Probiotics for improving female genital tract infection and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to probiotics for improving female genital tract infection and application. The probiotics provided by the invention comprise at least one of plant lactobacillus plantarum VM-2, lactobacillus gasseri VL-3 and lactobacillus crispatus VX-4. The three strains have the effects of prevention and adjuvant therapy of female genital tract infectious diseases, and have very high probiotic potential. The invention also provides a combined product of the three strains, and growth inhibition experiments among the component strains ensure that the three strains can fully exert probiotic functions.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a probiotic for improving female reproductive tract infection and an application thereof. Background Art

[0002] Female reproductive tract infections (RTIs) are a major public health and social problem worldwide. Common female RTIs include bacterial vaginitis (BV), candidal vaginitis (VVC), and trichomonas vaginitis (TV). Female RTIs are characterized by the replacement of lactobacilli by pathogens such as anaerobic bacteria, fungi, and parasitic protozoa, such as Gardnerella vaginalis (GV), Prevotella, and Candida albicans, which leads to a decrease in the normal dominant flora of the vagina and an increase in pathogenic flora. In addition to affecting the health of women themselves, female RTIs also threaten the health of their partners and offspring. Studies have shown that female RTIs may be related to sexually transmitted infections, infertility, and premature birth, which may increase children's health risks, such as the risk of lifelong cardiovascular, respiratory, and neurodevelopmental diseases. Despite this, there are few new treatments available, and antibiotics are still the main treatment, with commonly used antibiotics including metronidazole, clindamycin, and tinidazole. Although antibiotic treatment is short-term and effective, and can relieve symptoms in the short term, the cure rate is not high in the long run. According to statistics, more than 50% of women will experience repeated attacks of bacterial vaginosis within 1 year after using antibiotics for treatment. This is mainly because while antibiotics kill pathogens, they also inhibit the growth of probiotics in the vagina, making the vaginal environment still vulnerable to infection by pathogenic bacteria, and fundamentally failing to improve the vaginal microecological environment. Secondly, although antibiotics can enhance their lethality to bacteria by destroying biofilms, they lose some of their effectiveness because they bind to the substances that make up the biofilm before acting on the bacteria. In addition, the presence of biofilms makes pathogenic bacteria highly resistant to antibiotics, ultimately making it impossible for antibiotics to completely eradicate the bacteria. In addition, antibiotic treatment has large side effects and is not suitable for pregnant and lactating women. All these reasons pose hidden dangers to antibiotic therapy.

[0003] The lower genital tract microbial communities of women from different regions and ethnicities vary significantly, but healthy microbial communities are usually dominated by Lactobacillus. L. crispatus ) were associated with a healthy microbiome, whereas a flora dominated by Lactobacillus iners ( L. iners ) are associated with a potential risk of vaginitis. The vaginal microbial community of healthy women of childbearing age in China is dominated by Lactobacillus gasseri ( L. gasseri , also known as Lactobacillus gasseri, Lactobacillus crispatus ( L. crispatus ) and Lactobacillus iners ( L. iners ). In addition, the vaginal flora also includes some other lactobacilli, such as Lactobacillus jensenii ( L. jensenii )、Lactobacillus rhamnosus( L. rhamnosus )、Lactobacillus acidophilus( L. acidophilus )、Lactobacillus plantarum( Lactiplantibacillus plantarum , formerly known as Lactobacillus plantarum , Lactobacillus plantarum, etc. Lactobacillus can metabolize glycogen secreted by vaginal epithelial cells, produce organic acids (such as lactic acid), and maintain the normal acidic environment of the vagina (pH <4-4.5). The acidic environment of the vagina can inhibit the growth of potential pathogens. In addition, Lactobacillus can resist the colonization and growth of pathogens by secreting some antibacterial agents or through competitive exclusion.

[0004] With the accumulation and deepening of research on human microecology, microbiome research and understanding of human health, more and more probiotic preparations are being used to regulate intestinal microecology and treat intestinal inflammatory diseases. However, the number of commercially available probiotic drugs that can be used to treat female RTIs is scarce. In the 1980s, Professor Kang Bai's team at Dalian Medical University conducted research on microecological preparations for female reproductive tract diseases and screened out a strain of Lactobacillus delbrueckii (DM8909) for the treatment of bacterial vaginosis. This is also the only lactobacillus drug currently approved for clinical use in my country (Dingjunsheng). In vitro experimental studies and clinical data show that DM8909 has the characteristics of high acid production and certain adhesion advantages to vaginal epithelial cells. At the same time, it can inhibit the growth and reproduction of harmful pathogens, regulate the balance of vaginal flora, and effectively treat bacterial vaginitis. However, there are limitations in the discussion of the therapeutic effect of DM8909 on bacterial vaginitis, and its inhibitory effect on various pathogens of vaginitis, such as GV and Candida albicans, is unclear. GV and Candida albicans are the main pathogens that cause BV and VVC. In addition, Lactobacillus delbrueckii is not a common bacterium in the lower reproductive tract of healthy Chinese women, and it is unknown whether its relief effect is better than the treatment effect of dominant bacteria. There are many research reports on vaginal probiotics abroad, such as Lactobacillus crispatus, Lactobacillus delbrueckii and Lactobacillus plantarum that can be used to treat VVC in rat models, Lactobacillus crispatus and Lactobacillus rhamnosus BMX54 that are used to treat BV, Lactobacillus reuteri RC-14 and Lactobacillus salivarius CECT9145 that are used to reduce vaginal group B streptococci in pregnant women, and Lactobacillus paracasei LPS-S01 that can reduce vaginal Gardnerella in healthy women. There are few types of vaginal probiotic preparations in domestic literature.

[0005] There are many patents for the application of Lactobacillus plantarum in food, but fewer for the treatment of RTIs in women. The Lactobacillus plantarum mentioned in Chinese patents CN 116103189 A, CN 114717150 A, etc. has a certain degree of cell adhesion ability, acid production ability or inhibition ability, or has a certain ability to inhibit pathogens such as GV in vitro. However, the existing screening methods have certain shortcomings. First, the screening process for lactobacillus from female vagina is still insufficient. Secondly, the detection methods for antibacterial metabolites such as lactic acid and hydrogen peroxide mainly rely on instruments or kits, which will be limited to a certain extent by their quality and cost. Thirdly, the in vitro inhibition experiment of pathogens is mainly aimed at GV. Some patents have added pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Shigella dysenteriae, etc., but there are still many common pathogens in female vagina that are not included. In addition, the above patents lack the evaluation of the growth and proliferation ability of the applied strains in different pH environments, which is precisely one of the very important abilities of the strains, because the pH environment of the patient's vagina has often changed. Finally, the adhesion ability of strains is mainly tested based on in vitro experiments of Hela cell lines, and there is a lack of testing on the aggregation and hydrophilicity of the strains themselves, which can affect the colonization ability of the strains in the microenvironment.

[0006] Lactobacillus gasseri ( L. gasseri , also known as Lactobacillus gasseri) and Lactobacillus crispatus ( L. crispatus ) is the main lactobacillus in the vaginal microbial community of healthy Chinese women of childbearing age, and has great probiotic potential. Currently, many probiotic combination preparations on the market claim to contain a variety of lactic acid bacteria, but after laboratory separation and identification, these products often fail to isolate all the identified strains. This may be because in the actual cultivation process, the strains with growth advantages completely inhibit the growth of other component strains. Therefore, the art is in urgent need of a female vaginal probiotic combination that has excellent lactic acid and hydrogen peroxide production capabilities, good adhesion, can inhibit the growth of multiple vaginal pathogens, has excellent wide-range pH growth characteristics, and does not inhibit the growth of each other between strain components, so as to fully exert the probiotic function.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a probiotic for improving female reproductive tract infection and its application.

[0009] Specifically, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a probiotic for improving female reproductive tract infection, wherein the probiotic comprises Lactobacillus plantarum ( Lactiplantibacillus plantarum )VM-2, Lactobacillus gasseri ( Lactobacillus gasseri) VL-3 and Lactobacillus crispatus ( Lactobacillus crispatus ) VX-4; wherein the preservation number of Lactobacillus plantarum VM-2 is CGMCC No.30810; the preservation number of Lactobacillus gasseri VL-3 is CGMCC No.32745; the preservation number of Lactobacillus crispatus VX-4 is CGMCC No.32746.

[0010] Preferably, the probiotics include Lactobacillus plantarum VM-2, Lactobacillus gasseri VL-3 and Lactobacillus crispatus VX-4, and the ratio of the live bacteria counts of the three is (0.8~1.2):(8~12):(80~120); more preferably 1:10:100.

[0011] In a second aspect, the present invention provides a culture or a fermentation product, wherein the culture or the fermentation product is obtained by expanding the probiotics described in the first aspect.

[0012] In a third aspect, the present invention provides a bacterial agent, which includes the probiotics described in the first aspect or the culture or fermentation product described in the second aspect.

[0013] Preferably, in the bacterial agent, the effective viable count of Lactobacillus plantarum VM-2 is not less than 1×10 6 CFU / mL.

[0014] Preferably, in the bacterial agent, the effective viable count of Lactobacillus gasseri VL-3 is not less than 1×10 7 CFU / mL.

[0015] Preferably, in the bacterial agent, the effective viable count of Lactobacillus crispatus VX-4 is not less than 1×10 8 CFU / mL.

[0016] In a fourth aspect, the present invention provides the use of the aforementioned probiotics or the culture or fermentation product or the bacterial agent in the preparation of a drug for preventing and assisting in the treatment of female reproductive tract infectious diseases.

[0017] In a fifth aspect, the present invention provides the use of the aforementioned probiotics or the culture or fermentation product or the bacterial agent in the prevention and auxiliary treatment of female reproductive tract infectious diseases.

[0018] In a sixth aspect, the present invention provides a drug for preventing and assisting in the treatment of infectious diseases of the female reproductive tract, which contains the aforementioned probiotics or the culture or fermentation product or the bacterial agent.

[0019] Beneficial effects: The present invention provides a probiotic for improving female reproductive tract infection and its application. The probiotic provided by the present invention comprises at least one of Lactobacillus plantarum VM-2, Lactobacillus gasseri VL-3 and Lactobacillus crispatus VX-4. The above three strains all have better effects of preventing and assisting in the treatment of female reproductive tract infectious diseases, all come from the human body itself, will not cause additional health burden to the host, and have high probiotic potential. The present invention also provides a combination product of the above three strains, and ensures that the three strains can fully exert their probiotic functions through growth inhibition experiments between component strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0021] Figure 1 These are morphological photos of the Lactobacillus plantarum VM-2, Lactobacillus gasseri VL-3 and Lactobacillus crispatus VX-4 strains of the present invention, from left to right they are Lactobacillus plantarum VM-2, Lactobacillus gasseri VL-3 and Lactobacillus crispatus VX-4.

[0022] Figure 2 These are the experimental results of the growth and proliferation abilities of Lactobacillus plantarum VM-2, Lactobacillus gasseri VL-3 and Lactobacillus crispatus VX-4 of the present invention in different pH culture environments.

[0023] Figure 3 The results are experimental results comparing the growth ability of Lactobacillus crispatus VX-4 of the present invention with other Lactobacillus crispatus.

[0024] Figure 4 The results of the lactic acid production experiment of Lactobacillus plantarum VM-2 of the present invention are shown in FIG.

[0025] Figure 5 These are the experimental results of hydrogen peroxide production by Lactobacillus plantarum VM-2, Lactobacillus gasseri VL-3 and Lactobacillus crispatus VX-4 of the present invention.

[0026] Figure 6 These are the experimental results of the hydrophobicity of the cell surfaces of the Lactobacillus plantarum VM-2, Lactobacillus gasseri VL-3 and Lactobacillus crispatus VX-4 strains of the present invention.

[0027] Figure 7 The experimental results of the present invention's Lactobacillus plantarum VM-2 inhibiting common vaginal pathogens, wherein the left picture shows an example of co-culture with Klebsiella pneumoniae and the right picture shows an example of co-culture with Gardnerella vaginalis.

[0028] Figure 8These are the experimental results of the Lactobacillus plantarum VM-2, Lactobacillus gasseri VL-3 and Lactobacillus crispatus VX-4 strains of the present invention inhibiting Candida albicans.

[0029] Fig. 9 The results of the growth inhibition experiment of the mixed bacterial solution of three strains included in the examples of the present invention are shown. DETAILED DESCRIPTION

[0030] At present, the main treatment for genital tract infections is still oral or topical antibiotics, including metronidazole, clindamycin, tinidazole, etc. However, there is a generally high recurrence rate after the initial treatment. Another issue that needs to be considered is bacterial resistance. For recurrent vaginal infections, frequent use of antibiotics may cause microorganisms in the body to acquire antibiotic resistance under selective pressure, which poses a certain risk to host health.

[0031] In response to the current problems, the present invention collects bacteria from the reproductive tract of healthy women of childbearing age, and among the strains that have been collected and completed 16SrDNA sequencing, accurately screens potential lactic acid strains by evaluating their growth and proliferation ability, lactic acid production ability, hydrogen peroxide production ability, cell surface hydrophobic properties, and ability to inhibit common vaginal pathogens in different pH culture environments.

[0032] The present invention conducts an in vitro inhibition experiment on common pathogens in the female vagina to further evaluate the probiotic potential of the strain for preventing and treating female RTIs and maintaining the vaginal microecological environment. The selected pathogens are common pathogens of the urogenital system, including vaginal Gardnerella, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus agalactiae and Candida albicans. Among them, Escherichia coli includes common type (number 1029) and O104:H4 type (hemorrhagic Escherichia coli, number Fx30), Staphylococcus aureus includes model strains (ATCC 6538) and polyenterotoxin type (number S63), Klebsiella pneumoniae includes common type (number 95) and carbapenem-resistant type (number 5933, 7614), Streptococcus agalactiae includes six most common serotypes (Ⅰa, Ⅰb, Ⅱ, Ⅲ, Ⅳ, Ⅴ) strains, and Candida albicans is a common type (number GDMCC2.178).

[0033] Finally, the present invention screened and obtained three probiotics that can prevent and assist in the treatment of female reproductive tract infectious diseases. The three probiotics are Lactobacillus plantarum VM-2, Lactobacillus gasseri VL-3 and Lactobacillus crispatus VX-4.

[0034] The in vitro culture techniques of these three probiotics are simple and universal, with low acquisition costs. When used in the prevention and treatment of female reproductive tract infections, they will not cause excessive economic burden. Among them, Lactobacillus plantarum VM-2 has strong growth and reproduction capabilities under different pH environmental conditions, showing high universality, and can play a role in different stages of women's life, physiological cycles and health conditions; and Lactobacillus plantarum VM-2 can still show excellent acid production ability under insufficient nutritional conditions; Lactobacillus plantarum VM-2 can also effectively inhibit common vaginal pathogens such as Gardnerella vaginalis; Lactobacillus gasseri VL-3 has good hydrogen peroxide production ability; Lactobacillus crispatus VX-4 is the main lactobacillus in the vaginal microbial community of healthy women of childbearing age in China, has good cell adhesion ability, and has a growth ability significantly better than other Lactobacillus crispatus. The above three lactobacilli all have high probiotic potential.

[0035] The present invention preserves the above three probiotics. Among them, the preservation number of Lactobacillus plantarum VM-2 is: CGMCC No.30810, and the preservation date is: May 29, 2024; the preservation number of Lactobacillus gasseri VL-3 is: CGMCC No.32745, and the preservation date is: November 22, 2024; the preservation number of Lactobacillus crispatus VX-4 is: CGMCC No.32746, and the preservation date is: November 22, 2024. The preservation units of the above three probiotics are all: China General Microbiological Culture Collection Management Center; the preservation addresses are all: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0036] The present invention also provides a composition of the above three bacterial strains, and ensures that the three bacterial strains can fully exert their probiotic functions through growth inhibition experiments among the component strains.

[0037] The present invention provides an excellent path for preventing and treating female reproductive tract infections from a biological perspective, and uses probiotic strains to alleviate female RTIs. Lactobacillus plantarum, Lactobacillus gasseri and Lactobacillus crispatus are strains collected from the reproductive tract of healthy women. They use their own metabolites to regulate the internal environment of the reproductive tract and naturally inhibit the growth of pathogens. They are safer, more reliable and more efficient, and help to restore the imbalanced vaginal flora faster and better. They are more advantageous from the perspectives of microecology, biology and socioeconomics.

[0038] The technical solutions provided by the present invention are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used can be obtained from commercial sources.

[0039] Example 1 This embodiment screens and provides probiotics for preventing and assisting in the treatment of female reproductive tract infectious diseases, wherein the probiotics include at least one of Lactobacillus plantarum VM-2, Lactobacillus gasseri VL-3 and Lactobacillus crispatus VX-4. The above three strains are all derived from the human body, have the effect of preventing and assisting in the treatment of female reproductive tract infectious diseases, and have high probiotic potential.

[0040] The specific experimental process is as follows: 1. Functional screening of each strain in the probiotic combination By screening the preserved Lactobacillus plantarum, Lactobacillus gasseri and Lactobacillus crispatus for growth and proliferation ability in different pH culture environments, lactic acid production, hydrogen peroxide production, cell surface hydrophobicity, and inhibition of common vaginal pathogens, we obtained the best performing strains of Lactobacillus plantarum (VM-2), Lactobacillus gasseri (VL-3) and Lactobacillus crispatus (VX-4).

[0041] Results Figure 1 , from left to right are the strain morphologies of Lactobacillus plantarum VM-2, Lactobacillus gasseri VL-3 and Lactobacillus crispatus VX-4.

[0042] The specific method is as follows: (1) Experiment on growth and proliferation ability in different pH culture environments: Use hydrochloric acid and sodium hydroxide to adjust the pH value of MRS broth medium, and the pH value range is 3, 4, 5, 6, and 7. The preserved experimental strains were inoculated into MRS broth for activation (pH value is about 4.8-5.0) and cultured anaerobically at 37℃ for 18-20h. The concentration of the bacterial solution in the vigorous growth period after activation was adjusted to 0.5 McFarland units, that is, the approximate bacterial concentration was 1.5×10 8 / mL, and then the activated bacterial solution was transferred to MRS broth culture medium with different pH values ​​at a 1% inoculation rate. After anaerobic culture at 37℃ for 18-20h, the OD600nm value was measured to observe the growth and proliferation of the experimental strains under different pH conditions.

[0043] The experimental results are shown in Figure 2 It can be seen that the growth and proliferation ability of Lactobacillus plantarum VM-2 in various pH environments is significantly better than that of other control strains and other strains of Lactobacillus plantarum. In particular, it exhibits excellent growth and proliferation ability at pH 4-7.

[0044] (2) Lactobacillus crispatus growth ability test: The Lactobacillus crispatus to be screened was cultured anaerobically in MRS broth at 37°C for 18-20 h, and the concentration was adjusted to about 1×10 8CFU / mL. Inoculate 5 mL of MRS broth at a 1% (V / V, volume / volume) inoculation volume and incubate anaerobically at 37°C for 20-24 hours. Take 500 μL of bacterial suspension, add MRS broth to dilute by 10, take 1 mL to apply to MRS plates, so that the final plate count is between 3-300 colonies. Record the number of Lactobacillus crispatus colonies on the MRS plate, and the results are recorded in CFU / mL (the total number of bacterial colonies contained in each milliliter of the original sample).

[0045] The experimental results are shown in Figure 3 It can be seen that the growth ability of Lactobacillus crispatus VX-4 is significantly better than that of other strains of Lactobacillus crispatus.

[0046] (3) Lactic acid production experiment: Inoculate the experimental strain at a 1% (V / V, volume / volume) inoculation rate in MRS broth medium and oligonutrient medium (distilled water containing 1% glucose and 1% yeast extract), and culture anaerobically at 37°C for 66 hours. Place blank controls for both culture media at the same time. Pipette the upper bacterial liquid into a centrifuge tube and mix well. Pipette 2-3 mL of the mixed bacterial liquid and mix with twice the volume of carbon dioxide-free water. Add a titration indicator and use 0.1 M sodium hydroxide standard titration solution for acidity titration. Keep the color unchanged for 30 seconds after the color changes. Record the number of milliliters of sodium hydroxide titration solution consumed and substitute it into the formula for calculation: X=( V 1 - V 2 )×C NaOH ×0.09×100 / V S .

[0047] Where, X: acid production of the sample (in terms of lactic acid), in grams per 100 milliliters (g / 100mL); V 1 : The volume of sodium hydroxide standard solution consumed by the sample, in milliliters (mL); V 2 : The volume of sodium hydroxide standard solution consumed by blank culture medium, in milliliters (mL); C NaOH : The calibrated sodium hydroxide concentration, in grams per liter (g / L); 0.09: Conversion factor of lactic acid; V S : Sample solution volume, in milliliters (mL).

[0048] The experimental results are shown in Figure 4 It can be seen that Lactobacillus plantarum VM-2 has a better lactic acid production capacity than the control strain under normal nutritional conditions. It is worth noting that Lactobacillus plantarum also showed excellent lactic acid production capacity under oligotrophic conditions.

[0049] (4) Hydrogen peroxide production experiment: Resuscitate the experimental strain in MRS broth medium and culture anaerobically at 37°C for 18-20 hours. Use an inoculation needle to pick a single colony of bacteria in the logarithmic growth phase and place it on a clean glass slide. Add 3% H 2 O 2 Mix 1-2 drops of liquid with the colony. If a large number of bubbles are produced within 1 minute of standing, it is positive. If no bubbles are produced, it indicates a negative result. The positive control of the experiment is Staphylococcus aureus; the negative control is Streptococcus. Inoculate the culture into the culture medium at an inoculum of 1% (V / V, volume / volume) in MRS broth medium, and culture it anaerobically at 37°C for 24 hours. At the same time, place the culture medium as a blank control. Take 9mL of the culture and centrifuge it at 12000×g for 20 minutes at 4°C. Harvest the cells, wash them twice with 50mM PPB (pH 6.0), and then resuspend them in 9mL of the same PPB supplemented with 5mM glucose. Transfer 0.5mL of this mixture to 9.5mL of PPB containing glucose and culture it aerobically at 37°C for 24 hours. After centrifugation at 12000×g for 20 minutes at 4°C, perform H 2 O 2 Analysis. Add 1 mL of horseradish peroxidase solution and 0.1 mL of tetramethylbenzidine methanol solution to 5 mL of supernatant. Incubate the tube at 37°C for 10 minutes. Add 0.2 mL of 4 N HCl to stop the reaction. Measure the absorbance at 400 nm. Determine the H by comparing with the value of the standard curve generated under the same conditions. 2 O 2 concentration.

[0050] The experimental results are shown in Figure 5 It can be seen that the ability of Lactobacillus gasseri VL-3 to produce hydrogen peroxide is significantly better than that of the control strains of other species, and is also better than other strains of Lactobacillus gasseri.

[0051] (5) Cell surface hydrophobicity detection: Based on the hydrocarbon adsorption capacity method, organic substances are used to detect the cell surface hydrophobicity of bacteria. Hydrophobicity determines the nonspecific adsorption capacity of bacteria, indicating whether the bacteria can be adsorbed and reproduced in the vaginal environment. The proliferating bacteria were centrifuged at 4000 r / min for 15 min, the bacteria were collected, and the bacteria were washed three times with PBS. The concentration of the bacterial solution was adjusted with PBS to make its OD 600nm value about 0.5, and the initial OD value was recorded as A0. Take 3 mL of the adjusted bacterial solution and add two hydrophobic solvents: xylene and hexadecane, respectively. The control group did not add any, shake for 1 min, and then let it stand for 15 min to allow the layers to separate. Take the aqueous phase, use PBS buffer as the blank control, and detect its OD 600nm value as At. The hydrophobicity calculation formula is: hydrophobicity = (A0-At) / A0×100%.

[0052] The experimental results are shown in Figure 6 . Blue is hexadecane and orange is xylene. It can be seen that the hydrophobicity of Lactobacillus crispatus VX-4, Lactobacillus gasseri VL-3 and Lactobacillus plantarum VM-2 belongs to the higher range (60-100% is high; 30-59% is medium; 0-29% is low), among which Lactobacillus crispatus VX-4 has the highest hydrophobicity. The experimental results show that the three strains contained in the probiotic combination have excellent non-specific adsorption ability to biological surfaces.

[0053] (6) Experiment on inhibition of common vaginal pathogens: The method for inhibiting pathogens is to culture the experimental strains anaerobically at 37°C in MRS broth medium for 18-20 hours and adjust the concentration to about 1×10 8 CFU / mL. Resuscitate pathogens to blood plates or liquid culture media (such as BHI). Spot 2μL of bacterial suspension on the surface of MRS solid culture medium containing 1.5% (w / v) agar, and incubate anaerobically at 37°C for 24 hours. Transfer the pathogens to liquid culture medium, adjust the concentration to 0.5 McFarland turbidity, and incubate anaerobically at 37°C for 24 hours. Mix 1mL of pathogen suspension with 100mL of liquid culture medium containing agar (such as BHIs broth + 0.7% agar) (the final viable count is about 1×10 6 CFU / mL) and poured onto the solid plate spotted with the experimental strain. Incubate the plate anaerobically at 37°C for 48h. Measure the diameter of the inhibition zone (mm).

[0054] The method for inhibiting pathogenic fungi is as follows: Cultivate Candida albicans in TSB medium at 30°C. Cultivate the experimental strain anaerobically in MRS broth medium at 37°C. Adjust the concentration of Candida albicans suspension to 1×10 7 CFU / mL, inoculated into 5 mL MRS broth at a ratio of 1% (v / v). Adjust the concentration of the experimental strain suspension to 1×10 8 CFU / mL, diluted 10 times and 100 times, and the three concentrations of experimental strains were inoculated into MRS broth inoculated with Candida albicans at a ratio of 1% (v / v). The mixed solution was cultured anaerobically at 37°C for 24 h. The co-culture solution was diluted with PBS buffer, and three dilutions were selected. 100 μL was taken from each tube and coated on Sabouraud agar plates. The number of Candida albicans on the Sabouraud agar plates in the positive group was counted. The formula for calculating the inhibition rate is: inhibition rate = (number of live bacteria in the control group - number of live bacteria in the positive group) / number of live bacteria in the control group × 100%.

[0055] Figure 7 These are photos (part) of co-culture of experimental strains and control strains with pathogens. The pathogen in the left photo is Klebsiella pneumoniae; the pathogen in the right photo is Gardnerella vaginalis.

[0056] The inhibition zones of the experimental strains against different pathogens are shown in Table 1 below.

[0057] Table 1 Inhibition zones of experimental strains against different pathogens (mm) It can be seen that the inhibition zone of Lactobacillus plantarum VM-2 against a variety of common vaginal pathogens is significantly larger than that of other strains, indicating that the inhibitory effect of VM-2 is significantly better than that of other control strains.

[0058] The experimental results of inhibiting Candida albicans are shown in Figure 8 . At its own concentration of 1×10 6 CFU / mL, the inhibition rates of Lactobacillus plantarum VM-2, Lactobacillus gasseri VL-3 and Lactobacillus crispatus VX-4 against Candida albicans were 92.19%, 88.47% and 71.6%, respectively. The inhibition rate of the experimental strains against Candida albicans was basically the same as that of the control strains, and slightly better than the positive control strain DM8909.

[0059] 2. Combination ratio of each bacterial species in the probiotic combination This embodiment provides a composition comprising the above three strains, and through the growth inhibition experiment of the mixed bacterial liquid of the three strains, the optimal combination ratio of the three is obtained: the live bacterial count of Lactobacillus plantarum VM-2, Lactobacillus gasseri VL-3 and Lactobacillus crispatus VX-4 is 1:10:100.

[0060] Mixed bacterial liquid growth inhibition experiment: The experimental strains to be mixed (Lactobacillus plantarum VM-2, Lactobacillus gasseri VL-3, Lactobacillus crispatus VX-4) were inoculated in MRS broth medium and cultured anaerobically at 37°C for 20-24h. The bacterial liquid concentration was adjusted to about 1×10 8 CFU / mL. The three bacterial solutions were diluted in different gradients, inoculated into 2 L MRS broth medium in different proportions, mixed, and cultured anaerobically at 37°C for 24 hours. The experimental ratio was 1:1 to 1:1000 for each two bacterial solutions. Every 2 hours, 120 μL of the mixed bacterial solution was taken, added to MRS broth medium for 10-fold gradient dilution, 500 μL was taken to spread on MRS plates, and the plates were counted after anaerobically cultured at 37°C for 20-24 hours. The colony counts of the three bacteria on the plates were recorded separately, and the results were recorded in CFU / mL (the total number of bacterial colonies contained in each mL of the original sample).

[0061] The experimental results are shown in Fig. 9. The experiment finally obtained the optimal starting mixing ratio of Lactobacillus plantarum, Lactobacillus gasseri and Lactobacillus crispatus of 1:10:100. Within 24 hours, as time went on, Lactobacillus plantarum VM-2 showed a growth advantage in the mixed bacterial solution, but it did not completely inhibit the growth of other lactobacilli. In the end, the number of live bacteria of Lactobacillus gasseri VL-3 accounted for about 10%, and the number of live bacteria of Lactobacillus crispatus VX-4 accounted for about 20%. In the combined preparation, the growth of the three strains was not completely inhibited by the component strains, and they can play their respective probiotic functions.

[0062] The information of the control strains selected for the above experiments is shown in Table 2 below.

[0063] Table 2 Information of the control strains selected for the experiment In summary, the present invention provides a probiotic combination and its application. The probiotic combination of the present invention comprises Lactobacillus plantarum VM-2, Lactobacillus gasseri VL-3 and Lactobacillus crispatus VX-4 ， The ratio of live bacteria is 1:10:100. All three strains have the function of preventing and assisting in the treatment of female reproductive tract infectious diseases. They all come from the human body and will not cause additional health burden on the host. The in vitro culture techniques are simple and universal, and the acquisition cost is low. When applied to the prevention and treatment of female reproductive tract infections, it will not cause excessive economic burden. Among them, VM-2 has strong growth and reproduction ability under different pH environmental conditions, showing high universality, and can play a role in different life stages, physiological cycles and health conditions of women. VM-2 can still show excellent acid production ability under insufficient nutritional conditions; VL-3 shows better hydrogen peroxide production ability; VX-4 is the main lactobacillus of the vaginal microbial community of healthy women of childbearing age in China, has good cell adhesion ability, and has a growth ability significantly better than other Lactobacillus crispatus. The present invention provides an excellent path for the prevention and treatment of female reproductive tract infections from a biological perspective.

[0064] The above-described embodiments only express several implementation methods of the present invention, which are convenient for understanding the technical solution of the present invention in detail, but cannot be understood as limiting the scope of protection of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the scope of protection of the present invention.

Claims

1. A probiotic for improving female reproductive tract infection, characterized in that: The probiotics include Lactobacillus plantarum ( Lactiplantibacillus plantarum )VM-2, Lactobacillus gasseri( Lactobacillus gasseri ) VL-3 and Lactobacillus crispatus ( Lactobacillus crispatus ) VX-4; wherein the preservation number of Lactobacillus plantarum VM-2 is CGMCC No.30810; the preservation number of Lactobacillus gasseri VL-3 is CGMCC No.32745; the preservation number of Lactobacillus crispatus VX-4 is CGMCC No.32746.

2. The probiotic for improving female reproductive tract infection according to claim 1, characterized in that: The probiotics include Lactobacillus plantarum VM-2, Lactobacillus gasseri VL-3 and Lactobacillus crispatus VX-4, and the ratio of the number of live bacteria among the three is (0.8-1.2):(8-12):(80-120), preferably 1:10:

100.

3. A culture or fermentation product, characterized in that The culture or fermentation product is obtained by expanding the probiotics according to claim 1 or 2.

4. A bacterial agent, characterized in that The bacterial agent includes the probiotics according to claim 1 or 2 or the culture or fermentation product according to claim 3.

5. The bacterial agent according to claim 4, characterized in that: The effective viable count of Lactobacillus plantarum VM-2 is not less than 1×10 6 CFU / mL; and / or, the effective viable count of Lactobacillus gasseri VL-3 is not less than 1×10 7 CFU / mL; and / or, the effective viable count of Lactobacillus crispatus VX-4 is not less than 1×10 8 CFU / mL.

6. Use of the probiotics according to claim 1 or 2, the culture or fermentation product according to claim 3, or the bacterial agent according to claim 4 or 5 in the preparation of a medicament for preventing and assisting in the treatment of female reproductive tract infectious diseases.

7. Use of the probiotics according to claim 1 or 2, or the culture or fermentation product according to claim 3, or the bacterial agent according to claim 4 or 5 in the prevention and auxiliary treatment of female reproductive tract infectious diseases.

8. A drug for preventing and assisting in the treatment of female reproductive tract infectious diseases, characterized in that: It contains the probiotics according to claim 1 or 2, or the culture or fermentation product according to claim 3, or the bacterial agent according to claim 4 or 5.

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