Postbiotic with improved expression of host antimicrobial peptides and immunoglobulins prepared from lactobacillus salivarius and applications thereof

Abstract

The application discloses a postbiotic prepared from saliva combined lactobacillus and having the function of improving expression of host antibacterial peptide and immunoglobulin and application thereof, and belongs to the technical field of microorganisms. The saliva combined lactobacillus has the ability of regulating host immunity and improving host oral cavity anti-pathogenic bacteria infection, can significantly up-regulate expression of human oral cavity keratinocyte antibacterial peptide HBD-2 gene and protein in vitro, can significantly up-regulate expression of DEFB3 (homolog of human HBD-2) and secreted immunoglobulin SIgA in mice in vivo, and can be used for preparing a series of products for enhancing host oral cavity immunity and resisting oral cavity pathogenic bacteria infection, and has great application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a postbiotic prepared from a strain of Lactobacillus salivarius, which can improve the expression of host antibacterial peptides and immunoglobulins, and an application thereof, belonging to the field of microbial technology. BACKGROUND

[0002] Human oral microbiota is the second largest microbiota after intestinal microbiota. Oral microecology is a complex and complete system, mainly composed of host inherent oral characteristics and oral flora. The special anatomical morphology and tissue structure of the oral cavity participate in the composition of oral microecology. The anatomical structure of the oral cavity includes lips, cheeks, hard palate, soft palate, teeth, gums, tongue and tonsils, etc. 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.

[0003] Oral microorganisms interact with the host oral mucosal immune system, epithelial barrier and other factors to maintain the balance of oral microecology. The physical barrier of oral mucosa is the first line of defense between the body and the environment. Oral mucosal epithelial cells can produce antibacterial peptides and other antibacterial substances, and rapidly respond to foreign antigens in the environment, integrating the two defense systems of innate immunity and acquired immunity, and playing an important role in the early infection of pathogenic bacteria. Oral antibacterial peptides are an important part of the innate immune system. Oral epithelial cells, neutrophils and other cells can secrete defensins, LL-37, calprotectin and other antibacterial peptides. Among them, the microbial-related molecular model can up-regulate the secretion of β-defensin-2 (HBD-2) in oral epithelial cells, involving pathways such as TLR2-Myd88-IKBζ and IL-17-IKBζ, which play an indispensable role in regulating host innate immune response and preventing pathogenic bacterial infection.

[0004] Currently, the application of symbiotic bacteria in oral diseases mainly focuses on the alleviation of diseases in the presence of oral diseases, and the mechanism of action is mostly direct action on pathogenic bacteria, such as competition for binding sites, nutritional competition, production of antibacterial substances, etc. However, there are many anti-infection mechanisms in the host body, and the microbial-related molecular model can effectively activate the anti-infection pathway in the host body, regulate the production of downstream antibacterial substances, and thus play an anti-infection role. Taylor et al. indicated that the up-regulation of IKBzeta, a protein in the non-classical pathway of NF-kappa B, can regulate the expression of host antibacterial peptide DEFB3, thereby playing a role in anti-Candida albicans infection. There have been many reports on the interaction between probiotics and the host to prevent pathogenic bacterial infection in the intestinal and nasal cavity fields, but more research in the oral field is still focused on the direct inhibition of pathogenic bacteria by probiotics. Therefore, it is particularly important to link probiotics to host immunity to prevent oral pathogenic bacterial infection. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a postbiotic prepared from Ligilactobacillus salivarius, aiming to solve the lack of symbiotic bacteria in regulating the oral immunity of the host in the prior art. It can significantly up-regulate the expression of antibacterial peptides in the oral cavity of the host in vivo and in vitro, thereby improving the oral immunity of the host and resisting the infection of oral pathogenic bacteria.

[0006] The present application provides Ligilactobacillus salivarius CCFM1417, which is taxonomically named as Ligilactobacillus salivarius, and has been preserved in the Guangdong Microbial Culture Collection Center on August 2, 2024, with a preservation number of GDMCC No:64942 and a preservation address of No. 59 Building, 5th Floor, Guangzhou City, Guangzhou City, Guangdong Province.

[0007] The Ligilactobacillus salivarius CCFM1417 is a gram-positive bacterium, and the cells are short rods under a microscope. The colonies are generally white or yellowish convex after being inoculated on MRS solid medium and cultured for 48 h, with a diameter of 0.5-2 mm and a round shape.

[0008] The present application also provides a microbial preparation containing the Ligilactobacillus salivarius CCFM1417.

[0009] In one embodiment, the content of Ligilactobacillus salivarius CCFM1417 in the microbial preparation is ≥1×10 6 CFU / mL or 1×10 6 CFU / g.

[0010] The present application also provides a postbiotic prepared from the Lactobacillus salivarius CCFM1417.

[0011] In an embodiment, the postbiotic comprises a bacterial inactivant, a bacterial lysate, and / or an inactivated fermentation broth.

[0012] In an embodiment, the postbiotic is prepared by culturing the Lactobacillus salivarius CCFM1417 to the logarithmic growth phase, inactivating by heat treatment, centrifuging, collecting the precipitate to obtain the postbiotic, and freeze-drying to obtain the postbiotic freeze-dried powder.

[0013] In an embodiment, the postbiotic is prepared by culturing the Lactobacillus salivarius CCFM1417 to the logarithmic growth phase, inactivating by heat treatment, high-pressure homogenizing, and freeze-drying to obtain the postbiotic freeze-dried powder.

[0014] In an embodiment, the bacterial lysate is a lysate of the Lactobacillus salivarius CCFM1417 after high-pressure homogenization; the preparation method comprises culturing the Lactobacillus salivarius CCFM1417 in a fermentation medium, resuspending the bacterial slurry with sterile normal saline, inactivating the bacterial cells by heat treatment at 65°C for 30 min, and then high-pressure homogenizing to obtain the bacterial lysate.

[0015] The present application also provides a product containing the Lactobacillus salivarius CCFM1417 and / or the postbiotic thereof.

[0016] In an embodiment, the product is a food, a health product, a pharmaceutical product, or a daily-use product.

[0017] In an embodiment, the food contains the Lactobacillus salivarius CCFM1417 and / or the postbiotic prepared therefrom, and a conventional excipient.

[0018] In an embodiment, the conventional excipient comprises one or more of a filler, a flavoring agent, a binder, a disintegrant, a lubricant, an antacid, and a nutritional fortifier.

[0019] In an embodiment, the health product contains the Lactobacillus salivarius CCFM1417 and / or the postbiotic prepared therefrom, and a conventional excipient.

[0020] In an embodiment, the conventional excipient comprises one or more of a filler, a flavoring agent, a binder, a disintegrant, a lubricant, an antacid, and a nutritional fortifier.

[0021] In an embodiment, the pharmaceutical product contains the Lactobacillus salivarius CCFM1417 and / or the postbiotic prepared therefrom, and a pharmaceutically acceptable carrier.

[0022] In an embodiment, the pharmaceutically acceptable carrier comprises one or more of fillers, binders, humectants, disintegrants, lubricants, flavoring agents commonly used in medicine.

[0023] In an embodiment, the daily use product comprises toothpaste, mouthwash or oral spray.

[0024] The present application also provides the use of the L. salivarius CCFM1417 and / or its postbiotic in the preparation of a medicament for improving immunity and / or resisting oral pathogenic bacterial infection.

[0025] In an embodiment, the use comprises but is not limited to promoting the expression of oral epithelial cell antibacterial peptide HBD-2, and increasing the gene and protein expression level of antibacterial peptide HBD-2 in the individual's tissue.

[0026] Beneficial effects:

[0027] The present application provides a L. salivarius CCFM1417, which has the effect of enhancing the host's oral immunity against pathogenic bacterial infection, specifically embodied in:

[0028] (1) promoting the expression of oral epithelial cell antibacterial peptide HBD-2;

[0029] (2) increasing the gene and protein expression level of antibacterial peptide HBD-2 in the individual's tongue tissue;

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

[0031] (4) activating the IKBz anti-pathogenic bacterial infection pathway in the host's body;

[0032] (5) reducing the load of Candida albicans in the individual's tongue tissue;

[0033] (6) improving the pathological condition of the individual's tongue tissue;

[0034] (7) improving the invasion of oral pathogenic bacteria Candida albicans into the individual's tongue tissue;

[0035] Therefore, the postbiotic prepared from the L. salivarius CCFM1417 has great application prospects in products for regulating the host's oral immunity and resisting oral pathogenic bacterial infection.

[0036] Biological material preservation

[0037] Ligilactobacillus salivarius CCFM1417, 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: 64942 and a preservation address of No. 59, Building 5, 100, Martyrs' Road, Guangzhou. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Effects of different bacterial lysates on HBD-2 expression of oral epithelial cells (HOK-16B);

[0039] Figure 2 Flow chart of animal experiment design scheme; wherein, Control is a blank control group; Model is a model group; Ligilactobacillus salivarius CCFM1417-P is a bacterial lysate in vitro use group;

[0040] Figure 3 Changes of mouse tongue mucosa after intervention of Ligilactobacillus salivarius;

[0041] Figure 4 Effects of Ligilactobacillus salivarius on Candida albicans load in mouse tongue tissue;

[0042] Figure 5 Effects of Ligilactobacillus salivarius on activation of anti-infection pathways in mouse tongue tissue;

[0043] Figure 6 Effects of Ligilactobacillus salivarius on expression of DEFB3 and SIgA in mouse tongue tissue;

[0044] Figure 7 Pathological evaluation diagram of mouse tongue tissue;

[0045] Figure 8 PAS staining pathological evaluation diagram of mouse tongue tissue. DETAILED DESCRIPTION

[0046] For purposes of the present application, the technical solutions and advantages are more clearly and obviously understood, the following further details the present application with specific examples, and with reference to the accompanying drawings. The following examples involve strains, cells and animals 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). Lactobacillus salivarius CCFM1417, FWXBH242 from Jiangnan University Biotechnology Center Culture Collection. Candida albicans SC5314, purchased from Ningbo Mingzhou Technology Co., Ltd. Human oral keratinocytes HOK-16B purchased from Qingqi (Shanghai) Biotechnology Development Co., Ltd.

[0047] The culture medium involved in the following examples is as follows:

[0048] 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 diammonium 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.

[0049] 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 diammonium 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.

[0050] 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).

[0051] The preparation method of the bacterial lysate and bacterial suspension involved in the following examples is as follows:

[0052] Preparation of Lactobacillus suspension:

[0053] (1) Preparation of seed liquid:

[0054] A small amount of Lactobacillus salivarius CCFM1417, FWXBH242 bacterial liquid was taken with a sterile inoculation loop to activate in MRS solid medium, and cultured at 37℃ for 48h. Then a single colony was picked and inoculated in MRS liquid medium, and cultured in a 37℃ incubator for 18h to prepare the seed liquid.

[0055] (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 18h. The bacteria were collected by centrifugal concentration and the number of colonies was counted. 30% glycerol was preserved in a -80°C refrigerator for animal experiments. Before intervention, the glycerol was removed by centrifugation, and the concentration of the bacterial suspension was adjusted to 5×10 10 CFU / mL with sterile normal saline.

[0056] Lactobacillus cell lysate:

[0057] (1) The resuspended bacterial suspension (concentration about 1.5×10 9 CFU / mL) was homogenized (800-1200MPa) for 10 times in a high-pressure homogenizer, and then filtered with a 0.22μm filter membrane.

[0058] (2) Preparation of cell culture solution containing 5% (v / v) lactobacillus cell lysate:

[0059] The cell lysate obtained above was added to the cell culture medium at a proportion 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 10000U / mL, streptomycin concentration 10mg / mL) + 5% (v / v) lactobacillus cell lysate.

[0060] Candida albicans bacterial suspension:

[0061] (1) Preparation of seed liquid:

[0062] Candida albicans SC5314 was inoculated into YPD medium and cultured in a shaker at 28°C for 18h to prepare the seed liquid.

[0063] (2) The Candida albicans seed liquid was inoculated into YPD medium at an inoculation amount of 2%, and cultured in a shaker at 28°C for 18h. The bacteria were collected by centrifugation and resuspended with sterile normal saline to a concentration of 1×10 7 CFU / mL.

[0064] Example 1: Isolation and identification of saliva combined with Lactobacillus CCFM1417

[0065] Strain screening: 0.2mL of the sample collected from healthy people was taken in 1.8mL of sterile normal saline to obtain 10 -1 dilution liquid, and then 0.5mL of 10 -1 dilution liquid was taken in 4.5mL of normal saline to obtain 10 -2 dilution liquid, and so on to obtain 10 -3 , 10 -4 , 10-5 ,10 -6 Gradient dilution. Take 10 -4 ,10 -5 ,10 -6 Dilution 1 mL in each dish, pour into MRS solid medium, mix gently, after the medium solidification, at 37°C inverted culture for 48h.

[0066] Strain identification: a small amount of strain was taken with a sterile inoculation ring to activate MRS solid medium, cultured at 37°C for 48h. Then single colonies were picked and inoculated in MRS liquid medium, cultured in a 37°C incubator for 18h to obtain the corresponding strain liquid fermentation broth. 1.0 mL of cultured bacterial solution was centrifuged at 5000 r / min for 3 min, the supernatant was discarded, and 1.0 mL of sterile physiological saline was added for washing 3 times, then resuspended in 1.0 mL of sterile water for use as a template for strain identification. The PCR system with a volume of 20 μL was set, in which 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 were added. The primer information is shown in Table 1.

[0067] Table 1: Primer information table

[0068]

[0069] After sequencing, the 16S sequence is shown as SEQ ID NO. 1. The obtained 16S sequence is subjected to species identification by BLAST of NCBI (http: / / www.ncbi.nlm.nih.gov / BLAST). The closer Query Cover and Identification are to 100%, the more feasible it is. If there are multiple species in the alignment results, the Complete genome species is preferred in consideration of the numerical value; the results show that the above strain is Lactobacillus salivarius, named CCFM1417, and the next step experiment is carried out.

[0070] Example 2: Ability of Lactobacillus to promote the production of HBD-2 of oral epithelial cells

[0071] The oral epithelial cells HOK cells were resuscitated, and after three passages, the cell concentration was adjusted to 5×10 5The cells were inoculated in 6-well cell culture plates at a concentration of 2 mL / mL, and after 24 h of incubation at 37°C in 5% CO2, the intervention was added. Control and strain sample treatment groups were set up, and the control group was added with cell culture solution containing 5% PBS; the strain sample treatment group was added with cell culture solution containing 5% Lactobacillus cell lysate, and 3 replicate wells were set up in each group. After 24 h of intervention, the cell supernatant was collected to detect the expression amount of antibacterial peptide HBD-2 in the supernatant according to the ELISA kit instructions.

[0072] The results are shown in Figure 1 After 24 h of intervention, the content of HBD-2 in the supernatant of the blank group was 198.94 pg / mg, the content of HBD-2 in the saliva combined Lactobacillus CCFM 1417 cell lysate group (CCFM 1417-P) was 238.87 pg / mg, which could significantly up-regulate the expression of antibacterial peptide HBD-2 in oral epithelial cells (p<0.01); and the saliva combined Lactobacillus FWXBH242 cell lysate group (FWXBH242-P) had no significant difference compared with the blank control group. Therefore, at the cellular level, saliva combined Lactobacillus CCFM 1417-P can promote the expression of oral epithelial antibacterial peptide HBD-2, regulate host innate immunity, and enhance the ability of the host to resist pathogenic bacterial infection.

[0073] Example 3: Application of saliva combined Lactobacillus CCFM 1417 in regulating host immunity

[0074] 1. Preparation of Lactobacillus probiotics

[0075] The saliva combined Lactobacillus CCFM 1417 was inoculated into MRS culture medium to prepare a seed liquid at 37°C; 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 bacteria were collected by centrifugal concentration, and the bacteria were resuspended in sterile physiological saline to a concentration of 5×10 10 CFU / ml, inactivated at 65°C for 30 min, and the inactivation effect was checked by plate coating. After heat treatment, the bacteria were lysed by high-pressure homogenization to obtain the probiotics, which were freeze-dried for standby. Before intervention, the bacteria suspension was adjusted to a concentration of 5×10 10 CFU / ml with sterile physiological saline.

[0076] 2. Experimental animals and strains:

[0077] SPF grade BALB / c mice, female, 6 weeks old, body weight 15-18 g, purchased from Beijing Vantoll Life Science and Technology Co., Ltd. (Production License No. SCXK (Jing) 2012-0001). Lactobacillus salivarius CCFM1417 was from the Jiangnan University Biotechnology Center Culture Collection. Candida albicans was purchased from Ningbo Mingzhou Technology Co., Ltd. Figure 2 Table 2 is the animal experiment process.

[0078] Table 2: Animal experiment scheme and grouping

[0079]

[0080] Before the experiment, all mice were randomly divided into 3 groups according to body weight, and the mice were placed in a 22-24℃, 12-hour light-dark cycle environment for one week. Referring 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 (from day 0 to day 17), 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 of 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 intervention group was continuously intervened with postbiotic prepared from Lactobacillus salivarius CCFM1417 for 17 days (from day 0 to day 17), and the immunosuppressant was injected on the 14th and 16th days, and Candida albicans infection was performed on the 15th day. The specific operation method of all interventions was oral flushing with an 8-gauge gavage needle.

[0081] The specific steps are as follows:

[0082] (1) Intervention experiment: from day 0, the specific steps are as follows:

[0083] Blank control group: 30 μL of sterile saline was taken with an 8-gauge gavage needle for oral flushing, once a day.

[0084] Model group mice: 30 μL of sterile saline was taken with an 8-gauge gavage needle for oral flushing, once a day.

[0085] Lactobacillus salivarius CCFM1417 group: 30 μL of Lactobacillus salivarius CCFM1417 bacterial lysate was taken with an 8-gauge gavage needle for oral flushing, once a day.

[0086] (2) Immunosuppression: Mice in all groups underwent immunosuppression on days 14 and 16 by subcutaneous injection of 0.2 mL of cortisone acetate at a concentration of 225 mg / kg in the neck.

[0087] (3) Infection experiment (modeling period): Except for the control group, all other groups were anesthetized on day 15, and cotton balls of the same size were soaked in a solution with a concentration of 1×10⁻⁶. 7 The cotton ball was inoculated into a CFU / mL Candida albicans suspension and then inoculated under the tongue of mice for about 60 minutes before being removed.

[0088] After the intervention, samples were taken from the tongue tissue of mice for subsequent analysis of pathogenic bacterial load, HE histopathology, PAS pathogenic bacteria hyphae invasion, and the expression levels of relevant antibacterial indicators in the tongue tissue.

[0089] 3. Experimental Results:

[0090] (1) Changes in the tongue mucosa of mice

[0091] After the intervention, the surface of the tongue mucosa of mice in each group was observed to assess the degree of Candida albicans infection in the oral cavity. Figure 3 As shown, the tongue of the control group mice was pale red, smooth, and moist, with no colonization of the pathogenic Candida albicans. The tongue of the model group mice was covered with a thick pseudomembrane formed by Candida albicans, adhering in patches to the surface of the tongue. After intervention with saliva combined with Lactobacillus, the colonization of Candida albicans on the surface of the mouse tongue mucosa was significantly reduced compared to the model group, with only a few points showing the pseudomembrane. The appearance of the mouse tongue tissue was similar to that of the control group mice, indicating that the metabiotic prepared by saliva combined with Lactobacillus CCFM1417 can largely resist the colonization of Candida albicans in the oral cavity and play an anti-pathogenic role in the host's oral cavity.

[0092] (2) Candida albicans load in mouse tongue tissue

[0093] After the intervention, homogenate and count the tongue tissue of mice to assess the colonization of Candida albicans in the tongue tissue. Figure 4 As shown, the average colonization of Candida albicans in the tongue tissue of the model group mice was approximately 7.52 × 10⁻⁶. 5 CFU / g; The viral load of Candida albicans in the tongue tissue of the salivary lactobacillus CCFM1417-P group was approximately 8.67 × 10⁻⁶ CFU / g. 4 The CFU / g level showed a significant decreasing trend compared to the model group (P < 0.05). Therefore, the metabiotic prepared by combining saliva with Lactobacillus CCFM1417 can reduce the colonization of pathogenic Candida albicans in the oral cavity of mice and exert an anti-pathogenic bacterial infection effect.

[0094] (3) Expression of anti-infection pathways in mouse tongue tissue

[0095] Activation of the IL-17 / IKBζ pathway is crucial for the resistance of mice to pathogenic bacterial infections in the oral cavity. Upregulation of IL-17 activates the expression of the non-canonical NF-κB pathway protein IKBζ in oral epithelial cells, which in turn upregulates the expression of the downstream product antimicrobial peptide DEFB3, thus playing a role in preventing oropharyngeal Candida infection in the mouse oral cavity.

[0096] After intervention, the expression of IL-17 and IKBζ in each group was assessed by homogenizing mouse tongue tissue. Figure 5 As shown in Figure A, the IL-17 level in the blank group was approximately 17.69 pg / mg, and the IL-17 level in the model group was approximately 13.69 pg / mg. There was no significant difference between the two groups because both groups received immunosuppressants, while neither the blank group nor the model group received probiotic intervention; therefore, there was no significant difference in the indicators. In the saliva-combined Lactobacillus CCFM1417-P group, the IL-17 level in the tongue tissue after intervention was approximately 19.67 pg / mg, which was significantly different from the model group (P < 0.05). Similarly, as... Figure 5 As shown in Figure B, the IKBζ expression level in the blank group was approximately 81.11 pg / mg, while the IKBζ expression level in the model group was approximately 67.73 pg / mg, with no significant difference between the two. However, in the saliva-combined Lactobacillus CCFM1417-P group, the IKBζ expression level in the tongue tissue after intervention was approximately 119.35 pg / mg, which was significantly different from the model group (P < 0.05). Therefore, the postbiotic prepared by saliva combined with Lactobacillus CCFM1417 can activate the IL-17 / IKBζ anti-pathogenic infection pathway in mice, upregulating the expression of downstream antimicrobial peptides and exerting an anti-pathogenic infection effect.

[0097] (4) Expression of antimicrobial peptides and immunoglobulins in mouse tongue tissue

[0098] After the intervention, the expression levels of the antimicrobial peptide DEFB3 and secretory immunoglobulin SIgA in the tongue tissue of mice were assessed. Figure 6 As shown in Figure A, after the intervention, the average expression level of the antimicrobial peptide DEFB3 in the tongue tissue of the model group was approximately 13.64 pg / mg, while the average expression level of the antimicrobial peptide DEFB3 in the tongue tissue of the saliva combined with Lactobacillus CCFM1417-P group was approximately 30.69 pg / mg after the intervention, which was significantly higher than that of the model group (P < 0.01). Similarly, as... Figure 6As shown in Figure B, after the intervention, the average expression level of immunoglobulin SIgA in the tongue tissue of the model group was approximately 125.56 ng / mg, while the average expression level of immunoglobulin SIgA in the tongue tissue of the saliva combined with Lactobacillus CCFM1417-P group was approximately 243.12 ng / mg, which was significantly higher than that of the model group (P < 0.05). Therefore, the postbiotic prepared by saliva combined with Lactobacillus CCFM1417 can upregulate the expression of antimicrobial peptide DEFB3 and secretory immunoglobulin SIgA in mice, thereby enhancing the host's oral immunity and resisting infection by oral pathogens.

[0099] (5) Histopathological analysis of mouse tongue tissue

[0100] After the intervention, longitudinal sections of the mouse tongue tissue were observed. Figure 7 As shown, in the control group, the filiform papillae on the dorsum of the tongue of mice were neatly arranged, the epithelial stratum corneum was intact and smooth, the boundaries between the epithelial layers were clear, and there was no recruitment of inflammatory cells. In contrast, the epithelial stratum corneum of the model group was severely damaged, the filiform papillae on the dorsum of the tongue were almost absent, and there was a large recruitment of inflammatory cells and the formation of small abscesses in some locations. After intervention with postbiotics from saliva combined with Lactobacillus, the pathological sections of the mouse tongue tissue were similar to those of the control group, indicating that the postbiotics prepared from saliva combined with Lactobacillus CCFM1417 can resist the invasion of oral pathogens and protect the oral mucosa from damage to a certain extent.

[0101] (6) PAS staining analysis of mouse tongue tissue

[0102] 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 8 As shown, PAS staining in the control group did not reveal hyphae invading the tongue tissue; however, PAS staining in the model group showed a large number of hyphae invading the epithelial layer of the tongue tissue, accompanied by the recruitment of numerous inflammatory cells. These hyphae mostly invaded the deep layers of the epithelial tissue at vertical or oblique angles, causing tissue damage. After intervention with saliva combined with Lactobacillus postbiotics, the PAS staining of the mouse tongue tissue was similar to that of the control group, indicating that the postbiotic prepared with saliva combined with Lactobacillus CCFM1417 can, to a certain extent, resist the invasion of oral pathogen Candida albicans into the oral mucosa, thereby playing a role in resisting oral pathogen infection.

[0103] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Lactobacillus salivarius ( Ligilactobacillus salivarius CCFM1417 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 2, 2024, with accession number GDMCC No:64942.

2. A microbial preparation containing the Lactobacillus salivarius CCFM1417 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The content of *Lactobacillus saliva-associated* CCFM1417 in the microbial preparation is ≥1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

4. The postbiotic prepared using the saliva-based Lactobacillus CCFM1417 as described in claim 1, characterized in that, The method for preparing the metabiotic is as follows: *Lactobacillus saliva-associated* CCFM1417 is inoculated into MRS medium and a seed culture is prepared at 37 °C; the prepared seed culture is inoculated into MRS liquid medium at a 2% (v / v) inoculation rate and cultured at 37 °C for 18 h; the bacterial cells are collected by centrifugation and concentrated, and the bacterial cell concentration is resuspended in sterile physiological saline to a final concentration of 5 × 10⁻⁶. 10 The bacterial cell lysate was obtained by inactivation at 65℃ for 30 minutes using CFU / ml and plate coating to check the inactivation effect. After heat treatment, the lysate was obtained by high-pressure homogenization, and the metabiotic was obtained and lyophilized for later use.

5. A product containing *Lactobacillus salivarius* CCFM1417 as described in claim 1, characterized in that, The product is either food or medicine.

6. The product according to claim 5, characterized in that, The product contains the aforementioned Lactobacillus salivans CCFM1417 and conventional excipients.

7. The product according to claim 5 or 6, characterized in that, The products include microbial preparations, dietary supplements, or solid beverages.

8. A health product containing the Lactobacillus salivarius CCFM1417 as described in claim 1.

9. A daily chemical product containing the post-genetic agent as described in claim 4, characterized in that, The daily chemical products mentioned include toothpaste, mouthwash, or oral spray.

10. The use of the *Lactobacillus salivariae* CCFM1417 and / or its postbiotics as described in claim 1 in the preparation of a medicament against oral pathogenic bacterial infections, characterized in that... The pathogenic bacterium is Candida albicans.

11. The use of the Lactobacillus salivariae CCFM1417 and / or its postbiotics as described in claim 1 in the preparation of health products that enhance immunity.

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