Postbiotics prepared by combining saliva with lactobacillus and capable of improving expression of host antibacterial peptide and immune globulin and application thereof

Abstract

The invention discloses a metagen prepared by combining saliva with lactobacillus and capable of improving expression of host antibacterial peptide and immune globulin and application of the metagen, and belongs to the technical field of microorganisms. The combined lactobacillus salivarius disclosed by the invention has the capability of regulating and controlling host immunity and improving pathogenic bacterium infection resistance of a host oral cavity, can remarkably up-regulate expression of a human oral keratinocyte antibacterial peptide HBD-2 gene and protein in vitro, and can remarkably up-regulate expression of DEFB3 (homolog of human HBD-2) and secretory immune globulin SIgA in a mouse body; the compound can be used for preparing a series of products for enhancing the oral immunity of a host and resisting oral pathogenic bacteria infection, and has a huge application prospect.

Description

Technical Field

[0001] The invention relates to a postbiotic prepared by a saliva-combined lactobacillus and capable of improving the expression of host antimicrobial peptides and immunoglobulins and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] The human oral microbiome is the second largest microbiome after the intestine. 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 are involved in the composition of the oral microecology. The anatomical structure of the oral cavity includes the lips, cheeks, hard palate, soft palate, teeth, gums, tongue and tonsils. Different parts of the oral cavity 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 concentrations in different parts all affect the colonization of oral microorganisms. Therefore, different oral sites have their own unique and complex microbiome.

[0003] Oral microorganisms interact with the host oral mucosal immune system, epithelial barrier and other factors to jointly maintain the balance of oral microecology. The physical barrier of the oral mucosa is the first line of defense between the body and the environment. Oral mucosal epithelial cells can produce antimicrobial substances such as antimicrobial peptides, quickly respond to foreign antigens in the environment, integrate the two defense systems of innate immunity and acquired immunity, and play an important role in early resistance to pathogenic bacteria infection. Oral antimicrobial peptides are an important part of the innate immune system. Oral epithelial cells, neutrophils and other cells can secrete antimicrobial peptides such as defensins, LL-37, and calprotectin. Among them, microbial-related molecular models can upregulate 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 the host innate immune response and preventing infection by pathogenic bacteria.

[0004] At present, the application of commensal bacteria in oral diseases mainly focuses on the alleviation of diseases when oral diseases occur. Most of their mechanisms of action are directly acting on pathogenic bacteria, such as competing with pathogenic bacteria for binding sites, nutrient competition, and producing antibacterial substances. However, there are many anti-infection mechanisms in the host. Microbial-related molecular models can effectively activate anti-infection pathways in the host and regulate the production of downstream antibacterial substances, thereby playing an anti-infection role. Taylor et al. showed that the upregulation of NF-κB non-classical pathway protein IKBζ can regulate the expression of host antimicrobial peptide DEFB3, thereby playing an anti-Candida albicans infection role. There have been many reports on the interaction between probiotics and hosts to prevent pathogenic bacteria infection in the intestinal and nasal fields, but more research in the oral field is still focused on the direct inhibition of probiotics on pathogens. Therefore, it is particularly important to link probiotics with host immunity to prevent oral pathogenic bacteria infection. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a postbiotic prepared by saliva combined with lactobacillus, aiming to solve the problem that the prior art lacks the regulation of host oral immunity by symbiotic bacteria. It can significantly upregulate the expression of antimicrobial peptides in the host oral cavity in vivo and in vitro, thereby improving the host oral immunity and resisting infection by oral pathogens.

[0006] The present invention provides salivary lactobacillus (Ligilactobacillus salivarius) CCFM1417, which is taxonomically named Ligilactobacillus salivarius, has been deposited in Guangdong Provincial Microbiological Culture Collection Center on August 2, 2024, with a deposit number of GDMCC No: 64942, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] The salivary Lactobacillus (Ligilactobacillus salivarius) CCFM1417 is a Gram-positive bacterium, and the cells are short rod-shaped under a microscope. After being inoculated on an 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.

[0008] The invention also provides a microbial preparation containing the saliva-combined lactobacillus CCFM1417.

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

[0010] The present invention also provides a postbiotic prepared by combining the saliva with Lactobacillus CCFM1417.

[0011] In one embodiment, the postbiotics include inactivated bacteria, bacterial lysate and / or inactivated fermentation broth.

[0012] In one embodiment, the preparation method of the postbiotics is: culturing saliva and Lactobacillus CCFM1417 to the logarithmic growth phase, inactivating them by heat treatment and then centrifuging, collecting the precipitate to obtain the postbiotics, and freeze-drying to obtain the postbiotic freeze-dried powder.

[0013] In one embodiment, the preparation method of the postbiotics is: culturing saliva and Lactobacillus CCFM1417 to the logarithmic growth phase, inactivating them by heat treatment, homogenizing them under high pressure, and freeze-drying them to obtain postbiotic freeze-dried powder.

[0014] In one embodiment, the bacterial lysate is a lysate of saliva combined with Lactobacillus CCFM1417 after high-pressure homogenization; its preparation method is: culturing the saliva combined with Lactobacillus CCFM1417 in a fermentation medium, collecting the bacterial mud and resuspending it with sterile saline, heat-treating it at 65°C for 30 minutes to obtain inactivated bacterial cells, and then high-pressure homogenizing to obtain a bacterial lysate.

[0015] The present invention also provides a product containing the saliva-combined Lactobacillus CCFM1417 and / or its postbiotics.

[0016] In one embodiment, the product is food, health care product, medicine or daily chemical product.

[0017] In one embodiment, the food contains the saliva-associated Lactobacillus CCFM1417 and / or postbiotics prepared therefrom, and conventional auxiliary materials.

[0018] In one embodiment, the conventional auxiliary materials include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional enhancers.

[0019] In one embodiment, the health product contains the saliva-combined Lactobacillus CCFM1417 and / or postbiotics prepared therefrom, and conventional auxiliary materials.

[0020] In one embodiment, the conventional auxiliary materials include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional enhancers.

[0021] In one embodiment, the medicine contains the saliva-combined Lactobacillus CCFM1417 and / or postbiotics prepared therefrom, and a pharmaceutically acceptable carrier.

[0022] 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.

[0023] In one embodiment, the daily chemical product includes toothpaste, mouthwash or oral spray.

[0024] The present invention also provides the use of the saliva combined with Lactobacillus CCFM1417 and / or its postbiotics in the preparation of a medicine for improving immunity and / or resisting oral pathogenic bacteria infection.

[0025] In one embodiment, the application includes but is not limited to promoting the expression of antimicrobial peptide HBD-2 in oral epithelial cells and increasing the gene and protein expression levels of antimicrobial peptide HBD-2 in individual tissues.

[0026] Beneficial effects:

[0027] The present invention provides a strain of Ligilactobacillus salivarius CCFM1417, which has the function of enhancing the host's oral immunity to resist infection by pathogenic bacteria, which is specifically embodied in:

[0028] (1) Promote the expression of antimicrobial peptide HBD-2 in oral epithelial cells;

[0029] (2) increase the gene and protein expression levels of the antimicrobial peptide HBD-2 in individual tongue tissues;

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

[0031] (4) Activate the IKBζ pathway in the host to resist pathogenic bacteria infection;

[0032] (5) reduce the load of Candida albicans in the tongue tissue of individuals;

[0033] (6) Improve the pathological condition of individual tongue tissue;

[0034] (7) Improve the invasion of oral pathogen Candida albicans into individual tongue tissue;

[0035] Therefore, the postbiotics prepared by combining saliva with Lactobacillus CCFM1417 have great application prospects in products that regulate host oral immunity and resist oral pathogenic bacteria infections.

[0036] Biomaterial Deposit

[0037] Saliva Lactobacillus (Ligilactobacillus salivarius) CCFM1417, taxonomically named Ligilactobacillus salivarius, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 2, 2024, with the deposit number GDMCC No: 64942, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0039] Figure 2 The flowchart of the animal experiment design scheme; Control is the blank control group; Model is the model group; saliva combined with Lactobacillus CCFM1417-P is the bacterial lysate external application group;

[0040] Figure 3 The changes of tongue mucosa of mice after intervention with saliva and Lactobacillus.

[0041] Figure 4 To study the effect of saliva combined with lactobacillus on the load of Candida albicans in the tongue tissue of mice;

[0042] Figure 5 To investigate the effects of saliva combined with Lactobacillus on the activation of anti-infection pathways in the tongue tissue of mice;

[0043] Figure 6 To investigate the effects of saliva combined with Lactobacillus on the expression of DEFB3 and SIgA in the tongue tissue of mice;

[0044] Figure 7 This is a picture of the histopathological evaluation of mouse tongue;

[0045] Figure 8 This is a picture of the pathological evaluation of PAS staining of mouse tongue tissue. DETAILED DESCRIPTION

[0046] To make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. The strains, cells and animals involved in the following embodiments are as follows: SPF-level BALB / c mice, female, 6 weeks old, weighing 15-18g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (production license number SCXK (Beijing) 2012-0001). Saliva combined lactobacillus CCFM1417 and FWXBH242 are from the strain bank of the Biotechnology Center of Jiangnan University. 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.

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

[0048] MRS liquid culture 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.

[0049] MRS solid culture 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.

[0050] Complete cell culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100× penicillin and streptomycin mixed solution (the penicillin content in the mixed solution is 10000 U / mL, and the streptomycin concentration is 10 mg / mL).

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

[0052] Preparation of Lactobacillus suspension:

[0053] (1) Preparation of seed solution:

[0054] Use a sterile inoculation loop to dip a small amount of saliva and activate the bacterial liquid of Lactobacillus CCFM1417 and FWXBH242 in MRS solid medium, and culture at 37°C for 48 hours. Then pick a single colony and inoculate it in MRS liquid medium, and culture it in a 37°C incubator for 18 hours to prepare the seed liquid.

[0055] (2) The prepared seed solution was inoculated in 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 concentration and the colonies were counted. 30% glycerol was stored 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.

[0056] Lactobacillus cell lysate:

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

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

[0059] The bacterial lysate obtained above was added into a cell culture medium at a ratio of 5% (v / v), including 84% (v / v) DMEM culture medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100× penicillin and streptomycin mixed solution (penicillin content in the mixed solution was 10000 U / mL, and streptomycin concentration was 10 mg / mL) + 5% (v / v) lactobacillus bacterial lysate.

[0060] Candida albicans suspension:

[0061] (1) Preparation of seed solution:

[0062] 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.

[0063] (2) The Candida albicans seed solution was inoculated into YPD medium at a 2% inoculum volume, and 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.

[0064] Example 1: Isolation and identification of saliva-associated Lactobacillus CCFM1417

[0065] Screening of strains: Pipette 0.2 mL of samples collected from healthy people into 1.8 mL of sterile saline to obtain 10 -1 Dilution, then draw 0.5mL 10 -1 Dilute in 4.5 mL of saline to obtain 10 -2 Dilution, follow this procedure to obtain 10 -3 , 10 -4 , 10-5 , 10 -6 Gradient dilution. Take 10 -4 , 10 -5 , 10 -6 Place 1 mL of each dilution in a plate, pour into MRS solid culture medium, mix gently, and after the culture medium solidifies, invert and culture at 37°C for 48 h.

[0066] Identification of strains: Use a sterile inoculation loop to dip a small amount of strains into MRS solid medium for activation and culture at 37°C for 48h. Then pick a single colony and inoculate it into MRS liquid medium, and culture it in a 37°C incubator for 18h to obtain the liquid fermentation broth of the corresponding strain. Take 1.0mL of the cultured bacterial solution, centrifuge it at 5000r / min for 3min, discard the supernatant, add 1.0mL of sterile saline to wash 3 times, resuspend it in 1.0mL of sterile water, and use it as a template for strain identification. Set up a PCR system with a volume of 20μL, add 0.5μL of forward primer (10μM), 0.5μL of reverse primer (10μM), 10μL of 2×Taq Mixture, 0.5μL of bacterial suspension, and 8.5μL of double distilled water. Primer information is shown in Table 1.

[0067] Table 1: Primer information

[0068]

[0069] After sequencing, the 16S sequence is shown as SEQ ID NO.1. The obtained 16S sequence was confirmed by NCBI BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST). The closer the Query Cover and Identification are to 100%, the more feasible it is. If there are multiple species in the comparison results, the Complete genome species will be prioritized while taking into account the numerical value. The results showed that the above strain was Lactobacillus salivarius, named CCFM1417, and the next experiment was carried out.

[0070] Example 2: Ability of Lactobacillus to Promote HBD-2 Production in Oral Epithelial Cells

[0071] The oral epithelial HOK cells were revived and passaged three times, and the cell concentration was adjusted to 5×10 5 / mL, 2mL of cell suspension of this concentration was inoculated in a 6-well cell culture plate, and the sample was added after incubation for 24h under 5% CO2 and 37℃. A control group and a strain sample treatment group were set up. The control group was added with a cell culture solution containing 5% PBS; the strain sample treatment group was added with a cell culture solution containing 5% Lactobacillus cell lysate, and 3 replicates were set up in each group. After 24h of intervention, the cell supernatant of the 6-well plate was collected and the expression of antimicrobial peptide HBD-2 in the supernatant was detected according to the instructions of the ELISA kit.

[0072] The results are as follows Figure 1 As shown in the results, after 24 hours of intervention, the content of HBD-2 in the supernatant of the blank group was 198.94 pg / mg, and the content of HBD-2 in the saliva combined with Lactobacillus CCFM 1417 bacterial lysate group (CCFM 1417-P) was 238.87 pg / mg compared with the blank control group, which could significantly upregulate the expression of antimicrobial peptide HBD-2 in oral epithelial cells (p < 0.01); while there was no significant difference between the saliva combined with Lactobacillus FWXBH242 bacterial lysate group (FWXBH242-P) and the blank control group. Therefore, at the cellular level, saliva combined with Lactobacillus CCFM 1417-P can promote the expression of antimicrobial peptide HBD-2 in oral epithelial cells, regulate host innate immunity, and enhance the host's ability to resist pathogenic bacteria infection.

[0073] Example 3: Application of saliva combined with Lactobacillus CCFM1417 in regulating host immunity

[0074] 1. Preparation of Lactobacillus postbiotics

[0075] Saliva combined with Lactobacillus CCFM1417 was inoculated into MRS medium to prepare seed solution at 37°C; the prepared seed solution was inoculated into MRS liquid medium at a 2% (v / v) inoculation amount, cultured in a 37°C incubator for 18 h, centrifuged and concentrated to collect the cells, and resuspended with sterile saline to a concentration of 5×10 10 CFU / ml, inactivated at 65°C for 30 minutes, and the inactivation effect was checked by plate coating. After heat treatment, the bacterial lysate was obtained by high-pressure homogenization, and the postbiotics were obtained and freeze-dried for later use. Before intervention, the concentration of the bacterial suspension was adjusted to 5×10 with sterile saline. 10 CFU / ml.

[0076] 2. Experimental animals and strains:

[0077] SPF BALB / c mice, female, 6 weeks old, weighing 15-18 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (Production License No. SCXK (Beijing) 2012-0001). Saliva-combined Lactobacillus CCFM1417 was from the strain bank of the Biotechnology Center of Jiangnan University. Candida albicans was Candida albicans SC5314, purchased from Ningbo Mingzhou Technology Co., Ltd. Figure 2 Table 2 shows the animal experiment process.

[0078] Table 2: Animal experimental plan and grouping

[0079]

[0080] Before the experiment, all mice were randomly divided into 3 groups according to their body weight, and the mice were placed in an environment with a 22℃-24℃ and 12-hour light-dark cycle to adapt 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 after Candida albicans infection (the infection method of Candida albicans was: anesthetizing mice, soaking cotton balls of the same size in a concentration of 1×10 7 CFU / ml of Candida albicans suspension, and then inoculated under the tongue of mice, the action time is about 60min). The intervention group was continuously intervened for 17 days (0-17 days) with postbiotics prepared by saliva combined with Lactobacillus CCFM1417, and immunosuppressants were 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 irrigation with a No. 8 gavage needle.

[0081] The specific steps are as follows:

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

[0083] Blank control group: Use No. 8 elbow gavage needle to draw 30 μL of sterile saline for oral irrigation once a day.

[0084] Model group mice: Use a No. 8 elbow gavage needle to draw 30 μL of sterile saline for oral irrigation once a day.

[0085] Saliva combined with Lactobacillus CCFM1417 group: Use a No. 8 elbow gavage needle to draw 30 μL of saliva combined with Lactobacillus CCFM1417 bacterial lysate into the mouth and slowly inject it into the mouse mouth once a day.

[0086] (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.

[0087] (3) Infection experiment (modeling period): All groups except the blank group were anesthetized on the 15th day and soaked in 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.

[0088] 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 the expression levels of related 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 mucosal tissue of mice in each group was observed to evaluate the infection degree of Candida albicans in the oral cavity. Figure 3 As shown in the figure, the tongue dorsum of the mice in the control group was light red, the tongue was smooth and moist, and there was no colonization of pathogenic bacteria Candida albicans; the tongue of the mice in the model group was covered with a thick pseudomembrane formed by Candida albicans, which adhered to the surface of the mouse tongue in pieces. After the intervention of saliva combined with lactobacillus, the colonization of Candida albicans on the mucosal surface of the mouse tongue was greatly reduced compared with the model group, and only a few points had pseudomembranes of Candida albicans. The appearance of the mouse tongue tissue was similar to that of the blank group mice, indicating that the postbiotics prepared by saliva combined with Lactobacillus CCFM1417 can largely resist the colonization of Candida albicans in the oral cavity and play an anti-pathogenic bacterial infection role in the host's oral cavity.

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

[0093] After the intervention, the tongue tissues of mice were homogenized and counted to assess the colonization of Candida albicans in the tongue tissues. Figure 4 As shown in Figure 2, the average colonization of Candida albicans in the tongue tissue of mice in the model group was about 7.52×10 5 CFU / g; the load of pathogenic bacteria Candida albicans in tongue tissue of the saliva combined with Lactobacillus CCFM1417-P group after intervention was about 8.67×10 4 CFU / g showed a significant downward trend compared with the model group (P < 0.05). Therefore, the postbiotics prepared by saliva combined with Lactobacillus CCFM1417 can reduce the colonization of pathogenic bacteria Candida albicans in the mouse oral cavity and play an anti-pathogenic bacterial infection role.

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

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

[0096] After intervention, the tongue tissue homogenates of mice were processed to evaluate the expression of IL-17 and IKBζ in each group. Figure 5 As shown in A, the IL-17 content of the blank group was about 17.69pg / mg, and the IL-17 content of the model group was about 13.69pg / mg. There was no significant difference between the two, because all groups were injected with immunosuppressants, and neither the blank group nor the model group was intervened with probiotics, so there was no significant difference between the indicators. The IL-17 content in the tongue tissue of the saliva combined with Lactobacillus CCFM1417-P group after intervention was about 19.67pg / mg, which was significantly different from the model group (P < 0.05). Similarly, Figure 5 As shown in B, the expression of IKBζ in the blank group was about 81.11pg / mg, and the expression of IKBζ in the model group was about 67.73pg / mg, and there was no significant difference between the two. However, the expression of IKBζ in the tongue tissue of the saliva combined with Lactobacillus CCFM1417-P group after intervention was about 119.35pg / mg, which was significantly different from the model group (P < 0.05). Therefore, the postbiotics prepared by saliva combined with Lactobacillus CCFM1417 can activate the anti-pathogenic bacteria infection pathway IL-17 / IKBζ in mice, upregulate the expression of downstream antimicrobial peptides, and play an anti-pathogenic bacteria infection role.

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

[0098] After the intervention, the expression of antimicrobial peptide DEFB3 and secretory immunoglobulin SIgA in the tongue tissue of mice was evaluated. Figure 6 As shown in A, after the intervention, the average expression of antimicrobial peptide DEFB3 in the tongue tissue of the model group was about 13.64 pg / mg, and the average expression of antimicrobial peptide DEFB3 in the tongue tissue of the saliva combined with Lactobacillus CCFM1417-P group after the intervention was about 30.69 pg / mg, which was significantly higher than that of the model group (P < 0.01). Figure 6As shown in Figure B, after the intervention, the average expression of immunoglobulin SIgA in the tongue tissue of the model group was about 125.56 ng / mg, and the average expression of immunoglobulin SIgA in the tongue tissue of the saliva combined with Lactobacillus CCFM1417-P group after intervention was about 243.12 ng / mg, which was significantly higher than that of the model group (P < 0.05). Therefore, the postbiotics 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 oral immunity and resisting the infection of oral pathogens.

[0099] (5) Histopathological analysis of mouse tongue

[0100] After the intervention, longitudinal sections of the mouse tongue tissue were observed. Figure 7 As shown in the figure, the filiform papillae on the dorsum of the tongue tissue of the blank group 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 the model group, the epithelial stratum corneum was severely damaged, the filiform papillae on the dorsum of the tongue were basically non-existent, and there was a large number of inflammatory cells recruited and small abscesses formed in some locations. After the intervention of postbiotics of saliva combined with Lactobacillus, the pathological sections of the mouse tongue tissue were similar to those of the blank group, indicating that the postbiotics prepared by saliva combined with Lactobacillus CCFM1417 can resist the invasion of oral pathogens and protect the oral mucosal tissue 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, the PAS staining of the blank group did not observe the invasion of hyphae into the tongue tissue; the PAS staining of the model group showed that a large number of hyphae invaded the epithelial layer of the tongue tissue, and these hyphae were accompanied by a large number of inflammatory cells. Most of these hyphae invaded the deep layer of the epithelial tissue at a vertical or inclined angle to damage the tissue. After intervention with saliva combined with Lactobacillus postbiotics, the PAS staining of the mouse tongue tissue was similar to that of the blank group, indicating that the postbiotics prepared by saliva combined with Lactobacillus CCFM1417 can resist the invasion of oral pathogens Candida albicans into oral mucosal tissues to a certain extent, thereby playing a role in resisting oral pathogen infection.

[0103] Although the present invention has been disclosed as above in the form of a preferred embodiment, 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. Ligilactobacillus salivarius CCFM1417 was deposited in Guangdong Provincial Microbiological Culture Collection Center on August 2, 2024, with the deposit number GDMCC No: 64942.

2. A microbial preparation containing the saliva-combined Lactobacillus CCFM1417 according to claim 1.

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

4. The postbiotic prepared by combining saliva and Lactobacillus CCFM1417 according to claim 1.

5. A product containing the saliva combined with Lactobacillus CCFM1417 and / or its postbiotics according to claim 1, characterized in that: The products are foods, health products, medicines or daily chemical products.

6. The product according to claim 5, characterized in that The product contains the saliva-combined lactobacillus CCFM1417 and conventional auxiliary materials.

7. The product according to claim 5 or 6, characterized in that The products include but are not limited to microbial preparations, dietary supplements, solid beverages, toothpaste, mouthwash or oral spray.

8. Use of the saliva-combined Lactobacillus CCFM1417 and / or its postbiotics as claimed in claim 1 in the preparation of a medicament for improving immunity and / or resisting oral pathogenic bacteria infection.

9. The use according to claim 8, characterized in that: The pathogenic bacteria include but are not limited to Candida albicans.

10. Use of the saliva combined with Lactobacillus CCFM1417 and / or its postbiotics as claimed in claim 1 in preparing health products for improving immunity.

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