WO2022100632A1 - Lacticaseibacillus rhamnosus for preventing and/or treating diseases caused by reproductive tract flora disorder and/or bone loss - Google Patents

Abstract

Lacticaseibacillus rhamnosus for preventing and/or treating diseases caused by the reproductive tract flora disorder and/or bone loss and an application thereof. The Lacticaseibacillus rhamnosus is OF44-15Ph10T, and the accession number thereof is GDMCC No:60406. The strain is capable of producing secretions such as lactic acid and hydrogen peroxide to inhibit the growth of the reproductive tract infection pathogenic bacteria, and is used for preventing and/or treating the reproductive tract infection disease. The strain can also inhibit bone loss so as to treat and improve osteoporosis.

Description

Lactobacillus rhamnosus for the prevention and/or treatment of disorders of reproductive tract flora and/or bone loss

priority information

This application claims priority to and the benefit of the patent application No. 202011248842.0 filed with the State Intellectual Property Office of China on November 10, 2020, which is hereby incorporated by reference in its entirety.

technical field

The present disclosure relates to the field of biotechnology, in particular to a Lactobacillus rhamnosus for preventing and/or treating diseases caused by genital tract flora disorder and/or bone loss and applications thereof.

Background technique

The female reproductive tract environment is often affected by fluctuations in hormone levels, poor hygiene control, seasonal changes, and even unstable moods, resulting in flora disturbances, which in turn lead to the invasion of pathogenic bacteria and lead to bacterial, mold, virus and other microbial infections. Common There are Escherichia coli, Gardnerella, Candida albicans and HPV viruses. 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. According to statistics, 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. 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. Therefore, there is an urgent need to develop a strain or drug that can prevent or treat female reproductive tract infections without affecting the abundance of probiotics in the reproductive tract environment.

Osteoporosis is a systemic bone disease characterized by decreased bone mass, impaired bone quality and reduced bone strength, leading to increased brittle bone and susceptibility to fractures. Epidemiological surveys show that osteoporosis has become an important health problem for people over 50 years old in my country, with a prevalence rate of 19.2%, and a low bone mass population of 46.4%. Although osteoporosis does not directly lead to death in most cases, osteoporosis increases the chance of fracture, which affects the health and independent living ability of patients, and greatly increases the social medical burden. Existing drug treatments, including estrogen replacement therapy, etc., can treat osteoporosis, but have high potential risks and side effects, such as hormonal disorders in the human body.

SUMMARY OF THE INVENTION

Women's reproductive health is closely related to the microbial flora living in the genital tract, and many studies have shown that the flora disturbance of the genital tract is the direct cause of bacterial vaginosis, fungal vaginitis or HPV infection. The treatment of inflammatory diseases of the genital tract in modern medicine mainly focuses on drug treatment such as taking antibiotics and placing antibiotic suppositories. However, there are many problems in antibiotic drug treatment: First, antibiotics kill pathogenic bacteria and eliminate inflammation while inhibiting the growth of probiotics, reducing the abundance of probiotics, which is very unfavorable for the reproductive tract to maintain homeostasis and greatly increases repeatability. The risk of infection and recurrence; second, taking drugs cannot achieve the purpose of precise treatment, but can only have broad-spectrum systemic effects, especially easy to damage liver and kidney functions; third, frequent use of antibiotics can easily lead to drug resistance of pathogens, This is also an important cause of secondary infection.

In the vaginal flora, although there are individual differences, most of the dominant bacteria in the female genital tract are Lactobacillus, which mainly produces lactic acid to maintain a lower pH value of the vagina, 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. Once the flora structure is destroyed, pathogenic bacteria can infect the reproductive tract, cause inflammation, and form a competitive relationship with Lactobacillus, thereby disrupting the microbial homeostasis of the reproductive tract. At this time, supplementation with Lactobacillus to maintain a healthy reproductive tract flora is more effective than antibiotics alone.

Existing approaches to prevent and treat osteoporosis include: estrogen replacement therapy, calcitonin, selective estrogen receptor modulators, and bisphosphonates. Drugs used to treat and prevent the development of osteoporosis are divided into two categories. The first category is drugs that inhibit bone resorption, including calcium, vitamin D and active vitamin D, calcitonin, bisphosphonates, estrogens, and isoforms. Flavonoids; the second category is bone-promoting drugs, including fluoride, anabolic steroids, parathyroid hormone, and isoflavones.

For the treatment of osteoporosis, hormone replacement therapy is considered to be the best choice for the treatment of osteoporosis in postmenopausal women, and it is also the most effective treatment method. And hormone replacement therapy cannot be used in patients with breast disease, and those who cannot tolerate its side effects. Selective Estrogen Receptor Modulators These drugs have weak estrogen-like effects in some organs and estrogen antagonistic effects in others. SERMs prevent osteoporosis and also reduce the incidence of cardiovascular disease, breast cancer and endometrial cancer. Such drugs include raloxifene, a non-steroidal benzothiophene, an estrogen agonist that inhibits bone resorption, increases bone density in the spine and hip, and reduces the risk of vertebral fractures by 40% to 50%. %, but the efficacy is worse than that of estrogen, and it is contraindicated in premenopausal women. Although existing drug treatments can treat osteoporosis, they have high potential risks and side effects, such as hormonal disturbances in the body.

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. Screening out a novel Lactobacillus rhamnosus (Lactobacillus rhamnosus) for preventing and/or treating diseases related to reproductive tract flora disorders (especially referring to female reproductive tract flora disorders in humans) and/or related diseases caused by bone loss rhamnosus) OF44-15Ph10T, which can produce secretions such as lactic acid and hydrogen peroxide to inhibit the growth of pathogenic bacteria in reproductive tract infections, for the prevention and/or treatment of reproductive tract flora disorders related diseases, especially to reduce the recurrence rate, toxicity Side effects are small and the effect is long-lasting. At the same time, the bacteria can inhibit bone loss, thereby treating or improving osteoporosis.

To this end, one aspect of the present disclosure provides a strain of Lactobacillus rhamnosus OF44-15Ph10T, whose deposit number is GDMCC No: 60406.

The present disclosure selects a female reproductive tract probiotic Lactobacillus rhamnosus OF44-15Ph10T from a library of about 30,000 human symbiotic bacteria. On August 24, 2008, it was preserved 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 preservation number is GDMCC No: 60406.

The Lactobacillus rhamnosus OF44-15Ph10T 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 colony of OF44-15Ph10T was white, smooth, round, with neat edges, and the colony diameter was about 1-2 mm. Observed under a microscope at 1000 times, the cells were elongated and rod-shaped, Gram staining was positive, and no spores and flagella were produced.

Another aspect of the present disclosure provides the use of the Lactobacillus rhamnosus or its fermentation product or its bacterial suspension or its culture solution in preparing a product for preventing and/or treating genital tract flora disorders related to Disease and/or associated disease caused by bone loss.

The Lactobacillus rhamnosus OF44-15Ph10T provided by the present disclosure has been found to have a strong ability to produce L-lactic acid, D-lactic acid and hydrogen peroxide; strong growth ability and high acid and alkali resistance; Antibiotic sensitivity, no risk genes such as plasmids and transfer elements; at the same time, it also shows strong adhesion to human vaginal epithelial cells, and has strong bacteriostatic ability against common vaginal infection pathogens, which can be used for prevention and/or treatment of reproductive tract Microbial infection.

At the same time, the inventor found through large-scale population association studies and animal model experiments that Lactobacillus rhamnosus OF44-15Ph10T can effectively reduce bone loss and can be used to prevent and/or treat related diseases caused by osteopenia, such as bone loss. Psoriasis.

In some embodiments of the present disclosure, the disorder associated with a reproductive tract flora disorder comprises a reproductive tract infection.

In some embodiments of the present disclosure, the disease associated with a disorder of the reproductive tract flora is a reproductive tract infection.

In some embodiments of the present disclosure, the reproductive tract infection comprises at least one selected from bacterial vaginosis, fungal vaginitis, trichomonas vaginitis, aerobic vaginitis, senile vaginitis, and viral infection one.

In other embodiments of the present disclosure, the bacteria causing the bacterial vaginosis are common bacteria that cause female genital tract infections, including Escherichia coli (E.coli), Gardnerella vaginalis (BNCC337545), Corynebacterium , Haemophilus, Staphylococcus aureus, Pseudomonas aeruginosa and other Gram-negative anaerobic bacteria.

In some embodiments of the present disclosure, 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.

In other embodiments of the present disclosure, 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.

In some embodiments of the present disclosure, the related diseases caused by the bone loss include at least one selected from the group consisting of osteopenia, osteoporosis, and osteoporotic fractures.

Another aspect of the present disclosure provides the use of the Lactobacillus rhamnosus 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, _H2O2 production, and/or _inhibition of bone loss.

In some embodiments of the present disclosure, the product is a food, a drug, or a nutraceutical. Wherein, the food can be probiotic yogurt, probiotic tablet, probiotic solid drink and the like.

In other embodiments of the present disclosure, the product is a drug, and the drug is administered at a dose of 10 ⁵ -10 ¹² CFU/day.

In yet another aspect of the present disclosure, a food or health care product is provided, the food or health care product includes the Lactobacillus rhamnosus or its fermentation product or its bacterial suspension or its culture solution.

In some embodiments of the present disclosure, the food or health product is selected from the group consisting of products for preventing and/or treating disorders related to reproductive tract flora disorders, preventing and/or treating related diseases caused by bone loss, antibacterial products, At least one of a product of vaginal epithelial cells and/or cervical cells, a lactic acid _- producing product, and a H2O2 _- producing product.

Another aspect of the present disclosure 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 present disclosure, the pharmaceutical composition is in the form of a single dose, the pharmaceutical composition comprising a daily dose of ^105-1012 CFU ^of the Lactobacillus rhamnosus.

In other embodiments of the present disclosure, the pharmaceutical composition is in a dosage form suitable for topical or oral administration.

Another aspect of the present disclosure provides that the above-mentioned Lactobacillus rhamnosus 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 used in the prevention and/or treatment of genital tract flora disorder Use in related diseases and/or related diseases caused by bone loss.

In some embodiments of the present disclosure, the disorder associated with a reproductive tract flora disorder comprises a reproductive tract infection.

In some embodiments of the present disclosure, the reproductive tract infection comprises at least one selected from bacterial vaginosis, fungal vaginitis, trichomonas vaginitis, aerobic vaginitis, senile vaginitis, and viral infection one.

In some embodiments of the present disclosure, the related diseases caused by the bone loss include at least one selected from the group consisting of osteopenia, osteoporosis, and osteoporotic fractures.

Yet another aspect of the present disclosure provides a method for preventing and/or treating disorders related to reproductive tract flora disorders and/or related diseases caused by bone loss. The method includes:

Administer at least one of the following to a subject in need:

(1) above-mentioned Lactobacillus rhamnosus or its fermentation product or its bacterial suspension or its culture solution;

(2) the above-mentioned food or health products;

(3) The above-mentioned pharmaceutical composition.

In some embodiments of the present disclosure, the disorder associated with a reproductive tract flora disorder comprises a reproductive tract infection.

In some embodiments of the present disclosure, the reproductive tract infection comprises at least one selected from bacterial vaginosis, fungal vaginitis, trichomonas vaginitis, aerobic vaginitis, senile vaginitis, and viral infection one.

In some embodiments of the present disclosure, the related diseases caused by the bone loss include at least one selected from the group consisting of osteopenia, osteoporosis, and osteoporotic fractures.

As used in this disclosure, "reproductive tract infection" refers to a reproductive tract infection of a female in an animal or a female in a human.

At present, there are also reports of the use of 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. As 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. However, 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.

Treatment with antibiotics and suppositories has many disadvantages. For example, antibiotics such as metronidazole, tinidazole, and clindamycin are used to kill invading anaerobic pathogenic bacteria, thereby treating bacterial vaginosis. However, 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 intervenes in the intestinal flora to regulate the immune system, and local use to supplement the abundance of lactobacilli in the reproductive tract can make up for the shortcomings of traditional antibiotic treatment, and help the flora to restore its steady state while inhibiting or eliminating pathogenic bacteria. It can greatly improve the cure rate, reduce the recurrence rate, help HPV turn negative, effectively treat or prevent microbial infections in the reproductive tract, and solve a major clinical problem.

The inventors of the present disclosure screened out a female reproductive tract probiotic Lactobacillus rhamnosus OF44-15Ph10T from a library of about 30,000 human symbiotic bacteria. It has strong ability to produce L-lactic acid, D-lactic acid and hydrogen peroxide, is sensitive to most antibiotics, has strong growth ability and is highly resistant to acid and alkali, and also shows strong adhesion to human vaginal epithelial cells, and is resistant to common vaginal microbial infections. The pathogenic bacteria Lactobacillus rhamnosus OF44-15Ph10T 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. The Lactobacillus rhamnosus 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 recurrence rate, toxic and side effects Small, long-lasting potency. Meanwhile, the inventors also found that the Lactobacillus rhamnosus in the present disclosure can effectively reduce bone loss, so the bacteria can also be used to prevent and/or treat related diseases caused by bone loss, such as osteoporosis. Therefore, the existing drugs for the treatment of osteoporosis have large potential risks and large side effects, which may lead to the problem of hormonal disorders in the human body.

Additional aspects and advantages of the present disclosure will be set forth, in part, from the following description, and in part will become apparent from the following description, or may be learned by practice of the present disclosure.

Deposit information:

Strain name: Lactobacillus rhamnosus OF44-15Ph10T

Deposit date: August 24, 2018

Preservation unit: Guangdong Microbial Culture Collection Center (GDMCC)

Deposit number: GDMCC No: 60406

Preservation address: 5th Floor, Building 59, Yard, No. 100, Xianlie Middle Road, Guangzhou

Description of drawings

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

Figure 1: Genome map of Lactobacillus rhamnosus OF44-15Ph10T;

Figure 2: Annotation results of probiotic function genes of Lactobacillus rhamnosus OF44-15Ph10T;

Figure 3A: Comparison of Lactobacillus rhamnosus OF44-15Ph10T and commercially available strain L-lactic acid;

Figure 3B: Comparison of Lactobacillus rhamnosus OF44-15Ph10T and commercially available strain D-lactic acid;

In Figures 3A and 3B, GR-1 represents Lactobacillus rhamnosus GR-1, and OF44 represents Lactobacillus rhamnosus OF44-15Ph10T in the present disclosure;

Figure 4: Inhibition of growth of E. coli by Lactobacillus rhamnosus OF44-15Ph10T and Lactobacillus rhamnosus GR-1, E.coli in the figure represents E. coli, and OF44 represents rhamnolactide in the present disclosure Bacillus OF44-15Ph10T, GR-1 represents Lactobacillus rhamnosus GR-1;

Figure 5: Lactobacillus rhamnosus OF44-15Ph10T and Lactobacillus rhamnosus GR-1 inhibit the growth of Gardnerella vaginalis, GV in the figure represents Gardnerella, and OF44 represents the present disclosure In Lactobacillus rhamnosus OF44-15Ph10T, GR-1 represents Lactobacillus rhamnosus GR-1;

Figure 6: The bacterial count of Lactobacillus rhamnosus OF44-15Ph10T in the model group and the probiotic group in Example 9;

Figure 7: The bacterial count of Gardnerella in the model group and the probiotic group in Example 9;

Fig. 8: Comparison of bone-related indexes of mice in Sham (sham operation) control group, model group and probiotic group in Example 10;

Fig. 9: Comparison results of three-dimensional reconstruction images (micro-CT) of bone-related tissues of mice in the Sham (sham operation) control group, the model group and the probiotic group in Example 10.

Detailed description of the invention

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 rhamnosus OF44-15Ph10T

1. Sample collection

The isolated sample came from the feces of a healthy female. The feces were collected into sterile sample tubes and brought back to the laboratory for sorting within 1 hour.

2. Separation and purification

The collected fresh samples were immediately transferred to the anaerobic operation box, and 0.2 g of the samples were taken in 1 mL of sterile PBS (phosphate buffered saline), fully shaken and mixed, and then applied by gradient dilution. The culture medium was modified PYG medium. (purchased from Huankai Microorganism Technology Co., Ltd.), the specific formula is (1L): tryptone 8g, soybean peptone 2g, polyprotein 1g, casein 1g, yeast powder 10g, beef extract 5g, glucose 5g, K ₂ HPO ₄ 2g , Maltose 0.5g, Cellobiose 0.5g, Soluble Starch 0.5g, Sodium Sulfide 0.25g, Tween 80 0.5mL, Cysteine-HCl H ₂ O 0.5g, Glycerin 0.5mL, Sodium Acetate 5g, Heme 5mg, Vitamin K 11μL , inorganic salt solution (each L inorganic salt 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 pH to 6.8-7.0. The coated plate was placed at 37°C for anaerobic culture, and the anaerobic gas composition was N ₂ : CO ₂ : H ₂ =90:5:5. After 3 days of culture, single colonies were picked and streaked to obtain pure cultured strains of each single bacteria.

3. Culture preservation

The obtained pure cultured strain was cultured to a concentration of about 10 ⁹ CFU/mL, 400 μL of bacterial liquid was added with 400 μL 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: 60406.

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 physiological 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, placed in 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. After freeze-drying, take out the sample ampoule tube, and in accordance with the "Standard Operating Procedures for Vacuum Ampoule Melting and Sealing Machine", use a strong flame to thin the area below the tampon at the neck of the ampoule tube for melting and sealing.

4. 16S rDNA identification

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 μL; dNTP, 2.5 μL; 27F(5'-AGAGTTTGATCATGGCTCAG-3', as shown in SEQ ID NO:1 shown), 0.5 μL; 1492R (5′-TAGGGTTACCTTGTTACGACTT-3′, shown in SEQ ID NO: 2), 0.5 μL; Taq enzyme, 0.3 μL; template, 1 μL; ddH ₂ O, 18.2 μL. 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 1200 bp (SEQ ID NO: 3). This sequence was analyzed by blast in genebank, and the identification result of OF44-15Ph10T was obtained as Lactobacillus rhamnosus.

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

5. Physiological and biochemical characteristics of OF44-15Ph10T

Cultured in MRS medium (purchased from Huankai Microorganism Technology Co., Ltd.) for 48 hours, the colony of OF44-15Ph10T is white, smooth, round, with neat edges, and the colony diameter is about 1-2 mm. Observed under a microscope at 1000 times, the cells were elongated and rod-shaped, Gram staining was positive, and no spores and flagella were produced. The catalase reaction of OF44-15Ph10T was negative, oxidase negative, strictly anaerobic, and the carbon source utilization was detected by API 50CHL kit. The results are shown in Table 1 (+ means positive reaction; - means negative reaction; W means weak positive reaction).

Table 1: OF44-15Ph10T carbon source utilization results

Numbering	reaction	result	Numbering	reaction	result
1	control	+	26	Aesculus	+
2	glycerin	-	27	salicin	-
3	Erythritol	-	28	D-Cellobiose	+
4	D-arabinose	-	29	D-maltose	+
5	L-arabinose	+	30	D-Lactose	+
6	D-ribose	+	31	D-melibiose	-
7	D-xylose	-	32	D-Sucrose	+
8	L-Xylose	-	33	D-Trehalose	-
9	D-side calendulol	-	34	Inulin	-
10	Methyl-βD Xylopyranoside	-	35	D-Matsutose	+
11	D-galactose	+	36	D-Raffinose	+
12	D-glucose	+	37	starch	-
13	D-Fructose	+	38	Glycogen	-
14	D-Mannose	+	39	Xylitol	-
15	L-Sorbitose	-	40	D-gentiobiose	-
16	D-Rhamnose	-	41	D-Toulon Sugar	-
17	dulcitol	-	42	D-lyxose	-
18	Inositol	-	43	D-tagatose	-
19	Mannitol	+	44	D-fucose	-
20	Sorbitol	+	45	L-fucose	-
twenty one	Methyl-αD Mannopyranoside	-	46	D-Arabinol	-
twenty two	methyl-αD glucopyranoside	+	47	L-Arabinol	-
twenty three	N-Acetyl Glucosamine	+	48	Potassium Gluconate	+
twenty four	Amygdalin	+	49	Potassium 2-ketogluconate	-
25	Arbutin	+	50	Potassium 5-ketogluconate	-

Example 2: Genome sequencing, species classification and functional gene analysis of Lactobacillus rhamnosus OF44-15Ph10T

1. Genome sequencing

The overnight culture of OF44-15Ph10T was centrifuged at 7,227g for 10 minutes at 4°C, the resulting pellet was resuspended in 1mL Tris-EDTA, 50μL 10% SDS and 10μL proteinase K (20mg/mL) were added at 55°C In a water bath for 2 hours, cells were lysed and DNA was extracted by the phenol-chloroform method. The Illumina Hiseq 2000 platform was used for sequencing, and the sequencing length was 500 bp in both directions, and the reads were assembled with SOAPdenovo. After evaluation, GCskew was used to analyze genome-wide GC content and visualize genome-wide sequence and functional distribution (Figure 1).

2. Classification of strain genome species

The whole genome sequence was compared and analyzed using Checkm software, and the closest species information to the genome was found to be Firmicutes-Bacillus-Lactobacillus-Lactobacillus-Lactobacillus-Lactobacillus rhamnosus species , the number of annotated genomes is 31, the number of annotated markers is 586, the completeness of the genome is 99.46%, and the degree of contamination is 0.

3. Prebiotic functional genes and safety analysis

In order to examine whether the metabolic pathways related to the treatment of reproductive tract infections in OF44-15Ph10T are complete, we selected Lactic acid synthesis and Peroxide hydrogen production from the prokaryoties database of KEGG. A separate database is established for all enzymes related to short-chain fatty acid synthesis. Use blastx to align the whole gene sequences of OF44-15Ph10T with these databases, select the annotation results with e-value greater than or equal to 0.01 and identity greater than or equal to 60, the gene copy number of the enzyme in the relevant pathway is indicated by the shade of color, and the gene is annotated to the gene. It proves that the 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. .

Example 3: Bioactive substances of Lactobacillus rhamnosus OF44-15Ph10T

The bioactive substances of OF44-15Ph10T were mainly examined for L-lactic acid content, D-lactic acid content, and hydrogen peroxide production in metabolites.

1. Sample pretreatment

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

1 mL of each bacterial solution was taken and centrifuged at 8000 r/min for 5 min, and the supernatant was taken to detect the contents of L-lactic acid and D-lactic acid.

Then take 1 mL of bacterial solution and add lysozyme (final concentration is 1 mg/mL), let stand at 37 °C for 15 min, centrifuge at 8000 r/min for 5 min, take the supernatant, and test the hydrogen peroxide concentration.

2. Measurement method

L-lactic acid and D-lactic acid content were measured with L-Lactic Acid (L-Lactate) Assay Kit and D-Lactic Acid (D-Lactate) Assay Kit (purchased from Megazyme Inc. US) according to 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.

3. The experimental results are shown in Table 2.

Table 2 Determination of lactic acid and hydrogen peroxide yields of OF44-15Ph10T

4. Comparison with commercial strains

The present disclosure selects commercially available Lactobacillus rhamnosus GR-1 as a control test, the experimental method is the same as above, and the results show that under anaerobic conditions, L-lactic acid of Lactobacillus rhamnosus GR-1 and The yields of D-lactic acid were 5.59 g/L and 0.45 g/L, respectively. In contrast, the yields of L-lactic acid and D-lactic acid of OF44-15Ph10T were higher than those of the control strains. The results are shown in Figures 3A and 3B.

Example 4: Identification of the ability of Lactobacillus rhamnosus OF44-15Ph10T to inhibit reproductive tract infection pathogens

1. Identification of the ability of OF44-15Ph10T to inhibit Escherichia coli E.coli

Under anaerobic conditions, overnight cultured OF44-15Ph10T and Lactobacillus rhamnosus GR-1 (concentration up to 10 ⁸ CFU) were filtered with a 0.22 μm filter to obtain the supernatant. 100 μL of overnight cultured Escherichia coli (concentration up to 10 ⁸ CFU) was added to 1 mL of OF44-15Ph10T supernatant and MRS medium, respectively, and cultured at 37°C for 24 hours to obtain bacterial liquid, and its absorbance at OD595 was measured. The bacterial liquid obtained by adding Escherichia coli to MRS liquid medium was used as a positive control. As shown in Figure 4, E.coli represents the bacterial solution added to the MRS liquid medium, OF44 represents the bacterial solution added to the supernatant of OF44-15Ph10T, and GR-1 represents the bacterial solution added to Lactobacillus rhamnosus GR- 1) The bacterial liquid of the supernatant, the results show that the metabolites of OF44-15Ph10T and GR-1 in the present disclosure can significantly inhibit the growth of E. coli, and the inhibitory effect of OF44-15Ph10T is better than that of GR-1.

2. Identification of the ability of OF44-15Ph10T to inhibit Gardnerella vaginalis BNCC337545

Under anaerobic conditions, overnight cultured OF44-15Ph10T and Lactobacillus rhamnosus GR-1 (concentration reached 10 ⁸ CFU) were filtered with a 0.22 μm filter to obtain the supernatant, and 100 μL of The overnight cultured Gardnerella (at a concentration of 10 ⁸ CFU) was added to 1 mL of OF44-15Ph10T supernatant and MRS medium, respectively, and cultured at 37° C. for 24 h to obtain bacterial liquid, and its absorbance at OD595 was measured. The bacterial solution obtained by adding Gardnerella to MRS liquid medium was used as a positive control. As shown in Figure 5, GV represents the bacterial solution added to the MRS liquid medium, OF44 represents the bacterial solution added to the supernatant of OF44-15Ph10T, and GR-1 represents the bacterial solution added to the upper layer of Lactobacillus rhamnosus GR-1 The results show that the metabolites of OF44-15Ph10T and GR-1 in the present disclosure can significantly inhibit the growth of Gardnerella, and the inhibitory effect of OF44-15Ph10T is better than that of GR-1.

3. Identification of the ability of OF44-15Ph10T to inhibit Candida albicans SC5314

The overnight cultured OF44-15Ph10T and Lactobacillus rhamnosus GR-1 were inoculated into 1.5 mL of MRS medium, and then 100 uL of overnight cultured SC5314 (concentration of 10 ⁵ CFU) was inoculated into OF44- The mixed bacterial solution was obtained from 15Ph10T and Lactobacillus rhamnosus GR-1 bacterial solution, and cultured at 37°C for 24h under aerobic conditions. The mixed bacterial solution was diluted and spread on PDA solid medium (purchased from Huankai Microorganism Technology Co., Ltd.) and cultured at 37°C for 24 h, and the colonies were counted. At the same time, only inoculated with SC5314 was used as a positive control. The count results showed that the OF44-15Ph10T group was 9.4x10 ⁴ CFU/mL, the SC5314 group was 8.6x10 ⁵ CFU/mL, and the GR-1 group was 1.2x10 ⁵ CFU/mL. The results showed that OF44-15Ph10T and Lactobacillus rhamnosus GR-1 could inhibit the growth of Candida albicans, and OF44-15Ph10T had better inhibitory effect than GR-1.

Example 5: Antibiotic susceptibility of Lactobacillus rhamnosus OF44-15Ph10T

To investigate the susceptibility of OF44-15Ph10T to 15 common antibiotics, the drug susceptibility paper method was used for the experiment. 100 μL of the bacterial liquid of OF44-15Ph10T cultured to the log phase was used for flat coating, and the antibiotic susceptibility 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.

Table 3 Antibiotic susceptibility of OF44-15Ph10T

antibiotic	Inhibition zone diameter (cm)	antibiotic	Inhibition zone diameter (cm)
Ampicillin	3	Ceftriaxone	2
Bacitracin	0	Vancomycin	0
penicillin	3.6	oxacillin	1.4
kanamycin	1.5	amoxicillin	3
tetracycline	2.6	Azithromycin	2
guaracillin	3.5	Clindamycin	2.2
erythromycin	2.7	Gentamicin	1.4
Chloramphenicol	2.7

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

Example 6: Tolerance of Lactobacillus rhamnosus OF44-15Ph10T to acids and bile salts

Because human probiotics need to pass through the stomach and small intestine to reach the intestinal tract, they need to experience gastric acid around pH 2.5 and bile salts at a concentration of 0.3%. Only acid- and bile-salt-tolerant strains reach the gut for prebiotic effects. Therefore, the acid and bile salt tolerance of OF44-15Ph10T was investigated in this example.

1. Acid tolerance of OF44-15Ph10T

MRS mediums of pH 2, pH 3, pH 4, pH 4.5 and pH 7 were prepared respectively, and 100 μL of OF44-15Ph10T bacterial liquid with a concentration of 8.5E+09 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. The results showed that OF44-15Ph10T 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).

Table 4 Acid tolerance of OF44-15Ph10T

2. Bile salt tolerance of OF44-15Ph10T

MRS medium containing 0.05%, 0.1%, 0.2% and 0.3% bile salts was prepared respectively, and 100 μL of OF44-15Ph10T bacterial liquid with a concentration of 8.5E+09 overnight culture was inoculated into MRS medium with different bile salt contents, 37 After culturing at ℃ for 24 h, the bacterial liquid was counted by plate coating. The results showed that OF44-15Ph10T could not only survive but also grow under the conditions of 0.05%, 0.1%, 0.2%, and 0.3% bile salts (Table 5).

Table 5 Bile salt tolerance of OF44-15Ph10T

The results show that the Lactobacillus rhamnosus OF44-15Ph10T of the present disclosure has strong tolerance to acids and bile salts.

Example 7: Evaluation of the colonization ability of Lactobacillus rhamnosus OF44-15Ph10T

1. Evaluation of the self-agglutination ability of OF44-15Ph10T

The OF44-15Ph10T bacterial liquid cultured overnight was divided into 10 mL in a 15 mL test tube, the uppermost bacterial liquid was taken, and the absorbance at OD600 was measured with a UV 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. OF44-15Ph10T showed better self-aggregation ability (Table 6).

Table 6 Self-agglutination ability of OF44-15Ph10T

2. Evaluation of the adhesion ability of OF44-15Ph10T to human cervical cancer epithelial cells Hela

Digest the cultured Hela cells, dilute the cells with 1640 complete culture medium (GIBCO, purchased from Volkawei Beijing Biotechnology Co., Ltd.) without double antibody, and count the cells on a hemocytometer (see below for the method) to make the cell concentration To reach about 2×10 ⁵ cells/mL, drop 1 mL into a cell culture dish (12 or 6-well plate), and incubate at 37° C. in a 5% CO ₂ -95% air incubator until complete differentiation.

After the cells grow into a dense monolayer, rinse the cells twice with sterilized PBS buffer, then add 1 mL of 1640 culture medium and 1 mL of overnight culture to each well and adjust the bacterial suspension to 10 ⁸ CFU/mL, and shake gently to mix. , continue to incubate in an incubator at 37°C and 5% CO ₂ , and repeat three wells for each bacterial sample.

After incubation for 90 min, 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 34.59±4.63.

The present disclosure selects commercially widely used strains: Lactobacillus rhamnosus GR-1, Lactobacillus reuteri RC-14 and marketed medicinal strain Lactobacillus delbrueckii as control experiments. The experimental methods are the same as above, and the results show that each Hela cells adhered on average to 13.43±7.07 GR-1, 17.48±4.24 RC-14 and 25.23±2.12 Lactobacillus delbrueckii, indicating that the adhesion ability of OF44-15Ph10T to human cervical cancer cell Hela was higher than that of the two strains commercial strains. 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.

3. Evaluation of the adhesion ability of OF44-15Ph10T to human vaginal epithelial cells VK2E6/E7

Digest the cultured VK2E6/E7 cells (purchased from Beijing Beina Chuanglian Institute of Biotechnology), dilute the cells with 1640 complete culture medium without double antibody, and count them on a hemocytometer (see below for the method) to make the cell concentration To reach about 2×10 ⁵ cells/mL, drop 1 mL into a cell culture dish (12 or 6-well plate), and incubate at 37° C. in a 5% CO ₂ -95% air incubator until complete differentiation.

After the cells grew into a dense monolayer, rinse the cells twice with sterilized PBS buffer, then add 1 mL of 1640 culture medium and 1 mL of overnight culture to each well and adjust to 10 ⁸ CFU/mL of bacterial suspension, and shake gently to mix. , continue to incubate in an incubator at 37°C and 5% CO ₂ , and repeat three wells for each bacterial sample.

After incubation for 90 min, 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, it was 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 69.51±47.02.

The present disclosure selects commercially widely used strains: Lactobacillus rhamnosus GR-1, Lactobacillus reuteri RC-14 and marketed medicinal strain Lactobacillus delbrueckii as control experiments. The experimental methods are the same as above, and the results show that each The average number of VK2E6/E7 cells adhered to 27.4±8.11 GR-1, 14.5±4.63 RC-14 and 22.24±9.26 L. delbrueckii, indicating that OF44-15Ph10T has a high ability to adhere to human vaginal epithelial cells VK2E6/E7 Lactobacillus rhamnosus GR-1, Lactobacillus reuteri RC-14 and Lactobacillus delbrueckii. 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.

4. Cell count

Wipe the hemocytometer and the cover slip clean, and cover the cover slip on the counting plate; then aspirate a little of the cell suspension, drop it on the edge of the cover slip, fill the suspension between the cover slip and the counting plate, and let it stand. 3 minutes; at the end of the microscopic examination, the total number of cells in the four grids of the counting plate was calculated, and only the cells on the left and the upper side of the line were counted (the number of cells/mL=the total number of cells in 4 large grids/4×10000).

Example 8: Rat toxicity test of Lactobacillus rhamnosus OF44-15Ph10T

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, gavaged with different doses, each rat was orally gavaged with 0.5 mL of fresh bacterial solution (concentrations were 1×10 ⁵ , 1×10 ⁹ , 1×10 ¹² CFU/mL, respectively), Once a day for 3 consecutive days, from the first day of gavage to the seventh day, the rats were all healthy and survived and gained 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 (concentrations were 1×10 ⁵ , 1×10 ⁹ , 1×10 ¹² CFU/mL, respectively ) once a day for 3 consecutive days. From the lavage on the first day to the seventh day, the rats were all healthy and survived and gained weight.

The above experimental results show that the administration of Lactobacillus rhamnosus OF44-15Ph10T to rats at a dose of 0.2×10 ⁵ to 0.5×10 ¹² CFU/day will not affect the health of rats, and no toxic reaction is found. , indicating that it is relatively safe at this dose.

Example 9: The ability of Lactobacillus rhamnosus OF44-15Ph10T to treat genital tract infection in rats

The rat model selected in this example is a rat model infected with Gardnerella genitalium, SD female rats, 7 weeks old, body weight 250g ± 250g, the rat rearing environment is SPF grade, and the experimental animals are divided into 2 groups groups, the model group and the probiotic group, respectively. Each group of 10 animals, a total of 20 animals, were fed with a standard diet.

The experiment is divided into adaptation period, modeling period, intervention period and observation period. Adaptation period: Rats were fed a standard diet for 7 days. Three days before modeling, 0.5 mg β-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 is 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. On the 4th day, observe and record whether the vagina is red, swollen and discharge, whether there are phenotypic characteristics such as erythema and particulate matter, and select the rats that meet the modeling requirements. test. Intervention period: a total of 7 days, the rats in the model group were not treated; the rats in the probiotic group were washed with OF44-15Ph10T PBS bacterial solution, and the total viable bacteria each time was not less than 1×10 ⁸ CFU/mL, every day Rinse once for 7 consecutive days. Regularly observe and record whether the rat vagina is red, swollen and discharge, whether there are erythema and particulate phenotype. Observation period: 3 days after the intervention, observe and record whether the rat vagina is red, swollen and discharge, and whether there is erythema and particulate phenotype. While observing the rat phenotype each time, the vaginal washing fluid of the rat was taken, and quantitative PCR was used to detect the clearance of Gardnerella and the colonization of Lactobacillus gasseri

The results showed that Lactobacillus rhamnosus OF44-15Ph10T could colonize the vagina of rats (Fig. 6), and at the same time had the effect of improving the infection of Gardnerella vaginalis and inhibiting its recurrence (Fig. 7 and Table 7). (Table 8).

Table 7 Recording results of rat vaginal phenotype

Example 10: The effect of Lactobacillus rhamnosus OF44-15Ph10T on the bone loss model in mice

Bone loss model adopts C57BL/6 female mice (purchased from Guangdong Provincial Medical Experimental Animal Center), 8 weeks old, body weight 20±2g, and can eat and drink freely in SPF environment. Randomly divided into 3 groups with 9 to 10 animals in each group.

The first group of 9 mice was the Sham (sham operation) control group. Sham operation was performed, only the skin was incised and then sutured. During the experiment, the medium containing the target strain (OF44-15Ph10T) was fed, and the feeding amount and body weight were proportional;

The second group of 10 mice was used as the experimental group. The osteoporosis model was induced by the removal of ovarian organs. The target strain was given a saturated suspension by gavage, and the feeding amount was proportional to the body weight;

The third group of 10 mice was the negative control group, and the osteoporosis model was induced by the removal of ovarian organs.

During the experiment, the weight data of the mice were recorded weekly (Table 8); the mice were sacrificed after the experiment, and the changes in bone-related indexes were detected by micro-CT (shown in Table 9 and Figure 8), and three-dimensional reconstructed images of the tissues were established ( Figure 9).

The results showed that there was no significant difference between the probiotic group (the second group) and the Sham control group (the first group) (p>0.05); compared with the model group (the third group), a number of bone-related indicators were statistically significant The results showed that the intestinal probiotic Lactobacillus rhamnosus OF44-15Ph10T has the effect of inhibiting bone loss in the mouse model.

Table 8 Records of body weights of mice in each group before and after gavage

Table 9 Records of bone-related indexes of mice in each group

In the description of this specification, reference to the terms "one embodiment," "some embodiments," "example," "specific example," "some embodiments," or "some examples," or the like, is meant to incorporate the embodiment or example. A particular feature, structure, material, or characteristic described is included in at least one embodiment or example of the present disclosure. In this specification, schematic representations of the above terms are not necessarily directed 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, those skilled in the art may combine and combine the different embodiments or examples described in this specification, as well as the features of the different embodiments or examples, without conflicting each other.

Although the embodiments of the present disclosure have been shown and described above, it should be understood that the above-described embodiments are exemplary and should not be construed as limitations of the present disclosure, and those of ordinary skill in the art may interpret the above-described embodiments within the scope of the present disclosure. Embodiments are subject to variations, modifications, substitutions and variations.

Claims (21)

1. A strain of Lactobacillus rhamnosus OF44-15Ph10T, whose deposit number is GDMCC No: 60406.
2. Lactobacillus rhamnosus described in claim 1 or its fermentation product or its bacterial suspension or its application in the preparation of a product, wherein the product is used for the prevention and/or treatment of genital tract flora disorders related diseases and/or related diseases caused by bone loss.
3. The use according to claim 2, wherein the diseases related to the disorder of reproductive tract flora include reproductive tract infection.
4. The use according to claim 3, wherein the genital tract infection comprises a genital tract infection selected from the group consisting of bacterial vaginitis, fungal vaginitis, trichomonas vaginitis, aerobic vaginitis, senile vaginitis and viral infection at least one.
5. The use according to claim 2, wherein the related diseases caused by the bone loss include at least one selected from the group consisting of osteopenia, osteoporosis, and osteoporotic fractures.
6. Lactobacillus rhamnosus according to claim 1 or its fermentation product or its bacterial suspension or its application in the preparation of a product, wherein the product is used for antibacterial, adhesion to vaginal epithelial cells and/or cervical cells , lactate production, _H2O2 production, and/or _inhibition of bone loss.
7. The application according to any one of claims 2-6, wherein the product is food, medicine or health care product.
8. The use according to any one of claims 2-6, wherein the product is a drug, and the drug is administered at a dose of 10 ⁵ -10 ¹² CFU/day.
9. A food or health product, wherein the food or health product comprises the Lactobacillus rhamnosus or its fermentation product or its bacterial suspension or its culture solution according to claim 1.
10. The food or health product according to claim 9, wherein the food or health product is selected from the group consisting of products for preventing and/or treating diseases related to genital flora disorders, preventing and/or treating related diseases caused by bone loss, At least one of an antibacterial product, a product adhering to vaginal epithelial cells and/or cervical cells, a lactic acid _- producing product, and a H2O2 _- producing product.
11. A pharmaceutical composition, wherein the pharmaceutical composition comprises the Lactobacillus rhamnosus or its fermentation product or its bacterial suspension or its culture solution according to claim 1.
12. The pharmaceutical composition according to claim 11, wherein the pharmaceutical composition is in the form of a single dose, and the pharmaceutical composition contains the Lactobacillus rhamnosus in a daily dose of 10 ⁵ -10 ¹² CFU.
13. The pharmaceutical composition according to claim 11, wherein the pharmaceutical composition is in a dosage form suitable for external use or oral administration.
14. Lactobacillus rhamnosus described in claim 1 or its fermented product or bacterial suspension or its culture solution, food or health product described in claim 9 or 10, and any one of claims 11-13 Application of the pharmaceutical composition in preventing and/or treating diseases related to genital tract flora disorder and/or related diseases caused by bone loss.
15. The use according to claim 14, wherein the diseases related to disorders of reproductive tract flora include reproductive tract infections.
16. The use according to claim 15, wherein the reproductive tract infection comprises a genital tract infection selected from the group consisting of bacterial vaginitis, fungal vaginitis, trichomonas vaginitis, aerobic vaginitis, senile vaginitis and viral infection at least one.
17. The use according to claim 14, wherein the related diseases caused by the bone loss include at least one selected from the group consisting of osteopenia, osteoporosis, and osteoporotic fractures.
18. A method for preventing and/or treating disorders related to reproductive tract flora disorders and/or related diseases caused by bone loss, wherein the method comprises:

    Administer at least one of the following to a subject in need:

    (1) Lactobacillus rhamnosus described in claim 1 or its fermentation product or its bacterial suspension or its culture solution;

    (2) The food or health care product according to claim 9 or 10;

    (3) The pharmaceutical composition of any one of claims 11-13.
19. 19. The method of claim 18, wherein the genital tract flora disorder-related disease comprises a genital tract infection.
20. The method of claim 19, wherein the reproductive tract infection comprises a genital tract infection selected from the group consisting of bacterial vaginosis, fungal vaginitis, trichomonas vaginitis, aerobic vaginitis, senile vaginitis, and viral infections at least one.
21. The method according to claim 18, wherein the related disease caused by the bone loss comprises at least one selected from the group consisting of osteopenia, osteoporosis, and osteoporotic fracture.

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