Zinc oxide nanoflower and preparation method and application thereof

Abstract

The application belongs to the technical field of antibacterial materials, and particularly relates to zinc oxide nanoflower as well as a preparation method and application thereof. The method comprises the following steps: dissolving zinc acetate dihydrate in anhydrous ethanol to obtain a zinc acetate ethanol solution; mixing the zinc acetate ethanol solution and a lactobacillus P101 fermentation liquor, and then performing ultrasonic treatment; adding a sodium hydroxide solution to adjust the pH to 10-14; finally, placing the mixture in a microwave oven for irradiation until obvious white flocculation is observed, and then performing centrifugal separation, washing, and freeze-drying to obtain the zinc oxide nanoflower. The preparation method is simple in operation, mild in conditions, economical and energy-saving, and does not cause harm to the environment and human bodies in the synthesis process. The zinc oxide nanoflower is synthesized by using the lactobacillus P101 fermentation liquor, and has good biocompatibility. In addition, the zinc oxide nanoflower has excellent bacterial biofilm inhibition and removal capacity at a low concentration, and shows good antibacterial activity on staphylococcus aureus and escherichia coli.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial materials technology, specifically relating to a zinc oxide nanoflower, its preparation method, and its application. Background Technology

[0002] Food quality and safety have always been a focus of public concern. However, the contamination and spread of foodborne pathogens remain a significant hidden danger to food safety, posing a substantial threat to human health. Microbial contamination threatens both human health and environmental safety, with approximately one-third of the world's food waste occurring unnecessarily. Therefore, controlling pathogenic contamination and inhibiting their reproduction, suppressing bacterial biofilm formation, and developing materials with highly effective antibacterial properties and biosafety have become major directions in scientific research.

[0003] As is well known, bacterial infections can cause various diseases, posing a serious threat to humans and the environment. In the last century, antibiotics achieved their bactericidal purpose through mechanisms such as inhibiting bacterial cell wall synthesis, altering bacterial cell membrane permeability, inhibiting and suppressing bacterial cell protein synthesis, inhibiting bacterial nucleic acid synthesis, and hindering bacterial folic acid synthesis. Therefore, they have become a powerful weapon against bacterial infections. However, due to the widespread use of antibiotics, a large number of drug-resistant bacteria have emerged, causing unprecedented harm to humanity. Therefore, in recent years, many scholars have been researching how to utilize nano-metal oxides to combat bacterial infections, and even drug-resistant bacteria. Among many nano-metal oxides, nano-zinc oxide exhibits significant bactericidal activity against bacteria, fungi, and viruses. However, early nano-zinc oxide relied on chemical synthesis methods, using various organic reagents that caused certain environmental harms. Therefore, recent reports indicate that the synthesis of nano-zinc oxide is gradually shifting towards green biosynthesis methods.

[0004] According to existing literature, metals and metal oxides themselves are not typically a hot research topic. However, with the application of nanotechnology in various aspects of life, interest in nanomaterials is growing. Nanotechnology itself, on the other hand, yields remarkable research results when combined with metals and metal oxides. Several types of nano-metals and nano-metal oxides possess antibacterial activity, including silver, gold, titanium dioxide, copper oxide, iron oxide, and zinc oxide. Among these, zinc oxide nanoparticles have attracted significant attention due to their unique electronic, optical, and pharmaceutical properties. Their high biocompatibility makes them another promising antibacterial material after nano-silver. Nano-zinc oxide also has broad biological applications, including drug delivery, biosensing, and excellent antibacterial activity. Therefore, nano-zinc oxide stands out among many nano-metal oxides. Currently, nano-zinc oxide can be synthesized using chemical, physical, or biological methods. Chemical methods include precipitation, microemulsion, chemical reduction, sol-gel, and hydrothermal techniques, all of which require high pressure or high temperature conditions and thus high energy consumption. Physical synthesis methods for nano-zinc oxide include vapor deposition, plasma, and ultrasonic irradiation. However, these technologies typically require significant energy and sophisticated equipment, increasing the cost of synthesis. Existing reports mostly focus on spherical zinc oxide nanoparticles, while reports on flower-shaped zinc oxide nanoparticles are less common. Similar to flower-shaped zinc oxide nanoparticles, sheet-like zinc oxide nanoparticles exhibit stronger antibacterial activity compared to spherical ones. Therefore, developing a method to prepare zinc oxide with strong antibacterial activity is of great significance. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a zinc oxide nanoflower, its preparation method, and its application. Specifically, the following technical solution is adopted:

[0006] A method for preparing zinc oxide nanoflowers includes the following steps:

[0007] Zinc acetate dihydrate was dissolved in anhydrous ethanol to obtain a zinc acetate ethanol solution. The zinc acetate ethanol solution was mixed with the fermentation broth of Lactobacillus plantarum P101, and sonicated for 15 min-30 min. Then, sodium hydroxide solution was added to adjust the pH to 10-14. Finally, the mixture was irradiated in a microwave oven for 2 min-5 min until obvious white flocculent matter was observed. After centrifugation, washing, and freeze-drying, zinc oxide nanoflowers were finally obtained.

[0008] This invention uses Lactobacillus plantarum P101 fermentation broth as a stabilizer and reducing agent. The nano zinc oxide prepared by reacting with zinc acetate ethanol solution presents a flower shape composed of many needle-like substances, each needle-like substance being approximately 150 nm in length. Sodium hydroxide acts as a hydrolysant in the system to hydrolyze zinc acetate, while Lactobacillus plantarum P101 fermentation broth acts as a reducing agent and stabilizer to react with zinc in the system and reduce it to nano zinc oxide.

[0009] As a further preferred embodiment, the above-mentioned Lactobacillus plantarum P101 fermentation broth is obtained by the following steps:

[0010] Lactobacillus plantarum P101 was inoculated into MRS medium and cultured anaerobically at 30 ℃-40 ℃ for 12 h-30 h. After the bacterial culture gradually became turbid, it was centrifuged in a refrigerated centrifuge to obtain the Lactobacillus plantarum P101 fermentation broth. The Lactobacillus plantarum P101 fermentation broth obtained by this method has strong reducing ability, and it shows more economical and environmentally friendly advantages compared with chemical reagents with the same effect.

[0011] As a further preferred embodiment, the inoculum size of *Lactobacillus plantarum* P101 is 2%-5%; more preferably, the inoculum size of *Lactobacillus plantarum* P101 is 4%; and the bacterial concentration in the *Lactobacillus plantarum* P101 fermentation broth is 10. 9 CFU / mL. Among them, *Lactobacillus plantarum* P101 ( Lactobacillus plantarum P101 was deposited at the China Center for Type Culture Collection in Wuhan on January 19, 2021, with accession number CCTCC M 2021108.

[0012] As a further preferred embodiment, the ratio of the zinc acetate ethanol solution to the *Lactobacillus plantarum* P101 fermentation broth is 1:0.5-2; more preferably, the ratio is 1:1. The concentration of the zinc acetate ethanol solution is 0.05 mol / L-0.2 mol / L. Excessive addition of *Lactobacillus plantarum* P101 fermentation broth will result in excessively large synthesized nano-zinc oxide particles, which will reduce their antibacterial activity; insufficient addition may lead to incomplete reaction. Excessive concentration of the zinc acetate ethanol solution may cause severe aggregation, which will reduce its contact area with pathogenic bacteria and weaken its antibacterial activity.

[0013] As a further preferred embodiment, the temperature in the refrigerated centrifuge is 2 ℃-10 ℃, the centrifugation speed is 9600 r / min, and the centrifugation time is 5 min-10 min.

[0014] The present invention also provides a zinc oxide nanoflower prepared by the above preparation method, which can be used in the preparation of drugs for inhibiting Staphylococcus aureus; it can also be used in the preparation of drugs for inhibiting Escherichia coli.

[0015] The beneficial effects of this invention are as follows:

[0016] The preparation method of this invention is simple to operate, mild in conditions, economical and energy-saving, and the synthesis process will not cause harm to the environment and human body. This invention uses the fermentation broth of Lactobacillus plantarum P101 to synthesize zinc oxide nanoflowers, which have good biocompatibility. In addition, according to the data results of this embodiment, the zinc oxide nanoflowers prepared by this invention have excellent bacterial biofilm inhibition and removal capabilities at low concentrations, and show good antibacterial activity against Staphylococcus aureus and Escherichia coli. Attached Figure Description

[0017] Figure 1 The image shown is a scanning electron microscope (SEM) image of zinc oxide nanoflowers synthesized from the fermentation broth of Lactobacillus plantarum P101.

[0018] Figure 2 The figure shows the inhibition rate of zinc oxide nanoflowers synthesized from Lactobacillus plantarum P101 fermentation broth against Staphylococcus aureus and Escherichia coli.

[0019] Figure 3 The experiment shown is an evaluation of the inhibitory effect of zinc oxide nanoflowers synthesized from the fermentation broth of Lactobacillus plantarum P101 on the biofilm of Staphylococcus aureus and Escherichia coli.

[0020] Figure 4 The experiment shows the inhibition zone experiments of different concentrations of zinc oxide nanoflowers and 30 nm zinc oxide against Escherichia coli and Staphylococcus aureus. Detailed Implementation

[0021] The following will provide a clear and complete description of the concept, specific structure, and technical effects of the present invention in conjunction with the embodiments and accompanying drawings, so as to fully understand the purpose, solution, and effects of the present invention. It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0022] Example 1

[0023] A method for preparing zinc oxide nanoflowers specifically includes the following steps:

[0024] Step 1: Inoculate Lactobacillus plantarum P101 at 3% into 50 mL of MRS medium in a biosafety cabinet. Then, incubate the medium inoculated with Lactobacillus plantarum P101 under anaerobic conditions at 37 °C for 24 h. When the bacterial culture gradually becomes turbid, remove it from the incubator and dispense it into 50 mL centrifuge tubes. Then, place them in a refrigerated centrifuge and centrifuge at 4 °C at 9600 r / min for 5 min. Collect the supernatant and store it at 6 °C to obtain the fermentation broth of Lactobacillus plantarum P101.

[0025] Step 2: Dissolve 0.55 g of zinc acetate dihydrate in 50 mL of anhydrous ethanol. If it cannot be completely dissolved, it can be heated to completely dissolve it and prepare a 0.05 mol / L zinc acetate ethanol solution.

[0026] Step 3: Mix the zinc acetate ethanol solution obtained in Step 2 with the Lactobacillus plantarum P101 fermentation broth obtained in Step 1 at a volume ratio of 1:0.5. After mixing, place the mixture in an ultrasonic machine and sonicate for 15 min to ensure thorough mixing. After ultrasonic treatment, add 1 M sodium hydroxide to adjust the mixture to 10. Finally, transfer the mixture to a microwave oven and irradiate for 3 min. Obvious white flocculents can be observed. After centrifugation, a white precipitate is obtained, which is washed with anhydrous ethanol and freeze-dried to finally obtain zinc oxide nanoflowers.

[0027] Example 2

[0028] A method for preparing zinc oxide nanoflowers specifically includes the following steps:

[0029] Step 1: Inoculate 4% of Lactobacillus plantarum P101 into 50 mL of MRS medium in a biosafety cabinet. Then, incubate the medium inoculated with Lactobacillus plantarum P101 under anaerobic conditions at 40 °C for 36 h. When the bacterial culture gradually becomes turbid, remove it from the incubator and dispense it into 50 mL centrifuge tubes. Then, place them in a refrigerated centrifuge and centrifuge at 0 °C at 9600 r / min for 5 min. Collect the supernatant and store it at 8 °C to obtain the fermentation broth of Lactobacillus plantarum P101.

[0030] Step 2: Dissolve 1.1 g of zinc acetate dihydrate in 50 mL of anhydrous ethanol. If it cannot be completely dissolved, heat it to prepare a 0.1 mol / L zinc acetate ethanol solution.

[0031] Step 3: Mix the zinc acetate ethanol solution obtained in Step 2 with the Lactobacillus plantarum P101 fermentation broth obtained in Step 1 at a volume ratio of 1:1.5. Place the mixed solution in an ultrasonic machine and sonicate for 10 min to ensure thorough mixing. After ultrasonic treatment, add 1 M sodium hydroxide dropwise to adjust the mixed solution to 12. Finally, transfer the mixed solution to a microwave oven and irradiate for 3 min. Obvious white flocculents can be observed. After centrifugation, a white precipitate is obtained, which is washed with anhydrous ethanol and freeze-dried to finally obtain zinc oxide nanoflowers.

[0032] Example 3

[0033] A method for preparing zinc oxide nanoflowers specifically includes the following steps:

[0034] Step 1: Inoculate 5% of Lactobacillus plantarum P101 into 50 mL of MRS medium in a biosafety cabinet. Then, incubate the medium inoculated with Lactobacillus plantarum P101 under anaerobic conditions at 37 °C for 24 h. When the bacterial culture gradually becomes turbid, remove it from the incubator and dispense it into 50 mL centrifuge tubes. Then, place them in a refrigerated centrifuge and centrifuge at 9600 r / min for 5 min at 4 °C. Collect the supernatant and store it at 4 °C to obtain the fermentation broth of Lactobacillus plantarum P101.

[0035] Step 2: Dissolve 2.2 g of zinc acetate dihydrate in 50 mL of anhydrous ethanol. If it cannot be completely dissolved, heat it to prepare a 0.2 mol / L zinc acetate ethanol solution.

[0036] Step 3: Mix the zinc acetate ethanol solution obtained in Step 2 with the Lactobacillus plantarum P101 fermentation broth obtained in Step 1 at a volume ratio of 1:1. Place the mixture in an ultrasonic machine and sonicate for 20 min to ensure thorough mixing. After ultrasonic treatment, add 1 M sodium hydroxide dropwise to adjust the mixture to 14. Finally, transfer the mixture to a microwave oven and irradiate for 3 min. Obvious white flocculents can be observed. After centrifugation, a white precipitate is obtained, which is washed with anhydrous ethanol and freeze-dried to finally obtain zinc oxide nanoflowers.

[0037] Example 4

[0038] A method for preparing zinc oxide nanoflowers specifically includes the following steps:

[0039] Step 1: Inoculate Lactobacillus plantarum P101 at 3% into 50 mL of MRS medium in a biosafety cabinet. Then, incubate the medium inoculated with Lactobacillus plantarum P101 under anaerobic conditions at 37 °C for 24 h. When the bacterial culture gradually becomes turbid, remove it from the incubator and dispense it into 50 mL centrifuge tubes. Then, place them in a refrigerated centrifuge and centrifuge at 4 °C at 9600 r / min for 5 min. Collect the supernatant and store it at 4 °C to obtain the fermentation broth of Lactobacillus plantarum P101.

[0040] Step 2: Dissolve 1.1 g of zinc acetate dihydrate in 50 mL of anhydrous ethanol. If it cannot be completely dissolved, heat it to prepare a 0.1 mol / L zinc acetate ethanol solution.

[0041] Step 3: Mix the zinc acetate ethanol solution obtained in Step 2 with the Lactobacillus plantarum P101 fermentation broth obtained in Step 1 at a volume ratio of 1:1. Place the mixed solution in an ultrasonic machine and sonicate for 10 min to ensure thorough mixing. After ultrasonic treatment, add a certain concentration of sodium hydroxide to adjust the mixed solution to 12. Finally, transfer the mixed solution to a microwave oven and irradiate for 3 min. Obvious white flocculents can be observed. After centrifugation, a white precipitate is obtained, which is washed with anhydrous ethanol and freeze-dried to finally obtain zinc oxide nanoflowers.

[0042] The zinc oxide nanoflowers prepared above were examined by scanning electron microscopy (SEM), and the results are as follows: Figure 1 As shown, and by Figure 1 It is known that the nano zinc oxide prepared by using Lactobacillus plantarum P101 fermentation broth as a stabilizer and reducing agent exhibits a flower shape composed of many needle-like substances, each needle being approximately 150 nm in length. This shape differs from the shape of most nano zinc oxide reported in the literature, which is mostly spherical. This is one of the reasons why it has better antibacterial properties.

[0043] Example 5

[0044] The zinc oxide nanoflowers prepared in Example 1 were tested for antibacterial effects. The specific process is as follows:

[0045] (1) Bacterial culture: Single colonies of Staphylococcus aureus CMCC 26001 and Escherichia coli ATCC43888 on LB solid medium were picked and placed in fresh LB liquid medium. They were activated by culturing in a shaker at 37°C and 180 r / min for 12 h. They were then inoculated into LB liquid medium at a ratio of 1% and cultured in a shaker at 37°C and 180 r / min for 12 h for later use.

[0046] (2) Antibacterial effect of green synthesized zinc oxide nanoflowers against Escherichia coli and Staphylococcus aureus: Freshly cultured bacterial solutions were diluted to 10 μL using sterile 1×PBS buffer. 6 CFU / mL was set aside. Zinc oxide nanoflowers at concentrations of 0, 1 / 4 MIC, 1 / 2 MIC, and MIC were then mixed with 10... 6 CFU / mL bacterial suspension was co-incubated in a 37 ℃ incubator for a certain period of time. After incubation, the suspension was removed for plate counting and spreading, and a biofilm inhibition experiment was performed simultaneously. 6 CFU / mL bacterial suspension was mixed with an equal volume of zinc oxide nanoflower solution in a 96-well plate and incubated at 37 ℃ for 24 h and 48 h. After each incubation period, crystal violet staining was performed, and data were read using a microplate reader to evaluate the bacterial biofilm inhibition assay. This invention uses commercially available 30nm zinc oxide as a control. The specific experimental procedure is the same as described above, and the experimental results are as follows: Figures 2-4As shown:

[0047] Figure 2 The figure shows the antibacterial rate of LPF-ZnO NPs (zinc oxide nanoflowers) at different concentrations. Here, ad represents LPF-ZnO NPs at concentrations of 0, 1 / 4 MIC, 1 / 2 MIC, and MIC, respectively, after incubation with Staphylococcus aureus for 6 h and then plated; eh represents LPF-ZnO NPs at concentrations of 0, 1 / 4 MIC, 1 / 2 MIC, and MIC, respectively, after incubation with Escherichia coli for 6 h and then plated; ij represents the inhibition rate of Staphylococcus aureus and Escherichia coli, respectively. Figure 2 It can be seen that the antibacterial rate of LPF-ZnO NPs against Escherichia coli and Staphylococcus aureus is positively correlated with concentration. Until the MIC concentration, no colonies of Staphylococcus aureus and Escherichia coli appeared on the plates. This shows that LPF-ZnO NPs have excellent antibacterial activity.

[0048] Figure 3 The figures show the effects of LPF-ZnO NPs on biofilms of *Escherichia coli* and *Staphylococcus aureus*. (a1-d4) represent the inhibitory effects of different concentrations of LPF-ZnO NPs on biofilm formation in *Escherichia coli* and *Staphylococcus aureus* after 24 h and 48 h of treatment; (eh) represent the quantification of the biofilm formation index of *Escherichia coli* and *Staphylococcus aureus* after 24 h and 48 h of treatment with different concentrations of LPF-ZnONPs. Figure 3 It can be seen that, compared with the group without zinc oxide nanoflower treatment, LPF-ZnO NPs at a concentration of 1 / 2MIC have a certain biofilm removal ability. After treatment with LPF-ZnO NPs at a concentration of 2MIC for 24 hours, the BFI of Escherichia coli and Staphylococcus aureus is less than 0.35, that is, almost no sterile biofilm formation. After treatment with LPF-ZnO NPs for 48 hours, the BFI is still less than 0.35. Therefore, LPF-ZnO NPs have excellent bacterial biofilm inhibition and removal ability at low concentrations.

[0049] Figure 4 The figure shows the inhibition zone experiments of different concentrations of LPF-ZnO NPs (a) and 30 nm zinc oxide (b) against *Escherichia coli* and *Staphylococcus aureus*. As can be seen from the figure, the size of the inhibition zone is positively correlated with the concentration of both types of zinc oxide nanoparticles. The results indicate that although the inhibition zone size of LPF-ZnO NPs is similar to that of commercially available 30 nm zinc oxide nanoparticles, the inhibition zone size of LPF-ZnO NPs at different concentrations is more stable. Furthermore, the synthesis method of LPF-ZnO NPs is more environmentally friendly and economical than commercial synthesis methods.

[0050] In summary, this invention provides a method for preparing zinc oxide nanoflowers and an evaluation of their antibacterial effects. It has good application prospects in preventing pathogenic bacteria from contaminating food, and also has certain application prospects in the fields of antibacterial food packaging and antibacterial coatings on the surface of medical devices.

[0051] Although the description of the invention has been quite detailed and particularly of several described embodiments, it is not intended to limit it to any of these details or embodiments or any particular embodiment, but should be considered as providing a broad possible interpretation of the claims by referring to the appended claims and taking into account the prior art, thereby effectively covering the intended scope of the invention. Furthermore, the invention has been described above with respect to embodiments foreseeable by the inventors in order to provide a useful description, and non-substantial modifications to the invention that have not yet been foreseen may still represent equivalent modifications.

Claims

1. A method for preparing zinc oxide nanoflowers, characterized in that, Includes the following steps: Zinc acetate dihydrate was dissolved in anhydrous ethanol to obtain zinc acetate ethanol solution; the zinc acetate ethanol solution was mixed with Lactobacillus plantarum P101 fermentation broth, sonicated for 15 min-30 min, then sodium hydroxide solution was added to adjust the pH to 10-14, and finally placed in a microwave oven for 2 min-5 min until obvious white flocculents were observed, then centrifuged, washed, and freeze-dried to finally obtain zinc oxide nanoflowers. The fermentation broth of Lactobacillus plantarum P101 was obtained through the following steps: Lactobacillus plantarum P101 was inoculated into MRS medium and cultured under anaerobic conditions at 30 ℃-40 ℃ for 12 h-30 h. After the bacterial culture gradually became turbid, it was centrifuged in a refrigerated centrifuge to obtain the fermentation broth of Lactobacillus plantarum P101. The inoculum size of *Lactobacillus plantarum* P101 was 2%-5%; the bacterial concentration in the *Lactobacillus plantarum* P101 fermentation broth was 10. 9 CFU / mL; Lactobacillus plantarum P101 was deposited at the China Center for Type Culture Collection in Wuhan on January 19, 2021, with accession number CCTCC M 2021108. The volume ratio of zinc acetate ethanol solution to Lactobacillus plantarum P101 fermentation broth is 1:0.5-2; The concentration of the zinc acetate ethanol solution is 0.05 mol / L-0.2 mol / L.

2. The preparation method according to claim 1, characterized in that, The temperature in the refrigerated centrifuge is 2 ℃-10 ℃, the centrifugation speed is 9600 r / min, and the centrifugation time is 5 min-10 min.

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