A nano-enzyme-probiotic compound, a preparation method and application thereof

Abstract

The application discloses a nano-enzyme-probiotic compound with high safety, which is used for treating vaginitis, effectively reducing damage to vaginal mucosa tissue and cells, improving the ability of vagina to resist infection of various pathogenic microorganisms, reducing the recurrence rate of vaginitis, and the like. The application further discloses a preparation method of the nano-enzyme-probiotic compound, and the method has simple preparation process, low cost, and is convenient for industrial production and clinical transformation. The application further discloses application of the nano-enzyme-probiotic compound in production of medicines for treating, relieving and / or preventing vaginitis and reducing the recurrence rate of the disease.

Description

TECHNICAL FIELD

[0001] The present application relates to a medicine for treating vaginitis, in particular to a nano-enzyme-probiotic compound and a preparation method and application thereof, and belongs to the field of medicine and health. BACKGROUND

[0002] The dominant bacteria of the vaginal microenvironment of healthy women are mainly lactic acid bacteria. Once the number or proportion of lactic acid bacteria changes, the vaginal microenvironment will be destroyed, thereby causing various vaginal infectious diseases, including bacterial vaginitis, candidal vaginitis, trichomonal vaginitis, senile vaginitis, juvenile vaginitis, and mixed vaginitis. Among them, candidal vaginitis is a common and frequently-occurring inflammatory disease of the vulva and vagina caused by fungal infection, which is usually caused by Candida albicans. According to statistics, about 75% of women in the world have suffered from candidal vaginitis at least once in their lifetime; more seriously, 5-8% of women will experience recurrence of candidal vaginitis. Therefore, candidal vaginitis not only causes pathological changes and discomfort in women's bodies, but also seriously affects women's mental health.

[0003] At present, the antifungal drugs for treating candidal vaginitis in clinical practice mainly include clotrimazole, fluconazole, miconazole and nystatin. However, some of these drugs can only inhibit the growth of fungi, but cannot kill fungi. In addition, long-term use of these antifungal drugs not only damages the normal tissues and cells of the vaginal mucosa, but also destroys the vaginal microenvironment, leading to local flora imbalance, acid-base balance disorder, defense system damage, double infection, and even drug resistance, thereby increasing the recurrence rate and increasing the difficulty of clinical treatment.

[0004] Since candidal vaginitis is caused by a large number of estrogen leading to a large number of Candida albicans proliferation, an increase in vaginal pH and vaginal flora imbalance, the occurrence of candidal vaginitis has certain correlation with the imbalance of vaginal microenvironment. Therefore, the clinical demand for maintaining the balance of vaginal microenvironment has promoted the development of microecology in the treatment of candidal vaginitis. In principle, probiotics can be used to regulate the vaginal microenvironment, such as using clinical vaginal lactic acid bacteria, which can produce lactic acid (to maintain the vaginal pH in the normal range (less than 4.5)), target-specific bacteriocins and H2O2 with broad-spectrum bactericidal activity to achieve the purpose of protecting the vagina from external pathogenic microorganisms. However, this method is only suitable for bacterial vaginitis, and the treatment effect on candidal vaginitis is not good. The reason is that compared with bacteria, fungi have a more complex structure and belong to eukaryotic cells, which have hyphae and spores. The concentration of the antibacterial substances such as lactic acid, H2O2 and bacteriocins produced by lactic acid bacteria is low, and it is difficult to penetrate into the inside of the fungal cells, making it more difficult to kill fungi. Therefore, in order to effectively treat candidal vaginitis, it is necessary to use vaginal lactic acid bacteria to maintain the vaginal microenvironment, and at the same time, other strategies are needed to kill fungi.

[0005] Studies have shown that reactive oxygen species (ROS) and / or reactive nitrogen species (RNS) have been widely used in anti-infection research. For example, some ions (such as Fe 2+ ) and natural enzymes (such as myeloperoxidase-nicotinamide adenine dinucleotide) can catalyze H2O2 to produce a large amount of ROS and / or RNS, but the former causes certain damage to normal tissues and cells due to high ion concentration, and the latter is limited in further application due to problems such as instability, non-recyclability, and immunogenicity.

[0006] Nanoplasma (i.e., a nanomaterial with enzyme-like catalytic properties) has the ability to catalyze the production of ROS and / or RNS and has been widely used in the fields of biomedicine and the like. Some studies have shown that nanoplasma with peroxidase-like activity (POD) can produce ROS and / or RNS and has high antibacterial activity. Therefore, if a nanoplasma with POD-like activity can be designed and used in combination with probiotics, ROS and / or RNS can be produced by using H2O2 and / or lactic acid produced by probiotics, so that the problems faced by the currently used antifungal drugs can be solved, that is, the purposes of killing Candida albicans and regulating the vaginal microenvironment can be simultaneously achieved, which is expected to be popularized to clinical practical application. SUMMARY

[0007] The technical problem to be solved by the present application is to provide a nanoplasma-probiotic compound and a gel thereof with high safety, which can effectively reduce damage to vaginal mucosa and cells, improve the ability of the vagina to resist infection by various pathogenic microorganisms, reduce the recurrence rate of vaginitis, and treat vaginitis.

[0008] The technical problem to be solved by the present application is to provide a nanoplasma-probiotic compound and a gel thereof with high safety, which can effectively reduce damage to vaginal mucosa and cells, improve the ability of the vagina to resist infection by various pathogenic microorganisms, reduce the recurrence rate of vaginitis, and treat vaginitis.

[0009] The technical problem to be solved by the present application is to provide a nanoplasma-probiotic compound and a gel thereof with high safety, which can effectively reduce damage to vaginal mucosa and cells, improve the ability of the vagina to resist infection by various pathogenic microorganisms, reduce the recurrence rate of vaginitis, and treat vaginitis.

[0010] To solve the above technical problems, the present application provides the following technical solutions:

[0011] The present application provides a nanoplasma-probiotic compound, which contains nanoplasma and probiotics, wherein the nanoplasma is nanoplasma with peroxidase-like activity, and the probiotics are probiotics capable of producing H2O2 and / or lactic acid.

[0012] The nano-enzyme-probiotic compound further comprises an inert carrier.

[0013] The nano-enzyme comprises an oxygen compound nano-enzyme, a nitrogen compound nano-enzyme, a metal nano-enzyme, a metal-organic framework-based nano-enzyme, a covalent organic framework-based nano-enzyme, a carbon-based nano-enzyme, or a composite nano-enzyme.

[0014] The probiotic comprises one or more of lactic acid bacteria, yeast, spore bacteria, butyrate clostridium, bifidobacterium, and actinomycetes; and the concentration of the probiotic is 10 2 -10 4 CFU / mL.

[0015] The nano-enzyme-probiotic compound has a dosage form comprising a solution, an injection, a lotion, a liniment, a powder, a pill, a tablet, a film, an ointment, a gel, a suppository, a paste, an aerosol, or a spray.

[0016] The application further provides a preparation method of the nano-enzyme-probiotic compound, comprising the following steps: mixing the nano-enzyme and the probiotic, adding a dosage form matrix, uniformly mixing, and incubating to obtain the nano-enzyme-probiotic compound.

[0017] The application further provides a nano-enzyme-probiotic compound gel, which contains a nano-enzyme and a probiotic.

[0018] The application further provides a preparation method of the nano-enzyme-probiotic compound gel, comprising the following steps:

[0019] (1) a mixed solution of hyaluronic acid, N-hydroxysuccinimide, and 1-ethyl-(3-dimethylaminopropyl) carbodiimide is prepared; L-cysteine methyl ester hydrochloride is added to the mixed solution, and the solution is stirred in the dark, and the pH value is adjusted to obtain an acidic mixed solution; the mass ratio of the hyaluronic acid, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl) carbodiimide, and L-cysteine methyl ester hydrochloride is 1:1-5:1-5:1-5;

[0020] (2) the acidic mixed solution is dialyzed and freeze-dried to obtain a fluffy solid;

[0021] (3) dissolving the peroxygenase-like nanoscale enzyme and the probiotic solution producing H2O2 and / or lactic acid in a buffer salt solution, adding fluffy solids, uniformly mixing, incubating, and forming a nanoscale enzyme-probiotic complex; the concentration of the probiotic solution is 10 1 -10 5 CFU / mL, and the mass ratio of the peroxygenase-like nanoscale enzyme to the fluffy solids is 1:100-1000.

[0022] The application also provides the nanoscale enzyme-probiotic complex and the nanoscale enzyme-probiotic complex gel for treating, relieving, and / or preventing vaginitis and reducing the recurrence rate of the disease.

[0023] The vaginitis includes bacterial vaginitis, candidal vaginitis, trichomonal vaginitis, senile vaginitis, juvenile vaginitis, mixed vaginitis, and / or other vaginitis.

[0024] As other embodiments of the present application, the nanoscale enzyme of the present application also includes oxygen family compound nanoscale enzyme, nitrogen family compound nanoscale enzyme, metal nanoscale enzyme, metal-organic framework-based (MOF) nanoscale enzyme, covalent organic framework-based (COF) nanoscale enzyme, carbon-based nanoscale enzyme or composite nanoscale enzyme, etc. The oxygen family compound nanoscale enzyme includes but is not limited to one or more of cerium oxide-based nanoscale enzyme, manganese oxide-based nanoscale enzyme, copper oxide-based nanoscale enzyme, iron oxide-based nanoscale enzyme, nickel oxide-based nanoscale enzyme, cobalt oxide-based nanoscale enzyme, zirconium oxide-based nanoscale enzyme, hafnium oxide-based nanoscale enzyme, metal and / or non-metal doped oxide-based nanoscale enzyme, manganese sulfide-based nanoscale enzyme, iron sulfide-based nanoscale enzyme, cobalt sulfide-based nanoscale enzyme, nickel sulfide-based nanoscale enzyme, copper sulfide-based nanoscale enzyme, zinc sulfide-based nanoscale enzyme, metal and / or non-metal doped sulfide-based nanoscale enzyme, manganese selenide-based nanoscale enzyme, iron selenide-based nanoscale enzyme, cobalt selenide-based nanoscale enzyme, nickel selenide-based nanoscale enzyme, copper selenide-based nanoscale enzyme, zinc selenide-based nanoscale enzyme, metal and / or non-metal doped selenide-based nanoscale enzyme, ruthenium telluride-based nanoscale enzyme, rhodium telluride-based nanoscale enzyme, palladium telluride-based nanoscale enzyme, silver telluride-based nanoscale enzyme, cadmium telluride-based nanoscale enzyme, metal and / or non-metal doped telluride-based nanoscale enzyme; the nitrogen family compound nanoscale enzyme includes but is not limited to one or more of nitrogen element-containing nanoscale enzyme, phosphorus element-containing nanoscale enzyme, arsenic element-containing nanoscale enzyme, antimony element-containing nanoscale enzyme, bismuth element-containing nanoscale enzyme; the metal nanoscale enzyme includes but is not limited to one or more of manganese-based nanoscale enzyme, iron-based nanoscale enzyme, cobalt-based nanoscale enzyme, nickel-based nanoscale enzyme, copper-based nanoscale enzyme, zinc-based nanoscale enzyme, gold nanoscale enzyme, copper nanoscale enzyme, silver nanoscale enzyme, platinum nanoscale enzyme, palladium nanoscale enzyme, rhodium nanoscale enzyme, ruthenium nanoscale enzyme, alloy nanoscale enzyme, metal and / or noble metal-based nanoscale enzyme; the MOF-based nanoscale enzyme includes but is not limited to one or more of iron-based MOF nanoscale enzyme, zinc-based MOF nanoscale enzyme, copper-based MOF nanoscale enzyme, zirconium-based MOF nanoscale enzyme, hafnium-based MOF nanoscale enzyme, vanadium-based MOF nanoscale enzyme, metal-doped MOF nanoscale enzyme; the COF nanoscale enzyme includes but is not limited to one or more of boron-based COF nanoscale enzyme, imine-based COF nanoscale enzyme, triazine-based COF nanoscale enzyme, other types of COF nanoscale enzyme; the carbon-based nanoscale enzyme includes but is not limited to fullerene-based nanoscale enzyme, carbon fiber-based nanoscale enzyme, carbon nanotube-based nanoscale enzyme, graphene-based nanoscale enzyme, other carbon-based nanoscale enzyme.

[0025] The above nanoscale enzymes of the present application can catalyze superoxide anion, hydroxyl radical, peroxide radical and / or other substances with antibacterial function generated by endogenous and / or exogenous H2O2, and can realize treatment, relief and / or prevention of vaginitis and reduce the recurrence rate of the disease. Among the above nanomaterials with enzyme-like catalytic properties, sulfide nanoscale enzyme is a very representative type of nanoscale enzyme, and the present application selects ferrous disulfide nanoscale enzyme, platinum-iron alloy nanoscale enzyme, ferroferric oxide nanoscale enzyme and pyrite nanoscale enzyme for experiment.

[0026] For the poor dispersibility of nanoscale enzyme, inert carrier can be used to achieve the effect of dispersion, so as to better play the activity of nanoscale enzyme, wherein the inert carrier includes but is not limited to reduced graphene oxide, polylactic acid-hydroxyacetic acid copolymer (PLGA) or hydroxyapatite. The reduced graphene oxide is selected for experiment in the present application.

[0027] As other embodiments of the present application, the probiotics of the present application include but are not limited to yeast, spore bacteria, butyrate Clostridium, lactic acid bacteria, bifidobacterium, actinomycetes, etc.; the yeast includes but is not limited to one or more of Saccharomyces, Delbuccia, Candida, Williopsis, Pichia, Blastomyces, Torulopsis, Starmerella, Rhodotorula, Schizosaccharomyces, and Brettanomyces; the spore bacteria includes but is not limited to one or more of Bacillus cereus, Bacillus licheniformis, Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus coagulans; the lactic acid bacteria includes but is not limited to one or more of Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus casei subsp. casei, Lactobacillus reuteri, Lactobacillus helveticus, Lactobacillus crispatus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus salivarius; the bifidobacterium includes but is not limited to one or more of Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis, Bifidobacterium asteroides, Bifidobacterium bifidum, Bifidobacterium bovis, Bifidobacterium breve, Bifidobacterium dentium, Bifidobacterium infantis (Bifidobacterium longum subsp. infantis), Bifidobacterium lactis (Bifidobacterium lactis subsp. lactis), Bifidobacterium longum, Bifidobacterium pseudolongum, Bifidobacterium thermophilum, and Bifidobacterium acidophilum; and the actinomycetes includes but is not limited to one or more of Streptomyces, Nocardia, Micromonospora, Streptosporangium, and Actinoplanes.

[0028] The compound dosage form of the nanoscale enzyme and the probiotics of the present application can be all kinds of dosage forms of drugs on the market, including but not limited to solution, injection, lotion, liniment, powder, pill, tablet, film, ointment, gel, suppository, paste, aerosol, spray. The above dosage forms are all convenient for intravaginal administration and reduce omission.

[0029] The mechanism of action of the present application: the ROS and / or RNS generated by the nanoscale enzyme catalyzing the metabolic products (H2O2 and / or lactic acid) of the probiotics have super strong oxidation reaction on pathogenic microorganisms, which mainly produce anti-infection effect through two action modes: on the one hand, through hydrogen abstraction reaction to replace unsaturated phospholipid molecules, which further react with oxygen to form lipid peroxide, destroy the integrity of cell membrane structure, and increase the ion permeability of cell membrane; on the other hand, can directly cause oxidative damage to biological molecules (such as unsaturated lipids, polypeptides and enzymes, etc. cell components), thereby effectively killing pathogenic microorganisms.

[0030] Beneficial effects: Compared with the prior art, the present application has the following remarkable advantages:

[0031] 1. Nanoscale enzymes can catalyze the metabolic products (H2O2 and / or lactic acid) of probiotics to produce ROS and / or RNS for the treatment of vaginitis, which reveals the possibility of nanoscale enzymes with POD-like activity in the treatment of deep infectious diseases;

[0032] 2. The different dosage forms of the vaginal drug containing nanoscale enzymes and probiotics have the function of in-situ responsive release, and release nanoscale enzymes and probiotics in-situ at the infected site of the vagina to minimize the damage of ROS and / or RNS to normal tissues and cells of the vaginal mucosa;

[0033] 3. The different dosage forms of the vaginal drug containing nanoscale enzymes and probiotics can regulate the vaginal microenvironment, specifically by reducing the vaginal pH and regulating the balance of vaginal flora, which is suitable for various vaginitis and can improve the ability of the vagina to resist various pathogenic microbial infections;

[0034] 4. The different dosage forms of the vaginal drug containing nanoscale enzymes and probiotics can kill pathogenic microorganisms and regulate the vaginal microenvironment at the same time, which can significantly reduce the recurrence rate of vaginitis compared with traditional small molecule antifungal drugs (such as clotrimazole suppositories);

[0035] 5. The combination of nanoscale enzymes and clinically approved probiotics has high safety and meets the clinical needs;

[0036] 6. The preparation process is simple, the cost of raw materials is low, and it is convenient for industrial production and clinical transformation. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 Effect of lactic acid bacteria of different concentrations and fermentation time on the cell viability of Candida albicans (standard strain);

[0038] Figure 2 A is the standard curve detection result of different concentrations of H2O2; Figure 2 B is the detection result of the concentration of H2O2 produced by lactic acid bacteria; Figure 2 C is the effect of lactic acid bacteria on the pH value of MRS broth;

[0039] Figure 3 A is the electron microscope image of reduced graphene oxide; Figure 3 B is the distribution diagram of iron disulfide on the surface of reduced graphene oxide; Figure 3 C is the electron microscope image of iron disulfide;

[0040] Figure 4 X-ray diffraction (XRD) pattern of iron disulfide nanoscale enzyme with reduced graphene oxide as carrier;

[0041] Figure 5 A is the peroxidase-like (POD) activity detection result of iron disulfide nanoszyme; Figure 5 B is the peroxidase-like (POD) activity detection result of platinum-iron alloy nanoszyme, magnetite nanoszyme, and pyrite nanoszyme; Figure 5 C is the effect of iron disulfide nanoszyme mixed with hydrogen peroxide on the cell viability of Candida albicans (standard strain);

[0042] Figure 6 A is the effect of iron disulfide nanoszyme mixed with lactic acid bacteria on the cell viability of Candida albicans (standard strain); Figure 6 B is the effect of iron disulfide nanoszyme mixed with lactic acid bacteria on the cell viability of lactic acid bacteria; Figure 6 C is the effect of iron disulfide nanoszyme mixed with lactic acid bacteria on the pH value of MRS broth;

[0043] Figure 7 A is the effect of iron disulfide nanoszyme mixed with lactic acid bacteria on the cell viability of Candida albicans (clinical isolated strain); Figure 7 B is the effect of iron disulfide nanoszyme mixed with lactic acid bacteria on the cell viability of lactic acid bacteria; Figure 7 C is the effect of iron disulfide nanoszyme mixed with lactic acid bacteria on the pH value of MRS broth;

[0044] Figure 8 is the change of cumulative in vitro release rate of lactic acid bacteria gel;

[0045] Figure 9 is the schematic diagram of construction of vaginitis animal model and administration time;

[0046] Figure 10 is the effect evaluation of iron disulfide nanoszyme-lactic acid bacteria gel for treating, relieving, and / or preventing vaginitis;

[0047] Figure 11 is the schematic diagram of iron disulfide nanoszyme-lactic acid bacteria gel for treating, relieving, and / or preventing vaginitis and reducing the recurrence rate of the disease;

[0048] Figure 12 A is the effect of iron disulfide nanoszyme-lactic acid bacteria gel on the vaginal flora structure of mice; Figure 12 B is the effect of iron disulfide nanoszyme-lactic acid bacteria gel on the relative abundance of Firmicutes; Figure 12 C is the effect of iron disulfide nanoszyme-lactic acid bacteria gel on the relative abundance of Proteobacteria;

[0049] Figure 13 is the effect of iron disulfide nanoszyme-lactic acid bacteria gel and clotrimazole suppository on the cell viability of Candida albicans. DETAILED DESCRIPTION

[0050] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0051] Example 1: Anti-Candida albicans activity of different concentrations of lactic acid bacteria fermented for different times

[0052] 1. Cultivation of Lactobacillus strains

[0053] There are no special requirements for the selection of lactic acid bacteria; commercially available lactic acid bacteria can be used in this invention. However, in order to highlight the experimental results, ensure the effectiveness of the lactic acid bacteria, and avoid the influence of other drugs, the commercially available lactic acid bacteria were recultured in this embodiment.

[0054] (1) Take one vaginal lactobacillus capsule (Inner Mongolia Shuangqi Pharmaceutical Co., Ltd., S20030005), cut it open with sterile scissors, dissolve the white powder inside in 15 mL of MRS broth (5.2%), and culture it under microaerophilic conditions (O2 concentration 6%-12%; CO2 concentration 5-8%; N2 concentration 80-90%) for 24 h to obtain lactobacillus culture medium;

[0055] (2) The lactic acid bacteria culture was inoculated into MRS solid medium with a sterile inoculation loop and cultured under microaerophilic conditions for 24 h to obtain a single clonal strain of lactic acid bacteria.

[0056] (3) Select a single clone of lactic acid bacteria, put it into 15 mL of the same MRS broth under microaerophilic conditions, and continue to culture for 24 h to obtain lactic acid bacteria culture.

[0057] (4) The lactic acid bacteria culture was washed three times by centrifugation with 1 mL of sterile phosphate solution (prepared by dissolving 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 in 1000 mL of sterile distilled water) (pH 8.0, 0.01 M) (centrifugation speed 3500 rpm / min, centrifugation time 3 min). Finally, 10 lactic acid bacteria solutions of different concentrations were prepared. 1 CFU / mL, 10 2 CFU / mL, 10 3 CFU / mL, 10 4 CFU / mL, 10 5 (CFU / mL), for later use.

[0058] 2. Anti-Candida albicans activity of different concentrations of lactic acid bacteria fermented for different times

[0059] The anti-Candida albicans activity of different concentrations of lactic acid bacteria was investigated by plate dilution method. The detection method was as follows: (1) Candida albicans (10 6CFU / mL standard strain) and different concentrations of lactic acid bacteria were divided into six groups in 24-well plates: ① control group: Candida albicans + MRS broth; ② Candida albicans + lactic acid bacteria (10 1 CFU / mL) + MRS broth; ③ Candida albicans + lactic acid bacteria (10 2 CFU / mL) + MRS broth; ④ Candida albicans + lactic acid bacteria (10 3 CFU / mL) + MRS broth; ⑤ Candida albicans + lactic acid bacteria (10 4 CFU / mL) + MRS broth; ⑥ Candida albicans + lactic acid bacteria (10 5 CFU / mL) + MRS broth. (2) The above six groups were mixed with MRS broth and incubated at 37°C under microaerobic conditions for 12h, 24h, 36h and 48h, respectively. Then 100μL was taken and spread on MRS medium agar plates and incubated for 48h. Immediately afterwards, ImageJ software was used to quantitatively analyze the results of each group of Candida albicans as shown in Figure 1 . Different concentrations of lactic acid bacteria solution all had obvious anti-Candida albicans activity at 48h. Considering the role of vaginal microbiota balance on vaginal health, high concentrations of lactic acid bacteria would increase the killing of pathogenic bacteria, but would inevitably affect the growth of other probiotics.

[0060] Example 2 Detection of the concentration of H2O2 produced by lactic acid bacteria fermentation and the ability to reduce pH

[0061] The microaerobic gas production bag and gas production bag (microaerobic environment) (Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., HBYY008), MRS broth were used to simulate the microenvironment of candidal vaginitis. Since the pH would rise after vaginal infection with pathogenic bacteria, the pH of the MRS broth was adjusted to about 5.7. The standard curve of H2O2 was drawn as shown in Figure 2 A according to the instructions of the hydrogen peroxide detection kit (Shanghai Biyun Tian Biological Technology Co., Ltd., S0038), and the content of H2O2 produced by the lactic acid bacteria (10 3 CFU / mL) obtained in Example 1 was detected, and the results are shown in Figure 2 B. The concentration of H2O2 produced by lactic acid bacteria was 30-50μM. In addition, the lactic acid bacteria (10 3 CFU / mL) were re-dispersed in MRS broth, and the pH value of the MRS broth was detected by a pH meter. The results showed that the lactic acid bacteria could significantly reduce the pH value of the MRS broth. As shown in Figure 2 C, the pH value of the MRS broth could be reduced from 5.7 to 4.5 at 24h.

[0062] Example 3 Preparation of a gel containing iron disulfide nanoszyme and lactic acid bacteria

[0063] 1. Preparation of reduced graphene oxide as carrier of iron disulfide nanoszyme (rGO@FeS2)

[0064] Since sulfides are prone to aggregation, affecting their activity, a carrier needs to be selected to disperse them. In this embodiment, graphene oxide is selected as the raw material to prepare reduced graphene oxide, and the obtained reduced graphene oxide is used as a carrier to disperse iron disulfide, so as to ensure that its activity can be better exerted.

[0065] (1) 675 mg of ferric chloride (National Pharmaceutical Group Chemical Reagents Co., Ltd., 7705-08-0) and 190 mg of thiourea (National Pharmaceutical Group Chemical Reagents Co., Ltd., 62-56-6; both ferric chloride and thiourea are used as raw materials for synthesizing iron disulfide nanoszyme) were respectively dissolved in 10 mL of deionized water, and stirred at room temperature at 800 rpm / min for 0.5 h to obtain a mixed solution of ferric chloride and thiourea;

[0066] (2) 42 mg of graphene oxide (Jiangsu Xianfeng Nanometer Material Technology Co., Ltd., 7440-44-0) was placed in 7.5 mL of deionized water, and stirred at room temperature for 0.5 h to obtain a 5.6 g / L solution of reduced graphene oxide;

[0067] (3) The mixed solution of ferric chloride and thiourea was slowly added to the reduced graphene oxide solution by using a syringe pump, and 28 μL of concentrated ammonia water (National Pharmaceutical Group Chemical Reagents Co., Ltd., 1336-21-6) (25-28%) was added, and the stirring was continued at 800 rpm / min for 0.5 h to obtain a mixed solution;

[0068] (4) The mixed solution obtained in step (3) was transferred to a reaction kettle, and heated at 180°C for 12 h to obtain iron disulfide nanoszyme;

[0069] (5) After the iron disulfide nanoszyme was cooled to room temperature, it was washed three times by centrifugation (8000 rpm / min for 5 min) with deionized water and anhydrous ethanol alternately to remove residual reagents. Finally, the precipitate obtained by centrifugation was added to 2 mL of deionized water and dispersed thoroughly, and then freeze-dried by a freeze-drying machine (Beijing Yaxing Instrument and Science Technology Development Co., Ltd., LGJ-10N) to obtain an iron disulfide nanoszyme solid powder, which was ready for use.

[0070] 2. Preparation of iron disulfide nanoszyme-lactic acid bacteria gel (FeLab)

[0071] (1) 400 mg of hyaluronic acid (HA) (Shanghai Yuan Ye Biotechnology Co., Ltd., 9067-32-7), 575 mg of N-hydroxysuccinimide (NHS) (Shanghai Yuan Ye Biotechnology Co., Ltd., 106627-54-7) and 958 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) (Shanghai Yuan Ye Biotechnology Co., Ltd., 25952-53-8) were dissolved in 100 mL of deionized water and stirred at 800 rpm / min at room temperature for 1 h to obtain a mixed solution;

[0072] (2) 855 mg of L-cysteine methyl ester hydrochloride (Shanghai Yuan Ye Biotechnology Co., Ltd., 18598-63-5) was added to the mixed solution prepared in step (1) and stirred in the dark for 24 h to obtain a thiol-containing mixed solution;

[0073] (3) The pH value of the mixed solution obtained in step (2) was adjusted to 4.8 using NaOH (1.0 M) and HCl (1.0 M) solutions to obtain an acidic mixed solution;

[0074] (4) The acidic mixed solution obtained in step (3) was thoroughly dialyzed in a dialysis bag in a dilute HCl solution (concentration of 15%) to remove residual reagents; and finally freeze-dried into a fluffy solid;

[0075] (5) Iron disulfide nanoszyme (1 mg / mL, 50 μL) and lactic acid bacteria solution (10 3 CFU / mL, 100 μL) were dissolved in 400 μL of sterile phosphate buffered saline solution (pH 8.0, 0.01 M), and then 16 mg of fluffy solid was added, vortexed and mixed uniformly for 10 s, and then incubated at 37°C until the iron disulfide nanoszyme-lactic acid bacteria gel (FeLab) was formed.

[0076] Example 4 Preparation of platinum-iron alloy nanoszyme-probiotic compound gel (not using reduced graphene oxide as a carrier)

[0077] 1. Preparation of platinum-iron alloy nanoszyme (Pt3Fe)

[0078] (1) Take 16 mg of platinum acetylacetonate (Beijing Bailingwei Technology Co., Ltd., 15170-57-7), 21.4 mg of iron acetylacetonate (Beijing Bailingwei Technology Co., Ltd., 14024-18-1), 50 mg of benzoic acid (National Pharmaceutical Group Chemical Reagent Co., Ltd., 65-85-0) and 80 mg of polyvinylpyrrolidone (Shanghai Aladdin Biochem Technology Co., Ltd., 9003-39-8; platinum acetylacetonate, iron acetylacetonate, benzoic acid and polyvinylpyrrolidone are all raw materials for synthesizing platinum-iron alloy nanoscale enzyme) and disperse them in 5 mL of benzyl alcohol (National Pharmaceutical Group Chemical Reagent Co., Ltd., 100-51-6), stir at room temperature at 2000 rpm / min for 15 min, and obtain a mixed solution.

[0079] (2) Transfer the mixed solution obtained in step (1) to a reaction kettle, heat at 180°C for 12 h, and obtain a platinum-iron alloy nanoscale enzyme;

[0080] (3) After the platinum-iron alloy nanoscale enzyme is cooled to room temperature, it is washed 6 times by centrifugation (10000 rpm / min for 5 min) with acetone and anhydrous ethanol alternately to remove residual reagents, and obtain a precipitate. Finally, after adding 2 mL of deionized water to the precipitate and dispersing it thoroughly, freeze-drying is performed by a freeze-drying machine (Beijing Yaxing Instrument Technology Development Co., Ltd., LGJ-10N) to obtain a platinum-iron alloy nanoscale enzyme solid powder, which is ready for use.

[0081] 2. Preparation of platinum-iron alloy nanoscale enzyme-probiotic compound gel

[0082] Take 400 mg of hyaluronic acid (HA), 575 mg of N-hydroxysuccinimide (NHS), 958 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and 855 mg of L-cysteine methyl ester hydrochloride, and prepare a fluffy solid according to the procedure of Example 1; dissolve 1 mg / mL 50 μL of platinum-iron alloy nanoscale enzyme and 10 3 CFU / mL of lactic acid bacteria solution in 400 μL of sterile phosphate buffered saline solution (pH 8.0, 0.01 M), and then add 16 mg of fluffy solid, respectively. After vortex mixing for 10 s, incubate at 37°C until a platinum-iron alloy nanoscale enzyme-lactic acid bacteria gel is formed.

[0083] Example 5 Preparation of ferroferric oxide nanoscale enzyme-probiotic compound gel (without reduced graphene oxide as carrier)

[0084] 1. Preparation of ferroferric oxide nanoscale enzyme (Fe3O4)

[0085] (1) Take ferric trichloride (Chemical Reagent Co., Ltd. of China National Pharmaceutical Group, 7705-08-0) and ferrous sulfate monohydrate (Chemical Reagent Co., Ltd. of China National Pharmaceutical Group, 17375-41-6) respectively, and dissolve them in 10 mL of deionized water under nitrogen protection, to obtain a mixed solution.

[0086] (2) Add 5 mL of concentrated ammonia (Chemical Reagent Co., Ltd. of China National Pharmaceutical Group, 1336-21-6) (25-28%) to the mixed solution obtained in step (1), and stir at room temperature at 2000 rpm / min for 0.5 h to obtain a ferroferric oxide nanoscale enzyme.

[0087] (3) Add deionized water to the ferroferric oxide nanoscale enzyme obtained in step (2) and separate and wash with a magnet for 5 times to remove residual reagents. Finally, after adding 2 mL of deionized water to the obtained precipitate and dispersing it thoroughly, freeze-drying with a freeze-drying machine (Beijing Yaxing Instrument Technology Development Co., Ltd., LGJ-10N) to obtain a ferroferric oxide nanoscale enzyme solid powder, which is ready for use.

[0088] 2. Preparation of ferroferric oxide nanoscale enzyme-lactic acid bacteria gel

[0089] Take 400 mg of hyaluronic acid (HA), 575 mg of N-hydroxysulfosuccinimide (NHS), 958 mg of 1-ethyl-(3-dimethylaminopropyl) carbonyldiimide (EDC), and 855 mg of L-cysteine methyl ester hydrochloride, respectively, and prepare fluffy solids according to the procedure of Example 1; dissolve 50 μL of 1 mg / mL ferroferric oxide nanoscale enzyme and 100 μL of 10 CFU / mL lactic acid bacteria solution in 400 μL of sterile phosphate buffered saline solution (pH 8.0, 0.01 M), and then add 16 mg of fluffy solids, respectively, and vortex for 10 s to mix uniformly, and then incubate at 37°C until a ferroferric oxide nanoscale enzyme-lactic acid bacteria gel is formed. 3 CFU / mL of lactic acid bacteria solution in 400 μL of sterile phosphate buffered saline solution (pH 8.0, 0.01 M), and then add 16 mg of fluffy solids, respectively, and vortex for 10 s to mix uniformly, and then incubate at 37°C until a ferroferric oxide nanoscale enzyme-lactic acid bacteria gel is formed.

[0090] Example 6 Preparation of pyrite nanoscale enzyme-probiotic compound gel

[0091] 1. Preparation of pyrite nanoscale enzyme (Pyrite)

[0092] (1) 0.7 g of polyvinylpyrrolidone (Shanghai Aldrin Biochemical Technology Co., Ltd., 9003-39-8), 0.5 g of ferric chloride hexahydrate (National Pharmaceutical Group Chemical Reagent Co., Ltd., 10025-77-1) and 3.6 g of sodium acetate (National Pharmaceutical Group Chemical Reagent Co., Ltd., 2836-32-0) were dispersed in 30 mL of ethylene glycol (National Pharmaceutical Group Chemical Reagent Co., Ltd., 107-21-1), stirred at room temperature at 2000 rpm / min for 15 min to obtain a mixed solution.

[0093] (2) 0.4 g of sulfur powder (National Pharmaceutical Group Chemical Reagent Co., Ltd., 7704-34-9) was added to the mixed solution obtained in step (1), and stirring was continued at room temperature at 2000 rpm / min for 60 min to obtain a sulfur-containing mixed solution.

[0094] (3) The sulfur-containing mixed solution obtained in step (2) was transferred to a reaction kettle, heated at 200°C for 12 h to obtain pyrite nanoscale enzyme;

[0095] (4) After the pyrite nanoscale enzyme cooled to room temperature, it was washed 9 times by centrifugation (10000 rpm / min for 5 min) with chloroform, anhydrous ethanol and deionized water to remove residual reagents. Finally, the precipitate obtained by centrifugation was dispersed in 2 mL of deionized water, and then freeze-dried by a freeze-drying machine (Beijing Yaxing Instrument Technology Development Co., Ltd., LGJ-10N) to obtain pyrite nanoscale enzyme solid powder, which was ready for use.

[0096] 2. Preparation of pyrite nanoscale enzyme-probiotic compound gel

[0097] 400 mg of hyaluronic acid (HA), 575 mg of N-hydroxysulfosuccinimide (NHS), 958 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and 855 mg of L-cysteine methyl ester hydrochloride were taken respectively, and a fluffy solid was prepared according to the procedure of Example 1; 1 mg / mL of pyrite nanoscale enzyme 100 μL and 10 3 CFU / mL of lactic acid bacteria solution 100 μL were dissolved in 400 μL of sterile phosphate buffered saline solution (pH 8.0, 0.01 M), and then 16 mg of fluffy solid was added respectively, and vortexed uniformly for 10 s, and then incubated at 37°C until platinum-iron alloy nanoscale enzyme-lactic acid bacteria gel, ferroferric oxide nanoscale enzyme-lactic acid bacteria gel and pyrite nanoscale enzyme-lactic acid bacteria gel were formed.

[0098] Preparation of lactic acid bacteria gel (Lab) of Comparative Example 1

[0099] The lactic acid bacteria solution (10 3The lactic acid bacteria solution (1 mg / mL, 50 μL) and the hyaluronic acid solution (10 mg / mL, 100 μL) were dissolved in 400 μL of sterile phosphate buffered saline solution (pH 8.0, 0.01 M), and then 16 mg of the fluffy solid prepared in Example 3 was added. After being uniformly mixed by vortex for 10 s, the mixture was incubated at 37 °C until a lactic acid bacteria-hyaluronic acid gel (Lab@HA) was formed.

[0100] Preparation of a reduced graphene oxide-lactic acid bacteria gel (rGO Lab) in Comparative Example 2

[0101] The graphene oxide solution (1 mg / mL, 50 μL) and the lactic acid bacteria solution (1 mg / mL, 50 μL) in Example 1 were dissolved in 400 μL of sterile phosphate buffered saline solution (pH 8.0, 0.01 M), and then 16 mg of the fluffy solid prepared in Example 3 was added. After being uniformly mixed by vortex for 10 s, the mixture was incubated at 37 °C until a lactic acid bacteria-hyaluronic acid gel (Lab@HA) was formed.

[0102] The graphene oxide solution (1 mg / mL, 50 μL) and the lactic acid bacteria solution (1 mg / mL, 50 μL) in Example 1 were dissolved in 400 μL of sterile phosphate buffered saline solution (pH 8.0, 0.01 M), and then 16 mg of the fluffy solid prepared in Example 3 was added. After being uniformly mixed by vortex for 10 s, the mixture was incubated at 37 °C until a lactic acid bacteria-hyaluronic acid gel (Lab@HA) was formed. 3 The lactic acid bacteria solution (1 mg / mL, 50 μL) and the hyaluronic acid solution (10 mg / mL, 100 μL) were dissolved in 400 μL of sterile phosphate buffered saline solution (pH 8.0, 0.01 M), and then 16 mg of the fluffy solid prepared in Example 3 was added. After being uniformly mixed by vortex for 10 s, the mixture was incubated at 37 °C until a lactic acid bacteria-hyaluronic acid gel (Lab@HA) was formed.

[0103] Preparation of a reduced graphene oxide gel (rGO@HA) in Comparative Example 3

[0104] The graphene oxide solution (1 mg / mL, 50 μL) and the lactic acid bacteria solution (1 mg / mL, 50 μL) in Example 1 were dissolved in 400 μL of sterile phosphate buffered saline solution (pH 8.0, 0.01 M), and then 16 mg of the fluffy solid prepared in Example 3 was added. After being uniformly mixed by vortex for 10 s, the mixture was incubated at 37 °C until a lactic acid bacteria-hyaluronic acid gel (Lab@HA) was formed.

[0105] Preparation of a reduced graphene oxide-ferrous disulfide nanoszyme gel (rGO@FeS2@HA) in Comparative Example 4

[0106] The graphene oxide solution (1 mg / mL, 50 μL) and the lactic acid bacteria solution (1 mg / mL, 50 μL) in Example 1 were dissolved in 400 μL of sterile phosphate buffered saline solution (pH 8.0, 0.01 M), and then 16 mg of the fluffy solid prepared in Example 3 was added. After being uniformly mixed by vortex for 10 s, the mixture was incubated at 37 °C until a lactic acid bacteria-hyaluronic acid gel (Lab@HA) was formed.

[0107] Characterization, determination and analysis of different products in Experimental Example 1

[0108] The iron disulfide nanoszyme (rGO@FeS2) prepared in Example 3 was characterized by transmission electron microscopy (TEM) (JEOL Ltd., JEM-2100), and the results are shown in FIG. 3. Figure 3 As can be seen from FIG. 3, the reduced graphene oxide (rGO) has a two-dimensional layered structure, and the iron disulfide nanoszyme (rGO@FeS2) is uniformly distributed on the surface of the rGO. Figure 3

[0109] The iron disulfide nanoszyme (rGO@FeS2) prepared in Example 3 was diffracted by X-ray diffraction technology (XRD) with reduced graphene oxide as a control, and the diffraction spectrum was analyzed, and the results are shown in FIG. 4. Figure 4 The characteristic peaks around 25° and 43° in the spectrum correspond to reduced graphene oxide and marcasite (FeS2), respectively, indicating the successful synthesis of the iron disulfide nanoszyme (rGO@FeS2).

[0110] ​The peroxidase-like activity (POD) and anti-Candida albicans (standard strain) activity of rGO@FeS2 prepared in Example 3 were tested by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO, Dojin Chemical, 3317-61-1) and plate dilution method. The peroxidase-like activity (POD) assays were performed in the following groups: Control group: DMPO (100 mM); H2O2 group: H2O2 solution (80 μM) + DMPO (100 mM); rGO group: rGO solution (25 μg / mL) + DMPO (100 mM); rGO@FeS2 group: rGO@FeS2 solution (25 μg / mL) + DMPO (100 mM); rGO+H2O2 group: rGO solution (25 μg / mL) + H2O2 solution (80 μM) + DMPO (100 mM); rGO@FeS2+H2O2 group: rGO@FeS2 solution (25 μg / mL) + H2O2 solution (80 μM) + DMPO (100 mM); Pt3Fe group Pt3Fe group: Pt3Fe solution (25 μg / mL) + DMPO (100 mM); Fe3O4 group: Fe3O4 solution (25 μg / mL) + DMPO (100 mM); Pyrite group: Pyrite solution (25 μg / mL) + DMPO (100 mM); Pt3Fe + H2O2 group: Pt3Fe solution (25 μg / mL) + H2O2 solution (80 μM) + DMPO (100 mM); Fe3O4 + H2O2 group: Fe3O4 solution (25 μg / mL) + H2O2 solution (80 μM) + DMPO (100 mM); Pyrite + H2O2 group: Pyrite solution (25 μg / mL) + H2O2 solution (80 μM) + DMPO (100 mM). The anti-Candida albicans (standard strain) activity assays were performed in the following groups: Control group: Candida albicans (10 6 CFU / mL standard strain); H2O2 group: H2O2 solution (80μM) + Candida albicans (10 6 CFU / mL standard strain); rGO group: rGO solution (25 μg / mL) + Candida albicans (10 6 CFU / mL standard strain); rGO@FeS2 group: rGO@FeS2 solution (25 μg / mL) + Candida albicans (10 6 CFU / mL standard strain); rGO+H2O2 group: rGO solution (25 μg / mL) + H2O2 solution (80 μM) + Candida albicans (10 6 CFU / mL standard strain); rGO@FeS2+H2O2 group: rGO@FeS2 solution (25μg / mL) + H2O2 solution (80μM) + Candida albicans (106 CFU / mL standard strain); Pt3Fe group: Pt3Fe solution (25 pg / mL) + C. albicans (10 6 CFU / mL standard strain); Fe3O4 group: Fe3O4 solution (25 pg / mL) + C. albicans (10 6 CFU / mL standard strain); Pyrite group: Pyrite solution (25 pg / mL) + C. albicans (10 6 CFU / mL standard strain); Pt3Fe + H2O2 group: Pt3Fe solution (25 pg / mL) + H2O2 solution (80 mM) + C. albicans (10 6 CFU / mL standard strain); Fe3O4 + H2O2 group: Fe3O4 solution (25 pg / mL) + H2O2 solution (80 mM) + C. albicans (10 6 CFU / mL standard strain); Pyrite + H2O2 group: Pyrite solution (25 pg / mL) + H2O2 solution (80 mM) + C. albicans (10 6 CFU / mL standard strain). From Figure 5 It can be seen that rGO@FeS2 has the strongest POD-like activity, that is, even the same concentration of rGO, Pt3Fe, Fe3O4, and Pyrite solution can produce weak hydroxyl radical (one of ROS) signals, however, rGO@FeS2 can catalyze H2O2 to produce the strongest signal; at the same time, rGO, Pt3Fe, Fe3O4, and Pyrite solution can catalyze H2O2 to produce certain anti-C. albicans activity, but rGO@FeS2 has the strongest anti-C. albicans activity in cooperation with H2O2. Therefore, we selected rGO@FeS2 as the target nanoscale enzyme for exploration.

[0111] Experimental Example 2 Exploration of the Anti-C. albicans (standard strain, clinical isolated strain) activity of rGO@FeS2 solution and lactic acid bacteria solution in combination

[0112] The anti-C. albicans (standard strain) activity of rGO@FeS2 solution and lactic acid bacteria solution was explored by plate dilution method. The detection method was as follows: (1) C. albicans (standard strain) (10 6 CFU / mL) and lactic acid bacteria (10 3CFU / mL) were divided into six groups in 24-well plates: ① Control group: C. albicans (standard strain) + MRS broth; ② Lactic acid bacteria treatment group: C. albicans (standard strain) + lactic acid bacteria + MRS broth; ③ rGO treatment group: C. albicans (standard strain) + rGO solution (25 μg / mL) + MRS broth; ④ rGO@FeS2 treatment group: C. albicans (standard strain) + rGO@FeS2 solution (25 μg / mL) + MRS broth; ⑤ rGO + lactic acid bacteria treatment group: C. albicans (standard strain) + lactic acid bacteria + rGO solution (25 μg / mL) + MRS broth; ⑥ rGO@FeS2 + lactic acid bacteria treatment group: C. albicans (standard strain) + lactic acid bacteria + rGO@FeS2 solution (25 μg / mL) + MRS broth. (2) After mixing the above six groups with MRS broth and incubating at 37°C under microaerobic conditions for 24 h, 100 μL was taken and spread on MRS medium agar plates and incubated for 48 h, and then ImageJ software was used to quantitatively analyze the C. albicans (standard strain) (as shown in Figure 6 A) and lactic acid bacteria cell viability (as shown in Figure 6 B). At the same time, the pH values of the MRS broth containing lactic acid bacteria solutions were detected with a pH meter, and the results are shown in Figure 6 C. The lactic acid bacteria treatment group had little effect on the cell viability of C. albicans (standard strain); compared with other groups, the combined use of rGO@FeS2 solution and lactic acid bacteria solution had obvious anti-C. albicans (standard strain) activity and could maintain the viability of lactic acid bacteria, and also could reduce the pH value of the MRS broth mixed solution (as shown in Figure 6 ).

[0113] To further demonstrate the actual conversion possibility of the present application, the obtained rGO@FeS2 solution and lactic acid bacteria solution were used in combination to investigate their antibacterial activity against clinically isolated C. albicans and their pH-lowering ability. Similarly, the antibacterial activity of rGO@FeS2 solution and lactic acid bacteria solution (Lactobacillus) against C. albicans (clinically isolated strain) was investigated by the plate dilution method. The detection method was: (1) C. albicans (clinically isolated strain) (10 6 CFU / mL) and lactic acid bacteria solution (10 3CFU / mL) were divided into six groups in 24-well plates: ① Control group: C. albicans (clinical isolate) + MRS broth; ② Lactic acid bacteria treatment group: C. albicans (clinical isolate) + lactic acid bacteria + MRS broth; ③ rGO treatment group: C. albicans (clinical isolate) + rGO solution (25 pg / mL) + MRS broth; ④ rGO@FeS2 treatment group: C. albicans (clinical isolate) + rGO@FeS2 solution (25 pg / mL) + MRS broth; ⑤ rGO + lactic acid bacteria treatment group: C. albicans (clinical isolate) + lactic acid bacteria + rGO solution (25 pg / mL) + MRS broth; ⑥ rGO@FeS2 + lactic acid bacteria treatment group: C. albicans (clinical isolate) + lactic acid bacteria + rGO@FeS2 solution (25 pg / mL) + MRS broth. (2) After mixing the above six groups with MRS broth solution, incubate at 37°C under microaerobic conditions for 24 h, take 100 pL and disperse on MRS medium agar plates and incubate for 48 h, then immediately use ImageJ software to quantitatively analyze the C. albicans (clinical isolate) (such as Figure 7 A shows) and lactic acid bacteria cell viability (such as Figure 7 B shows). At the same time, use a pH meter to detect the pH value of the MRS broth containing lactic acid bacteria solution, and the results are shown in Figure 7 C. The effect of lactic acid bacteria treatment group on the cell viability of clinically isolated C. albicans is very small; compared with other groups, the combined use of rGO@FeS2 solution and lactic acid bacteria solution also has obvious antifungal ability for clinically isolated C. albicans and can maintain the viability of lactic acid bacteria, and can also reduce the pH value of the mixed MRS broth solution (as shown in Figure 7 ).

[0114] Investigation of gel agent responsive release behavior

[0115] Since the hyaluronidase (HAase) secreted by bacteria and fungi can hydrolyze hyaluronic acid, thereby releasing nanoscale enzymes and probiotics in the gel agent, we investigated the release behavior of the gel agent obtained in Comparative Example 1. The specific implementation method is: each take 10 mL of lactic acid bacteria gel agent (Lab) and immerse it in sterile phosphate buffered saline solution (0.1M pH 5.0) containing no HAase and sterile phosphate buffered saline solution (0.1M pH 5.0) containing 150 U / mg HAase, and continuously oscillate. At 0, 2h, 4h, 8h, 12h, 24h, take 100 pL of the surrounding medium, and use an enzyme marker (Meiguoyi instrument (Shanghai) Co., Ltd., SpectraMax M2e) to measure the optical density (OD) value at 600 nm to calculate the cumulative release rate of lactic acid bacteria. The results are shown in Figure 8As shown, the cumulative release rate of lactic acid bacteria in the gel after adding HAase is 100% within 24h, while the cumulative release rate of the gel without HAase is only about 20%, indicating that the prepared gel has excellent responsive release behavior, so the gel will not affect the exertion of drug efficacy.

[0116] Experimental Example 4 Mouse experiment

[0117] The mouse candidal vaginitis was treated with the iron disulfide nanoszyme-lactic acid bacteria gel (FeLab) obtained in Example 3, and the recurrence was evaluated. Since Balb / C mice are the most commonly used animals in the fields of tumor, inflammation and autoimmune, we selected Balb / C as the model animal to construct vaginitis, and according to the method of Example 1, the mice were injected with estradiol benzoate solution (2mg / mL, 100μL) in the back of the neck every two days for a total of 3 times; 10 Figure 9 As shown, the mice were injected with Candida albicans (10 9 CFU / mL20μL) to establish a model of vaginitis infected with Candida albicans. Subsequently, the preparations obtained in Example 3 and Comparative Examples 1-4 were administered intravaginally five times (0.4mg / kg) as controls for the healthy group and the group infected with Candida albicans. As shown in Figure 10 As shown, the Lab treatment group also had an effect on the cell viability of Candida albicans, but could not cause Candida albicans to completely lose cell viability, while the number of Candida albicans colonies in the FeLab treatment group was the least, which could effectively kill Candida albicans, indicating that FeLab has a significant effect on the treatment of vaginitis.

[0118] The mice with Candida albicans infected vaginitis were treated with clotrimazole suppository in the same way. The flora structure in the vaginal lavage fluid was investigated by 16S rRNA gene sequencing technology, and it was found that the flora structure of the mouse vagina infected with Candida albicans was seriously disordered, which was specifically manifested in that the number of Firmicutes was relatively reduced, and the number of Proteobacteria was relatively increased. On the contrary, as shown in Figure 11 As shown, the number of Firmicutes in the vaginal flora of the mice treated with FeLab was relatively increased, and the number of Proteobacteria was relatively reduced (as shown in Figure 12 This result indicates that compared with clotrimazole suppository, FeLab treatment of vaginitis can achieve the regulation of vaginal flora structure, and has greater treatment advantages. In addition, when Candida albicans was injected into the vagina of the mice again, the number of Candida albicans colonies in the vaginal lavage fluid after FeLab treatment was significantly reduced (as shown in Figure 13 Thus, it can be seen that FeLab of the present application can kill Candida albicans and regulate the vaginal microenvironment at the same time, so as to relieve candidal vaginitis and reduce the recurrence rate.

[0119] The products prepared in Examples 4-6 were respectively subjected to mouse tests in the same manner, and the effects of the platinum-iron alloy nanoscale enzyme-probiotic composite, the ferroferric oxide nanoscale enzyme-lactic acid bacteria composite and the pyrite nanoscale enzyme-probiotic composite on the treatment, relief and / or prevention of vaginitis were similar to that of the iron disulfide nanoscale enzyme-lactic acid bacteria composite, with only differences in the generation capacity of hydroxyl radicals and the effect on anti-Candida albicans.

[0120] In summary, the nanoscale enzyme-probiotic medicine for vaginas prepared by the present application, when injected in situ into the vaginitis lesion, releases nanoscale enzymes in situ in response to the lesion environment, which can use the H2O2 and / or lactic acid produced by the probiotics to generate a large amount of ROS and / or RNS, thereby successfully killing the pathogenic bacteria in the lesion site and minimizing damage to the normal tissues and cells of the vaginal mucosa; at the same time, under the microaerobic conditions in the vagina, the large-scale reproduction of probiotics is conducive to the regulation of the vaginal microenvironment (reducing the pH of the vagina and maintaining the balance of vaginal flora) to reduce the recurrence rate of vaginitis. This complementary and efficient design makes the present application have many characteristics, can realize the treatment, relief and / or prevention of vaginitis and reduce the recurrence rate of the disease, fully indicating its potential for clinical transformation.

Claims

1. A nano-enzyme-probiotic complex, characterized in that, The nano-enzyme-probiotic complex contains a nano-enzyme and lactic acid bacteria, and the nano-enzyme is a ferrous disulfide nano-enzyme or a pyrite nano-enzyme.

2. The nano-enzyme-probiotic complex according to claim 1, wherein, The nano-enzyme-probiotic complex further comprises an inert carrier.

3. The nano-enzyme-probiotic complex according to claim 2, wherein, The inert carrier comprises reduced graphene oxide, polylactic acid-glycolic acid copolymer or hydroxyapatite.

4. The nano-enzyme-probiotic complex of claim 1, wherein, The concentration of the lactic acid bacteria is 10 2 -10 4 CFU / mL.

5. The nano-enzyme-probiotic complex of claim 1, wherein, The dosage form of the nano-enzyme-probiotic complex comprises an injection, a lotion, a liniment, a powder, a pill, a tablet, a film, an ointment, a gel, a suppository, a paste, an aerosol or a spray.

6. A method of producing the nano-enzyme-probiotic complex according to any one of claims 1 to 5, characterized in that, The method comprises the following steps: The nano-enzyme is mixed with lactic acid bacteria, a dosage form base is added, and then uniform mixing and incubation are performed to obtain the nano-enzyme-probiotic complex.

7. A nano-enzyme-probiotic complex gel, characterized in that, The nano-enzyme-probiotic complex gel contains the nano-enzyme-probiotic complex of any one of claims 1-5, and the nano-enzyme and the lactic acid bacteria are combined by the gel.

8. A method of preparing the nanozyme-probiotic complex gel of claim 7, characterized in that, The method comprises the following steps: (1) A mixed solution of hyaluronic acid, N-hydroxysulfosuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole is prepared, L-cysteine methyl ester hydrochloride is added to the mixed solution, stirring is performed in the dark, the pH value is adjusted, and an acidic mixed solution is obtained; the mass ratio of the hyaluronic acid, N-hydroxysulfosuccinimide, 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole and L-cysteine methyl ester hydrochloride is 1:1-5:1-5:1-5; (2) The acidic mixed solution is dialyzed and freeze-dried to obtain a fluffy solid; (3) the peroxidase-like nano-enzyme and the lactic acid bacteria solution are dissolved in a buffer salt solution, and then fluffy solids are added, mixed uniformly, incubated, and a nano-enzyme-probiotic compound gel is formed; the concentration of the lactic acid bacteria solution is 10 1 -10 5 CFU / mL, and the mass ratio of the peroxidase-like nano-enzyme to the fluffy solids is 1:100-1000.

9. Use of the nano-enzyme-probiotic complex of any one of claims 1-5 or the nano-enzyme-probiotic complex gel of claim 7 in the preparation of a medicament for treating, relieving and / or preventing vaginitis and reducing the recurrence rate of the disease.

10. Use according to claim 9, characterized in that, The vaginitis comprises bacterial vaginitis, candidal vaginitis, trichomonal vaginitis, senile vaginitis, juvenile vaginitis, mixed vaginitis and / or other vaginitis.

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