Lactobacillus mucilaginosus CQPC202403 and application thereof in preparation of oral preparation for improving periodontitis

Abstract

The invention relates to the technical field of biology, and discloses fermented lactobacillus mucus CQPC202403 and application of the fermented lactobacillus mucus CQPC202403 in preparation of an oral preparation for improving periodontitis, the preservation number of the fermented lactobacillus mucus CQPC202403 in the China Center for Type Culture Collection is CGMCC NO.31106, and the preservation date is June 27, 2024. The fermented lactobacillus mucus CQPC202403 disclosed by the invention can be used for improving the intestinal flora structure, so that the fermented lactobacillus mucus CQPC202403 has a treatment effect on periodontitis. As a probiotic preparation, the traditional Chinese medicine composition can further avoid the problem of drug resistance caused by long-term use of antibiotics, and pathological sections show that the traditional Chinese medicine composition can reduce periodontal tissue inflammatory cell infiltration and promote regular arrangement of collagenous fibers and alveolar bone neogenesis.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to fermented Lactobacillus mucinus CQPC202403 and its application in the preparation of oral preparations for improving periodontitis. Background Technology

[0002] Periodontitis is a common, chronic, and destructive oral disease, a complex chronic inflammatory disease of the periodontal tissues caused by Gram-negative bacterial plaque. The main clinical manifestations of periodontitis include gingival bleeding, periodontal pockets, alveolar bone resorption, and tooth mobility. Periodontal pathogens not only cause irreversible damage such as destruction of periodontal supporting tissues and tooth loosening and loss, but can also have systemic effects through various mechanisms, such as bacteremia caused by the transfer of periodontal pathogens into the systemic circulation, and endotoxemia caused by the lipopolysaccharides of periodontal pathogens. Furthermore, interleukins produced due to chronic inflammation are closely related to various systemic diseases such as inflammatory bowel disease, diabetes, atherosclerotic vascular diseases, and cardiovascular and cerebrovascular diseases. With socioeconomic development, the prevalence of periodontitis in children and adults is constantly increasing, and the periodontal health of middle-aged and elderly people is worrying. With further population aging, the disease and socioeconomic burden caused by periodontal disease may become increasingly serious. Currently, the standard treatment for chronic periodontitis is mechanical debridement of periodontal pockets, while antibiotics are used as routine adjunctive therapy to remove periodontal pathogens and promote tissue recovery. Although antibiotics have been widely used as a traditional adjunctive treatment, the increasing bacterial resistance to antibiotics and their limited effectiveness have led clinicians and researchers to search for feasible alternative treatments.

[0003] Metagenomic studies have shown that the periodontal microbiota may be involved in the disease process. Dysbiosis in dental plaque can trigger inflammation and immune responses, ultimately leading to direct or indirect damage to periodontal tissues by periodontal pathogens and virulence factors. *Porphyromonas gingivalis*, *Treponema denticulata*, *Daniloxylon ammodendron*, and *Actinomyces actinomycetes* are all considered major pathogenic players in periodontitis. The frequent re-establishment of periodontal pathogens at treatment sites and the emergence of antibiotic resistance have led to calls for new treatment methods to manage periodontal disease. One recently popular approach is probiotics as an alternative biological therapy that can modify the periodontal plaque biofilm and help control periodontitis.

[0004] Probiotics are live microorganisms that, when given in sufficient quantities, provide health benefits to the host. The most common probiotic genera are *Lactobacillus* and *Bifidobacterium*. In dentistry, probiotics have been used as a useful adjunct in reducing tooth decay, inhibiting oral candidiasis, and controlling halitosis. Recent studies have demonstrated the potential benefits of probiotics in managing periodontal disease, particularly periodontitis. Evidence suggests that *Lactobacillus reuteri*, *Lactobacillus salivarius*, *Lactobacillus brevis*, and *Lactobacillus rhamnosus* can all alleviate the inflammatory response of periodontitis to varying degrees and reduce tissue damage. *Lactobacillus acidophilus* and *Lactobacillus rhamnosus* are associated with antibacterial and antifungal effects, while *Lactobacillus reuteri* and *Lactobacillus salivarius* can simultaneously inhibit cariogenic and periodontal pathogens. However, research on the effects of *Lactobacillus casei* in preventing and regulating periodontitis is relatively limited. Furthermore, no more effective treatments have been found for periodontitis induced by a high-sugar, high-viscosity diet. Summary of the Invention

[0005] In view of this, the present invention proposes a fermented Lactobacillus mucinus CQPC202403 and its application in the preparation of oral preparations for improving periodontitis, aiming to solve the problem that current technologies cannot effectively treat periodontitis caused by high-sugar and high-viscosity diets.

[0006] This invention proposes a fermentation Lactobacillus mucinus CQPC202403, which has the accession number CGMCC NO.31106 at the China General Microbiological Culture Collection Center and the accession date is June 27, 2024.

[0007] The present invention also provides the application of the aforementioned *Lactobacillus fermentatus* CQPC202403 in the preparation of oral preparations for improving periodontitis. The application method is to prepare *Lactobacillus fermentatus* CQPC202403 into a bacterial agent and then add it to the oral preparation for improving periodontitis.

[0008] Preferably, the preparation method of the fermented Lactobacillus mucinus CQPC202403 inoculum includes the following steps: 1) The bacterial strain is activated and then cultured to obtain a bacterial solution; 2) Centrifuge the bacterial solution to obtain bacterial precipitate; 3) The bacterial precipitate was resuspended in a freeze-drying protectant, and an encapsulating agent and excipients were added to obtain bacterial slurry; 4) The bacterial slurry was freeze-dried to obtain fermented Lactobacillus mucinus CQPC202403 bacterial agent.

[0009] Preferably, the oral preparation for improving periodontitis can be a mouthwash, gel, oral liquid, capsule, tablet, dispersant or granule.

[0010] Preferably, the freeze-drying protectant is an aqueous solution containing trehalose and glycerol; The lyophilization protectant contains 5-10% trehalose and 3-7% glycerol by mass. The mass ratio of the bacterial precipitate to the freeze-drying protectant is 1:3~5.

[0011] Preferably, the encapsulating agent is chitosan and the excipient is anhydrous lactose.

[0012] Preferably, the amount of the encapsulating agent added is 0.5-3% of the total amount of the lyophilization protectant; The amount of excipient added is 1-5% of the total amount of freeze-drying protectant.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: The fermented Lactobacillus mucinus CQPC202403 described in this invention can improve the intestinal flora structure, thereby producing a therapeutic effect on periodontitis. As a probiotic preparation, it can further avoid the drug resistance problem caused by long-term use of antibiotics. Pathological sections show that it can reduce the infiltration of inflammatory cells in periodontal tissues and promote the orderly arrangement of collagen fibers and alveolar bone regeneration. Attached Figure Description

[0014] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 This is a statistical chart showing the serum levels of inflammatory factors IL-6, IL-12, TNF-α, IFN-γ, and IL-10 in mice after the experiment, including normal control group, periodontitis model group, positive control group after oral antibiotic administration, experimental group after oral high-concentration bacterial solution administration, and experimental group after oral low-concentration bacterial solution administration. Figure 2 HE-stained bone tissue sections from mice in the normal control group, periodontitis model group, antibiotic-positive control group, high-concentration bacterial solution experimental group, and low-concentration bacterial solution experimental group after the experiment. Figure 2 In the figure, A represents a 2000x magnified histopathological section of the maxilla of each group of mice, stained with hematoxylin and eosin (HE). Figure 2 In the image, B represents a 5000x magnified HE-stained pathological section of the maxillae of each group of mice. Figure 2 In the image, C represents a 2000x magnified HE-stained pathological section of the mandible of each group of mice. Figure 2 D in the image represents a HE staining image of a bone tissue section of the mandible of each group of mice magnified 5000 times. Figure 3The images show the bone tissue analysis results of mice after the experiment, including the normal control group, the periodontitis model group, the positive control group treated with antibiotics by gavage, the experimental group treated with high-concentration bacterial solution by gavage, and the experimental group treated with low-concentration bacterial solution by gavage. Figure 3 In the image, A represents a Micro-CT scan of the maxilla. Figure 3 B in the figure represents the bone mineral density test result of the maxillary periodontal bone in mice; Figure 4 The results are the morphological parameters of the microstructure of the trabecular bone in the maxillary periodontal bone. Figure 4 In this context, A represents the bone volume fraction (BV / TV) test result. Figure 4 In the figure, B represents the result of the trabecular bone number (Tb.N) test. Figure 4 In the figure, C represents the trabecular bone thickness (Tb.Th) measurement result. Figure 4 In the figure, D represents the trabecular separation (Tb.Sp) test result. Figure 4 In this context, E represents the Structural Pattern Index (SMI) test result and... Figure 4 In this context, F represents the detection result of trabecular connectivity density (Conn.Dn); Figure 5 The results of cortical morphology measurements were obtained from mice in the normal control group, periodontitis model group, antibiotic-positive control group, high-concentration bacterial solution experimental group, and low-concentration bacterial solution experimental group after the experiment. Figure 5 In the figure, A represents the porosity test results. Figure 5 In the image, B represents the pore volume detection result. Figure 6 The results show the expression of inflammatory factor genes in the gingiva of mice after the experiment, including the normal control group, periodontitis model group, positive control group after oral antibiotic administration, experimental group after oral high-concentration bacterial solution administration, and experimental group after oral low-concentration bacterial solution administration. Detailed Implementation

[0015] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0016] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0017] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0018] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0019] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0020] This invention proposes a fermentation Lactobacillus mucinus CQPC202403, which has the accession number CGMCC NO.31106 at the China General Microbiological Culture Collection Center and the accession date is June 27, 2024.

[0021] The present invention also provides the application of the aforementioned *Lactobacillus fermentatus* CQPC202403 in the preparation of oral preparations for improving periodontitis. The application method is to prepare *Lactobacillus fermentatus* CQPC202403 into a bacterial agent and then add it to the oral preparation for improving periodontitis.

[0022] In this invention, the preparation method of the fermented Lactobacillus mucinus CQPC202403 inoculum includes the following steps: 1) The bacterial strain is activated and then cultured to obtain a bacterial solution; 2) Centrifuge the bacterial solution to obtain bacterial precipitate; 3) The bacterial precipitate was resuspended in a freeze-drying protectant, and an encapsulating agent and excipients were added to obtain bacterial slurry; 4) The bacterial slurry was freeze-dried to obtain fermented Lactobacillus mucinus CQPC202403 bacterial agent.

[0023] In this invention, the oral preparation for improving periodontitis may be a mouthwash, gel, oral liquid, capsule, tablet, dispersant or granule.

[0024] In this invention, the freeze-drying protectant is an aqueous solution containing trehalose and glycerol.

[0025] In this invention, the lyophilization protectant contains 5-10% trehalose and 3-7% glycerol by mass.

[0026] In this invention, the mass ratio of the bacterial precipitate to the freeze-drying protectant is 1:3~5.

[0027] In this invention, the encapsulating agent is chitosan and the excipient is anhydrous lactose.

[0028] In this invention, the amount of the encapsulating agent added is 0.5 to 3% of the total amount of the freeze-drying protectant.

[0029] In this invention, the amount of excipient added is 1 to 5% of the total amount of freeze-drying protectant.

[0030] Example 1 Isolation and purification of Lactobacillus fermentans CQPC202403: The pickled vegetables were made by a family in Nan'an District, Chongqing. After the contents of the pickling jar were thoroughly stirred with a sterile spoon, 50mL of pickling water was drawn into a sterilized centrifuge tube using a sterile syringe. The tube was then placed in a low-temperature food sampling box and brought back to the laboratory for freezing and storage in an ultra-low temperature freezer at -80℃ for later use.

[0031] Take 1 mL of each kimchi brine sample and dilute it 10-fold with sterile physiological saline to a final concentration of 10. -6 Then take 10 -4 10 -5 10 -6 Three different gradients of bacterial suspension (100 μL each) were plated and incubated at 37°C for 24–48 h. Colony morphology was observed and recorded. Colonies of different morphologies were picked from the plates and streaked for isolation. After incubation at 37°C for 48 h, single colonies of different morphologies were picked from the plates again and streaked for isolation. This process was repeated multiple times until pure single colonies with consistent morphology were obtained.

[0032] The purified suspected target strain was inoculated into MRS broth and incubated at 37°C for 18–24 h. DNA was then extracted using a bacterial genomic DNA extraction kit. The extracted DNA was numbered and stored at -20°C for later use.

[0033] The extracted DNA was subjected to PCR amplification. The mixture consisted of 1 μL of upstream primer 27F (5'-AGA GTT TGA TCC TGGCTCAG-3'), 1 μL of downstream primer 1495R (5'-CTA CGG CTA CCTTGT TAC GA-3'), 12.5 μL of 2×Taq plus Buffer, and 1 μL of template DNA. The volume was brought to 25 μL with sterile dd H2O. Sterile ultrapure water was used as a negative control instead of template DNA. The amplification conditions were: 94℃ for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, for a total of 29 cycles, with a final extension at 72℃ for 5 min.

[0034] The PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The successfully sequenced sequences were compared and analyzed using the BLAST (Basic Local Alignment Search Tool) program in NCBI.

[0035] GeneBank analysis of lactic acid bacteria species The strain was successfully sequenced. BLAST analysis confirmed that the strain is *Lactobacillus fermentans*. The sequencing results are as follows: The efficacy of *Lactobacillus mucinus* CQPC202403 isolated in this embodiment in the treatment of periodontitis was tested using an animal model. The specific test methods and results are as follows: 1. Preparation of bacterial culture The bacteria were activated using MRS medium and incubated at 37°C for 48 hours until the logarithmic growth phase. The number of colony-forming units (CFU) was standardized by measuring optical density (wavelength 600 nm). After centrifugation, the CFU were divided into 10 groups. 9 High concentration of CFU / (kg·mb·d) and 10 8 A low concentration of CFU / (kg·mb·d) was prepared as a bacterial suspension using PBS for later use.

[0036] 2. Animal Model Construction Fifty male SPF-grade Balb / C mice, aged 8 weeks and weighing 30-35g, were selected. The experimental protocol was approved by the Ethics Committee of the Collaborative Innovation Center for Child Nutrition and Health Development, Chongqing Second Normal University (Approval No.: 2023032701B), and all procedures were performed in accordance with the requirements of the Laboratory Animal Ethics Committee. The experiment was divided into 5 groups, with 10 mice in each group: normal control group (NC group), periodontitis model group (PD group), antibiotic positive control group by gavage (Antibiotic group), and high-concentration bacterial solution by gavage (10 9 CFU / (kg·mb·d)) experimental group (CQPC202403-H group) and low concentration bacterial solution administered by gavage (10 8 CFU / (kg·mb·d)) experimental group (CQPC202403-L).

[0037] Except for the NC normal group mice, which were fed a normal maintenance diet, all other groups were fed Keyes 2000 formula diet (fructose 56%, degraded milk powder 28%, flour 6%, yeast 4%, alfalfa meal 3%, animal liver meal 1%, salt 2%) and a small amount of vegetables for 5 weeks, combined with a single oral injection of LPS to establish the model. During this period, the NC group and PD model group mice were administered 200 μL of physiological saline by gavage daily, while the other groups were administered 200 μL of the corresponding antibiotic mixture (metronidazole, ampicillin, vancomycin, and neomycin in a 1:1:1:1 ratio, 50 mg / kg) or CQPC202403 live bacterial solution by gavage until the end of the experiment.

[0038] 3. Serum collection and ELISA detection At the end of the experiment, peripheral blood from mice was collected in 1.5 ml sterile centrifuge tubes using the eye-binding method. The tubes were incubated at room temperature for 2 hours and then overnight at 4°C. Afterward, the tubes were centrifuged at 3000 rpm for 10 minutes, and the supernatant serum was collected and stored at -20°C for later analysis. The levels of relevant inflammatory factors IL-6, IL-12, TNF-α, IFN-γ, and IL-10 in mouse serum were detected using ELISA. The procedure was followed according to the commercial kit instructions. Specifically, the anti-mouse monoclonal antibody of the target indicator was coated onto an ELISA plate, the complex was attached to the plate, horseradish peroxidase-labeled streptavidin was conjugated with biotin, and the enzyme substrate tetramethylbenzidine was added. After enzyme action, the plate turned sky blue, and after adding sulfuric acid as a stop solution, it turned yellow. The reaction system was placed in an ELISA reader, and after zeroing the blank wells, the absorbance (A) was measured at 450 nm. The levels of the measured indicators were obtained by plotting a standard curve. The results are shown below. Figure 1 As shown in the figure, compared with the normal NC group, the serum levels of inflammatory factors IL-6, IL-12, TNF-α, and IFN-γ in the PD model group were significantly increased (p<0.05). Compared with the PD group, the CQPC202403-H group and the Antibiotic group showed the same effect, significantly preventing the increase of these pro-inflammatory factors (p<0.05), and restoring the levels of IFN-γ and IL-12 to levels comparable to the normal group (p>0.05). Regarding IL-10, compared with the normal NC group, the PD model group showed a significantly decreased level of the anti-inflammatory factor IL-10 (p<0.05). CQPC202403 intervention significantly increased the serum IL-10 level in the PD mouse model, and there was no significant difference in effect among the three intervention groups (p>0.05). These results indicate that gavage administration of the probiotic CQPC202403 can downregulate the levels of systemic pro-inflammatory factors and increase the levels of anti-inflammatory factors in PD mice, thereby playing a role in inflammation regulation.

[0039] 4. Histological observation Mice were euthanized by cervical dislocation after blood collection. Periodontal tissue specimens of the maxilla and mandible were collected and fixed in paraformaldehyde for at least 48 hours. The specimens were trimmed, retaining only the molar segment of teeth and periodontal tissue. Decalcification was performed using a mixed decalcification solution. Then, 5μm thick combined sections of tooth and periodontal tissue were longitudinally sectioned, stained with hematoxylin and eosin (HE), and observed under a light microscope for junctional epithelium, subepithelial inflammatory cell infiltration, and alveolar bone resorption. Specific details are as follows: Figure 2 As shown, Figure 2From top to bottom, the images show HE-stained histopathological sections of the maxillae (2000x magnification), mandible (5000x magnification), and mandible (2000x magnification), representing bone tissue pathology in each group of mice. The images show that the gingival epithelium and junctional epithelium in the normal NC group are intact, the periodontal ligament collagen fibers are neatly arranged, and no large number of obvious inflammatory cells are observed. In the PD periodontitis model group, significant loss of gingival junctional epithelium is visible in the mandible. Figure 2 (As indicated by the black arrow in D) The periodontal ligament collagen fibers are disordered, dissolved, and degenerated, with most being replaced by inflammatory cells. Figure 2 (As indicated by the yellow arrows and circles in B and D) Alveolar bone crest resorption, osteoclast and bone resorption lacunae formation, pulp congestion and increased trabecular bone spacing, significant bone resorption, and cavities appearing in the crown and dentin. Figure 2 (The part circled in black in D in the text); The antibiotic and CQPC202403-H intervention groups showed epithelial edema, hyperplasia, mild lymphoblastic cell infiltration in the subepithelial and connective tissues, regular periodontal ligament fiber arrangement, significantly reduced inflammatory cells in the alveolar bone compared to the PD group, disappearance of bone resorption lacunae, and signs of alveolar bone regeneration. The effect of CQPC202403-L was second best.

[0040] 5. Mouse maxilla examination After sampling, the mouse maxillae were fixed in a tissue fixation solution. The bone tissue samples were then scanned using a Bruker SkyScan 1176 small animal micro-CT imaging system (Germany), with a resolution of 18 μm. The main parameters for the micro-CT scan were: voltage 50 kV, current 455 μA, and exposure time 265 ms. The scanned images are shown below. Figure 3 As shown in A; and the bone mineral density of the maxillary periodontal bone in mice was measured, with the results as follows. Figure 3 As shown in Figure B, compared with the normal NC group, the bone mineral density (BMD) of the PD model group was significantly decreased. Compared with the PD group, the BMD of the Antibiotic group and the CQPC202403-H intervention group was significantly increased (P < 0.05). There was no significant difference in BMD between the CQPC202403-H group and the NC group. Although the BMD of the CQPC202403-L group was increased, there was no statistically significant difference compared with the PD model group.

[0041] Figure 4The figures show morphological indices of the trabecular bone microstructure in the maxillary periodontal bone. Compared to the normal NC group, the PD model group exhibited significantly lower bone volume fraction (BV / TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) (P < 0.05). This indicates a decrease in trabecular bone mass, increased osteolysis, and a greater osteolysis than osteosynthesis, suggesting an imbalance in bone metabolism. In contrast, the antibiotic and CQPC202403 intervention groups showed no difference compared to the normal group (P < 0.05), but were significantly higher than the PD model group (P < 0.05). This indicates that antibiotics and CQPC202403 significantly inhibited the decrease in BV / TV, Tb.N, and Tb.Th caused by periodontitis. Although the data showed that the high-concentration CQPC202403 group was superior to the low-concentration group, there was no statistically significant difference in the intervention effects among the three groups (P > 0.05). Trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) can be used to evaluate the spatial morphology of trabecular bone. Furthermore, trabecular separation (Tb.Sp) was significantly increased in the PD model group (P < 0.05). The antibiotic group and CQPC202403-H intervention could reduce Tb.Sp to some extent, but the reduction effect was not significant (P > 0.05). These findings demonstrate that high-concentration CQPC202403, like antibiotics, can prevent and delay the breakdown and loss of periodontal bone tissue.

[0042] The structure model index (SMI) is a parameter describing the ratio of lamellar to rod-like structures in the trabecular bone structure. In osteoporosis, the trabeculae transform from lamellar to rod-like structures, and this value increases. Results showed that compared to the normal NC group, the PD model group had a significantly increased SMI (P>0.05). Antibiotic intervention significantly inhibited the increase in the SMI index, while the effect of CQPC202403 intervention was less than that of antibiotics. Furthermore, high concentrations of CQPC202403 were more effective than low concentrations.

[0043] Connectivity density (Conn.Dn) represents the number of connections between the trabecular meshwork per cubic millimeter of volume, such as... Figure 4As shown in F, compared with the normal NC group, Conn.Dn was significantly reduced in the PD group (P < 0.05), but significantly higher in the Antibiotic group and CQPC202403-H group than in the PD model group. The CQPC202403-L group showed no difference from the PD group (P > 0.05), demonstrating that CQPC202403-H, like antibiotics, has the effect of inhibiting and alleviating the reduction in the number of trabecular connections.

[0044] 6. Morphological parameters of mouse cortex Test results as follows Figure 5 As shown, Figure 5 In this context, A represents the porosity test result. Figure 5 B in the figure represents the pore volume measurement result. From Figure 5 As can be seen, compared with the normal NC group, the PD model group showed significantly increased total porosity (percent, Po(tot)) and total pore space (Po.V(tot)) (P < 0.05). Antibiotic and CQPC202403 interventions significantly inhibited the increase of these two parameters in cortical bone caused by periodontitis. Data from the high-concentration CQPC202403 group showed a slight improvement over the low-concentration group, but the difference was not statistically significant. This suggests that CQPC202403 can achieve effects similar to antibiotics, significantly inhibiting and slowing down the porosity changes in cortical bone caused by inflammation-induced osteoporosis.

[0045] 7. Expression of inflammatory factor genes in mouse gingiva The gene expression of intragingival inflammatory factors was detected by RT-qPCR, and the results are as follows: Figure 6 As shown, compared with the normal NC group, the mRNA expression of IL-6, IL-17A, TNF-α, IFN-γ, and IL-1β in the maxillary periodontal tissue of PD model mice was significantly increased (P<0.05), indicating an inflammatory response in the periodontal tissue of PD mice. Antibiotic, high-concentration CQPC202403, and low-concentration CQPC202403 interventions significantly reduced the gene expression levels of IL-6, TNF-α, and IL-1β in the periodontal tissue of PD mice (P<0.05), and there was no significant difference in the effects among the three groups (P>0.05), with the most significant inhibitory effect on IL-6 gene expression. However, the inhibitory effect of CQPC202403-L group on IL-17A and IFN-γ gene expression was not significant (P>0.05), suggesting that gavage administration of CQPC202403 can significantly downregulate the level of inflammation in the periodontal tissue of PD mice, with the high-concentration group being more effective than the low-concentration group.

[0046] The above experimental results indicate that high-concentration CQPC202403 possesses good anti-inflammatory and immunomodulatory activities, and can exert anti-inflammatory effects and alleviate periodontal tissue lesions in experimental periodontitis. Lactobacillus paracasei CQPC202403 can be used in the field of probiotic adjuvant therapy for periodontitis.

[0047] Example 2 The preserved freeze-dried strain of *Lactobacillus fermentum* CQPC202403 was inoculated into MRS anaerobic liquid medium (formula: peptone 15.83 g / L, yeast extract 4.0 g / L, beef extract 5.0 g / L, glucose 30.72 g / L, Tween 80 1.0 mL / L, dipotassium hydrogen phosphate 5.16 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, ammonium citrate 2.0 g / L, pH adjusted to 6.8 ± 0.2), and cultured anaerobicly at 37℃ for 24 h to gradually restore bacterial activity.

[0048] The revived bacterial culture was transferred to a solid slant culture medium and incubated at 37°C for 48 hours to form uniform colonies to ensure purity. The culture was then washed with sterile physiological saline to prepare a bacterial suspension (OD600≥1.2).

[0049] Inoculate the bacterial suspension into the seed tank at an inoculum rate of 2.9%, maintain an anaerobic environment at 37°C, stir at 100 rpm, and incubate for 18 hours until the logarithmic growth phase, with a viable count ≥ 1 × 10⁻⁶. 9 CFU / mL.

[0050] The seed culture was transferred to the fermenter at a ratio of 8%. The culture medium was the same as the MRS anaerobic liquid culture medium formula mentioned above, with an initial pH of 6.8±0.2 and the tank pressure maintained at 0.05MPa.

[0051] The entire culture process was anaerobic (air was replaced with sterile nitrogen gas) using a stepped temperature strategy: 37℃ for the first 24 hours, and 35℃ for the next 24 hours. Carbon source concentration was maintained by pulsed addition of 20% glucose solution, and pH was adjusted to 6.5±0.3 in real time using 25% NaOH. Dissolved oxygen saturation was controlled at ≤5%, and the stirring speed was 150 rpm to avoid cell damage. After 48 hours of culture, when the OD600 value was ≥3.5 and the viable cell count was ≥1×10⁻⁶, the culture was considered complete. 10 Fermentation was terminated when CFU / mL was reached.

[0052] After fermentation, the fermentation broth was processed by a disc centrifuge at 4°C and 12,000 r / min for 10 min. The bacterial precipitate was collected, and the supernatant containing metabolic waste was removed.

[0053] The bacteria were washed three times by centrifugation with sterile phosphate buffer (pH 7.0). After each wash, the bacterial cell recovery rate was ≥90% and the detection rate of contaminating bacteria was ≤10 CFU / g.

[0054] The bacterial precipitate was resuspended in a freeze-drying protectant (containing 10% trehalose, 5% glycerol, and 2% yeast extract), with a bacterial to protectant volume ratio of 1:3. The mixture was then allowed to stand at room temperature for 30 minutes to allow for complete binding.

[0055] Add 0.5% chitosan and 1% anhydrous lactose, mix using a two-way spiral stirrer at 200 r / min, with a mixing coefficient of variation of <5%, to form a homogeneous bacterial slurry.

[0056] After the bacterial slurry is dispensed, it is placed in an ultra-low temperature freezer at -80℃ for 5 hours to ensure that ice crystals form evenly.

[0057] The product is transferred to a vacuum freeze dryer, where the vacuum level is maintained at 10~20Pa. It is first dried at -40℃ for 8 hours, and then heated to 25℃ for 4 hours. The final product has a water activity ≤0.25.

[0058] After aseptic pulverization, the freeze-dried blocks were passed through an 80-mesh sieve to obtain a powdered bacterial agent with a viable count ≥2×10⁻⁶. 10 CFU / g.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A fermented Lactobacillus muci napus CQPC202403, characterized in that, The preservation number of the fermented Lactobacillus muiis CQPC202403 in China General Microbiological Culture Collection Center is CGMCC NO.31106, and the preservation date is June 27, 2024.

2. Use of the fermented Lactobacillus muci genosus CQPC202403 according to claim 1 for the preparation of an oral preparation for improving periodontitis, characterized in that, The application method is to add the fermented Lactobacillus muiis CQPC202403 agent into the oral preparation for improving periodontitis after the fermented Lactobacillus muiis CQPC202403 is prepared into the fermented Lactobacillus muiis CQPC202403 agent.

3. Use of the Lactobacillus muci genosus CQPC202403 of claim 2 for the preparation of an oral preparation for improving periodontitis, characterized in that, The preparation method of the fermented Lactobacillus muiis CQPC202403 agent comprises the following steps: 1) activating the strain and then culturing to obtain a bacterial liquid; 2) centrifuging the bacterial liquid to obtain a bacterial precipitate; 3) resuspending the bacterial precipitate in a freeze-drying protective agent, adding an embedding agent and an excipient to obtain a bacterial slurry; 4) freeze-drying the bacterial slurry to obtain the fermented Lactobacillus muiis CQPC202403 agent.

4. Use of the Lactobacillus muci genosus CQPC202403 of claim 3 for the preparation of an oral preparation for improving periodontitis, characterized in that, The oral preparation for improving periodontitis can be a gargle, a gel, an oral liquid, a capsule, a tablet, a dispersant or a granule.

5. Use of the Lactobacillus muci genosus CQPC202403 of claim 4 for the preparation of an oral preparation for improving periodontitis, characterized in that, The freeze-drying protective agent is an aqueous solution containing trehalose and glycerol; The mass fraction of trehalose in the freeze-drying protective agent is 5-10%, and the mass fraction of glycerol is 3-7%; The mass ratio of the bacterial precipitate to the freeze-drying protective agent is 1:3-5.

6. Use of the Lactobacillus muci genosus CQPC202403 of claim 5 for the preparation of an oral preparation for improving periodontitis, characterized in that, The embedding agent is chitosan, and the excipient is anhydrous lactose.

7. Use of the Lactobacillus muci genosus CQPC202403 of claim 6 for the preparation of an oral preparation for improving periodontitis, characterized in that, The added amount of the embedding agent is 0.5-3% of the total amount of the freeze-drying protective agent; The added amount of the excipient is 1-5% of the total amount of the freeze-drying protective agent.

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