A strain of Lactobacillus salivariae CCFM1332 with multi-target regulatory effects and its application in periodontitis

Abstract

This invention discloses a strain of *Lactobacillus salivarius* CCFM1332 with multi-target regulatory effects and its application in periodontitis, belonging to the field of microbial technology. This invention yields a strain of *Lactobacillus salivarius* CCFM1332, which not only has vaginal immunomodulatory effects but also alleviates periodontitis. Specifically, it can: promote the secretion of antimicrobial peptides by vaginal epithelial cells VK2 / E6E7; reduce the periodontal probing depth in rats with periodontitis; reduce the number of pathogenic bacteria in the oral cavity of rats with periodontitis; downregulate the levels of serum inflammatory factors IL-1β, IL-6, and IL-17A; and reduce alveolar bone loss in rats with periodontitis, effectively alleviating alveolar bone resorption in rats.

Description

Technical Field

[0001] This invention relates to a strain of Lactobacillus salivarius CCFM1332 with multi-target regulatory effects and its application in periodontitis, belonging to the field of microbial technology. Background Technology

[0002] Probiotics, as a class of live microorganisms with beneficial effects on host health, have become an important biological intervention for regulating the microecological balance in multiple sites such as the gut, oral cavity, and vagina, thanks to their antibacterial, anti-inflammatory, and immunomodulatory functions. These microorganisms play a crucial role in maintaining mucosal barrier homeostasis and systemic immune balance by precisely regulating the microbial community structure and its metabolites (such as short-chain fatty acids and antimicrobial peptides). In recent years, probiotic-based microecological regulation therapy has received widespread attention due to its multidimensional intervention potential.

[0003] Taking *Ligilactobacillus salivarius* as an example, this Gram-positive facultative anaerobic bacterium lacks catalase and oxidase activity. Its core metabolic characteristics include lactate production and bacteriocin secretion. Studies have found that *Ligilactobacillus salivarius* can improve host health through multiple pathways, including regulating host flora composition, inhibiting pathogen colonization, and activating protective immune responses (such as enhancing sIgA secretion). However, current probiotic research still generally suffers from problems of single-mechanism limitations and limited effects. For example, while patent CN119120279A discloses *Ligilactobacillus salivarius* CCFM1417, which can upregulate the expression of antimicrobial peptide (LL37) and immunoglobulins, its mechanism focuses on immune activation; patents CN115960741B and CN117683696B focus on the direct antibacterial function of this bacterium in the oral microenvironment. It is worth noting that the occurrence and development of human diseases often involve multi-organ interaction networks, and probiotics with single-target effects are difficult to meet the needs of complex pathological regulation.

[0004] This contradiction is particularly prominent in the treatment of periodontitis. As a chronic inflammatory disease characterized by progressive destruction of periodontal supporting tissues and alveolar bone resorption, periodontitis not only causes local symptoms such as halitosis, gingival bleeding, tooth loosening, and even tooth loss, but also has a significant association with systemic diseases (such as cardiovascular disease and diabetes). Recent research reveals that oral pathogens can migrate across ecological niches through the "microbe-immune axis"—for example, the ectopic colonization of periodontal pathogens in the vaginal mucosa leads to a significantly increased incidence of periodontitis in patients with bacterial vaginosis. Such cross-border microbiota reshape the host microenvironment by regulating local pH and interfering with the expression of antimicrobial peptides (such as LL37 / HBD2), while the shared immune cell migration pathways between the vaginal, oral, and intestinal mucosa further enhance the synergy of multi-organ immune responses.

[0005] Based on the above mechanisms, developing probiotic formulations with multi-target regulatory functions has become an inevitable trend. Such multifunctional probiotics may provide a breakthrough prevention and treatment strategy for periodontitis and related systemic diseases. Summary of the Invention

[0006] This invention provides a Ligilactobacillus salivarius CCFM1332, wherein the Ligilactobacillus salivarius is Ligilactobacillus salivarius GDMCC NO: 63788.

[0007] The *Ligilactobacillus salivarius* CCFM1332 strain was derived from a female herdsman in Hulunbuir, Inner Mongolia. Sequencing analysis revealed its 16S rDNA sequence to be shown in SEQ ID NO.1. Nucleic acid sequence comparison was performed using NCBI, confirming the strain as *Ligilactobacillus salivarius* with 100% homology; it was named *Ligilactobacillus salivarius* CCFM1332.

[0008] The colonies of the salivary lactobacillus CCFM1332 on MRS medium are small, white, and round.

[0009] This invention provides a microbial preparation containing *Lactobacillus salivarius* CCFM1332, wherein the bacterial count of *Lactobacillus salivarius* CCFM1332 in the microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0010] The present invention provides a postbiotic prepared from the Lactobacillus salivarius CCFM1332, wherein the postbiotic is obtained by inactivating the fermentation broth of the Lactobacillus salivarius CCFM1332 and then crushing it under high pressure.

[0011] The present invention provides a product containing the aforementioned Lactobacillus salivarius CCFM1332 or the aforementioned microbial preparation or the aforementioned postbiotic.

[0012] In one embodiment, the product includes food, medicine, or daily chemical products.

[0013] In one embodiment, the dosage form of the drug includes granules, capsules, tablets, pills, and oral liquids.

[0014] In one embodiment, the daily chemical product includes mouthwash, mouthwash pellets, toothpaste, dental powder, whitening strips, periodontal gel, dental gel, or tooth wax.

[0015] This invention provides the use of the aforementioned *Lactobacillus salivarius* CCFM1332, the aforementioned microbial preparation, or the aforementioned postbiotic in the preparation of products for relieving oral inflammation, including periodontitis or gingivitis, and the products having any one or more of the following uses:

[0016] (a) Reduce the number of Porphyromonas gingivalis in the oral cavity of individuals with periodontitis;

[0017] (b) Reduce the level of inflammatory factors in the serum of individuals with periodontitis;

[0018] (c) Reduce probe depth in individuals with periodontitis;

[0019] (d) Reduce alveolar bone height in individuals with periodontitis.

[0020] In one embodiment, the product includes pharmaceuticals or daily chemical products.

[0021] This invention provides the use of the aforementioned Lactobacillus salivariae CCFM1332, the aforementioned microbial preparation, or the aforementioned postbiotic in the preparation of products that enhance vaginal immunity.

[0022] In one embodiment, the product is used to promote the secretion of antimicrobial peptides by vaginal epithelial cells VK2 / E6E7.

[0023] This invention provides the application of the aforementioned Lactobacillus salivarius CCFM1332 or the aforementioned microbial preparation in the preparation of antimicrobial peptides, wherein the antimicrobial peptides are LL37 and HBD2.

[0024] Beneficial effects:

[0025] 1. This invention yields a strain of *Lactobacillus salivarius* CCFM1332, which promotes the secretion of antimicrobial peptides by vaginal epithelial cells and alleviates periodontitis, specifically in the following ways:

[0026] (1) The postbiotic effect of Lactobacillus salivariae CCFM1332 can promote the secretion of antimicrobial peptides by vaginal epithelial cells VK2 / E6E7. The effect is reflected in: increasing the content of LL37 secreted by vaginal epithelial cells VK2 / E6E7 from 17.80 to 24.77 ng / L; and increasing the content of HBD2 from 57.88 to 71.01 ng / L.

[0027] (2) Saliva combined with live Lactobacillus CCFM1332 or post-biotics can directly reduce periodontitis-related indicators. The effect is reflected in the reduction of probing depth of the maxillary left molar in rats from 1.24 mm to 0.85 mm and 0.92 mm, respectively.

[0028] (3) The combination of live Lactobacillus CCFM1332 in saliva or postbiotics can effectively inhibit the proliferation and growth of periodontal pathogens in the oral cavity of rats. The effect is reflected in the reduction of the number of Porphyromonas gingivalis in the oral cavity of rats from 5.58 and 5.40 log (CFU / mL) at the first sampling to 4.44 and 4.40 log (CFU / mL) at the end of the experiment.

[0029] (4) The combination of live Lactobacillus CCFM1332 in saliva or post-biotics can also slow down the progression of periodontitis from the perspective of regulating the body's immune response, reduce the secretion of pro-inflammatory factors in the serum, and thus reduce alveolar bone loss caused by persistent inflammation. The effects were as follows: Saliva combined with live Lactobacillus CCFM1332 reduced the serum levels of IL-1β, IL-6, and IL-17A in rats with periodontitis from 28.51, 87.70, and 11.70 ng / L to 23.58, 74.77, and 7.10 ng / L, respectively; saliva combined with live Lactobacillus CCFM1332 or postbiotics reduced the serum levels of IL-1β, IL-6, and IL-17A in rats with periodontitis from 28.51, 87.70, and 11.70 ng / L to 20.00, 70.39, and 7.63 ng / L, respectively; saliva combined with live Lactobacillus CCFM1332 or postbiotics reduced alveolar bone resorption in rats with periodontitis from 1.13 mm to 0.79 mm and 0.76 mm, respectively.

[0030] Therefore, this saliva-associated Lactobacillus CCFM1332 has a very broad application prospect in the preparation of daily chemical products (such as mouthwash, toothpaste, etc.) and pharmaceuticals for relieving periodontitis. Attached Figure Description

[0031] Figure 1 Changes in the content of antimicrobial peptides secreted by vaginal epithelial cells VK2 / E6E7.

[0032] Figure 2 This is a schematic diagram of periodontal ligation in rats.

[0033] Figure 3 This is a flowchart for animal experiments on periodontitis.

[0034] Figure 4 The change in Porphyromonas gingivalis in the oral cavity of rats with periodontitis.

[0035] Figure 5 Changes in inflammatory factors in the serum of rats with a periodontitis model.

[0036] Figure 6 Micro-CT image of the maxilla in a rat model of periodontitis.

[0037] Figure 7 This study describes the changes in maxillary alveolar bone resorption in rats with a periodontitis model.

[0038] Biological Material Deposit

[0039] A strain of *Ligilactobacillus salivarius* CCFM1332, with the taxonomic designation *Ligilactobacillus salivarius*, was deposited with the Guangdong Provincial Microbial Culture Collection Center on September 12, 2023, with the deposit number GDMCC No: 63788. The deposit address is 5th Floor, Building 59, No. 100 Courtyard, Xianlie Middle Road, Guangzhou, Guangdong Province, China. Detailed Description of Embodiments

[0040] The present invention is further described below with reference to specific examples and the accompanying drawings.

[0041] *Porphyromonas gingivalis* involved in the following examples was purchased from Guangdong Provincial Microbial Culture Collection Center, with the product number GDM 1.851; SPF-grade Wistar rats involved in the following examples were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (production license No. SCXK (Jing) 2012-0001). ELISA kits involved in the following examples were purchased from Nanjing Senbeijia Biotechnology Co., Ltd.; other reagents involved in the following examples were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0042] The media involved in the following examples are as follows:

[0043] MRS solid medium (per L): 10 g / L peptone, 10 g / L beef extract, 20 g / L glucose, 2 g / L sodium acetate, 5 g / L yeast powder, 2 g / L diammonium hydrogen citrate, 2.6 g / L K₂PO₄·3H₂O, 0.1 g / L MgSO₄·7H₂O, 0.05 g / L MnSO₄, 1 mL / L Tween 80, 20 g / L agar.

[0044] MRS liquid medium (per L): 10 g / L peptone, 10 g / L beef extract, 20 g / L glucose, 2 g / L sodium acetate, 5 g / L yeast powder, 2 g / L diammonium hydrogen citrate, 2.6 g / L K₂PO₄·3H₂O, 0.1 g / L MgSO₄·7H₂O, 0.05 g / L MnSO₄, 1 mL / L Tween 80.

[0045] BHI solid medium (per L): 10.0 g tryptone, 17.5 g beef heart infusion powder, 5.0 g sodium chloride, 5.0 g yeast extract, 2.0 g glucose, 2.5 g disodium hydrogen phosphate dodecahydrate, 1 mL / L 0.5% vitamin K₁ hemin, 20 g / L agar, 50 mL / L sterile defibrinated sheep blood, pH 7.2-7.4.

[0046] BHI liquid culture medium ( / L): tryptone 10.0g, beef heart extract 17.5g, sodium chloride 5.0g, yeast extract 5.0g, glucose 2.0g, disodium hydrogen phosphate dodecahydrate 2.5g, 0.5% vitamin K1 heme chloride 1mL / L, pH 7.2~7.4.

[0047] The preparation method of the bacterial suspension involved in the following examples is as follows:

[0048] Porphyromonas gingivalis bacterial suspension: Single colonies were obtained by anaerobic culture at 37°C for more than 48 hours on BHI solid medium. These single colonies were then inoculated into BHI liquid medium and anaerobically cultured at 37°C for 48 hours to obtain culture medium 1. Culture medium 1 was then inoculated into BHI liquid medium at an inoculation rate of 4% of the total volume of BHI medium. After anaerobic culture at 37°C for 48 hours, the concentration was adjusted to 1×10⁻⁶ BHI medium. 9 CFU / mL.

[0049] Saliment-associated Lactobacillus suspension: Single colonies were obtained by culturing on MRS solid medium at 37°C for 20 h. A single colony was picked and inoculated into MRS liquid medium, and cultured at 37°C for 20 h to obtain culture medium 1. Culture medium 1 was inoculated into MRS medium at an inoculation rate of 2% of the total volume of the medium, and cultured at 37°C for 20 h. The bacterial cells were then collected, washed twice with PBS, and the concentration was adjusted to 1×10⁻⁶ PBS. 9 CFU / mL.

[0050] The preparation method of the postgenetic agent involved in the following embodiments is as follows:

[0051] Postbiotics of *Lactobacillus salivarius*: After culturing *Lactobacillus salivarius* in MRS liquid medium for 20 h as described above, the bacterial concentration was adjusted to 1 × 10⁻⁶ using MRS medium. 9 The fermentation broth, including the bacterial cells, was pasteurized at CFU / mL. The completely inactivated saliva combined with the Lactobacillus bacterial culture was then subjected to high-pressure homogenization to obtain the metabiotic.

[0052] The periodontitis modeling method involved in the following embodiments is as follows:

[0053] Periodontal ligation method: Male, 5-week-old SPF-grade Wistar rats weighing 150-170g were selected. The rats were anesthetized by intramuscular injection of 200mg / kg ketamine hydrochloride. Two 0.22mm sterile orthodontic sutures were passed through the interdental spaces of the first and second molars and the second and third molars on the right maxilla of the rat, respectively, and knotted on both sides, placing the orthodontic sutures as close to the gingival sulcus as possible. After periodontal ligation, the rats entered a 7-day recovery period. During the first 3 days of the recovery period, they were fed purified water containing 125ug / mL ampicillin sodium daily. The ligation effect is shown in [the table below]. Figure 2 The experimental procedure is as follows: Figure 3 .

[0054] Method of pathogen infection: After a 7-day recovery period, use a sterile syringe to draw a concentration of 1×10 9 A suspension of 0.50 mL of Porphyromonas gingivalis CFU / mL was slowly rinsed at the ligation site of the maxilla in rats, and the rats were kept fasted and deprived of water for half an hour. The infection frequency was 5 times a week (5 days of infection, 2 days of interval) for 2 weeks.

[0055] The prevention and control methods involved in the following embodiments are as follows:

[0056] Model group: 0.50 mL of sterile MRS liquid culture medium was drawn with a sterile syringe and slowly rinsed at the ligation site of the rat's maxilla. The rat was then fasted and deprived of water for half an hour. The treatment frequency was 6 times / week for 2 weeks.

[0057] CCFM1332 live bacteria group: Aspirate a concentration of 1×10⁻⁶ using a sterile syringe. 9 Slowly rinse the ligated site of the rat's maxilla with 0.50 mL of a CFU / mL saliva-containing Lactobacillus CCFM1332 suspension, and withhold food and water for half an hour. The treatment frequency is 6 times a week (6 days of treatment, 1 day interval) for 2 weeks.

[0058] CCFM1332 post-biotic group: 0.50 mL of saliva combined with Lactobacillus CCFM1332 post-biotic was drawn with a sterile syringe and slowly rinsed at the ligation site of the rat's maxilla. The rat was then fasted and deprived of water for half an hour. The treatment frequency was 6 times a week (6 days of treatment, 1 day interval) for 2 weeks.

[0059] The detection methods involved in the following embodiments are as follows:

[0060] Detection of probing depth (PD):

[0061] Before sacrificing the rats, the probing depth (PD) at the maxillary model site was measured using a periodontal probe.

[0062] The probing depth (PD) at the maxillary model site in rats was measured using a periodontal probe. The specific procedure was as follows: The periodontal probe was used to detect the distance from the gingival margin to the bottom of the pocket or gingival sulcus at three points on the palatal side of the right maxillary second molar: distal, median, and mesial.

[0063] Detection of Porphyromonas gingivalis:

[0064] During the experiment, disposable sterile cotton swabs were used to gently wipe the ligation site of the rat's maxilla. The collected samples were diluted and smeared for counting. Three samplings were performed during the experiment: the first sampling was conducted on day 21, after the pathogenic bacteria were rinsed, as a test for *Porphyromonas gingivalis* colonization; the second sampling was conducted one week after probiotic intervention, on day 28; and the third sampling was conducted on day 35, the final sampling before the rats were sacrificed. *Porphyromonas gingivalis* colony counting was performed using BHI blood agar plates.

[0065] Methods for detecting serum markers:

[0066] Blood was collected from the abdominal aorta of rats and placed in serum tubes. The serum was centrifuged at 3000 rpm for 10 min and stored at -80°C until use. The levels of IL-1β, IL-6, and IL-17A in the serum were determined using an enzyme-linked immunosorbent assay (ELISA) kit.

[0067] Methods for measuring alveolar bone resorption (ABL):

[0068] Rat maxillae were immersed in 4% paraformaldehyde for 48 hours, then washed with PBS buffer and air-dried. Rat maxillae were scanned using a micro-computed tomography (Micro-CT) system. Samples were scanned using the following settings: 90 kV, 88 μA, 18 μm pixel size, and 180° rotation. Alveolar bone resorption (ABL) was the distance between the cementoenamel junction (CEJ) and the alveolar ridge (ABC). ABL was measured at six locations: distal, mesial, and centric on the buccal and palatal sides of the second molar.

[0069] Example 1: Regulating the secretion of VK2 / E6E7 antimicrobial peptides from vaginal epithelial cells by saliva in conjunction with Lactobacillus CCFM1332

[0070] 1. Experimental Methods

[0071] VK2 / E6E7 vaginal epithelial cells in the logarithmic growth phase were used at a concentration of 2×10⁻⁶. 5 1 mL of DMEM was seeded into each well of a 6-well plate. The culture medium consisted of DMEM and 10% fetal bovine serum. After adhering to the cell culture vessel for 24 hours, 5% (v / v) postbiotic was added. 9 The sample was prepared by combining CFU / mL of saliva with Lactobacillus CCFM1332. After culturing for 24 hours, the supernatant was collected by centrifugation and measured according to the instructions of the LL37 and HBD2 detection kits.

[0072] 2. Experimental Results

[0073] The results are as follows Figure 1 As shown, after intervention with saliva combined with Lactobacillus CCFM1332, the levels of LL37 and HBD2 secreted by VK2 / E6E7 vaginal epithelial cells were significantly increased. The secretion levels of LL37 and HBD2 increased from baseline values ​​of 17.80 ng / L and 57.88 ng / L to 24.77 (P<0.05) and 71.01 ng / L (P<0.05), respectively. As core effector molecules of the innate immune system, the synergistic upregulation of LL37 and HBD2 suggests that CCFM1332 can enhance the pathogen clearance capacity of the mucosal barrier by activating the host epithelial immune response, and can serve as a candidate probiotic strain for regulating the vaginal immune barrier.

[0074] Example 2: In vitro inhibition of Porphyromonas gingivalis by Lactobacillus salivaria CCFM1332

[0075] 1. Experimental Methods

[0076] To prepare BHI blood agar plates, a concentration of 1×10⁻⁶ was used. 6 A CFU / mL *Porphyromonas gingivalis* bacterial suspension was spread onto a BHI blood agar plate. After air-drying, four sterile Oxford cups were evenly placed on the plate. Then, 100 μL of a 1×10⁻⁶ CFU / mL solution was added to each well of the Oxford cup. 9 Postbiotics prepared by combining CFU / mL saliva with Lactobacillus CCFM1332 were used as a negative control in MRS liquid medium. The mixture was diffused at 4°C for 4 h, followed by anaerobic culture at 37°C for 48 h. The size of the inhibition zone was then observed and measured.

[0077] 2. Experimental Results

[0078] Table 1. Diameter of inhibition zones of different strains against *Porphyromonas gingivalis*.

[0079]

[0080] The results, as shown in Table 1, indicate that *Lactobacillus salivarius* CCFM1332 has a significant antibacterial effect against *Porphyromonas gingivalis*, with an inhibition zone diameter of 20.15 mm. This demonstrates that *Lactobacillus salivarius* CCFM1332 can effectively inhibit the growth of oral pathogens, improve the oral flora environment, prevent periodontal disease, and can be considered as a high-quality probiotic candidate strain for improving periodontitis.

[0081] Example 3: Saliva combined with Lactobacillus CCFM1332 improves periodontitis symptoms

[0082] 1. Effect of salivary-associated Lactobacillus CCFM1332 probe depth on rat probe depth

[0083] (1) Experimental methods

[0084] Experimental procedure as follows Figure 3 As shown:

[0085] Twenty-four male Wistar rats were randomly divided into four groups (n=6 per group): control group, model group, CCFM1332 live bacteria group, and CCFM1332 postbiotic group. Before the experiment, all rats were placed in a constant temperature environment of 22–24°C with alternating 12-hour light and 12-hour dark cycles for one week to acclimatize. The experiment lasted for 5 weeks, a total of 35 days. Except for the control group, all other groups underwent periodontitis modeling (periodontal ligation + pathogenic bacterial infection). After periodontal ligation, a 7-day recovery period was allowed. At the end of the recovery period, a 1×10⁻⁶ live bacteria concentration was aspirated using a sterile syringe. 9 0.5 mL of a CFU / mL *Porphyromonas gingivalis* suspension was slowly rinsed at the maxillary ligation site in rats, who were then fasted and deprived of water for half an hour. This was repeated 5 times per week for 2 weeks. After model establishment, 0.5 mL of a 1×10⁻⁶ CFU / mL suspension was administered daily. 9 CFU / mL of saliva combined with a suspension of Lactobacillus CCFM1332 (CCFM1332 live bacteria group), or 0.5 mL of the equivalent concentration of 1×10 9 A CFU / mL solution of saliva combined with a postbiotic prepared from Lactobacillus CCFM1332 (CCFM1332 postbiotic group) was used to slowly rinse the ligated site of the maxilla in rats, followed by fasting and water restriction for half an hour. This treatment was performed 6 times per week for 2 weeks. After the experiment, rats in each group were sacrificed.

[0086] (2) Experimental Results

[0087] Before sacrificing the rats, the probing depth (PD) at the maxillary model site was measured using a periodontal probe, and the results are shown in Table 2.

[0088] Table 2. Probing depth of the maxilla in each group of rats

[0089]

[0090] The results showed that the probing depth at the model site in the rats was 1.24 mm, which was significantly increased compared with the control group. After two weeks of intervention with saliva combined with live Lactobacillus CCFM1332 or postbiotics, the probing depth of the periodontal tissues decreased to 0.85 mm and 0.92 mm, respectively.

[0091] In summary, the combination of saliva with live Lactobacillus CCFM1332 and post-natal bacteria can reduce the formation of periodontal pockets in rats to some extent.

[0092] 2. Effects of saliva-associated Lactobacillus CCFM1332 on the number of pathogenic bacteria in the oral cavity of rats with periodontitis

[0093] (1) Experimental methods

[0094] The specific implementation method is the same as step 1. Samples were collected three times during the experiment: the first time was on day 21 after the pathogen infection ended; the second time was on day 28 one week after probiotic intervention; and the third time was on day 35 two weeks after probiotic intervention. The experimental results are as follows: Figure 4 .

[0095] (2) Experimental Results

[0096] Depend on Figure 4 It was found that *Porphyromonas gingivalis* was not detected in the control group during the three samplings. At the end of the pathogenic bacterial infection, approximately 5.50 log (CFU / mL) of *Porphyromonas gingivalis* colonization was detected in the saliva of both the model and experimental groups, indicating successful colonization of *Porphyromonas gingivalis* in the rat oral cavity. After two weeks of intervention with saliva combined with live *Lactobacillus CCFM1332* or postbiotics, the number of *Porphyromonas gingivalis* in the rat oral cavity decreased significantly (P<0.01), decreasing from 5.58 and 5.40 log (CFU / mL) at the first sampling to 4.44 and 4.40 log (CFU / mL) at the end of the experiment, respectively. This indicates that saliva combined with live *Lactobacillus CCFM1332* and postbiotics had a significant inhibitory effect on *Porphyromonas gingivalis* in the oral cavity of rats with periodontitis.

[0097] 3. Effects of saliva combined with Lactobacillus CCFM1332 on serum inflammatory factors in rats with periodontitis

[0098] (1) Experimental methods

[0099] The specific implementation method is the same as step 1. After the experiment, serum was collected from each group of rats. The levels of IL-1β, IL-6, and IL-17A in the serum were measured according to the instructions of the enzyme-linked immunosorbent assay (ELISA) kit. The experimental results are as follows. Figure 5 As shown.

[0100] (2) Experimental Results

[0101] according to Figure 5 The results showed that the serum levels of IL-1β, IL-6, and IL-17A in the model group were significantly higher than those in the control group (P<0.01), indicating that periodontitis can cause systemic inflammation in rats. After two weeks of intervention with saliva combined with live Lactobacillus CCFM1332 or postbiotics, the serum levels of IL-1β, IL-6, and IL-17A all decreased significantly. Specifically, in the live bacteria group, the levels decreased from 28.51, 87.70, and 11.70 ng / L to 23.58, 74.77, and 7.10 ng / L, respectively; in the postbiotic group, they decreased to 20.00, 70.39, and 7.63 ng / L, respectively. This indicates that oral administration of saliva combined with live Lactobacillus CCFM1332 and postbiotics can reduce systemic inflammation caused by periodontitis.

[0102] 4. Effects of saliva combined with Lactobacillus CCFM1332 on alveolar bone in rats with periodontitis

[0103] (1) Experimental methods

[0104] The specific implementation method is the same as step 1 of Example 2. After the experiment, the rat maxilla was taken, and the alveolar bone resorption (ABL) was measured. The results are as follows: Figures 6-7 As shown.

[0105] (2) Experimental Results

[0106] according to Figure 6 Micro-CT of the maxilla and Figure 7 It was found that after the periodontitis model was established, the amount of alveolar bone resorption in rats increased significantly, from 0.39 mm in the control group to 1.13 mm in the model group (P<0.01). Furthermore, the alveolar bone resorption in the model group was obvious, and the bifurcation of the second molar root was exposed, indicating successful modeling. After two weeks of intervention with saliva combined with live lactobacillus CCFM1332 or post-biotics, the alveolar bone density (ABL) in rats with periodontitis decreased to 0.79 mm and 0.76 mm, respectively. This indicates that saliva combined with live lactobacillus CCFM1332 and post-biotics can protect the alveolar bone in rats, alleviate alveolar bone resorption in rats with periodontitis, and reduce bone loss.

[0107] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person 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. A strain of *Lactobacillus salivarius* ( Ligilactobacillus salivarius CCFM1332, characterized in that, The Lactobacillus salivans CCFM1332 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 12, 2023, with accession number GDMCC No: 63788.

2. A microbial preparation containing the *Lactobacillus salivarius* CCFM1332 as described in claim 1, characterized in that, In the microbial preparation, the bacterial count of the *Lactobacillus saliva-associated* CCFM1332 is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

3. The postbiotic prepared by combining saliva with Lactobacillus CCFM1332 as described in claim 1, characterized in that, The metabiotic is obtained by inactivating the fermentation broth of the Lactobacillus salivarius CCFM1332 and then crushing it under high pressure.

4. A product containing the *Lactobacillus salivarius* CCFM1332 of claim 1, the microbial preparation of claim 2, or the postbiotic of claim 3, characterized in that, The products mentioned are food, medicine, or daily chemical products.

5. The product as described in claim 4, characterized in that, The dosage forms of the medicine include granules, capsules, tablets, pills, and oral liquids.

6. The product as described in claim 5, characterized in that, The aforementioned daily chemical products include mouthwash, mouthwash pellets, toothpaste, tooth powder, teeth whitening strips, periodontal gel, dental gel, or tooth wax.

7. The use of the *Lactobacillus salivariae* CCFM1332 of claim 1, the microbial preparation of claim 2, or the postbiotic of claim 3 in the preparation of a product for relieving oral inflammation, characterized in that, The oral inflammation is periodontitis or gingivitis; the product is a medicine or daily chemical product.

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