Multifunctional lactobacillus crispatus and its use in the preparation of health products

Abstract

This invention belongs to the field of microbial technology, specifically relating to the multifunctional Lactobacillus curvatureii and its application in the preparation of health products. The Lactobacillus curvatureii (… Lactobacillus crispatus Lactobacillus crispatus The accession number for MCOlact00102 is CGMCC No. 34381. The described *Lactobacillus curvatureensis* (MCOlact00102) is... Lactobacillus crispatus ​ The two strains, MCOlact00126 (CGMCC No. 34382), exhibit significant inhibitory effects against bacterial vaginosis (BV) and infections caused by Gardnerella vaginalis, Group B Streptococcus (GBS), Escherichia coli, or Candida albicans. The safety of these strains was evaluated using a 3D vaginal epithelial organoid model, showing no irritant effect on cells. Furthermore, hemolytic activity tests, antibiotic resistance gene detection, and functional validation all demonstrated their good safety profile. In addition, the aerobic acclimation process significantly improved the stability and bioactivity of these two strains in actual production and clinical applications, laying a solid foundation for their industrialization and application.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the multifunctional lactobacillus curvatureii and its application in the preparation of health products. Background Technology

[0002] Reproductive tract infections and inflammation pose a significant threat to the health of women and newborns. Globally, approximately 20%-50% of women suffer from reproductive tract infections, with higher rates among married and menopausal women, which is related to an imbalance in the vaginal microbiota. The vaginal microecology of healthy women is colonized by various microorganisms, including lactobacilli, streptococci, staphylococci, corynebacteria, enterobacilli, enterococci, Candida albicans, mycoplasma, and viruses, collectively forming the normal vaginal flora. In a lactobacillus-dominated vaginal microecology, various microorganisms interact to form a dynamically balanced micro-ecosystem, maintaining a healthy vagina. Lactobacilli can utilize glycogen stored in shed vaginal epithelial cells as energy to grow and reproduce in the vaginal environment. They can inhibit the invasion and overgrowth of pathogenic microorganisms and regulate the vaginal microecological balance by competing with pathogens for nutrients; competing for adhesion receptors on the surface of vaginal epithelial cells; stimulating local immune responses; and producing hydrogen peroxide, antibiotics, and lactic acid.

[0003] Bacterial vaginosis is one of the most common vaginal infections in women, characterized by typical vaginal microecological dysbiosis, increased pathogenic or aerobic bacterial load, and increased production of volatile amines. If left untreated, it can ascend and cause pelvic inflammatory disease, infertility or ectopic pregnancy, and increase the risk of premature birth during pregnancy. It also disrupts the vaginal environment, increasing the likelihood of sexually transmitted infections (such as HPV and HIV). Bacterial vaginosis is more prevalent in sexually active women, as well as menopausal and elderly women. Antibiotic treatment is currently the main treatment for bacterial vaginosis, but the recurrence rate within one year of antibiotic treatment is 50%-100%. Furthermore, with the widespread development of antibiotic resistance, treatment for antibiotic-resistant bacterial vaginosis and Group B streptococcal infections urgently needs to be addressed. Treatment of gynecological infections or inflammations, especially bacterial vaginosis, by altering the female gut or reproductive tract flora through oral or vaginal administration has been attempted, but the results have not been significant due to differences in the functions of healthy vaginal flora and strains.

[0004] In addition, Group B Streptococcus, Escherichia coli, and Candida albicans are four common opportunistic pathogens that are widely present on the surface of human skin or mucous membranes. When the body's immunity is weakened or the mucosal barrier is damaged, they can invade different sites and cause a variety of infections. For example, Group B Streptococcus is an important pathogen of neonatal sepsis and meningitis, and can also cause perinatal infections in pregnant women. Staphylococcus aureus is adept at causing systemic purulent infections ranging from skin boils to pneumonia, osteomyelitis, and even sepsis, and drug-resistant strains are increasing. Escherichia coli, as a normal intestinal flora, is the most common pathogen of urinary tract infections and one of the main culprits of abdominal infections and neonatal meningitis. Candida albicans, as a fungus, mainly affects mucous membranes (such as oral thrush and vaginitis) and skin folds, and can be life-threatening when it invades deep into the body.

[0005] Treating diseases through the microecological system demonstrates multiple advantages: it can effectively disrupt the biofilm structure of pathogenic bacteria, reduce their colonization ability in the vaginal environment, inhibit and reduce the risk of recurrence, and rebuild the vaginal acidic environment through immunomodulation, promoting microecological balance and thus avoiding drug resistance problems caused by antibiotic abuse. This therapy provides a safer and more sustainable treatment option for bacterial vaginosis and related diseases. However, current gynecological probiotic and drug products on the market have significant limitations. Most product strains have unknown origins and are mainly isolated from the gut microbiota, while the female vagina and gut microbiota differ significantly in niche, genes, and function—the female reproductive tract typically does not contain gut bacteria. Furthermore, even strains labeled as the same species may exhibit significant heterogeneity in gene sequences and functional characteristics, further limiting the precision and effectiveness of their clinical application. Currently available patents and literature related to vaginal microbiota show that the discovery process of existing strains lacks a rigorous screening mechanism. In contrast, conducting microbiome analysis on large cohorts of healthy individuals, combined with in vitro culture and multidimensional functional screening (such as colonization ability, immunomodulatory activity, and metabolite detection), can systematically verify the effectiveness of strains and provide more reliable experimental evidence for functional realization. Summary of the Invention

[0006] In view of the defects and deficiencies in the existing technology, the present invention provides two novel multifunctional vaginal curly lactobacilli and their applications in the preparation of health products.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a multifunctional vaginal curly lactobacillus, said curly lactobacillus ( Lactobacillus crispatusMCOlact00102 was deposited on April 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34381.

[0008] Or, the curly Lactobacillus ( Lactobacillus crispatus MCOlact00126 was deposited on April 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34382.

[0009] In a second aspect, the present invention provides a composition comprising live, inactivated, fermentation broth, or cell-free supernatant of the multifunctional lactobacillus described in the first aspect.

[0010] In a third aspect, the present invention provides the use of the multifunctional vaginal curly lactobacillus described in the first aspect or the composition described in the second aspect in the preparation of health products.

[0011] Alternatively, in the above applications, the health product can inhibit pathogens.

[0012] Alternatively, in the above applications, the health product can reduce the load of pathogenic bacteria.

[0013] Alternatively, in the above applications, the pathogenic bacteria include Gardnerella vaginalis, Group B Streptococcus, Escherichia coli, or Candida albicans.

[0014] Alternatively, in the above applications, the health product can be used to prevent or treat bacterial vaginosis.

[0015] Alternatively, in the above applications, the product may be a drug, health product, functional food, food supplement, food for special medical purposes, medical device, or hygiene product.

[0016] Alternatively, in the above applications, the product may also contain a pharmaceutically, health-promoting, or food-grade carrier.

[0017] Preferably, the carrier includes fillers, binders, wetting agents, disintegrants, lubricants, or flavoring agents known in the art.

[0018] Alternatively, in the above applications, the dosage form of the product includes pills, tablets, lozenges, lyophilized powders, granules, capsules, aqueous solutions, alcoholic solutions, oil solutions, syrups, emulsions, suspensions, suppositories, solutions for injection or infusion, ointments, gels, tinctures, creams, patches, lotions, sprays, aerosols, powder sprays, effervescent tablets, transdermal therapy systems, microcapsules, or implants.

[0019] Preferably, the drug is for oral, topical, or vaginal administration. The carrier in the drug is a conventionally used carrier suitable for preparing oral, topical, or vaginal forms.

[0020] Preferably, the term "external use" refers to application to the skin, mucous membranes, or vulva.

[0021] More preferably, the daily dose of the drug depends on the method of administration (oral, topical, or vaginal) and the type of treatment (therapeutic or prophylactic).

[0022] Preferably, the health products, functional foods, food supplements, and foods for special medical purposes are for oral administration.

[0023] Alternatively, in the above applications, the sanitary products include sanitary wipes, sanitary napkins, panty liners, sanitary tampons, sanitary napkins, vaginal washes, and feminine antibacterial / bacteriostatic washes.

[0024] Alternatively, in the above applications, the product may also contain a second component.

[0025] Preferably, the second component includes probiotics, postbiotics, prebiotics, antibacterial agents, or immunomodulators.

[0026] Compared with the prior art, the present invention has the following advantages: This invention is based on 1,400 strains of lactobacillus derived from healthy women of reproductive age in 29 countries worldwide. Through systematic culturomics methods, 468 strains capable of growing under different conditions were screened. Further, whole-genome sequencing and comprehensive evaluation of 13 key functional indicators were performed on 200 of these strains. Using aerobic environment acclimation technology, two multifunctional lactobacillus strains derived from the vaginal flora of healthy women were ultimately selected. These two strains exhibit significant inhibitory effects on bacterial vaginosis (BV) and infections caused by Gardnerella vaginalis, Group B Streptococcus, Escherichia coli, or Candida albicans. The safety of this invention was evaluated using an FDA-approved 3D vaginal epithelial cell model. The results showed that these two strains had no irritant effect on cells, and their good safety was further demonstrated through hemolysis tests, antibiotic resistance gene detection, and functional verification. Furthermore, the aerobic environment acclimation process significantly improved the stability and bioactivity of these two strains in actual production and clinical applications, laying a solid foundation for their industrialization and application. Attached Figure Description

[0027] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 Blood agar hemolysis test for 34381 and 34382.

[0028] Figure 2 The results of long-read whole-genome sequencing of 34381.

[0029] Figure 3 The results of long-read whole-genome sequencing of 34382.

[0030] Figure 4 Comparison of D-lactic acid secretion concentrations of 34381 and 34382 with market products.

[0031] Figure 5 Tracking the inhibitory effects of 34381, 34382, and other market products on Gardnerella vaginalis over 800 minutes (40 cycles).

[0032] Figure 6 The inhibitory effects of 34381, 34382 and other market products on Gardnerella vaginalis at 800 minutes (40 cycles).

[0033] Figure 7 Tracking the inhibitory effects of 34381, 34382 and other market products on Group B Streptococcus for 24 hours (77 cycles).

[0034] Figure 8 The inhibitory effect of 34381, 34382 and other market products on Group B Streptococcus within 24 hours.

[0035] Figure 9 Tracking the inhibitory effects of 34381, 34382 and other market products on Candida albicans over 24 hours (77 cycles).

[0036] Figure 10 The inhibitory effect of 34381, 34382 and other market products on Candida albicans within 24 hours.

[0037] Figure 11 Tracking the inhibitory effects of 34381, 34382 and other market products on Escherichia coli over 24 hours (77 cycles).

[0038] Figure 12 The inhibitory effect of 34381, 34382 and other market products on Escherichia coli within 24 hours.

[0039] Figure 13 The inhibitory effect of co-culturing 34381 and 34382 with cells on the translocation rate of Group B Streptococcus infection.

[0040] Figure 14 Comparison of growth of 34381 after oxygen acclimation. Figure A shows the comparison of anaerobic growth of 34381 before and after oxygen acclimation (right). Figure B shows the 24-hour growth tracking of 34381 before and after oxygen acclimation in an aerobic environment (top) and the 24-hour growth tracking of 34381 before and after oxygen acclimation in an anaerobic environment (bottom).

[0041] Figure 15 Comparison of the growth of 34382 after oxygen acclimatization. Figure A shows the comparison of the anaerobic growth of 34382 after unacclimatization (left) and oxygen acclimatization (right). Figure B shows the 24-hour growth tracking of 34382 under aerobic environment (top) and 24-hour growth tracking of 34382 under anaerobic environment (bottom).

[0042] Figure 16 The effects of vaginal epithelial 3D organoids 34381, 34382 and other marketed products on cell stimulation were detected. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. The following are the experimental methods and results used in the implementation examples.

[0044] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0045] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0046] Example: In this invention, 1400 strains of bacteria were cultured from the vaginal microbiome of healthy reproductive-age women under aerobic and anaerobic environments using different culture media, including 468 strains of Lactobacillus. Metagenomic sequencing was performed on 200 of these strains. Subsequently, the following functions were verified and stratified through in vitro experiments: hemolysis, lactic acid production, inhibition of pathogenic bacteria, degree of adhesion to vaginal cells, long-read sequencing of the whole genome and antibiotic resistance genes, growth rate (under aerobic and anaerobic conditions), carbohydrate metabolism, hydrogen peroxide concentration, autoaggregation, antibiotic sensitivity, GBS transcellular transport, cell adhesion, and cytotoxicity verified using an in vitro 3D model. A systematic scoring system was established for these 13 functions and their importance. Overall, the experiments showed that two strains of *Lactobacillus curvature* were dominant in different functions and received the highest overall scores. The two selected vaginal *Lactobacillus curvature* strains are: *Lactobacillus curvature* (… Lactobacillus crispatus MCOlact00102 was deposited on April 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34381, along with Lactobacillus curlis (…). Lactobacillus crispatus MCOlact00126 was deposited on April 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34382.

[0047] Since there are not many Lactobacillus curvature extract products available on the market from healthy vaginal individuals, we purchased Shufuyin Lactobacillus health tablets and used them as a market product to compare with Lactobacillus curvature.

[0048] The experimental methods and results for each experiment are shown below: 1. Blood agar hemolysis test This experiment aims to determine whether a bacterial strain can lyse red blood cells, thus indicating its virulence or cytotoxicity. The *Lactobacillus curvatureii* bacterial suspension of this invention was streaked onto blood agar plates containing 5% defibrinated sheep blood. After 24 hours of anaerobic incubation, the hemolytic activity of the strain was observed visually. The type of hemolysis was determined based on the phenomena surrounding the colonies: a clear zone indicated β-hemolysis, a green halo indicated α-hemolysis, and no change indicated γ-hemolysis. *Staphylococcus aureus* was used as a positive control.

[0049] 1.1 Experimental Methods: (1) Inoculate the bacterial strain onto a blood agar plate.

[0050] (2) Anaerobic culture at 37°C for 24 hours.

[0051] (3) Observe the type of hemolysis around the colony.

[0052] 1.2 Experimental Results: Figure 1 The results showed that strains 34381 and 34382 did not have obvious lysis zones or hemolysis, proving the safety of the two strains of Lactobacillus curvularia vaginalis for human use.

[0053] 2. Long-read sequencing of the entire genome and antibiotic resistance genes 2.1 Experimental Methods: This invention utilizes whole genome sequencing to detect antimicrobial resistance genes. The specific steps are as follows: (1) Sample preparation and sequencing: Collect genomic DNA from the samples to be tested and obtain their genomic sequences using long-chain nanopore sequencing. Ensure that the genomic sequences of the samples are saved in FASTA format to obtain complete genomic data for subsequent analysis.

[0054] (2) Data processing: Perform quality control and filtering on the raw data obtained from sequencing to remove low-quality sequences.

[0055] (3) Genome alignment: The query genome is compared with the current reference genome ( Lactobacillus crispatus Compare with DC21.1.

[0056] (4) Circular plot display: A circular plot is used to display the comparison results between the query genome and the reference genome. The query genome is marked in orange and the reference genome is marked in green.

[0057] (5) Annotation: Annotate the functions of the queried genome, and identify genes, functional regions, etc.

[0058] 2.2 Experimental Results: Long-read sequencing whole-genome sequencing results are as follows Figure 2and Figure 3 As shown, no known antibiotic resistance genes were detected in the potential plasmids identified by sequencing.

[0059] 3. Antibiotic susceptibility testing Using a sterile inoculation loop, the *Lactobacillus curvatureii* bacterial suspension of this invention was taken from the cryopreservation tube and streaked onto an MRS plate, which was then incubated in an anaerobic flask at 37°C for 24 hours. *Lactobacillus* colonies from the MRS plate were scraped off with a 1 μL inoculation loop and resuspended in 10 mL of LSM (a culture medium composed of IST medium (90%) and MRS medium (10%), pH adjusted to 6.7 and then sterilized) broth, and incubated in an anaerobic flask at 37°C for 24 hours. After thoroughly mixing the bacterial suspension, 100 μL was spread onto an LSM plate and incubated anaerobically at 37°C for 24 hours. Subsequently, the bacteria were resuspended in sterile 0.85% NaCl solution, and the turbidity was adjusted to 1 McFarland turbidity (OD). 600 Approximately 0.25 g / L. Using a sterile cotton swab, apply the prepared bacterial suspension evenly to the entire surface of an LSM plate, repeating three times to form a bacterial colony. After the plate has slightly dried, carefully place the Etest strip onto the agar surface with the graduated side facing up using sterile forceps, pressing gently to ensure complete contact with the agar. Two replicates were performed for each antibiotic. The plates were incubated in an anaerobic jar at 37°C for 48 hours, and the minimum inhibitory concentration (MIC) of each antibiotic against the tested Lactobacillus was recorded. The results are shown in Table 1.

[0060] Table 1: Antibiotic sensitivity was tested, and there was no significant difference in antibiotic resistance compared to other wild-type lactobacilli. 4. Lactic acid production 4.1 Experimental Methods: D-Lactate Production Assay: The D-lactate colorimetric assay kit was used, strictly following the manufacturer's instructions. The supernatant consisted of two strains of *Lactobacillus curvature* from this invention and a commercially available *Lactobacillus curvature* product. After anaerobic culture in fresh NYCIII medium for 24 hours, the OD value was adjusted to 1, and the mixture was filtered through a 0.2 μm filter to obtain a cell-free supernatant. To ensure that the sample readings were within the detection range of the standard curve, the culture supernatant was diluted 1000-fold with ultrapure water.

[0061] 4.2 Experimental Results: Experimental results are as follows Figure 4 As shown, under the same culture conditions, both strains 34381 and 34382 secreted high concentrations of D-lactic acid, especially strain 34381, which secreted significantly higher levels of D-lactic acid than the commercially available product (SFY Lactobacillus Health Tablets). This demonstrates that these two strains have a superior ability to control the acidic environment and inhibit the growth of other bacteria. Each experiment had three replicates, and the experiments were repeated three times. The following figures show the common trends across the three experiments. The statistical method used was one-way ANOVA. p<0.1, p<0.01, p<0.001, p<0.0001.

[0062] 5. Inhibition of pathogenic bacteria 5.1 Inhibition of pathogenic Gardnerella vaginalis (CCUG 44114) Inhibition assays against Gardnerella vaginalis were performed in 96-well plates, with a final volume of 200 μL per well. Gardnerella vaginalis was streaked from cryopreserved tubes onto NYCIII plates and anaerobically incubated at 37°C for 24 hours. Colonies were scraped from the plates using a 1 μL inoculation loop and resuspended in 5 mL of NYCIII broth, and anaerobically incubated at 37°C for 24 hours. The final Gardnerella vaginalis culture was centrifuged (10,000 g, 10 min), resuspended in fresh NYCIII medium, and OD was adjusted. 600 To a concentration of 0.1, add 50 μL of cell-free supernatant of the tested lactobacillus to 150 μL of *Gardnerella vaginalis* suspension with adjusted OD values. The supernatant consisted of two strains of *Lactobacillus curvature* and a commercially available *Lactobacillus curvature*, cultured overnight in fresh NYCIII medium. After adjusting the OD value to 1, the supernatant was filtered through a 0.2 μm filter, and 50 μL of the supernatant was added to 150 μL of *Gardnerella vaginalis* suspension. 50 μL of sterile medium was used as a control. The 96-well plate was placed in an anaerobic workstation and incubated at 37°C for 800 minutes for monitoring. OD values ​​were read every 20 minutes. 600 After incubation, bacterial growth was quantified by measuring the absorbance at 600 nm in each well. Each experiment had three replicates, and the experiments were repeated three times. The following figures illustrate the common trends across the three experiments. Statistical analysis was performed using one-way ANOVA. p<0.1, p<0.01, p<0.001, p<0.0001.

[0063] like Figure 5 and Figure 6As shown, two strains of *Lactobacillus curlis* and a commercially available product (*Lactobacillus sophia*) were compared. Compared to the control group which had no lactobacillus supernatant, the supernatants of the two *Lactobacillus curlis* strains showed significant inhibition of *Gardnerella vaginalis* after approximately 160 minutes (cycle 8), and this inhibition lasted until 800 minutes (40 cycles) at the end of the experiment. The two *Lactobacillus curlis* strains of this invention also showed significantly better inhibitory effects on *Gardnerella vaginalis* than the commercially available product over 40 cycles.

[0064] 5.2 Inhibition of other pathogenic bacteria 5.2.1 Experimental Methods: Commonly infectious strains of Group B Streptococcus (CCUG 47293), Candida albicans (CCUG 44135), and Escherichia coli (ST131) were purchased and cultured overnight in LB aerobic media. The strains were then cultured overnight in liquid LB aerobic medium at 37°C. The final cultures of the above strains were centrifuged and resuspended in fresh NYCIII medium, and the OD was adjusted. 600 To adjust the OD value to 0.1, add 50 μL of cell-free supernatant of the Lactobacillus strain to be tested to 150 μL of the strain with adjusted OD value. The supernatant consisted of the two Lactobacillus curvature strains of this invention and the commercially available Lactobacillus curvature, cultured overnight in fresh NYCIII medium, and then the OD value was adjusted to 1. The supernatant was then filtered through a 0.2 μm filter membrane. 50 μL of sterile medium was used as a control. The 96-well plate was placed in an ELISA reader in an aerobic workstation and incubated at 37°C for 24 hours for monitoring. OD values ​​were read every 20 minutes. 600 After cultivation, bacterial growth was quantified by measuring the absorbance at 600 nm in each well. Each experiment had three replicates, and the experiment was repeated three times. The following figures illustrate the common trends across the three experiments. Statistical analysis was performed using one-way ANOVA. p<0.1, p<0.01, p<0.001, p<0.0001.

[0065] 5.2.2 Experimental Results: like Figure 7 and Figure 8As shown, Group B Streptococci are common pathogens causing skin, vaginal, and intestinal infections, especially dangerous in premature infants. A comparison was made using the two *Lactobacillus curvatureii* strains of this invention and a commercially available product (*Lactobacillus succinate*). Compared to the control group which had no lactobacillus supernatant, the supernatant from the two *Lactobacillus curvatureii* strains showed significant inhibition of Group B Streptococci upon addition. This inhibition persisted for 24 hours after the end of the experiment. No growth of Group B Streptococci was observed. The two *Lactobacillus curvatureii* strains of this invention exhibited significant inhibition of Group B Streptococci, almost completely suppressing their growth, and their effect over 77 cycles was significantly superior to the commercially available product. No growth of Group B Streptococci was observed throughout the entire cycle.

[0066] like Figure 9 and Figure 10 As shown, Candida albicans is a common skin, vaginal, and nosocomial infection, and is a WHO key pathogen of antibiotic resistance concern. A comparison was made using the two strains of *Lactobacillus curvularis* from this invention and a commercially available product (*Lactobacillus suis*). Compared to the control group which had no lactobacillus supernatant, the supernatant of *Lactobacillus curvularis* 34382 showed significant inhibition of Candida albicans upon addition. This inhibitory effect persisted for 24 hours after the end of the experiment. The inhibitory effect of this bacterium against Candida albicans was also significantly superior to the commercially available product over 77 cycles.

[0067] like Figure 11 and Figure 12 As shown, Escherichia coli infection, especially ST131, is very common in urethritis, vaginitis, and other inflammatory infections. A comparison was made using the two *Lactobacillus curvaturei* strains of this invention and a commercially available product (*Lactobacillus schoensis*). Compared to the control group which had no lactobacillus supernatant, the supernatant from the two *Lactobacillus curvaturei* strains showed significant inhibition of *E. coli* upon addition. This inhibitory effect persisted for 24 hours after the end of the experiment. The inhibitory effect of this bacterium on *E. coli* was also significantly superior to the commercially available product over 77 cycles, with strain 34381 being significantly superior to strain 34382.

[0068] 6. The degree of automatic aggregation and adhesion to vaginal cells 6.1 Experimental Methods: 6.1.1 Automated aggregation experimental method: Take 5 mL of bacterial culture that has been incubated in MRS medium at 37°C for 24 hours, collect the bacterial cells by centrifugation (10,000 g, 15 minutes), wash twice, resuspend in PBS buffer, and adjust the viable count to approximately 10⁻⁶. 8CFU / mL. For the determination, take 10 mL of bacterial suspension and vortex for 10 seconds. Under static conditions at room temperature, take 1 mL of the suspension from the upper layer at 0, 6, 12, and 24 hours and measure its absorbance at 600 nm. The spontaneous aggregation rate is calculated using the following formula: Spontaneous aggregation rate (%) = [1 - (At / A0)] × 100%, where At is the absorbance value at time t (6, 12, and 24 hours), and A0 is the absorbance value at time 0.

[0069] 6.1.2 Adhesion test method for vaginal cells: VK2 / E6E7 cell line was planted at 5 × 10⁶ cells per well. 4 Cells were seeded at a density of 100% in 24-well plates and cultured in a CO2 incubator until 80%-90% confluence. 24 hours before the adhesion assay, the old culture medium was discarded, and the cells were gently washed twice with pre-warmed PBS. 1 mL of antibiotic-free cell culture medium was added to each well. Simultaneously, the target *Lactobacillus* strain was cultured anaerobically at 37°C for 24 hours. For the adhesion assay, cells from 3 wells were resuspended in 1 mL of fresh, pre-warmed antibiotic-free culture medium, and cell counts were performed to determine the multiple of infection. When the cells reached 100% confluence, the number of cells per well was approximately 5 × 10⁶. 5 Each lactobacillus was washed with 1 mL PBS, resuspended in antibiotic-free cell culture medium, and OD was adjusted. 600 Up to 1.0 (at which point the bacterial concentration is approximately 10). 9 CFU / mL). The bacterial concentration was adjusted according to the multiplicity of infection (cells:bacteria = 1:100), and added to the cell wells. Each sample was prepared in triplicate, with a negative control (no bacteria added). After inoculation, the 24-well plate was centrifuged at 1000 rpm for 10 minutes, and then incubated in a CO2 incubator for 4 hours. After incubation, the cells were washed twice with pre-warmed PBS to remove unattached bacteria. 300 μL of TrypLE Express digestion solution was added to each well, and digestion was performed at 37°C for 5 minutes, followed by the addition of 700 μL of PBS to terminate the digestion. 1 mL of cell suspension was collected from each well and serially diluted with PBS (10-10). - ¹ to 10 -6 ), and 10 μL of each dilution was taken to seed MRS plates for viable count. MRS plates were incubated at 37℃ under anaerobic conditions for 24-48 hours, and appropriate dilutions with colony counts between 30-300 were selected for counting to determine the number of adherent lactobacilli.

[0070] 6.2 Experimental Results: Table 2: Self-aggregation and adhesion to VK2 cells As shown in Table 2, the experiment showed that at 6 hours, strain 34381 produced 57% self-aggregation, while strain 34382 produced 91% self-aggregation. The cell adhesion of the two strains at 12 hours was 75% and 95%, respectively.

[0071] 7. GBS transcellular transport assay 7.1 Experimental Methods: The VK2 / E6E7 cell line was cultured in cell-SFM complete medium (containing 0.1 ng / mL EGF, 0.5 mg / mL bovine extract, 0.4 mM calcium chloride, and 1% penicillin-streptomycin). After the cells reached 85-95% confluence, they were digested and resuspended at 6.67 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 300 μL / well on the top side of the Transwell insert in 24-well plates, and 600 μL of culture medium was added to the bottom side. The plates were incubated at 37°C and 5% CO2 for 48 hours, and the medium was changed every 2 days thereafter. Transendothelial resistance (TEER) was measured daily using a Millicell ERS-2 resistivity meter to monitor cell monolayer formation until the resistance value reached a peak of 15 Ω·cm². Before the experiment, the cell culture medium was replaced with antibiotic-free medium and incubated for at least 12 hours. Then, the *Lactobacillus curvatureii* suspension (OD=1) of the present invention was added to the top side of the insert and incubated for 2 hours. After washing with PBS, GBS suspension (OD=5) was added and incubated for 4 hours. The culture medium from the bottom side was collected and serially diluted for plating. The bacterial translocation ability was assessed by colony counting.

[0072] 7.2 Experimental Results: like Figure 13 As shown, the results indicate that pretreatment of cells with 34381 and 34382 significantly reduced GBS penetration. In particular, 34382 showed a relative translocation rate of less than 40%, indicating that it provided superior protection for the integrity of the monolayer cell membrane.

[0073] 8. Carbohydrate fermentation characteristic test 8.1 Experimental Methods: Carbohydrate utilization profiling was performed using the API 50 CHL identification system, strictly following the manufacturer's instructions. Results were recorded for the two *Lactobacillus curvature* strains of this invention at 24 and 48 hours of incubation. In addition, experimental groups were added that underwent final incubation under both aerobic and anaerobic conditions.

[0074] 8.2 Experimental Results: Table 3: Characteristics of carbohydrate fermentation 9. Hydrogen peroxide generation test 9.1 Experimental Methods: This test was performed using a commercial kit. The culture media of the two *Lactobacillus curvatureii* strains of this invention, after 16 hours of culture at OD=1 and in a uniform growth phase, were collected and filtered through a 0.2 μm filter membrane to obtain sterile supernatant. To ensure that the sample readings were within the detection range of the standard curve, the culture supernatant was diluted 10-fold with ultrapure water.

[0075] 9.2 Experimental Results: Table 4: Hydrogen peroxide production As shown in Table 4, the experiment showed that both strains had a high ability to produce hydrogen peroxide, with 34381 producing 1.26 mg / L and 34382 producing 1.95 mg / L.

[0076] 10. Research on aerobic environment acclimatization technology 10.1 Experimental Methods: The growth capacity of each strain under aerobic conditions was evaluated. In short, the two *Lactobacillus curvature* strains of this invention were streaked onto MRS agar plates containing L-cysteine, and all operations were performed in an anaerobic environment. Subsequently, the strains were transferred to MRS plates without L-cysteine ​​and removed from the anaerobic incubator, and cultured for more than 50 generations under continuous aerobic exposure to obtain aerobic-acclimated strains 34381 and 34282. After two passages under anaerobic and aerobic conditions, all strains were monitored using a Spectromax microplate reader. The OD value of each bacterial suspension was adjusted to 1.0, and aerobic culture was performed in a spectrophotometer. Two experimental groups were set up: one with and one without 0.5% L-cysteine. Monitoring was conducted continuously for 24-48 hours to observe the strains' resistance to oxidative stress. The results are shown in the figure.

[0077] 10.2 Experimental Results: like Figure 14 A- Figure 14 B and Figure 15 A- Figure 15 As shown in Figure B, the experimental results indicate that the growth rate of the two Lactobacillus curvature strains, 34381 and 34282, after aerobic acclimation did not decrease significantly. This demonstrates that the Lactobacillus curvature strains 34381 and 34282 of this invention possess stronger resistance to oxidative stress.

[0078] 11. Safety evaluation of 3D vaginal epithelial organoid models This invention utilizes the EpiVaginal™ (VEC-100) tissue model to assess vaginal irritation, specifically comprising the following steps: First, 5.0 mL of preheated VEC-100-MM medium is added to each well of a 12-well plate, and the EpiVaginal tissue sample is placed in the plate and incubated overnight at 37°C in a CO2 incubator; the next day, after changing the medium, 60 μL of the test bacterial suspension (such as OD) is applied to the tissue surface. 600 =0.1, approximately 10 8 The tissue was incubated for 4 hours with either a CFU / mL solution, a negative control (PBS), or a positive control (1% Triton X-100). Subsequently, the tissue was rinsed 15 times with DPBS and completely immersed in 150 mL of DPBS three times to remove residual bacteria. Next, the tissue was transferred to a 24-well plate containing 300 μL of MTT solution (1 mg / mL) and incubated in a CO2 incubator for 3 hours. After the reaction, the tissue was washed with DPBS, transferred to a new plate, and 2 mL of isopropanol extraction buffer was added to each well. The plate was incubated overnight at room temperature in the dark to extract formazan. Finally, the OD of the extract was measured in a 96-well plate. 570 nm, and relative cell viability (%) was calculated using a negative control as a baseline. This method can be used to evaluate the potential irritant effects of different bacterial strains on vaginal tissue.

[0079] like Figure 16 As shown, experiments indicate that the two strains of *Lactobacillus curvature* of this invention and the commercially available products, at the concentrations used, had no significant stimulating effect on the 3D organoid model of vaginal epithelium, and the cell survival rate was not significantly different from that of the negative control.

[0080] Obviously, the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the invention. Therefore, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention also intends to include these modifications and variations.

Claims

1. A multifunctional vaginal curly lactobacillus, characterized in that: The curly lactobacillus ( Lactobacillus crispatus MCOlact00102 was deposited on April 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34381.

2. A multifunctional vaginal curly lactobacillus, characterized in that: The curly lactobacillus ( Lactobacillus crispatus MCOlact00126 was deposited on April 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34382.

3. A composition, characterized in that: It contains live bacteria, inactivated bacteria, fermentation broth, or cell-free supernatant of the multifunctional vaginal curly lactobacillus as described in claim 1 or claim 2.

4. The use of the multifunctional vaginal curly lactobacillus of claim 1 or claim 2, or the composition of claim 3, in the preparation of health products.

5. The application according to claim 4, characterized in that: The health product can inhibit pathogenic bacteria, eliminate the biofilm of pathogenic bacteria, and / or reduce the load of pathogenic bacteria. Preferably, the health product can prevent or treat bacterial vaginosis.

6. The application according to claim 5, characterized in that: The pathogens include Gardnerella vaginalis, Group B Streptococcus, Escherichia coli, or Candida albicans.

7. The application according to claim 4, characterized in that: The product is a drug, health product, functional food, food supplement, food for special medical purposes, medical device or hygiene product. Preferably, the hygiene product includes sanitary wipes, sanitary napkins, panty liners, sanitary tampons, sanitary cotton, vaginal wash, and feminine antibacterial / bacteriostatic wash.

8. The application according to claim 7, characterized in that: The product also contains pharmaceutically, health-promoting, or food-grade carriers.

9. The application according to claim 7, characterized in that: The dosage forms of the products include pills, tablets, lozenges, lyophilized powders, granules, capsules, aqueous solutions, alcoholic solutions, oil solutions, syrups, emulsions, suspensions, suppositories, solutions for injection or infusion, ointments, gels, tinctures, creams, patches, lotions, sprays, aerosols, powder sprays, effervescent tablets, transdermal therapy systems, microcapsules, or implants.

10. The application according to claim 4, characterized in that: The product also contains a second component, which includes probiotics, postbiotics, prebiotics, antimicrobial agents, or immunomodulators.

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