A salivarius-associated lactobacillus strain capable of increasing host secretory immunoglobulin expression and enhancing its anti-infection properties and its postbiotics

Abstract

The present invention discloses a saliva-associated lactobacillus and its postbiotics that can increase the expression of host secretory immunoglobulin and enhance its anti-infection ability, belonging to the field of microbial technology. The saliva-associated lactobacillus live bacteria and its postbiotics provided by the present invention have the ability to regulate host immunity and improve the host's ability to resist oral pathogen infection. They can significantly upregulate the expression of PIGR in the basal part of oral mucosal epithelial cells in vitro and in vivo, and significantly upregulate the expression of SIgA in animals, thereby improving the host's oral immunity and resisting infection by oral pathogens. They have great application prospects in regulating the host's oral immunity and preparing products that improve the host's oral anti-infection ability.

Description

Technical Field

[0001] The invention relates to a saliva-associated lactobacillus capable of increasing the expression of host secretory immunoglobulin and enhancing the host's anti-infection ability and a postbiotic thereof, belonging to the technical field of microorganisms. Background Art

[0002] The human oral cavity's anti-infection system primarily comprises the physical barrier of the oral mucosa, immune cell activation, and the production of antimicrobial substances such as antimicrobial peptides and secretory immunoglobulins. The oral epithelial barrier serves as a crucial information relay station for maintaining oral environmental homeostasis, and the gradual maturation of the stratified squamous epithelium is essential for its normal function. The polymeric immunoglobulin receptor (PIGR) expressed by oral mucosal epithelial cells, along with various antimicrobial substances secreted by oral mucosal epithelial cells, salivary gland epithelial cells, and neutrophils, form an effective multi-molecular defense network to resist invading pathogens and maintain a normal oral microbiome.

[0003] The oral microbiome is a complex and complete system, primarily composed of the host's inherent oral characteristics and oral flora. The unique anatomical morphology and tissue structure of the oral cavity contribute to the composition of the oral microbiome. Different oral sites have unique microenvironments, providing distinct binding sites for the adhesion and colonization of a variety of oral microorganisms. Oral temperature, humidity, pH conditions, nutrient metabolism, and oxygen concentrations in different sites all affect the colonization of oral microorganisms, resulting in different oral sites having their own unique and complex microbial communities. In addition, maintenance of the host's oral health can only be achieved when there is a balance between microbial colonization and the host's immune defense mechanisms. The secretory immunoglobulin SIgA of the oral mucosa is the first line of defense against pathogens and plays an important role in resisting pathogen infection in humans and animal models.

[0004] Immunoglobulin A (IgA) in the oral cavity is primarily produced by plasma cells in tissues such as salivary gland ducts, near acini, and in the oral mucosa. IgA secreted by plasma cells possesses a J chain that specifically binds to the polymeric immunoglobulin receptor (PIGR) expressed at the basal level of epithelial cells, forming an IgA-PIGR complex. The complex is then internalized and hydrolyzed and released at the apical level of the epithelial cell to form secretory immunoglobulin SIgA. Because each transport consumes one pIgR molecule, upregulation of pIgR expression increases the ability of mucosal epithelial cells to transport dimeric IgA. The resulting mucosal SIgA can restrict the adhesion of pathogens to epithelial and dental surfaces, regulate host immunity by neutralizing enzymes, toxins, and viruses, or synergize with other antimicrobial factors (such as lysozyme, lactoferrin, salivary peroxidase, and mucins), thereby enhancing the host's ability to resist infection in the oral cavity.

[0005] Currently, the application of commensal bacteria in oral diseases primarily focuses on their mitigating effects in the presence of oral diseases, such as the mitigating effects of commensal bacteria on dental caries, periodontitis, halitosis, and oral mucosal diseases. These mechanisms of action are primarily through direct action on pathogenic bacteria. However, numerous anti-infective mechanisms exist within the host, and commensal bacteria can effectively activate these pathways and regulate the production of downstream antimicrobial substances, thereby exerting their anti-infective effects. Lin et al. demonstrated that Lactobacillus rhamnosus supernatant could activate intestinal PIGR transport of IgA, thereby reversing liver damage in mice with autoimmune hepatitis. Fan et al. demonstrated that intestinal commensal bacteria could activate HIF-1α and LL-37, inhibiting Candida albicans colonization in the gastrointestinal tract. The regulation of host immunity by commensal bacteria has been widely reported in fields such as the intestinal tract. Similarly, focusing on the oral cavity, it is particularly important to link commensal bacteria with the prevention of oral anti-infective measures. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a saliva-associated Lactobacillus and its postbiotics, aiming to solve the problem in the prior art that symbiotic bacteria lack the regulation of host oral immunity.

[0007] The present invention provides saliva-associated Lactobacillus (Ligilactobacillus salivarius) CCFM1418, which is taxonomically named Ligilactobacillus salivarius and has been deposited in Guangdong Provincial Microbiological Culture Collection Center on August 2, 2024, with a deposit number of GDMCC No: 64943, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] The salivary Lactobacillus (Ligilactobacillus salivarius) CCFM1418 is a Gram-positive bacterium. The cells are short rod-shaped under a microscope. After being inoculated on MRS solid culture medium and cultured for 48 hours, the colonies are generally milky white or light yellow protrusions and are circular with a diameter of 0.5-2 mm.

[0009] The present invention also provides a postbiotic prepared by combining saliva with Lactobacillus CCFM1418. The postbiotic prepared by combining saliva with Lactobacillus CCFM1418 comprises an inactivated bacterial cell of saliva with Lactobacillus CCFM1418 and / or a lysate thereof.

[0010] In one embodiment, the preparation method of the postbiotic is: culturing saliva and Lactobacillus CCFM1418 to the logarithmic growth phase, inactivating the saliva by heat treatment, centrifuging the saliva, discarding the supernatant, and freeze-drying to obtain a freeze-dried powder of the postbiotic.

[0011] In one embodiment, the preparation method of the postbiotic is: culturing saliva and Lactobacillus CCFM1418 to the logarithmic growth phase, inactivating the saliva by heat treatment, and then crushing the saliva by high-pressure homogenization and freeze-drying to obtain a postbiotic freeze-dried powder.

[0012] In one embodiment, the lysate is a lysate obtained by high-pressure homogenization of saliva combined with Lactobacillus CCFM1418; the saliva combined with Lactobacillus CCFM1418 is inoculated into a fermentation medium for culture, the bacterial sludge is collected and resuspended with sterile saline, and heat-treated at 65°C for 30 minutes to obtain inactivated bacterial cells, and then high-pressure homogenized to obtain a bacterial lysate.

[0013] The present invention also provides a microbial preparation, which contains the saliva-associated Lactobacillus CCFM1418 and / or its postbiotics.

[0014] In one embodiment, the content of Lactobacillus saliva CCFM1418 in the microbial preparation is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0015] The present invention also provides a medicine containing the saliva-combined Lactobacillus CCFM1418 and / or its postbiotics.

[0016] In one embodiment, the medicine contains the saliva-associated Lactobacillus CCFM1418 and / or its postbiotics, and a pharmaceutically acceptable carrier.

[0017] In one embodiment, the pharmaceutically acceptable carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0018] In one embodiment, the content of saliva-associated Lactobacillus CCFM1418 in the drug is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0019] The present invention also provides daily chemical products containing the postbiotics, and the daily chemical products include toothpaste, mouthwash or oral spray.

[0020] The present invention also provides the use of saliva combined with Lactobacillus CCFM1418 and / or its postbiotics in the preparation of a medicine for improving immunity and / or resisting oral pathogen infection.

[0021] In one embodiment, the use comprises upregulating the expression of basal PIGR and / or upregulating the expression of SIgA in the host oral mucosal epithelial cells.

[0022] The present invention also provides the use of saliva combined with Lactobacillus CCFM1418 and / or its postbiotics in the preparation of health products for improving immunity.

[0023] Beneficial effects:

[0024] The present invention provides a strain of Ligilactobacillus salivarius CCFM1418, which has the effect of enhancing the host's oral immunity against pathogenic bacteria infection, specifically embodied in:

[0025] (1) Promote the expression level of polymeric immunoglobulin receptor (PIGR) protein in oral epithelial cells;

[0026] (2) increasing the gene and protein expression levels of polymeric immunoglobulin receptor (PIGR) in tongue tissue of individuals;

[0027] (3) Increase the expression level of secretory immunoglobulin SIgA in individual tongue tissue;

[0028] (4) reduce the load of Candida albicans in the tongue tissue of individuals;

[0029] (5) Improve the pathological condition of individual tongue tissue;

[0030] (6) Improve the invasion of oral pathogen Candida albicans into individual tongue tissue;

[0031] Therefore, saliva combined with Lactobacillus CCFM1418 and / or its postbiotics has great application prospects in products that regulate host oral immunity and resist oral pathogenic bacteria infections.

[0032] Biomaterial Deposit

[0033] Saliva Lactobacillus (Ligilactobacillus salivarius) CCFM1418, taxonomically named Ligilactobacillus salivarius, was deposited in the Guangdong Provincial Microbiological Culture Collection on August 2, 2024, with the deposit number GDMCC No: 64943, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The effect of different bacterial lysates on PIGR expression in oral epithelial cells (HOK-16B).

[0035] Figure 2This is a flow chart for the animal experiment design plan; among them, Control is the blank control group; Model is the model group; saliva combined with Lactobacillus CCFM1418-L is the live bacteria external application group; saliva combined with Lactobacillus CCFM1418-P is the bacterial lysate external application group.

[0036] Figure 3 The changes in the tongue mucosa of mice after saliva combined with Lactobacillus intervention.

[0037] Figure 4 The effect of saliva combined with Lactobacillus on the load of Candida albicans in mouse tongue tissue.

[0038] Figure 5 The effect of saliva combined with Lactobacillus on the expression of PIGR and SIgA in mouse tongue tissue.

[0039] Figure 6 Figure 2 shows the histopathological evaluation of mouse tongue.

[0040] Figure 7 This is a picture of the pathological evaluation of PAS staining of mouse tongue tissue. DETAILED DESCRIPTION

[0041] To make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be described in further detail below in conjunction with specific embodiments and with reference to the accompanying drawings. The bacterial strains, cells and animals involved in the following examples are as follows: SPF-grade BALB / c mice, female, 6 weeks old, weighing 15-18 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (production license number SCXK (Beijing) 2012-0001). Saliva-derived lactobacilli CCFM1418 and FGSYC2M4 were from the strain bank of the Biotechnology Center of Jiangnan University. Candida albicans SC5314 was purchased from Ningbo Mingzhou Technology Co., Ltd. Human oral keratinocytes HOK-16B were purchased from Qingqi (Shanghai) Biotechnology Development Co., Ltd.

[0042] The culture medium involved in the following examples is as follows:

[0043] MRS liquid medium: yeast powder 5.0 g / L, beef extract 10.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, Tween-80 1 mL, pH 6.2-6.4.

[0044] MRS solid medium: yeast powder 5.0 g / L, beef extract 10.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, Tween-80 1 mL, agar 20.0 g / L, pH 6.2-6.4.

[0045] Complete cell culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100× mixed solution of penicillin and streptomycin (the penicillin content in the mixed solution is 10000 U / mL, and the streptomycin concentration is 10 mg / mL).

[0046] The preparation methods of the bacterial suspension and bacterial lysate involved in the following examples are as follows:

[0047] Preparation of Lactobacillus suspension:

[0048] (1) Preparation of seed solution:

[0049] Use a sterile inoculating loop to sip a small amount of saliva and activate the Lactobacillus CCFM1418 and FGSYC2M4 bacterial cultures in MRS solid medium. Incubate at 37°C for 48 hours. Then, pick a single colony and inoculate it into MRS liquid medium. Incubate in a 37°C incubator for 18 hours to prepare the seed solution.

[0050] (2) The prepared seed liquid was inoculated into MRS liquid medium at a 2% (v / v) inoculation rate and cultured in a 37°C incubator for 18 h. The bacteria were collected by centrifugation and concentrated, and the colonies were counted. 30% glycerol was stored in a -80°C refrigerator for later use. Before intervention, the glycerol was removed by centrifugation and the concentration of the bacterial suspension was adjusted to 5×10 10 CFU / mL.

[0051] Lactobacillus cell lysate:

[0052] (1) According to the preparation method of Lactobacillus culture liquid, the culture liquid was centrifuged to obtain bacterial mud, and the suspension concentration was adjusted to 1.5×10 9 CFU / mL, and after homogenization in a high-pressure homogenizer (800-1200 MPa) for 10 times, the cells were filtered through a 0.22 μm filter membrane to obtain the bacterial lysate prepared from the bacterial suspension of the corresponding concentration.

[0053] (2) Preparation of cell culture medium containing 5% (v / v) Lactobacillus cell lysate:

[0054] The bacterial lysate obtained above was added to a cell culture medium at a ratio of 5% (v / v), including 84% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100× penicillin and streptomycin mixed solution (penicillin content in the mixed solution was 10,000 U / mL, and streptomycin concentration was 10 mg / mL) + 5% (v / v) Lactobacillus bacterial lysate.

[0055] Candida albicans suspension:

[0056] (1) Preparation of seed solution:

[0057] Candida albicans SC5314 was inoculated into YPD medium and cultured in a shaking incubator at 28°C for 18 h to prepare a seed solution.

[0058] (2) The Candida albicans seed solution was inoculated into YPD medium at a 2% inoculum volume. After incubation at 28°C for 18 h, the cells were collected by centrifugation and resuspended in sterile saline to a concentration of 1×10 7 CFU / mL.

[0059] Example 1: Screening and identification of saliva-associated Lactobacillus CCFM1418

[0060] Screening of strains: 0.2 mL of samples collected from healthy people were added to 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 operation 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 dish, pour into MRS solid culture medium, mix gently, wait for the culture medium to solidify, and culture inverted at 37°C for 48 h.

[0061] Strain Identification: Use a sterile inoculating loop to dip a small amount of the strain into MRS solid medium for activation and incubate at 37°C for 48 hours. Subsequently, pick a single colony and inoculate it into MRS liquid medium. Incubate in a 37°C incubator for 18 hours to obtain a liquid fermentation broth of the corresponding strain. Take 1.0 mL of the cultured bacterial broth, centrifuge at 5000 rpm for 3 minutes, discard the supernatant, add 1.0 mL of sterile saline, wash three times, and resuspend in 1.0 mL of sterile water to use as a template for strain identification. Set up a 20 μL PCR system, add 0.5 μL of forward primer (10 μM), 0.5 μL of reverse primer (10 μM), 10 μL of 2× Taq Mixture, 0.5 μL of bacterial suspension, and 8.5 μL of double-distilled water. Primer information is shown in Table 1.

[0062] Table 1: Primer information

[0063]

[0064] The 16S sequence obtained by sequencing is shown in SEQ ID NO. 1. The resulting 16S sequence was confirmed for species identity using NCBI's BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST). The closer the Query Cover and Identification values ​​are to 100%, the more reliable the identification. If multiple species were identified in the alignment, the Complete Genome species was prioritized, taking into account the numerical values. The results showed that the strain was Lactobacillus salivarius, designated CCFM 1418, and proceeded to the next step of the experiment.

[0065] Example 2: Ability of Lactobacillus to Promote PIGR Production in Oral Epithelial Cells

[0066] Oral epithelial HOK cells were revived and passaged three times, and the cell concentration was adjusted to 5×10 5 2 mL of this cell suspension was inoculated into a 6-well cell culture plate and incubated at 5% CO₂ and 37°C for 24 hours before the intervention. A control group received cell culture medium containing 5% PBS, while the strain-treated groups received cell culture medium containing 5% (v / v) Lactobacillus cell lysate. Three replicates were set up in each group. After 24 hours, the cell supernatant was collected and the secretory component (SC) content in the supernatant was assayed according to the ELISA kit instructions to characterize PIGR expression.

[0067] The results are as follows Figure 1As shown in the results, after 24 hours of intervention, the PIGR content in the blank group supernatant was 252.65 pg / mg. Compared with the blank control group, the PIGR content in the saliva-Lactobacillus CCFM 1418 bacterial lysate (CCFM 1418-P) was 339.54 pg / mg, which significantly upregulated the expression of the polymeric immunoglobulin receptor (PIGR) in oral epithelial cells (p < 0.01). However, the PIGR content in the saliva-Lactobacillus FGSYC2M4-P group supernatant was 283.52 pg / mg, which was not significantly different from the control group. Therefore, at the cellular level, saliva-Lactobacillus CCFM 1418-P can promote the expression of PIGR in oral epithelial cells, which is expected to increase the transport of IgA to form SIgA in the body, thereby exerting an anti-infective effect in the host.

[0068] Example 3: Application of saliva combined with Lactobacillus CCFM1418 in regulating host immunity

[0069] 1. Preparation of Lactobacillus suspension

[0070] Saliva-Lactobacillus CCFM1418 live bacteria group: A small amount of saliva-Lactobacillus CCFM1418 bacterial suspension was dipped into a sterile inoculating loop and activated on an MRS solid plate, incubated at 37°C for 48 hours. Subsequently, a single colony was picked and inoculated into MRS liquid medium and incubated in a 37°C incubator for 18 hours to prepare a seed solution. The prepared seed solution was inoculated into MRS liquid medium at a 2% (v / v) inoculum volume and incubated in a 37°C incubator for 18 hours. The bacteria were collected by centrifugation and the colonies were counted. The cells were stored in a -80°C refrigerator with 30% glycerol until use. Before intervention, the glycerol was removed by centrifugation and the concentration of the bacterial suspension was adjusted to 5×10 10 CFU / mL.

[0071] Saliva combined with Lactobacillus CCFM1418 dead bacteria group: The preparation method and concentration of the dead bacteria group were the same as those of the live bacteria. The difference was that the dead bacteria group was inactivated at 65°C for 30 minutes, and the inactivation effect was checked by plate coating. After heat treatment, the supernatant was removed by centrifugation, resuspended in sterile saline, and then homogenized under high pressure to obtain a bacterial lysate. The postbiotics were obtained and freeze-dried for later use. Before intervention, the dead bacteria group was resuspended with sterile saline to a concentration corresponding to that of the live bacteria group suspension.

[0072] 2. Experimental animals and strains:

[0073] SPF-grade BALB / c female mice, 6 weeks old, weighing 15-18 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (Production License No. SCXK(Jing)2012-0001). Saliva-conjugated Lactobacillus CCFM1418 was obtained from the strain bank of the Biotechnology Center of Jiangnan University. Candida albicans SC5314 was used (purchased from Ningbo Mingzhou Technology Co., Ltd.). Figure 2Table 2 shows the animal experiment process.

[0074] Table 2: Animal experimental plan and grouping

[0075]

[0076] Before the experiment began, all mice were randomly divided into 4 groups according to their body weight, and the mice were placed in an environment with a light-dark cycle of 22°C-24°C and 12 hours for one week. Referring to Table 2, all groups of mice were fed normally throughout the experiment. The blank group mice were treated with normal saline instead of probiotics throughout the experiment, and immunosuppressants were injected on the 14th and 16th days. The Candida albicans model group was treated with normal saline instead of probiotics on days 0-17, and immunosuppressants were injected on the day before and the day after Candida albicans infection (the infection method of Candida albicans was: anesthetize the mice, soak them in a cotton ball of the same size with a concentration of 1×10 7 CFU / ml of Candida albicans suspension was then inoculated sublingually in mice for approximately 60 minutes. The probiotic intervention group received saliva combined with live Lactobacillus CCFM1418 bacteria and bacterial lysate for 17 consecutive days (days 0-17). Immunosuppressants were injected on days 14 and 16, and Candida albicans infection was performed on day 15. All interventions involved oral irrigation with an 8-gavage needle. (The dead bacteria group had the same bacterial concentration as the live bacteria, and the bacteria were inactivated at 65°C for 30 minutes, with the inactivation effect checked by plate smearing.)

[0077] The specific steps are as follows:

[0078] (1) Intervention experiment: Days 1-17,

[0079] Blank control group: Use No. 8 elbow gavage needle to draw 30 μL of sterile saline for oral irrigation once a day.

[0080] Model group mice: Use a No. 8 elbow gavage needle to draw 30 μL of sterile saline for oral irrigation once a day.

[0081] Saliva combined with live Lactobacillus CCFM1418 bacteria group: Use a No. 8 elbow gavage needle to draw 30 μL of saliva combined with live Lactobacillus CCFM1418 bacteria suspension for oral irrigation, and slowly inject it into the mouse oral cavity once a day.

[0082] Saliva combined with killed Lactobacillus CCFM1418 group: Use a No. 8 elbow gavage needle to draw 30 μL of saliva combined with Lactobacillus CCFM1418 bacterial lysate for oral irrigation, and slowly inject it into the mouse oral cavity once a day.

[0083] (2) Immunosuppression: All groups of mice were immunosuppressed on days 14 and 16 by subcutaneous injection of 0.2 mL of 225 mg / kg cortisone acetate.

[0084] (3) Infection experiment (modeling period): Except for the blank group, the other groups were anesthetized on the 15th day and soaked with cotton balls of the same size in a concentration of 1×10 7 CFU / mL of Candida albicans suspension was then inoculated sublingually in mice for about 60 minutes, and then the cotton ball was removed.

[0085] After the intervention, the tongue tissue of mice was sampled for subsequent tissue pathogen load, HE tissue pathology analysis, PAS pathogen hyphae invasion analysis, and detection of PIGR and secretory immunoglobulin (SIgA) expression levels in tongue tissue.

[0086] 3. Experimental results:

[0087] (1) Changes in the tongue mucosa of mice

[0088] After the intervention, the tongue mucosal tissue of the mice was observed to assess the infection degree of Candida albicans in the oral cavity. Figure 3 As shown, the tongue dorsum of the control mice was light red and smooth and moist, while the tongue of the model group mice was covered with a thick pseudomembrane formed by Candida albicans, and the tongue was atrophic. After the saliva combined with Lactobacillus and its postbiotics intervention, the appearance of the tongue tissue of each group of mice was similar to that of the control group, indicating that Lactobacillus can significantly resist the colonization of Candida albicans in the oral cavity and exert its anti-pathogenic effect in the host's oral cavity.

[0089] (2) Candida albicans load in mouse tongue tissue

[0090] After the intervention, the number of Candida albicans attached to the tongue of the mice was used to assess the degree of infection. Figure 4 As shown, Log 10 CFU / g is the vertical axis. After the intervention, the model group had the highest load of pathogenic bacteria in the tongue tissue, which was about 7.52×10 5 CFU / g; the load of Candida albicans in the tongue tissue of the saliva combined with Lactobacillus CCFM1418-L group after intervention was approximately 1.0×10 5 CFU / g, which was significantly different from that in the model group (P < 0.05); similarly, the load of pathogenic bacteria Candida albicans in the tongue tissue of the saliva combined with Lactobacillus CCFM1418-P group after intervention was approximately 5.33×10 4The CFU / g showed a significant decrease compared with the model group (P < 0.05). Therefore, saliva combined with live bacteria and bacterial lysate of Lactobacillus CCFM1418 can reduce the colonization of pathogenic bacteria Candida albicans in the mouse oral cavity, playing an anti-pathogenic bacterial infection role.

[0091] (3) Expression of PIGR and SIgA in mouse tongue tissue

[0092] After the intervention, the expression levels of PIGR and SIgA in the tongue tissue of mice were evaluated. Figure 5 As shown in Figure A, the average expression levels of PIGR in the blank group and the model group were 47.11 pg / mg and 40.40 pg / mg, respectively, with no significant difference between the two groups. This is because all groups were injected with immunosuppressants, and neither the blank group nor the model group received probiotic intervention, so there was no significant difference between the indicators. After intervention, the saliva combined with Lactobacillus CCFM1418-L group expressed 70.06 pg / mg of PIGR in the tongue tissue of mice, a significant increase compared to the model group (P < 0.01). Similarly, after intervention, the saliva combined with Lactobacillus CCFM1418-P group expressed 60.65 pg / mg of PIGR in the tongue tissue, a significant increase compared to the model group (P < 0.05). Therefore, both the live bacteria and bacterial lysate of saliva combined with Lactobacillus CCFM1418 can upregulate the expression of the polymeric immunoglobulin receptor PIGR that transports IgA in tongue tissue, thereby potentially further increasing the expression of mucosal SIgA in vivo and enhancing the anti-pathogenic bacterial infection effect in the mouse oral cavity.

[0093] Upregulation of PIGR increases the binding of IgA within tissues, thereby increasing the production of SIgA on mucosal surfaces. Therefore, the expression level of SIgA in mouse tongue tissue was further evaluated in vivo. Figure 5 As shown in Figure B, the average SIgA expression levels in the blank and model groups were 180.95 ng / mg and 125.56 ng / mg, respectively, with no significant difference between the two groups. In the saliva-Lactobacillus CCFM1418-L group, SIgA expression in tongue tissue was 266.94 ng / mg after intervention, significantly higher than that in the model group (P < 0.01). Similarly, in the saliva-Lactobacillus CCFM1418-P group, SIgA expression in tongue tissue was 210.38 ng / mg after intervention, also significantly higher than that in the model group (P < 0.05). In summary, both live bacteria and bacterial lysates of saliva-Lactobacillus CCFM1418 can upregulate the expression of secretory immunoglobulin (SIgA) on the tongue mucosal surface, thereby preventing the invasion of mucosal pathogens and enhancing the host's oral anti-infection ability.

[0094] (4) Histopathological analysis of mouse tongue

[0095] After the intervention, longitudinal sections of the mouse tongue tissue were observed. Figure 6 As shown, the dorsum of the tongue tissue of mice in the blank group had neatly arranged filiform papillae, an intact and smooth epithelial stratum corneum, clear boundaries between the epithelial layers, and no inflammatory cell recruitment. In contrast, the epithelial stratum corneum of the model group was severely damaged, with virtually no filiform papillae on the dorsum of the tongue. Furthermore, there was a large recruitment of inflammatory cells and the formation of small abscesses in some locations. After intervention with saliva combined with Lactobacillus CCFM1418-L and CCFM1418-P, pathological sections of the mouse tongue tissue were similar to those in the blank group, indicating that both live bacteria and bacterial lysates of saliva combined with Lactobacillus CCFM1418 can resist the invasion of oral pathogens and, to a certain extent, protect the oral mucosal tissue from damage.

[0096] (5) Analysis of hyphae invasion in mouse tongue

[0097] After the intervention, PAS staining was performed on the tongue tissue of mice to observe the invasion of Candida albicans hyphae into the tongue tissue. Figure 7 As shown, PAS staining of the blank group showed no hyphae invading the tongue tissue; PAS staining of the model group revealed a large number of hyphae invading the epithelial layer of the tongue tissue, surrounded by a large number of inflammatory cells. These hyphae mostly invaded the deep layers of the epithelial tissue at a vertical or oblique angle, causing tissue damage. After intervention with saliva combined with Lactobacillus CCFM1418-L and CCFM1418-P, PAS staining of the mouse tongue tissue was similar to that of the blank group, indicating that the live bacteria and bacterial lysate of saliva combined with Lactobacillus CCFM1418 can, to a certain extent, prevent the invasion of the oral pathogen Candida albicans into the oral mucosal tissue, thereby exerting a role in resisting oral pathogen infection.

[0098] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Saliva combined with Lactobacillus ( Ligilactobacillus salivarius )CCFM1418 was deposited in Guangdong Provincial Microbiological Culture Collection on August 2, 2024, with the deposit number GDMCC No: 64943.

2. A postbiotic prepared by combining saliva with Lactobacillus CCFM1418, characterized in that: The postbiotics include the inactivated bacteria of saliva-associated Lactobacillus CCFM1418 and / or its lysate according to claim 1.

3. A method for preparing saliva-combined Lactobacillus CCFM1418 postbiotics, characterized in that: Including (a) or (b): (a) culturing the saliva-associated Lactobacillus CCFM1418 of claim 1 to the logarithmic growth phase, inactivating the saliva by heat treatment, centrifuging, discarding the supernatant, and lyophilizing to obtain a postbiotic lyophilized powder; (b) Cultivating the saliva-associated Lactobacillus CCFM1418 of claim 1 to the logarithmic growth phase, inactivating the culture by heat treatment, crushing the culture by high-pressure homogenization, and then freeze-drying to obtain a postbiotic freeze-dried powder.

4. A microbial preparation, characterized in that Contains the saliva-associated Lactobacillus CCFM1418 according to claim 1, and / or its postbiotics.

5. The microbial preparation according to claim 4, characterized in that The content of saliva-associated Lactobacillus CCFM1418 in the microbial preparation is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

6. A medicine containing the saliva-combined Lactobacillus CCFM1418 and / or its postbiotics according to claim 1, characterized in that: The medicine is a medicine for resisting oral pathogenic bacteria infection; the pathogenic bacteria is Candida albicans.

7. The medicine according to claim 6, characterized in that The content of saliva combined Lactobacillus CCFM1418 in the medicine is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

8. A daily chemical product containing the postbiotic according to claim 2, characterized in that: The daily chemical products include toothpaste, mouthwash or oral spray.

9. Use of the saliva-combined Lactobacillus CCFM1418 and / or its postbiotics according to claim 1 in the preparation of a medicament for resisting oral pathogen infection, characterized in that: The pathogenic bacteria is Candida albicans.

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