CN112760247A

CN112760247A - Lactobacillus rhamnosus for preventing and/or treating diseases caused by genital tract flora disorder and/or bone loss

Info

Publication number: CN112760247A
Application number: CN202011248842.0A
Authority: CN
Inventors: 张笑薇; 吕金丽; 赵少伟; 高蒙玙; 邹远强; 肖亮
Original assignee: BGI Shenzhen Co Ltd
Current assignee: Bgi Precision Nutrition Shenzhen Technology Co ltd; BGI Shenzhen Co Ltd
Priority date: 2020-11-10
Filing date: 2020-11-10
Publication date: 2021-05-07
Anticipated expiration: 2040-11-10
Also published as: WO2022100632A1; CN112760247B

Abstract

The invention relates to the technical field of biology, in particular to lactobacillus rhamnosus for preventing and/or treating diseases caused by genital tract flora disorder and/or bone loss and application thereof. The invention aims to solve the technical problems that the existing medicines for treating genital tract infection have side effects with different degrees, high recurrence rate or large wound and the like, screens out a new Lactobacillus rhamnosus OF44-15Ph10T for preventing and/or treating diseases related to genital tract flora disorder and/or related diseases caused by bone loss, can generate secretion such as lactic acid, hydrogen peroxide and the like to inhibit the growth OF genital tract infection pathogenic bacteria, is used for preventing and/or treating genital tract infection diseases, particularly reduces the recurrence rate, has small toxic and side effects, and is used as a vaginal dominant Lactobacillus to restore flora disorder and restore vaginal homeostasis. Meanwhile, the lactobacillus rhamnosus can inhibit bone loss, so that the osteoporosis can be treated and improved.

Description

Lactobacillus rhamnosus for preventing and/or treating diseases caused by genital tract flora disorder and/or bone loss

Technical Field

The invention relates to the technical field of biology, in particular to lactobacillus rhamnosus for preventing and/or treating diseases caused by genital tract flora disorder and/or bone loss and application thereof.

Background

The female genital tract environment is often affected by various influences such as hormone level fluctuation, poor sanitary condition control, seasonal variation, even unstable mood and the like to cause flora disturbance, so as to further cause pathogen invasion and cause microbial infection such as bacteria, mold, viruses and the like, wherein common microorganisms include escherichia coli, gardnerella, candida albicans, HPV viruses and the like. The clinical symptoms of genital tract infection diseases are represented by: itching and burning vagina, abnormal secretion, frequent micturition and odynuria, which can lead to premature delivery of pregnant women, cervical cancer and the like in severe cases. According to statistics, the reproductive tract microbial infection rate of women of childbearing age in China is up to 80%, and most of patients suffering from bacterial or fungal infection receive a clinical treatment means of using antibiotics. The antibiotics can reduce the abundance of probiotics while resisting pathogenic bacteria, and are not beneficial to maintaining the stable state of genital tract flora, thereby increasing the recurrence risk. However, for patients infected with HPV virus, there is no perfect treatment method, and a regular screening and follow-up method is mostly adopted, and surgery treatment is performed if the patient is serious. Therefore, there is a need to develop a strain or a medicament capable of preventing or treating female genital tract infection without affecting the abundance of probiotic bacteria in the genital tract environment.

Osteoporosis is a systemic bone disease characterized by a decrease in bone mass, a loss of bone mass, and a decrease in bone strength, leading to an increase in brittle bone mass and susceptibility to fracture. Epidemiological investigation shows that osteoporosis becomes an important health problem for people over 50 years old in China, the prevalence rate is as high as 19.2%, and the population with low bone mass is as high as 46.4%. Although osteoporosis does not directly lead to death in most cases, osteoporosis increases the chances of fracture, thereby affecting the health and the independent living ability of patients and greatly increasing the social medical burden. The existing drug therapy includes estrogen replacement therapy and the like, although osteoporosis can be treated, the existing drug therapy has high potential risk and large side effect, and for example, hormone disorder of a human body can be caused.

Disclosure of Invention

Female reproductive health is closely related to the microbial flora living in the reproductive tract, and a plurality of studies show that flora disturbance in the reproductive tract is a direct cause of bacterial vaginitis, mycotic vaginitis or HPV infection. Modern medicine mainly focuses on taking antibiotics, placing antibiotic suppositories and other medicines for treating genital tract inflammatory diseases. However, antibiotic drug therapy has many problems: firstly, the antibiotics kill pathogenic bacteria, eliminate inflammation, inhibit the growth of probiotics, reduce the abundance of the probiotics, are very unfavorable for maintaining the stable state of the reproductive tract, and greatly increase the risk of repeated infection and relapse; secondly, the purpose of accurate treatment cannot be achieved by taking the medicine, the medicine only can act in a broad-spectrum whole body range, and particularly the liver and kidney functions are easy to be damaged; thirdly, the frequent use of antibiotics is likely to cause drug resistance in pathogenic bacteria, which is also a significant cause of secondary infections.

Although there are individual differences in vaginal flora, most of the dominant species in the female genital tract are lactobacilli, which produce lactic acid to maintain the vaginal pH low, thereby inhibiting the invasion and growth of pathogenic bacteria, and which synthesize hydrogen peroxide to ensure the anaerobic environment in the vagina to prevent the growth of aerobic bacteria. Once the flora structure is destroyed, pathogenic bacteria infect the reproductive tract, causing inflammation and competing with lactobacilli, thereby disrupting reproductive tract microbial homeostasis. At this time, supplementation with lactobacilli to maintain a healthy reproductive tract flora structure is more effective than antibiotic use alone.

Existing methods for preventing and treating osteoporosis include: estrogen replacement therapy, calcitonin, selective estrogen receptor modulators and bisphosphonates. Drugs used to treat and prevent the development of osteoporosis fall into two broad categories, the first being bone resorption inhibiting drugs including calcium agents, vitamin D and active vitamin D, calcitonin, bisphosphonates, estrogens and isoflavones; the second category is osteogenic agents, including fluoride, synthetic steroids, parathyroid hormone, and isoflavones.

Hormone replacement therapy is considered to be the best choice for the treatment of osteoporosis in postmenopausal women and is also the most effective treatment for osteoporosis, however, there is a problem in that hormone replacement therapy may cause other systemic adverse effects. And hormone replacement therapy cannot be applied to patients suffering from breast diseases and those who cannot tolerate the side effects thereof. Selective estrogen receptor modulators this class of drugs has a weak estrogen-like effect in certain organs, while they act as estrogen antagonists in other organs. SERMs prevent osteoporosis and reduce the incidence of cardiovascular disease, breast cancer and endometrial cancer. The medicine is raloxifene, is nonsteroidal benzothiophene, is an agonist of estrogen, can inhibit bone absorption, increase bone density of vertebral column and hip, reduce risk of vertebral fracture by 40-50%, but has poor curative effect compared with estrogen, and is forbidden for premenopausal women. Although the existing drug therapy can treat osteoporosis, the potential risk is high, and side effects are large, such as hormone disorder of a human body.

The invention aims to solve the technical problems that the medicines in the prior art have side effects of different degrees, high recurrence rate or large wound and the like. A new Lactobacillus rhamnosus OF44-15Ph10T which can produce lactic acid and hydrogen peroxide and other secretion substances to inhibit the growth OF pathogenic bacteria infected by the genital tract and be used for preventing and/or treating diseases related to genital tract flora disorder (especially female genital tract flora disorder in human) and/or related diseases caused by bone loss is screened out, and the Lactobacillus rhamnosus OF44-15Ph10T has the advantages OF low recurrence rate, low toxic and side effects and lasting efficacy. Meanwhile, the strain can inhibit bone loss, thereby treating or improving osteoporosis.

Therefore, the invention provides a Lactobacillus rhamnosus OF44-15Ph10T with the preservation number OF GDMCC No. 60406.

The invention screens a female genital tract probiotic Lactobacillus rhamnosus (Lactobacillus rhamnosus) OF44-15Ph10T from a human symbiotic single bacterium library OF about 30,000 strains, the strain is a brand-new isolate, the strain is preserved in Guangdong province microbial strain preservation center (GDMCC, Junyowa Useoul No. 100 college laboratory building 5 in Guangdong province, Guangzhou City, Cross district, with the preservation number OF GDMCC No. 60406 in 2018, 24 months.

The Lactobacillus rhamnosus OF44-15Ph10T is separated by adopting an improved PYG culture medium, and is subjected to subculture by using an MRS culture medium after being identified. When cultured in MRS medium for 48 hours, the colonies OF44-15Ph10T are white, smooth, round, and have neat edges, and the diameter OF the colonies is about 1-2 mm. When observed under a microscope with 1000 times, the thalli presents a slender rod shape, gram staining is positive, and no spores and flagella are generated.

The invention also provides application of the lactobacillus rhamnosus or a fermentation product or a bacterial suspension or a culture solution thereof in preparing a product for preventing and/or treating diseases related to genital tract flora disorder and/or diseases related to bone loss.

Research shows that the Lactobacillus rhamnosus OF44-15Ph10T has strong capacity OF producing L-lactic acid, D-lactic acid and hydrogen peroxide; strong growth ability and high acid and alkali resistance; sensitive to most antibiotics and has no risk genes such as plasmids, transfer elements and the like; meanwhile, the vaginal gel also shows strong adhesion capability to human vaginal epithelial cells, has strong bacteriostatic capability to common pathogenic bacteria of vaginal infection, and can be used for preventing and/or treating genital tract microbial infection.

Meanwhile, the inventor discovers that the Lactobacillus rhamnosus OF44-15Ph10T can effectively reduce the bone loss through large-scale population correlation research and animal model experiments, and can be used for preventing and/or treating related diseases caused by bone mass reduction, such as osteoporosis.

In some embodiments of the invention, the disease associated with a disturbance of the genital tract flora is a genital tract infection.

In some embodiments of the invention, the genital tract infection comprises at least one selected from the group consisting of bacterial vaginitis, mycotic vaginitis, trichomonas vaginitis, aerobic vaginitis, senile vaginitis, and viral infection.

In other embodiments of the invention, the bacteria causing the bacterial vaginitis are common bacteria causing infections of female genital tract, including gram-negative anaerobic bacteria such as escherichia coli (e.coli), Gardnerella vaginalis BNCC337545, corynebacterium, haemophilus, staphylococcus aureus, pseudomonas aeruginosa, etc.

In some embodiments of the invention, the mold causing the mycotic vaginitis is common Candida causing infection of female genital tract, including Candida albicans (Candida albicans SC5314), Candida tropicalis, Candida parapsilosis, Candida dublin, and the like.

In other embodiments of the invention, the virus causing the genital tract viral infection is a common virus causing female genital tract infection, including HPV, herpes simplex virus, cytomegalovirus, and the like.

In some embodiments of the invention, the disease associated with bone loss comprises at least one selected from osteopenia, osteoporosis, and osteoporotic fractures.

The invention also provides the application of the lactobacillus rhamnosus or the fermentation product or the bacterial suspension or the culture solution thereof in preparing products for resisting bacteria, adhering to vaginal epithelial cells and/or cervical cells, producing lactic acid and producing H₂O₂And/or inhibiting bone loss.

In some embodiments of the invention, the product is a food, pharmaceutical or nutraceutical. Wherein the food can be probiotic yogurt, probiotic tablets, probiotic solid beverage and the like.

In other embodiments of the invention, the product is a medicament administered at a dose of 10⁵-10¹²CFU/day.

In still another aspect of the present invention, a food, a medicine or a health product is provided, which comprises the lactobacillus rhamnosus or its fermentation product or its bacterial suspension or its culture solution.

In some embodiments of the invention, the food, pharmaceutical or nutraceutical is selected from a product for the prevention and/or treatment of diseases associated with disorders of the genital tract flora, a product for the prevention and/or treatment of diseases associated with bone loss, an antibacterial product, a product adhering to vaginal epithelial cells and/or cervical cells, a lactic acid producing product, an H producing product₂O₂At least one of the products of (1).

In another aspect, the invention provides a pharmaceutical composition comprising the lactobacillus rhamnosus or its fermentation product or its bacterial suspension or its culture solution.

In some embodiments of the invention, the pharmaceutical composition is in a single dose form, the pharmaceutical composition containing a daily dose of 10⁵-10¹²CFU of said Lactobacillus rhamnosus.

In other embodiments of the present invention, the pharmaceutical composition is in a dosage form suitable for topical or oral administration. As used herein, "genital tract infection" refers to a genital tract infection in a female of an animal or in a female of a human.

Currently, there are also reports of the use of lactobacilli for the prevention or treatment of 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 as probiotic strains for maintaining female genital tract health. Products are available in many brands, including Jarrow Formulas, Blackmores, Renew life and clinicals. But the strain is not the dominant strain of the reproductive tract, and the related clinical research is limited to European and American people, and the curative effect evaluation of Asian people is lacked.

There are a number of disadvantages associated with treatment with antibiotics and suppositories. For example, antibiotics such as metronidazole, tinidazole, clindamycin, etc., act to kill invading anaerobic pathogenic bacteria, thereby treating bacterial vaginitis. But also inhibits the growth of vaginal probiotic lactobacillus and cannot reconstruct a healthy flora structure. The suppository of miconazole, clotrimazole, etc. is used mainly in mycotic vaginitis, and has the action mechanism of inhibiting the sterol synthesis of candida and other fungi cell membrane, affecting cell membrane permeability, inhibiting fungi growth and resulting in death. The medicine is commonly used for suppository, has certain use limitation, causes a plurality of inconveniences, and causes secondary infection when being improperly used.

The method of orally taking probiotics intervenes intestinal flora to regulate an immune system, supplements the abundance of lactobacillus in the genital tract by local use, can make up the defects brought by the traditional antibiotic treatment, helps the flora to recover the steady state while inhibiting or eliminating pathogenic bacteria, greatly improves the cure rate, reduces the recurrence rate, helps HPV to turn negative, effectively treats or prevents genital tract microbial infection diseases, and solves a great clinical problem.

The inventor screens a female genital tract probiotic Lactobacillus rhamnosus OF44-15Ph10T from about 30,000 human symbiotic single bacteria libraries, the strain is a brand new isolate, has strong L-lactic acid, D-lactic acid and hydrogen peroxide production capacity, is sensitive to most antibiotics, has strong growth capacity and high acid and alkali resistance, simultaneously has strong adhesion capacity to human vaginal epithelial cells, has strong bacteriostatic capacity to common pathogenic bacteria infected by vaginal microorganisms, and can be used as food, health care products, external or oral medicaments for development to prevent, treat or assist in treating the symptoms OF female genital tract infection. The lactobacillus rhamnosus can inhibit the growth of genital tract infection pathogenic bacteria by generating lactic acid, hydrogen peroxide and other exudates, is used for preventing and/or treating genital tract infection diseases, and particularly reduces the recurrence rate, and has small toxic and side effects and lasting efficacy. Meanwhile, the inventor also finds that the lactobacillus rhamnosus provided by the invention can effectively reduce the bone loss, so that the lactobacillus rhamnosus can be also used for preventing and/or treating related diseases caused by the bone loss, such as osteoporosis. Thereby solving the problems that the prior medicine for treating osteoporosis has larger potential risk and side effect and can cause hormone disorder of human body.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Preservation information:

the strain name: lactobacillus rhamnosus OF44-15Ph10T

The preservation date is as follows: 24 days 8 month in 2018

The preservation unit: guangdong province microbial culture Collection center (GDMCC)

The preservation number is: GDMCC No. 60406

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1: a genomic map OF Lactobacillus rhamnosus (Lactobacillus rhamnosus) OF44-15Ph 10T;

FIG. 2: results OF probiotic functional gene annotation by Lactobacillus rhamnosus OF44-15Ph 10T;

FIG. 3A: comparison OF Lactobacillus rhamnosus OF44-15Ph10T with the commercially available strain L-lactic acid;

FIG. 3B: comparison OF Lactobacillus rhamnosus OF44-15Ph10T with the commercial strain D-lactic acid;

in FIGS. 3A and 3B, GR-1 denotes Lactobacillus rhamnosus GR-1, OF44 denotes Lactobacillus rhamnosus OF44-15Ph 10T;

FIG. 4: lactobacillus rhamnosus (Lactobacillus rhamnosus) OF44-15Ph10T and Lactobacillus rhamnosus GR-1 inhibit growth OF Escherichia coli, wherein E.coli represents Escherichia coli, OF44 represents Lactobacillus rhamnosus OF44-15Ph10T, and GR-1 represents Lactobacillus rhamnosus GR-1;

FIG. 5: lactobacillus rhamnosus (Lactobacillus rhamnosus) OF44-15Ph10T and Lactobacillus rhamnosus GR-1 inhibit the growth OF Gardnerella (Gardnerella vagianalis), wherein GV represents the Gardner bacillus, OF44 represents Lactobacillus rhamnosus OF44-15Ph10T and GR-1 represents Lactobacillus rhamnosus GR-1;

FIG. 6: the number OF lactobacillus rhamnosus OF44-15Ph10T OF the model group and the probiotic group in example 9;

FIG. 7: the numbers of bacteria of the model group and the probiotic group gardnerella in example 9;

FIG. 8: comparison of bone-related indices in Sham control, model and probiotic mice in example 10;

FIG. 9: comparison of bone-related tissue three-dimensional reconstructed images (micro-CT) of mice in Sham control, model and probiotic groups in example 10.

Detailed Description

The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.

Reagents used in the experiments of examples are commercially available unless otherwise specified.

Example 1: isolation and identification OF Lactobacillus rhamnosus OF44-15Ph10T

1. Sample collection

The isolated sample was obtained from the feces of a healthy female, collected into sterile sample tubes, and brought back to the laboratory for sorting within 1 h.

2. Separating and purifying

The collected fresh samples are immediately transferred to an anaerobic operation box, 0.2g of samples are taken in 1mL of sterile PBS (phosphate buffer solution), the samples are fully shaken and uniformly mixed, then gradient dilution coating is carried out, the culture medium adopts a modified PYG culture medium (purchased from Kyork Microbiology technologies Co., Ltd.), and the specific formula is (1L): 8g of tryptone, 2g of soybean peptone, 1g of polyprotein, 1g of casein, 10g of yeast powder, 5g of beef extract, 5g of glucose and K₂HPO₄2g, maltose 0.5g, cellobiose 0.5g, soluble starch 0.5g, sodium sulfide 0.25g, Tween 800.5 mL, Cysteine-HCl. H₂0.5g of O, 0.5mL of glycerol, 5g of sodium acetate, 5mg of heme, 11 mu L of vitamin K and inorganic salt solution (each L of inorganic salt solution contains CaCl₂·2H₂O0.25g，MgSO₄·7H₂O 0.5g，K₂HPO₄ 1g，KH₂PO₄ 1g，NaHCO₃10g of NaCl 2g) and 1mg of resazurin, adding distilled water to 1L, and adjusting the pH value to 6.8-7.0. The coated plate is placed at 37 ℃ for anaerobic culture, and the anaerobic gas component is N₂：CO₂：H₂When the ratio is 90: 5: 5. after 3 days of culture, single colonies were picked and streaked to obtain pure culture strains of each individual strain.

3. Strain preservation

The obtained pure culture strain was cultured to a concentration of about 10⁹CFU/mL, 400 μ L of bacteria solution is taken, added with 400 μ L of 40% glycerol to make the glycerol concentration reach 20%, and then is preserved at the ultralow temperature of minus 80 ℃.

The strain vacuum freeze-dried powder is prepared according to the following operation steps and is preserved to GDMCC No. 60406 of Guangdong province microbial strain preservation center.

The ampoule tube and the protective agent are sterilized by high pressure for standby, the bacteria liquid cultured overnight is streaked, the bacteria liquid is cultured for 24 hours at 37 ℃, and the following operations are carried out after no mixed bacteria pollution is observed. Centrifugally collecting bacterial liquid, washing with sterilized normal saline, adding 2-3 mL of skim milk protective agent, and suspending to obtain 10 colony counts⁸～10¹⁰The bacterial suspension is packaged in a sterile ampoule tube and is pre-frozen for 1 to 2 hours in a refrigerator at the temperature of minus 80 DEG CThen (c) is performed. And (4) carrying out freeze drying in a freeze dryer for 8-20 h according to standard operating procedures of the freeze dryer until freeze drying. Taking out the sample ampoule tube after freeze-drying, and performing melt-sealing by drawing down the tampon at the neck part of the ampoule tube with strong flame according to standard operating procedures of vacuum ampoule melt-sealing machines.

4. 16S rDNA identification

Culturing the obtained separated strain in a liquid PYG culture medium for 24h, centrifuging 1mL of bacterial liquid at 10000r/min for 5min, collecting the thallus, and extracting the genome DNA. Using genome DNA as a template and using 16S rDNA universal primers to carry out PCR amplification, wherein the amplification system is as follows: 10 × PCR buffer, 3 μ L; dNTP, 2.5 μ L; 27F (5'-AGAGTTTGATCATGGCTCAG-3', shown as SEQ ID NO: 1), 0.5. mu.L; 1492R (5'-TAGGGTTACCTTGTTACGACTT-3', shown in SEQ ID NO: 2), 0.5. mu.L; taq enzyme, 0.3. mu.L; template, 1 μ L; ddH₂O, 18.2. mu.L. The PCR amplification conditions were: pre-denaturation at 95 ℃ for 4min, followed by denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 40s, and extension at 72 ℃ for 1min for 30s, for 30 cycles. The obtained 16S rDNA amplification product is subjected to electrophoresis detection, purification and 3730 sequencing to obtain a 16S rDNA sequence (SEQ ID NO:3) with the length of 1200 bp. The sequence is subjected to blast alignment analysis in genebank, and OF44-15Ph10T is obtained and identified as Lactobacillus rhamnosus (Lactobacillus rhamnosus).

The 16S rDNA sequence OF Lactobacillus rhamnosus OF44-15Ph10T is as follows:

5. physiological and biochemical characteristics OF OF44-15Ph10T

When cultured in MRS medium (available from Cinchaku Microbiology technologies) for 48 hours, the colonies OF OF44-15Ph10T were white, smooth, round, and neat in edge, and had a diameter OF about 1-2 mm. When observed under a microscope with 1000 times, the thalli presents a slender rod shape, gram staining is positive, and no spores and flagella are generated. OF44-15Ph10T was negative for catalase reaction, negative for oxidase, strictly anaerobic, and carbon source utilization was determined using API 50CHL kit. The results are shown in Table 1(+, positive reaction; -, negative reaction; and W, weak positive reaction).

Table 1: OF44-15Ph10T carbon source utilization results

Example 2: genome sequencing, species classification and functional gene analysis OF Lactobacillus rhamnosus (Lactobacillus rhamnosus) OF44-15Ph10T

1. Genome sequencing

The overnight-cultured OF44-15Ph10T bacterial solution was centrifuged at 7,227g at 4 ℃ for 10 minutes, the resulting precipitate was resuspended in 1mL OF Tris-EDTA, 50. mu.L OF 10% SDS and 10. mu.L OF proteinase K (20mg/mL) were added, and the solution was incubated at 55 ℃ for 2 hours to lyse the cells, and the DNA was extracted by the phenol-chloroform method. And sequencing the DNA with Illumina Hiseq 2000 platform, wherein the sequencing length is bidirectional 500bp, and assembling reads with SOAPdenovo. After evaluation, GCskew was used to analyze whole genome GC content and visualize whole genome sequence and functional distribution (fig. 1).

2. Strain genomic species classification

The whole genome sequence was analyzed by comparison using the Checkm software, and the species information most similar to the genome was 31 in terms of the number of annotated genome, 586 in terms of the number of annotated markers, 99.46% in terms of the integrity of the genome, and 0 in terms of the degree of contamination.

3. Probiotic functional gene and safety analysis

In order to examine whether or not the metabolic pathways involved in the treatment OF reproductive tract infection diseases were completed in OF44-15Ph10T, enzymes involved in all OF Lactic acid synthesis (Lactic acid synthesis), hydrogen Peroxide production (Peroxide hydrocarbon production) and short-chain fatty acid synthesis (short-chain fatty acid synthesis) were selected from databases OF prokaryoites OF KEGG, respectively, and separate databases were created, respectively. The whole gene sequences OF OF44-15Ph10T are respectively compared with the databases by using blastx, the annotation results OF e-value being more than or equal to 0.01 and identity being more than or equal to 60 are selected, the gene copy number OF the enzyme OF the related pathway is indicated by light color, the bacterial strain is proved to have the function when the gene is annotated, the function is proved to be stronger when the gene copy number is increased (figure 2), and the antibiotic resistance gene, virulence factors, plasmids, transfer elements, phages and viruses are not annotated in the genome, so that the bacterial strain can be safely used.

Example 3: bioactive substance OF Lactobacillus rhamnosus OF44-15Ph10T

The bioactive substance OF OF44-15Ph10T was mainly used to examine the L-lactic acid content, D-lactic acid content, and hydrogen peroxide production in the metabolites.

1. Sample pretreatment

The strain OF44-15Ph10T was inoculated into MRS medium and cultured at 37 ℃ for 24h under aerobic and anaerobic conditions, respectively.

Respectively taking 1mL of bacterial liquid, centrifuging at 8000r/min for 5min, taking supernatant, and detecting the contents of L-lactic acid and D-lactic acid.

Adding lysozyme (final concentration of 1mg/mL) into 1mL of bacterial liquid, standing at 37 deg.C for 15min, centrifuging at 8000r/min for 5min, collecting supernatant, and detecting hydrogen peroxide concentration.

2. Measurement method

L-Lactic Acid and D-Lactic Acid contents were determined using the L-Lactic Acid (L-Lactate) Assay Kit and the D-Lactic Acid (D-Lactate) Assay Kit (available from Megazyme Inc. US) according to standard operating manuals.

The hydrogen peroxide content was determined using a hydrogen peroxide assay kit (colorimetric method) (purchased from Nanjing, Biotech institute for Biotechnology) according to standard operating manual.

3. The results are shown in Table 2.

TABLE 2OF44-15Ph10T determination OF lactic acid and hydrogen peroxide yields

4. Comparison with commercial strains

According to the invention, the commercial Lactobacillus rhamnosus GR-1 is selected as a control test, the experimental method is the same as that OF the control test, and the results show that the L-lactic acid yield and the D-lactic acid yield OF the Lactobacillus rhamnosus GR-1 are respectively 5.59g/L and 0.45g/L under the anaerobic condition, compared with the yield OF44-15Ph10T which is higher than that OF the control strain, the results are shown in FIGS. 3A and 3B.

Example 4: identification OF capacity OF Lactobacillus rhamnosus OF44-15Ph10T in inhibiting pathogenic bacteria in genital tract infection

1. Identification OF OF44-15Ph10T ability to inhibit E.coli

Under anaerobic conditions, overnight cultured OF44-15Ph10T and Lactobacillus rhamnosus GR-1 (concentration 10) were added⁸CFU) was filtered through a 0.22 μm filter to obtain a supernatant, and 100. mu.L of E.coli (concentration 10) was cultured overnight⁸CFU) was added to 1mL OF OF44-15Ph10T supernatant and MRS medium, respectively, and cultured at 37 ℃ for 24 hours to obtain a bacterial solution, and the absorbance OF the bacterial solution at OD595 was measured. The bacteria liquid obtained by adding Escherichia coli into MRS liquid culture medium is used as positive control. As shown in FIG. 4, E.coli represents a bacterial liquid added to an MRS liquid culture medium, OF44 represents a bacterial liquid added to an OF44-15Ph10T supernatant, and GR-1 represents a bacterial liquid added to an Lactobacillus rhamnosus GR-1 supernatant, the results show that metabolites OF OF44-15Ph10T and GR-1 in the invention can obviously inhibit the growth OF Escherichia coli, and the inhibiting effect OF OF44-15Ph10T is better than that OF GR-1.

2. OF44-15Ph10T characterization OF ability to inhibit Gardnerella varginalis BNCC337545

Under anaerobic conditions, overnight cultured OF44-15Ph10T and Lactobacillus rhamnosus (Lactobacillus rhamnosus)s GR-1) (concentration up to 10⁸CFU) was filtered through a 0.22 μm filter to obtain a supernatant, and 100. mu.L of overnight-cultured Gardnerella (concentration: 10) was added⁸CFU) was added to 1mL OF OF44-15Ph10T supernatant and MRS medium, respectively, and cultured at 37 ℃ for 24 hours to obtain a bacterial solution, and the absorbance OF the bacterial solution at OD595 was measured. The bacterial liquid obtained by adding Gardnerella into MRS liquid culture medium is used as positive control. As shown in FIG. 5, GV represents a bacterial liquid added to an MRS liquid culture medium, OF44 represents a bacterial liquid added to an OF44-15Ph10T supernatant, and GR-1 represents a bacterial liquid added to a Lactobacillus rhamnosus GR-1 supernatant, the results show that metabolites OF OF44-15Ph10T and GR-1 in the invention can obviously inhibit the growth OF Gardnerella and that the inhibiting effect OF OF44-15Ph10T is better than that OF GR-1.

3. OF44-15Ph10T characterization OF ability to inhibit Candida albicans SC5314

Overnight-cultured OF44-15Ph10T and Lactobacillus rhamnosus GR-1 were inoculated into 1.5mL OF MRS medium, respectively, and 100uL OF overnight-cultured SC5314 (10 concentration)⁵CFU) is respectively inoculated into OF44-15Ph10T and Lactobacillus rhamnosus GR-1 bacterial liquid to obtain mixed bacterial liquid, and the mixed bacterial liquid is cultured for 24 hours at 37 ℃ under aerobic condition. The mixed bacterial liquid is diluted and coated on PDA solid medium (purchased from Kyork microbial technology company) in a gradient way, and cultured for 24h at 37 ℃, and colonies are counted. At the same time, SC5314 inoculated only was used as a positive control. The counting result shows that OF44-15Ph10T is 9.4x10⁴CFU/mL, SC5314 group 8.6X10⁵CFU/mL, 1.2x10 for GR-1 group⁵CFU/mL. The results show that OF44-15Ph10T and Lactobacillus rhamnosus GR-1 can inhibit the growth OF Candida albicans, and the inhibiting effect OF OF44-15Ph10T is better than that OF GR-1.

Example 5: antibiotic sensitivity OF Lactobacillus rhamnosus OF44-15Ph10T

Considering the sensitivity OF OF44-15Ph10T to 15 common antibiotics, a drug sensitive paper method is adopted for experiment, 100 mu L OF bacterial liquid cultured to logarithmic phase OF44-15Ph10T is taken for plate coating, antibiotic drug sensitive pieces are attached to the surface OF a plate and cultured at 37 ℃ for 48h, and the size OF a bacteriostatic circle is measured, and the result is shown in Table 3.

TABLE 3OF44-15Ph10T antibiotic susceptibility

Antibiotic	Diameter of bacteriostatic circle (cm)	Antibiotic	Diameter of bacteriostatic circle (cm)
				Ampicillin	3	Ceftriaxone	2
Bacitracin	0	Vancomycin	0
				Penicillin	3.6	Oxacillin	1.4
Kanamycin	1.5	Amoxicillin	3
				Tetracycline derivatives	2.6	Azithromycin	2
Gulalaxin	3.5	Clindamycin	2.2
				Erythromycin	2.7	Gentamicin	1.4
Chloromycetin	2.7

The results show that OF44-15Ph10T is resistant to bacitracin and vancomycin, sensitive to other 13 antibiotics and safe to use.

Example 6: tolerance OF Lactobacillus rhamnosus OF44-15Ph10T to acid and bile salts

Since the human probiotics need to pass through the stomach and small intestine to reach the intestine, it needs to experience gastric acid at a ph around 2.5 and bile salts at a concentration of 0.3%. Only acid and bile salt tolerant strains reach the intestine to exert a probiotic effect. Therefore, this example was examined for the acid and bile salt tolerance OF OF44-15Ph 10T.

1. OF44-15Ph10T acid tolerance

MRS culture media with pH2, pH 3, pH 4, pH 4.5 and pH 7 are prepared respectively, 100 mu L OF OF44-15Ph10T bacterial liquid cultured overnight at the concentration OF 8.5E +09 is inoculated into the MRS culture media with different pH values, and after the bacterial liquid is cultured for 24 hours at 37 ℃, the bacterial liquid is subjected to plate coating counting. The results show that OF44-15Ph10T can not only survive but also grow under conditions OF pH2, pH 3, pH 4, pH 4.5, pH 7 (Table 4).

TABLE 4OF44-15Ph10T acid tolerance

2. OF44-15Ph10T bile salt tolerance

MRS culture media containing 0.05%, 0.1%, 0.2% and 0.3% OF cholate are prepared respectively, 100 mu L OF OF44-15Ph10T bacterial liquid cultured overnight at the concentration OF 8.5E +09 is inoculated into the MRS culture media with different cholate contents, and the bacterial liquid is subjected to plate coating counting after being cultured for 24 hours at 37 ℃. The results show that OF44-15Ph10T can not only survive but also grow under conditions OF 0.05%, 0.1%, 0.2%, 0.3% bile salts (Table 5).

TABLE 5OF44-15Ph10T bile salt tolerance

The results show that: the Lactobacillus rhamnosus OF44-15Ph10T has strong acid and bile salt tolerance.

Example 7: evaluation OF colonization ability OF Lactobacillus rhamnosus OF44-15Ph10T

1. Evaluation OF OF44-15Ph10T self-agglutination ability

And subpackaging 10mL OF overnight cultured OF44-15Ph10T bacterial liquid into 15mL test tubes, taking the uppermost bacterial liquid, and measuring the absorbance value at OD600 by using an ultraviolet spectrophotometer. Standing at room temperature for 30min, taking the uppermost layer bacterial liquid, and measuring the light absorption value at OD600 position with an ultraviolet spectrophotometer. Comparing the absorbance values before and after the comparison, the larger the difference is, the stronger the self-agglutination ability is. OF44-15Ph10T showed better self-aggregation ability (Table 6).

TABLE 6OF44-15Ph10T self-aggregation capability

2. OF44-15Ph10T evaluation OF adhesion ability to human cervical carcinoma epithelial cells Hela

The cultured Hela cells were digested, diluted with a double-antibody-free 1640 complete medium (GIBCO, available from Warcawei Beijing Biotechnology Ltd.), and counted on a hemocytometer (see below) to a cell concentration of about 2X10⁵cell/mL, 1mL drop wise in cell culture dishes (12 or 6 well plates) in 5% CO₂Incubate at 37 ℃ in-95% air incubator until complete differentiation.

After the cells had grown into a dense monolayer, the cells were rinsed 2 times with sterile PBS buffer, and lmL1640 medium and 1mL of overnight culture were added to each well and adjusted to 10⁸CFU/mL bacterial suspension, gently shaking and mixing, at 37 deg.C and 5% CO₂The incubators continued to incubate, repeating three wells for each strain sample.

After incubation for 90min, the six-well plate was removed, the bacterial suspension was discarded, the cell was washed 5 times with sterilized PBS buffer for the monolayer to remove non-adherent bacteria, and then absolute methanol was added for fixation for 20 min. The cell slide fixed by the absolute methanol is taken for gram staining. The number of adhered bacteria on 100 cells in 20 random fields was counted by observation under the dry lens, and the average adhesion of 34.59. + -. 4.63 per cell was calculated.

The invention selects the strains widely used in commerce: lactobacillus rhamnosus GR-1, Lactobacillus reuteri RC-14 and the already marketed medicinal strain Lactobacillus delbrueckii (Lactobacillus delbrueckii) were used as control experiments, and the experimental method is as above, and the results show that the average adhesion OF 13.43 +/-7.07 GR-1, 17.48 +/-4.24 RC-14 and 25.23 +/-2.12 Lactobacillus delbrueckii per Hela cell is higher than that OF the OF44-15Ph10T and human cervical carcinoma cell Hela. The stronger the adhesion capacity, the stronger the colonization capacity of the bacterial strain is proved, the bacterial strain can be more easily remained in the vaginal environment for reproduction, and then the functions of inhibiting pathogenic bacteria, protecting vaginal mucosa and recovering vaginal flora are achieved, and the curative effect of treating or preventing vaginal infection symptoms is achieved.

3. OF44-15Ph10T evaluation OF ability to adhere to human vaginal epithelial cells VK2E6/E7

Cultured VK2E6/E7 cells (purchased from the institute of Biotechnology, Chuanglian, Beijing) were digested, diluted with 1640 complete medium without double antibody, and counted on a hemocytometer (see below) to a cell concentration of about 2X10⁵cell/mL, 1mL drop wise in cell culture dishes (12 or 6 well plates) in 5% CO₂Incubate at 37 ℃ in-95% air incubator until complete differentiation.

After incubation for 90min, the six-well plate was removed, the bacterial suspension was discarded, the cell was washed 5 times with sterilized PBS buffer for the monolayer to remove non-adherent bacteria, and then absolute methanol was added for fixation for 20 min. The cell slide fixed by the absolute methanol is taken for gram staining. The number of adhered bacteria on 100 cells in 20 random fields was counted by observation under the dry mirror, and the average adhesion of 69.51 + -47.02 cells per cell was calculated.

The invention selects the strains widely used in commerce: lactobacillus rhamnosus GR-1, Lactobacillus reuteri RC-14 and the already marketed medicinal strain Lactobacillus delbrueckii (Lactobacillus delbrueckii) were used as control experiments, the experimental methods are as above, and the results show that the average adhesion OF 27.4 + -8.11 GR-1, 14.5 + -4.63 RC-14 and 22.24 + -9.26 Lactobacillus delbrueckii per VK2E6/E7 cell is higher than that OF Lactobacillus rhamnosus GR-1, Lactobacillus reuteri RC-14 and Lactobacillus delbrueckii, indicating that OF44-15Ph10T has higher adhesion to human vaginal epithelial cells VK2E 6/E7. The stronger the adhesion capacity, the stronger the colonization capacity of the bacterial strain is proved, the bacterial strain can be more easily remained in the vaginal environment for reproduction, and then the functions of inhibiting pathogenic bacteria, protecting vaginal mucosa and recovering vaginal flora are achieved, and the curative effect of treating or preventing vaginal infection symptoms is achieved.

4. Cell counting

Cleaning the blood counting plate and the cover plate by wiping, and covering the cover plate on the counting plate; then sucking a little cell suspension out, dripping the cell suspension on the edge of the cover plate to fill the suspension between the cover plate and the counting plate, and standing for 3 minutes; finally, microscopic examination is carried out, the total number of the four big grids of the plate is counted, and the line pressing cells are counted only on the left side and the upper side (the cell number/mL is 4 big grids of the total number of the cells/4 multiplied by 10000).

Example 8: rat toxicity test OF Lactobacillus rhamnosus OF44-15Ph10T

The rats selected in this example were SD female rats aged 7 weeks and weighed 250g ± 50g, the breeding environment of the rats was SPF grade, and the experimental animals were divided into 6 groups, 3 groups of gavage groups and 3 groups of vaginal irrigation groups. Each group was fed with 10, 60 total, standard diets.

And (3) gastric lavage group: dividing into 3 groups, and intragastrically administering with different dosages, wherein each rat orally administers 0.5mL fresh bacterial liquid (concentration is 1 × 10 respectively)⁵、1×10⁹、1×10¹²CFU/mL), 1 time per day for 3 consecutive days, and rats were kept alive and gained weight from gavage on day 1 to day 7.

Vaginal irrigation group: divided into 3 groups, and the vagina was irrigated with different doses, and each rat was irrigated with 0.2mL of fresh bacterial solution (concentration 1X 10 each)⁵、1×10⁹、1×10¹²CFU/mL) 1 time per day for 3 consecutive days, and rats were all healthy and alive and gained weight from lavage day 1 to day 7.

The results of the above experiments show that 0.2X 10 administration to rats⁵～0.5×10¹²The CFU/day dose OF Lactobacillus rhamnosus OF44-15Ph10T has no influence on the health OF rats, no toxic reaction is found, and the dose is safe.

Example 9: the ability OF Lactobacillus rhamnosus OF44-15Ph10T to treat genital tract infections in rats

The rat model selected in this example is a rat model infected with gardnerella vaginalis in genital tract, SD female rats are used, the rat is 7 weeks old, the weight is 250g ± 250g, the breeding environment of the rats is SPF grade, and the experimental animals are divided into 2 groups, namely a model group and a probiotic group. Each group was fed with 10, a total of 20, and a standard diet.

The experiment is divided into an adaptation period, a modeling period, a dry prediction period and an observation period. An adaptation period: rats were fed a standard diet for 7 days. The rat false oestrus phenomenon was maintained by subcutaneous injection of 0.5mg of beta-estradiol-3-benzoate three days before molding. Observing and recording whether the vagina of a rat is red and swollen, and overflowing, and whether erythema and particle phenotypic characteristics exist before modeling; a modeling period: the rat vagina is washed by gardnerella vaginalis (purchased from Beijing Beinanna institute of Biotechnology) (PBS bacterial liquid) which is cultured overnight, the rat vagina is continuously washed for 3 days once a day, and whether the vagina is red and swollen, overflow, erythema, particulate matter and other phenotypic characteristics exist or not is observed and recorded on the 4 th day, and the rat which meets the modeling requirement is selected for testing. Dry expect: for a total of 7 days, model group rats were not treated; probiotic group rats use OF44-15Ph10T PBS liquid to wash vagina OF rats, and the total viable bacteria amount is not less than 1 × 10⁸CFU/mL, washing 1 time per day for 7 days. The rats were observed periodically and recorded for redness and swelling of the vagina, fluid discharge, erythema and particulate phenotypic characteristics. And (3) an observation period: 3 days after the end of the intervention, the rats were observed and recorded for redness and swelling of the vagina, discharge, erythema and granular phenotypic characteristics. While observing the phenotype of the rat each time, taking vaginal washing liquid of the rat, and detecting the removal condition of the gardnerella and the permanent planting condition of the lactobacillus gasseri by a quantitative PCR method

The results show that lactobacillus rhamnosus OF44-15Ph10T can colonize the vagina OF rats (fig. 6), while improving the effect OF gardnerella vaginalis infection and inhibiting the recurrence thereof in rats (fig. 7 and table 7). (Table 8).

TABLE 7 vaginal phenotype of rats the results are recorded

Example 10: effect OF Lactobacillus rhamnosus OF44-15Ph10T on mouse bone loss model

The bone loss model used C57BL/6 female mice (purchased from Guangdong provincial animal center) 8 weeks old and weighing 20 + -2 g freely eating and drinking water under SPF environment. The samples were randomly divided into 3 groups of 9-10 samples each.

The first group OF 9 mice was Sham control, Sham operated, skin dissected only and sutured, and the experiment was performed by feeding medium containing the target strain (OF44-15Ph10T) in a proportional amount to body weight ratio;

a second group of 10 mice is an experimental group, an osteoporosis model is caused by the extirpation induction of ovarian organs, a saturated suspension of a target strain is perfused into the stomach, and the feeding amount is in direct proportion to the body weight;

the third group of 10 mice is a negative control group, an osteoporosis model is caused by the extirpation induction of ovarian organs, the culture medium is perfused into the stomach, and the feeding amount is proportional to the weight.

During the experiment, weekly mouse weight data were recorded (table 8); after the experiment, the mice were sacrificed, and the change of the bone-related index was detected using micro-CT (shown in table 9 and fig. 8), and a three-dimensional reconstructed image of the tissue was created (fig. 9).

The results showed no significant difference (p >0.05) between the probiotic group (second group) and the Sham control group (first group); a number of bone related indicators were statistically significantly different (p <0.05) compared to the model group (third group); the results show that the intestinal probiotic Lactobacillus rhamnous OF44-15Ph10T has the effect OF inhibiting bone loss in a mouse model.

TABLE 8 weight records of groups of mice before and after gavage

Numbering	Group of	20190722	20190805	20190819	20190902	20190913
							GG_20864	Second group	20.2	22.3	22.2	23.5	23.8
GG_20874	Second group	19.1	23.7	22.8	22.7	24
							GG_20845	Second group	18.5	24.5	22.2	22.6	24.4
GG_20855	Second group	18.5	22.4	22	21.9	22.5
							GG_20899	Second group	18.7	22.5	21.1	20.8	22.4
GG_20857	Second group	17.7	22.6	21.4	21.3	22.3
							GG_20872	Second group	18.4	21.5	21.8	21	21.8
GG_20853	Second group	18.3	20	20.4	20	22.1
							GG_20868	Second group	17.8	21.7	21.4	21.8	22.1
GG_20866	Second group	15.2	19.3	20.1	20.2	20.8
							OVX_20800	Third group	18.5	21.7	22.3	22.1	24
OVX_20885	Third group	18.9	22.6	21.4	22.5	22.6
							OVX_20871	Third group	20.1	22.1	22.7	22.7	23.7
OVX_20861	Third group	18.7	20.6	21	21.2	22.9
							OVX_20881	Third group	17.6	23.6	21.2	21.6	21.9
OVX_20878	Third group	18.2	22.9	22	21.2	22.8
							OVX_20896	Third group	18.3	21.3	20.7	20.4	21.5
OVX_20849	Third group	15.7	22.2	21.5	21.8	22.9
							OVX_20882	Third group	17	20.2	21.4	20.2	21.9
OVX_20851	Third group	17.1	16.6	18.4	21.5	22.6
							Sham_20804	First group	18.7	20.9	20.5	20.5	21.6
Sham_20806	First group	18.6	19.4	19.9	20.3	21.1
							Sham_20809	First group	17.3	18.8	19.4	19.8	20.9
Sham_20811	First group	18.4	19.1	19	20	20.8
							Sham_20813	First group	18.8	20	20.2	20.2	22.2
Sham_20814	First group	17	17.8	19.1	19.1	20.5
							Sham_20816	First group	18	19.5	20.2	20	21.7
Sham_20817	First group	18.6	19.9	20.3	19.4	20.3
							Sham_20818	First group	19	20.1	20.8	19.9	21.6

TABLE 9 bone-related index profiles for groups of mice

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," "some embodiments," or "some examples" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Sequence listing

<110> Shenzhen Huashengshengsciences institute

<120> Lactobacillus rhamnosus for preventing and/or treating diseases caused by disorders of genital tract flora and/or bone loss

<130> PIDC3202923

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

agagtttgat catggctcag 20

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<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tagggttacc ttgttacgac tt 22

<210> 3

<211> 1200

<212> DNA

<213> Lactobacillus rhamnosus OF44-15Ph10T (Lactobacillus rhamnosus OF44-15Ph10T)

<220>

<221> misc_feature

<223> 16S rDNA sequence

<400> 3

gtgcctatac atgcaagtcg aacgagttct gattattgaa aggtgcttgc atcttgattt 60

aattttgaac gagtggcgga cgggtgagta acacgtgggt aacctgccct taagtggggg 120

ataacatttg gaaacagatg ctaataccgc ataaatccaa gaaccgcatg gttcttggct 180

gaaagatggc gtaagctatc gcttttggat ggacccgcgg cgtattagct agttggtgag 240

gtaacggctc accaaggcaa tgatacgtag ccgaactgag aggttgatcg gccacattgg 300

gactgagaca cggcccaaac tcctacggga ggcagcagta gggaatcttc cacaatggac 360

gcaagtctga tggagcaacg ccgcgtgagt gaagaaggct ttcgggtcgt aaaactctgt 420

tgttggagaa gaatggtcgg cagagtaact gttgtcggcg tgacggtatc caaccagaaa 480

gccacggcta actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttatccgga 540

tttattgggc gtaaagcgag cgcaggcggt tttttaagtc tgatgtgaaa gccctcggct 600

taaccgagga agtgcatcgg aaactgggaa acttgagtgc agaagaggac agtggaactc 660

catgtgtagc ggtgaaatgc gtagatatat ggaagaacac cagtggcgaa ggcggctgtc 720

tggtctgtaa ctgacgctga ggctcgaaag catgggtagc gaacaggatt agataccctg 780

gtagtccatg ccgtaaacga tgaatgctag gtgttggagg gtttccgccc ttcagtgccg 840

cagctaacgc attaagcatt ccgcctgggg agtacgaccg caaggttgaa actcaaggaa 900

ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgagaacc 960

ttaccaggtc ttgacatctt tttgatcacc tgagagatca agtttctcct tcgggggcaa 1020

atgacagtgt gcatgcttgt cgtcagctcg tgttcgtgag atgttggtta agttccgcac 1080

gagcgcaccc tatgactagg tgctagcatt tagtggtcac tctagtaaga actgcgtgac 1140

catcgagatg gtgggtatga cgtcatcatc atggccttat gacctggcta caacgtgtct 1200

Claims

1. Lactobacillus rhamnosus OF44-15Ph10T with the accession number GDMCC No. 60406.

2. Use of lactobacillus rhamnosus or its fermentation product or its bacterial suspension or its culture solution according to claim 1 for the preparation of a product for the prevention and/or treatment of diseases associated with disorders of the genital tract flora and/or related diseases caused by bone loss.

3. The use according to claim 2, wherein the disease associated with a disturbance of the genital tract flora is an infection of the genital tract; optionally, the genital tract infection includes at least one selected from bacterial vaginitis, mycotic vaginitis, trichomonas vaginitis, aerobic vaginitis, senile vaginitis, and viral infection;

optionally, the disease associated with bone loss comprises at least one selected from osteopenia, osteoporosis, and osteoporotic fractures.

4. Use of lactobacillus rhamnosus or its fermentation product or its bacterial suspension or its culture solution according to claim 1 for the preparation of a product for antibacterial, adhesion to vaginal epithelial cells and/or cervical cells, lactic acid production, H production₂O₂And/or inhibiting bone loss.

5. Use according to any one of claims 2 to 4, wherein the product is a food, a pharmaceutical or a nutraceutical product.

6. Use according to any one of claims 2 to 4, wherein the product is a medicament to be administered in a dose of 10⁵-10¹²CFU/day.

7. A food, pharmaceutical or nutraceutical product comprising lactobacillus rhamnosus or its fermentation product or its bacterial suspension or its culture solution according to claim 1.

8. The food, pharmaceutical or nutraceutical of claim 7, wherein the food, pharmaceutical or nutraceutical is selected from the group consisting of a product for preventing and/or treating diseases associated with disorders of the genital tract flora, a product for preventing and/or treating diseases associated with bone loss, an antibacterial product, a product adhering to vaginal epithelial cells and/or cervical cells, a lactic acid producing product, a H producing product₂O₂At least one of the products of (1).

9. A pharmaceutical composition, which comprises the Lactobacillus rhamnosus or its fermentation product or its bacterial suspension or its culture solution of claim 1,

optionally, the pharmaceutical composition is in a single dose form, the pharmaceutical composition containing a daily dose of 10⁵-10¹²CFU of said Lactobacillus rhamnosus.

10. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition is in a dosage form suitable for topical or oral administration.