350 rpm) to ensure uniform dispersion, thus obtaining the lysozyme amyloid fiber coating liquid, i.e., LAFs.

[0032] (2) The lysozyme amyloid fiber coating solution prepared in step (1) is sonicated for 5 min at room temperature to remove any air bubbles, and the working solution is obtained. The cherry tomatoes are washed with cool water and dried. Then, the stems of the cherry tomatoes are picked up with tweezers and immersed in the working solution. After 5 seconds, the cherry tomatoes soaked in the working solution are taken out and dried at room temperature.

[0033] Example 2: LAFs / TA coating liquid treatment group (1) Preparation of lysozyme amyloid fiber / tannic acid coating solution A certain amount of lysozyme powder (animal source: egg white; manufacturer: Beijing Boao Tuoda Technology Co., Ltd.) was weighed and added to a certain volume of ultrapure water to prepare a lysozyme solution with a mass percentage concentration of 8%. Then, 0.1 M hydrochloric acid solution was added to adjust the pH of the 8% lysozyme solution to 2. The mixture was then reacted for 12 hours under 90℃ water bath heating and 350 rpm magnetic stirring. After the reaction, it was naturally cooled to room temperature to obtain a solution of lysozyme amyloid fibers (LAFs). Tannic acid powder (specifically, tannic acid) was added to the prepared lysozyme amyloid fiber solution at a cross-linking agent concentration of 30 mM. The mixture was then magnetically stirred under 50℃ water bath heating until homogeneous (30 min, 350 rpm). Then stop heating and stirring and allow it to cool naturally to room temperature to obtain the lysozyme amyloid fiber / tannic acid complex; add glycerol to the prepared lysozyme amyloid fiber / tannic acid complex at a plasticizer (specifically glycerol) concentration of 35% (w / w), and then magnetically stir at room temperature for 30 min (350 rpm) to ensure uniform dispersion to obtain the lysozyme amyloid fiber / tannic acid, i.e., LAFs / TA coating solution.

[0034] (2) The lysozyme amyloid fiber / tannic acid coating solution prepared in step (1) is sonicated for 5 min at room temperature to remove any air bubbles, and the working solution is obtained. The cherry tomatoes are washed with cool water and dried. Then, the fruit stems of the cherry tomatoes are picked up with tweezers and immersed in the working solution. After 5 seconds, the cherry tomatoes soaked in the working solution are taken out and dried at room temperature.

[0035] Example 3: LAFs / TA / ZnO-1 coating liquid treatment group (1) Preparation of lysozyme amyloid fiber / tannic acid / zinc oxide coating solution A certain amount of lysozyme powder (animal source: egg white; manufacturer: Beijing Boao Tuoda Technology Co., Ltd.) was weighed and added to a certain volume of ultrapure water to prepare a lysozyme solution with a mass percentage concentration of 8%. Then, 0.1 M hydrochloric acid solution was added to adjust the pH of the 8% lysozyme solution to 2. The mixture was then reacted for 12 hours under 90℃ water bath heating and 350 rpm magnetic stirring. After the reaction, it was naturally cooled to room temperature to obtain a solution of lysozyme amyloid fibers (LAFs). Tannic acid powder (60 mM) was added to the prepared lysozyme amyloid fiber solution, and then the mixture was magnetically stirred under 50℃ water bath heating until homogeneous (30 min, 350 rpm). The mixture was heated to 350 rpm, then heating and stirring were stopped and allowed to cool naturally to room temperature to obtain a lysozyme amyloid cellulose / tannic acid complex. Glycerin (specifically glycerol) was added to the prepared lysozyme amyloid cellulose / tannic acid complex at a plasticizer concentration of 35% (w / w), and then magnetically stirred at room temperature for 30 min (350 rpm) to ensure uniform dispersion. Zinc oxide powder was dissolved in 0.1 M hydrochloric acid solution to obtain a 10 mM zinc oxide solution, and the pH was adjusted to 4 with sodium hydroxide. Then, glycerin at a feed concentration of 35% (w / w) was added, and magnetically stirred at room temperature for 30 min (350 rpm) to ensure uniform dispersion. The zinc oxide solution containing glycerin was slowly added dropwise (to avoid the formation of flocculent matter due to excessive addition) to the lysozyme amyloid cellulose / tannic acid complex containing glycerin (the volume ratio of the zinc oxide solution system containing glycerin to the LAFs / TA system containing glycerin was 1:1, i.e., the tannic acid concentration was diluted to 30 mM), and then slowly stirred at room temperature for 15 minutes. The lysozyme amyloid cellulose / tannic acid / zinc oxide coating solution was obtained by stirring magnetically at 100 rpm for 1 minute. This solution was denoted as LAFs / TA / ZnO-1.

[0036] (2) The lysozyme amyloid fiber / tannic acid / zinc oxide coating solution (i.e. LAFs / TA / ZnO-1 coating solution) prepared in step (1) is sonicated for 5 min at room temperature to remove any air bubbles and obtain the impregnation working solution; the cherry tomatoes are washed with cool water and dried, and then the fruit stems of the cherry tomatoes are picked up with tweezers and immersed in the impregnation working solution. After 5 seconds, the cherry tomatoes soaked in the impregnation working solution are taken out and dried at room temperature.

[0037] Example 4: LAFs / TA / ZnO-2 coating liquid treatment group (1) Preparation of lysozyme amyloid fiber / tannic acid / zinc oxide coating solution A certain amount of lysozyme powder (animal source: egg white; manufacturer: Beijing Boao Tuoda Technology Co., Ltd.) was weighed and added to a certain volume of ultrapure water to prepare a lysozyme solution with a mass percentage concentration of 8%. Then, 0.1 M hydrochloric acid solution was added to adjust the pH of the 8% lysozyme solution to 2. The mixture was then reacted for 12 hours under 90℃ water bath heating and 350 rpm magnetic stirring. After the reaction, it was naturally cooled to room temperature to obtain a solution of lysozyme amyloid fibers (LAFs). Tannic acid powder (60 mM) was added to the prepared lysozyme amyloid fiber solution, and then the mixture was magnetically stirred under 50℃ water bath heating until homogeneous (30 min, 350 rpm). The mixture was heated to 100 rpm, then heating and stirring were stopped and allowed to cool naturally to room temperature to obtain a lysozyme amyloid cellulose / tannic acid complex. Glycerin (specifically glycerol) was added to the prepared lysozyme amyloid cellulose / tannic acid complex at a plasticizer concentration of 35% (w / w), and then magnetically stirred for 30 min (350 rpm) at room temperature to ensure uniform dispersion. Zinc oxide powder was dissolved in 0.1 M hydrochloric acid solution to obtain a 20 mM zinc oxide solution, and the pH was adjusted to 4 with sodium hydroxide. Then, glycerin at a concentration of 35% (w / w) was added, and magnetically stirred for 30 min (350 rpm) at room temperature to ensure uniform dispersion. The zinc oxide solution containing glycerin was slowly added dropwise (to avoid the formation of flocculent material due to excessive addition) to the lysozyme amyloid cellulose / tannic acid complex containing glycerin (the volume ratio of the zinc oxide solution system containing glycerin to the LAFs / TA system containing glycerin was 1:1), and then slowly stirred for 15 min (magnetic stirring, 100 rpm) at room temperature. (rpm), to obtain lysozyme amyloid fiber / tannic acid / zinc oxide coating solution, denoted as LAFs / TA / ZnO-2.

[0038] (2) The lysozyme amyloid fiber / tannic acid / zinc oxide coating solution (i.e. LAFs / TA / ZnO-2 coating solution) prepared in step (1) is sonicated for 5 min at room temperature to remove any air bubbles and obtain the impregnation working solution; the cherry tomatoes are washed with cool water and dried, and then the fruit stems of the cherry tomatoes are picked up with tweezers and immersed in the impregnation working solution. After 5 seconds, the cherry tomatoes soaked in the impregnation working solution are taken out and dried at room temperature.

[0039] Example 5: LAFs / TA / ZnO-3 coating liquid treatment group (1) Preparation of lysozyme amyloid fiber / tannic acid / zinc oxide coating solution A certain amount of lysozyme powder (animal source: egg white; manufacturer: Beijing Boao Tuoda Technology Co., Ltd.) was weighed and added to a certain volume of ultrapure water to prepare a lysozyme solution with a mass percentage concentration of 8%. Then, 0.1 M hydrochloric acid solution was added to adjust the pH of the 8% lysozyme solution to 2. The mixture was then reacted for 12 hours under a 90℃ water bath and magnetic stirring at 350 rpm. After the reaction, it was naturally cooled to room temperature to obtain a solution of lysozyme amyloid fibers (LAFs). Tannic acid powder (60 mM) was added to the prepared lysozyme amyloid fiber solution, and then the mixture was heated in a 50℃ water bath and magnetically stirred until homogeneous (30 min, 350 rpm). The mixture was heated to 100 rpm, then heating and stirring were stopped and allowed to cool naturally to room temperature to obtain a lysozyme amyloid cellulose / tannic acid complex. Glycerin (specifically glycerol) was added to the prepared lysozyme amyloid cellulose / tannic acid complex at a plasticizer concentration of 35% (w / w), and then magnetically stirred for 30 min (350 rpm) at room temperature to ensure uniform dispersion. Zinc oxide powder was dissolved in 0.1 M hydrochloric acid solution to obtain a 30 mM zinc oxide solution, and the pH was adjusted to 4 with sodium hydroxide. Then, glycerin at a feed concentration of 35% (dry weight, w / w) was added, and magnetically stirred for 30 min (350 rpm) at room temperature to ensure uniform dispersion. The zinc oxide solution containing glycerin was slowly added dropwise (to avoid the formation of flocculent material due to excessive addition) to the lysozyme amyloid cellulose / tannic acid complex containing glycerin (the volume ratio of the zinc oxide solution system containing glycerin to the LAFs / TA system containing glycerin was 1:1), and then slowly stirred for 15 min (magnetic stirring, 100 rpm) at room temperature. (rpm), to obtain lysozyme amyloid cellulose / tannic acid / zinc oxide coating solution, denoted as LAFs / TA / ZnO-3.

[0040] (2) The lysozyme amyloid fiber / tannic acid / zinc oxide coating solution (i.e. LAFs / TA / ZnO-3 coating solution) prepared in step (1) is sonicated for 5 min at room temperature to remove any air bubbles, and the working solution is obtained. The cherry tomatoes are washed with cool water and dried. Then, the fruit stems of the cherry tomatoes are picked up with tweezers and immersed in the working solution. After 5 seconds, the cherry tomatoes soaked in the working solution are taken out and dried at room temperature.

[0041] Example 6: LAFs / TA / ZnO-4 coating liquid treatment group (1) Preparation of lysozyme amyloid fiber / tannic acid / zinc oxide coating solution A certain amount of lysozyme powder (animal source: egg white; manufacturer: Beijing Boao Tuoda Technology Co., Ltd.) was weighed and added to a certain volume of ultrapure water to prepare a lysozyme solution with a mass percentage concentration of 8%. Then, 0.1 M hydrochloric acid solution was added to adjust the pH of the 8% lysozyme solution to 2. The mixture was then reacted for 12 hours under 90℃ water bath heating and 350 rpm magnetic stirring. After the reaction, it was naturally cooled to room temperature to obtain a solution of lysozyme amyloid fibers (LAFs). Tannic acid powder (60 mM) was added to the prepared lysozyme amyloid fiber solution, and then the mixture was magnetically stirred under 50℃ water bath heating until homogeneous (30 min, 350 rpm). The mixture was heated to 100 rpm, then heating and stirring were stopped and allowed to cool naturally to room temperature to obtain a lysozyme amyloid cellulose / tannic acid complex. Glycerin (specifically glycerol) was added to the prepared lysozyme amyloid cellulose / tannic acid complex at a plasticizer concentration of 35% (w / w), and then magnetically stirred for 30 min (350 rpm) at room temperature to ensure uniform dispersion. Zinc oxide powder was dissolved in 0.1 M hydrochloric acid solution to obtain a 40 mM zinc oxide solution, and the pH was adjusted to 4 with sodium hydroxide. Then, glycerin at a feed concentration of 35% (dry weight, w / w) was added, and magnetically stirred for 30 min (350 rpm) at room temperature to ensure uniform dispersion. The zinc oxide solution containing glycerin was slowly added dropwise (to avoid the formation of flocculent material due to excessive addition) to the lysozyme amyloid cellulose / tannic acid complex containing glycerin (the volume ratio of the zinc oxide solution system containing glycerin to the LAFs / TA system containing glycerin was 1:1), and then slowly stirred for 15 min (magnetic stirring, 100 rpm) at room temperature. (rpm), to obtain lysozyme amyloid fiber / tannic acid / zinc oxide coating solution, denoted as LAFs / TA / ZnO-4.

[0042] (2) The lysozyme amyloid fiber / tannic acid / zinc oxide coating solution (i.e. LAFs / TA / ZnO-4 coating solution) prepared in step (1) is sonicated for 5 min at room temperature to remove any air bubbles and obtain the impregnation working solution; the cherry tomatoes are washed with cool water and dried, and then the fruit stems of the cherry tomatoes are picked up with tweezers and immersed in the impregnation working solution. After 5 seconds, the cherry tomatoes soaked in the impregnation working solution are taken out and dried at room temperature.

[0043] (III) Coating Structure and Performance Analysis 1. Preparation of coating samples In addition, 2 mL or more of the coating liquid from each example was taken and dropped into a 5 cm × 5 cm silicone mold, and then placed in a 37°C oven for 24 h to dry (to facilitate the peeling of the coated sample, and the thickness of the coated sample was slightly greater than the film thickness of the coating liquid on the cherry tomato surface). After drying, the corresponding coated samples were peeled off for analysis and testing. The film thickness and color of the peeled coating were described.

[0044] The results are shown in Table 1. The thickness of the coating sample formed by the LAFs coating solution (hereinafter referred to as the LAFs coating) was 0.0826 mm. After the addition of tannic acid, the thickness of the coating sample formed by the LAFs / TA coating solution (hereinafter referred to as the LAFs / TA coating) increased, which was due to the increase in dry matter content. In addition, the thickness of the coating samples formed by the LAFs / TA / ZnO-1 and LAFs / TA / ZnO-2 coating solutions (hereinafter referred to as the LAFs / TA / ZnO-1 coating and the LAFs / TA / ZnO-2 coating) increased slightly compared with the LAFs / TA coating. This may be because, with the increase of concentration, zinc oxide forms metal coordination bonds with the phenolic hydroxyl groups of tannic acid, thereby forming a dense structure. The coating samples formed by LAFs / TA / ZnO-3 and LAFs / TA / ZnO-4 coating solutions (hereinafter referred to as LAFs / TA / ZnO-3 coating and LAFs / TA / ZnO-4 coating) showed a significant increase in thickness (p<0.05), which may be attributed to excessive cross-linking of excess zinc oxide and the formation of particle agglomerates, leading to a looser structure. The LAFs coating's thickness... The value was the highest (93.73), indicating that the LAF coating was the clearest and most transparent film among the seven coatings. After adding tannic acid, L... The value reached its lowest point (76.23), a and b The values ​​reached the highest (8.54 and 26.62), indicating that the LAFs / TA coating was the least transparent of the seven coatings, which may be related to the color of tannic acid itself. Compared with the LAFs / TA coating, the ΔE values ​​of the four LAFs / TA / ZnO coatings were... The values ​​are relatively low because the addition of zinc oxide significantly enhances the stability and uniformity of these coatings. Furthermore, with increasing zinc oxide concentration, the LA value of ... Value increases, a and b The values ​​are all reduced. More zinc oxide will enhance the coating's ability to reflect and scatter visible light, reduce light absorption, and thus significantly improve the overall transparency of the coating.

[0045] Table 1. Thickness and color characteristics of antibacterial coating samples prepared with coating solutions from different treatment groups

[0046] 2. Infrared spectroscopy analysis Using an FT-IR spectrometer at 4000-500 cm⁻¹ -1 Within the specified range, coating samples prepared using coating solutions from the LAFs coating solution treatment group, LAFs / TA coating solution treatment group, LAFs / TA / ZnO-1 coating solution treatment group, LAFs / TA / ZnO-2 coating solution treatment group, LAFs / TA / ZnO-3 coating solution treatment group, and LAFs / TA / ZnO-4 coating solution treatment group were subjected to full-wavelength scanning.

[0047] The results are as follows Figure 1 As shown, compared with the coating samples treated with LAFs coating liquid, the infrared spectra of the coating samples treated with LAFs / TA coating liquid are in the range of 3000-3500 cm⁻¹. -1 Interval peak broadening and 2962 cm -1 The decrease in peak intensity caused by the -CH2 stretching vibration indicates that tannic acid and lysozyme-amyloid fibers interact through cross-linking. Compared with the coating samples treated with LAFs / TA coating solution, the infrared spectra of the coating samples treated with LAFs / TA / ZnO-1, LAFs / TA / ZnO-2, LAFs / TA / ZnO-3, and LAFs / TA / ZnO-4 coating solutions showed further changes in peak shape and intensity, indicating that the introduction of zinc oxide altered the chemical environment of the coating. In the corresponding LAFs / TA / ZnO coatings, the peak intensity at 1709 cm⁻¹... -1 The carbonyl peaks at these locations become more pronounced, and the CO peaks (1191, 1086, 1024 cm⁻¹) are also observed. -1 The red shift in these spectroscopic features indicates that zinc oxide coordinates with the carboxyl and hydroxyl groups of tannic acid, forming a stable metal-polyphenol network structure.

[0048] 3. Microstructure observation Coated samples prepared using coating solutions from the LAFs coating solution treatment group, LAFs / TA coating solution treatment group, LAFs / TA / ZnO-1 coating solution treatment group, LAFs / TA / ZnO-2 coating solution treatment group, LAFs / TA / ZnO-3 coating solution treatment group, and LAFs / TA / ZnO-4 coating solution treatment group were respectively attached to conductive adhesives and then sputtered with gold. Images of each coated sample were then obtained using a scanning electron microscope under vacuum conditions and an accelerating voltage of 5.0 kV.

[0049] The results are as follows Figure 2 As shown, the surface of the coated samples in the LAFs / TA coating liquid treatment group was slightly smoother than that in the LAFs coating liquid treatment group. The coated samples in the LAFs / TA / ZnO-1 and LAFs / TA / ZnO-2 coating liquid treatment groups had dense and uniform structures, which may be due to the zinc oxide being embedded between lysozyme amyloid fibers and tannic acid, affecting the molecular interaction and arrangement, and enhancing the density of the coating structure. However, the surfaces of the coated samples in the LAFs / TA / ZnO-3 and LAFs / TA / ZnO-4 coating liquid treatment groups became rough and uneven, which may be due to the increased zinc oxide concentration.

[0050] 4. Analysis of water vapor transmission rate and oxygen transmission rate Coated samples (5 cm × 5 cm) prepared using coating solutions from four groups (LAFs, LAFs / TA, LAFs / TA / ZnO-1, LAFs / TA / ZnO-2, LAFs / TA / ZnO-3, and LAFs / TA / ZnO-4) were placed over the openings of centrifuge tubes filled with distilled water and sealed tightly. The initial weight of each centrifuge tube was weighed and recorded. After 12 hours, the tubes were placed in a desiccator, and the final weight was recorded. The water vapor transmission rate was calculated using the formula shown below. WVP [g·(m·s·Pa] -1 ] =

[0051] Where Δm is the weight difference of the centrifuge tubes (g); d is the coating thickness (mm); and A is the effective area of ​​the coating (mm²). 2 ); t is time (s); ΔP is the water vapor pressure difference across the coating (3.168 × 10⁻⁶). 3 Pa).

[0052] Take another set of coated samples (5 cm × 5 cm) and cover the opening of a centrifuge tube containing 3 g of deoxidizer, then seal tightly. Weigh and record the initial weight of each centrifuge tube, and place them in a 25°C incubator for equilibration. After 48 h, weigh and record the final weight of each centrifuge tube, and calculate the oxygen transmission rate (according to the transmission rate calculation formula shown below): ROT [g·(m 2 ·s) -1 ] =

[0053] in, m is the weight difference (g) of the centrifuge tube before and after 48 hours; t is the time (s); A is the effective area of ​​the coating (mm²). 2 ).

[0054] The results are as follows Figure 3 As shown, the coating samples of the LAFs coating liquid treatment group and the LAFs / TA coating liquid treatment group have high oxygen permeability and water vapor permeability values. This may be due to their relatively loose structure, which increases the channels for water molecules and oxygen to permeate through the coating. The coating samples of the LAFs / TA / ZnO-1 coating liquid treatment group and the LAFs / TA / ZnO-2 coating liquid treatment group have low water vapor permeability and oxygen permeability values ​​(with the decrease in water vapor permeability value being more significant). This may be due to the metal coordination effect of zinc oxide and tannic acid, which makes the coating structure more compact and reduces the migration paths of water molecules and oxygen. The coating samples of the LAFs / TA / ZnO-3 coating liquid treatment group and the LAFs / TA / ZnO4 coating liquid treatment group show a rebound in water vapor permeability and oxygen permeability values. The increase in water vapor permeability and oxygen permeability of the coating samples of these two groups may be due to the excessive cross-linking caused by the increased zinc oxide addition, which makes the coating rougher and thus provides more permeation pathways for water molecules and oxygen.

[0055] 5. Adhesion Analysis Coated samples prepared using coating solutions from four groups—LAFs, LAFs / TA, LAFs / TA / ZnO-1, LAFs / TA / ZnO-2, LAFs / TA / ZnO-3, and LAFs / TA / ZnO-4—were cut to 20 mm × 20 mm. A peel test was then conducted to assess the adhesion of each group of samples. Specifically, each cut sample was adhered to two glass plates, and a peel test was performed at a tensile rate of 20 mm / min to determine the adhesion of the coating samples.

[0056] The results are as follows Figure 4As shown, compared to the LAFs coating liquid treatment group (0.048 MPa), the adhesion strength of the LAFs / TA coating liquid treatment group (0.066 MPa) was significantly improved. This is mainly attributed to the stronger network structure formed by the interaction and cross-linking of tannic acid and lysozyme amyloid fibers. However, compared to the LAFs / TA coating liquid treatment group, the adhesion strength of the LAFs / TA / ZnO-1 and LAFs / TA / ZnO-2 coating liquid treatment groups (0.101 MPa and 0.162 MPa, respectively) was significantly lower. The adhesion strength (MPa) was further improved, mainly due to the formation of a metal-phenolic network between zinc oxide and tannic acid, and the formation of a double-network interpenetrating gel network structure with lysozyme amyloid fibers, resulting in a more uniform gel network structure. Compared with the LAFs / TA / ZnO-2 coating liquid treatment group, the adhesion strength of the coated samples in the LAFs / TA / ZnO-3 and LAFs / TA / ZnO-4 coating liquid treatment groups decreased (0.136 MPa and 0.074 MPa, respectively), which may be due to excessive cross-linking caused by the increased zinc oxide content.

[0057] 6. Cytotoxicity analysis For the LAFs coating liquid treatment group, LAFs / TA coating liquid treatment group, LAFs / TA / ZnO-1 coating liquid treatment group, LAFs / TA / ZnO-2 coating liquid treatment group, LAFs / TA / ZnO-3 coating liquid treatment group, and LAFs / TA / ZnO-4 coating liquid treatment group, 0.8, 1.6, 2.4, 3.2, and 4.0 mg of coating samples were added to 4 mL of DMEM (37℃) and extracted for 24 h. After extraction, the supernatant was collected and clarified by centrifugation and other methods. The resulting liquid was the coating extract and was used for later use.

[0058] The cell viability of mouse intestinal epithelial cells (Wuhan Yunclone Technology Co., Ltd.) under the influence of various experimental materials was determined using the thiazolyl blue method. Specifically, cells were seeded in 96-well plates (1 × 10⁻⁶). 5 Cells were incubated at 37°C and 5% CO2 for 24 h. Then, 100 μL of different concentrations (0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL) of coating extract were added, and the cells were incubated at 37°C and 5% CO2 for another 24 h. After that, 10 μL of thiazolyl blue solution was added to each well, and the cells were incubated for 4 h. Then, 200 μL of dimethyl sulfoxide was added, and the absorbance was measured at 570 nm after 15 minutes to determine cell viability.

[0059] The results are as follows Figure 5As shown, the cell survival rate was highest in the LAFs coating solution treatment group after treatment with the coating extract. In addition, compared with the LAFs coating solution treatment group, the cell survival rate was slightly lower in the LAFs / TA coating solution treatment group with added tannic acid and the LAFs / TA / ZnO-1, LAFs / TA / ZnO-2, LAFs / TA / ZnO-3, and LAFs / TA / ZnO-4 coating solutions treatment groups with added tannic acid and zinc oxide, but the cell viability was greater than 80% in all groups (with cell viability greater than 85% in some groups). This indicates that the lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating formed by the lysozyme amyloid fiber / tannic acid / zinc oxide coating solution is a cell-compatible material.

[0060] 7. Antibacterial Analysis The antibacterial activity of the coating solutions against *Escherichia coli* and *Staphylococcus aureus* in each group was evaluated using the agar diffusion method. Specifically, 100 μL of bacterial suspension was first added to an agar plate. After placing an Oxford cup on the agar plate, 200 μL of the coating solution sample was added to the Oxford cup. Then, the space between the sample solution and the agar plate was removed by gentle pressure. The plate was incubated at 37°C for 24 h, and the diameter of the inhibition zone was recorded. An equal volume of PBS solution was included as a blank control group.

[0061] The results are as follows Figure 6 As shown, compared with the LAF coating liquid treatment group, the antibacterial coating material of the LAFs / TA coating liquid treatment group showed a significant increase in antibacterial effect, which is attributed to the synergistic antibacterial effect of tannic acid and lysozyme amyloid fibers; moreover, the antibacterial performance of the lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating material of the LAFs / TA / ZnO-1 coating liquid treatment group, LAFs / TA / ZnO-2 coating liquid treatment group, LAFs / TA / ZnO-3 coating liquid treatment group, and LAFs / TA / ZnO-4 coating liquid treatment group was further increased.

[0062] Other experimental results show that the lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating material has an inhibition rate of ≥95% against Staphylococcus aureus and Escherichia coli.

[0063] (iv) Fruit preservation effect Cherry tomatoes were immersed in a coating solution and dried (see Examples 1 to 6 above for specific procedures) before being stored. The storage conditions were as follows: placed in a food-grade plastic basket at a temperature of 25°C and a humidity of 85%–90%, in a cool and ventilated place. The condition of the cherry tomatoes (including dehydration, shrinkage, rotting, etc.) was recorded every three days for a total of 19 days.

[0064] The results are as follows Figure 7As shown, during the initial storage period (0-3 days), the cherry tomatoes in all groups appeared bright and plump, indicating similar freshness. With increasing storage time, the cherry tomatoes in the uncoated group (Control) and the LAFs-coated group became dull and wrinkled, while the cherry tomatoes in the LAFs / TA / ZnO-1, LAFs / TA / ZnO-2, LAFs / TA / ZnO-3, and LAFs / TA / ZnO-4 coated groups still retained a glossy appearance. By 9 days of storage, the cherry tomatoes in the uncoated group had already begun to rot (see...). Figure 7 (The part circled in "9d"), the cherry tomatoes in the other groups remained in good condition, and after 15 days of storage, only the cherry tomatoes in the LAFs / TA / ZnO-1 coating liquid treatment group, LAFs / TA / ZnO-2 coating liquid treatment group, LAFs / TA / ZnO-3 coating liquid treatment group, and LAFs / TA / ZnO-4 coating liquid treatment group did not show rot or large-area shrinkage of the surface. This indicates that the corresponding lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating formed on the surface of the cherry tomatoes can effectively extend the storage time of the cherry tomatoes. This is not only due to the good antibacterial properties of the coating, but more importantly, the coating also has good moisture retention and air permeability. Although the LAFs / TA / ZnO-3 and LAFs / TA / ZnO-4 coating solutions resulted in coating peeling off the cherry tomatoes after 15 days of storage (possibly due to excessive zinc oxide causing a rougher coating surface), the final experimental results showed that, compared to the uncoated cherry tomatoes, the antibacterial coatings formed on the surface of the cherry tomatoes in the LAFs / TA / ZnO-1, LAFs / TA / ZnO-2, LAFs / TA / ZnO-3, and LAFs / TA / ZnO-4 coating solutions extended the storage period of the cherry tomatoes to 18 days. Figure 7 The part circled in "18d" is mainly wrinkled.

[0065] (v) Advantages of the present invention (1) The lysozyme, tannic acid and zinc oxide used in the preparation of the antibacterial coating of this invention are industrial products, which are abundant, environmentally friendly, meet food-grade standards and have good biocompatibility.

[0066] (2) The antibacterial coating prepared by the present invention has good mechanical properties (such as adhesion properties), biocompatibility, moisturizing and breathable properties and antibacterial properties.

[0067] (3) The antibacterial coating prepared by the present invention can extend the shelf life of fruits at room temperature (for example, cherry tomatoes can be stored for more than 18 days).

[0068] (4) The preparation method of the antibacterial coating of the present invention is simple and easy to operate, which is conducive to improving food production efficiency and reducing production costs, and is suitable for promotion and application in industrial production.

Claims

1. A method for preparing a lysozyme amyloid fibril / tannic acid / zinc oxide antibacterial coating solution, characterized by: Includes the following steps: 1) Lysozyme amyloid cellulose solution was prepared by hydrothermal method using lysozyme as raw material; 2) Add tannic acid to the lysozyme amyloid cellulose solution to a final concentration of 20-80 mM, then stir at 30-50℃. After stirring until the mixture is homogeneous, stop heating and let stand to obtain the lysozyme amyloid cellulose / tannic acid complex. 3) Add zinc oxide to an acidic aqueous solution to a final concentration of 10-60 mM and adjust the pH to 3.5-5 to obtain a zinc oxide solution. Mix the zinc oxide solution with the lysozyme amyloid fiber / tannic acid complex and plasticizer to obtain a lysozyme amyloid fiber / tannic acid / zinc oxide coating solution.

2. The method for preparing the lysozyme amyloid cellulose / tannic acid / zinc oxide antibacterial coating liquid according to claim 1, characterized in that: In step 1, the conditions for the hydrothermal method include: a lysozyme solution mass fraction of 6% to 10%, a reaction temperature of 60 to 90°C, and a reaction time of 12 to 16 h.

3. The method for preparing a lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating liquid according to claim 1, characterized in that: Step 1 specifically includes the following steps: lysozyme is added to water to prepare a solution with a lysozyme mass fraction of 8%, and then 0.1~0.2 M hydrochloric acid solution is added to adjust the pH to 1.6~2. The solution is then reacted at 90℃ with stirring for 12~14 hours. After the reaction is completed, the solution is cooled to obtain a lysozyme amyloid cellulose solution.

4. The method for preparing a lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating liquid according to claim 1, characterized in that: In step 3, the acidic aqueous solution is selected from 0.1~0.2 M hydrochloric acid solution.

5. The method for preparing a lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating liquid according to claim 1, characterized in that: In step 3, the plasticizer is selected from glycerin.

6. The method for preparing a lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating liquid according to claim 5, characterized in that: In step 3, glycerol is added to the zinc oxide solution and the lysozyme amyloid cellulose / tannic acid complex at a final concentration of 30% to 35% before mixing, and the glycerol is uniformly dispersed. The zinc oxide solution containing glycerol is added to the lysozyme amyloid cellulose / tannic acid complex containing glycerol at a volume of 1 to 2 times, and then stirred for 10 to 30 minutes.

7. A method for preparing a lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating, characterized in that: Includes the following steps: The lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating liquid prepared by any one of the preparation methods described in claims 1 to 6 is subjected to ultrasonic treatment to remove air bubbles, and then the food is impregnated and dried to form a lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating on the surface of the food.

8. The method for preparing a lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating according to claim 7, characterized in that: The conditions for soaking include: soaking the food for 5 to 10 seconds and then removing it.

9. The method for preparing a lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating according to claim 7, characterized in that: The drying conditions are: air drying at 20~30℃.

10. The application of a method for preparing a lysozyme amyloid fiber / tannic acid / zinc oxide antibacterial coating as described in claim 7 in extending the shelf life of food or preserving food freshness.

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