Ligilactobacillus salivarius strain capable of improving expression of secretory immunoglobulins in host and enhancing resistance thereof to infections, and postbiotic thereof

Abstract

The present invention relates to the technical field of microorganisms. Provided are a Ligilactobacillus salivarius strain capable of improving the expression of secretory immunoglobulins in a host and enhancing the resistance thereof to infections, and a postbiotic thereof. The provided viable Ligilactobacillus salivarius strain and the postbiotic thereof are capable of modulating host immunity and improving the resistance of a host to oral pathogenic bacterial infections, can significantly up-regulate the expression of PIGR in the basal membrane of oral mucosal epithelial cells both in vitro and in vivo in animals, and significantly up-regulate the expression of SIgA in animals, thereby improving the oral immunity of the host and resisting infections from oral pathogenic bacteria; and have wide application prospects in the modulation of the oral immunity of a host, the preparation of a product for improving the resistance of a host to oral infections, etc.

Description

Salivaria lactis with improved expression of host secretory immunoglobulin to enhance its anti-infection and its postbiotic TECHNICAL FIELD

[0001] The present application relates to a Salivaria lactis with improved expression of host secretory immunoglobulin to enhance its anti-infection and its postbiotic, belonging to the field of microbial technology. BACKGROUND

[0002] The anti-infection system of human oral cavity mainly includes: physical barrier of oral mucosa, immune cell activation and production of antibacterial peptides and secretory immunoglobulin and other antibacterial substances. The oral epithelial barrier is an important information transfer station for maintaining the homeostasis of the oral environment, and the gradual maturation of stratified squamous epithelium is a necessary condition for it to perform normal functions. The polymeric immunoglobulin receptor PIGR expressed by oral mucosal epithelial cells and various antibacterial substances secreted by oral mucosal epithelial cells, salivary gland epithelial cells and neutrophils form an effective multi-molecular defense network to resist invading pathogenic microorganisms and maintain normal oral microecology.

[0003] The oral microecology is a complex and complete system, mainly composed of the host's inherent oral characteristics and oral flora. The special anatomical morphology and tissue structure of the oral cavity participate in the composition of the oral microecology. Different oral sites have unique microenvironments, providing different binding sites for the adhesion and colonization of various microorganisms in the oral cavity. Oral temperature, humidity, pH conditions, metabolism of nutrients, and oxygen concentration in different parts all affect the colonization of oral microorganisms, so different oral sites have their unique and complex microbial flora. In addition, only when the balance between microbial colonization and host immune defense mechanisms is achieved, can the maintenance of the host's oral health status be achieved. Secretory immunoglobulin SIgA of 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 mainly produced by plasma cells in tissues such as salivary ducts, acinar near and oral mucosa. IgA secreted by plasma cells has a J chain, which can be specifically combined with polymeric immunoglobulin receptor PIGR expressed on the basal surface of epithelial cells to form an IgA-PIGR complex. The complex is then endocytosed into the cell and hydrolyzed at the top of the epithelial cell to form secretory immunoglobulin SIgA. Since one pIgR molecule is consumed per transport, upregulation of pIgR expression will increase the ability of mucosal epithelial cells to transport dimeric IgA. The formed mucosal SIgA can limit the adhesion of pathogens to the epithelial and dental surface, regulate host immunity by neutralizing enzymes, toxins and viruses, or synergistically acting with other antibacterial factors (such as lysozyme, lactoferrin, salivary peroxidase and mucin), and improve the host's anti-infection ability in the oral cavity.

[0005] At present, the application of symbiotic bacteria in oral diseases mainly focuses on the alleviation of diseases in the occurrence of oral diseases, such as the alleviation of symbiotic bacteria on caries, periodontitis, halitosis and oral mucosa and the like, and the mechanism of action is mostly to directly act on pathogenic bacteria to play a role. However, there are many anti-infection mechanisms in the body of the host, and the symbiotic bacteria in the body of the host can effectively activate the anti-infection pathway in the body of the host, regulate the production of downstream antibacterial substances, and thus play an anti-infection role. Lin et al. showed that the supernatant of Lactobacillus rhamnosus can activate the transport of IgA in the intestinal tract, thereby reversing the liver damage of autoimmune hepatitis mice. Fan et al. indicated that intestinal symbiotic bacteria can activate HIF-1α and LL-37 to inhibit the colonization of Candida albicans in the gastrointestinal tract. There are many reports on the regulation of host immunity by symbiotic bacteria in the field of intestinal tract, and similarly, it is particularly important to focus on the oral field and link symbiotic bacteria with the prevention of host oral anti-infection. SUMMARY

[0006] Therefore, the purpose of the present application is to provide a strain of Ligilactobacillus salivarius and its postbiotic, aiming to solve the lack of symbiotic bacteria in the prior art for regulating the oral immunity of the host.

[0007] The present application provides Ligilactobacillus salivarius CCFM1418, which is taxonomically named as Ligilactobacillus salivarius, and has been preserved in Guangdong Microbial Culture Collection Center on August 2, 2024, with a preservation number of GDMCC No: 64943 and a preservation address of No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100 Courtyard.

[0008] The Ligilactobacillus salivarius CCFM1418 is a gram-positive bacterium, and under a microscope, the cells are short rods. After being inoculated on MRS solid medium and cultured for 48 h, the colonies are generally milky white or light yellow protrusions with a diameter of 0.5-2 mm.

[0009] The present application also provides a postbiotic prepared from Ligilactobacillus salivarius CCFM1418, which comprises inactivated bacteria of Ligilactobacillus salivarius CCFM1418 and / or its lysate.

[0010] In one embodiment, the preparation method of the postbiotic is as follows: Ligilactobacillus salivarius CCFM1418 is cultured to the logarithmic growth phase, inactivated by heat treatment, centrifuged, the supernatant is discarded, and then freeze-dried to obtain a postbiotic freeze-dried powder.

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

[0012] In an embodiment, the lysate is a lysate of Lactobacillus salivarius CCFM1418 after high-pressure homogenization; the Lactobacillus salivarius CCFM1418 is inoculated into a fermentation medium for culture, the bacterial slurry is collected and resuspended with sterile normal saline, inactivated bacterial cells are obtained after heat treatment at 65℃ for 30 min, and then bacterial lysate is obtained by high-pressure homogenization.

[0013] The application also provides a microbial preparation containing the Lactobacillus salivarius CCFM1418 and / or probiotic thereof.

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

[0015] The application also provides a product containing the Lactobacillus salivarius CCFM1418 and / or probiotic thereof, wherein the product is a food, health product, medicine or daily chemical product.

[0016] In an embodiment, the food contains the Lactobacillus salivarius CCFM1418 and / or probiotic thereof, and conventional adjuvants.

[0017] In an embodiment, the conventional adjuvants include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids and nutritional fortifiers.

[0018] In an embodiment, the health product contains the Lactobacillus salivarius CCFM1418 and / or probiotic thereof, and conventional adjuvants.

[0019] In an embodiment, the conventional adjuvants include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids and nutritional fortifiers.

[0020] In an embodiment, the medicine contains the Lactobacillus salivarius CCFM1418 and / or probiotic thereof, and a pharmaceutically acceptable carrier.

[0021] In an embodiment, the pharmaceutically acceptable carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, flavoring agents commonly used in medicine.

[0022] In an embodiment, the daily use product comprises toothpaste, mouthwash or mouth spray.

[0023] In an embodiment, the content of the L. salivarius CCFM1418 in the product is not less than 1x10 6 CFU / mL or 1x10 6 CFU / g.

[0024] The present application also provides the use of L. salivarius CCFM1418 and / or its postbiotic in the preparation of a medicament for improving immunity and / or resisting oral pathogenic bacteria infection.

[0025] In an embodiment, the use comprises up-regulating the expression of PIGR of the host oral mucosal epithelial cells and / or up-regulating the expression of SIgA.

[0026] The present application also provides the use of L. salivarius CCFM1418 and / or its postbiotic in the preparation of a health product for improving immunity. Beneficial effects:

[0027] The present application provides a L. salivarius CCFM1418, which has the effect of enhancing the host's oral immunity against pathogenic bacteria infection, specifically embodied in:

[0028] (1) promoting the expression level of PIGR protein of oral epithelial cells;

[0029] (2) increasing the gene and protein expression level of PIGR in the tongue tissue of the individual;

[0030] (3) increasing the expression level of SIgA in the tongue tissue of the individual;

[0031] (4) reducing the load of Candida albicans in the tongue tissue of the individual;

[0032] (5) improving the pathological condition of the tongue tissue of the individual;

[0033] (6) improving the invasion of oral pathogenic bacteria Candida albicans to the tongue tissue of the individual;

[0034] Therefore, L. salivarius CCFM1418 and / or its postbiotic have great application prospects in products for regulating the host's oral immunity and resisting oral pathogenic bacteria infection.

[0035] Biological material preservation

[0036] Ligilactobacillus salivarius CCFM1418, taxonomically named as Ligilactobacillus salivarius, has been preserved in Guangdong Microbial Culture Collection Center on August 2, 2024, with a preservation number of GDMCC No: 64943 and a preservation address of No. 59, Building 5, 100, Martyrs' Avenue, Guangzhou. BRIEF DESCRIPTION OF DRAWINGS

[0037] Fig. 1 is an effect of different bacterial lysates on PIGR expression of oral epithelial cells (HOK-16B).

[0038] Fig. 2 is a flowchart of an animal experiment design scheme; wherein, Control is a blank control group; Model is a model group; Ligilactobacillus salivarius CCFM1418-L is a live bacteria external use group; and Ligilactobacillus salivarius CCFM1418-P is a bacterial lysate external use group.

[0039] Fig. 3 is a change of mouse tongue mucosa after intervention of Ligilactobacillus salivarius.

[0040] Fig. 4 is an effect of Ligilactobacillus salivarius on Candida albicans load in mouse tongue tissue.

[0041] Fig. 5 is an effect of Ligilactobacillus salivarius on PIGR and SIgA expression in mouse tongue tissue.

[0042] Fig. 6 is a histopathological evaluation diagram of mouse tongue tissue.

[0043] Fig. 7 is a PAS staining histopathological evaluation diagram of mouse tongue tissue. DETAILED DESCRIPTION

[0044] To make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with specific examples and with reference to the drawings. The strains, cells and animals involved in the following examples are as follows: SPF BALB / c mice, female, 6 weeks old, weighing 15-18 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Production License No. SCXK (Jing) 2012-0001). Ligilactobacillus salivarius CCFM1418 and FGSYC2M4 are from the Jiangnan University Biotechnology Center Culture Collection. Candida albicans SC5314 is purchased from Ningbo Mingzhou Technology Co., Ltd. Human oral keratinocytes HOK-16B are purchased from Qingqi (Shanghai) Biotechnology Development Co., Ltd.

[0045] The culture media involved in the following examples are as follows:

[0046] 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, sodium acetate anhydrous 2.0 g / L, citric acid hydrogen diamine 2.0 g / L, potassium phosphate dibasic 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.

[0047] 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, sodium acetate anhydrous 2.0 g / L, citric acid hydrogen diamine 2.0 g / L, potassium phosphate dibasic 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.

[0048] Cell complete medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100 x penicillin and streptomycin mixed solution (penicillin content 10000 U / mL, streptomycin concentration 10 mg / mL in the mixed solution).

[0049] The preparation method of the bacterial suspension and the bacterial lysate involved in the following examples is as follows:

[0050] Preparation of lactobacillus bacterial suspension:

[0051] (1) Preparation of seed liquid:

[0052] A small amount of saliva combined with lactobacillus CCFM1418 and FGSYC2M4 bacterial liquid was taken with a sterile inoculation loop to activate in MRS solid medium, and cultured at 37°C for 48 h. Then a single colony was picked and inoculated in MRS liquid medium, and cultured in a 37°C incubator for 18 h to prepare the seed liquid.

[0053] (2) The prepared seed liquid was inoculated in MRS liquid medium at an inoculation amount of 2% (v / v), and cultured in a 37°C incubator for 18 h. The bacterial cells were concentrated and collected by centrifugation, and the number of bacterial colonies was counted. 30% glycerol was preserved in a-80°C refrigerator for standby, and the glycerol was removed by centrifugation before intervention, and the bacterial suspension concentration was adjusted to 5 x 10 10 CFU / mL with sterile normal saline.

[0054] Lactobacillus bacterial lysate:

[0055] (1) According to the preparation method of lactobacillus bacterial liquid, the bacterial slurry was obtained by centrifugation of the bacterial liquid, and the suspension concentration was adjusted to 1.5 x 10 9CFU / mL, and the bacterial lysate was prepared by filtering the bacterial suspension with a concentration corresponding to the bacterial lysate prepared by 10 times of homogenization (800-1200 MPa) in a high-pressure homogenizer and 0.22 μm filter membrane.

[0056] (2) Preparation of cell culture solution containing 5% (v / v) of Lactobacillus bacterial lysate:

[0057] The bacterial lysate obtained above was added into the cell culture medium at a proportion of 5% (v / v), including 84% (v / v) of DMEM medium + 10% (v / v) of fetal bovine serum + 1% (v / v) of 100 x penicillin and streptomycin mixed solution (penicillin content 10000 U / mL, streptomycin concentration 10 mg / mL) + 5% (v / v) of Lactobacillus bacterial lysate.

[0058] Candida albicans bacterial suspension:

[0059] (1) Preparation of seed solution:

[0060] Candida albicans SC5314 was inoculated into YPD medium, and seed solution was prepared by incubation at 28°C for 18 h on a shaker.

[0061] (2) The Candida albicans seed solution was inoculated into YPD medium at an inoculation amount of 2%, and after incubation at 28°C on a shaker for 18 h, the bacterial cells were collected by centrifugation and resuspended in sterile normal saline to a concentration of 1 x 10 7 CFU / mL.

[0062] Example 1: Screening and identification of saliva combined with Lactobacillus CCFM1418

[0063] Strain screening: 0.2 mL of sample collected from healthy people was taken in 1.8 mL of sterile normal saline to obtain 10 -1 dilution, and 0.5 mL of 10 -1 dilution was taken in 4.5 mL of normal saline to obtain 10 -2 dilution, and this operation was repeated to obtain 10 -3 , 10 -4 , 10 -5 , 10 -6 gradient dilutions. 1 mL of 10 -4 , 10 -5 , 10 -6 dilution was taken in a plate and poured into MRS solid medium, and after the medium was solidified, it was incubated at 37°C for 48 h.

[0064] Strain identification: A small amount of the strain was activated in MRS solid medium using a sterile inoculation loop and cultured at 37°C for 48 h. A single colony was then picked and inoculated into MRS liquid medium and cultured at 37°C for 18 h to obtain the liquid fermentation broth of the corresponding strain. 1.0 mL of the cultured bacterial suspension was centrifuged at 5000 rpm for 3 min, the supernatant was discarded, and the suspension was washed three times with 1.0 mL of sterile physiological saline and resuspended in 1.0 mL of sterile water as a template for strain identification. A 20 μL PCR system was prepared, containing 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.

[0065] Table 1: Primer Information Table

[0066] The 16S sequence obtained from sequencing is shown in SEQ ID NO.1. The obtained 16S sequence was then used for species identification via NCBI's BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST). Query Cover and Identification scores closer to 100% were preferred. If multiple species were identified in the alignment results, the species with the Complete genome label was prioritized, taking into account both numerical values. The results showed that the strain was *Lactobacillus salivarius*, named CCFM 1418, and further experiments were conducted.

[0067] Example 2: The ability of Lactobacillus to promote PIGR generation in oral epithelial cells

[0068] Oral epithelial cells (HOK cells) were revived, passaged three times, and then the cell concentration was adjusted to 5 × 10⁻⁶. 5 Cells / mL were seeded into 2 mL of this concentration of cell suspension in 6-well cell culture plates and incubated at 37°C with 5% CO2 for 24 h before sample addition. A control group and a strain sample treatment group were set up. The control group received cell culture medium containing 5% PBS; the strain sample treatment groups received cell culture medium containing 5% (v / v) Lactobacillus cell lysis buffer, with 3 replicates per group. After 24 h, cell supernatant was collected, and the content of secreted component SC in the supernatant was detected according to the ELISA kit instructions to characterize the expression level of PIGR.

[0069] The results are shown in Figure 1. After 24 hours of intervention, the content of PIGR in the supernatant of the blank group was 252.65 pg / mg, the content of PIGR in the supernatant of the Lactobacillus salivarius CCFM 1418 cell lysate (CCFM 1418-P) group was 339.54 pg / mg, which could significantly up-regulate the expression of the polymeric immunoglobulin receptor PIGR in the oral epithelial cells (p<0.01), and the content of PIGR in the supernatant of the Lactobacillus salivarius FGSYC2M4-P group was 283.52 pg / mg, which had no significant difference compared with the control group. Therefore, at the cellular level, Lactobacillus salivarius CCFM 1418-P can promote the expression of PIGR in the oral epithelium, and is expected to increase the transport of IgA to form SIgA in vivo, thereby playing an anti-infection role in the host body.

[0070] Example 3: Application of Lactobacillus salivarius CCFM 1418 in regulating host immunity

[0071] 1. Preparation of Lactobacillus suspension

[0072] Lactobacillus salivarius CCFM 1418 live bacteria group: A small amount of Lactobacillus salivarius CCFM 1418 bacterial solution was taken with a sterile inoculation loop to activate on an MRS solid plate, and cultured at 37°C for 48 hours. Then a single colony was picked and inoculated in MRS liquid medium, and cultured in a 37°C incubator for 18 hours to prepare a seed solution. The prepared seed solution was inoculated in MRS liquid medium at a 2% (v / v) inoculation amount, and cultured in a 37°C incubator for 18 hours. The bacteria were collected by centrifugation, and the number of bacteria was counted. The bacteria were preserved in 30% glycerol at -80°C refrigerator, and before intervention, the glycerol was removed by centrifugation, and the bacterial suspension was adjusted to a concentration of 5×10 10 CFU / mL.

[0073] Lactobacillus salivarius CCFM 1418 dead bacteria group: The preparation method and concentration of the dead bacteria group were the same as those of the live bacteria group, except that the bacteria were 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, and the cell lysate was resuspended in sterile physiological saline and homogenized under high pressure to obtain the probiotic, which was freeze-dried for use. Before intervention, the bacterial suspension was resuspended in sterile physiological saline to a concentration corresponding to that of the live bacteria group.

[0074] 2. Experimental animals and strains:

[0075] SPF grade BALB / c mice, female, 6 weeks old, body weight 15-18 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Production License No. SCXK (Jing) 2012-0001). Lactobacillus salivarius CCFM1418 was from the Jiangnan University Biotechnology Center Culture Collection. Candida albicans was Candida albicans SC5314 (purchased from Ningbo Mingzhou Technology Co., Ltd.). Figure 2 and Table 2 are the animal experiment procedures.

[0076] Table 2: Animal experiment scheme and grouping

[0077] Before the experiment, all mice were randomly divided into 4 groups according to body weight, and the mice were placed in a 22-24℃, 12-hour light-dark cycle environment for one week. According to Table 2, all groups of mice were normally fed throughout the experiment. The blank group mice were replaced with saline throughout the experiment, and the immunosuppressant was injected on the 14th and 16th days. The Candida albicans model group was replaced with saline for 17 days, and the immunosuppressant was injected on the day before and the day after Candida albicans infection (the infection method of Candida albicans was: the mice were anesthetized, and the same size cotton ball was immersed in Candida albicans suspension with a concentration of 1×10 7 CFU / ml, and then inoculated sublingually in mice, with an action time of about 60 min). The probiotic intervention group was continuously intervened with Lactobacillus salivarius CCFM1418 live bacteria and bacterial lysate for 17 days (0-17 days), and the immunosuppressant was injected on the 14th and 16th days, and the Candida albicans infection was performed on the 15th day. The specific operation method of all interventions was oral cavity flushing with an 8-gauge gavage needle (the concentration of dead bacteria group was the same as that of live bacteria, and the bacteria were inactivated at 65℃ for 30 minutes, and the inactivation effect was checked by plate coating).

[0078] The specific steps are as follows:

[0079] (1) Intervention experiment: days 1-17,

[0080] Blank control group: 30μL of sterile saline was taken with an 8-gauge gavage needle for oral cavity flushing, once a day.

[0081] Model group mice: 30μL of sterile saline was taken with an 8-gauge gavage needle for oral cavity flushing, once a day.

[0082] Lactobacillus salivarius CCFM1418 live bacteria group: 30μL of Lactobacillus salivarius CCFM1418 live bacteria suspension was taken with an 8-gauge gavage needle for oral cavity flushing, and slowly injected into the mouse oral cavity, once a day.

[0083] Salivaria lactis CCFM1418 dead bacteria group: 30 μL of Salivaria lactis CCFM1418 cell lysate was taken by a 8-bend gavage needle and used for oral cavity flushing, and was slowly injected into the oral cavity of the mouse, once a day.

[0084] (2) Immunosuppression: All groups of mice were immunosuppressed on day 14 and day 16, and 0.2 mL of cortisone acetate with a concentration of 225 mg / kg was injected subcutaneously on the neck.

[0085] (3) Infection experiment (modeling period): Except for the blank group, other groups were anesthetized on day 15, and a cotton ball of the same size was soaked in a Candida albicans suspension with a concentration of 1 × 10 7 CFU / mL, and then inoculated under the tongue of the mouse, with an action time of about 60 min, and then the cotton ball was removed.

[0086] After the intervention ended, the tongue tissue of the mouse was sampled, and subsequent tissue pathogenic bacteria load, HE histopathology analysis, PAS pathogenic fungus hypha invasion analysis, and PIGR and secreted immunoglobulin (SIgA) expression level detection in the tongue tissue were performed.

[0087] 3. Experimental results:

[0088] (1) Changes in the tongue mucosa of the mouse

[0089] After the intervention ended, the tongue mucosa tissue of the mouse was observed to evaluate the degree of Candida albicans infection in the oral cavity. As shown in FIG. 3, the tongue dorsum of the control group mouse was light red, and the tongue was smooth and moist; the tongue of the model group mouse was covered with a thick pseudomembrane formed by Candida albicans, and the tongue body was atrophic. After the intervention of Salivaria lactis and its postbiotic, the appearance of the tongue tissue of each group of mice was similar to that of the blank group of mice, indicating that the lactobacillus could resist the colonization of Candida albicans in the oral cavity to a great extent and played a role in resisting pathogenic bacteria infection in the host oral cavity.

[0090] (2) Candida albicans load in the tongue tissue of the mouse

[0091] After the intervention ended, the number of Candida albicans attached to the tongue of the mouse was used to evaluate the degree of infection of the mouse. As shown in FIG. 4, the vertical coordinate was Log 10 CFU / g, and the pathogenic bacteria load in the tongue tissue of the model group after the intervention was the most, about 7.52 × 10 5 CFU / g; the Candida albicans load in the tongue tissue of the Salivaria lactis CCFM1418-L group after the intervention was about 1.0 × 10 5CFU / g, which was significantly different from the model group (P<0.05); similarly, the amount of C. albicans in the tongue tissue of the L. salivarius CCFM1418-P group after intervention was about 5.33 x 10 4 CFU / g, which was significantly different from the model group (P<0.05); similarly, the amount of C. albicans in the tongue tissue of the L. salivarius CCFM1418-P group after intervention was about 5.33 x 10

[0092] (3) Expression of PIGR and SIgA in the tongue tissue of mice

[0093] The expression of PIGR and SIgA in the tongue tissue of mice after intervention was evaluated. As shown in FIG. 5A, the average expression of PIGR in the blank group and the model group was 47.11 pg / mg and 40.40 pg / mg, respectively, and there was no significant difference between the two groups, because immunosuppressants were injected in each group, and the blank group and the model group were not intervened by probiotics, so there was no significant difference between the indicators. The expression of PIGR in the tongue tissue of mice after intervention in the L. salivarius CCFM1418-L group was 70.06 pg / mg, which was significantly higher than that in the model group (P<0.01); similarly, the expression of PIGR in the tongue tissue of mice after intervention in the L. salivarius CCFM1418-P group was 60.65 pg / mg, which was significantly higher than that in the model group (P<0.05). Therefore, the live bacteria and the cell lysate of L. salivarius CCFM1418 can up-regulate the expression of PIGR, the polymeric immunoglobulin receptor that transports IgA, in the tongue tissue, which can further increase the expression of mucosal SIgA in vivo and enhance the anti-pathogenic bacteria infection effect in the oral cavity of mice.

[0094] The up-regulation of PIGR increases the binding of IgA in the tissue, thereby increasing the production of SIgA on the mucosal surface. Therefore, the expression level of SIgA in the mouse tongue tissue was further evaluated in vivo. As shown in FIG. 5B, the average expression of SIgA in the blank group and the model group was 180.95 ng / mg and 125.56 ng / mg, respectively, and there was no significant difference between the two groups. The expression of SIgA in the mouse tongue tissue after intervention in the Lactobacillus salivarius CCFM1418-L group was 266.94 ng / mg, which was significantly higher than that in the model group (P<0.01). Similarly, the expression of SIgA in the tongue tissue after intervention in the Lactobacillus salivarius CCFM1418-P group was 210.38 ng / mg, which was also significantly up-regulated compared with the model group (P<0.05). In summary, the live bacteria and the cell lysate of Lactobacillus salivarius CCFM1418 can up-regulate the expression of secreted immunoglobulin SIgA on the tongue mucosal surface, thereby preventing the invasion of pathogens on the mucosal surface and enhancing the anti-infection ability of the host oral cavity.

[0095] (4) Histopathological analysis of mouse tongue tissue

[0096] After the intervention, the longitudinal section of the mouse tongue tissue was observed. As shown in FIG. 6, the filiform papillae on the dorsal tongue of the blank group were arranged in order, the epithelial keratin layer was complete and smooth, the boundaries between the epithelial layers were clear, and there was no inflammatory cell recruitment. In the model group, the epithelial keratin layer was severely damaged, the filiform papillae on the dorsal tongue basically did not exist, and there were a large number of inflammatory cell recruitment and the formation of individual locations of microabscesses. After the intervention of Lactobacillus salivarius CCFM1418-L and CCFM1418-P, the pathological section of the mouse tongue tissue was similar to that of the blank group, indicating that the live bacteria and the cell lysate of Lactobacillus salivarius CCFM1418 can resist the invasion of oral pathogenic bacteria and protect the oral mucosal tissue from damage to some extent.

[0097] (5) Analysis of the invasion of mouse tongue mycelium

[0098] After the intervention, the tongue tissue of the mice was subjected to PAS staining to observe the invasion of Candida albicans hyphae into the tongue tissue. As shown in Figure 7, no hyphae invasion into the tongue tissue was observed in the PAS staining of the blank group; the PAS staining of the model group showed that a large number of hyphae invaded the epithelial layer of the tongue tissue, accompanied by a large number of inflammatory cell recruitment around the hyphae, and most of the hyphae invaded the deep layer of the epithelial tissue at a vertical or inclined angle to destroy the tissue. After the intervention of saliva combined with Lactobacillus CCFM1418-L and CCFM1418-P, the PAS staining of the tongue tissue of the mice was similar to that of the blank group, indicating that the viable bacteria and bacterial lysates of saliva combined with Lactobacillus CCFM1418 can resist the invasion of oral pathogenic bacteria Candida albicans into oral mucosal tissue to a certain extent, thereby playing a role in resisting oral pathogenic bacterial infection.

[0099] Example 4: Preparation of saliva combined Lactobacillus CCFM1418 microbial preparation

[0100] Freeze-drying protective agent formula: maltose 100 g / L, trehalose 50 g / L, skim milk powder 100 g / L, mannitol 10 g / L, proline 5 g / L, L-cysteine hydrochloride 0.5 g / L, Tween 1 g / L.

[0101] The saliva combined Lactobacillus CCFM1418 was prepared into a microbial preparation, and the specific operation method was as follows: the saliva combined Lactobacillus CCFM1418 was cultured in a 37℃ incubator for 18 h, then centrifuged at 8000 g for 15 min at 4℃, and the bacterial slurry was taken. The bacterial slurry obtained by centrifugation was mixed with the freeze-drying protective agent at a mass volume ratio of 1:1 to prepare an emulsion, and then vortexed and dispersed for sufficient mixing, and then subjected to freeze-drying according to the following procedure: the temperature of the plate was reduced to -4℃ within 10 min, and then reduced to -50℃ for pre-freezing, and maintained for 1 h; during the first drying, the vacuum degree was reduced, the plate was heated to -30℃ within 1 h, and in order to remove free water, it was operated at a vacuum of 20 Pa for 18 h; during the second drying, the temperature of the plate was controlled to be heated to 25℃ within 1 h, and continuously controlled at 2 Pa for 16 h.

[0102] Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and anyone skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. Ligilactobacillus salivarius CCFM1418 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 2, 2024, with accession number GDMCC No: 64943.

2. A postbiotic prepared using saliva in combination with Lactobacillus CCFM1418, characterized in that, The metabiotic includes the cell inactivation product of Lactobacillus salivarius CCFM1418 as described in claim 1 and / or its lysate.

3. A method for preparing postbiotics from *Lactobacillus salivans* CCFM1418, characterized in that... Including (a) or (b): (a) The *Lactobacillus salivarius* CCFM1418 of claim 1 was cultured to the logarithmic growth phase, inactivated by heat treatment, centrifuged, the supernatant was discarded and then freeze-dried to obtain post-biotic freeze-dried powder. (b) The *Lactobacillus salivariae* CCFM1418 of claim 1 is cultured to the logarithmic growth phase, inactivated by heat treatment, homogenized under high pressure, and then freeze-dried to obtain post-biotic freeze-dried powder.

4. A microbial preparation, characterized in that, Contains the Lactobacillus salivarius CCFM1418 as described in claim 1, and / or its postgenes.

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

6. A product containing *Lactobacillus salivarius* CCFM1418 as described in claim 1 and / or its postbiotic, characterized in that, The products mentioned are food, health products, medicines, or daily chemical products.

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

8. The product according to claim 6, characterized in that, The products include, but are not limited to, microbial preparations, dietary supplements, solid beverages, toothpaste, mouthwash, or oral sprays.

9. The product according to claim 6, characterized in that, The product also includes conventional excipients; the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

10. The use of the Lactobacillus salivariae CCFM1418 and / or its postbiotics as described in claim 1 in the preparation of a medicament for enhancing immunity and / or resisting oral pathogenic bacterial infections.

11. The application according to claim 10, characterized in that, The pathogenic bacteria include, but are not limited to, Candida albicans; the application includes upregulating the expression of PIGR in the basal layer of host oral mucosal epithelial cells and / or upregulating the expression of SIgA.

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