Bacteriostatic enhanced two-dimensional zinc oxide nano-composite as well as preparation method and application thereof

Abstract

The invention discloses a bacteriostatic enhanced two-dimensional zinc oxide nano-composite and a preparation method and application thereof, and relates to the field of bacteriostatic materials, the preparation method comprises the following steps: dispersing dimethylimidazole in DMF to obtain a score dispersion A, dispersing a Zn-GL precursor in DMF to obtain a score dispersion B, dispersing zinc oxide nano-particles in DMF to obtain a score dispersion C; mixing the dispersoid B and the dispersoid C, continuously stirring and dropwise adding the dispersoid A to obtain a mixed solution; the mixed solution is subjected to a closed heating reaction at the temperature of 150-170 DEG C, after the reaction is finished, the mixed solution is subjected to annealing treatment, generated precipitates are separated out, then the precipitates are freeze-dried, and the zinc oxide nanocomposite is obtained. The bacteriostatic enhanced two-dimensional zinc oxide nano-composite constructed by the invention has stronger bacteriostatic activity, and has a wider application prospect in the fields of grain storage, long-distance transportation, carrier mildew prevention and the like.

Description

Technical Field

[0001] The present invention relates to the field of antibacterial materials, and specifically to an antibacterial-enhanced two-dimensional zinc oxide nanocomposite, a preparation method thereof, and an application thereof. Background Art

[0002] The embryo part of corn kernels is relatively large, and it is rich in its own nutrients, has strong hygroscopicity, a large amount of bacteria, and a relatively high moisture content at the time of harvest. Therefore, it is easily invaded by microorganisms and undergoes heating and mildew during storage. The heating of the grain pile caused by the growth and reproduction of microorganisms will further exacerbate the deterioration process of corn quality, and the metabolized mycotoxins will directly threaten people's life and health. During the storage of corn, with the continuous improvement of requirements such as "green and healthy" and "quality improvement and efficiency increase", it is urgent to develop an efficient and stable mildew-proof agent.

[0003] As an antibacterial and mildew-proof agent with great application potential, the antibacterial zinc oxide nano material has been verified to have the effects of inhibiting the growth of microorganisms and inhibiting the metabolism of mycotoxins during the long-term storage of grains, and at the same time shows the application potential in maintaining the stability of grain storage quality. However, due to the high specific surface area and low surface potential of zinc oxide nanoparticles, it is easy for particles to agglomerate with each other, forming larger particle clusters, resulting in the weakening of the nano effect; and sedimentation or aggregation occurs during storage or use, resulting in poor dispersion stability. These properties will all lead to a reduction in the antibacterial ability of zinc oxide nanoparticles during the storage of corn.

[0004] Reference 1: Chinese patent document with the publication number of CN 111034720 A.

[0005] Reference 1 discloses a preparation method of a zinc oxide-metal organic framework composite antibacterial material, which uses lanthanide MOFs with good biocompatibility and easy preparation as the framework material; MOFs-Ln is prepared by using thiodi acetic acid (TDA) and lanthanide nitrate. Through two-step exfoliation, the particle size and porosity of MOFs-Ln can be adjusted to obtain MOFs structures of different sizes; subsequently, in an in-situ growth manner, zinc oxide NPs are grown on the surface and within the pores of MOFs-Ln by using zinc nitrate, sodium hydroxide, and sodium chloride, and then the ZnO NPs@MOFs-Ln material with good antibacterial performance can be obtained. The realization of the high-efficiency antibacterial effect of this invention is mainly based on the growth of a large number of ZnO NPs in the pores and on the surface of MOFs, which improves the ZnO loading efficiency of the composite material.

[0006] Reference 2: Chinese patent document with the publication number of CN111972493B.

[0007] Reference 2 discloses an application, preparation method and recovery method of a zinc oxide nano antibacterial composite in grain storage. The preparation method includes the preparation of zinc oxide nanoparticles, the modification of mesoporous silica on the surface of zinc oxide nanoparticles and the modification of surface amphoteric groups. Specifically, by modifying mesoporous silica on the surface of zinc oxide nanoparticles, the antibacterial effect of zinc oxide nanoparticles can be exerted, and the pollution caused by the direct contact between the antibacterial core of zinc oxide nanoparticles and grains can be avoided. Moreover, more importantly, the designed amphoteric group C16CA enables the zinc oxide nano composite to have the characteristics of "acid precipitation and alkali dissolution", which can be removed by rinsing and recycled, completely eliminating the potential food safety hazards brought by the zinc oxide nano antibacterial composite. Summary of the Invention

[0008] The present invention aims to provide an antibacterial enhanced two-dimensional zinc oxide nano composite, its preparation method and application to obtain a highly efficient antibacterial agent based on zinc oxide nanoparticles.

[0009] To achieve the above object, the specific solution adopted by the present invention is as follows: A preparation method of an antibacterial enhanced two-dimensional zinc oxide nano composite, comprising the following steps:

[0010] Disperse dimethylimidazole in DMF to obtain dispersion A, disperse the Zn-GL precursor in DMF to obtain dispersion B, and disperse zinc oxide nanoparticles in DMF to obtain dispersion C;

[0011] Mix dispersion B and dispersion C, continuously stir and dropwise add dispersion A to obtain a mixed solution;

[0012] Heat the mixed solution in a sealed manner at 150-170 °C for reaction. After the reaction is completed, cool the mixed solution to room temperature, separate the generated precipitate, and then freeze-dry the precipitate to obtain the zinc oxide nano composite.

[0013] As a further optimization of the above technical solution: The preparation method of the Zn-GL precursor is: Add anhydrous zinc acetate to glycerol, heat to 120 °C and keep for 30 min, then heat to 150 °C and keep for 1 h, cool, separate the obtained precipitate, and vacuum freeze-dry to obtain the Zn-GL precursor.

[0014] As a further optimization of the above technical solution: When dropping dispersion A into the solution after mixing dispersion B and dispersion C, the stirring speed is 200 rpm.

[0015] As a further optimization of the above technical solution: The molar ratio of dimethylimidazole, Zn-GL precursor and zinc oxide nanoparticles in the mixed solution is 8:2:1.

[0016] As a further optimization of the above technical solution: the concentration of 2-methylimidazole in Dispersion A is 0.25 - 0.30 mmol / mL; the concentration of Zn-GL precursor in Dispersion B is 0.06 - 0.08 mmol / mL; the concentration of zinc oxide nanoparticles in Dispersion C is 2.30 - 2.70 mg / mL.

[0017] As a further optimization of the above technical solution: the time for sealed heating is 15 - 17 h.

[0018] An antibacterial enhanced two-dimensional zinc oxide nanocomposite is prepared by the method described in any one of the above.

[0019] Application of an antibacterial enhanced two-dimensional zinc oxide nanocomposite as a bacterial growth inhibitor or a mold growth inhibitor.

[0020] As a further optimization of the above technical solution: the bacteria are Staphylococcus aureus or Escherichia coli, and the molds are Aspergillus niger, Aspergillus flavus or Penicillium citrinum.

[0021] Application of an antibacterial enhanced two-dimensional zinc oxide nanocomposite in food storage.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention uses a top-down template method in the self-assembly mechanism. By introducing a specific template and controlling the temperature, solvent and reaction time, a composite material with specific morphology, size and function is accurately synthesized.

[0024] The antibacterial enhanced two-dimensional zinc oxide nanocomposite of the present invention has a better inhibitory effect on the selected model bacteria Staphylococcus aureus and the model mold Aspergillus niger than zinc oxide nanoparticles at the same concentration; when simulating the storage of corn, the antibacterial enhanced two-dimensional zinc oxide nanocomposite can inhibit the growth and reproduction of microorganisms during the storage of corn, and is an ideal mildew-proof preparation.

[0025] The antibacterial enhanced two-dimensional zinc oxide nanocomposite constructed by the present invention is based on the application of ZnO-NPs in the growth and reproduction of microorganisms and the simulation of corn storage during corn storage. Through a top-down template method in the self-assembly effect, with glycerol-zinc with a flake structure as the precursor, while adding zinc oxide nanoparticles, 2-methylimidazole replaces the glycerol group in the precursor to form a new MOF structure embedding zinc oxide nanoparticles on the micron-scale flake structure. The constructed antibacterial enhanced two-dimensional zinc oxide nanocomposite not only has the broad-spectrum antibacterial activity of ZnO nanoparticles, but also the surface-modified new MOF framework structure has a loose pore structure and can achieve ordered self-assembly to form a micron-scale flake structure.

[0026] The antibacterial-enhanced two-dimensional zinc oxide nanocomposite constructed by the present invention has stronger antibacterial activity and has broader application prospects in the fields of grain storage, long-distance transportation, and vehicle mildew prevention. The MOF structure and ZnO nanoparticles form a synergistic antibacterial effect, with a lethality rate of up to 100% against Staphylococcus aureus and a minimum inhibitory concentration of 0.075 mg / mL; the lethality rate against Aspergillus niger can reach 86.2%, and the minimum inhibitory concentration is 0.3 mg / mL. Description of the Drawings

[0027] Figure 1 Schematic diagram of the preparation principle of the antibacterial-enhanced two-dimensional zinc oxide nanocomposite;

[0028] Figure 2 Characterization of 2D ZnO@MOF: (A) Sample picture, (B) SEM image, (C) TEM image, (D) Element mapping composite spectrum, (E) EDS element spectrum, (F) Particle size distribution of 2D ZnO@MOF and ZnO, (G) Comparison of XRD patterns of 2D ZnO@MOF and ZnO with the standard PDF card, (H) Nitrogen adsorption and desorption isotherms of 2D ZnO@MOF and ZnO, (I) Pore size distribution of 2D ZnO@MOF and ZnO;

[0029] Figure 3 Antibacterial effects of different antibacterial particles against Staphylococcus aureus and Aspergillus niger;

[0030] Figure 4 Situation of lethality rate of different antibacterial particles against Staphylococcus aureus (A) and inhibition rate of Aspergillus niger hyphal growth (B);

[0031] Figure 5 Effects of ZnO and 2D ZnO@MOF on the ROS content in Aspergillus niger, Penicillium citrinum, and Aspergillus flavus;

[0032] Figure 6 Inhibition of different concentrations of 2D ZnO@MOF against bacteria (Staphylococcus aureus and Escherichia coli);

[0033] Figure 7 Inhibition of different concentrations of 2D ZnO@MOF against molds (Aspergillus flavus, Penicillium citrinum, and Aspergillus niger);

[0034] Figure 8 Inhibition of spore germination of different concentrations of 2D ZnO@MOF and microscopic images (AD Aspergillus niger, BE Penicillium citrinum, CF Aspergillus flavus);

[0035] Figure 9 Effects of different concentrations of 2D ZnO@MOF on the zinc element accumulation in corn;

[0036] Figure 10 Effect of 2D ZnO@MOF with different concentrations on the total number of fungal colonies on the surface of corn. Detailed implementation manners

[0037] The technical solutions of the present invention will be further elaborated in detail below in combination with specific embodiments. For parts not detailedly recorded and disclosed in the following embodiments of the present invention, they should all be understood as the prior art known or should be known to those skilled in the art.

[0038] The present invention discloses a preparation method of an antibacterial enhanced two-dimensional zinc oxide nano-composite, comprising the following steps:

[0039] Add anhydrous zinc acetate to glycerol under continuous magnetic stirring, heat the solution to 120 °C at a heating rate of 3 °C / min and keep it for 30 min, and keep it in an oil bath at 120 °C for 30 min. At this time, the solution gradually becomes clear and transparent; then increase the temperature to 150 °C at a heating rate of 2 °C / min -1 and keep it at this temperature for 1 h. At this time, the solution gradually becomes turbid and forms a white suspension precipitate. After naturally cooling to room temperature, the white precipitate is separated, and finally vacuum freeze-dried to obtain the Zn-GL precursor. -1 Disperse dimethylimidazole in DMF (full name: N,N-dimethylformamide), control the concentration of dimethylimidazole to be 0.25 - 0.30 mmol / mL to obtain dispersion A, disperse the Zn-GL precursor in DMF, control the concentration of the Zn-GL precursor to be 0.06 - 0.08 mmol / mL to obtain dispersion B, and disperse zinc oxide nanoparticles in DMF, control the concentration of zinc oxide nanoparticles to be 2.30 - 2.70 mg / mL to obtain dispersion C.

[0040] Mix dispersion B and dispersion C at a stirring speed of 200 rpm, continuously stir and dropwise add dispersion A to obtain a mixed solution. The molar ratio of dimethylimidazole, Zn-GL precursor and zinc oxide nanoparticles in the mixed solution is 8:2:1. Seal and heat the mixed solution at 150 - 170 °C for 15 - 17 h, cool to room temperature, separate the obtained precipitate, and freeze-dry to obtain the zinc oxide nano-composite.

[0041] The zinc oxide nano-composite disclosed by the present invention, through a top-down template method in the self-assembly effect, uses glycerol-zinc with a sheet structure as a precursor. While adding zinc oxide nanoparticles, dimethylimidazole replaces the glycerol group in the precursor to form a new MOF structure embedding zinc oxide nanoparticles on the micron-scale sheet structure, also known as 2D ZnO@MOF nano-composite.

[0042]

[0043] Example 1

[0044] A preparation method of an antibacterial enhanced two-dimensional zinc oxide nanocomposite is as follows:

[0045] 1) Referring to the method disclosed in "Zhang Dongdong, Hu Si, Hayat Zakir, et al. Inhibitory effect of zinc oxide nanoparticles on microorganisms during maize storage [J]. Food Science, 2021, 42(21): 137-144.", zinc oxide nanoparticles are prepared.

[0046] 2) Add 100 mL of glycerol (GL) into a 250 mL round-bottom flask, and add 5.0 g of anhydrous zinc acetate under continuous magnetic stirring. Heat the solution to 120 °C at a heating rate of about 3 °C min -1 and keep it in an oil bath at 120 °C for 30 min. The solution gradually becomes clear and transparent; heat the temperature to 150 °C at a heating rate of 2 °C min -1 and keep it at this temperature for 1 h. As the temperature rises to 150 °C, the solution gradually becomes turbid and forms a white suspended precipitate. After the solution is naturally cooled to room temperature, wash it 4 times with absolute ethanol, centrifuge and separate the precipitate at 4000 rpm, and finally vacuum freeze-dry the precipitate to obtain the Zn-GL precursor.

[0047] 3) Dissolve 8 mmol of 2-methylimidazole (2-MIM) in 30 mL of DMF to obtain dispersion A; dissolve 2 mmol of the Zn-GL precursor in another 30 mL of DMF to obtain dispersion B; dissolve zinc oxide nanoparticles in DMF to prepare a suspension with a concentration of 2.5 mg / mL to obtain dispersion C; treat dispersions A, B, and C in ultrasonic for 30 min to make them uniformly dispersed;

[0048] At room temperature, under the condition of a stirring speed of 200 rpm, first mix 30 mL of dispersion B and 30 mL of dispersion C evenly, and then dropwise add 30 mL of dispersion A to the mixture of dispersion B + dispersion C drop by drop. The whole process lasts for 30 min to obtain a mixed dispersion. Finally, transfer the mixed dispersion to a Teflon reaction kettle, close it and heat it at 160 °C for 16 h. After naturally cooling to room temperature, wash it 3 times with absolute ethanol, centrifuge and recycle the solid precipitate in the mixed dispersion at 4000 rpm, and finally form a soft white powder by freeze-drying the separated solid precipitate, which is the antibacterial enhanced two-dimensional zinc oxide nanocomposite.

[0049] It should be noted that this antibacterial enhanced two-dimensional zinc oxide nanocomposite is 2D ZnO@MOF.

[0050] Example 2

[0051] A preparation method of an antibacterial-enhanced two-dimensional zinc oxide nanocomposite is as follows:

[0052] This example is the same as steps 1) and 2) of the embodiment, the difference is that:

[0053] Step 3): Dissolve 2-methylimidazole (2-MIM) in DMF to obtain dispersion A; dissolve the Zn-GL precursor in another DMF to obtain dispersion B; dissolve zinc oxide nanoparticles in DMF to prepare a suspension with a concentration of 2.5 mg / mL to obtain dispersion C; adjust the addition amounts of 2-methylimidazole, the Zn-GL precursor, and zinc oxide nanoparticles so that the concentration of 2-methylimidazole in dispersion A is 0.25 mmol / mL; the concentration of the Zn-GL precursor in dispersion B is 0.06 mmol / mL; the concentration of zinc oxide nanoparticles in dispersion C is 2.70 mg / mL; Treat dispersions A, B, and C in ultrasonic waves for 30 min to make them uniformly dispersed;

[0054] Under the conditions of room temperature and a stirring speed of 200 rpm, first mix 30 mL of dispersion B and 30 mL of dispersion C evenly, and then dropwise add 30 mL of dispersion A to the mixture of dispersion B + dispersion C drop by drop. The whole process lasts for 30 min to obtain a mixed dispersion. Finally, transfer the mixed dispersion to a Teflon reactor, seal it and heat it at 150 °C for 17 h. After naturally cooling to room temperature, wash it 3 times with absolute ethanol, centrifuge and circulate to separate the solid precipitate in the mixed dispersion at 4000 rpm. Finally, the separated solid precipitate is freeze-dried to form a soft white powder, which is the antibacterial-enhanced two-dimensional zinc oxide nanocomposite.

[0055] Example 3

[0056] A preparation method of an antibacterial-enhanced two-dimensional zinc oxide nanocomposite is as follows:

[0057] This example is the same as steps 1) and 2) of Example 1, the difference is that:

[0058] Step 3): Dissolve 2-methylimidazole (2-MIM) in DMF to obtain dispersion A; dissolve the Zn-GL precursor in another DMF to obtain dispersion B; dissolve zinc oxide nanoparticles in DMF to prepare a suspension with a concentration of 2.5 mg / mL to obtain dispersion C; adjust the addition amounts of 2-methylimidazole, the Zn-GL precursor, and zinc oxide nanoparticles so that the concentration of 2-methylimidazole in dispersion A is 0.30 mmol / mL; the concentration of the Zn-GL precursor in dispersion B is 0.08 mmol / mL; the concentration of zinc oxide nanoparticles in dispersion C is 2.30 mg / mL; Treat dispersions A, B, and C in ultrasonic waves for 30 min to make them uniformly dispersed;

[0059] Under the conditions of room temperature and a stirring speed of 200 rpm, first mix 30 mL of dispersion B and 30 mL of dispersion C evenly, and then dropwise add 30 mL of dispersion A to the mixture of dispersion B + dispersion C. The whole process lasts for 30 min to obtain a mixed dispersion. Finally, transfer the mixed dispersion to a Teflon reactor, seal it, and heat it at 170 °C for 15 h. After naturally cooling to room temperature, wash it 3 times with absolute ethanol, centrifuge and circulate to separate the solid precipitate in the mixed dispersion at 4000 rpm. Finally, the separated solid precipitate is freeze-dried to form a soft white powder, which is the antibacterial-enhanced two-dimensional zinc oxide nanocomposite.

[0060] Comparative Example 1

[0061] According to the method in step 1) of Example 1, that is, referring to the method disclosed in "Zhang Dongdong, Hu Si, Hayat Zakir, et al. Inhibitory effect of zinc oxide nanoparticles on microorganisms during corn storage [J]. Food Science, 2021, 42(21): 137-144.", zinc oxide nanoparticles were prepared.

[0062] Comparative Example 2

[0063] Prepare the MOF framework. The specific method is as follows:

[0064] According to the method in step 2) of Example 1, prepare the Zn-GL precursor;

[0065] Then dissolve 8 mmol of dimethylimidazole (2-MIM) in 30 mL of DMF to obtain dispersion A; dissolve 2 mmol of the Zn-GL precursor in another 30 mL of DMF to obtain dispersion B; treat dispersions A and B in ultrasonic for 30 min to make them evenly dispersed;

[0066] Under the conditions of room temperature and a stirring speed of 200 rpm, mix 30 mL of dispersion B and 30 mL of dispersion C evenly, then transfer the mixed dispersion to a Teflon reactor, seal it, and heat it at 160 °C for 16 h. After naturally cooling to room temperature, wash it 4 times with absolute ethanol, centrifuge and circulate to separate the solid in the mixed dispersion at 4000 rpm. Finally, the separated solid is freeze-dried to form a white powder, which is the MOF framework.

[0067] Next, the performance of the zinc oxide nanocomposite prepared in Example 1 was detected as follows.

[0068] I. Microstructure and Component Identification of Zinc Oxide Nanocomposite

[0069] As Figure 1As shown in the figure, the present invention adopts a "top-down" template method to form a novel two-dimensional nano-scale MOF framework material (2D ZnO@MOF) that can uniformly embed zinc oxide nanoparticles through aggregation self-assembly in only one dimension.

[0070] It is known from Figure 2 that 2D ZnO@MOF is a white, soft-textured powder, which is assembled by the aggregation of 12-sided MOF small particles in only one direction, and has a flake structure with an overall particle size distribution in the range of 1990 - 2670 nm. It can be confirmed by transmission electron microscopy and EDS spectroscopy that zinc oxide nanoparticles are successfully embedded in the MOF framework. According to the analysis of the results of the elemental mapping, the 2D ZnO@MOF nanocomposite only contains C, O, Zn, and N elements, among which the proportion of C element is 53.7%, the proportion of O element is 4.43%, the proportion of Zn element is 28.95%, and the proportion of N element is 12.92%. The X-ray diffraction pattern shows that the characteristic diffraction peaks of the 2D ZnO@MOF nanocomposite are located at 2θ = 7.297, 10.338, 12.678, 14.658, 16.4, 17.981, 22.08, 31.7, 34.042, 36.18, 47.679, 56.561, 66.17, 67.938, 69.097, 79.023, which is basically consistent with the zinc blende phase of ZnO nanoparticles. However, characteristic diffraction peaks of MOF appear in the nanocomposite, and MOF has no influence on the crystal structure of ZnO; it is in good agreement with the diffraction peaks of (011), (002), (112), (022), (013), (222), (114), (013), (014), (100), (101), (102), (110), (103), (200), (201), (004), (202), (104), (203), indicating that the 2D ZnO@MOF nanocomposite has a good crystal plane matching with the standard zinc blende phase XRD pattern (JCPDS 5-0664) of ZnO and the crystal plane of MOF. Through nitrogen adsorption (BET) method testing, the specific surface area of the 2D ZnO@MOF nanocomposite reaches 1124.50 m 2 / g, which is much higher than that of unmodified ZnO nanoparticles. At the same time, the porosity was analyzed, and the average pore diameter of the 2D ZnO@MOF nanocomposite was calculated to be 1.58 nm, which is a microporous structure.

[0071] II. Inhibitory effect of zinc oxide nanocomposite on dominant microorganisms in corn

[0072] According to the research of Zhang Dongdong et al., specifically referring to "Zhang Dongdong, Zhao Jinfeng, Xie Siyuan, et al. Analysis of Microbial Diversity in Maize Based on High-Throughput Sequencing [J]. Journal of Chinese Institute of Food Science and Technology, 2023, 23(10): 305-314", Staphylococcus aureus, Escherichia coli, Aspergillus flavus, Aspergillus niger, and Penicillium citrinum, which have relatively high abundances and seriously affect the quality in maize, were selected as model microorganisms to study the antibacterial effect of 2D ZnO@MOF. All the strains used in the experiment were extracted from stored maize.

[0073] <Detection of Antibacterial Performance of 2D ZnO@MOF>

[0074] Staphylococcus aureus (S. aureus) and Aspergillus niger (A. niger) were selected as model microorganisms. For bacteria, the antibacterial enhancement characteristics of 2D ZnO@MOF were verified by the plate colony counting method and the minimum inhibitory concentration (MIC) determination; for molds, the agar punching method and the minimum inhibitory concentration (MIC) determination were selected to verify the antibacterial enhancement characteristics of 2D ZnO@MOF.

[0075] 1. Plate Colony Counting Method

[0076] In order to avoid affecting the test results due to different dispersion degrees of the inhibitor, during the preparation of the following suspensions Ⅰ, Ⅱ, Ⅲ, and Ⅳ in the present invention, the dispersion degree of the suspension was detected to ensure that the dispersion of the inhibitor in each group of suspensions was similar.

[0077] 1) The 2D ZnO@MOF prepared in Example 1 was suspended in distilled water to prepare a 2D ZnO@MOF suspension with a concentration of 0.25 mg / mL (i.e., suspension Ⅰ); the suspension Ⅰ was ultrasonically treated for 15 min under the condition of an ultrasonic power of 240 W. After the 2D ZnO@MOF was dispersed evenly, dynamic light scattering test (DLS) was carried out, and the polymer dispersity index (PDI) of the suspension Ⅰ was 0.041, indicating that the dispersity of the 2D ZnO@MOF suspension was similar to that of suspensions Ⅱ, Ⅲ, and Ⅳ.

[0078] The ZnO prepared in Comparative Example 1 was suspended in distilled water to prepare suspension Ⅱ with a concentration of 0.25 mg / mL; the suspension Ⅱ was ultrasonically treated for 15 min under the condition of an ultrasonic power of 240 W. After the ZnO was dispersed evenly, dynamic light scattering test (DLS) was carried out, and the polymer dispersity index (PDI) of the suspension Ⅱ was 0.038, indicating that the dispersity of the suspension Ⅱ was similar to that of suspensions Ⅰ, Ⅲ, and Ⅳ.

[0079] The MOF prepared in Comparative Example 2 was suspended in distilled water to prepare suspension III with a concentration of 0.25 mg / mL. Suspension III was sonicated for 15 min at an ultrasonic power of 240 W. After the MOF was evenly dispersed, dynamic light scattering (DLS) measurement was carried out, and the polymer dispersity index (PDI) of suspension III was 0.04, indicating that the dispersity of suspension III was similar to that of suspensions I, II, and IV.

[0080] Theoretically, the mass ratio of ZnO to MOF in 2D ZnO@MOF is 1:4. Therefore, ZnO prepared in Comparative Example 1 and MOF prepared in Comparative Example 2 were mixed in a ratio of 1:4 to prepare suspension IV with a ZnO+MOF concentration of 0.25 mg / mL. Suspension IV was sonicated for 15 min at an ultrasonic power of 240 W. After the mass ratio of ZnO to MOF of 1:4 was evenly dispersed, dynamic light scattering (DLS) measurement was carried out, and the dispersity index (PDI) of suspension IV was 0.037, which was similar to the dispersity of suspensions I, II, and III.

[0081] Suspensions I, II, III, and IV were respectively mixed evenly with nutrient agar medium (LB). The Staphylococcus aureus bacterial solution was serially diluted to a concentration at which plate counting could be performed (the number of colonies on each plate was within 30 - 300). 100 μL of the Staphylococcus aureus bacterial suspension was respectively and evenly spread on the medium without nanoparticles (i.e., the control group), the medium containing suspension I (i.e., 0.25 mg / mL of 2D ZnO@MOF), the medium containing suspension II (i.e., 0.25 mg / mL of ZnO NPs), the medium containing suspension III (i.e., 0.25 mg / mL of MOF), and the medium containing suspension IV (i.e., 0.25 mg / mL of ZnO+MOF), and this step was repeated three times to ensure the accuracy of the experiment. The spread media were placed in a constant temperature and humidity incubator and cultured at 37 °C for 24 h.

[0082] 2. Determination of minimum inhibitory concentration (MIC) of bacteria

[0083] The minimum inhibitory concentration (MIC) is measured by determining the lowest concentration of a sample at which no bacterial growth is observed. Staphylococcus aureus (S. aureus) purified from stored corn was selected as the experimental strain. ZnO NPs, 2D ZnO@MOF nanocomposites, and ZnO@mSiO 2 were serially diluted to a concentration range from 0.01 mg / mL to 500 mg / mL. It should be noted that the preparation process of ZnO@mSiO 2 refers to the patent document with the publication number CN111972493B in the prior art.

[0084] Culture Staphylococcus aureus to a concentration of 1×10 6 CFU / mL. When performing the MIC test, first add 100 μL of the Staphylococcus aureus bacterial solution to each well of a 96-well plate, and then add 100 μL of the LB liquid medium of the nanomaterial that has been gradient-diluted. After that, use a microplate reader to measure the initial absorbance at 595 nm. Then, place the 96-well plate in a shaker at 37 °C and incubate it gently for 12 h. Finally, use the microplate reader to measure the final absorbance at 595 nm again to complete the determination of MIC.

[0085] 3. Agar well diffusion method

[0086] 1) Take the 2D ZnO@MOF prepared in Example 1 and suspend it in distilled water to prepare suspension I with a concentration of 1.5 mg / mL; place suspension I under an ultrasonic power of 240 W and ultrasonically treat it for 15 min. After uniformly dispersing 2D ZnO@MOF, perform dynamic light scattering test (DLS). The polymer dispersity index (PDI) of suspension II is 0.12, indicating that the dispersibility of the 2D ZnO@MOF suspension is similar to that of suspensions II, III, and IV.

[0087] Take the ZnO prepared in Comparative Example 1 and suspend it in distilled water to prepare suspension II with a concentration of 1.5 mg / mL; place suspension II under an ultrasonic power of 240 W and ultrasonically treat it for 15 min. After uniformly dispersing ZnO, perform dynamic light scattering test (DLS). The polymer dispersity index (PDI) of suspension II is 0.12, indicating that the dispersibility of suspension II is similar to that in suspensions I, III, and IV.

[0088] Take the MOF prepared in Comparative Example 2 and suspend it in distilled water to prepare suspension III with a concentration of 1.5 mg / mL; place suspension III under an ultrasonic power of 240 W and ultrasonically treat it for 15 min. After uniformly dispersing MOF, perform dynamic light scattering test (DLS). The polymer dispersity index (PDI) of suspension III is 0.11, indicating that the dispersibility of suspension III is similar to that in suspensions I, II, and IV.

[0089] Theoretically, the mass ratio of ZnO to MOF in 2D ZnO@MOF is 1:4. Therefore, take the ZnO prepared in Comparative Example 1 and the MOF prepared in Comparative Example 2 and mix them in a ratio of 1:4 to prepare suspension IV with a ZnO + MOF concentration of 1.5 mg / mL; place suspension IV under an ultrasonic power of 240 W and ultrasonically treat it for 15 min. After uniformly dispersing the ZnO and MOF with a mass ratio of 1:4, perform dynamic light scattering test (DLS). The ZnO polymer dispersity index (PDI) of suspension IV is 0.12, which is similar to the dispersibility of suspensions I, II, and III.

[0090] Suspension I, Suspension II, Suspension III, and Suspension IV were respectively mixed evenly with potato dextrose agar (PDA) medium. A small hole with a diameter of 8 mm was made in the center of the PDA medium without nanoparticles (i.e., the control group), the PDA medium containing Suspension I (i.e., 2D ZnO@MOF at 1.5 mg / mL), the PDA medium containing Suspension II (i.e., ZnO NPs at 1.5 mg / mL), the PDA medium containing Suspension III (i.e., MOF at 1.5 mg / mL), and the PDA medium containing Suspension IV (i.e., ZnO + MOF at 1.5 mg / mL). 100 μL of Aspergillus niger spore suspension with a concentration of 1×10 6 CFU / mL was inoculated into each of them, and each group was repeated three times to ensure the accuracy of the experiment. The inoculated medium was placed in a constant temperature environment at 28 °C for 5 days, and the hyphal growth area was observed.

[0091] 4. Determination of the minimum inhibitory concentration (MIC) of molds

[0092] Aspergillus niger (A. niger) purified from stored corn was selected as the experimental strain. ZnO NPs, 2D ZnO@MOF, and ZnO@mSiO 2 were respectively gradient-diluted to make their concentration range from 100 mg / mL to 1000 mg / mL. It should be noted that the preparation process of ZnO@mSiO 2 refers to the patent document with the publication number CN111972493B in the prior art.

[0093] The Aspergillus niger spore suspension was cultured to a concentration of 1×10 6 CFU / mL. When performing the MIC determination, first, 100 μL of the Aspergillus niger spore suspension was added to each well of a 96-well plate, and then 100 μL of the PDB liquid medium of the nanoparticles that had been gradient-diluted was added. After that, the initial absorbance was measured at 595 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Then, the 96-well plate was placed at 28 °C and gently shaken and incubated for 36 hours. Finally, the final absorbance was measured again at 595 nm using the ELISA reader to complete the MIC determination.

[0094] Experimental results and data analysis:

[0095] Table 1. Minimum inhibitory concentration (MIC) of different antibacterial materials

[0096] As shown in Table 1, the minimum inhibitory concentration of the 2D ZnO@MOF nanocomposite against Staphylococcus aureus is 0.075 mg / mL, and the minimum inhibitory concentration against Aspergillus niger is 0.3 mg / mL. The minimum inhibitory concentration of ZnO nanoparticles against Staphylococcus aureus is 0.2 mg / mL, and the minimum inhibitory concentration against Aspergillus niger is 0.5 mg / mL. ZnO@mSiO 2 The minimum inhibitory concentration against Staphylococcus aureus is 0.5 mg / mL, and the minimum inhibitory concentration against Aspergillus niger is 0.4 mg / mL. This indicates that for both bacteria and molds, the antibacterial performance of the 2D ZnO@MOF nanocomposite is significantly higher than that of ZnO nanoparticles.

[0097] To avoid misleading in the determination of antibacterial performance caused by the unformed composite structure of MOF and ZnO, in the present invention, the MOF framework material, uncomposite MOF, and ZnO are adjusted to the same concentration and added to the culture medium. The results are as Figure 3 、 Figure 4 shown: Compared with the control group, the inhibition rate of ZnO nanoparticles against Staphylococcus aureus is 79.35%, the inhibition rate of the MOF framework against Staphylococcus aureus is 24.23%, the inhibition rate of uncomposite MOF and ZnO nanoparticles against Staphylococcus aureus is 36.92%, while the inhibition rate of the 2D ZnO@MOF nanocomposite against Staphylococcus aureus is 100%. It can be seen that compared with ZnO nanoparticles, MOF framework materials, uncomposite MOF and ZnO nanoparticles, the antibacterial property of 2D ZnO@MOF against Staphylococcus aureus has been greatly improved, and it can be used as an effective antibacterial agent against Staphylococcus aureus.

[0098] The inhibition rate of ZnO nanoparticles against the growth of Aspergillus niger hyphae is 42.26%, the inhibition rate of the MOF framework material against the growth of Aspergillus niger hyphae is 20.49%, the inhibition rate of uncomposite MOF and ZnO materials against the growth of Aspergillus niger hyphae is 36.39%, while the inhibition rate of 2D ZnO@MOF against the growth of Aspergillus niger hyphae is 86.20%, which is much greater than the sum of the inhibition rate of ZnO nanoparticles against the growth of Aspergillus niger hyphae and the inhibition rate of the MOF framework material against the growth of Aspergillus niger hyphae (42.26% + 20.49% = 62.75%), and also much greater than uncomposite MOF and ZnO nanoparticles at the same concentration. It can be shown that after the MOF and ZnO nanoparticles are combined by the method of the present invention to obtain a novel nanocomposite, a synergistic antibacterial effect is produced.

[0099] The antibacterial performance of 2D ZnO@MOF is speculated to be enhanced compared with ZnO nanoparticles due to the role of its morphological structure and the synergistic antibacterial properties. In view of the fact that the antibacterial performance of the 2D ZnO@MOF nanocomposite is significantly higher than that of ZnO nanoparticles, the present invention proposes an enhanced synergistic antibacterial property of ZnO and the self-assembled MOF structure. During the antibacterial process, 2D ZnO@MOF can induce the generation of more ROS, thereby causing damage to microbial cells and leading to their death. In the present invention, the method of observation by laser confocal microscopy was used to clearly observe the green fluorescence generated by ROS at the same concentration of ZnO nanoparticles and 2D ZnO@MOF addition. In the 2D ZnO@MOF addition group, more intense green fluorescence was generated in the hyphae of three strains of molds, Aspergillus niger, Penicillium citrinum, and Aspergillus flavus, under the field of view of the laser confocal microscope, proving that more ROS were generated in the hyphae, as Figure 5 shown, which would thus damage the structure of the cell membrane and disrupt the normal cell metabolism in the cells, causing the decline and death of the cells. At the same time, during the action process of 2D ZnO@MOF, more Zn 2+ ions are also released into the cells of microorganisms ( Figure 9 ), thereby affecting the activity of some enzymes in the cells and disturbing the normal metabolic process of microbial cells. Finally, the special two-dimensional structure of 2D ZnO@MOF ( Figure 2 BD), during the contact with microbial cells, like a sharp blade, there is also an obvious physical contact effect, causing damage to the microbial cell membrane and even leakage of cell fluid, thereby affecting the normal physiological functions of the cells and causing the decline and death of microorganisms.

[0100] <Inhibitory effect of 2D ZnO@MOF on bacteria>

[0101] Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were selected as model bacteria, and the inhibitory effect of the 2D ZnO@MOF nanocomposite on bacteria was judged by the plate colony counting method, determination of the minimum inhibitory concentration (MIC), and determination of the minimum bactericidal concentration (MBC).

[0102] 1. Observation of the inhibitory situation of bacteria by the plate colony counting method

[0103] 2D ZnO@MOF was mixed evenly with LB medium at concentrations of 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL. 100 μL of the bacterial suspension was taken and evenly spread on the LB agar plate. Three parallel experiments were carried out, and then it was placed in an incubator at 37 °C for 24 h.

[0104] 2. Determination of the minimum inhibitory concentration (MIC) of bacteria

[0105] The specific method is the same as the determination of the minimum inhibitory concentration (MIC) of bacteria described in <Antibacterial Activity Detection of 2D ZnO@MOF>.

[0106] 3. Determination of the minimum bactericidal concentration (MBC) of bacteria

[0107] The minimum bactericidal concentration (MBC) refers to the lowest drug concentration that can kill 99.9% of the tested bacteria. According to the method for determining the minimum inhibitory concentration (MIC) of bacteria described in <Antibacterial Activity Detection of 2DZnO@MOF>, after determining the minimum inhibitory concentration (MIC) of bacteria, several concentration wells near the MIC concentration (1 / 2MIC, MIC, 2×MIC, 4×MIC, etc.) are selected, and 100 μL of the bacterial solution is aspirated from each well and spread on the LB medium plate, which is then placed in an incubator at 37 °C and cultured for another 24 h. After the culture is completed, the number of colonies on the plate is counted, and the lowest concentration with the number of colonies less than 0.1% of the initial inoculum amount is the minimum bactericidal concentration (MBC).

[0108] Experimental results and data analysis:

[0109] Table 2. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 2D ZnO@MOF against bacteria

[0110] As can be seen from Figure 6 Table 2, low-concentration 2D ZnO@MOF has a significant inhibitory effect on both Staphylococcus aureus and Escherichia coli. Among them, the minimum inhibitory concentration of 2D ZnO@MOF against Staphylococcus aureus is 0.075 mg / mL, and the minimum bactericidal concentration is 0.20 mg / mL; the minimum inhibitory concentration against Escherichia coli is 0.15 mg / mL, and the minimum bactericidal concentration is 0.40 mg / mL.

[0111] <Inhibitory Effect of 2D ZnO@MOF on Molds>

[0112] Aspergillus flavus (A. flavus), Aspergillus niger (A. niger) and Penicillium citrinum (P. citrinum) are selected as model molds, and the inhibitory effect of 2D ZnO@MOF on molds is judged by the agar punching method, spore germination rate determination, minimum inhibitory concentration (MIC) determination and minimum bactericidal concentration (MBC) determination.

[0113] 1. Agar punching method

[0114] The 2D ZnO@MOF nanocomposite is mixed evenly with PDA medium at concentration gradients of 0, 0.5, 1.0, 1.5, 2.0, 2.5 mg / mL, a small hole with a diameter of 8 mm is punched in the center of the medium, and the inoculation concentration is 1×106 A spore suspension of CFU / mL, and each group was repeated three times to ensure the accuracy of the experiment. The inoculated medium was cultured in a constant temperature environment of 28 °C for 7 d, and the mycelial growth area was observed.

[0115] 2. Spore germination rate

[0116] Mix 2D ZnO@MOF with PDB liquid medium to make its concentration 0, 0.1, 0.2, 0.3, 0.4, 0.5 mg / mL. Then, add 100 μL of Aspergillus flavus spore suspension (1×10 6 CFU / mL) respectively, and place it in a constant temperature shaker at 28 °C for 12 h. Use sterile water treatment as a control, and each treatment was repeated 3 times. After the culture, use an optical microscope to observe and count the number of germinated spores of the three molds in each treatment group. The total number of spores observed for each parallel was not less than 100.

[0117] 3. Determination of the minimum inhibitory concentration (MIC) of molds

[0118] The specific method is the same as the determination of the minimum inhibitory concentration (MIC) of molds in <Antibacterial properties of 2D ZnO@MOF>.

[0119] 4. Determination of the minimum bactericidal concentration (MBC) of molds

[0120] The specific method is the same as the determination of the minimum bactericidal concentration (MBC) of bacteria in <Inhibitory effect of 2D ZnO@MOF on bacteria>.

[0121] Experimental results and data analysis:

[0122] Table 3. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 2D ZnO@MOF against molds

[0123] As Figure 7 and Table 3 show, when the addition of 2D ZnO@MOF reaches a certain concentration, it can inhibit the growth of Aspergillus flavus, Penicillium citrinum, and Aspergillus niger for a long time and significantly. Among them, the minimum inhibitory concentration of 2D ZnO@MOF against Aspergillus flavus is 0.4 mg / mL, and the minimum bactericidal concentration is 2.5 mg / mL; the minimum inhibitory concentration against Penicillium citrinum is 0.4 mg / mL, and the minimum bactericidal concentration is 2.0 mg / mL; the minimum inhibitory concentration against Aspergillus niger is 0.3 mg / mL, and the minimum bactericidal concentration is 3.0 mg / mL. As Figure 8 shows, when the concentration reaches 0.5 mg / mL, the spore germination inhibition rates of 2D ZnO@MOF against Aspergillus flavus, Penicillium citrinum, and Aspergillus niger reach 82.17%, 96.23%, and 88.78% respectively.

[0124] <Effect of 2D ZnO@MOF with Different Concentrations on Zinc Element Accumulation in Maize>

[0125] During the 42-day storage period, every 7 days, the zinc element accumulation in maize with 2D ZnO@MOF added at concentrations of 0 g / kg (control group), 0.5 g / kg, 1.5 g / kg, and 2.5 g / kg was measured by an atomic absorption spectrophotometer.

[0126] As Figure 9 known, in the early stage of simulated storage, there was no significant change in the zinc ion content in maize. As the storage time extended, there was no significant change in the zinc content of maize in the control group; the zinc contents of maize with 2D ZnO@MOF added at concentrations of 0.5 g / kg, 1.5 g / kg, and 2.5 g / kg increased by 8.92, 15.98, and 43.13 mg / kg respectively, and their zinc element accumulations all met the maximum limit of 110 mg / kg of zinc element stipulated in the Announcement No. 2625 of the Ministry of Agriculture and Rural Affairs of China. The research data show that the addition of low-concentration 2D ZnO@MOF can effectively supply the micronutrient zinc to maize, which can not only prevent mildew but also improve the nutritional quality of stored maize.

[0127] <Effect of 2D ZnO@MOF on the Total Number of Fungal Colonies in Stored Maize>

[0128] The maize was screened and impurity-removed, and then randomly divided into 2 equal parts, each weighing 2 kg. During the process of adjusting the moisture of maize, different concentrations of 2D ZnO@MOF suspension were evenly sprayed on the maize in 4 times, so that the final concentrations of 2D ZnO@MOF in each part of maize reached 0 g / kg (control group), 0.5 g / kg, 1.5 g / kg, and 2.5 g / kg respectively. The treated samples were sealed in bags and placed in a constant temperature and humidity incubator at 30 °C and 80% relative humidity to simulate storage, and the changes of microorganisms on the maize surface were monitored every 7 days. The total number of colonies and molds were selected as microbial detection indicators and carried out with reference to GB / T4789.15-2016 "National Food Safety Standard Food Microbiology Examination - Enumeration of Molds and Yeasts".

[0129] As Figure 10 known, after storage at 30 °C and 80% relative humidity for 28 days, the total number of fungal colonies on the surface of maize in the 2D ZnO@MOF addition group was relatively stable during storage, fluctuating around 1.2×10 4 CFU / mL. However, the total number of fungal colonies in the control group increased significantly, and the total number of fungal colonies increased from the initial 2000 CFU / mL to 1.9×10 6CFU / mL. This indicates that the addition of 2D ZnO@MOF can significantly inhibit the growth of fungi during the storage of corn. At the same time, within the 42-day storage period, the total number of fungal colonies in the group with the addition of 2D ZnO@MOF was always lower than that of the control group and remained at a low level, indicating that the addition of 2D ZnO@MOF has a good long-term inhibitory effect on the total number of fungal colonies.

[0130] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing an antibacterial enhanced two-dimensional zinc oxide nanocomposite, characterized in that: The following steps are involved: Dispersing dimethylimidazole in DMF to obtain dispersion A, dispersing Zn-GL precursor in DMF to obtain dispersion B, and dispersing zinc oxide nanoparticles in DMF to obtain dispersion C; Dispersion B and dispersion C are mixed, stirred and dispersion A is added dropwise to obtain a mixed solution; The mixed solution is heated in a sealed state at 150-170° C. for reaction. After the reaction is completed, the mixed solution is cooled to room temperature, the generated precipitate is separated, and the precipitate is freeze-dried to obtain a zinc oxide nanocomposite.

2. The method for preparing an antibacterial enhanced two-dimensional zinc oxide nanocomposite according to claim 1, characterized in that: The preparation method of the Zn-GL precursor is as follows: adding anhydrous zinc acetate to glycerol, heating to 120° C. for 30 minutes, then heating to 150° C. for 1 hour, cooling, separating the obtained precipitate, and vacuum freeze-drying to obtain the Zn-GL precursor.

3. The method for preparing an antibacterial enhanced two-dimensional zinc oxide nanocomposite according to claim 1, characterized in that: When dispersion A was added dropwise to the solution obtained by mixing dispersion B and dispersion C, the stirring speed was 200 rpm.

4. The method for preparing an antibacterial enhanced two-dimensional zinc oxide nanocomposite according to claim 1, characterized in that: The molar ratio of dimethylimidazole, Zn-GL precursor and zinc oxide nanoparticles in the mixed solution is 8:2:

1.

5. The method for preparing an antibacterial enhanced two-dimensional zinc oxide nanocomposite according to claim 1, characterized in that: The concentration of dimethylimidazole in dispersion A is 0.25-0.30 mmol / mL; the concentration of Zn-GL precursor in dispersion B is 0.06-0.08 mmol / mL; and the concentration of zinc oxide nanoparticles in dispersion C is 2.30-2.70 mg / mL.

6. The method for preparing an antibacterial enhanced two-dimensional zinc oxide nanocomposite according to claim 1, characterized in that: The closed heating time is 15-17h.

7. An antibacterial enhanced two-dimensional zinc oxide nanocomposite, characterized in that: The invention is prepared by the method described in any one of claims 1 to 6.

8. Use of the antibacterial enhanced two-dimensional zinc oxide nanocomposite as claimed in claim 7 as a bacterial growth and reproduction inhibitor or a mold growth and reproduction inhibitor.

9. Use of the antibacterial enhanced two-dimensional zinc oxide nanocomposite according to claim 8 as a bacterial growth and reproduction inhibitor or a mold growth and reproduction inhibitor, characterized in that: The bacteria are Staphylococcus aureus or Escherichia coli, and the molds are Aspergillus niger, Aspergillus flavus or Penicillium citrinum.

10. Use of the antibacterial enhanced two-dimensional zinc oxide nanocomposite according to claim 7 in food storage.

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