Lactobacillus crispatus lcr5-6 and use thereof

Abstract

The present application relates to the technical field of microorganisms, in particular to a lactobacillus crispatus Lcr5-6 and application thereof, the lactobacillus crispatus Lcr5-6 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.38486;The adhesion rate of the lactobacillus crispatus Lcr5-6 to Hela cells is as high as 61.11%, which is significantly higher than that of the control strain lactobacillus delbrueckii DM8909 (12.13%), has excellent vaginal epithelial cell adhesion and colonization ability, and can be long-term colonization in the vagina to play a probiotic role.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to Lactobacillus curvatureis Lcr5-6 and its applications. Background Technology

[0002] Bacterial vaginosis (BV) is a common clinical syndrome caused by vaginal microecological dysbiosis. It is characterized by a significant reduction or absence of beneficial lactobacilli producing hydrogen peroxide, while various anaerobic bacteria (such as Molecularcuroides and Prevotella) and facultative anaerobic bacteria (such as Gardnerella vaginalis and Atobacillus vaginalis) proliferate abnormally, forming a mixed biofilm infection. Epidemiological data show that BV has a high prevalence among women of reproductive age, posing a continuing burden on women's reproductive health worldwide. More importantly, BV is not merely a local inflammation; its presence disrupts the integrity of the vaginal mucosal barrier, significantly increasing the risk of pelvic inflammatory disease, tubal infertility, and increasing susceptibility to sexually transmitted pathogens such as human papillomavirus (HPV) and human immunodeficiency virus (HIV). For pregnant women, BV is a clearly defined independent risk factor for adverse pregnancy outcomes such as preterm birth, miscarriage, and low birth weight infants.

[0003] In the vaginal microecology of healthy women, *Lactobacillus curvatureii* (… Lactobacillus crispatus Lactobacillus curvatureii is one of the most common and dominant bacteria in the vagina. It maintains the acidic environment of the vagina by producing lactic acid, secretes hydrogen peroxide to inhibit the proliferation of pathogenic bacteria, and forms a biofilm barrier on the surface of vaginal epithelial cells with its strong adhesion ability, preventing the colonization and invasion of pathogens. However, while antibiotic treatment for vaginal vaginitis (BV) inhibits pathogenic bacteria, it also non-selectively kills beneficial bacteria such as Lactobacillus curvatureii, disrupting the vaginal microecological balance and leading to a persistently high recurrence rate. Therefore, restoring the vaginal microecology through exogenous supplementation with Lactobacillus curvatureii strains possessing excellent probiotic properties has become an important direction in BV treatment.

[0004] Currently, the variety of vaginal probiotic products on the market is limited, and the adhesion and colonization capabilities and antibacterial activity of existing strains still need to be improved. There is an urgent need to discover new strains of Lactobacillus curvature with stronger probiotic properties. Therefore, developing a new strain of Lactobacillus curvature with high adhesion and colonization capabilities, broad-spectrum antibacterial activity, and excellent acid and hydrogen peroxide production capabilities is of great significance for the prevention and treatment of bacterial vaginosis. Summary of the Invention

[0005] This invention provides a Lactobacillus curvature Lcr5-6 strain and its application, which solves the problems of insufficient adhesion and colonization ability, limited antibacterial activity, and single function of vaginal probiotic strains in the prior art.

[0006] The technical solution adopted is as follows:

[0007] This invention provides a curly Lactobacillus ( Lactobacillus crispatus Lcr5-6, the Lactobacillus curvature Lcr5-6, was deposited on April 29, 2026 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38486, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0008] As a further technical solution, the adhesion rate of the Lactobacillus curvature Lcr5-6 to HeLa cells is ≥60%.

[0009] The present invention also proposes a fermentation supernatant of Lactobacillus curvature Lcr5-6 for inhibiting Gardnerella vaginalis, which is obtained by centrifugation after fermentation culture of Lactobacillus curvature Lcr5-6. The fermentation supernatant contains 0.12-0.14 mM of D-lactic acid and 0.07-0.08 mM of hydrogen peroxide.

[0010] This invention also proposes a method for preparing freeze-dried Lactobacillus curvaturei Lcr5-6 bacterial powder, comprising the following steps:

[0011] (1) The Lactobacillus curlyis Lcr5-6 described in claim 1 or 2 is inoculated into MRS liquid culture medium and cultured anaerobically at 37°C for 18-24 h to obtain activated strains;

[0012] (2) The activated strain from step (1) was inoculated into the optimized culture medium and cultured anaerobically at 37°C for 24 hours to obtain the seed culture.

[0013] (3) The seed liquid from step (2) was inoculated into the fermentation medium at an inoculation rate of 5%, and fermentation was carried out at a temperature of 38℃, pH 6.5 and a stirring speed of 150 rpm to obtain a high-density fermentation liquid.

[0014] (4) Centrifuge the fermentation broth from step (3) to concentrate the bacterial cells, add a protectant and freeze dry to obtain freeze-dried bacterial powder.

[0015] As a further technical solution, the protective agent in step (4) includes mannitol, trehalose, and sodium ascorbate, and the mass ratio of the bacterial sludge to the protective agent is 1:0.8. The viable count of Lactobacillus criniformis Lcr5-6 in the freeze-dried bacterial powder is ≥1×10⁻⁶. 9 CFU / g.

[0016] This invention also proposes the application of the aforementioned Lactobacillus curvature Lcr5-6 in the preparation of a vaginal microecological balance regulator.

[0017] The present invention also proposes the use of the fermentation supernatant in the preparation of antibacterial products for inhibiting Gardnerella vaginalis and / or Staphylococcus aureus and / or Escherichia coli.

[0018] This invention also proposes a screening method for Lactobacillus curvature Lcr5-6, comprising the following steps: isolating lactic acid bacteria from vaginal secretions of healthy women, screening for antibacterial activity using Gardnerella vaginalis as an indicator bacterium, identifying strains with antibacterial activity by 16S rDNA sequencing, and selecting strains identified as Lactobacillus curvature and with an adhesion rate of ≥60% to HeLa cells as target strains.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] (1) The Lactobacillus curvature Lcr5-6 of this invention was isolated from the vagina of healthy Chinese women. It is a dominant vaginal strain in the Chinese population and has good adaptability to the regulation of vaginal microecology in Chinese women. Whole genome sequencing analysis showed that the strain has no virulence factor genes and has high safety.

[0021] (2) The Lactobacillus criniformis Lcr5-6 of the present invention has a significant in vitro inhibitory effect on Gardnerella vaginalis, Escherichia coli and Staphylococcus aureus, with a broad antibacterial spectrum; the D-lactic acid content in the fermentation supernatant reaches 0.12-0.14mM and the hydrogen peroxide content reaches 0.07-0.08mM, with excellent acid production and hydrogen peroxide production capabilities, which can effectively maintain the acidic environment of the vagina and inhibit the growth of pathogenic bacteria.

[0022] (3) The adhesion rate of the Lactobacillus curvature Lcr5-6 of this invention to HeLa cells is as high as 61.11%, which is significantly higher than that of the control strain Lactobacillus delbrueckii DM8909 (12.13%). It has excellent vaginal epithelial cell adhesion and colonization ability and can colonize in the vagina for a long time to play a probiotic role.

[0023] (4) The method for preparing freeze-dried Lactobacillus criniformis Lcr5-6 bacterial powder provided by the present invention has a stable process, and the number of viable bacteria after freeze-drying can reach 1×10⁻⁶. 9 With a CFU / g or higher, the product quality is controllable and suitable for industrial production. This strain can be used alone or as a probiotic agent to prepare vaginal microecological regulators, showing good application prospects. Attached Figure Description

[0024] Figure 1 This is a colony morphology diagram of Lactobacillus curvature Lcr5-6 of the present invention;

[0025] Figure 2 A schematic diagram of the structure of Lactobacillus curvature Lcr5-6 under Gram staining microscopic examination;

[0026] Figure 3 The first set of experimental diagrams shows the inhibition of Gardnerella vaginalis by Lactobacillus curvature Lcr5-6 and Lactobacillus delbrueckii DM8909.

[0027] Figure 4The second set of experimental diagrams shows the inhibition of Gardnerella vaginalis by Lactobacillus curvature Lcr5-6 and Lactobacillus delbrueckii DM8909.

[0028] Figure 5 This is the third set of experimental diagrams showing the inhibition of Gardnerella vaginalis by Lactobacillus curvature Lcr5-6 and Lactobacillus delbrueckii DM8909.

[0029] Figure 6 The first set of experimental diagrams shows the inhibition of Escherichia coli by Lactobacillus curvature Lcr5-6.

[0030] Figure 7 This is the second set of experimental diagrams showing the inhibition of Escherichia coli by Lactobacillus curvature Lcr5-6.

[0031] Figure 8 This is the third set of experimental diagrams showing the inhibition of Escherichia coli by Lactobacillus curvature Lcr5-6;

[0032] Figure 9 The first set of experimental diagrams shows the inhibition of Staphylococcus aureus by Lactobacillus curvature Lcr5-6.

[0033] Figure 10 The second set of experimental diagrams shows the inhibition of Staphylococcus aureus by Lactobacillus curvature Lcr5-6.

[0034] Figure 11 This is the third set of experimental diagrams showing the inhibition of Staphylococcus aureus by Lactobacillus curvature Lcr5-6. Detailed Implementation

[0035] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0036] Example 1

[0037] 1. Isolation and purification of Lactobacillus curvature Lcr5-6

[0038] Vaginal secretions from healthy women were placed in centrifuge tubes containing 3 mL of liquid MRS culture medium and vortexed to mix. The mixture was then serially diluted to 10⁻⁶. -6 From 10 respectively -4 10 -5 10 -6100 μL of each of the three gradients was evenly spread onto MRS solid medium and incubated in an anaerobic environment at 37°C for 48 hours. Colony morphology was observed and recorded. Single colonies exhibiting typical lactic acid bacteria characteristics were picked up with an inoculation loop and streaked onto MRS solid medium, then anaerobically incubated at 37°C for 48 hours to obtain pure colonies. The pure colonies obtained from the plates were inoculated into MRS liquid medium and anaerobically incubated at 37°C for 18 hours. Then, the pure bacterial strain was mixed with a 30%-50% (v / v) glycerol solution in equal proportions and stored at -80°C. 3 mL of liquid MRS medium was added to each sample, vortexed, and 100 μL was taken out. The remaining sample was incubated overnight in an anaerobic environment for enrichment and preservation. The remaining 100 μL was diluted with physiological saline and spread onto MRS solid medium, incubated anaerobically at 37°C for 48 hours.

[0039] 2. Screening and identification of Lactobacillus curvature Lcr5-6

[0040] Finally, multiple bacterial strains were isolated from the vaginas of healthy women. The isolated strains were then tested for their inhibitory effects on *Escherichia coli* ATCC 25922, *Staphylococcus aureus* ATCC 29213, and *Gardnerella vaginalis* ATCC 14018. *Lactobacillus curvatureii* Lcr5-6 was found to have the best inhibitory effect. The bacterial suspension was amplified using the universal 16S rDNA primers 27F and 1522R, followed by sequencing to identify the strain species. The primer sequences for the universal 16S rDNA primers 27F and 1522R are as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1522R: 5'-AAGGAGGTGATCCAGCCGCA-3'; PCR system components: 1 μL bacterial suspension, 1 μL 27F, 1 μL 1522R, 7 μL sterile water, 10 μL 2× ApexHF PCR Master Mix (PCR premix).

[0041] The PCR program was as follows: pre-denaturation at 95℃ for 5 min, denaturation at 98℃ for 10 s, annealing at 55℃ for 10 s, extension at 72℃ for 10 s, 35 cycles; final extension at 72℃ for 5 min. The amplified PCR product was removed, and 3 μL of PCR product was added to an agarose gel. The electrophoresis apparatus voltage was set to 140V, and the gel was run for 30 min. After the gel was removed, the DNA bands were observed using a gel imaging system, and the target fragment length was determined to be 1500 bp.

[0042] 16S rDNA sequencing: The validated PCR products were sent to Shanghai Bioengineering Technology Co., Ltd. for sequencing. The obtained 16S rDNA sequence was BLASTed on NCBI and compared with the sequencing information of known strains for homology to identify the strains.

[0043] The selected strain Lcr5-6 was identified as *Lactobacillus curvatureensis* by morphological and 16S rDNA analysis, and its 16S rDNA sequence is shown in SEQ ID NO.1. Details are as follows:

[0044]

[0045] The above-mentioned strains were deposited in a cultural heritage institution. Information is as follows:

[0046] Lactobacillus curvature Lcr5-6 strain was deposited on April 29, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38486. The depository institution is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0047] 3. Biochemical identification experiment of Lactobacillus curvature Lcr5-6

[0048] (1) Experimental preparation: Take out a biochemical identification strip from the packaging box, open the cap, and make a hole with a punch or directly tear off the membrane. If it is contaminated or discolored, it cannot be used and a new strip should be taken. Note: Wipe the punch with 75% alcohol before use.

[0049] (2) Inoculation method: Pick the purified culture, make a bacterial suspension, and inoculate it into the following culture media: aescin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, and lactose.

[0050] Bacterial suspension method: Take a test tube containing 2 mL of sterile physiological saline, pick a single Lactobacillus curvature Lcr5-6 from the purified culture plate with an inoculation needle, grind it carefully in sterile physiological saline to prepare a homogeneous bacterial suspension, and add 100 μL of bacterial suspension to each well.

[0051] (3) After inoculation, mark the inoculation, cover it, place it in the tray, and incubate at 36±1℃. The results are shown in Table 1.

[0052] Table 1: Biochemical identification results of Lactobacillus curvature Lcr5-6

[0053]

[0054] (4) The results showed that in this biochemical identification, Lactobacillus curvature Lcr5-6 was negative for the biochemical reactions corresponding to aescin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin and lactose.

[0055] Example 2

[0056] Strain genome sequencing

[0057] Lactobacillus curvature ( Lactobacillus crispatus The Lcr5-6 strain was inoculated into 5 mL of anaerobic MRS broth (sealed with liquid paraffin), cultured to the late logarithmic growth stage, and the whole genome DNA of the strain was extracted for next-generation whole genome sequencing.

[0058] Based on the genome assembly and annotation results, *Lactobacillus curvature* (… Lactobacillus crispatus The total length of the genome sequence was 1,770,482 bp, with a GC content of 37.09%, containing 1,798 predicted protein-coding sequences (CDSs) and 61 structural RNA genes (59 tRNAs and 2 rRNAs). After assembly and annotation, the protein sequences were entered into the KEGG database for gene function analysis, genome mapping analysis, and the Virulence Factor Databases (VFDB) and the Comprehensive Antibiotic Resistance Database (CARD) for virulence factor and drug resistance gene analysis, respectively.

[0059] COG database functional annotations show that most annotations are related to amino acid transport and metabolism (188 genes), followed by carbohydrate transport and metabolism (164 genes), translation, ribosome structure and biosynthesis (127 genes), transcription (103 genes), and nucleotide transport and metabolism (93 genes). KEGG annotation results show that 801 genes are enriched in 32 secondary pathways under the metabolic category, with carbohydrate metabolism (184 genes) indicating that this *Lactobacillus curvature* possesses extremely active and diverse carbohydrate breakdown and conversion capabilities, providing the core metabolic basis for its high lactic acid production. Furthermore, related pathways such as amino acid metabolism (72 genes) and energy metabolism (71 genes) are also highly developed.

[0060] Annotation analysis of the VFDB database revealed that *Lactobacillus curlis* (…) Lactobacillus crispatus A total of 161 genes associated with potential virulence factors were predicted in strain Lcr5-6. However, the vast majority of the predicted genes had low sequence similarity to known virulence factors in the database. Only a few genes had a similarity of more than 50%, and only 4 genes had a similarity of more than 70%, with the highest similarity not exceeding 80%.

[0061] A total of 108 genes encoding carbohydrate CAZy family protein domains were identified. These included 38 glycoside hydrolases (GHs), 27 glycosyltransferases (GTs), 10 carbohydrate binding modules (CBMs), 21 carbohydrate esterases (CEs), 9 accessory activities (AAs), and 3 polysaccharide lyases (PLs).

[0062] After the whole genome sequence of Lactobacillus curvature Lcr5-6 was uploaded to JSpeciesWS, it was compared with the whole genome sequences of all the same species that could be linked to. The average nucleotide identity (ANI) ratios are shown in Table 2, which proves that it is a new Lactobacillus strain.

[0063] Table 2: Comparison of genome-wide ANI between Lactobacillus curvature Lcr5-6 and different Lactobacillus curvature strains

[0064]

[0065] Example 3

[0066] I. Antibacterial ability test

[0067] 1. Comparative experiment on the ability of Lactobacillus curvature Lcr5-6 and Lactobacillus delbrueckii DM8909 to inhibit Gardnerella vaginalis.

[0068] (1) Preparation of instruments and reagents

[0069] Freeze dryer: Shin-Zhi;

[0070] Petri dish: PETRI DISHES 90mm*15mm;

[0071] Centrifuge: CT15RE;

[0072] Spectrophotometer: UV-1100 (serial number UEB 1812032);

[0073] MRS medium: Haibo Biotechnology (No. HB0384-1);

[0074] BHI medium: Daily Water Biology (Catalogue No. 10805).

[0075] (2) Preparation of Lactobacillus supernatant

[0076] Previously preserved Lactobacillus curvature Lcr5-6 and Lactobacillus delbrueckii DM8909 glycerol tubes were inoculated into MRS liquid medium at a 1% inoculation rate and cultured in an anaerobic incubator at 37°C for 22 h. Then, the inoculum was transferred to new MRS liquid medium at a 1% inoculation rate and cultured in an anaerobic incubator at 37°C for another 22 h. The bacterial culture in the stable period was centrifuged at 4000 rpm for 5 min, and 2 mL of the supernatant was collected. The supernatant was then frozen at -80°C and freeze-dried. The freeze-dried powder was prepared for use and dissolved in 200 μL of sterile water before use.

[0077] (3) Preparation of vaginal Gardnerella vaginalis culture dishes

[0078] Add 10% horse serum to sterilized BHI solid medium and pour into plates. Place the plates in a clean bench and air dry. Inoculate Gardnerella vaginalis (ATCC14018) at a 2% inoculum volume into liquid BHI culture containing 10% horse serum and incubate overnight (24 h). Measure OD600 = 0.9, then dilute 2 times and add 250 μL of bacterial culture to plates. Spread evenly on the prepared BHI plates.

[0079] (4) Place the Oxford cup and add lactobacillus supernatant.

[0080] Using forceps, sterile Oxford cups were placed into Gardnerella vaginalis culture dishes. 200 μL of concentrated supernatant of *Lactobacillus curvatureii* Lcr5-6 and *Lactobacillus delbrueckii* DM8909 was added to each dish. The dishes were incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the size of the inhibition zone was recorded and photographed. Three parallel samples were used for each specimen. The results are shown in Table 3.

[0081] Table 3. The ability of Lactobacillus curvature Lcr5-6 and Lactobacillus delbrueckii DM8909 to inhibit Gardnerella vaginalis.

[0082]

[0083] (5) Experimental results

[0084] The results showed that the average inhibition zone diameter of *Lactobacillus curvaturei* Lcr5-6 against *Gardnerella vaginalis* was 1.9 cm, and the average inhibition zone diameter of *Lactobacillus delbrueckii* DM8909 against *Gardnerella vaginalis* was 2.03 cm. Both strains showed clear in vitro inhibitory activity against *Gardnerella vaginalis*, and the inhibitory activity of *Lactobacillus curvaturei* Lcr5-6 was similar to that of *Lactobacillus delbrueckii* DM8909, achieving similar in vitro inhibitory effects against *Gardnerella vaginalis*, confirming that this strain has good antagonistic activity against *Gardnerella vaginalis*.

[0085] 2. Experiment on the ability of Lactobacillus curvature Lcr5-6 to inhibit Escherichia coli and Staphylococcus aureus

[0086] (1) Preparation of instruments and reagents

[0087] Spectrophotometer: UV-1100, model number: UEB 1812032;

[0088] Escherichia coli (ATCC25922);

[0089] Staphylococcus aureus (ATCC29213);

[0090] Petri dish: PETRI DISHES 90mm*15mm;

[0091] Centrifuge: CT15RE;

[0092] Cotton swabs: medical cotton swabs;

[0093] MRS culture medium: Haibo Biotechnology product number: HB0384-1;

[0094] LB medium: Tryptone OXOID (Catalog No.: LP0042B), Yeast extract OXOID (Catalog No.: LP0021B), Sodium chloride (Shanghai test batch No.: 20241028).

[0095] (2) Preparation of Lactobacillus supernatant

[0096] The previously preserved Lactobacillus curvature Lcr5-6 glycerol tubes were inoculated into MRS liquid medium at a 1% inoculation rate and cultured in an anaerobic incubator at 37°C for 22 hours. Then, the inoculum was transferred to a new MRS liquid medium at a 1% inoculation rate and cultured in an anaerobic incubator at 37°C for another 22 hours. The bacterial culture in the stable period was then centrifuged at 4000 rpm for 5 minutes and the supernatant was collected for later use.

[0097] (3) Preparation of culture dishes for Escherichia coli and Staphylococcus aureus

[0098] Pour the sterilized LB solid medium into plates and place them in a clean bench to dry. Inoculate Escherichia coli (ATCC25922) and Staphylococcus aureus (ATCC29213) at a 1% inoculum volume into LB liquid medium and incubate overnight. Adjust the OD600 to 0.5 with sterile physiological saline, moisten with a sterile cotton swab, and spread evenly on LB plates.

[0099] (4) Place the Oxford cup and add lactobacillus supernatant.

[0100] Using tweezers, place the sterile Oxford cup into the prepared Escherichia coli and Staphylococcus aureus culture dishes, add 200 μL of the prepared Lactobacillus criniformis Lcr5-6 supernatant to each, and incubate overnight at room temperature. Once a clear inhibition zone is observed, photograph the size and record the results. The results are shown in Table 4.

[0101] Table 4: Inhibitory ability of Lactobacillus curvature Lcr5-6 against Escherichia coli and Staphylococcus aureus

[0102]

[0103] (5) Experimental results

[0104] The results showed that the average diameter of the inhibition zone of Lactobacillus curvature Lcr5-6 against Escherichia coli was 1.5 cm and the average diameter of the inhibition zone of Lactobacillus curvature Lcr5-6 against Staphylococcus aureus was 1.6 cm, indicating that the strain also has a clear in vitro inhibitory effect on these two common pathogens, further confirming that Lactobacillus curvature Lcr5-6 has a broad-spectrum in vitro inhibitory ability against pathogens.

[0105] II. Comparative Experiment on the Lactic Acid Production Capacity of Lactobacillus curvature Lcr5-6 and Lactobacillus delbrueckii DM8909

[0106] (1) Preparation of instruments and reagents

[0107] Spectrophotometer: UV-1100, model number: UEB 1812032;

[0108] Centrifuge: CT15RE;

[0109] D-Lactate Assay Kit (WST-8 method, Beyotime, Lot No. A259250902).

[0110] (2) Preparation of the reagent kit

[0111] a. Dissolve BeyoLysis™ Buffer A for Metabolic Assay and Lactate Assay Buffer, equilibrate to room temperature, mix well and set aside. Store other reagents on ice. After use, store immediately according to the conditions required by the kit.

[0112] b. Preparation of WST-8 Working Solution: Prepare an appropriate amount of working solution for each 50 μL test reaction. Mix 44 μL Lactate Assay Buffer, 2 μL Enzyme Solution, 2 μL Substrate, and 2 μL WST-8 evenly to prepare 50 μL of WST-8 working solution. Prepare an appropriate amount of WST-8 working solution according to the number of samples (including standards) to be tested; refer to the instruction manual for specific preparation methods. The prepared WST-8 working solution can be used on the same day if stored at 4ºC or in an ice bath protected from light, but it is recommended to prepare and use it immediately.

[0113] (3) Inoculate L. crimped L. 5-6 and L. delta DM8909 glycerol tubes at a 1% inoculation rate into MRS medium and culture in an anaerobic incubator at 37℃ for 22h. Then transfer to fresh MRS medium and culture in an anaerobic incubator at 37℃ for 22h. Take out the fermentation broth, centrifuge at 4000rpm for 5min and take the supernatant for later use.

[0114] (4) Standard preparation: Test according to the kit steps.

[0115] A. Setting up the D-lactic acid standard curve:

[0116] Take 1 μL of D-Lactate Standard (100 mM) and add 99 μL of BeyoLysis™ Buffer A for Metabolic Assay or Lactate Assay Buffer. Mix well to prepare 100 μL of 1 mM D-Lactate Standard. Add 0, 1, 2.5, 5, 10, 25, and 50 μL of 1 mM D-Lactate Standard to the wells of a 96-well plate, and bring the volume to 50 μL with the corresponding BeyoLysis™ Buffer A for Metabolic Assay or Lactate Assay Buffer. At this point, the D-lactic acid concentration and amount of substance in each well of the standard curve are 0, 0.02, 0.05, 0.1, 0.2, 0.5, 1 mM and 0, 1, 2.5, 5, 10, 25, 50 nmol, respectively.

[0117] B. Sample determination:

[0118] a. Transfer 50 μL of the sample prepared in step 1 to the wells of a 96-well plate. Simultaneously, set up wells containing only BeyoLysis™ Buffer A for Metabolic Assay or Lactate Assay Buffer as blank control wells. Note: To ensure values ​​are within the standard curve range, it is recommended to set multiple dilution factors for the sample. This allows for preliminary experiments to determine the approximate sample concentration. If the values ​​are not within the standard curve range, the dilution factor can be adjusted. When the sample is a lysed cell or tissue sample, BeyoLysis™ Buffer A for Metabolic Assay should be used for dilution. When the sample is blood or other samples, Lactate Assay Buffer is recommended. The sample dilution factor here is denoted as n.

[0119] b. Add 50 μL of WST-8 colorimetric working solution to each well and mix well. Incubate at 37ºC in the dark for 30 minutes. Note: If the absorbance is low, i.e. the color of the plate is light, the reaction time can be extended to 45 or 60 minutes; if the D-lactic acid content is high, i.e. the color of the plate is dark, the incubation time can be shortened to 15 or 20 minutes.

[0120] c. Measure absorbance at 450 nm.

[0121] d. Subtract the signal value of the zero-concentration standard well from the signal value of each well in the standard curve. Establish the standard curve and calculate the concentration (A) of D-lactic acid in the sample. The signal value of the sample well should be subtracted from the signal value of the blank control well. If the background control well signal of the sample is relatively high, the sample signal value needs to be subtracted from the sample background control value. A D-lactic acid standard curve can be referenced. Figure 2 A good linear relationship is observed within the concentration range of 0.02-1 mM. The formula for calculating D-lactic acid concentration is as follows:

[0122] C(mM) = A × n

[0123] Note 1: A is the D-lactic acid concentration (mM) of the diluted sample determined according to the standard curve in step 4d; n is the sample dilution factor in step a.

[0124] Note 2: The calculated D-lactic acid concentration includes the molar concentrations of D-lactic acid and D-lactic acid ions, and can also be understood as including the molar concentrations of D-lactic acid and D-lactic acid salt. For ease of description only, the above description focuses on D-lactic acid. If necessary, the mass concentration of D-lactic acid ions in the sample (μg / mL) can be calculated based on the molecular weight of D-lactic acid ions (89.07) = C × 0.08907.

[0125] (5) The results of lactic acid production capacity of Lactobacillus curvature Lcr5-6 and Lactobacillus delbrueckii DM8909 are shown in Table 5.

[0126] Table 5:

[0127]

[0128] (6) Experimental results

[0129] The results showed that the D-lactic acid concentrations in the fermentation broths of *Lactobacillus curvaturei* Lcr5-6 and *Lactobacillus delbrueckii* DM8909 were similar, and there was no significant difference in their total D-lactic acid production capacity. *Lactobacillus curvaturei* Lcr5-6 even showed better stability in producing D-lactic acid than *Lactobacillus delbrueckii* DM8909, demonstrating good acid-producing characteristics.

[0130] III. Comparative Experiment on the Hydrogen Peroxide Production Capacity of Lactobacillus curvature Lcr5-6 and Lactobacillus delbrueckii DM8909

[0131] (1) Preparation of instruments and reagents

[0132] Spectrophotometer: UV-1100, model number: UEB 1812032;

[0133] Centrifuge: CT15RE;

[0134] Hydrogen peroxide detection kit (titanium sulfate colorimetric method, Yuan Ye, LOT KR374244).

[0135] (2) Standard product configuration

[0136] A. Preparation of 10mM H2O2 standard solution: Since hydrogen peroxide is not very stable, the actual concentration of hydrogen peroxide needs to be determined before use. The H2O2 base solution provided by this kit has an H2O2 concentration of approximately 1M. Dilute it 100 times with distilled water to make the H2O2 concentration approximately 10mM. Zero the instrument with distilled water and measure the A240 using a spectrophotometer. Calculate the actual concentration of H2O2 in the H2O2 base solution using the formula H2O2 concentration (mM) = 22.94 × A240. Then dilute the H2O2 base solution with acetone to prepare a 10mM H2O2-acetone standard solution (generally, the A240 of a freshly prepared 10mM H2O2 base solution is 0.4~0.45, and the A240 after 3 months is 0.35~0.42).

[0137] B. Preparation of titanium sulfate solution: Add 0.3g of titanium sulfate to 6mL of distilled water to obtain a 5% titanium sulfate solution, and store at 4℃.

[0138] (3) Inoculate L. crimped Lcr5-6 and L. delta DM8909 with 1% glycerol tubes into MRS medium and culture in an anaerobic incubator at 37℃ for 22h. Transfer to fresh MRS medium and culture in an anaerobic incubator at 37℃ for 22h. Centrifuge at 4000rpm for 5min and collect the supernatant for later use.

[0139] (4) Detect hydrogen peroxide concentration according to the kit instructions.

[0140] A. H2O2 addition: Set up blank tubes, standard tubes, and test tubes according to the table below. Add the solution sequentially, taking care to avoid generating bubbles. If the H2O2 concentration in the sample is too high, reduce the sample volume or dilute it appropriately before measurement. Parallel tubes are recommended for sample detection. Use a hydrogen peroxide detection kit (titanium sulfate colorimetric method) for the determination. Specific sample addition methods are shown in Table 6.

[0141] Table 6:

[0142]

[0143] B. H2O2 determination: Mix well, let stand at room temperature for 5 min, centrifuge at 12000g for 15 min, discard the supernatant, keep the precipitate, and if necessary, add pre-cooled acetone to wash the precipitate repeatedly. Add 2 mL of acidic base solution to the precipitate in each tube, shake to completely dissolve the precipitate; the optical path of the cuvette is 1 cm, the blank tube is zeroed, and the absorbance of each standard tube and the test tube at 412 nm is measured by spectrophotometer. If there is no spectrophotometer, an enzyme-linked immunosorbent assay (ELISA) reader can also be used for detection.

[0144] Calculation: Plot a standard curve with a series of acetone-H2O2 standards (0.3, 0.5, 0.8, 1, 3, 5, 8 mM) as the x-axis and the corresponding absorbance as the y-axis, and obtain the regression equation; substitute the absorbance of the test tube into the regression equation to obtain the concentration of H2O2 in the sample to be tested.

[0145] H2O2 in tissue sample (mmol / g) = (C0XVTXN) / m

[0146] Liquid sample H2O 2 mmol / L) = C0 X N

[0147] In the formula: C0 = H2O2 concentration (mM) obtained from the absorbance of the sample on the standard curve.

[0148] VT = Total volume of the sample to be tested (L)

[0149] m = sample mass (g)

[0150] N = Sample dilution factor

[0151] (5) The results of hydrogen peroxide concentration determination of fermentation supernatant of Lactobacillus curvature Lcr5-6 and Lactobacillus delbrueckii DM8909 are shown in Table 7.

[0152] Table 7:

[0153]

[0154] (6) Experimental results

[0155] The results showed that the hydrogen peroxide concentration in the fermentation supernatant of Lactobacillus curvature Lcr5-6 and Lactobacillus delbrueckii DM8909 was similar, and there was no significant difference in the total hydrogen peroxide production capacity. The hydrogen peroxide production stability of Lactobacillus curvature Lcr5-6 was similar to that of Lactobacillus delbrueckii DM8909, and it also had a stable hydrogen peroxide production capacity.

[0156] IV. Comparative Experiment on the Adhesion Ability of Lactobacillus curvature Lcr5-6 and Lactobacillus delbrueckii DM8909 to HeLa Cells

[0157] (1) Preparation of instruments and reagents

[0158] Cell incubator: Model: RC03000T-5-VBC;

[0159] Cell culture medium: DMEM gibco batch number: 6125387, serum excell;

[0160] Cell culture dish: NEST 100mm*20mm;

[0161] PBS: Seville batch number: 251080014403;

[0162] 0.1% Triton: Beyotime Triton X-100 Solution, Lot No.: 071423240402;

[0163] MRS culture medium: Haibo Biotechnology product number: HB0384-1;

[0164] Hela cell line;

[0165] HeLa culture medium: DMEM + 10% FBS + 1% penicillin-streptomycin;

[0166] Culture medium for Lactobacillus: MRS broth (anaerobic, 37 ℃).

[0167] (2) Cell resuscitation

[0168] A. Preparation: Preheat the water bath to 37°C, and prepare the complete culture medium (DMEM + 10% FBS + 1% penicillin-streptomycin), centrifuge tubes, and culture dishes.

[0169] B. Rapid thawing: Remove the cryovial from the -80℃ freezer and immediately place it in a 37℃ water bath. Gently shake it to thaw completely within 1-2 minutes.

[0170] C. Transfer and dilution: Wipe the surface of the cryovial with an alcohol swab, and transfer the cell suspension to a centrifuge tube containing 8 mL of complete culture medium in a clean bench, and mix gently.

[0171] D. Centrifugation: Centrifuge at 1000 rpm for 3 minutes and discard the supernatant.

[0172] E. Resuspension: Resuspend the cells in fresh complete culture medium, inoculate them into a culture dish, and add 8 mL of sufficient culture medium.

[0173] F. Culture and observation: Place in a 37℃, 5% CO2 incubator, change the culture medium the next day, and observe cell adhesion and growth.

[0174] (3) Cell passage

[0175] Passage the cells when the cell density reaches 80%-90%. Preheat PBS, trypsin (containing EDTA), and complete culture medium.

[0176] A. Washing: Discard the old culture medium, add PBS and gently wash the cells 1-2 times, then discard the PBS.

[0177] B. Digestion: Add 1 mL of trypsin to cover the cell layer and digest at 37°C for 1-3 minutes (adjust the time as needed). Stop digestion when the cells become rounded and the gaps between them increase under a microscope.

[0178] C. Pipe centrifugation: Gently pipette the digested cells to detach them into a 1.5mL centrifuge tube, centrifuge at 1000rpm for 3 minutes, discard the supernatant, add it to a culture dish, and add 8mL of sufficient culture medium.

[0179] (4) Adhesion test

[0180] The cells have been passaged 2-3 times and are in good condition, making them suitable for adhesion testing.

[0181] A. Add 1.5 × 10⁻⁶ ppm to each well of a 24-well plate. 5 Cells / 500μL of complete culture medium were incubated at 37℃ and 5% CO2 for 24 hours to form a monolayer. One hour before the experiment, the cells were washed three times with PBS and then replaced with antibiotic-free DMEM to reduce the impact of antibiotics on the bacteria.

[0182] B. Wash the overnight cultured Lactobacillus with PBS, adjust OD600 to 0.5 with DMEM medium, and the bacterial concentration is approximately 1×10⁻⁶. 8 CFU / mL.

[0183] C. Adhesion co-incubation

[0184] Add 500 μL of bacterial suspension (OD600=0.5) to each well of the HeLa monolayer and dilute it 100 times.

[0185] Incubate at 37℃ with 5% CO2 for 2 hours.

[0186] Wash to remove unadhered bacteria, then rinse 4 times with PBS.

[0187] D. Recovery and counting of adhesive bacteria

[0188] (5) Colony counting method (quantitative)

[0189] Add 500 μL of 0.1% Triton X-100-PBS to each well and incubate for 5 min at room temperature. Mix thoroughly by pipetting, take 30 μL of the lysis buffer, serially dilute 10-fold, and spot onto MRS agar. Incubate anaerobically at 37℃ for 48 h, and count CFU.

[0190] Calculation: Adhesion rate (%) = (N1 - N2 / N0) × 100

[0191] N1: Viable bacterial count in lysis buffer (CFU / mL) (bacterial solution with HeLa monolayer pores added)

[0192] N2: Viable bacterial count in lysis buffer (CFU / mL) (bacterial solution added to cell-free blank wells)

[0193] N0: Initial number of viable bacteria added (CFU / mL).

[0194] (6) The results of the assay of the adhesion ability of Lactobacillus curvature Lcr5-6 and Lactobacillus delbrueckii DM8909 to HeLa cells are shown in Table 8.

[0195] Table 8:

[0196]

[0197] (7) The results showed that the adhesion rate of Lactobacillus curvature Lcr5-6 was significantly higher than that of Lactobacillus delbrueckii DM8909, with an average adhesion rate of 61.11%, which was much higher than the 12.13% of Lactobacillus delbrueckii DM8909. Moreover, the coefficient of variation was lower, indicating that Lactobacillus curvature Lcr5-6 had a stronger adhesion ability to HeLa cells and better adhesion stability, and had excellent adhesion and colonization characteristics.

[0198] Example 4

[0199] Antimicrobial susceptibility testing of Lactobacillus curvature Lcr5-6 to different antibiotics

[0200] (1) Preparation of instruments and reagents

[0201] Spectrophotometer: UV-1100, model number: UEB 1812032;

[0202] MRS liquid culture medium (Qingdao Haibo): Sterilize at 118℃ for 15 min.

[0203] Antibiotic standards: Ampicillin, Erythromycin, Gentamicin, Kanamycin, Tetracycline, Vancomycin, Clindamycin, Levofloxacin, Metronidazole.

[0204] (2) Preparation of the mother liquor of the test sample

[0205] Ampicillin (dissolved in DMSO), erythromycin (dissolved in anhydrous ethanol), tetracycline (dissolved in DMSO), levofloxacin (dissolved in DMSO), metronidazole (dissolved in DMSO), and the others are all soluble in water. The stock solution concentration is 2.56 mg / mL.

[0206] (3) Preparation of concentration gradient of test sample

[0207] Two-fold dilution of the test sample solution: Aseptically, take a sterile 96-well plate, add 150 μL of the stock solution obtained above to well 1, and add 150 μL of water to wells 1-12 respectively. Add 150 μL from well 1 to well 2 and mix well. Add 150 μL from well 2 to well 3, and repeat this process up to well 11. Finally, add only 150 μL of water to one well as a growth control. This yields a series of gradient concentrations of the test sample solution: 1280, 640, 320, 160, 80, 40, 20, 10, 5, 2.5, 1.25, and 0 μg / mL.

[0208] (4) Determination of MIC

[0209] A. MIC determination

[0210] Lactobacillus curvature Lcr5-6 strain was inoculated into 5 mL of MRS liquid medium and anaerobic cultured at 37 ℃ for 22 h. The bacterial concentration was then diluted with MRS to obtain approximately 10⁻⁶. 6 CFU / mL bacterial suspension. After OD600=0.5, 1% of MRS medium was added. Take a sterile 96-well culture plate, add 10 μL of the test sample dilution prepared according to (3) at different gradient concentrations to each well, and then add 90 μL of each bacterial suspension. After mixing, place in an anaerobic incubator at 37 ℃ and incubate for 24 h to interpret the results. The test concentrations were 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0 μg / mL.

[0211] B. Result Interpretation

[0212] After 24 hours of incubation, the OD600 was measured. An OD value greater than 0.1 was considered as indicating the growth of the test bacteria.

[0213] (5) The results of the drug sensitivity test of Lactobacillus curvature Lcr5-6 to different antibiotics are shown in Table 9.

[0214] Table 9:

[0215]

[0216] (6) Experimental results

[0217] The results showed that *Lactobacillus curvature* Lcr5-6 exhibited natural resistance to kanamycin and metronidazole; it was highly sensitive to ampicillin, erythromycin, tetracycline, vancomycin, and clindamycin, with the MIC values ​​of erythromycin and clindamycin not exceeding 0.125 μg / mL; the MIC values ​​of gentamicin and levofloxacin were both 32 μg / mL. The overall drug resistance characteristics met the requirements for drug resistance characteristics of lactic acid bacteria as probiotics and complied with the relevant standards for the safe use of probiotics.

[0218] Example 5

[0219] Method for preparing mycelium powder:

[0220] (1) Activation of bacterial strain: Lactobacillus curvature Lcr5-6, which was frozen at -80℃, was inoculated into MRS liquid medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.) and cultured anaerobically at 37℃ for 18-24h. This process was repeated once to obtain activated bacterial strain.

[0221] (2) Optimize the preparation of culture medium

[0222] Prepare the optimized culture medium according to the specified proportions, mix thoroughly, adjust the pH to 6.8, and sterilize at 115℃ for 30 minutes.

[0223] The modified MRS liquid culture medium consists of the following components: 20g peptone, 10g yeast extract, 22g glucose, 6g sodium acetate, 0.5g magnesium sulfate, 0.15g manganese sulfate, and 1L purified water.

[0224] (3) Preparation of seed liquid

[0225] Take the activated strain from step (1) and inoculate it into the optimized culture medium prepared in step (2), and culture it anaerobically at 37°C for 24 hours.

[0226] (4) Inoculation and fermentation

[0227] The seed culture from step (3) was inoculated at 5% into the optimized culture medium prepared in step (2), with the temperature controlled at 38°C, the pH at 6.5, and the stirring speed at 150 rpm. The pH was controlled by adding neutralizing agents, namely NaCO3 and NaOH, during the above process.

[0228] (5) Termination of fermentation

[0229] Fermentation was terminated when acid production by the bacteria ceased, resulting in a high-density fermentation broth with a viable cell count of 1 × 10⁻⁶. 10 CFU / mL or higher. The cessation of acid production can be determined by whether the pH stops decreasing and the addition of the neutralizing agent ceases.

[0230] (6) Freeze-drying

[0231] a. Cell centrifugation: The high-density fermentation broth was centrifuged at 12,000 rpm to concentrate the cells; the concentrated bacterial sludge was resuspended in sterile PBS buffer solution and centrifuged again at 12,000 rpm to obtain the fermentation strain bacterial sludge;

[0232] b. Addition of a preservative: The preservative solution consists of the following components: mannitol 2.4g, trehalose 1.36g, sodium ascorbate 0.24g, and water 4g. The mass ratio of the bacterial sludge to the freeze-drying preservative is approximately 1:0.8.

[0233] c. Drying: The bacterial suspension with added protectant was placed in a tray to a thickness of 1 cm and freeze-dried to obtain freeze-dried powder. The powders of each strain were mixed in a certain proportion, and the total number of viable bacteria in the mixed powder was controlled to reach 1 × 10⁻⁶. 9CFU / g. Method for detecting viable bacteria count: Dissolve 0.5g of bacterial powder in 4.5mL of sterile physiological saline, then perform 10-fold serial dilutions. Pour 100μL of each appropriate dilution onto a sterile Petri dish, and then pour in MRS solid medium containing 1.5% agar at 45℃. After cooling, place in an anaerobic incubator and incubate at 37℃ for 48 hours. Count the colonies and calculate the viable bacteria count for each sample.

[0234] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention.

Claims

1. A type of curly lactobacillus ( Lactobacillus crispatus Lcr5-6, characterized in that, The Lactobacillus curvature Lcr5-6 was deposited on April 29, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38486, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The *Lactobacillus curvatureii* Lcr5-6 according to claim 1, characterized in that, The adhesion rate of the Lactobacillus criniformis Lcr5-6 to HeLa cells is ≥60%.

3. A fermentation supernatant of *Lactobacillus curvatureii* Lcr5-6 for inhibiting *Gardnerella vaginalis*, characterized in that, The fermentation supernatant is obtained by centrifugation after fermentation culture of Lactobacillus criniformis Lcr5-6 as described in claim 1 or 2, wherein the fermentation supernatant contains 0.12-0.14 mM of D-lactic acid and 0.07-0.08 mM of hydrogen peroxide.

4. A method for preparing freeze-dried Lactobacillus criniformis Lcr5-6 bacterial powder, characterized in that, Includes the following steps: (1) The Lactobacillus curlyis Lcr5-6 described in claim 1 or 2 is inoculated into MRS liquid culture medium and cultured anaerobically at 37°C for 18-24 h to obtain activated strains; (2) The activated strain from step (1) was inoculated into the optimized culture medium and cultured anaerobically at 37°C for 24 hours to obtain the seed culture. (3) The seed liquid from step (2) was inoculated into the fermentation medium at an inoculation rate of 5%, and fermentation was carried out at a temperature of 38℃, pH 6.5 and a stirring speed of 150 rpm to obtain a high-density fermentation liquid. (4) Centrifuge the fermentation broth from step (3) to concentrate the bacterial cells, add a protectant and freeze dry to obtain freeze-dried bacterial powder.

5. The method for preparing *Lactobacillus crinoides* Lcr5-6 lyophilized bacterial powder according to claim 4, characterized in that, The preservative mentioned in step (4) includes mannitol, trehalose, and sodium ascorbate. The mass ratio of the bacterial sludge to the preservative is 1:0.

8. The viable count of Lactobacillus criniformis Lcr5-6 in the freeze-dried bacterial powder is ≥1×10⁻⁶. 9 CFU / g.

6. The use of Lactobacillus curvature Lcr5-6 as described in claim 1 or 2 in the preparation of a vaginal microecological balance regulator.

7. The use of the fermentation supernatant of claim 3 in the preparation of antibacterial products for inhibiting Gardnerella vaginalis and / or Staphylococcus aureus and / or Escherichia coli.

8. A screening method for Lactobacillus curvature Lcr5-6, characterized in that, Includes the following steps: Lactobacilli were isolated from vaginal secretions of healthy women. Gardnerella vaginalis was used as an indicator bacterium for screening antibacterial activity. Strains with antibacterial activity were identified by 16S rDNA sequencing. Strains identified as Lactobacillus curvature and with an adhesion rate of ≥60% to HeLa cells were selected as target strains.

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