Bacterial strain for improving micro-ecology of vagina of human body and application of bacterial strain

Abstract

The invention discloses an application of a bacterial strain in production of an external product for improving the micro-ecology of the vagina of a human body, and the bacterial strain is Lactobacillus crispatus SPR005 and has 16S rDNA (ribosomal deoxyribonucleic acid) as shown in SEQ ID NO.1. The invention also discloses an application of the bacterial strain in production of the external product for improving the micro-ecology of the vagina of the human body. The preservation number is GDMCC NO: 66665; the strain produces H2O2 and only produces D-lactic acid, and no drug-resistant gene is found; the external product is suitable for improving changes, caused by vaginal pathogenic bacteria, of micro-ecology of the vagina of the human body. Compared with other lactobacillus crispatus of the same type, the strain disclosed by the invention can generate a strong bacteriostatic effect on staphylococcus aureus when the concentration of the strain is about 1 * 10 < 7 > CFU / ml, and the bacteriostatic rate reaches 99.78 or above. In addition, the strain disclosed by the invention is relatively high in acid production capacity, and the total amount of D-lactic acid produced in 48 hours is 16.28 mM; the D-lactic acid not only can effectively reduce the pH value of the vagina and maintain the pH value of the vagina between 3.8 and 4.0, but also can enhance the recognition and removal capability of pathogens by adjusting the immune response of the host. D-lactic acid can inhibit the growth of fungi, such as candida albicans; the D-lactic acid level is also positively correlated with the vaginal homeostasis, so that the treatment of BV patients is facilitated.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and in particular to a strain that improves the human vaginal microecology and its application. Background Technology

[0002] The female vagina is composed of the inherent anatomical structure, immune system, and microecological flora. The number and types of microecological flora are numerous and are affected by a variety of factors, including internal and external factors. Dominant bacteria such as lactobacilli can secrete and produce enzymes, lactic acid, and hydrogen peroxide, which maximize the maintenance of vaginal ecological balance and acidic environment [Huang Fei, et al. Correlation between high-risk human papillomavirus infection and vaginal microecology and local cellular immunity of the cervix [J]. Chinese Journal of Microecology, 2022, 34(4): 463-466.].

[0003] A balanced vaginal flora is characterized by a microbial community dominated by beneficial lactobacilli. In stark contrast, vaginal flora imbalance, such as in bacterial vaginosis (BV), is characterized by the overgrowth of various anaerobic bacteria, significantly increasing the risk of urogenital and reproductive health problems. The main difference between balanced and imbalanced vaginal flora lies in lactic acid concentration: in a balanced state, the high concentration of lactic acid produced by lactobacilli acidifies the vagina, while in an imbalance, lactic acid concentration drops sharply and pH rises. When the vaginal microecological balance is disrupted, the incidence of gynecological diseases, including gynecological tumors, increases.

[0004] Cervical cancer is a common gynecological malignancy, primarily associated with persistent infection by high-risk human papillomavirus (HPV). A recent study [Ke Peng, et al. mSystems. 2025 Sep 23;10(9):e0068325. doi:10.1128 / msystems.00683-25.] found that extracellular vesicles (nCEVs) derived from *Lactobacillus curvatureensis* are crucial for cervical-vaginal homeostasis; nCEVs deliver the key bioactive component D-lactic acid, promoting wound healing and blocking HPV infection through macrophage polarization. (Note: *mSystems* is an open-access journal under the American Society for Microbiology (ASM), focusing on high-level research in microbiome and systems biology. Its current impact factor is 5.0, placing it in JCR Q1 and the Chinese Academy of Sciences Biology Q2 journals.)

[0005] Existing strains used to improve the vaginal microecology of humans have problems such as low total amount or concentration of D-lactic acid produced in the short term, insufficient acid production capacity, and inability to produce H2O2. They cannot exert a strong killing effect on harmful anaerobic bacteria, especially the antibacterial effect against Staphylococcus aureus is poor. Application content

[0006] To address the shortcomings of existing technologies, this application provides a strain for improving the human vaginal microecology and its application, thereby solving the problems that existing strains used to improve the human vaginal microecology cannot produce D-lactic acid, have insufficient acid production capacity, and cannot produce H2O2, resulting in poor antibacterial effects against Staphylococcus aureus.

[0007] To solve the above-mentioned technical problems, this application adopts the following solution:

[0008] The application of a strain in the production of topical products that improve the human vaginal microecology, said strain being Lactobacillus crispatus SPR005, having 16S rDNA as shown in SEQ ID NO.1;

[0009] The strain produces H2O2, generates only D-lactic acid, and no drug resistance genes were found.

[0010] The external product is intended to improve changes in the human vaginal microecology caused by vaginal pathogens.

[0011] Furthermore, the external product includes at least one of the following: disinfectant, disposable hygiene product, external medicine, and external medical device.

[0012] Furthermore, the external product includes a gynecological gel product used for the adjustment, maintenance, and treatment of the female vaginal microecology.

[0013] Furthermore, the gynecological gel product includes therapeutic gels and nursing gels.

[0014] Furthermore, the vaginal pathogens are anaerobic bacteria, including at least one of the following: Prevotella, Peptostreptococcus, Molybditis, Gardnerella, Atobacillus, Staphylococcus, and Candida.

[0015] Specifically, the Prevotella genus includes at least one of Prevotella disiens, Prevotella buccae, and Prevotella bivia;

[0016] The Gardnerella genus includes at least one of Gardnerella leopoldii, Gardnerella piotii, Gardnerella phoenicis, and Gardnerella swidsinskii;

[0017] The Staphylococcus genus includes Staphylococcus aureus;

[0018] The Candida genus includes Candida albicans.

[0019] Furthermore, the topical product is suitable for maintaining the pH value of the human vaginal microecology between 3.8 and 4.0.

[0020] This application also provides a microbial agent, the active ingredient of which includes the fermentation broth obtained from the above-mentioned strain Lactobacilluscrispatus SPR005 or the fermentation strain Lactobacillus crispatus SPR005.

[0021] Biological Preservation Information:

[0022] The Lactobacillus crispatus (SPR005) strain of this application was deposited on July 11, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) (address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou), with accession number GDMCC NO: 66665.

[0023] Compared with existing technologies, this application has the following advantages:

[0024] 1. Compared to other Lactobacillus species of the same kind, the strain described in this application exhibits a strong inhibitory effect on Staphylococcus aureus at relatively low bacterial concentrations. The bacterial concentration is 1×10⁻⁶. 7 At a concentration of CFU / mL, it can exert a strong antibacterial effect against Staphylococcus aureus, with an inhibition rate of 99.78%; the bacterial concentration at 1×10⁻⁶ CFU / mL can also inhibit the growth of Staphylococcus aureus. 7 At CFU / mL, SPR005 achieved an inhibition rate of 75.7% against Candida albicans.

[0025] 2. Compared to other Lactobacillus species of the same type, the strain described in this application can produce H2O2. With a standard inoculum of 1%, the hydrogen peroxide concentration can reach 130-150 μM / L after 12 hours of cultivation. H2O2 is a strong oxidant that can directly kill or inhibit various pathogenic and opportunistic pathogens, such as Gardnerella vaginalis, Molecularcurb, Prevotella, and other BV-related anaerobic bacteria. H2O2 has a strong killing effect on harmful anaerobic bacteria, while having a relatively small impact on Lactobacillus itself, thus helping to maintain its dominant bacterial population.

[0026] 3. The strains of this application, after being prepared into gynecological gel products, can be used in various external products for industrial applications, and can also be added to other products for mixed use.

[0027] 4. Compared to other Lactobacillus sp. species, the strain described in this application produces only D-lactic acid, and its acid-producing capacity is high, with a total D-lactic acid production of 16.28 mM over 48 hours. D-lactic acid not only effectively lowers vaginal pH, maintaining it between 3.8 and 4.0, but also enhances the recognition and clearance of pathogens by regulating the host's immune response. D-lactic acid can inhibit fungal growth, such as Candida albicans; D-lactic acid levels are also positively correlated with vaginal homeostasis, which is beneficial for the treatment of BV patients. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a growth curve of the strain described in this application.

[0030] Figure 2 This is a 48-hour acid production curve of the strain described in this application.

[0031] Figure 3 This is a bar graph showing the total acid production of the strain in this application over 48 hours.

[0032] Figure 4 This is a 48-hour H2O2 production curve of the strain in this application.

[0033] Figure 5 The results of CFU plating for the strains of this application in the 1:1 group of the antibacterial test - Staphylococcus aureus test group;

[0034] Figure 6 The results of CFU plating for the strains of this application in the 10:1 group of the antibacterial test-Staphylococcus aureus test group;

[0035] Figure 7 The results of CFU plating for the strain of this application in the 30:1 group of the antibacterial test-Staphylococcus aureus test;

[0036] Figure 8 The results of CFU plating for the strain of this application in the blank control group of the antibacterial test - Staphylococcus aureus test group;

[0037] Figure 9 The results of CFU plating for the strain of this application in the 1:1 group of the antibacterial test - Candida albicans test group;

[0038] Figure 10The results of CFU plating for the strain of this application in the 10:1 group of the antibacterial test-Candida albicans test group;

[0039] Figure 11 The results of CFU plating for the strain of this application in the 30:1 group of the antibacterial test - Candida albicans test;

[0040] Figure 12 The results of CFU plating for the blank control group in the antibacterial test - Candida albicans test group of the strain of this application are shown.

[0041] Figure 13 This is an electrophoresis image of the strain in this application during the amplification and initial screening process. Detailed Implementation

[0042] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings, so that the advantages and features of this application can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of this application. Obviously, the embodiments described in this application are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of this application.

[0043] Furthermore, the technical features involved in the different embodiments of this application described below can be combined with each other as long as they do not conflict with each other.

[0044] (I) Preparation of strains

[0045] The Lactobacillus crispatus SPR005 of this application was obtained by screening on MRS plates and comparing with 16S rDNA sequences. It should be noted that SPR005 appears as slightly curved rod-shaped bacteria under a microscope. Most of the bacteria are "curled" and mostly aggregated into clusters. The individual bacteria are relatively small, and many bacteria aggregate in series to form thin, long rod-shaped chains.

[0046] The specific steps are as follows:

[0047] Vaginal secretions from healthy individuals were collected using disposable sterile swabs, inoculated onto MRS medium using the direct spread method, and anaerobically cultured at 37°C for 18-27 hours to obtain multiple colonies.

[0048] DNA was extracted from multiple single colonies, and the 16S rDNA sequence of each single colony was amplified and measured. The measurement results were compared with the 16S rDNA sequence of Lactobacillus. After a series of functional tests, the strain of this application was obtained. This strain is Lactobacillus crispatus, SPR005, which can improve the vaginal environment of the human body. Its 16S rDNA sequence is shown in SEQ ID NO.1.

[0049] 1) Specimen collection: Vaginal secretions from over 100 healthy women. The procedure is as follows:

[0050] Subject selection criteria: Inclusion criteria were healthy women aged 18-45 years who had not used antibiotics or vaginal topical medications in the past 3 months, had no symptoms of reproductive tract infection, and had regular menstrual cycles. Patients with pregnancy, immunodeficiency, diabetes, or other systemic diseases were excluded.

[0051] Sampling method: Subjects were placed in the lithotomy position, and the cervix was exposed using a sterile speculum. Secretions were collected from the lower 1 / 3 of the vaginal wall using a sterile swab, avoiding contact with the cervix and urethral opening to prevent contamination. Each sample was immediately transferred to an EP tube containing 5 mL of sterile PBS phosphate buffer and gently vortexed to prevent bacterial rupture.

[0052] Sample preservation and transportation: Place the sample in an ice box and deliver it to the laboratory within 2 hours. Store at 4°C for no more than 24 hours and proceed with subsequent processing immediately.

[0053] Transfer process: After collection, the samples are numbered, the basic information of the subjects is recorded, anonymized, and coded, and then transferred to the biosafety cabinet in the microbiology laboratory for processing.

[0054] 2) Initial screening: After serially diluting the sample, single colonies are obtained by plate-plating. The procedure is as follows:

[0055] Sample pretreatment: Take 1 mL of sample suspension, centrifuge at 3000 rpm for 10 minutes, discard the supernatant, and resuspend the precipitate in 1 mL of sterile physiological saline to remove impurities.

[0056] Serial dilution: Take 100 μL of the resuspension and perform serial dilutions of 10-fold each time. -1 Up to 10 -6 Use a new pipette tip for each dilution to prevent cross-contamination.

[0057] Spreading on plates: Take 100 μL of bacterial suspension at each dilution and spread it evenly on the surface of an MRS agar plate, which is suitable for the growth of lactic acid bacteria. Use a sterile spreader and proceed sequentially from low to high concentration, making 3 replicates for each dilution.

[0058] Culture conditions: Invert the plate and place it in an anaerobic incubator. Incubate at 37°C with 85% N2, 10% CO2, and 5% H2 for 48–72 hours, and observe the colony growth.

[0059] Transfer process: After the culture is completed, transfer the plates to the clean bench and select plates with appropriate dilution for the next step of single colony collection, 30-300 colonies / plate.

[0060] 3) Collect single colonies, culture them, and then extract the bacterial genome. The procedure is as follows:

[0061] Single colony selection: In a clean bench, use a sterile inoculation needle to pick up a typical, isolated single colony, such as a round, milky white colony with neat edges, and inoculate it into 5 mL of MRS liquid culture medium.

[0062] Liquid culture: Anaerobic culture at 37℃ for 24 hours until the logarithmic growth phase, OD600 ≈ 0.6.

[0063] Genomic DNA extraction: Take 1.5 mL of culture, centrifuge at 12000 rpm for 5 minutes, and discard the supernatant. Use a bacterial genomic DNA extraction kit, such as TIANGEN or Qiagen, and follow the instructions: treat with lysozyme at 37°C for 30 minutes, digest with proteinase K, purify by column binding, and elute to obtain genomic DNA. Detect DNA concentration and purity using a UV spectrophotometer; A260 / A280 ≈ 1.8.

[0064] Transfer process: The extracted genomic DNA was aliquoted into sterile EP tubes and stored at -20°C for subsequent PCR detection; the original strain was inoculated onto slant agar and stored at 4°C for a short period.

[0065] 4) Design two pairs of PCR primers for amplification and initial screening. The procedure is as follows:

[0066] Strains: L. crispatus; Target gene: Hypothetical protein;

[0067] Primer design: First pair: Universal primers for the 16S rRNA gene, used to confirm whether it is bacterial.

[0068] F: 5'-TGGCGAAGAGACACCAATATC-3';

[0069] R: 5'-TGACGTAACGCATGATGAAT-3'.

[0070] The second pair: Primers specific to the target bacterial genus, used for preliminary identification of whether it is the target lactic acid bacteria:

[0071] F: 5'-ATTGATCGGAAGCGCAGTCT-3';

[0072] R: 5'-CAGTTGGAGTGCTGAAAGG-3'.

[0073] The PCR reaction system of 25 μL is shown in Table 1:

[0074] Template DNA: 1 μL;

[0075] Primers (20 μM each): 0.5 μL;

[0076] 2× TaqPlus MasterMix: 12.5μL;

[0077] ddH2O: 10.5 μL.

[0078]

[0079] Table 1

[0080] Amplification program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 20 s, 63℃ annealing for 30 s, 72℃ extension for 1.5 min, 40 cycles of amplification; 72℃ extension for 10 min.

[0081] Electrophoresis detection: Take 5 μL of PCR product, perform electrophoresis on a 1.2% agarose gel, and observe the band size using a UV imaging system. Figure 13 As shown.

[0082] Transfer process: PCR-positive strains enter the next round of acid-producing screening; negative or non-specific banded strains are eliminated.

[0083] 5) The initially obtained strains are subjected to preliminary screening for acid production. The procedure is as follows:

[0084] Acid production capacity test: Candidate strains were inoculated into MRS liquid medium containing 0.2% glucose and anaerobically cultured at 37°C for 24 hours. 1 mL of culture supernatant was collected, centrifuged at 12000 rpm for 5 minutes, and the supernatant was collected. The pH of the culture medium was measured using a pH meter and compared with the uninoculated control group. Alternatively, bromocresol purple indicator was used: the color changed from purple to yellow when pH < 5.2, indicating acid production capacity.

[0085] Lactic acid quantification: Lactic acid concentration is determined by lactate oxidase method or HPLC method.

[0086] Transfer process: Strains that produce significant acid proceed to the next stage, i.e., pH ≤ 4.5 or lactic acid > 10 g / L; the rest are eliminated.

[0087] 6) Qualified strains undergo initial screening for yield and genetic stability. The procedure is as follows:

[0088] Initial screening of yield: Three batches were cultured repeatedly, and the lactic acid yield at the 48-hour endpoint was measured. The average value was taken.

[0089] High yield standard: lactic acid yield ≥ 15 g / L.

[0090] Initial screening for genetic stability: The strain was passaged five times consecutively, starting with a single colony each time. The genome of each generation was extracted and subjected to RAPD-PCR or BOX-PCR fingerprint analysis to observe whether the bands were consistent. Strains with stable fingerprints were considered genetically stable.

[0091] Transfer process: Strains that simultaneously meet the criteria of high acid production, high yield, and genetic stability enter the sequencing and identification stage.

[0092] 7) Sequencing was performed, yielding 8 target strains. The procedure is as follows:

[0093] 16S rRNA gene sequencing: PCR products were sent to a sequencing company for Sanger sequencing. The sequencing results were compared with the NCBI BLAST database to determine the bacterial species classification.

[0094] Screening criteria: Select strains that are highly homologous to known probiotics or have a defined function and ≥99% similarity.

[0095] Final confirmation: A total of 8 target strains were obtained, numbered LC-01 to LC-08.

[0096] Transfer process: The eight strains confirmed by sequencing were inoculated onto MRS slant, stored at 4°C, and glycerol strains were prepared with a final concentration of 15% glycerol and stored at -80°C for long-term storage.

[0097] 8) The obtained 8 strains were continuously passaged for 2 months. The procedure is as follows:

[0098] Subculturing protocol: Each strain was subcultured twice a week for 8 weeks, for a total of 16 generations. Each time, a single colony was picked from the slant and inoculated onto MRS liquid medium, and anaerobically cultured at 37°C for 24 hours. 100 μL of the bacterial culture was then transferred to fresh MRS medium and cultured further.

[0099] Records: Record the growth rate, turbidity changes, and presence or absence of contamination for each generation.

[0100] Transfer process: Samples from each generation of culture were retained for subsequent monitoring; after passage, the 1st, 8th, and 16th generations were selected for systematic testing.

[0101] 9) Monitor colony growth using a plating test. The procedure is as follows:

[0102] Regular streaking: At the 1st, 8th and 16th generations, take 100 μL of bacterial culture and streak it onto MRS agar.

[0103] Observation indicators: Consistency of colony morphology, including size, color, edge, and protrusion. The presence of abnormal colonies indicates variation or contamination, such as coarse colonies or satellite colonies. Calculate colony-forming units (CFU / mL) to assess growth activity.

[0104] Transfer process: Abnormal strains are terminated from the experiment; morphologically stable strains are used for yield monitoring.

[0105] 10) Monitor the yield of the strain after 48 hours, repeating twice. The procedure is as follows:

[0106] Yield determination: At generations 1, 8, and 16, bacterial strains were inoculated into MRS medium and anaerobic incubated at 37°C for 48 hours. Samples were centrifuged, and the lactic acid concentration in the supernatant was determined using HPLC. Each sample was tested in triplicate, and the average value was taken.

[0107] Stability assessment: If the lactic acid yield fluctuates by ≤ 10% for three consecutive generations, the yield is considered stable. Fluctuations > 15% are considered unstable.

[0108] Transfer process: Strains with stable yields are retained; strains with large fluctuations are eliminated.

[0109] 11) Eliminate strains with poor genetic stability. The procedure is as follows:

[0110] Final genetic stability assessment: Genomic DNA was extracted from generations 1 and 16. Whole-genome resequencing or core gene sequencing, such as recA and pheS, was performed, and SNP / Indel variants were compared. Alternatively, PFGE pulsed-field gel electrophoresis was used for genomic fingerprinting.

[0111] Elimination criteria: Strains exhibiting significant genetic variation, decreased yield, or altered colony morphology will be eliminated.

[0112] (II) Physicochemical characteristics of the strain

[0113] The original bacterial strain was streaked onto a plate, and a single colony was inoculated into liquid culture medium and cultured to the late logarithmic growth phase. 1% of the bacterial suspension was then inoculated into 5 ml of culture medium and cultured to the late logarithmic growth phase. This process was repeated to subculture and activate the bacteria, obtaining strains for detection and production. After CFU determination, 1% was inoculated each time, and stable strains were selected for functional testing, including the following tests:

[0114] 1) Growth curve of the strain

[0115] like Figure 1As shown, the culture time is plotted on the x-axis, and the OD600 value on the y-axis. OD600 refers to the optical density or absorbance of a liquid culture medium containing microorganisms, such as bacteria and yeast, measured using a spectrophotometer when illuminated with light at a wavelength of 600 nm. It reflects the turbidity of the microbial cells in the culture medium, and turbidity is positively correlated with cell concentration. The OD600 value reflects the relative number of viable bacteria per unit volume.

[0116] Specifically, the bacterial growth of the strain of this application was scraped from the MRS solid medium using a sterile inoculation loop and transferred to another MRS liquid medium. After gentle mixing, the medium was placed in an incubator at 37°C. At the beginning of the culture, samples were taken from the medium and the number of the strain was measured and recorded. After 4 hours of culture, samples were taken and tested every two hours. It should be noted that after 24 hours of culture, samples were taken and tested every two to four hours to create a growth curve until the end of the culture.

[0117] During the culture of this strain, the OD600 value increased slowly between 0 and 6 hours. This indicates that the strain was adapting to the new environment and had not yet begun rapid division. From approximately 6 hours onwards, the OD600 value rose rapidly, especially between 6 and 24 hours, exhibiting an exponential growth trend. This indicates that the strain entered a rapid reproduction phase, with a dramatic increase in cell number. After approximately 24 hours, the OD600 value tended to plateau, reaching its maximum value of approximately 1.7. This indicates that nutrients were depleted, metabolic waste accumulated, the strain's growth rate slowed, and it entered a stable state.

[0118] 2) 48h output

[0119] The strain described in this application was cultured in MRS medium at 37°C under anaerobic conditions with shaking at 80 RPM for 48 hours. The strain exhibited good growth performance, with a final OD600 of 1.80, indicating high biomass production potential. It should be noted that 37°C is close to human body temperature and is the optimal growth temperature for most intestinal probiotics; therefore, the 37°C culture environment was set to simulate the human vaginal microecological temperature. *Lactobacillus curvatureis* is an anaerobic bacterium, and an anaerobic environment is more conducive to its growth and metabolism. Cultivation at 80 RPM with moderate mixing of the culture medium promotes nutrient distribution and gas exchange.

[0120] No obvious decay period was observed within 48 hours; the OD600 value remained at a high level without significant decrease, and remained at 1.7 × 10⁻⁶. 8 Approximately CFU. This may be due to the short experimental time or favorable culture conditions, meaning the strain has not yet entered a mass mortality phase. This demonstrates that the strain in this application is easy to culture and grows rapidly. It should be noted that OD600 = 1.0 corresponds to 1.0 × 10⁻⁶ CFU. 8CFU / mL.

[0121] 3) 48h acid production curve

[0122] like Figure 2 As shown, the culture time is plotted on the x-axis, and the pH value of the culture medium on the y-axis. Samples were taken every 2 or 4 hours from 0 to 48 hours to create a growth curve, which was maintained until the end of the culture. This acid production curve clearly shows the dynamic changes in acid production of strain SPR005 over 24 hours, including three main stages: initial slow acid production, rapid acid production, and stable acid production. These data can be used to assess the strain's acid production capacity, the suitability of optimal fermentation time and culture conditions.

[0123] At 0 hours, the pH was approximately 6.4, then dropped to approximately 6.2 at 4 hours. This indicates that the strain began producing small amounts of acidic substances, but at a slow rate. From 6 to 10 hours, the pH dropped rapidly, from approximately 6.0 to around 4.2. This indicates that the strain entered a rapid acid-producing phase, with increased metabolic activity and the production of large amounts of organic acids. After approximately 14 hours, the pH stabilized, remaining at around 4.0 until the end of 48 hours.

[0124] 4) Final pH value after 48 hours

[0125] Since the pH value did not change significantly for a long period of time after 14 hours, this indicates that after the strain reached a certain level of acid production, the acid production rate slowed down, and the pH value no longer decreased significantly, eventually remaining at around 4.0.

[0126] 5) Acid type: D-lactic acid, no L-lactic acid, as shown in Table 2 and Appendix Figure 3 As shown, the total D-lactic acid produced in 48 hours was 16.28 mM.

[0127]

[0128] Table 2

[0129] It should be noted that D-lactic acid has a stronger antibacterial effect than L-lactic acid. It not only inhibits pathogens by lowering the pH value, but also penetrates the cell membrane of pathogens in cationic form, acidifying their interior and directly causing their death. The concentration of D-lactic acid in the vagina is several times higher than that of L-lactic acid, making it the main force in maintaining the acidic environment of the vagina.

[0130] D-lactic acid can precisely regulate the immune response of vaginal epithelial cells. It can inhibit the production of pro-inflammatory factors, such as IL-6 and IL-8, while promoting the release of anti-inflammatory factors, thereby reducing unnecessary inflammatory damage. This regulatory effect is particularly important during pregnancy, helping to maintain maternal and fetal immune tolerance and reducing the risk of premature birth. D-lactic acid can directly act on cervical stem cells, inhibiting their abnormal proliferation, thereby effectively blocking the precancerous lesion process of cervical cancer. This is a very important new discovery in maintaining reproductive health.

[0131] 6) Test the hydrogen peroxide (H2O2) production every 12 hours until 48 hours.

[0132] like Figure 4 As shown, the horizontal axis represents time, in hours.

[0133] Vertical axis: Represents the concentration of hydrogen peroxide, in micromoles per liter, μM / L, ranging from 0.00 to 250.00 μM / L.

[0134] From 0 hours to 48 hours, 1 mL of liquid was sampled from 1 L of culture medium every 12 hours for testing, thereby creating a growth curve until the end of the culture.

[0135] This hydrogen peroxide production curve clearly shows the dynamic changes in hydrogen peroxide concentration of the strain in this application over 48 hours, including two main stages: initial rapid generation and subsequent stabilization.

[0136] Initial phase, 0-12 hours:

[0137] At 0 hours, the hydrogen peroxide concentration was close to 0 μM / L. Subsequently, within 12 hours, the concentration rapidly increased to approximately 130 μM / L. This indicates that the rate of hydrogen peroxide formation was extremely rapid in the initial 12 hours.

[0138] Plateau period, 12-48 hours:

[0139] From 12 hours onwards, the hydrogen peroxide concentration tended to stabilize, remaining at around 140 μM / L until the end of 48 hours. Although the concentration increased slightly at 24, 36, and 48 hours, the overall change was not significant, remaining at a relatively stable level.

[0140] An approximation can be made using the trapezoidal method or by simply multiplying the average concentration over each time interval by the length of that time interval.

[0141] For example, for the first 12 hours, from 0 to 12 hours, the average concentration at the beginning and end can be used: (0 + 130) / 2 = 65 μM / L, and this average concentration persists for 12 hours. Therefore, the amount of hydrogen peroxide produced during these 12 hours is approximately 65 μM. Since the hydrogen peroxide concentration tends to stabilize from 24 hours onwards, maintaining around 140 μM / L until the end of 48 hours, the total amount of hydrogen peroxide produced by the strain in this application is approximately 140 μM over 48 hours. Furthermore, although the hydrogen peroxide concentration tends to stabilize after 12 hours, it still shows a slight upward trend, approaching 150 μM / L at the 48-hour mark.

[0142] 7) Preparation for antibacterial experiment

[0143] Preparation of selective culture medium:

[0144] Sabouraud broth: Weigh 10g peptone, 40g glucose, and 20g agar using a balance, bring the volume to 1000 mL, and sterilize in an autoclave at 115℃ for 20 min.

[0145] LB medium: Weigh 5g yeast powder, 10g tryptone, and 10g NaCl using a balance, bring the volume to 1000 mL, and sterilize in an autoclave at 121℃ for 15 min.

[0146] MRS medium:

[0147] Dissolve the following ingredients in 1 liter of distilled water: 10 g peptone, 10 g beef extract, 5 g yeast extract, 2 g dipotassium hydrogen phosphate, 2 g diammonium citrate, 5 g sodium acetate, 20 g glucose, 1 ml Tween 80, 0.5 g magnesium sulfate, 0.25 g manganese sulfate, and 15 g agar powder; then adjust the pH to 6.2-6.4; place in an autoclave at 101 kPa and 121°C for 15 minutes for sterilization.

[0148] NA medium: Dissolve the following ingredients in 1 liter of distilled water: 10 g peptone, 3 g beef extract powder, 5 g sodium chloride, and 15 g agar. Then adjust the pH to 7.3 ± 0.1. Bring the volume to 1000 mL with deionized water.

[0149] NB medium: Dissolve the following ingredients in 1 liter of distilled water: 10 g peptone, 3 g beef extract powder, and 5 g sodium chloride. Then adjust the pH to 7.2 ± 0.2. Add deionized water to a final volume of 1000 mL.

[0150] YM liquid culture medium:

[0151] Dissolve the following ingredients in 1 liter of distilled water: 5 g casein peptone, 2 g malt extract, 10 g glucose, 3 g yeast extract, and 20 g agar; then adjust the pH to 6.0 ± 6.4; autoclave at 121°C for 15 minutes and set aside.

[0152] Lactobacillus: Passaged Lactobacillus #5 was inoculated into 30 mL of MRS liquid medium and cultured in a shaker at 37°C for 48 hours.

[0153] For passage of lactic acid bacteria, use 1 mL of bacterial culture solution + 30 mL of liquid culture medium;

[0154] Candida albicans and Staphylococcus aureus were re-inoculated and shaken for 24 hours, with 1 mL of bacterial suspension + 10 mL of liquid culture medium.

[0155] Candida albicans: YM liquid culture medium;

[0156] Staphylococcus aureus: NB liquid medium;

[0157] The colony-forming units (CFU) of Candida albicans, Staphylococcus aureus, and Lactobacillus were simultaneously detected using the plate coating method.

[0158] Candida albicans was plated on YM plates, Staphylococcus aureus on NB plates, and Lactobacillus on MRS plates.

[0159] CFU assay results: Plate colony counts are shown in Table 3.

[0160]

[0161] Table 3

[0162] It should be noted that the data obtained in the table above were obtained by randomly selecting one or two experimental groups with different dilution ratios from multiple dilution groups to count the number of Candida albicans, Staphylococcus aureus, and strain #5.

[0163] Calculation formula:

[0164] CFU / mL (or CFU / g) = Average colony count × Dilution factor × 1 / Inoculation volume (mL)

[0165] Example: Dilution of 10 -6 The average colony count on the plate is 150. If 0.1 mL is inoculated, then:

[0166] CFU / mL = 150 × 10 6 ×10=1.5×10 9 CFU / mL.

[0167] The following can be calculated from the data in Table 3:

[0168] Candida albicans CFU: 52×10 5 ×10=5.2×10 7 CFU / mL;

[0169] Staphylococcus aureus CFU: 531 × 10 7 ×10=5.31×10 10 CFU / mL;

[0170] 5# strain CFU: 28×10 5 ×10=2.8×10 7 CFU / mL.

[0171] Total co-culture volume 10 mL, 1 × 10 7 CFU sampling amount:

[0172] Take 192 μL of Candida albicans stock solution, which is 1 × 10⁻⁶. 7 / (5.2×10 7 =0.192 mL = 192 μL;

[0173] Take 100x:19μL of Staphylococcus aureus dilution; dilute the original solution 100 times = 5.31×10 8 ;

[0174] 1×10 7 / (5.31×10 8 ) = 0.019 mL = 19 μL;

[0175] 5#: 1×10 7 / (2.8×10 7 =0.357mL = 357μL.

[0176] 8) Antibacterial test - Staphylococcus aureus

[0177] Experimental Groups:

[0178] (1) Blank control group, Staphylococcus aureus positive control, such as Figure 8 As shown;

[0179] (2) 1:1 group, i.e., 1×10 7 Lactobacillus + 1×10 7 Staphylococcus aureus, such as Figure 5 As shown;

[0180] (3) 10:1 group, i.e., 1×10 8 Lactobacillus + 1×10 7 Staphylococcus aureus, such as Figure 6 As shown;

[0181] (4) 30:1 group, i.e., 3×10 8 Lactobacillus + 1×10 7 Staphylococcus aureus, such as Figure 7 As shown;

[0182] As shown in Table 4:

[0183]

[0184] Table 4

[0185] Experimental procedure:

[0186] (1) Place Lactobacillus and diluted Staphylococcus aureus in MRS+NB mixed medium and co-culture for 12 h;

[0187] (2) Dilute the bacterial culture and spread it on LB medium plates. Dilute to 1 mL, take 100 μL and spread it on the plate. Incubate at 37°C for 2 days.

[0188] (3) Count the number of Staphylococcus aureus colonies. The results of the Staphylococcus aureus inhibition test plate count are shown in Table 5:

[0189]

[0190] Table 5

[0191] (4) Calculate the inhibition rate. Inhibition rate (%) = [(average number of viable bacteria in the control group - average number of viable bacteria in the treatment group) / average number of viable bacteria in the control group] × 100%.

[0192] Antibacterial effect calculation, unit: CFU / mL:

[0193] Staphylococcus aureus, positive control: 59 / 0.1×10 6 =590×10 6 ;

[0194] In the 5# / Staphylococcus aureus (1:1) treatment group, the average viable count of Staphylococcus aureus was 13 / 0.1×10⁻⁶. 4 =1.3×10 6 ;

[0195] Antibacterial rate (%) = [(590 × 10)] 6 -1.3×10 6 ) / 590×10 6 ] × 100% = 99.78%.

[0196] (5) Take a photo on the tablet and save it.

[0197] 9) Antibacterial test - Candida albicans

[0198] Experimental Groups:

[0199] (1) Blank control group, Candida albicans positive control, such as Figure 12 As shown;

[0200] (2) 1:1 group, i.e., 1×10 7 Lactobacillus + 1×10 7 Candida albicans, such as Figure 9 As shown;

[0201] (3) 10:1 group, i.e., 1×10 8 Lactobacillus + 1×10 7 Candida albicans, such as Figure 10 As shown;

[0202] (4) 30:1 group, i.e., 3×10 8 Lactobacillus + 1×10 7 Candida albicans, such as Figure 11 As shown;

[0203] As shown in Table 6:

[0204]

[0205] Table 6

[0206] Experimental procedure:

[0207] (1) Lactobacillus and Candida albicans were co-cultured in 10 mL MRS+YM mixed medium for 12 h;

[0208] (2) Dilute the bacterial suspension and spread it on Sabouraud dextrose agar plates. Dilute to 1 mL, take 100 μL and spread it on the plate. Incubate at 30°C for 2 days.

[0209] (3) Count the number of Candida albicans colonies. The results of the Candida albicans antibacterial plate count are shown in Table 7:

[0210]

[0211] Table 7

[0212] (4) Calculate the inhibition rate. Inhibition rate (%) = [(average number of viable bacteria in the control group - average number of viable bacteria in the treatment group) / average number of viable bacteria in the control group] × 100%.

[0213] Antibacterial effect calculation, unit: CFU / mL:

[0214] Candida albicans, positive control: 7 / 0.1×10 5 =7×10 6 ;

[0215] In the 5# / Candida albicans (1:1) treatment group, the average viable count of Candida albicans was 17 / 0.1×10⁻⁶.4 =1.7×10 6 ,

[0216] Antibacterial rate (%) = [(7×10)] 6 -1.7×10 6 ) / 7×10 6 ] × 100% = 75.7%;

[0217] In the 5# / Candida albicans (10:1) treatment group, the average viable count of Candida albicans was 15 / 0.1×10⁻⁶. 4 =1.5×10 6 ,

[0218] Antibacterial rate (%) = [(7×10)] 6 -1.5×10 6 ) / 7×10 6 ] × 100% = 78.6%;

[0219] In the 5# / Candida albicans (30:1) treatment group, the average viable count of Candida albicans was 10 / 0.1×10⁻⁶. 4 =1.0×10 6 ,

[0220] Antibacterial rate (%) = [(7×10)] 6 -1.0×10 6 ) / 7×10 6 ] × 100% = 85.7%.

[0221] (5) Take a photo on the tablet and save it.

[0222] In summary, the strain SPR005 of this application, at a strain concentration of 1×10⁻⁶, [achieved optimal results]. 7 At a concentration of CFU / mL, it can exert a strong antibacterial effect against Staphylococcus aureus, with an inhibition rate of 99.78%; at a strain concentration of 1×10⁻⁶, it can also inhibit the growth of Staphylococcus aureus. 7 At CFU / mL, the inhibition rate against Candida albicans can reach 75.7%; with a conventional inoculum of 1%, the concentration of hydrogen peroxide can reach 130-150 μM / L after 12 hours of culture.

[0223] It should be noted that the strain provided in this application has broad-spectrum antibacterial activity, and its activity is not limited to Staphylococcus aureus and Candida albicans. In this embodiment, only representative bacteria such as Staphylococcus aureus and fungi such as Candida albicans were selected as experimental subjects to verify the antibacterial effect of the strain. In fact, this strain exhibits inhibitory effects against most bacteria and fungi, possessing both antibacterial and antifungal activities. Given the vast number of microorganisms, it is impossible to conduct experiments to verify each one individually; therefore, the experiments listed in this application are only representative examples and are insufficient to exhaust all possible antibacterial spectra.

[0224] (III) Application of strains

[0225] This strain can be developed into various external products such as gels, suppositories, douches, and hygiene products. Administered locally through the vagina or vulva, it utilizes its H2O2 and D-lactic acid-producing properties to safely and effectively inhibit various vaginal pathogens, such as Gardnerella vaginalis, Prevotella, Staphylococcus aureus, and Candida albicans, thereby treating and preventing vaginal infections and maintaining women's reproductive health. Product designs can cover all scenarios from treatment to daily care. Several specific application product types are listed below:

[0226] Gynecological probiotic gels / suppositories / capsules are external medications or medical devices. They are formed by mixing live bacteria with a gel matrix and a preservative, such as carbomer or hyaluronic acid, and trehalose, as the preservative. They can also be formulated as vaginal suppositories or hard capsules. They are suitable for adjunctive treatment or recurrence prevention of bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC); or for daily microecological maintenance and flora reconstruction after antibiotic use. Their main function is to directly replenish dominant vaginal probiotics, inhibiting pathogenic bacteria such as Gardnerella vaginalis, Prevotella, and Candida albicans by producing H2O2 and D-lactic acid, restoring an acidic environment, and rebuilding microecological balance.

[0227] Probiotic rinse / wash solution is a type of cleansing agent or hygiene product. It is formulated by mixing bacterial strains or their metabolites with physiological saline, buffer solution, and mild surfactants. It is suitable for auxiliary cleaning and maintenance before and after menstruation, after sexual activity, or when feeling unwell. Its main function is to cleanse while replenishing probiotics, regulating pH, and inhibiting the colonization of pathogenic bacteria.

[0228] Probiotic tampons / panty liners are disposable hygiene products. They consist of probiotics and their nutrient substrate embedded in the surface or inner layer of the tampon or panty liner. Suitable for menstruation or daily use. Their main function is to continuously release probiotics during menstruation, a time when the gut microbiota is prone to imbalance, inhibiting the overgrowth of harmful anaerobic bacteria and yeasts, and reducing odor and the risk of infection.

[0229] Probiotic sprays are topical medications or hygiene products, formulated as a vaginal spray containing probiotic powder or liquid. They are suitable for daily care when experiencing vulvar itching, dampness, or discomfort. Their main function is to quickly relieve vulvar discomfort and inhibit the growth of pathogenic bacteria in the vulvar area, such as Staphylococcus aureus or Candida.

[0230] Lyophilized probiotic powder, which can be used as an excipient in medical devices or pharmaceuticals, is made by freeze-drying bacterial strains into powder. It can be used directly or after reconstitution. It can be flexibly applied in various scenarios, such as for irrigation after reconstitution or for use in combination with other products. Its main function is to provide a highly active source of bacteria for customized treatment or scientific research.

[0231] It exerts its inhibitory effect through the following mechanisms:

[0232] It produces H2O2, and hydrogen peroxide has broad-spectrum antibacterial activity, which can effectively kill or inhibit Gardnerella spp., Prevotella spp., Peptostreptococcus spp., Molecularbacterium spp., Atobacillus spp., and Staphylococcus aureus, etc.

[0233] It produces D-lactic acid, which lowers the local pH of the vagina, creating an acidic environment unfavorable to the growth of the aforementioned anaerobic pathogens and Candida albicans. D-lactic acid is a typical metabolite of lactobacilli in the human vagina and has good biocompatibility.

[0234] Competitive exclusion: It competes with pathogens for adhesion sites and nutrients, thus preventing them from colonizing.

[0235] The above description is only a preferred embodiment of this application and does not limit the patent scope of this application. Any equivalent structural or procedural transformations made using the content of this application's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of this application.

[0236] SEQUENCE LISTING

[0237] <110> Shenzhen Sipu Ruixin Biotechnology Co., Ltd.

[0238] <120> A strain that improves the human vaginal microecology and its application

[0239] <160> 1

[0240] <210> 1

[0241] <211> 1595

[0242] <212> DNA

[0243] <213> Lactobacillus crispatus SPR005

[0244] <220>

[0245] <221> 16S rDNA

[0246] <223> native sequence

[0247] <400> 1

[0248] GAAAATTATTCTTTCCCCAAGAGCCTCGCTTTACGCCCCATTAAATCCGGAAATCATTTG 60

[0249] CCCCCTCCCCCAGACTACCAAAGTCATGACGAACGCTGGCGGCGTGCCTAATACATGCAA 120

[0250] GTCGAGCGAGCGGAACTAACAGATTTACTTCGGTAATGACGTTAGGAAAGCGAGCGGCGG 180

[0251] ATGGGTGAGTAACACGTGGGGAACCTGCCCCATAGTCTGGGATACCACTTGGAAACAGGT 240

[0252] GCTAATACCGGATAAGAAAGCAGATCGCATGATCAGCTTTTAAAAGGCGGCGTAAGCTGT 300

[0253] CGCTATGGGATGGCCCCGCGGTGCATTAGCTAGTTGGTAAGGTAAAGGCTTACCAAGGCG 360

[0254] ATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAA 420

[0255] CTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAAC 480

[0256] GCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAG 540

[0257] AGGTAGTAACTGGCCTTTATTTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGTGC 600

[0258] CAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGA 660

[0259] GCGCAGGCGGAAGAATAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAACTGCATCG 720

[0260] GAAACTGTTTTTCTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATG 780

[0261] CGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAACTGACGCTG 840

[0262] AGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACG 900

[0263] ATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCAC 960

[0264] TCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACA 1020

[0265] AGCGGTGGAGCATGTGGTTTAATTCGACGCAACGCGAAGAACCTTACCAGGTCTTGACAT 1080

[0266] CTAGTGCCATTTGTAGAGATACAAAGTTCCCTTCGGGGACGCTAAGACAGGTGGTGCATG 1140

[0267] GCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTG 1200

[0268] TTATTAGTTGCCAGCATTAAGTTGGGCACTCTAATGAGACTGCCGGTGACAAACCGGAGG 1260

[0269] AAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACA 1320

[0270] ATGGGCAGTACAACGAGAAGCGAGCCTGCGAAGGCAAGCGAATCTCTGAAAGCTGTTCTC 1380

[0271] AGTTCGGACTGCAGTCTGCAACTCGACTGCACGAAGCTGGAATCGCTAGTAATCGCGGAT 1440

[0272] CAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGA 1500

[0273] GTCTGCAATGCCCAAAGCCGGTGGCCTAACCTTCGGGAAGGAGCCGTCTAAGGCAGGGCA 1560

[0274] GATGACTGGGGTGAAGTCGAAAAGGGGGTTAAATC 1595。

Claims

1. The application of a bacterial strain in the production of topical products that improve the human vaginal microecology, characterized in that, The strain is Lactobacillus crispatus SPR005, which has 16S rDNA as shown in SEQ ID NO.1; The accession number is GDMCC NO: 66665; The strain produces H2O2, generates only D-lactic acid, and no drug resistance genes were found. The external product is suitable for improving changes in the human vaginal microecology caused by vaginal pathogens.

2. The application as described in claim 1, characterized in that, The external products include at least one of the following: disinfectants, disposable hygiene products, external medications, and external medical devices.

3. The application as described in claim 1, characterized in that, The external products include gynecological gel products used for the adjustment, maintenance, and treatment of the vaginal microecology in women.

4. The application as described in claim 3, characterized in that, The gynecological gel products include therapeutic gels and nursing gels.

5. The application as described in claim 1, characterized in that, The vaginal pathogens mentioned are anaerobic bacteria, including at least one of the following: Prevotella, Peptostreptococcus, Molybditis, Gardnerella, Atobacillus, Staphylococcus, and Candida.

6. The application as described in claim 5, characterized in that, The Prevotella genus includes at least one of Prevotella disiens, Prevotella buccae, and Prevotella bivia; The Gardnerella genus includes at least one of Gardnerella leopoldii, Gardnerella piotii, Gardnerella phoenicis, and Gardnerella swidsinskii; The Staphylococcus genus includes Staphylococcus aureus; The Candida genus includes Candida albicans.

7. The application as described in claim 1, characterized in that, The topical product is suitable for maintaining the pH value of the human vaginal microecology between 3.8 and 4.

0.

8. A microbial agent, characterized in that, The active ingredient of the microbial agent includes the fermentation broth obtained from the strain Lactobacilluscrispatus SPR005 as described in claim 1 or the fermentation strain Lactobacillus crispatus SPR005.

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