Agents for preventing or improving periodontal disease

Abstract

To provide materials that inhibit infection by periodontal pathogens and are useful for preventing or improving periodontal disease.SOLUTION: There is provided a periodontal pathogen infection inhibitor containing Neisseria mucosa bacterial cells or cell cultures as an active ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an agent for preventing or ameliorating periodontal disease. [Background technology]

[0002] Periodontal disease is caused by periodontal pathogens, which cause chronic inflammatory diseases among the more than 600 species of bacteria present in the oral cavity.Periodontal pathogens infect periodontal tissues and invade cells, causing gingivitis and even periodontal tissue destruction.

[0003] Existing techniques for preventing and improving periodontal disease include the use of bactericides and antibiotics against periodontal pathogens. However, these techniques have strong bactericidal and bacteriostatic effects, which can destroy the normal oral flora and cause the emergence of resistant bacteria, potentially leading to serious illness.

[0004] Recently, techniques have been reported for inhibiting the growth of periodontal pathogens using lactic acid bacteria and bifidobacteria, which have attracted attention as probiotics. For example, it has been reported that certain strains of Lactococcus lactis, Lactobacillus rhamnosus, and Lactobacillus curvatus have the ability to inhibit oral biofilm formation and the growth of Porphyromonas gingivalis and Fusobacterium nucleatum (Patent Document 1), that lactic acid bacteria of the Enterococcus faecium species have the effect of inhibiting the growth of Porphyromonas gingivalis (Patent Document 2), and that Leuconostoc mesenteroides has the ability to inhibit the formation of biofilms caused by Porphyromonas gingivalis and other bacteria (Patent Document 3).

[0005] On the other hand, it is known that Neisseria bacteria such as Neisseria mucosa, Neisseria sicca, Neisseria flava, Neisseria subflava, Neisseria flavescens, and Neisseria elongata exist as non-pathogenic normal bacteria in the oral cavity, but their functions remain unclear. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2010-124772 [Patent Document 2] Japanese Patent Application Laid-Open No. 2006-328052 [Patent Document 3] Japanese Patent Application Laid-Open No. 2010-53062 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to providing a material that inhibits infection by periodontal pathogens and is useful for preventing or ameliorating periodontal disease. [Means for solving the problem]

[0008] After extensive research into oral microorganisms, we discovered that Neisseria mucosa, a non-pathogenic oral indigenous bacterium, or its cultures, can suppress infection of gingival cells by periodontal pathogens and are useful as preventive or therapeutic agents for periodontal disease.

[0009] That is, the present invention relates to the following 1) to 7). 1) A periodontal pathogen infection inhibitor containing Neisseria mucosa bacteria or a culture of bacteria as the active ingredient. 2) An alveolar bone resorption inhibitor containing Neisseria mucosa bacteria or a culture of bacteria as the active ingredient. 3) A preventive or ameliorative agent for periodontal disease, containing Neisseria mucosa bacteria or a culture of bacteria as the active ingredient. 4) A food for inhibiting periodontal pathogenic bacterial infections, containing Neisseria mucosa bacteria or bacterial cultures as the active ingredient. 5) A food for inhibiting alveolar bone resorption, containing Neisseria mucosa bacteria or a culture of bacteria as the active ingredient. 6) A food for preventing or improving periodontal disease, containing Neisseria mucosa bacteria or a culture of bacteria as an active ingredient. 7) A method for producing an EPS-containing culture of Neisseria mucosa, which comprises adding a culture supernatant of Porphyromonas gingivalis to a culture solution of Neisseria mucosa, culturing the culture, and then recovering the culture after removing the bacterial cells and medium components. [Effects of the Invention]

[0010] Since the Neisseria mucosa of the present invention is a non-pathogenic oral indigenous bacterium, it can provide an agent for preventing or improving periodontal disease that can be taken over a long period of time by healthy individuals as well as the elderly and those recovering from illness. [Brief explanation of the drawings]

[0011] [Figure 1] The inhibitory effect of Neisseria mucosa on periodontal pathogen infection is dependent on the number of bacteria. The MOI (Multiplicity of infection) indicates the number of bacteria added. [Figure 2] Inhibitory effect of Neisseria bacteria on periodontal pathogen infection. [Figure 3] Inhibitory effect of Neisseria mucosa on infections caused by various periodontal pathogens. [Figure 4] The effect of adding Porphyromonas gingivalis culture supernatant on increasing the yield of Neisseria mucosa culture. [Figure 5] Photograph of EPS produced by Neisseria mucosa, which increased with the addition of Porphyromonas gingivalis culture supernatant. [Figure 6] Inhibitory effect of Neisseria mucosa culture on periodontal pathogen infection. [Figure 7] Inhibitory effect of Neisseria mucosa on alveolar bone resorption (amount of alveolar bone resorption). [Figure 8] (A) Inhibitory effect of Neisseria mucosa on alveolar bone resorption (photograph of mouse maxilla after test), (B) Measurement site of alveolar bone resorption (arrow: measurement site). DETAILED DESCRIPTION OF THE INVENTION

[0012] Neisseria mucosa used in the present invention is a gram-negative cocci belonging to the genus Neisseria in the family Neisseria, and is a non-pathogenic normal bacterium inhabiting the oral cavity. Neisseria mucosa can be isolated from human saliva, dental plaque, etc. It is also available from JCM (Neisseria mucosa JCM12992 strain (ATCC19696 strain)).

[0013] In the present invention, the "cells" of Neisseria mucosa may be live, wet, or dry cells of Neisseria mucosa. They may be live or dead cells. Furthermore, the cells also include cell components such as cytoplasm and cell wall fractions obtained by treating cells with enzymes or physical means. Examples of "cell cultures" of Neisseria mucosa include cultures containing medium components, cells, and extracellular polymeric substances (EPS), as well as cultures containing cells and EPS from which the medium components have been removed, cultures containing medium components and EPS from which the cells have been removed, and cultures containing EPS from which the medium components and cells have been removed. The bacterial cells or bacterial cell culture of the present invention can be prepared in any form depending on the intended use, such as freeze-dried powder, spray-dried powder, or suspension in liquid, as desired.

[0014] The medium for culturing Neisseria mucosa of the present invention is not particularly limited as long as it contains peptone or the like as a nutrient source, and examples thereof include BHI medium (manufactured by BD Japan). The culture method is not particularly limited as long as the conditions are such that Neisseria mucosa cells grow well. For example, the culture may be performed at 20 to 40°C, preferably 35 to 38°C, under aerobic conditions, preferably in the presence of 5% CO2, for 16 to 72 hours.

[0015] In the present invention, when a bacterial culture of Neisseria mucosa is used, the culture of Neisseria mucosa is preferably carried out at 37°C in the presence of 5% CO2 for 24 hours or more, using the culture supernatant of a periodontal pathogen such as Porphyromonas gingivalis, for example, by culturing Porphyromonas gingivalis under anaerobism, centrifuging the resulting culture, and then adding the resulting supernatant after filter sterilization, from the viewpoint of EPS production.

[0016] As shown in the Examples below, Neisseria mucosa cells or cell cultures have the effect of suppressing infection of gingival epithelial cells by periodontal pathogens such as Porphyromonas gingivalis, and also suppress alveolar bone resorption. Therefore, Neisseria mucosa bacteria or bacterial cell cultures can be used as agents for inhibiting periodontal pathogen infection, agents for inhibiting alveolar bone resorption, or agents for preventing or improving periodontal disease, and Neisseria mucosa bacteria or bacterial cell cultures can be used to manufacture agents for inhibiting periodontal pathogen infection, agents for inhibiting alveolar bone resorption, or agents for preventing or improving periodontal disease. Furthermore, Neisseria mucosa cells or cell cultures can be used to inhibit infection by periodontal pathogens, inhibit alveolar bone resorption, and prevent or ameliorate periodontal disease. Such uses may be administered to humans or non-human animals, or to specimens derived therefrom, and may be therapeutic or non-therapeutic. Non-human animals include non-human mammals, amphibians, and cartilaginous fish, including apes, other primates, mice, rats, horses, cows, pigs, sheep, dogs, cats, hamsters, and companion animals.

[0017] In the present invention, "periodontal disease" refers to an inflammatory disease in which inflammation of the gums is caused by bacteria in dental plaque, and includes gingivitis and periodontitis. Gingivitis is a pathological condition in which periodontal disease bacteria in dental plaque infect periodontal tissues, causing inflammation of the gums. If this gingivitis pathological condition is left untreated and the inflammation worsens, not only will the gums swell and bleed, but the periodontal pockets will deepen and progress to periodontitis. The progression of this periodontitis will cause gingival recession and resorption of the alveolar bone that supports the tooth roots, ultimately leading to tooth loss. Furthermore, "suppression of periodontal pathogen infection" means suppressing infection of gingival cells by periodontal pathogens, and examples of periodontal pathogens include Porphyromonas gingivalis, as well as Fusobacterium nucleatum, Prevotella intermedia, Treponema denticola, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, Campylobacter rectus, and Selenomonas sputigena. Furthermore, "inhibition of alveolar bone resorption" means inhibiting the destruction (resorption) of the alveolar bone, which is the bone that supports the teeth.

[0018] As used herein, "prevention" refers to preventing or delaying the onset of a disease or condition in an individual, or reducing an individual's risk of developing a disease or condition. Furthermore, "improvement" means improvement of a disease, symptom or condition, prevention or delay of worsening of a disease, symptom or condition, or reversal, prevention or delay of progression of a disease or symptom, and includes the concept of so-called "treatment."

[0019] The periodontal pathogen infection inhibitor, alveolar bone resorption inhibitor, and periodontal disease prevention or amelioration agent of the present invention can be used by itself as a pharmaceutical, quasi-drug, food, or oral composition for inhibiting infection by periodontal pathogens, inhibiting alveolar bone resorption, or preventing or ameliorating periodontal disease, or can be used as a material or preparation to be incorporated into such pharmaceutical, quasi-drug, food, or oral composition. The foods mentioned include foods that are based on the concept of preventing or improving periodontal disease or bad breath, and that are labeled as such as necessary, functional foods, foods for specified health uses, foods for the sick, and supplements.

[0020] When used as a pharmaceutical, oral administration is preferred, and the dosage forms include, for example, liquid preparations; solid preparations such as tablets, granules, fine granules, powders, and tablets; or various forms containing the liquid or solid preparations, such as capsules, oral sprays, and troches. These various dosage forms of pharmaceutical preparations can be prepared by appropriately combining other pharmaceutically acceptable excipients, binders, fillers, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, flavoring agents, flavorings, coating agents, carriers, diluents, and the like, as long as they do not interfere with the activity of Neisseria mucosa bacteria or bacterial cell cultures.

[0021] When used as a food, the form of the product may include beverages such as fruit or vegetable juice drinks, carbonated drinks, tea drinks, dairy drinks, fermented milk, fermented fruit juice, fermented vegetable juice, alcoholic drinks, and soft drinks, as well as various foods such as jelly foods, various snacks, baked goods, cakes, chocolate, jam, bread, gum, candy, soups, pickles, and tsukudani (foods boiled in soy sauce), as well as supplements in the same form as the oral dosage forms described above (tablets, capsules, syrup, etc.). Such foods can be prepared according to standard methods by appropriately combining Neisseria mucosa cells or a culture of the cells with any food material, other active ingredient, or food-acceptable additive (e.g., solvents, softeners, oils, emulsifiers, preservatives, acidulants, sweeteners, bittering agents, pH adjusters, stabilizers, colorants, UV absorbers, antioxidants, humectants, thickeners, adhesives, dispersants, flow improvers, humectants, aromatics, seasonings, flavor adjusters, etc.).

[0022] Specific forms of oral compositions include mouthwashes, mouthwashes, toothpastes, powder toothpastes, dentifrices, oral ointments, gels, tablets, granules, fine granules, gummy jelly, troches, tablets, capsules, candies, chewing gums, etc., and preferably toothpastes, mouthwashes, gummy jelly, and troches. Oral compositions can be prepared according to conventional methods by appropriately blending, for example, binders, humectants, abrasives, sweeteners, preservatives, surfactants, flavorings, etc.

[0023] The amount of Neisseria mucosa cells contained in the above-mentioned medicines, foods, and oral compositions is not particularly limited, and may be adjusted appropriately according to the daily dose, etc. For example, 5 × 10 Neisseria mucosa cells may be contained. 8 1×10 or more, preferably 1×10 10 1×10 or more 13 or less, more preferably 1×10 11 1×10 or more 12 The amount of the Neisseria mucosa bacterial cell culture contained is not particularly limited and may be adjusted appropriately depending on the daily dose, etc., and may be, for example, 0.1 mg or more, preferably 100 mg or more, more preferably 500 mg or more, and 5000 mg or less, preferably 2500 mg or less, more preferably 1000 mg or less.

[0024] In the above-mentioned pharmaceuticals, foods, and oral compositions, the dosage of Neisseria mucosa bacteria can be determined appropriately depending on various conditions such as the patient's weight, age, sex, and symptoms. For example, when expressed in terms of the number of Neisseria mucosa bacteria, the dosage per day is 1 × 10 8 1×10 or more, preferably 1×10 10 1×10 or more 13 or less, more preferably 1×10 11 1×10 or more 12 The dosage of the Neisseria mucosa bacterial cell culture per adult per day is, in terms of dry weight when freeze-dried, 0.1 mg or more, preferably 100 mg or more, more preferably 500 mg or more, and 5000 mg or less, preferably 2500 mg or less, more preferably 1000 mg or less.

[0025] Subjects to be administered or ingested with the periodontal pathogen infection inhibitor, food for inhibiting periodontal pathogen infection, alveolar bone resorption inhibitor, food for inhibiting alveolar bone resorption, agent for preventing or ameliorating periodontal disease, and food for preventing or ameliorating periodontal disease of the present invention preferably include humans infected with periodontal pathogens, humans suffering from gingival inflammation such as gingivitis or periodontitis, and even alveolar bone resorption, and humans wishing to prevent the onset of gingival inflammation or alveolar bone resorption, such as humans who have or are at risk of gingival swelling or bleeding, and humans who have or are at risk of gingival recession or alveolar bone resorption.

[0026] In relation to the above-described embodiment, the present invention further discloses the following aspects. <1> An agent for inhibiting periodontal pathogen infection, containing Neisseria mucosa bacteria or bacterial culture as the active ingredient. <2> An alveolar bone resorption inhibitor containing Neisseria mucosa bacteria or a culture of bacteria as the active ingredient. <3> A preventive or ameliorative agent for periodontal disease, containing Neisseria mucosa bacteria or a culture of bacteria as the active ingredient. <4> A food product for inhibiting periodontal pathogenic bacterial infections, containing Neisseria mucosa bacteria or bacterial cultures as the active ingredient. <5> A food for inhibiting alveolar bone resorption, containing Neisseria mucosa bacteria or bacterial culture as the active ingredient. <6> A food for preventing or improving periodontal disease, containing Neisseria mucosa bacteria or a culture of bacteria as the active ingredient.

[0027] <7> Use of Neisseria mucosa cells or a culture of cells for producing an agent for inhibiting periodontal pathogen infection. <8> Use of Neisseria mucosa cells or a culture of cells for producing an alveolar bone resorption inhibitor. <9> Use of Neisseria mucosa cells or a culture of cells for producing an agent for preventing or ameliorating periodontal disease. <10> Use of Neisseria mucosa cells or a culture of cells for producing a food for inhibiting periodontal pathogen infection. <11> Use of Neisseria mucosa cells or a culture of cells for producing a food for inhibiting alveolar bone resorption. <12> Use of Neisseria mucosa cells or a culture of cells for producing a food for preventing or improving periodontal disease.

[0028] <13> A Neisseria mucosa cell or cell culture for use in inhibiting periodontal pathogen infection. <14> A bacterium or bacterium culture of Neisseria mucosa for use in inhibiting alveolar bone resorption. <15> A bacterium or bacterium culture of Neisseria mucosa for use in preventing or ameliorating periodontal disease.

[0029] <16> Non-therapeutic use of Neisseria mucosa cells or cell cultures to inhibit periodontal pathogenic bacterial infections. <17> Non-therapeutic use of Neisseria mucosa cells or cell cultures to inhibit alveolar bone resorption. <18> Non-therapeutic use of Neisseria mucosa cells or cell cultures for preventing or ameliorating periodontal disease.

[0030] <19> A method for inhibiting periodontal pathogen infection, comprising administering or ingesting an effective amount of Neisseria mucosa cells or a culture of cells to a subject in need thereof. <20> A method for inhibiting alveolar bone resorption, comprising administering or ingesting an effective amount of Neisseria mucosa cells or a culture of cells to a subject in need thereof. <21> A method for preventing or improving periodontal disease, comprising administering or ingesting an effective amount of Neisseria mucosa bacteria or a culture of bacteria to a subject in need thereof.

[0031] <22> <1> , <4> , <7> , <10> , <13> , <16> or <19> In the method, the periodontal pathogen is preferably one or more species selected from Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Treponema denticola, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, Campylobacter rectus, and Selenomonas sputigena. <23> <1> ~ <22> In the method, the bacterial cell culture is preferably a culture obtained by adding a culture supernatant of Porphyromonas gingivalis to a culture medium of Neisseria mucosa and culturing it, or an EPS-containing culture obtained by adding a culture supernatant of Porphyromonas gingivalis to a culture medium of Neisseria mucosa and culturing it, and then removing the bacterial cells and medium components. <24> A method for producing an EPS-containing culture of Neisseria mucosa, comprising adding a culture supernatant of Porphyromonas gingivalis to a culture solution of Neisseria mucosa, culturing the culture, and then removing the bacterial cells and medium components and recovering the culture. [Example]

[0032] Example 1: Inhibitory effect of Neisseria mucosa on infection with periodontal pathogens (Porphyromonas gingivalis, Fusobacterium nucleatum, Treponella denticola, and Prevotella intermedia) (1)Cell culture conditions Human gingival epithelial cells (Ca9-22) were obtained from the JCRB Cell Bank (JCRB0625). Ca9-22 was cultured in DMEM (Gibco, containing 10% v / v FBS, 1% v / v Penicillin-Streptomycin) at 37°C and 5% CO2.

[0033] (2) Bacterial culture conditions Porphyromonas gingivalis and Fusobacterium nucleatum were obtained from ATCC, and Treponella denticola and Prevotella intermedia were obtained from the Japan Microbial Research Center (JCM) (Table 1). Colony cultures were grown in AnaeroColumbia medium (Becton Dickinson Japan [BD Japan]). Liquid cultures were grown in GAM broth (Nissui Pharmaceutical, containing 5.0 μg / mL hemin, 17.4 μg / mL KHPO, and 1.0 μg / mL vitamin K) at 37°C under anaerobic conditions using Anaeropack Kenki (Mitsubishi Gas Chemical). Six species of Neisseria bacteria were obtained from ATCC or JCM (Table 1). Colony cultures were performed using Brain Heart Infusion (BHI) Agar medium (BD Japan), and liquid cultures were performed using BHI medium (BD Japan) at 37°C.

[0034] Porphyromonas gingivalis was cultured for 24 hours, then diluted 100-fold and cultured in liquid for another 24 hours. Fusobacterium nucleatum, Treponema denticola, and Prevotella intermedia were cultured for 24 hours, then diluted 1000-fold and cultured in liquid for another 24 hours. Neisseria bacteria were cultured for 24 hours, then diluted 100-fold and cultured in liquid for another 48 hours.

[0035] [Table 1]

[0036] (3) Reagents The labeling reagent, Carboxyfluorescein diacetate succinimidyl ester (CFSE), was purchased from Dojindo Chemical Industries, Ltd., dissolved in DMSO, and used at a final concentration of 10 μM.

[0037] (4) Fluorescent labeling method for bacterial cells Cultures of P. gingivalis, Fusobacterium nucleatum, Treponema denticola, and Prevotella intermedia were centrifuged at 3,500 g for 10 minutes at 4°C and the precipitated cells were collected. The collected cells were washed with PBS(-) and then with DMEM (Gibco) at 3,500 g for 5 minutes at 4°C, adjusted to an OD600 of 1.0, and then incubated in 10 μM CFSE solution (dissolving DMEM) at 37°C for 30 minutes in the dark for fluorescent labeling. The fluorescently labeled cells were washed twice with DMEM (Gibco) at 3,500 g for 5 minutes at 4°C, and the turbidity of the cell suspension was adjusted to produce CFSE-labeled P. gingivalis, Fusobacterium nucleatum, Treponema denticola, and Prevotella intermedia.

[0038] (5) Infection method Ca9-22 cells cultured for 24 hours (seeding at 0.3 × 10 6 After removing the medium from the culture plates (24 cells / well in 12-well plates), the plates were washed with PBS(-), and various Neisseria species or Neisseria mucosa suspended in DMEM (Gibco) were added at MOIs of 20 to 500. CFSE-labeled Porphyromonas gingivalis, Fusobacterium nucleatum, Treponema denticola, or Prevotella intermedia was then added at MOIs of 500 and cultured for 2 hours for infection.

[0039] (6) Analysis method of infected cells The medium was removed from Ca9-22 cells infected with CFSE-labeled Porphyromonas gingivalis, Fusobacterium nucleatum, Treponema denticola, or Prevotella intermedia. After washing twice with PBS(-), infected cells were detached using 0.25% Trypsin-EDTA (Gibco) and collected in a 1.5 mL tube. The collected cells were fixed with 400 μL of 4% paraformaldehyde-phosphate buffer (Wako) by pipetting and centrifugation at 4°C for 30 minutes. 800 μL of PBS (containing 2% FBS) was added, pipetting, and centrifuged at 600 g for 3 minutes at 4°C. The cells were then suspended in 400 μL of PBS (containing 2% FBS) for analysis. Flow cytometry (BD, FACSVerse) was used for analysis. The mean fluorescence intensity (MFI) was calculated from the fluorescence intensity in the FITC channel of 500,000 cells gated based on forward scatter (FSC) and side scatter (SSC). Cell infection was assessed by detecting cells attached or infected with fluorescently labeled Porphyromonas gingivalis, Fusobacterium nucleatum, Treponema denticola, and Prevotella intermedia.

[0040] (7) Statistical analysis The results were expressed as mean ± standard error (SE). Differences in mean values were tested using the statistical analysis software SPSS (IBM) with Dunnett's test or Tukey-Kramer test, and a P value of less than 0.05 (P<0.05) was considered statistically significant.

[0041] (8) Results 1) The inhibitory effect of Neisseria mucosa on Porphyromonas gingivalis infection is shown in Figure 1. Figure 1 confirms that Neisseria mucosa inhibits Porphyromonas gingivalis infection of gingival epithelial cells in a manner dependent on the bacterial cell number. 2) The inhibitory effect of Neisseria bacteria on Porphyromonas gingivalis infection is shown in Figure 2. Figure 2 confirms that, among Neisseria bacteria, Neisseria mucosa specifically inhibits infection by Porphyromonas gingivalis. 3) The inhibitory effect of Neisseria mucosa on infections with Fusobacterium nucleatum, Treponema denticola, and Prevotella intermedia is shown in Figure 3. Figure 3 confirms that Neisseria mucosa also inhibits infection by periodontal pathogens other than Porphyromonas gingivalis (Fusobacterium nucleatum, Treponema denticola, and Prevotella intermedia).

[0042] Example 2: Inhibitory effect of Neisseria mucosa cell culture on infection with periodontal pathogens (Porphyromonas gingivalis, Fusobacterium nucleatum, Treponella denticola, and Prevotella intermedia) (1) Preparation of Neisseria mucosa culture Neisseria mucosa was cultured in 300 mL of BHI medium at 37°C for 48 hours to prepare a Neisseria mucosa culture solution. In the group without P. gingivalis culture supernatant, GAM broth was added to the Neisseria mucosa culture medium to a final concentration of 5%. After 48 hours of incubation at 37°C, 20 mL of the Neisseria mucosa culture medium was collected in a 50 mL tube. In the group with P. gingivalis culture supernatant, P. gingivalis culture supernatant adjusted to a final concentration of 5% was added to the Neisseria mucosa culture medium. After 48 hours of incubation at 37°C, 20 mL of the Neisseria mucosa culture medium was collected in a 50 mL tube. The P. gingivalis culture supernatant was prepared by adding a P. gingivalis preculture solution to GAM broth medium (Nissui Pharmaceutical Co., Ltd.) to a final concentration of 1%, culturing the culture solution at 37°C for 24 hours under anaerobic conditions, centrifuging the resulting culture solution at 13,000 g for 15 minutes at 4°C to remove the bacterial cells, and sterilizing the supernatant fraction through a 0.22 μm filter. To remove medium components from the Neisseria mucosa culture, the mixture was centrifuged at 16,000 g for 30 minutes at 4°C. The supernatant was discarded and 20 mL of ultrapure water was added for washing. This washing process was repeated six times. After washing, the mixture was centrifuged again, and the precipitated bacterial culture containing the bacterial cells and EPS was dispersed in ultrapure water and then sonicated. Ultrasonic disruption conditions: After disruption using a BRANSON SONIFIER 150 (power 4, disruption for 20 seconds followed by cooling for 20 seconds, 5 sets), the cells were precipitated at 16,000 g, 4°C, and 30 minutes, and the supernatant was collected in a new 50 mL tube. To completely remove the bacterial cells, the collected supernatant was centrifuged again at 16,000 g at 4°C for 30 minutes, and the supernatant was collected. The collected supernatant was freeze-dried to obtain Neisseria mucosa cultures from which the bacteria and medium components had been removed (groups with and without P. gingivalis culture supernatant added). Figures 4 and 5 show that the addition of P. gingivalis culture supernatant increased the yield of the cultured bacteria.

[0043] (2) The Neisseria mucosa cell culture prepared in (1) was dissolved in DMEM (Gibco) to a final concentration of 0.1%, and the inhibitory effect on infection of gingival epithelial cells by Porphyromonas gingivalis, Fusobacterium nucleatum, Denticola, Prevotella intermedia, and Treponema denticola was confirmed using the same method as in Example 1. The results are shown in Figure 6.

[0044] (3) Results As shown in Figure 6, the bacterial culture of Neisseria mucosa was found to have an inhibitory effect on infection of gingival epithelial cells by periodontal pathogens including Porphyromonas gingivalis, Fusobacterium nucleatum, Treponema denticola, and Prevotella intermedia.

[0045] Example 3: Inhibitory effect of Neisseria mucosa on alveolar bone resorption in a mouse model of periodontal disease (1) Target animals Four-week-old female BALB / c mice (Japan SLC) were used as subjects. Mice were housed at room temperature of 23±2°C, 55±10% humidity, and a 12-hour light-dark cycle (light period: AM7-PM7). During the one-week pre-breeding period, mice were allowed to drink tap water ad libitum and were given CE-2 solid animal food (Japan CLEA) ad libitum.

[0046] (2) Bacterial culture conditions Bacterial culture was carried out in the same manner as in Example 1 using the same strains of Porphyromonas gingivalis as in Example 1, as well as Neisseria mucosa and Neisseria flavescens.

[0047] (3) Porphyromonas gingivalis-infected mouse periodontitis model Evaluation was performed using a Porphyromonas gingivalis-infected periodontitis model based on the method of Kobayashi et al. (R. Kobayashi, et al. J Dent Res (2011) 90(5):653-658). After one week of preliminary breeding, the mice were allowed to drink tap water containing 2 mg / mL sulfamethoxazole (Fujifilm Wako Pure Chemical Industries, Ltd.) and 0.4 mg / mL trimethoprim (Toronto Research Chemicals) for 10 days. After four days of free access to tap water, the mice were assigned to (1) an uninfected group, (2) a Porphyromonas gingivalis monoinfected group, (3) a Porphyromonas gingivalis and Neisseria mucosa co-infected group, or (4) a Porphyromonas gingivalis and Neisseria flavescens co-infected group, so that the mean weights were equal among the groups. After assignment, Porphyromonas gingivalis was administered for 10 days. 9 CFU, Neisseria mucosa and Neisseria flavescens 10 8 The virus was suspended in 100 μL of 2% sodium carboxymethylcellulose (SIGMA) / PBS (Fujifilm Wako Pure Chemical Industries, Ltd.) so that each CFU was equal to 100 μL. Under isoflurane inhalation anesthesia, the virus was instilled into the oral cavity of mice once daily for a total of 15 times (five times per week for a total of three weeks) to induce periodontitis. Similarly, 100 μL of 2% CMC / PBS was instilled daily into the sham group, in which no periodontitis was induced. Twenty-seven days after the final infection, the mice were euthanized by exsanguination under isoflurane inhalation anesthesia, and the maxillae were collected.

[0048] (4) Measurement of alveolar bone resorption The measurement of alveolar bone resorption was performed according to the method of Jeong et al. (Jeong SH, et al. Exp Mol Med (2018) 23; 50 (3): e460). Excess tissue was removed from the mouse jawbone and transferred to a 24-well plate. The tissue was then defleshed by heating in a microwave oven. 1 mL of 3% hydrogen peroxide (Fujifilm Wako Pure Chemical Corporation) was added to the 24-well plate containing the heated jawbone tissue and immersed overnight at room temperature. The plate was washed with pure water, stained with 1% Methylene Blue solution (Fujifilm Wako Pure Chemical Corporation) for 1 minute, and then thoroughly washed with pure water. After staining, the samples were photographed using a stereomicroscope, and the distance from the cementoenamel junction (CEJ) of the maxillary second molar to the alveolar bone crest (ABC) was measured at two points using Image J (arrows in Figure 8(B)). The amount of alveolar bone resorption was evaluated by calculating the average length.

[0049] (5) Statistical analysis The results were expressed as mean ± standard error (SE). Differences in mean values were tested using the Tukey-Kramer test with statistical analysis software SPSS (IBM), and a P value of less than 0.05 (P<0.05) was considered statistically significant.

[0050] (6) Results The inhibitory effect of Neisseria mucosa on alveolar bone resorption is shown in Figure 7, and a photograph of the mouse maxilla after the test is shown in Figure 8(A). Figure 7 confirms that co-infection with Neisseria mucosa suppresses alveolar bone resorption induced by Porphyromonas gingivalis infection. On the other hand, co-infection with Neisseria flavescens, another Neisseria bacterium, did not have any inhibitory effect on alveolar bone resorption.

Claims

1. A periodontal pathogen infection inhibitor containing a bacterial culture of Neisseria mucosa as an active ingredient, wherein the bacterial culture is an EPS-containing culture obtained by adding a culture supernatant of Porphyromonas gingivalis to a culture medium of Neisseria mucosa and culturing the culture medium, and then removing the bacterial cells and medium components.

2. An alveolar bone resorption inhibitor containing a bacterial culture of Neisseria mucosa as an active ingredient, wherein the bacterial culture is an EPS-containing culture obtained by adding a culture supernatant of Porphyromonas gingivalis to a culture medium of Neisseria mucosa and culturing the culture medium, and then removing the bacterial cells and medium components.

3. A preventive or ameliorating agent for periodontal disease, which contains a bacterial culture of Neisseria mucosa as an active ingredient, wherein the bacterial culture is an EPS-containing culture obtained by adding a culture supernatant of Porphyromonas gingivalis to a culture medium of Neisseria mucosa and culturing the culture medium, and then removing the bacterial cells and medium components.

4. A food for suppressing infection with periodontal pathogens, which contains a culture of Neisseria mucosa bacteria as an active ingredient, wherein the culture of the bacteria is a culture obtained by adding a culture supernatant of Porphyromonas gingivalis to a culture solution of Neisseria mucosa, or an EPS-containing culture obtained by adding a culture supernatant of Porphyromonas gingivalis to a culture solution of Neisseria mucosa and culturing it, and then removing the bacteria and medium components. 。

5. A food for inhibiting alveolar bone resorption that contains a bacterial culture of Neisseria mucosa as an active ingredient, wherein the bacterial culture is an EPS-containing culture obtained by adding a culture supernatant of Porphyromonas gingivalis to a culture medium of Neisseria mucosa and culturing it, and then removing the bacterial cells and medium components.

6. A food for preventing or improving periodontal disease, which contains a bacterial culture of Neisseria mucosa as an active ingredient, wherein the bacterial culture is an EPS-containing culture obtained by adding a culture supernatant of Porphyromonas gingivalis to a culture solution of Neisseria mucosa and culturing it, and then removing the bacterial cells and medium components.

7. Periodontal pathogens include Porphyromonas gingivalis and Fusobacterium nucleatum , Prevotella intermedia, Treponema denticola, Tannerella forsa Ishii, Aggregatibacter actinomycetemcomitans, Campylobacter The agent according to claim 1 or the food according to claim 4, wherein the agent is one or more species selected from the group consisting of S. sputigena and S. sputigena.

8. A method for producing an EPS-containing culture of Neisseria mucosa, comprising adding a culture supernatant of Porphyromonas gingivalis to a culture solution of Neisseria mucosa, culturing the culture, and then removing the bacterial cells and medium components and recovering the culture.

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