Lactobacillus crispatus with reproductive tract microecological regulation function and application thereof

Abstract

The application discloses a lactobacillus crispatus with a reproductive tract microecological regulation function and an application thereof, and belongs to the technical field of functional microorganism screening and application. Lactobacillus crispatus The strain is a lactobacillus crispatus (B2726) from a vagina, and the preservation number is CGMCC No.39574. The strain and its postbiotic have strong inhibition and killing activities on perinatal important pathogenic bacteria, such as streptococcus agalactiae, and core pathogenic bacteria of bacterial vaginosis, including gardnerella vaginalis, haeserella vaginalis and prevotella bivia. The strain is obtained from 1089 lactobacillus strains from vaginas of 186 healthy pregnant women through multiple rounds of in-vitro bacteriostatic activity system evaluation screening based on continuous monitoring data of vaginal microecology of 692 pregnant women, has strong acid production capacity, excellent biological safety, strong vaginal environment adaptability, can be prepared into a microecological preparation, and is suitable for the regulation of the vaginal microecological environment of women including pregnant women.

Description

Technical Field

[0001] This invention relates to the field of functional microbial screening and application technology, specifically to a strain of *Lactobacillus curvatureii* isolated from the vaginal microecology of a healthy pregnant woman. Lactobacillus crispatus B2726, and its application in the preparation of formulations for regulating the vaginal microecological environment of women, including pregnant and postpartum women. Background Technology

[0002] The vaginal microecological environment in women is a complex, dynamically balanced ecosystem, in which lactobacilli are the dominant flora in a healthy state. Lactobacilli maintain a low vaginal pH environment (pH < 4.5) through lactic acid production, produce antibacterial substances such as hydrogen peroxide and bacteriocins, and competitively occupy vaginal epithelial adhesion sites, forming a natural microecological barrier against pathogenic microorganism colonization. When the abundance of lactobacilli decreases and pathogens proliferate excessively, the vaginal microecological balance is disrupted, leading to various vaginal infections such as bacterial vaginosis (BV) and aerobic vaginitis (AV), seriously affecting women's reproductive health. Especially for pregnant and postpartum women, vaginal microecological imbalance not only causes local discomfort symptoms but is also closely related to adverse pregnancy outcomes such as premature rupture of membranes, premature birth, postpartum endometritis, and neonatal infections. Therefore, maintaining and regulating the vaginal microecological environment in women, restoring it to a healthy state dominated by lactobacilli, is an important strategy for preventing and treating vaginal infections, ensuring women's reproductive health, and protecting the safety of mothers and infants during the perinatal period.

[0003] Currently, clinical interventions for vaginal microecological imbalance primarily rely on antibiotics. While drugs such as metronidazole and clindamycin can suppress pathogenic bacteria in the short term, their broad-spectrum bactericidal effects also interfere with the colonization of lactobacilli, which are crucial for maintaining vaginal microecological balance. This intervention cannot fundamentally address the vaginal microecological imbalance, directly leading to a relapse rate of over 50% within 12 months of antibiotic treatment for vaginal vaginitis (BV). Even more concerning is that repeated exposure to antibiotics is increasingly fostering drug-resistant strains. Therefore, there is an urgent need to develop a microecological regulator that can restore vaginal microecological balance, restore the dominant position of lactobacilli, and effectively inhibit various vaginal pathogens.

[0004] Supplementing with exogenous vaginal lactobacilli is one feasible approach to restoring vaginal microecological balance. Ideal microecological regulating strains should possess the following characteristics: originating from a healthy vaginal microecological environment with a good safety profile; exhibiting natural adaptability and colonization to the vaginal environment; capable of assisting in restoring the low pH environment of the vagina through acid production; and possessing broad-spectrum anti-pathogen activity to reduce pathogen load and create conditions for the re-establishment of lactobacillus dominance. However, most reported vaginal lactobacillus strains currently available only show inhibitory activity against one or two types of pathogens. A single strain is less effective at simultaneously covering multiple core perinatal vaginal pathogens, including *Streptococcus agalactiae*. Streptococcus agalactiaeGBS), Gardnerella vaginalis ( Gardnerella vaginalis GV), Fanny Hesse (GV), vaginal Fanny Hesse (GV) Fannyhessea vaginae FV) and Prevotella 2 ( Prevotella bivia There are very few strains available for use in perinatal microecological regulation (PB) preparations. In particular, most strains reported in existing studies originate from commercially available strain banks or healthy non-pregnant individuals, lacking supporting evidence of a long-term healthy background for maintaining vaginal microecological homeostasis. Furthermore, there is a lack of systematic tracking and dynamic evaluation of the temporal composition of the donor's vaginal flora. This results in insufficient information on the clinical relevance, vaginal colonization adaptability, and safety of these strains, directly limiting their application in perinatal microecological regulation preparations and making it difficult to guarantee colonization efficacy and medication safety in special populations.

[0005] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing microecological regulation strains, such as narrow antibacterial spectrum and unclear origin background, and to provide a strain of Lactobacillus curvature (… Lactobacillus crispatus This study describes strain B2726 and its application in the preparation of formulations for regulating the vaginal microecological environment in women, including pregnant and postpartum women. The strain was isolated from vaginal secretions of a pregnant woman whose vaginal microecology remained consistently healthy throughout pregnancy, with *Lactobacillus curvularia* as the dominant bacteria and a vaginal pH consistently at 3.8. It is a vaginal-native lactobacillus. The vaginal flora composition of the individual from whom this strain originated has been systematically evaluated and confirmed using 16S rRNA sequencing and culture methods, demonstrating a clear background of sustained vaginal microecological health, natural adaptability and colonization ability to the vaginal environment, and excellent biosafety.

[0007] The *Lactobacillus curvatureii* B2726 provided by this invention effectively inhibits and kills four core perinatal vaginal pathogens: *Streptococcus agalactiae*, *Gardnerella vaginalis*, *Fanny Hercetella vaginalis*, and *Prevotella urticaria*, exhibiting stable and broad-spectrum anti-pathogen activity. Simultaneously, this strain continuously produces acid during fermentation, which helps lower the environmental pH, creating conditions for restoring the vaginal microecological balance dominated by lactobacilli. This strain simultaneously covers the core pathogen spectrum of perinatal GBS and BV at the single-strain level, possessing the dual functions of inhibiting pathogens and assisting in restoring the vaginal acidic environment. This invention provides a strain material basis with a clear source background, reliable safety, and broad-spectrum anti-pathogen activity and microecological regulation function for preparing formulations for regulating the vaginal microecological environment of women, including pregnant and postpartum women.

[0008] Based on the above findings, the present invention provides the following technical solution: In a first aspect, the present invention provides the aforementioned *Lactobacillus curvatureii* (… Lactobacillus crispatusLactobacillus curlis B2726, was deposited on July 16, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 39574. 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 curlis (…). Lactobacillus crispatus The sequence of the sample showed the highest similarity, so it was identified as Lactobacillus curvature and named Lactobacillus curvature B2726.

[0009] The present invention provides *Lactobacillus curvatureii* ( Lactobacillus crispatus B2726 was isolated from a pregnant woman whose vaginal microecology remained consistently healthy throughout pregnancy. This strain is a vaginal lactobacillus, possessing natural adaptability and colonization ability to the vaginal environment. In vitro experiments showed that it has potent inhibitory and bactericidal activity against four core perinatal vaginal pathogens: GBS, GV, FV, and PB. This invention provides a new strain selection and technical solution for preparing formulations used to regulate the vaginal microecological environment of women, including pregnant and postpartum women.

[0010] Secondly, the present invention provides a bacterial preparation comprising the aforementioned Lactobacillus curvature B2726.

[0011] The bacterial cells in the bacterial preparation are live bacteria, inactivated bacteria, or a combination thereof; the physical form of the bacterial preparation is a liquid preparation (such as a solution or suspension) or a solid preparation (such as a lyophilized powder, tablet, or granule).

[0012] Thirdly, the present invention provides a metabiotic, the metabiotic comprising metabolites obtained by fermenting the Lactobacillus curvature B2726 in a culture medium.

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

[0014] The metabolites may be present in the fermentation broth containing the cells and all their metabolites; or in the fermentation supernatant rich in metabolites after the cells have been removed by centrifugation and / or filtration; or in the powder prepared by drying the fermentation broth or the fermentation supernatant after removing the cells.

[0015] Fourthly, the present invention provides the use of the Lactobacillus curvature B2726, the bacterial preparation, or the postbiotic in the preparation of products for regulating the vaginal microecological environment of women, including pregnant and postpartum women.

[0016] The product of this invention regulates the vaginal microecological environment by inhibiting vaginal pathogens and / or assisting in restoring the vaginal acidic environment; wherein the vaginal pathogens include one or more of Streptococcus agalactiae, Gardnerella vaginalis, Fannie Herzegovina vaginalis, and Prevotella 2. The product includes, but is not limited to, vaginal microecological preparations, vaginal gels, suppositories, dressings, lubricants, disinfectants, antibacterial agents, oral medications, or health foods.

[0017] Fifthly, the present invention provides a product for regulating the vaginal microecological environment of women, including pregnant and postpartum women, comprising the Lactobacillus curvaturei B2726, the bacterial preparation, or the postbiotic.

[0018] The product may be a vaginal health product (including vaginal health care products, vaginal cleansing products, vaginal care products, vaginal cosmetics, and vaginal hygiene products), 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, tablets, suppositories, microcapsules, ampoules, and liquid sprays. Furthermore, the product may also contain one or more pharmaceutically or cosmetically acceptable additives, such as excipients, preservatives, diluents, fillers, absorption enhancers, and sweeteners.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The *Lactobacillus curvatureii* B2726 described in this invention was isolated from vaginal secretions of pregnant women with consistently healthy vaginal microecology throughout pregnancy. The composition of the vaginal flora of the source individuals throughout pregnancy has been systematically evaluated and confirmed by 16S rRNA sequencing and culture methods, demonstrating a clear healthy vaginal microecological background. No known antibiotic resistance genes or virulence factors were detected by whole-genome sequencing. This strain is a vaginal native lactobacillus with natural adaptability and colonization ability to the vaginal environment, making it particularly suitable for the safety needs of local vaginal application in women, including pregnant and postpartum women.

[0020] (2) This strain simultaneously covers four core perinatal vaginal pathogens, namely GBS and BV key pathogens GV, FV and PB, at the single strain level. At the same time, this strain produces acid during fermentation culture, which can help reduce the pH value of the environment and create favorable conditions for restoring the vaginal microecological balance dominated by lactobacilli.

[0021] (3) This invention constructs a full-form product system from live bacteria, inactivated bacteria to postbiotics, which can be prepared into various dosage forms such as vaginal microecological preparations, gels, suppositories, and dressings. It expands the application scenarios of microecological regulation strategies in the maintenance and regulation of vaginal microecology in perinatal women, and provides a safe and sustainable new microecological regulation solution for reducing the risk of maternal and infant infection during the perinatal period. Attached Figure Description

[0022] Figure 1The images show the colony morphology of pure cultures of Lactobacillus curvature B2726 (CGMCC No. 39574) on Columbia blood agar (a), MRS solid medium (b), and LBS solid medium (c).

[0023] Figure 2 Gram staining images of pure culture of Lactobacillus curvature B2726 (CGMCC No. 39574) under microscopic examination; (a) and (b) show the bacterial morphology under two different fields of view in oil immersion (100X).

[0024] Figure 3 This is an overview of the vaginal flora of pregnant woman S420, from whom Lactobacillus curvature B2726 (CGMCC No. 39574) 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 B2726 are marked with asterisks.

[0025] Figure 4 These are original microecological images (oil immersion, 100X field of view) of pregnant women with Lactobacillus curlis B2726 (CGMCC No. 39574) throughout their pregnancy. Among them, (a) is a sampled image taken at 59 days of the first trimester, (b) is a sampled image taken at 163 days of the second trimester, (c) is a sampled image taken at 183 days of the second trimester, (d) is a sampled image taken at 213 days of the second trimester, and (e) is a sampled image taken at 245 days of the third trimester.

[0026] Figure 5 Images show the original culture isolation time of pregnant women from Lactobacillus curvature B2726 (CGMCC No. 39574) at 183 days of gestation; (a) is on Columbia blood agar medium and (b) is on LBS solid medium. Detailed Implementation

[0027] 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.

[0028] 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.

[0029] Unless otherwise stated, the experimental methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in this technical field.

[0030] 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.

[0031] 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.

[0032] 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 selected and identified as lactobacilli by MALDI-TOF-MS protein fingerprinting. Pure cultures were obtained through repeated streak purification. A total of 1089 vaginal lactobacilli isolates were obtained, including *Lactobacillus curvatureii* (…). Lactobacillus crispatus Lactobacillus japonicus (), Lactobacillus jensenii Lactobacillus gasseri ( Lactobacillus gasseri 21 types of lactobacilli, including 1000.

[0033] The LBS medium formula 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.

[0034] The aforementioned Columbia blood agar medium was purchased from Guangzhou Dijing Microbial Technology Co., Ltd., with 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 above-mentioned MRS liquid culture 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 culture medium, 15.0 g agar needs to be added, and the medium is 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 modified NYC Ⅲ broth medium formula 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 medium 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 NYCⅢ broth formula was prepared in the same manner as in step (2).

[0042] (4) Preparation of Prevotella ileiflora CGMCC No. 59002 pathogen suspension The selected strain was *Prevotella iliforme*, isolated from vaginal secretions of pregnant women and deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 20, 2026, with accession number CGMCCNO.59002. 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 iliforme* 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, and after static incubation at 37 ℃ and CO2 for 48 h, the bacterial cells were collected by centrifugation at 6000 rpm for 5 min and 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 curvatureensis* strain B2726 was ultimately confirmed to possess stable antibacterial activity against four vaginal pathogens simultaneously, exhibiting strong acid-producing ability and no known antibiotic resistance genes or virulence factors detected, indicating good biosafety and microecological regulation capabilities. Therefore, it was selected as the target strain for this invention. The strain was then activated from the 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 curvatureii* B2726. The Gram-stained microscopic image of the strain is shown below. Figure 2 As shown. The full-length genome of this strain is 2,374,024 bp, with a GC content of 37.04%. 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 strain B2726 throughout their entire pregnancy. The vaginal flora profile during the entire pregnancy is as follows: Figure 3 As shown, images of the original gut microbiota throughout pregnancy are as follows: Figure 4 As shown, the vaginal pH remained at 3.8. Original images at the isolation and culture time points are shown below. Figure 5 As shown in the image, 16S rRNA sequencing results indicated that *Lactobacillus curvularia* was the absolutely dominant bacteria in the pregnant woman's vaginal flora throughout pregnancy (relative abundance >90%), with no dominant growth of vaginal pathogens such as *Streptococcus agalactiae*, *Gardnerella vaginalis*, *Fanny Hesse*, or *Prevotella urticaria*. ITS sequencing and culture results both showed no vaginal fungal colonization throughout the pregnancy. These results confirm that the pregnant woman's vaginal flora remained healthy throughout pregnancy, and that *Lactobacillus curvularia* B2726, derived from this bacteria, has a good foundation for vaginal adaptability and safety.

[0050] Example 2: Identification and biological characteristics of Lactobacillus curvature B2726 The monoclonal pure culture of Lactobacillus curvature B2726 obtained by isolation and purification in Example 1 was used for the following identification experiments.

[0051] A single colony of strain B2726 was picked from the plate using a disposable 1 μL inoculation loop and spread evenly in the designated wells. 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. Next, 1 μL of mass spectrometry sample processing matrix solution was added to cover the sample for pretreatment according to the protein spectrometry kit instructions. The plate was then allowed to air dry at room temperature. After drying, the mass spectrometry plate was placed on a MALDI-TOF-MS protein spectrometer for testing and identification. The strain B2726 was identified as *Lactobacillus curlis*.

[0052] Genomic DNA was extracted from *Lactobacillus curvature* B2726, and 16S rRNA gene amplification was performed by PCR and Sanger sequencing. The sequence was compared with *Lactobacillus curvature* (*Lactobacillus curvature*) using the NCBI database BLAST algorithm. Lactobacillus crispatus The strain showed the highest similarity and was identified as Lactobacillus curvaturei.

[0053] The optimal growth temperature for Lactobacillus curvature B2726 in MRS medium is 37 ℃. After static incubation at 37 ℃ with CO2, the absorbance of the bacterial solution at OD600 is 1.282, and the pH value is 3.6.

[0054] The aforementioned *Lactobacillus curlis* B2726 was deposited on July 16, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 39574. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The classification and nomenclature are: *Lactobacillus curlis* (…). Lactobacillus crispatus ).

[0055] Example 3: Inhibitory effect of Lactobacillus curvature B2726 on Streptococcus agalactiae, Gardnerella vaginalis, Fannie Herzegovina vaginalis, and Prevotella micrantha. 1. Preparation of Lactobacillus curvatureus B2726 mycelial cake A single colony of *Lactobacillus curlii* B2726 (CGMCC No. 39574) from Example 1 was inoculated into fresh MRS liquid medium and incubated statically 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 statically 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 obtained by punching holes in the agar medium using a 6 mm inner diameter punch. The MRS liquid medium formulation was the same as in Example 1.

[0056] 2. Preparation of suspensions of pathogens including Streptococcus agalactiae, Gardnerella vaginalis, Fannie Herzegovina vaginalis, and Prevotella micranthae. The selected test strains were laboratory standard strains: *Streptococcus agalactiae* ATCC12386, *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 (*Streptococcus agalactiae* 37 ℃, CO2 incubation for 24 h; *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 Streptococcus agalactiae, Gardnerella vaginalis, Fannie H. Neisseria vaginalis, and Prevotella bisporus were significantly inhibited in the experimental group, indicating that the Lactobacillus curvature B2726 of the present invention has good inhibitory activity against the growth of Streptococcus agalactiae, Gardnerella vaginalis, Fannie H. Neisseria vaginalis, and Prevotella bisporus.

[0062] Example 4: Killing effect of cell-free modified NYC III fermentation supernatant of Lactobacillus curvularia B2726 on Streptococcus agalactiae, Gardnerella vaginalis, Fannie Herzegovina vaginalis, and Prevotella micranthae. A single colony of *Lactobacillus curvatureii* B2726 (CGMCC No. 39574) from Example 1 was inoculated into fresh modified NYCⅢ liquid medium and incubated statically at 37 °C with CO2 for 48 h to obtain the seed culture. The seed culture was then transferred to fresh modified NYCⅢ liquid medium at a 2% (v / v) inoculation rate and incubated statically 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 NYCⅢ fermentation supernatant, which was stored at -80 °C for later use. The formulation of the modified NYCⅢ liquid medium was the same as in Example 1.

[0063] The GBS, GV, and FV pathogen stock solutions prepared in Example 1 were diluted to 10 μL each. 6 CFU / mL, and another 10 CFU / mL of the PB pathogen stock solution prepared in Example 1. 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. Co-culture was performed under different pathogen culture conditions (Streptococcus agalactiae 37 ℃, CO2 incubation for 24 h; 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 then serially diluted and inoculated onto Columbia blood agar. Pathogen culture was performed under the same conditions as above, and counts were made. The experiment was repeated three times, and the live CFU of Streptococcus agalactiae, 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 agalactococci in the culture medium of the control group was 10. 6 CFU / mL, vaginal Gardnerella vaginalis concentration was 10. 6 CFU / 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 Streptococcus agalactiae, Gardnerella vaginalis, Fannie Herzegovina vaginalis, and Prevotella ilex.

[0066] In summary, this invention provides a *Lactobacillus curvaturei* strain with reproductive tract microecological regulatory effects and its applications. This strain is a vaginal *Lactobacillus curvaturei* strain (… Lactobacillus crispatus B2726, with accession number CGMCC No. 39574. This strain and its metabolites are effective against Streptococcus agalactiae, an important perinatal pathogen. Streptococcus agalactiae ) and the core pathogens of bacterial vaginosis include Gardnerella vaginalis ( Gardnerella vaginalis ), Fanny Hesse (vaginis) Fannyhessea vaginae ) and Prevotella 2 Prevotella bivia All of them possess potent inhibitory and bactericidal activities. The strains of this invention are based on continuous monitoring data of the vaginal microecology of 692 pregnant women. They were obtained from 1089 strains of Lactobacillus derived from the vagina of 186 continuously healthy individuals through multiple rounds of in vitro antibacterial activity systematic evaluation and screening. They have strong acid-producing capacity, excellent biosafety, and strong adaptability to the vaginal environment. They can be prepared as microecological preparations and are suitable for regulating the vaginal microecological environment of women, including pregnant and postpartum women.

Claims

1. A strain of Lactobacillus curvature ( Lactobacillus crispatus B2726, characterized in that, The preservation number of the *Lactobacillus curvature* is CGMCC No. 39574.

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

1.

3. A bacterial preparation, characterized in that, It contains the curly Lactobacillus 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 curvature 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 the *Lactobacillus curvature* as described in claim 1 or 2, the bacterial preparation as described in claim 3 or 4, or the postbiotic as described in claim 5 or 6 in the preparation of products for regulating the vaginal microecological environment of women, including pregnant and postpartum women.

8. The application according to claim 7, characterized in that, The products are microecological preparations, oral medicines, health foods, feed additives, dressings, gels, lubricants, disinfectants, or antibacterial agents.

9. A product for regulating the vaginal microecological environment of women, including pregnant and postpartum women, characterized in that, It contains Lactobacillus curvatureus 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.

10. The product according to claim 9, characterized in that, The product is one or more of the following: vaginal health products, vaginal medicines, and vaginal medical devices; 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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