Self-assembly preparation method and application of multi-enzyme active nano-zinc oxide for treating salmonella enteritis

Abstract

The invention discloses a self-assembly preparation method and application of multi-enzyme active nano-zinc oxide for treating salmonella enteritis, a trace amount of oleylamine is added as a protective agent, a self-assembly nano-enzyme is synthesized by a simple method, and the direct antibacterial effect of ZnO is improved; the minimal inhibitory concentration of ZnO synthesized by the same method in vitro is 250 [mu] g / mL, the minimal inhibitory concentration of ZnO doped with trace Cu and Mn is about 30 [mu] g / mL, and the bacteriostatic efficiency is improved by about 8 times; trace elements of copper (Cu) and manganese (Mn) are doped into a ZnO nano material, so that the ZnO nano material is endowed with various enzyme activities, peroxidase activity is exerted under the acidic condition of the stomach to generate bactericidal active oxygen, and superoxide dismutase and catalase activity for resisting oxidative stress are exerted when the ZnO nano material reaches neutral intestinal tracts, so that inflammation of the intestinal tracts is relieved; the traditional Chinese medicine composition can play a role in the acidic state of the stomach, can relieve inflammation of the intestinal tract in the intestinal tract, and can completely adapt to the environment of the gastrointestinal tract.

Description

Technical Field

[0001] The invention belongs to the technical field of biological nanomaterials, and in particular relates to a self-assembly preparation method and application of multi-enzyme active nano zinc oxide for treating salmonella enteritis. Background Art

[0002] Salmonella is the main cause of foodborne gastroenteritis. Its infection causes symptoms such as fever and vomiting. Its unique virulence, such as typhoid toxin, can cause serious complications. Salmonella enters macrophages and spreads throughout the reticuloendothelial system, causing systemic infection. This disease has become a serious problem in the global health and agricultural fields, especially in the livestock and poultry farming sector.

[0003] Currently, the initial treatment of the disease uses third-generation cephalosporins (such as ceftriaxone) for at least 7-10 days, and the treatment time may be longer for some focal infections such as meningitis (4 weeks) or osteomyelitis (4 to 6 weeks); chronic carriers of the bacteria require four weeks of fluoroquinolone treatment to resolve the carrier state, and existing treatment drugs generally have long treatment times and unsatisfactory effects. From 2020, my country's Ministry of Agriculture and Rural Affairs has stipulated that antibiotics are completely prohibited from being added to feed to reduce the harm caused by the abuse of antibiotics. In livestock and poultry farming, the treatment of Salmonella infection is a very difficult problem.

[0004] In the prior art, for example, Patent No. CN201911142226.4 uses the metabolites of Lactobacillus plantarum ZS2058 and eugenol combination components to inhibit the growth of Salmonella by 90.4%; Patent No. CN202010321258.7 uses a Chinese medicine composition to treat poultry salmonellosis, which has a good effect; Patent No. CN201610103682.8 uses the Chinese medicine compound sanguinarine hydrochloride to directly inhibit the function of the type III secretion system of Salmonella and play an anti-infection role, and is not easy to induce the production of drug resistance, and is used to treat Salmonella infection. These patents all use natural compounds as raw materials to prevent or treat infections caused by Salmonella, which are not suitable for large-scale industrial production and application.

[0005] Zinc oxide is a safe and effective antibacterial agent, which is widely used in the pharmaceutical and feed industries. Pure zinc oxide has a low antibacterial effect and is not capable of treating Salmonella. In the published patents, zinc oxide is doped with other metal ions to improve its antibacterial ability. For example, the patent with publication number CN111296481A discloses a long-acting disinfectant of a modified chitosan-coated nano-Ag / ZnO composite material, which utilizes the surface plasmon resonance effect between Ag and ZnO for synergistic antibacterial effect, and achieves adsorption of bacterial cells so that the bacteria are in close contact with ZnO and Ag nanoparticles. The minimum inhibitory concentration for Escherichia coli is 50 μg / mL, and the minimum inhibitory concentration for Staphylococcus aureus is 100 μg / mL. However, Ag nanoparticles have poor stability in the environment and are prone to aggregation and precipitation, resulting in a decrease in the antibacterial effect. At the same time, Ag is a toxic heavy metal. When Ag nanoparticles come into contact with organisms, they may penetrate the cell membrane, enter the cell, and interact with the biological molecules (such as DNA, proteins, etc.) in the cell, resulting in damage to cell function or even cell death, and their biosafety is low. In addition, the patent with the publication number CN110521733A discloses a Co-doped ZnO quantum dot / attapulgite nanocomposite antibacterial agent and its preparation method. The nanocomposite antibacterial agent obtained by the method using Co-doped ZnO quantum dots has the advantages of safety, environmental protection, strong drug resistance, etc., but its biocompatibility needs to be improved, and its inhibition rate on Escherichia coli is only 85.71%. In addition, the patent with the publication number CN116235903A discloses a feed enteric zinc oxide additive and its preparation method; the method covers the surface of ZnO particles with stearic acid, cellulose and lauric acid monoglyceride layer by layer, so that after entering the intestine, the surface modification material disintegrates under the action of digestive juice, and ZnO is quickly released and exists in the intestine in a molecular state, which can play a role in preventing and controlling piglet diarrhea. Finally, the surface of the modified ZnO particles is modified with white carbon black, and the surface adsorbs white carbon black particles to improve its fluidity. However, the particle size of ZnO prepared by this method is too large, the absorption and utilization rate of poultry is low, and the preparation process is complicated, making it difficult to promote and use on a large scale.

[0006] Although the antibacterial ability of these doped zinc oxides has been improved, they cannot inhibit the growth of Salmonella and treat infections caused by Salmonella. Therefore, it is necessary to improve the doping form of zinc oxide, improve the degree of antibacterial (Salmonella), help to remove harmful active oxygen in cells, reduce cell oxidative damage, improve the ability to resist bacterial invasion, and enable large-scale production and application. Summary of the invention

[0007] In order to solve the above technical problems, the present invention designs a self-assembly preparation method and application of multi-enzyme active nano zinc oxide for treating salmonella enteritis. The method of preparing doped nano zinc oxide by this method is suitable for industrial large-scale production, and the antibacterial effect is improved by 8 times compared with pure zinc oxide. In addition, the acidic conditions of the foregut in the body can produce hydroxyl radicals (·OH), which are toxic to bacteria and thus assist in sterilization. In the neutral environment of gastrointestinal inflammation, hydrogen peroxide is catalyzed to decompose into water and oxygen, which is an important antioxidant reaction. This activity helps to remove harmful active oxygen in cells, reduce cell oxidative damage, and improve the ability to resist bacterial invasion; and its sterilization efficiency against salmonella can reach 99.85%.

[0008] In order to achieve the above technical effects, the present invention discloses a self-assembly preparation method of multi-enzyme active nano zinc oxide for treating Salmonella enteritis, comprising the following steps:

[0009] S1: Dissolve zinc chloride and reflux at 60°C for 100 min under magnetic stirring until the solution becomes clear and completely dissolved;

[0010] S2: Add amines to the solution of S1, stir magnetically for 1 h at room temperature, then add copper chloride and manganese chloride and stir until completely dissolved;

[0011] S3: The mixture is transferred into a polytetrafluoroethylene reactor, subjected to hydrothermal high pressure reaction for a period of time, and then cooled to room temperature; thereafter, the zinc oxide-doped ZnO-CM / ZnO nanozyme is obtained after centrifugation, washing, and drying.

[0012] Further, the molar mass of copper chloride used in S2 is 1%-3%, and the molar mass of manganese chloride is 1%-2%;

[0013] Further, the molar mass of copper chloride used in S2 is 1%, and the molar mass of manganese chloride is 1%;

[0014] Furthermore, the hydrothermal high pressure reaction temperature in S3 is 150° C. and the reaction time is 2 h;

[0015] Furthermore, the centrifugal speed in S3 is 4200 rpm and the time is 10 min;

[0016] Furthermore, the washing in S3 is performed with ethanol for 3 times; the drying temperature is 60° C. and the drying time is 24 hours;

[0017] On the other hand, the present invention provides a use of multi-enzyme active self-assembled nano zinc oxide in the preparation of a Salmonella enteritis drug.

[0018] The medicine contains the multi-enzyme active self-assembled nano zinc oxide prepared by the method.

[0019] Furthermore, the multi-enzyme active self-assembled nano zinc oxide is applied by intragastric administration at a concentration of 20 mg / kg-50 mg / kg.

[0020] The beneficial effects of the present invention are:

[0021] 1. The present invention improves the direct antibacterial effect of ZnO by synthesizing doped zinc oxide by adding trace amine compounds as protective agents in a simple manner; the minimum inhibitory concentration of ZnO synthesized by the same method in vitro is 250 μg / mL, while the minimum inhibitory concentration of ZnO doped with trace Cu and Mn is about 30 μg / mL, and the antibacterial efficiency is improved by about 8 times;

[0022] 2. Trace elements copper (Cu) and manganese (Mn) are doped into ZnO nanomaterials to give ZnO multiple enzyme activities. Under the acidic conditions of the foregut, peroxidase activity is exerted to generate bactericidal reactive oxygen, and in the neutral hindgut, superoxide dismutase and catalase activities are exerted to resist oxidative stress, thereby alleviating intestinal inflammation; that is, it can exert a bactericidal effect in the acidic state of the foregut, and an anti-inflammatory effect in the hindgut.

[0023] 3. Copper-doped ZnO has peroxidase and catalase activities. It can produce hydroxyl radicals (·OH) in the acidic conditions of the foregut, which are toxic to bacteria and thus assist in sterilization. In the neutral environment of inflammation in the hindgut, it catalyzes the decomposition of hydrogen peroxide into water and oxygen, which helps to remove harmful reactive oxygen species in cells, reduce cell oxidative damage, and improve the ability to resist bacterial invasion. By doping with manganese, ZnO nanozymes acquire superoxide dismutase activity. Excessive accumulation of superoxide radicals in cells can cause cell damage, so the enhancement of superoxide dismutase activity helps protect cells from oxidative damage. The nanozyme synthesized by this method has a bactericidal efficiency of 99.85% against Salmonella at a concentration of 20 μg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings required for describing the embodiment are briefly introduced below.

[0025] Figure 1 It is a characterization diagram of Cu and Mn doped ZnO in the present invention; Figure 1A is a scanning electron microscope (SEM) image; BD are transmission electron microscope (TEM) measurements of ZnO-CM morphology and lattice constant at different magnifications; E is a high-angle annular dark field scanning transmission electron microscope (HAADF-STEM) image and element mapping; F is a Zeta potential diagram of ZnO and ZnO- CM; G is an XRD diffraction spectrum of ZnO and ZnO-CM (compared with the standard card PDF#79-2205); HK is the XPS spectrum of Zn, Cu, Mn, and O of ZnO-CM.

[0026] Figure 2 The in vitro antibacterial activity of the ZnO composite nanozyme prepared by the present invention; A in the figure is a simulation diagram of the ZnO-CM enzyme activity reaction; B is a scanning of ZnO and different doped ZnO wavelengths to represent the peroxidase enzyme activity; C is the ESR of hydroxyl radicals ( . OH) generation; D is the peroxidase activity determination of ZnO-CM at different pH; E is the comparison of SOD enzyme activity of 100 μg / ml ZnO and different doped ZnO; F is the SOD enzyme activity determination at different concentrations of ZnO-CM; G is the CAT enzyme activity determination of 100 μg / ml ZnO and different doped ZnO; H is the CAT enzyme activity determination of ZnO-CM at different concentrations.

[0027] Figure 3 It is a diagram showing the in vitro broad-spectrum antibacterial effect of the present invention and a diagram showing the in vitro minimum inhibitory concentration effect of Salmonella;

[0028] Figure 4 is the intracellular bacterial CFU after the infected cell material of the present invention is treated, Figure 4 A is a specific flow chart of the antibacterial experiment of ZnO-CM on RAW 264.7 cells; B and C are the results of the plate experiment; D is the same treatment, the cell nucleus is stained with DAPI, and the red fluorescence distribution expressed by Salmonella is observed by immunofluorescence (IF); G is the RFP in different groups of RAW 264.7 cells infected with ST and treated with ST + Flow cytometry signal diagram; F is a quantitative bar graph of immunofluorescence signal; G is flow cytometry RFP + Quantitative bar graph of signal data.

[0029] Figure 5 is the visceral bacterial load after the Salmonella infection mouse model of the present invention; Figure 5 A is a diagram of the treatment process; B is the death curve of mice; C is a curve of weight changes of mice in each group after treatment; D is the quantification of CFU on plate coating of small intestine and colon of mice; E is in vitro visceral imaging showing bacterial concentration and distribution signals; F is a photo of bacterial plate coating of small intestine and colon after treatment of each group; G is immunofluorescence observation of bacterial distribution in small intestine and colon tissue sections. DETAILED DESCRIPTION

[0030] Example 1

[0031] The present invention discloses a self-assembly preparation method and application of multi-enzyme active nano zinc oxide for treating salmonella enteritis, comprising the following steps:

[0032] S1: Dissolve zinc chloride and reflux at 60°C for 100 min under magnetic stirring until the solution becomes clear and completely dissolved;

[0033] S2: Add amine substances (oleylamine) as a protective agent to the solution of S1, and add 1% molar weight copper chloride and 1% molar weight manganese chloride after magnetic stirring at room temperature for 1 hour, and stir until completely dissolved;

[0034] S3: The mixture was transferred into a polytetrafluoroethylene reactor, reacted at 150°C for 3 hours, and then cooled to room temperature; thereafter, it was centrifuged at 4200 rpm for 10 minutes and washed with ethanol three times, and then dried in a vacuum oven at 60°C for 24 hours to obtain ZnO-CM / ZnO nanozyme.

[0035] In this example, the ZnO-CM / ZnO nanozyme prepared by the above preparation method was first judged by specific characterization. The specific results are as follows: Figure 1 As shown by Figure 1 It can be seen that the ZnO-CM / ZnO nanozyme prepared by this method is spherical, with uniform particle distribution and a diameter of 430 nm, and is composed of a large number of small ZnO nanocrystals; Figure 1 It can be seen from the BD that the characteristic ZnO lattice fringes with a crystal spacing of 0.28 nm indicate that the doping of Cu and Mn does not change the crystal structure; Figure 1 Figure E shows that Zn, O, Cu, and Mn are uniformly distributed throughout the ZnO-CM lattice, further indicating the successful synthesis of ZnO-CM nanospheres; Figure 1 From Figure 1, we can see that the Zeta potentials of ZnO and ZnO-CM are -19 mV and -4.63 mV, respectively. Compared with ZnO, the Zeta potential value of ZnO-CM increases significantly, indicating that Cu and Mn are successfully co-doped. According to the estimation in Table 1, the contents of Cu and Mn in ZnO-CM nanospheres are 4.7 wt% and 7.6 wt%, respectively.

[0036] Table 1 Inductively coupled plasma mass spectrometry (ICP-MS) analysis

[0037] Element Converted content Units Mass fraction Cu 47451.0 mg / kg 4.7451% Zn 291458.4 mg / kg 29.1458% Mn 75647.0 mg / kg 7.5647%

[0038] Example 2

[0039] In this example, based on Example 1, the activity of ZnO-CM / ZnO nanozymes under different pH environments was further analyzed; specifically, colorless 3,3',5,5'-tetramethylaniline (TMB) was used as an indicator, which can be oxidized into blue oxTMB with a characteristic UV-visible absorption peak at 652 nm, such as Figure 2 As shown in Figure B, co-doped ZnO-CM and single-doped ZnO-Cu and ZnO-Mn can oxidize colorless TMB to blue oxTMB, indicating that both Cu / Mn single-doped and co-doped ZnO have peroxidase activity. Cu / Mn co-doped ZnO exhibits higher peroxidase catalytic activity than Cu or Mn single-doped ZnO. Figure 2 C in the figure is the electron spin resonance (ESR) measurement to prove the generation of OH under acidic conditions. As can be seen from the figure, four DMPO-OH signals (intensity of 1:2:2:1) appear in both single-doped and co-doped ZnO. The signal intensity of Cu / Mn co-doped ZnO is significantly higher than that of Cu or Mn single-doped ZnO, indicating that ZnO-CM produces more OH species. Figure 2 D under different pH conditions; As can be seen from the figure, oxTMB blue can only be observed at pH = 3-6.5, while no color change was observed at pH = 7 and 8, indicating that the peroxidase-like activity of ZnO-CM only occurs under acidic conditions, and relatively low pH is conducive to the peroxidase activity of ZnO-CM. In summary, ZnO-CM exhibits good peroxidase enzyme activity under acidic microenvironment, so it can produce antibacterial active ·OH in the acidic foregut without affecting the antioxidant process in the normal physiological hindgut (pH 7 ~ 7.8).

[0040] Example 3

[0041] In this example, based on the ZnO-CM / ZnO nanozyme prepared by the preparation method of Example 1, the inhibitory effect of Salmonella was further explored, and the specific process was as follows:

[0042] First, a Salmonella infection cell model was constructed in RAW 264.7 cells using ST-RFP (the establishment process of this model is prior art and will not be described in detail here); then ZnO-CM nanozymes were used for intervention for 24 h at concentrations of 0 μg / mL, 20 μg / mL, and 40 μg / mL; the specific experimental process is as follows Figure 4 As shown in A; the detection of intracellular bacterial survival counts, whether it is the result of the plate experiment ( Figure 4 B and C), or RFP signal by flow cytometry and immunofluorescence ( Figure 4DG), both indicate that 10 μg / mL Cu-doped ZnO has better antibacterial effect than pure ZnO. When the concentration is 40 μg / mL, the antibacterial effect of ZnO-Cu is significantly enhanced, and the bacteria in the cells are almost completely eliminated. Mn-doped ZnO only shows a slight bactericidal effect, and its bactericidal effect does not increase significantly with the increase of concentration. ZnO doped with Cu and Mn at the same time has the best bactericidal effect at low concentrations. When the concentration is 20 μg / mL, the bactericidal efficiency reaches 98% in the state where RAW 264.7 survives.

[0043] Example 4

[0044] In this example, a mouse model was further used to explore the synergistic therapeutic effect of the ZnO-CM / ZnO nanozyme prepared by this method on Salmonella in vivo.

[0045] The specific experiment was divided into 5 groups: control group, model group, ZnO (50µg / mL) group, ZnO-CM-low dose (25µg / mL) group and ZnO-CM-high dose (50µg / mL) group. Except for the control group, the other groups were infected with Salmonella. The process was as follows Figure 5 As shown in A. Figure 5 As shown in B, mice died continuously from the first day after infection, and the mortality rate of mice in the model group was the highest, at 45%. At the same time, the zinc oxide group also showed a high mortality rate of 10% within two days of the start of treatment. At the same time, during the 4 days after medication, neither high-dose nor low-dose ZnO-CM caused further death. In addition, Figure 5 As shown in C, Salmonella infection significantly reduced the body weight of mice (by about 30%). The addition of ZnO partially alleviated this weight loss (by about 20%), and ZnO-CM significantly alleviated this weight loss (by only about 5%). Mice were infected with Salmonella labeled with red fluorescent protein (ST-RFP), and the distribution and bactericidal effect of ZnO-CM in mice were observed; the results of in vivo imaging of mice showed that Salmonella was widely distributed in various intestinal segments and significantly aggregated in the cecum and colon. ZnO can significantly reduce the number of Salmonella in the intestine (98% reduction in the small intestine and 96% reduction in the colon). Compared with ZnO, the effect of ZnO-CM was more significant, and the number of Salmonella in the small intestine and colon was significantly reduced (>99%). In particular, there was almost no Salmonella infection in the ZnO-CM-H group ( Figure 5 EG). Subsequently, the immunofluorescence results of small intestine and colon Salmonella further showed that ZnO-CM can effectively kill Salmonella in vivo ( Figure 5 G).

[0046] In summary, the nano zinc oxide prepared by this method is doped with trace amounts of Cu and Mn to make the minimum inhibitory concentration about 30 μg / mL, and the antibacterial efficiency is increased by about 8 times; at the same time, the trace elements copper (Cu) and manganese (Mn) are doped into the ZnO nanomaterial to give it a variety of enzyme activities, and the peroxidase activity is exerted under the acidic conditions of the stomach to generate bactericidal active oxygen, and the superoxide dismutase and catalase activity that resist oxidative stress in the neutral intestines are exerted to relieve inflammation in the intestines; and it can play a role in the acidic state of the stomach, and can relieve inflammation in the intestines in the intestines, and can fully adapt to the environment of the gastrointestinal tract. And the zinc oxide nanozyme synthesized by this method has a significant inhibitory effect on salmonella in vitro and in vivo (under in vitro conditions, at a concentration of 20 μg / mL, its bactericidal efficiency reaches 99.85%; the antibacterial efficiency in vivo is greater than 96%), and can be widely used in food safety, livestock and poultry health breeding and feed additives.

[0047] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to only the specific implementation methods described.

Claims

1. A self-assembly preparation method of multi-enzyme active nano zinc oxide for treating salmonella enteritis, characterized in that: The following steps are involved: S1: Dissolve zinc chloride and reflux at 60°C for 100 min under magnetic stirring until the solution becomes clear and completely dissolved; S2: Add amines to the solution of S1, stir magnetically for 1 h at room temperature, then add copper chloride and manganese chloride and stir until completely dissolved; S3: The mixture is transferred into a polytetrafluoroethylene reactor, subjected to hydrothermal high pressure reaction for a period of time, and then cooled to room temperature; thereafter, the zinc oxide-doped ZnO-CM / ZnO nanozyme is obtained after centrifugation, washing, and drying.

2. The self-assembly preparation method of multi-enzyme active nano zinc oxide for treating Salmonella enteritis according to claim 1, characterized in that: The amine substance added in S1 includes but is not limited to oleylamine.

3. The self-assembly preparation method of multi-enzyme active nano zinc oxide for treating Salmonella enteritis according to claim 1, characterized in that: The molar mass of the copper chloride used in the S2 is 1%-3%, and the molar mass of the manganese chloride is 1%-2%.

4. The self-assembly preparation method of multi-enzyme active nano zinc oxide for treating Salmonella enteritis according to claim 2, characterized in that: The molar mass of the copper chloride used in the S2 is 1%, and the molar mass of the manganese chloride is 1%.

5. The self-assembly preparation method of multi-enzyme active nano zinc oxide for treating Salmonella enteritis according to claim 1, characterized in that: The hydrothermal high-pressure reaction temperature in S3 is 150° C. and the reaction time is 2 h.

6. The self-assembly preparation method of multi-enzyme active nano zinc oxide for treating Salmonella enteritis according to claim 1, characterized in that: The centrifugal speed in S3 is 4200 rpm and the time is 10 min.

7. The self-assembly preparation method of multi-enzyme active nano zinc oxide for treating Salmonella enteritis according to claim 1, characterized in that: In the S3, ethanol is used for washing, and the number of washing times is 3 times; the drying temperature is 60° C., and the drying time is 24 hours.

8. Use of the self-assembled nano zinc oxide with multi-enzyme activity prepared by the self-assembled preparation method of nano zinc oxide with multi-enzyme activity for treating salmonella enteritis according to any one of claims 1 to 7 in preparing a drug for treating salmonella enteritis.

9. The use according to claim 8, characterized in that The medicine contains the multi-enzyme active self-assembled nano zinc oxide prepared by the method.

10. The use according to claim 8, wherein the multi-enzyme active self-assembled nano zinc oxide is administered by intragastric administration at a concentration of 20 mg / kg-50 mg / kg.

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