Application of weissella confuse LHJY in periodontitis treatment

Abstract

In view of the current situation of probiotics for periodontitis treatment in the prior art, the application provides a Weissella confusa LHJY. The Weissella confusa LHJY strain is named as Weissella confusa LHJY strain, is preserved in the China Center for Type Culture Collection, and has a preservation number of CCTCC NO: M 20251930 and a preservation date of September 1, 2025. The inventors isolate and purify the probiotic Weissella confusa LHJY from a pickled vegetable sample; and find that the Weissella confusa LHJY has a significant inhibitory effect on P. gingivalis and F. nucleatum, which are important periodontal pathogenic bacteria, through a bacteriostatic experiment and a biofilm removal experiment, thereby filling the blank of application of the Weissella confusa to periodontitis treatment in the prior art. Moreover, the Weissella confusa LHJY can tolerate a high concentration of lysozyme, effectively reduces the expression of interleukin 1β, tumor necrosis factor α and interleukin 6 inflammatory factors in an inflammatory cell model, and has an important application prospect in inhibiting periodontal disease pathogenic bacteria and regulating immune responses.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and relates to probiotics that inhibit oral pathogens, specifically involving the isolation and identification of a strain of Weissella micrantha LHJY and its evaluation of its therapeutic effect on experimental periodontitis. Background Technology

[0002] Periodontitis is one of the most common oral diseases, a chronic inflammation caused by periodontal pathogens that leads to the gradual loss of periodontal supporting tissues. It not only causes inflammation of periodontal tissues and induces alveolar bone resorption leading to tooth loss, but can also seriously affect overall health. Studies show that periodontitis is closely related to the oral microbiota. These microorganisms coexist and counterbalance each other, working together to maintain the balance of the oral microecology. Once this balance is disrupted, periodontitis can occur. Among them, *Fusobacterium nucleatum* and *Porphyromonas gingivalis* are important periodontal pathogens. *Fusobacterium nucleatum* is an invasive Gram-negative anaerobic bacterium, and it is the most abundant bacterium in infection sites such as supragingival plaque, subgingival plaque, and periodontal pockets in periodontitis. *P. gingivalis*, on the other hand, is a Gram-negative oral anaerobic bacterium and one of the most dominant bacteria in the lesions of chronic periodontitis. It participates in the pathogenesis of periodontitis and is currently recognized as a periodontal pathogen.

[0003] The treatment of periodontitis primarily aims to control plaque, eliminate inflammation, restore periodontal tissue function, halt disease progression, and prevent recurrence. Currently, the most common methods are mechanical therapy and adjunctive drug therapy, but both have inherent limitations. Mechanical therapy alone cannot completely remove plaque from specific areas such as root furcations, leading to recolonization of periodontal pathogens after treatment. Furthermore, it is difficult to perform, thus drug therapy is often used as an adjunct. Drug therapy commonly employs antibiotics such as amoxicillin, cephalexin, and metronidazole to eliminate periodontal pathogens; however, antibiotics can reduce the colonization of symbiotic bacteria, disrupt the normal oral microbiota, and contribute to increasing bacterial resistance. Even worse, maintaining effective drug concentrations in periodontal tissues requires high-dose systemic antibiotic administration, which may lead to microbial imbalance, drug resistance, and risks of liver and kidney toxicity. Therefore, developing a safe, efficient, non-invasive, and antibiotic-free treatment strategy for periodontitis is of great significance.

[0004] Probiotics are defined as "live microorganisms that, when ingested in sufficient quantities, are beneficial to the health of the host." In recent years, probiotics have shown great promise in the prevention and treatment of oral diseases as a potential bioregulator. This is because probiotics usually achieve their inhibitory effect on pathogens based on multiple interaction mechanisms, including (1) competitively colonizing and occupying the host's mucosal binding sites and inhibiting the growth of pathogenic bacteria; (2) synthesizing antimicrobial metabolites; (3) regulating the host's immune homeostasis; and (4) upregulating the expression of tight junction proteins, promoting mucin secretion, and strengthening the mucosal physical barrier. Weissella confusa (also known as Fusion Weissella) is a type of Gram-positive, facultative anaerobic rod-shaped bacterium commonly found in high-acid or fermented environments such as dairy products, grains, and kimchi. This strain is highly acid-resistant, with an optimal growth pH of 5.5-5.8, and is slightly aerobic at 30°C. However, there are no reports of Weissella confusa being used for the treatment of periodontitis. Summary of the Invention

[0005] In view of the current situation regarding the use of probiotics in the treatment of periodontitis, this invention provides a strain of *Weissella confusa* LHJY. This *Weissella confusa* LHJY exhibits inhibitory effects on important periodontal pathogens, *Porphyromonas gingivalis* and *Fusobacterium nucleatum*, and can tolerate high concentrations of lysozyme. It effectively reduces the expression of interleukin-1β, tumor necrosis factor-α, and interleukin-6 inflammatory factors in inflammatory cell models, showing significant potential for application in inhibiting periodontal pathogens and regulating immune responses.

[0006] The technical solution of the present invention:

[0007] A strain of *Weissella confusa* that inhibits periodontal pathogens, named *Weissella confusa* LHJY, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251930 and deposit date of September 1, 2025. The inventors isolated and purified the probiotic *Weissella confusa* LHJY from kimchi samples. Antibacterial and biofilm removal experiments revealed that *Weissella confusa* LHJY exhibits significant inhibitory effects against important periodontal pathogens *Porphyromonas gingivalis* and *Fusobacterium nucleatum*, filling a gap in the existing technology for the application of *Weissella confusa* in the treatment of periodontitis. The technology demonstrates significant efficacy and broad application prospects.

[0008] A fermentation culture of *Westernella confoundingii*, wherein the fermentation culture is a bacterial culture system obtained by culturing *Westernella confoundingii* as described above in a microbial culture medium.

[0009] A fermentation supernatant of *Weissella asiatica*, wherein the fermentation supernatant is obtained by centrifuging the fermentation culture as described above to remove bacterial cells.

[0010] An inoculum, comprising the previously described *Westernella*, fermentation culture, or fermentation supernatant.

[0011] The previously described application of *L. humilis*, fermentation cultures, or fermentation supernatants in the preparation of products inhibiting important periodontal pathogens. The important periodontal pathogens are *Porphyromonas gingivalis* and / or *Fusobacterium nucleatum*. The products include pharmaceuticals, oral care products, and animal feed. Through animal model experiments, the inventors found that *L. humilis* LHJY increased the body weight of rats, reduced periodontal probing depth, gingival bleeding index, and the degree of inflammatory infiltration; Micro-CT results showed a reduction in bone resorption. This indicates that *L. humilis* LHJY shows good effects in inhibiting periodontal pathogens and regulating immune responses, and can be used to prepare products for the prevention, relief, or treatment of oral diseases caused by periodontal pathogens, possessing considerable market potential and economic value.

[0012] Preferably, the product is in solid, liquid, semi-solid, gel, or aerosol form.

[0013] A pharmaceutical composition comprising, as previously described, *Westernella* confounding bacteria, a fermentation culture or fermentation supernatant, and a pharmaceutically acceptable carrier. The pharmaceutical composition is in various pharmaceutically acceptable dosage forms, including tablets, capsules, oral liquids, sprays, ointments, or lyophilized powders.

[0014] The beneficial effects of this invention are:

[0015] (1) This application provides an innovative strain of Weissella alias LHJY that can inhibit periodontal pathogens, filling the gap in the application of Weissella alias in the treatment of periodontitis in the prior art. It has outstanding substantive features and has achieved significant progress.

[0016] (2) The *Weissella hystericus* LHJY described in this application not only has a significant inhibitory effect on important periodontal pathogens (*Porphyromonas gingivalis* and *Fusobacterium nucleatum*), but also can tolerate high concentrations (3 mg / mL) of lysozyme and can colonize and survive in the oral environment.

[0017] (3) The *Westernella* LHJY described in this application reduces the expression of interleukin-1β, tumor necrosis factor-α, and interleukin-6 inflammatory factors in the inflammatory cell model; at the same time, it effectively increases the body weight of rats in animal experiments and reduces clinical indicators such as periodontal probing depth, gingival bleeding index, and degree of inflammatory infiltration, which fully demonstrates that *Westernella* LHJY has good effects in inhibiting periodontal pathogens and regulating immune responses. Attached Figure Description

[0018] Appendix Figure 1 To avoid confusion between morphological observation (A), 16S rDNA identification results (B), and SEM images of the bacterial cells (C) of Weissella LHJY.

[0019] Appendix Figure 2 To confuse the growth curve (A) and acid production curve (B) of Weissella LHJY.

[0020] Appendix Figure 3 Determination of the antibacterial activity of Weissella and its metabolites against F. nucleatum Y (A) and P. gingivalis Y (B); wherein A consists of ① chlorhexidine, ② Weissella cells, ③ MRS medium, ④ Weissella supernatant, and ⑤ Weissella bacterial suspension.

[0021] Appendix Figure 4 The biofilm formation rate was measured (A) and the lysozyme tolerance was evaluated (B).

[0022] Appendix Figure 5 This study aimed to determine the expression of cellular inflammatory factors by *Westernella*. Specifically, (A) the effect of *Westernella* on the relative expression level of the IL-6 gene in RAW264.7 cells; (B) the effect of *Westernella* on the relative expression level of the IL-1β gene in RAW264.7 cells; and (C) the effect of *Westernella* on the relative expression level of the TNF-α gene in RAW264.7 cells.

[0023] Appendix Figure 6 The results of experimental periodontitis model construction and clinical index detection include: (A) Observation of gingival tissue in the blank group, periodontitis group and Weissella group of rats constructed by ligating bilateral maxillary second molars with silk thread; (B) Changes in body weight of rats in each group 1-5 weeks after modeling; (C) Gingival bleeding index of rats in each group; (D) Changes in periodontal probing depth of rats in each group.

[0024] Appendix Figure 7 Periodontal histological evaluation and alveolar bone Micro-CT analysis for each group of rats; (A) Alveolar bone Micro-CT analysis. (B) CEJ-ABC (cementoenamel junction-alveolar crest) distance analysis. Detailed Implementation

[0025] The present invention will be further described below with reference to the embodiments.

[0026] Example 1: Isolation, purification and identification of strains

[0027] (1) Isolation and purification of probiotic strains

[0028] Take kimchi samples, dissolve them in PBS, and perform serial dilutions to 10. -4 After dilution, 100 μL of the diluted sample was evenly spread onto CaCO3 MRS agar plates under sterile conditions. The plates were inverted and incubated at 37°C for 48-72 h. Single colonies exhibiting the morphology of *Westernella* were selected based on colony morphology and the size of the clear zone, and continuously transferred to MRS agar plates until microscopic examination revealed pure colonies. The purified strain was inoculated into MRS broth and incubated at 37°C for 24 h, then stored in cryovials at -80°C with 20% glycerol for later use.

[0029] (2) Identification of the isolated and purified strains

[0030] First, morphological observation was performed: the isolated and purified strain was inoculated into MRS broth medium and cultured at 37 ℃ for 12 h. A 1 μL sterile inoculation stick was then used to inoculate the strain onto MRS solid medium and streaked. After culturing at 37 ℃ for 24 h, colony morphology was observed. Figure 1 A). By Figure 1 As can be seen, the colonies of Weissella LHJY are white, round, 3-4 mm in diameter, with a raised center and a smooth, dense surface. Figure 1 C represents the cell morphology of *Weisseria gonorrhoeae* under SEM. Figure 1 As can be seen, Weissella cells exhibit typical spherical, nearly spherical, or rod-shaped morphology; most cells are arranged in unipolar or bipolar pairs, with occasional tetrads.

[0031] Next, after activating the bacterial strain, bacterial cells were collected, and total DNA was extracted using a bacterial DNA extraction kit. Then, PCR amplification and cloning of the strain's 16S rRNA sequence were performed. Specifically, after activating the strain, bacterial cells were collected, and DNA was extracted using a Tiangen bacterial DNA extraction kit. After successful extraction, the DNA was detected and its concentration was determined by 1% agarose gel electrophoresis. After extracting DNA from the sample, PCR amplification and cloning of the strain's 16S rRNA sequence were performed. The PCR amplification system was: 12.5 μL of 2×Phanta Flash Master Mix (DyePlus), 1.0 μL of upstream primer, 1.0 μL of downstream primer, 1.0 μL of DNA template, and ddH2O to a final volume of 25 μL. The variable regions of the bacterial 16S rRNA gene V3~V4 were determined as follows: upstream primer 38F: 5'-TGACGGGCGGTGTGTACAAG-3', downstream primer 38R: 5'-TGACGGGCGGTGTGTACAAG-3'. PCR amplification conditions: 98 ℃ pre-denaturation for 3 s, 35 cycles (98 ℃ denaturation for 10 s, 55 ℃ annealing for 5 s, 72 ℃ extension for 10 s, 72 ℃ stable extension for 1 min), and finally stored at 4 ℃.

[0032] The PCR products were subjected to 1% agarose gel electrophoresis and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequenced sequences were compared with known sequences in the NCBI database, with *Escherichia coli* (a non-corresponding species) as the outgroup. A 16S rDNA phylogenetic tree of strain *Escherichia coli* LHJY was constructed using MEGA 11 software. Figure 1 B). By Figure 1 As shown in Figure B, the isolated strain forms an independent branch in the phylogenetic tree and is closely related to *Weissella confusa* NWAFU 8011. Therefore, this strain is named *Weissella confusa* LHJY and deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251930 and deposit date of September 1, 2025.

[0033] Example 2: Morphological observation, growth curve and pH determination of Weissella LHJY

[0034] The test strain was inoculated into MRS broth medium at a 1% inoculum and cultured in a shaker at 37°C for 24 h. Samples were taken every 2 h to measure the pH value and the OD value was measured using a UV spectrophotometer. 600nm Values, and to create growth curves ( Figure 2 A) and acid production curve ( Figure 2 B).

[0035] Depend on Figure 2 As shown in A, the strain was in a slow growth phase from 0 to 4 hours, exhibiting a slow growth trend; from 4 to 10 hours, it was in the logarithmic growth phase, with the growth curve rising rapidly; after 10 hours, it entered a stationary phase, and the OD value tended to stabilize. Figure 2 As shown in section B, the strain produces relatively little acid and the pH value decreases only slightly during the first 0-4 hours. As the strain enters the logarithmic growth phase, it begins to secrete large amounts of acidic substances, causing a sharp drop in pH. Once the strain reaches a stationary phase, the pH value also tends to stabilize. This indicates that after 10 hours of cultivation, the strain's growth and metabolic activities tend to stabilize, which is beneficial for controlling the fermentation process and ensuring stable product quality.

[0036] Example 3: Antibacterial activity of Weissella LHJY and its metabolites

[0037] Periodontitis is a mixed infection caused by multiple microorganisms, among which *Fusobacterium nucleatum* and *Porphyromonas gingivalis* are important periodontal pathogens. This example uses the Oxford cup diffusion method to detect the antibacterial activity of *Westernella* LHJY and its metabolites against *F. nucleatum* and *P. gingivalis*. The specific procedure is as follows:

[0038] Preparation of Weissella LHJY fermentation broth and fermentation supernatant: (1) The frozen Weissella LHJY was streaked on MRS solid medium for two generations. Single colonies were picked and inoculated into MRS liquid medium. The culture was incubated at 37℃ for 24h to obtain the fermentation culture, i.e., the fermentation broth. (2) The fermentation culture was centrifuged at 12000 r / min for 10 min at 4℃ to remove the cells. The supernatant was filtered through a 0.22μm sterile filter membrane to obtain cell-free supernatant, which is the Weissella LHJY fermentation supernatant. It was stored at -20℃ for later use.

[0039] Antibacterial activity assay: *F. nucleatum* and *P. gingivalis*, preserved in 30% glycerol, were streaked twice on BHI solid medium. Single colonies were picked and inoculated into BHI liquid medium and cultured anaerobically at 37 °C for 48 h. The concentration of the indicator bacterial suspension was adjusted to 10... 7CFU / mL. Pour 10 mL of 1.5% pure agar into a plate. After solidification, place four autoclaved Oxford cups at equal intervals. Then add BHI solid medium containing 5% indicator bacteria suspension. After solidification, remove the Oxford cups and add 150 µL of *Westernella* supernatant, *Westernella* cells, uninoculated MRS liquid medium, and chlorhexidine solution to each well. Incubate anaerobicly at 37 °C for 24 h, then measure the diameter of the inhibition zone. Results are accurate to 0.1 mm. Perform three replicates per group. See Table 1 for detailed results. Figure 3 A (for F. nucleatum), Table 2 and Figure 3 B (to P. gingivalis).

[0040] Table 1. Antibacterial activity of Weissella metabolites against F. nucleatum

[0041]

[0042] Note: The diameter of the inhibition zone includes the outer diameter of the Oxford cup (8mm); "-" indicates no inhibition zone;

[0043] * indicates a difference of P < 0.001 between groups compared to the MRS culture medium group.

[0044] Table 2. Antibacterial activity of Weissella metabolites against P. gingivalis

[0045]

[0046] Note: The diameter of the inhibition zone includes the outer diameter of the Oxford cup (8mm); "-" indicates no inhibition zone;

[0047] * indicates a difference of P < 0.0001 between groups compared to the MRS culture medium group.

[0048] Figure 3 A represents the antibacterial activity of *Westernella* LHJY and its metabolites against *F. nucleatum*. Figure 3 As shown in Table A and Table 1, *Westernella* LHJY cells showed no inhibition zone against the periodontal pathogen *F. nucleatum*, indicating no inhibitory effect. However, the supernatant of *Westernella* LHJY showed an inhibition zone of 13.37±1.00 mm against *F. nucleatum*, while the bacterial suspension showed an inhibition zone of 14.30±0.85 mm, indicating that both the supernatant and bacterial suspension of *Westernella* LHJY had significant inhibitory effects on *F. nucleatum*. However, compared with the positive control group chlorhexidine (inhibition zone 29.3±2.47 mm), the antibacterial effect was weaker.

[0049] Figure 3B represents the antibacterial activity of *Westernella* LHJY and its metabolites against *P. gingivalis*. Figure 3 As shown in B and Table 2, *Westernella* LHJY cells also showed no inhibition zone against the periodontal pathogen *P. gingivalis*, indicating no inhibitory effect. However, the inhibition zone of *Westernella* LHJY supernatant against *P. gingivalis* was 12.61±0.43 mm, and the inhibition zone of the bacterial suspension against *P. gingivalis* was 14.03±0.28 mm, indicating that both the supernatant and the bacterial suspension of *Westernella* LHJY had significant inhibitory effects on *P. gingivalis*. Similarly, compared with the positive control group chlorhexidine (inhibition zone 27.30±2.23 mm), the antibacterial effect was weaker.

[0050] In summary: (1) The supernatant and bacterial culture of Weissella LHJY described in this application both have significant inhibitory effects on periodontal pathogens F. nucleatum and P. gingivalis, indicating that the main antibacterial component in Weissella LHJY is in its metabolites. The inventors speculate that the lack of antibacterial activity in the bacterial cells is related to their colonization ability and can only be manifested in vivo. (2) Weissella LHJY is a probiotic that can be used in food. Although the antibacterial effect of Weissella LHJY is weaker than that of chlorhexidine, an antibiotic, it also solves the technical problems of antibiotics reducing the colonization of symbiotic bacteria, destroying the oral microecology, and causing bacterial resistance, thus producing significant technical effects.

[0051] Example 4: Determination of biofilm content

[0052] To further determine the antibacterial effect of the *Westernella* LHJY supernatant, this example measured the amount of biofilm formed by *Fusobacterium nucleatum* and *Porphyromonas gingivalis*. The specific procedure was as follows:

[0053] Add 10 to each of the 24-well plates 780 µL each of CFU / mL *F. nucleatum* and *P. gingivalis* suspensions were added, followed by 160 µL of filtered *Westernella* LHJY supernatant. The mixture was anaerobic incubated at 37 °C for 48 h. The negative control group used the same volume of blank MRS liquid medium instead of the *Westernella* LHJY supernatant, while the positive control group used the same volume of chlorhexidine instead. After incubation, the biofilm was washed twice with PBS, fixed with 99% methanol for 15 min, dried at room temperature for 10 min, and then allowed to air dry at room temperature. 500 μL of 0.1% crystal violet solution was added to each well to stain the biofilm for 30 min. After staining, the biofilm was washed twice with PBS and photographed. 100 μL of 95% ethanol was added to dissolve the biofilm, and the mixture was shaken on a shaker for 30 min. OD values ​​were then read. 450nm Value. See results for details. Figure 4 A.

[0054] Figure 4 A represents the results of the assay for inhibiting dental plaque biofilm formation. (From...) Figure 4 As can be seen in Figure A, the OD of the control group 450nm The value was 0.994, while the OD of the Weissella LHJY supernatant group was... 450nm The value was 0.401; compared with the control group, the biofilm formation in the Weissella supernatant group was significantly reduced. This indicates that the antibacterial substances in the metabolites of Weissella LHJY supernatant can effectively inhibit the formation of biofilms from Fusobacterium nucleatum and Porphyromonas gingivalis (P < 0.0001).

[0055] Example 5: Lysozyme tolerance

[0056] Colonization of bacteria in the oral cavity is the first step in achieving therapeutic effects. Due to the complexity of the oral environment, the mouth contains substances such as lysozyme, which, while protecting the oral system from pathogens, can also kill beneficial bacteria. Saliva contains a certain concentration of lysozyme, which can inhibit or even kill some bacteria, including Escherichia coli. As a probiotic strain in the oral cavity, it must be able to tolerate a certain concentration of lysozyme. To test the survival ability of *Weisseria gonorrhoeae* LHJY in the oral environment, this embodiment uses a spectrophotometer to measure the tolerance of *Weisseria gonorrhoeae* LHJY to lysozyme. Specifically, the bacterial suspension was inoculated into a 96-well plate, and different concentrations of lysozyme were added to each well to achieve a final concentration of 0–3 mg / ml. After incubation at 37°C for 24 hours, the OD was measured. 450nm The value is [value]. See details for the results. Figure 4 B.

[0057] Figure 4 B represents the growth density of *Westernella* LHJY at different lysozyme concentrations. Figure 4As shown in B, when the lysozyme concentration is zero, the cell growth density OD... 450nm The value was 2.190; with increasing lysozyme concentration, the cell growth density OD... 450nm The value gradually decreased. Specifically, when the lysozyme concentration was 2.0 mg / mL, the cell growth density OD... 450nm The value was 2.04; when the lysozyme concentration was 2.8 mg / mL, the cell growth density OD was... 450nm The value was 1.992. When the lysozyme concentration was 3.2 mg / mL, the cell growth density OD was... 450nm The value was 1.967. Therefore, it can be concluded that: (1) when the lysozyme concentration was between 0 and 2.0 mg / mL, the growth of *Westernella* LHJY was almost unaffected; (2) when the concentration was >2.8 mg / mL, although the absorbance value decreased, the maximum tolerance range was still greater than 60%, which is far greater than the concentration of lysozyme in the human oral cavity (1-57 μg / mL). This indicates that *Westernella* LHJY has the ability to colonize in the oral environment and can survive and colonize well in the oral cavity. Therefore, *Westernella* LHJY may compete with pathogenic bacteria for nutrients, thereby inhibiting the growth and adhesion of pathogenic bacteria.

[0058] Example 6: Measurement of cellular inflammatory factors

[0059] This embodiment constructs a RAW264.7 cell inflammation model to detect the effects of Weissella LHJY on the gene expression of pro-inflammatory factors interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). The specific procedures are as follows:

[0060] The activated RAW264.7 cells were evenly seeded into 6-well plates, with 2 ml added to each well, resulting in a concentration of 2 × 10⁻⁶ cells. 5 After culturing for 2 hours, lipopolysaccharide (LPS) at a concentration of 1 μg / ml was added to induce an inflammatory cell model. Simultaneously, 60 μL of *Westernella* LHJY supernatant was added to each well for further culturing for 12 hours. The blank control group received neither LPS nor *Westernella* LHJY supernatant; the model group received only LPS without *Westernella* LHJY supernatant; and the experimental groups received LPS followed by single-strain supernatant. At the end of the culture, total RNA was extracted from the cells using a cell RNA extraction kit, and the concentration of the extracted RNA was measured. RNA was reverse transcribed into cDNA using the FasKing gDNADispelling RT SuperMix reverse transcription kit (KR118, Tiangen Biotech Co., Ltd., Beijing), and then amplified using a 2×HQ SYBR qPCR Mix (No Rox) quantitative kit. β-actin was used as an internal control, and 2... -ΔΔCtThe expression of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) mRNA was detected by a specific method. See details for the results. Figure 5 .

[0061] Figure 5 A represents the effect of Weissella LHJY on the relative expression level of the IL-6 gene in RAW264.7 cells. (From...) Figure 5 As shown in Figure A, compared with the inflammation model group, the expression levels of inflammatory factors were significantly downregulated after treatment with Weissella LHJY supernatant, and the difference was statistically significant (p<0.0001). Figure 5 B represents the effect of Weissella LHJY on the relative expression level of the IL-1β gene in RAW264.7 cells. Figure 5 As shown in B, compared with the inflammation model group, the expression levels of inflammatory factors were significantly downregulated after treatment with Weissella LHJY supernatant, and the difference was statistically significant (p<0.0001). Figure 5 C represents the effect of Weissella LHJY on the relative expression level of the TNF-α gene in RAW264.7 cells. Figure 5 As shown in C, compared with the inflammation model group, the expression levels of inflammatory factors were significantly downregulated after treatment with Weissella LHJY supernatant, and the difference was statistically significant (p<0.0001).

[0062] In summary, Weissella LHJY has the ability to downregulate the gene expression of pro-inflammatory factors interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). This indicates that Weissella LHJY has the ability to regulate related immune responses, reduce the secretion of inflammatory factors, and reduce the damage of inflammatory factors to periodontal tissues, thus playing a positive role in the treatment of periodontitis. It can be inferred that after Weissella LHJY colonizes in the oral cavity, it inhibits the growth and adhesion of pathogenic bacteria and reduces the production of pathogenic virulence factors such as LPS.

[0063] Example 7: Construction of a rat experimental periodontitis model and group intervention

[0064] (1) An experimental rat periodontitis animal model was constructed using the silk ligation method.

[0065] Fifteen male SD rats were randomly divided into three groups (n=5 per group): a blank control group (normal control group), a periodontitis group, and a Weissella group. Rats were anesthetized with an intraperitoneal injection of 2.5% aphthylamine at a dose of 1.2 mL / 100 g. The gingiva of the bilateral second molars was separated mesially and distally using a dental probe. 4-0 silk sutures were used to ligate the gingival sulcus, extending the sutures as far as possible into the gingival sulcus, and fixing the sutures to the cervical region of the right maxillary second molar. The sutures were checked for detachment every two days. One week after modeling, the probiotic group underwent daily inoculation using a sterile syringe to slowly rinse the ligation sites with bacterial solution for four weeks. Figure 6 A). Record your weight changes weekly; see details below. Figure 6 .

[0066] Figure 6 A shows the gingival tissue observations in animal models of bilateral maxillary second molars constructed by wire ligation, including the blank group, periodontitis group, and Weisslerella group. Figure 6 As can be seen, (1) in the blank group, the gingival tissue was pink, thin at the edges and tightly attached to the tooth surface, and dense and tough in texture; (2) in the periodontitis group, the gingival tissue was red and swollen, soft in texture, thickened at the edges, and spontaneous bleeding occurred. (3) after treatment with Weissella esculenta, the redness and swelling of the gingiva were reduced compared with the periodontitis group. It can be concluded that after treatment with Weissella esculenta LHJY, the clinical manifestations of periodontal inflammation in rats were improved, and the treatment effect was significant.

[0067] Figure 6 B represents the changes in body weight of rats in each group 1-5 weeks after modeling. Figure 6 As shown in Figure B, the rate of weight recovery in the Weisslerella group was significantly higher than that in the periodontitis group in the first week after modeling, and also significantly higher in the fourth week. By the end of the modeling periodontitis periodontitis periodontitis periodontitis group, the weight had returned to the control group level (no significant difference in weight, p>0.05). This indicates that the Weisslerella group reduced weight loss in rats compared to the periodontitis model group; this suggests that Weisslerella LHJY treatment can alleviate the symptoms of periodontitis, thereby increasing food intake in rats.

[0068] Figure 6 C represents the gingival bleeding index of rats in each group. Figure 6 C indicates that the gingival bleeding index of the *Westernella* group was 1.56, while that of the periodontitis group was 2.56, and the gingival bleeding index of the normal control group (without probing bleeding) was 0.306. Therefore, the gingival bleeding index of the *Westernella* group was 40% lower than that of the periodontitis group, and the bleeding was significantly reduced; the difference was statistically significant (p<0.0001). Figure 6 D represents the change in periodontal probing depth in each group of rats. Figure 6As shown in Figure D, the periodontal probing depth in the *Westernella* group was 1.014, while that in the periodontitis group was 1.558, and in the normal control group without periodontitis was 0.306. This indicates that the periodontal probing depth in the *Westernella* group was 35% lower than that in the periodontitis group, and the degree of periodontal tissue damage was significantly reduced, with a statistically significant difference (p<0.0001). In summary, although the periodontal probing depth and gingival bleeding index increased in both the periodontitis and *Westernella* groups compared to the normal control group, both indicators showed significant improvement after *Westernella* intervention, and the differences were statistically significant (p<0.0001). This demonstrates that after treatment with *Westernella* LHJY, the overall periodontal health was significantly improved, and the periodontal tissues are recovering to a healthy state, indicating a significant treatment effect.

[0069] (2) Observe and test the periodontal clinical indicators of rats in each group.

[0070] After modeling, rats were anesthetized with 2.5% aphthylamine (1.2 mL / 100 g) via intraperitoneal injection and then sacrificed. Hematoxylin-eosin (HE) staining was performed, and samples of the right maxilla were separated for Micro-CT analysis. Gingival tissue from the second molar was also collected for periodontal histopathological evaluation. Specifically, periodontal clinical indicators, the degree of inflammatory infiltration, and the amount of bone resorption were observed and measured in each group of rats. Detailed results can be found in [link to results]. Figure 7 .

[0071] Figure 7 A shows the results of micro-computed tomography (Micro-CT) scans of the maxillary alveolar bone in each group of rats. Figure 7 The red line in A represents the distance from the cementoenamel junction to the alveolar ridge crest. The longer the red line, the greater the distance and the more bone resorption occurs in the alveolar bone. Figure 7 B represents the distance analysis of CEJ-ABC (cementoenamel junction - alveolar ridge crest). Figure 7 B shows that the CEJ-ABC distance in the normal control group was 0.568, the CEJ-ABC distance in the periodontitis group was 1.077, and the distance in the Weissella group was 0.857, slightly higher than the normal group but significantly lower than the periodontitis group. Combined with... Figure 7 A and Figure 7As shown in Figure B, compared with the normal control group, the distance of CEJ-ABC (cementoenamel-alveolar ridge crest) in the periodontitis group was significantly increased, with a statistically significant difference (p<0.0001), indicating significant bone resorption and the successful establishment of the rat experimental periodontitis model. Conversely, the distance of CEJ-ABC in the Weisslerella group was significantly decreased compared with the periodontitis group, with a statistically significant difference (p<0.001), indicating reduced bone resorption. This suggests that Weisslerella LHJY effectively reduced alveolar bone resorption and prevented bone loss, which also demonstrates its therapeutic effect on periodontitis from another perspective. Based on the aforementioned therapeutic effects, it can also be inferred that after Weisslerella LHJY colonizes the oral cavity, it inhibits the growth and adhesion of pathogenic bacteria, which corroborates the results of Example 6.

[0072] In summary, this application innovatively provides a probiotic strain, *Lactobacillus plantarum* LHJY, for the treatment of periodontitis. This is because: firstly, it exhibits significant inhibitory effects on both *Fusobacterium nucleatum* and *Porphyromonas gingivalis*, both periodontal pathogens; secondly, *Lactobacillus plantarum* LHJY has the ability to regulate related immune responses, reduce the secretion of inflammatory factors, and decrease the damage of inflammatory factors to periodontal tissues; thirdly, *Lactobacillus plantarum* LHJY effectively reduces alveolar bone resorption and prevents bone loss. Furthermore, *Lactobacillus plantarum* LHJY tolerates high concentrations (3 mg / mL) of lysozyme and can colonize and survive in the oral environment. Based on the foregoing reasons, the probiotic *Lactobacillus plantarum* LHJY fills the gap in the existing technology for the application of *Lactobacillus plantarum* in the treatment of periodontitis, possessing outstanding substantive characteristics and achieving significant progress.

Claims

1. A strain of Weissella confusa that inhibits periodontal pathogenic bacteria, characterized in that: The Weissella confusa is named as Weissella confusa LHJY strain, which is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20251930, and the preservation date is September 1, 2025.

2. A fermented culture of Weissella confusa, characterized in that: The fermentation culture is obtained by culturing the Weissella confusa in a microbial culture medium.

3. A fermentation supernatant of Weissella confusa, characterized in that: The fermentation supernatant is obtained by removing the bacteria bodies from the fermentation culture by centrifugation.

4. An inoculant characterized in that: The product comprises the Weissella confusa, the fermentation culture, or the fermentation supernatant.

5. The use of the Weissella confusa, the fermentation culture, or the fermentation supernatant in the preparation of a product for inhibiting periodontal important pathogenic bacteria.

6. Use according to claim 5, characterized in that: The periodontal important pathogenic bacteria are Porphyromonas gingivalis and / or Fusobacterium nucleatum.

7. Use according to claim 5 or 6, characterized in that: The product comprises a medicine or an oral care product.

8. Use according to claim 7, characterized in that: The product adopts a solid form, a liquid form, a semi-solid form, a gel, or an aerosol form.

9. A pharmaceutical composition, characterized by: The product comprises the Weissella confusa, the fermentation culture, or the fermentation supernatant and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition of claim 9, wherein: The pharmaceutical composition adopts various pharmaceutically acceptable dosage forms, such as tablets, capsules, oral liquids, sprays, ointments, or lyophilized powders.

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