Novel lactobacillus strains, and composition and application thereof

Abstract

Disclosed are novel Lactobacillus strains, relating to a new Lactobacillus crispatus V435 strain and a new Lactobacillus jensenii V216 strain, and a composition comprising the strains used in the prevention or treatment of vaginitis. The Lactobacillus crispatus V435 strain is capable of producing lactic acid at a high yield, and the Lactobacillus jensenii V216 strain is capable of producing lactic acid and hydrogen peroxide at a high yield. Both strains have an excellent ability to inhibit clinically isolated Gardnerella, clinically isolated Atopobium, and fungi, a good ability to inhibit mixed vaginal pathogenic flora, and can be stably colonized in vaginal environments to become a dominant flora therein. The two strains exhibit a mutually beneficial symbiotic relationship without generating an antagonistic effect, and also exhibit a synergistic effect, resulting in stronger probiotic capabilities than a single dominant strain.

Description

Novel Lactobacillus strains, their compositions, and applications

[0001] Cross-references to related applications

[0002] This invention claims priority to two earlier applications: Patent Application No. 202410783465.2, filed with the China National Intellectual Property Administration on June 17, 2024, entitled "Novel Lactobacillus Strains and Compositions Thereof and Applications," and Patent Application No. 202510787214.6, filed with the China National Intellectual Property Administration on June 12, 2025, also entitled "Novel Lactobacillus Strains and Compositions Thereof and Applications." The full text of these two earlier applications is incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of microbiology, specifically relating to two novel strains of Lactobacillus, which can be used alone or in combination as probiotics. In particular, this invention relates to the use of these two strains, alone or in combination, for the prevention or treatment of vaginal infections or genitourinary system infections. Background Technology

[0004] The female vaginal microecological system is composed of the vaginal microbiota, immune regulation system, and vaginal anatomical structure. In normal women, the vaginal microbiota is dominated by Lactobacillus, maintaining vaginal microecological balance. However, in patients with bacterial vaginosis (BV) and mixed vaginitis, pathogenic bacteria proliferate in the vaginal microecology, leading to a decrease in the number of Lactobacillus. According to the "Guidelines for the Diagnosis and Treatment of BV (2021 Revised Edition)" and the "Expert Consensus on the Diagnosis and Treatment of Mixed Vaginitis (2021 Edition)," antibiotics are used for treatment. However, antibiotic treatment can further reduce the number of Lactobacillus in the vaginal microecology, exacerbating the disruption of the vaginal microecology and causing recurrent vaginal inflammation.

[0005] Studies have shown that lactic acid produced by lactobacilli can lower the pH of the vaginal microecology, disrupt the growth environment of pathogenic bacteria, and enhance the growth advantage of lactobacilli. H2O2 metabolized by lactobacilli also has the effect of inhibiting the growth of pathogenic bacteria. In addition, the large-scale growth of lactobacilli plays a role in occupying space in the microecological environment, which can squeeze out the physical space for the growth and survival of pathogenic bacteria, thereby inhibiting the growth of pathogenic bacteria.

[0006] Currently, only two vaginal probiotic preparations are marketed in China. One is a commercially available drug called "Yanhua" produced by Xi'an Zhenghao Biopharmaceutical Co., Ltd., which contains *Streptococcus faecalis*, a species that is not a dominant vaginal flora and whose bacteria are opportunistically pathogenic. The other is a commercially available drug called "Wanze Shuangqi" produced by Inner Mongolia Shuangqi Pharmaceutical Co., Ltd., which contains one lactobacillus species—*Lactobacillus delbrueckii*, a species that is not a dominant vaginal flora in Chinese women. Therefore, using dominant lactobacillus species to prepare vaginal probiotic preparations for the treatment of bacterial vaginosis has a greater therapeutic advantage.

[0007] Chinese patent document CN102851248A discloses a *Lactobacillus japonicus* for placement in the vagina of healthy Chinese women, which is a dominant lactobacillus isolated from the vagina of healthy Chinese women. Chinese patent document CN 107794236A discloses a *Lactobacillus curvatureii* and its application, which is a dominant lactobacillus isolated from the vagina of healthy Chinese women for the treatment of bacterial vaginosis. Chinese patent document CN 108004187A discloses a *Lactobacillus gasseri* and its application in the preparation of vaginal antibacterial drugs, wherein the *Lactobacillus gasseri* is a dominant bacterium isolated from the vagina of healthy Chinese women. Besides the three Chinese patent documents listed above, there are many other similar patent documents related to the treatment of bacterial vaginosis using dominant lactobacilli isolated from the vagina of healthy Chinese women. These all employ single lactobacilli screened from the vaginas of healthy Chinese women to treat bacterial vaginosis.

[0008] The female reproductive tract of healthy women contains various types of lactobacilli. Differences in lactobacillus species lead to variations in their antibacterial effects. Therefore, it is necessary to investigate and screen strains from multiple perspectives to identify those with strong acid-producing and H2O2-producing abilities and robust colonization capabilities for development. Simultaneously, it is essential to comprehensively consider the types of lactobacilli and the probiotic capabilities of different species, and to prepare vaginal microecological preparations using a compounding method with dominant lactobacillus species for the treatment of bacterial vaginosis. Summary of the Invention

[0009] The purpose of this invention is to address the shortcomings of existing technologies by providing two strains of vaginal lactobacillus and their combination, which exhibit broad-spectrum antibacterial effects against various genera of vaginal pathogens. This treatment addresses bacterial vaginosis through antibacterial, anti-inflammatory, and colonization mechanisms, and also treats recurrence of bacterial vaginosis after antibiotic intervention. The two strains exhibit a mutually beneficial symbiotic relationship without antagonism and possess a synergistic effect, resulting in a stronger probiotic capacity than a single dominant strain.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] In a first aspect, the present invention provides an isolated lactobacillus strain selected from Lactobacillus crispatus V435, which has the accession number CGMCC NO.30732.

[0012] Secondly, the present invention provides an isolated lactobacillus strain selected from Lactobacillus jensenii V216, which has the accession number CGMCCNO.30731.

[0013] Thirdly, the present invention provides a composition in which the active ingredient comprises at least one selected from Lactobacillus curlis V435 or Lactobacillus janniae V216.

[0014] In one embodiment, the active ingredients of the composition comprise Lactobacillus curvatureii V435 and Lactobacillus janniae V216.

[0015] Fourthly, the present invention provides the use of the isolated lactobacilli described in the first or second aspect or the composition described in the third aspect in the preparation of medicines for the prevention and treatment of vaginitis, general foods or health products.

[0016] In one implementation, the vaginitis is an infectious vaginitis, such as bacterial vaginitis, fungal vaginitis, mixed vaginitis, aerobic vaginitis, trichomonal vaginitis, and others; or a non-infectious vaginitis, such as atrophic vaginitis and others.

[0017] In one embodiment, the drug is administered orally, vaginally, or rectally, or instilled into the bladder, and preferably the carrier material is at least one pharmaceutically acceptable carrier.

[0018] In another embodiment, the food is selected from oatmeal porridge, lactic acid fermented foods, resistant starch, dietary fiber, sugars, proteins, and carrier materials of glycosylated proteins; in yet another embodiment, the food is selected from bread, cheese, yogurt, juice, health bars, spreads, biscuits, and cereals.

[0019] Fifthly, the present invention provides the use of the isolated lactobacilli described in the first or second aspect or the composition described in the third aspect in the preparation of genital hygiene products, such as sanitary napkins, tampons, or genital hygiene solutions.

[0020] In a sixth aspect, the present invention provides the use of the isolated lactobacilli described in the first or second aspect or the composition described in the third aspect in the preparation of pharmaceuticals or health care products for regulating vaginal flora balance.

[0021] In a seventh aspect, the present invention provides the use of the isolated lactobacilli described in the first or second aspect or the composition described in the third aspect in the preparation of a pharmaceutical or health care product having vaginal epithelial cell adhesion function.

[0022] Eighthly, the present invention provides the use of the isolated lactobacilli described in the first or second aspect or the composition described in the third aspect in the preparation of pharmaceuticals, health products, or food additives having the function of preventing or treating pathogenic bacteria, wherein the pathogenic bacteria include, but are not limited to, any one or more of Gardnerella vaginalis (Gv), Atopobium vaginae (Av), Prevotella bivia (Pb), or Candida albicans.

[0023] Ninthly, the present invention provides the use of the isolated lactobacilli described in the first or second aspect or the composition described in the third aspect in the preparation of topical care products for infants born via cesarean section. Since infants born via cesarean section do not pass through the woman's vagina, they do not receive exogenous probiotics during their delivery. Therefore, probiotics screened from the woman's vagina can be used to make topical care products for application to the infant's body or for bathing.

[0024] In a tenth aspect, the present invention provides the use of the isolated lactobacilli described in the first or second aspect or the composition described in the third aspect in the preparation of a vaginal medical device.

[0025] In one embodiment, the medical device according to the invention can be formulated as a suspension, spray, gel, cream, lotion, powder, capsule, ointment, oil, irrigation solution, ovule, vaginal plug, suppository, lozenge, tablet, or microcapsule product.

[0026] The beneficial effects of this invention are:

[0027] 1. The two strains of Lactobacillus curlis V435 and Lactobacillus janniae V216 provided by the present invention are sensitive to linezolid, penicillin, erythromycin, vancomycin, daptomycin and clindamycin, and are intermediate strains sensitive to imipenem, thus exhibiting good biosafety.

[0028] 2. The lactic acid production capacity of the two lactobacillus strains, *Lactobacillus curvaturei* V435 and *Lactobacillus janniae* V216, provided by this invention is higher than that of *Lactobacillus delbrueckii* DM8909. The lactic acid production capacity of *Lactobacillus curvaturei* V435 is 11728.92 μg / mL, and that of *Lactobacillus janniae* V216 is 16696.59 μg / mL.

[0029] 3. The hydrogen peroxide production capacity of Lactobacillus curli V435 provided by the present invention is comparable to that of Lactobacillus delbrueckii DM8909 (2605.08 μmol / L VS 2637.41 μmol / L), and the hydrogen peroxide production level of Lactobacillus janniae V216 is higher than that of DM8909 (2867.49 μmol / L VS 2637.41 μmol / L).

[0030] 4. The cell-free supernatant of *Lactobacillus curvatureii* V435 and *Lactobacillus janniae* V216 provided by this invention significantly inhibits the growth of *Gardnerella vaginalis* compared to the blank control and *Lactobacillus delbrueckii* DM8909. The antibacterial effects (inhibition zone diameter) of *Lactobacillus curvatureii* V435 and *Lactobacillus janniae* V216 against nine clinical isolates of *Gardnerella vaginalis* and standard strains ATCC14018T, *Atopobacterium*, or *Candida albicans* were all greater than those of *Lactobacillus delbrueckii* DM8909.

[0031] 5. Compared with the control group Lactobacillus delbrueckii DM8909, the Lactobacillus curli V435 and Lactobacillus janniae V216 provided by this invention can significantly promote the secretion of IL-10 and significantly reduce the IL-6 / IL-10 ratio, indicating that under inflammatory conditions, Lactobacillus curli V435 and Lactobacillus janniae V216 can effectively inhibit the immune response.

[0032] 6. The single bacteria Lactobacillus curvaturee V435 and Lactobacillus janniae V216 provided by the present invention have a significant killing effect on Gardnerella vaginalis in the vaginal pathogenic flora, and can significantly increase in mixed culture with vaginal pathogenic flora.

[0033] 7. The combination of *Lactobacillus curvatureii* V435 and *Lactobacillus janniae* V216 provided by this invention, compared with *Lactobacillus delbrueckii* DM8909 (reducing Gardnerella vaginalis by 1.24 times), *Lactobacillus curvatureii* V435 (reducing Gardnerella vaginalis by 2.65 times), and *Lactobacillus janniae* V216 (reducing Gardnerella vaginalis by 3.68 times) as single bacteria, showed a significant enhancement in the bactericidal ability against Gardnerella vaginalis in the vaginal pathogenic flora (reducing Gardnerella vaginalis by 5.69 times), indicating that the combination of lactobacilli provided by this invention can significantly enhance the antibacterial ability of single bacteria.

[0034] Preservation Information

[0035] The preservation information for the two types of Lactobacillus involved in this invention is as follows:

[0036] (1) Lactobacillus crispatus V435: deposited on May 22, 2024 at the China General Microbiological Culture Collection Center, with accession number CGMCC No.30732.

[0037] (2) Lactobacillus jensenii V216: deposited on May 22, 2024, at the China General Microbiological Culture Collection Center, with accession number CGMCC No.30731. Attached Figure Description

[0038] Figure 1. Antibacterial effect of cell-free supernatant of vaginal lactobacilli isolated against standard strain 14018T of Gardnerella vaginalis, clinically isolated Gardnerella vaginalis 52-2-1, and 45-3-1; where A represents the effect against standard strain 14018T of Gardnerella vaginalis, B represents the effect against Gardnerella vaginalis 52-2-1, and C represents the effect against Gardnerella vaginalis 45-3-1.

[0039] Figure 2. Standard curve of hydrogen peroxide;

[0040] Figure 3. Effects of different treatments on IL-10 secretion by cells;

[0041] Figure 4. Results of IL-6 / IL-10 ratio under different treatments;

[0042] Figure 5. Results of bactericidal activity of Lactobacillus curvaturei V435 and Lactobacillus janniae V216 against vaginal pathogenic bacteria;

[0043] Figure 6. Growth rate of Lactobacillus curvatureis V435 and Lactobacillus janniae V216 in mixed culture with vaginal pathogens;

[0044] Figure 7. Results of bactericidal activity of Lactobacillus curvaturei V435, Lactobacillus janniae V216 and their complex against vaginal pathogens. Detailed Implementation

[0045] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0046] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. % in the following examples refers to mass percentages.

[0047] The bacterial culture media or reagents used in the following examples can be prepared as follows:

[0048] 1. MRS liquid culture medium: Weigh 26g of MRS Broth medium, dissolve it in 500mL of ultrapure water, mix well, sterilize at 121℃ for 15min, and store at 4℃.

[0049] 2. MRS plate preparation: Add 1.5% agar to the prepared liquid culture medium, autoclave at 121℃ for 15 min, let stand until about 50℃, pour 15-20 mL of culture medium into each plate, then let stand until solidified, and store in a refrigerator at 4℃.

[0050] 3. BHI liquid medium ( / L): Weigh 18.5g of BHI broth medium, mix it evenly with 500mL of ultrapure water, autoclave at 121℃ for 15min, and store at 4℃.

[0051] 4. BHI semi-solid culture medium: Add 0.75-0.8% agar to the prepared liquid culture medium and autoclave at 115℃ for 20 minutes.

[0052] 5. Preparation of NYC III culture medium:

[0053] (1) Dissolve 3.8g Yeast extract, 2.4g Hepes, 5g NaCl, and 15g peptone in 900mL of ultrapure water. After thorough mixing, sterilize at 121℃ for 15min. Before using the sterilized culture medium, add 100mL horse serum and 20mL filtered 25% glucose.

[0054] (2) 25% glucose: Weigh 50g of anhydrous glucose and dissolve it in 200mL of ultrapure water. After the glucose is fully dissolved, filter it through a 0.22μm filter membrane to obtain a sterile glucose solution. It should not be sterilized.

[0055] 6. Preparation of NYC III semi-solid culture medium: Add 0.75% agar to the prepared liquid culture medium and autoclave at 115℃ for 20 min.

[0056] 7. Preparation of Columbia blood culture medium: Weigh 17.5g of Columbia broth powder culture medium, dissolve it in 500mL of ultrapure water, mix well, sterilize at 121℃ for 15min, and store at 4℃.

[0057] 8. Preparation of Columbia blood agar plates: Weigh 17.5g of Columbia agar basal medium, dissolve it in 500mL of ultrapure water, sterilize at 121℃ for 15min, let stand until about 50℃, add sterile defibrinated sheep blood at a dosage of 5%, mix well, pour into plates, and then let stand until coagulation.

[0058] 9. Preparation of 20 mmol / L tetramethylbenzidine (TMB) solution: Weigh 84 mg TMB and dissolve it in 17.4 ml of dimethyl sulfoxide solution. Store in the dark.

[0059] 10. Preparation of 100 mmol / L piperazine-N,N'-di-ethanesulfonic acid (PIPES) solution: Take 5 mL of 1 mol / L PIPES solution and add 45 mL of sterile water for later use.

[0060] 11. Preparation of 100 μmol / L hydrogen peroxide solution: Pipette 1 mL of 30% v / v hydrogen peroxide solution (approximately 9.128 mol / L), add 8.128 mL of 100 mM / L PIPES solution and dissolve thoroughly to obtain a 1 mol / L hydrogen peroxide solution. Then, pipette 100 μL of the 1 mol / L hydrogen peroxide solution and add 9900 μL of 100 mM / L PIPES solution to obtain a 10 mmol / L hydrogen peroxide solution. Repeat the previous step to obtain a 100-fold dilution to obtain a 100 μmol / L hydrogen peroxide solution.

[0061] 12. Preparation of horseradish peroxidase solution: Take one vial of 25 mg / mL peroxidase solution, pipette 100 μL of the enzyme solution, add 2.4 mL of sterile water and dissolve evenly. Then, store it in the dark with aluminum foil and place it on ice for later use.

[0062] 13. Preparation of 3 mol / L sodium hydroxide solution: Weigh 12 g of sodium hydroxide and dissolve it evenly in 100 mL of ultrapure water.

[0063] Example 1. Isolation and identification of Lactobacillus curvatureii V435 and Lactobacillus janniae V216

[0064] 1.1 Vaginal swab sample:

[0065] Volunteer sampling requirements:

[0066] (1) Before collection, ensure that you have not taken orally or topically any antibiotics for at least one week;

[0067] (2) No oral or topical use of estrogen or progesterone in the past month;

[0068] (3) No sexual activity, tub bath, vaginal douching or vaginal medication in the past 3 days;

[0069] (4) Not during menstruation.

[0070] Sampling procedure: Before sampling, soak a sterile swab in sterile saline solution. Wipe the sampling site 5-10 times, rotating the swab during the wiping process to ensure that microorganisms at the sampling site are evenly adhered to the swab head. After collection, break off the swab head and place it in a storage tube containing 1 mL of MRS liquid. Manually shake to mix 10-20 times, then freeze and store at -80°C.

[0071] 1.2 Strain isolation and purification:

[0072] After organizing the sample numbers, add 1 mL of MRS liquid to each tube, vortex to mix, discard the sample swab, and transfer 300 μL of sample solution to 270 μL of MRS liquid medium, marking it as 10⁻¹. Then, sequentially transfer 300 μL of bacterial suspension to 2.7 mL of MRS liquid for stepwise dilution to 10⁻³. For each corresponding sample, transfer 100 μL of bacterial suspension to MRS solid medium and Columbia solid medium. After spreading, transfer to an anaerobic incubator for anaerobic culture.

[0073] 1.3 Culture of Lactobacillus

[0074] All strains were stored in a -80°C freezer with 30% glycerol before activation. The Lactobacillus glycerol tubes were removed, and after thawing, they were inoculated into MRS medium at a 2% inoculum and cultured at 37°C for 24 hours. Then, they were inoculated into MRS medium again at a 2% inoculum and cultured at 37°C for 24 hours to activate the third generation.

[0075] The product "Dingjunsheng" was purchased from Inner Mongolia Shuangqi Pharmaceutical Co., Ltd. The packaging box was opened in a biosafety cabinet, one capsule was taken and the capsule shell was opened. The bacterial powder inside the capsule shell was poured into 10 mL of sterile PBS solution, vortexed, and recorded as a 10⁻¹ dilution. Then, a four-zone streak was performed on an MRS plate using an inoculation loop. After anaerobic incubation for 24-48 h, a single colony was picked from the plate and transferred to MRS liquid medium, and anaerobic incubated at 37℃ for 16-24 h. After incubation, the supernatant was collected by centrifugation to obtain bacterial cells. An equal volume of 20% glycerol was added, vortexed to mix, and aliquoted into cryovials to obtain the control strain Lactobacillus delbrueckii DM8909.

[0076] 1.4 Strain Identification:

[0077] (1) 16S sequencing:

[0078] PCR amplification was performed using a kit with primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'). The PCR products were subjected to gel electrophoresis to identify the 16S rRNA gene fragment. If the gel electrophoresis results indicated successful PCR, the PCR samples were sent to a gene sequencing company for 16S rRNA sequencing. The sequenced sequences were compared with data in the NCBI database using BLAST sequence similarity analysis. Based on the highest homology score >97%, 12 isolates were identified as *Lactobacillus crenata*, and 6 isolates were identified as *Lactobacillus janniae*.

[0079] The 16S rRNA of Lactobacillus curvature V435 is Seq.ID No.:1.

[0080] The 16S rRNA of Lactobacillus janniae V216 is Seq.ID No.:2.

[0081] (2) Physiological and biochemical identification:

[0082] Streaking: Second-generation Lactobacillus culture was streaked onto a plate and incubated at 37°C for 24 hours in an anaerobic incubator. McFarland turbidity adjustment: The streaked Lactobacillus agar was scraped into physiological saline using a cotton swab. The culture was centrifuged at 4000 rpm for 5 minutes, the supernatant was discarded, and 20 mL of saline solution was added. After dissolving 50 CHL Medium evenly, adjust the McFarland turbidity value of the bacterial solution to 2 using a McFarland turbidimeter.

[0083] Physiological and biochemical experiments: Add approximately 10 mL of sterile water to the bottom of the incubation box, and place all the test strips into the bottom of the incubation box. Take the dissolved bacterial solution and add it sequentially to the wells of the 0-49 test strips, adding 100 μL of bacterial solution to each well, with three replicates per strain. After closing the incubation box, place it in an anaerobic chamber, along with 3 M / L sodium hydroxide solution, and incubate at 37℃. The identification results are shown in Tables 1 and 2.

[0084] Table 1. Physiological and biochemical identification results of Lactobacillus curvatureii V435

[0085] Table 2. Physiological and biochemical identification results of Lactobacillus janniae V216

[0086] Example 2. Antibiotic sensitivity of Lactobacillus curvatureii V435 and Lactobacillus janniae V216

[0087] After activating the bacterial culture to the third generation, take 1 mL of the bacterial culture, centrifuge at 4000 r / min for 5 min, resuspend in MH liquid medium, and adjust the McFarland turbidity to 1.0 (after deducting the medium).

[0088] Bacterial suspension plating: Pipette 100 μL of bacterial suspension onto MH + 5% defibrinated sheep blood agar plates, plating a total of 4 MH agar plates, and then incubate in a carbon dioxide incubator.

[0089] Antimicrobial susceptibility testing: After the bacterial suspension has been fully absorbed, erythromycin and vancomycin susceptibility test strips were respectively affixed to the center of a 1.0 McFarland turbidity agar plate. The plate was then incubated at 37°C in a CO2 incubator. Readings were taken after 24 hours based on the bacterial growth. *Streptococcus pneumoniae* ATCC49619 was used as a quality control bacterium, and the antibiotic susceptibility of the control bacterium was within the control range. The antibiotic susceptibility results for *Lactobacillus curvatureii* V435 and *Lactobacillus japonicus* V216 are as follows: both strains were sensitive to linezolid, penicillin, erythromycin, vancomycin, daptomycin, and clindamycin; their sensitivity to imipenem was intermediate.

[0090] Table 3. Antibiotic susceptibility of Lactobacillus curvatureii V435 and Lactobacillus japonicus V216

[0091] Example 3. Antibacterial effect of cell-free supernatant from vaginal lactobacillus isolation

[0092] Twelve *Lactobacillus curvature* strains and six *Lactobacillus jannulata* strains screened from vaginal swabs of healthy individuals were tested for antibacterial activity against the control strain *Lactobacillus delbrueckii* DM8909. The antibacterial effect of cell-free supernatant from 19 *Lactobacillus* strains against *Gardnerella vaginalis* standard strain 14018T, clinically isolated *Gardnerella vaginalis* strains 52-2-1 and 45-3-1 was also tested. After activating the 19 *Lactobacillus* strains for three generations, the supernatant of the *Lactobacillus* strains cultured for 24 hours was collected, centrifuged at 8000g for 15 min at 4℃, and the supernatant was filtered through a 0.22μm sterile filter membrane to obtain cell-free supernatant.

[0093] 50 μL of GV bacteria were added to each well of a 96-well plate, with three parallel wells for each Lactobacillus strain. 150 μL of cell-free Lactobacillus supernatant was added. MRS medium was selected as a blank control group. The plates were co-cultured in an anaerobic chamber at 37°C. The absorbance at 595 nm was measured at 0 h and 24 h, and the OD was calculated. 595nm The rate of change.

[0094] Using Lactobacillus V716, which had no antibacterial activity during the screening process, as a negative control, the results showed that both strains had significant antibacterial effects against three Gardnerella vaginalis strains (ATCC14018T, 51-2-1, and 45-3-1). The inhibition rates of V435 were -18.42%, -21.18%, and -7.67%, respectively; and the inhibition rates of V216 were -15.82%, -14.84%, and -8.86%, respectively.

[0095] The inhibition rates of Lactobacillus delbrueckii DM8909 against three Gardnerella vaginalis strains (ATCC14018T, 51-2-1, and 45-3-1) were 23.26%, 25.65%, and 19.49%, respectively; while the inhibition rates of the blank control group were -0.94%, 8.66%, and -5.07%, respectively.

[0096] The antibacterial results are shown in Figure 1. Compared with the blank control and Lactobacillus delbrueckii DM8909, the cell-free supernatant of Lactobacillus curvaturei V435 and Lactobacillus janniae V216 significantly inhibited the growth of Gardnerella vaginalis.

[0097] Example 4. Lactic acid production capacity of lactobacilli isolated from the vagina

[0098] Lactobacillus japonicus V216 and Lactobacillus curvatureus V435, whose cell-free supernatants showed good antibacterial activity, were further tested to determine their lactic acid production capacity. The three Lactobacillus strains were inoculated into MRS liquid medium and cultured at 37°C for 24 hours, activating for three generations. The lactic acid content of the supernatant from the Lactobacillus strains after 24 hours of activation and three generations was measured, with Lactobacillus delbrueckii DM8909 used as a positive control and MRS medium as a negative control.

[0099] The results are shown in Table 4: The lactic acid production capacity of both lactobacilli strains was higher than that of DM8909. The lactic acid production capacity of Lactobacillus curvaturei V435 was 11728.92 μg / mL, and that of Lactobacillus janniae V216 was 16696.59 μg / mL.

[0100] Table 4. Results of lactic acid production determination of three strains of vaginal lactobacillus

[0101] Example 5. Hydrogen peroxide production experiment of lactobacilli isolated from the vagina

[0102] To prepare the standard curve: First, dilute the 30% (v / v) H2O2 (equivalent to 9.128 mol / L) stock solution to 1 mol / L using 100 mmol / L PIPES. Then, dilute the 1 mol / L H2O2 to 0, 20, 40, 60, 80, and 100 μmol / L working solutions using 100 mmol / L PIPES. Next, mix 100 μL of the above H2O2 working solution with 100 μL of 20 mmol / L TMB solution (three replicates for each concentration). Finally, add 2 μL of horseradish peroxidase (1 mg / mL) to the mixture and mix well. Incubate at 16°C for 10 min and measure OD600 to plot the corresponding standard curve. Perform the same operation on 100 μL of Lactobacillus supernatant. Calculate the hydrogen peroxide concentration in the supernatant based on the standard curve. The standard curve is shown in Figure 2. 2 =0.9996.

[0103] Based on the antibacterial effect and lactic acid production capacity of cell-free supernatant, the hydrogen peroxide production capacity of *Lactobacillus curvatureii* V435 and *Lactobacillus japonicus* V216 was determined. *Lactobacillus delbrueckii* DM8909 was used as a positive control, and MRS as a negative control. The hydrogen peroxide content of the target *Lactobacillus* strains was determined as follows: 100 μL of bacterial culture was added to an EP tube containing 100 μL of 20 mmol / L TMB. After mixing, 2 μL of horseradish peroxidase was added to catalyze a colorimetric reaction between tetramethylbenzidine and H2O2. The OD was then rapidly measured. 595nm Calculate the concentration of H2O2 by taking the OD value.

[0104] The results are shown in Table 5. The hydrogen peroxide production capacity of Lactobacillus curvature V435 was comparable to that of Lactobacillus delbrueckii DM8909 (2605.08 μmol / L vs. 2637.41 μmol / L), while the hydrogen peroxide production level of Lactobacillus janniae V216 was higher than that of DM8909 (2867.49 μmol / L vs. 2637.41 μmol / L).

[0105] Table 5. Results of hydrogen peroxide production assay for three strains of vaginal lactobacillus

[0106] Example 6. Immunomodulatory effects of Lactobacillus curvatureii V435 and Lactobacillus janniae V216

[0107] To measure the effects of promoting IL-10 secretion and inhibiting IL-6 secretion, human dendritic cells were co-cultured with Lactobacillus V216 and V435, and the secretion levels of IL-10 and IL-6 were measured to determine their anti-inflammatory effect on the human immune system, or whether they promoted inflammation. The experimental procedures are as follows:

[0108] Approximately 200 mL of human blood was drawn, and a mononuclear cell layer from the peripheral blood was obtained. The cells were washed 2-3 times with buffer solution, and then suspended in an appropriate culture medium. Lactobacillus was activated to the 3rd generation. After washing away the culture medium, the cells were resuspended in an appropriate buffer solution. Dendritic cells and resuspended cells were co-cultured for 24 h. The levels of IL-6 and IL-10 were measured by ELISA. The experimental background containing only cells was used as a blank control, the LPS-treated cell group was used as a negative control, and Lactobacillus delbrueckii DM8909 was used as a positive control. The experimental results are shown in Figures 3 and 4.

[0109] The experimental results showed that, compared with the control group Lactobacillus delbrueckii DM8909, Lactobacillus curvaturei V435 and Lactobacillus janniae V216 significantly promoted the secretion of IL-10 and significantly reduced the IL-6 / IL-10 ratio, indicating that under inflammatory conditions, Lactobacillus curvaturei V435 and Lactobacillus janniae V216 can effectively inhibit the immune response.

[0110] Example 7. Inhibition of Gardnerella vaginalis isolates by Lactobacillus curvaturei V435 and Lactobacillus janniae V216.

[0111] Based on the above in vitro results, *Lactobacillus curlis* V435 and *Lactobacillus japonicus* V216 were finally selected for further testing of their inhibitory spectrum against nine clinical isolates of *Gardnerella vaginalis* and the *Gardnerella vaginalis* standard strain ATCC14018T. 2 μL of overnight cultured *Lactobacillus* (approximately 1 × 10⁻⁶) was used. 8 A bacterial suspension of CFU / mL was spotted onto the surface of MRS solid medium containing 1.5% (w / v) agar and incubated at 37°C for 24 h. Subsequently, 1 mL of the 24-h Gardnerella vaginalis suspension was mixed with 100 mL of BHIs broth (0.75% agar) (final viable count approximately 1 × 10⁻⁶ CFU / mL). 6 The concentration (CFU / mL) was poured onto pre-inoculated Lactobacillus-containing solid plates, and the plates were anaerobically incubated at 37°C for 48 h. The diameter of the inhibition zone was then measured. Uninoculated MRS agar solid medium was used as a negative control, and Lactobacillus delbrueckii DM8909 was used as a positive control.

[0112] As shown in Table 6, Lactobacillus curvaturei V435 and Lactobacillus janniae V216 showed better antibacterial effects than Lactobacillus delbrueckii DM8909 against nine clinical isolates of Gardnerella vaginalis and the standard strain ATCC14018T.

[0113] Table 6. Diameter of inhibition zones (mm) against clinical Gardnerella vaginalis.

[0114] Example 8. The ability of Lactobacillus curvaturei V435 and Lactobacillus janniae V216 to inhibit clinically isolated Atobacillus.

[0115] Further verification of the antibacterial spectrum of *Lactobacillus curlis* V435 and *Lactobacillus japonicus* V216 against four clinical isolates of *Atopobacterium* and the standard strain CCμG 38953 was performed using 2 μL of overnight cultured *Lactobacillus* (approximately 1 × 10⁻⁶). 8 A bacterial suspension (CFU / mL) was spotted onto the surface of MRS solid medium containing 1.5% (w / v) agar and incubated at 37°C for 24 h. Subsequently, 1 mL of the 24-h incubated Atobacillus suspension was mixed with 100 mL of NYC III semi-solid medium (0.75% agar) and poured onto the spotted Lactobacillus solid plate. The plate was then anaerobically incubated at 37°C for 48 h, and the diameter of the inhibition zone was measured. Uninoculated MRS agar solid medium served as a negative control, and Lactobacillus delbrueckii DM8909 served as a positive control.

[0116] As shown in Table 7, Lactobacillus curvaturei V435 and Lactobacillus janniae V216 had better antibacterial effects against four clinical isolates of Atobacillus and CCμG 38953 than Lactobacillus delbrueckii DM8909.

[0117] Table 7. Diameter of inhibition zone against Atobacillus tumefaciens

[0118] Example 9. Inhibition of Candida albicans by Lactobacillus curvaturei V435 and Lactobacillus janniae V216

[0119] Take 2 μL of overnight cultured Lactobacillus (approximately 1 × 10⁻⁶). 8 A bacterial suspension (CFU / mL) was spotted onto the surface of MRS solid medium containing 1.5% (w / v) agar and incubated at 37°C for 24 h. Subsequently, 1 mL of Candida albicans cultured for 24 h was mixed with 100 mL of YPD semi-solid medium (0.75% agar) and poured onto the spotted Lactobacillus solid plate. The plate was then anaerobically incubated at 37°C for 48 h, and the diameter of the inhibition zone was measured. Uninoculated MRS agar solid medium served as a negative control, and Lactobacillus delbrueckii DM8909 served as a positive control.

[0120] The results are shown in Table 8. Lactobacillus curvaturei V435 and Lactobacillus janniae 216 showed obvious inhibition zones, and their antibacterial ability was better than that of DM8909.

[0121] Table 8. Diameter of inhibition zone against Candida albicans

[0122] Example 10. Bactericidal ability of Lactobacillus curvatureii V435 and Lactobacillus janniae V216 against vaginal pathogenic bacteria

[0123] The cultured clinical isolates of Gv 22-3, Av 27-6, and Pb standard strain Prevotella bivia CCμG 9557T were adjusted to McFarland turbidity of 0.5 using NYC III medium and a McFarland turbidimeter. The cultured Lj (Lactobacillus curvatureis V435) / Lc (Lactobacillus janniae V216) cultured on MRS medium was adjusted to McFarland turbidity of 0.5 using a McFarland turbidimeter. Then, Gv 22-3:Av 27-6:Pb standard strain:Lj / Lc were added to a 15mL centrifuge tube in a ratio of 13:3:4:3 and mixed thoroughly to obtain a mixed bacterial solution. MRS was used as a negative control. 1mL of the mixed bacterial solution was taken at 0 and 24h for q-PCR.

[0124] Primer sequence

[0125] As shown in Figures 5 and 6, single bacteria of Lactobacillus curvatureis V435 and Lactobacillus janniae V216 have a significant killing effect on Gardnerella vaginalis, a pathogenic bacteria in the vagina, and can grow significantly in mixed culture with vaginal pathogens.

[0126] Example 11. The bactericidal ability of Lactobacillus curvaturei V435 and Lactobacillus janniae V216 against vaginal pathogenic bacteria.

[0127] According to literature reports, patients with bacterial vaginosis experience vaginal flora imbalance, shifting from a lactobacillus-dominated vaginal environment to one dominated by pathogenic bacteria such as Gardnerella vaginalis, Atopobium vaginae, and Prevotella bivia. After this imbalance, the approximate ratio of Gardnerella vaginalis, Atopobium vaginae, Prevotella bivia, and lactobacillus in the vaginal environment is 13:3:4:3. Therefore, clinically isolated Gardnerella vaginalis, Atopobium vaginae, Prevotella bivia, and lactobacillus were mixed in a 13:3:4:3 ratio to simulate the vaginal flora of BV patients. The specific experimental method is as follows.

[0128] The cultured Lj V216 / Lc V435 was adjusted to 0.5 turbidity using a McFarland turbidimeter with MRS medium. Then, *Lactobacillus curvatureii* V435 and *Lactobacillus janniae* V216 were mixed at a 1:1 volume ratio, ensuring the total viable count matched the single-cell viable count, thus preparing a *Lactobacillus* composite culture. Subsequently, the cultured Gv 22-3, Av 27-6, and Pb standard strains were adjusted to the same McFarland turbidimeter level using NYC III medium. A mixture of Gv 22-3:Av 27-6:Pb standard strains (1:1) or single strains (V216 / V435) at a ratio of 13:3:4:3 was added to a 15 mL centrifuge tube and thoroughly mixed to obtain a mixed culture. MRS was used as a blank control, and DM8909 as a positive control. 1 mL of the mixed culture at 0 and 24 h was used for q-PCR.

[0129] As shown in Figure 7, after 24 hours of mixed culture, Gardnerella vaginalis increased by 19.66 times in the blank control group, decreased by 1.24 times in the DM8909 group, decreased by 3.68 times in Lactobacillus janniae V216, and decreased by 2.65 times in Lactobacillus curvature V435. Compared with DM8909, or Lactobacillus curvature V435 and Lactobacillus janniae V216, the mixed bacteria (1:1) showed a significant enhancement (5.69 times) in the bactericidal ability of Gardnerella vaginalis in the vaginal pathogenic flora, indicating that the combination of lactobacilli can significantly enhance the antibacterial ability of single bacteria.

Claims

1. An isolated lactobacillus, characterized in that, The strain was selected from Lactobacillus crispatus V435, with accession number CGMCC NO.30732.

2. An isolated lactobacillus, characterized in that, The strain was selected from Lactobacillus jensenii V216, with accession number CGMCC NO.30731.

3. A composition, characterized in that, The active ingredient of the composition comprises at least one lactobacillus selected from claims 1 or 2.

4. The composition according to claim 3, characterized in that, The active ingredient of the composition comprises the lactobacillus as described in claims 1 and 2.

5. The use of the isolated lactobacillus as described in claim 1 or 2, or the composition as described in claim 3 or 4, in the preparation of medicines, general foods, or health products for the prevention or treatment of vaginitis.

6. The application as described in claim 5, characterized in that, The drug is administered orally, vaginally, or rectally, or by instillation into the bladder; preferably, the drug comprises a carrier material, which is at least one pharmaceutically acceptable carrier; preferably, the food is selected from oatmeal porridge, lactic acid fermented foods, resistant starch, dietary fiber, sugars, proteins, and glycosylated protein carrier materials; more preferably, the food is selected from bread, cheese, yogurt, juice, health bars, spreads, biscuits, and cereals.

7. The use of the isolated lactobacillus of claim 1 or 2 or the composition of claim 3 or 4 in the preparation of genital hygiene products, such as sanitary napkins, tampons or genital hygiene solutions.

8. The use of the isolated lactobacillus as described in claim 1 or 2 or the composition as described in claim 3 or 4 in the preparation of a pharmaceutical or health care product for regulating vaginal flora balance.

9. The use of the isolated lactobacillus of claim 1 or 2 or the composition of claim 3 or 4 in the preparation of pharmaceuticals, health products, or food additives that prevent or treat pathogenic bacteria, wherein the pathogenic bacteria include, but are not limited to, any one or more of Gardnerella vaginalis, Atopobium vaginae, Prevotella bivia, or Candida albicans.

10. The use of the isolated lactobacillus of claim 1 or 2 or the composition of claim 3 or 4 in the preparation of a vaginal medical device.

Images