CN112760247B

CN112760247B - Lactobacillus rhamnosus for preventing and/or treating diseases caused by genital tract flora disorder and/or bone loss

Info

Publication number: CN112760247B
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 Precision Nutrition Shenzhen Technology Co ltd; 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: 2022-10-25
Anticipated expiration: 2040-11-10
Also published as: WO2022100632A1; CN112760247A

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 which is used 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 urination and painful urination, which can lead to premature delivery of pregnant women, cervical cancer and the like. 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 flora of the genital tract, thereby increasing the risk of relapse. 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

The reproductive health of women is closely related to the microbial flora living in the reproductive tract, and a plurality of researches indicate that the flora disturbance of 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 genital 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 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 some organs and an estrogen-antagonistic effect in others. 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 high, 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 for preventing and/or treating diseases related to genital tract flora disorder (especially female genital tract flora disorder in human beings) and/or diseases related to bone loss is screened, and the new Lactobacillus rhamnosus OF44-15Ph10T can generate lactic acid and hydrogen peroxide and other secretions to inhibit the growth OF pathogenic bacteria infected by genital tract, is used for preventing and/or treating diseases related to genital tract flora disorder, and especially reduces recurrence rate, has small 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 (Lactobacillus rhamnosus) OF44-15Ph10T with the accession number 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, institute OF Mass, no. 100, university 5 building in Fuxiu district, guangzhou City, guangzhou province) 24 days in 2018, and the preservation number is GDMCC No. 60406.

The Lactobacillus rhamnosus (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 identification. When cultured in MRS medium for 48 hours, OF44-15Ph10T colonies are white, smooth, round, and neat in edges, and the diameter OF the colonies is about 1-2mm. When observed under a microscope at 1000 times, the thalli are in a slender rod shape, gram-positive, and no spores and flagella are generated.

The invention also provides application of the lactobacillus rhamnosus or a fermentation product thereof or a bacterial suspension thereof or a culture solution thereof in preparing a product, wherein the product is used 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 (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 human correlation research and animal model experiments, and can be used for preventing and/or treating related diseases caused by osteopenia, 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 the female genital tract, including e.coli (e.coli), gardnerella vaginalis (BNCC 337545), corynebacteria, haemophilus, staphylococcus aureus, pseudomonas aeruginosa, and the like gram-negative anaerobes.

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 SC 5314), 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 H ₂ O ₂ And/or inhibiting bone loss.

In some embodiments of the invention, the product is a medicament.

In other embodiments of the invention, the product is a medicament administered at a dose of 10 ⁵ -10 ¹² CFU/day.

In yet another aspect of the present invention, a medicament 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 drug is selected from the group consisting of a drug for preventing and/or treating a disease associated with a disorder of the genital tract flora, a drug for preventing and/or treating a disease associated with bone loss, an antibacterial drug, a drug that adheres to vaginal epithelial cells and/or cervical cells, a lactic acid-producing drug, an H-producing drug, and combinations thereof ₂ O ₂ At least one of the medicaments 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 10 ⁵ -10 ¹² CFU 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 such as miconazole, clotrimazole and the like is mainly used for mycotic vaginitis, and the action mechanism of the suppository is to inhibit sterol synthesis of cell membranes of fungi such as candida and the like, influence the permeability of the cell membranes, inhibit the growth of the fungi and cause 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 genital tract by local use, can make up the defects brought by the traditional antibiotic treatment, helps the flora to recover stable 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 shows 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 an external or oral medicine for development to prevent, treat or assist in treating female genital tract infection diseases. 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 rhamnous OF44-15Ph10T

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

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 genome map OF Lactobacillus rhamnosus (Lactobacillus rhamnosus) OF44-15Ph10T;

FIG. 2: the probiotic functional gene annotation result OF Lactobacillus rhamnosus (Lactobacillus rhamnosus) OF44-15Ph10T;

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 OF44-15Ph10T OF the present invention;

FIG. 4 is a schematic view of: 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 in the invention;

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

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

FIG. 7: the numbers of the model group and 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: separation and identification OF Lactobacillus rhamnosus OF44-15Ph10T

1. Sample collection

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

2. Separating and purifying

Fresh samples collected were immediately transferred to an anaerobic chamber and 0.2g of sample was taken in 1mL sterile PBS (phosphorus)Acid buffer solution), then performing gradient dilution and coating, wherein the culture medium adopts an improved PYG culture medium (purchased from Kyork Microbiology technologies), 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, K ₂ HPO ₄ 2g, maltose 0.5g, cellobiose 0.5g, soluble starch 0.5g, sodium sulfide 0.25g, tween 80.5 mL, cysteine-HCl. H ₂ 0.5g 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 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, 1mg of resazurin, distilled water to 1L, and adjusting the pH to 6.8-7.0. The coated plate is placed at 37 ℃ for anaerobic culture, and the anaerobic gas component is N ₂ ：CO ₂ ：H ₂ =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 10 ⁹ CFU/mL, 400 μ L of 40% glycerol was added to 400 μ L of the bacterial solution to make the glycerol concentration reach 20%, and then the mixture was stored at-80 deg.C.

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.

And (3) autoclaving the ampoule tube and the protective agent for standby, streaking the overnight culture bacterial liquid, culturing at 37 ℃ for 24 hours, and carrying out the following operations after observing that no mixed bacteria pollution is found. Centrifugally collecting bacterial liquid, washing with sterilized normal saline, adding 2-3 mL of skimmed milk protective agent, and suspending to obtain 10-numbered bacterial colonies ⁸ ～10 ¹⁰ The bacterial suspension is divided into individual/mL, subpackaged in a sterile ampoule tube and pre-frozen in a refrigerator at the temperature of 80 ℃ below zero for 1 to 2 hours. 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 melting the sample ampoule tube according to the specificationStandard practice of sealing machine-the ampoule neck is drawn down by a strong flame to seal it.

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. Taking genome DNA as a template, and carrying out PCR amplification by using a 16S rDNA universal primer, wherein the amplification system is as follows: 10 × PCR buffer,3 μ L; dNTP,2.5 μ L;27F (5; 1492R (5 'TAGGGTTACCTGTTACCTGTTACGACTT-3' as shown in SEQ ID NO: 2), 0.5 mu L; taq enzyme, 0.3. Mu.L; template, 1 μ L; ddH ₂ O, 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, extension at 72 ℃ for 1min30s,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 the identification result OF OF44-15Ph10T is obtained and is Lactobacillus rhamnosus (Lactobacillus rhamnosus).

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

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

5. physiological and biochemical characteristics OF OF44-15Ph10T

When cultured in MRS medium (available from Cinchaku Microbiology technologies) for 48 hours, OF44-15Ph10T colonies are white, smooth, round, and neat in edge, and the diameter OF the colonies is about 1-2mm. 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 OF44-15Ph10T catalase reaction is negative, the oxidase reaction is negative, strict anaerobism is realized, and the carbon source utilization condition is detected by using an API 50 CHL 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

Numbering	Reaction of	Results	Number of	Reaction of	Results
						1	Control	+	26	Qiyeling (medicine for treating gynecopathy)	+
2	Glycerol	-	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-adonitol	-	34	Inulin powder	-
10	Methyl-beta D xylopyranosides	-	35	D-melezitose	+
						11	D-galactose	+	36	D-raffinose	+
12	D-glucose	+	37	Starch	-
						13	D-fructose	+	38	Glycogen	-
14	D-mannose	+	39	Xylitol, its preparation method and use	-
						15	L-sorbose	-	40	D-gentiobiose	-
16	D-rhamnose	-	41	D-Turentang	-
						17	Dulcitol	-	42	D-lyxose	-
18	Inositol	-	43	D-tagatose	-
						19	Mannitol	+	44	D-fucose	-
20	Sorbitol	+	45	L-fucose	-
						21	Methyl-alpha D mannopyranosides	-	46	D-arabitol	-
22	Methyl-alpha D glucopyranoside	+	47	L-arabitol	-
						23	N-acetylglucosamine	+	48	Potassium gluconate	+
24	Amygdalin	+	49	2-Keto-Potassium gluconate	-
						25	Arbutin	+	50	5-Keto-Potassium gluconate	-

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 4 ℃ for 10 minutes, the resulting precipitate was suspended in 1mL OF Tris-EDTA, 50. Mu.L OF 10% SDS and 10. Mu.L OF proteinase K (20 mg/mL) were added, and the mixture was subjected to a water bath 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% 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 diseases associated with genital tract infections in OF44-15Ph10T are completed, enzymes associated with 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 established, respectively. The whole gene sequences OF OF44-15Ph10T are respectively compared with the databases by using blastx, the annotation result that e-value is more than or equal to 0.01 and identity is more than or equal to 60 is 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 larger (figure 2), and the antibiotic resistance gene, virulence factor, plasmid, transfer element, bacteriophage and virus are not annotated in the genome, so that the bacterial strain is proved to be safe to use.

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

OF44-15Ph10T is a bioactive substance mainly used for examining the L-lactic acid content, D-lactic acid content and hydrogen peroxide generation in metabolites.

1. Sample pretreatment

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

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

Adding lysozyme (the final concentration is 1 mg/mL) into 1mL of the bacterial solution, standing at 37 ℃ for 15min, centrifuging at 8000r/min for 5min, taking the supernatant, and detecting the concentration of hydrogen peroxide.

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 2 determination of the production of OF44-15Ph10T lactic acid and hydrogen peroxide

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 as above, and the result shows that the yield OF L-lactic acid and D-lactic acid OF the Lactobacillus rhamnosus GR-1 is 5.59g/L and 0.45g/L respectively under the anaerobic condition, compared with the yield OF L-lactic acid and D-lactic acid OF the comparative strain, the yield OF OF44-15Ph10T is higher than that OF the comparative strain, and the result is shown in FIGS. 3A and 3B.

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

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

Under anaerobic condition, overnight cultured OF44-15Ph10T and Lactobacillus rhamnosus GR-1 (with a concentration OF 10) ⁸ CFU) was filtered through a 0.22 μm filter to obtain a supernatant, and 100. Mu.L of overnight-cultured E.coli (concentration: 10) was added ⁸ CFU) is added into 1mL OF OF44-15Ph10T supernatant and MRS culture medium respectively, and cultured for 24h at 37 ℃ to obtain bacterial liquid, and the absorbance value OF the bacterial liquid under OD595 is measured. The bacteria liquid obtained by adding Escherichia coli into MRS liquid culture medium is used as positive control. As shown in FIG. 4, 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 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 Escherichia coli, and the inhibiting effect OF OF44-15Ph10T is better than that OF GR-1.

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

Under anaerobic condition, overnight cultured OF44-15Ph10T and Lactobacillus rhamnosus GR-1 (with a concentration OF 10) ⁸ CFU) was filtered through a 0.22 μm filter to obtain a supernatant, and 100. Mu.L of overnight-cultured Gardnerella (concentration: 10) was added ⁸ CFU) is added into 1mL OF OF44-15Ph10T supernatant and MRS culture medium respectively, and cultured for 24h at 37 ℃ to obtain bacterial liquid, and the absorbance value OF the bacterial liquid under OD595 is 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 the bacterial liquid added to MRS liquid medium, OF44 represents the bacterial liquid added to the supernatant OF OF44-15Ph10T, and GR-1 represents the bacterial liquid added to the supernatant OF MRS liquid mediumThe result OF the bacterial liquid OF the supernatant OF Lactobacillus rhamnosus (Lactobacillus rhamnosus GR-1) shows that the metabolites OF OF44-15Ph10T and GR-1 in the invention can obviously inhibit the growth OF Gardnerella, and the inhibiting effect OF OF44-15Ph10T is better than that OF GR-1.

3. Identification OF OF44-15Ph10T 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) ⁵ CFU) 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. SC5314 inoculated only was also used as a positive control. The counting result shows that the OF44-15Ph10T group is 9.4x10 ⁴ CFU/mL, with 8.6x10 for the SC5314 group ⁵ CFU/mL, 1.2x10 for GR-1 group ⁵ CFU/mL. The result shows 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

And (3) investigating the sensitivity OF OF44-15Ph10T to 15 common antibiotics, performing an experiment by adopting a drug sensitive paper sheet method, taking 100 mu L OF bacterial liquid OF OF44-15Ph10T cultured to a logarithmic phase to perform plate coating, attaching an antibiotic drug sensitive sheet on the surface OF a plate, culturing for 48h at 37 ℃, measuring the size OF an inhibition zone, and obtaining the result shown in Table 3.

TABLE 3 antibiotic sensitivity of OF44-15Ph10T

Antibiotics	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 compounds	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 (Lactobacillus rhamnosus) OF44-15Ph10T to acid and bile salt

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 strains with acid and bile salt tolerance can reach the intestine to exert a probiotic effect. Therefore, this example was examined on 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 microliter OF OF44-15Ph10T bacterial solution cultured overnight with the concentration OF 8.5E +09 is inoculated into the MRS culture media with different pH values, and after the bacterial solution is cultured for 24 hours at 37 ℃, the bacterial solution is subjected to plate coating counting. The results show that OF44-15Ph10T can not only survive but also grow under the conditions OF pH2, pH 3, pH 4, pH 4.5, pH 7 (Table 4).

TABLE 4 acid tolerance of OF44-15Ph10T

2. Bile salt tolerance OF OF44-15Ph10T

MRS culture media containing 0.05%, 0.1%, 0.2% and 0.3% bile salts 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 bile salt contents, and after 24 hours OF culture at 37 ℃, plate coating counting is carried out on the bacterial liquid. The results show that OF44-15Ph10T can not only survive but also grow under the conditions OF 0.05%, 0.1%, 0.2%, 0.3% bile salt (Table 5).

TABLE 5 bile salt tolerance of OF44-15Ph10T

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 capability

And subpackaging 10mL OF the overnight cultured OF44-15Ph10T bacterial liquid in a 15mL test tube, taking the bacterial liquid on the uppermost layer, 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 6 self-aggregation capability of OF44-15Ph10T

2. Evaluation OF OF44-15Ph10T and human cervical carcinoma epithelial cell Hela adhesion capability

The cultured Hela cells were digested, diluted with a 1640 complete medium (GIBCO, from Warcaway Beijing Biotechnology Ltd.) containing no double antibody, and counted on a hemocytometer (see below) to a cell concentration of about 2X10 ⁵ cell/mL, 1mL drop in cell culture dishes (12 or 6 well plates), 5% CO ₂ Incubate at 37 ℃ in-95% air incubator until complete differentiation.

After the cells have grown to a dense monolayer, the cells are rinsed 2 times with sterile PBS buffer, and then lmL1640 medium and 1mL of the buffer are added to each well, incubated overnight and adjusted to 10 ⁸ CFU/mL suspension, gently shaking and mixing, at 37 deg.C, 5% CO ₂ The 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 20min. 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 commercially widely used strains: 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 method is the same 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 adhesion OF OF44-15Ph10T and human cervical cancer cells Hela in two commercial strains. 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. Assessment OF OF44-15Ph10T adhesion Capacity to human vaginal epithelial cells VK2E6/E7

The cultured VK2E6/E7 cells (purchased from Beijing Beiner institute of Biotechnology, innovation and Biotechnology) were digested, diluted with 1640 complete medium without double antibody, and counted on a hemocytometer (see below) to a cell concentration of about 2X10 ⁵ cell/mL, 1mL drop in cell culture dishes (12 or 6 well plates), 5% CO ₂ Incubate at 37 ℃ in-95% air incubator until complete differentiation.

After the cells have grown into a dense monolayer, the cells are sterilizedThe cells were rinsed 2 times with PBS buffer, and then lmL1640 medium and 1mL of overnight culture were added to each well and adjusted to 10 ⁸ CFU/mL suspension, gently shaking and mixing, at 37 deg.C, 5% CO ₂ The 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 monolayers were washed 5 times with sterile PBS buffer to remove non-adherent bacteria, and then fixed with anhydrous methanol for 20min. The cell slide fixed by the absolute methanol is taken for gram staining. After drying, counting was observed under the mirror, and the number of adhered bacteria on 100 cells in 20 random fields was counted, and an 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 indicates that OF44-15Ph10T has higher adhesion to human vaginal epithelial cells VK2E6/E7 than Lactobacillus rhamnosus GR-1, lactobacillus reuteri RC-14 and Lactobacillus delbrueckii. 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

Wiping the blood counting chamber and the cover plate and covering the cover plate on the counting chamber; then sucking out a little cell suspension, dripping the cell suspension on the edge of the cover plate to ensure that the suspension is filled between the cover plate and the counting plate, and standing for 3 minutes; finally, microscopic examination was performed, and the total number of four large grids of cells on the plate was counted, and the line-pressed cells were counted only on the left side and the upper side (cell number/mL =4 large grids of cells/4 × 10000).

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

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

And (3) intragastric administration 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 、1×10 ⁹ 、1×10 ¹² CFU/mL) 1 time per day for 3 consecutive days, and rats survived healthily 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 、1×10 ⁹ 、1×10 ¹² CFU/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 rats ⁵ ～0.5×10 ¹² The 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: ability OF Lactobacillus rhamnosus OF44-15Ph10T to treat genital tract infection in rats

The rat model selected in this example is a genital tract gardnerella infection rat model, SD female rats are used, the rat age is 7 weeks, 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. 0.5mg of beta-estradiol-3-benzoate is injected subcutaneously three days before molding, and the false estrus phenomenon of the rat is kept. Observing and recording whether the vagina of a rat is red and swollen, and has effusion, erythema and particle phenotypic characteristics before modeling; a modeling period: the vagina of the rat is washed once a day for 3 days continuously by overnight cultured gardnerella vaginalis (purchased from Beijing BeiNai Chuanglian Biotechnology research institute) (PBS bacterial liquid),and observing and recording whether the vagina is inflamed and spilled or not and whether phenotypic characteristics such as erythema and particles exist or not on the 4 th day, and selecting rats meeting the modeling requirements for testing. Dry prediction: for a total of 7 days, model group rats were not treated; probiotic group rats use OF44-15Ph10T PBS liquid to wash rat vagina, and the total viable bacteria amount is not less than 1 x10 ⁸ CFU/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 discharge of the vagina, presence of erythema and phenotypic characteristics of the particulate matter. And (3) taking a vaginal washing liquid of the rat while observing the phenotype of the rat every time, and detecting the removal condition of the gardnerella and the colonization condition of the lactobacillus rhamnosus by using a quantitative PCR method.

The results showed that Lactobacillus rhamnosus OF44-15Ph10T could colonize the vagina OF rats (FIG. 6) while having the effect OF improving and inhibiting the recurrence OF Gardnerella vaginalis infection in rats (FIG. 7 and Table 7). (Table 8).

Table 7 vaginal phenotype recording results in rats

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

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

The first group OF 9 mice was Sham control group, which was Sham operated, skin was dissected only and sutured, and the experiment was performed by feeding medium containing the target strain (OF 44-15Ph 10T) in a proportional amount to body weight;

the second group of 10 mice is an experimental group, an osteoporosis model is caused by the removal induction of ovarian organs, the stomach is irrigated by saturated suspension of the target strain, 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 in bone-related indices (shown in table 9 and fig. 8) was examined using micro-CT, and a three-dimensional reconstructed image of the tissue (fig. 9) was created.

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. Moreover, various embodiments or examples and features of various embodiments or examples described in this specification can be combined and combined by one skilled in the art without being mutually inconsistent.

Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and that changes, 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 Hua Dasheng Life science 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

<210> 2

<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 OF44-15Ph 10T)

<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 collection number GDMCC No. 60406.

2. Use of lactobacillus rhamnosus or its bacterial suspension according to claim 1 for the preparation of a medicament for the prevention and/or treatment of diseases associated with disorders of the genital tract flora and/or diseases associated with 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.

4. The use according to claim 3, wherein 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.

5. The use according to claim 2, wherein the disease associated with bone loss comprises at least one selected from osteopenia, osteoporosis, and osteoporotic fractures.

6. Use of Lactobacillus rhamnosus or its bacterial suspension according to claim 1 for the preparation of a medicament for antibacteria, adhesion to vaginal epithelial cells and/or cervical cells, lactic acid production, H production ₂ O ₂ And/or inhibiting bone loss.

7. The use according to any one of claims 2 to 6, wherein the medicament is administered at a dose of 10 ⁵ -10 ¹² CFU/day.

8. A medicament comprising the lactobacillus rhamnosus or a bacterial suspension thereof according to claim 1.

9. The medicament according to claim 8, wherein the medicament is selected from the group consisting of a medicament for preventing and/or treating a disease associated with a disturbance of the genital tract flora, a medicament for preventing and/or treating a disease associated with bone loss, an antibacterial medicament, a medicament for adhering to vaginal epithelial cells and/or cervical cells, a lactic acid-producing medicament, an H-producing medicament ₂ O ₂ At least one of the medicaments of (1).

10. A pharmaceutical composition comprising the lactobacillus rhamnosus or a bacterial suspension thereof according to claim 1.

11. Pharmaceutical composition according to claim 10, in single dose form, containing a daily dose of 10 ⁵ -10 ¹² CFU of said Lactobacillus rhamnosus.

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