A strain of Lactobacillus plantarum and its application in the treatment of periodontitis

Abstract

The present invention provides a strain of Lactobacillus plantarum SLH. The plant lactobacillus is named as the plant lactobacillus (Lactobacillus plantarum) SLH strain, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20242817 and a deposit date of December 16, 2024. The plant lactobacillus SLH has a significant inhibitory effect on the important periodontal pathogens Porphyromonas gingivalis and Fusobacterium nucleatum, filling the gap in the prior art for the application of plant lactobacillus in the treatment of periodontitis, with significant technical effects and broad application prospects. Not only that, the plant lactobacillus described in the present application can tolerate higher concentrations of lysozyme and can colonize and survive in the oral environment. Furthermore, animal model experiments confirmed that the Lactobacillus plantarum SLH showed good effects in inhibiting periodontal pathogens and regulating immune responses, and can be used to prepare products for preventing, alleviating or treating oral diseases caused by periodontal pathogens, and has considerable market prospects and economic value.

Description

Technical Field

[0001] The present invention belongs to the field of microbiology and relates to probiotics for inhibiting oral pathogens, and particularly relates to a strain of Lactobacillus plantarum SLH and an evaluation of its therapeutic effect on experimental periodontitis. Background Art

[0002] Periodontitis is one of the most common oral diseases. It is a chronic inflammatory disease caused by periodontal pathogens, resulting in the gradual loss of periodontal supporting tissues. This not only causes inflammation of the periodontal tissues and induces alveolar bone resorption, leading to tooth loss, but can also severely impact overall health. Studies have shown that periodontitis is closely linked to the oral microbiome. These microorganisms coexist and balance each other, maintaining a balanced oral microecology. Disturbances in this microbial ecosystem can lead to periodontitis. Fusobacterium nucleatum and Porphyromonas gingivalis are important periodontal pathogens. Fusobacterium nucleatum is an aggressive, Gram-negative, anaerobic fusobacterium that is most abundant in infected sites such as supragingival and subgingival plaques and periodontal pockets. Porphyromonas gingivalis (P. gingivalis), a Gram-negative oral anaerobe, is one of the most dominant bacteria in chronic periodontitis lesions, contributing to the pathogenesis of periodontitis and is a recognized 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 commonly used approaches are mechanical therapy and drug-assisted therapy, but both have their limitations. Mechanical therapy alone cannot completely remove plaque from specific areas, such as the furcation zone, can lead to post-treatment recolonization by periodontal pathogens, and is difficult to perform. Therefore, drug therapy is often used as an adjunct. Drug therapy often uses antibiotics such as amoxicillin, cephalexin, and metronidazole to eliminate periodontal pathogens. However, antibiotics can reduce the colonization of commensal bacteria, disrupt the normal oral microbiome, and contribute to the increasing prevalence of drug resistance. Furthermore, maintaining effective drug concentrations in periodontal tissues requires systemic administration of large doses of antibiotics, which can lead to microbial imbalance, drug resistance, and the risk of hepatotoxicity and renal toxicity. Therefore, the development of safe, effective, non-invasive, and non-antibiotic treatment strategies for periodontitis is of vital importance.

[0004] Probiotics are a class of microorganisms that, when consumed in appropriate amounts, can positively impact the host's health. As a biological therapy that "treats bacteria with bacteria," probiotic therapy is widely recognized for its safety. Consequently, there is growing interest in probiotics as an alternative adjunctive treatment and prevention for periodontitis. Lactobacillus plantarum, a Gram-positive bacterium belonging to the genus Lactobacillus, can be used in food. Its optimal growth temperature is 30-37°C, it is facultatively anaerobic, and its optimal pH is approximately 6.5. Currently, Lactobacillus has been added to foods and daily necessities such as milk, yogurt, lozenges, chewing gum, toothpaste, and mouthwash. However, Lactobacillus plantarum has not yet been reported for the treatment of periodontitis. Summary of the Invention

[0005] In response to the current state of probiotic use for periodontitis treatment, the present invention provides a strain of Lactobacillus plantarum SLH. This Lactobacillus plantarum has inhibitory effects on the important periodontal pathogens Porphyromonas gingivalis and Fusobacterium nucleatum, and can tolerate high concentrations of lysozyme. This strain has significant potential for inhibiting periodontal pathogens and regulating immune responses.

[0006] The technical solution of the present invention:

[0007] A strain of Lactobacillus plantarum that inhibits periodontal pathogens is named Lactobacillus plantarum SLH, deposited with the China Center for Type Culture Collection with a deposit number of CCTCCNO: M 20242817 and a deposit date of December 16, 2024. The inventors isolated and purified the probiotic Lactobacillus plantarum SLH from fecal samples of healthy infants. Antibacterial and biofilm removal experiments revealed that the Lactobacillus plantarum SLH exhibited significant inhibitory effects against the important periodontal pathogens Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum), filling a gap in the prior art for the application of Lactobacillus plantarum in the treatment of periodontitis. The invention has significant technical effects and broad application prospects.

[0008] A culture of Lactobacillus plantarum, wherein the culture is a system obtained by culturing the aforementioned Lactobacillus plantarum SLH in a microbial culture medium; or a supernatant obtained by further solid-liquid separation thereof.

[0009] A bacterial agent comprises the aforementioned Lactobacillus plantarum SLH or culture.

[0010] The use of Lactobacillus plantarum SLH or culture as described above in the preparation of products for preventing, alleviating or treating oral diseases caused by periodontal pathogens. Wherein, the periodontal pathogens are Porphyromonas gingivalis (P.gingivalis) and / or Fusobacterium nucleatum (F.nucleatum); the oral disease is periodontitis; the products include medicines, oral care products, feed and probiotics. The inventors found through animal model experiments that the Lactobacillus plantarum SLH can increase the weight of rats, reduce periodontal probing depth, gingival bleeding index and degree of inflammatory infiltration, and Micro-CT results showed that the amount of bone resorption was reduced. This shows that the Lactobacillus plantarum SLH shows good effects in inhibiting periodontal pathogens and regulating immune responses, and can be used to prepare products for preventing, alleviating or treating oral diseases caused by periodontal pathogens, and has considerable market prospects and economic value.

[0011] Preferably, the product may be in solid form, liquid form, semi-solid form, gel, aerosol form, or the like.

[0012] A pharmaceutical composition comprises the aforementioned Lactobacillus plantarum SLH or culture and a pharmaceutically acceptable carrier.

[0013] Preferably, the pharmaceutical composition is in various pharmaceutically acceptable dosage forms, such as tablets, capsules, oral liquids, sprays, ointments or freeze-dried powders.

[0014] Beneficial effects of the present invention:

[0015] (1) This application pioneered the provision of a strain of Lactobacillus plantarum SLH that can inhibit periodontal pathogens, filling the gap in the prior art in the application of Lactobacillus plantarum in the treatment of periodontitis, possessing outstanding substantive characteristics and achieving significant progress.

[0016] (2) The plant lactobacillus SLH described in the present application not only has a significant inhibitory effect on important periodontal pathogens (Porphyromonas gingivalis and Fusobacterium nucleatum), but also can tolerate a higher concentration (3 mg / mL) of lysozyme and can colonize and survive in the oral environment.

[0017] (3) The Lactobacillus plantarum SLH described in the present application can also reduce the expression of interleukin-1β, tumor necrosis factor α and interleukin-6 inflammatory factors in the inflammatory cell model, indicating that Lactobacillus plantarum SLH may have the effect of regulating related immune responses and reducing the damage of inflammatory responses to periodontal tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Attachment Figure 1Morphological observation and 16S rDNA identification results of the strain. (A) Colony morphology of L. plantarum SLH; (B) Gram staining (10×100) of L. plantarum SLH under oil immersion microscope. (C) Phylogenetic tree of the 16S rDNA of L. plantarum SLH.

[0019] Attachment Figure 2 The growth curves and acid production capacity of the strains. (A) Growth curve of L. plantarum SLH; (B) Acid production curve of L. plantarum SLH.

[0020] Attachment Figure 3 Antibacterial activity assay of L. plantarum SLH and its metabolites. Ingredients: ① Chlorhexidine ② L. plantarum SLH supernatant ③ MRS medium ④ L. plantarum SLH cells. (A) Antibacterial activity of L. plantarum SLH and its metabolites against F. nucleatum; (B) Antibacterial activity of L. plantarum SLH and its metabolites against P. gingivalis.

[0021] Attachment Figure 4 The biofilm formation capacity was measured and the lysozyme tolerance was evaluated. (A) Determination of the biofilm formation capacity of dental plaque; (B) Growth density of L. plantarum SLH at different lysozyme concentrations.

[0022] Attachment Figure 5 The effect of L. plantarum SLH on the expression of inflammatory factors in cells. (A) Effect of L. plantarum SLH on the relative expression of IL-6 gene in RAW264.7 cells; (B) Effect of L. plantarum SLH on the relative expression of IL-1β gene in RAW264.7 cells; (C) Effect of L. plantarum SLH on the relative expression of TNF-α gene in RAW264.7 cells.

[0023] Attachment Figure 6 Figure 2: Experimental periodontitis model construction and clinical index testing results. (A) An experimental periodontitis model was established by silk thread ligature on the right maxillary second molar of rats; (B) Gross observation of gingival tissue in the normal control group; (C) Gross observation of gingival tissue in the periodontitis model group; (D) Gross observation of gingival tissue after Lactobacillus plantarum treatment; (E) Body weight changes of rats in each group 1-8 weeks after modeling; (F) Changes in gingival bleeding index and periodontal probing depth of rats in each group.

[0024] Attachment Figure 7Periodontal histological evaluation and alveolar bone Micro-CT analysis of rats in each group. (A) HE staining results; (B) Micro-CT analysis of alveolar bone. (C) CEJ-ABC (cementoenamel junction-alveolar crest) distance analysis. DETAILED DESCRIPTION

[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] Healthy infant fecal samples collected from clinic were dissolved in PBS and diluted to 10 -4 After quantification, 100 μL of each diluted stool sample was evenly spread onto a CaCO₃MRS solid plate in a sterile environment. The plate was inverted and incubated at 37°C for 48-72 hours. Single colonies with lactic acid bacteria were selected based on their morphological characteristics and the size of the clearing zone. They were continuously transferred to MRS solid plates until pure colonies were observed under microscopic examination. The purified strains were inoculated into MRS broth and incubated at 37°C for 24 hours. They were then stored in cryovials with 20% glycerol by volume and stored at -80°C until further use.

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

[0030] First, morphological observation was performed: the strain isolated and purified in step (1) was inoculated on MRS solid medium for streak culture, and the colony morphology was observed ( Figure 1 A), and pick a single colony for Gram staining and microscopic observation ( Figure 1 B) By Figure 1 As shown in A, the colony of L. plantarum SLH is milky white, round, 3-4 mm in diameter, convex in the middle, and smooth and dense on the surface. Figure 1 B shows that the Gram staining (10×100) of L. plantarum SLH is blue-purple under oil microscope, which is G + Bacillus.

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

[0032] The PCR products were subjected to 1% agarose gel electrophoresis and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequence was aligned with the known sequences in the NCBI database, and the 16srDNA phylogenetic tree of the strain L. plantarum SLH was constructed ( Figure 1 C) By Figure 1 As shown in C, the isolated strain formed an independent branch in the evolutionary tree and was closely related to Lactobacillus Plantarum (OQ410491). Therefore, the strain was named Lactobacillus plantarum SLH (L. plantarum SLH). It was deposited with the China Center for Type Culture Collection with the deposit number CCTCC NO: M 20242817 and the deposit date was December 16, 2024.

[0033] Example 2: Determination of L. plantarum SLH Growth Curve and pH Value

[0034] The test strain L. plantarum SLH was inoculated into MRS broth medium and cultured at 37°C for 12 h. The fermentation strain was inoculated into MRS broth medium at a 1% inoculum and cultured at 37°C in a shaker for 24 h. Samples were taken every 2 h to measure the pH value and the OD was measured using a UV spectrophotometer. 600 Values, and make growth curves ( Figure 2 A) and acid production curve ( Figure 2 B) By Figure 2From A, we can see that the strain is in the growth retardation phase from 0 to 4 hours, with a slow growth trend; from 4 to 12 hours, it is in the growth logarithmic phase, with the growth curve rising rapidly; after 12 hours, it is in the stable phase, with the OD value tending to be stable. Figure 2 As shown in Figure B, the strain produced less acid and had a smaller pH drop between 0 and 4 hours. As the strain entered the logarithmic growth phase, it began to secrete large amounts of acidic substances, causing a sharp drop in pH. Once the strain reached the stationary phase, the pH also stabilized. This suggests that after 12 hours of culture, the strain's growth and metabolic activity reached a stable state, which is beneficial for fermentation process control and product quality stability.

[0035] Example 3: Antibacterial activity of L. plantarum SLH and its metabolites

[0036] Periodontitis is a mixed infection caused by multiple microorganisms, of which Fusobacterium nucleatum (F. nucleatum) and Porphyromonas gingivalis (P. gingivalis) are important periodontal pathogens. This example uses the Oxford cup diffusion method to detect the antibacterial activity of L. plantarum SLH and its metabolites against F. nucleatum and P. gingivalis. The specific procedures are as follows:

[0037] Preparation of L. plantarum SLH fermentation supernatant: Frozen Lactobacillus plantarum was streaked onto MRS solid medium for the second generation. A single colony was selected and inoculated into MRS liquid medium. The culture was incubated at 37°C for 24 hours to obtain a fermentation culture. The fermentation culture was centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was filtered through a 0.22 μm sterile filter to obtain the cell-free supernatant, which was the L. plantarum SLH fermentation supernatant and stored at -20°C until needed.

[0038] F. nucleatum and P. gingivalis stored in 30% glycerol were streaked on BHI solid medium for two generations, and single colonies were picked and inoculated into BHI liquid medium and cultured at 37°C under anaerobic conditions for 48 hours. The concentration of indicator bacteria suspension was adjusted to 10 7 CFU / mL. Pour 10mL of 1.5% plain agar into the plate, place 4 high-temperature sterilized Oxford cups at equal intervals after solidification, and then add BHI solid culture medium containing 5% indicator bacteria suspension; after the culture medium solidifies, remove the Oxford cup, add 150μL of L. plantarum SLH supernatant, L. plantarum SLH bacteria, uninoculated MRS liquid culture medium and chlorhexidine solution into the wells respectively, and measure the diameter of the inhibition zone after anaerobic culture at 37℃ for 24h. The results are accurate to 0.1mm, and three repeated experiments are performed for each group. The results are shown in Table 1 and Figure 3A (for F. nucleatum), Table 2 and Figure 3 B (for P.gingivalis).

[0039] Table 1 Antibacterial activity of L. plantarum SLH cells and their supernatants against F. nucleatum

[0040]

[0041] Note: The diameter of the inhibition zone includes the outer diameter of the Oxford cup (8 mm); “-” indicates no inhibition zone; * indicates the difference between the two groups compared with the MRS medium group is P < 0.001.

[0042] Table 2 Antibacterial activity of L. plantarum SLH cells and their supernatants against P. gingivalis

[0043]

[0044] Note: The diameter of the inhibition zone includes the outer diameter of the Oxford cup (8 mm); “-” indicates no inhibition zone; * indicates the difference between the two groups compared with the MRS medium group is P < 0.0001.

[0045] Figure 3 A is the antibacterial activity of L. plantarum SLH and its metabolites against F. nucleatum. Figure 3 As shown in Table 1, L. plantarum SLH cells showed no inhibition zone against the periodontal pathogen F. nucleatum, indicating no inhibitory effect. However, the L. plantarum SLH supernatant exhibited a significant inhibitory effect on F. nucleatum, with a zone of inhibition of 26.20 ± 1.98 mm. However, this inhibition was weaker than that of the positive control group containing chlorhexidine (zone of inhibition of 32.43 ± 2.53 mm).

[0046] Figure 3 B is the antibacterial activity of L. plantarum SLH and its metabolites against P. gingivalis. Figure 3 As shown in Table 2 and Table 3, L. plantarum SLH cells also showed no inhibition zone against the periodontal pathogen P. gingivalis, indicating no inhibitory effect. However, the L. plantarum SLH supernatant had an inhibition zone of 31.77 ± 2.63 mm against P. gingivalis, demonstrating a significant inhibitory effect. Similarly, compared with the positive control group of chlorhexidine (inhibition zone of 35.75 ± 1.23), the inhibitory effect was weaker.

[0047] In summary, it can be seen that: (1) The supernatant of Lactobacillus plantarum SLH described in the present application has a significant inhibitory effect on periodontal pathogens F. nucleatum and P. gingivalis, indicating that the component that produces the main antibacterial effect in L. plantarum SLH is in the metabolites. The inventors speculate that the reason why the bacteria do not show antibacterial activity is related to its colonization ability, which needs to be manifested in the body. (2) Lactobacillus plantarum SLH is a probiotic that can be used in food, and chlorhexidine is an antibiotic drug. Although the antibacterial effect of Lactobacillus plantarum is weaker than that of chlorhexidine, it also solves the technical problems of antibiotics reducing the colonization of symbiotic bacteria, destroying the oral flora microecology, and causing bacterial resistance, and produces significant technical effects.

[0048] Example 4: Biofilm Amount Determination

[0049] To further determine the antibacterial effect of the Lactobacillus plantarum SLH supernatant, this example measured the amount of biofilm formed by Fusobacterium nucleatum and Porphyromonas gingivalis. The specific operation was as follows:

[0050] 10 were added to each well of a 24-well plate. 7 80 μL of each F.nucleatum and P.gingivalis bacterial suspension at CFU / mL was added, and then 160 μL of filtered L.plantarum SLH supernatant was added and cultured anaerobically at 37°C for 48 hours. The negative control group used the same volume of blank MRS liquid culture medium instead of the Lactobacillus supernatant, and the positive control group used the same volume of chlorhexidine instead of the Lactobacillus supernatant. After the culture was completed, the biofilm was washed twice with PBS and fixed with 99% methanol for 15 minutes, dried at room temperature for 10 minutes, and allowed to stand at room temperature to dry. 500 μL of 0.1% crystal violet solution was added to each well to stain the biofilm for 30 minutes. After staining, it was washed twice with PBS and photographed. Then 100 μL of 95% ethanol was added to dissolve it, and the plate was placed on a shaker for 30 minutes, and the OD was read. 600 The results are shown in Figure 4 A.

[0051] Depend on Figure 4 A shows that the OD of the negative control group 600 The value was 3.25±0.103, and the OD of the positive control group (chlorhexidine group) 600 The value was 0.513±0.013; while the OD of the supernatant of L. plantarum SLH group was 600The value was 1.461±0.046, which was significantly lower than that of the negative control group, but higher than that of the positive control group. It can be seen from this that: (1) Compared with the negative control group, the antibacterial substances in the metabolites of the supernatant of L. plantarum SLH can effectively inhibit the formation of the dual biofilm of Fusobacterium nucleatum and Porphyromonas gingivalis (P < 0.0001); (2) Compared with the positive control group (chlorhexidine), the antibacterial effect on periodontal pathogens is weaker, which is consistent with the test results of Example 3 (P < 0.0001). It shows that the supernatant of L. plantarum SLH has a significant inhibitory effect on the formation of the dual biofilm of Fusobacterium nucleatum and Porphyromonas gingivalis.

[0052] Example 5: Detection of lysozyme tolerance

[0053] The colonization of bacteria in the oral cavity is the first step to exert therapeutic effects. Due to the complexity of the oral environment, the oral cavity contains substances such as lysozyme, which can kill beneficial bacteria while protecting the oral system from pathogenic bacteria. The saliva in the mouth contains a certain concentration of lysozyme, which can inhibit or even kill some bacteria including Escherichia coli. As an oral probiotic strain, it must be able to tolerate a certain concentration of lysozyme. In order to test the viability of Lactobacillus plantarum SLH in the oral environment, this embodiment uses a spectrophotometer to measure the tolerance of L. plantarum SLH to lysozyme. The specific operation is: inoculate the bacterial suspension into a 96-well plate, and add different mass concentrations of lysozyme so that the final concentration of each well is 0-3 mg / ml. Measure the OD after culturing at 37°C for 24 hours. 600 The results are shown in Figure 4 B.

[0054] Figure 4 B is the growth density of L. plantarum SLH under different lysozyme concentrations. Figure 4 B can be seen that when the lysozyme concentration is zero, the bacterial growth density OD 600 The value was 1.811±0.001; with the increase of lysozyme concentration, the bacterial growth density OD 600 The value gradually decreased. Among them, when the lysozyme concentration was 2.0 mg / mL, the bacterial growth density OD 600 was 1.53±0.030; when the lysozyme concentration was 2.4 mg / mL, the bacterial growth density OD 600 When the lysozyme concentration was 3.0 mg / mL, the bacterial growth density OD 600The results show that: (1) when the lysozyme concentration is between 0 and 2.0 mg / mL, the growth of L. plantarum SLH is almost unaffected; (2) when the concentration is greater than 2.4 mg / mL, the absorbance value decreases, but the maximum tolerance range is greater than 60%, which is much greater than the human oral lysozyme concentration (1 to 57 μg / mL). This shows that L. plantarum SLH has the ability to colonize in the oral environment and can survive and colonize well in the oral cavity. Therefore, L. plantarum SLH may compete with pathogens for nutrients, thereby inhibiting the growth and adhesion of pathogens.

[0055] Example 6: Determination of cellular inflammatory factors

[0056] In this example, a RAW264.7 cell inflammation model was constructed to detect the effect of L. plantarum SLH on the gene expression of the pro-inflammatory factors interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6). The specific operation was as follows:

[0057] The activated RAW264.7 cells were evenly plated in a 6-well plate, and 2 ml was added to each well at a concentration of 2×10 5 / ml, after culturing for 2 hours, lipopolysaccharide (LPS) at a concentration of 1μg / ml was added to induce an inflammatory cell model. At the same time, 60μL L.plantarum SLH supernatant was added to each well and cultured for 12 hours. The blank control group did not add LPS and L.plantarum SLH supernatant; the model group only added LPS without L.plantarum SLH supernatant. After the culture was completed, the total cell RNA was extracted using a cell RNA extraction kit and the concentration of the extracted RNA was determined. The RNA was reverse transcribed into cDNA using the Tiangen FasKing gDNA Dispelling RT SuperMix Reverse Transcription Kit (KR118, Tiangen Biochemical Technology Co., Ltd., Beijing), and then the amplification reaction was performed using the 2×HQSYBR qPCR Mix (No Rox) quantitative kit. β-actin was used as an internal reference and 2 -ΔΔCt The mRNA expressions of interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), and interleukin-6 (IL-6) were detected by ELISA. Figure 5 .

[0058] Figure 5 A shows the effect of L. plantarum SLH on the relative expression of IL-6 gene in RAW264.7 cells. Figure 5A shows that compared with the inflammation model group, the expression of inflammatory factors after treatment with L. plantarum SLH supernatant was significantly downregulated, and there was a significant statistical difference (p<0.0001). Figure 5 B shows the effect of L. plantarum SLH on the relative expression of IL-1β gene in RAW264.7 cells. Figure 5 B shows that compared with the inflammation model group, the expression of inflammatory factors after treatment with L. plantarum SLH supernatant was significantly downregulated, and there was a significant statistical difference (p<0.0001). Figure 5 C is the effect of L. plantarum SLH on the relative expression of TNF-α gene in RAW264.7 cells. Figure 5 C shows that compared with the inflammation model group, the expression of inflammatory factors after treatment with L. plantarum SLH supernatant was significantly downregulated, and there was a significant statistical difference (p<0.0001).

[0059] In summary, L. plantarum SLH has the ability to downregulate the gene expression of proinflammatory factors interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6). This indicates that L. plantarum SLH has the ability to regulate related immune responses, reduce the secretion of inflammatory factors, and reduce the damage of inflammatory factors to periodontal tissues, and has a positive effect on the treatment of periodontitis. It can be inferred that after L. plantarum SLH is colonized in the oral cavity, it inhibits the growth and adhesion of pathogens and reduces the production of pathogenic virulence factors such as LPS.

[0060] Example 7: Construction of experimental periodontitis model in rats and group intervention

[0061] (1) An experimental rat periodontitis model was established using the silk thread ligation method.

[0062] Twenty-four male SD rats were randomly divided into a normal control group, a periodontitis group, and a Lactobacillus plantarum group according to a random number table, with 8 rats in each group. The rats were anesthetized with an intraperitoneal injection of 2.5% Avertin (1.2 mL / 100 g). The gingivae of the second molar mesiodistal to the mesiodistal space were separated using a dental probe. A 4-0 silk suture was placed in the mesiodiscal space, and the suture was tied into the gingival sulcus as much as possible. The suture was then fixed at the cervical region of the right maxillary second molar ( Figure 6 A). Check the ligature thread every 2 days to see if it has fallen off. One week after modeling, the Lactobacillus plantarum group was slowly injected into the ligature periodontal pocket with a sterile syringe every day for 7 weeks. Figure 6 C) and record the changes in body weight every week. The results are shown in Table 3 and Figure 6 .

[0063] Figure 6 A: An experimental periodontitis model was established by ligating the right maxillary second molar of rats with silk thread. Figure 6 B is the general observation of gingival tissue in the normal control group. Figure 6 B shows that the gums are pink, with thin edges that adhere closely to the tooth surface and a dense and tough texture. Figure 6 C is the general observation of gingival tissue in the periodontitis model group. Figure 6 C shows that after the modeling was completed, the gums of the rats in the periodontitis group were red and swollen, with a soft texture, thickened edges, and bleeding when lightly probed. Figure 6 D is the gross observation of gingival tissue after Lactobacillus plantarum treatment. Figure 6 As can be seen in D, the redness and swelling of the gums were less severe than in the periodontitis group. This indicates that after treatment with Lactobacillus plantarum SLH, the clinical manifestations of periodontal inflammation in rats were improved, and the treatment effect was significant.

[0064] Figure 6 E is the weight change of rats in each group from 1 to 8 weeks after model establishment. Figure 6 E As can be seen, the L. plantarum SLH intervention group reduced weight loss compared to the periodontitis model group, indicating that L. plantarum SLH treatment can alleviate periodontitis symptoms and increase food intake in rats. This indicates that the L. plantarum intervention group had a significantly higher rate of weight recovery than the periodontitis group after the fourth week, returning to the control group level by the end of modeling (no significant difference in weight, p>0.05). Figure 6 F represents the changes in the gingival bleeding index and periodontal probing depth of rats in each group (plotted based on the data in Table 3). As shown in Table 3, the gingival bleeding index of the Lactobacillus plantarum group was 1.160±0.183, and the periodontal probing depth was 0.945±0.094, while the gingival bleeding index of the periodontitis group was 3.500±0.245, and the periodontal probing depth was 1.420±0.072. The gingival bleeding index of the control group was 0.333±0.111, and the periodontal probing depth was 0.298±0.013. This indicates that the normal control group had no periodontal pockets or bleeding on probing, while the periodontitis group and the Lactobacillus plantarum group had increased periodontal probing depth and gingival bleeding index compared with the normal control group, and the differences were statistically significant (p<0.0001). However, the periodontal probing depth and gingival bleeding in the Lactobacillus plantarum group were reduced compared with the periodontitis group, and the differences were statistically significant (p<0.0001). This shows that after treatment with Lactobacillus plantarum SLH, the overall periodontal health has been significantly improved, the periodontal tissue is recovering to a healthy state, and the treatment effect is significant.

[0065] Table 3 Gingival bleeding index and periodontal probing depth of rats in each group after modeling

[0066]

[0067] Note: * indicates that the gingival bleeding index and periodontal probing depth of the periodontitis group were different from those of the Lactobacillus plantarum group, with differences of P < 0.001.

[0068] (2) Observe and detect the periodontal clinical indicators of rats in each group.

[0069] After modeling, rats were anesthetized with an intraperitoneal injection of 2.5% avertin (1.2 mL / 100 g) and then sacrificed. Hematoxylin-eosin (HE) staining was performed, and right maxillary bone samples were isolated for Micro-CT analysis. Gingival tissue from the second molar was collected for periodontal histopathological evaluation. The specific results included observation and testing of periodontal clinical indicators, inflammatory infiltration, and bone resorption in each group of rats. Figure 7 .

[0070] Figure 7 A is the result of hematoxylin-eosin (HE) staining of gingival tissue. Figure 7 A shows that compared with the normal control group, the periodontitis group had thickening of the gingival epithelium, hyperplasia of the spinous layer, elongation of the epithelial pegs, destruction and disorder of the reticular fiber connective tissue, hyperplasia of blood vessels, and infiltration of inflammatory cells around the teeth; compared with the periodontitis group, the inflammation of the gingival epithelium in the Lactobacillus plantarum group was milder, the degree of inflammatory cell infiltration was reduced, the reticular fiber structure was less damaged, and the treatment effect was significant.

[0071] Figure 7 B is the micro-computed tomography (Micro-CT) results of the maxillary alveolar bones of rats in each group. Figure 7 The red line in B represents the distance from the cementoenamel junction to the alveolar ridge. The longer the red line, the longer the distance, and the greater the amount of bone absorption in the alveolar bone. Figure 7 C is the distance analysis of CEJ-ABC (cementoenamel junction-alveolar crest). Figure 7 C shows that the distance between CEJ-ABC in the normal control group was 0.707±0.060, that in the periodontitis group was 1.157±0.032, and that in the Lactobacillus plantarum group was 0.853±0.032, which was slightly higher than that in the normal group but significantly lower than that in the periodontitis group. Figure 7 B and Figure 7C shows that, compared with the normal control group, the distance between CEJ-ABC (cementum enamel junction-alveolar ridge) in the periodontitis group increased significantly, and the difference was statistically significant (p < 0.0001), and bone resorption was obvious, indicating that the rat experimental periodontitis model was successfully constructed. The distance between CEJ-ABC in the plantarum group and the periodontitis group was significantly reduced, and the difference was statistically significant (p < 0.001), and bone resorption was reduced, indicating that plantarum Lactobacillus SLH effectively reduced the absorption of alveolar bone and prevented the loss of bone, which also embodied its therapeutic effect on periodontitis from another angle. According to the aforementioned therapeutic effect, it can be inferred that L.plantarum SLH inhibited the growth and adhesion of pathogens after oral colonization, which is confirmed by the results of Example 6.

[0072] In summary, the present application provides a probiotic Lactobacillus plantarum SLH that can be used for the treatment of periodontitis. This is because: first, it has a significant inhibitory effect on periodontal pathogens Fusobacterium nucleatum and Porphyromonas gingivalis; secondly, Lactobacillus plantarum SLH has the ability to regulate related immune responses, reduce the secretion of inflammatory factors, and reduce the damage of inflammatory factors to periodontal tissues; third, Lactobacillus plantarum SLH effectively reduces the absorption of alveolar bone and prevents bone loss. Not only that, the Lactobacillus plantarum SLH tolerates lysozyme at a higher concentration (3mg / mL) and can colonize and survive in the oral environment. Based on the foregoing reasons, the probiotic Lactobacillus plantarum SLH fills the gap in the prior art for the treatment of periodontitis by Lactobacillus plantarum, has outstanding substantive characteristics, and has achieved significant progress.

Claims

1. A strain of Lactobacillus plantarum that inhibits periodontal pathogens, characterized by: The plant lactobacillus is named plant lactobacillus ( Lactobacillus plantarum ) SLH strain, deposited in China Center for Type Culture Collection, with the deposit number CCTCC NO: M 20242817 and the deposit date being December 16, 2024.

2. A fermentation culture of Lactobacillus plantarum, characterized in that: The culture is a system obtained by culturing the plant lactobacillus according to claim 1 in a microbial culture medium.

3. A bacterial agent, characterized in that: The method comprises the plant lactobacillus according to claim 1 or the culture according to claim 2.

4. Use of the plantarum Lactobacillus according to claim 1 or the culture according to claim 2 in the preparation of a product for inhibiting important periodontal pathogens; the important periodontal pathogens are Porphyromonas gingivalis ( Porphyromonas gingivalis ) and / or Fusobacterium nucleatum ( Fusobacterium nucleatum ).

5. The use according to claim 4, characterized in that: The products include pharmaceuticals, oral care products and feed.

6. The use according to claim 4, characterized in that: The product is in solid form, liquid form, semi-solid form, gel or aerosol form.

7. A pharmaceutical composition, characterized in that: The method comprises the plant lactobacillus according to claim 1 or the culture according to claim 2 and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that: The pharmaceutical composition adopts various pharmaceutically acceptable dosage forms, such as tablets, capsules, oral liquids, sprays, ointments or freeze-dried powders.

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