WO2024032452A1 - 一株植物乳杆菌及其应用 - Google Patents

一株植物乳杆菌及其应用 Download PDF

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WO2024032452A1
WO2024032452A1 PCT/CN2023/110924 CN2023110924W WO2024032452A1 WO 2024032452 A1 WO2024032452 A1 WO 2024032452A1 CN 2023110924 W CN2023110924 W CN 2023110924W WO 2024032452 A1 WO2024032452 A1 WO 2024032452A1
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lactobacillus plantarum
plantarum
product
vaginal
lactobacillus
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PCT/CN2023/110924
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English (en)
French (fr)
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黄浩喜
崔阳文
刘佳丽
刘武
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成都倍特生物制药有限公司
成都倍特药业股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/02—Drugs for genital or sexual disorders; Contraceptives for disorders of the vagina
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/02—Local antiseptics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/25—Lactobacillus plantarum

Definitions

  • the invention belongs to the field of microbial technology, and in particular relates to a strain of Lactobacillus plantarum and its application.
  • Lactobacillus In the vagina of healthy women of childbearing age, Lactobacillus is the dominant flora, accounting for more than 70% of the vaginal flora. Studies have shown that Lactobacilli in the female reproductive tract mainly inhibit pathogenic microorganisms by producing lactic acid and bacteriostatin.
  • Reproductive tract infections account for 40.2%-55.6% of obstetrics and gynecology outpatients in my country. At least 200 million people suffer from diseases related to reproductive tract infections, of which 100 million are recurrent patients. The resulting medical expenses are as high as more than 20 billion yuan every year. The survey shows that 99.3% of symptomatic patients with vaginal infection in gynecological outpatient clinics have vaginal microecological imbalance.
  • vaginal microecological imbalance the essence of vaginal infection is "vaginal microecological imbalance.” If the vaginal microecology is in an abnormal state for a long time, the vaginal resistance to pathogenic microorganisms is reduced, which is often an important reason for the recurrence of vaginal infections or secondary new infections.
  • vaginal infections include: bacterial vaginosis (BV), vulvovaginal candidiasis (VVC), trichomonas vaginitis (TV), aerobic vaginitis (AV), etc., all of which are accompanied by Lactobacillus decrease or disappear.
  • Conventional vaginal infection treatment options are mainly "antibacterial". After treatment with antibiotics such as clindamycin, bacterial vaginal pathogenic bacteria are inhibited and the growth of lactobacilli in the vagina is inhibited.
  • antibiotics such as clindamycin
  • metronidazole is administered at a certain dose It will not inhibit the growth of Lactobacilli, but it will not promote the recovery of Lactobacilli. Studies have shown that the metronidazole resistance rate is 63.8% and the clindamycin resistance rate is 24.1% to 67%.
  • the recurrence rate and drug resistance rate after using antibiotics are very high.
  • Antibiotics can only inhibit the floating Gardnerella vaginalis and temporarily control symptoms, but cannot completely remove the biofilm and Gardnerella vaginalis within it. Once treatment is stopped, , the bacteria in the biofilm will revive, multiply, and spread again, leading to the recurrence of bacterial vaginosis.
  • Biofilms can reduce bacterial sensitivity to antimicrobial drugs and increase resistance to antimicrobial drugs, and resistance to pathogenic strains can be acquired through mobile genetic elements, thus causing antibiotics to become less and less effective or even ineffective. .
  • vaginal microecological preparations can be used to restore the weakly acidic environment dominated by functional lactobacilli, promote the balance of vaginal microecology and immune regulation, and reduce the recurrence of vaginal infections.
  • vaginal microecological preparations there are only two types of vaginal microecological preparations on the market in China.
  • One is a commercially available product named "Yanhua” produced by Xi'an Zhenghao Biopharmaceutical Co., Ltd., which contains Streptococcus faecalis. It is not the dominant bacterial species in the vagina, and the bacteria under this species are conditionally pathogenic.
  • the other is a commercially available product named "Wanze Shuangqi” produced by Inner Mongolia Shuangqi Pharmaceutical Co., Ltd., which contains 1 species of Lactobacillus - Lactobacillus delbrueckii.
  • vaginal microecological preparations currently on the market are far from meeting clinical needs, and there is still a lot of room for optimization. Therefore, if Lactobacillus strains with stronger probiotic capabilities can be developed and used to prevent and/or treat vaginal infections, it will have great application value.
  • the object of the present invention is to provide a strain of Lactobacillus plantarum (Lactiplantibacillus plantarum) 1-D1, which strain can inhibit the growth of pathogenic bacteria in the vagina, regulate the balance of vaginal microecology, has the effect of preventing and treating vaginitis, and can be used for preparations to prevent and treat vaginitis. in medicines, food, and hygiene products.
  • the present invention provides the following technical solutions:
  • a strain of Lactobacillus plantarum (Lactiplantibacillus plantarum) 1-D1 the deposit number of this strain is: CCTCC NO: M 20221191.
  • Lactobacillus plantarum (Lactiplantibacillus plantarum) 1-D1 provided by the invention in the preparation of antibacterial products;
  • antibacterial products include but are not limited to bacterial inhibitors, feed additives, antimicrobial peptides, etc.
  • the bacteriostatic product is a product that inhibits one or more of Gardnerella, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella, and Shigella dysentery.
  • the invention provides the application of Lactobacillus plantarum (Lactiplantibacillus plantarum) 1-D1 in the preparation of products for preventing and/or treating vaginal infectious diseases.
  • treatment refers to any administration of a therapeutic agent according to a therapeutic regimen that achieves the desired effect, i.e. Partially or completely alleviate, ameliorate, alleviate, inhibit, delay onset, reduce severity, and/or reduce one or more symptoms or characteristics of a particular disease, disorder, and/or condition (e.g., improve vaginal flora structure and function, improve vaginal flora diversity, reducing the abundance of inflammation-associated flora, treating the incidence of vaginitis); in some embodiments, administration of a therapeutic agent according to a therapeutic regimen is associated with the achievement of a desired effect.
  • Such treatment may be in subjects who are not exhibiting the relevant disease, disorder and/or condition and/or in subjects who are exhibiting only early signs of the disease, disorder and/or condition. Alternatively or additionally, such treatment may be directed to subjects exhibiting one or more identified signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be directed to a subject who has been diagnosed with a relevant disease, disorder, and/or condition. In some embodiments, treatment may be directed to subjects known to have one or more susceptibility factors that are statistically associated with an increased risk of development of the relevant disease, disorder, and/or condition.
  • the products include but are not limited to medicines, food, health products, hygiene products, etc.
  • the strains are active strains or inactivated strains.
  • drug covers drugs for use in both humans and animals in human and veterinary medicine, as well as drugs for incorporation into animal feed, such as livestock feed and/or pet food.
  • drug as used herein means any substance that provides therapeutic, prophylactic and/or beneficial effects. in this article
  • drug as used is not necessarily limited to substances requiring a Marketing Approval, but includes substances that can be used in cosmetics, nutraceuticals, food (including, for example, feeds and beverages), probiotic cultures and dietary supplements substance.
  • Lactobacillus plantarum (Lactiplantibacillus plantarum) 1-D1 of the present invention can be added as a beneficial ingredient in fermented foods, immunity-enhancing health foods, female personal care solid drinks and other foods, and can enhance human immunity, especially female reproductive tract immunity. effect.
  • the vaginal infectious disease is caused by one or more of bacterial vaginal pathogens, vulvovaginal Candida pathogens, trichomonas vaginitis, and aerobic vaginitis;
  • pathogenic bacteria of aerobic vaginitis include but are not limited to Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Shigella dysentery or Salmonella, etc.;
  • the bacterial vaginosis pathogen is Gardnerella vaginalis.
  • the product is a product that lowers vaginal pH.
  • the product is a product that is metabolized in the vaginal environment to produce H 2 O 2 , organic acids and/or bacteriocins.
  • the invention provides the application of Lactobacillus plantarum (Lactiplantibacillus plantarum) 1-D1 in the preparation of products that regulate the microecological balance of bacterial flora.
  • the invention provides the application of Lactobacillus plantarum (Lactiplantibacillus plantarum) 1-D1 in the production of sanitary products.
  • the present invention also protects the application of Lactobacillus plantarum (Lactiplantibacillus plantarum) 1-D1 as a probiotic.
  • the present invention also provides a product whose active ingredient contains Lactobacillus plantarum; the Lactobacillus plantarum is such as the aforementioned Lactobacillus plantarum (Lactiplantibacillus plantarum) 1-D1.
  • the products include but are not limited to food, health products, medicines, daily necessities, etc.;
  • the product is a product for preventing and/or treating vaginal infectious diseases.
  • the products of the present invention may also include pharmaceutically acceptable excipients or diluents.
  • Lactobacillus plantarum (Lactiplantibacillus plantarum) 1-D1 of the present invention has genetic stability, no pathogenic/virulence genes, good safety, and is effective against Gardnerella vaginalis, Staphylococcus aureus, and Pseudomonas aeruginosa. , Escherichia coli, Salmonella paratyphi B and Shigella dysenteriae all showed good inhibitory capabilities.
  • Lactiplantibacillus plantarum 1-D1 provided by the present invention has a strong ability to produce lactic acid, can reduce vaginal pH, maintain a healthy vaginal acidic environment, and improve the balance of vaginal flora. At the same time, it produces antibacterial substances, such as Hydrogen peroxide, bacteriocins, organic acids, etc. can inhibit the growth and reproduction of pathogenic bacteria, improve mucosal immunity, and enhance the host's anti-infection effect.
  • Lactiplantibacillus plantarum 1-D1 provided by the present invention has excellent The strong cell adhesion allows it to adhere to vaginal epithelial cells, forming a microecological barrier to prevent pathogens from colonizing or competing for epithelial cell receptors.
  • Lactiplantibacillus plantarum 1-D1 provided by the present invention are significantly better than commercially available products, such as Wanze Shuangqi and Metronidazole, and have good industrial value and promotion value.
  • Lactiplantibacillus plantarum 1-D1 has been deposited in the China Type Culture Collection Center on July 28, 2022.
  • the strain preservation number is: CCTCC NO: M 20221191, address: Wuhan, China.
  • Figure 1 is a frontal photo of the colony morphology of Lactobacillus plantarum 1-D1;
  • Figure 2 is a Gram stain observation picture of Lactobacillus plantarum 1-D1;
  • Figure 3 shows the comparison of the whole genome ANI of Lactobacillus plantarum 1-D1 and the standard strain
  • FIG. 4 shows the hemolytic test results (left: Staphylococcus aureus ATCC 25923; right: Lactobacillus plantarum 1-D);
  • Figure 5 shows the body weight change trend in the mouse toxicity test
  • Figure 6 shows the vaginal Lactobacillus colonization score of SD rats.
  • the present invention uses Lactobacillus delbrueckii isolated from Dingjunsheng (hereinafter collectively referred to as DJS-H3) as a positive control at each stage, and rapidly isolates the bacteria through low pH medium screening, amplified growth ability and calcium dissolution zone screening. Lactobacillus that tolerates low pH, has strong reproductive capacity and strong lactic acid production capacity. Then, the dominant Lactobacilli with strong colonization ability and strong ability to inhibit the growth of pathogenic bacteria were screened out through the ability to adhere to Hela cells and antibacterial test.
  • Lactobacillus plantarum 1-D1 provided by the present invention, examine its genetic stability and safety, and provide further support for its commercial application. Representative specific examples are as follows:
  • DMEM medium Gibco; cat: 11995065
  • MRS liquid culture medium Huankai Biotechnology, cat: 1110151
  • HeLa cells Beina Biotech, product number: BNCC342189
  • VK2/E6E7 cells Qingqi (Shanghai) Biotechnology
  • vaginal secretions of healthy female volunteers of childbearing age were subjected to Gram staining microscopy and Nugent score, and 29 healthy female volunteers of childbearing age were selected. Place vaginal cotton swabs from healthy volunteers in MRS acidic liquid culture medium and amplify bacteria overnight at 37°C. After 10-fold gradient dilution of the enrichment solution, apply the appropriate gradient on 2% calcium carbonate-0.8% MRS agar. On the culture medium, culture for 36-48 hours at 37°C; pick single colonies with obvious transparent circles on the cultured calcium carbonate-MRS culture medium and place them in the MRS liquid culture medium.
  • nucleotide sequences (16S rRNA sequences) of four Lactobacillus plantarum strains 1-D1, 16-B12, 53-D2, and 51S-H2 are shown in SEQ ID No. 1 to 4 respectively. .
  • Lactiplantibacillus plantarum 1-D1 was deposited in the China Type Culture Collection Center on July 28, 2022.
  • the strain preservation number is: CCTCC NO: M 20221191, address: Wuhan, China;
  • Lactobacillus crispatus 51S-H2 was deposited at the China Type Culture Collection Center on July 28, 2022.
  • the deposit number is: CCTCC NO: M 20221194, address: Wuhan, China;
  • Lactobacillus paragasseri 16-B12 was deposited in the China Type Culture Collection Center on July 28, 2022.
  • the deposit number is: CCTCC NO: M 20221193, address: Wuhan, China;
  • Lactobacillus jensenii 53-D2 was deposited at the China Type Culture Collection Center on July 28, 2022.
  • the deposit number is: CCTCC NO: M 20221192, address: Wuhan, China.
  • Lactobacillus delbrueckii was isolated from Dingjunsheng according to the above method for isolating Lactobacillus plantarum.
  • the growth curve data of Lactobacillus isolation stage showed that most Lactobacilli entered the late logarithmic phase at 12 h. Therefore, the viable bacterial count of each strain was determined using the agar pouring method 12 h after inoculation. at the same vaccination Under time conditions, strains with high viable bacterial counts have relatively obvious advantages in their growth performance. The top 25 strains with growth advantages were selected to enter the next phase of the test. The data showed that Lactobacillus plantarum 1-D1 had excellent growth performance and strong reproductive ability. The viable bacterial count in 12 hours reached 6.37 ⁇ 10 9 CFU/ml, which was beneficial to High-density fermentation in commercial transformation processes.
  • Lactobacilli The ability of Lactobacilli to adhere to HeLa cells and VK2/E6E7 cells was used to evaluate the colonization ability of Lactobacilli.
  • activate the lactobacilli take 50 ⁇ L of each lactobacillus and insert it into 5 ml MRS liquid medium, culture it at 37°C for 18-24 hours, then take it out and prepare it for transfer.
  • Lactobacillus transfer Take 50 ⁇ L of each Lactobacillus activation solution and insert it into 5 ml MRS liquid medium, culture it at 37°C for 18 to 24 hours, 12000 rpm, centrifuge for 2 minutes, take it out, resuspend in DMEM medium, and adjust to 0.5 McFarland turbidity.
  • Carry out cell distribution take 500 ⁇ l of recovered cells with a concentration of 10 5 cells/mL and inoculate them into a 24-well culture plate, place it in a carbon dioxide incubator, and culture it overnight at 37°C and 5% CO 2 . Discard the DMEM stock solution, wash 3 times with PBS and add 500 ⁇ L DMEM culture medium.
  • Lactobacillus plantarum 1-D1 The ability of Lactobacillus plantarum 1-D1 to adhere to cells is shown in Tables 1 and 2 below. Through Fisher LSD mean comparison analysis, it was found that the adhesion ability of Lactobacillus plantarum 1-D1 is much greater than that of DJS-H3 isolated from Dingjunsheng. The differences in adhesion numbers between groups are extremely significant. Lactobacillus plantarum 1-D1 strain The number of adhesion to Hela cells in 4 hours is more than 100 times that of Dingjunsheng, and the number of adhesion to VK2/E6E7 cells in 4 hours is more than 10 times that of Dingjunsheng.
  • Bacteriostatic tests were used to evaluate the ability of Lactobacillus plantarum to inhibit different pathogenic bacteria.
  • the lactobacilli were mixed with 0.8% MRS agar medium, and then a 10 mm lactobacilli cake was made.
  • Different pathogenic bacteria Gardnerella vaginalis, Staphylococcus aureus, etc.
  • Bacteria plates Place the Lactobacillus bacteria cake gently on the surface of the pathogenic bacteria plate, incubate at 37°C for 18-24 hours, take it out, and measure the size of the inhibition zone with a vernier caliper to evaluate the ability of each Lactobacillus to inhibit pathogenic bacteria. Specific steps are as follows:
  • Gardnerella vaginalis and Atropobacter vaginalis were respectively inoculated into 5% horse serum-anaerobic BHI liquid. After activation and transfer, 0.9% anaerobic saline was diluted 10 times, and 1 ml of diluent was mixed with 10 ml containing Mix 5% horse serum and anaerobic BHI agar, pour it into a 9cm dish, wait until it is completely solidified, and set aside.
  • Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella paratyphi B and Shigella dysenteriae were respectively inoculated into the nutrient broth culture medium. After activation and transfer, they were inoculated with 0.9% physiological saline 10 times. Gradient, dilute the pathogenic bacteria liquid to 100 times, take 1ml of diluent and mix with 10ml of nutrient agar, pour it into a 9cm dish, wait until it is completely solidified, and set aside.
  • Lactobacillus bacteria cake gently on the surface of each pathogenic bacteria plate, place 4 bacteria cakes symmetrically on each dish, and 3 of each strain in parallel.
  • the antibacterial abilities of different Lactobacilli are as shown in Table 3.
  • VFDB virulence gene database
  • Hemolysin and Hemolysin III were 100% consistent in Lactobacillus plantarum.
  • the Hemolysin III gene has been confirmed to be widely present in Lactobacillus plantarum (including commercial Lactobacillus plantarum L. plantarum 299 V, L. plantarum JDM1 and L.
  • Lactobacillus plantarum 1-D1 has good safety at the genetic level.
  • Lactobacillus plantarum 1-D1 was cultured anaerobically on a blood agar plate (Chengdu Ruiqi; Sichuan Medical Instrument No. 20152400001) for 48 hours, and cultured with ⁇ -hemolytic Staphylococcus aureus ATCC 25923 (Shanghai Baolu Biotech) was used as a positive control and cultured aerobically for 24 hours.
  • Determine whether Lactobacillus plantarum 1-D1 is hemolytic by observing whether Lactobacillus plantarum 1-D1 forms a hemolytic ring on the blood agar plate after culture.
  • mice Use 5 mice weighing 18-22g. Each mouse is orally gavaged with 0.5ml of fresh Lactobacillus plantarum 1-D1 bacterial solution (not less than 1.0 ⁇ 10 9 CFU/0.5ml), once a day for 3 consecutive days. , continuous observation from the first day of intragastric administration to the seventh day.
  • the agar diffusion disk method was used to determine the sensitivity of strains to antibiotics, and the sensitivity level of strains to antibiotics was determined based on the size of the inhibition zone.
  • the standard sensitive strains Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 are used as quality control bacteria. Analyzes are conducted with reference to the American Clinical and Laboratory Standards Institute (CLSI) international standards and the health industry standards of the People's Republic of China. The quality control strains are used to judge.
  • the standard is based on the formulation of the sensitivity level determination standard of Lactobacillus plantarum 1-D1.
  • the sensitivity level is divided into sensitive, intermediate and resistant, represented by S, I and R respectively.
  • Lactobacillus plantarum 1-D1 was resistant to penicillin, oxacillin, azithromycin, gentamicin, kanamycin, vancomycin, enrofloxacin, clindamycin and metronidazole It is sensitive to ampicillin, imipenem, ceftriaxone, amoxicillin, piperacillin, and tetracycline, and moderately sensitive to erythromycin.
  • metronidazole and clindamycin are commonly used in clinical treatment of bacterial vaginosis. The results of this experiment suggest that Lactobacillus plantarum 1-D1 can theoretically be used in combination with metronidazole and clindamycin.

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Abstract

提供了一株植物乳杆菌(Lactiplantibacillus plantarum)1-D1菌株及其应用，该菌株具有遗传稳定性，无致病/毒力基因、安全性好，对致病菌的抑菌能力强，能够降低阴道pH，维持健康阴道酸性环境，同时具有突出的Hela细胞粘附能力，能够粘附于阴道上皮细胞，形成微生态屏障，防止病原体定植或竞争上皮细胞受体。该菌株可用于制备防治阴道炎的药物或卫生用品中。

Description

一株植物乳杆菌及其应用 技术领域
本发明属于微生物技术领域，特别是涉及一株植物乳杆菌及其应用。
背景技术
在健康育龄女性阴道中，乳杆菌是优势菌群，占阴道菌群的70％以上。研究表明，女性生殖道内的乳杆菌主要通过产乳酸、产抑菌素来抑制病原微生物。
我国妇产科门诊患者中，生殖道感染占40.2％-55.6％。至少有2亿人次患生殖道感染相关疾病，其中复发患者有1亿人次，由此产生的医疗费用每年高达200多亿元。调查显示，99.3％的妇科门诊阴道感染有症状患者存在阴道微生态失衡。2016年，中华医学会妇产科分会感染协作组提出阴道感染的本质是“阴道微生态失衡”。阴道微生态如果长期处于异常状态，阴道对致病微生物的抵抗力降低，往往是阴道感染反复发作或继发新感染的重要原因。
常见的阴道感染有：细菌性阴道病(BV)、外阴阴道假丝酵母菌病(VVC)、滴虫阴道炎(TV)、需氧菌性阴道炎(AV)等，这些均伴随着乳杆菌减少或消失。常规的阴道感染治疗方案以“抗菌”为主，抗生素如克林霉素治疗后，细菌性阴道致病菌得到抑制的同时也抑制了阴道内乳杆菌的生长；甲硝唑虽在给药剂量下不会抑制乳杆菌生长，但也无法促进乳杆菌的恢复。且有研究表明，甲硝唑耐药率63.8％，克林霉素耐药率24.1％～67％。
另外，使用抗生素后的复发率和耐药率很高，抗生素仅能抑制浮游的阴道加德纳菌，短暂控制症状，无法完全清除生物膜及在其内的阴道加德纳菌，一旦停止治疗，生物膜内的菌将再次复苏、繁殖、扩散，导致细菌性阴道病复发。生物膜会降低细菌对抗菌药物的敏感性，增强对抗菌药物的耐药性，且致病菌株耐药性可以通过移动遗传元件获得，因此会导致抗生素的治疗效果越来越弱甚至变得无效。
那日苏等人的研究显示，阴道内乳杆菌可干扰阴道加德纳菌生物膜的形成。现阶段，中华医学会妇产科分会感染协作组指出可应用阴道微生态制剂恢复以有功能的乳杆菌为主的弱酸性环境，促进阴道微生态的平衡和免疫调节，减少阴道感染的反复发作(那日苏,等.阴道内乳杆菌对阴道加德纳菌生物膜影响的初步研究[J].现代妇产科进展,2015,24(09):641-645.)。目前国内上市的阴道微生态制剂仅有2种，一种是西安正浩生物制药有限公司生产的商品名为“延华”的市售产品，包含一种肠链球菌(Streptococcus faecalis)，该菌种不是阴道优势菌种，而且这个物种下的细菌具有条件致病性。另一种是内蒙古双奇药业股份有限公司生产的商品名为“万泽双奇”的市售产品，包含1种乳杆菌——德氏乳杆菌(Lactobacillus delbrueckii)。
目前上市的阴道微生态制剂远不能满足临床需求，且还有很大的优化空间。因此，若能开发出益生能力更强的乳杆菌菌种运用于预防和/或治疗阴道感染，将有巨大的应用价值。
发明内容
本发明的目的在于提供一株植物乳杆菌(Lactiplantibacillus plantarum)1-D1，该菌株能够抑制阴道内致病菌的生长，调节阴道微生态平衡，具有防治阴道炎的作用，可用于制备防治阴道炎的药物、食品、卫生用品中。
为了实现本发明的目的，本发明提供了以下技术方案：
一株植物乳杆菌(Lactiplantibacillus plantarum)1-D1，该菌株保藏号为：CCTCC NO：M 20221191。
本发明提供的植物乳杆菌(Lactiplantibacillus plantarum)1-D1在制备抑菌产品中的应用；
进一步地，所述抑菌产品包括但不限于细菌抑制剂、饲料添加剂和抗菌肽等。
进一步地，所述抑菌产品是抑制加德纳菌、金黄色葡萄球菌、绿脓假单胞菌、大肠杆菌、沙门氏菌、痢疾志贺菌中的一种或几种菌的产品。
本发明提供的植物乳杆菌(Lactiplantibacillus plantarum)1-D1在制备预防和/或治疗阴道感染性疾病产品中的应用。
术语“治疗(treatment)”(也称为“治疗(treat)”或“治疗(treating)”)是指根据治疗性方案的治疗性试剂的任何施用，所述治疗性方案达到所需效果，即部分或完全减轻、改善、缓解、抑制、延迟发作、降低严重程度和/或降低特定疾病、障碍和/或病症的一种或多种症状或特征(例如改善阴道菌群结构和功能、提高阴道菌群多样性、降低炎症相关菌群丰度、治疗阴道炎)的发生率；在一些实施方式中，根据治疗性方案的治疗性试剂的施用与所需效果的实现相关。这种治疗可以针对没有表现出相关疾病、障碍和/或病症的受试者和/或针对仅表现出疾病、障碍和/或病症的早期迹象的受试者。替代地或另外地，这种治疗可以针对表现出相关疾病、障碍和/或病症的一种或多种所确定迹象的受试者。在一些实施方式中，治疗可以针对已被诊断患有相关疾病、障碍和/或病症的受试者。在一些实施方式中，治疗可以针对已知具有一种或多种易感因素的受试者，所述易感因素在统计学上与相关疾病、障碍和/或病症发展的风险增加相关。
所述产品包括但不限于药物、食品、保健品、卫生用品等，在产品中应用时，菌株为活性菌株或灭活性菌株。
术语“药物”涵盖了人类医学和兽医学中供人类和动物两者使用的药物，同时涵盖了用于掺入动物饲料(例如牲畜饲料和/或宠物食物)中的药物。此外，本文中所使用的术语“药物”意指提供治疗、预防和/或有益效果的任何物质。本文中 所使用的术语“药物”不一定限于需要上市许可证(Marketing Approval)的物质，而是包括可以用于化妆品、保健品、食物(包括例如饲料和饮料)、益生菌培养物和膳食补充剂的物质。
本发明植物乳杆菌(Lactiplantibacillus plantarum)1-D1可作为有益成分添加在发酵食品、增强免疫力保健食品、女性私护固体饮料等食品中，能够提升人体免疫力，尤其能够增强女性生殖道免疫力作用。
所述阴道感染性疾病是由细菌性阴道致病菌、外阴阴道假丝酵母菌致病菌、滴虫阴道炎、需氧菌性阴道炎中的一种或几种引起的；
进一步地，所述需氧性阴道炎致病菌包括但不限于金黄色葡萄球菌、大肠杆菌、绿脓假单胞菌、痢疾志贺菌或沙门氏菌等；
进一步地，所述细菌性阴道病致病菌是阴道加德纳氏菌。
进一步地，所述产品是降低阴道pH值的产品。
进一步地，所述产品是在阴道环境中代谢产H2O2、有机酸和/或细菌素的产品。
本发明提供的植物乳杆菌(Lactiplantibacillus plantarum)1-D1在制备调节菌群微生态平衡的产品的应用。
本发明提供的植物乳杆菌(Lactiplantibacillus plantarum)1-D1在生产卫生用品中的应用。
本发明还保护植物乳杆菌(Lactiplantibacillus plantarum)1-D1作为益生菌的应用。
本发明还提供一种产品，其活性成分含有植物乳杆菌；所述植物乳杆菌如前述的植物乳杆菌(Lactiplantibacillus plantarum)1-D1。
进一步地，所述产品包括但不限于食品、保健品、药品、日用品等；
进一步地，所述产品为预防和/或治疗阴道感染性疾病的产品。
本发明所述产品还可包括药学上可接受的赋形剂或者稀释剂。
有益效果：
(1)本发明植物乳杆菌(Lactiplantibacillus plantarum)1-D1具有遗传稳定性，无致病/毒力基因、安全性好，对阴道加德纳菌、金黄色葡萄球菌、绿脓假单胞菌、大肠杆菌、乙型副伤寒沙门氏菌和痢疾志贺菌均表现出较好的抑制能力。
(2)本发明提供的植物乳杆菌(Lactiplantibacillus plantarum)1-D1产乳酸的能力强，能够降低阴道pH，维持健康阴道酸性环境，改善阴道菌群平衡，同时，其产生的抑菌物质，如过氧化氢、细菌素、有机酸等，能够抑制致病菌生长繁殖，提高黏膜免疫力，增强宿主抗感染作用。
(3)本发明提供的植物乳杆菌(Lactiplantibacillus plantarum)1-D1具有优 势的细胞粘附力，使其能够粘附于阴道上皮细胞，形成微生态屏障，防止病原体定植或竞争上皮细胞受体。
(4)本发明提供的植物乳杆菌1-D1(Lactiplantibacillus plantarum 1-D1)各项评价指标显著优于市售产品，如万泽双奇、甲硝唑，具有良好的产业价值和推广价值。
菌株保藏信息：
植物乳杆菌(Lactiplantibacillus plantarum)1-D1已于2022年7月28日保藏于中国典型培养物保藏中心，该菌株保藏号为：CCTCC NO：M 20221191，地址：中国武汉。
附图说明
图1为植物乳杆菌1-D1菌落形态正面照片；
图2为植物乳杆菌1-D1革兰氏染色观察图片；
图3为植物乳杆菌1-D1与标准菌株全基因组ANI比较；
图4为溶血性检测结果(左：金黄色葡萄球菌ATCC 25923；右：植物乳杆菌1-D)；
图5为小鼠毒性试验体重变化趋势图；
图6为SD大鼠阴道乳杆菌定植评分。
具体实施方式
为了使本领域技术人员更好的理解本发明，下面结合具体实施例对本发明作进一步的详细说明。本领域技术人员应当理解的是，这不应被理解为对本发明权利要求范围的限定。除有定义外，以下实施例中所用的技术和科学术语具有与本发明所属领域技术人员普遍理解的相同含义。同时需要注意的是，本发明中的试剂或仪器无特别说明的均可通过市售购买得到。以下实施例中所用的培养基制备方法均为公知的方法。
本发明以从定君生中分离出的德氏乳杆菌(以下统称为DJS-H3)作为各阶段的阳性对照，通过低pH培养基筛选、扩增生长能力及钙溶圈筛选，快速分离出耐受低pH，繁殖能力强及产乳酸能力强的乳杆菌。再通过粘附Hela细胞的能力和抑菌试验筛选出定植能力强及抑制致病菌生长能力强的优势乳杆菌。对本发明提供的植物乳杆菌1-D1进行进一步的深入研究，考察其遗传稳定性和安全性，为其商业化应用做进一步的支撑。其中有代表性的具体实施例如下：
DMEM培养基:Gibco；cat:11995065
MRS液体培养基:环凯生物，cat:1110151
2％碳酸钙-0.8％MRS:生工，H122BA0030
琼脂:生工生物，A505255-0250
HeLa细胞:北纳生物，货号：BNCC342189
VK2/E6E7细胞：青旗(上海)生物
实施例1乳杆菌的分离
对健康育龄女性志愿者阴道分泌物进行革兰氏染色镜检及Nugent评分，选取29名健康育龄女性志愿者。将健康志愿者的阴道棉拭子置于MRS酸性液体培养基，37℃过夜扩培增菌；取增菌液10倍梯度稀释后，取适宜梯度涂布于2％碳酸钙-0.8％MRS琼脂培养基上，37℃，培养36-48h；于培养好的碳酸钙-MRS培养基上挑取具有明显透明圈的单菌落于MRS液体培养基中，37℃过夜培养后，对培养后的1816株菌株进行PCR扩增后送16S rRNA测序，剔除其余产酸的非乳杆菌后，选择不同样品中的乳杆菌共72株进行后续筛选试验。
其中，分离得到的菌株中，植物乳杆菌1-D1、16-B12、53-D2、51S-H2四株菌株的核苷酸序列(16S rRNA序列)分别如SEQ ID No.1～4所示。
初步结果显示，植物乳杆菌1-D1在碳酸钙-MRS培养基上的钙溶圈远远大于从定君生中分离的德氏乳杆菌DJS-H3，证明其产乳酸能力具有显著优势。将植物乳杆菌1-D1进行16S rRNA测序。
菌株保藏信息：
植物乳杆菌(Lactiplantibacillus plantarum)1-D1于2022年7月28日保藏于中国典型培养物保藏中心，该菌株保藏号为：CCTCC NO：M 20221191，地址：中国武汉；
卷曲乳杆菌(Lactobacillus crispatus)51S-H2，于2022年7月28日保藏于中国典型培养物保藏中心，保藏编号为：CCTCC NO：M 20221194，地址：中国武汉；
格氏乳杆菌(Lactobacillus paragasseri)16-B12，于2022年7月28日保藏于中国典型培养物保藏中心，保藏编号为：CCTCC NO：M 20221193，地址：中国武汉；
詹氏乳杆菌(Lactobacillus jensenii)53-D2，于2022年7月28日保藏于中国典型培养物保藏中心，保藏编号为：CCTCC NO：M 20221192，地址：中国武汉。
按照上述分离植物乳杆菌的方法从定君生中分离出德氏乳杆菌(DJS-H3)。
实施例2植物乳杆菌1-D1生长性能测定
乳杆菌分离阶段生长曲线数据表明，大多数乳杆菌在12h时进入对数末期。因此，在接种12h时采用琼脂倾注法对各菌株进行活菌数测定。在相同接种时 间下，活菌数高的菌株其生长性能具有相对明显优势。选择排名前25株具有生长优势的菌株进入下一阶段试验，数据显示：植物乳杆菌1-D1具有优秀的生长性能，繁殖能力强，12h活菌数达到6.37×109CFU/ml，有利于商业化转化过程中的高密度发酵。
对植物乳杆菌1-D1进行培养形态和镜检特征鉴定，结果如图1、图2。
实施例3植物乳杆菌1-D1粘附能力
用乳杆菌粘附Hela细胞、VK2/E6E7细胞的能力来评估乳杆菌的定植能力。首先，将乳杆菌活化：取各乳杆菌50μL接入5ml MRS液体培养基中，37℃培养18-24h后取出，备转接用。乳杆菌转接：取各乳杆菌活化液50μL接入5ml MRS液体培养基中，37℃培养18～24h，12000rpm，离心2min后取出，DMEM培养基重悬，调至0.5麦氏浊度。进行细胞布板：取复苏好的细胞500μl浓度为105个/mL细胞接种于24孔培养板中，置于二氧化碳培养箱，37℃、5％CO2条件下过夜培养。弃去DMEM原液，PBS清洗3次后加入500μL DMEM培养基。最后细菌与细胞互作：取500μL浓度为108CFU/mL活化过夜培养的、经DMEM培养基重悬的乳杆菌加入上述含有细胞的24孔培养板中，置于二氧化碳培养箱，37℃、5％CO2条件下分别孵育2h、4h后，PBS清洗4次以除去未粘附的乳杆菌。加入500μL 0.25％的胰酶消化2min，加入600μl完全培养基终止消化，吹打混匀。无菌PBS对细胞-菌悬液进行10倍梯度稀释，选择适当梯度稀释液100μl加入一次性无菌平皿中，0.8％MRS琼脂培养基倾注混匀，凝固后将平板倒置。倒置的平板在37℃下培养48h后取出计数，每组3个平行，取平均值作为最终黏附数。乳杆菌数量越多，证明其粘附细胞的能力越强。
植物乳杆菌1-D1的粘附细胞的能力如下表1～2。通过Fisher LSD均值比较分析后发现，植物乳杆菌1-D1的粘附能力远远大于从定君生中分离的DJS-H3，组间黏附数差异均为极显著，植物乳杆菌1-D1菌株对Hela细胞4h黏附数是定君生的100倍以上，对VK2/E6E7细胞4h黏附数是定君生的10倍以上。
表1植物乳杆菌1-D1不同时间对Hela细胞的粘附能力

注：不同字母表示差异显著，a:P≤0.05；b:P≤0.01。
表2植物乳杆菌1-D1不同时间对VK2/E6E7细胞的粘附能力
实施例4植物乳杆菌抑菌能力
用抑菌试验来评估植物乳杆菌抑制不同致病菌的能力。
将乳杆菌与0.8％MRS琼脂培养基混匀，随后制成10mm的乳杆菌菌饼。将不同的致病菌(阴道加德纳菌、金黄色葡萄球菌等)分别采用适宜其生长的培养基和生长条件活化转接后，与对应的半固体培养基混匀，分别制成致病菌平皿。将乳杆菌菌饼轻放在致病菌平皿表面，37℃正置培养18-24h后取出，游标卡尺测量抑菌圈大小，从而评估各乳杆菌的抑制致病菌的能力。具体步骤如下：
分别将植物乳杆菌和德氏乳杆菌(DJS-H3)活化后，取0.1ml菌液与MRS固体培养基混匀，倾注入6cm平皿内，完全凝固后，在37℃下培养48h，取出平皿，用内径10mm的打孔器在琼脂培养基上打孔，获得乳杆菌菌饼，备用。
将阴道加德纳菌和阴道阿托波菌分别接种至5％马血清-厌氧BHI液体中，经活化转接后，0.9％厌氧生理盐水进行10倍稀释，取1ml稀释液与10ml含5％马血清的无氧BHI琼脂混合，倾注入9cm平皿内，待其完全凝固，备用。
将金黄色葡萄球菌、绿脓假单胞菌、大肠杆菌、乙型副伤寒沙门氏菌和痢疾志贺菌分别接种至营养肉汤培养基中，经活化转接后，用0.9％生理盐水以10倍梯度，将致病菌菌液稀释至100倍，取1ml稀释液与10ml营养琼脂混匀，倾注入9cm平皿内，待其完全凝固，备用。
将乳杆菌菌饼轻放在各致病菌平皿表面，每皿对称放4个菌饼，每株菌3个平行。将阴道加德纳菌和阴道阿托波菌分别平皿正置放入放有厌氧产气袋的厌氧密封盒中，在37℃下培养48h，将其他致病菌平皿正置放入培养箱，在37℃培养24h，最后用游标卡尺测量各平皿的抑菌圈大小。不同乳杆菌的抑菌能力如下表3。
表3植物乳杆菌对不同致病菌的抑菌圈大小(mm)
结果显示，植物乳杆菌1-D1的综合抑菌能力远远大于从定君生中分离的德氏乳杆菌DJS-H3和甲硝唑。
实施例5植物乳杆菌1-D1全基因组序列分析
对植物乳杆菌1-D1进行全基因组测序。将植物乳杆菌1-D1的全基因组序列上传到EzBiocloud后，与其标准菌株比较，得到平均核苷酸一致性(average nucleotide identity，ANI)比值如图3。经全基因组测序后证实为Lactiplantibacillus plantarum；中文名：植物乳杆菌。
使用毒力基因数据库VFDB对全基因组序列中的毒力基因进行分析(identity>70％，coverage length>70％)(A defined commensal consortium elicits CD8 T cells and anti-cancer immunity[J].Nature,2019,565(7741):1)
，并通过NCBI数据库进行人工Blast，发现两个溶血相关基因(Hemolysin及HemolysinⅢ)在植物乳杆菌中具有100％一致性。但HemolysinⅢ基因已被证实广泛存在于植物乳杆菌中(包含商用植物乳杆菌L.plantarum 299 V，L.plantarum JDM1及L.plantarum ST-III等)(Chokesajjawatee N,Santiyanont P,Chantarasakha K,et al.Safety Assessment of a Nham Starter Culture Lactobacillus plantarum BCC9546 via Whole-genome Analysis[J].Scientific Reports,2020,10(1))，且该基因仅可对细菌或者真菌裂解，没有发现在哺乳动物细胞裂解。因此，认为携带HemolysinⅢ基因的菌株不会引起安全问题，而Hemolysin则需要多个基因同时存在才能表现出溶血性(Karina,Arellano,Jorge,et al.Safety Evaluation and Whole-Genome Annotation of Lactobacillus plantarum Strains from Different Sources with Special Focus on Isolates from Green Tea.[J].Probiotics and antimicrobial proteins,2019)，而1-D1中不存在与Hemolysin互作产生溶血性的基因。同时，植物乳杆菌1-D1无质粒，且基因组中的可转移元件无耐药基因，因此不存在耐药基因转移风险。综上所述，植物乳杆菌1-D1在基因层面具有良好的安全性。
实施例6植物乳杆菌1-D1的溶血性试验
为了进一步确认植物乳杆菌1-D1是否具有溶血性，在血琼脂平板(成都瑞琦；川械注准20152400001)上对植物乳杆菌1-D1进行划线厌氧培养48h，采用具有β溶血的金黄色葡萄球菌ATCC 25923(上海宝录生物)作为阳性对照好氧培养24h。通过培养后观察植物乳杆菌1-D1是否在血琼脂平板上形成溶血环，判断植物乳杆菌1-D1是否具有溶血性。
下图4结果显示，阳性对照平皿ATCC 25923菌落周围可见明显透明圈，呈典型β溶血特征，植物乳杆菌1-D1无任何溶血环产生，说明植物乳杆菌1-D1不 产生溶血素溶解红细胞，即不具有溶血性。
实施例7植物乳杆菌1-D1的小鼠毒性试验
用体重18～22g小鼠5只，每只小鼠经口灌胃0.5ml植物乳杆菌1-D1新鲜菌液(不少于1.0×109CFU/0.5ml)，每天1次，连续3天，从第1天灌胃起连续观察至第7天。
经小鼠毒性试验证明，小鼠体态观察正常，四肢活动正常，行动灵活，毛色顺滑，正常进食和饮水，没有发现排泄物和分泌物异常，没有发现明显的中毒情况，实验期间也没有动物异常死亡且小鼠体重均增加。
实施例8植物乳杆菌1-D1的抗生素敏感试验
按照2020年版药典第三部微生态活菌制品总论中抗生素敏感性试验的要求，采用琼脂扩散纸片法测定菌株对抗生素的敏感性，根据抑菌圈的大小判断菌株对抗生素敏感性级别。
采用标准敏感菌株大肠埃希菌ATCC 25922和金黄色葡萄球菌ATCC 25923作为质控菌，参照美国临床和实验室标准协会(CLSI)国际标准和中华人民共和国卫生行业标准进行分析，以质控菌株判断标准为依据制订植物乳杆菌1-D1敏感程度判定标准，其敏感程度分为敏感、中介度和耐药，分别以S、I、R表示。
抗生素敏感性试验结果显示，植物乳杆菌1-D1对青霉素、苯唑西林、阿奇霉素、庆大霉素、卡那霉素、万古霉素、恩诺沙星、克林霉素和甲硝唑耐药，对氨苄西林、亚胺培南、头孢曲松、阿莫西林、哌拉西林、四环素敏感，对红霉素中度敏感。另一方面，临床常用甲硝唑、克林霉素治疗细菌性阴道病，本实验结果提示，植物乳杆菌1-D1理论上可与甲硝唑、克林霉素联合使用。
表5植物乳杆菌1-D1药敏试验抑菌圈直径及判定结果
实施例9植物乳杆菌1-D1在大鼠阴道内的定植试验
对SD大鼠通过阴道给药，每天1次给予植物乳杆菌1-D1，连续给药5天，再观察10天。在每次给药前采集阴道分泌物并进行革兰氏染色镜检(D1的分泌物为初始给药前采集)，判定植物乳杆菌1-D1的定植情况。试验方案见下表6。
革兰氏染色镜检显示，初始给药前绝大部分大鼠阴道分泌物中无任何微生物，极少数大鼠阴道分泌物中偶见革兰氏阴性杆菌、革兰氏阳性球菌。持续给药后，相比于空白对照组，给予植物乳杆菌1-D1的大鼠，其阴道分泌物中革兰氏阳性杆菌较给药前及空白对照组明显增多，表明植物乳杆菌1-D1可成功在SD大鼠 阴道内定植。停止给药5天后(D10)，视野中仍可见革兰氏阳性杆菌，停止给药10天后(D15)，大鼠阴道微环境恢复到给药前状态。
表6植物乳杆菌1-D1在大鼠阴道内的定植实验方案
表7 SD大鼠阴道乳杆菌定植评分标准

注：评分＜0，表示乳杆菌定植成功；评分≥0，则乳杆菌定植失败。
本领域的技术人员应理解，上述描述及附图中所示的本发明的实施例只作为举例而并不限制本发明。本发明的目的已经完整并有效地实现。本发明的功能及结构原理已在实施例中展示和说明，在没有背离所述原理下，本发明的实施方式可以有任何变形或修改。

Claims (10)

  1. 一株植物乳杆菌(Lactiplantibacillus plantarum)1-D1，其特征在于，该菌株保藏号为：CCTCC NO：M 20221191。
  2. 权利要求1所述的植物乳杆菌(Lactiplantibacillus plantarum)1-D1在制备抑菌产品中的应用；
    进一步地，所述抑菌产品是抑制加德纳菌、金黄色葡萄球菌、绿脓假单胞菌、大肠杆菌、沙门氏菌、痢疾志贺菌中的一种或几种菌的产品。
  3. 权利要求1所述的植物乳杆菌(Lactiplantibacillus plantarum)1-D1在制备预防和/或治疗阴道感染性疾病产品中的应用。
  4. 根据权利要求3所述的应用，其特征在于，所述阴道感染性疾病是由细菌性阴道致病菌、外阴阴道假丝酵母菌致病菌、滴虫阴道炎、需氧菌性阴道炎中的一种或几种引起的；
    进一步地，所述需氧性阴道炎致病菌是金黄色葡萄球菌、大肠杆菌、绿脓假单胞菌、痢疾志贺菌或沙门氏菌；
    进一步地，所述细菌性阴道病致病菌是阴道加德纳氏菌。
  5. 根据权利要求3所述的应用，其特征在于，所述产品是降低阴道pH值的产品。
  6. 根据权利要求3所述的应用，其特征在于，所述产品是在阴道环境中代谢产H2O2、有机酸和/或细菌素的产品。
  7. 权利要求1所述的植物乳杆菌(Lactiplantibacillus plantarum)1-D1在制备调节菌群微生态平衡的产品的应用。
  8. 权利要求1所述的植物乳杆菌(Lactiplantibacillus plantarum)1-D1在生产卫生用品中的应用。
  9. 一种产品，其特征在于，其活性成分含有植物乳杆菌；所述植物乳杆菌为权利要求1所述的植物乳杆菌(Lactiplantibacillus plantarum)1-D1。
  10. 根据权利要求9所述的产品，其特征在于，所述产品为食品、保健品、药品或日用品；
    进一步地，所述产品为预防和/或治疗阴道感染性疾病的产品。
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