Composition containing lactobacillus strain and lactobacillus and use thereof

Abstract

The present invention relates to a composition, which contains lactobacillus and lactobacillus, wherein the number of viable lactobacillus in the composition is not less than 1×10 6 CFU, the composition contains 1×10 6 ~1×10 10 The composition of the present invention can be used to treat and / or prevent vaginal infections such as bacterial vaginosis and aerobic bacterial vaginitis, and can be used in the pharmaceutical field.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, in particular to a composition containing a lactobacillus strain and lactobacillus and use thereof. Background Art

[0002] In the vagina of healthy women of childbearing age, Lactobacillus is the dominant flora, accounting for over 70% of the vaginal flora. Studies have shown that Lactobacillus in the female reproductive tract primarily inhibits pathogenic microorganisms by producing lactic acid and antibacterial agents.

[0003] Among outpatients in obstetrics and gynecology clinics in my country, reproductive tract infections (RTIs) account for 40.2%-55.6%. At least 200 million patients suffer from RTI-related illnesses, including 100 million with recurrent illnesses, resulting in medical costs exceeding 20 billion yuan annually. Surveys show that 99.3% of patients with symptomatic vaginal infections in gynecological clinics experience an imbalance in the vaginal microbiome. In 2016, the Infection Collaboration Group of the Chinese Medical Association's Obstetrics and Gynecology Branch proposed that the root cause of vaginal infection is an imbalance in the vaginal microbiome. Chronic abnormalities in the vaginal microbiome reduce the vagina's resistance to pathogenic microorganisms, often leading to recurrent vaginal infections or the development of new secondary infections.

[0004] Common vaginal infections include bacterial vaginosis (BV), vulvovaginal candidiasis (VVC), Trichomonas vaginitis (TV), and aerobic vaginitis (AV), all of which are associated with a decrease or disappearance of Lactobacilli. Conventional treatment for vaginal infections focuses on antimicrobial therapy. Antibiotics, such as clindamycin, suppress BV pathogens while also inhibiting the growth of Lactobacilli within the vagina. Although metronidazole has been reported to not inhibit Lactobacilli growth at doses administered, it also fails to promote Lactobacilli recovery. Furthermore, relapse rates and drug resistance are high after antibiotic use. Antibiotics only inhibit planktonic Gardnerella vaginalis, providing temporary symptom control but are unable to completely eliminate biofilms and the G. vaginalis within them. Once treatment is discontinued, the bacteria within the biofilms will reactivate, multiply, and spread, leading to BV recurrence (Sui Long et al. Study on the Role of Gardnerella Biofilm in Recurrent Bacterial Vaginosis [C] / / Chinese Women's Reproductive Tract Infections Summit, 2014). A multicenter, prospective, open-label, controlled trial showed that even extended antibiotic treatment failed to reduce recurrences and instead increased the incidence of VVC and other adverse reactions (Sobel, Jack D, Ferris, Daron, Schwebke, Jane, et al. Am. J. Obstet. Gynecol., 2006, 194:1283-9). A study by Sui Long et al. (2014) showed a metronidazole resistance rate of 63.8% and a clindamycin resistance rate of 24.1% (compared to 67% in international studies). Biofilms reduce bacterial susceptibility to antimicrobial drugs and enhance antimicrobial resistance. Resistance can be acquired by pathogenic strains through mobile genetic elements, thus diminishing the effectiveness of antibiotics or even rendering them ineffective.

[0005] In 2016, the Infection Collaborative Group of the Chinese Medical Association's Obstetrics and Gynecology Branch proposed that the essence of vaginal infection is "an imbalance in the vaginal microecology." They advocated shifting the goal of vaginal infection treatment from "symptomatic treatment" to "restoring vaginal microecological balance," and from simply "killing pathogens" to a "fighting pathogens—repairing the mucosa—restoring lactobacilli" treatment model. Vaginal lactobacilli can interfere with the formation of vaginal Gardnerella biofilms. The use of vaginal microecological preparations can restore a weakly acidic environment dominated by functional lactobacilli, promote vaginal microecological balance and immune regulation, and reduce recurrence of vaginal infections. However, exogenously added lactobacilli are administered in a freeze-dried, dormant state and require recovery, reproduction, and colonization in the vagina to exert their antibacterial effects. The amount of antibacterial agents and lactic acid produced by vaginal freeze-dried lactobacilli does not achieve optimal antibacterial efficacy, making it difficult to cure vaginal infections.

[0006] Clinical research on the current use of probiotics to treat bacterial vaginosis by the School of Pharmacy of Shanghai Jiao Tong University showed that in the past 10 years, 4 of the 5 experiments showed that probiotics were effective in reducing BV recurrence, and only 1 experiment showed that Lactobacillus had a therapeutic effect on BV. In the 8 clinical trials of probiotics combined with antibiotics for the treatment of BV, the results of 2 experiments showed that adding probiotics to traditional antibiotic treatment had no significant therapeutic effect, 5 experiments showed that adding probiotics to traditional antibiotic treatment had a significant effect on improving the cure rate of BV, and 1 experiment showed that whether there was a significant difference was related to the selected strain (Qian Zhixiang, Chen Daijie. Clinical research on probiotics for the treatment of bacterial vaginosis and its related mechanisms [J]. Chinese Journal of Antibiotics, 2020, 45(10): 974-981. DOI: 10.13461 / j.cnki.cja.007024.).

[0007] Currently available vaginal probiotic preparations, such as Dingjunsheng, fall far short of meeting clinical needs, and there is significant room for development. Combining antibiotics with vaginal probiotics has some benefits for BV, but this also carries with it the potential side effects of antibiotics. Therefore, developing Lactobacillus strains with enhanced probiotic potential and alternative treatments to antibiotics for the prevention and / or treatment of vaginal infections would improve cure rates and possess significant clinical value. Summary of the Invention

[0008] The present invention aims to provide a composition containing a lactobacillus strain and lactobacillus, which can treat and / or prevent vaginal and urinary tract infections and improve vaginal health.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] Provided is a composition containing lactobacillus strains and lactobacillus, wherein common dominant vaginal lactobacillus and / or other lactobacillus with strong growth ability and lactic acid production ability are isolated from the secretions of healthy women of childbearing age, and lactobacillus resistant to lactobacillus is selected through a stress growth test under high concentration of lactobacillus.

[0011] The inventors discovered that lactobacillus alone cannot quickly colonize and exert its antibacterial effect in the pathological environment of microecological imbalance to treat vaginal infections; lactobacillus can quickly lower the pH value of the vagina, directly inhibit pathogenic bacteria, and provide a favorable growth and reproduction environment for exogenously supplemented lactobacillus. The combination of lactobacillus and lactobacillus can maximize the synergistic antibacterial effect, which is beneficial to improving the efficiency of treating vaginal infections.

[0012] Furthermore, the viable count of Lactobacillus is not less than 1×10 6 CFU; further, the number of viable lactobacilli does not exceed 1×10 10The average vaginal secretion of a healthy woman of childbearing age is about 1~3 mL, and the number of live Lactobacillus bacteria is about 10 7 ~10 8 CFU / mL, generally no more than 1×10 10 CFU / mL. As a live bacterial preparation, it will colonize and grow and multiply after application, maintaining a certain level.

[0013] In the composition, the number of viable lactobacilli may be 1×10 6 ~1×10 8 CFU, 1×10 8 ~1×10 10 CFU, etc., wherein the number of viable lactobacilli can be any value within the range, including the endpoint values ​​at both ends, for example, the number of viable lactobacilli can be 1×10 6 CFU, 1×10 7 CFU, 1×10 8 CFU, 1×10 9 CFU, 1×10 10 CFU, etc.

[0014] Further, the composition comprises at least 20 mg of lactobacillus;

[0015] Furthermore, the composition comprises at least 20 to 200 mg of lactobacillus;

[0016] Furthermore, the composition comprises at least 40 to 150 mg of lactobacillus;

[0017] Furthermore, the composition contains at least 40-100 mg of lactobacillus.

[0018] In the composition, the content of lactobacillus may be 20-40 mg, 40-60 mg, 60-80 mg, 80-100 mg, 100-120 mg, 120-150 mg, 150-200 mg, 200-250 mg, etc., wherein the content of the lactobacillus may be any value in the range, including the endpoint values ​​at both ends. For example, in the composition, the lactobacillus may be 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 150 mg, 170 mg, 200 mg, 220 mg, 250 mg, 300 mg, etc.

[0019] In the present invention, the composition comprises 1×10 6~ 1×10 10The lactobacillus has basic probiotic functions and reproductive capacity and can tolerate an effective dose of lactobacillus, that is, the lactobacillus and the lactobacillus composition containing 40-100 mg of lactobacillus can still maintain the survival of the lactobacillus when inoculated and cultured.

[0020] Furthermore, the Lactobacillus plantarum strain is active, inactivated, inhibited, genetically modified or killed.

[0021] The lactobacillus of the present invention includes but is not limited to at least one of the following isolated strains or variants thereof: Lactobacillus plantarum ( Lactiplantibacillus plantarum )1-D1, the deposit number is: CCTCC NO: M 20221191, Lactobacillus crispatus ( Lactobacillus crispatus ) 51S-H2, the deposit number is: CCTCC NO: M 20221194, Lactobacillus gasseri ( Lactobacillus paragasseri )16-B12, the deposit number is: CCTCC NO: M 20221193, Lactobacillus jensenii ( Lactobacillus jensenii ) 53-D2, deposit number: CCTCC NO: M 20221192. For example, the lactobacillus of the present invention may include the following combinations:

[0022] It can be Lactobacillus plantarum ( Lactiplantibacillus plantarum )1-D1, Lactobacillus crispatus 51S-H2, Lactobacillus gasseri ( Lactobacillus paragasseri ) 16-B12, Lactobacillus jensenii ( Lactobacillus jensenii ) Any strain of 53-D2;

[0023] or Lactobacillus plantarum ( Lactiplantibacillus plantarum )1-D1, Lactobacillus crispatus ( Lactobacillus crispatus ) 51S-H2, Lactobacillus gasseri ( Lactobacillus paragasseri ) 16-B12, Lactobacillus jensenii ( Lactobacillus jensenii ) Any two strains of 53-D2, such as, Lactobacillus plantarum 1-D1 and Lactobacillus gasseri 16-B12, Lactobacillus plantarum 1-D1 and Lactobacillus jensenii 53-D2, Lactobacillus plantarum 1-D1 and Lactobacillus crispatus 51S-H2, Lactobacillus gasseri 16-B12 and Lactobacillus jensenii 53-D2, Lactobacillus gasseri 16-B12 and Lactobacillus crispatus 51S-H2, Lactobacillus crispatus 51S-H2 and Lactobacillus jensenii 53-D2.

[0024] or Lactobacillus plantarum ( Lactiplantibacillus plantarum )1-D1, Lactobacillus crispatus ( Lactobacillus crispatus ) 51S-H2, Lactobacillus gasseri ( Lactobacillus paragasseri) 16-B12, Lactobacillus jensenii ( Lactobacillus jensenii ) Any three strains of 53-D2, for example, can be Lactobacillus plantarum 1-D1, Lactobacillus gasseri 16-B12 and Lactobacillus jensenii 53-D2, can be Lactobacillus plantarum 1-D1, Lactobacillus gasseri 16-B12 and Lactobacillus crispatus 51S-H2, can be Lactobacillus gasseri 16-B12, Lactobacillus jensenii 53-D2 and Lactobacillus crispatus 51S-H2;

[0025] or Lactobacillus plantarum ( Lactiplantibacillus plantarum )1-D1, Lactobacillus crispatus ( Lactobacillus crispatus ) 51S-H2, Lactobacillus gasseri ( Lactobacillus paragasseri ) 16-B12, Lactobacillus jensenii ( Lactobacillus jensenii ) Lactobacillus combination composed of 53-D2.

[0026] Provided is a use of the aforementioned composition in preparing a product for treating and / or preventing infections in the vagina and urinary tract.

[0027] Furthermore, the vaginal infection includes but is not limited to bacterial vaginosis, yeast vaginitis, Trichomonas vaginitis, aerobic vaginitis, viral vaginitis, especially bacterial vaginosis and aerobic bacterial vaginitis;

[0028] The infections in the vagina also include sexually transmitted diseases such as HIV and chlamydia, infections that endanger the fetus in pregnant women, premature births, and the like.

[0029] Furthermore, the bacterial vaginosis is mainly caused by Gardnerella vaginalis; the aerobic vaginitis is caused by pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteriae, Gardnerella or Salmonella.

[0030] The term "treatment" (also referred to as "treat" or "treating") refers to any administration of a therapeutic agent according to a therapeutic regimen that achieves a desired effect, i.e., partially or completely alleviates, ameliorates, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition (e.g., improving vaginal flora structure and function, increasing vaginal flora diversity, reducing the abundance of inflammation-associated flora, treating vaginitis); in some embodiments, the administration of a therapeutic agent according to a therapeutic regimen is associated with the achievement of a desired effect. Such treatment may be directed to subjects who do not exhibit the relevant disease, disorder, and / or condition and / or to subjects who only exhibit early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be directed to subjects who exhibit one or more established signs of the relevant disease, disorder, and / or condition. In some embodiments, treatment may be directed to subjects who have been diagnosed with the relevant disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically associated with an increased risk of developing the relevant disease, disorder, and / or condition.

[0031] The product is a drug.

[0032] Furthermore, the composition further comprises a pharmaceutically acceptable carrier; the “pharmaceutically acceptable carrier” includes but is not limited to physiologically acceptable excipients and diluents.

[0033] Furthermore, the composition is administered in the form of tablets, suckable tablets, capsules, enteric-coated tablets and capsules, suppositories, mini-enemas, vaginal tablets, vaginal gelatin capsules, vaginal lozenges, creams, gels, ointments, lotions, melting strips, vaginal suppositories, and sprays.

[0034] Lactobacillus is a natural antibacterial agent and a raw material drug with national standards. It is widely used and has similar effects to antibiotics, but with a different mechanism of action. It is specific and does not produce drug resistance or toxicity at all. However, it is currently mainly used orally and has not been used for vaginal administration and / or for vaginal infections.

[0035] Furthermore, the composition includes but is not limited to oral, vaginal, rectal administration, and instillation into the bladder, and can also be a combination of two or more administration methods for therapeutic administration.

[0036] A method for treating and / or preventing an inflammatory response is provided, comprising the step of administering to a subject in need thereof a pharmaceutically effective amount of a composition as defined above.

[0037] A method for treating and / or preventing vaginitis is provided, comprising the step of administering to a subject in need thereof a pharmaceutically effective amount of a composition as defined above.

[0038] A kit for treating and / or preventing inflammatory reactions is provided, which comprises at least a container containing the composition as described above.

[0039] A kit for treating and / or preventing vaginitis is provided, which comprises at least a container containing the above composition.

[0040] As used herein, the term "comprises" and variations of the term are not intended to exclude other additives, components or steps.

[0041] Beneficial effects:

[0042] (1) The lactobacillus and lactobacillus composition provided by the present invention has a synergistic antibacterial effect that is superior to that of using lactobacillus alone, and can achieve the effect of rapidly inhibiting pathogenic bacteria.

[0043] (2) Lactobacillus alone cannot colonize quickly in a pathological environment with an unbalanced microecology and thus exert its antibacterial effect in treating vaginal infections. Lactobacillus can quickly lower the pH value of the vagina, directly inhibit pathogenic bacteria, and provide a favorable growth and reproduction environment for exogenously supplemented Lactobacillus. This maximizes the antibacterial synergistic effect of Lactobacillus and Lactobacillus, which is beneficial to improving the efficacy of treating vaginal infections.

[0044] (3) The application of lactobacilli is also beneficial to promote the growth of common dominant lactobacilli in the patient's vaginal background and promote their proliferation to restore to a healthy microecological state.

[0045] (4) Exogenously supplemented Lactobacillus can quickly replenish Lactobacillus for patients who are deficient in Lactobacillus and maintain vaginal health.

[0046] (5) The compositions provided by the present invention complement each other and cannot achieve the therapeutic purpose of inhibiting pathogenic bacteria and restoring vaginal microecology when used alone.

[0047] (6) The composition provided by the present invention has good safety and no drug resistance, and is expected to replace antibiotic therapy for the prevention and / or treatment of vaginal infections, with the potential to improve cure rates and recurrence rates, and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Vaginal Lactobacillus colonization score of SD rats. DETAILED DESCRIPTION

[0049] In order to enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below with reference to specific examples. It should be understood by those skilled in the art that this should not be construed as limiting the scope of the claims of the present invention. Unless otherwise defined, the technical and scientific terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. It should also be noted that the reagents or instruments in the present invention, unless otherwise specified, are all conventional biochemical reagents or instruments and can be purchased commercially.

[0050] Strain deposit information:

[0051] Lactobacillus plantarum ( Lactiplantibacillus plantarum ) 1-D1 was deposited in China Center for Type Culture Collection on July 27, 2022. The deposit number of the strain is: CCTCC NO: M 20221191. Address: Wuhan, China;

[0052] Lactobacillus crispatus ( Lactobacillus crispatus ) 51S-H2, deposited in China Center for Type Culture Collection on July 27, 2022, with the deposit number: CCTCC NO: M 20221194, address: Wuhan, China;

[0053] Lactobacillus gasseri ( Lactobacillus paragasseri ) 16-B12, deposited in China Center for Type Culture Collection on July 27, 2022, with the deposit number: CCTCC NO: M 20221193, address: Wuhan, China;

[0054] Lactobacillus jensenii ( Lactobacillus jensenii ) 53-D2, deposited in China Center for Type Culture Collection on July 27, 2022, with the deposit number: CCTCC NO: M 20221192, address: Wuhan, China;

[0055] Lactobacillus (Xingcheng Tiger Pharmaceutical Co., Ltd., batch number: 210226).

[0056] The composition of the present invention comprises 1×10 6 ~1×10 10 CFU lactobacillus and 40-100 mg lactobacillus, that is, the composition has probiotic function and reproduction ability, and the number of viable lactobacillus that can tolerate an effective dose of 40-100 mg lactobacillus is not less than 1×10 10 CFU / g.

[0057] The present invention uses Lactobacillus delbrueckii (hereinafter referred to as DJS-H3), isolated from the commercially available product Dingjunsheng (Inner Mongolia Shuangqi Pharmaceutical Co., Ltd., National Medicine Approval No. S20030005, Batch No. 202110110), as a positive control at each stage. Through low-pH medium screening, growth amplification, and calcium-solubility zone screening, lactobacilli with low pH tolerance, strong reproductive capacity, and high lactic acid production are rapidly isolated. HeLa cell adhesion and antibacterial tests are then used to screen for dominant lactobacilli with strong colonization and pathogenic bacteria growth inhibition abilities. Finally, the efficacy of the dominant lactobacilli and lactobacillus combination is evaluated. Representative examples are as follows:

[0058] DMEM medium: Gibco; cat:11995065

[0059] MRS liquid culture medium: Huankai Biotechnology, cat:1110151

[0060] 2% calcium carbonate-0.8% MRS: Shenggong, H122BA0030

[0061] Agar: Sangon Biotech, A505255-0250

[0062] HeLa cells: Beina Biotechnology, Catalog Number: BNCC342189

[0063] VK2 / E6E7 cells: Qingqi (Shanghai) Biotechnology

[0064] Example 1 Isolation of Lactobacillus

[0065] Vaginal secretions from healthy female volunteers of childbearing age were examined by Gram staining and Nugent scoring. Twenty-nine healthy female volunteers were enrolled. Vaginal swabs were placed in MRS acidic (pH 5.0) liquid medium and cultured overnight at 37°C. The culture solution was diluted 10-fold and plated onto 2% calcium carbonate-0.8% MRS agar (Sangong Biotechnology, H122BA0030) (Agar from Sangon Biotechnology, A505255-0250) at 37°C for 36-48 hours. Single colonies with distinct clear zones on the calcium carbonate-MRS medium were picked and transferred to MRS liquid medium for overnight culture at 37°C. 1816 strains were amplified by PCR and sent for 16S rRNA sequencing. After eliminating acid-producing non-lactobacilli, 72 Lactobacilli from different samples were selected for subsequent screening.

[0066] Among the isolated strains, the nucleotide sequences (16S rRNA sequences) of the four strains of Lactobacillus plantarum 1-D1, 16-B12, 53-D2, and 51S-H2 are shown in SEQ ID No. 1 to 4, respectively.

[0067] Example 2 Lactobacillus growth performance determination

[0068] Growth curve data from the Lactobacillus isolation phase indicate that most Lactobacillus enter the late logarithmic phase after 12 hours. Therefore, viable counts of each strain were determined using the agar pour method 12 hours after inoculation. At the same inoculation time, strains with higher viable counts exhibited significantly superior growth performance. The strains with the best growth advantage, along with previously isolated, commonly dominant vaginal bacteria with relatively excellent growth capabilities, were selected for further study.

[0069] The growth curve data of the lactobacillus isolation stage showed that most lactobacilli entered the late logarithmic phase at 12 h. Therefore, the viable count of each strain was determined by the agar pouring method at 12 h of inoculation. At the same inoculation time, the strains with high viable counts have a relatively obvious advantage in growth performance. In this example, the top 25 strains with growth advantages were selected to enter the 12-h viable count test. The data showed that among the 25 plant lactobacilli, plant lactobacillus 1-D1 had excellent growth performance and strong reproductive ability, and the 12-h viable count could reach 6.37×10 9 CFU / mL, which is conducive to high-density fermentation during commercial transformation.

[0070] Four strains with the best growth advantage and common dominant bacteria in the vagina were selected for subsequent research. The information of these four strains and the number of viable bacteria at 12 hours are shown in Table 1.

[0071] Table 1 Information on dominant bacterial strains

[0072]

[0073] Example 3 Adhesion ability of Lactobacillus plantarum 1-D1

[0074] The colonization ability of lactobacilli was evaluated by the ability of lactobacilli to adhere to Hela and VK2 / E6E7 cells.

[0075] First, activate the Lactobacillus: take 50 μL of each Lactobacillus and inoculate it into 5 mL of MRS liquid medium. Incubate at 37°C for 18-24 hours, then remove it for transfer. Lactobacillus transfer: take 50 μL of each Lactobacillus activation solution and inoculate it into 5 mL of MRS liquid medium. Incubate it at 37°C for 18-24 hours, centrifuge it at 12000 rpm for 2 minutes, remove it, and resuspend it in DMEM medium (Gibco; cat:11995065) and adjust the turbidity to 0.5. Perform cell plating: take 500 μL of the revived cells and inoculate them at a concentration of 10 5 Cells were seeded into 24-well culture plates at a concentration of 10 μg / mL and cultured overnight in a CO2 incubator at 37°C and 5% CO2. The DMEM stock solution was discarded, and the cells were washed three times with PBS before adding 500 μL of DMEM medium. 8 Lactobacilli cultured overnight (CFU / mL) and resuspended in DMEM were added to the 24-well culture plate containing the cells described above. The plate was incubated at 37°C, 5% CO2 for 2 and 4 hours, respectively. The plate was then washed four times with PBS to remove any unadhered lactobacilli. Digestion was then performed with 200 μL of 0.25% trypsin for 2 minutes. Digestion was terminated by adding 600 μL of complete culture medium and the plate was mixed thoroughly by pipetting. The cell-bacteria suspension was serially diluted 10-fold with sterile PBS. 100 μL of each appropriate serial dilution was added to a disposable sterile plate. 0.8% MRS agar was then poured over the plate to mix thoroughly. Once solidified, the plate was inverted. The inverted plate was incubated at 37°C for 48 hours, then counted. Three replicates were performed for each group, and the average of the adhered lactobacilli was used as the final count. A greater number of lactobacilli indicates a stronger ability to adhere to cells.

[0076] The cell adhesion ability of Lactobacillus plantarum 1-D1 is shown in Tables 2 and 3. Fisher LSD mean comparison analysis revealed that the adhesion ability of Lactobacillus plantarum 1-D1 was significantly greater than that of Lactobacillus delbrueckii isolated from Dingjunsheng. The differences in adhesion counts between the groups were highly significant. The adhesion count of Lactobacillus plantarum 1-D1 on Hela cells over a 4-hour period was over 100 times that of Dingjunsheng (DJS-H3) and over 10 times that of Dingjunsheng on VK2 / E6E7 cells over a 4-hour period.

[0077] Table 2 Adhesion ability of Lactobacillus plantarum 1-D1 to Hela cells at different times

[0078]

[0079] Note: Different letters indicate significant differences, a: P≤0.01; b: P≤0.05.

[0080] Table 3 Adhesion ability of Lactobacillus plantarum 1-D1 to VK2 / E6E7 cells at different times

[0081]

[0082] Example 4 Effect of lactobacillus on the growth ability of Lactobacillus

[0083] The survival rates of different lactobacilli isolated from the vagina were explored under the stress of 250 mg high-dose lactobacillus (in 5 mL system).

[0084] Each lactobacillus isolated in Example 1 was inoculated at a 1% inoculum into MRS broth containing 250 mg of lactobacillus (250 mg of lactobacillus + 4.75 mL of MRS broth). The culture was shaken at 37°C for 12 hours and then removed. The co-culture liquid was diluted 10-fold in a series. 100 μl of the appropriate dilution was added to a disposable sterile plate and poured onto the agar plate for counting. Three replicates were prepared for each group. After solidification, the cells were incubated inverted at 37°C for 36-48 hours. The cells were removed and counted. The survival rate of lactobacilli at high lactobacillus concentrations was investigated. Simultaneously, the cells were inoculated into MRS broth without lactobacillus, and all procedures were repeated as above. This served as a control. The survival rate of each lactobacillus is detailed in Table 4.

[0085] The experimental results showed that under the stress of high concentration of lactobacillus, all lactobacilli still grew and survived, and the common dominant vaginal bacteria (Lactobacillus gasseri, Lactobacillus crispatus, and Lactobacillus jensenii) showed higher growth rates than those in the control group without lactobacillus, indicating that lactobacillus did not affect the growth of different lactobacilli, but could promote the growth of common dominant vaginal bacteria.

[0086] Table 4 Survival rate of each lactobacillus

[0087]

[0088] Example 5 Antibacterial ability of different lactobacilli and different concentrations of lactobacilli

[0089] Antibacterial ability of different lactobacilli: An antibacterial test is used to evaluate the ability of Lactobacillus plantarum to inhibit different pathogens. Mix 0.1 ml of lactobacilli with 0.8% MRS agar medium, and then make a 10 mm lactobacillus cake. Different pathogenic bacteria (Gardnerella vaginalis, Staphylococcus aureus, etc.) are activated and transferred using culture media and growth conditions suitable for their growth, and then mixed with the corresponding semi-solid culture medium to make pathogenic bacteria plates. Gently place the lactobacillus cake on the surface of the pathogenic bacteria plate, incubate it upright at 37°C for 18-24 hours, then take it out and use a vernier caliper to measure the size of the inhibition zone to evaluate the ability of each lactobacillus to inhibit pathogens. The specific steps are as follows:

[0090] After activating Lactobacillus plantarum and Lactobacillus delbrueckii DJS-H3 respectively, 0.1 mL of bacterial solution was mixed with MRS solid medium and poured into a 6 cm plate. After complete solidification, it was cultured at 37°C for 48 h. The plate was removed and holes were punched on the agar medium with a puncher with an inner diameter of 10 mm to obtain lactobacillus cakes for later use.

[0091] Gardnerella vaginalis was inoculated into 5% horse serum-anaerobic BHI liquid medium. After activation and transfer, a 10-fold dilution gradient was performed with anaerobic and sterile 0.9% anaerobic saline. 1 mL of the dilution was mixed with 10 mL of anaerobic BHI agar containing 5% horse serum, and poured into a 9 cm plate. After it completely solidified, it was set aside.

[0092] Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella Paratyphi B, and Shigella dysenteriae were inoculated into nutrient broth respectively. After activation and transfer, the pathogenic bacteria liquid was diluted to 100 times with 0.9% saline. 1 mL of the dilution was mixed with 10 mL of nutrient agar semi-solid medium, poured into a 9 cm plate, and allowed to solidify completely for later use.

[0093] Gently place a Lactobacillus cake on the surface of each pathogenic bacteria plate, placing four cakes symmetrically per plate, with three plates per strain in parallel. Place the Gardnerella vaginalis plate upright in an anaerobic sealed box containing an anaerobic gas production bag and incubate at 37°C for 48 hours. Place the other pathogenic bacteria plates upright in an incubator and incubate at 37°C for 24 hours. Measure the size of the inhibition zone on each plate with a vernier caliper. The antibacterial activity of different Lactobacilli is shown in Table 4.

[0094] Antibacterial activity of different lactobacilli: Weigh 0.8 g of lactobacilli and add 1 mL of normal saline, vortex and mix to a concentration of 0.8 g / mL. Serially dilute by twofold.

[0095] 100 μl of vaginal Gardnerella was inoculated into 5% horse serum-BHI liquid, and 100 μl of the remaining pathogenic bacteria except vaginal Gardnerella was inoculated into nutrient broth. The vaginal Gardnerella was cultured at 37°C for 48 hours and then removed. The other strains were cultured at 37°C for 18-24 hours and then removed.

[0096] Mix the activated solutions of strains 16-B12, 53-D2, and 51S-H2 in a 1:1:1 ratio to create a mixed culture. Dilute each aerobic pathogen 100-fold, add 1 mL of the dilution to a disposable plate, pour over 20 mL of nutrient agar, and allow to completely solidify before punching. Dilute Gardnerella vaginalis 10-fold, add 1 mL of the dilution to a disposable plate, pour over 20 mL of 5% horse serum-BHI agar, and allow to completely solidify before punching.

[0097] Add 100 μl of lactobacillus solution of varying concentrations to each well. Set up three replicates per group. Incubate at 37°C for 18–24 hours, then remove the cells. Measure the size of the inhibition zone using a vernier caliper.

[0098] The results in Table 5 show that lactobacilli can inhibit aerobic bacteria that are resistant to metronidazole. Different lactobacilli and mixed bacteria exhibit comparable antibacterial activity against aerobic bacteria and Gardnerella vaginalis as pure lactobacilli. However, the inhibitory effect of pure lactobacilli is short-lived, unlike lactobacilli, which can achieve long-term antibacterial effects through proliferation and colonization.

[0099] Table 5 Antibacterial ability of different lactobacilli and different concentrations of lactobacilli (mm)

[0100]

[0101] Example 6 Antibacterial ability of different lactobacilli and lactobacillus compositions

[0102] The specific procedure was as follows: 1-D1 and Ding Junsheng's Lactobacillus delbrueckii were inoculated into MRS liquid medium at a 1% inoculum, cultured overnight at 37°C, and then removed for later use. Except for Gardnerella vaginalis, which was inoculated into 5% horse serum-BHI broth, all other pathogens were inoculated into nutrient broth.

[0103] Mix the activated solutions of strains 16-B12, 53-D2, and 51S-H2 in a 1:1:1 ratio to create a mixed culture. Prepare lactobacillus by centrifuging the supernatant from each lactobacillus strain. Prepare lactobacillus at a concentration of 0.8 g / mL and then dilute it two-fold to 0.4 g / mL and 0.2 g / mL.

[0104] Dilute each aerobic pathogen 100-fold. Add 1 mL of the dilution to a disposable plate. Pour 20 mL of nutrient agar over the plate. After complete solidification, punch a well. Add 100 μL of the bacteria + lactobacillus solution to each well. Set up three replicates per group. Incubate at 37°C for 18-24 hours, then remove the wells. Measure the size of the inhibition zone.

[0105] Dilute the Gardnerella vaginalis culture 10-fold. Add 1 mL of the dilution to a 90 mm disposable plate. Pour 5% horse serum-BHI agar. After complete solidification, punch a well. Add 100 μl of the bacteria plus lactobacillus solution to each well. Set up three replicates for each group. Incubate at 37°C for 48 hours, then remove the plate. Measure the size of the inhibition zone using a vernier caliper.

[0106] The results showed that the antibacterial ability of the combination of different lactobacilli and lactobacilli was better than that of the lactobacilli alone in Example 5 in terms of inhibiting aerobic bacteria and Gardnerella vaginalis, and the antibacterial ability of the combination was dose-dependent on lactobacilli.

[0107] Table 6 Antibacterial ability of different lactobacilli and lactobacillus compositions (mm)

[0108]

[0109] The results in Table 6 show that the comprehensive antibacterial ability of the combination of Lactobacillus plantarum 1-D1 and lactobacillus is significantly better than that of single bacteria and / or lactobacillus, and the comprehensive antibacterial ability of the combination of Lactobacillus plantarum 1-D1 and lactobacillus is better than that of mixed bacteria of other Lactobacillus plantarum.

[0110] Example 7 Colonization test of a composition containing lactobacillus and lactobacillus in rat vagina

[0111] SD rats were vaginally administered Lactobacillus plantarum 1-D1 and / or lactobacillus once daily for 5 consecutive days, followed by 10 days of observation. Vaginal secretions were collected and Gram-stained before each administration (D1 secretions were collected before the initial administration) to assess Lactobacillus plantarum 1-D1 colonization and the effects of lactobacillus on this colonization. The experimental plan is shown in Table 7 below.

[0112] Gram staining microscopy showed that before the initial administration, there were no microorganisms in the vaginal secretions of most rats, and Gram-negative rods and Gram-positive cocci were occasionally found in the vaginal secretions of a very small number of rats. After continuous administration, compared with the blank control group, the number of Gram-positive rods in the vaginal secretions of rats given Lactobacillus plantarum 1-D1 was significantly increased compared with before administration and the blank control group, indicating that Lactobacillus plantarum 1-D1 can successfully colonize in the vagina of SD rats. Compared with the Lactobacillus plantarum 1-D1 group, there was no significant difference in the number of lactobacilli in the vaginal secretions of rats in the low-dose group and the high-dose group of the composition, indicating that lactobacillus has no effect on the colonization of Lactobacillus plantarum 1-D1 and does not affect its survival. Five days after stopping the administration (D10), Gram-positive rods were still visible in the field of vision, and 10 days after stopping the administration (D15), the vaginal microenvironment of the rats returned to the state before administration (experimental results are shown in Figure 2). Figure 1 shown).

[0113] Table 7 Experimental plan for colonization in rat vagina

[0114]

[0115] Table 8 Scoring criteria for vaginal Lactobacillus colonization in SD rats

[0116]

[0117] Note: A score of <0 indicates successful Lactobacillus colonization; a score of ≥0 indicates a failure of Lactobacillus colonization.

[0118] Those skilled in the art will appreciate that the embodiments of the present invention described above and shown in the accompanying drawings are intended to be illustrative only and are not intended to limit the present invention. The objectives of the present invention have been fully and effectively achieved. The functional and structural principles of the present invention have been demonstrated and illustrated in the embodiments. Any variations or modifications may be made to the embodiments of the present invention without departing from the principles described.

Claims

1. A composition of lactobacillus and lactobacillus, characterized in that The viable lactobacillus count is not less than 1×106 CFU; The composition contains no less than 20 mg of lactobacillus, which is sourced from Xingcheng Tiger Pharmaceutical Co., Ltd., batch number: 210226; The lactobacillus is selected from one of the following lactobacilli: (1) Lactobacillus plantarum ( Lactiplantibacillusplantarum )1-D1, the deposit number is: CCTCC NO: M20221191; (2) Lactobacillus crispatus ( Lactobacillus crispatus )51S-H2, the deposit number is: CCTCC NO: M20221194; (3) Lactobacillus gasseri ( Lactobacillus paragasseri )16-B12, the deposit number is: CCTCC NO: M20221193; (4) Lactobacillus jensenii ( Lactobacillus jensenii ) 53-D2, deposit number: CCTCC NO: M20221192; and (5) Lactobacillus crispatus ( Lactobacillus crispatus ) 51S-H2, deposit number: CCTCC NO: M20221194, Lactobacillus gasseri ( Lactobacillus paragasseri )16-B12, the deposit number is: CCTCC NO: M20221193, Lactobacillus jensenii ( Lactobacillus jensenii )53-D2, the deposit number is: CCTCC NO: M20221192.

2. The composition according to claim 1, characterized in that The number of viable lactobacilli is 1×10 6 ~1×10 10 CFU; The composition contains 20-150 mg of lactobacillus.

3. The composition according to claim 2, characterized in that The composition contains 40-100 mg of lactobacillus.

4. Use of the composition according to any one of claims 1 to 3 in the preparation of a medicament for treating and / or preventing vaginal infection; characterized in that: The vaginal infection is bacterial vaginosis or aerobic bacterial vaginitis; The bacterial vaginosis is caused by Gardnerella vaginalis; the aerobic bacterial vaginitis is caused by Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteriae or Salmonella paratyphi B.

5. The use according to claim 4, characterized in that The composition further comprises a pharmaceutically acceptable carrier; the composition is administered in the form of a vaginal tablet, a vaginal gelatin capsule, a vaginal lozenge or a vaginal suppository.

6. Use of the composition according to any one of claims 1 to 3 in preparing a kit for treating and / or preventing vaginal infection, characterized in that: The kit comprises at least a container containing the composition according to any one of claims 1 to 3, wherein the vaginal infection is bacterial vaginosis or aerobic vaginitis; The bacterial vaginosis is caused by Gardnerella vaginalis; the aerobic bacterial vaginitis is caused by Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteriae or Salmonella paratyphi B.

Images