Lactobacillus rhamnosus of vaginal origin and its application in antagonizing core pathogenic bacteria of bacterial vaginosis

Abstract

The application discloses a vaginal Lactobacillus rhamnosus and application thereof in antagonizing core pathogenic bacteria of bacterial vaginosis, and belongs to the technical field of functional microorganism screening and application. Lacticaseibacillus rhamnosus The application provides the Lactobacillus rhamnosus (Lactobacillus rhamnosus) with a preservation number of CGMCC No.39572. The strain is isolated from vaginal secretion of a pregnant woman with a healthy vaginal micro-ecosystem throughout the whole pregnancy period, is screened from 1089 Lactobacillus strains from vaginas of 186 healthy women through multiple rounds of in-vitro bacteriostatic activity system evaluation based on continuous monitoring data of vaginal micro-ecosystems of 692 pregnant women in early, middle and late pregnancy periods, and is identified as the Lactobacillus rhamnosus through 16S rRNA gene sequencing. In-vitro experiments show that the strain has inhibiting and killing activities on Gardnerella vaginalis, Haemophilus vaginalis and Prevotella bivia. The three pathogenic bacteria are key bacteria species causing bacterial vaginosis. The strain can simultaneously cover the three pathogenic bacteria under in-vitro conditions, and provides a candidate strain material for further evaluation for development of a vaginal micro-ecosystem preparation focusing on a core pathogenic spectrum of BV.

Description

Technical Field

[0001] This invention relates to the field of functional microbial screening and application technology, specifically to a strain of *Lactobacillus rhamnosus* derived from the vagina of a pregnant woman with a consistently healthy vaginal microecology throughout pregnancy, which can simultaneously antagonize the core pathogens of bacterial vaginosis. Lacticaseibacillus rhamnosus B1893, and its use in the preparation of products for antagonizing the core pathogens of bacterial vaginosis. Background Technology

[0002] Bacterial vaginosis (BV) is not caused by a single pathogen, but rather by Gardnerella vaginalis (… Gardnerella vaginalis GV), Fanny Hesse (GV), vaginal Fanny Hesse (GV) Fannyhessea vaginae FV) and Prevotella 2 ( Prevotella bivia Vaginal vaginitis (BV) is caused by the synergistic involvement of multiple anaerobic bacteria, including PB (Propionibacterium arachnoides). These three pathogenic bacteria exhibit metabolic complementarity and synergistic pathogenicity, forming a structured biofilm on the vaginal epithelial surface, enhancing their survival in the vaginal environment and their tolerance to drugs. This dual mechanism of multi-bacterial synergy and biofilm barrier makes intervention strategies targeting only one or two pathogenic bacteria insufficient to fundamentally break the synergistic pathogenic network, a significant reason for the persistently high recurrence rate of BV after conventional antimicrobial treatment.

[0003] Current clinical treatment for vaginal vaginitis (BV) primarily involves oral or topical administration of anti-anaerobic antibiotics such as metronidazole and clindamycin. While this approach can suppress some pathogens in the short term, it also affects the colonization of protective lactobacilli in the vagina. After discontinuation of medication, residual pathogens can proliferate again and reform biofilms, leading to recurrent infections. Against this backdrop, supplementing with vaginal lactobacilli to help restore normal flora and reduce the risk of recurrence has become an exploratory approach in BV management.

[0004] Current studies have reported the in vitro inhibitory activity of some lactobacilli against BV-related pathogens, but most focus on a few vaginal flora such as *Lactobacillus curvularia*, and the background of these strains is unclear. In contrast, *Lactobacillus rhamnosus* (… Lacticaseibacillus rhamnosus Although *Lactobacillus rhamnosus* has been widely used in the food and gut probiotic fields, data on its systemic inhibitory efficacy against the core pathogens of bacterial vulgaris (BV), namely GV, FV, and PB, are currently insufficient. Screening for a vaginal-derived *Lactobacillus rhamnosus* strain with a well-defined background that simultaneously antagonizes these three core BV pathogens would help provide candidate materials for the diversified development of BV probiotic preparations. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of *Lactobacillus rhamnosus* isolated from the vagina of a pregnant woman with a consistently healthy vaginal microecology throughout pregnancy, which exhibits inhibitory and bactericidal activity against the three core pathogenic bacteria of bacterial vaginosis.Lacticaseibacillus rhamnosus B1893. This strain was identified as *Lactobacillus rhamnosus* by 16S rRNA gene sequencing. It is a vaginal lactobacillus with good safety.

[0006] This invention is based on continuous vaginal microecological monitoring data from 692 pregnant women in the first, second, and third trimesters of pregnancy. It also includes 186 volunteers with consistently healthy vaginal microecological profiles throughout pregnancy. From their vaginal secretion samples, 1089 strains of lactobacilli, encompassing 21 species, were isolated. An in vitro model of co-culturing cell-free fermentation supernatant with pathogens was used to systematically evaluate the resistance of each strain to *Streptococcus agalactiae* (…). Streptococcus agalactiae GBS), Gardnerella vaginalis ( Gardnerella vaginalis GV), Fanny Hesse (GV), vaginal Fanny Hesse (GV) Fannyhessea vaginae FV), Prevotella 2 ( Prevotella bivia PB ） After multiple rounds of screening and comparison of the inhibitory activities against four bacterial pathogens, *Lactobacillus rhamnosus* B1893, as described in this invention, was finally selected. In vitro experiments showed that this strain exhibits inhibitory and bactericidal activity against GV, FV, and PB. These three pathogens are core members of the BV synergistic pathogenicity network. This strain can simultaneously target all three pathogens under in vitro conditions, providing a candidate strain material for further evaluation in the development of vaginal microecological products focusing on the core pathogen spectrum of BV.

[0007] Lactobacillus rhamnosus B1893 was deposited on July 6, 2026, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, accession number: CGMCC No. 39572. Its 16S rRNA gene sequence is shown in SEQ ID No. 1. BLAST homology comparison in the GenBank database shows that this strain is similar to Lactobacillus rhamnosus (…). Lacticaseibacillus rhamnosus The sequence of the bacterium with the highest similarity was identified as Lactobacillus rhamnosus, and it was named Lactobacillus rhamnosus B1893.

[0008] Based on the above findings, the present invention provides the following technical solution: In a first aspect, the present invention provides the Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus B1893, the Lactobacillus rhamnosus isolated from vaginal secretions of pregnant women with a consistently healthy vaginal microecology throughout pregnancy, was deposited on July 6, 2026 at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 39572.

[0009] The 16S rDNA sequence of *Lactobacillus rhamnosus* is shown in SEQ ID NO.1.

[0010] Secondly, the present invention provides a bacterial preparation comprising the Lactobacillus rhamnosus B1893.

[0011] The bacterial cells in the bacterial preparation are in the form of live bacteria, inactivated bacteria, or a combination thereof, and the physical form of the bacterial preparation is a liquid preparation or a solid preparation.

[0012] Thirdly, the present invention provides an epigenetic agent, which includes metabolites obtained by fermenting and culturing Lactobacillus rhamnosus B1893 in a culture medium as described in the present invention.

[0013] The culture medium can be MRS medium or modified NYC III medium.

[0014] The metabolites may be present in the whole fermentation liquid, or in the fermentation supernatant after centrifugation and / or filtration to remove the cells, or in a powder made by drying the whole fermentation liquid or the fermentation supernatant after removing the cells.

[0015] Fourthly, the present invention provides the use of the Lactobacillus rhamnosus B1893, the bacterial preparation, or the postbiotic in the preparation of products for antagonizing the core pathogens of bacterial vaginosis.

[0016] The application involves Gardnerella vaginalis (Gardnerella vaginalis) Gardnerella vaginalis ), Fanny Hesse (vaginis) Fannyhessea vaginae ) and Prevotella 2 Prevotella bivia One or more of the following inhibitors. The products include oral medications, health foods, feed additives, dressings, gels, lubricants, or disinfectants / antibacterial agents.

[0017] Fifthly, the present invention provides a product for antagonizing the core pathogens of bacterial vaginosis, comprising the Lactobacillus rhamnosus B1893, the bacterial preparation, or the postbiotic.

[0018] The product may be a vaginal health product, a vaginal medicine, or a vaginal medical device. The dosage form or form of the product may be one or more of the following: powder, tablet, suppository, microcapsule, ampoule, or liquid spray. The product may also contain one or more pharmaceutically or cosmetically acceptable additives, including excipients, preservatives, diluents, fillers, absorption enhancers, sweeteners, or combinations thereof.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The strain was derived from the vagina of a pregnant woman with a consistently healthy vaginal microecology throughout the entire pregnancy. It is a vaginal Lactobacillus rhamnosus. No known antibiotic resistance genes or virulence factors were detected by whole-genome screening, indicating a good safety basis.

[0020] (2) The strain screening was based on continuous monitoring data of vaginal microecology throughout pregnancy from a large-scale pregnant cohort (692 cases). The target strain was obtained from 1089 strains of lactobacillus from 186 continuously healthy individuals through multiple rounds of in vitro efficacy evaluation system screening. It is scientific and advanced, and has a clear strain background.

[0021] (3) In vitro experiments showed that the strain had inhibitory and killing activities against three core pathogenic bacteria of BV: Gardnerella vaginalis, Fanny Hesse vaginalis and Prevotella 2. It can simultaneously cover the key bacterial groups of the BV synergistic pathogenic network, providing candidate strain materials for further evaluation for the development of microecological products focusing on the core pathogen spectrum of BV.

[0022] (4) This strain can be prepared into various forms such as live bacterial preparations, inactivated bacterial cells or postbiotics, providing a relatively complete material basis for product development in different application scenarios, and has clinical value and industrial transformation prospects. Attached Figure Description

[0023] Figure 1 The images show the colony morphology of pure cultures of Lactobacillus rhamnosus B1893 (CGMCC No. 39572) on Columbia blood agar (a), MRS solid medium (b), and LBS solid medium (c).

[0024] Figure 2 The images show Gram staining morphology of pure culture of Lactobacillus rhamnosus B1893 (CGMCC No. 39572), with (a) and (b) showing the bacterial morphology under different fields of view in oil immersion (100X).

[0025] Figure 3 Original images (oil immersion, 100X field of view) of the vaginal microecology of pregnant women with Lactobacillus rhamnosus B1893 (CGMCC No. 39572) throughout the entire pregnancy, including (a) image taken at 64 days of the first trimester, (b) image taken at 179 days of the second trimester, (c) image taken at 214 days of the second trimester, and (d) image taken at 243 days of the third trimester.

[0026] Figure 4This is an overview of the vaginal flora of pregnant woman S060, from whom Lactobacillus rhamnosus B1893 (CGMCC No. 39572) was derived. It shows the results of vaginal microecological diagnosis (a), 16S rRNA sequencing (b), and vaginal bacterial and fungal culture (c) at each sampling point. The specific isolation time points of B1893 are marked with asterisks.

[0027] Figure 5 MALDI-TOF-MS protein fingerprint of Lactobacillus rhamnosus B1893 (CGMCC No. 39572). Detailed Implementation

[0028] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0029] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and reagents used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, equipment, and materials similar to or equivalent to those described in the embodiments of this invention may be used to implement this invention.

[0030] Unless otherwise stated, the experimental methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in this technical field.

[0031] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art.

[0032] Example 1: Clinical cohort origin, high-throughput screening, and isolation and purification of the target strain 1. Clinical cohort and strain library construction This embodiment is based on a prospective longitudinal cohort study of vaginal microbiota, which included 692 pregnant women. Vaginal secretion samples were collected in the first, second, and third trimesters of pregnancy for analysis of vaginal flora throughout pregnancy. Based on the vaginal microbiota diagnosis throughout pregnancy, 186 pregnant volunteers with consistently healthy vaginal microbiota throughout pregnancy were selected as the source population for probiotic strains.

[0033] Vaginal lactobacilli were isolated from vaginal secretion samples of 186 pregnant women with consistently healthy vaginal microecology throughout pregnancy. Samples were spread onto LBS solid medium and Columbia blood agar and incubated at 37 ℃ in a 5% CO2 incubator for 48 h. Morphologically typical single colonies were picked and identified as lactobacilli by MALDI-TOF-MS protein fingerprinting. Monoclonal pure cultures were obtained through repeated streak purification. A total of 1089 vaginal lactobacilli isolates were obtained, including *Lactobacillus rhamnosus* (…). Lacticaseibacillus rhamnosus Lactobacillus japonicus (), Lactobacillus jensenii Lactobacillus gasseri ( Lactobacillus gasseri 21 types of lactobacilli, including 1000.

[0034] The formulation of the LBS medium is as follows: 10.0 g peptone, 5.0 g yeast extract, 20.0 g D-glucose, 6.0 g dipotassium hydrogen phosphate, 15.0 g sodium acetate trihydrate, 2.0 g triammonium citrate, 0.575 g magnesium sulfate, 0.12 g manganese sulfate, 0.034 g ferrous sulfate, 1.0 g Tween 80, and distilled water to a final volume of 1000 mL. When preparing the solid medium, 15.0 g agar should be added, and the medium should be sterilized at 121 °C for 15 min. The Columbia blood agar medium was purchased from Guangzhou Dijing Microbial Technology Co., Ltd., catalog number LS0109-1.

[0035] 2. Preparation of cell-free MRS fermentation supernatant The 1089 Lactobacillus strains were inoculated into fresh MRS liquid medium and incubated at 37 °C with 5% CO2 for 48 h to obtain seed culture. Each strain's seed culture was then transferred to fresh MRS liquid medium at a 2% (v / v) inoculation rate and incubated under the same conditions for another 48 h. The Lactobacillus cultures were collected, centrifuged at 6000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter for sterilization. The resulting filtrate was the cell-free MRS fermentation supernatant for each strain, aliquoted, and stored at -80 °C for later use.

[0036] The formula for the MRS medium is as follows: 10.0 g peptone, 8.0 g beef extract, 4.0 g yeast extract, 20.0 g glucose, 2.0 g dipotassium hydrogen phosphate, 2.0 g diammonium hydrogen citrate, 5.0 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 1.0 g Tween 80, and distilled water to a final volume of 1000 mL. When preparing the solid medium, 15.0 g agar should be added, and the medium should be sterilized at 121 °C for 15 min.

[0037] 3. Preparation of pathogen suspension (1) Preparation of suspension of Streptococcus agalactiae ATCC 12386 pathogen The selected test strain was the laboratory standard strain ATCC12386, which was revived on Columbia blood agar medium and incubated at 37°C with CO2 for 24 h. Single colonies were picked and inoculated into fresh streptococcal broth medium and incubated at 37°C with CO2 for 8 h until the early stage of bacterial growth plateau, yielding the Streptococcus agalactiae stock solution at a growth concentration of 10. 8 CFU / mL; dilute to 10 CFU / mL with fresh streptococcal broth. 7 CFU / mL was used to obtain a suspension of agalactococci.

[0038] The streptococcal broth culture medium mentioned above was purchased from Jiangmen Kailin Trading Co., Ltd., and is a 5 mL broth culture medium (streptococcal broth type), with the product number T0526L.

[0039] (2) Preparation of Gardnerella vaginalis ATCC 14018 pathogen suspension The selected test strain was the laboratory standard strain ATCC14018, which was revived on Columbia blood agar medium and incubated at 37°C with CO2 for 48 h. Single colonies were picked and inoculated into modified NYC III broth medium and incubated at 37°C with CO2 for 40 h until the early stage of bacterial growth plateau, yielding Gardnerella vaginalis stock solution with a growth concentration of 10. 8 CFU / mL; dilute with fresh modified NYC III broth to 10. 7 CFU / mL was used to obtain a vaginal Gardnerella vaginalis suspension.

[0040] The formula for the modified NYC Ⅲ broth is as follows: HEPES 4.0 g, Peptone No. 3 15.0 g, sodium chloride 5.0 g, glucose 5.0 g, add distilled water to make up to 1000 mL, sterilize at 121 ℃ for 15 min, cool and then add 10% (v / v) heat-inactivated fresh horse serum.

[0041] (3) Preparation of vaginal Fanny Hesse BNCC 382351 pathogen suspension The selected test strain was the laboratory standard strain BNCC382351, which was revived on Columbia blood agar medium and anaerobically cultured at 37 ℃ for 48 h. Single colonies were picked and inoculated into modified NYC III broth, and anaerobically cultured at 37 ℃ for 40 h until the early stage of bacterial growth plateau, yielding the *Fanny Hesse* stock solution at a concentration of 10⁻⁶. 8 CFU / mL; dilute with fresh modified NYC III broth to 10. 7 CFU / mL was used to obtain a vaginal suspension of Fanny Hercet's bacterium. The modified NYC Ⅲ broth formula was prepared in the same manner as in step (2).

[0042] (4) Preparation of suspension of Prevotella ileiflora CGMCC No. 59002 pathogen The selected strain was *Prevotella bivia*, isolated from vaginal secretions of pregnant women and deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 6, 2026 (CGMCC NO. 59002), and classified as *Prevotella bivia*. The strain was revived on Columbia blood agar and anaerobically cultured at 37 °C for 48 h. Single colonies were picked and inoculated into modified BHI broth, and anaerobically cultured at 37 °C for 48 h until the early stage of bacterial growth plateau, yielding a *Prevotella bivia* suspension with a growth concentration of 10⁻⁶. 8 CFU / mL.

[0043] The modified BHI broth culture medium formula is as follows: 10.0 g peptone, 12.5 g dehydrated calf brain extract, 5.0 g dehydrated calf heart extract, 5.0 g sodium chloride, 2.0 g glucose, 2.5 g disodium hydrogen phosphate, and distilled water to a final volume of 1000 mL. The mixture is sterilized at 121 °C for 15 min, cooled, and then vitamin K3 (final concentration 1 mg / L) and heme (final concentration 5 mg / L) are added.

[0044] 4. High-throughput in vitro primary screening for antibacterial activity of Lactobacillus The suspensions of the above pathogens (Streptococcus agalactiae, Gardnerella vaginalis, and Fanny H. necrosis vulgaris) were prepared at a ratio of 10... 7 CFU / mL, Prevotella 2 was 10 8 10 μL of the CFU / mL solution was inoculated into each well of a 96-well plate containing 75 μL of the corresponding fresh liquid culture medium. Subsequently, 75 μL of the cell-free MRS fermentation supernatant of the 1089 Lactobacillus strains was added to the experimental group, while the control group received an equal volume of fresh MRS liquid culture medium. Co-culture was performed under different pathogen culture conditions (Streptococcus agalactiae and Gardnerella vaginalis at 37 ℃, 5% CO2 incubation; Fannie Herzegovina vaginalis and Prevotella vaginalis at 37 ℃, anaerobic incubation). The clarity of the liquid in the co-culture wells was visually observed; a clarity similar to that of the negative control was considered indicative of antibacterial activity. Results showed that 84.4% (919 / 1089) of the tested Lactobacillus strains exhibited significant antibacterial activity against at least one pathogenic bacterium; among them, 25 Lactobacillus strains showed good antibacterial activity against all tested vaginal pathogenic bacteria.

[0045] 5. Candidate strain rescreening and biosafety assessment The 25 candidate lactobacilli strains were repeatedly validated, and the inhibitory activity and stability of each strain against the pathogen were compared. At the same time, the 25 candidate lactobacilli strains were inoculated into MRS liquid medium, incubated at 37 ℃ and CO2 for 48 h, and then centrifuged at 6000 rpm for 5 min to collect the bacterial cells. The bacterial cells were sent to the sequencing center for whole genome sequencing.

[0046] The genome was screened for resistance genes and virulence factors using CARD (The Comprehensive Antibiotic Resistance Database), ResFinder, VFDB, and VirulenceFinder (The virulence factor database). The screening criteria were a 90% redundancy removal sequence identity threshold for resistance genes and pident>80 and evalue<1e-5 for virulence factors.

[0047] The *Lactobacillus rhamnosus* strain B1893 was ultimately confirmed to exhibit the most stable and strongest antibacterial activity against the three core pathogens of bacterial vaginosis: *Gardnerella vaginalis*, *Fanny Hesse*, and *Prevotella cirrhosa*. Furthermore, no known antibiotic resistance genes or virulence factors were detected, indicating good biosafety, and this strain was selected as the target strain for this invention. The strain was then activated from its original glycerol culture tube and streaked onto Columbia blood agar, MRS solid medium, and LBS solid medium for isolation. Typical single colonies were picked, such as... Figure 1 As shown, a monoclonal pure culture was obtained after repeated purification and named *Lactobacillus rhamnosus* B1893. The Gram-stained microscopic image of the strain is shown below. Figure 2 As shown. The full-length genome of this strain is 2,965,912 bp, with a GC content of 46.61%. GC content refers to the percentage of guanine (G) and cytosine (C) bases in a nucleic acid molecule relative to the total number of bases.

[0048] In addition, this strain is derived from vaginal secretions of pregnant women with consistently normal vaginal flora throughout pregnancy. It is a native vaginal bacterium and is also one of the edible Lactobacillus species approved in my country's current "List of Bacterial Strains that Can Be Used in Food," which ensures the safety of the strain.

[0049] 6. Retrospective confirmation of vaginal flora throughout pregnancy of pregnant women to determine the source of the strains. A retrospective analysis was conducted on vaginal secretion samples from pregnant women with Lactobacillus rhamnosus B1893 throughout their pregnancy. Original images of the vaginal microecology throughout the entire pregnancy are shown below. Figure 3 As shown, the overview of the microbial community analysis is as follows: Figure 4As shown in the figure, sequencing and culture results showed that no dominant growth of BV-related pathogens such as Gardnerella vaginalis, Fannie H. necrosis vaginalis, and Prevotella ergium was observed throughout the pregnant woman's pregnancy, and no vaginal fungal colonization was also observed. These results confirm that the vaginal flora of this pregnant woman remained healthy throughout her pregnancy, and that Lactobacillus rhamnosus B1893, from which it was derived, has a good foundation for vaginal adaptability and safety.

[0050] Example 2: Identification and biological characteristics of Lactobacillus rhamnosus B1893 The monoclonal pure culture of Lactobacillus rhamnosus B1893 obtained by isolation and purification in Example 1 was used for the following identification experiments.

[0051] A single colony of strain B1893 was picked from the plate using a disposable 1 μL inoculation loop and evenly spread onto the protein fingerprint target plate. 1 μL of 70% (v / v) formic acid solution was added to lyse the bacteria. The plate was then allowed to air dry at room temperature. Following the protein fingerprint kit instructions, 1 μL of mass spectrometry sample processing matrix solution was added to cover the sample for pretreatment. The plate was then allowed to air dry at room temperature. After drying, the target plate was placed on a MALDI-TOF-MS protein spectrometer for testing and identification. The MALDI-TOF-MS protein fingerprint is shown below. Figure 5 As shown, strain B1893 was identified as Lactobacillus rhamnosus.

[0052] Genomic DNA was extracted from *Lactobacillus rhamnosus* B1893, and 16S rRNA gene amplification was performed by PCR and Sanger sequencing. BLAST alignment with the NCBI database showed that its sequence was similar to that of *Lactobacillus rhamnosus* (…). Lacticaseibacillus rhamnosus The strain showed the highest similarity and was identified as Lactobacillus rhamnosus.

[0053] The optimal growth temperature for *Lactobacillus rhamnosus* B1893 in MRS medium was 37 °C. After static incubation at 37 °C with CO2, the absorbance of the bacterial culture at OD600 was 1.445, and the pH was 3.5.

[0054] The aforementioned *Lactobacillus rhamnosus* B1893 was deposited on July 6, 2026, at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 39572, address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and its classification and nomenclature: *Lactobacillus rhamnosus* (…). Lacticaseibacillus rhamnosus ).

[0055] Example 3: Inhibitory effect of Lactobacillus rhamnosus B1893 on Gardnerella vaginalis, Fannie Hercetella vaginalis, and Prevotella 2 vaginalis. 1. Preparation of Lactobacillus rhamnosus B1893 mycelial cake A single colony of *Lactobacillus rhamnosus* B1893 (CGMCC No. 39572) isolated and purified in Example 1 was inoculated into fresh MRS liquid medium and incubated at 37 °C with CO2 for 48 h to obtain a seed culture. The seed culture was then transferred to fresh MRS liquid medium at a 2% (v / v) inoculation rate and incubated for another 12 h under the same conditions. The mixture was then thoroughly combined with MRS solid medium at a 1:100 (v / v) ratio and poured into 6 cm Petri dishes. After complete solidification, the dishes were incubated at 37 °C with CO2 for 48 h. The resulting bacterial pellets were then punched into agar medium using a 6 mm inner diameter punch to obtain the same culture medium formulation as in Example 1.

[0056] 2. Preparation of suspensions of Gardnerella vaginalis, Fannie H. N. vaginalis, and Prevotella 2 vaginalis pathogens. The selected test strains were laboratory standard strains: *Gardnerella vaginalis* ATCC14018, *Fanny Hesse* BNCC382351, and *Prevotella ilex* CGMCC No. 59002, which were revived on Columbia blood agar. They were cultured under different conditions (*Gardnerella vaginalis* 37 ℃, CO2 incubation for 48 h; *Fanny Hesse* and *Prevotella ilex* 37 ℃, anaerobic incubation for 48 h). Colonies were scraped from agar plates and transferred to 0.9% sterile sodium chloride solution, adjusted to a McFarland turbidity of 0.5 (i.e., 10-10). 8 CFU / mL, then diluted 10 times to 10 7 CFU / mL was used to obtain a suspension of pathogens.

[0057] 3. Determination of inhibition zones using the agar plate method In the experimental group, the pathogen suspension was mixed with fresh Brucella blood agar at a ratio of 1:10 (v / v) and poured into 9cm Petri dishes. In the control group, an equal volume of fresh Brucella blood agar was added. After complete solidification, the prepared Lactobacillus bacterial cakes were placed on the surface of the pathogen agar, with 4 cakes placed symmetrically on each dish, and 3 replicates were set up for each group. The Petri dishes were placed upright and incubated for 24 h under different pathogen culture conditions. The diameter of the inhibition zone (mm) was measured using calipers. The results are shown in Table 1.

[0058] The formula for the above-mentioned Brucella blood agar medium is as follows: 10.0 g peptone, 2.0 g yeast extract, 10.0 g casein peptone, 5.0 g sodium chloride, 1.0 g glucose, 0.1 g sodium bisulfite, 15.0 g agar, add distilled water to make up to 1000 mL, sterilize at 121 ℃ for 15 min, cool and then add 5% (v / v) sterile defibrinated sheep blood.

[0059] Table 1

[0060] The control group data was the diameter of the mycelium cake, while the experimental group data was the measured diameter of the inhibition zone, which included the diameter of the mycelium cake.

[0061] As shown in Table 1, compared with the control group, the growth of Gardnerella vaginalis, Fannie H. Neisseria vaginalis, and Prevotella bisporus in the experimental group was significantly inhibited, indicating that Lactobacillus rhamnosus B1893 of the present invention has good inhibitory activity against the growth of Gardnerella vaginalis, Fannie H. Neisseria vaginalis, and Prevotella bisporus.

[0062] Example 4: Killing effect of cell-free modified NYC III fermentation supernatant of Lactobacillus rhamnosus B1893 on Gardnerella vaginalis, Fannie Hercetella vaginalis, and Prevotella ilex. A single colony of *Lactobacillus rhamnosus* B1893 (CGMCC No. 39572) was inoculated into fresh modified NYC III liquid medium and incubated at 37 °C with CO2 for 48 h as the seed culture. The seed culture was then transferred to fresh modified NYC III liquid medium at a 2% (v / v) inoculation rate and incubated for another 48 h under the same conditions. The *Lactobacillus* culture was collected and centrifuged at 6000 rpm for 5 min. The supernatant was filtered through a 0.22 μm filter for sterilization. The resulting filtrate was the cell-free modified NYC III fermentation supernatant, which was stored at -80 °C for later use. The formulation of the modified NYC III medium was the same as in Example 1.

[0063] Dilute the GV and FV pathogen stock solutions prepared in Example 1 to 10. 6 CFU / mL, and another PB pathogen suspension prepared in Example 1 (10 8 10 μL of each of the following was inoculated into 96-well plates containing 75 μL of fresh liquid culture medium corresponding to each pathogen culture (CFU / mL). Then, 75 μL of cell-free modified NYC III fermentation supernatant was added. For the control group, an equal volume of fresh modified NYC III liquid culture medium was added. After co-culturing under different pathogen culture conditions (Gardnerella vaginalis 37 ℃, CO2 incubation for 48 h; Fannie Herzegovina vaginalis and Prevotella vaginalis 37 ℃, anaerobic incubation for 48 h), the co-culture solutions were serially diluted and inoculated onto Columbia blood agar. The pathogens were cultured and counted under the same conditions as above. Three replicate experiments were performed, and the live CFU of Gardnerella vaginalis, Fannie Herzegovina vaginalis, and Prevotella vaginalis were counted. The results are shown in Table 2.

[0064] Table 2

[0065] The results showed that the concentration of Gardnerella vaginalis in the culture medium of the control group was 10. 6CFU / mL, vaginal Fanny Hesse concentration was 10. 8 CFU / mL, Prevotella divaricata concentration was 10. 8 The CFU / mL concentration was significantly higher than that of the experimental groups, and no pathogens were observed in the experimental groups. This indicates that the fermentation broth has the activity of directly killing Gardnerella vaginalis, Fannie Herzegovina vaginalis, and Prevotella ilex.

Claims

1. A strain of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus B1893, characterized in that, The preservation number of the Lactobacillus rhamnosus is CGMCC No. 39572.

2. The *Lactobacillus rhamnosus* according to claim 1, characterized in that, The 16S rDNA sequence of *Lactobacillus rhamnosus* is shown in SEQ ID NO.

1.

3. A bacterial preparation, characterized in that, It contains Lactobacillus rhamnosus as described in claim 1 or 2.

4. The bacterial preparation according to claim 3, characterized in that, The bacterial cells in the bacterial preparation are either live or inactivated, and the bacterial preparation can be a liquid or a solid formulation.

5. An epigenetic agent, characterized in that, The metabolite includes metabolites obtained by fermenting *Lactobacillus rhamnosus* as described in claim 1 or 2 in a culture medium.

6. The epigenetic agent according to claim 5, characterized in that, The metabolites are present in the whole fermentation broth, or in the fermentation supernatant after centrifugation and / or filtration to remove the cells, or in a powder prepared by drying the whole fermentation broth or the fermentation supernatant after removing the cells.

7. The use of Lactobacillus rhamnosus of claim 1 or 2, the bacterial preparation of claim 3 or 4, or the metabiotic of claim 5 or 6 in the preparation of products for antagonizing the core pathogens of bacterial vaginosis.

8. The application according to claim 7, characterized in that, The bacteria is Gardnerella vaginalis (… Gardnerella vaginalis ), Fanny Hesse ( Fannyhessea vaginae ), Prevotella 2 Prevotella bivia One or more of the following; The product is an oral medicine, health food, or feed additive; The product is a dressing, gel, lubricant, disinfectant, or antibacterial agent.

9. A product for antagonizing the core pathogens of bacterial vaginosis, characterized in that, It contains Lactobacillus rhamnosus as described in claim 1 or 2; or, it contains the bacterial preparation as described in claim 3 or 4; or, it contains the metabiotic as described in claim 5 or 6.

10. The product according to claim 9, characterized in that, The product is one or more of vaginal health products, vaginal medicines, and vaginal medical devices; the dosage form or form of the product is one or more of powder, tablets, suppositories, microcapsules, ampoules, liquid sprays, gels, or dressings. The product also contains one or more pharmaceutically or cosmetically acceptable additives, including excipients, preservatives, diluents, fillers, absorption enhancers, sweeteners, or combinations thereof.

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