A strain of Lactobacillus crispatus NCU-13 and its application

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to a strain of Lactobacillus crispatus NCU‑13 and its application. The name of the Lactobacillus crispatus NCU‑13 is Lactobacillus crispatus NCU‑13, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on July 17, 2023, with a deposit number of CGMCC No.27918. The present invention evenly places the freeze-dried Lactobacillus crispatus powder in the absorption layer of the sanitary napkin, and when in use, utilizes the suitable environment of the human body and the moist environment formed during menstruation to activate and promote the activation and proliferation of probiotics attached to the sanitary napkin, so that it enters the vagina. The Lactobacillus crispatus and its beneficial metabolites can not only promote the health of the vaginal environment, but also improve the barrier function of the vaginal mucosa, and may help restore the balance of vaginal microecology through its anti-inflammatory and immunomodulatory effects, while optimizing female reproductive health.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Lactobacillus crispatus NCU-13 and an application thereof. Background Art

[0002] The present invention relates to the field of gynecological health, in particular to a probiotic sanitary napkin for treating bacterial vaginitis (BV) and a preparation method thereof. BV is a common disease caused by an imbalance in vaginal flora, which is manifested by a decrease in the number of lactobacilli and an increase in anaerobic bacteria such as vaginal Gardnerella and vaginal Atopibrio. This flora imbalance not only affects women's reproductive health, but may also lead to a variety of complications.

[0003] Traditional BV treatments rely mainly on antibiotics, but these methods often lead to high recurrence rates and drug resistance problems, and fail to restore the normal microecological environment of the vagina. In recent years, prebiotics, as a supplement and "advanced alternative" to probiotics, have shown great potential in the application of the digestive system, but there are relatively few studies on the reproductive system.

[0004] Therefore, considering the above research status and significance, seeking a safe, effective method that can maintain the balance of vaginal microecology has become the focus of research. Summary of the invention

[0005] The purpose of the present invention is to solve the deficiencies in the prior art and provide a strain of Lactobacillus crispatus NCU-13 and its application, which specifically adopts the following technical scheme:

[0006] In a first aspect of the present invention, a strain of Lactobacillus crispatus NCU-13 is provided. The strain of Lactobacillus crispatus NCU-13 is named Lactobacillus crispatus NCU-13, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on July 17, 2023, with a deposit number of CGMCC No. 27918. The address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] Furthermore, the 16S rDNA gene sequence of Lactobacillus crispatus is shown in SEQ ID No: 1:

[0008] GGGGGGGGCAGCTATACATGCAGTCGAGCGAGCGGAACTAACAGATTTACTTCGGTAATGACGTTAGGAAAGCGAGCGGCGGATGGGTGAGTAACACGTGGGGAACCTGCCCCATAGTCTGGGATACCACTTGGAAACAGGTGCTAATACCGGATAAGAAAGCAGATCGCATGATCAGCTTTTAAAAGGCGGCGTAAGCTGTCGCTATGGGATGGCCCCGCGGTGCATTAGCTAGTTGGTAAGGTAAAGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGTAGTAACTGGCCTTTATTTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAAGAATAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAACTGCATCGGAAACTGTTTTTCTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGGGAGGTTTCCGCCTCTCACTGCTGCAGCTAACGCATTACCACTCCGCCTGGGGAGTACGACCGCAAGTTGAAACTCAAAGAATTGACGGGGCCCGCACAACCGTGGAGCATGTGGTTTATTCAAGCAACACGAAGAACCTTACCAGTCTTGACATCTAGTGCATTTGTAGAGATACAAGT。

[0009] The second aspect of the present invention also provides the use of the above-mentioned Lactobacillus curvature NCU-13 in the preparation of a drug for preventing or treating vaginitis caused by bacteria or fungi. The bacteria are one or more of Salmonella, Shigella dysenteriae, Shigella flexneri, Listeria monocytogenes, Gardnerella vaginalis, Escherichia coli, Atopobium vaginalis and Pseudomonas aeruginosa; the fungi are Candida albicans. Lactobacillus curvature can colonize in the vagina, inhibit the growth of pathogenic microorganisms by secreting hydrogen peroxide, bacteriocins and other antimicrobial factors, and is an important component of the natural vaginal flora.

[0010] The third aspect of the present invention also provides the use of the above-mentioned Lactobacillus crispatus NCU-13 in the preparation of vaginal probiotic sanitary napkins.

[0011] In a fourth aspect, the present invention also provides a vaginal probiotic sanitary napkin containing Lactobacillus crispatus, wherein the vaginal probiotic sanitary napkin contains the above-mentioned Lactobacillus crispatus NCU-13 and also contains postbiotic short-chain fatty acids, which are metabolites of Lactobacillus crispatus. Adding short-chain fatty acids, a metabolite of Lactobacillus crispatus, to the sanitary napkin can adjust the pH value of the vagina and promote local immune balance.

[0012] The fifth aspect of the present invention also provides a method for preparing the above-mentioned vaginal probiotic sanitary napkin containing Lactobacillus crispatus, comprising the following steps:

[0013] First, Lactobacillus crispatus is cultured in a liquid culture medium, and after the culture is completed, it is mixed with skim milk and stored in a -80°C refrigerator for 24 hours, and then made into freeze-dried powder in a freeze dryer; then the freeze-dried powder is evenly placed on the inner absorption layer of the sanitary napkin to obtain a vaginal probiotic sanitary napkin containing Lactobacillus crispatus.

[0014] Lactobacillus crispatus produces a variety of metabolites during its growth, including beneficial acids (such as lactic acid), antibacterial substances, enzymes, etc. When these strains are grown in liquid culture media, their metabolites accumulate in the culture media. When the strains are mixed with skim milk and freeze-dried, these metabolites are also included in the final freeze-dried powder.

[0015] When the vaginal probiotic sanitary napkin prepared by the above method is used, the probiotics are activated and proliferated by utilizing the suitable environment of the human body and the moist environment during menstruation, so as to achieve the effect of preventing and treating gynecological diseases. The method aims to utilize the dual probiotic properties of the live bacteria of Lactobacillus crispatus and its metabolites, which can not only directly inhibit the growth of pathogenic bacteria, but also maintain the healthy state of the vagina for a long time by means of vaginal microbiome transplant (VMT). In addition, the sanitary napkin of the present invention can also restore the vaginal microecological balance of BV patients through the metabolites of Lactobacillus crispatus, such as short-chain fatty acids, providing a safe, effective and easy-to-use treatment plan.

[0016] The sixth aspect of the present invention also provides a pharmaceutical composition for vaginal use, which comprises the live bacteria, inactivated bacteria or fermentation products of the above-mentioned Lactobacillus crispatus NCU-13.

[0017] The beneficial effects of the present invention are as follows: the present invention provides a strain of Lactobacillus crispatus NCU-13 and the application of Lactobacillus crispatus NCU-13. The present invention also provides a vaginal probiotic sanitary napkin and a preparation method, wherein freeze-dried Lactobacillus crispatus powder is evenly placed in the absorption layer of the sanitary napkin, and when in use, the suitable environment of the human body and the moist environment formed during menstruation are utilized to activate and promote the activation and proliferation of the probiotics attached to the sanitary napkin, so that the probiotics enter the vagina. Lactobacillus crispatus and its beneficial metabolites, such as short-chain fatty acids, can not only promote the health of the vaginal environment, but also improve the barrier function of the vaginal mucosa, and may help restore the balance of vaginal microecology through its anti-inflammatory and immunomodulatory effects. In addition, the present invention adds postbiotics to the sanitary napkin, including short-chain fatty acids and metabolites of probiotics, such as lactic acid, cell surface components and bioactive peptides, which have multiple benefits for human health and have higher safety and stability than living microorganisms.

[0018] The present invention uses the concept of "treating bacteria with bacteria" to activate and multiply probiotics in large quantities during the use of sanitary napkins, thereby inhibiting pathogenic bacteria inside and outside the vagina, and effectively preventing and treating bacterial vaginitis, fungal vaginitis and trichomonas vaginitis. Compared with traditional drug treatment, this probiotic treatment is a green therapy without drug side effects. In addition, the sanitary napkin is easy to use, efficient and economical, suitable for wide application, and aims to provide an effective method for preventing and treating diseases related to vaginal microecological imbalance, while optimizing female reproductive health. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shown is a growth curve diagram of Lactobacillus crispatus and Lactobacillus acidophilus in Example 2 of the present invention;

[0020] Figure 2The figure shows the number of Lactobacillus crispatus and Lactobacillus acidophilus at different pH values ​​in the acid resistance experiment in Example 3 of the present invention;

[0021] Figure 3 The figure shows the hydroxyl radical scavenging rates of Lactobacillus crispatus and Lactobacillus acidophilus in the antioxidant test in Example 4 of the present invention;

[0022] Figure 4 The figure shows the superoxide radical scavenging rates of Lactobacillus crispatus and Lactobacillus acidophilus in the antioxidant test in Example 4 of the present invention;

[0023] Figure 5 The results of the antibacterial experiment of Lactobacillus crispatus and Lactobacillus acidophilus against Salmonella, Shigella dysenteriae, Shigella flexneri, Listeria monocytogenes, Candida albicans, Gardnerella vaginalis, Escherichia coli, Atopobium vaginalis and Pseudomonas aeruginosa in Example 5 of the present invention are shown;

[0024] Figure 6 The figure shows a comparison of the efficacy of the product, the traditional product and a commercially available probiotic product detected by the qRT-PCR method in Example 7 of the present invention, specifically the transcription level detection of six common vaginal pathogens, namely Salmonella, Candida albicans, Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus and Atopobium vaginalis. DETAILED DESCRIPTION

[0025] The following will be combined with the embodiments and drawings to clearly and completely describe the concept, specific structure and technical effects of the present invention, so as to fully understand the purpose, scheme and effect of the present invention. It should be noted that the embodiments and features in the embodiments of this application can be combined with each other without conflict.

[0026] In the following examples, vaginal secretion samples were collected from 15 women of childbearing age (healthy group) who had normal physical examinations at the outpatient clinic of the Second Affiliated Hospital of Nanchang University starting from October 20, 2022. The inclusion criteria for healthy women were: luteinizing hormone (LH) 2-7U / L, age 20-40 years old, normal menstrual cycle, and normal uterine cavity. Exclusion criteria included: (1) cognitive dysfunction or mental illness; (2) heart, liver, or kidney dysfunction; (3) surgical contraindications; (4) drug contraindications; (5) failure to meet follow-up conditions.

[0027] In the following examples, patients with bacterial vaginitis are 15 patients (patient group) who were treated at the Second Affiliated Hospital of Nanchang University from October 20, 2022 to October 20, 2023. The inclusion criteria for patients with bacterial vaginitis are: (1) meeting the diagnostic criteria for bacterial vaginitis; (2) excluding other obvious gynecological diseases; (3) aged 20-45 years; (4) not using antibiotics or hormone drugs to treat vaginitis. Exclusion criteria include: (1) cognitive dysfunction or mental illness; (2) heart, liver, or kidney dysfunction; (3) surgical contraindications; (4) drug contraindications; (5) unable to meet follow-up conditions.

[0028] Vaginal secretions were collected from women in both the healthy and patient groups during the mid-luteal phase of menstruation (7-9 days after ovulation). The collected vaginal secretion specimens were placed in sterile PBS at 4°C, brought back to the laboratory within 1 hour, and frozen at -80°C.

[0029] Prior to participation, all prospective participants were given a detailed participant information sheet and the rationale for the study was explained to them. All participants provided written informed consent. All biospecimens were assigned anonymous participant identification numbers, which were matched with the participants’ personal information in a securely stored linked file, recording the participant’s name, age, sex, contact information, pathological diagnosis, histological type, surgery time, surgery name, and primary or recurrent disease.

[0030] Example 1

[0031] Cultivation, Isolation, Screening, Purification, Identification and Preservation of Lactobacillus crispatus NCU-13

[0032] (1) Cultivation and isolation of Lactobacillus crispatus NCU-13:

[0033] In cooperation with the Department of Obstetrics and Gynecology of the Affiliated Hospital of Nanchang University, vaginal secretions from healthy women were collected within 3 to 7 days after the end of menstruation using standardized gynecological examination and vaginal secretion collection methods. Vaginal secretions were collected with a sterile cotton swab in a sterile centrifuge tube containing 5 ml of 50% glycerol, and the pH value was immediately measured. Routine leucorrhea examinations were performed, and vaginal secretion samples from healthy women were stored in a -80°C freezer. Vaginal secretions from healthy women were screened for subsequent experiments.

[0034] Take an appropriate amount of vaginal secretion into a sterile 1.5 mL EP tube and dilute with sterile PBS at 10 1 ~10 6 For serial dilution, MRS, LB, BSM were diluted to 10 2 , 10 4 Dilution: BHI, BHI+DMS according to 10 3 , 10 530 μL of the dilution was spread on solid culture medium and cultured under anaerobic and aerobic conditions for 72 h; all strains were recorded and analyzed, and each strain was counted and classified. The results are shown in Table 1:

[0035] Table 1

[0036]

[0037] (2) Screening and purification of Lactobacillus crispatus NCU-13

[0038] Initial screening:

[0039] Inoculate the sample into a selective medium that promotes the growth of the target strain while inhibiting the growth of other microorganisms. For Lactobacillus crispatus, MRS medium (suitable for the cultivation of lactic acid bacteria) can be used.

[0040] In a sterile clean bench, observe the morphology of the colonies on the plate, including size, color, whether the edges are neat, whether they are raised, and other characteristics. Based on these characteristics, select single colonies that look similar to Lactobacillus crispatus.

[0041] Purification culture:

[0042] The selected single colony was inoculated onto a new MRS solid culture medium for purification and culture, and the process was repeated several times to ensure that a single strain of bacteria was obtained; the purified strain was inoculated into an MRS liquid culture medium again for proliferation.

[0043] Morphological and biochemical characterization:

[0044] Use a microscope to observe the morphological characteristics of the purified strains, such as cell shape and arrangement.

[0045] A series of biochemical tests (such as catalytic enzyme test, sugar fermentation test, etc.) were performed to determine the biochemical characteristics of the strain, which helped to further confirm that it was Lactobacillus crispatus.

[0046] Molecular Biology Identification:

[0047] Extract the genomic DNA of the strain and perform PCR amplification of specific genes (such as 16S rRNA gene).

[0048] The PCR products were sequenced and compared with known Lactobacillus crispatus sequences to confirm their identity.

[0049] Strain storage:

[0050] Once the purified strain is confirmed to be Lactobacillus crispatus NCU-13, it can be stored for a long time by low temperature preservation (such as -80°C refrigerator) or freeze-drying.

[0051] (3) Morphological and physiological and biochemical characteristics of Lactobacillus crispatus NCU-13:

[0052] Morphological characteristics

[0053] Shape: Lactobacillus crispatus is usually rod-shaped, longer than it is wide, with rounded ends.

[0054] Arrangement: These bacteria usually occur alone or in pairs, and less commonly in chains.

[0055] Gram Stain: Gram-positive, which means they remain purple when Gram-stained.

[0056] Physiological and biochemical characteristics

[0057] Fermentation Type: Lactobacillus crispatus is a lactic acid fermentation bacterium that produces lactic acid by fermenting sugars.

[0058] Oxygen requirement: These strains are usually facultative anaerobes, meaning they can grow in both aerobic and anaerobic conditions, but grow better in anaerobic conditions.

[0059] Temperature range: The growth temperature range of Lactobacillus crispatus is generally between 30 ℃ and 40 ℃, and the optimal growth temperature is about 37 ℃.

[0060] pH Range: They can grow over a wide pH range, but the optimum is usually between 5.5 and 6.2.

[0061] Salt tolerance: Some Lactobacillus crispatus strains show tolerance to salt and can survive in environments with higher salt concentrations.

[0062] Sugar fermentation: Able to ferment a variety of sugars, such as glucose, fructose, galactose, etc., to produce lactic acid. Some strains may also produce small amounts of acetic acid or other secondary metabolites.

[0063] Biochemical tests

[0064] Catalytic enzyme test: usually negative.

[0065] VP (Voges-Proskauer) test: generally negative, no propylene glycol is produced.

[0066] Gas production: No gas is produced.

[0067] Tolerance: Tolerance to antibiotics and other growth inhibitors can vary among strains.

[0068] (4) Molecular biological identification of Lactobacillus crispatus NCU-13

[0069] 1 mL was inoculated into 100 mL of MRS liquid culture medium and cultured anaerobically at 37 °C. Molecular biological methods (such as 16S rRNA gene sequencing) were used to accurately identify the specific strain. According to the 16S determination results, the nucleotide sequence of the 16S rDNA of the strain was shown in SEQ ID NO: 1. Then, based on the morphological characteristics of the strain and the sequencing results, the strain was identified as Lactobacillus crispatus NCU-13 strain, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on July 17, 2023, with the deposit number CGMCC No. 27918.

[0070] SEQ ID NO: 1:

[0071] GGGGGGGGCAGCTATACATGCAGTCGAGCGAGCGGAACTAACAGATTTACTTCGGTAATGACGTTAGGAAAGCGAGCGGCGGATGGGTGAGTAACACGTGGGGAACCTGCCCCATAGTCTGGGATACCACTTGGAAACAGGTGCTAATACCGGATAAGAAAGCAGATCGCATGATCAGCTTTTAAAAGGCGGCGTAAGCTGTCGCTATGGGATGGCCCCGCGGTGCATTAGCTAGTTGGTAAGGTAAAGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGTAGTAACTGGCCTTTATTTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAAGAATAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAACTGCATCGGAAACTGTTTTTCTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGGGAGGTTTCCGCCTCTCACTGCTGCAGCTAACGCATTACCACTCCGCCTGGGGAGTACGACCGCAAGTTGAAACTCAAAGAATTGACGGGGCCCGCACAACCGTGGAGCATGTGGTTTATTCAAGCAACACGAAGAACCTTACCAGTCTTGACATCTAGTGCATTTGTAGAGATACAAGT。

[0072] Example 2

[0073] Study on the growth curve of Lactobacillus crispatus NCU-13

[0074] (1) activating the Lactobacillus crispatus and the control strain Lactobacillus acidophilus isolated in Example 1 overnight;

[0075] (2) Take 1 mL and inoculate it into 100 mL of MRS liquid culture medium, culture it anaerobically at 37 °C, and measure its optical density at a wavelength of 600 nm at 0, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, and 24 h.

[0076] (3) Draw a growth curve with time as the horizontal axis and optical density as the vertical axis. The result is as follows: Figure 1 shown by Figure 1 It can be seen that the growth ability of Lactobacillus crispatus provided by the present invention is stronger than that of Lactobacillus acidophilus.

[0077] Example 3

[0078] Acid resistance test of Lactobacillus crispatus NCU-13

[0079] (1) The Lactobacillus crispatus isolated in Example 1 and the control strain Lactobacillus acidophilus were cultured in a liquid culture medium at 37°C in a constant temperature incubator for 72 h, and the strains were retained for later use;

[0080] (2) Take out 100 μL of the cultured strain in step (1) and dilute it with PBS buffer for 10 min. 1 , 10 3 , 10 5 times, centrifuge at 4000rpm for 3 min and discard the supernatant; add PBS buffer with pH=3, 4, 5, 7, let stand for 4 h, mix well, take 10 μL to plate, culture in a 37℃ constant temperature incubator or anaerobic incubator for 72 h, and count the viable bacteria;

[0081] (3) Culture the culture medium containing 0.0% to 0.3% ox bile salt in a 37°C constant temperature incubator for 48 h, mix well, take 10 μL and spread on a plate, culture in a 37°C constant temperature incubator or anaerobic incubator for 48 h, and count the viable bacteria. The results are shown in Table 1. Figure 2 shown by Figure 2 It can be seen that the acid resistance of Lactobacillus crispatus is stronger than that of Lactobacillus acidophilus.

[0082] Example 4

[0083] Experimental evaluation of the antioxidant activity of Lactobacillus crispatus NCU-13

[0084] (1) Determination of DPPH free radical scavenging ability: Take 2 mL of the supernatant of Lactobacillus crispatus culture and add 2 mL (0.2 mmol / L) DPPH methanol solution. Incubate at room temperature in the dark for 30 min. Take the supernatant and measure the OD at 517 nm. The control is 2 mL of deionized water plus DPPH methanol solution:

[0085] DPPH free radical scavenging rate = [1-A517 (sample) / A517 (blank)] × 100%

[0086] Sample: Lactobacillus crispatus and DPPH methanol solution

[0087] Blank: deionized water and DPPH methanol solution

[0088] (2) Determination of hydroxyl radical scavenging ability: Take 1 mL of 2 mmol / L FeSO4 solution, 1 mL of 6 mmol / L H2O2, and 1 mL of 6 mmol / L salicylic acid, add 1 mL of the supernatant of Lactobacillus crispatus culture solution, let it stand at room temperature for 30 min, use deionized water as a blank control, measure the absorbance at 510 nm, and calculate the scavenging rate of hydroxyl radicals:

[0089] Hydroxyl radical scavenging rate = [1-A510 (sample) / A510 (blank)] × 100%

[0090] (3) Determination of superoxide free radical scavenging ability: 2 mL of 150 mmol / L Tris-HCl solution (pH=8.0) and 1 mL of 1.2 mmol / L pyrogallol solution were added to 0.5 mL of Lactobacillus crispatus culture medium, respectively. The mixture was reacted at room temperature for 30 min and the absorbance at 330 nm was measured.

[0091] Superoxide radical scavenging rate = [1-(A11-A10) / (A01-A00) × 100%

[0092] A00: without sample and pyrogallol; A01: without sample and pyrogallol

[0093] A10: Contains sample but not pyrogallol; A11: Contains sample and pyrogallol

[0094] (4) Fe 2+Determination of chelating ability: add 0.1 mL of 0.4% ferrous sulfate solution to 0.5 mL of sample solution, gently invert and mix, then add 0.1 mL of 1% VC and 1 mL of 0.2 mol / L NaOH solution, react at room temperature for 20 min, then add 1 ml of 10% trichloroacetic acid, centrifuge at 6 000 rpm at 4°C for 10 min to remove protein, take 0.4 mL of the above solution and add 4 mL of 0.1% o-phenanthroline, react at room temperature for 10 min, and measure the absorbance at 536 nm:

[0095] Fe 2+ Chelating capacity = [A blank - A sample / A blank] × 100%

[0096] (5) Determination of total reducing power: Take 1 mL of Lactobacillus crispatus culture medium, add 1 mL of phosphate buffer (pH 6.6) and 1 mL of 1% K3[Fe(CN)6] solution, mix thoroughly by inverting, keep warm at 50 °C in a constant temperature box for 2 min, add 1 mL of 10% trichloroacetic acid solution, shake and mix, take 1 mL of the mixed solution, add 4 mL of deionized water and 0.4 mL of 0.1% FeCl3 solution, and let stand for 10 min. Then use deionized water as a blank and measure the absorbance at 700 nm. The larger the absorbance value, the stronger the reducing power of the analyte.

[0097] In this experiment, Lactobacillus acidophilus was used as a comparative strain. The antioxidant activity test was the same as that of Lactobacillus crispatus. The results are shown in Figure 3 and Figure 4 shown by Figure 3 and Figure 4 It can be seen that the antioxidant capacity of Lactobacillus crispatus is stronger than that of Lactobacillus acidophilus

[0098] Example 5

[0099] Antibacterial experiment of Lactobacillus crispatus NCU-13

[0100] (1) Lactobacillus crispatus and Lactobacillus acidophilus were inoculated into the corresponding liquid culture medium, cultured in a carbon dioxide incubator at 37°C for 72 h, and centrifuged at 6000 g for 5 min.

[0101] (2) Salmonella, Shigella dysenteriae, Shigella flexneri, Listeria monocytogenes, Candida albicans, Gardnerella vaginalis, Escherichia coli, Atopobium vaginalis and Pseudomonas aeruginosa were spread on LB solid culture medium respectively, and the Oxford cup was gently placed on the plate. 250 μL of the culture supernatant of Lactobacillus crispatus NCU-13 was aspirated into the Oxford cup (three replicates were performed for each group), and the culture was kept at 37°C. The size of the inhibition zone was observed every 2 h, and the diameter of the inhibition zone was measured after 8 h.

[0102] (3) The measured diameters of the inhibition zones were tabulated to compare the antibacterial abilities of Lactobacillus crispatus and Lactobacillus acidophilus. The results are shown in Table 1. Figure 5 shown by Figure 5 It can be seen that the antibacterial ability of Lactobacillus crispatus against various opportunistic pathogens is generally stronger than that of Lactobacillus acidophilus.

[0103] Example 6

[0104] A vaginal probiotic sanitary napkin containing Lactobacillus crispatus, the preparation method of which comprises the following steps:

[0105] (1) The Lactobacillus crispatus isolated in Example 1 was cultured in a liquid culture medium, then mixed with skim milk and stored in a -80°C refrigerator for 24 hours, and then prepared into a freeze-dried powder in a freeze dryer;

[0106] (2) Cut the inspected raw materials including the surface layer material, the absorption layer material and the bottom layer material according to the designed size of the sanitary napkin, then evenly place the freeze-dried powder prepared in step (1) on the absorption layer of the sanitary napkin, and use glue to merge the surface layer, the absorption layer and the bottom layer together to obtain a vaginal probiotic sanitary napkin containing Lactobacillus crispatus.

[0107] Example 7

[0108] qRT-PCR experiments

[0109] (1) Experimental design: 9 volunteers with BV were selected from the Second Affiliated Hospital of Nanchang University. It was ensured that they did not use antibiotics or other drugs that may affect the experimental results before the experiment. The participants were randomly assigned to three experimental groups: ordinary sanitary napkins, commercially available sanitary napkins containing vaginal probiotics (Lactobacillus acidophilus), and sanitary napkins containing Lactobacillus crispatus used in the product of the present invention.

[0110] (2) Sample collection and storage: Vaginal secretion samples were collected within 48 hours after the end of the volunteers' menstrual cycle. A standardized sterile sampling kit was then used, and the sampling time was recorded to ensure the reproducibility of the results. The samples were immediately frozen and stored at -80 °C until DNA extraction.

[0111] (3) DNA extraction: Use a DNA extraction kit suitable for viscous samples, such as a kit optimized for vaginal secretions. Follow the manufacturer's instructions and use a quantitative standard curve to assess the purity and concentration of the extracted DNA.

[0112] (4) qPCR primer and probe design: Design specific primers and probes for the following pathogenic bacteria: Salmonella, Candida albicans, Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Atopobium vaginalis. Validate each primer and probe to ensure amplification efficiency and specificity.

[0113] (5) qPCR experiment: set up internal reference genes, such as human GAPDH, to standardize the expression levels between samples. Perform three technical replicates on all samples to ensure the reliability of the data. Include positive and negative controls, with the positive control using a known amount of bacterial DNA and the negative control being a no-template control;

[0114] (6) Data analysis: The ΔΔCT method was used to compare the relative expression between groups. For absolute quantity calculation, a standard curve with known copy numbers was used to determine the exact copy number of each pathogen. Statistical analysis, such as ANOVA, was performed to determine the significant differences between different groups.

[0115] The results are as follows Figure 6 As shown, compared with the group using traditional sanitary napkins, the level of opportunistic pathogens in the vagina of participants who chose sanitary napkins containing vaginal probiotics was significantly reduced. More noteworthy is that compared with the existing vaginal probiotic sanitary napkins on the market, the group using the product of the present invention showed lower levels of opportunistic pathogens. This result strongly indicates that this product (a probiotic formula containing Lactobacillus crispatus and its postbiotics) has a significant effect on improving the vaginal microecological environment of BV patients, thereby highlighting its unique therapeutic potential in regulating vaginal health. In addition, this finding also provides strong scientific support for this product in the field of women's personal hygiene, highlighting its superiority in promoting vaginal health and preventing bacterial vaginitis.

[0116] Although the description of the present invention has been quite detailed and specifically describes several described embodiments, it is not intended to be limited to any of these details or embodiments or any particular embodiment, but should be regarded as providing a broad possible interpretation of these claims in view of the prior art by reference to the appended claims, thereby effectively covering the intended scope of the present invention. In addition, the above description of the present invention is based on the embodiments foreseeable by the inventor, and its purpose is to provide a useful description, and those non-substantial changes to the present invention that have not yet been foreseen may still represent equivalent changes to the present invention.

Claims

1. Use of Lactobacillus crispatus NCU-13 in the preparation of a drug for preventing or treating vaginitis caused by bacteria and fungi, characterized in that: The name of the Lactobacillus crispatus NCU-13 is Lactobacillus crispatus NCU-13 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 17, 2023, with the deposit number CGMCC No.27918; The bacteria are Salmonella, Shigella dysenteriae, Shigella flexneri, Listeria monocytogenes, Gardnerella vaginalis, Escherichia coli, Atopobium vaginalis and Pseudomonas aeruginosa; The mold is Candida albicans.

2. The application of Lactobacillus crispatus NCU-13 in the preparation of vaginal probiotic sanitary napkins, characterized in that: The name of the Lactobacillus crispatus NCU-13 is Lactobacillus crispatus NCU-13 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 17, 2023, with the deposit number CGMCC No.27918.

3. A vaginal probiotic sanitary napkin containing Lactobacillus crispatus NCU-13, characterized in that: The name of the Lactobacillus crispatus NCU-13 is Lactobacillus crispatus NCU-13 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 17, 2023, with the deposit number CGMCC No.27918.

4. The vaginal probiotic sanitary napkin containing Lactobacillus crispatus NCU-13 according to claim 3, characterized in that: It also includes postbiotics; the postbiotics are short-chain fatty acids; the short-chain fatty acids are metabolites of Lactobacillus crispatus NCU-13.

5. A method for preparing a vaginal probiotic sanitary napkin containing Lactobacillus crispatus NCU-13, characterized in that: The following steps are involved: First, the Lactobacillus crispatus NCU-13 is cultured in a liquid culture medium, and after the culture is completed, it is mixed with skim milk and stored in a -80°C refrigerator for 24 hours, and then freeze-dried in a freeze dryer; and then the freeze-dried powder is evenly placed on the inner absorption layer of the sanitary napkin to obtain a vaginal probiotic sanitary napkin containing Lactobacillus crispatus; The name of the Lactobacillus crispatus NCU-13 is Lactobacillus crispatus NCU-13 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 17, 2023, with the deposit number CGMCC No.27918.

6. A pharmaceutical composition for vaginal administration, characterized in that: Contains live Lactobacillus crispatus NCU-13 bacteria, the name of the Lactobacillus crispatus NCU-13 is Lactobacillus crispatus NCU-13 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 17, 2023, with the deposit number CGMCC No.27918.

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