Nanoenzyme, antibacterial film, preparation method and application thereof

Abstract

The present application belongs to the field of polymer material technology, and is specifically related to a kind of nanozyme, antibacterial film and preparation method and application. The present application uses ZIF-67 as a template, introduces chitosan as an in-situ growth matrix, and doped with copper ions, and the ZIF-67 structure is modified while etching, and then prepared by a solvent thermal method to produce CS / ZIF-67-Cu-based nanozymes that are not easy to aggregate, have enzyme-like activity and good antibacterial properties. The nanozyme obtained by the method is uniform and stable, simple to prepare, has excellent oxidase-like and peroxidase-like activities, can accelerate the production of reactive oxygen species during the catalytic reaction, and can effectively capture and kill typical Gram-negative bacteria and Gram-positive bacteria such as Escherichia coli and Staphylococcus aureus at lower concentrations, with a broad-spectrum antibacterial effect. In addition, the nanozyme of the present application can be well dispersed in carboxymethyl cellulose film materials, and the antibacterial film prepared has good mechanical properties and antibacterial properties, and can effectively maintain the color, moisture and nutrients of fresh fruits.

Description

Technical Field

[0001] The present application belongs to the technical field of polymer materials, and specifically relates to a CS / ZIF-67-Cu nanozyme, and an antibacterial film prepared using the nanozyme material. Background Art

[0002] Fresh food, such as fruits and vegetables, suffer from short storage times and perishability. This not only leads to food waste but also causes economic losses for agricultural producers and consumers. To improve food preservation technology and extend shelf life, antimicrobial films have emerged. These films incorporate antimicrobial agents into plastic wrap, achieving both antimicrobial and freshness-preserving properties through sustained release and photocatalytic effects. In recent years, with the advancement of nanomaterials, metal-organic frameworks (MOFs), due to their ultrahigh porosity and excellent thermal and chemical stability, have become a key material for the preparation of antimicrobial films, often serving as templates for loading and immobilizing nanoantimicrobial agents. Patent CN 116655970 A discloses a composite material, Ag@ZIF-67, using dimethylimidazolium cobalt (ZIF-67) as the metal-organic framework and loaded with silver ions. This material has been used to prepare a new type of plastic wrap. Fruit preservation experiments showed that when wrapped in cling film made from the Ag@ZIF-67 composite material, the fruit showed no mold growth during a six-day storage period, demonstrating that this cling film can, to a certain extent, slow the decay of food and effectively inhibit bacteria. However, the applicant discovered that the Ag@ZIF-67 nanozyme in the aforementioned patent relies solely on the release of silver ions to kill microorganisms and achieve antibacterial effects, resulting in poor antibacterial effects. Summary of the Invention

[0003] The purpose of this application is to provide a nanozyme with oxidase- and peroxidase-like activities. This nanozyme can not only kill microorganisms by releasing metal ions, but more importantly, it can disinfect microorganisms by generating reactive oxygen species (ROS) during the catalytic reaction, thereby achieving a better antibacterial effect. This is achieved specifically through the following technical solutions:

[0004] A method for preparing a nanozyme, wherein the nanozyme is prepared from modified dimethylimidazole cobalt and copper salt using a solvothermal method; the modified dimethylimidazole cobalt is chitosan-modified dimethylimidazole cobalt; and the molar ratio of cobalt ions in the chitosan-modified dimethylimidazole cobalt to copper ions in the copper salt is 1:(0.25~1).

[0005] Preferably, the preparation method of chitosan-modified cobalt dimethylimidazole comprises the following steps: dissolving cobalt salt in chitosan solution to obtain solution A; dissolving dimethylimidazole in sodium hydroxide solution to obtain solution B; and mixing solution B with solution A to react to obtain modified cobalt dimethylimidazole.

[0006] Preferably, the cobalt salt is cobalt nitrate hexahydrate; the chitosan concentration in the chitosan solution is 0.5-1.5%; and the amount of cobalt nitrate hexahydrate added to solution A is 0.02-0.04 g / mL.

[0007] Preferably, the chitosan solution is a chitosan acetic acid solution, and the concentration of the acetic acid is 1-2%.

[0008] Preferably, the concentration of the sodium hydroxide solution is 4-6%, and the amount of dimethylimidazole added to solution B is 0.11-0.27 g / mL.

[0009] Preferably, the conditions of the solvothermal method are: temperature 80-120° C., time 0.5-2 h.

[0010] A nanozyme is prepared using any of the preparation methods described above.

[0011] Application of any of the above nanozymes in the preparation of antibacterial materials.

[0012] An antibacterial film, wherein the raw materials for preparing the antibacterial film include any of the nanozymes described above.

[0013] Preferably, the raw materials for preparing the antibacterial film further include sodium carboxymethyl cellulose and a plasticizer.

[0014] Compared with the prior art, this application has the following beneficial effects:

[0015] The present application uses ZIF-67 as a template, introduces chitosan as an in-situ growth matrix, and dopes it with copper ions. The ZIF-67 structure is modified while etching, and then a simple one-step solvent thermal method is used to prepare a CS / ZIF-67-Cu-based nanozyme that is not easy to aggregate, has enzyme-like activity and good antibacterial properties. The nanozyme obtained by this method is uniform and stable, simple to prepare, has excellent oxidase-like and peroxidase-like activities, can accelerate the production of reactive oxygen species during the catalytic reaction, and can effectively capture and kill typical Gram-negative and Gram-positive bacteria such as Escherichia coli and Staphylococcus aureus at lower concentrations, with a broad-spectrum antibacterial effect. In addition, the nanozyme of the present application can be well dispersed in carboxymethyl cellulose film materials, and the prepared antibacterial film has good mechanical properties and antibacterial properties, which can effectively maintain the color, moisture and nutrients of fresh fruits. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] To clearly illustrate the embodiments, the following briefly introduces the drawings:

[0017] Figure 1The results of the oxidase-like and peroxidase-like catalytic activity determinations in Performance Test 1 of the present invention are as follows;

[0018] Figure 2 Comparison of the antibacterial effects of the CS / ZIF-67-Cu nanozyme according to the present invention and other antibacterial materials;

[0019] Figure 3 The antibacterial effect results of the CS / ZIF-67-Cu nanozyme antibacterial film according to the embodiment of the present invention are shown;

[0020] Figure 4 These are photos of the appearance of the CS / ZIF-67-Cu nanozyme antibacterial film of the present invention and the cherry tomatoes of the control group during storage;

[0021] Figure 5 This is a comparison of the changes in the total colony count of cherry tomatoes before and after storage in the CS / ZIF-67-Cu nanozyme antibacterial film of the embodiment of the present invention and the control group. DETAILED DESCRIPTION

[0022] The present application will be further described below in the form of specific embodiments. Those skilled in the art will be able to implement the present application based on these descriptions. In addition, the embodiments of the present application involved in the following description are generally only embodiments of a portion of the present application, rather than all embodiments. Therefore, based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making any creative efforts should fall within the scope of protection of the present application.

[0023] Example 1

[0024] This embodiment discloses a method for preparing a nanozyme, comprising the following steps:

[0025] S1. Synthesis of CS / ZIF-67 by room temperature stirring method: 0.4 g Co(NO3)2·6H2O was dissolved in 20 mL of 0.5% chitosan acetic acid solution (acetic acid concentration was 1%) and ultrasonicated for 10 min to obtain a uniform pink mixed solution A; 2.4 g dimethylimidazole was dissolved in 22 mL of 4% sodium hydroxide solution to obtain a transparent solution B, which was then slowly added dropwise to solution A until the pink color in solution A was completely converted into a purple precipitate. The mixture was then aged at room temperature for 12 h to allow the Co-N coordination bond to fully form and ensure the uniform growth of ZIF-67 on chitosan, thus obtaining a CS / ZIF-67 aging solution.

[0026] S2. Synthesis of CS / ZIF-67-Cu nanozyme by solvothermal method: Cu(NO3)2 was added to the aging solution prepared in S1 at a molar ratio of 1:1. After ultrasonic mixing for 30 minutes, the mixture was transferred to a reactor and incubated in an oven at 80°C for 2 hours. After cooling to room temperature, the mixture was filtered through a 0.45 μm microporous filter to obtain a solid powder. The powder was then washed three times with methanol and deionized water, respectively, and filtered again. The resulting solid powder was dried in a vacuum oven at 55°C for 12 hours to obtain CS / ZIF-67-Cu nanozyme powder.

[0027] Example 2

[0028] This embodiment discloses a method for preparing a nanozyme, comprising the following steps:

[0029] S1. Synthesize CS / ZIF-67 by room temperature stirring method: dissolve 0.6 g Co(NO3)2·6H2O in 15 mL of 1.5% chitosan acetic acid solution (acetic acid concentration is 2%), and ultrasonicate for 15 min to obtain a uniform pink mixed solution A; dissolve 4.8 g dimethylimidazole in 18 mL of 6% sodium hydroxide solution to obtain a transparent solution B, and then slowly add solution B dropwise to solution A until the pink in solution A is completely converted into a purple precipitate. Then age at room temperature for 24 h to fully form the Co-N coordination bond and ensure uniform growth of ZIF-67 on chitosan, thus obtaining CS / ZIF-67 aging solution.

[0030] S2. Synthesis of CS / ZIF-67-Cu nanozyme by solvothermal method: CuSO4 was added to the aging solution prepared in S1 at a molar ratio of 1:0.25. After ultrasonic mixing for 30 minutes, the mixture was transferred to a reactor and incubated in an oven at 80°C for 2 hours. After cooling to room temperature, the mixture was filtered through a 0.45 μm microporous filter to obtain a solid powder. The solid powder was then washed three times with methanol and deionized water, respectively, and filtered again. The resulting solid powder was dried in a vacuum oven at 60°C for 8 hours to obtain CS / ZIF-67-Cu nanozyme powder.

[0031] Example 3

[0032] This embodiment discloses a method for preparing an antibacterial film, comprising the following steps:

[0033] 7.5 mg of the CS / ZIF-67-Cu nanozyme powder prepared in Example 1 was weighed and dispersed in 1 mL of deionized water. Ultrasonication was performed for 5 min to uniformly disperse the nanozyme powder dispersion. 0.15 g of sodium carboxymethyl cellulose, 0.3 g of glycerol plasticizer, and 30 mL of water were mixed, and the nanozyme powder dispersion was added thereto. After stirring, the mixture was allowed to stand for 24 h to defoam. The film was then cast onto a mold using a casting method, air-dried at room temperature, and peeled off after drying to obtain the CS / ZIF-67-Cu nanozyme-based antibacterial film.

[0034] Example 4

[0035] This embodiment discloses a method for preparing an antibacterial film, comprising the following steps:

[0036] 23.5 mg of the CS / ZIF-67-Cu nanozyme powder prepared in Example 2 was weighed and dispersed in 1.5 mL of deionized water. Ultrasonication was performed for 10 min to uniformly disperse the nanozyme powder dispersion. 0.9 g of sodium carboxymethyl cellulose, 0.1 g of glycerol plasticizer, and 25 mL of water were mixed, and the nanozyme powder dispersion was added thereto. After stirring, the mixture was allowed to stand for 48 h to defoam. The film was then cast onto a mold using a casting method, air-dried at room temperature, and peeled off after drying to obtain a CS / ZIF-67-Cu nanozyme-based antibacterial film.

[0037] Comparative Example 1

[0038] In this comparative example, ZIF-67 was used as the nanozyme.

[0039] Comparative Example 2

[0040] The only difference between the CS / ZIF-67 nanozyme in this comparative example and Example 1 is that copper ions are not doped.

[0041] Comparative Example 3

[0042] The difference between the ZIF-67-Cu nanozyme in this comparative example and Example 1 is that the dimethylimidazole cobalt is not modified with chitosan.

[0043] Comparative Example 4

[0044] The difference between the ZIF-67-Ag nanozyme in this comparative example and comparative example 3 is that silver nitrate is used instead of copper nitrate for doping.

[0045] Comparative Example 5

[0046] The difference between the CS / ZIF-67-Ag nanozyme in this comparative example and comparative example 4 is that dimethylimidazole cobalt is modified with chitosan.

[0047] Comparative Example 6

[0048] The difference between the ZIF-67-Fe nanozyme in this comparative example and comparative example 3 is that iron nitrate is used instead of copper nitrate for doping.

[0049] Comparative Example 7

[0050] The difference between the CS / ZIF-67-Fe nanozyme in this comparative example and comparative example 6 is that dimethylimidazole cobalt is modified with chitosan.

[0051] Performance test 1, oxidase and peroxidase activity test

[0052] This performance test evaluated the oxidase- and peroxidase-like catalytic activities of the nanozymes of Example 1 and Comparative Examples 1-7. The oxidase-like activity was determined by adding 20 µL of 1.0 mg / mL nanozyme to a centrifuge tube, then adding 40 µL of 10 mM TMB (concentration in the system is mM) and 140 µL of acetic acid-sodium acetate buffer (pH = 4.5, 20 mM), mixing thoroughly, and then shaking at room temperature for 30 minutes. Finally, the change in the solution's absorbance at 652 nm was recorded. The peroxidase-like activity was determined by adding 20 µL of 1.0 mg / mL nanozyme to a centrifuge tube, then adding 40 µL of 10 mM TMB (concentration in the system is mM), 40 µL of 1 M H₂O₂ (final concentration 250 mM), and 100 µL of acetic acid-sodium acetate buffer (pH = 4.5, 20 mM), mixing thoroughly, and then shaking at room temperature for 30 minutes. Finally, record the change in the absorption intensity of the solution at 652 nm. Figure 1 It is known that the present application uses ZIF-67 as a template, introduces chitosan as an in-situ growth matrix, and selects doped copper ions, utilizes the coordination between copper ions and chitosan to etch and modify the ZIF-67 structure, and the prepared CS / ZIF-67-Cu nanozyme exhibits extremely strong class oxidase and class peroxidase activity. Although iron ions also have the function of catalyzing the production of reactive oxygen species, the experimental results show that under the CS / ZIF-67-M system prepared by the present application, the catalytic efficiency of CS / ZIF-67-Fe cannot rival the present application CS / ZIF-67-Cu. In addition, the applicant has also carried out nickel ion and zinc ion doping experiments, but its enzyme activity is also not as good as the present application. This also confirms from the opposite side that copper ion doping can be coordinated with chitosan modification to jointly improve class oxidase and class peroxidase activity.

[0053] Performance test 2, antibacterial performance test

[0054] This performance test was conducted on the antibacterial activity of the nanozymes of Example 1, Comparative Examples 1 and 2. The test method was as follows: For the inhibition zone experiment of the nanozyme, the concentration used was about 10 8CFU / mL of Escherichia coli suspension, draw 100μL of bacterial suspension on sterilized and condensed nutrient agar medium, and spread it evenly. Use the punching method to punch holes with a diameter of 6mm on the agar surface, use sterile water to disperse each nanozyme sample into a concentration of 20mg / mL, take 50μL and add it to the hole, then place the culture dish upright in a 37℃ incubator and culture for 18h to observe whether the nanozyme can form a clear inhibition zone on the agar gel. Figure 2 As shown in the results, CS / ZIF-67-Cu has a significantly larger inhibition zone than chitosan and other intermediate products ZIF-67 and CS / ZIF-67, indicating that CS / ZF-67-Cu has significantly enhanced antibacterial activity.

[0055] Performance test 3, antibacterial effect test

[0056] In this performance test, the antibacterial film was subjected to an antibacterial experiment. The co-culture dilution coating plate method was used. 500 mg of the composite film with 0%, 0.5%, 1.0%, and 1.5% CS / ZIF-67-Cu content was cut and added to 20 mL of 10 6 CFU / mL of bacterial suspension, inhibit bacteria at 25℃ for 0, 0.5, 1.0, 1.5, 2.0, and 2.5h, draw 100μL of bacterial suspension treated with antibacterial composite film onto sterilized and condensed nutrient agar medium, spread evenly, then place the culture dish upside down in a 37℃ incubator for 24h, and record the number of surviving colonies. Figure 3 As shown in the figure, the antibacterial film showed excellent antibacterial effect after 0.5 h, and the antibacterial effect of more than 99% was achieved in 0.5 h by relying solely on CMC@0.5%MOF (the mass of CS / ZIF-67-Cu accounted for 0.5% of the mass of sodium carboxymethyl cellulose used).

[0057] Performance test 4: Antibacterial film preservation effect test

[0058] This performance test used the CS / ZIF-67-Cu nanozyme-based antibacterial film prepared in Example 3 to package cherry tomatoes and verify its preservation effect. The specific experimental plan is as follows:

[0059] S1. Fresh-keeping treatment: 200 mature cherry tomatoes of uniform size, free of rot, pests, and mechanical damage were selected from the same batch. After washing with deionized water, they were dried at room temperature and treated with the following four methods: ① No treatment; ② PE film packaging; ③ CMC film packaging; ④ CMC@0.5% MOF film packaging; ⑤ CMC@1.0% MOF film packaging; ⑥ CMC@2.0% MOF film packaging.

[0060] S2. The treated cherry tomatoes were stored in a ventilated storage box at 25°C. The total colony count and appearance changes of the cherry tomatoes in each group were detected on the 0th, 3rd, 6th, 9th and 12th days to study the quality changes of cherry tomatoes during the storage process. Figure 4 As shown in the figure, from the changes in appearance, the cherry tomatoes in the untreated group and the PE ziplock bag group showed obvious shrinkage and mold on the 9th day, among which the untreated cherry tomatoes were obviously more severely rotten, while there was no obvious change in the other groups.

[0061] With reference to GB4789.2-2022, the changes in the total colony count of cherry tomatoes during storage were determined. Four small tomatoes were placed in a sterile sampling bag, crushed with a slapping homogenizer, 5 g was weighed and transferred to another sterile sampling bag, 45 mL of sterile saline was added and slapped evenly to make a 1:10 sample solution. Prepare three gradients of 10-fold dilutions, draw 1 mL of the sample solution into a sterile petri dish, and make two petri dishes for each dilution. At the same time, draw 1 mL of blank dilution into two sterile petri dishes as blank controls. Pour 15 mL - 20 mL of plate count agar culture cooled to 46 ° C ~ 50 ° C into the petri dish in time, and turn the petri dish to mix it evenly. Place horizontally and wait for the agar to solidify, then turn the plate over and incubate at 37 ° C for 48 hours. If the sample may contain colonies spreading and growing on the surface of the agar culture medium, a thin layer of plate count agar culture medium (about 4 mL) can be covered on the surface of the solidified agar culture medium, and the plate can be turned over after solidification for incubation. As Figure 5 As shown in the figure, the antibacterial film containing the nanozyme of the present invention can significantly inhibit the growth of microorganisms in cherry tomatoes, judging by the changes in the total colony count. As the concentration of the nanozyme increases, the inhibitory effect gradually increases. In summary, the antibacterial film of the present invention can effectively inhibit microbial contamination during the storage of cherry tomatoes and extend the storage period of cherry tomatoes.

Claims

1. A method for preparing a nanozyme, characterized in that: The nanozyme is prepared by a solvent thermal method using modified dimethylimidazole cobalt and copper salt; the modified dimethylimidazole cobalt is chitosan-modified dimethylimidazole cobalt; the molar ratio of cobalt ions in the chitosan-modified dimethylimidazole cobalt to copper ions in the copper salt is 1:(0.25~1).

2. The method for preparing a nanozyme according to claim 1, wherein: The preparation method of chitosan-modified cobalt dimethylimidazole comprises the following steps: dissolving cobalt salt in chitosan solution to obtain solution A; dissolving dimethylimidazole in sodium hydroxide solution to obtain solution B; and mixing solution B with solution A to react and obtain modified cobalt dimethylimidazole.

3. The method for preparing a nanozyme according to claim 2, wherein: The cobalt salt is cobalt nitrate hexahydrate; the chitosan concentration in the chitosan solution is 0.5-1.5%; and the amount of cobalt nitrate hexahydrate added to solution A is 0.02-0.04 g / mL.

4. The method for preparing a nanozyme according to claim 3, wherein: The chitosan solution is a chitosan acetic acid solution, and the concentration of the acetic acid is 1-2%.

5. The method for preparing a nanozyme according to claim 2, wherein: The concentration of the sodium hydroxide solution is 4-6%, and the amount of dimethylimidazole added to solution B is 0.11-0.27 g / mL.

6. The method for preparing a nanozyme according to any one of claims 1 to 5, characterized in that: The conditions of the solvothermal method are: temperature 80-120° C., time 0.5-2 h.

7. A nanozyme, characterized in that It is prepared using the preparation method according to any one of claims 1 to 6.

8. Use of the nanozyme according to claim 7 in the preparation of antibacterial materials.

9. An antibacterial film, characterized in that: The raw materials for preparing the antibacterial film include the nanozyme according to claim 7.

10. The antibacterial film according to claim 9, characterized in that: The raw materials for preparing the antibacterial film also include sodium carboxymethyl cellulose and a plasticizer.

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