Lactobacillus gasseri and its application in preparing products for improving reproductive tract health

Abstract

The present invention discloses a Lactobacillus gasseri and its application in preparing a product for improving reproductive tract health, and relates to the technical field of microorganisms. The present invention discloses a Lactobacillus gasseri strain, which is Lactobacillus gasseri ( Lactobacillus gasseri ) NHNK‑607, which was deposited in the China Center for Type Culture Collection on March 7, 2024, and its deposit number is CCTCC NO:M 2024420. Experiments have shown that NHNK‑607 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. It can be used to prepare medicines or products to improve vaginitis, etc.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Lactobacillus gasseri and an application thereof in preparing a product for improving reproductive tract health. 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] Under normal conditions, lactobacilli are the dominant bacteria in the vaginal flora, including Lactobacillus jensenii ( Lactobacillus jensenii ) and Lactobacillus crispatus ( Lactobacillus crispatus ), etc. Lactobacillus competitively adheres to vaginal epithelial cells, metabolizes and secretes lactic acid, hydrogen peroxide (H2O2), bacteriocins and other substances, which 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 related to 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 gasseri and applications thereof.

[0009] The present invention provides Lactobacillus gasseri ( Lactobacillus gasseri ), which is Lactobacillus gasseri ( Lactobacillus gasser ) NHNK-607, 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 2024420).

[0010] The present invention provides the use of the above-mentioned Lactobacillus gasseri in preparing a product for improving reproductive tract health, wherein the improvement of reproductive tract health comprises inhibiting the proliferation of vaginal pathogens, and the inhibition of the proliferation of vaginal pathogens comprises inhibiting the growth of atypical Veillonella and Venereella Montpellier.

[0011] Further, the improving reproductive tract health includes regulating the virulence genes of vaginal pathogens, and the regulating 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 At least one of .

[0012] Furthermore, the improving reproductive tract health comprises competitively binding to vaginal pathogenic fungal hyphae, and the competitively binding to vaginal pathogenic fungal hyphae is binding to Candida albicans hyphae.

[0013] Furthermore, the improvement of reproductive tract health includes agglutinating vaginal pathogens, and the agglutinating vaginal pathogens include agglutinating Candida albicans, atypical Veillonella, and Gardnerella vaginalis.

[0014] Furthermore, the improving reproductive tract health includes promoting the proliferation of beneficial vaginal bacteria, and the promoting the proliferation of beneficial vaginal bacteria includes promoting the proliferation of Lactobacillus jensenii and Lactobacillus crispatus.

[0015] Furthermore, the Lactobacillus gasseri in the product for improving reproductive tract health includes one or two of the following (1) or (2):

[0016] (1) the above-mentioned strains and / or inactivated bacteria of Lactobacillus gasseri;

[0017] (2) The fermentation and / or secretion products of the above-mentioned Lactobacillus gasseri.

[0018] The Lactobacillus gasseri disclosed in the present invention ( Lactobacillus gasseri ) NHNK-607, whose deposit number is CCTCC NO: M 2024420. Experiments have shown that NHNK-607 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 to improve reproductive tract health.

[0019] Biological Deposit Description

[0020] Lactobacillus gasseri ( Lactobacillus gasseri ) NHNK-607, 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 2024420. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Lactobacillus gasseri ( Lactobacillus gasseri ) MRS plate colony image and Gram staining image of NHNK-607;

[0022] Figure 2 Lactobacillus gasseri (Lactobacillus gasseri ) Coaggregation of NHNK-607 inactivated bacteria and pathogens;

[0023] Figure 3 Lactobacillus gasseri ( Lactobacillus gasseri ) Rapid coaggregation of NHNK-607 live bacteria and Candida albicans;

[0024] Figure 4 Lactobacillus gasseri ( Lactobacillus gasseri ) NHNK-607 inactivated bacteria combined with Candida albicans mycelium;

[0025] Figure 5 Lactobacillus gasseri ( Lactobacillus gasseri ) Gram staining of NHNK-607 inactivated bacteria combined with Candida albicans mycelium.

[0026] The following specific implementation manner will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0027] The present invention provides Lactobacillus gasseri and its application. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. 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 by preferred embodiments, and the relevant personnel can obviously change 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.

[0028] The Lactobacillus gasseri NHNK-607 of the present invention is derived from the intestinal tract of sea cucumber and is identified as Lactobacillus gasseri ( Lactobacillus gasseri ). This strain is Gram-positive, rod-shaped and slightly curved under a microscope; it is suitable for static growth in MRS liquid medium at 37°C, and the bacteria present white precipitation. It can form flat, diffuse colonies with regular edges when grown on MRS plates.

[0029] Lactobacillus gasseri ( Lactobacillus gasseri )NHNK-607, 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 2024420.

[0030] Further, the Lactobacillus gasseri provided by the present invention ( Lactobacillus gasseri) In the application described in the present invention, NHNK-607 exists in the form of live bacteria or inactivated bacteria, or in the form of fermentation products (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, live bacteria, and inactivated bacteria.

[0031] In vitro experiments show that the Lactobacillus gasseri of the present invention ( Lactobacillus gasseri ) NHNK-607 has the effect of inhibiting the growth of vaginal pathogens atypical Veillonella and Pseudomonas aeruginosa, with an inhibition rate of 27.27%~78.39%.

[0032] In vitro experiments show that the Lactobacillus gasseri of the present invention ( Lactobacillus gasseri NHNK-607 downregulates 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 The relative expression of genes was downregulated to 0.04~0.81 times.

[0033] In vitro experiments show that the Lactobacillus gasseri of the present invention ( Lactobacillus gasseri NHNK-607 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 0.06~0.91 times.

[0034] In vitro experiments show that the Lactobacillus gasseri of the present invention ( Lactobacillus gasseri ) NHNK-607 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 31.41%~94.41%.

[0035] In vitro experiments show that the Lactobacillus gasseri of the present invention ( Lactobacillus gasseri ) NHNK-607 has the function of agglutinating vaginal pathogens Candida albicans, atypical Veillonella and Gardnerella vaginalis, and the agglutination rate can reach 23.98%~43.66%.

[0036] In vitro experiments show that the Lactobacillus gasseri of the present invention ( Lactobacillus gasseri) NHNK-607 fermentation products can promote the proliferation of beneficial vaginal bacteria Lactobacillus jensenii and Lactobacillus crispatus, and the proliferation rate can reach 13.92%~44.19%.

[0037] 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:

[0038] Example 1 Isolation of NHNK-607

[0039] 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-607.

[0040] Gram staining microscopy: strain NHNK-607 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.

[0041] Example 2 Nucleic acid identification of NHNK-607

[0042] 1. 16S rDNA gene sequence analysis:

[0043] 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.

[0044] 2. Results

[0045] The PCR product was sequenced and compared with the standard sequences published in the GenBank database (BLASTN), and the NHNK-607 strain was identified as Lactobacillus gasseri ( Lactobacillus gasseri ).

[0046] Example 3: Experiment on the inhibition of vaginal pathogen growth by NHNK-607

[0047] 1. Preparation of NHNK-607 fermentation products

[0048] Pick out Lactobacillus gasseri ( Lactobacillus gasseri ) NHNK-607 single colony was cultured in MRS liquid medium at 37℃ for 48h and then diluted with MRS to adjust 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.

[0049] 2. Preparation of pathogenic bacteria solution

[0050] 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.

[0051] 3. Experiment on inhibiting the growth of pathogens

[0052] 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%

[0053] The results are shown in Table 1:

[0054] Table 1

[0055]

[0056] The results showed that the cell-free fermentation product of NHNK-607 could effectively inhibit the growth of atypical Veillonella and Pseudomonas aeruginosa vaginal pathogens.

[0057] Example 4 Experiment on NHNK-607 regulating the expression of virulence genes of Gardnerella vaginalis

[0058] 1. Preparation of NHNK-607 fermentation products and inactivated bacteria

[0059] Pick out Lactobacillus gasseri ( Lactobacillus gasseri ) NHNK-607 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.

[0060] 2. Experiment on regulating the expression of virulence genes of Gardnerella vaginalis

[0061] Gardnerella vaginalis isolated from vaginal secretions Gardnerella vaginalis ) BNCC354890 cryovials 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-607 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 cultured 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 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.

[0062] Formula: F=2 -ΔΔCT ,in:

[0063] △CT 实验 =CT 实验 -CT 内参（实验）;

[0064] △CT 对照 =CT 对照 -CT 内参（对照）;

[0065] △△CT=△CT 实验 -△CT 对照。

[0066] The results are shown in Tables 2 and 3 below:

[0067] Table 2

[0068]

[0069] Table 3

[0070]

[0071] The results showed that NHNK-607 had the effect of down-regulating the expression of virulence genes of Gardnerella vaginalis.

[0072] Example 5 Experiment on regulating the expression of virulence genes of Candida albicans by NHNK-607

[0073] 1. Preparation of NHNK-607 fermentation products and inactivated bacteria

[0074] The preparation method is as in Example 4.

[0075] 2. Experiment on regulating the expression of virulence genes of Candida albicans

[0076] 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-607 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.

[0077] The results are shown in Tables 4 and 5 below:

[0078] Table 4

[0079]

[0080] Table 5

[0081]

[0082] The results showed that the fermentation products and inactivated bacteria of NHNK-607 had the effect of down-regulating the expression of virulence genes of Candida albicans.

[0083] Example 6 Experiment on the inhibition of pathogenic bacteria biofilm formation by NHNK-607

[0084] 1. Preparation of NHNK-607 fermentation products and inactivated bacteria

[0085] The preparation method is as in Example 4.

[0086] 2. Preparation of pathogenic bacteria solution

[0087] 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.

[0088] 3. Experiment on inhibition of pathogenic bacteria biofilm

[0089] Take 100μL of pathogenic bacteria solution and add it to a 96-well plate. Add 100μL of NHNK-607 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 is completed, 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.

[0090] The calculation formula and results are shown in Tables 6 and 7 below:

[0091] Table 6: NHNK-607 fermentation products inhibit pathogenic bacterial biofilm formation

[0092]

[0093] Table 7: NHNK-607 inactivated bacteria inhibits pathogenic bacteria biofilm formation

[0094]

[0095] The results showed that the fermentation products and inactivated bacteria of NHNK-607 had the effect of inhibiting the biofilm formation of pathogenic bacteria.

[0096] Example 7 Experiment on the inhibition of pathogenic bacteria-fungus composite biofilm formation by NHNK-607

[0097] 1. Preparation of NHNK-607 fermentation products and inactivated bacteria

[0098] The preparation method is as in Example 4.

[0099] 2. Preparation of pathogenic bacteria solution

[0100] The preparation method is similar to that of Example 6.

[0101] 3. Experiment on inhibition of pathogenic bacteria-fungus composite biofilm

[0102] 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-607 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.

[0103] The calculation formula and results are shown in Tables 8 and 9 below:

[0104] Table 8: NHNK-607 fermentation products inhibit bacterial-fungal composite biofilm formation

[0105]

[0106] Table 9: NHNK-607 inactivated bacteria inhibits bacterial-fungal composite biofilm formation

[0107]

[0108] The results showed that NHNK-607 fermentation products / inactivated bacteria inhibited the formation of pathogenic bacteria-fungus composite biofilm.

[0109] Example 8 NHNK-607 Agglutination Pathogen Experiment

[0110] 1. Preparation of NHNK-607 inactivated bacteria

[0111] The preparation method is as in Example 4.

[0112] 2. Preparation of pathogenic bacteria solution

[0113] 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.

[0114] 3. Agglutination

[0115] The pathogen solution was mixed with the NHNK-607 inactivated bacteria solution at a volume ratio of 1:1. The pathogen plus PBS was used as a control. The mixture was allowed to stand for 30 minutes and the flocculation status was observed.

[0116] 4. Index determination

[0117] After 30 minutes of reaction, samples of NHNK-607 alone, pathogens, and the mixed reaction liquid of NHNK-607 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=600nm was measured. At the same time, the agglutination precipitate was stained with Gram stain and its bacterial agglutination state was observed. Figure 2 shown.

[0118] Calculation formula:

[0119] Agglutination rate (%) = [(Ax+Ay)-2Amix] / (Ax+Ay)×100%;

[0120] Note: Ax: OD measured by NHNK-607 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-607 and pathogens are mixed 600 Numeric value.

[0121] Table 10

[0122]

[0123] The results showed that NHNK-607 inactivated bacteria can agglutinate and capture Candida albicans, atypical Veillonella, and Gardnerella vaginalis, thereby achieving physical contact with the pathogens, thereby enabling NHNK-607 to downregulate the virulence genes of pathogens, reduce their pathogenicity, inhibit the formation of pathogenic biofilms, and reduce the colonization ability of pathogens.

[0124] Example 9 Rapid Agglutination of Candida albicans by Living Cells of NHNK-607

[0125] 1. Activation of NHNK-607 and preparation of bacterial suspension

[0126] NHNK-607 glycerol bacteria were inoculated into fresh MRS medium at a ratio of 2% and cultured at 37°C for 24 h. After the culture, live cells were obtained by centrifugation at 5000 rpm for 10 min, washed twice with sterile PBS, and then the OD was adjusted with PBS. 600 =1.0.

[0127] 2. Preparation of Candida albicans Liquid

[0128] The glycerol culture of Candida albicans was inoculated into fresh Sabouraud medium at a ratio of 2% and cultured at 37°C for 24 h. After the culture, the cells were centrifuged at 5000 rpm for 10 min to obtain the cells, washed twice with sterile PBS, and then adjusted to OD with PBS. 600 =1.0 standby.

[0129] 3. Determination of the time curve of NHNK-607 agglutination of Candida albicans

[0130] Coagulation: OD 600 =1.0 NHNK-607 and Candida albicans suspension were mixed evenly in a ratio of 1:1 (v / v), and an equal volume of PBS was added to Candida albicans as a control. After standing for 5 minutes, the flocculation state was observed.

[0131] Index determination: Take 50 μL of the upper layer of NHNK-607, Candida albicans, and NHNK-607 mixed with Candida albicans at 10s, 30s, 1min, 5min, and 10min after mixing, and measure its absorbance at 600nm. Finally, stain the agglutination precipitate with Gram stain and observe its bacterial agglutination state. Figure 3 shown.

[0132] Calculation formula:

[0133] Agglutination ability percentage (%) = [(Ax+Ay)-2Amix] / (Ax+Ay)×100%;

[0134] Note: Ax: the value measured at 600nm for NHNK-607 alone; Ay: the value measured at 600nm for Candida albicans alone; Amix: the value measured at 600nm for the reaction time after NHNK-607 and Candida albicans were mixed.

[0135] The agglutination rates of NHNK-607 on Candida albicans at different times are shown in Table 11 below:

[0136] Table 11

[0137]

[0138] The results showed that the live bacteria of NHNK-607 rapidly agglutinated Candida albicans within 30s-1min, and gradually entered the plateau phase in about 1min-5min.

[0139] Example 10 Agglutination of Candida albicans with living cells of NHNK-607 at different ratios

[0140] 1. Activation of NHNK-607 and preparation of bacterial suspension

[0141] NHNK-607 glycerol bacteria were inoculated into fresh MRS medium at a ratio of 2% and cultured at 37°C for 24 h. After the culture, the bacteria were centrifuged at 5000 rpm for 10 min, washed twice with sterile PBS, and then the OD was adjusted with PBS. 600 =1.0 standby.

[0142] 2. Preparation of Candida albicans Liquid

[0143] The glycerol culture of Candida albicans was inoculated into fresh Sabouraud medium at a ratio of 2% and cultured at 37°C for 24 h. After the culture, the cells were centrifuged at 5000 rpm for 10 min to obtain the cells, washed twice with sterile PBS, and then adjusted to OD with PBS. 600 =1.0 standby.

[0144] 3. NHNK-607 agglutination test of Candida albicans

[0145] Coaggregation: When the ratio of NHNK-607 to Candida albicans was 1:1, the OD 600 = 1.0 for NHNK-607 and OD 600 =1.0, 0.25 ml of each suspension of Candida albicans was added to a centrifuge tube at a ratio of 1:1 (v / v), and the total volume was supplemented with PBS to 1 ml of the bacterial suspension and mixed evenly. An equal volume of PBS was added to Candida albicans as a control, and the flocculation state was observed after standing for 1 min. When the ratio of NHNK-607 to Candida albicans was 1:2, the OD 600 =1.0 NHNK-607 and Candida albicans suspension in a ratio of 1:2 (v / v), 0.25 ml of NHNK-607 and 0.5 ml of Candida albicans were added to the centrifuge tube, and the total volume was made up to 1 ml with sterile PBS. Then the bacterial suspension was mixed evenly, and an equal volume of PBS was added to Candida albicans as a control. After standing for 1 minute, the flocculation status was observed. Figure 4 shown.

[0146] Index determination: After 1 min of reaction, 50 μL of the upper layer liquid of NHNK-607, Candida albicans, and NHNK-607 mixed with Candida albicans was taken to determine its absorbance at 600 nm. The calculation formula was based on the formula of Example 9.

[0147] The experimental results are shown in Table 12:

[0148] Table 12

[0149]

[0150] The experimental results showed that NHNK-607: Candida albicans had similar agglutination rates at 1:1 and 1:2, ranging from 59.32% to 64.45%. NHNK-607 was able to rapidly coagulate twice its own amount of Candida albicans in one minute. Figure 4 shown.

[0151] Example 9: Experiment on the inhibition of Candida albicans hyphae formation by NHNK-607

[0152] 1. Preparation of NHNK-607 fermentation products and inactivated bacteria

[0153] The preparation method is as in Example 4.

[0154] 2. Induction of hyphae morphology of Candida albicans

[0155] 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.

[0156] 3. Experiment on the inhibition of Candida albicans hyphae formation by NHNK-607

[0157] In the experimental group, 100 μL of FBS-resuspended Candida albicans was added to a 96-well plate, and 100 μL of NHNK-607 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 13 below:

[0158]

[0159] The results showed that NHNK-607 fermentation products and inactivated bacteria could effectively inhibit the hyphae formation of Candida albicans.

[0160] Example 10 Experiment on the combination of NHNK-607 inactivated bacteria and Candida albicans hyphae

[0161] 1. Preparation of NHNK-607 inactivated bacteria

[0162] The preparation method is similar to that of Example 3.

[0163] 2. Preparation of Candida albicans mycelium

[0164] 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.

[0165] 3. Experiment on inactivated bacteria combined with Candida albicans mycelium

[0166] Add 0.7 ml of Candida albicans mycelium and 0.7 ml of NHNK-607 inactivated bacteria to 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 5 shown.

[0167] There is a significant size difference between the inactivated NHNK-607 bacteria and the mycelia of Candida albicans, and the inactivated NHNK-607 bacteria and the mycelia of Candida albicans can be distinguished by observation. The results showed that the inactivated NHNK-607 bacteria can significantly bind to the mycelial part of the mycelia of Candida albicans, and has a competitive inhibition effect on the binding of other pathogens to the mycelia of Candida albicans.

[0168] Example 11 Experiment on promoting the proliferation of beneficial vaginal bacteria by NHNK-607

[0169] 1. Preparation of NHNK-607 fermentation products

[0170] The preparation method is as in Example 3.

[0171] 2. Collection and cultivation of beneficial vaginal bacteria

[0172] Lactobacillus jensenii ( Lactobacillus jensenii )ATCC 25258, Lactobacillus crispatus ( Lactobacillus crispatus ) A single colony of CICC 24879 was inoculated into MRS broth and cultured anaerobically at 37°C for 48 h.

[0173] 3. Experiment on promoting the proliferation of beneficial vaginal bacteria by NHNK-607 fermentation products

[0174] In the experimental group, 4 ml of MRS and 1 ml of NHNK-607 fermentation product were added to a centrifuge tube, and in the control group, an equal volume of MRS liquid culture medium was added, and vaginal beneficial bacteria liquid 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 14 below:

[0175] Table 14: NHNK-607 fermentation products promote the proliferation of Lactobacillus

[0176]

[0177] The results showed that the fermentation product of NHNK-607 has the effect of promoting the proliferation of beneficial vaginal bacteria Lactobacillus jensenii and Lactobacillus crispatus, with the proliferation rate reaching 27.09%~30.87%.

[0178] 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 gasseri ( Lactobacillus gasseri ), the strain is Lactobacillus gasseri NHNK-607, which was deposited in the China Center for Type Culture Collection on March 7, 2024, and its deposit number is CCTCC NO: M 2024420.

2. The use of Lactobacillus gasseri in preparing a product for improving reproductive tract health according to claim 1, characterized in that: The improving of reproductive tract health includes inhibiting the proliferation of vaginal pathogens, and the inhibiting of the proliferation of vaginal pathogens includes inhibiting atypical Veillonella ( Veillonella Atypica ) and Venereobacterium montpellierii ( Veillonella montpellierensis ) growth.

3. The use according to claim 2, characterized in that: The improving of reproductive tract health comprises regulating the virulence genes of vaginal pathogens, and the regulating of the virulence genes of vaginal pathogens comprises down-regulating the mycelial cell wall protein gene in the virulence genes of Candida albicans HWP1 , cell surface glycoprotein genes ALS1 , cell surface glycoprotein genes ALS3 and down-regulation of vaginal lysozyme gene in the virulence 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 At least one of .

4. The use according to claim 2, characterized in that: The improving of reproductive tract health comprises competitively binding vaginal pathogenic fungal hyphae, wherein the competitively binding vaginal pathogenic fungal hyphae is binding to Candida albicans ( Candida albicans ) mycelium.

5. The use according to claim 2, characterized in that: The improving of reproductive tract health includes agglutinating vaginal pathogens, wherein the agglutinating vaginal pathogens include agglutinating Candida albicans, atypical Veillonella, and Gardnerella vaginalis ( Gardnerella vaginalis ).

6. The use according to claim 2, characterized in that: The improving of reproductive tract health includes promoting the proliferation of beneficial vaginal bacteria, and the promoting of the proliferation of beneficial vaginal bacteria includes promoting Lactobacillus jensenii ( Lactobacillus jensenii )、Lactobacillus crispatus( Lactobacillus crispatus )proliferation.

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

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