Lactobacillus crispatus and its application in preparing product for improving reproductive tract flora

Abstract

The present invention discloses a Lactobacillus crispatus and its application in preparing a product for improving reproductive tract flora, and relates to the technical field of microorganisms. The present invention discloses a Lactobacillus crispatus, which is a strain of Lactobacillus crispatus ( Lactobacillus crispatus ) NHNK‑608, which was deposited in the China Center for Type Culture Collection on March 7, 2024, and its deposit number is CCTCC NO:M 2024421. Experiments have shown that NHNK‑608 has the functions of inhibiting the growth of vaginal pathogens, regulating the virulence genes of vaginal pathogens, inhibiting the bacterial-fungal composite biofilm of vaginal pathogens, competitively binding to vaginal pathogen fungal hyphae, and agglutinating vaginal pathogens, and can be used to prepare products to improve the genital tract flora.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Lactobacillus crispatus and application thereof in preparing a product for improving reproductive tract flora. Background Art

[0002] Sea cucumber is an aquatic species with extremely high nutritional and economic value. The rapid development of sea cucumber farming has become a very important part of marine aquaculture. Edible species usually contain rich symbiotic bacteria, which are involved in the host's nutrient absorption, energy metabolism, growth and health. The study found that the imitation sea cucumber ( Apostichopus japonicus ) had the highest richness in the gut bacterial community. Marine microbes have developed complex molecular adaptations to cope with these harsh conditions, affecting their primary and secondary metabolic pathways. This has led to the evolution of unique physiological traits and metabolic processes, and marine microbes are more likely to synthesize structurally unique enzymes and secondary metabolites than terrestrial microbes.

[0003] Virulence refers to the severity of the disease caused by the pathogen, and is the strength of the pathogen's pathogenicity, also translated as toxicity. Virulence factors refer to the structure and physiological characteristics of pathogens that cause diseases, usually referring to a class of effector molecules, such as proteins, lipid molecules and their combined functional units, which play an important role in the pathogenicity of pathogens. Accurate detection of virulence factor genes (VFGs) is essential for precise treatment and prognosis management of infections caused by highly virulent pathogens. Pathogens are usually also one of the normal symbiotic flora of the vagina, and only show pathogenicity under specific circumstances. Virulence factors have become an important indicator for medical testing of pathogen toxicity.

[0004] Normally, lactobacilli are the dominant bacteria in the vaginal flora, including Lactobacillus jensenii ( Lactobacillus jensenii ) and Lactobacillus gasseri ( Lactobacillus gasseri ), etc. Lactobacillus competitively adheres to vaginal epithelial cells and metabolizes to secrete lactic acid and hydrogen peroxide (H 2 O 2 ), bacteriocins and other substances can effectively inhibit the growth of pathogens and maintain an acidic and healthy environment in the vagina.

[0005] Bacterial vaginosis is a common gynecological disease in women, which seriously threatens women's reproductive health. Common harmful bacteria include vaginal Gardnerella ( Gardnerella vaginalis ) are represented by the Vagina strain, which can not only secrete a variety of virulence factors, but also form biofilms to adhere to vaginal epithelial cells, while being able to evade immune attacks and increase the ability to resist the body's immune system.

[0006] The current study found that three types of bacteria have a harmful effect on the cervical microenvironment, namely atypical Veillonella ( Veillonella atypica ), Venereobacteria montpellierii ( Veillonella montpellierensis ) and Megasphaera ( Megasphaera micronuciformis ). Atypical Veillonella and Pseudomonas aeruginosa reduce lactic acid in the vagina, providing conditions for pathogen infection. Giant sphaerocephala further promotes disease progression by increasing inflammation and promoting cell death by producing certain fat molecules. When the vaginal environment of women changes or their immunity is low, they are prone to concurrent infection with Candida albicans ( Candida albicans ), the pathogenicity of Candida albicans is associated with several virulence factors, and during the course of the disease, it sprouts to form hyphae that attach to the vaginal mucosal epithelial cells, increasing the damage to the mucosal cells. Vaginal pathogens can attach to any part of Candida albicans, including the Candida albicans itself, or even to the polysaccharides on the surface of the bacteria. In addition to its wide range of movement, this intricate cell mass is also very resilient, and studies have also confirmed that the composite biofilm produced by bacterial-fungal collaboration is larger and more numerous than the biofilm formed by a single species.

[0007] Marine microorganisms are one of the earliest sources of marine bioactive substances known to people, including algae, bacteria, fungi, protozoa and other microorganisms. These bioactive substances have important application value in various fields such as medicine, food, and cosmetics. Therefore, it is of great practical significance to provide a marine industry-related product developed using microbial technology. Summary of the invention

[0008] In view of this, the present invention provides a Lactobacillus crispatus and applications thereof.

[0009] The present invention provides Lactobacillus crispatus ( Lactobacillus crispatus ), which is Lactobacillus crispatus ( Lactobacillus crispatus ) NHNK-608, which was deposited in the China Center for Type Culture Collection (CCTCC for short, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, Postal Code 430072) on March 7, 2024, and its preservation number is CCTCC NO: M 2024421 of Lactobacillus crispatus).

[0010] The present invention provides the use of the above-mentioned Lactobacillus crispatus in the preparation of a product for inhibiting the proliferation of vaginal pathogens, wherein the inhibition of the proliferation of vaginal pathogens includes inhibiting the growth of atypical Veillonella and Venereella Montpellier.

[0011] Furthermore, the present invention provides the use of the above-mentioned Lactobacillus crispatus in the preparation of a product for improving genital tract flora, wherein the improvement of genital tract flora includes inhibiting the proliferation of vaginal pathogens, and the inhibition of the proliferation of vaginal pathogens includes inhibiting the growth of atypical Veillonella and Pseudomonas aeruginosa.

[0012] Further, the improvement of the reproductive tract flora includes regulating the virulence genes of vaginal pathogens, and the regulation of the virulence genes of vaginal pathogens includes downregulating the virulence genes of Candida albicans. HWP1 , ALS1 , ALS3 and downregulation of virulence genes of Gardnerella vaginalis vly , sld , pat , gtf , atm , stp At least one of .

[0013] Furthermore, the improvement of reproductive tract flora includes competitive binding to vaginal pathogenic fungal hyphae, and the competitive binding to vaginal pathogenic fungal hyphae is binding to Candida albicans hyphae.

[0014] Furthermore, the improvement of reproductive tract flora includes agglutinating vaginal pathogens, and the agglutinating vaginal pathogens include agglutinating Candida albicans, atypical Veillonella, and Gardnerella vaginalis.

[0015] Furthermore, the improving of reproductive tract flora includes promoting the proliferation of beneficial vaginal bacteria, and the promoting the proliferation of beneficial vaginal bacteria includes promoting the proliferation of Lactobacillus jensenii.

[0016] Furthermore, the Lactobacillus crispatus in the product for improving reproductive tract flora includes one or two of the following (1) or (2):

[0017] (1) the above-mentioned strains and / or inactivated bacteria of Lactobacillus crispatus;

[0018] (2) The fermentation and / or secretion products of the above-mentioned Lactobacillus crispatus.

[0019] The Lactobacillus crispatus disclosed in the present invention ( Lactobacillus crispatus ) NHNK-608, whose deposit number is CCTCC NO: M 2024421. Experiments have shown that NHNK-608 has the functions of inhibiting the proliferation of vaginal pathogens, regulating the virulence genes of vaginal pathogens, inhibiting the bacterial-fungal composite biofilm of vaginal pathogens, competitively binding to vaginal pathogen fungal hyphae, agglutinating vaginal pathogens, and promoting the proliferation of vaginal beneficial bacteria, and can be used to prepare products for improving reproductive tract flora, etc.

[0020] Biological Deposit Description

[0021] Lactobacillus crispatus ( Lactobacillus crispatus ) NHNK-608, was deposited in the China Center for Type Culture Collection (CCTCC for short, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, Postal Code 430072) on March 7, 2024, and its deposit number is CCTCC NO: M 2024421. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 For the Lactobacillus crispatus ( Lactobacillus crispatus ) MRS plate colony image and Gram staining image of NHNK-608;

[0023] Figure 2 For the Lactobacillus crispatus ( Lactobacillus crispatus ) NHNK-608 inactivates bacteria and agglutinates pathogens;

[0024] Figure 3 For the Lactobacillus crispatus ( Lactobacillus crispatus ) NHNK-608 inactivated bacteria combined with Candida albicans mycelium.

[0025] The following specific implementation manner will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0026] The present invention provides Lactobacillus crispatus and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0027] The Lactobacillus crispatus NHNK-608 of the present invention is derived from the intestinal tract of sea cucumber and is identified as Lactobacillus crispatus ( Lactobacillus crispatus ). This strain is Gram-positive and slightly curved in rod shape under a microscope. It is suitable for static growth in MRS liquid medium at 37°C, and the bacteria present white precipitation. When grown on MRS plates, it can form smooth white semi-transparent circular colonies with neat surface edges.

[0028] Lactobacillus crispatus ( Lactobacillus crispatus )NHNK-608, depository unit: China Center for Type Culture Collection, address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, deposit date: March 7, 2024, deposit number: CCTCC NO: M 2024421.

[0029] Further, the Lactobacillus crispatus provided by the present invention ( Lactobacillus crispatus) In the application described in the present invention, NHNK-608 is in the form of sterilization or fermentation product (i.e., supernatant), and the derivative form is preferably selected from: metabolites, metabolic biological products, prebiotics, cell walls and their components, extracellular polysaccharides and compounds containing immunogenic components, preferably selected from: fermentation products, inactivated bacteria.

[0030] In vitro experiments show that the Lactobacillus crispatus of the present invention ( Lactobacillus crispatus ) NHNK-608 has the effect of inhibiting the growth of vaginal pathogens atypical Veillonella and Pseudomonas aeruginosa, with an inhibition rate of 11.47%~79.08%.

[0031] In vitro experiments show that the Lactobacillus crispatus of the present invention ( Lactobacillus crispatus NHNK-608 down-regulates the lysozyme gene of Gardnerella vaginalis vly , sialidase gene sld , vaginal epithelial cell adhesion-related TadE-like protein gene pat , glycosyltransferase genes gtf , ABC transporter permease genes related to self-metabolism atm and stp The relative expression of the gene was downregulated to 0.09~0.91 times.

[0032] In vitro experiments show that the Lactobacillus crispatus of the present invention ( Lactobacillus crispatus NHNK-608 has the ability to inhibit the cell surface glycoprotein gene associated with Candida albicans ALS1 / ALS3 , hyphal cell wall protein genes HWP1 The relative expression of genes was downregulated to 0.04~0.62 times.

[0033] In vitro experiments show that the Lactobacillus crispatus of the present invention ( Lactobacillus crispatus ) NHNK-608 has the effect of inhibiting the formation of biofilms of Gardnerella vaginalis, Venereella montpellierii, atypical Veillonella, and Candida albicans, as well as the composite biofilms of Gardnerella vaginalis-Candida albicans, Venereella montpellierii-Candida albicans, and atypical Veillonella albicans-Candida albicans, with an inhibition rate of 17.14%~92.81%.

[0034] In vitro experiments show that the Lactobacillus crispatus of the present invention ( Lactobacillus crispatus NHNK-608 has the function of agglutinating vaginal pathogens such as Candida albicans, atypical Veillonella and Gardnerella vaginalis, and the agglutination rate can reach 18.11%~28.79%.

[0035] In vitro experiments show that the Lactobacillus crispatus of the present invention ( Lactobacillus crispatus) NHNK-608 fermentation products have the effect of promoting the proliferation of vaginal beneficial bacteria Lactobacillus jensenii, and the proliferation rate can reach 27.09%~30.87%.

[0036] The reagents and consumables used in the present invention are all common commercially available products. The present invention is further described below in conjunction with the embodiments:

[0037] Example 1 Separation of NHNK-608

[0038] Fresh imitation sea cucumbers were sampled from the Qingdao market. The surface of the sea cucumber was wiped with a cotton ball soaked in sterile saline. The sea cucumber intestines were removed and cut into pieces with sterile surgical scissors and scalpels. The sea cucumber intestines were rinsed 2-3 times with 0.9% sterile saline. The rinsed and cut sea cucumber intestines were placed in a triangular bottle containing MRS liquid culture medium containing small glass beads for enrichment culture. The culture was carried out at 37℃ and 160r / min on a shaking table for 24h. An appropriate amount of culture solution was applied to MRS solid culture medium and placed in an anaerobic bag. After constant temperature culture at 37℃ for 48h, white colonies were picked and repeatedly streaked and screened until a uniform single colony was obtained, which was named NHNK-608.

[0039] Gram staining microscopy: strain NHNK-608 is a Gram-positive colony, which is rod-shaped and slightly curved under a microscope; it grows on an MRS plate and forms white, smooth, rounded, semi-transparent round colonies with neat edges; it grows evenly in MRS liquid culture medium and becomes turbid and white precipitates after long-term storage. Figure 1 shown.

[0040] Example 2 Nucleic acid identification of NHNK-608

[0041] 1. 16S rDNA gene sequence analysis:

[0042] Pick a single colony in MRS liquid medium, culture overnight at 37℃, centrifuge at 8000 rpm for 1min to collect the bacteria, and operate according to the instructions of the Gram-positive bacteria DNA extraction kit. The primers used were bacterial 16S sequencing universal primers 27F and 1492R, and the PCR amplification system was 20μL. The PCR amplification program was 95℃ pre-denaturation for 5min, 94℃ for 15s, 58℃ for 15s, 72℃ for 1min, 35 cycles; 72℃ extension for 10min.

[0043] 2. Results

[0044] The PCR product was sequenced and compared with the standard sequences published in the GenBank database (BLASTN), and the NHNK-608 strain was identified as Lactobacillus crispatus ( Lactobacillus crispatus ).

[0045] Example 3: Experiment on the inhibition of vaginal pathogen growth by NHNK-608

[0046] 1. Preparation of NHNK-608 fermentation products

[0047] Pick out Lactobacillus crispatus ( Lactobacillus crispatus ) NHNK-608 single colony was cultured in MRS liquid medium at 37℃ for 48h and then diluted with MRS to adjust the OD 600 = 0.2, centrifuged at 5000 rpm for 10 min, took the supernatant and filtered it at 0.22 μM, and filtered the supernatant with a 0.22 μm filter membrane to obtain the cell-free fermentation product.

[0048] 2. Preparation of pathogenic bacteria solution

[0049] Vaginal pathogens: atypical Veillonella Veillonella Atypica ) DSM 20739, Venereum montpellierii ( Veillonella montpellierensis ) Single colonies of DSM 17217 were picked and inoculated into Veillonella liquid culture medium and PYG liquid culture medium respectively, and cultured at 37°C overnight until the stable phase.

[0050] 3. Experiment on inhibiting the growth of pathogens

[0051] Atypical Veillonella and Venereella Montpellier were inoculated with pathogenic bacteria at a ratio of 2%, and the fermentation products were added to the pathogenic bacteria at a ratio of 5% (V / V). The same volume of MRS liquid medium was added as a control, and the culture was carried out at 37°C for 24 hours. The concentration of the bacterial solution (OD 600 ) and calculate the pathogen inhibition rate. Calculation formula: Inhibition rate = (1-absorbance value of experimental group / absorbance value of control group) × 100%

[0052] The results are shown in Table 1:

[0053] Table 1

[0054]

[0055] The results showed that the cell-free fermentation product of NHNK-608 could effectively inhibit the growth of atypical Veillonella and Pseudomonas aeruginosa vaginal pathogens.

[0056] Example 4 Experiment on NHNK-608 regulating the expression of virulence genes of Gardnerella vaginalis

[0057] 1. Preparation of NHNK-608 fermentation products and inactivated bacteria

[0058] Pick out Lactobacillus crispatus ( Lactobacillus crispatus ) NHNK-608 single colony was cultured in MRS liquid medium at 37℃ for 48h, and then the bacterial solution was adjusted to OD 600= 1.0, centrifuged at 5000 rpm for 10 min, and the supernatant was filtered with a 0.22 μm filter membrane to obtain a sterile fermentation product; then the bacterial precipitate was adjusted to OD 600 = 1.0, washed twice with PBS solution, and resuspended to OD 600 =1.0. Autoclave at 121℃ for 15 min to obtain inactivated bacteria.

[0059] 2. Experiment on regulating the expression of virulence genes of Gardnerella vaginalis

[0060] Gardnerella vaginalis isolated from vaginal secretions Gardnerella vaginalis ) BNCC354890 cryopreserved tubes were inoculated into fresh Columbia liquid basal medium at a ratio of 2%, and cultured anaerobically at 37°C for 48 h. Take 3 mL of cultured vaginal Gardnerella liquid, add 1 mL of NHNK-608 fermentation product or inactivated bacteria (the control group added an equal volume of MRS or PBS), 1 mL of fresh Columbia liquid basal medium, and culture anaerobically at 37°C for 2 h. After the culture, centrifuge at 8000 rpm for 1 min to obtain the bacteria, extract total RNA according to the kit instructions, detect RNA concentration and purity, and reverse transcribe it into cDNA. 16S As the internal reference gene, qPCR fluorescence quantitative technology was used to detect vly , sld , pat , gtf , atm , stp The relative expression of the gene in the control group was F=1, and the expression of -ΔΔCT The F value of each sample was calculated by this method.

[0061] Formula: F=2 -ΔΔCT ,in:

[0062] △CT 实验 =CT 实验 -CT 内参（实验）;

[0063] △CT 对照 =CT 对照 -CT 内参（对照）;

[0064] △△CT=△CT 实验 -△CT 对照。

[0065] The results are shown in Tables 2 and 3 below:

[0066] Table 2

[0067]

[0068] Table 3

[0069]

[0070] The results showed that NHNK-608 had the effect of down-regulating the expression of virulence genes of Gardnerella vaginalis.

[0071] Example 5 Experiment on NHNK-608 regulating the expression of virulence genes of Candida albicans

[0072] 1. Preparation of NHNK-608 fermentation products and inactivated bacteria

[0073] The preparation method is as in Example 4.

[0074] 2. Experiment on regulating the expression of virulence genes of Candida albicans

[0075] Candida albicans ( Candida albicans ) CGMCC 2.4550 cryovials were inoculated into fresh Sabouraud medium at a ratio of 2%, and incubated at 37°C for 24 hours. 3 mL of cultured Candida albicans was added with 1 mL of NHNK-608 fermentation product or inactivated bacteria (the control group added an equal volume of MRS or PBS) and 1 mL of fresh Sabouraud medium, and incubated at 37°C for 24 hours. After the incubation, the cells were centrifuged at 8000 rpm for 1 minute to extract the total RNA according to the kit instructions. After testing the RNA concentration and purity, the RNA was reverse transcribed into cDNA. TDH1 As the internal reference gene, qPCR detection was performed HWP1 , ALS1 and ALS3 The relative expression of the gene in the control group was F=1, and the expression of -ΔΔCT The F value of each sample was calculated by the method described in Example 3.

[0076] The results are shown in Tables 4 and 5 below:

[0077] Table 4

[0078]

[0079] Table 5

[0080]

[0081] The results showed that NHNK-608 fermentation products and inactivated bacteria had the effect of down-regulating the expression of virulence genes of Candida albicans.

[0082] Example 6 Experiment on the inhibition of pathogenic bacteria biofilm formation by NHNK-608

[0083] 1. Preparation of NHNK-608 fermentation products and inactivated bacteria

[0084] The preparation method is as in Example 4.

[0085] 2. Preparation of pathogenic bacteria solution

[0086] Single colonies of pathogens: vaginal Gardnerella BNCC354890, atypical Veillonella DSM 20739, Pseudomonas montpellierii DSM 17217, and Candida albicans CGMCC 2.4550 were picked and inoculated into Columbia liquid basal medium, Veillonella liquid medium, PYG liquid medium, and Sabouraud medium, respectively, and cultured at 37°C overnight. After the culture was completed, the OD 600 =0.3.

[0087] 3. Experiment on inhibition of pathogenic bacteria biofilm

[0088] Take 100μL of pathogenic bacteria solution and add it to a 96-well plate. Add 100μL of NHNK-608 fermentation product or inactivated bacteria to the experimental group, and add an equal volume of MRS medium or PBS solution to the control group. Set up 3 parallels for each group and culture at 37℃ for 24h. After the culture, discard the supernatant, add 100μL of sterile PBS to each well and wash twice, then add 100μL of 4% paraformaldehyde fixative to each well and fix at room temperature for 30min. Discard the fixative, add 100μL of crystal violet to each well, and stain at room temperature for 30min. After staining, wash twice with sterile PBS and dry, add 100μL of anhydrous ethanol to each well, let it stand for 1min, measure the absorbance at 600nm, and calculate the inhibition rate according to the formula.

[0089] The calculation formula and results are shown in Tables 6 and 7 below:

[0090] Table 6: NHNK-608 fermentation products inhibit pathogenic bacterial biofilm formation

[0091]

[0092] Table 7: NHNK-608 inactivated bacteria inhibited pathogen biofilm formation

[0093]

[0094] The results showed that the fermentation products and inactivated bacteria of NHNK-608 had the effect of inhibiting the formation of biofilm of pathogenic bacteria.

[0095] Example 7 Experiment on the inhibition of pathogenic bacteria-fungus composite biofilm formation by NHNK-608

[0096] 1. Preparation of NHNK-608 fermentation products and inactivated bacteria

[0097] The preparation method is as in Example 4.

[0098] 2. Preparation of pathogenic bacteria solution

[0099] The preparation method is similar to that of Example 6.

[0100] 3. Experiment on inhibition of pathogenic bacteria-fungus composite biofilm

[0101] 100 μL of Candida albicans liquid was mixed with 100 μL of other bacterial pathogen liquid and added to 96-well plates. The experimental group was added with 100 μL of NHNK-608 fermentation product or inactivated bacteria, and the control group was added with an equal volume of MRS medium or PBS solution. Each group was set up in parallel for 3 times and cultured at 37°C for 24 hours. The fixation, staining and determination methods of the 96-well plate were referred to Example 5.

[0102] The calculation formula and results are shown in Tables 8 and 9 below:

[0103] Table 8: NHNK-608 fermentation products inhibit bacterial-fungal composite biofilm formation

[0104]

[0105] Table 9: NHNK-608 inactivated bacteria inhibits bacterial-fungal composite biofilm formation

[0106]

[0107] The results showed that NHNK-608 fermentation products / inactivated bacteria inhibited the formation of pathogenic bacteria-fungus composite biofilm.

[0108] Example 7 NHNK-608 Agglutination Pathogen Experiment

[0109] 1. Preparation of NHNK-608 inactivated bacteria

[0110] The preparation method is as in Example 4.

[0111] 2. Preparation of pathogenic bacteria solution

[0112] The culture method of Candida albicans, atypical Veillonella, and Gardnerella vaginalis was referred to Example 6. After the culture was completed, the supernatant was discarded by centrifugation at 5000 rpm for 10 min, and then washed twice with sterile PBS buffer, and then resuspended with sterile PBS buffer to adjust the OD 600 =1.0.

[0113] 3. Agglutination

[0114] The pathogen solution was mixed with the NHNK-608 inactivated bacteria solution in a volume ratio of 1:1. The pathogen was added with PBS as a control. After shaking at a constant speed for 2 hours on a shaker, the mixture was allowed to stand for 15 minutes to observe the flocculation condition.

[0115] 4. Index determination

[0116] After 2 hours of reaction, samples of lactic acid bacteria, pathogens, and the mixed reaction liquid of lactic acid bacteria and pathogens were collected. The sampling range was the top 50 μL of the liquid surface. After aspiration, the samples were transferred to a 96-well plate and the absorbance at OD = 600 nm was measured. At the same time, the agglutination precipitate was stained with Gram stain and its bacterial agglutination state was observed. Figure 2 shown.

[0117] Calculation formula:

[0118] Agglutination rate (%) = [(Ax+Ay)-2Amix] / (Ax+Ay)×100%;

[0119] Note: Ax: OD measured by NHNK-608 alone at the reaction time 600 Value; Ay: OD of a single pathogen measured at the reaction time 600 Value; Amix: OD measured at the reaction time after NHNK-608 and pathogens are mixed 600 Numeric value.

[0120] Table 10

[0121]

[0122] The results showed that NHNK-608 inactivated bacteria can agglutinate and capture albicans yeast, atypical Veillonella, and Gardnerella vaginalis, thereby achieving physical contact with the pathogens, thereby playing the role of NHNK-608 in downregulating the virulence genes of pathogens, reducing their pathogenicity, and inhibiting the formation of pathogen biofilms, thereby reducing the colonization ability of pathogens.

[0123] Example 8 Experiment on the inhibition of Candida albicans hyphae formation by NHNK-608

[0124] 1. Preparation of NHNK-608 fermentation products and inactivated bacteria

[0125] The preparation method is as in Example 4.

[0126] 2. Induction of hyphae morphology of Candida albicans

[0127] The method for culturing Candida albicans is described in Example 5. After the culture is completed, the cells are centrifuged at 5000 rpm for 10 min to obtain the cells. The cells are washed twice with PBS and then centrifuged. The cells are resuspended with fetal bovine serum (FBS) and the OD is adjusted. 600 =0.15.

[0128] 3. Experiment on the inhibition of Candida albicans hyphae formation by NHNK-608

[0129] In the experimental group, 100 μL of FBS-resuspended Candida albicans was added to a 96-well plate, and 100 μL of NHNK-608 fermentation product was added. In the control group, an equal amount of MRS liquid culture medium was added. Three parallels were set up for each group and cultured at 37°C for 2 hours. After the culture, the culture medium was discarded, washed once with 70% alcohol, washed once with 0.25% SDS solution, washed 3 times with sterile water, and then stained with 0.1% crystal violet solution for 30 minutes. After staining, it was washed once with 0.25% SDS solution and washed 3 times with sterile water. After the well plate was naturally dried, the biofilm at the bottom was observed. 200 μL of 40 mmol / L HCL-isopropanol solution and 50 μL of 0.25% SDS solution were added to each well, and the absorbance at OD=600nm was measured after standing at room temperature for 1 minute. The calculation formula and results are shown in Table 11 below:

[0130]

[0131] The results showed that NHNK-608 fermentation products and inactivated bacteria could effectively inhibit the hyphae formation of Candida albicans.

[0132] Example 8 Experiment on the combination of NHNK-608 inactivated bacteria and Candida albicans hyphae

[0133] 1. Preparation of NHNK-608 inactivated bacteria

[0134] The preparation method is similar to that of Example 3.

[0135] 2. Preparation of Candida albicans mycelium

[0136] The method for culturing Candida albicans is referred to Example 6. After the culture is completed, the cells are centrifuged at 5000 rpm for 10 min to obtain the mycelium. The cells are resuspended with fetal bovine serum (FBS) and adjusted to OD = 0.1, and cultured at 37°C for 3 h to obtain mycelium.

[0137] 3. Experiment on inactivated bacteria combined with Candida albicans mycelium

[0138] Add 0.7 ml of Candida albicans mycelium and 0.7 ml of lactic acid bacteria into the test tube, vortex thoroughly and let it stand. After 1 hour, a white precipitate will appear in the test tube. Gently aspirate the precipitate and stain it with Gram stain. Observe and take pictures with a microscope at 1000 times oil lens. Figure 3 shown.

[0139] There is a significant size difference between NHNK-608 inactivated bacteria and Candida albicans mycelia, and NHNK-608 inactivated bacteria and Candida albicans mycelia can be distinguished with the naked eye. The results showed that NHNK-608 inactivated bacteria can significantly bind to the hyphae of Candida albicans mycelia, and has a competitive inhibition effect on other pathogens binding to Candida albicans mycelia.

[0140] Example 10 Experiment on promoting the proliferation of beneficial vaginal bacteria by NHNK-608

[0141] 1. Preparation of NHNK-608 fermentation products

[0142] The preparation method is as in Example 3.

[0143] 2. Collection and cultivation of beneficial vaginal bacteria

[0144] Pick out Lactobacillus jensenii ( Lactobacillus jensenii ) A single colony of ATCC 25258 was inoculated into MRS broth and cultured anaerobically at 37°C for 48 h.

[0145] 3. Experiment on promoting the proliferation of beneficial vaginal bacteria by NHNK-608 fermentation products

[0146] In the experimental group, 4 ml of MRS and 1 ml of NHNK-608 fermentation product were added to a centrifuge tube, and in the control group, an equal volume of MRS liquid culture medium was added, and Lactobacillus jensenii was inoculated at a 1% inoculation rate, and cultured at 37°C for 24 hours, and the absorbance at OD=600nm was measured. The results are shown in Table 12 below:

[0147] Table 12: NHNK-608 fermentation products promote the proliferation of beneficial vaginal bacteria

[0148]

[0149] The results showed that the fermentation product of NHNK-608 has the effect of promoting the proliferation of vaginal lactic acid bacteria Lactobacillus jensenii, with the proliferation rate reaching 27.09%~30.87%.

[0150] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Lactobacillus crispatus ( Lactobacillus crispatus ), which is Lactobacillus crispatus ( Lactobacillus crispatus ) NHNK-608 was deposited in the China Center for Type Culture Collection on March 7, 2024, and its deposit number is CCTCC NO: M 2024421.

2. The use of Lactobacillus crispatus according to claim 1 in preparing a product for improving reproductive tract flora, characterized in that: The improvement of the reproductive tract flora includes inhibiting the proliferation of vaginal pathogens, and the inhibition of the proliferation of vaginal pathogens is to inhibit atypical Veillonella ( Veillonella atypica ) and Venereobacterium montpellierii ( Veillonella montpellierensis ) growth.

3. The use of Lactobacillus crispatus according to claim 2 in preparing a product for improving reproductive tract flora, characterized in that: The improvement of the reproductive tract flora includes regulating the virulence genes of vaginal pathogens, and the regulation of the virulence genes of vaginal pathogens is down-regulating the virulence genes of Candida albicans HWP1 , ALS1 , ALS3 and downregulation of virulence genes of Gardnerella vaginalis vly , sld , Pat , gtf , atm , stp At least one of .

4. The use of Lactobacillus crispatus according to claim 2 in preparing a product for improving reproductive tract flora, characterized in that: The method for improving the genital tract flora comprises competitively binding vaginal pathogenic fungal hyphae, wherein the competitively binding vaginal pathogenic fungal hyphae is binding Candida albicans hyphae ( Candida albicans ).

5. The use of Lactobacillus crispatus in the preparation of a product for improving reproductive tract flora according to claim 2, characterized in that: The improved reproductive tract flora includes agglutinating vaginal pathogens, wherein the agglutinating vaginal pathogens are agglutinating Candida albicans, atypical Veillonella, and Gardnerella vaginalis ( Gardnerella vaginalis ).

6. The use of Lactobacillus crispatus in the preparation of a product for improving reproductive tract flora according to claim 2, characterized in that: The improving of the reproductive tract flora includes promoting the proliferation of beneficial vaginal bacteria, and the promoting of the proliferation of beneficial vaginal bacteria is promoting Lactobacillus jensenii ( Lactobacillus jensenii )proliferation.

7. The use according to any one of claims 2 to 6, characterized in that: The Lactobacillus crispatus in the product for improving reproductive tract flora is one or both of the following (1) or (2): (1) The strain and / or inactivated bacteria of Lactobacillus crispatus according to claim 1; (2) The fermentation product of Lactobacillus crispatus according to claim 1.

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