Use of microorganisms secreting mesaconic acid for inhibiting growth of periodontal pathogens and / or for inhibiting progression of periodontitis

Abstract

The invention relates to the technical field of probiotics, in particular to application of microorganisms secreting mesaconic acid to inhibition of growth of periodontal pathogenic bacteria and / or inhibition of periodontitis progress. The invention finds that the bacillus subtilis R0179 has the effect of inhibiting the growth of periodontal pathogenic bacteria, namely porphyromonas gingivalis ATCC 3327 (P.g), and also inhibits the periodontal tissue loss of mouse periodontitis. A supernatant secreted by the bacillus subtilis R0179 is found to be a main effective component which plays a role in inhibiting the growth of P.g, and mesaconic acid in the secretion of the bacillus subtilis R0179 is found to have the effect of inhibiting the growth of P.g; the mesaconic acid is also found to be capable of inhibiting the quantity of P.g in the oral cavity of the mouse, inhibiting the loss of periodontium of periodontitis of the mouse and increasing the bone density of alveolar bones, and the beneficial effects of the mesaconic acid are gradually enhanced along with the increase of the concentration of the mesaconic acid.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, and particularly to the application of microorganisms secreting mesaconic acid in inhibiting the growth of periodontal pathogenic bacteria and / or inhibiting the progression of periodontitis. Background Art

[0002] Periodontitis is a common oral disease caused by oral pathogenic microorganisms, which is prone to occur in people over 35 years old. In severe cases, it can lead to the absorption of alveolar bone around the teeth, resulting in tooth loosening and loss. Dental plaque biofilm is the initiating factor of periodontal disease, and Porphyromonas gingivalis is the main pathogenic bacterium leading to the occurrence and development of periodontitis. Porphyromonas gingivalis is an obligate Gram-negative anaerobe and is part of the normal human oral flora. During the occurrence of periodontitis, the oral flora is dysregulated, the beneficial bacteria gradually decrease, while the pathogenic bacteria (mainly Porphyromonas gingivalis) increase continuously. They can aggregate and adhere to each other to form a biofilm on the surface of periodontal tissues, and then mature plaque adheres to the surface of periodontal tissues. Subsequently, the pathogenic bacteria in the biofilm gradually damage the periodontal tissues through their own virulence factors, leading to the formation and development of periodontitis, and even possibly resulting in tooth loosening and exfoliation. So far, the initial treatment plan for periodontitis is oral scaling and root planing to remove pathogenic bacteria and their biofilms, and combined with chlorhexidine or antibiotics for treatment. Among these antibiotics, metronidazole is the most effective. However, the principle of all these methods is to transform the pathogenic bacteria in the biofilm into beneficial bacteria that are more relevant to health. Due to the relatively short effective time of antibiotic drugs, periodontal pathogenic bacteria will re-colonize the periodontal tissues again. Therefore, these methods have little effect on long-term effective oral maintenance. In addition, these antibiotics including metronidazole will increase the drug resistance of pathogenic bacteria. Therefore, the development of alternative "drugs" that antagonize periodontal pathogenic bacteria has received more and more attention.

[0003] Due to the deficiencies of the above-mentioned antibiotic-assisted treatment of periodontitis, there is an urgent need to develop more natural and harmless treatment methods, such as the application of probiotics. Probiotics are defined by the World Health Organization as live microorganisms that are beneficial to the host when applied in appropriate amounts. Recent studies have shown that they have the potential ability to treat diseases caused by bacterial disorders and inhibit the formation and maturation of oral pathogen biofilms. In addition, they also compete with pathogens for nutrients and space, and even regulate the inflammatory response of body tissues. Although there is evidence of the beneficial effects of probiotics, the effects of probiotics are strain-specific, and even the same probiotic genus may produce different effects. The types of existing probiotic strains for treating periodontal diseases are not comprehensive. Most of them only conduct preliminary isolation to find the supernatant or inactivated cells of probiotics with the effect of treating periodontitis, and do not further purify and confirm the active ingredients that play an anti-periodontitis role, so it is impossible to screen and optimize the strains targeted. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide the application of microorganisms secreting itaconic acid in inhibiting the growth of periodontal pathogenic bacteria and / or inhibiting the progression of periodontitis.

[0005] The present invention provides the application of microorganisms secreting itaconic acid in the preparation of products for preventing, treating and / or improving periodontitis.

[0006] In the present invention, the microorganisms secreting itaconic acid are Bacillus subtilis secreting itaconic acid.

[0007] In some embodiments, the Bacillus subtilis is Bacillus subtilis R0179.

[0008] In the present invention, the prevention, treatment and / or improvement of periodontitis includes: inhibiting periodontal pathogenic bacteria, improving bone loss, improving periodontal attachment loss and / or increasing jaw bone density.

[0009] In some embodiments, the periodontal pathogenic bacteria are Porphyromonas gingivalis.

[0010] In some embodiments, the inhibition includes inhibiting the growth of Porphyromonas gingivalis, inhibiting the formation of Porphyromonas gingivalis biofilm and / or inhibiting the maturation of Porphyromonas gingivalis biofilm.

[0011] In the present invention, the inhibition of the formation of Porphyromonas gingivalis biofilm and / or the inhibition of Porphyromonas gingivalis biofilm include inhibiting the expression of genes related to biofilm formation and maturation. Genes related to biofilm formation and / or maturation include at least one of hagA, hagB, kgpA, rgpA, ragB or fimA.

[0012] The present invention also provides a product for preventing, treating and / or improving periodontitis, which comprises microorganisms secreting itaconic acid or their fermentation products.

[0013] In some embodiments, the product comprises the fermentation supernatant of Bacillus subtilis R0179.

[0014] In some specific embodiments, in the product, the concentration of itaconic acid is not less than 0.313 mg / ml, or the volume fraction of the fermentation supernatant is not less than 10%.

[0015] In the present invention, the product is toothpaste, mouthwash, oral cleanser, oral spray or dental floss.

[0016] Furthermore, the present invention also provides a method for preventing, treating and / or improving periodontitis, which comprises administering the product of the present invention.

[0017] In the present invention, the mode of administration includes brushing teeth, gargling and / or coating, as well as oral proximal surface cleaning.

[0018] As a probiotic, Bacillus subtilis R0179 mainly treats intestinal inflammation, and there is no research showing its antagonistic effect against periodontal pathogenic bacteria and periodontal diseases. First, by constructing an animal model of periodontitis and conducting relevant experiments, it was found that Bacillus subtilis R0179 has the effect of inhibiting the growth of the periodontal pathogenic bacterium Porphyromonas gingivalis ATCC 33277 (P. g), and inhibiting the loss of periodontal tissue in murine periodontitis. Secondly, by initially separating the secreted supernatant and heat-inactivated cells of Bacillus subtilis R0179, it was found that the secreted supernatant is the main effective component for exerting the inhibitory effect on the growth of P. g, and the minimum concentration of the secreted supernatant of Bacillus subtilis R0179 for inhibiting the growth of P. g and its mechanism for inhibiting the formation of P. g biofilm were found; Thirdly, through non-targeted metabolomics and proteomics, it was found that a kind of aconitic acid in the secreted supernatant of Bacillus subtilis R0179 has the effect of inhibiting the growth of P. g; Finally, by topically applying three different concentrations of low / middle / high aconitic acid to the oral cavity of mice with periodontitis infected with P. g, it was found that aconitic acid can inhibit the number of P. g in the oral cavity of mice, inhibit the loss of periodontal tissue in murine periodontitis, and increase the alveolar bone mineral density, and with the increase of the aconitic acid concentration, its beneficial effect is gradually enhanced. Description of the Drawings

[0019] Figure 1 Showing the construction of the murine periodontitis model;

[0020] Figure 2 Showing the relative quantification results of the number of P. g in the oral cavity of mice in the blank control group, periodontitis group and probiotic group;

[0021] Figure 3 Showing the three-dimensional and two-dimensional models constructed after micro-ct scanning of the maxilla of mice in the blank control group, periodontitis group and probiotic group, and the statistical results of ABL, BV / TV, BMD;

[0022] Figure 4 Showing the morphology and size of the inhibition zones of the blank group, metronidazole group and probiotic group against P. g;

[0023] Figure 5 Showing the inhibitory effects of the blank control group, heat-inactivated cell group and supernatant group of probiotics on P. g;

[0024] Figure 6 Showing the inhibitory effects of the blank control group and the supernatant of Bacillus subtilis R0179 with different volume fractions on P. g;

[0025] Figure 7 Showing the inhibitory effects of the blank control group and the supernatant of Bacillus subtilis R0179 with the minimum inhibitory volume fraction on the bacterial number and biofilm formation of P. g;

[0026] Figure 8 Show the inhibitory effects of the supernatant of Bacillus subtilis R0179 in the control group and the minimum inhibitory volume fraction on the viable and dead cell morphology and quantity of P.g, as well as on the thickness of the P.g biofilm;

[0027] Figure 9 Show the inhibitory effects of the supernatant of Bacillus subtilis R0179 in the control group and the minimum inhibitory volume fraction on the gene expression related to P.g biofilm formation (day 2) and maturation (day 5 + 2 days);

[0028] Figure 10 Show the proteomics (10-a) of the supernatant of Bacillus subtilis R0179, the metabolic pathway enrichment map (10-b), and the C5-Branched dibasic acid metabolism pathway map (10-c);

[0029] Figure 11 Show the morphology and size of the inhibition zones of the blank group, metronidazole group, citraconic acid group, and mesaconic acid group on P.g;

[0030] Figure 12 Show the inhibitory effects of the dimethyl sulfoxide control group and mesaconic acid at different gradient concentrations on P.g;

[0031] Figure 13 Show the safety experiments of the dimethyl sulfoxide control group and mesaconic acid at the minimum inhibitory concentration on mouse RAW 264.7 cells;

[0032] Figure 14 Show the relative quantification results of the quantity of P.g in the oral cavity of mice in the periodontitis group and those treated with low-concentration, medium-concentration, and high-concentration mesaconic acid;

[0033] Figure 15 Show the three-dimensional (figure a) and two-dimensional (figure b) models constructed after micro-CT scanning of the maxilla of mice in the periodontitis group and those treated with low-concentration, medium-concentration, and high-concentration mesaconic acid, as well as the statistical results of ABL, BV / TV, and BMD (figure c);

[0034] Figure 16 Show the experimental flow chart of the present invention. Detailed implementation manners

[0035] The present invention provides the use of microorganisms secreting itaconic acid in inhibiting the growth of periodontal pathogenic bacteria and / or inhibiting the progression of periodontitis. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate modifications and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0036] Unless otherwise defined in the present invention, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.

[0037] In the present invention, the term "and / or" describes the association relationship of associated objects and indicates that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, and B exists alone. Wherein A and B can be singular or plural.

[0038] In the present invention, "comprising", "including" and "having" can be used interchangeably, aiming to express the inclusiveness of the solution, meaning that the solution may have other elements in addition to the listed elements. At the same time, it should be understood that when using "comprising", "including" and "having" to describe in this article, the solution of "consisting of..." is also provided.

[0039] In the present invention, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following" or its similar expression means any combination of these items, including any combination of single item (s) or plural items (s).

[0040] In the present invention, "periodontitis" is also known as periodontal disease. It is a chronic inflammation caused by bacteria in dental plaque invading the periodontal tissue, which can lead to the destruction of periodontal supporting tissues (gingiva, periodontal ligament, alveolar bone and cementum), the formation of periodontal pockets (the junctional epithelium proliferates apically, and its coronal part separates from the root surface to deepen the gingival sulcus), attachment loss and alveolar bone resorption. As the disease progresses, the teeth will gradually become loose and may eventually lead to tooth loss.

[0041] In the present invention, the prevention and treatment include prevention and / or treatment. The prevention refers to taking a series of measures to reduce the risk of periodontitis. The treatment includes taking a series of measures to relieve or eliminate the symptoms of periodontitis and restore the health of teeth and periodontal tissues. The measures include but are not limited to: using an effective amount of the product of the present invention to maintain oral hygiene, such as brushing teeth, rinsing mouth, etc., to remove bacteria and dental plaque on the tooth surface.

[0042] In the present invention, the "effective amount" of the therapeutic agent refers to the amount of the therapeutic agent that can effectively prevent or alleviate periodontitis or the progression of periodontitis when administered alone or in combination with another therapeutic agent to cells, tissues or subjects. The "effective amount" also refers to the amount sufficient to alleviate symptoms, such as treating, curing, preventing or alleviating periodontitis, or increasing the rate of treating, curing, preventing or alleviating the conditions of periodontitis. When the active ingredient is administered alone to an individual, the therapeutically effective dose refers only to that ingredient. When applying a certain combination, the therapeutically effective dose refers to the combined amount of the active ingredients that produce a therapeutic effect, whether administered in combination, continuously or simultaneously.

[0043] In the present invention, the "subject" to be prevented and treated refers to an organism that receives treatment for periodontitis as described in the present invention. Exemplarily, the "subject" includes mammals that receive treatment for periodontitis, such as humans, primates (e.g., monkeys) or non - primate mammals.

[0044] In one embodiment of the present invention, a mouse periodontitis model was constructed by silk ligation and infection with P.g, and Bacillus subtilis R0179 was topically applied to the oral cavity of mice. On the 14th day after modeling, the content of P.g in the oral cavity of mice was measured, and then the bone mass of their alveolar bone was quantified by micro - ct. The key conclusion was obtained: Bacillus subtilis R0179 can inhibit the number of P.g in the oral cavity of mice and the periodontal tissue loss caused by periodontitis.

[0045] In one embodiment of the present invention, the supernatant secreted by Bacillus subtilis R0179 and its inactivated cells were isolated. After co - culturing with P.g for 24 h, the absorbance value and Luminescence Assay of the co - cultured bacterial solution were measured, and through experiments such as scanning electron microscopy, laser confocal microscopy observation and real - time fluorescence quantitative PCR, the key conclusion was obtained: The component of Bacillus subtilis R0179 that inhibits the growth, biofilm formation and maturation of P.g is its secreted supernatant rather than its inactivated cells.

[0046] In one embodiment of the present invention, through proteomics and non - targeted metabolomics analysis, the effective antibacterial components in the supernatant secreted by Bacillus subtilis R0179 may be mesaconic acid and citraconic acid. Further, the two compounds of mesaconic acid and citraconic acid were respectively subjected to the inhibition zone experiment, and the key conclusion was obtained: The compound with the effect of inhibiting the growth of P.g is mesaconic acid (linear molecular formula is HOOCCH=C(CH3)COOH) rather than citraconic acid.

[0047] In one embodiment of the present invention, mesaconic acid with gradient concentrations was co-cultured with P.g for 24 h. Again, the absorbance value and Luminescence Assay of the co-culture were detected, and mesaconic acid with the minimum inhibitory concentration was co-cultured with mouse RAW264.7 cells. The cell proliferation was detected by the CCK8 experiment. The key conclusion was obtained: the minimum inhibitory concentration of mesaconic acid inhibiting the growth of P.g was 0.313 mg / ml, and 0.313 mg / ml of mesaconic acid had no obvious cytotoxicity to mouse RAW 264.7 cells and had no obvious effect on cell proliferation.

[0048] In one embodiment of the present invention, low / middle / high three different concentrations of mesaconic acid were locally applied to the periodontitis of mice infected with P.g. The content of P.g in the oral cavity of mice was detected by real-time fluorescence quantitative PCR, and the content of alveolar bone was quantified by micro-ct. The key conclusion was obtained: as the concentration of applied mesaconic acid increased, its inhibitory effect on the content of P.g in the oral cavity of periodontitis mice and the loss of periodontal tissue of mice became stronger.

[0049] In the present invention, the product can be added with excipients arbitrarily selected according to the product formula or the purpose of use, but it is not limited thereto. For example, pure water, oil, surfactant, humectant, higher alcohol, thickener, chelating agent, pigment, fatty acid, antioxidant, preservative, wax, pH regulator, fragrance, etc. can be added.

[0050] The test materials used in the present invention are all ordinary commercially available products and can be purchased in the market.

[0051] The Bacillus subtilis involved in the embodiment is Bacillus subtilis R0179 extracted from Medilac-Vita. The specific extraction operation process refers to the previous published paper by the inventor (Immunomodulatory mechanism of Bacillus subtilis R0179 in RAW 264.7 cells against Candida albicans challenge).

[0052] The Porphyromonas gingivalis ATCC 33277 (P.g) involved in the embodiment was donated by the American Type Culture Collection (ATCC).

[0053] It should be understood that in various embodiments of the present invention, the size of the serial numbers of the above processes does not mean the order of execution. Some or all steps can be executed in parallel or sequentially. The execution order of each process should be determined according to its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of the present application. The present invention will be further described below in conjunction with the embodiments:

[0054] Example 1. Bacillus subtilis R0179 can inhibit the quantity of P.g in the oral cavity of mice and relieve the periodontitis phenotype.

[0055] In this invention, a mouse periodontitis model was constructed. Male C57BL / 6 mice (mouse strain) at 8 weeks old with a body weight of nearly 23.5 g per mouse were selected, and 5 mice were included in each group. The blank control group was not treated. The periodontitis group and the probiotic group were modeled for periodontitis according to previous reports. The bilateral maxillary second molars were ligated and infected with P.g ( Figure 1 ).

[0056] Mice in the probiotic group and the periodontitis group were simultaneously given P.g infection on the 0th day, 1st day, 4th day, 7th day, 10th day, and 13th day of silk ligation (1×10 6 cfu each time, cfu refers to the number of colony forming units). On the 4th day, 7th day, 10th day, and 13th day of ligation, the probiotic group was coated with Bacillus subtilis R0179 bacterial solution containing 1×10 6 cfu around the ligated teeth. Then, samples were collected on the 14th day. First, oral microorganisms near the cervical part of the second molars of each group of mice were collected by absorbing the moisture at the fingertips (#40), and their corresponding DNA was extracted using a DNA extraction kit. The relative content of 16s rDNA of P.g in the oral cavity of mice was detected by real-time fluorescence quantitative PCR means, and the conclusion was drawn that the number of Porphyromonas gingivalis in the periodontitis group was significantly increased compared with the blank control group ( Figure 2 ); at the same time, compared with the periodontitis group, the application of Bacillus subtilis R0179 in the probiotic group could significantly reduce the quantity of P.g in the oral cavity of mice ( Figure 2 ).

[0057] After that, the left maxilla of the mice was scanned by micro-ct (micro-computed tomography technology). The bone mineral density (BMD) of the alveolar bone around the roots of the second molars was measured by computer. The distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) was used as the alveolar bone loss (ABL), and the ratio of bone volume to tissue volume (BV / TV) of the mice was used to quantitatively analyze the bone mass of the alveolar bone with the above three indicators. The conclusion was drawn that compared with the blank control group, there was bone resorption around the roots in the periodontitis group, with a decrease in BMD and BV / TV, and an increase in ABL ( Figure 3);whereas the local application of Bacillus subtilis R0179 in the probiotic group increased the BMD and BV / TV in periodontitis mice and inhibited ABL caused by periodontitis in mice( Figure 3 )。

[0058] Example 2. Bacillus subtilis R0179 has antibacterial activity against P.g.

[0059] Mix 5*10^8 cfu of P.g with solid medium containing sheep blood agar before it solidifies, and place it in a sterile petri dish with a diameter of 10 cm. Place 3 sterile blank round filter papers with a diameter of 6 mm on each solidified medium, divided into three groups. On the filter papers in the blank group( Figure 4 a) add 12 μl of sterile filtered water, and on the filter papers in the metronidazole group( Figure 4 c) add 12 μl of metronidazole with a concentration of 8 μg / ml as a positive control. On the filter papers in the probiotic group( Figure 4 b) add 12 μl of the bacterial solution of Bacillus subtilis R0179 (concentration 6×10 8 cfu / ml). After incubating for 48 h, observe whether a clear zone appears around the filter paper as the inhibition zone diameter (except for the 6 mm size of the filter paper itself, a clear zone diameter > 6 mm is considered effective). It is concluded that Bacillus subtilis R0179 produced an effective inhibition zone against P.g, and its diameter size is similar to that of the inhibition zone produced by 8 μg / ml of metronidazole.

[0060] Example 3. The effective component of Bacillus subtilis R0179 in inhibiting P.g is the supernatant it secretes.

[0061] After amplifying the bacterial solution of Bacillus subtilis R0179 to a density of 6*10^8 cfu / ml, prepare the supernatant for this step and steps 4 to 6 after centrifugation (6000 g, 10 min) and filtration (0.22 μm membrane). The bacterial cells are inactivated by autoclaving at 120 °C for 15 minutes to prepare inactivated bacterial cells without supernatant, and then the inactivated bacterial cells of Bacillus subtilis R0179 are resuspended to 5×10 7 cfu / ml. In a 96-well plate with a total volume of 100 μl in the system, where the number of P.g is 10^ 6 cfu / well, and the co-culture system contains different gradient volume percentages of the supernatant of Bacillus subtilis R0179. After co-culturing for 24 hours, measure the absorbance value and luminescence assay (RLU: relative light units corresponding to the number of viable bacteria in the co-culture bacterial solution) of the co-culture bacterial solution. It is concluded that compared with the control group, the supernatant secreted by Bacillus subtilis R0179 rather than its inactivated bacterial cells can inhibit the number of P.g bacteria( Figure 5 )。

[0062] Example 4. The minimum volume ratio of the supernatant of Bacillus subtilis R0179 that inhibits the growth of P. g is 10%.

[0063] In a 96-well plate (total volume 100 μl), the absorbance value of the co-cultured bacterial solution was measured after co-culturing the gradient-diluted supernatant of Bacillus subtilis R0179 with P. g (number 10 6 cfu / well) for 24 h, and the conclusion was drawn that the minimum volume ratio of the supernatant of Bacillus subtilis R0179 that inhibits the growth of P. g is 10% ( Figure 6 ).

[0064] Example 5. 10% volume of the supernatant of Bacillus subtilis R0179 can inhibit the bacterial count and biofilm formation of P. g.

[0065] In a 96-well plate, the cells after co-culturing 10% volume ratio (total volume 100 μl) of the supernatant of Bacillus subtilis R0179 and P. g (number 10 6 cfu / well) for 5 days were stained with crystal violet. The bacterial solutions of the control group and the supernatant group were equally diluted at appropriate gradients and then evenly spread on an agar solid medium suitable for the growth of P. g and cultured for 7 days. By counting the colony number of P. g and observing the biofilm through the thickness of crystal violet staining, the conclusion was drawn that 10% volume of the supernatant of Bacillus subtilis R0179 can inhibit the bacterial count and biofilm formation of P. g ( Figure 7 ).

[0066] Example 6. The supernatant of Bacillus subtilis R0179 can inhibit the formation and maturation of P. g biofilm and the expression of its related genes.

[0067] In a 24-well plate (total system 500 μl), 10% volume of the supernatant of Bacillus subtilis R0179 was co-cultured with P. g (10 6 cfu / well) for 2 days, and then the samples were collected. The bacterial count and biofilm morphology of P. g were observed by scanning electron microscopy for all samples; and in a confocal dish with a diameter of 30 mm (total volume 2 ml), P. g (10 6 cfu) was first cultured for 5 days, then 10% volume of the supernatant of Bacillus subtilis R0179 was added and incubated for another 2 days, and then the samples were collected. All samples were stained with live and dead bacteria and then observed by laser confocal microscopy for the viability of P. g and the thickness of its biofilm; the conclusion was drawn that 10% volume ratio of the supernatant of Bacillus subtilis R0179 can inhibit the viable bacterial count of P. g, increase the dead bacterial count, and reduce the thickness of the P. g biofilm ( Figure 8 ).

[0068] The DNA of bacteria was extracted using a bacterial genomic DNA extraction kit, and then the expression of genes related to P.g biofilm formation and maturation (haA, hagB, kgpA, rgpA, rgpB, fimA) was detected by real-time fluorescence quantitative PCR. The conclusion was drawn that the supernatant of Bacillus subtilis R0179 at a volume ratio of 10% could inhibit the expression of genes related to P.g biofilm formation (on the 2nd day) and maturation (on the 5th day + 2 days) ( Figure 9 ).

[0069] Example 7. The metabolite that plays an antibacterial role in the supernatant of Bacillus subtilis R0179 may be mesaconic acid.

[0070] First, in a 10-cm-diameter bacterial culture dish, Bacillus subtilis R0179 (4×10 9 cfu) and P.g (10 8 cfu) were mixed in a liquid to form a culture solution with a total volume of 20 ml. After culturing for 5 days, it was centrifuged at 6000 g for 10 min, and then filtered through a 0.22-μm filter membrane to obtain a cell-free supernatant. It was detected by non-targeted metabolomics and proteomics, and combined with bioinformatics analysis. The conclusion was drawn that after enrichment analysis by proteomics and non-targeted metabolomics, a common signaling pathway was obtained - namely: C5-Branched dibasic acid metabolism( Figure 10 mesa and Figure 10 mesb), and there were two compounds in this metabolic pathway present in the supernatant of Bacillus subtilis R0179, namely mesaconic acid and citraconate( Figure 10 mesc). Therefore, mesaconic acid and citraconate may be the metabolites that play an antibacterial role in the supernatant of Bacillus subtilis R0179.

[0071] Secondly, mesaconic acid and citraconate were diluted to 40 mg / ml, and the antibacterial ring experiment was carried out by the above-mentioned paper disk diffusion method. It was divided into three groups. The blank group: containing the diluent used to dilute the two compounds (10% v / v dimethyl sulfoxide - dmso)- Figure 11 mesa and Figure 11 mesd, the metronidazole group (with the same concentration and dose as above)- Figure 11 mesb and Figure 11 mese, the 40 mg / ml citraconate group( Figure 11 mesc) and the 40 mg / ml mesaconic acid group( Figure 11 f). The conclusion was drawn that 40 mg / ml mesaconic acid (the linear molecular formula is HOOCCH=C(CH3)COOH) had the effect of inhibiting the growth of P.g, while 40 mg / ml citraconate did not have this inhibitory effect.

[0072] In Example 8, the minimum inhibitory concentration of itaconic acid against the growth of P. g is 0.313 mg / ml.

[0073] Firstly, gradient concentrations of itaconic acid (maximum concentration 40 mg / ml) were co-cultured with P. g (10 6 cfu / well) in a 96-well plate (total volume 100 μl) for 24 h. Dimethyl sulfoxide at the corresponding concentration was used as the control group. By detecting the absorbance value of the co-cultured bacterial solution ( Figure 12 a) and Luminescence Assay ( Figure 12 b), it was concluded that the minimum concentration of itaconic acid inhibiting the growth of P. g is 0.313 mg / ml (containing 0.07825% v / v of dimethyl sulfoxide).

[0074] Secondly, itaconic acid at a final concentration of 0.313 mg / ml was co-cultured with mouse mononuclear macrophages RAW 264.7 in a 96-well plate (total volume 100 μl). The control group was the dimethyl sulfoxide solution group with a final volume fraction of 0.07825% v / v. The CCK8 assay was used to detect cell proliferation. It was concluded that compared with the control group, 0.313 mg / ml of itaconic acid has no obvious cytotoxicity to mouse RAW 264.7 cells and has no obvious effect on their proliferation ( Figure 13 ).

[0075] In Example 9, the inhibitory effect of itaconic acid on the number of P. g in the mouse oral cavity and periodontal loss of periodontitis shows a dose-dependent relationship.

[0076] In the periodontitis model constructed in the present invention, mice were given itaconic acid at low concentration of 100 mg / kg body weight / day, medium concentration of 200 mg / kg body weight / day, and high concentration of 300 mg / kg body weight / day. The selection criteria for mice were the same as in point 1, with 5 mice in each group. Mice in the periodontitis group and the itaconic acid application group were simultaneously given an infection of P. g (1×10 6 cfu each time, cfu refers to the number of colonies) on the 0th, 1st, 4th, 7th, 10th, and 13th days after silk ligation. At the same time, the itaconic acid drug application group began to coat the corresponding low / medium / high concentration of itaconic acid around the second maxillary molar of the mice every day starting from the 4th day after ligation. Then, samples were collected on the 14th day, and the relative content of 16s rDNA of P. g in the mouse oral cavity was detected by real-time fluorescence quantitative PCR. It was concluded that the application of different concentrations of itaconic acid can effectively reduce the number of P. g in the mouse oral cavity; and as the concentration of itaconic acid increases, its ability to reduce the number of P. g in the mouse oral cavity becomes stronger. Figure 14 )

[0077] And by micro-CT scanning the left maxilla of the mice, through constructing three-dimensional (Figure 15 in a) and two-dimensional ( Figure 15 the scanned graph in b), and calculate the ABL, BV / TV and BMD of the periodontal tissues around the maxillary second molar Figure 15 in c). The conclusion is drawn that compared with the periodontitis group, mesaconic acid at three concentrations can reduce the degree of bone loss and periodontal attachment loss caused by periodontitis in mice, that is, mesaconic acid at three concentrations can all reduce the degree of ABL and increase the ratio of BV / TV Figure 15 in c). At the same time, mesaconic acid at medium and high concentrations can increase the bone density of the maxilla in periodontitis mice, that is, increase the BMD index, but low-concentration mesaconic acid has no obvious effect on increasing the bone density (BMD) of periodontitis in mice Figure 15 in c).

[0078] The above is only the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of a microorganism secreting itaconic acid in the preparation of a product for preventing and / or improving periodontitis.

2. The application according to claim 1, wherein The microorganism secreting itaconic acid is Bacillus subtilis secreting itaconic acid.

3. The application according to claim 2, characterized in that The Bacillus subtilis is Bacillus subtilis R0179.

4. The application according to claim 1, characterized in that, The prevention and / or improvement of periodontitis includes: inhibiting periodontal pathogenic bacteria, improving bone loss, improving periodontal attachment loss and / or increasing the bone density of the jawbone.

5. The application according to claim 4, wherein, The periodontal pathogenic bacteria is Porphyromonas gingivalis.

6. The application according to claim 4, characterized in that The inhibition includes inhibiting the growth of Porphyromonas gingivalis, inhibiting the formation of Porphyromonas gingivalis biofilm and / or inhibiting the maturation of Porphyromonas gingivalis biofilm.

7. A product for preventing and / or improving periodontitis, characterized in that, It includes a microorganism secreting itaconic acid or its fermentation product.

8. The product according to claim 7, wherein, It includes the fermentation supernatant of Bacillus subtilis R0179.

9. The product according to claim 7 or 8, characterized in that, Among them, the concentration of itaconic acid is not less than 0.313 mg / ml, or the volume fraction of the fermentation supernatant is not less than 10%.

10. The product according to any one of claims 7 to 9, characterized in that, It is toothpaste, mouthwash, oral cleaner, oral spray or dental floss.

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