Micro-ecological viable bacterium preparation and preparation method and application thereof

Abstract

The invention discloses a micro-ecological viable bacterium preparation as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) preparing a micro-nutrient solution; (2) pretreating a micro nutrient solution; (3) preparing target probiotic bacterial sludge; (4) preparing a micro-ecological viable bacteria solution; (5) adding a freeze-drying protective agent into the micro-ecological viable bacteria solution to prepare a micro-ecological viable bacteria solution before freeze-drying; and (6) freeze-drying the micro-ecological viable bacteria solution before freeze-drying to obtain the micro-ecological viable bacteria preparation. According to the method disclosed by the invention, nutritional ingredients for promoting the probiotics to inhibit pathogenic bacteria and the probiotics are jointly freeze-dried to prepare the micro-ecological living bacteria preparation, nutrients in the preparation can be used for the probiotics to rapidly proliferate after administration and generate functional substances so as to play a role, the antibacterial rate reaches 90% or above within 8 hours and reaches 100% within 24 hours, and the treatment effect is safely and rapidly achieved; meanwhile, the dependence of probiotics on environmental nutrition is reduced. The micro-ecological living bacteria preparation prepared by the process can be applied to prevention and treatment of gynecological genital tract inflammation such as colpitis mycotica.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a microecological live bacteria preparation, a preparation method and use thereof. Background Art

[0002] Microecological live bacteria preparations are a type of live bacteria biological products used to regulate the body's microecological balance and improve the host's health level. The application of human microecological live bacteria preparations is currently mainly concentrated in two categories. One is intestinal live bacteria preparations, which are used to regulate the balance of intestinal flora, prevent or treat diarrhea, constipation, and improve immunity; the other is vaginal live bacteria preparations, which are used to supplement vaginal probiotics, inhibit pathogens, regulate the vaginal pH environment, and prevent or treat various vaginitis. Microecological live bacteria preparations use live probiotics that are beneficial to the human body as the main raw materials. These probiotics can grow and colonize in the digestive tract or reproductive tract, and attach to the mucosa with other beneficial bacteria to form a protective biological barrier. During its metabolism, it can produce small molecules such as short-chain fatty acids and antimicrobial peptides to inhibit the proliferation and attachment of pathogens. It can produce nutrients such as vitamins to participate in the body's metabolism. In addition, it can activate the immune system and induce antibody production. At present, human microecological live bacteria preparations have been used in the fields of medicine, health care, and food. Taking medicine as an example, Jinshuangbi contains three probiotics derived from human body, namely Bifidobacterium longum, Lactobacillus bulgaricus and Streptococcus thermophilus; Siliankang tablets contain Bifidobacterium, Enterococcus faecalis, Lactobacillus and Bacillus cereus; Lactobacillus live bacteria capsules for vaginal use (Dingjunsheng) contain Lactobacillus delbrueckii. Yanhua (Lactobacillus vaginal capsules) contain Streptococcus enterica.

[0003] Whether live bacterial preparations can play a significant role in the human body depends on many factors, such as the activity of the bacteria, the number of live bacteria, and whether the human environment is suitable for bacterial growth and colonization. Among them, appropriate nutritional supply is crucial to the proliferation and function of probiotics. When the nutrition, pH, etc. of the internal environment are suitable, the probiotics produce some functional substances during the process of rapid proliferation and metabolism, thereby playing the role of antibacterial and colonization. Many studies at home and abroad have found that the functions of probiotics, such as antibacterial ability, can be enhanced by providing appropriate nutrients.

[0004] However, the current preparation process of live bacterial preparations often focuses on the survival of bacteria in the preparations, ignoring whether the preparations can survive and function well after entering the body. In general, live bacterial preparations are often composed of bacterial powder and excipients (such as fillers and flow aids), and there are few nutrients for bacterial growth. After administration, the live bacteria in the preparation can only maintain their activity with the help of nutrients from the human environment. When the internal environment is not suitable for the growth of the bacteria, even if a sufficient amount of live bacteria is given, its efficacy may still be unsatisfactory. This may explain why the efficacy of probiotic preparations varies from person to person and is not ideal for some people.

[0005] Patent CN 117618395 A discloses a composite microecological preparation for treating intestinal diseases for animals and its preparation method. The composite microecological preparation for treating intestinal diseases for animals includes a nutrient layer, and it is believed that the nutrient layer wrapped inside can provide sufficient nutrition for the composite microorganisms. The preparation process consists of multiple steps such as granulation, drying, spraying, and drying. The preparation process is complicated, the production time required for preparing the product is long, and the production efficiency is low; and the formed preparation particles are divided into five layers, which are the nutrient layer, the inner shell layer, the bacterial layer, the middle layer, and the outer shell layer from the inside to the outside. The nutrient layer and the bacterial layer are not closely connected, which is not conducive to the maintenance of bacterial activity and the function of the function. Summary of the invention

[0006] In order to solve the above technical problems, the first purpose of the present invention is to provide a method for preparing a microecological live bacteria preparation, by adding nutrients suitable for the proliferation and efficacy of the probiotics into the live bacteria preparation, so that the probiotics can rapidly proliferate and maintain their abundance after administration, and quickly produce functional substances to exert their efficacy. The antibacterial rate reaches 90% or more within 8 hours, and the antibacterial rate reaches 100% within 24 hours, so as to achieve the therapeutic effect safely and quickly; at the same time, the nutritional requirements of the probiotics for the administration environment are reduced, and the environmental adaptability is enhanced.

[0007] The second object of the present invention is to provide a microecological live bacteria preparation.

[0008] The third object of the present invention is to provide a use of a microecological live bacteria preparation.

[0009] The first object of the present invention is implemented by the following technical scheme: a method for preparing a microecological live bacteria preparation, comprising the following steps: (1) preparing a micronutrient solution; (2) pretreating the micronutrient solution; (3) preparing a target probiotic slurry; (4) preparing a microecological live bacteria solution; (5) adding a freeze-drying protective agent to the microecological live bacteria solution to prepare a pre-freeze-dried microecological live bacteria solution; (6) freeze-drying the pre-freeze-dried microecological live bacteria solution to obtain a microecological live bacteria preparation; wherein, (1) Preparation of micronutrient solution: Optimize the culture medium of the target probiotics to obtain a micronutrient solution that is conducive to the proliferation of the target probiotics while inhibiting pathogenic bacteria; (2) pretreatment of the micronutrient solution: pretreatment of the micronutrient solution to obtain a pretreated micronutrient solution; (3) Preparing the target probiotic slurry: The target probiotics are inoculated and fermented step by step, and the probiotic fermentation liquid that reaches the end of the fermentation is centrifuged, and the supernatant is discarded, and the slurry is retained; (4) Preparing a microecological live bacterial solution: Mix the bacterial sludge and the pretreated micronutrient solution at a mass ratio of 1:5 to 1:40, and stir evenly to obtain a microecological live bacterial solution; (5) Adding a freeze-drying protective agent to the microecological live bacteria liquid to prepare a pre-freeze-dried microecological live bacteria liquid: adding the sterilized freeze-drying protective agent to the obtained microecological live bacteria liquid, stirring well to obtain a pre-freeze-dried microecological live bacteria liquid; wherein the mass ratio of the microecological live bacteria liquid to the freeze-drying protective agent is 1:1 to 1:10; (6) The pre-lyophilized probiotic liquid is freeze-dried to obtain a probiotic preparation: the pre-lyophilized probiotic liquid is freeze-dried and crushed by vacuum freeze drying, and then sterile excipients are added and mixed evenly to obtain a probiotic preparation.

[0010] Furthermore, the method for optimizing the culture medium of the target probiotics to obtain a micronutrient solution that is beneficial to the proliferation of the target probiotics while inhibiting pathogenic bacteria specifically includes: S1: Based on the culture medium of the target probiotics, several groups of different micronutrient liquid culture media are prepared and sterilized, each group of the micronutrient liquid culture medium comprises the following components in mass percentage concentrations: carbon source 1.0% to 6.0%, nitrogen source 0.2% to 8.0%, inorganic salt 0.2% to 5.0%, trace elements 0.001% to 1.5%, emulsifier 0 to 0.5%; the solvent of the micronutrient liquid culture medium is purified water; the pH of the micronutrient liquid culture medium is 6.5 to 7.5; S2: Add the target probiotics and pathogenic bacteria to each group of micronutrient liquid culture medium, with the live bacteria ratio of the target probiotics to the pathogenic bacteria being 1:1 to 10000:1; co-culture at 35°C to 38°C for 24 hours in an aerobic, anaerobic or facultative anaerobic environment; S3: Take samples at the 8-24h culture time point, detect the concentration changes of pathogenic bacteria in each group, compare and screen out a group of micronutrient liquid culture media, i.e., micronutrient solution, with the fastest bacteriostasis and an inhibition rate of 90% or more in 8h and 100% in 24h.

[0011] Furthermore, the carbon source is one of lactose and glucose or a combination of the two; the nitrogen source is one of beef extract powder, beef extract, peptone, beef peptone, polyvalent peptone or yeast powder or a combination of more than one; the inorganic salt is one of anhydrous sodium acetate, potassium acetate, dipotassium hydrogen phosphate or ammonium citrate or a combination of more than one; the trace element is one of magnesium sulfate, manganese sulfate, L-cysteine ​​hydrochloride, calcium chloride or sodium chloride or a combination of more than one; the emulsifier is Tween-80.

[0012] Furthermore, the specific method of pretreatment of the micronutrient solution is: The micronutrient solution obtained in step (1) is prepared at a concentration of 1 to 20 times concentrated, the pH is adjusted to 6.5 to 7.5, and then sterilized by moist heat at 121° C. for 15 to 30 minutes to obtain a pretreated micronutrient solution. The micronutrient solution is prepared at a concentration of 1 to 20 times concentrated, which can reduce the water content, improve the freeze-drying effect, and increase the dry matter mass of the nutrients after freeze-drying.

[0013] Furthermore, the target probiotics are of the genus Lactobacillus.

[0014] Furthermore, the Lactobacillus genus is a mixture of one or more species of Lactobacillus delbrueckii, Lactobacillus crispatus or Lactobacillus plantarum.

[0015] Furthermore, the pathogenic bacteria is Candida.

[0016] Furthermore, the micronutrient solution includes the following components in mass percentage concentrations: carbon source 1.0% to 4.0%, beef extract powder 0 to 2.0%, beef extract 0 to 2.0%, peptone 0 to 2.0%, beef peptone 0 to 1.0%, polyvalent peptone 0 to 2.0%, yeast powder 0.2% to 1.5%, anhydrous sodium acetate 0.1% to 1.0%, potassium acetate 0 to 1.0%, dipotassium hydrogen phosphate 0 to 1.0%, ammonium citrate 0 to 1.0%, magnesium sulfate 0.01% to 0.1%, manganese sulfate 0 to 0.2%, L-cysteine ​​hydrochloride 0 to 1.0%, and Tween-80 0.01% to 0.2%; the solvent of the micronutrient solution is purified water, and the pH is adjusted to 6.5 to 7.5.

[0017] Furthermore, the carbon source is lactose or glucose or a combination of the two.

[0018] Furthermore, when the target probiotic is Lactobacillus delbrueckii and the pathogenic bacteria is Candida, the micronutrient solution includes the following components in mass percentage concentrations: 2.0% lactose, 0.5% polyvalent peptone, 1.0% yeast powder, 1.0% beef extract, 0.7% anhydrous sodium acetate, 0.7% potassium acetate, 0.02% magnesium sulfate, 0.05% L-cysteine ​​hydrochloride, and 0.1% Tween-80; the pH of the micronutrient solution is adjusted to 7.4, and the solvent is purified water.

[0019] Furthermore, when the target probiotic is Lactobacillus crispatus or Lactobacillus plantarum and the pathogenic bacteria is Candida, the micronutrient solution includes the following components in mass percentage concentrations: 2.0% glucose, 1.0% peptone, 1.0% yeast powder, 0.7% anhydrous sodium acetate, 0.7% potassium acetate, 0.01% magnesium sulfate, 0.01% manganese sulfate, and 0.1% Tween-80; the pH of the micronutrient solution is adjusted to 7.2, and the solvent is purified water.

[0020] The second object of the present invention is implemented by the following technical scheme: a probiotic preparation is prepared by the method for preparing a probiotic preparation according to the first object of the present invention.

[0021] The third purpose of the present invention is implemented by the following technical scheme: the microecological live bacteria preparation described in the second purpose of the present invention is used to produce medicines, health products, detergents or private care products.

[0022] Furthermore, the microecological live bacteria preparation is used to produce medicines for preventing or treating gynecological genital tract inflammation. The gynecological genital tract inflammation of the present invention is candidal vaginitis.

[0023] Advantages of the present invention: (1) The method of the present invention freeze-dries the nutrients that promote probiotics to inhibit pathogenic bacteria together with the probiotics to prepare a microecological live bacteria preparation. The nutrients in the preparation enable the probiotics to rapidly proliferate and maintain their abundance after administration, and quickly produce functional substances to exert their efficacy. The antibacterial rate reaches 90% or more within 8 hours, and the antibacterial rate reaches 100% within 24 hours, achieving a safe and rapid therapeutic effect; at the same time, the probiotics' nutritional requirements for the administration environment are reduced, thereby enhancing environmental adaptability.

[0024] (2) The present invention wraps the bacteria with the optimized nutritional ingredients and then freeze-dries them, so that the bacteria are fully in a nutritional environment, which is beneficial to maintaining the activity of the bacteria and exerting their functions.

[0025] (3) The preparation method of the microecological live bacteria preparation of the present invention is simple, the production time required for preparing the product is short, the production efficiency is high, and it is more suitable for industrial production.

[0026] (4) The present invention forms a powder by mixing the bacteria, nutrients and protective agents one by one and then freeze-drying them in vacuum, which is beneficial to the storage stability of the probiotics, the efficacy stability of the preparation, and long-term storage and transportation. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 This is a sample image of the Lactobacillus delbrueckii microecological live bacteria preparation prepared in Example 3; Figure 2 This is a comparison chart of the residual concentration of Candida albicans ATCC10231 at different time points in the experiment of Example 4; Figure 3This is a comparison chart of the residual concentration of Escherichia coli at different time points in the experiment of Example 5; Figure 4 This is a comparison chart of the residual concentration of Staphylococcus aureus ATCC6538 at different time points in the experiment of Example 6; Figure 5 This is a comparison chart of the antibacterial effect of the composite microecological live bacteria preparation in Example 14. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. In the following examples, the pathogenic bacteria strains: Escherichia coli 8099, Staphylococcus aureus ATCC6538, Candida albicans ATCC10231, Candida albicans 2.4159 and Candida glabrata 2.3961 were all purchased from the China General Microbiological Culture Collection Administration Center.

[0031] The Lactobacillus delbrueckii, Lactobacillus crispatus and Lactobacillus plantarum involved in the following examples are Lactobacillus delbrueckii DM8909, Lactobacillus crispatus SQ1505 and Lactobacillus plantarum SQ1631, respectively. All three are isolated from the reproductive tract of healthy women, are dominant lactobacilli in the female reproductive tract, and are all derived from Inner Mongolia Shuangqi Pharmaceutical Co., Ltd.

[0032] Example 1: Optimization of micronutrient solution to promote the antibacterial effect of Lactobacillus delbrueckii (1) Prepare 9 micronutrient solutions with different ingredients. The specific ingredients are shown in Table 1 and are numbered 1# to 9#. Then sterilize them by moist heat sterilization at 121°C for 15 min.

[0033] (2) Sterile LBS liquid culture medium (10#) and sterile physiological saline (11#) were used as the control group and blank group, respectively (Note: Sterile LBS liquid culture medium is a commonly used culture medium for Lactobacillus delbrueckii, and its composition is shown in Table 1).

[0034] (3) Take 11 sterile test tubes and number them 1#-11#. Add 9.5mL of the corresponding culture medium to each test tube 1#-10#, and add 9.5mL of sterile saline to 11#. Add 0.5g of 2×10 9 CFU / g Lactobacillus delbrueckii powder, 0.5mL 2×10 6CFU / mL Candida albicans (ATCC10231) bacterial solution, mix thoroughly. Then place it in facultative anaerobic culture at 37℃. Detect the number of live Candida albicans in each test tube at 8h and 24h of culture.

[0035] Results: The antibacterial effect of Lactobacillus delbrueckii in 8# micronutrient solution was the best, and its antibacterial effect was significantly better than that of other groups (see Table 2).

[0036] Table 1: Composition of various micronutrient solutions for co-cultivation of Lactobacillus delbrueckii and Candida albicans

[0037] Table 2: Antibacterial ability of Lactobacillus delbrueckii against Candida albicans under different conditions

[0038] Example 2: Pretreatment of micronutrient solution and preparation of Lactobacillus delbrueckii microecological live bacteria preparation (1) Prepare 200 g of the 8# micronutrient solution in Example 1 according to a 10-fold concentrated formula, adjust the pH to 7.4, and then sterilize with moist heat at 121°C for 15 min to obtain the pretreated micronutrient solution. Prepare 100 g of a lyophilization protective agent with a mass percentage concentration of 20%.

[0039] (2) Take the Lactobacillus delbrueckii fermentation liquid that has been fermented to the end point and collect the bacterial sludge by centrifugation.

[0040] (3) Take 10g of bacterial sludge and add 100g of pretreated micronutrient solution, stir and mix thoroughly to make the nutrients completely cover the bacteria, then add 110g of freeze-drying protective agent, stir and mix thoroughly, pour into freeze-drying tray for freeze drying. After drying, grind through a 40-mesh sieve, add auxiliary materials, and package at 1g / bag, which is the Lactobacillus delbrueckii microecological live bacteria preparation.

[0041] Example 3: Properties, viable counts and water activity of Lactobacillus delbrueckii microecological live bacteria preparation The Lactobacillus delbrueckii microecological live bacteria preparation obtained in Example 2 was a light yellow powder. The number of live bacteria was detected to be 9.03×10 9 CFU / g, water activity is 0.053, preparation samples such as Figure 1 .

[0042] Example 4: Efficacy verification of Lactobacillus delbrueckii microecological live bacteria preparation - inhibition of Candida albicans ATCC10231 Experimental methods: The Lactobacillus delbrueckii microecological live bacteria preparation prepared in Example 2 and the conventional freeze-dried Lactobacillus delbrueckii powder were adjusted to a live bacterial count of 2.00×10 9CFU / g for later use. Take 3 test tubes and number them 1#-3#. Add 9.5mL of normal saline and 0.5mL of Candida albicans solution to each test tube, then add 0.5g of Lactobacillus delbrueckii microecological live bacteria preparation to 1#; add 0.5g of conventional Lactobacillus delbrueckii powder to 2#. Place the test tubes in facultative anaerobic culture at 37℃. Detect the number of live bacteria of Lactobacillus delbrueckii and Candida albicans in each group at 8h and 24h of culture.

[0043] Results: Figure 2 As shown, the Lactobacillus delbrueckii microecological live bacteria preparation has a more significant inhibitory effect on Candida albicans than the conventional freeze-dried Lactobacillus delbrueckii powder.

[0044] Example 5: Efficacy verification of Lactobacillus delbrueckii microecological live bacteria preparation - inhibition of Escherichia coli 8099 Experimental method: Same as Example 4, wherein the pathogenic bacteria liquid is Escherichia coli liquid.

[0045] Results: Figure 3 As shown, the Lactobacillus delbrueckii microecological live bacteria preparation has a more significant inhibitory effect on Escherichia coli than the conventional freeze-dried Lactobacillus delbrueckii powder.

[0046] Example 6: Efficacy verification of Lactobacillus delbrueckii microecological live bacteria preparation - inhibition of Staphylococcus aureus ATCC6538 Experimental method: Same as Example 4, wherein the pathogenic bacteria liquid is Staphylococcus aureus liquid.

[0047] Results: Figure 4 As shown, the Lactobacillus delbrueckii microecological live bacteria preparation has a more significant inhibitory effect on Staphylococcus aureus than the conventional freeze-dried Lactobacillus delbrueckii powder.

[0048] Example 7: Safety evaluation of Lactobacillus delbrueckii microecological live bacteria preparation (vaginal mucosal irritation test) According to the safety test method of vaginal microecological live bacteria products in the 2020 edition of the "Chinese Pharmacopoeia", 5 healthy female mice weighing 24-26g were used, and there was no damage, congestion or edema in the vaginal opening. 10mg of Lactobacillus delbrueckii microecological live bacteria preparation was inserted into the vagina of each mouse once a day for 3 consecutive days. From the first day of administration, the mice were observed for 7 consecutive days. If they survived healthily, gained weight, and had no local redness, swelling, secretions and other symptoms in the vagina, they were qualified.

[0049] Results: Each mouse was given the drug for 3 consecutive days, and then observed and weighed for 7 consecutive days. The weight of each mouse gradually increased, and they ate normally and were in good condition. There was no redness, swelling, secretion, or congestion in the vagina of the mouse. This indicates that the vaginal administration of the Lactobacillus delbrueckii microecological live bacteria preparation is safe.

[0050] Table 3: Changes in mouse body weight and local vaginal conditions after administration of Lactobacillus delbrueckii microecological live bacteria preparation

[0051] Example 8: Evaluation of the therapeutic effect of Lactobacillus delbrueckii microecological live bacteria preparation on candidal vaginitis (VVC) in rats method: (1) Adaptive feeding of rats: 30 SPF-grade SD female rats aged 7-9 weeks were raised normally in a clean animal breeding room at 22°C for one week. Six rats were randomly selected and raised normally as a blank control group.

[0052] (2) Rat VVC modeling: The remaining 24 rats were subcutaneously injected with 0.5 mg of estradiol benzoate in the hind legs, once every 2 days. After 6 days, the rats were placed in a pseudo-estrus state. The pseudo-estrus state of the rats was maintained by injecting estradiol benzoate once every 2 days until the end of the experiment. Take the activated Candida albicans lawn and adjust it to 1×10 8 CFU / mL concentration, 0.1mL / time / day was injected into the vagina of each rat, and the rat was hung upside down for 2 minutes immediately after each injection to prevent the outflow of bacterial solution. After continuous injection for one week, the rats were observed for 6 days. On the 6th day, a sterile cotton swab was used to gently take samples from the rat vagina for smear microscopy. If there were more Candida albicans and hyphae, the rat VVC model was successfully established.

[0053] (3) Treatment and effect evaluation: 18 rats with successful modeling were randomly divided into 3 groups, 6 rats in each group, namely, negative control group, LBS-Lactobacillus delbrueckii preparation group, and Lactobacillus delbrueckii microecological live bacteria preparation group. Negative control group: sterile saline; LBS-Lactobacillus delbrueckii preparation group: 2 g of LBS-Lactobacillus delbrueckii preparation (about 9.03×10 9 CFU / g) was fully dissolved in 10 mL of sterile saline to a concentration of 1.80×10 9 CFU / mL; Lactobacillus delbrueckii microecological live bacteria preparation group: 2g of Lactobacillus delbrueckii microecological live bacteria preparation (9.03×10 9 CFU / g) was fully dissolved in 10 mL of sterile saline to a concentration of 1.80×10 9 CFU / mL.

[0054] Each group of rats was vaginally irrigated with the corresponding sample at a rate of 0.1 mL / time / day using a sterile applicator for 3 consecutive days. The redness and swelling of the vagina and the secretions of each rat were observed and recorded. On the 4th day, each rat was fully irrigated with 5 mL of sterile saline in multiple times and the irrigating fluid was collected to detect the Candida albicans content in the irrigating fluid and analyze it.

[0055] Results: The vagina of the rats with successful modeling had more white secretions, and the vaginal opening was obviously red, swollen and congested. After 3 days of administration of each sample, the vagina of the rats in the saline negative control group was obviously red and swollen, and more secretions were visible. After 3 days of administration, one of the rats in the LBS-Lactobacillus delbrueckii preparation group improved significantly, and 3 of them improved slightly. The amount of live bacteria of Candida albicans was lower than that of the control group, indicating that continuous administration of Lactobacillus delbrueckii for 3 days can inhibit Candida albicans to a certain extent. After 3 days of administration of the Lactobacillus delbrueckii microecological live bacteria preparation group, the vagina of 6 rats had returned to normal, the redness, swelling and secretions disappeared, and the amount of Candida albicans detected was extremely low, indicating that appropriate micro-nutrients can greatly promote the antibacterial effect of Lactobacillus delbrueckii and have a significant therapeutic effect on rat VVC.

[0056] Table 4: Record of local vaginal conditions in rats after administration of Lactobacillus delbrueckii microecological live bacteria preparation

[0057] Table 5: Comparison of Candida albicans concentrations in rat vaginal lavage fluid after administration of different samples for 3 days

[0058] Example 9: Optimization of micronutrient solution to promote Lactobacillus crispatus to inhibit Candida albicans (1) Prepare 8 different experimental groups of micronutrient solution and conventional MRS culture medium (control group). The composition of each group is shown in Table 6. They are numbered 1#-9# and sterilized at 121℃ for 15 min. Sterile saline (10#) was used as the blank group.

[0059] (2) Take 10 100mL triangular flasks (numbered 1#-10#), add 50mL of the corresponding numbered culture medium to each triangular flask, then add Candida albicans liquid and Lactobacillus crispatus powder to each group and mix thoroughly. Make sure that the initial concentration of Candida albicans in each group is the same and is 10 5 CFU / mL order of magnitude, the concentration of live Lactobacillus crispatus in each group was the same and was 1×10 8 CFU / mL.

[0060] (3) Each group was cultured facultatively anaerobically at 37°C. The number of viable Candida albicans in each group was detected at 0 h, 8 h, and 24 h of culture.

[0061] Results: In 7# micronutrient solution, Lactobacillus crispatus had the best antibacterial effect (see Table 7).

[0062] Table 6: Table of components of various micronutrient solutions for co-cultivation of Lactobacillus crispatus and Candida albicans

[0063] Table 7 Antibacterial ability of Lactobacillus crispatus against Candida albicans under different environments

[0064] Example 10: Optimization of micronutrient solution to promote Lactobacillus plantarum to inhibit Candida albicans The method is the same as that of Example 9. The target probiotic is Lactobacillus plantarum.

[0065] (1) Prepare 8 different experimental groups of micronutrient solution and conventional MRS culture medium (control group). The composition of each group is shown in Table 6. They are numbered 1# to 9# respectively and sterilized at 121℃ for 15 min.

[0066] Sterile saline (10#) was used as the blank group.

[0067] (2) Take 10 100mL triangular flasks (numbered 1#-10#), add 50mL of the corresponding numbered culture medium to each triangular flask, then add Candida albicans liquid and Lactobacillus plantarum powder to each group and mix thoroughly. Make sure that the initial concentration of Candida albicans in each group is the same and is 10 5 CFU / mL order of magnitude, the concentration of live Lactobacillus plantarum in each group was the same and was 1×10 8 CFU / mL.

[0068] (3) Each group was cultured facultatively anaerobically at 37°C. The number of viable Candida albicans in each group was detected at 0 h, 8 h, and 24 h of culture.

[0069] Results: In the 7# micronutrient solution, Lactobacillus plantarum had the best antibacterial effect (see Table 8). The optimal medium for Lactobacillus plantarum to inhibit Candida albicans was the same as that for Lactobacillus crispatus, both of which were 7# micronutrient medium.

[0070] Table 8 Antibacterial ability of Lactobacillus plantarum against Candida albicans under different environments

[0071] Example 11: Preparation of Lactobacillus crispatus microecological live bacteria preparation and Lactobacillus plantarum microecological live bacteria preparation It can be seen from Examples 9 and 10 that the preferred culture medium for Lactobacillus crispatus and Lactobacillus plantarum to inhibit Candida albicans is 7# micronutrient solution. Therefore, the same method can be used to prepare the microecological preparation.

[0072] (1) Prepare 200 g of the 7# micronutrient solution in Example 9 according to the formula of 3-fold concentration, adjust the pH to 7.2, and then sterilize it with moist heat at 121°C for 15 min to obtain the pretreated micronutrient solution. Then prepare 400 g of a lyophilization protective agent with a mass percentage concentration of 20%.

[0073] (2) Take the fermented Lactobacillus crispatus liquid to the end point, centrifuge, discard the supernatant, and collect the bacterial sludge. Then weigh 15g of the bacterial sludge, add 100g of the pretreated micronutrient solution and stir thoroughly to allow the nutrients to fully wrap the bacteria, then add 200g of freeze-drying protective agent and mix thoroughly, and freeze-dry. After drying, crush and pass through a 40-mesh sieve. After drying, crush and pass through a 40-mesh sieve, add auxiliary materials and mix well, and the Lactobacillus crispatus microecological live bacteria preparation is obtained.

[0074] (3) Take the Lactobacillus plantarum bacterial liquid that has been fermented to the end point, centrifuge, discard the supernatant, and collect the bacterial sludge. Then weigh 15g of the bacterial sludge, add 100g of the pretreated micronutrient solution and stir thoroughly to allow the nutrients to fully wrap the bacteria, then add 200g of freeze-drying protective agent and mix thoroughly, and freeze-dry. After drying, crush it through a 40-mesh sieve. After drying, crush it through a 40-mesh sieve, add auxiliary materials (auxiliary materials are the same as those of the Lactobacillus crispatus microecological live bacteria preparation), and mix thoroughly to obtain the Lactobacillus plantarum microecological live bacteria preparation.

[0075] Example 12: Detection of viable bacteria count of Lactobacillus crispatus microecological live bacteria preparation and Lactobacillus plantarum microecological live bacteria preparation and characteristics of aqueous solution (1) Detection of viable bacteria count The viable counts of Lactobacillus crispatus microecological live bacteria preparation and Lactobacillus plantarum microecological live bacteria preparation were detected by MRS solid culture medium, and the viable counts of Lactobacillus crispatus microecological live bacteria preparation were 6.30×10 9 CFU / g, Lactobacillus plantarum microecological live bacteria preparation is 8.17×10 9 CFU / g.

[0076] (2) Characteristics of aqueous solution of preparation Take 2g of Lactobacillus crispatus microecological live bacteria preparation, add 10mL of sterile water and fully dissolve. The aqueous solution is light yellow, the pH is 5.80, and the number of live bacteria is 1.26×10 9 cfu / mL. After the aqueous solution of the preparation was placed at 37°C for facultative anaerobic culture for 8 hours, the pH was 4.50 and the number of viable bacteria was 2.05×10 9 cfu / mL. Therefore, under the facultative anaerobic environment of 37°C, the nutrients contained in the preparation can provide Lactobacillus crispatus for continuous proliferation and acid production.

[0077] Take 2g of plant lactobacillus microecological live bacteria preparation, add 10mL of sterile water and fully dissolve. The aqueous solution is light yellow, the pH is 5.51, and the number of live bacteria is 1.63×10 9 cfu / mL. The aqueous solution of the preparation was placed at 37°C for facultative anaerobic culture for 8 hours, and its pH was 4.36, with a viable bacterial count of 2.12×10 9 cfu / mL. Therefore, in a facultative anaerobic environment at 37°C, the nutrients contained in the preparation can provide Lactobacillus plantarum for continuous proliferation and acid production.

[0078] Example 13: Preparation of Lactobacillus crispatus-Lactobacillus plantarum composite microecological live bacteria preparation In the preparation process, the Lactobacillus crispatus microecological live bacteria preparation and the Lactobacillus plantarum microecological live bacteria preparation prepared in Example 11 have the same nutritional ingredients and protective agent ratios, and are further prepared into a dual-strain preparation. The Lactobacillus crispatus microecological live bacteria preparation and the Lactobacillus plantarum microecological live bacteria preparation are mixed at a mass ratio of 1.3:1 to prepare a composite microecological live bacteria preparation with a live bacteria ratio of about 1:1 (live bacteria count 7.11×10 9 cfu / g).

[0079] Example 14: Study on the antibacterial activity of the composite microecological live bacteria preparation Test group samples: composite microecological live bacteria preparation prepared in Example 13 (live bacteria count 7.11×10 9 cfu / g); The control group sample was prepared with Lactobacillus crispatus preparation and Lactobacillus plantarum preparation without micronutrients in a ratio of 1:1, with a total live bacterial count of 7.11×10 9 cfu / g of composite live bacteria preparation.

[0080] Blank group sample: The 7# micronutrient solution in Example 9 was prepared according to a 3-fold concentrated formula, the pH was adjusted to 7.2, and then sterilized with wet heat at 121°C for 15 minutes to obtain 200g of pretreated micronutrient solution. Then 400g of lyophilized protective agent with a mass percentage concentration of 20% was prepared. 100g of the pretreated micronutrient solution was taken, and then 200g of lyophilized protective agent was added and mixed thoroughly, and freeze-dried to prepare a micronutrient lyophilized powder preparation without lactobacillus.

[0081] method: (1) Take 2.4159 of fresh Candida albicans and 2.3961 of Candida glabrata, and adjust the concentration to about 10% with sterile water. 6 CFU / mL.

[0082] (2) Take 6 50mL sterile beakers. Add 1g of compound microecological live bacteria preparation to test group 1# and 2# respectively; add 1g of compound live bacteria preparation without micronutrient ingredients to control group 3# and 4# respectively; blank group: add 1g of micronutrient freeze-dried powder preparation without lactobacillus to blank group 5# and 6# respectively.

[0083] (3) Add 3 mL of sterile water to each sample, stir thoroughly into a paste and spread evenly. Take 50 mL of sterile Sabouraud agar medium, cool to 40°C, add 5 mL of Candida albicans or Candida glabrata, mix evenly, use a pipette to take 5 mL and spread gently on the surface of each sample, and let it stand until it solidifies. Cover the beaker and invert it at 37°C for facultative anaerobic culture for 24 to 48 hours, and observe the growth of Candida in each group.

[0084] (4) Add 20 mL of sterile water to each beaker, blow off the Candida on the surface of the culture medium, transfer it to a 250 mL pearl delta flask, shake it on a shaker for 15 min, then detect the concentration of Candida and make a comparison.

[0085] Results: Figure 5 As shown in Table 9, no Candida colonies were found on the surface of the Sabouraud agar medium in the test group, some Candida colonies grew on the surface of the Sabouraud agar medium in the control group, and Candida colonies in the blank group covered the surface of the medium. The Candida in each group was washed with 20 mL of sterile water and its concentration was detected. The data are shown in Table 9. The concentration of Candida in the blank group was much higher than that in the control group, and the concentration of Candida in the experimental group was extremely low. Therefore, the composite microecological live bacteria preparation can significantly inhibit the growth of Candida albicans 2.4159 and Candida glabrata 2.3961.

[0086] Table 9 Comparison of Candida concentrations in each group

[0087] Example 15: Safety evaluation of Lactobacillus crispatus-Lactobacillus plantarum composite microecological live bacteria preparation (vaginal mucosal irritation test) According to the safety test method of vaginal microecological live bacteria products in the 2020 edition of the Chinese Pharmacopoeia, 5 healthy female mice weighing 24-26g were used, with no damage, congestion or edema in the vaginal opening. 10mg of the composite microecological live bacteria preparation was injected into the vagina of each mouse once a day for 3 consecutive days. From the first day of administration, the mice were observed for 7 consecutive days. If they survived healthily, gained weight, and had no local redness, swelling, secretions, etc. in the vagina, they were qualified.

[0088] Results: Each mouse was administered for 3 consecutive days, observed for 7 consecutive days and weighed. The weight of each mouse gradually increased, ate normally, and was in good condition. No redness, swelling, secretions, or congestion were observed in the vagina of the mouse. The data are shown in Table 10. This shows that the compound microecological live bacteria preparation is safe for vaginal administration.

[0089] Table 10 Changes in mouse body weight and local vaginal conditions after administration of the compound microecological live bacteria preparation

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a microecological live bacteria preparation, characterized in that: The method comprises the following steps: (1) preparing micronutrient solution; (2) pre-treating the micronutrient solution; (3) preparing target probiotic slurry; (4) preparing microecological live bacterial solution; (5) adding a freeze-drying protective agent to the microecological live bacterial solution to prepare a pre-freeze-dried microecological live bacterial solution; (6) freeze-drying the pre-freeze-dried microecological live bacterial solution to obtain a microecological live bacterial preparation; wherein: (1) Preparation of micronutrient solution: Optimizing the culture medium of the target probiotics to obtain a micronutrient solution that is beneficial to the proliferation of the target probiotics while inhibiting pathogenic bacteria, wherein the antibacterial rate of the micronutrient solution reaches 90% or more within 8 hours and 100% within 24 hours; (2) pretreatment of the micronutrient solution: pretreatment of the micronutrient solution to obtain a pretreated micronutrient solution; (3) Preparing the target probiotic slurry: The target probiotics are inoculated and fermented step by step, and the probiotic fermentation liquid that reaches the end of the fermentation is centrifuged, and the supernatant is discarded, and the slurry is retained; (4) Preparing a microecological live bacterial liquid: mixing the bacterial sludge and the pretreated micronutrient solution at a mass ratio of 1:5 to 1:40, and stirring evenly to obtain a microecological live bacterial liquid; (5) Adding a freeze-drying protective agent to the microecological live bacteria liquid to prepare a pre-freeze-dried microecological live bacteria liquid: adding the sterilized freeze-drying protective agent to the obtained microecological live bacteria liquid, stirring well to obtain a pre-freeze-dried microecological live bacteria liquid; wherein the mass ratio of the microecological live bacteria liquid to the freeze-drying protective agent is 1:1 to 1:10; (6) The pre-lyophilized probiotic liquid is freeze-dried to obtain a probiotic preparation: the pre-lyophilized probiotic liquid is freeze-dried and crushed by vacuum freeze drying, and then sterile excipients are added and mixed evenly to obtain a probiotic preparation.

2. The method for preparing a microecological live bacteria preparation according to claim 1, characterized in that: The method for optimizing the culture medium of the target probiotics to obtain a micronutrient solution that is beneficial to the proliferation of the target probiotics and inhibits pathogenic bacteria specifically includes: S1: Based on the culture medium of the target probiotics, several groups of different micronutrient liquid culture media are prepared and sterilized, each group of the micronutrient liquid culture medium comprises the following components in mass percentage concentrations: carbon source 1.0% to 6.0%, nitrogen source 0.2% to 8.0%, inorganic salt 0.2% to 5.0%, trace elements 0.001% to 1.5%, emulsifier 0 to 0.5%; the solvent of the micronutrient liquid culture medium is purified water; the pH of the micronutrient liquid culture medium is 6.5 to 7.5; S2: Add the target probiotics and pathogenic bacteria to each group of micronutrient liquid culture medium, with the live bacteria ratio of the target probiotics to the pathogenic bacteria being 1:1 to 10000:1; co-culture at 35°C to 38°C for 24 hours in an aerobic, anaerobic or facultative anaerobic environment; S3: Take samples at the 8-24h culture time point to detect the concentration changes of pathogenic bacteria in each group, compare and screen out a group of micronutrient liquid culture media with the fastest antibacterial effect, namely micronutrient solution.

3. The method for preparing a microecological live bacteria preparation according to claim 2, characterized in that: The carbon source is one of lactose and glucose or a combination of the two; the nitrogen source is one of beef extract powder, beef extract, peptone, beef peptone, polyvalent peptone or yeast powder or a combination of more than one; the inorganic salt is one of anhydrous sodium acetate, potassium acetate, dipotassium hydrogen phosphate or ammonium citrate or a combination of more than one; the trace element is one of magnesium sulfate, manganese sulfate, L-cysteine ​​hydrochloride, calcium chloride or sodium chloride or a combination of more than one; the emulsifier is Tween-80.

4. The method for preparing a microecological live bacteria preparation according to claim 1, characterized in that: The specific method of the micro-nutrient solution pretreatment is: The micronutrient solution obtained in step (1) is prepared according to a concentration of 1 to 20 times, the pH is adjusted to 6.5 to 7.5, and then sterilized to obtain a pretreated micronutrient solution.

5. The method for preparing a microecological live bacteria preparation according to claim 1, characterized in that: The target probiotics are of the genus Lactobacillus.

6. The method for preparing a microecological live bacteria preparation according to claim 5, characterized in that: The Lactobacillus genus is a mixture of one or more of Lactobacillus delbrueckii, Lactobacillus crispatus or Lactobacillus plantarum.

7. The method for preparing a microecological live bacteria preparation according to claim 1, characterized in that: The pathogenic bacteria is Candida.

8. The method for preparing a microecological live bacteria preparation according to claim 1, characterized in that: The micronutrient solution in step (1) comprises the following components in mass percentage concentrations: 1.0% to 4.0% carbon source, 0 to 2.0% beef extract powder, 0 to 1.5% beef extract, 0 to 1.5% peptone, 0 to 1.0% beef peptone, 0 to 2.0% polyvalent peptone, 0.2% to 1.5% yeast powder, 0.1% to 1.0% anhydrous sodium acetate, 0 to 1.0% potassium acetate, 0 to 1.0% dipotassium hydrogen phosphate, 0 to 1.0% ammonium citrate, 0.01% to 0.1% magnesium sulfate, 0 to 0.2% manganese sulfate, 0 to 1.0% L-cysteine ​​hydrochloride, and 0.01% to 0.2% Tween-80; the solvent of the micronutrient solution is purified water, and the pH is adjusted to 6.5 to 7.

5.

9. The method for preparing a microecological live bacteria preparation according to claim 8, characterized in that: The carbon source is lactose or glucose or a combination of the two.

10. The method for preparing a microecological live bacteria preparation according to claim 9, characterized in that: The target probiotic is Lactobacillus delbrueckii, the pathogenic bacteria is Candida, and the micronutrient solution includes the following components in mass percentage concentrations: 2.0% lactose, 0.5% polyvalent peptone, 1.0% yeast powder, 1.0% beef extract, 0.7% anhydrous sodium acetate, 0.7% potassium acetate, 0.02% magnesium sulfate, 0.05% L-cysteine ​​hydrochloride, and 0.1% Tween-80; the pH of the micronutrient solution is adjusted to 7.4, and the solvent is purified water.

11. The method for preparing a probiotic preparation according to claim 9, characterized in that: When the target probiotic is Lactobacillus crispatus or Lactobacillus plantarum and the pathogenic bacteria is Candida, the micronutrient solution includes the following components in mass percentage concentrations: 2.0% glucose, 1.0% peptone, 1.0% yeast powder, 0.7% anhydrous sodium acetate, 0.7% potassium acetate, 0.01% magnesium sulfate, 0.01% manganese sulfate, and 0.1% Tween-80; the pH of the micronutrient solution is adjusted to 7.2, and the solvent is purified water.

12. A probiotic preparation prepared by the method for preparing a probiotic preparation according to any one of claims 1 to 11.

13. The microecological live bacteria preparation as described in claim 12 is used to produce medicines or private care products.

14. The probiotic preparation as claimed in claim 13 is used for producing medicines for preventing or treating gynecological reproductive tract inflammation.

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