A probiotic live bacteria preparation, its preparation method and uses

Abstract

The present invention discloses a probiotic microecological preparation, its preparation method and uses. The preparation method includes the following steps: (1) preparing a micro-nutrient solution; (2) pre-treating the micro-nutrient solution; (3) preparing the target probiotic bacterial sludge; (4) preparing the probiotic microecological liquid; (5) adding a freeze-drying protectant to the probiotic microecological liquid to prepare the pre-freeze-drying probiotic microecological liquid; (6) freeze-drying the pre-freeze-drying probiotic microecological liquid to obtain the probiotic microecological preparation. The method of the present invention combines the nutrients that promote the inhibition of pathogenic bacteria by probiotics and the probiotics to be made into a probiotic microecological preparation through freeze-drying. The nutrients in the preparation can be used by the probiotics to rapidly proliferate and produce functional substances after administration so as to exert efficacy. The antibacterial rate reaches 90% or above at 8 h and 100% at 24 h, achieving the treatment effect safely and rapidly; at the same time, it reduces the dependence of probiotics on environmental nutrients. The probiotic microecological preparation prepared by this process may be applied to the prevention and treatment of gynecological reproductive tract inflammations such as mycotic vaginitis.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a probiotic live bacteria preparation, a preparation method and uses thereof. Background Art

[0002] Probiotic live bacteria preparations are a class of live bacteria biological products used to regulate the body's microecological balance and improve the health level of the host. The application of human probiotic live bacteria preparations mainly focuses on two categories at present. 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 pathogenic bacteria, regulate the vaginal pH environment, and prevent or treat various vaginitis. Probiotic live bacteria preparations use beneficial live probiotics to the human body as the main raw materials. These probiotics can grow and colonize in the digestive tract or reproductive tract, and together with other beneficial bacteria, adhere to the mucosa to form a protective biological barrier. During their metabolic process, they can produce small molecule substances such as short-chain fatty acids and antimicrobial peptides to inhibit the proliferation and adhesion of pathogenic bacteria, and can produce nutrients such as vitamins to participate in the body's metabolism. In addition, they can also activate the immune system and induce the production of antibodies. At present, human probiotic live bacteria preparations have been applied in the fields of medicine, health care, and food. Taking drugs as an example, Golden Bifid adds three probiotics derived from the human body, namely Bifidobacterium longum, Lactobacillus bulgaricus, and Streptococcus thermophilus; Siliankan tablets contain Bifidobacterium, Enterococcus faecalis, Lactobacillus, and Bacillus cereus; Lactobacillus vaginal capsules (Ding Junsheng) for vaginal use contain Lactobacillus delbrueckii. Yanhua (Lactic acid bacteria vaginal capsules) contains Enterococcus faecalis.

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

[0004] However, the current focus in the preparation process of live bacteria preparations often lies in the survival of the bacterial cells in the preparation, ignoring whether the preparation can survive well and play a role after entering the body. Generally, live bacteria preparations often consist of bacterial powder and excipients (such as fillers and glidants), and there is rarely any nutrition for the growth of the bacterial cells. After administration, the live bacteria in the preparation can only maintain their activity by relying on the nutrients in the human body environment. When the body environment is not suitable for the growth of the bacteria, even if a sufficient amount of live bacteria is given, the efficacy may still be unsatisfactory. This may explain the individual differences in the efficacy of microecological preparations, and the effect is not ideal for some people.

[0005] Patent CN 117618395 A discloses a compound probiotic preparation for treating intestinal diseases in animals and its preparation method. The compound probiotic preparation for treating intestinal diseases in animals includes a nutrient layer, and it is believed that the encapsulated nutrient layer can provide sufficient nutrients for the compound microorganisms. Its preparation process consists of multiple steps such as granulation, drying, spraying, and drying. The preparation process is complex, the production time required for preparing the product is long, and the production efficiency is low. Moreover, the formed preparation particles are divided into five layers, which are, from the inside to the outside, the nutrient layer, the inner shell layer, the bacterial layer, the intermediate layer, and the outer shell layer. The nutrient layer and the bacterial layer are not closely connected, which is not conducive to maintaining the activity and function of the bacteria. Summary of the Invention

[0006] To solve the above technical problems, the first object of the present invention is to provide a preparation method of a probiotic live bacteria preparation. By adding nutrient components suitable for the proliferation and efficacy of the probiotics to the live bacteria preparation, the probiotics can rapidly proliferate after administration to maintain their abundance, rapidly produce functional substances to exert their efficacy, and the antibacterial rate reaches 90% or more within 8 hours and 100% within 24 hours, safely and rapidly achieving the treatment effect. At the same time, it reduces the nutrient requirements of the probiotics for the administration environment and enhances the environmental adaptability.

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

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

[0009] The first object of the present invention is implemented by the following technical solutions: A preparation method of a probiotic live bacteria preparation, which includes the following steps: (1) Prepare a micro-nutrient solution; (2) Pretreat the micro-nutrient solution; (3) Prepare the target probiotic bacterial sludge; (4) Prepare the probiotic live bacteria liquid; (5) Add a freeze-drying protective agent to the probiotic live bacteria liquid to prepare the pre-freeze-drying probiotic live bacteria liquid; (6) Freeze-dry the pre-freeze-drying probiotic live bacteria liquid to obtain the probiotic live bacteria preparation; wherein,

[0010] (1) Prepare a micro-nutrient solution: Optimize the culture medium of the target probiotics to obtain a micro-nutrient solution that is conducive to the proliferation of the target probiotics and inhibits pathogenic bacteria;

[0011] (2) Pretreat the micro-nutrient solution: Pretreat the micro-nutrient solution to obtain the pretreated micro-nutrient solution;

[0012] (3) Prepare the target probiotic bacterial sludge: Gradually inoculate and ferment the target probiotics, centrifuge the probiotic fermentation broth after reaching the fermentation end point, discard the supernatant, and retain the bacterial sludge;

[0013] (4) Prepare the probiotic live bacteria liquid: Mix the bacterial sludge and the pretreated micro-nutrient solution at a mass ratio of 1:5 to 1:40 and stir evenly to obtain the probiotic live bacteria liquid;

[0014] (5) Preparing the pre-freeze microecological viable bacteria liquid by adding a freeze-drying protectant to the microecological viable bacteria liquid: adding the sterilized freeze-drying protectant to the obtained microecological viable bacteria liquid and stirring evenly to obtain the pre-freeze microecological viable bacteria liquid; wherein, the mass ratio of the microecological viable bacteria liquid to the freeze-drying protectant is 1:1 to 1:10;

[0015] (6) Preparing the microecological viable bacteria preparation by freeze-drying the pre-freeze microecological viable bacteria liquid: after the pre-freeze microecological viable bacteria liquid is vacuum freeze-dried and pulverized, adding sterile auxiliary materials and mixing evenly to obtain the microecological viable bacteria preparation.

[0016] Furthermore, the method for obtaining the micro-nutrient solution that is beneficial to the proliferation of the target probiotics and inhibits pathogenic bacteria by optimizing the culture medium of the target probiotics specifically includes:

[0017] S1: Based on the culture medium of the target probiotics, preparing several groups of different micro-nutrient liquid culture media and sterilizing them. Each group of the micro-nutrient liquid culture media includes components with the following mass percentage concentrations: carbon source 1.0% - 6.0%, nitrogen source 0.2% - 8.0%, inorganic salts 0.2% - 5.0%, trace elements 0.001% - 1.5%, emulsifier 0 - 0.5%; the solvent of the micro-nutrient liquid culture media is purified water; the pH of the micro-nutrient liquid culture media is 6.5 - 7.5;

[0018] S2: Adding the target probiotics and pathogenic bacteria to each group of the micro-nutrient liquid culture media, and the viable bacteria ratio of the target probiotics to the pathogenic bacteria is 1:1 to 10000:1; co-culturing at 35°C - 38°C for 24h in an aerobic or anaerobic or facultative anaerobic environment;

[0019] S3: Sampling at the 8 - 24h culture time point, detecting the concentration changes of the pathogenic bacteria in each group, comparing and screening out a group of micro-nutrient liquid culture media with the fastest antibacterial effect and an antibacterial rate reaching 90% or more at 8h and 100% at 24h, which is the micro-nutrient solution.

[0020] Furthermore, the carbon source is one or a combination of lactose and glucose; the nitrogen source is one or a combination of beef extract powder, beef extract, peptone, beef peptone, multivalent peptone or yeast powder; the inorganic salts are one or a combination of sodium acetate anhydrous, potassium acetate, dipotassium hydrogen phosphate or ammonium citrate; the trace elements are one or a combination of magnesium sulfate, manganese sulfate, L-cysteine hydrochloride, calcium chloride or sodium chloride; the emulsifier is Tween-80.

[0021] Furthermore, the specific method for pre-treating the micro-nutrient solution is:

[0022] Prepare the micro-nutrient solution obtained in step (1) at a concentration concentrated by 1 to 20 times, adjust the pH to 6.5 - 7.5, and then perform moist heat sterilization at 121 °C for 15 - 30 min to obtain the pretreated micro-nutrient solution. Preparing the micro-nutrient solution at a concentration concentrated by 1 to 20 times can reduce the water proportion, improve the freeze-drying effect, and increase the dry matter mass of the nutrient components after freeze-drying.

[0023] Further, the target probiotic bacteria belong to the genus Lactobacillus.

[0024] Further, the genus Lactobacillus is one or a mixture of one or more of Lactobacillus delbrueckii, Lactobacillus crispatus, or Lactobacillus plantarum.

[0025] Further, the pathogenic bacteria are Candida.

[0026] Further, the micro-nutrient solution includes the following components in mass percentage concentration: carbon source 1.0% - 4.0%, beef extract powder 0 - 2.0%, beef extract 0 - 2.0%, peptone 0 - 2.0%, beef peptone 0 - 1.0%, polyvalent peptone 0 - 2.0%, yeast powder 0.2% - 1.5%, anhydrous sodium acetate 0.1% - 1.0%, potassium acetate 0 - 1.0%, dipotassium hydrogen phosphate 0 - 1.0%, ammonium citrate 0 - 1.0%, magnesium sulfate 0.01% - 0.1%, manganese sulfate 0 - 0.2%, L-cysteine hydrochloride 0 - 1.0%, Tween-80 0.01% - 0.2%; the solvent of the micro-nutrient solution is purified water, and the pH is adjusted to 6.5 - 7.5.

[0027] Further, the carbon source is one or a combination of lactose and glucose.

[0028] Further, when the target probiotic bacteria are Lactobacillus delbrueckii and the pathogenic bacteria are Candida, the micro-nutrient solution includes the following components in mass percentage concentration: lactose 2.0%, polyvalent peptone 0.5%, yeast powder 1.0%, beef extract 1.0%, anhydrous sodium acetate 0.7%, potassium acetate 0.7%, magnesium sulfate 0.02%, L-cysteine hydrochloride 0.05%, Tween-80 0.1%; the pH of the micro-nutrient solution is adjusted to 7.4, and the solvent is purified water.

[0029] Further, when the target probiotic bacteria are Lactobacillus crispatus or Lactobacillus plantarum and the pathogenic bacteria are Candida, the micro-nutrient solution includes the following components in mass percentage concentration: glucose 2.0%, peptone 1.0%, yeast powder 1.0%, anhydrous sodium acetate 0.7%, potassium acetate 0.7%, magnesium sulfate 0.01%, manganese sulfate 0.01%, Tween-80 0.1%; the pH of the micro-nutrient solution is adjusted to 7.2, and the solvent is purified water.

[0030] The second object of the present invention is implemented by the following technical solution: a microecological viable bacteria preparation prepared by using the preparation method of a microecological viable bacteria preparation described in the first object of the present invention.

[0031] The third object of the present invention is implemented by the following technical solution: the microecological viable bacteria preparation described in the second object of the present invention is used for producing medicines, health products, disinfectants or personal care products.

[0032] Furthermore, the microecological viable bacteria preparation is used for producing medicines for preventing or treating gynecological reproductive tract inflammation. The gynecological reproductive tract inflammation in the present invention is mycotic vaginitis.

[0033] Advantages of the present invention:

[0034] (1) The method of the present invention combines the nutrients that promote probiotics to inhibit pathogenic bacteria and the probiotics to be made into a microecological viable bacteria preparation through freeze-drying. The nutrients in the preparation enable the probiotics to rapidly proliferate after administration to maintain their abundance, rapidly produce functional substances to exert their effects, and the antibacterial rate reaches 90% or more within 8 hours and 100% within 24 hours, safely and rapidly achieving the therapeutic effect; at the same time, it reduces the nutritional requirements of probiotics for the administration environment and enhances the environmental adaptability.

[0035] (2) The present invention wraps the optimized formula of nutritional components around the bacterial cells and then performs freeze-drying, enabling the bacterial cells to be fully in a nutritional environment, which is conducive to maintaining the activity of the bacterial cells and exerting their functions.

[0036] (3) The preparation method of the microecological viable 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.

[0037] (4) The present invention mixes the bacterial cells, nutrients, and protectants one by one and then performs vacuum freeze-drying to finally form a powder, which is conducive to the preservation stability of probiotics, the efficacy stability of the preparation, and long-term storage, transportation, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0039] Figure 1 It is a sample diagram of the Lactobacillus delbrueckii microecological viable bacteria preparation prepared in Example 3;

[0040] Figure 2 It is a comparison diagram of the residual concentration of Candida albicans ATCC10231 at different time points in the experiment of Example 4;

[0041] Figure 3 Graph showing the comparison of the residual concentrations of Escherichia coli at different time points for the experiment of Example 5;

[0042] Figure 4 Graph showing the comparison of the residual concentrations of Staphylococcus aureus ATCC6538 at different time points for the experiment of Example 6;

[0043] Figure 5 Graph showing the comparison of the antibacterial effects of the compound probiotic preparation in Example 14. Detailed implementation manners

[0044] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without any creative efforts shall fall within the protection scope of the present invention.

[0045] Unless otherwise specified, the materials and reagents used in the following embodiments can be obtained from commercial channels. In the following embodiments, the pathogenic bacteria strains: Escherichia coli 8099, Staphylococcus aureus ATCC6538, Candida albicans ATCC10231, Candida albicans 2.4159, and Candida glabrata 2.3961 are all purchased from the China General Microbiological Culture Collection Center.

[0046] The Lactobacillus delbrueckii, Lactobacillus crispatus, and Lactobacillus plantarum involved in the following embodiments are Lactobacillus delbrueckii DM8909, Lactobacillus crispatus SQ1505, and Lactobacillus plantarum SQ1631 respectively. The three are all isolated from the healthy female reproductive tract and are the dominant lactobacilli in the female reproductive tract, and all are from Inner Mongolia Shuangqi Pharmaceutical Co., Ltd.

[0047] Example 1: Optimization of the micro - nutrient solution for promoting the antibacterial effect of Lactobacillus delbrueckii

[0048] (1) Prepare 9 micro - nutrient solutions with different compositions. The specific compositions are shown in Table 1, numbered 1# - 9#, and then sterilize them at 121 °C by moist heat for 15 min.

[0049] (2) Use the sterile LBS liquid medium (10#) and sterile normal saline (11#) as the control group and the blank group respectively (Note: The sterile LBS liquid medium is a common medium for Lactobacillus delbrueckii, and the composition is shown in Table 1).

[0050] (3) Take 11 sterile test tubes and number them 1# - 11# respectively. Add 9.5 mL of the culture medium corresponding to the number to each of the test tubes numbered 1# - 10#, and add 9.5 mL of sterile normal saline to the 11# test tube. Add 0.5 g of Lactobacillus delbrueckii powder with 2×10 9 CFU / g to each test tube, and 0.5 mL of Candida albicans (ATCC10231) bacterial liquid with 2×10 6 CFU / mL, and mix well. Then place them in facultative anaerobic culture at 37°C. Detect the viable count of Candida albicans in each test tube at 8 h and 24 h of culture respectively.

[0051] Result: The antibacterial effect of Lactobacillus delbrueckii in the 8# micro - nutrient solution is the best, and the antibacterial effect is significantly better than that of other groups (see Table 2).

[0052] Table 1: Composition table of each micro - nutrient solution for co - culture of Lactobacillus delbrueckii and Candida albicans

[0053]

[0054] Table 2: Antibacterial ability of Lactobacillus delbrueckii against Candida albicans in different environments

[0055]

[0056] Example 2: Pretreatment of micro - nutrient solution and preparation of Lactobacillus delbrueckii micro - ecological viable bacteria preparation

[0057] (1) Prepare 200 g of the 8# micro - nutrient solution in Example 1 according to the formula concentrated 10 times, adjust the pH to 7.4, and then perform moist heat sterilization at 121°C for 15 min to obtain the pretreated micro - nutrient solution. Prepare 100 g of freeze - drying protectant with a mass percentage concentration of 20%.

[0058] (2) Take the Lactobacillus delbrueckii fermentation broth at the end of fermentation, and centrifuge to collect the bacterial sludge.

[0059] (3) Take 10 g of the bacterial sludge and add it to 100 g of the pretreated micro - nutrient solution, stir well to make the nutrients completely wrap the bacterial cells, then add 110 g of the freeze - drying protectant, stir well, pour it into the freeze - drying tray for freeze - drying. After drying, crush it through a 40 - mesh sieve, add excipients, and package it into 1 g / bag, which is the Lactobacillus delbrueckii micro - ecological viable bacteria preparation.

[0060] Example 3: Characteristics, viable count and water activity of Lactobacillus delbrueckii micro - ecological viable bacteria preparation

[0061] The Lactobacillus delbrueckii micro - ecological viable bacteria preparation obtained in Example 2 is a light yellow powder. After detection, its viable count is 9.03×10 9 CFU / g, and the water activity is 0.053. The preparation sample is asFigure 1 。

[0062] Example 4: Efficacy Verification of the Microecological Live Bacteria Preparation of Lactobacillus delbrueckii — Inhibiting Candida albicans ATCC10231

[0063] Experimental method:

[0064] Adjust the microecological live bacteria preparation of Lactobacillus delbrueckii prepared in Example 2 and the conventional freeze-dried Lactobacillus delbrueckii powder to a live bacteria count of 2.00×10 9 CFU / g for standby. Take 3 test tubes, numbered 1# - 3# respectively. Add 9.5 mL of normal saline and 0.5 mL of Candida albicans liquid to each test tube, and then: Tube 1#: Add 0.5 g of the microecological live bacteria preparation of Lactobacillus delbrueckii; Tube 2#: Add 0.5 g of the conventional Lactobacillus delbrueckii powder. Place the test tubes in facultative anaerobic culture at 37°C. Detect the live bacteria counts of Lactobacillus delbrueckii and Candida albicans in each group at 8 h and 24 h of culture respectively.

[0065] Results: As Figure 2 shown, the microecological live bacteria preparation of Lactobacillus delbrueckii has a more significant inhibitory effect on Candida albicans than the conventional freeze-dried Lactobacillus delbrueckii powder.

[0066] Example 5: Efficacy Verification of the Microecological Live Bacteria Preparation of Lactobacillus delbrueckii — Inhibiting Escherichia coli 8099

[0067] Experimental method: The same as Example 4, where the pathogenic bacteria liquid is Escherichia coli liquid.

[0068] Results: As Figure 3 shown, the microecological live bacteria preparation of Lactobacillus delbrueckii has a more significant inhibitory effect on Escherichia coli than the conventional freeze-dried Lactobacillus delbrueckii powder.

[0069] Example 6: Efficacy Verification of the Microecological Live Bacteria Preparation of Lactobacillus delbrueckii — Inhibiting Staphylococcus aureus ATCC6538

[0070] Experimental method: The same as Example 4, where the pathogenic bacteria liquid is Staphylococcus aureus liquid.

[0071] Results: As Figure 4 shown, the microecological live bacteria preparation of Lactobacillus delbrueckii has a more significant inhibitory effect on Staphylococcus aureus than the conventional freeze-dried Lactobacillus delbrueckii powder.

[0072] Example 7: Safety Evaluation of the Microecological Live Bacteria Preparation of Lactobacillus delbrueckii (Vaginal Mucosa Irritation Test)

[0073] 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.

[0074] 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.

[0075] Table 3: Changes in mouse body weight and local vaginal conditions after administration of Lactobacillus delbrueckii microecological live bacteria preparation

[0076]

[0077] Example 8: Evaluation of the therapeutic effect of Lactobacillus delbrueckii microecological live bacteria preparation on fungal vaginitis (VVC) in rats

[0078] method:

[0079] (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.

[0080] (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.

[0081] (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.

[0082] Negative control group: sterile saline;

[0083] LBS-Lactobacillus delbrueckii preparation group: Take 2 g of LBS-Lactobacillus delbrueckii preparation (about 9.03×10 9 CFU / g) and dissolve it thoroughly with 10 mL of sterile normal saline, with a concentration of 1.80×10 9 CFU / mL;

[0084] Lactobacillus delbrueckii microecological viable bacteria preparation group: Take 2 g of Lactobacillus delbrueckii microecological viable bacteria preparation (9.03×10 9 CFU / g) and dissolve it thoroughly with 10 mL of sterile normal saline, with a concentration of 1.80×10 9 CFU / mL.

[0085] Each group of rats was intravaginally instilled with 0.1 mL of the corresponding sample per time per day using a sterile syringe for 3 consecutive days. Observe and record whether the vagina of each rat was red and swollen and the condition of the secretions. On the 4th day, each rat was lavaged with 5 mL of sterile normal saline in multiple times to wash the vagina thoroughly and the lavage fluid was collected. Detect the content of Candida albicans in the lavage fluid and analyze it.

[0086] Results: The vagina of the rats with successful modeling had more white secretions, and the vaginal orifice was significantly red, swollen and congested. After 3 days of administration of each sample, the vaginas of the rats in the normal saline negative control group were significantly red and swollen, and more secretions could be seen. One of the rats in the LBS-Lactobacillus delbrueckii preparation group improved significantly after 3 days of administration, and 3 of them improved slightly. The viable bacteria count of Candida albicans was lower than that of the control group, indicating that continuous administration of Lactobacillus delbrueckii for 3 days could inhibit Candida albicans to a certain extent. After 3 days of administration of the Lactobacillus delbrueckii microecological viable bacteria preparation group, the vaginas of 6 rats had returned to normal, the redness, swelling and secretions disappeared, and the detection amount of Candida albicans was extremely low, indicating that appropriate micronutrient components could greatly promote the antibacterial effect of Lactobacillus delbrueckii and had a significant therapeutic effect on rat VVC.

[0087] Table 4: Record of the local conditions of the vaginas of rats after administration of Lactobacillus delbrueckii microecological viable bacteria preparation

[0088]

[0089] Table 5: Comparison of the concentrations of Candida albicans in the vaginal lavage fluid of rats after administration of different samples for 3 days

[0090]

[0091] Example 9: Optimization of the micronutrient solution for promoting Lactobacillus crispatus to inhibit Candida albicans

[0092] (1) Prepare 8 groups of different test group micronutrient solutions and conventional MRS medium (control group) respectively. The components of each group are shown in Table 6, numbered 1# - 9# respectively, and sterilized at 121 °C for 15 min. Use sterile normal saline (10#) as the blank group.

[0093] (2) Take 10 Erlenmeyer flasks of 100 mL (numbered 1# - 10#), add 50 mL of the culture solution with the corresponding number to each Erlenmeyer flask, and then add Candida albicans solution and Lactobacillus crispatus powder to each group and mix well. Make the initial concentration of Candida albicans in each group the same and all at the order of magnitude of 10 5 CFU / mL, and the viable concentration of Lactobacillus crispatus in each group is the same and all at 1×10 8 CFU / mL.

[0094] (3) Incubate each group under facultative anaerobic conditions at 37°C. Detect the viable count of Candida albicans in each group at 0 h, 8 h, and 24 h of incubation respectively.

[0095] Result: The antibacterial effect of Lactobacillus crispatus in the 7# micro-nutrient solution is the best (see Table 7).

[0096] Table 6: Composition table of each micro-nutrient solution for co-culturing Lactobacillus crispatus and Candida albicans

[0097]

[0098] Table 7 Antibacterial ability of Lactobacillus crispatus against Candida albicans in different environments

[0099]

[0100] Example 10: Optimization of micro-nutrient solution for promoting the inhibition of Candida albicans by Lactobacillus plantarum

[0101] The method is the same as that in Example 9. The target probiotic is Lactobacillus plantarum.

[0102] (1) Prepare 8 different experimental groups of micro-nutrient solutions and a conventional MRS medium (control group) respectively. The components of each group are shown in Table 6, numbered 1# - 9# respectively, and sterilize at 121°C for 15 min.

[0103] Use sterile normal saline (10#) as the blank group.

[0104] (2) Take 10 Erlenmeyer flasks of 100 mL (numbered 1# - 10#), add 50 mL of the culture solution with the corresponding number to each Erlenmeyer flask, and then add Candida albicans solution and Lactobacillus plantarum powder to each group and mix well. Make the initial concentration of Candida albicans in each group the same and all at the order of magnitude of 10 5 CFU / mL, and the viable concentration of Lactobacillus plantarum in each group is the same and all at 1×10 8 CFU / mL.

[0105] (3) Incubate each group under facultative anaerobic conditions at 37°C. Detect the viable count of Candida albicans in each group at 0 h, 8 h, and 24 h of incubation respectively.

[0106] Result: The antibacterial effect of Lactobacillus plantarum in the 7# micro-nutrient solution was the best (see Table 8). The optimal culture medium for Lactobacillus plantarum to inhibit Candida albicans was the same as that for Lactobacillus crispatus, both being the 7# micro-nutrient medium.

[0107] Table 8 Antibacterial ability of Lactobacillus plantarum against Candida albicans in different environments

[0108]

[0109] Example 11: Preparation of Lactobacillus crispatus microecological viable bacteria preparation and Lactobacillus plantarum microecological viable bacteria preparation

[0110] As can be seen from Examples 9 and 10, the preferred culture medium for both Lactobacillus crispatus and Lactobacillus plantarum to inhibit Candida albicans was the 7# micro-nutrient solution. Therefore, the microecological preparation can be prepared by the same method.

[0111] (1) Prepare 200 g of the 7# micro-nutrient solution in Example 9 according to the formula concentrated 3 times, adjust the pH to 7.2, and then perform moist heat sterilization at 121 °C for 15 min to obtain the pretreated micro-nutrient solution. Then prepare 400 g of freeze-drying protectant with a mass percentage concentration of 20%.

[0112] (2) Take the Lactobacillus crispatus bacterial liquid fermented to the end point, centrifuge, discard the supernatant, and collect the bacterial sludge. Then weigh 15 g of the bacterial sludge, add 100 g of the pretreated micro-nutrient solution, stir well to mix evenly, so that the nutrients fully wrap the bacterial cells, and then add 200 g of the freeze-drying protectant, mix well, and perform freeze-drying. 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 evenly to obtain the Lactobacillus crispatus microecological viable bacteria preparation.

[0113] (3) Take the Lactobacillus plantarum bacterial liquid fermented to the end point, centrifuge, discard the supernatant, and collect the bacterial sludge. Then weigh 15 g of the bacterial sludge, add 100 g of the pretreated micro-nutrient solution, stir well to mix evenly, so that the nutrients fully wrap the bacterial cells, and then add 200 g of the freeze-drying protectant, mix well, and perform freeze-drying. After drying, crush and pass through a 40-mesh sieve. After drying, crush and pass through a 40-mesh sieve, add auxiliary materials (the same auxiliary materials as those for the Lactobacillus crispatus microecological viable bacteria preparation), and mix evenly to obtain the Lactobacillus plantarum microecological viable bacteria preparation.

[0114] Example 12: Viable bacteria count detection of Lactobacillus crispatus microecological viable bacteria preparation and Lactobacillus plantarum microecological viable bacteria preparation and characteristics of the aqueous solution

[0115] (1) Viable bacteria count detection

[0116] The viable cell counts of the Lactobacillus crispatus microecological viable bacteria preparation and the Lactobacillus plantarum microecological viable bacteria preparation were respectively detected using MRS solid medium. The viable cell count of the Lactobacillus crispatus microecological viable bacteria preparation was 6.30×10 9 CFU / g, and that of the Lactobacillus plantarum microecological viable bacteria preparation was 8.17×10 9 CFU / g.

[0117] (2) Characteristics of the preparation aqueous solution

[0118] Take 2 g of the Lactobacillus crispatus microecological viable bacteria preparation and add 10 mL of sterile water, and dissolve it thoroughly. Its aqueous solution is light yellow, with a pH of 5.80, and its viable cell count is 1.26×10 9 cfu / mL. After placing the aqueous solution of the preparation in a facultative anaerobic culture at 37°C for 8 h, the pH is 4.50, and the viable cell count is 2.05×10 9 cfu / mL. Therefore, in a facultative anaerobic environment at 37°C, the nutrients contained in this preparation can support the continuous proliferation and acid production of Lactobacillus crispatus.

[0119] Take 2 g of the Lactobacillus plantarum microecological viable bacteria preparation and add 10 mL of sterile water, and dissolve it thoroughly. Its aqueous solution is light yellow, with a pH of 5.51, and its viable cell count is 1.63×10 9 cfu / mL. After placing the aqueous solution of the preparation in a facultative anaerobic culture at 37°C for 8 h, its pH is 4.36, and the viable cell count is 2.12×10 9 cfu / mL. Therefore, in a facultative anaerobic environment at 37°C, the nutrients contained in this preparation can support the continuous proliferation and acid production of Lactobacillus plantarum.

[0120] Example 13: Preparation of the Lactobacillus crispatus - Lactobacillus plantarum complex microecological viable bacteria preparation

[0121] During the preparation of the Lactobacillus crispatus microecological viable bacteria preparation and the Lactobacillus plantarum microecological viable bacteria preparation prepared in Example 11, their nutrient components and the proportion of protective agents were exactly the same, and they were further formulated into a dual - strain preparation. The Lactobacillus crispatus microecological viable bacteria preparation and the Lactobacillus plantarum microecological viable bacteria preparation were mixed according to a mass ratio of 1.3:1 to make a complex microecological viable bacteria preparation with a viable bacteria ratio of Lactobacillus crispatus to Lactobacillus plantarum of about 1:1 (the viable cell count is 7.11×10 9 cfu / g).

[0122] Example 14: Antibacterial study of the complex microecological viable bacteria preparation

[0123] Test group sample: The complex microecological viable bacteria preparation prepared in Example 13 (the viable cell count is 7.11×10 9 cfu / g);

[0124] Control group samples: A compound viable bacteria preparation with a viable bacteria ratio of 1:1 of Lactobacillus crispatus preparation and Lactobacillus plantarum preparation without micronutrients, and the total viable bacteria count is 7.11×10 9 cfu / g.

[0125] Blank group samples: The 7# micronutrient solution in Example 9 was prepared according to the formula concentrated 3 times, the pH was adjusted to 7.2, and then it was sterilized by moist heat at 121°C for 15 min to obtain 200 g of pretreated micronutrient solution. Then, 400 g of freeze-drying protectant with a mass percentage concentration of 20% was prepared. 100 g of the pretreated micronutrient solution was taken, and then 200 g of the freeze-drying protectant was added and mixed well, followed by freeze-drying to prepare a micronutrient freeze-dried powder preparation without Lactobacillus.

[0126] Method:

[0127] (1) Take fresh Candida albicans 2.4159 and Candida glabrata 2.3961, and adjust them to a concentration of about 10 6 CFU / mL with sterile water.

[0128] (2) Take 6 sterile 50 mL beakers. Add 1 g of the compound microecological viable bacteria preparation to Test groups 1# and 2#; add 1 g of the compound viable bacteria preparation without micronutrients to Control groups 3# and 4#; Blank group: Add 1 g of the micronutrient freeze-dried powder preparation without Lactobacillus to 5# and 6#.

[0129] (3) Add 3 mL of sterile water to each group of samples, stir well to form a paste and spread it flat. Take one portion of 50 mL of sterile Sabouraud agar medium, cool it to 40°C, add 5 mL of Candida albicans or Candida glabrata respectively, mix well, and use a pipette to aspirate 5 mL and gently spread it on the surface of each sample, and let it stand until solidified. Cover the beaker and invert it for facultative anaerobic culture at 37°C for 24 - 48 h, and observe the growth of Candida in each group.

[0130] (4) Add 20 mL of sterile water to each beaker, blow and beat the Candida on the surface of the medium thoroughly, transfer it into a 250 mL Erlenmeyer flask with a triangular bottom, shake it on a shaker for 15 min, then detect the concentration of Candida and make a comparison.

[0131] Results: As Figure 5 shown, no Candida colonies were seen on the surface of the Sabouraud agar medium in the test groups, some Candida colonies grew on the surface of the Sabouraud agar medium in the control groups, and the Candida colonies covered the surface of the medium in the blank group. Wash the Candida in each group with 20 mL of sterile water, detect its concentration, and the data are shown in Table 9. The concentration of Candida in the blank group is much higher than that in the control group, and the concentration of Candida in the experimental group is extremely low. Therefore, the compound microecological viable bacteria preparation can significantly inhibit the growth of Candida albicans 2.4159 and Candida glabrata 2.3961.

[0132] Comparison of Concentrations of Candida spp. in Each Group

[0133]

[0134] Example 15: Safety Evaluation of the Composite Probiotic Preparation of Lactobacillus crispatus - Lactobacillus plantarum (Vaginal Mucosa Irritation Test)

[0135] According to the safety test method for vaginal probiotic products in the 2020 edition of the Chinese Pharmacopoeia, 5 healthy female mice weighing 24 - 26 g with no damage, congestion, or edema at the vaginal orifice were used. 10 mg of the composite probiotic preparation was inserted into the vagina of each mouse once a day for 3 consecutive days. Starting from the first day of administration, the mice were observed continuously for 7 days. If the mice survived healthily, gained weight, and showed no symptoms such as local swelling, secretions, etc. in the vagina, it was considered qualified.

[0136] Results: Each mouse was administered the drug continuously for 3 days, observed continuously for 7 days, and weighed. The body weights of each mouse gradually increased, they ate normally, and their conditions were good. No local swelling or secretions were observed in the vagina of the mice, and no congestion was seen. The data are shown in Table 10. This indicates that the composite probiotic preparation is safe when administered vaginally.

[0137] Table 10 Records of Changes in Body Weight of Mice and Local Vaginal Conditions after Administration of the Composite Probiotic Preparation

[0138]

[0139] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within 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) Freeze-drying the pre-freeze-dried microecological live bacteria liquid to obtain a microecological live bacteria preparation: the pre-freeze-dried microecological live bacteria liquid is vacuum freeze-dried and crushed, and then sterile excipients are added and mixed evenly to obtain a microecological live bacteria preparation; 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 includes the following components in mass percentage concentration: carbon source 1.0%-4.0%, beef extract powder 0-2.0%, beef extract 0-1.5%, peptone 0-1.5%, beef peptone 0-1.0%, polyvalent peptone 0-2.0%, yeast powder 0.2%-1.5%, anhydrous sodium acetate 0.1%-1.0%, potassium acetate 0-1.0%, dipotassium hydrogen phosphate 0-1.0%, ammonium citrate 0-1.0%, magnesium sulfate 0.01%-0.1%, manganese sulfate 0-0.2%, L-cysteine ​​hydrochloride 0-1.0%, Tween-80 0.01%-0.2%; the solvent of the micronutrient solution is purified water, and the pH is adjusted to 6.5-7.5; the carbon source is one of lactose or glucose or a combination of the two; 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 under an aerobic, anaerobic or facultative anaerobic environment; S3: Take samples at 8 to 24 hours of culture time, detect the concentration changes of pathogenic bacteria in each group, and compare and screen out a group of micronutrient liquid culture media with the fastest antibacterial effect, i.e., micronutrient solution; 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; The target probiotics are selected from Lactobacillus delbrueckii DM8909, Lactobacillus crispatus SQ1505 or Lactobacillus plantarum SQ1631; When the target probiotic is Lactobacillus delbrueckii DM8909, and the pathogenic bacteria is one or more of Candida albicans ATCC10231, Escherichia coli 8099 or Staphylococcus aureus ATCC6538, the micronutrient solution includes the following components in mass percentage concentrations: lactose 2.0%, polyvalent peptone 0.5%, yeast powder 1.0%, beef extract 1.0%, anhydrous sodium acetate 0.7%, potassium acetate 0.7%, magnesium sulfate 0.02%, L-cysteine ​​hydrochloride 0.05%, and Tween-80 0.1%; the pH of the micronutrient solution is adjusted to 7.4, and the solvent is purified water; When the target probiotics are Lactobacillus crispatus SQ1505 or Lactobacillus plantarum SQ1631, and the pathogenic bacteria are one or more of Candida albicans ATCC10231, Candida albicans 2.4159 or Candida glabrata 2.3961, 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.

2. A microecological live bacteria preparation prepared by the method for preparing a microecological live bacteria preparation according to claim 1.

3. The use of the microecological live bacteria preparation as described in claim 2 in the production of medicines or private care products; when the target probiotic is Lactobacillus delbrueckii DM8909, the medicine or the private care product is the medicine or the private care product that inhibits the pathogenic bacteria from being one or more of Candida albicans ATCC10231, Escherichia coli 8099 or Staphylococcus aureus ATCC6538; when the target probiotic is Lactobacillus crispatus SQ1505 or Lactobacillus plantarum SQ1631, the medicine or the private care product is the medicine or the private care product that inhibits the pathogenic bacteria from being one or more of Candida albicans ATCC10231, Candida albicans 2.4159 or Candida glabrata 2.3961.

4. The use of the microecological live bacteria preparation according to claim 3 in the production of medicines, characterized in that: The medicine is a medicine for preventing or treating gynecological reproductive tract inflammation.

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