WO2022100633A1 - 用于预防和/或治疗生殖道菌群紊乱相关疾病的格氏乳杆菌 - Google Patents

用于预防和/或治疗生殖道菌群紊乱相关疾病的格氏乳杆菌 Download PDF

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WO2022100633A1
WO2022100633A1 PCT/CN2021/129910 CN2021129910W WO2022100633A1 WO 2022100633 A1 WO2022100633 A1 WO 2022100633A1 CN 2021129910 W CN2021129910 W CN 2021129910W WO 2022100633 A1 WO2022100633 A1 WO 2022100633A1
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lactobacillus gasseri
vaginitis
reproductive tract
bacterial
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English (en)
French (fr)
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张笑薇
吕金丽
赵少伟
邹远强
肖亮
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深圳华大生命科学研究院
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • 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
    • A61P31/04—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
    • 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
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • 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
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/145—Gasseri

Definitions

  • the present disclosure relates to the field of biotechnology, in particular to a novel Lactobacillus gasseri strain for preventing and/or treating reproductive tract infections and applications thereof.
  • the female reproductive tract environment is often affected by fluctuations in hormone levels, poor hygiene control, seasonal changes, and even unstable moods, resulting in bacterial flora disorders, which in turn lead to the invasion of pathogenic bacteria and lead to bacterial, mold, virus and other microbial infections.
  • the clinical symptoms of reproductive tract infections are: vaginal itching and burning, abnormal secretions, frequent urination and painful urination, and severe cases can lead to premature birth and cervical cancer.
  • the genital tract microbial infection rate among women of childbearing age in my country is as high as 80%, and the clinical treatment for most patients with bacterial or fungal infections is antibiotics.
  • Antibiotics can reduce the abundance of probiotics while fighting pathogens, which is not conducive to maintaining the homeostasis of reproductive tract flora, thereby increasing the risk of recurrence.
  • pathogens which is not conducive to maintaining the homeostasis of reproductive tract flora, thereby increasing the risk of recurrence.
  • For patients with HPV virus infection there is currently no perfect treatment method. Regular screening and follow-up are mostly used. In severe cases, surgical treatment is performed.
  • Lactobacillus In the vaginal flora, although there are individual differences, most of the dominant bacteria in the female genital tract are Lactobacillus, and mainly concentrated in four types: Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii and Lactobacillus inerts. . These lactobacilli mainly maintain a lower pH value of the vagina by producing lactic acid, thereby inhibiting the invasion and growth of pathogenic bacteria. In addition, Lactobacillus can also synthesize hydrogen peroxide to ensure an anaerobic environment in the vagina to prevent the growth of aerobic bacteria .
  • the present disclosure is to solve the technical problems of the drugs in the prior art having different degrees of side effects, high recurrence rate or large trauma.
  • one aspect of the present disclosure provides a strain of Lactobacillus gasseri TM13-16, whose deposit number is GDMCC No: 60405.
  • the present disclosure screened out a female reproductive tract probiotic Lactobacillus gasseri TM13-16 from a library of about 30,000 human symbiotic bacteria. On August 24, it was deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC, 5th Floor, Laboratory Building, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province), and its deposit number is GDMCC No: 60405.
  • GDMCC Guangdong Provincial Microbial Culture Collection Center
  • the Lactobacillus gasseri TM13-16 described in the present disclosure is isolated by using a modified version of PYG medium, and is passaged and preserved in MRS medium after identification. After culturing in MRS medium for 48 hours, the colonies of TM13-16 were white, opaque, smooth, round, with irregular edges and white protrusions in the middle, and the colony diameter was about 0.5-1.5 mm. Observation under the microscope of 1000 times showed that the cells were elongated and rod-shaped, and the Gram staining was negative, and no spores and flagella were produced.
  • the Lactobacillus gasseri TM13-16 provided by the present disclosure has no risk genes such as plasmids and transfer elements, and it has been found to have no risk genes such as plasmids and transfer elements. Strong ability to produce L-lactic acid, D-lactic acid and hydrogen peroxide; strong growth ability and high acid and alkali resistance; sensitive to most antibiotics, strong bacteriostatic ability against common vaginal infection pathogens, can be used for prevention and/or Or treat microbial infections of the reproductive tract.
  • Another aspect of the present disclosure provides the use of the Lactobacillus gasseri or its fermentation product or its bacterial suspension or its culture solution in preparing a product for preventing and/or treating diseases related to genital tract flora disorder .
  • the disorder associated with a reproductive tract flora disorder comprises a reproductive tract infection.
  • the disease associated with a disorder of the reproductive tract flora is a reproductive tract infection.
  • the reproductive tract infection comprises at least one selected from bacterial vaginosis, fungal vaginitis, trichomonas vaginitis, aerobic vaginitis, senile vaginitis, and viral infection one.
  • the bacteria causing the bacterial vaginosis are common bacteria causing female genital tract infections, including Escherichia coli (E.coli ATCC35218), Gardnerella vaginalis BNCC337545, Corynebacterium Bacillus, Haemophilus, Staphylococcus aureus, Pseudomonas aeruginosa and other Gram-negative anaerobic bacteria.
  • the fungal vaginitis-causing mold is a common Candida fungus that causes female genital tract infections, including Candida albicans (Candida albicans SC5314), Candida tropicalis bacteria, Candida parapsilosis, and Candida dublini.
  • the virus causing the genital tract viral infection is a common virus that causes female genital tract infection, including HPV, herpes simplex virus, cytomegalovirus, and the like.
  • Another aspect of the present disclosure provides the use of the Lactobacillus gasseri or its fermentation product or its bacterial suspension or its culture solution in preparing a product for antibacterial, adhesion to vaginal epithelial cells and/or cervix Cells, lactate production, H 2 O 2 production.
  • the product is a food, a drug, or a nutraceutical.
  • the food can be probiotic yogurt, probiotic tablet, probiotic solid drink and the like.
  • the Lactobacillus gasseri is administered at a dose of 105-1012 CFU/day.
  • Another aspect of the present disclosure provides a food or health product, the food or health product comprising the Lactobacillus gasseri or its fermentation product or its bacterial suspension or its culture solution.
  • the food or health product is selected from the group consisting of products for preventing and/or treating genital flora disorders related diseases, antibacterial products, products adhering to vaginal epithelial cells and/or cervical cells, products At least one of a product of lactic acid and a product of H 2 O 2 production.
  • Another aspect of the present disclosure provides a pharmaceutical composition comprising the Lactobacillus gasseri or its fermentation product or its bacterial suspension or its culture solution.
  • the pharmaceutical composition is in a single dose form, the pharmaceutical composition comprising a daily dose of 105-1012 CFU of the Lactobacillus gasseri.
  • the pharmaceutical composition is in a dosage form suitable for topical or oral administration.
  • Lactobacillus gasseri or its fermentation product or its bacterial suspension or its culture solution the above-mentioned food or health product, and the above-mentioned pharmaceutical composition are related to the prevention and/or treatment of genital tract flora disorder application in disease.
  • the disorder associated with a reproductive tract flora disorder comprises a reproductive tract infection.
  • the reproductive tract infection comprises at least one selected from bacterial vaginosis, fungal vaginitis, trichomonas vaginitis, aerobic vaginitis, senile vaginitis, and viral infection one.
  • Yet another aspect of the present disclosure provides a method for preventing and/or treating disorders related to reproductive tract flora disorders.
  • the method includes:
  • Lactobacillus gasseri or its fermentation product or its bacterial suspension or its culture solution
  • the disorder associated with a reproductive tract flora disorder comprises a reproductive tract infection.
  • the reproductive tract infection comprises at least one selected from bacterial vaginosis, fungal vaginitis, trichomonas vaginitis, aerobic vaginitis, senile vaginitis, and viral infection one.
  • reproductive tract infection refers to a reproductive tract infection of a female in an animal or a female in a human.
  • Lactobacillus to prevent or treat female genital tract infections, such as the combination of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14, which have been recognized and widely commercialized.
  • a probiotic strain for maintaining the health of the female reproductive tract There are many brands of products, including Jarrow Formulas, Blackmores, Renew life and Clinicans.
  • Jarrow Formulas Blackmores
  • Renew life and Clinicans As a probiotic strain for maintaining the health of the female reproductive tract.
  • its strains are not dominant strains of the reproductive tract, and related clinical studies are limited to European and American populations, and there is a lack of efficacy evaluations for Asian populations.
  • antibiotics and suppositories have many disadvantages.
  • antibiotics such as metronidazole, tinidazole, and clindamycin are used to kill invading anaerobic pathogenic bacteria, thereby treating bacterial vaginosis.
  • it also inhibits the growth of the vaginal probiotic Lactobacillus, unable to rebuild a healthy flora structure.
  • Miconazole, clotrimazole and other suppositories are mainly used for fungal vaginitis, and their mechanism of action is to inhibit the sterol synthesis of the fungal cell membrane such as Candida, affect the permeability of the cell membrane, inhibit the growth of fungi, and lead to death.
  • This drug is often used in suppositories, which has certain limitations in use, causing a lot of inconvenience, and improper use can cause secondary infection.
  • the method of oral probiotics interferes with the intestinal flora to regulate the immune system, and local use supplements the abundance of lactobacilli in the genital tract, reduces the drawbacks caused by traditional antibiotic treatment, and helps the flora to restore its steady state while inhibiting or eliminating pathogenic bacteria. Improve the cure rate, reduce the recurrence rate, help HPV turn negative, effectively treat or prevent reproductive tract microbial infections, and solve a major clinical problem.
  • the inventors of the present disclosure screened out a female reproductive tract probiotic Lactobacillus gasseri TM13-16 from many human symbiotic monobacteria libraries.
  • This strain is a brand-new isolate with strong Lactobacillus -The ability of lactic acid, D-lactic acid and hydrogen peroxide, strong acid and alkali resistance, sensitive to most antibiotics, and strong co-agglutination ability, also showed strong adhesion to human vaginal epithelial cells, in addition, to common Lactobacillus gasseri TM13-16, which is a pathogen of vaginal infection with strong bacteriostatic ability, can be developed as a food, health product, topical or oral drug to prevent, treat or assist in the treatment of female reproductive tract infections.
  • Lactobacillus gasseri in the present disclosure inhibits the growth of pathogenic bacteria of reproductive tract infection by producing secretions such as lactic acid and hydrogen peroxide, and is used for preventing and/or treating reproductive tract infection diseases, especially reducing the recurrence rate and having less toxic and side effects , the effect is lasting.
  • GDMCC Guangdong Microbial Culture Collection Center
  • Figure 1 Genome map of Lactobacillus gasseri TM13-16
  • Figure 2 Probiotic pathway annotation results of Lactobacillus gasseri TM13-16;
  • Figure 3A Comparison of Lactobacillus gasseri TM13-16 with commercial strain L-lactic acid
  • Figure 3B Comparison of Lactobacillus gasseri TM13-16 with commercial strain D-lactic acid
  • RC-14 represents Lactobacillus reuteri RC-14
  • GR-1 represents Lactobacillus rhamnosus GR-1
  • D represents Lactobacillus delbrueckii
  • TM13-16 represents Lactobacillus gasseri TM13-16 in the present disclosure
  • Figure 4 the bacterial count of Lactobacillus gasseri in each experimental group in Example 9;
  • Figure 5 The bacterial count of Gardnerella in each experimental group in Example 9;
  • Figure 6 The results of adjuvant treatment of bacterial vaginosis after the subjects in Example 10 took Lactobacillus gasseri TM13-16.
  • Embodiments of the present disclosure are described in detail below.
  • the embodiments described below are exemplary only for explaining the present disclosure and should not be construed as limiting the present disclosure. If no specific technique or condition is indicated in the examples, the technique or condition described in the literature in the field or the product specification is used.
  • the reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.
  • Example 1 Isolation and identification of Lactobacillus gasseri TM13-16
  • the isolated sample came from the feces of a healthy person.
  • the feces were collected into sterile sample tubes and brought back to the laboratory for sorting within 1 hour.
  • the collected fresh samples were immediately transferred to the anaerobic operation box, and 0.2 g of the sample was taken in 1 ml of sterile PBS (phosphate buffered saline), fully shaken and mixed, and then subjected to gradient dilution coating, and the medium was modified PYG medium.
  • sterile PBS phosphate buffered saline
  • the specific formula is (1L): tryptone 8g, soy peptone 2g, polyprotein 1g, casein 1g, yeast powder 10g, beef extract 5g, glucose 5g, K 2 HPO 4 2g, maltose 0.5g, cellobiose 0.5 g, soluble starch 0.5g, sodium sulfide 0.25g, Tween 80 0.5mL, Cysteine-HCl ⁇ H 2 O 0.5g, glycerol 0.5mL, sodium acetate 5g, heme 5mg, vitamin K 11 ⁇ L, inorganic salt solution (per liter inorganic salt The solution contains CaCl 2 ⁇ 2H 2 O 0.25g, MgSO 4 ⁇ 7H 2 O 0.5g, K 2 HPO 4 1g, KH 2 PO 4 1g, NaHCO 3 10g, NaCl 2g) 40mL, resazurin 1mg, add distilled water to 1L , adjust the pH to 6.8-7.0.
  • the obtained pure cultured strain was cultured to a concentration of about 10 9 CFU/mL, 400 ⁇ L of bacterial liquid was added with 400 ⁇ L of 40% glycerol to make the glycerol concentration reach 20%, and then cryopreserved at -80°C.
  • the vacuum freeze-dried powder of the strain was prepared according to the following operation steps, and was stored in the Guangdong Provincial Microorganism Culture Collection Center GDMCC No: 60405.
  • the ampoule tube and protective agent were sterilized by autoclaving for later use, streaked the bacterial liquid cultured overnight, incubated at 37°C for 24 hours, and performed the following operations after observing that no bacterial contamination was found.
  • the bacterial liquid was collected by centrifugation and washed with sterilized normal saline, added with 2-3 mL of skimmed milk protective agent, suspended to prepare a bacterial suspension with a colony count of 10 8 to 10 10 /mL, and packed in sterile ampoules. Pre-freeze in -80°C refrigerator for 1-2 hours. According to the "Standard Operating Procedure of Freeze Dryer", freeze-dry in freeze-drier for 8-20h until freeze-drying.
  • the obtained isolated strains were cultured in liquid PYG medium for 24 hours, 1 mL of bacterial liquid was taken and centrifuged at 10,000 r/min for 5 minutes, the bacteria were collected, and genomic DNA was extracted. Using genomic DNA as a template, 16S rDNA universal primers were used for PCR amplification.
  • the amplification system was: 10 ⁇ PCR buffer, 3 ⁇ L; dNTP, 2.5 ⁇ L; 27F(5'-AGAGTTTGATCATGGCTCAG-3', as shown in SEQ ID NO:1 shown), 0.5 ⁇ L; 1492R (5′-TAGGGTTACCTTGTTACGACTT-3′, shown in SEQ ID NO: 2), 0.5 ⁇ L; Taq enzyme, 0.3 ⁇ L; template, 1 ⁇ L; ddH 2 O, 18.2 ⁇ L.
  • PCR amplification conditions were: pre-denaturation at 95°C for 4 min, followed by 30 cycles of denaturation at 95°C for 30s, annealing at 57°C for 40s, and extension at 72°C for 1 min for 30s.
  • the obtained 16S rDNA amplification product was detected by electrophoresis, purified, and sequenced at 3730 to obtain a 16S rDNA sequence with a length of 1569 bp (SEQ ID No: 3). This sequence was analyzed by blast in genebank, and the identification result of TM13-16 was obtained as Lactobacillus gasseri.
  • the 16S rDNA sequence of Lactobacillus gasseri TM13-16 is as follows:
  • TM13-16 After culturing in MRS medium (purchased from Huankai Microorganism Technology Co., Ltd.) for 48 hours, the colonies of TM13-16 were white, opaque, smooth, round, with irregular edges, with white protrusions in the middle, and the colony diameter was about 0.5-1.5 mm. Observation under the microscope of 1000 times showed that the cells were elongated and rod-shaped, and the Gram staining was negative, and no spores and flagella were produced.
  • the catalase reaction of TM13-16 was positive, the fermentation of sugar alcohol was positive, and it was facultatively anaerobic.
  • the carbon source utilization was detected by API 50 CHL kit. The results are shown in Table 1 (+ means positive reaction; - means negative reaction; W means weak positive reaction).
  • Example 2 Genome sequencing and species classification and functional gene analysis of Lactobacillus gasseri TM13-16
  • strain has this function, and the higher the number of gene copies, the stronger the function ( Figure 2).
  • the genome is not annotated with antibiotic resistance genes, virulence factors, plasmids, transfer elements, bacteriophages and viruses, which proves that the strain can be used safely. .
  • the bioactive substances of TM13-16 were mainly examined for L-lactic acid content, D-lactic acid content and hydrogen peroxide production in metabolites.
  • strain TM13-16 was inoculated into MRS medium and cultured at 37°C for 24h under aerobic and anaerobic conditions, respectively.
  • L-lactic acid and D-lactic acid content were measured using L-Lactic Acid (L-Lactate) Assay Kit and D-Lactic Acid (D-Lactate) Assay Kit (purchased from Megazyme Inc. US) according to the standard operating manual.
  • the hydrogen peroxide content was measured with a hydrogen peroxide assay kit (colorimetric method) (purchased from Nanjing Jiancheng Bioengineering Institute) according to the standard operation manual.
  • the present disclosure selects Lactobacillus gasseri GDMCC 60092 and commercially available Lactobacillus rhamnosus GR-1, Lactobacillus reuteri RC-14 and Lactobacillus delbrueckii as control experiments,
  • the experimental method was the same as above, and the results showed that under anaerobic conditions, the lactic acid production of TM13-16 was significantly higher than that of other strains, especially L-lactic acid.
  • the results are shown in Figures 3A and 3B.
  • TM13-16 (7.9844g/L total amount of lactic acid) is higher than the lactic acid output (5.8280g/L total amount of lactic acid) of Lactobacillus gasseri GDMCC 60092 .
  • Example 4 Identification of the ability of Lactobacillus gasseri TM13-16 to inhibit reproductive tract infection pathogens
  • TM13-16 and Lactobacillus gasseri GDMCC 60092 bacterial solutions were seeded on MRS solid medium, and cultured at 37 °C for 24 h under anaerobic conditions to obtain bacterial plaques.
  • TM13-16 and Lactobacillus gasseri GDMCC 60092 bacteria were seeded on MRS solid medium, and cultured at 37 °C for 24 h under anaerobic conditions to obtain plaques.
  • the Gardnerella Columbia blood plate medium containing 0.5% agar was prepared, and after sterilization at 121°C for 20 minutes, the temperature was lowered to 50-60°C, and then 500 ⁇ L of 10 7 CFU/mL Gardnerella vaginalis BNCC337545 was mixed with 10 mL of the semi-solid The medium was mixed and poured onto a plate with TM13-16 plaques.
  • the diameter of the inhibition zone was observed and measured to be 0.74 ⁇ 0.26 cm, the diameter of the inhibition zone of Lactobacillus gasseri GDMCC 60092 is 0.59 ⁇ 0.22 cm, indicating that the Lactobacillus gasseri TM13-16 in the present disclosure has better inhibitory ability on Lactobacillus gasseri GDMCC 60092 than Lactobacillus gasseri GDMCC 60092. .
  • TM13-16 and Lactobacillus gasseri GDMCC 60092 were co-inoculated with SC5314 into modified PYG medium, and cultured at 37°C for 24h under anaerobic conditions to obtain a mixed bacterial liquid. It was serially diluted and spread on PDA fungal culture plates, and the colonies were counted after culturing at 37°C for 24 hours under aerobic conditions. At the same time, only inoculated with TM13-16 was used as a negative control, and only inoculated with SC5314 was used as a positive control.
  • TM13-16 was 8.21x10 3 CFU/mL; Lactobacillus gasseri GDMCC 60092 was 1.02x10 4 CFU/m; positive control group was 8.60x10 5 CFU/mL; negative control was 0 CFU/mL.
  • the results showed that Lactobacillus gasseri TM13-16 could effectively inhibit the growth of Candida albicans SC5314, and the inhibitory effect was significantly better than that of Lactobacillus gasseri GDMCC 60092.
  • TM13-16 The sensitivity of TM13-16 to 15 common antibiotics was investigated, and the drug sensitive paper method was used for the experiment. 100 ⁇ L of the bacterial solution of TM13-16 cultured to the logarithmic phase was used for flat coating, and the antibiotic drug sensitive sheet was pasted on the surface of the flat plate. , cultured at 37°C for 48h, and measured the size of the inhibition zone. The results are shown in Table 3.
  • antibiotic Inhibition zone diameter (cm) antibiotic Inhibition zone diameter (cm) Ampicillin 3 Ceftriaxone 3 Bacitracin 0 Vancomycin 1.9 penicillin 3.5 oxacillin 1.5 kanamycin 1.5 amoxicillin 2.5 tetracycline 3 Azithromycin 1.8 guaracillin 3.1 Clindamycin 0 erythromycin 2.8 Gentamicin 0 Chloramphenicol 2.7
  • TM13-16 was resistant to bacitracin, clindamycin and gentamicin, and sensitive to 12 other antibiotics, and could be used safely.
  • Example 6 Tolerance of Lactobacillus gasseri TM13-16 to acids and bile salts
  • MRS mediums of pH 2, pH 3, pH 4, pH 4.5 and pH 7 were prepared respectively, and 100 ⁇ L of TM13-16 bacterial liquid with a concentration of 1.2E+9 overnight culture was inoculated into MRS medium of different pH, and cultured at 37°C After 24h, the bacterial liquid was counted by plate coating.
  • TM13-16 could not only survive, but also grow under the conditions of pH 2, pH 3, pH 4, pH 4.5, and pH 7 (Table 4).
  • MRS medium containing 0.05%, 0.1%, 0.2%, and 0.3% bile salts was prepared respectively, and 100 ⁇ L of TM13-16 bacteria cultured at a concentration of 1.2E+9 overnight was inoculated into MRS medium with different bile salt contents, 37 After culturing at °C for 24 h, the bacterial liquid was counted by plate coating. The results showed that TM13-16 could not only survive but also grow under the conditions of 0.05%, 0.1%, 0.2%, and 0.3% bile salts (Table 5).
  • Lactobacillus gasseri TM13-16 of the present disclosure has strong tolerance to acids and bile salts.
  • Example 7 Evaluation of the colonization ability of Lactobacillus gasseri TM13-16
  • TM13-16 bacterial solution was divided into 10 mL in 15 mL test tubes, the uppermost bacterial solution was taken, and the absorbance value at OD600 was measured with an ultraviolet spectrophotometer. Let stand at room temperature for 30min, then take the uppermost bacterial liquid, and measure the absorbance value at OD600 with an ultraviolet spectrophotometer. The absorbance values before and after were compared, and the larger the difference, the stronger the self-aggregation ability. TM13-16 showed better self-aggregation ability (Table 6).
  • the six-well culture plate was taken out, the bacterial suspension was discarded, and the monolayer was washed 5 times with sterilized PBS buffer to remove unadhered bacteria, and then fixed with anhydrous methanol for 20 min.
  • the above cell slides fixed with anhydrous methanol were taken for Gram staining. After drying, the bacteria were observed and counted under a microscope, and the number of bacteria adhered to 100 cells in 20 random fields of view was calculated, and the average adhesion of each cell was calculated to be 46.25 ⁇ 22.41.
  • the present disclosure selects Lactobacillus gasseri GDMCC 60092 as a control test, and the experimental method is the same as above.
  • the results show that each Hela cell adheres to an average of 25.23 ⁇ 2.12 Lactobacillus gasseri GDMCC 60092, indicating the adhesion of TM13-16 to human cervical cancer cells Hela more capable.
  • the six-well culture plate was taken out, the bacterial suspension was discarded, and the monolayer was washed 5 times with sterilized PBS buffer to remove unadhered bacteria, and then fixed with anhydrous methanol for 20 min.
  • the above cell slides fixed with anhydrous methanol were taken for Gram staining. After drying, the cells were observed and counted under a microscope, and the number of bacteria adhered to 100 cells in 20 random fields of view was calculated, and the average adhesion of each cell was calculated to be 107.35 ⁇ 61.88.
  • the present disclosure selects Lactobacillus gasseri GDMCC 60092) as a control test, the experimental method is the same as above, the results show that each VK2E6/E7 cell adheres to an average of 31.2 ⁇ 4.25 Lactobacillus gasseri GDMCC 60092, indicating that TM13-16 and human vaginal epithelial cells VK2E6
  • the adhesion ability of /E7 is stronger. The stronger the adhesion ability, the stronger the colonization ability of the strain, the easier it is to survive in the vaginal environment for reproduction, and then play the function of inhibiting pathogenic bacteria, protecting the vaginal mucosa, restoring the vaginal flora, and achieving the treatment or prevention of vaginal infection symptoms. Efficacy.
  • Example 8 Rat toxicity test of Lactobacillus gasseri TM13-16
  • the rats selected in this example are SD female rats, 7 weeks old, body weight 250g ⁇ 50g, the rat rearing environment is SPF grade, and the experimental animals are divided into 6 groups, which are respectively 3 groups of gavage groups and 3 groups of vaginal irrigation groups. Wash group. Each group of 10 animals, a total of 60 animals, were fed with a standard diet.
  • Oral gavage group divided into 3 groups, gavage with different doses, each rat was orally gavaged with 0.5ml of fresh bacterial solution, the concentrations were 1 ⁇ 10 5 , 1 ⁇ 10 9 , 1 ⁇ 10 12 CFU/mL, every day Once for 3 consecutive days, from the first day of gavage to the seventh day, the rats should be healthy and survive and gain weight.
  • Vaginal lavage group divided into 3 groups, the vagina was lavaged with different doses, each rat was lavaged with 0.2 mL of fresh bacterial solution, the concentrations were 1 ⁇ 10 5 , 1 ⁇ 10 9 , 1 ⁇ 10 12 CFU/mL, respectively , once a day for 3 consecutive days, from the 1st day of lavage to the 7th day, the rats should survive healthy and gain weight.
  • Example 9 The ability of Lactobacillus gasseri TM13-16 to treat genital tract infection in rats
  • the rat model selected in this example is a mouse model infected with Gardnerella genitalium, SD female rats, 7 weeks old, body weight 250g ⁇ 250g, and the rats are reared in an SPF grade experimental animal and divided into 3 groups , respectively model group, Lactobacillus gasseri TM13-16 group and Lactobacillus gasseri GDMCC 60092 group. Each group of 10 animals, a total of 30 animals, were fed with a standard diet.
  • Adaptation period Rats were fed a standard diet for 7 days. Three days before modeling, 0.5 mg ⁇ -estradiol-3-benzoate was subcutaneously injected to keep the rat estrus phenomenon. Before modeling, observe and record whether the rat vagina is red, swollen and discharge, whether there are erythema and particulate phenotype; modeling period: use overnight cultured Gardnerella vaginalis (purchased from Beijing Beina Chuanglian Institute of Biotechnology) (PBS bacterial solution) rinse the rat vagina once a day for 3 consecutive days.
  • Gardnerella vaginalis purchasedd from Beijing Beina Chuanglian Institute of Biotechnology
  • Intervention period A total of 7 days, the rats in the model group were not treated; the rats in the Lactobacillus gasseri TM13-16 group and the Lactobacillus gasseri GDMCC 60092 group were washed with the PBS bacterial solution of the two strains respectively, each time.
  • the total amount of viable bacteria was not less than 1 ⁇ 10 8 CFU/mL, and the cells were washed once a day for 7 consecutive days.
  • Example 10 The ability of Lactobacillus gasseri TM13-16 adjuvant therapy to treat reproductive tract infectious diseases
  • Lactobacillus gasseri TM13-16 as one of the main active ingredients of the intervention product, a single-center, randomized controlled clinical study was carried out, and a 6-month intervention study was carried out according to the intervention plan (Figure 6).
  • the inclusion criteria of the subjects were women of childbearing age with simple bacterial vaginosis (diagnosed by Amsel criteria).
  • azole group 30 cases
  • intervention group metalronidazole group + probiotic group
  • a total of 67 subjects met the inclusion criteria, including 34 in the control group and 33 in the intervention group.
  • a total of 59 subjects were finally included in the analysis, including 27 in the control group and 32 in the intervention group.
  • the analysis results are as follows, the probiotic group can improve the 14-day cure rate (Table 8), and reduce the 3-month recurrence rate (Table 9).
  • Lactobacillus gasseri TM13-16 can be combined with other antibacterial drugs (such as metronidazole) to treat bacterial vaginosis (BV), compared with single drug treatment, the combination treatment It can significantly improve the cure rate and reduce the recurrence rate, indicating that Lactobacillus gasseri TM13-16 can assist in the treatment of reproductive tract infectious diseases.
  • other antibacterial drugs such as metronidazole
  • BV bacterial vaginosis

Abstract

一种用于预防和/或治疗生殖道感染和/或生殖道菌群紊乱的格氏乳杆菌菌株及其应用，该格氏乳杆菌(Lactobacillus gasseri)为TM13-16，保藏号为GDMCC No:60405，该菌株能够产生乳酸和过氧化氢等分泌物，以抑制生殖道感染致病菌的生长，用于预防和/或治疗生殖道感染疾病。

Description

用于预防和/或治疗生殖道菌群紊乱相关疾病的格氏乳杆菌
优先权信息
本申请请求2020年11月10日向中国国家知识产权局提交的、专利申请号为202011250419.4的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本公开涉及生物技术领域，具体涉及一种新的用于预防和/或治疗生殖道感染的格氏乳杆菌菌株及其应用。
背景技术
女性生殖道环境常会受到激素水平波动、卫生情况控制不佳、季节变化、甚至心情不稳定等各种影响而造成菌群紊乱，进而导致病原菌入侵，引发细菌、霉菌、病毒等微生物感染，常见的有大肠杆菌、加德纳菌、白色念珠菌和HPV病毒等。生殖道感染疾病的临床症状表现为：阴道瘙痒灼热，分泌物异常，尿频尿痛，严重则会导致孕妇早产，宫颈癌等。据统计，我国育龄女性中生殖道微生物感染率高达80％，绝大多数细菌性或真菌性感染的患者接受的临床治疗手段是使用抗生素。抗生素在对抗病原菌的同时也会降低益生菌的丰度，不利于维持生殖道菌群稳态，从而增高复发风险。而针对HPV病毒感染患者，现在暂无完善的治疗方法，多采用定期筛查和随访的方式，严重则会进行手术治疗。
因此，亟需开发一种能够预防或治疗女性生殖道感染的同时，又不会影响生殖道环境益生菌的丰度的菌种或药物。
发明内容
女性的生殖健康和生殖道内生存的微生物菌群息息相关，也已有多项研究表明生殖道的菌群紊乱是导致细菌性阴道炎、霉菌性阴道炎或HPV感染的直接原因。现代医学对生殖道炎症疾病的治疗主要集中在服用抗生素、放置抗生素栓剂等药物治疗。但抗生素药物治疗存在很多问题：第一，抗生素在杀死病原菌，消除炎症的同时也抑制了益生菌的生长，降低了益生菌的丰度，非常不利于生殖道维持稳态，大大增加了重复感染和复发的风险；第二，服用药物不能达到精准治疗的目的，只能广谱性的全身范围内作用，尤其容易损伤肝肾功能；第三，经常使用抗生素容易导致病原菌产生耐药性，这也是造成二次感染的重要原因。
在阴道菌群中，虽然存在个体差异，但绝大多数女性生殖道内的优势菌种是乳酸杆菌， 且主要集中于四种：卷曲乳杆菌、格氏乳杆菌、詹氏乳杆菌和惰性乳杆菌。这些乳杆菌主要通过产生乳酸来维持阴道较低的pH值，从而抑制病原菌的入侵和生长，另外，乳杆菌还可合成过氧化氢以保证阴道内的厌氧环境，以防止好氧细菌的滋生。一旦菌群结构被破坏，病原菌便会感染生殖道，引发炎症，并且和乳杆菌形成竞争关系，从而破坏生殖道微生物稳态。此时，补充乳杆菌以维持健康的生殖道菌群结构比单一使用抗生素更有效。
本公开为解决现有技术中的药物具有不同程度副作用、复发率高或创伤大等技术问题。筛选出一种新的用于预防和/或治疗生殖道感染(尤其指人类中女性生殖道感染)的格氏乳杆菌(Lactobacillus gasseri)TM13-16，其能够产生乳酸和过氧化氢等分泌物，以抑制生殖道感染致病菌的生长，用于预防和/或治疗生殖道感染疾病，尤其降低复发率，毒副作用小，效力持久。
为此，本公开一方面提供一株格氏乳杆菌(Lactobacillus gasseri)TM13-16，其保藏号为GDMCC No:60405。
本公开从约30,000株的人体共生单菌库里筛选出一株女性生殖道益生菌格氏乳杆菌(Lactobacillus gasseri)TM13-16，该菌株是一株全新的分离株，该菌株已于2018年8月24日在广东省微生物菌种保藏中心(GDMCC，广东省广州市越秀区先烈中路100号大院实验大楼5楼)保藏，其保藏编号为GDMCC No:60405。
本公开所述格氏乳杆菌(Lactobacillus gasseri)TM13-16采用改良版PYG培养基进行分离，鉴定后用MRS培养基传代保藏。在MRS培养基中培养48小时，TM13-16的菌落是白色不透明、光滑、圆形、边缘不规整，中间有白色凸起，菌落直径约0.5-1.5mm。在1000倍的显微镜下观察，菌体呈现细长杆状，革兰氏染色为阴性，没有芽孢和鞭毛产生。
本公开提供的格氏乳杆菌(Lactobacillus gasseri)TM13-16，经研究发现其无质粒和转移元件等风险基因；同时表现出强共凝集能力和对人阴道上皮细胞的强粘附能力，有较强产L-乳酸、D-乳酸和过氧化氢的能力；生长能力强且高度耐酸碱；对大多抗生素敏感，对常见的阴道感染的致病菌有强抑菌能力，可用于预防和/或治疗生殖道微生物感染。
本公开另一方面提供所述的格氏乳杆菌或其发酵产物或其菌悬液或其培养液在制备产品中的应用，所述产品用于预防和/或治疗生殖道菌群紊乱相关疾病。
在本公开的一些实施方案中，所述生殖道菌群紊乱相关疾病包括生殖道感染。
在本公开的一些实施方案中，所述生殖道菌群紊乱相关疾病为生殖道感染。
在本公开的一些实施方案中，所述生殖道感染包括选自细菌性阴道炎、霉菌性阴道炎、滴虫性阴道炎、好氧性阴道炎、老年性阴道炎和病毒感染中的至少之一。
在本公开的另一些实施方案中，引起所述细菌性阴道炎的细菌为引起女性生殖道感染的常见细菌，包括大肠杆菌(E.coli ATCC35218)、加德纳杆菌(Gardnerella vaginalis  BNCC337545)、棒状杆菌、嗜血杆菌、金黄色葡萄球菌、铜绿假单胞菌等革兰氏阴性厌氧菌。
在本公开的一些实施方案中，引起所述霉菌性阴道炎的霉菌为引起女性生殖道感染的常见假丝酵母菌，包括白假丝酵母菌(白色念珠菌Candida albicans SC5314)、热带假丝酵母菌、近平滑假丝酵母菌和都柏林假丝酵母菌等。
在本公开的另一些实施方案中，引起所述生殖道病毒感染的病毒为引起女性生殖道感染的常见病毒，包括HPV、单纯疱疹病毒、巨细胞病毒等。
在本公开又一方面提供所述的格氏乳杆菌或其发酵产物或其菌悬液或其培养液在制备产品中的应用，所述产品用于抗菌、粘附阴道上皮细胞和/或宫颈细胞、产乳酸、产H 2O 2。
在本公开的一些实施方案中，所述产品为食品、药物或者保健品。其中，所述食品可以是益生菌酸奶、益生菌片、益生菌固体饮料等。
在本公开的一些实施方案中，所述格氏乳杆菌的给药剂量为10 5-10 12CFU/天。
本公开又一方面提供一种食品或保健品，所述食品或保健品包括所述的格氏乳杆菌或其发酵产物或其菌悬液或其培养液。
在本公开的一些实施方案中，所述食品或保健品选自预防和/或治疗生殖道菌群紊乱相关疾病的产品、抗菌的产品、粘附阴道上皮细胞和/或宫颈细胞的产品、产乳酸的产品、产H 2O 2的产品中的至少之一。
本公开又一方面提供一种药物组合物，所述药物组合物包括所述的格氏乳杆菌或其发酵产物或其菌悬液或其培养液。
在本公开的一些实施方案中，所述药物组合物呈单剂量形式，所述药物组合物含有日剂量为10 5-10 12CFU的所述的格氏乳杆菌。
在本公开的另一些实施方案中，所述药物组合物呈适于外用或者口服的剂型。
本公开又一方面提供上述的格氏乳杆菌或其发酵产物或其菌悬液或其培养液、上述的食品或保健品、上述的药物组合物在预防和/或治疗生殖道菌群紊乱相关疾病中的应用。
在本公开的一些实施方案中，所述生殖道菌群紊乱相关疾病包括生殖道感染。
在本公开的一些实施方案中，所述生殖道感染包括选自细菌性阴道炎、霉菌性阴道炎、滴虫性阴道炎、好氧性阴道炎、老年性阴道炎和病毒感染中的至少之一。
本公开又一方面提供一种预防和/或治疗生殖道菌群紊乱相关疾病的方法。在本公开的一些实施方案中，所述方法包括：
向有需要的受试者施用以下中的至少之一：
(1)上述的格氏乳杆菌或其发酵产物或其菌悬液或其培养液；
(2)上述的食品或保健品；
(3)上述的药物组合物。
在本公开的一些实施方案中，所述生殖道菌群紊乱相关疾病包括生殖道感染。
在本公开的一些实施方案中，所述生殖道感染包括选自细菌性阴道炎、霉菌性阴道炎、滴虫性阴道炎、好氧性阴道炎、老年性阴道炎和病毒感染中的至少之一。
本公开中所用的“生殖道感染”是指动物中的雌性或人类中女性的生殖道感染。
目前，也有报道利用乳杆菌预防或治疗女性生殖道感染，例如鼠李糖乳杆菌GR-1和罗伊氏乳杆菌RC-14的组合菌，这两株菌已经被公认且被广泛商业化，作为维护女性生殖道健康的益生菌菌株。产品的品牌很多，有Jarrow Formulas,Blackmores，Renew life和Clinicans。但其菌种非生殖道优势菌种，且相关临床研究仅限于欧美人群，缺少亚洲人群疗效评估。
利用抗生素和栓剂进行治疗又存在诸多缺点。例如，甲硝唑、替硝唑、克林霉素等抗生素，其作用是杀死入侵的厌氧病原细菌，从而治疗细菌性阴道炎。但同时也抑制了阴道益生菌乳杆菌的生长，无法重建健康的菌群结构。咪康唑、克霉唑等栓剂，主要用于霉菌性阴道炎，其作用机制是抑制念珠菌等真菌细胞膜的固醇合成，影响细胞膜通透性，抑制真菌生长，导致死亡。该药物常用于栓剂，具有一定使用限制性，造成诸多不便，且不正当使用会造成二次感染。
而口服益生菌的方法干预肠道菌群调节免疫系统，局部使用补充生殖道乳酸杆菌的丰度，减少传统抗生素治疗带来的弊端，在抑制或清除病原菌的同时帮助菌群恢复稳态，大大提高治愈率，降低复发率，帮助HPV转阴，有效治疗或预防生殖道微生物感染疾病，解决临床一大难题。
本公开的发明人从诸多人体共生单菌库里筛选出一株女性生殖道益生菌格氏乳杆菌(Lactobacillus gasseri)TM13-16，该菌株是一株全新的分离株，其具有较强产L-乳酸、D-乳酸和过氧化氢的能力，强耐酸碱，并对大多抗生素敏感，同时有很强的共凝集能力，对人阴道上皮细胞也表现出强粘附能力，此外，对常见的阴道感染的致病菌有强抑菌能力的格氏乳杆菌TM13-16，可作为食品、保健品、外用或口服药品开发以预防、治疗或辅助治疗女性生殖道感染的病症。本公开中的格氏乳杆菌通过产生乳酸和过氧化氢等分泌物，以抑制生殖道感染致病菌的生长，用于预防和/或治疗生殖道感染疾病，尤其降低复发率，毒副作用小，效力持久。
本公开的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本公开的实践了解到。
保藏信息：
菌株名称：Lactobacillus gasseri TM13-16
保藏日期：2018年8月24日
保藏单位：广东省微生物菌种保藏中心(GDMCC)
保藏编号：GDMCC No:60405
保藏地址：广州市先烈中路100号大院59号楼5楼
附图说明
本公开的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1：格氏乳杆菌(Lactobacillus gasseri)TM13-16的基因组图谱；
图2：格氏乳杆菌(Lactobacillus gasseri)TM13-16的益生通路注释结果；
图3A：格氏乳杆菌(Lactobacillus gasseri)TM13-16与商业菌株L-乳酸的比较；
图3B：格氏乳杆菌(Lactobacillus gasseri)TM13-16与商业菌株D-乳酸的比较；
在图中3A和3B中，RC-14表示罗伊氏乳杆菌(Lactobacillus reuteri)RC-14，GR-1表示鼠李糖乳杆菌(Lactobacillus rhamnosus)GR-1，D表示德氏乳杆菌(Lactobacillus delbrueckii)，TM13-16表示本公开中的格氏乳杆菌(Lactobacillus gasseri)TM13-16；
图4：实施例9中各实验组中格氏乳杆菌的菌数；
图5：实施例9中个实验组中加德纳杆菌的菌数；
图6：实施例10中受试者服用格氏乳杆菌(Lactobacillus gasseri)TM13-16后辅助治疗细菌性阴道病的结果。
发明详细描述
下面详细描述本公开的实施例。下面描述的实施例是示例性的，仅用于解释本公开，而不能理解为对本公开的限制。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：格氏乳杆菌(Lactobacillus gasseri)TM13-16的分离鉴定
1、样品采集
分离样品来自于一位健康人粪便，将粪便采集至无菌的样品管中，1h内带回实验室进行分选。
2、分离纯化
收集的新鲜样品立刻转移至厌氧操作箱中，取0.2g样品于1ml无菌的PBS(磷酸缓冲液)中，充分震荡混匀，然后进行梯度稀释涂布，培养基采用改良版PYG培养基，具体配方是(1L)：胰蛋白胨8g，大豆蛋白胨2g，多聚蛋白1g，干酪素1g，酵母粉10g，牛肉膏5g，葡萄糖5g，K 2HPO 4 2g，麦芽糖0.5g，纤维二糖0.5g，可溶性淀粉0.5g，硫化钠0.25g，Tween 80 0.5mL，Cysteine-HCl·H 2O 0.5g，甘油0.5mL，乙酸钠5g，血红素5mg，维生素K11μL，无机盐溶液(每升无机盐溶液含有CaCl 2·2H 2O 0.25g，MgSO 4·7H 2O 0.5g，K 2HPO 41g，KH 2PO 4 1g，NaHCO 3 10g，NaCl 2g)40mL，刃天青1mg，加入蒸馏水至1L，调节pH至6.8～7.0。涂布的平板置于37℃厌氧培养，厌氧的气体组分为N 2：CO 2：H 2＝90：5：5。培养3天后，挑取单菌落进行划线分离，获得每株单菌的纯培养菌株。
3、菌种保藏
对获得的纯培养菌株进行培养，至浓度约为10 9CFU/mL，取400μL菌液添加40％甘油400μL，使其甘油浓度达到20％，然后进行-80℃超低温保藏。
按照如下操作步骤制作菌株真空冻干粉，并保藏至广东省微生物菌种保藏中心GDMCC No:60405。
将安瓿管和保护剂经高压灭菌待用，对过夜培养的菌液进行划线，37℃培养24h，观察未发现杂菌污染后进行以下操作。菌液经离心收集并用灭菌生理盐水洗涤，加入2～3mL脱脂牛奶保护剂，悬浮制成菌落数为10 8～10 10个/mL菌悬液，并分装在无菌安瓿管中，置于-80℃冰箱中预冻1～2小时。按照《冷冻干燥机标准操作规程》，在冷冻干燥机中进行冷冻干燥8～20h，直至冻干为止。冻干后取出样品安瓿管，按照《真空安瓿熔封机标准操作规程》，将安瓿管颈部棉塞以下处用强火焰拉细进行熔封。
4、16S rDNA鉴定
将获得的分离菌株在液体PYG培养基中培养24h，取1mL菌液进行10000r/min离心5min，收集菌体，提取基因组DNA。以基因组DNA作为模板，使用16S rDNA通用引物进行PCR扩增，扩增体系为：10×PCR buffer，3μL；dNTP，2.5μL；27F(5’-AGAGTTTGATCATGGCTCAG-3’，如SEQ ID NO:1所示)，0.5μL；1492R(5’-TAGGGTTACCTTGTTACGACTT-3’，如SEQ ID NO:2所示)，0.5μL；Taq酶，0.3μL；模板，1μL；ddH 2O，18.2μL。PCR扩增条件为：95℃预变性4min，然后95℃变性30s，57℃退火40s，72℃延伸1min30s，30个循环。将获得的16S rDNA扩增产物进行电泳检测、纯化、3730测序，获得长度为1569bp的16S rDNA序列(SEQ ID No:3)。将这段序列在genebank中进行blast比对分析，获得TM13-16的鉴定结果为格氏乳杆菌(Lactobacillus gasseri)。
格氏乳杆菌(Lactobacillus gasseri)TM13-16的16S rDNA序列如下：
Figure PCTCN2021129910-appb-000001
5、TM13-16的生理生化特征
在MRS培养基(购自环凯微生物科技公司)中培养48小时，TM13-16的菌落是白色不透明、光滑、圆形、边缘不规整，中间有白色凸起，菌落直径约0.5-1.5mm。在1000倍的显微镜下观察，菌体呈现细长杆状，革兰氏染色为阴性，没有芽孢和鞭毛产生。TM13-16的过氧化氢酶反应为阳性，糖醇发酵阳性，兼性厌氧，碳源利用情况使用API 50 CHL试剂盒进行检测。结果如表1(+，表示阳性反应；-，表示阴性反应；W表示弱阳性反应)。
表1 TM13-16碳源利用结果
编号 反应 结果 编号 反应 结果
1 对照 + 26 七叶灵 +
2 甘油 - 27 水杨苷 +
3 赤藻糖醇 - 28 D-纤维二糖 +
4 D-阿拉伯糖 - 29 D-麦芽糖 +
5 L-阿拉伯糖 - 30 D-乳糖 +
6 D-核糖 - 31 D-蜜二糖 W
7 D-木糖 - 32 D-蔗糖 +
8 L-木糖 - 33 D-海藻糖 +
9 D-侧金盏花醇 - 34 菊粉 W
10 甲基-βD吡喃木糖苷 - 35 D-松三糖 W
11 D-半乳糖 + 36 D-棉子糖 W
12 D-葡萄糖 + 37 淀粉 W
13 D-果糖 + 38 糖原 -
14 D-甘露糖 + 39 木糖醇 -
15 L-山梨糖 W 40 D-龙胆二糖 +
16 L-鼠李糖 - 41 D-土伦糖 -
17 卫矛醇 - 42 D-来苏糖 -
18 肌醇 - 43 D-塔格糖 +
19 甘露醇 - 44 D-岩藻糖 -
20 山梨醇 + 45 L-岩藻糖 -
21 甲基-αD吡喃甘露糖苷 - 46 D-阿拉伯醇 -
22 甲基-αD吡喃葡萄糖苷 - 47 L-阿拉伯醇 -
23 N-乙酰葡萄糖胺 + 48 葡萄糖酸钾 -
24 苦杏仁苷 + 49 2-酮基葡萄糖酸钾 -
25 熊果苷 W 50 5-酮基葡萄糖酸钾 -
实施例2：格氏乳杆菌(Lactobacillus gasseri)TM13-16的基因组测序及物种分类和功能基因分析
1、基因组测序
将过夜培养的TM13-16菌液，在7,227g 4℃下离心10分钟，将得到的沉淀重悬于1mL Tris-EDTA中，加入50μL 10％SDS和10μL蛋白酶K(20mg/mL)在55℃下水浴2小时，使细胞裂解，并用酚-氯仿法提取DNA。并用Illumina Hiseq 2000平台对其进行测序，测序长度为双向500bp，并用SOAPdenovo对reads进行组装。经过评估后，用GCskew分析全基因组GC含量及可视化全基因组序列及功能分布(图1)。
2、菌株基因组物种分类
使用Checkm软件对全基因组序列进行比对分析，得出与该基因组最相近的物种信息为厚壁菌门-芽孢杆菌纲-乳杆菌目-乳杆菌科-乳杆菌属-格氏乳杆菌种，注释到的基因组数量为 58，注释到的标记数量为449，基因组的完整度为99.22％，污染程度为0.78％。
3、益生功能基因及安全性分析
为了检查在TM13-16中与治疗生殖道感染疾病相关的代谢通路是否完善，我们分别从KEGG的prokaryoties的数据库中，选出了与乳酸合成(Lactic acid synthesis)、过氧化氢产生(Peroxide hydrocarbon production)和短链脂肪酸合成(short-chain fatty acid synthesis)所有相关的酶，分别建立单独的数据库。使用blastx将TM13-16的全基因序列分别与这些数据库比对，选择e-value大于等于0.01，identity大于等于60的注释结果，相关通路的酶的基因拷贝数多少用颜色深浅表示，注释到基因便证明菌株有该功能，基因拷贝数越多则证明功能越强(图2)，基因组未注释到抗生素抗性基因、毒力因子、质粒、转移元件、噬菌体及病毒，证明该菌株可安全使用。
实施例3：格氏乳杆菌(Lactobacillus gasseri)TM13-16产生的生物活性物质
TM13-16的生物活性物质主要考察代谢产物中的L-乳酸含量，D-乳酸含量和过氧化氢产生情况。
1、样品预处理
将菌株TM13-16接种至MRS培养基中，分别在好氧和厌氧条件下，于37℃培养24h。
各取1mL菌液进行8000r/min离心5min，取上清，待检测L-乳酸和D-乳酸含量。
再各取1mL菌液加入溶菌酶(终浓度为1mg/mL)，37℃静置15min，8000r/min离心5min，取上清，待检测过氧化氢浓度。
2、测定方法
用L-Lactic Acid(L-Lactate)Assay Kit和D-Lactic Acid(D-Lactate)Assay Kit(购自Megazyme Inc.US)按照标准操作手册测得L-乳酸和D-乳酸含量。
用过氧化氢测定试剂盒(比色法)(购自南京建成生物工程研究所)按照标准操作手册测得过氧化氢含量。
3、实验结果见表2。
表2 TM13-16乳酸及过氧化氢产量测定
Figure PCTCN2021129910-appb-000002
4、与商业菌株比较
本公开选择格氏乳杆菌GDMCC 60092以及市售鼠李糖乳杆菌(Lactobacillus rhamnosus  GR-1)、罗伊氏乳杆菌(Lactobacillus reuteri RC-14)和德氏乳杆菌(Lactobacillus delbrueckii)作为对照试验，实验方法同上，结果表明在厌氧条件下，TM13-16的乳酸产量显著高于其他菌株，尤其是L-乳酸，结果见图3A和3B。同时，本公开与格氏乳杆菌GDMCC 60092的产酸能力进行比较，TM13-16(7.9844g/L乳酸总量)高于格氏乳杆菌GDMCC 60092的乳酸产量(5.8280g/L乳酸总量)。
实施例4：格氏乳杆菌(Lactobacillus gasseri)TM13-16抑制生殖道感染致病菌的能力鉴定
1、TM13-16抑制大肠杆菌E.coli的能力鉴定
首先将2.5μL过夜培养的TM13-16和格氏乳杆菌GDMCC 60092的菌液分别点种在MRS固体培养基上，在厌氧条件下，于37℃培养24h，获得菌斑。配制含有0.5％琼脂的LB培养基，121℃高温高压灭菌20min后，待温度降至50-60℃，再将500μL 10 7CFU/mL的E.coli与10mL该半固体培养基混合，并倒入点种有TM13-16菌斑的平板上，待培养基凝固后，在厌氧条件下，于37℃培养24h后，观察并测量得TM13-16抑菌圈直径大小为1.37±0.17cm，格氏乳杆菌GDMCC60092抑菌圈直径大小为0.73±0.21cm，说明本公开中的格氏乳杆菌TM13-16对大肠杆菌的抑制能力优于格氏乳杆菌GDMCC 60092。
2、TM13-16抑制加德纳杆菌Gardnerella vaginalis BNCC337545的能力鉴定
首先将2.5μL过夜培养的TM13-16和格氏乳杆菌GDMCC 60092菌液分别点种在MRS固体培养基上，在厌氧条件下，于37℃培养24h，获得菌斑。配制含有0.5％琼脂的加德纳杆菌哥伦比亚血平板培养基，121℃高温高压灭菌20min后，待温度降至50-60℃，再将500μL10 7CFU/mL的Gardnerella vaginalis BNCC337545与10mL该半固体培养基混合，并倒入点种有TM13-16菌斑的平板上，待培养基凝固后，在厌氧条件下，于37℃培养24h后，观察并测量得抑菌圈直径大小为0.74±0.26cm，格氏乳杆菌GDMCC 60092抑菌圈直径大小为0.59±0.22cm，说明本公开中的格氏乳杆菌TM13-16对加德纳杆菌的抑制能力优于格氏乳杆菌GDMCC 60092。。
3、TM13-16抑制白色念珠菌Candida albicans SC5314的能力鉴定
将过夜活化的TM13-16和格氏乳杆菌GDMCC 60092分别与SC5314共同接种至改良PYG培养基中，在厌氧条件下，于37℃培养24h，获得混合菌液。将其连续稀释并涂布于PDA真菌培养平板，在好氧条件下，于37℃培养24h后，菌落计数。同时用只接种了TM13-16的作为阴性对照，用只接种了SC5314的作为阳性对照。计数结果显示TM13-16为8.21x10 3CFU/mL；格氏乳杆菌GDMCC 60092为1.02x10 4CFU/m；阳性对照组为8.60x10 5CFU/mL；阴性对照为0CFU/mL。结果表明格氏乳杆菌TM13-16可以有效抑制白色念珠菌Candida albicans SC5314的生长，并且抑制效果明显优于格氏乳杆菌GDMCC 60092。
实施例5：格氏乳杆菌(Lactobacillus gasseri)TM13-16的抗生素敏感情况
考察TM13-16对常见15种抗生素的敏感情况，采用药敏纸片法进行实验，取培养至对数期的TM13-16的菌液100μL进行平板涂布，将抗生素药敏片贴在平板表面，37℃培养48h，测量抑菌圈大小，其结果如表3。
表3 TM13-16的抗生素敏感情况
抗生素 抑菌圈直径(cm) 抗生素 抑菌圈直径(cm)
氨苄西林 3 头孢曲松 3
杆菌肽 0 万古霉素 1.9
青霉素 3.5 苯唑西林 1.5
卡那霉素 1.5 阿莫西林 2.5
四环素 3 阿奇霉素 1.8
呱啦西林 3.1 克林霉素 0
红霉素 2.8 庆大霉素 0
氯霉素 2.7    
结果显示，TM13-16对杆菌肽、克林霉素和庆大霉素有抗性，对其他12种抗生素敏感，可以安全使用。
实施例6：格氏乳杆菌(Lactobacillus gasseri)TM13-16对酸和胆盐的耐受能力
因人益生菌到达肠道需要经过胃和小肠，因此需要经历pH2.5左右的胃酸和0.3％浓度的胆盐。只有具有酸和胆盐耐受的菌株才能到达肠道发挥益生作用。所以本实施例对TM13-16的酸和胆盐耐受情况作了考察。
1、TM13-16的酸耐受情况
分别配制pH 2、pH 3、pH 4、pH 4.5、pH 7的MRS培养基，将100μL浓度为1.2E+9过夜培养的TM13-16菌液接种至不同pH的MRS培养基中，37℃培养24h后对菌液进行平板涂布计数。结果显示TM13-16在pH 2、pH 3、pH 4、pH 4.5、pH 7的条件下不仅可以存活，而且可以生长(表4)。
表4 TM13-16的酸耐受情况
Figure PCTCN2021129910-appb-000003
2、TM13-16的胆盐耐受情况
分别配制含有0.05％、0.1％、0.2％、0.3％胆盐的MRS培养基，将100μL浓度为1.2E+9过夜培养的TM13-16菌液接种至不同胆盐含量的MRS培养基中，37℃培养24h后对菌液进行平板涂布计数。结果显示TM13-16在0.05％、0.1％、0.2％、0.3％胆盐的条件下不仅可以存活，而且可以生长(表5)。
表5 TM13-16的胆盐耐受情况
Figure PCTCN2021129910-appb-000004
结果表明：本公开的格氏乳杆菌(Lactobacillus gasseri)TM13-16对酸和胆盐耐受能力强。
实施例7：格氏乳杆菌(Lactobacillus gasseri)TM13-16定植能力的评估
1、TM13-16自凝集能力的评估
将过夜培养的TM13-16菌液分装10mL在15mL试管中，取最上层菌液，用紫外分光光度计测得OD600处的吸光值。室温静置30min，再取最上层菌液，用紫外分光光度计测得OD600处的吸光值。比较前后的吸光值，差值越大表示自凝集能力越强。TM13-16表现出了较好的自凝集能力(表6)。
表6 TM13-16的自凝集能力
Figure PCTCN2021129910-appb-000005
2、TM13-16与人宫颈癌细胞Hela粘附能力的评估
将培养好的Hela细胞进行消化，用不含双抗的1640完全培养液(GIBCO，购自于沃卡威北京生物技术有限公司)稀释细胞，血球计数板计数(方法见下)，使细胞浓度达到约2×10 5cell/mL，滴加1mL于细胞培养皿(12或6孔板)中，于5％CO 2-95％空气培养箱中37℃孵育至完全分化。
待细胞长成致密单层后，用灭菌的PBS缓冲液漂洗细胞2次，然后每孔加入lmL1640培养液和1mL过夜培养并调整至10 8CFU/mL的菌悬液，轻轻摇晃混匀，于37℃、5％CO 2的孵箱继续孵育，每株菌样品重复三孔。
孵育90min后，取出六孔培养板，弃细菌悬液，单细胞层用灭菌的PBS缓冲液洗涤细胞5次，除去未粘附的细菌，然后加入无水甲醇固定20min。取上述经无水甲醇固定的细胞玻片，进行革兰氏染色。干燥后镜下观察计数，计算20个随机视野内100个细胞上粘附的 细菌数，计算得每个细胞平均粘附46.25±22.41个。
本公开选择格氏乳杆菌GDMCC 60092作为对照试验，实验方法同上，结果显示每个Hela细胞平均粘附25.23±2.12个格氏乳杆菌GDMCC 60092，表明TM13-16与人宫颈癌细胞Hela的粘附能力更强。粘附能力越强，证明菌株的定植能力更强，更容易存留在阴道环境内进行繁殖，进而起到抑制致病菌、保护阴道粘膜、恢复阴道菌群的功能，达到治疗或预防阴道感染症状的疗效。
3、TM13-16与人阴道上皮细胞VK2E6/E7粘附能力的评估
将培养好的VK2E6/E7细胞(购于 北京北纳创联生物技术研究院)进行消化，用不含双抗的1640完全培养液稀释细胞，血球计数板计数(方法见下)，使细胞浓度达到约2×10 5cell/mL，滴加1mL于细胞培养皿(12或6孔板)中，于5％CO 2-95％空气培养箱中37℃孵育至完全分化。
待细胞长成致密单层后，用灭菌的PBS缓冲液漂洗细胞2次，然后每孔加入lmL1640培养液和1mL过夜培养并调整至10 8CFU/mL的菌悬液，轻轻摇晃混匀，于37℃、5％CO 2的孵箱继续孵育，每株菌样品重复三孔。
孵育90min后，取出六孔培养板，弃细菌悬液，单细胞层用灭菌的PBS缓冲液洗涤细胞5次，除去未粘附的细菌，然后加入无水甲醇固定20min。取上述经无水甲醇固定的细胞玻片，进行革兰氏染色。干燥后镜下观察计数，计算20个随机视野内100个细胞上粘附的细菌数，计算得每个细胞平均粘附107.35±61.88个。
本公开选择格氏乳杆菌GDMCC 60092)作为对照试验，实验方法同上，结果显示每个VK2E6/E7细胞平均粘附31.2±4.25个格氏乳杆菌GDMCC 60092，表明TM13-16与人阴道上皮细胞VK2E6/E7的粘附能力更强。粘附能力越强，证明菌株的定植能力更强，更容易存留在阴道环境内进行繁殖，进而起到抑制致病菌、保护阴道粘膜、恢复阴道菌群的功能，达到治疗或预防阴道感染症状的疗效。
4、细胞计数
将血球计数板及盖片用擦试干净，并将盖片盖在计数板上；然后吸出细胞悬液少许，滴加在盖片边缘，使悬液充满盖片和计数板之间，静置3分钟；最后镜检，计算计数板四大格细胞总数，压线细胞只计左侧和上方的(细胞数/mL＝4大格细胞总数/4×10000)。
实施例8：格氏乳杆菌(Lactobacillus gasseri)TM13-16的大鼠毒性试验
本实施例所选取的大鼠为SD雌性大鼠，7周龄，体重250g±50g，大鼠饲养环境为SPF级，实验动物分为6组，分别为3组灌胃组和3组阴道灌洗组。每组10只，共60只，均用标准饮食饲喂。
灌胃组：分3组，以不同剂量灌胃，每只大鼠经口灌胃0.5ml新鲜菌液，浓度分别为1×10 5、1×10 9、1×10 12CFU/mL，每天1次，连续3天，从第1天灌胃起连续观察至第7天，大鼠均应健康存活、体重增加。
阴道灌洗组：分3组，以不同剂量灌洗阴道，每只大鼠用0.2mL新鲜菌液灌洗阴道，浓度分别为1×10 5、1×10 9、1×10 12CFU/mL，每天1次，连续3天，从第1天灌洗起连续观察至第7天，大鼠均应健康存活、体重增加。
上述实验结果表明，向大鼠施用0.2×10 5～0.5×10 12CFU天剂量的格氏乳杆菌(Lactobacillus gasseri)TM13-16，不会对大鼠的健康造成影响，未发现毒性反应，表明在该剂量下是较为安全的。
实施例9：格氏乳杆菌(Lactobacillus gasseri)TM13-16治疗大鼠生殖道感染的能力
本实施例所选取的大鼠模型为生殖道加德纳菌感染的小鼠模型，采用SD雌性大鼠，7周龄，体重250g±250g，大鼠饲养环境为SPF级实验动物分为3组，分别为模型组、格氏乳杆菌TM13-16组、格氏乳杆菌GDMCC 60092组。每组10只，共30只，均用标准饮食饲喂。
实验分为适应期，建模期，干预期和观察期。适应期：标准饮食饲喂大鼠7天。在造模前三天皮下注射0.5mgβ-estradiol-3-benzoate，保持大鼠假发情现象。造模前观察并记录大鼠阴道是否红肿和溢液，是否有红斑和颗粒物表型特征；建模期：用过夜培养的阴道加德纳菌(购于北京北纳创联生物技术研究院)(PBS菌液)冲洗大鼠阴道，每天一次，连续冲洗3天，第4天观察并记录阴道是否红肿和溢液，是否有红斑和颗粒物等表型特征，选择符合建模要求的大鼠进行试验。干预期：共7天，模型组大鼠不作处理；格氏乳杆菌TM13-16组和格氏乳杆菌GDMCC 60092组大鼠用分别用这两种菌株的PBS菌液冲洗大鼠阴道，每次总活菌量不低于1×10 8CFU/mL，每天冲洗1次，连续冲洗7天。定时观察并记录大鼠阴道是否红肿和溢液，是否有红斑和颗粒物表型特征。观察期：干预结束后3天，观察并记录大鼠阴道是否红肿和溢液，是否有红斑和颗粒物表型特征。在每次观察大鼠表型的同时，取大鼠阴道冲洗液，用定量PCR的方法检测加德纳菌的清除情况及格氏乳杆菌的定植情况。
结果显示，格氏乳杆菌TM13-16组可以定植大鼠阴道内，且定植能力优于格氏乳杆菌GDMCC 60092组(图4)，同时有改善大鼠阴道加德纳杆菌感染并抑制其复发的效果(图5和表7)。
表7大鼠阴道表型记录结果
Figure PCTCN2021129910-appb-000006
Figure PCTCN2021129910-appb-000007
实施例10：格氏乳杆菌(Lactobacillus gasseri)TM13-16辅助治疗治疗生殖道感染性疾病的能力
以格氏乳杆菌(Lactobacillus gasseri)TM13-16为干预产品的主要有效成分之一，开展了一项单中心、随机对照临床研究，按照干预方案(图6)开展为期6个月的干预研究。受试者入组标准为单纯性细菌性阴道病(Amsel标准诊断)的育龄妇女，计划样本量为60例，采用SPSS生成随机数表按1:1比例随机化分组，即对照组(甲硝唑组)30例，干预组(甲硝唑组+益生菌组)30例。符合入组标准的受试者共计67例，其中对照组34例，干预组33例，按照符合方案集分析最终纳入分析的受试者共计59例，其中对照组27例，干预组32例。分析结果如下，益生菌组可以提高14天治愈率(表8)，和降低3个月的复发率(表9)。
表8益生菌干预BV患者治愈率
  BV阴性 BV阳性 治愈率
对照组 19 8 70.37％
干预组 25 7 78.12％
表9益生菌干预BV患者复发率
  BV阴性 BV阳性 复发率
对照组 9 10 52.63％
干预组 18 7 28％
图6以及表8、9结果表明，格氏乳杆菌(Lactobacillus gasseri)TM13-16能够与其他抗菌药物(如甲硝唑)组合治疗细菌性阴道病(BV)，相比单一药物治疗，组合治疗能够显著提高治愈率，同时降低复发率，表明格氏乳杆菌TM13-16能够辅助治疗生殖道感染性疾病。
本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、“一些实施方案”或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本公开的至少一个实施例或示例中。在本说明书中，对上 述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本公开的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本公开的限制，本领域的普通技术人员在本公开的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (18)

  1. 一株格氏乳杆菌(Lactobacillus gasseri)TM13-16，其保藏号为GDMCC No:60405。
  2. 权利要求1所述的格氏乳杆菌或其发酵产物或其菌悬液或其培养液在制备产品中的应用，其中，所述产品用于预防和/或治疗生殖道菌群紊乱相关疾病。
  3. 根据权利要求2所述的应用，其中，所述生殖道菌群紊乱相关疾病包括生殖道感染。
  4. 根据权利要求3所述的应用，其中，所述生殖道感染包括选自细菌性阴道炎、霉菌性阴道炎、滴虫性阴道炎、好氧性阴道炎、老年性阴道炎和病毒感染中的至少之一。
  5. 权利要求1所述的格氏乳杆菌或其发酵产物或其菌悬液或其培养液在制备产品中的应用，其中，所述产品用于抗菌、粘附阴道上皮细胞和/或宫颈细胞、产乳酸、产H 2O 2。
  6. 根据权利要求2-5中任一项所述的应用，其中，所述产品为食品、药物或者保健品。
  7. 根据权利要求2-5中任一项所述的应用，其中，所述产品为药物，所述药物的给药剂量为10 5-10 12CFU/天。
  8. 一种食品或保健品，其中，所述食品或保健品包括权利要求1所述的格氏乳杆菌或其发酵产物或其菌悬液或其培养液。
  9. 根据权利要求8所述的食品或保健品，其中，所述食品或保健品选自预防和/或治疗生殖道菌群紊乱相关疾病的产品、抗菌的产品、粘附阴道上皮细胞和/或宫颈细胞的产品、产乳酸的产品、产H 2O 2的产品中的至少之一。
  10. 一种药物组合物，其中，所述药物组合物包括权利要求1所述的格氏乳杆菌或其发酵产物或其菌悬液或其培养液。
  11. 根据权利要求10所述的药物组合物，其中，所述药物组合物呈单剂量形式，所述药物组合物含有日剂量为10 5-10 12CFU的所述的格氏乳杆菌。
  12. 根据权利要求10所述的药物组合物，其中，所述药物组合物呈适于外用或者口服的剂型。
  13. 权利要求1所述的格氏乳杆菌或其发酵产物或其菌悬液或其培养液、权利要求8或9所述的食品或保健品、权利要求10-12中任一项所述的药物组合物在预防和/或治疗生殖道菌群紊乱相关疾病中的应用。
  14. 根据权利要求13所述的应用，其中，所述生殖道菌群紊乱相关疾病包括生殖道感染。
  15. 根据权利要求14所述的应用，其中，所述生殖道感染包括选自细菌性阴道炎、霉菌性阴道炎、滴虫性阴道炎、好氧性阴道炎、老年性阴道炎和病毒感染中的至少之一。
  16. 一种预防和/或治疗生殖道菌群紊乱相关疾病的方法，其中，所述方法包括：
    向有需要的受试者施用以下中的至少之一：
    (1)权利要求1所述的格氏乳杆菌或其发酵产物或其菌悬液或其培养液；
    (2)权利要求8或9所述的食品或保健品；
    (3)权利要求10-12中任一项所述的药物组合物。
  17. 根据权利要求16所述的方法，其中，所述生殖道菌群紊乱相关疾病包括生殖道感染。
  18. 根据权利要求17所述的方法，其中，所述生殖道感染包括选自细菌性阴道炎、霉菌性阴道炎、滴虫性阴道炎、好氧性阴道炎、老年性阴道炎和病毒感染中的至少之一。
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CN116574629A (zh) * 2022-11-16 2023-08-11 润盈生物工程（上海）有限公司 一种有利于肥胖男性生殖健康的益生菌及其应用
CN116751705A (zh) * 2023-05-17 2023-09-15 上海华朴生命健康科技有限公司 一种具有改善阴道炎症的罗伊氏乳杆菌及其应用
CN116751705B (zh) * 2023-05-17 2024-03-15 上海华朴生命健康科技有限公司 一种具有改善阴道炎症的罗伊氏乳杆菌及其应用
CN117327628A (zh) * 2023-11-16 2024-01-02 江苏科荣生物医药有限公司 一株可发酵中草药抗HPV病毒的格氏乳杆菌Mia及其应用
CN117327628B (zh) * 2023-11-16 2024-03-22 江苏科荣生物医药有限公司 一株可发酵中草药抗HPV病毒的格氏乳杆菌Mia及其应用
CN117535207A (zh) * 2024-01-04 2024-02-09 四川厌氧生物科技有限责任公司 一种格氏乳杆菌及其应用

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