JP6921655B2 - Protease inhibitors, biofilm formation inhibitors, and oral compositions - Google Patents

Description

The present invention relates to protease inhibitors, biofilm formation inhibitors, and oral compositions.

Porphyromonas gingivalis (hereinafter, also referred to as “P.g”), which is a periodontopathogenic bacterium, produces a potent protease called gingipain.
Gingipain is described in P.I. It is considered to be the main virulence factor of g, and by decomposing collagen that constitutes the gingiva, it directly destroys the periodontal tissue and induces inflammation. In addition, gingipain also degrades immune components such as antibodies to exacerbate inflammation.

P. Sterilization of g can suppress the production of new proteases, but the proteases already produced remain. Therefore, P.I. In addition to sterilization of g, controlling gingipain is considered to be effective in the prevention and treatment of periodontal disease.
As a method for controlling gingipain, a method for inhibiting the activity of the protease gingipain and a method for producing gingipain and gingipain are used. There are a method of suppressing the formation of a biofilm in which g stays and a method of dispersing and removing the formed biofilm.

In addition, in periodontal disease, which is an infectious disease caused by biofilm, P.I. In addition to g, bacteria such as Prevotella intermedia and Fusobacterium nucleatum are also considered to be the causative bacteria associated with periodontal disease. Therefore, P.I. It is considered that suppressing the formation of biofilms by these bacteria as well as g is more effective and important for the prevention and treatment of periodontal disease.
Conventionally, various gingipain inhibitors and biform inhibitors have been developed (see, for example, Patent Document 1).

Japanese Unexamined Patent Publication No. 2014-0000039

Since existing drugs are diluted and washed with saliva, there is a problem in the sustainability of the action. Further, Patent Document 1 discloses a technique for suppressing caries-causing bacteria and periodontopathic bacteria by lactic acid bacteria, and the lactic acid bacteria use lactic acid bacteria isolated from food. On the other hand, lactic acid bacteria found in the oral cavity have the ability to colonize the oral cavity, so that the action is expected to be sustained. However, while lactic acid bacteria usually have an antibacterial action against the causative bacteria of caries and periodontal disease, they do not show protease inhibitory activity. In addition, since lactic acid bacteria usually have a high acid-producing ability to produce acid by assimilating sugar, there is a risk of causing caries.
On the other hand, it is known that oral bacteria include many types of bacteria whose actions are unknown, in addition to pathogenic bacteria whose actions have been elucidated to some extent, such as periodontal disease pathogens.

An object of the present invention is to provide a protease inhibitor, a biofilm formation inhibitor, and an oral composition, which are expected to improve the sustainability of action, have a low risk of dental caries, and are useful for the prevention and treatment of periodontal disease. It is to be.

As a result of diligent studies on the above-mentioned problems, the present inventors focused on bacteria having extremely low acid-producing ability and no concern about caries risk, and have an effect of suppressing collagen decomposition and periodontal disease, which are effective for prevention and treatment of periodontal disease. We have found that the above problems can be solved by finding a specific bacterium having a biofilm formation inhibitory effect on the bacterium, and have completed the present invention.
That is, the present inventors provide the following [1] to [14].
[1] At least one selected from the group consisting of Corynebacterium spp., Catnera spp., And Russian spp. In cultures, culture supernatants, and cultures or culture supernatants of these bacteria. An inhibitor of a protease containing at least one selected from the group consisting of Japanese products as an active ingredient.
[2] The agent according to the above [1], wherein the protease is derived from a periodontal disease pathogen.
[3] The agent according to the above [2], wherein the periodontal disease pathogen is Porphyromonas gingivalis.
[4] The agent according to the above [3], wherein the protease is gingipain.
[5] At least one selected from the group consisting of Corinebacterium spp., Catnera spp., And Russian spp. In cultures, culture supernatants, and cultures or culture supernatants of these bacteria. A biofilm formation inhibitor containing at least one selected from the group consisting of Japanese products as an active ingredient.
[6] The agent according to the above [5], wherein the biofilm is derived from at least one pathogen selected from the group consisting of Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucriatum.
[7] The agent according to any one of the above [1] to [6], wherein the bacterium belonging to the genus Corynebacterium is Corynebacterium matrix.
[8] The agent according to any one of the above [1] to [7], wherein the bacterium of the genus Katonella is Katonella morphi.
[9] The agent according to any one of the above [1] to [8], wherein the Russian bacterium is at least one of Russian Dentocalysa and Russian Mushiraginosa.
[10] At least one selected from the group consisting of at least one of Corynebacterium spp. And Catonella spp., A culture of these bacteria, a culture supernatant, and a culture thereof or a neutralized product of the culture supernatant. An oral composition containing seeds as an active ingredient.
[11] The oral composition according to the above [10], wherein the Corynebacterium genus bacterium is Corynebacterium matrix.
[12] The oral composition according to the above [10] or [11], wherein the bacterium of the genus Katonella is Katonella morphi.
[13] The oral composition according to any one of [10] to [12], which is a dentifrice, a mouthwash, a mouthwash, an oral spray, an oral coating agent, or a mouthwash.
[14] The oral composition according to any one of the above [10] to [12], which is a tablet, gum, gummies, candy, or beverage.

According to the present invention, there are provided a protease inhibitor, a biofilm formation inhibitor, and an oral composition, which are expected to improve the sustainability of action, have a low risk of dental caries, and are useful for the prevention and treatment of periodontal disease. can do.

Hereinafter, the present invention will be described in detail according to the preferred embodiment thereof.

[1. Active ingredient]
In the protease inhibitor or biofilm formation inhibitor of the present invention, the active ingredient is at least one selected from the group consisting of Corinebacterium spp., Catonella spp., And Russian spp., Culture of these bacteria. At least one selected from the group consisting of a substance, a culture supernatant, and the culture or a neutralized product of the culture supernatant.
Further, in the oral composition of the present invention, the active ingredient is at least one of Corynebacterium spp. And Catonella spp., A culture of these bacteria, a culture supernatant, and the culture or the culture supernatant. At least one selected from the group consisting of neutralized products.

The dose of the active ingredient (total dose when used in combination) is ^{preferably 1 × 10 4} to 1 × 10 ¹² per use, and 1 × 10 ⁶ to 1 × 10 ¹¹ per use. Is more preferable, and 1 × 10 ⁷ pieces to 1 × 10 ¹⁰ pieces are even more preferable.

Bacteria that exist as oral bacteria are preferable because they are less toxic even when orally administered. Since the bacterium already exists as an oral bacterium, it is considered that the administration of the bacterium does not work effectively. However, regarding the balance of the proportion of bacteria in the oral cavity, the proportion of bacteria that can suppress periodontal disease can be increased regularly or temporarily to inhibit the activity of proteases, and bacteria can be transmitted from the outside to the entire oral cavity. It is considered that the treatment or prevention of periodontal disease can be expected by suppressing the formation of biofilm in the site where the proportion of the bacterium is low in the oral cavity by administration. The details of the active ingredient will be described below.

[1-1. Corynebacterium spp.]
Species of Corynebacterium spp. Are, for example, Corynebacterium matrusshoti, Corynebacterium durum, Corynebacterium algentratens, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium carnae. , Corynebacterium glutamicum, Corynebacterium meracecola, Corynebacterium ammoniagenes, Corynebacterium efficience.
Of these, Corynebacterium mattleshoti and Corynebacterium durum are preferred.
As these bacteria, those isolated from the oral cavity or commercially available products can be used, and the American Type Culture Collection (hereinafter abbreviated as "ATCC";"ATCC" is a registered trademark) corinebacterium. Matrusshoti ATCC 33806, the same "ATCC 43832", the same "ATCC 14266", the Corinebacterium Durum ATCC 33822 and the like can be used.

When Corynebacterium spp. Are used, the dose ^{is preferably 1 × 10 4} to 1 × 10 ^{12 and} more preferably 1 × 10 ⁶ to 1 × 10 ¹¹ per use. , 1 × 10 ⁷ to 1 × 10 ¹⁰ are even more preferred.

[1-2. Bacteria of the genus Catonella]
Examples of the bacterial species of the genus Katonella include Katonella morbi.
As the Katonella morphi, those separated from the oral cavity or a commercially available product can be used, and the Katonella morphi ATCC 51271 or the like can be used.

When a bacterium of the genus Catonella is used, the dose ^{is preferably 1 × 10 4} to 1 × 10 ¹² per use, more preferably 1 × 10 ⁶ to 1 × 10 ^11. × 10 ⁷ to 1 × 10 ¹⁰ are more preferable.

[1-3. Russian bacteria]
Species of Russian bacteria include, for example, Russian Dentocalysa, Russian Mushiraginosa, and Russian Aeria.
Among these, Russian dentocalysa and Russian mucilaginosa, which also have antibacterial activity against periodontal pathogens and caries pathogens in the presence of nitric acid, are preferable.
For Russian Dentkariosa and Russian Mushiraginosa, those separated from the oral cavity or commercially available products can be used. Russian Dentkariosa ATCC 17931, "ATCC BAA-907", "ATCC 14191", or Russia. Mushiraginosa ATCC 25296, the same "ATCC BAA-2381", the same "ATCC 49042" and the like can be used.

When Russian bacteria are used, the dose ^{is preferably 1 × 10 4} to 1 × 10 ^{12 and} more preferably 1 × 10 ⁶ to 1 × 10 ¹¹ per use. × 10 ⁷ to 1 × 10 ¹⁰ are more preferable.

[1-4. Culture]
The culture is obtained by culturing the above-mentioned bacteria under arbitrary conditions according to a conventional method of culturing using a medium or a nutrient source, and then may be collected by a separation means such as centrifugation or filtration. The collected cells may be washed with physiological saline, antibacterial water, or the like, or the bacteria may be dried by freeze-drying, spray-drying, or the like.

[1-5. Culture supernatant]
The culture supernatant is a supernatant obtained by removing bacteria from the above culture by a separation means such as centrifugation or filtration. The culture supernatant may be concentrated or dried by a method such as concentration under reduced pressure, freeze-drying, or spray-drying.

[1-6. Neutralizer]
The neutralized product is obtained by adjusting the pH of the culture or the culture supernatant to a neutral range (5.6 to 8.5 (temperature 10 to 30 ° C.)) using sodium hydroxide or the like.

[2. Protease inhibitor]
The protease inhibitor of the present invention is described in [1. The active ingredient is the bacterium described in [Active Ingredient] or a culture thereof. The bacteria and their cultures can inhibit the activity of proteases produced by periodontal disease pathogens and suppress collagen decomposition of gingiva and the like. Therefore, the protease inhibitor of the present invention is useful for the treatment and prevention of periodontal disease.

Periodontal disease pathogens include Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigresens, Eikenella corrodence, Treponema denticola, Tanerella forsysensis, Agrigate bacter Actinomycetemcomitans, Fusobacterium nucriatum, Campylobacter.

The protease inhibitor of the present invention decomposes collagen by a protease produced by Porphyromonas gingivalis, which is the most important pathogenic bacterium in the onset and progression of periodontitis among the above-mentioned periodontal disease pathogens. Suppression is observed in the examples described below. Therefore, the protease inhibitor of the present invention is useful for the treatment and prevention of periodontal disease.

Porphyromonas gingipain is a major protease produced by gingipain.
Gingipine is thought to cause decomposition of biological proteins such as collagen and produce pathological conditions such as periodontal disease. In addition, gingipain is known to eliminate the adhesiveness of gingival fibroblasts and vascular endothelial cells and induce cell death. Therefore, the target protease that inhibits the activity is preferably gingipain.

The form of the formulation of the protease inhibitor of the present invention is not particularly limited, and may be appropriately determined according to the purpose of use, such as freeze-dried powder, spray-dried powder, suspension in liquid, capsule, and the like. In addition, the amount of the active ingredient of the protease inhibitor can be appropriately set so as to satisfy the above-mentioned dose according to the form of the final product and the amount used.

[3. Biofilm formation inhibitor]
The biofilm formation inhibitor of the present invention is described in [1. Bacteria described in [Active Ingredients] and their cultures are used as active ingredients to suppress the formation of biofilms formed by pathogenic bacteria. The biofilm formation inhibitor of the present invention is not found to have antibacterial properties against pathogens that form biofilms. Therefore, the biofilm formation inhibitor of the present invention chemically or physically suppresses the adhesion factor to the solid surface corresponding to the biofilm formation site of the pathogenic bacterium forming the biofilm, thereby forming the biofilm. It is presumed that it exerts a formation-suppressing effect, and the biofilm formation-suppressing effect is observed in the examples described later.

Biofilms are strongly constructed by oral bacterial coaggregates and bacterial extracorporeal polysaccharides. In the present invention, the biofilm is a general term for plaque, tartar, tongue coating and the like. When the biofilm is formed, it becomes difficult to sufficiently kill the bacteria inside the biofilm, which leads to the progression of the condition of periodontal disease. Therefore, it is preferable to suppress it at the stage of forming the biofilm.

The pathogens that form biofilms include Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, Streptococcus mutans, Streptococcus sovereignus, Prevotella nigresence, Treponema denticola, Tanerella forsythes, Fusobacterium nucleatum. Examples include Nucleatum and Candida.

The biofilm formation inhibitor of the present invention can suppress the formation of a biofilm by at least one pathogen selected from the group consisting of Porphyromonas gingivalis, Prevotella intermedia, and Fuzobacterium nucriatum among the above pathogens. , It is recognized in the examples described later. Therefore, the biofilm formation inhibitor of the present invention is useful for the treatment and prevention of periodontal disease.

The form of the biofilm formation inhibitor of the present invention is not particularly limited, and may be appropriately determined according to the purpose of use, such as freeze-dried powder, spray-dried powder, suspension in a liquid, or capsule, if desired. In addition, the amount of the active ingredient of the biofilm formation inhibitor can be appropriately set so as to satisfy the above-mentioned dose according to the form of the final product and the amount used.

[4. Oral composition]
The oral composition of the present invention is described in [1. Among the bacteria described in [Active Ingredient], bacteria derived from at least one of Corynebacterium and Catonella, and their cultures, etc. are used as active ingredients.
[2. Protease Inhibitor], the active ingredient can inhibit the activity of protease produced by periodontal disease pathogens and suppress collagen decomposition of gingiva and the like. In addition, [3. As described in [Biofilm formation inhibitor], the active ingredient also has a biofilm formation inhibitory effect. Therefore, it can be said that the oral composition of the present invention is particularly useful for the treatment and prevention of periodontal disease.

The oral composition of the present invention is a toothpaste (toothpaste, powdered toothpaste, liquid toothpaste), mouthwash, mouth refreshing agent, artificial tooth cleaning agent, gargling agent, gingival massage agent, oral coating agent, oral spray agent. It can be used in such a form as.
When the oral composition of the present invention is used in the above form, the blending amount of the active ingredient is preferably 0.001 to 10% by mass with respect to the total amount of the composition.

When the oral composition is used in the above-mentioned form, an appropriate arbitrary ingredient according to various forms can be blended. Optional ingredients include abrasives, surfactants, binders, thickeners, sweeteners, preservatives, flavors, colorants, pH regulators, solvents, solvents, excipients, various medicinal ingredients and the like. ..
In addition, the optional component may be blended in an appropriate amount by one type alone or by appropriately combining two or more types.

(Abrasive)
Examples of the polishing agent include silicic anhydride (hereinafter, silicic acid anhydride as a polishing agent is also referred to as "polishable silica" or "silicic anhydride (polishable)"), crystalline silica, amorphous silica, silica gel, and alumino. Silica-based abrasives such as silicate, zeolite, calcium hydrogen phosphate anhydride, calcium hydrogen phosphate dihydrate, calcium pyrophosphate, calcium carbonate, aluminum hydroxide, alumina, magnesium carbonate, magnesium ferric phosphate, silicic acid Examples thereof include zirconium, calcium tertiary phosphate, hydroxyapatite, calcium tetraphosphate, and synthetic resin-based abrasives.

When the oral composition contains an abrasive, the content of the dentifrice is preferably 2 to 40% by mass, more preferably 5 to 20% by mass of the entire composition.
The abrasive may be used alone or in combination of two or more.

(Surfactant)
As the surfactant, an anionic surfactant, a nonionic surfactant, an amphoteric surfactant and the like can be used.

Examples of the anionic surfactant include N-acylamino acid salt, α-olefin sulfonate, N-acyl sulfonate, alkyl sulfate, sulfate of glycerin fatty acid ester, and the like. Of these, N-acylamino acid salts, α-olefin sulfonates, alkyl sulfates and the like are preferable in terms of versatility, and sodium lauroyl sarcosin and carbon chain lengths of alkyl chains are preferable in terms of foamability and water resistance. As a result, sodium α-olefin sulfonate, sodium lauryl sulfate and the like having 10 to 16 carbon atoms are more preferable.
Examples of the N-acylamino acid salt include an N-acyltaurine salt and the like, which have a linear or branched saturated or unsaturated hydrocarbon group having 8 to 18 carbon atoms, preferably 12 to 16 carbon atoms. Methyl taurine salts such as lauroyl methyl taurine salt, coconut oil fatty acid methyl taurine salt and myristyl methyl taurine salt; methyl alanine salts such as lauroyl methyl alanine salt; glutamates such as lauroyl glutamate and myristoyl glutamate may also be used.

Nonionic surfactants include polyoxyethylene alkyl ether, polyoxyethylene-polyoxypropylene block copolymer, polyoxyethylene hydrogenated castor oil, glycerin ester polyoxyethylene ether, sucrose fatty acid ester, alcoholylamide, and glycerin. Examples include fatty acid esters. Of these, polyoxyethylene alkyl ether, polyoxyethylene hydrogenated castor oil, alquilolamide, sorbitan fatty acid ester and the like are preferably used in terms of versatility. In the polyoxyethylene alkyl ether, the carbon chain length of the alkyl chain is preferably 14 to 18 in terms of the number of carbon atoms. The polyoxyethylene alkyl ether preferably has an average number of moles of ethylene oxide added of 15 to 30. The polyoxyethylene hydrogenated castor oil preferably has an average number of moles of ethylene oxide added (average addition EO) of 20 to 100. The alkylolamide preferably has an alkyl chain having a carbon chain length of 12 to 14 carbon atoms. The sorbitan fatty acid ester preferably has 12 to 18 carbon atoms in the fatty acid. The polyoxyethylene sorbitan fatty acid ester preferably has 16 to 18 carbon atoms in the fatty acid. Further, the polyoxyethylene sorbitan fatty acid ester preferably has an average number of moles of ethylene oxide added of 10 to 40.

Examples of amphoteric surfactants include alkyl betaine-based surfactants, amine oxide-based surfactants, and imidazolinium betaine-based surfactants. Examples of the amphoteric tenside include coconut oil fatty acid amide alkyl betaine, and coconut oil fatty acid amide propyl betaine is preferable.

When the oral composition contains a surfactant, the content thereof is usually 10% by mass or less, preferably 0.01 to 5% by mass, based on the entire oral composition.
The surfactant may be used alone or in combination of two or more.

(Binder)
Examples of the binder include an organic binder and an inorganic binder.

Organic binders include sodium polyacrylate, carrageenan, carboxymethyl cellulose, sodium alginate, xanthan gum, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, propylene glycol alginate, purulan, gelatin, arabic gum, guar gum, etc. Locust bean gum, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer and the like can be mentioned.
Examples of the inorganic binder include silicic anhydride (hereinafter, silicic anhydride as a binder is also referred to as "viscosity silica" or "silicic anhydride (viscosity)"), bentonite and the like. Of these, silicic anhydride (thickening) is preferable.

The organic binder and the inorganic binder may be one kind or a combination of two or more kinds, respectively. The binder may be a combination of an organic binder and an inorganic binder.

When the oral composition contains an organic binder, the content thereof is usually 0.01 to 3.5% by mass with respect to the entire oral composition. When the oral composition contains an inorganic binder, the content thereof is preferably 1 to 10% by mass, more preferably 1 to 7% by mass, and 2 to 6% by mass with respect to the entire oral composition. % Is more preferable.
The binder may be used alone or in combination of two or more.

(Viscosant)
Examples of the viscous agent include propylene glycol, butylene glycol; sugar alcohols such as glycerin, sorbitol (sorbitol), xylitol, erythritol, and maltitol; polyethylene glycol and the like.
When the oral composition contains a viscous agent, the content thereof is usually 5 to 70% by mass with respect to the entire oral composition.

(sweetener)
Examples of the sweetener include sodium saccharin, stebioside, neohesperidin hydrochalcone, glycyrrhizin, perillartine, p-methoxycinnamic aldehyde, thaumatin, palatinose, maltitol, xylitol, arabitol and the like.

When the oral composition contains a sweetener, the content thereof can be appropriately determined as long as the effect of the present invention is not impaired.
The sweetener may be used alone or in combination of two or more.

(Preservative)
Examples of the preservative include paraoxybenzoic acid esters such as sodium benzoate, methylparaben, ethylparaben and butylparaben, ethylenediaminetetraacetic acid salt, benzalkonium chloride and the like.

When the oral composition contains a preservative, the content thereof can be appropriately determined as long as the effect of the present invention is not impaired.
The preservative may be used alone or in combination of two or more.

(Fragrance)
Examples of the fragrance include natural fragrances, synthetic fragrances (single fragrances), blended fragrances (oil-based fragrances (oil-based fragrances), powdered fragrances, etc.).
The fragrance may be used alone or in combination of two or more.

Natural fragrances include musty oil, parsley oil, anis oil, eucalyptus oil, winter green oil, cassia oil, menthol oil, sparemint oil, peppermint oil, lemon oil, coriander oil, orange oil, mandarin oil, lime oil and lavender. Oil, laurel oil, chamomile oil, cardamon oil, caraway oil, bay oil, lemongrass oil, pine needle oil, neroli oil, rose oil, jasmine oil, iris concrete, peppermint absolute, rose absolute, orange flower, citrus oil, Mixed fruit oil, strawberry oil, cinnamon oil, clove oil, grape oil, thyme oil, sage oil, peppermint oil, rosemary oil, perilla oil, majorum oil, origanum oil, grapefruit oil, sweetie oil, yuzu oil, mango absolute, Examples include orange flower absolute, capsicum extract, ginger ole olesin, pepper ole oledin, capsicum ole oledin and the like.

Single fragrances include 1-menthol, carboxylic, eugenol, anator, methyl salicylate, timol, cinnamaldehyde, linalol, linalyl acetate, limonene, osimene, n-decyl alcohol, citronellol, varinine, menthon, menthyl acetate, pinene, octyl aldehyde. , Citral, Pregon, Calvia Acetate, Anisaldehyde, Ethyl Acetate, Ethyl Butyrate, Allyl Cyclohexane Propionate, Methyl Anthranilate, Ethyl Methyl Anthranilate, Vanillin, Undecalactone (γ-Undecalactone, δ- Undecalactone, etc.), Hexanal (trans-2-hexenal, etc.), ethinone alcohol, propyl alcohol, butanol, isoamyl alcohol, hexenol (cis-3-hexenol, etc.), dimethylsulfade, cycloten, furfural, trimethylpyrazine, ethyl Lactate, ethylrioacetate, cineole (1,8-cineole, etc.), mensoflan, linalol oxide, vanillylbutyl ether, isopregol, flaneol, ethylcyclopentenolone, 2-methylbutyric acid, propionic acid, decalactone (γ-) Decalactone, δ-decalactone, etc.), nonalactone (γ-nonalactone, δ-nonalactone, etc.), hexalactone (γ-hexalactone, δ-hexalactone, etc.), isoamyl acetate, benzaldehyde, hexyl acetate, ethyl-2-methylbutyrate, etc. , Lactone alcohol, α-terpineol, phenylethyl glycide, phenylethyl alcohol, allyl hexanoate, methyl cinnamate, ethyl β-methylthiopropionate, cis-6-nonenor, caron, methyl jasmonate and the like. ..

The single fragrance may be a cooling sensation agent. Examples of the cooling agent include menthol, N-ethyl-p-menthan-3-carboxyamide, N- (ethoxycarbonylmethyl) -3-p-menthancarboxyamide, N, 2,3-trimethyl-2-isopropylbutaneamide. , 3- (L-methoxy) Propane-1,2-diol, Menthyl Lactate (Mentyl Lactate), Monomenthol Succinate, Menthone Glycerin Acetal, 3-l-Mentoxy Propane-1,2-diol, Menthone Glycerin Ether, Spirantol , Monomenthol succinate and the like.

The blended fragrance is a fragrance made by blending a single fragrance and / or a natural fragrance. Mental micron, strawberry flavor, apple flavor, banana flavor, pineapple flavor, grape flavor, mango flavor, tropical fruit flavor, butter flavor, milk flavor, yogurt flavor, fruit mix flavor, herb mint flavor and the like can be mentioned.

Fragrances include essential oils such as peppermint, spearmint, rosemary oil, sage oil, perilla oil, lemon oil and orange oil, fruit-based essences such as lemon and strawberry, 1-menthol, carboxylic, eugenol, anator, linalol and limonene. , Osimen, cineol, n-decyl alcohol, citronellol, varinine, α-terpineol, methyl salicylate, timole and other fragrance materials are preferable.

The form of the fragrance is not limited, and it may be an essential oil, an extract, a solid substance, or a powder obtained by spray-drying any of these. When the oral composition contains a perfume material, the content thereof is preferably 0.000001 to 1.5% by mass with respect to the entire oral composition. When a perfume for perfume using the above perfume material is contained, the content thereof is preferably 0.1 to 2.0% by mass with respect to the entire oral composition.

(Colorant)
Coloring agents include natural pigments such as Benibana red pigment, Kuchinashi yellow pigment, Kuchinashi blue pigment, perilla pigment, red koji pigment, red cabbage pigment, carrot pigment, hibiscus pigment, cacao pigment, spirulina blue pigment, and tamarind pigment, and red. Legal dyes such as No. 3, Red No. 104, Red No. 105, Red No. 106, Yellow No. 4, Yellow No. 5, Green No. 3, Blue No. 1, riboflavin, sodium copper chlorophyllin, titanium dioxide and the like can be mentioned.
When the oral composition contains a colorant, the content thereof is preferably 0.00000001 to 3% by mass with respect to the entire oral composition.

(PH regulator)
Examples of the pH adjuster include phosphoric acid or a salt thereof (sodium phosphate, sodium dihydrogen phosphate, etc.), citric acid or a salt thereof (sodium citrate, etc.), malic acid or a salt thereof, gluconic acid or a salt thereof, maleic acid. Or a salt thereof, succinic acid or a salt thereof, glutamic acid or a salt thereof, lactic acid, hydrochloric acid, acetic acid, nitrate, sodium hydroxide, potassium hydroxide, sodium acetate, sodium carbonate and the like.
When the oral composition contains a pH adjuster, the content thereof can be appropriately determined as long as the effects of the present invention are not impaired.

(Solvent, solvent)
Examples of the solvent and solvent include water, ethanol, monohydric lower alcohols such as propanol, propylene glycol, polyethylene glycol and the like.

(Excipient)
Examples of excipients include sugars such as sucrose, glucose, dextrose, converted sugar, fructose, and dextrin, sugar alcohols such as xylitol, erythritol, sorbitol, mannitol, martitol, lactitol, reduced palatinose, and reduced candy, palatinose, trehalose, and oligos. Examples include sugar.

(Medicinal ingredient)
As medicinal ingredients, bacteriostatic or antibacterial agents such as chlorohexidine, triclosan, isopropylmethylphenol, cetylpyridinium chloride, zinc gluconate, zinc citrate; and dentin preventive agents such as condensed phosphate and ethanehydroxydiphosphonate: Anti-inflammatory agents such as tranexamic acid, glycyrrhizin dipotassium salt, ε-aminocaproic acid, aubaku extract; coating agents such as hydroxyethyl cellulosedimethyldialylammonium chloride; allantinchlorohydroxyaluminum, vitamin C, lysozyme chloride, glycyrrhetinic acid and its salts, chloride Astringents such as sodium and allantin; antihypersensitivity agents such as strontium chloride; stain removers such as sodium polyphosphate and sodium metaphosphate; enzyme agents such as dextranase and mutanase can be mentioned.

When the oral composition contains medicinal ingredients, the content can be used within a pharmaceutically acceptable range for each medicinal ingredient.
The medicinal ingredient may be used alone or in combination of two or more.

In addition, the oral composition of the present invention can be applied to foods [1. Active Ingredients] contains the active ingredients listed in Health foods, functional foods, nutritional supplements (supplements), foods for specified health uses, foods for medical use, foods for the sick, foods for infants, foods for nursing care, elderly people. It may be a food or drink composition such as a food for food (hereinafter, also simply referred to as a “food or drink composition”).

The form of the food and drink composition is not particularly limited, and beverages (soft beverages, carbonated beverages, nutritional beverages, powdered beverages, fruit beverages, dairy beverages, jelly beverages, tea, coffee, etc.), confectionery (candy, cookies, chocolate, etc.) Cakes, gums, candies, tablets, gummy, buns, snacks, biscuits, sheepskin, pudding, jelly, ice cream, sherbets, shaved ice, etc., processed marine products (kamaboko, chikuwa, hampen, etc.), processed livestock products (hamburger, ham, etc.) , Sauce, Wiener, cheese, butter, yogurt, cream, margarine, fermented milk, etc.), soup (powdered soup, liquid soup, etc.), main foods (rice, noodles (dried noodles, raw noodles), bread, cereals, etc. ), Seasonings (mayonnaise, shortening, dressing, sauce, sauce, soy sauce, etc.), retort pouch foods (curry, stew, parent and child bowl, porridge, miscellaneous dishes, Chinese bowl, katsudon, tempura bowl, una bowl, hayashi rice, oden, marbo dough , Beef bowl, meat sauce, egg soup, omelet rice, dumplings, shumai, hamburger, meat balls, etc.). It also includes pharmaceuticals such as tablets, capsules, drinks, troches, and oral granules, and quasi-drugs.
When the food and drink composition is used in the above form, the blending amount of the active ingredient is preferably 0.001% to 10% by mass with respect to the total amount of the composition.

The food and drink composition may contain other components as long as the effects of the present invention are not impaired. The other components are not particularly limited and may be appropriately selected depending on the intended purpose. For example, auxiliary raw materials or additives usually used in producing foods and drinks can be mentioned.
The auxiliary raw material or additive is not particularly limited and may be appropriately selected depending on the intended purpose. Frux, granulated sugar, sucrose, sugar, glucose, maltose, sorbitol, maltitol, dextrin, xylitol, gum base, stebioside, rubusoside, corn syrup, lactate, water candy, reduced water candy, corn starch, starch decomposition products, citric acid, tartrate, Malic acid, succinic acid, lactic acid, L-ascorbic acid, l-menthol, dl-α-tocopherol, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, Polyvinylpyrrolidone, Arabic gum, carrageenan, casein, gelatin, pectin, agar, vitamin Bs, vitamin C, nicotinic acid amide, calcium pantothenate, amino acids, calcium salts, crystalline cellulose, gum base, flavors, pigments, fragrances, preservatives , Fruit juice, fine silicon dioxide and the like.

Hereinafter, the present invention will be described in detail with reference to Examples. The following examples are for the purpose of preferably explaining the present invention, and do not limit the present invention.

[1. Collagen decomposition suppression test]
The inhibitory effect of protease was evaluated using the collagen-degrading action of periodontopathic bacteria as an index by the method described below.
200 μL of bacterial solution suspended in 10 mM phosphate buffer (pH 7.4) was added to a 96-well plate containing a collagen sponge (manufactured by KOKEN) under anaerobic conditions (80 vol% nitrogen, 10 vol% carbon dioxide,). 10 vol% hydrogen), allowed to stand at 37 ° C. for about 40 hours. The bacterial solution is added by adjusting the bacterial solution so that the periodontal disease bacteria and the evaluated bacteria are each about 1 × 10 ⁷ cells / 200 μL, and the collagen sponge is decomposed and disappears by the bacterial solution and the periodontal disease bacteria. Was visually observed.
In addition, as a control test, a control sample to which a bacterial solution containing no evaluation bacteria (that is, a bacterial solution containing only periodontal disease bacteria) was prepared, and the decomposition and disappearance of the collagen sponge were visually observed. There was no change in the number of pathogenic bacteria due to the addition of the evaluated bacteria. The evaluation criteria are described below.

(Evaluation criteria)
A: Collagen sponge remains almost completely B: Collagen sponge remains C: Collagen sponge remains slightly D: Collagen sponge does not remain

The bacterial solution was adjusted as follows. Tryptic soy medium (TS medium, manufactured by Becton and Dickinson) supplemented with 0.0001% menadione (manufactured by Wako Pure Chemical Industries, Ltd.) and 0.0005% hemin (manufactured by Sigma Aldrich) at 37 ° C. Under anaerobic conditions (80 vol% nitrogen, 10 vol% carbon dioxide, 10 vol% hydrogen), the following bacteria were cultured to a steady state. The cells were washed twice with 10 mM phosphate buffer (pH 7.4), and the bacteria cultured in the buffer were suspended so that the ^{number of bacteria was about 1 × 10 8 cells / mL.} The bacteria were washed by centrifuging at 10000 G for 10 minutes, discarding the supernatant, and redispersing with the same buffer.

The periodontal disease bacteria and evaluation bacteria used in the test are shown below, and the test results are shown in Table 1.
(Periodontal disease bacteria)
Porphyromonas gingivalis ATCC33277
(Evaluation bacteria)
Corynebacterium matruchotti ATCC33806
Catonella morbi ATCC51271
Russia Dentocariosa ATCC17931
Russian Musilaginosa ATCC15673

From Table 1, when Russian Dentocalysa or Russian Mushiraginosa was added as an evaluation bacterium, the evaluation was B, and the effect of suppressing the decomposition of collagen sponge was good. In addition, when Corynebacterium matrusshoti or Katonella morbi was added as an evaluation bacterium, the evaluation was A, and the effect of suppressing the decomposition of collagen sponge was excellent.
From these results, it can be seen that the evaluation bacterium inhibits the activity of gingipain, which is a protease produced by Porphyromonas gingivalis, which is a periodontal disease bacterium, and is useful for the treatment and prevention of periodontal disease.

[2. Biofilm formation suppression test]
Tryptic soy medium (TS medium, manufactured by Becton and Dickinson) supplemented with 0.0001% menadione (manufactured by Wako Pure Chemical Industries, Ltd.) and 0.0005% hemin (manufactured by Sigma Aldrich) at 37 ° C. Under anaerobic conditions (80 vol% nitrogen, 10 vol% carbon dioxide, 10 vol% hydrogen), the evaluation bacteria and pathogens shown below were cultured to a steady state. The solution suspended in BM medium was subjected to the test so that the number of bacteria was about 1 × 10 ^{8 cells / mL.} The adherent carrier was a hydroxyapatite (HA) plate (manufactured by Asahi Optical Co., Ltd.) having a diameter of 7 mm and a thickness of 3.5 mm, and was immersed in human non-irritating saliva filtered through a 0.45 μm filter for 1 hour (37 ° C.). The surface of the HA plate was coated with saliva components and used for the test.
The BM medium was volumetrically adjusted to 1 L and autoclaved at 121 ° C. for 20 minutes. The composition of the BM medium is shown below.

(Composition of BM medium: expressed by mass in 1 liter)
Proteose peptone (manufactured by Becton and Dickinson): 4 g / L
Tryptone (manufactured by Becton and Dickinson): 2 g / L
Yeast extract (manufactured by Becton and Dickinson): 2 g / L
Hemin (manufactured by Sigma): 2.5 mg / L
Vitamin K (manufactured by Wako Pure Chemical Industries, Ltd.): 0.5 mg / L
KCl (manufactured by Wako Pure Chemical Industries, Ltd.): 1 g / L
Cysteine (manufactured by Wako Pure Chemical Industries, Ltd.): 0.2 g / L
Distilled water: residue

The saliva-coated HA plate was washed once with 10 mM phosphate buffer (pH 7.4), and then a bacterial solution in which 3 pathogens and 1 evaluated bacterium were mixed at a ratio of 1: 1: 1: 1 was added. , 37 ° C., under anaerobic conditions (80 vol% nitrogen, 10 vol% carbon dioxide, 10 vol% hydrogen), and cultured for about 20 hours. After culturing, the HA plate was washed 3 times with 1 mL of 10 mM phosphate buffer (pH 7.4). The biofilm was dispersed by ultrasonic treatment (200 μA, 10 seconds) in 4 mL of the same buffer, and then subjected to 10-fold serial dilution using TS medium. This was smeared on a blood agar plate containing 10% cotton sheep defibrillated blood in an amount of 50 μL, and cultured under anaerobic conditions for about 1 week.
The blood agar plate medium was adjusted to a total volume of 1 L and autoclaved at 121 ° C. for 20 minutes. The composition of the blood agar plate medium is shown below.

(Composition of blood agar plate medium: expressed by mass in 1 liter)
Todd Hewitt Broth (Becton and Dickinson): 30g / L
Agar (manufactured by Becton and Dickinson): 15 g / L
Hemin (manufactured by Sigma-Aldrich): 5 mg / L
Vitamin K (manufactured by Wako Pure Chemical Industries, Ltd.): 1 mg / L
Distilled water: Residual cotton sheep defibrillated blood (manufactured by Japan Biotest Research Institute): 100 mL

From the number of colonies grown in the culture for about 1 week, the number of biofilm bacteria was calculated as cfu (colony forming unit) / HA plate, and the biofilm formation suppression rate was calculated by the following formula.
In addition, a control sample containing no evaluated bacteria was prepared as a bacterial solution, and the target test was carried out in the same manner to calculate the number of control bacteria.
Biofilm formation inhibition rate (%) = ((Number of control bacteria-Number of bacteria added to evaluation bacteria) / (Number of control bacteria)) x 100

The pathogens used in the test, the evaluated bacteria, and the criteria for determining the effect are shown below, and the test results are shown in Table 2. In Table 2, Pg indicates Porphyromonas gingivalis, Pi indicates Prevotella intermedia, and Fn indicates Fusobacterium nucriatum.
(Pathogen)
Porphyromonas gingivalis ATCC33277,
Prevotella intermedia ATCC43146,
Fusobacterium nucleatum ATCC23726
(Evaluation bacteria)
Corynebacterium matruchotti ATCC33806
Catonella morbi ATCC51271
Russia Dentocariosa ATCC17931
Russian Musilaginosa ATCC15673
(Evaluation criteria)
A: Biofilm formation inhibition rate is 75% or more B: Biofilm formation inhibition rate is 50% or more and less than 75% C: Biofilm formation inhibition rate is 25% or more and less than 50% D: Biofilm formation inhibition rate is less than 25%

From Table 2, it can be seen that the formation of the biofilm is suppressed by the addition of the evaluation bacteria. Catnera morbi had a good rating of B against pathogens other than Prevotella intermedia. Russia Dent Caliosa had a rating of B against all pathogens and was uniformly showing good effects. Russian Mushiraginosa was rated A against pathogens other than Porphyromonas gingivalis, and an excellent effect was observed. Corinebacterium matrusshoti was rated A against all pathogens and was uniformly excellent.
From these results, it can be seen that the evaluated bacteria suppress the formation of biofilms caused by pathogenic bacteria and are useful for the treatment and prevention of periodontal disease.

[3. Oral composition]
An example of formulation of the oral composition is shown below.

(Formulation example 1: Toothpaste)
Silicon dioxide (abrasive) 15.0
Sodium lauryl sulfate 1.0
Xanthan gum 0.5
Sodium alginate 0.5
Saccharin sodium 0.1
Sorbitol 10.0
Xylitol 5.0
Corynebacterium mattleshoti (ATCC 33806, approx. 2 x 10 ⁹ pieces / g) 0.2
Fragrance 1.0
Water balance
Total 100.0%

(Formulation example 2: mouthwash)
Propylene glycol 3.0
Glycenrin 15.0
Xylitol 5.0
Polyoxyethylene (60) hardened castor oil 1.0
Corynebacterium mattleshoti (ATCC 33806, approx. 1 x 10 ⁸ pieces / g) 0.01
Saccharin sodium 0.2
Sodium citrate 0.3
Citric acid 0.1
Fragrance 0.5
Water balance
Total 100.0%

(Formulation example 3: mouth refreshing agent)
Ethanol 10.0
Glycenrin 5.0
Xylitol 20.0
Katonera Morbi
(ATCC 51271, about 5 x 10 ⁸ pieces / g) 0.01
Sodium citrate 0.3
Citric acid 0.05
Fragrance 1.0
Water balance
Total 100.0%

(Formulation example 4: Oral spray)
Menthol 0.1
Glycenrin 40.0
Xylitol 5.0
Polyoxyethylene (60) hardened castor oil 5.0
Russian Mushiraginosa (ATCC 25296, approx. 8 x 10 ⁸ pieces / g) 0.02
Sodium citrate 0.25
Citric acid 0.02
Fragrance 0.2
Water balance
Total 100.0%

(Formulation Example 5: Oral coating agent)
Hirodoxy Ethyl Cellulose 2.0
Glycerin 20.0
Russian Mushiraginosa (ATCC 25296, approx. 4 x 10 ⁹ pieces / g) 0.1
Sucrose stearic acid ester 0.5
POE monostearate (20) sorbitan 0.2
Ethyl paraoxybenzoate 0.03
Water balance
Total 100.0%

(Formulation example 6: Gargle)
Glycerin 10.0
Hydroxyethyl cellulose 0.3
Propylene glycol 5.0
Russian Mushiraginosa (ATCC 25296, approx. 4 x 10 ⁸ pieces / g) 0.01
Fragrance 0.5
Water balance
Total 100.0%

(Formulation example 7: Tablet)
Sorbitol 70.0
Maltitol 25.0
Corynebacterium mattleshoti (ATCC 33806, approx. 1 x 10 ⁹ pieces / g) 0.1
Dextrin 0.4
Fragrance 4.0
Fine Silicon Dioxide 0.5
Total 100.0%

(Formulation example 8: gum)
Xylitol 49.0
Maltitol 20.0
Gum base 20.0
Russian Mushiraginosa (ATCC 25296, approx. 4 x 10 ⁹ pieces / g) 0.1
Dextrin 0.5
Gum arabic 9.0
Fragrance 1.0
Total 100.0%

(Formulation example 9: Gummy)
Sugar 30.0
Syrup 30.0
Gelatin 10.0
Russian Dent Caliosa (ATCC 17931, approx. 4 x 10 ⁹ pieces / g) 0.1
Fragrance 0.2
Water balance
Total 100.0%

(Formulation example 10: candy)
Sugar 40.0
Syrup 40.0
Citric acid 2.0
Katonera Morbi
(ATCC 51271, about 5 x 10 ⁸ pieces / g) 0.01
Dextrin 0.1
l-menthol 0.5
Fragrance 0.2
Water balance
Total 100.0%

(Formulation Example 11: Beverage)
Russian Mushiraginosa (ATCC 25296, approx. 4 x 10 ⁸ pieces / g) 0.01
Citric acid 0.5
Vitamin C 0.1
Fragrance 0.5
Water balance
Total 100.0%

Claims (7)

At least one selected from the group consisting of Corinebacterium Matrusotti, Corinebacterium Durum, Catnera morbi, Russian Dentocalysa, Russian Mushiraginosa, and Russian Aeria , cultures of these bacteria, culture supernatants, and the like. An inhibitor of a protease containing at least one selected from the group consisting of a culture or a neutralized product of the culture supernatant as an active ingredient. The agent according to claim 1, wherein the protease is derived from a periodontal disease pathogen. The agent according to claim 2, wherein the periodontal disease pathogen is Porphyromonas gingivalis. The agent according to claim 3, wherein the protease is gingipain. An oral composition for preventing or treating periodontal disease, which comprises the agent according to any one of claims 1 to 4 as an active ingredient. The oral composition according to claim 5 , which is a dentifrice, a mouthwash, a mouthwash, an oral spray, an oral coating, or a mouthwash. The oral composition according to claim 5 or 6 , which is a tablet, gum, gummies, candy, or beverage.