A probiotic composition for inhibiting periodontal pathogens and its application

Abstract

In view of the current state of probiotics used in the treatment of periodontitis, this invention provides a probiotic composition that inhibits periodontal pathogens. The probiotic composition comprises *Weissella confusa* LHJY and *Lacticaseibacillus rhamnosus* LHJY. Through the synergistic effect of the two probiotic strains, the composition significantly enhances the antibacterial effect and significantly reduces the expression of inflammatory factors, demonstrating significant technical efficacy and broad application prospects. Furthermore, the probiotic composition can tolerate high concentrations (3 mg / mL) of lysozyme and can colonize and survive in the oral environment. Animal model experiments have shown that the probiotic composition can increase the body weight of rats, reduce periodontal probing depth, gingival bleeding index, and the degree of inflammatory infiltration; Micro-CT results show a reduction in bone resorption. This indicates that the probiotic composition 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, with considerable market prospects and economic value.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and relates to probiotics that inhibit oral pathogens, specifically to a probiotic composition consisting of a strain of Weissella micrantha LHJY and Rhamnosus rhamnosus and its application. 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. Lactobacillus rhamnosus and Weissella pustulata are both probiotics. According to records, Lactobacillus rhamnosus can inhibit the growth of oral pathogenic bacteria such as Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus mutans, and Actinobacillus actinomycetii. Currently, there are no reports on Weissella pustulata, nor are there any reports on the synergistic improvement of oral health by Lactobacillus rhamnosus and Weissella pustulata. Summary of the Invention

[0005] In view of the current state of probiotics used in the treatment of periodontitis, this invention provides a probiotic composition for inhibiting periodontal pathogens. The probiotic composition comprises *Weissella confusa* LHJY and *Lacticaseibacillus rhamnosus* LHJY. Through the synergistic effect of these two bacteria, the probiotic composition not only inhibits important periodontal pathogens such as *Porphyromonas gingivalis* and *Fusobacterium nucleatum*, and can tolerate high concentrations of lysozyme, but also shows significant efficacy in reducing the expression of interleukin-1β, tumor necrosis factor-α, and interleukin-6 inflammatory factors in an inflammatory cell model. It has important application prospects in inhibiting periodontal pathogens and regulating immune responses.

[0006] The technical solution of the present invention:

[0007] A probiotic composition for inhibiting periodontal pathogens includes *Weissella confusa* LHJY and *Lactobacillus rhamnosus* LHJY. The live bacteria ratio of *Weissella confusa* LHJY to *Lactobacillus rhamnosus* in the probiotic composition is 1:1 to 1:4. Through the synergistic effect of the two probiotic strains, the probiotic composition achieves a significant enhancement in antibacterial efficacy and a significant reduction in the expression of inflammatory factors, demonstrating significant technical effectiveness and broad application prospects.

[0008] The *Westernia hysterophylla* LHJY is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 20251930 and accession date September 1, 2025. The inventors isolated and purified the probiotic *Westernia hysterophylla* LHJY from kimchi samples. Antibacterial and biofilm removal experiments revealed that *Westernia hysterophylla* 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 *Westernia hysterophylla* in the treatment of periodontitis. The *Rhamnosus rhamnosus* LHJY is also deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 20251929 and accession date September 1, 2025. The inventors isolated and purified the probiotic Lactobacillus rhamnosus LHJY from the feces of healthy infants collected clinically. Through antibacterial experiments and biofilm clearance experiments, it was found that Lactobacillus rhamnosus LHJY also has a significant inhibitory effect on important periodontal pathogens Porphyromonas gingivalis and Fusobacterium nucleatum.

[0009] A fermentation culture of a probiotic composition, wherein the fermentation culture is a bacterial culture system obtained by culturing the probiotic composition as described above in a microbial culture medium.

[0010] A fermentation supernatant of a probiotic composition, wherein the fermentation supernatant is obtained by centrifuging the fermentation culture as described above to remove bacterial cells.

[0011] A probiotic agent includes the probiotic composition, fermentation culture, or fermentation supernatant as described above. The dosage form of the probiotic agent includes lyophilized powder and / or aqueous solution.

[0012] The aforementioned probiotic composition, fermentation culture, or fermentation supernatant is used in the preparation of products that inhibit important periodontal pathogens. The important periodontal pathogens are *Porphyromonas gingivalis* and / or *Fusobacterium nucleatum*. The products include pharmaceuticals and oral care products; the products are in solid, liquid, semi-solid, gel, or aerosol form. The inventors found through animal model experiments that the probiotic composition can increase the body weight of rats, reduce periodontal probing depth, gingival bleeding index, and the degree of inflammatory infiltration; Micro-CT results showed a reduction in bone resorption. This indicates that the probiotic composition 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 prospects and economic value.

[0013] A pharmaceutical composition comprising a probiotic composition as described above, a fermentation culture or fermentation supernatant, and a pharmaceutically acceptable carrier. The pharmaceutical composition is in a pharmaceutically acceptable dosage form, such as tablets, capsules, oral liquids, sprays, ointments, or lyophilized powders.

[0014] The beneficial effects of this invention are:

[0015] (1) This application first provides a probiotic composition composed of Weissella micrantha LHJY and Lactobacillus rhamnosus. The probiotic composition achieves a significant improvement in antibacterial effect and a significant reduction in the expression of inflammatory factors through the synergistic effect of the two probiotic strains. The technical effect is significant and the application prospects are broad.

[0016] (2) The probiotic composition 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 probiotic composition described in this application not only 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 the probiotic composition shows good effects in inhibiting periodontal pathogens and regulating immune response. Attached Figure Description

[0018] Appendix Figure 1 Morphological observation (A, B), 16S rDNA identification results (C, D), and SEM images of Lactobacillus rhamnosus LHJY and Lactobacillus sieboldii LHJY (E) are shown.

[0019] Appendix Figure 2 The growth curves (A) and acid production curves (B) of Lactobacillus rhamnosus LHJY and Weissella fussula LHJY are shown.

[0020] Appendix Figure 3 The antibacterial activities of Lactobacillus rhamnosus LHJY, L. Weissella asiatica LHJY, and their combined metabolites against F. nucleatum (A) and P. gingivalis (B) were determined; among which: Weissella bacterial suspension ③ Combined bacterial suspension of Lactobacillus rhamnosus and Weissella spp. ③ Lactobacillus rhamnosus bacterial suspension chlorhexidine Weissella supernatant Combined supernatant of Lactobacillus rhamnosus and Weissella Lactobacillus rhamnosus supernatant Weissella cells Combined cells of Lactobacillus rhamnosus and Weissella Lactobacillus rhamnosus cells ⑪ MRS medium.

[0021] Appendix Figure 4 The biofilm formation of Lactobacillus rhamnosus LHJY, L. Weissella salina LHJY and the combined group was measured (A), the lysozyme tolerance was evaluated (B), and the effect of the ratio of Lactobacillus rhamnosus and L. Weissella salina in the combined group on antibacterial activity was investigated (C).

[0022] Appendix Figure 5 The study aimed to determine the expression of cellular inflammatory factors by *Lactobacillus rhamnosus* LHJY, *Westernella* LHJY, and their combination. Specifically, (A) the effect on the relative expression level of the IL-6 gene in RAW264.7 cells; (B) the effect on the relative expression level of the IL-1β gene in RAW264.7 cells; and (C) the effect 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, Lactobacillus rhamnosus group, Weissella group and combined group of rats with bilateral maxillary second molars ligated with silk thread; (B) changes in body weight of rats in each group 1-5 weeks after modeling; (C) changes in periodontal probing depth of rats in each group; and (D) gingival bleeding index 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. -4After 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 I 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] Clinically collected stool samples from healthy infants were dissolved in PBS and serially diluted to 10⁻⁶. -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 lactic acid bacteria morphology 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 II 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.

[0030] (2) Identification of the isolated and purified strains

[0031] 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 and 1B). Single colonies were picked, Gram-stained, and observed under a microscope. The fermentation strain was inoculated at a 1% inoculum into MRS broth medium and cultured at 37°C in a shaker for 24 h. Samples were taken every 2 h to measure the pH value and the OD600nm value using a UV spectrophotometer. Figure 1 As can be seen, the colonies of *Westernella* LHJY are white, round, 3-4 mm in diameter, with a raised center and a smooth, dense surface. Figure 1 As can be seen from B, the colony morphology of Lactobacillus rhamnosus LHJY is milky white, round, 3-4 mm in diameter, with a raised center and a smooth and dense surface. Figure 1 E represents the cell morphology of *Lactobacillus rhamnosus* and *Weisseria gonorrhoeae* under SEM. Figure 1 As can be seen, Weissella cells exhibit typical spherical, nearly spherical, and short rod-shaped morphologies, with most cells arranged in unipolar or bipolar pairs, and occasionally in tetrads. Lactobacillus rhamnosus cells are rod-shaped or slightly oval rod-shaped, with rounded and relatively regular ends, and often appear as single rods or in pairs; no flagella are observed, and they are non-motile.

[0032] 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 ℃.

[0033] 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 *Lactobacillus rhamnosus* LHJY and *Weissella vesiculosus* LHJY strains was constructed using MEGA 11 software. Figure 1 (C and 1D). By Figure 1 As shown in Figure C, the isolated strain forms an independent branch in the phylogenetic tree and is closely related to LC705412.2 *Lacticaseibacillus rhamnosus*. Therefore, this strain is named *Lacticaseibacillus rhamnosus* LHJY. It is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20251929, on September 1, 2025. Figure 1 As shown in Figure D, 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. It is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251930 and deposit date of September 1, 2025.

[0034] Example 2: Growth curves and pH determination of Lactobacillus rhamnosus LHJY and L. Weissella rhamnosus LHJY

[0035] 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, 2B) and acid production curves ( Figure 2 C, 2D).

[0036] Figure 2 A represents the growth curve of Lactobacillus rhamnosus. Figure 2 B represents the growth curve of *Westernella*. Figure 2 As shown in A and 2B, the growth curves of Lactobacillus rhamnosus and Weissella are basically the same; from 0 to 4 hours, they are in the lag phase with a slow growth trend; from 4 to 10 hours, they are in the logarithmic growth phase with a rapid increase in the growth curve; after 10 hours, they are in the stationary phase with the OD value stabilizing.

[0037] Figure 2 C represents the acid production curve of Lactobacillus rhamnosus. Figure 2 D represents the acid production curve of *Weisseria gonorrhoeae*. (From...) Figure 2 As shown in C and 2D, the acid production curves of Lactobacillus rhamnosus and Weissella are basically the same; the amount of acid produced is small in the first 0-4 hours, and the pH value decreases less; as the strain enters the logarithmic growth phase, it begins to secrete a large amount of acidic substances, and the pH value drops sharply; after the strain enters the stationary phase, the pH value also tends to stabilize.

[0038] In summary, the Lactobacillus rhamnosus and Weissella bacteria described in this application reach a stable state of growth and metabolic activity after 10 hours of cultivation, which is beneficial for controlling the fermentation process and ensuring product quality stability.

[0039] Example 3: Antibacterial activity of Lactobacillus rhamnosus LHJY, L. Weissella asiatica LHJY and their combined metabolites

[0040] Periodontitis is a mixed infection caused by multiple microorganisms, among which *Fusobacterium nucleatum* and *Porphyromonas gingivalis* are important periodontal pathogens. This embodiment uses the Oxford cup diffusion method to detect the antibacterial activity of *Lactobacillus rhamnosus* LHJY, *Weissella vesca* LHJY, and their combined metabolites against *F. nucleatum* and *P. gingivalis*. The specific procedures are as follows:

[0041] Preparation of fermentation broth and fermentation supernatant of Lactobacillus rhamnosus LHJY, L. Weissella LHJY and their combination (1:1): (1) The frozen Lactobacillus rhamnosus LHJY, L. Weissella LHJY and their combination were 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 24 h 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 fermentation supernatant. It was stored at -20℃ for later use.

[0042] 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... 7 CFU / 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 *Lactobacillus rhamnosus* and *Westernella* supernatant and their combined supernatant, *Lactobacillus rhamnosus* and *Westernella* cells and their combined cells, uninoculated MRS liquid medium, and chlorhexidine solution to each well. After anaerobic static incubation at 37 °C for 24 h, measure the diameter of the inhibition zone. The results are accurate to 0.1 mm. Each group is repeated three times. See Table 1 for detailed results. Figure 3 A (for F. nucleatum), Table 2 and Figure 3 B (to P. gingivalis).

[0043] Table 1. Antibacterial activity of Lactobacillus rhamnosus and Weissella metabolites against F. nucleatum.

[0044]

[0045] Note: The diameter of the inhibition zone includes the outer diameter of the Oxford cup (8mm); "-" indicates no inhibition zone;

[0046] * indicates a difference of P < 0.001 between groups compared to the MRS culture medium group.

[0047] Table 2. Antibacterial activity of Weissella metabolites against P. gingivalis

[0048]

[0049] Note: The diameter of the inhibition zone includes the outer diameter of the Oxford cup (8mm); "-" indicates no inhibition zone;

[0050] * indicates a difference of P < 0.0001 between groups compared to the MRS culture medium group.

[0051] Figure 3 A represents the antibacterial activity of *Lactobacillus rhamnosus* LHJY, *Weissella vesicularis* LHJY, and their combined metabolites (fermentation supernatant, bacterial cells, and bacterial culture) against *F. nucleatum*. Figure 3 As shown in Table A and Table 1, the bacterial cells of *Lactobacillus rhamnosus* LHJY, *Westernella* LHJY, and the combined group showed no inhibition zone against the periodontal pathogen *F. nucleatum*, indicating no inhibitory effect. However, the supernatants of *Lactobacillus rhamnosus* LHJY, *Westernella* LHJY, and the combined group showed inhibition zones of 16.76±0.48 mm, 13.37±1.00 mm, and 17.40±1.02 mm against *F. nucleatum*, respectively, while the bacterial suspensions showed inhibition zones of 15.98±0.64 mm, 14.03±0.85 mm, and 18.16±0.90 mm, respectively. This indicates that both the supernatants and bacterial suspensions of *Lactobacillus rhamnosus* LHJY, *Westernella* LHJY, and the combined group had significant inhibitory effects on *F. nucleatum*, with the bacterial suspensions showing better efficacy than the supernatants. However, compared with the positive control group chlorhexidine (inhibition zone of 29.3±2.47mm), the antibacterial effect was weaker.

[0052] Figure 3 B represents the antibacterial activity of *Lactobacillus rhamnosus* LHJY, *Weissella vesca* LHJY, and their combined metabolites against *P. gingivalis*. Figure 3 As shown in B and Table 2, the bacterial cells of *Lactobacillus rhamnosus* LHJY, *Westernella* LHJY, and the combined group showed no inhibition zone against the periodontal pathogen *P. gingivalis*, indicating no inhibitory effect. However, the supernatants of *Lactobacillus rhamnosus* LHJY, *Westernella* LHJY, and the combined group showed inhibition zones of 11.96±0.24 mm, 12.61±0.43 mm, and 13.48±0.30 mm against *P. gingivalis*, while the bacterial suspensions showed inhibition zones of 13.83±0.62 mm, 14.30±0.28 mm, and 16.01±0.79 mm. This indicates that both the supernatants and bacterial suspensions of *Lactobacillus rhamnosus* LHJY, *Westernella* LHJY, and the combined group had significant inhibitory effects on *P. gingivalis*, with the bacterial suspensions showing better effects than the supernatants. Similarly, compared with the positive control group chlorhexidine (inhibition zone 27.30±2.23), the antibacterial effect was weaker.

[0053] In summary: (1) The supernatant and bacterial culture of Lactobacillus rhamnosus LHJY, L. Weissleriana, and the combined group described in this application all have significant inhibitory effects on periodontal pathogens F. nucleatum and P. gingivalis, indicating that the main antibacterial component in L. Weissleriana LHJY is in the 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) Lactobacillus rhamnosus LHJY and L. Weissleriana LHJY are probiotics that can be used in food. Although the antibacterial effects of Lactobacillus rhamnosus LHJY and L. Weissleriana LHJY are weaker than those of chlorhexidine, chlorhexidine, as an antibiotic, has solved the technical problems of antibiotics reducing the colonization of symbiotic bacteria, destroying the oral microecology, and causing bacterial resistance, thus producing significant technical effects. (3) The combined group showed better antibacterial effect than Lactobacillus rhamnosus LHJY and L. Weissl bacillus LHJY.

[0054] Example 4: Determination of biofilm content in supernatants of Lactobacillus rhamnosus LHJY, L. Weisslerella rhamnosus LHJY, and combined groups

[0055] To further determine the antibacterial effects of *Lactobacillus rhamnosus* LHJY, *Weisseria gonorrhoeae* LHJY, and the supernatant of the combined group, this example measured the amount of biofilm formed by *Fusobacterium nucleatum* and *Porphyromonas gingivalis*. The specific procedure was as follows:

[0056] Add 10 to each of the 24-well plates 7 80 µL each of CFU / mL *F. nucleatum* and *P. gingivalis* suspensions were added, followed by 160 µL of filtered *Lactobacillus rhamnosus*, *Weisseria gonorrhoeae* LHJY, and supernatant from the combined group. The mixture was anaerobically cultured at 37 °C for 48 h. The negative control group used the same volume of blank MRS liquid medium instead of the *Lactobacillus* supernatant, while the positive control group used the same volume of chlorhexidine instead of the *Lactobacillus* supernatant. After culture, 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. 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 Lactobacillus rhamnosus supernatant group was... 450nm The OD value was 0.456 for the *Westernella* LHJY supernatant group.450nm The value was 0.401, and the OD of the combined group supernatant group was... 450nm The value was 0.143. Compared with the control group, the biofilm formation in the *Lactobacillus rhamnosus* supernatant group, *Westernella* supernatant group, and combined supernatant group was significantly reduced. This indicates that the antibacterial substances in the metabolites of the *Lactobacillus rhamnosus* LHJY supernatant group, *Westernella* LHJY supernatant group, and combined supernatant group can effectively inhibit the formation of biofilms by *Fusobacterium nucleatum* and *Porphyromonas gingivalis*, with the combined group showing better results than the single-strain group (P < 0.0001).

[0057] Example 5: Lysozyme tolerance of Lactobacillus rhamnosus and LHJY

[0058] Colonization of bacteria in the oral cavity is the first step in achieving therapeutic effects. Due to the complexity of the oral environment, which contains substances such as lysozyme, beneficial bacteria can be killed while protecting the oral system from pathogens. Saliva contains a certain concentration of lysozyme, which can inhibit or even kill some bacteria, including Escherichia coli. As oral probiotic strains, they must be able to tolerate a certain concentration of lysozyme. To test the survival ability of *Lactobacillus rhamnosus* LHJY and *Weisseria gonorrhoeae* LHJY in the oral environment, this embodiment uses a spectrophotometer to measure their tolerance 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. 600nm The value is [value]. See details for the results. Figure 4 B.

[0059] Figure 4 B represents the growth density of *Lactobacillus rhamnosus* LHJY and *Weissella vesicularis* LHJY at different lysozyme concentrations. Figure 4 As shown in Figure B, when the lysozyme concentration is zero, the cell growth density OD of Lactobacillus rhamnosus and Weissella asiatica is... 450nm The values ​​were 2.356 and 2.190, respectively; with the increase of lysozyme concentration, the cell growth density OD... 450nm The values ​​gradually decreased. Specifically, when the lysozyme concentration was 2.0 mg / mL, the cell growth density OD of *Lactobacillus rhamnosus* and *Westernella* was [value missing]. 450nm The values ​​were 2.234 and 2.04, respectively; when the lysozyme concentration was 2.8 mg / mL, the cell growth density OD of Lactobacillus rhamnosus and Weissella were... 450nm The values ​​were 1.942 and 1.992, respectively. When the lysozyme concentration was 3.2 mg / mL, the cell growth density OD of *Lactobacillus rhamnosus* and *Weisseria gonorrhoeae* was... 450nmThe values ​​were 1.859 and 1.967, respectively. Therefore, it can be concluded that: (1) when the lysozyme concentration is between 0 and 2.0 mg / mL, the growth of *Lactobacillus rhamnosus* and *Westernella* LHJY is almost unaffected; (2) when the concentration is >2.8 mg / mL, although the absorbance value decreases, the maximum tolerance range is greater than 60%, which is far greater than the concentration of lysozyme in the human oral cavity (1-57 μg / mL). This indicates that *Lactobacillus rhamnosus* LHJY and *Westernella* LHJY have the ability to colonize in the oral environment and can survive and colonize well in the oral cavity. Therefore, *Lactobacillus rhamnosus* and *Westernella* LHJY may compete with pathogenic bacteria for nutrients, thereby inhibiting the growth and adhesion of pathogenic bacteria.

[0060] Example 6: Effect of the ratio of Lactobacillus rhamnosus and Weissella asiatica in the combined group on the antibacterial effect

[0061] To further determine the effect of the ratio of *Lactobacillus rhamnosus* and *Westernella* LHJY in the combined group on the antibacterial effect, this example measured the amount of biofilm formed by *Fusobacterium nucleatum* and *Porphyromonas gingivalis*. The specific operation was the same as in Example 4. In the combined group, the ratios of *Lactobacillus rhamnosus* LHJY and *Westernella* LHJY were 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, and 1:7, respectively, and the experiment was conducted according to the biofilm inhibition method. The results were consistent. *Porphyromonas gingivalis* is used as an example for further explanation. Figure 4 C). By Figure 4 As shown in C, when the mixing ratio of the two bacteria is between 1:1 and 1:4, the antibacterial effect of the combined group is significantly better than that of the single strains (see Example 4 for detailed data). This indicates that there is a synergistic effect between Lactobacillus rhamnosus and Weissella in the combined group; and the effect is best when the mixing ratio is 1:1.

[0062] Example 7: Measurement of cellular inflammatory factors

[0063] 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:

[0064] 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. 5After 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 supernatant from *Lactobacillus rhamnosus* LHJY, *Weisseria gonorrhoeae* LHJY, and a combined group (1:1 ratio) was added to each well for further culturing for 12 hours. The blank control group received no LPS or supernatant; the model group received only LPS without supernatant; and the experimental groups received LPS followed by single-strain supernatant and combined supernatant. At the end of culture, total RNA was extracted using a cell RNA extraction kit, and the concentration of the extracted RNA was measured. RNA was reverse transcribed into cDNA using the FasKing gDNA Dispelling 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... -ΔΔCt The 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 .

[0065] Figure 5 A represents the effect of *Lactobacillus rhamnosus* LHJY, *Weissella vesicularis* LHJY, and their combined group on the relative expression level of the IL-6 gene in RAW264.7 cells. 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 supernatants of Lactobacillus rhamnosus LHJY, L. Weissella salina LHJY, and the combined group. The combined group showed better results than the single-bacterial group, and the difference was statistically significant (p<0.0001). Figure 5 B represents the effect of *Lactobacillus rhamnosus* LHJY, *Weissella vesicularis* LHJY, and their combined group 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 the supernatant of Lactobacillus rhamnosus LHJY, L. Weissella salina LHJY and the combined group. The combined group was more effective than the single-bacterial group, and the difference was statistically significant (p<0.0001). Figure 5 C represents the effect of *Lactobacillus rhamnosus* LHJY, *Weissella vesicularis* LHJY, and the combined group 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 the supernatant of Lactobacillus rhamnosus LHJY, L. Weissella salina LHJY and the combined group. The combined group was more effective than the single-bacterial group, and the difference was statistically significant (p<0.0001).

[0066] In summary, *Lactobacillus rhamnosus* LHJY, *Westernella vesicularis* LHJY, and their combination all demonstrated the ability to downregulate the gene expression of pro-inflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), with the combination group showing better efficacy than the single-strain group. This indicates that *Lactobacillus rhamnosus* LHJY, *Westernella vesicularis* LHJY, and their combination have the ability to regulate related immune responses, reduce the secretion of inflammatory factors, and decrease the damage of inflammatory factors to periodontal tissues, thus playing a positive role in the treatment of periodontitis; moreover, the combination group exhibits a synergistic effect, which is better than the single-strain group. It can be inferred that after colonization in the oral cavity, *Lactobacillus rhamnosus* LHJY, *Westernella vesicularis* LHJY, and their combination inhibit the growth and adhesion of pathogenic bacteria and reduce the production of pathogenic virulence factors such as LPS; with the combination group showing the best efficacy.

[0067] Example 8: Construction of a rat experimental periodontitis model and group intervention

[0068] (1) An experimental rat periodontitis animal model was constructed using the silk ligation method.

[0069] Twenty-five male SD rats were randomly divided into four groups (n=5 per group): a blank control group (normal control group), a periodontitis group, a *Lactobacillus rhamnosus* group, a *Westernella* group, and a combined group (1:1 ratio). Rats were anesthetized with an intraperitoneal injection of 2.5% aphthylamine at a ratio 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 *Lactobacillus rhamnosus* and *Westernella* groups were inoculated daily with bacterial solution aspirated using a sterile syringe to slowly rinse the ligation sites for six weeks. Figure 6 A). Record weight changes weekly; results are detailed below. Figure 6 .

[0070] Figure 6 A shows the gingival tissue observations in animal models constructed by ligating the bilateral maxillary second molars of rats with silk ligation, including a blank group, a periodontitis group, a Lactobacillus rhamnosus group, a Weissella group, and a combined group. Figure 6 As can be seen from A, (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 Lactobacillus rhamnosus, Weissella, and the combined group, the redness and swelling of the gingiva were all reduced compared with the periodontitis group; among them, the combined group had a better effect than the single-bacterial group, with the least bleeding after probing. It can be seen that after treatment with Lactobacillus rhamnosus, Weissella, and the combined group, the clinical manifestations of periodontal tissue inflammation in rats were improved, and the combined group had a particularly significant effect.

[0071] Figure 6 B represents the changes in body weight of rats in each group 1-5 weeks after modeling. Figure 6 B shows that the probiotic group (Lactobacillus rhamnosus, Weissella vesicles, and the combined group) were basically consistent: (1) Body weight change trend: From week 1 to week 5, the body weight of rats gradually increased, the difference with the periodontitis group became larger and larger, and the difference with the blank group became smaller and smaller. (2) Weight recovery rate change trend: The weight recovery rate was significantly higher than that of the periodontitis group in the first to fourth weeks after modeling. At the end of the fifth week of modeling, the weight recovered to the control group level (no significant difference in weight, p>0.05). It can be seen that the probiotic group reduced the weight loss of rats compared with the periodontitis model group; the combined group was almost the same as the two single bacteria groups. This shows that the treatment with Lactobacillus rhamnosus LHJY, Weissella vesicles LHJY, and the combined group can alleviate the symptoms of periodontitis and increase the food intake of rats. Figure 6 C represents the change in periodontal probing depth in each group of rats. Figure 6 C shows that the periodontal probing depth was 1.008 in the *Lactobacillus rhamnosus* group, 1.014 in the *Westernella* group, and 0.894 in the combined group; while the periodontal probing depth was 1.558 in the periodontitis group and 0.306 in the normal control group without periodontitis. Therefore, the periodontal probing depth in the *Westernella* group was 35% lower than that in the periodontitis group, indicating a significant reduction in periodontal tissue damage (p<0.0001). The periodontal probing depths in the *Lactobacillus rhamnosus*, *Westernella*, and combined groups were all reduced compared to the periodontitis group, with the combined group showing the best effect (p<0.0001). Figure 6 C represents the gingival bleeding index of rats in each group. Figure 6C shows that the gingival bleeding index was 1.54 in the *Lactobacillus rhamnosus* group, 1.56 in the *Westernella* group, and 1.08 in the combined group; while the gingival bleeding index was 2.56 in the periodontitis group, and 0.306 in the normal control group (without probing bleeding). Therefore, the gingival bleeding index was 40% lower in the *Lactobacillus rhamnosus* group than in the periodontitis group, 40% lower in the *Westernella* group than in the periodontitis group, and 58% lower in the combined group than in the periodontitis group; bleeding was significantly reduced in all three groups, with the combined group showing the most significant reduction (p<0.0001). In conclusion, although the periodontal probing depth and gingival bleeding index increased in the periodontitis group and the probiotic groups (*Lactobacillus rhamnosus*, *Westernella*, and the combined group) compared to the normal control group, both indicators showed significant improvement after probiotic intervention, and the differences were statistically significant (p<0.0001). This indicates that after probiotic treatment, the overall periodontal health was significantly improved, the periodontal tissues are recovering to a healthy state, and the treatment effect is significant; moreover, the combined group is significantly more effective than the single-bacterial group.

[0072] (2) Observe and test the periodontal clinical indicators of rats in each group.

[0073] 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 . 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, while the distance in the *Lactobacillus rhamnosus* group was 0.913, the distance in the *Westernella* group was 0.857, and the distance in the combined group was 0.723. This indicates that the probiotic groups (*Lactobacillus rhamnosus* group, *Westernella* group, and combined group) were all slightly higher than the normal group but significantly lower than the periodontitis group. Figure 7 A and Figure 7As shown in Figure B, compared with the normal control group, the CEJ-ABC distance (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. After probiotic intervention, the CEJ-ABC distance in the probiotic group was significantly reduced compared with the periodontitis group, with the combined probiotic group showing the best effect and a statistically significant difference (p<0.001), indicating reduced bone resorption.

[0074] The distance of CEJ-ABC in the probiotic group (Lactobacillus rhamnosus, Weissella group, and combined group) was significantly reduced compared to the periodontitis group, with a statistically significant difference (p<0.001). Reduced bone resorption indicates that the Lactobacillus rhamnosus, Weissella group, and combined group all effectively reduced alveolar bone resorption and prevented bone loss, which also demonstrates their therapeutic effect on periodontitis from another perspective. Based on the aforementioned therapeutic effects, it can also be inferred that after colonization in the oral cavity, the Lactobacillus rhamnosus, Weissella group, and combined group inhibited the growth and adhesion of pathogenic bacteria, which corroborates the results of Example 6.

[0075] In summary, this application innovatively provides a probiotic composition for inhibiting periodontal pathogens. The probiotic composition comprises *Weissella confusa* LHJY and *Lacticaseibacillus rhamnosus* LHJY. Through the synergistic effect of these two bacteria, the probiotic composition not only inhibits important periodontal pathogens such as *Porphyromonas gingivalis* and *Fusobacterium nucleatum*, and can tolerate high concentrations of lysozyme, but also significantly reduces the expression of interleukin-1β, tumor necrosis factor-α, and interleukin-6 inflammatory factors in an inflammatory cell model. It shows significant application potential in inhibiting periodontal pathogens and regulating immune responses.

Claims

1. A probiotic composition, characterized in that: The probiotic composition includes *Weissella confusa* LHJY and *Lactobacillus rhamnosus* LHJY; the *Weissella confusa* LHJY is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251930 and accession date September 1, 2025; the *Lactobacillus rhamnosus* LHJY is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20251929 and accession date September 1, 2025; the live bacteria ratio of *Weissella confusa* LHJY and *Lactobacillus rhamnosus* in the probiotic composition is 1:1 to 1:

4.

2. A fermentation culture of a probiotic composition, characterized in that: The fermentation culture is a bacterial culture system obtained by culturing the probiotic composition of claim 1 in a microbial culture medium.

3. A fermentation supernatant of a probiotic composition, characterized in that: The fermentation supernatant is obtained by centrifuging the fermentation culture according to claim 2 to remove the bacterial cells.

4. A probiotic agent, characterized in that: It includes the probiotic composition of claim 1, the fermentation culture of claim 2, or the fermentation supernatant of claim 3.

5. The probiotic agent according to claim 4, characterized in that: The probiotic agent can be in the form of lyophilized powder and / or aqueous solution.

6. The application of the fermentation culture as described in claim 2 in the preparation of products that inhibit important periodontal pathogens, characterized in that: The important periodontal pathogens mentioned are Porphyromonas gingivalis and / or Fusobacterium nucleatum.

7. The application according to claim 6, characterized in that: The product includes a drug; the product may be in solid, liquid, gel, or aerosol form.

8. The application of the fermentation supernatant as described in claim 3 in the preparation of products that inhibit important periodontal pathogens, characterized in that: The important periodontal pathogens mentioned are Porphyromonas gingivalis and / or Fusobacterium nucleatum.

9. The application according to claim 8, characterized in that: The products include pharmaceuticals and oral care products; the products are in solid, liquid, gel, or aerosol form.

10. A pharmaceutical composition, characterized in that: The pharmaceutical composition includes the probiotic composition of claim 1, the fermentation culture of claim 2, or the fermentation supernatant of claim 3, and a pharmaceutically acceptable carrier; the dosage form of the pharmaceutical composition is tablets, capsules, oral liquid, spray, ointment, or lyophilized powder.

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