Lactobacillus jensenii for regulating and controlling expression of host defense peptide and interferon to reduce host infection risk and metagen of lactobacillus jensenii

Abstract

The invention discloses a Lactobacillus jensenii strain for regulating and controlling expression of host defense peptide and interferon to reduce the risk of host infection and a metagen thereof, and belongs to the technical field of microorganisms. The lactobacillus jensenii disclosed by the invention is separated from the vagina of a healthy female, and has the effects of improving the immunocompetence of the vagina and enhancing the self-defense of a host, which are specifically embodied in that: the expression and secretion of host defense peptide of vaginal tissues of an organism are promoted; the generation of body interferon is reduced; the level of myeloperoxidase is reduced; characterizing the fungal infection resistance of the organism through periodic acid Schiff staining; the pathological characterization of the body vagina is improved. Therefore, the lactobacillus jensenii has a huge application prospect in products for improving vaginal immunity, enhancing host defense and preventing infection.

Description

Technical Field

[0001] The invention relates to a Lactobacillus jensenii strain capable of regulating the expression of host defense peptides and interferons to reduce the risk of host infection and a postbiotic thereof, belonging to the technical field of microorganisms. Background Art

[0002] In recent years, the incidence of vaginal infectious diseases such as bacterial vaginosis and fungal vaginitis has been on the rise. Such infections not only have a serious impact on women's reproductive health, but may also further cause a series of complications, such as pelvic inflammatory disease, infertility, etc. Therefore, finding effective prevention and treatment methods is of great clinical significance.

[0003] Traditional methods of treating vaginal infections mainly rely on antibiotics or antifungal drugs. However, long-term use of antibiotics may lead to the development of drug-resistant strains and destroy the host's normal vaginal microecological system, further aggravating the condition. Therefore, preventing and treating vaginal infections by regulating the host immune system or restoring the balance of the vaginal microecological system is the key to current research. In addition to probiotics themselves, postbiotics (i.e., probiotic metabolites or inactivated probiotics) also have significant potential in regulating host immunity and anti-infection. Postbiotics do not rely on living cells and have good stability. Postbiotics do not need to be refrigerated, which can reduce costs and avoid the safety risks that may be caused by live bacteria, while retaining the ability to regulate immunity and inhibit pathogens.

[0004] Azole antifungal drugs (such as fluconazole, itraconazole, etc.) are the first choice for the treatment of candidal vaginitis. These drugs can be taken orally or applied topically, and they work mainly by inhibiting the biosynthetic pathway of fungi. With the emergence of drug resistance, especially the increase in Candida strains resistant to the commonly used fluconazole, researchers are developing a new generation of antifungal drugs. Clinical studies have shown that oral use of probiotics, such as Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14, helps reduce the recurrence of vaginal infections, especially bacterial vaginosis and infections caused by Candida albicans. However, the efficacy of probiotics may be affected by individual differences, strain selection and application methods. Therefore, the development of more stable and effective probiotic products has become a future research direction. Lactobacillus jensenii belongs to the community state of vaginal flora CST V, which is a relatively healthy state of vaginal microbial community, which can maintain the acidic environment of the vagina and has a lower risk of infection. The patent (CN117771306A) mentions a probiotic agent and a preparation method thereof, which contains Lactobacillus jensenii as one of the active ingredients. The main effects of the probiotic agent are to regulate intestinal function, treat diarrhea and relieve alcohol, but Lactobacillus jensenii that can effectively prevent and / or treat vaginal Candida albicans infection has not yet been found. Summary of the invention

[0005] In view of the deficiencies of the above-mentioned prior art, the present invention provides a strain of Lactobacillus jensenii and its postbiotics that regulate the secretion of host interferon and host defense peptides to enhance anti-infection ability, aiming to solve the technical problems of the prior art in preventing vaginal infection, reducing the occurrence and development of diseases, and research on reducing the risk of host infection, and Lactobacillus jensenii and its postbiotics that can enhance the host defense ability.

[0006] The first technical solution provided by the present invention is a strain of Lactobacillus jensenii CCFM1431, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 20, 2024, with a deposit number of GDMCC No: 65157. The Lactobacillus jensenii CCFM1431 was isolated from the vagina of a healthy woman.

[0007] The second technical solution provided by the present invention is a microbial preparation containing the Lactobacillus jensenii CCFM1431.

[0008] In some embodiments, the content of Lactobacillus jensenii CCFM1431 in the microbial preparation is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0009] The third technical solution provided by the present invention is a postbiotic containing the Lactobacillus jensenii CCFM1431.

[0010] In certain embodiments, the postbiotics include but are not limited to the lysate of the Lactobacillus jensenii CCFM1431.

[0011] In certain embodiments, the method for preparing the postbiotics is to culture the Lactobacillus jensenii CCFM1431 to a stable period, homogenize the bacterial solution under high pressure, and then sterilize it to obtain the postbiotics.

[0012] In certain embodiments, the postbiotics may be used in liquid form or after freeze-drying.

[0013] The fourth technical solution provided by the present invention is a product containing the Lactobacillus jensenii CCFM1431 or the microbial preparation or the postbiotic.

[0014] In certain embodiments, the product is a medicine or a hygiene product.

[0015] Furthermore, the medicine comprises the above-mentioned strain and / or preparation, and a pharmaceutically acceptable carrier.

[0016] Furthermore, the carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0017] Furthermore, the dosage form of the drug includes granules, capsules, tablets, pills, suppositories or oral liquids.

[0018] Furthermore, the medicines include oral enteric-coated tablets and capsules, oral liquids; vaginal suppositories, tablets, gelatin capsules, sprays, creams, and gels.

[0019] Furthermore, the sanitary products include sanitary wet wipes, sanitary napkins, sanitary pads, sanitary tampons, sanitary napkins, vaginal washes, and women's antibacterial / antibacterial washes.

[0020] The fifth technical solution provided by the present invention is the use of the Lactobacillus jensenii CCFM1431 or the microbial preparation in the preparation of a product for preventing and / or treating vaginal Candida albicans infection.

[0021] In certain embodiments, the product has at least one of the following effects: reducing the expression of virulence factors of pathogenic bacteria; promoting the expression of animal host defense peptide β-defensin and CRAMP genes; reducing the production of animal interferon IFN-γ; and improving mouse vaginal flora and vaginal pathological manifestations.

[0022] In certain embodiments, the product is a medicine or a hygiene product.

[0023] Furthermore, the medicine comprises the above-mentioned strain and / or preparation, and a pharmaceutically acceptable carrier.

[0024] Furthermore, the carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0025] Furthermore, the dosage form of the drug includes granules, capsules, tablets, pills, suppositories or oral liquids.

[0026] Furthermore, the medicines include oral enteric-coated tablets and capsules, oral liquids; vaginal suppositories, tablets, gelatin capsules, sprays, creams, and gels.

[0027] Furthermore, the sanitary products include sanitary wet wipes, sanitary napkins, sanitary pads, sanitary tampons, sanitary napkins, vaginal washes, and women's antibacterial / antibacterial washes.

[0028] Beneficial effects:

[0029] The present invention provides a strain of Lactobacillus jensenii CCFM1431, which is isolated from the vagina of a healthy woman and has the effects of improving the immunity of the vagina and strengthening the self-defense of the host, which is specifically embodied in: (1) reducing the expression of virulence factors of pathogenic bacteria; (2) improving the expression of animal host defense peptide β-defensin and CRAMP genes; (3) reducing the production of animal interferon IFN-γ; (4) improving the vaginal flora of mice; and (5) pathological manifestations. Therefore, the Lactobacillus jensenii has a huge application prospect in products for improving the immunity of the vagina and strengthening the self-defense of the host.

[0030] Biomaterial Deposit

[0031] The Lactobacillus jensenii CCFM1431 provided by the present invention was isolated from the vagina of a healthy woman and was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 20, 2024. The deposit number is GDMCC No: 65157, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : Flowchart of animal experiments.

[0033] Figure 2 :The effect of topical Lactobacillus jensenii CCFM1431 on the load of Candida albicans in the vaginal tissue lavage fluid of animals, the expression of Candida albicans virulence factor ALS3 and the host defense peptide CRAMP gene.

[0034] Figure 3 :The effect of oral Lactobacillus jensenii CCFM1431 on the load of Candida albicans in the vaginal lavage fluid of animals and the expression of Candida albicans virulence factor ALS3 in vaginal tissue.

[0035] Figure 4 :The effect of oral administration of Lactobacillus jensenii CCFM1431 on the expression of host defense peptide β-defensin and CRAMP gene in animal vaginal tissue.

[0036] Figure 5 :The effect of oral administration of Lactobacillus jensenii CCFM1431 on the production of interferon IFN-γ in animal serum.

[0037] Figure 6 :The effect of oral administration of Lactobacillus jensenii CCFM1431 on the production of myeloperoxidase (MPO) in animal tissues.

[0038] Figure 7 : Effects of oral administration of Lactobacillus jensenii CCFM1431 on vaginal flora in animals.

[0039] Figure 8 :The effect of oral administration of Lactobacillus jensenii CCFM1431 on periodic acid-Schiff staining of vaginal tissue in mice.

[0040] Fig. 9 :The effect of oral administration of Lactobacillus jensenii CCFM1431 on vaginal pathological characteristics in mice.

[0041] (Different lowercase letters in the figure indicate significant differences between the groups, p < 0.05) DETAILED DESCRIPTION

[0042] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0043] The culture medium involved in the following examples is as follows:

[0044] MRS solid medium ( / L): 5g peptone, 5g yeast extract powder, 15g glucose, 3g disodium hydrogen phosphate, 1mL Tween-80, 3g dipotassium hydrogen phosphate, 3g diammonium hydrogen citrate, 0.1g magnesium sulfate heptahydrate, 0.05g manganese sulfate monohydrate, 20g agar, pH 6.2-6.4.

[0045] MRS liquid medium ( / L): 5g peptone, 5g yeast extract powder, 15g glucose, 3g disodium hydrogen phosphate, 1mL Tween-80, 3g dipotassium hydrogen phosphate, 3g diammonium hydrogen citrate, 0.1g magnesium sulfate heptahydrate, 0.05g manganese sulfate monohydrate, pH 6.2-6.4.

[0046] SDA medium ( / L): animal tissue pepsin hydrolysate and trypsin casein 10g, glucose 40g, agar 15g, pH 5.6±0.2.

[0047] SDB medium ( / L): animal tissue pepsin hydrolysate and trypsin casein 10g, glucose 20g, pH 5.6±0.2.

[0048] SDA medium (containing chloramphenicol) ( / L): 10 g of a mixture of equal amounts of animal tissue pepsin hydrolysate and trypticase, 40 g of glucose, 15 g of agar, 0.1 g of chloramphenicol, pH 5.6±0.2.

[0049] The strains and animals involved in the following examples are as follows:

[0050] Lactobacillus delbrueckii DM8909 was isolated from Dingjunsheng vaginal lactobacillus capsules and stored in the strain bank of Food Biotechnology Center of Jiangnan University.

[0051] Lactobacillus jensenii CCFM1431 was isolated from female vagina and stored in the strain bank of Food Biotechnology Center of Jiangnan University.

[0052] 7-week-old female SPF BALB / c mice, weighing 18-20 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (production license number SCXK (Beijing) 2012-0001).

[0053] Candida albicans SC5314 was purchased from Guangdong Institute of Microbiology Culture Collection Center (GDMCC).

[0054] The preparation method of the bacterial solution involved in the following examples is as follows:

[0055] Live Lactobacillus jensenii CCFM1431 (CCFM1431-L): Lactobacillus jensenii CCFM1431 was streaked and isolated in MRS solid medium, cultured in an anaerobic incubator at 37°C for 36 h, single colonies were picked and placed in 5 mL of MRS liquid medium, cultured in an anaerobic incubator at 37°C for 20 h, inoculated into 1 L of culture system at 4% inoculum, cultured in an incubator at 37°C for 20 h, and centrifuged to obtain bacterial sludge, and the bacterial solution concentration was adjusted to 5×10 9 CFU / mL is a suspension of live bacteria of Lactobacillus jensenii CCFM1431. Lactobacillus delbrueckii DM8909 was prepared as above and is recorded as DM8909-L.

[0056] Lactobacillus jensenii CCFM1431 postbiotics (CCFM1431-D): 5×10 9 The live bacterial suspension of Lactobacillus jensenii CCFM1431 at CFU / mL was homogenized 10 times (800-1200 MPa) and pasteurized (65°C for 30 min) in a high-pressure homogenizer, then freeze-dried and stored in a -80°C refrigerator. It was resuspended to the original volume with sterile saline before the experiment.

[0057] Candida albicans suspension: SC5314 was streaked on SDA medium and cultured in a 28°C incubator for 48 h. A single colony was picked and inoculated into SDB medium for expansion. The final concentration of the bacterial suspension was adjusted to 5 × 10 8 CFU / mL.

[0058] Estradiol: 0.1 mg of β-estradiol is dissolved in 0.05 mL of sesame oil and is ready for use.

[0059] Example 1 Isolation and identification of Lactobacillus jensenii CCFM1431

[0060] The specific steps are as follows:

[0061] Collect vaginal swab samples from healthy women and place them in an EP tube containing 1 mL of sterile saline. Pipette 0.2 mL into 1.8 mL of sterile saline to obtain 10 -1 Dilution, then draw 0.5mL 10-1 Dilute in 4.5 mL of saline to obtain 10 -2 Dilution, follow this procedure to obtain 10 -3 , 10 -4 , 10 -5 , 10 -6 Gradient dilution. Take 10 -4 , 10 -5 , 10 -6 Place 1 mL of each dilution in a plate, pour into MRS solid culture medium, mix gently, and after the culture medium solidifies, invert and culture at 37°C for 48 h.

[0062] Select colonies of different morphologies and streak them on MRS plates for purification. Pick the purified single colony and inoculate it into 5mL liquid culture medium and culture it at 37℃ for 48h. Take 1.5mL of cultured bacterial solution, centrifuge it at 6000r / min for 3min, discard the supernatant, add 1.5mL of sterile water to wash 3 times, resuspend it in 1.5mL of sterile water, and use it as a template for bacterial identification. Set up a PCR system with a volume of 20μL, add 0.5μL forward primer (10μM), 0.5μL reverse primer (10μM), 10μL 2×Taq Mixture, 0.5μL bacterial suspension, and 8.5μL double distilled water. Primer information is shown in Table 1.

[0063] Table 1: Primer information

[0064]

[0065] PCR conditions: 95°C, 5min; 95°C, 10s; 55°C, 30s; 72°C, 30s; step2-4 30×; 72°C, 5min; 12°C, 2min. The PCR product was sent to a professional sequencing company, and the sequencing results were searched and compared with similarity in GeneBank using BLAST, and it was identified as Lactobacillus jensenii.

[0066] For the correctly identified strains, take 1.5 mL of bacterial solution into a 2 mL strain storage tube, centrifuge at 6000 r / min for 3 min, remove the supernatant in a clean bench, add 1 mL of 30% sterile glycerol, mix thoroughly with a vortex oscillator, and store in a -80°C refrigerator.

[0067] 16S sequence information (SEQ ID No.1):

[0068]

[0069] Example 2 Application of external use of bacterial sludge lysate of Lactobacillus jensenii CCFM1431 in improving mice's resistance to pathogenic bacteria infection Experimental animals and strains:

[0070] Twenty-five 7-week-old SPF female BALB / c mice, weighing 18-20 g, were randomly divided into 5 cages, with 5 mice in each cage.

[0071] Candida albicans SC5314 was purchased from Guangdong Institute of Microbiology Culture Collection Center (GDMCC). Figure 1 Flow chart of animal experiments.

[0072] Table 2: Animal experimental plan and grouping

[0073]

[0074] The specific steps are as follows:

[0075] The vaginal infection prevention experiment adopted the method of intervention first and then modeling. The blank group and the model group were required to be vaginally inoculated with 20 μL of normal saline for 17 consecutive days, and the experimental group was vaginally inoculated with 20 μL of 5×10 9 CFU / mL live bacterial liquid was used to prepare the bacterial lysate of Lactobacillus delbrueckii DM8909 and the bacterial lysate of Lactobacillus jensenii CCFM1431. The mice were inverted for 1-2 minutes. On days 12-15, the blank group was subcutaneously injected with 50 μL of normal saline, and the model group and the experimental group were subcutaneously injected with 50 μL of estradiol to induce estrus (subcutaneous injection on days 12 and 15). On days 15-17, the model group and the experimental group used a pipette to draw 20 μL of 5×10 8 CFU / mL of Candida albicans suspension was slowly injected into the vagina of mice, and the mice were inverted for 1-2 minutes. The blank group was inoculated with normal saline in the same way once a day. The experimental period was 17 days. At the end of the intervention (day 18), 50 μL of normal saline was sucked out of the vagina of the mice with a gun tip each time to take samples, and finally 300 μL of vaginal lavage fluid was collected for subsequent analysis of the Candida albicans load and flora in the vagina of mice. At the same time, on day 18, all experimental mice were killed and the vaginal tissue was stripped for detection of Candida albicans virulence factors and host defense peptide CRAMP in vaginal tissue, as well as subsequent experimental tissue pathology analysis and periodic acid Schiff staining analysis, and animal serum was used for interferon (IFN-γ) determination.

[0076] Determination method: After the animals were killed, blood was collected from the eyeballs and animal serum was obtained by centrifugation for subsequent interferon (IFN-γ) determination. After the mice were killed, vaginal tissues were dissected: a portion of the tissues were homogenized with pre-cooled RIPA lysis buffer (Biyuntian Biotechnology Co., Ltd.) and a mixture of protease inhibitors. The samples were centrifuged at 12000r / min for 15min at 4°C, and the vaginal tissue supernatant was taken for mBD3, CRAMP, and MPO content determination according to the kit instructions (Nanjing Senbeijia Biotechnology Co., Ltd.). A portion of the tissues were ground and reverse transcribed to obtain cDNA for gene expression determination of host defense peptides mBD3 and CRAMP. The primers are shown in Table 3 below. Another portion of the vaginal tissue was placed in a 4% polyformaldehyde solution for periodic acid Schiff staining and histopathological examination. DNA was extracted from the collected vaginal lavage fluid, and the purified DNA fragments were sequenced by 16S RNAV3-V4. The overall structural changes of the mouse vaginal flora were analyzed based on the sequencing results. Alpha diversity was used to analyze the effects of each intervention group on the richness, uniformity and community structure of the vaginal flora in mice.

[0077] Table 3 Animal experiment primer information

[0078]

[0079] Periodic acid Schiff staining and histopathological observation: Vaginal tissue was fixed with 4% paraformaldehyde solution, embedded in paraffin, sliced ​​into 5 mm thick sections, and stained with periodic acid Schiff (PAS) and hematoxylin and eosin (H&E). Vaginal tissue samples were observed under a pathological section scanner (Panoramic MIDI, 3DHistech Ltd, Budapest, Hungary) at a magnification of 20 times.

[0080] Experimental results:

[0081] (1) Candida albicans load in mouse vaginal lavage fluid and expression of Candida albicans virulence factor (ALS3) in tissues

[0082] In the mouse vaginal Candida albicans infection model, the detection load can quantitatively assess the extent of infection ( Figure 2 A) Pipette 0.1 mL of the collected vaginal lavage fluid into 0.9 mL of sterile saline to obtain 10 -1 Dilution, then draw 0.1mL10 -1 Dilute in 0.9 mL of saline to obtain 10 -2 Dilution, follow this procedure to obtain 10 -3 , 10 -4 , 10 -5 , 10 -6Gradient dilution. Take 0.1 ml of each of the above gradient dilutions in a plate, pour in the chloramphenicol SDA medium, mix gently, wait for the medium to solidify, invert and culture at 28°C for 48 hours, and record the number of live bacteria. Chloramphenicol has an inhibitory effect on both Gram-positive and Gram-negative bacteria and has a stronger effect on the latter. It is often used for the selective separation of fungi and the growth and development of antibacterial bacteria. We divide the calculated number of Candida albicans by the logarithmic function to calculate the result. The average load of Candida albicans in the model group was 4.18 after taking the logarithm, while Candida albicans decreased to 3.38 after intervention with Lactobacillus delbrueckii DM8909 (p < 0.001), and the reduction of Candida albicans after intervention with topical Lactobacillus gasseri live bacteria and inactivated bacteria solution was more significant, 2.22 and 3.24 respectively (p < 0.0001). The reduction in the load of Candida albicans may mean that after the intervention of Lactobacillus, the immune system was regulated to successfully control the infection.

[0083] ALS3 (Agglutinin-like sequence 3) is an important virulence factor of Candida albicans and is closely related to its pathogenicity in host tissues. It is an adhesion protein of Candida albicans and is involved in the interaction between Candida albicans and host cells, especially in the infection process in vaginal tissues, where it plays a vital role. By detecting the expression of ALS3 mRNA, it can be seen that ( Figure 2 B), the model group highly expressed virulence factor ALS3 of 2.05, the Lactobacillus delbrueckii DM8909 sludge lysate group (DM8909-sub) expressed virulence factor ALS3 of 1.71, which was not significantly different from the model group, and the Lactobacillus jensenii sludge lysate group (CCFM1431-sub) expressed virulence factor ALS3 of 0.79 (61.5% lower than the model group). The Lactobacillus jensenii CCFM1431 sludge lysate group was able to significantly reduce the expression of Candida albicans virulence factors, and the effect was better than that of the Lactobacillus delbrueckii DM8909 sludge lysate group. Therefore, Lactobacillus jensenii CCFM1431 can effectively reduce the expression of Candida albicans virulence factor ALS3 and reduce the adhesion of pathogenic bacteria.

[0084] (2) Host defense peptide CRAMP gene expression in mouse vaginal tissue

[0085] Host defense peptide CRAMP (Cathelicidin-related antimicrobial peptide) is an important innate immune factor that plays a key antibacterial role. CRAMP significantly improves the host's ability to resist infection by directly killing pathogenic microorganisms, regulating inflammatory responses, and enhancing barrier function. By detecting the expression of host defense peptide CRAMP mRNA, it can be seen that ( Figure 2C), the gene expression of host defense peptide CRAMP in the blank group was 1.26, the model group had low expression of host defense peptide CRAMP of 0.75, the Lactobacillus delbrueckii DM8909 sludge lysate group (DM8909-sub) expressed host defense peptide CRAMP of 1.72 (129.3% higher than the model group), and the Lactobacillus jenseni sludge lysate group (CCFM1431-sub) expressed host defense peptide CRAMP of 1.75 (133.3% higher than the model group). Both the Lactobacillus delbrueckii DM8909 sludge lysate group and the Lactobacillus jenseni CCFM1431 sludge lysate group were able to significantly increase the expression of host defense peptide CRAMP. Therefore, external use of Lactobacillus jenseni CCFM1431 sludge lysate can upregulate the gene expression level of CRAMP, activate the host immune response, thereby enhancing the local antibacterial activity and improving the host's self-defense ability.

[0086] Example 3 Application of oral Lactobacillus jensenii CCFM1431 and its postbiotics in improving mice's resistance to pathogenic bacterial infection

[0087] Experimental animals and strains:

[0088] Twenty-five 7-week-old SPF female BALB / c mice, weighing 18-20 g, were randomly divided into 5 cages, with 5 mice in each cage.

[0089] Candida albicans SC5314 was purchased from Guangdong Institute of Microbiology Culture Collection Center (GDMCC). Figure 1 Flow chart of animal experiments.

[0090] Table 4: Animal experimental plan and grouping

[0091]

[0092] The specific steps are as follows:

[0093] The vaginal infection prevention experiment adopted the method of intervention first and then modeling. The blank group and the model group were gavaged with 200 μL of normal saline for 17 consecutive days, and the experimental group was gavaged with 200 μL of 5×10 9 CFU / mL of live bacteria of Lactobacillus delbrueckii DM8909, live bacteria of Lactobacillus jensenii CCFM1431, 5×10 9 CFU / mL bacterial suspension prepared from Lactobacillus jensenii CCFM1431 postbiotics. On days 12-15, the blank group was subcutaneously injected with 50 μL of saline, and the model group and the experimental group were subcutaneously injected with 50 μL of estradiol to induce estrus (subcutaneous injection on days 12 and 15). On days 15-17, the model group and the experimental group used a pipette to draw 20 μL of 5×10 8CFU / mL of Candida albicans suspension was slowly injected into the vagina of mice, and the mice were inverted for 1-2 minutes. The blank group was inoculated with normal saline in the same way once a day. The experimental period was 17 days. At the end of the intervention (day 18), 50 μL of normal saline was sucked out of the vagina of the mice with a gun tip each time to take samples, and 300 μL of vaginal lavage fluid was finally collected for subsequent analysis of the vaginal flora of mice. At the same time, on day 18, all experimental mice were killed and vaginal tissues were stripped for detection of the expression of Candida albicans virulence factors, myeloperoxidase MPO, host defense peptides CRAMP and mBD3 in vaginal tissues, as well as subsequent experimental tissue pathology analysis and periodic acid Schiff staining analysis, and animal serum was used for interferon (IFN-γ) determination.

[0094] Determination method: After the animals were killed, blood was collected from the eyeballs and animal serum was obtained by centrifugation for subsequent interferon (IFN-γ) determination. After the mice were killed, vaginal tissues were dissected: part of the tissue was homogenized with pre-cooled RIPA lysis buffer (Biyuntian Biotechnology Co., Ltd.) and a mixture of protease inhibitors. The samples were centrifuged at 12000r / min for 15min at 4°C, and the vaginal tissue supernatant was taken for MPO content determination according to the kit instructions. Part of the tissue was ground and reverse transcribed to obtain cDNA for gene expression determination of host defense peptides mBD3 and CRAMP. The primers are shown in Table 5. Another part of the vaginal tissue was placed in 4% paraformaldehyde solution for periodic acid Schiff staining and histopathological examination. DNA was extracted from the collected vaginal lavage fluid, and the purified DNA fragments were sequenced by 16S RNAV3-V4. The overall structural changes of the mouse vaginal flora were analyzed based on the sequencing results. Alpha diversity was used to analyze the effects of each group of interventions on the richness, uniformity and community structure of the mouse vaginal flora.

[0095] Table 5 Animal experiment primer information

[0096]

[0097] Periodic acid Schiff staining and histopathological observation: Vaginal tissue was fixed with 4% paraformaldehyde solution, embedded in paraffin, sliced ​​into 5 mm thick sections, and stained with periodic acid Schiff (PAS) and hematoxylin and eosin (H&E). Vaginal tissue samples were observed under a pathological section scanner (Panoramic MIDI, 3DHistech Ltd, Budapest, Hungary) at a magnification of 20 times.

[0098] Experimental results:

[0099] (1) Candida albicans load in mouse vaginal lavage fluid and expression of virulence factor (ALS3) in tissues

[0100] Quantitative detection of the load of Candida albicans in the vagina of mice can help us evaluate the severity of Candida albicans infection, monitor the progression of infection, and the effectiveness of our use of probiotics to prevent fungal infections. The load of Candida albicans was detected by Sabouraud dextrose solid medium containing chloramphenicol ( Figure 3 A), the logarithm of the average load of Candida albicans in the model group was 4.18, while after intervention with Lactobacillus delbrueckii DM8909, the Candida albicans load decreased to 3.65 (p < 0.01), and after intervention with oral Lactobacillus gasseri live bacteria and inactivated bacteria solution, the Candida albicans load decreased more significantly, to 3.20 (p < 0.0001) and 3.58 (p < 0.01), respectively.

[0101] ALS3 (Adhesin-like protein 3) is an important virulence factor in Candida albicans. It plays a key role in the adhesion, invasion and biofilm formation of the strain. ALS3 promotes the adhesion of Candida albicans by binding to the surface receptors of host cells, thereby enhancing its pathogenicity. In addition, ALS3 may also play a role in immune escape, helping the fungus resist the host's immune response. By detecting the expression of ALS3 mRNA, it can be seen that ( Figure 3 B), the model group highly expressed the virulence factor ALS3, which was 2.26, the Lactobacillus delbrueckii DM8909 live bacteria group (DM8909-L) expressed the virulence factor ALS3 of 0.64 (71.7% lower than the model group), the Lactobacillus jenseni live bacteria group (CCFM1431-L) expressed the virulence factor ALS3 of 0.58 (74.3% lower than the model group), and the Lactobacillus jenseni postbiotic group (CCFM1431-D) expressed the virulence factor ALS3 of 0.57 (74.8% lower than the model group). The Lactobacillus jenseni CCFM1431 live bacteria group and the postbiotic group can significantly reduce the expression of Candida albicans virulence factors, and the effect is better than that of the Lactobacillus delbrueckii DM8909 live bacteria group. Therefore, oral administration of Lactobacillus jenseni CCFM1431 can effectively reduce the expression of Candida albicans virulence factor ALS3 and inhibit the adhesion of pathogenic bacteria. (2) Gene expression of host defense peptide mBD3 and CRAMP in mouse vaginal tissue

[0102] Host Defense Peptides (HDPs), also known as host defense peptides, are usually small molecule polypeptides composed of 7 to 100 amino acids. They are an important part of the natural immune defense system of organisms and have multiple biological functions such as antibacterial, antiviral, antiparasitic, and immunomodulatory. The two main host defense peptides in mice are defensins and cathelicidins, of which mBD3 and CRAMP are representatives of the two major types of host defense peptides. Studies have shown that these two host defense peptides have better antibacterial defense effects.

[0103] Defensins can effectively fight bacteria, fungi and viruses by destroying cell membranes and regulating immune responses; CRAMP is a mouse-specific cathelicidin with a wide range of antibacterial activities. These host defense peptides are not only directly involved in the elimination of pathogens, but also play an important role in regulating immune responses and promoting wound healing. The expression of defensins and CRAMP is regulated by multiple factors, including pathogenic bacteria infection, cytokines and other immune stimuli.

[0104] By detecting the expression of mBD3 mRNA, we found that ( Figure 4 A), Lactobacillus jensenii CCFM1431-L and its prepared postbiotic CCFM1431-D can significantly increase the expression of mBD3 mRNA to 1.06 and 3.71 (125.7% and 439.1% higher than the model group 0.845, respectively); Lactobacillus delbrueckii DM8909 increases the expression of mBD3 mRNA to 1.025, 121.3% higher than the model group. Lactobacillus jensenii CCFM1431 is better than Lactobacillus delbrueckii DM8909 in increasing the expression of defensins.

[0105] By detecting the expression of CRAMP mRNA, it was found that ( Figure 4 B), Lactobacillus jensenii CCFM1431-L and its prepared postbiotic CCFM1431-D can significantly increase the expression of CRAMP mRNA to 1.87 and 1.83 (239.7% and 234.6% higher than the model group 0.78 respectively); Lactobacillus delbrueckii DM8909 increases the expression of CRAMP mRNA to 1.40, which is 179.5% higher than the model group. Both the live bacteria and postbiotic groups of Lactobacillus jensenii CCFM1431 can significantly increase the expression of host CRAMP mRNA. That is, oral administration of Lactobacillus jensenii CCFM1431 and its postbiotics can resist infection caused by Candida albicans by increasing the expression of host defense peptides mBD3 and CRAMP.

[0106] (3) Interferon (IFN-γ) secretion level in mouse serum

[0107] When fungal infection occurs, interferon levels usually increase, promoting local immune response, enhancing leukocyte activity and improving antifungal effects. At the same time, interferon further enhances host defense by inducing the expression of host defense peptides. However, high levels of interferon can trigger persistent inflammatory responses and may inhibit the proliferation and function of normal cells, affecting cell repair and regeneration processes. IFN-γ is an important regulatory factor in T cell-mediated immune responses, mainly produced by activated Th1 lymphocytes, CTLs and NK cells; the mechanism of action of IFN-γ in the body's resistance to systemic Candida albicans infection is very complex and may be related to the following mechanisms: (1) Activation of macrophages and neutrophils; (2) Reduction of vascular endothelial cell damage; (3) Regulating the balance of Th1 / Th2 cells.

[0108] like Figure 5 As shown, the serum interferon (IFN-γ) content of the blank control group was 884.49 ng / L, the content of the model group was 1092.65 ng / L, the content of oral live bacteria Lactobacillus delbrueckii DM8909 was 1006.94 ng / L (7.8% lower than the model group), and the oral Lactobacillus jenseni CCFM1431 live bacteria group and postbiotic group secreted IFN-γ of 890.95 ng / L (18.5% lower than the model group) and 952.86 ng / L (12.8% lower than the model group), which was significantly lower than the model group. At the same time, the ability of the Lactobacillus jenseni CCFM1431 live bacteria group and postbiotic group to reduce interferon (IFN-γ) secretion was better than that of the Lactobacillus delbrueckii DM8909 live bacteria group. In summary, oral Lactobacillus jenseni CCFM1431 can significantly reduce the secretion of interferon (IFN-γ) in mouse serum, inhibit the further development of body damage, and accelerate inflammation repair.

[0109] (4) Myeloperoxidase (MPO) secretion level in mouse serum

[0110] Myeloperoxidase (MPO) is a heme-containing peroxidase that is expressed in a variety of inflammatory cells, including neutrophils, activated microglia, monocytes / macrophages, astrocytes, and neurons, and plays an important role in immune surveillance and host defense mechanisms. However, the secretion level of myeloperoxidase in mouse vaginal tissue has a potent pro-inflammatory effect and may be directly involved in tissue damage.

[0111] like Figure 6As shown, the content of myeloperoxidase (MPO) in the blank control group was 15.55 ng / L, that in the model group was 24.05 ng / L, that in the oral Lactobacillus delbrueckii DM8909 live bacteria group was 20.82 ng / L (13.4% lower than that in the model group), that in the oral Lactobacillus jenseni CCFM1431 live bacteria and postbiotic group was 15.90 ng / L (33.9% lower than that in the model group) and 18.68 ng / L (22.3% lower than that in the model group), and the MPO level after probiotic intervention was significantly lower than that in the model group, while the oral Lactobacillus jenseni CCFM1431 live bacteria group and postbiotic group were better than the Lactobacillus delbrueckii DM8909 live bacteria group in reducing the secretion of MPO. In summary, oral Lactobacillus jenseni CCFM1431 can significantly reduce the secretion of myeloperoxidase (MPO) in the vaginal tissue of mice, thereby reducing tissue damage and defending against pathogenic bacteria infection.

[0112] (5) Diversity of bacterial flora in mouse vaginal lavage fluid

[0113] The surface of the female reproductive tract is colonized by numerous microorganisms, and the healthy vaginal flora dominated by Lactobacillus plays a key protective role in the human body. Lactobacillus (Lactobacilus) is a group of Gram-positive anaerobic bacteria. It is a standard symbiotic flora of the human gastrointestinal tract and urogenital tract, and has natural resistance to potential pathogenic microorganisms. As the dominant bacteria in the normal vaginal flora, Lactobacillus can bind to the epithelial cell receptors of the vaginal mucosa to produce a space-occupying effect, and prevent the adhesion of pathogenic microorganisms through a competitive mechanism. In addition, Lactobacillus can also produce self-regulatory factors such as organic acids, hydrogen peroxide and bacteriocins, thereby regulating nonspecific host immune responses and resisting the invasion of pathogenic bacteria.

[0114] like Figure 7As shown in the figure, the α diversity of this study is mainly reflected by the richness (Chao1 index) and uniformity (Shannon index) of the vaginal flora. After Candida albicans infection, the Chaol (p < 0.05) and Shannon (p < 0.05) indices of the vaginal flora of mice were lower than those of the blank control group. There was no significant difference between the Chao1 and Shannon of the Lactobacillus delbrueckii DM8909 group and the model group, indicating that Lactobacillus delbrueckii DM8909 cannot significantly regulate the flora and cannot change the vaginal flora after pathogenic bacteria infection. The Chao1 index of the live bacteria group of Lactobacillus jensenii CCFM1431 was not significantly different from that of the model group and the blank group, indicating that Lactobacillus jensenii CCFM1431 can change the vaginal flora, but it is still subject to the infection of pathogenic bacteria, and its effect is better than that of Lactobacillus delbrueckii DM8909; the postbiotic group of Lactobacillus jensenii CCFM1431 showed a better ability to promote the development of vaginal flora towards a balanced and stable direction, and it had obvious differences with pathogenic bacteria. It was better than the live bacteria group of Lactobacillus jensenii CCFM1431 and Lactobacillus delbrueckii DM8909 in the ability to restore various indexes. This may be due to the co-aggregation of the lactobacillus used with the original vaginal flora of mice, which increased the abundance of health-related vaginal symbiotic bacteria and antagonized the growth of pathogenic bacteria, resulting in a decrease in the number of pathogenic bacteria in the vaginal flora. Therefore, oral administration of Lactobacillus jensenii CCFM1431 can regulate the vaginal flora and increase the richness and uniformity of the vaginal flora.

[0115] (6) Periodic acid-Schiff staining analysis of mouse vaginal tissue

[0116] PAS staining, also known as periodic acid-Schiff staining or glycogen staining, can specifically stain carbohydrates, including polysaccharides and glycoproteins. PAS staining is very important in the diagnosis of fungal infections because the cell wall of fungi is mainly composed of polysaccharides, especially chitin and β-glucan. When tissues are infected with fungi, PAS staining can effectively reveal the presence and distribution of fungi. Figure 8As shown in the figure, the vaginal tissue surface of mice in the normal group showed a relatively uniform staining pattern, in which polysaccharides such as glycogen showed uniform purple-red in the vaginal epithelial cells, indicating the healthy state of the vaginal mucosa; fungal spores appeared in the vaginal epithelial cells of mice in the model group, forming a characteristic "pseudofilamentous" structure, and the glycogen content decreased, resulting in a lighter staining result. Compared with the model group, the vaginal tissue of mice in the oral Lactobacillus delbrueckii DM8909, Lactobacillus jensenii CCFM1431 live bacteria and postbiotic groups was keratinized, the surface glycogen content was reduced, but the fungal invasion was greatly reduced, indicating that the early intervention of probiotics can prevent Candida albicans infection and enhance the host's ability to resist infection. The mucus layer on the surface of oral Lactobacillus delbrueckii DM8909 disappeared, and a large number of inflammatory cells appeared in the submucosal layer, while there was a small amount of mucous substance on the surface of Lactobacillus jensenii CCFM1431, and the inflammatory cells in the submucosal layer were evenly distributed. Therefore, oral administration of Lactobacillus jensenii CCFM1431 showed a stronger defense ability on the recovery of the vaginal tissue of mice than the model group, and effectively alleviated the symptoms of vaginal infection in mice.

[0117] (7) Mouse vaginal histopathological analysis

[0118] HE staining was used to observe the vaginal tissue of mice, which could clearly evaluate the protective status and inflammation degree of the vaginal mucosa of each group of mice. Fig. 9 As shown in the figure, in the mice in the blank control group, the vaginal mucosa maintained an intact structure, and its surface was covered with a normal keratinized layer. In contrast, the vaginal mucosal epithelium of the mice in the model group showed continuous destruction, accompanied by superficial erosion and hole formation, and congestion and infiltration of a large number of inflammatory cells appeared in the submucosal layer. The vaginal tissue of mice intervened by probiotics had a good preventive effect. The inflammatory cell infiltration in the oral Lactobacillus delbrueckii DM8909 group was less than that in the model group, and the improvement effect on the vaginal tissue of mice was not obvious. Compared with the model group, there were still superficial erosions and discontinuous holes in the epithelium; oral Lactobacillus jensenii CCFM1431 showed a stronger ability to promote the recovery of vaginal mucosa in mice than in the model group, effectively alleviated the inflammatory symptoms of the vagina of mice, and improved the damage to the vaginal structure.

[0119] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. The present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by technicians in this technical field within the essential scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus jensenii CCFM1431, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 20, 2024, with a deposit number of GDMCC No: 65157.

2. A microbial preparation containing the Lactobacillus jensenii CCFM1431 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The content of Lactobacillus jensenii CCFM1431 in the microbial preparation is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

4. The postbiotic prepared by Lactobacillus jensenii CCFM1431 according to claim 1, characterized in that The postbiotics include but are not limited to the lysate of the Lactobacillus jensenii CCFM1431.

5. A product containing the Lactobacillus jensenii CCFM1431 according to claim 1, the microbial preparation according to claim 2 or 3, or the postbiotic according to claim 4.

6. The product according to claim 5, characterized in that The products include medicines or hygiene products.

7. Use of the Lactobacillus jensenii CCFM1431 according to claim 1, the microbial preparation according to claim 2 or 3, or the postbiotic according to claim 4 in the preparation of a product for preventing and / or treating vaginal Candida albicans infection.

8. The use according to claim 7, characterized in that: The product has at least one of the following effects: (1) reducing the expression of Candida albicans virulence factors after infection; (2) promoting the expression of host defense peptide β-defensin and CRAMP genes; (3) reducing the production of interferon IFN-γ; (4) reducing the level of myeloperoxidase MPO; (5) regulating vaginal flora; (6) improving staining after vaginal fungal infection; and (7) improving vaginal pathological manifestations.

9. The use according to claim 8, characterized in that: The product is a medicine or a sanitary product.

10. The use according to claim 9, characterized in that: The sanitary products include sanitary wet wipes, sanitary napkins, sanitary pads, sanitary tampons, sanitary napkins, vaginal washes, and women's antibacterial / antibacterial washes.

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