Lactobacillus paracasei for regulating local secretory immunoglobulin and systemic immunoglobulin of host to enhance anti-infection ability

Abstract

The invention discloses a Lactobacillus paracasei strain for regulating local secretory immune globulin and systemic immune globulin of a host to enhance infection resistance, and belongs to the technical field of microorganisms. The lactobacillus paracasei provided by the invention has the effects of reducing vaginal mucosa injury and inhibiting vagina inflammation, and the effects are specifically reflected as follows: (1) the expression of a polymeric immune globulin receptor gene of an organism is improved; (2) promoting the secretion of secretory immune globulin sIgA of the body; (3) promoting secretion of immune globulin IgG of the body; (4) the generation of a chemotactic factor CCL20 of an organism is reduced; (5) reducing secretion of granulocyte colony stimulating factors of the body; (6) the secretion of TNF-alpha is inhibited, and the secretion of IL-10 is promoted; (7) improving pathological characterization of the organism; and (8) improving glycogen staining characterization after fungal infection of the organism. Therefore, the lactobacillus paracasei has a huge application prospect in products for improving vaginal mucosa injury and inhibiting vagina inflammation.

Description

Technical Field

[0001] The invention relates to a strain of Lactobacillus paracasei which can regulate local secretory immunoglobulin and systemic immunoglobulin of a host to enhance anti-infection ability, and belongs to the technical field of microorganisms. Background Art

[0002] Candida albicans is a common opportunistic fungus that is widely found in the human body's oral cavity, intestines, vagina, and other parts. When the host's immune system function is suppressed or the flora is unbalanced, Candida albicans overgrows and causes local or systemic infection. Clinical treatment for Candida albicans infection mainly relies on antifungal drugs, but long-term use of antifungal drugs may cause vaginal microecological imbalance, mucosal barrier damage, and extremely high recurrence rate after stopping medication. With the long-term use of these drugs, fungal resistance is greatly increased, making the treatment of candidal vaginitis more difficult.

[0003] The host's immune system plays a vital role in resisting the invasion of external pathogens, especially in the defense of local tissues, where immune response is the key to resisting infection. Secretory immunoglobulin A is an antibody that plays a key role in mucosal immunity. It can neutralize toxins produced by the body after pathogen intervention and has anti-inflammatory effects. In systemic immunity, IgG also plays an important role. It can recognize and bind to specific antigens, thereby exerting its antibacterial, antiviral and toxin neutralizing functions. Compared with traditional direct antibacterial methods, preventing vaginal pathogen infection by enhancing host immunity is to regulate the host's own immunity, enhance the host's ability to identify and eliminate pathogens, provide a lasting defense effect, and greatly reduce the recurrence of the disease. Compared with treatment with probiotics, prevention is significantly different in mechanism. Treatment usually refers to measures taken for diseases that have already occurred, with the aim of eliminating pathogens and alleviating symptoms, while prevention is to take measures before the disease occurs to reduce the risk of infection.

[0004] However, there are many anti-infective effects in the host, and studies have shown that commensal bacteria can effectively activate anti-infective pathways in the host and regulate the production of antibacterial substances to resist the invasion of pathogenic bacteria. Andrew et al. proposed that by repeatedly giving mice oral commensal bacteria, IgA production can be selectively induced in the mucosa and serum. Adriana et al. showed that Bacteroides fragilis can promote type I interferon (IFN) signaling to maintain immune tolerance in mice. However, research on commensal bacteria regulating host immunity mainly focuses on the intestine, while research in the vagina is less. Therefore, by activating the anti-infective effect in the host through vaginal commensal bacteria, maintaining the balance of local microecology, reducing the chance of infection and recurrence, and preventing the spread of infection from the source. This strategy can not only reduce the harm of infection to the host, but also reduce dependence on traditional drug treatment, avoid the generation of drug resistance and side effects. It is worth noting that there are significant differences between prevention and treatment in dealing with Candida albicans infection. Treatment is usually carried out after the infection has occurred, with the aim of eliminating existing pathogens and alleviating related symptoms, but the treatment effect is often limited by factors such as pathogen resistance, infection site and host immune status. In contrast, prevention is to prevent infection by improving host immunity and reducing the chance of pathogen colonization. Davar et al. compared the relapse rate within 6 months between probiotics and placebo in patients with vaginal candidiasis receiving preventive treatment and found that the relapse rate of patients using probiotics was significantly lower than that of patients using placebo.

[0005] Therefore, regulating the host's local and systemic immunity through symbiotic bacteria to enhance the host's anti-infection ability and restore the vagina's natural defense mechanism are the most important strategies to resist the invasion of pathogens and prevent the aggravation of the disease. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a strain of Lactobacillus paracasei that regulates host local secretory immunoglobulin and systemic immunoglobulin to enhance anti-infection ability, the purpose of which is to solve the problems in the prior art in preventing vaginal infection and reducing the occurrence and development of diseases. The present invention aims to improve the host's immunity and the ability to resist infection by pathogenic bacteria, and to enhance the technical problem of Lactobacillus paracasei that can enhance the host's defense ability.

[0007] The first technical solution provided by the present invention is a strain of Lactobacillus paracasei CCFM1433, which was deposited in Guangdong Microbiological Culture Collection Center on September 20, 2024, with a deposit number of GDMCC No: 65159. The Lactobacillus paracasei CCFM1433 was isolated from the vagina of a healthy woman.

[0008] The second technical solution provided by the present invention is a microbial preparation containing the Lactobacillus paracasei CCFM1433 described in the first technical solution.

[0009] In certain embodiments, the content of Lactobacillus paracasei CCFM1433 in the microbial preparation is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0010] The third technical solution provided by the present invention is a product containing the Lactobacillus paracasei CCFM1433 described in the first technical solution or the microbial preparation described in the second technical solution.

[0011] In certain embodiments, the product is a food, a medicine, or a hygiene product.

[0012] Furthermore, the medicine comprises the above-mentioned strain and / or preparation, and a pharmaceutically acceptable carrier.

[0013] Furthermore, the carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0014] Furthermore, the dosage form of the drug includes granules, capsules, tablets, pills, suppositories or oral liquids.

[0015] Furthermore, the medicines include oral enteric-coated tablets and capsules, oral liquids; vaginal suppositories, tablets, gelatin capsules, sprays, creams, and gels.

[0016] Furthermore, the sanitary products include sanitary wet wipes, sanitary napkins, sanitary pads, sanitary tampons, sanitary napkins, vaginal washes, and women's antibacterial / antibacterial washes.

[0017] In some embodiments, the content of Lactobacillus paracasei CCFM1433 in the product is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0018] The fourth technical solution provided by the present invention is the use of Lactobacillus paracasei CCFM1433 described in the first technical solution or the microbial preparation described in the second technical solution in the preparation of a product for enhancing vaginal immunity.

[0019] In certain embodiments, the effect of enhancing vaginal immunity includes increasing the secretion of secretory immunoglobulins sIgA and IgG, reducing the production of chemokines CCL20 and granulocyte colony stimulating factor after intervention, inhibiting the secretion of TNF-α, increasing the secretion of IL-10 and improving the pathological characteristics of the body after infection.

[0020] In certain embodiments, the product is a medicine or a hygiene product.

[0021] Furthermore, the medicine comprises the above-mentioned strain and / or preparation, and a pharmaceutically acceptable carrier.

[0022] Furthermore, the carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0023] Furthermore, the dosage form of the drug includes granules, capsules, tablets, pills, suppositories or oral liquids.

[0024] Furthermore, the medicines include oral enteric-coated tablets and capsules, oral liquids; vaginal suppositories, tablets, gelatin capsules, sprays, creams, and gels.

[0025] Furthermore, the sanitary products include sanitary wet wipes, sanitary napkins, sanitary pads, sanitary tampons, sanitary napkins, vaginal washes, and women's antibacterial / antibacterial washes.

[0026] The fifth technical solution provided by the present invention is the use of Lactobacillus paracasei CCFM1433 described in the first technical solution or the microbial preparation described in the second technical solution in the preparation of a product for preventing vaginal pathogenic bacteria infection.

[0027] In certain embodiments, the effects of the product include reducing TNF-α and increasing IL-10 secretion in mouse spleen tissue.

[0028] In certain embodiments, the product is a medicine or a hygiene product.

[0029] Furthermore, the medicine comprises the above-mentioned strain and / or preparation, and a pharmaceutically acceptable carrier.

[0030] Furthermore, the carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0031] Furthermore, the dosage form of the drug includes granules, capsules, tablets, pills, suppositories or oral liquids.

[0032] Furthermore, the medicines include oral enteric-coated tablets and capsules, oral liquids; vaginal suppositories, tablets, gelatin capsules, sprays, creams, and gels.

[0033] Furthermore, the sanitary products include sanitary wet wipes, sanitary napkins, sanitary pads, sanitary tampons, sanitary napkins, vaginal washes, and women's antibacterial / antibacterial washes.

[0034] Beneficial effects:

[0035] The present invention provides a strain of Lactobacillus paracasei CCFM1433, which is isolated from the vagina of a healthy woman. The strain has the effects of enhancing the ability of the vagina to resist infection, strengthening the host immune defense and inhibiting vaginal inflammation, and is specifically manifested as follows: (1) improving the expression of the polymeric immunoglobulin receptor gene of the body; (2) promoting the secretion of secretory immunoglobulin sIgA of the body; (3) promoting the secretion of immunoglobulin IgG of the body; (4) reducing the production of chemokine CCL20 by the body after intervention; (5) reducing the secretion of granulocyte colony stimulating factor of the body; (6) inhibiting the secretion of TNF-α and increasing the secretion of IL-10; (7) improving the pathological manifestations of the vagina; and (8) improving the glycogen staining manifestations after fungal infection of the body. Therefore, the Lactobacillus paracasei has a huge application prospect in products for enhancing local immunity and regulating vaginal inflammation.

[0036] Biomaterial Deposit

[0037] The Lactobacillus paracasei CCFM1433 provided by the present invention was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 20, 2024, with a deposit number of GDMCC No: 65159, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 : Streak results of Lactobacillus paracasei CCFM1433.

[0039] Figure 2 : Flowchart of animal experiments.

[0040] Figure 3 :The effect of Lactobacillus paracasei on the gene expression of polymeric immunoglobulin receptor pIgR in animals.

[0041] Figure 4 :The effect of Lactobacillus paracasei on the secretion of secretory immunoglobulin sIgA in animals.

[0042] Figure 5 :The effect of Lactobacillus paracasei on the secretion of immunoglobulin IgG in animals.

[0043] Figure 6 :The effect of Lactobacillus paracasei on the secretion of chemokine CCL20 in animals after intervention.

[0044] Figure 7 :The effect of Lactobacillus paracasei on the secretion of granulocyte colony stimulating factor in animals.

[0045] Figure 8 :The effect of Lactobacillus paracasei on the expression of animal cytokines TNF-α and IL-10.

[0046] Fig. 9 : Figure 3 histopathological evaluation of vaginal tissue in mice.

[0047] Fig.10 : Periodic acid-Schiff staining of mouse vaginal tissue.

[0048] (Different lowercase letters in the figure indicate significant differences among the groups, p < 0.05). DETAILED DESCRIPTION

[0049] 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.

[0050] The culture medium involved in the following examples is as follows:

[0051] LBS solid culture medium ( / L): 5g tryptic peptone, 5g yeast extract powder, 6g potassium dihydrogen phosphate, 0.034g potassium dihydrogen phosphate, 0.575g magnesium sulfate, 20g glucose, 25g sodium acetate, 2g ammonium citrate, 0.12g manganese sulfate, 15g agar, 1mL Tween-80, 1.3mL glacial acetic acid, pH 5.3-5.7.

[0052] 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.

[0053] 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.

[0054] SDA medium ( / L): animal tissue pepsin hydrolysate and trypsin casein 10g, glucose 40g, agar 15g, pH 5.6±0.2.

[0055] SDB medium ( / L): animal tissue pepsin hydrolysate and trypsin casein 10g, glucose 20g, pH 5.6±0.2.

[0056] The strains and animals involved in the following examples are as follows:

[0057] The bacterial culture of Lactobacillus paracasei CCFM1433 was isolated from female vagina and stored in the bacterial strain bank of Food Biotechnology Center of Jiangnan University.

[0058] Lactobacillus delbrueckii DM8909 was isolated from Dingjunsheng vaginal lactobacillus capsules and stored in the strain bank of Food Biotechnology Center of Jiangnan University.

[0059] Candida albicans SC5314 was purchased from Guangdong Institute of Microbiology Culture Collection Center (GDMCC).

[0060] 7-week-old SPF female BALB / c mice, weighing 18-20 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (production license number SCXK (Beijing) 2012-0001).

[0061] The bacterial solution preparation method involved in the following examples is as follows:

[0062] Lactobacillus paracasei CCFM1433 (CCFM1433): Lactobacillus paracasei CCFM1433 was streaked and isolated on 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. The preparation method of Lactobacillus delbrueckii DM8909 is the same as above, and is recorded as DM8909.

[0063] 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.

[0064] Estradiol: 0.1 mg of β-estradiol is dissolved in 0.05 mL of sesame oil and is ready for use.

[0065] Example 1 Isolation and identification of Lactobacillus paracasei CCFM1433

[0066] 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.

[0067] Select colonies of different morphologies and perform streak purification on MRS plates ( Figure 1 ), pick the purified single colony and inoculate it into 5mL liquid culture medium, and culture it at 37℃ for 48h. Take 1.5mL of the 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 of forward primer (10μM), 0.5μL of reverse primer (10μM), 10μL2×Taq Mixture, 0.5μL of bacterial suspension, and 8.5μL of double distilled water. Primer information is shown in Table 1.

[0068] Table 1: Primer information

[0069]

[0070] PCR conditions: 95℃5min; 95℃10s; 55℃30s; 72℃30s; step2-4 30×; 72℃5min; 12℃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 paracasei.

[0071] 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.

[0072] 16S sequence information (SEQ ID No.1):

[0073]

[0074] Example 2 Application of Lactobacillus paracasei CCFM1433 in improving resistance of mice to pathogenic bacteria infection

[0075] Twenty 7-week-old SPF female BALB / c mice weighing 18-20 g were randomly divided into 4 cages, with 5 mice in each cage. Figure 2 Flow chart of animal experiments.

[0076] Table 2: Animal experimental plan and grouping

[0077]

[0078] The mice were randomly divided into 4 groups according to their body weight. The specific grouping information is shown in Table 2. All mice in the groups were fed normally during the whole experiment. 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 Lactobacillus delbrueckii DM8909 or Lactobacillus paracasei CCFM1433. 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 vagina once a day. On the 18th day, all experimental mice were killed and serum was separated and vaginal tissue was peeled off for detection of polymerized immunoglobulin receptor gene expression, immunoglobulin secretion level, TNF-α, IL-10, CCL20, G-CSF content and subsequent experimental tissue pathology staining.

[0079] Determination method: At the end of the experiment, mice were killed and vaginal tissue was dissected. A portion of the tissue was homogenized with pre-cooled RIPA lysis buffer (Biyuntian Biotechnology Co., Ltd.) and a mixture of protease inhibitors. The sample was centrifuged at 12000r / min for 15min at 4°C, and the vaginal tissue supernatant was taken for determination of pIgR, sIgA, TNF-α, and IL-10 content according to the instructions of the kit (Nanjing Senbeijia Biotechnology Co., Ltd.). Another portion of the vaginal tissue was placed in 4% paraformaldehyde solution for histopathological examination.

[0080] Histopathological observation: Vaginal tissue was fixed with 4% paraformaldehyde solution, embedded in paraffin, sliced ​​into 5 mm thick sections, stained with hematoxylin and eosin (H&E) and periodic acid-Schiff staining. Vaginal tissue samples were observed under a pathological section scanner (PanoramicMIDI, 3DHistech Ltd, Budapest, Hungary) with a magnification of 20 times.

[0081] Experimental results:

[0082] (1) Polymeric immunoglobulin receptor pIgR

[0083] pIgR (Polymeric Immunoglobulin Receptor) is a transmembrane protein that is mainly expressed on the surface of epithelial cells. Its main function is to mediate the transport of secretory immunoglobulins (such as IgA) and participate in mucosal immune responses.

[0084] Table 3: Primers

[0085]

[0086] like Figure 3 As shown, compared with the blank group (promoting human pIgR expression to 1.07), the relative mRNA expression of pIgR in the model group was significantly reduced to 0.43, oral Lactobacillus delbrueckii DM8909 promoted human pIgR expression to 0.8 (increased 186.0%), and Lactobacillus paracasei CCFM1433 promoted human pIgR expression to 0.84 (increased 195.3%). Probiotics can effectively increase the relative mRNA expression of pIgR (p>0.05), and Lactobacillus paracasei CCFM1433 has a more significant effect in increasing the relative mRNA expression of pIgR.

[0087] (2) Secretory immunoglobulin receptor sIgA

[0088] Secretory immunoglobulin A (sIgA) is an antibody that plays a key role in mucosal immunity. The production of sIgA after intervention involves B cells in mucosa-associated lymphoid tissues, which differentiate into plasma cells under antigen stimulation and secrete polymeric IgA (pIgA). pIgA is transported across mucosal epithelial cells through polymeric immunoglobulin receptors (pIgR), eventually forming sIgA and releasing it to the mucosal surface. sIgA can prevent pathogen adhesion and play an immune clearance role.

[0089] like Figure 4As shown, compared with the blank group (0.84 μg / mL), the content of sIgA produced by the body after intervention in the model group was significantly reduced to 0.47 μg / mL, and the body produced sIgA of 0.66 μg / mL (increased by 140.4%) after oral Lactobacillus delbrueckii DM8909 intervention, which was significantly different from the model group, and the content of sIgA produced by the body after intervention of Lactobacillus paracasei CCFM1433 was 0.72 μg / mL (increased by 153.2%), which was significantly different from the model group before, and the level of sIgA was higher, close to the blank group. Therefore, Lactobacillus paracasei CCFM1433 has a good ability to promote the secretion of sIgA.

[0090] (3) Immunoglobulin IgG

[0091] Immunoglobulin G (IgG) is the most important type of antibody in human serum, accounting for about 75% of the total immunoglobulin. IgG can recognize and bind to specific antigens, such as bacteria and viruses, thereby exerting a neutralizing effect and is a key antibody for the human body to resist infection.

[0092] like Figure 5 As shown, compared with the blank group (7.17 μg / mL), the IgG content produced by the model group after intervention was significantly reduced to 6.23 μg / mL, and the IgG produced by the body after oral Lactobacillus delbrueckii DM8909 intervention was 6.53 μg / mL (increased by 104.8%), but there was no significant difference compared with the model group; the IgG content produced by the body after intervention with Lactobacillus casei CCFM1433 was 7.01 μg / mL (increased by 112.5%), and there was a significant difference in the IgG level increased by Lactobacillus paracasei CCFM1433 compared with the model group (p>0.05), with a better effect, indicating that Lactobacillus paracasei CCFM1433 can significantly enhance the host systemic immunity to resist pathogenic bacteria infection.

[0093] (4) Chemokine CCL20

[0094] CCL20 (Chemokine CC MotifLigand 20), also known as MIP-3α (MacrophageInflammatory Protein-3α), is a small molecule chemokine that belongs to the CC chemokine family. It mainly exerts its effects by binding to its receptor CCR6. Elevated CCL20 content is an important biomarker that can reflect a variety of disease states.

[0095] like Figure 6As shown, compared with the blank group (364.07ng / L), the content of chemokine CCL20 produced by the body in the model group after intervention was significantly increased to 413.46ng / L, the content of chemokine CCL20 produced by the body after oral Lactobacillus delbrueckii DM8909 intervention was 377.65ng / L (a decrease of 8.7% compared with the model group), and the content of chemokine CCL20 produced by the body after intervention with Lactobacillus paracasei CCFM1433 was 365.00ng / L (a decrease of 11.7% compared with the model group). There were significant differences between the probiotic interventions and the model group, but Lactobacillus paracasei CCFM1433 could significantly reduce the level of chemokine CCL20, so that the body tended to normal levels and thus resisted a series of inflammations caused by pathogenic bacteria infection.

[0096] (5) Granulocyte colony stimulating factor (G-CSF)

[0097] Granulocyte colony-stimulating factor (G-CSF) is a proinflammatory cytokine whose main role is to promote the survival, proliferation, differentiation and function of neutrophil progenitors and mature neutrophils.

[0098] like Figure 7 As shown, compared with the blank group (364.07 ng / L), the content of G-CSF produced by the model group after intervention was significantly increased to 419.63 ng / L, the content of G-CSF produced by the body after oral Lactobacillus delbrueckii DM8909 intervention was 372.72 ng / L (a decrease of 11.2% compared with the model group), and the content of G-CSF produced by the body after intervention with Lactobacillus paracasei CCFM1433 was 350.49 ng / L (a decrease of 16.5% compared with the model group). Both can significantly reduce the level of G-CSF, and the effect of Lactobacillus paracasei CCFM1433 in reducing G-CSF is more significant, so that the body tends to normalize the level and thus resists a series of inflammations caused by pathogenic bacteria infection.

[0099] (6) Secretion of tumor necrosis factor-α (TNF-α) and interleukin IL-10 in mouse spleen tissue

[0100] TNF-α plays a promoting role in inflammatory response and is a small molecule protein mainly secreted by monocytes and macrophages; IL-10 is a multi-functional cytokine with strong anti-inflammatory properties, which is mainly secreted by activated T cells, monocytes, B cells and macrophages.

[0101] like Figure 8As shown in (A), the TNF-α content of the blank control group was 243.70 ng / L, and that of the model group was 408.91 ng / L. Compared with the model group, the TNF-α produced by the body after oral intervention with live bacteria of Lactobacillus delbrueckii DM8909 was 352.93 ng / L (a decrease of 13.7% compared with the model group), and the TNF-α produced by the body after oral intervention with live bacteria of Lactobacillus paracasei CCFM1433 was 325.76 ng / L (a decrease of 20.3% compared with the model group). Compared with the model group, both groups were able to significantly reduce the TNF-α level, but Lactobacillus paracasei CCFM1433 was better than the Lactobacillus delbrueckii DM8909 group in reducing the TNF-α level.

[0102] like Figure 8 As shown in (B), the secretion of IL-10 in the blank control group was 572.42 pg / mL, and the secretion of IL-10 in the model group was 413.82 pg / mL. Compared with the model group, the IL-10 secreted by oral Lactobacillus delbrueckii DM8909 was 497.77 pg / mL (increased by 120.3%), and the IL-10 secreted by oral Lactobacillus paracasei CCFM1433 was 512.82 pg / mL (increased by 123.9%). Compared with the model group, both of them could significantly increase the IL-10 level. The ability of the Lactobacillus paracasei CCFM1433 group to increase IL-10 was significantly better than that of the Lactobacillus delbrueckii DM8909 group.

[0103] In summary, Lactobacillus paracasei CCFM1433 can significantly reduce TNF-α and increase the secretion of IL-10 in the vaginal tissue of mice, inhibit the further development of inflammation, and accelerate the body's immune regulation.

[0104] (7) Mouse vaginal histopathological analysis

[0105] HE staining, or Hematoxylin-Eosin staining, is the most commonly used staining technique in histopathology and histology research. This staining method can clearly show the morphological structure of cells and tissues, and is the basis for disease diagnosis and research.

[0106] like Fig. 9As shown in the figure, in the blank control group, the vaginal epithelium of mice was smooth and continuous, with intact tissue structure, and no obvious inflammatory cell infiltration was observed. In the model group, the continuity of the vaginal mucosal epithelium was poor, the surface cells were eroded, the submucosal stroma was congested, and a large number of inflammatory cells infiltrated into the epithelium and stroma. The inflammatory cell infiltration in the Lactobacillus delbrueckii DM8909 group was less than that in the model group, but there was obvious squamous epithelial hyperplasia, and the surface of the vaginal tissue was slightly damaged; after treatment with Lactobacillus paracasei CCFM1433, the epithelial layer of mice infected with the infection was less damaged, the inflammatory cells were significantly reduced, and the keratinized layer reappeared on the mucosal surface. Lactobacillus paracasei CCFM1433 helps to repair the vaginal mucosa, and the degree of damage to the vaginal tissue of mice after its intervention is lower than that of the Lactobacillus delbrueckii DM8909 group.

[0107] (8) Periodic Acid Schiff Staining Analysis of Mouse Vaginal Tissue

[0108] Periodic Acid-Schiff (PAS) staining is a commonly used histochemical staining technique, mainly used to show polysaccharides in tissues, such as glycogen, mucopolysaccharides and fungi. In PAS staining, polysaccharides appear purple or magenta, while cell nuclei are stained blue by hematoxylin.

[0109] like Fig.10 As shown in the figure, the PAS staining results of the vaginal tissue of the normal group mice showed a uniform purple-red color, reflecting the normal distribution of glycogen in the vaginal epithelial cells, which is a manifestation of mucosal health. Purple-red fungal spore-like structures were observed in the vaginal epithelial cells of the model group mice, indicating fungal invasion. In contrast, the mice gavaged with live bacteria of Lactobacillus delbrueckii DM8909 and Lactobacillus paracasei CCFM1433 had a significant decrease in glycogen content on the surface of the vaginal tissue, and at the same time, fungal invasion was significantly reduced, indicating that the preventive intervention of probiotics has a positive effect on resisting Candida albicans infection and enhancing the host's prevention mechanism. However, a small amount of glycogen aggregated on the surface of Lactobacillus paracasei CCFM1433, indicating that after infection, Lactobacillus paracasei CCFM1433 regulated the host's anti-infection ability and enhanced the host's defense ability, while after oral administration of Lactobacillus delbrueckii DM8909, the glycogen on the surface of the vaginal tissue disappeared and the mucosa was slightly damaged. Therefore, Lactobacillus paracasei CCFM1433 can enhance the host's defense ability and reduce fungal invasion, thereby playing a positive role in preventing candidal vaginitis.

[0110] 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 paracasei CCFM1433, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 20, 2024, with a deposit number of GDMCCNo:65159.

2. a microbial preparation containing the Lactobacillus paracasei CCFM1433 of claim 1.

3. The microbial preparation according to claim 2, characterized in that The content of Lactobacillus paracasei CCFM1433 in the microbial preparation is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

4. A product containing the Lactobacillus paracasei CCFM1433 of claim 1 or the microbial preparation of claim 2 or 3.

5. The product according to claim 4, characterized in that The product is food, medicine or sanitary product.

6. The product according to claim 4, characterized in that The content of Lactobacillus paracasei CCFM1433 in the product is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

7. Use of the Lactobacillus paracasei CCFM1433 according to claim 1 or the microbial preparation according to claim 2 or 3 in preparing a product for enhancing vaginal immunity, characterized in that: The enhancement of vaginal immunity includes at least one of the following functions: (1) Increase the expression of polymeric immunoglobulin receptor genes; (2) Promote the secretion of local secretory immunoglobulin sIgA in the vagina; (3) Promote the secretion of immunoglobulin IgG; (4) Reduce the production of chemokine CCL20; (5) Reduce the body's secretion of granulocyte colony-stimulating factor G-CSF; (6) Inhibit the secretion of TNF-α and increase the secretion of IL-10; (7) Improve vaginal pathology manifestations; (8) Improve glycogen staining after fungal infection.

8. The use according to claim 7, characterized in that: The product is a medicine or a sanitary product; preferably, the sanitary product includes sanitary wet wipes, sanitary napkins, sanitary pads, sanitary tampons, sanitary napkins, vaginal washes, women's antibacterial washes or antibacterial washes.

9. Use of the Lactobacillus paracasei CCFM1433 described in claim 1 or the microbial preparation described in claim 2 or 3 in preparing a product for preventing vaginal pathogenic bacteria infection.

10. The use according to claim 9, characterized in that: The pathogenic bacteria include but are not limited to Candida albicans; the product is a medicine or a sanitary product; preferably, the sanitary product includes sanitary wet wipes, sanitary napkins, sanitary pads, sanitary tampons, sanitary napkins, vaginal washes, women's antibacterial washes or antibacterial washes.

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