WO2023282356A1 - Oral composition and prophylactic or treatment method for diseases of the oral cavity - Google Patents

Oral composition and prophylactic or treatment method for diseases of the oral cavity Download PDF

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WO2023282356A1
WO2023282356A1 PCT/JP2022/027147 JP2022027147W WO2023282356A1 WO 2023282356 A1 WO2023282356 A1 WO 2023282356A1 JP 2022027147 W JP2022027147 W JP 2022027147W WO 2023282356 A1 WO2023282356 A1 WO 2023282356A1
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nite
oral
bacteria
isolate
lactobacillus plantarum
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PCT/JP2022/027147
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French (fr)
Japanese (ja)
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幹雄 青木
和樹 味方
貴子 清水
敏裕 甲斐
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住友化学株式会社
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Priority to JP2023533205A priority Critical patent/JPWO2023282356A1/ja
Publication of WO2023282356A1 publication Critical patent/WO2023282356A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • the present invention relates to an oral composition and a method for preventing or treating oral diseases.
  • Periodontal disease is caused by pathogenic microorganisms such as periodontal disease bacteria, and is an infectious disease that affects many humans. Compositions containing ingredients with antibacterial activity against pathogenic bacteria have been developed to control periodontal disease.
  • Patent Document 1 one or more bacterial cells or bacterial cell cultures selected from Lactobacillus rhamnosus KO3 strain, Lactobacillus casei YU3 strain and Lactobacillus paracasei YU4 strain A prophylactic or therapeutic agent for oral diseases is disclosed, which contains a substance, etc. as an active ingredient.
  • Patent Document 2 discloses a composition for preventing or treating oral diseases containing Kog1 or Kog3 produced from Lactobacillus rhamnosus KO1 strain and Lactobacillus rhamnosus KO3 strain. ing.
  • Patent Document 3 discloses a toothpaste containing nisin.
  • Patent Document 4 discloses an oral antibacterial composition comprising nisin and polylysine.
  • German Patent Application Publication No. 102011116325 discloses a composition for oral care comprising cells of bacteria belonging to Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus acidophilus or parts thereof. is disclosed.
  • An object of the present invention is to provide a novel composition capable of suppressing oral diseases.
  • the present invention relates to items exemplified below.
  • the Lactobacillus plantarum is at least one selected from NITE-BP-03198, NITE-BP-03199, NITE-BP-03200, NITE-BP-03201 and NITE-BP-03202, The composition according to any one of [1] to [5].
  • a method for preventing or treating oral diseases which comprises applying Lactobacillus plantarum cells, cell cultures, or extracts thereof to the oral cavity of an animal.
  • the method of [9] wherein the oral disease is periodontal disease.
  • a growth inhibitor or bactericidal agent for periodontal disease-causing bacteria containing Lactobacillus plantarum cells, cell cultures, or extracts thereof.
  • a method for inhibiting the growth of or sterilizing periodontal disease bacteria which comprises contacting Lactobacillus plantarum cells, cell cultures, or extracts thereof with periodontal disease-causing bacteria.
  • FIG. 2 shows (A) colony shape and (B) Gram staining results of NITE BP-03198 in Experiment 1.
  • FIG. Fig. 2 shows (A) colony shape and (B) Gram staining results of NITE BP-03199 in Experiment 2.
  • FIG. 10 shows (A) colony shape and (B) Gram staining results of NITE BP-03200 in Experiment 3.
  • FIG. 10 shows (A) colony shape and (B) Gram staining results of NITE BP-03201 in Experiment 4.
  • FIG. FIG. 10 shows (A) colony shape and (B) Gram staining results of NITE BP-03202 in Experiment 5.
  • FIG. FIG. 10 is a diagram illustrating an antibacterial activity evaluation test against bacteria in Experiments 6 to 8.
  • composition for oral cavity An oral composition according to one embodiment of the present invention comprises cells or cell cultures of Lactobacillus plantarum or extracts thereof.
  • the composition according to the present invention has antibacterial activity against periodontal bacteria.
  • Lactobacillus plantarum is preferably at least one selected from NITE-BP-03198, NITE-BP-03199, NITE-BP-03200, NITE-BP-03201 and NITE-BP-03202.
  • NITE-BP-03198, NITE-BP-03199, NITE-BP-03200, NITE-BP-03201 and NITE-BP-03202 are respectively accession number NITE BP-03198 (original deposit date: April 9, 2020) , Accession No. NITE BP-03199 (Original Deposit Date: April 9, 2020), Accession No. NITE BP-03200 (Original Deposit Date: April 9, 2020), Accession No.
  • NITE BP-03201 (Original Deposit Date: April 9, 2020), under the accession number NITE BP-03202 (original deposit date: April 9, 2020), National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (NPMD, Address: 292-0818 Chiba It is a bacterium that has been internationally deposited under the Budapest Treaty at Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Prefecture. All of the above bacteria belong to Lactobacillus plantarum, which is a kind of lactic acid bacteria. The mycological properties of the bacteria are shown in Tables 1 to 10 and FIGS. 1 to 5, which will be described later.
  • NITE-BP-03198 exists in fermented foods, and NITE-BP-03199, NITE-BP-03200, NITE-BP-03201 and NITE-BP-03202 exist in the natural environment, they are highly safe to humans. it is conceivable that.
  • the lactic acid bacteria may be isolated bacteria.
  • the oral composition according to one embodiment of the present invention is at least one selected from NITE-BP-03198, NITE-BP-03199, NITE-BP-03200, NITE-BP-03201 and NITE-BP-03202 bacteria or bacterial cell cultures or extracts thereof.
  • the fungus may be isolated from the fermented food or the environment, or may be cultured.
  • the cells may be dead cells or live cells.
  • the cells may exist in a culture medium, buffer solution, or the like, or may be concentrated to remove the liquid, a lyophilized product thereof, or a frozen stock.
  • the bacterial cell culture may contain bacterial secretions, metabolites, and the like.
  • Cell cultures can contain peptides, proteins, sugars, enzymes, organic acids produced by bacteria, and media containing these (liquid media and solid media).
  • the cell culture may be the supernatant after culturing bacteria.
  • the culture supernatant can be obtained, for example, by removing bacteria from a liquid medium in which bacteria have been cultured, by centrifugation, filtration, or the like.
  • NITE-BP-03198, NITE-BP-03199, NITE-BP-03200, NITE-BP-03201 and NITE-BP-03202 can be cultured according to a normal culture method for lactic acid bacteria.
  • a typical culture method includes a method of culturing at a temperature of 30° C. using MRS (de Mann, Rogosa and Sharpe) liquid medium or MRS agar medium.
  • the bacterial cell or bacterial cell culture extract is prepared so as not to lose the antibacterial activity against periodontal disease bacteria possessed by the lactic acid bacteria cell or bacterial cell culture. Extracts can be obtained, for example, by subjecting bacterial cells or cell cultures to ultrasonic disruption, bead grinding, freeze-thawing, chemical lysis, or the like. The extract may be obtained by salting-out, ultrafiltration, ion-exchange chromatography, liquid-phase extraction using an organic solvent, or the like, on the cells or cell culture. These treatments can be performed in combination as appropriate.
  • the extract may contain bacterial cell fragments, nucleic acids, peptides, proteins, sugars and enzymes. In the present specification, bacterial cells, bacterial cell cultures, or extracts thereof are also referred to as "microbial cell preparations.”
  • the oral composition may be an oral composition that is taken into the body through the oral cavity.
  • the oral composition may be a food product and may be a non-human animal food or feed.
  • the oral composition may be a composition that is used in the oral cavity and is discharged from the oral cavity after use.
  • the oral composition may be a composition for extra-oral use.
  • the composition for extraoral use may be, for example, a denture cleanser.
  • the oral composition can be, for example, an oral care product.
  • the oral composition preferably the oral composition, can contain additive components within a range that does not impair the effects of the present invention.
  • Additives suitable for oral intake include solvents such as water, carbohydrates, proteins, lipids, vitamins, minerals, trace metals essential for living organisms (manganese sulfate, zinc sulfate, magnesium chloride, potassium carbonate, etc.), fragrances, and foods. sanitary or pharmaceutically acceptable carriers, food additives and the like.
  • Additive components can be added as needed by appropriately selecting one or a combination of two or more.
  • Carbohydrates include saccharides, processed starch (dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like.
  • Proteins include whole milk powder, skimmed milk powder, partially skimmed milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, ⁇ -casein, ⁇ -casein, ⁇ -casein, ⁇ -lactoglobulin, Animal and plant proteins such as ⁇ -lactalbumin, lactoferrin, soy protein, chicken egg protein, and meat protein, and their hydrolysates, as well as butter, milk minerals, cream, whey, non-protein nitrogen, sialic acid, and phosphorus Examples include lipids and various milk-derived components such as lactose.
  • Lipids include animal oils such as lard, fish oil, fractionated oils thereof, hydrogenated oils thereof, transesterified oils thereof, palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, Vegetable oils and fats such as these fractionated oils, these hydrogenated oils, and these interesterified oils are included.
  • Vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline, and folic acid. etc.
  • Minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, and selenium.
  • the oral composition may be in an orally ingestible form such as solution, suspension, emulsion, powder, paste, semi-solid molding, or solid molding.
  • Foods include, for example, milk, vegetable milk, milk beverages, soft drinks, fermented milk, lactic acid beverages, lactic beverages, infant formula, liquid milk, liquid diets, foods for the sick, frozen foods.
  • fermented foods processed foods, confectionery, seasonings and other commercially available foods. More specific examples include yoghurt, cheese, ice cream, ice cream, chocolate, tablets, gummies, candies, jellies, gums, breads, biscuits, crackers, pizza crusts, and the like.
  • the oral composition may be supplements, health foods, functional foods, foods for special dietary uses, foods with health claims, foods for specified health uses, foods with nutrient function claims, foods with function claims, quasi-drugs, cosmetics, and the like. , tablets, capsules, powders, granules, jelly and the like.
  • the oral composition preferably the oral care product, can contain additive components within a range that does not impair the effects of the present invention.
  • additive components include fluorine compounds, medicinal ingredients, abrasives, binders, thickening agents, surfactants, corrigents, preservatives, fragrances, coloring agents, pH adjusters, solvents, solubilizers, and bases. , detergents, adsorbents, and the like.
  • the additive component can be appropriately selected according to the dosage form of the composition. Additive components can be added as needed by appropriately selecting one or a combination of two or more.
  • fluorine compounds include sodium fluoride, potassium fluoride, ammonium fluoride, tin fluoride, amine fluoride, sodium monofluorophosphate, potassium monofluorophosphate, sodium silicon fluoride, and calcium silicon fluoride. Sodium fluoride or sodium monofluorophosphate is preferred. Fluorine compounds can inhibit caries.
  • the fluorine compound is used in an amount such that the fluorine concentration in the composition is, for example, 1001 ppm to 3000 ppm, preferably 1001 ppm to 2000 ppm, more preferably 1001 ppm to 1500 ppm.
  • medicinal ingredients bactericides, anti-inflammatory agents, blood circulation promoters, tartar deposition inhibitors, stain removers, hypersensitivity inhibitors, vitamins, crude drug extracts, plaque-degrading enzymes, etc. are added to the oral composition. can do.
  • These medicinal ingredients are not particularly limited as long as they can be used for pharmaceuticals and the like.
  • Bactericides include cationic fungicides such as cetylpyridinium chloride, benzalkonium chloride, benzethonium chloride, chlorhexidine hydrochloride, and chlorhexidine gluconate; amphoteric fungicides such as dodecyldiaminoethylglycine; ionic fungicides; and hinokitiol and the like.
  • Anti-inflammatory agents include ⁇ -glycyrrhetinic acid, glycyrrhetinic acid, glycyrrhizic acid, diammonium glycyrrhizinate, disodium glycyrrhizinate, trisodium glycyrrhizinate, dipotassium glycyrrhizinate, monoammonium glycyrrhizinate, ⁇ -aminocaproic acid, azulene sulfonic acid.
  • Examples of blood circulation promoting agents include sodium chloride.
  • zeolite As a tartar deposition inhibitor, zeolite, disodium hydrogen phosphate, disodium dihydrogen pyrophosphate, sodium pyrophosphate, anhydrous sodium pyrophosphate, tetrasodium pyrophosphate (anhydrous), disodium monohydrogen phosphate, sodium hydrogen phosphate hydrate, disodium hydrogen phosphate (crystal), trisodium phosphate, sodium polyphosphate and the like.
  • macrogol As a stain remover, macrogol (Macrogol 200, Macrogol 300, Macrogol 400, Macrogol 600, Macrogol 1000, Macrogol 1500, Macrogol 1540, Macrogol 4000, Macrogol 6000, Macrogol 20000, etc.) , sodium polyphosphate, polyvinylpyrrolidone, and the like.
  • antihypersensitivity agents examples include potassium nitrate and aluminum lactate.
  • Vitamin preparations include ascorbic acid, L-ascorbic acid, sodium ascorbate, sodium L-ascorbate, pyridoxine hydrochloride, DL- ⁇ -tocopherol acetate, tocopherol acetate, dl- ⁇ -tocopherol nicotinate, tocopherol nicotinate etc.
  • abrasives examples include silicic anhydride, silica (crystalline silica or amorphous silica), silica gel, silica-based abrasives such as aluminosilicate, zeolite, calcium hydrogen phosphate anhydrate, and calcium hydrogen phosphate dihydrate. substances, calcium pyrophosphate, calcium carbonate, aluminum hydroxide, alumina, magnesium carbonate, magnesium phosphate tertiary, zirconium silicate, calcium phosphate tertiary, hydroxyapatite, calcium tetraphosphate, and synthetic resin abrasives.
  • Binders include pullulan, gelatin, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, sodium carboxymethylcellulose, carrageenan, sodium alginate, xanthan gum, sodium polyacrylate, gum arabic, guar gum, locust bean gum, polyvinyl alcohol, polyvinylpyrrolidone, and organic binders such as carboxyvinyl polymer, and inorganic binders such as thickening silicic anhydride and bentonite.
  • thickening agents include polyhydric alcohols (more specifically, sorbitol, glycerin, concentrated glycerin, ethylene glycol, propylene glycol, 1,3-butylene glycol, propanediol (1,3-propanediol), polyethylene glycol, polypropylene glycol, xylitol, maltitol, lactitol, etc.), trehalose, sodium hyaluronate, hydrolyzed collagen, and the like.
  • polyhydric alcohols more specifically, sorbitol, glycerin, concentrated glycerin, ethylene glycol, propylene glycol, 1,3-butylene glycol, propanediol (1,3-propanediol), polyethylene glycol, polypropylene glycol, xylitol, maltitol, lactitol, etc.
  • trehalose sodium hyaluronate
  • hydrolyzed collagen and the like.
  • Surfactants include anionic surfactants such as N-acyl amino acid salts, ⁇ -olefin sulfonates, N-acyl sulfonates, alkyl sulfates (eg, sodium lauryl sulfate, etc.), sulfates of glycerin fatty acid esters. etc.
  • Nonionic surfactants include polyoxyethylene alkyl ether, polyoxyethylene-polyoxypropylene block copolymer, polyoxyethylene hydrogenated castor oil, polyoxyethylene ether of glycerin ester, sucrose fatty acid ester, alkylolamide, glycerin.
  • Amphoteric surfactants include alkyl betaine surfactants, amine oxide surfactants, and imidazolinium betaine surfactants. Specific examples of these include 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine, lauryldimethylaminoacetic acid betaine, coconut oil alkylbetaine (coconut alkyldimethylaminoacetic acid betaine), stearyldimethylaminoacetic acid betaine. .
  • flavoring agents include sodium L-glutamate, saccharin, sodium saccharin, disodium glycyrrhizinate, trisodium glycyrrhizinate, sucrose, glucose, fructose, lactose, honey, aspartame, stevia, sucralose, xylitol, inositol, and D-sorbitol.
  • D-mannitol arabitol, raffinose, lactulose, lactitol, erythritol, reduced palatinose, palatinose, palatinit, acesulfame K, maltose, maltosyltrehalose, maltitol, neohesperidin dihydrochalcone, perillartine, p-methoxycinnamic aldehyde, thaumatin, etc. is mentioned.
  • antiseptics examples include glycine, sodium benzoate, paraoxybenzoic acid esters such as methylparaben, ethylparaben, butylparaben, isopropylparaben, propylparaben, isobutylparaben, and benzylparaben; alcohols such as phenoxyethanol and ethanol; acid, benzoic acid, dehydroacetic acid, propionic acid and salts thereof, ethylenediaminetetraacetate, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, and alkyldiaminoethylglycine hydrochloride.
  • paraoxybenzoic acid esters such as methylparaben, ethylparaben, butylparaben, isopropylparaben, propylparaben, isobutylparaben, and benzylparaben
  • alcohols such as phenoxyethanol and
  • Flavors include, for example, L-menthol, peppermint, spearmint, fruit flavors, and peppermint oil. Perfumes also have the advantage of stimulating salivation.
  • Coloring agents include safflower red pigment, gardenia yellow pigment, gardenia blue pigment, perilla pigment, monascus pigment, red cabbage pigment, carrot pigment, hibiscus pigment, cacao pigment, spirulina blue pigment, and natural pigment such as coumarind pigment, red Legal pigments such as No. 3, Red No. 104, Red No. 105, Red No. 106, Yellow No. 4, Yellow No. 5, Green No. 3, and Blue No. 1, riboflavin, sodium copper chlorophyllin, and titanium dioxide. .
  • pH adjusters include formic acid, lactic acid, acetic acid, hydrochloric acid, sulfuric acid, nitric acid, citric acid, phosphoric acid, malic acid, gluconic acid, maleic acid, succinic acid, glutamic acid, pyrophosphoric acid, tartaric acid, sodium acetate hydroxide, hydroxide Acids, alkalis, and buffers such as potassium, sodium acetate, sodium carbonate, sodium citrate, sodium hydrogen citrate, phosphoric acid, sodium phosphate, sodium monohydrogen phosphate, and sodium dihydrogen phosphate potassium dihydrogen phosphate etc.
  • solvents examples include water and lower alcohols such as ethanol and propanol.
  • a solubilizer may be added to promote the dissolution of the above additives or medicinal ingredients in water.
  • solubilizers include polyhydric alcohols such as propylene glycol, dipropylene glycol, butylene glycol, and polyethylene glycol.
  • bases examples include sodium hydrogen carbonate.
  • cleaning agents examples include sodium polyphosphate.
  • adsorbents examples include ⁇ -cyclodextrin.
  • Oral compositions include, for example, mouthwashes, dentifrices (toothpaste, liquid toothpaste, toothpaste, etc.), mouth fresheners, gums, lozenges, buccal tablets, gingival adhesive tape preparations, intraoral oral gel, oral ointment, oral spray, oral paste, oral paste, oral pill, oral tablet, oral powder, oral powder, oral liquid , intraoral suspensions, intraoral emulsions, intraoral granules, intraoral capsules, and denture cleaners.
  • the oral composition is preferably a mouthwash or dentifrice.
  • Oral compositions may be quasi-drugs, hygiene products or cosmetics.
  • the manufacturing method of the oral composition is not particularly limited.
  • the oral composition may be manufactured by any manufacturing method by which common oral compositions, oral care products, and the like are manufactured.
  • a method for producing an oral composition may include a step of adding the cell preparation of the lactic acid bacterium to any step of a general oral composition, an oral care composition, or the like.
  • the method for producing an oral composition may include, for example, a step of adding the cell preparation of lactic acid bacteria to the oral composition, oral care product and the like produced.
  • the composition for oral cavity may be the bacterial cell preparation itself of the above-mentioned lactic acid bacteria.
  • the oral composition may contain liquids, spreading agents, and the like to facilitate the attachment, absorption, or mixing of the lactic acid bacteria cell preparation with the oral composition, oral care products, and the like.
  • the amount of the lactic acid bacteria cell preparation contained in the oral cavity composition is not particularly limited as long as it exhibits an antibacterial activity against periodontal disease bacteria.
  • the total amount of the cell preparation of lactic acid bacteria in the oral composition may be, for example, 1 ⁇ 10 3 to 1 ⁇ 10 12 cfu (colony forming units)/g. 12 cfu/g, or 1 ⁇ 10 5 to 1 ⁇ 10 12 cfu/g.
  • the colony-forming unit can be determined by an agar plate culture method.
  • the total amount (content ratio) of the bacterial cell preparation of lactic acid bacteria in the oral composition is 0.0001% by mass to 30% by mass, preferably 0.001% by mass to 10% by mass relative to the weight of the oral composition. %.
  • the total concentration of the lactic acid bacteria cell preparation in the oral composition may be 0.1 to 100,000 ppm, may be 1 to 100,000 ppm, or may be 10 to 100,000 ppm. may The total amount can be weighed with a balance or the like.
  • the oral composition can be applied to animals.
  • the animal may be a human or non-human animal.
  • Animals include mammals. Mammals may include Rodentia, Lagomorpha, Carnivora, Cetatodactyla, Perissodactyla, Primate. More specifically, the above mammals include mice, rats, hamsters, guinea pigs, hedgehogs, ferrets, rabbits, dogs, cats, cows, pigs, goats, horses, sheep, monkeys, orangutans, chimpanzees and the like. Animals may be pets, farm animals or laboratory animals.
  • One embodiment of the present invention is the use of Lactobacillus plantarum cells or cell cultures or extracts thereof in the production of oral compositions.
  • the oral composition according to the present invention may be a preventive or therapeutic composition for oral diseases.
  • treatment includes alleviation of symptoms, amelioration of symptoms, and complete cure.
  • the prophylactic or therapeutic composition may be pharmaceuticals or veterinary drugs, quasi-drugs, cosmetics, and the like.
  • Lactobacillus plantarum exhibits antimicrobial activity against causative bacteria of oral diseases
  • Lactobacillus plantarum can be used to prevent or treat oral diseases.
  • Oral diseases include periodontal diseases such as gingivitis and periodontitis; dental caries; oral candidiasis; mucositis; glossitis; Bacteria that cause periodontal disease include Porphyromonas gingivalis (hereinafter also referred to as "Pg bacteria”) and Tannerella forsythia (hereinafter also referred to as Tf bacteria). ), Treponema denticola (hereinafter also referred to as "T.d.
  • Caries-causing bacteria include Streptococcus mutans, Streptococcus sobrinus, and the like.
  • Candida fungi include Candida albicans, Candida glabrata, Candida tropicalis, and the like.
  • compositions containing a bacterial cell preparation of Lactobacillus plantarum are effective in preventing or treating oral diseases.
  • compositions containing bacterial cell preparations of Lactobacillus plantarum are more effective than antibiotics (minocycline, etc.) and chemical fungicides (cetylpyridinium chloride (CPC), isopropylmethylphenol (IPMP), etc.) on oral flora. Less is. For this reason, compositions containing a cell preparation of Lactobacillus plantarum are less likely to unbalance the oral microflora. Cell preparations of Lactobacillus plantarum can also inhibit the growth of bacteria that promote plaque formation. Bacteria that promote plaque formation include, for example, Streptococcus gordonii (hereinafter also referred to as “Gordonii”).
  • the preventive or therapeutic composition may or may not contain other antibacterial agents and antibiotics against causative bacteria of oral diseases.
  • the dosage form and manufacturing method of the preventive or therapeutic composition may be the same as the dosage form and manufacturing method of the composition described above.
  • the content of the bacterial cell preparation of Lactobacillus plantarum in the preventive or therapeutic composition is not particularly limited as long as it exhibits antibacterial activity against periodontal disease bacteria, and is the same range as the content in the oral composition. can be
  • One embodiment of the present invention is the use of Lactobacillus plantarum cells or cell cultures or extracts thereof in the manufacture of compositions for the prevention or treatment of oral diseases.
  • a method for preventing or treating oral diseases includes applying Lactobacillus plantarum cells or cell cultures or extracts thereof to the oral cavity of an animal.
  • the animal may be a human or non-human animal.
  • Oral diseases include periodontal diseases such as gingivitis and periodontitis; dental caries; oral candidiasis; mucositis; glossitis;
  • the bacterial cell preparation of Lactobacillus plantarum may be orally ingested, or may be excreted out of the oral cavity after being used in the oral cavity.
  • Cells or cell cultures of Lactobacillus plantarum or extracts thereof may be supplied as the intraoral composition.
  • One embodiment of the present invention is the use of Lactobacillus plantarum cells or cell cultures or extracts thereof for the prevention or treatment of oral diseases.
  • a growth inhibitor or fungicide for periodontal disease-causing bacteria contains Lactobacillus plantarum cells, cell cultures, or extracts thereof.
  • Periodontal disease-causing bacteria include, for example, at least one selected from Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola.
  • Lactobacillus plantarum cells, cell cultures, or extracts thereof can inhibit the growth of or kill periodontal disease-causing bacteria, and particularly have antibacterial activity against all of the above three causative bacteria.
  • the antiproliferative agent or bactericidal agent may be in the same form as the oral composition.
  • Antiproliferative agents or antiseptics may or may not include other antibacterial agents and antibiotics against causative agents of oral disease.
  • the content of the lactic acid bacteria cell preparation contained in the growth inhibitor or bactericide is not particularly limited, but may be, for example, 1 ⁇ 10 3 to 1 ⁇ 10 12 cfu/mL, or 1 ⁇ 10 5 to 1 ⁇ 10 cfu/mL. It may be x10 12 cfu/mL, or may be from 1 x 10 7 to 1 x 10 12 cfu/mL.
  • the colony-forming unit can be determined by an agar plate culture method.
  • the total concentration of the lactic acid bacteria cell preparation contained in the growth inhibitor or disinfectant is not particularly limited, and may be 0.1 to 100,000 ppm, or 1 to 100,000 ppm. , from 10 to 100,000 ppm. The total concentration can be weighed with a balance or the like.
  • a method for inhibiting or sterilizing the growth of periodontal disease-causing bacteria comprises contacting Lactobacillus plantarum bacteria or bacterial cell cultures or extracts thereof with periodontal disease-causing bacteria. Including.
  • the method of contacting the bacterial cell preparation of Lactobacillus plantarum with the causative bacteria of periodontal disease is not particularly limited. may be added, an object in which periodontal disease-causing bacteria are present may be immersed in a liquid containing a bacterial cell preparation of Lactobacillus plantarum, and a host infected with periodontal disease-causing bacteria may be lacto Compositions containing the bacterial cell preparation of Bacillus plantarum may be administered orally.
  • the bacterial cell preparation of Lactobacillus plantarum may be supplied in the form of a periodontal disease-causing bacteria growth inhibitor or bactericide.
  • One embodiment of the present invention is the use of Lactobacillus plantarum cells or cell cultures or extracts thereof in the production of growth inhibitors or fungicides for periodontal disease-causing bacteria.
  • One embodiment of the present invention is the use of Lactobacillus plantarum cells or cell cultures or extracts thereof for growth inhibition or sterilization of periodontal disease-causing bacteria.
  • Isolate A was identified by 16S rRNA gene analysis, morphological observation, and physiological and biochemical characterization tests.
  • 16S rRNA gene analysis Genomic DNA was extracted from the isolate A, and the resulting genomic DNA was used as a template for cloning forward primer 9F and cloning reverse primer 1510R (Yoshiyoshi Nakagawa et al.: Genetic analysis method of 16S rRNA gene.
  • PCR amplification of the 16S rRNA gene was performed using a nucleotide sequencing method, Actinomycetes Society of Japan, Classification and Identification of Actinomycetes, 88-117 pp. Japan Society Office, 2001).
  • PCR amplification was performed using Tks Gflex DNA polymerase (manufactured by Takara Bio Inc.), and the amplified product after PCR was purified.
  • a cycle sequencing reaction was performed using the purified amplified product after PCR.
  • the cycle sequencing reaction was performed using BigDye Terminator v3.1 Cycle Sequencing Kit.
  • the resulting reaction solution was purified, and the purified solution was subjected to DNA sequence analysis (3130xl DNA Analyzer) to determine the base sequence of the 16S rRNA gene of the template DNA extracted from isolate A.
  • Sequence analysis primers include 9F, 515F, 1099F, 536R, 926R, and 1510R (Yoshiyoshi Nakagawa et al.: Gene analysis method 16S rRNA gene sequencing method, Actinomycetes Society of Japan, Classification and Identification of Actinomycetes, 88 -117 pp. Japan Society Administrative Center, 2001) was used.
  • the base sequence of the 16S rRNA gene of isolate A was identified as a microorganism identification database DB-BA15.0 (manufactured by Techno Suruga Lab), an international base sequence database A BLAST homology search was performed against (DDBJ/ENA(EMBL)/GenBank).
  • the nucleotide sequence of the 16S rRNA gene of isolate A has 99.87% identity to the nucleotide sequence of the 16S rRNA gene of Lactobacillus pentosus (JCM1558), Lactobacillus plantarum subsp.
  • isolate A formed circular colonies.
  • isolate A was positive for Gram staining.
  • Tables 1 and 2 show the results of physiological/biochemical property tests and fermentability tests of isolate A. Isolate A was a non-motile Gram-positive bacillus, did not form spores, was negative in catalase and oxidase reactions, and fermented glucose. These properties were consistent with those of the genus Lactobacillus, for which the possibility of attribution was shown as a result of 16S rDNA partial nucleotide sequence analysis.
  • isolate A fermented galactose, fructose, melezitose, etc., but did not ferment glycerol, D-xylose, etc.
  • Isolate A showed no arginine dihydrolase activity and grew at 15°C. These properties were attributed to L. cerevisiae, whose attribution was suggested as a result of 16S rDNA partial nucleotide sequence analysis.
  • pentosus and L. Among L. plantarum, L. plantarum shows no fermentation of glycerol and D-xylose. Unlike L. pentosus, L. It matched the properties of plantarum. Therefore, isolate A was found to be a novel isolate belonging to Lactobacillus plantarum. The isolate A was internationally deposited as NITE BP-03198.
  • Isolate B was isolated by the same method as Experiment 1, except that Lembu was used as the isolation source. Identification of isolate B was performed by the same method as in Experiment 1.
  • the base sequence of the 16S rRNA gene of isolate B has 100.0% identity to the base sequence of the 16S rRNA gene of Lactobacillus pentosus (JCM1558), Lactobacillus plantarum subsp. It showed 100.0% identity to the nucleotide sequence of the 16S rRNA gene of Lactobacillus (JCM1149) and 99.80% identity to the nucleotide sequence of the 16S rRNA gene of Lactobacillus paraplantarum (DSM10667).
  • isolate B formed circular colonies. As shown in FIG. 2B, isolate B was positive for Gram staining. Tables 3 and 4 show the results of physiological and biochemical property tests and fermentability tests of isolate B. Isolate B was a non-motile Gram-positive bacillus, did not form spores, was negative in catalase and oxidase reactions, and fermented glucose. These properties were consistent with those of the genus Lactobacillus, for which the possibility of attribution was shown as a result of 16S rDNA partial nucleotide sequence analysis.
  • isolate B fermented galactose, fructose, ⁇ -methyl-D-mannoside and melezitose, etc., but did not ferment glycerol, D-xylose, etc.
  • Isolate B showed no arginine dihydrolase activity and grew at 15°C. These properties were attributed to L. cerevisiae, whose attribution was suggested as a result of 16S rDNA partial nucleotide sequence analysis.
  • pentosus and L Among L. plantarum, L. plantarum shows no fermentation of glycerol and D-xylose. Unlike L. pentosus, L. It matched the properties of plantarum. Therefore, isolate B was found to be a novel isolate belonging to Lactobacillus plantarum. Isolate B was internationally deposited as NITE BP-03199.
  • the base sequence of the 16S rRNA gene of isolate C has 99.87% identity to the base sequence of the 16S rRNA gene of Lactobacillus pentosus (JCM1558), Lactobacillus plantarum subsp. It showed 99.87% identity to the nucleotide sequence of the 16S rRNA gene of Lactobacillus (JCM1149) and 99.66% identity to the nucleotide sequence of the 16S rRNA gene of Lactobacillus paraplantarum (DSM10667). However, there was no microorganism with a 16S rRNA gene that completely matched the base sequence of the isolate C 16S rRNA gene.
  • isolate C formed circular colonies. As shown in FIG. 3B, isolate C was positive for Gram staining. Tables 5 and 6 show the results of physiological and biochemical property tests and fermentability tests of isolate C. Isolate C was a non-motile Gram-positive bacillus, did not form spores, was negative in catalase and oxidase reactions, and fermented glucose. These properties were consistent with those of the genus Lactobacillus, for which the possibility of attribution was shown as a result of 16S rDNA partial nucleotide sequence analysis.
  • isolate C fermented galactose, fructose, ⁇ -methyl-D-mannoside and melezitose, etc., but did not ferment glycerol, D-xylose, etc.
  • Isolate C showed no arginine dihydrolase activity and grew at 15°C. These properties were shown to be closely related as a result of 16S rDNA partial nucleotide sequence analysis.
  • pentosus and L Among L. plantarum, L. plantarum shows no fermentation of glycerol and D-xylose. Unlike L. pentosus, L. It matched the properties of plantarum. Therefore, isolate C was found to be a novel isolate belonging to Lactobacillus plantarum. Isolate C was internationally deposited as NITE BP-03200.
  • the base sequence of the 16S rRNA gene of isolate D has 99.93% identity to the base sequence of the 16S rRNA gene of Lactobacillus pentosus (JCM1558), Lactobacillus plantarum subsp. It showed 99.93% identity to the nucleotide sequence of the 16S rRNA gene of Lactobacillus (JCM1149) and 99.73% identity to the nucleotide sequence of the 16S rRNA gene of Lactobacillus paraplantarum (DSM10667). However, no microorganism with a 16S rRNA gene that completely matches the base sequence of the 16S rRNA gene of isolate D was found.
  • isolate D formed circular colonies.
  • isolate D was positive for Gram staining.
  • Tables 5 and 6 show the results of physiological/biochemical property tests and fermentability tests of isolate D. Isolate D was a non-motile Gram-positive bacillus, did not form spores, was negative in catalase and oxidase reactions, and fermented glucose. These properties were consistent with those of the genus Lactobacillus, for which the possibility of attribution was shown as a result of 16S rDNA partial nucleotide sequence analysis.
  • isolate D fermented galactose, fructose and melezitose, etc., but did not ferment glycerol, D-xylose, etc.
  • Isolate D showed no arginine dihydrolase activity and grew at 15°C. These properties were attributed to L. cerevisiae, whose attribution was suggested as a result of 16S rDNA partial nucleotide sequence analysis.
  • pentosus and L. Among L. plantarum, L. plantarum shows no fermentation of glycerol and D-xylose. Unlike L. pentosus, L. It matched the properties of plantarum. Therefore, isolate D was found to be a novel isolate belonging to Lactobacillus plantarum. Isolate D was internationally deposited as NITE BP-03201.
  • Isolate E was isolated by the same method as in Experiment 1, except that Pinus chinensis was used as the isolation source. Identification of isolate E was performed by the same method as in Experiment 1.
  • the nucleotide sequence of the 16S rRNA gene of isolate E has 99.93% identity to the nucleotide sequence of the 16S rRNA gene of Lactobacillus pentosus (JCM1558), Lactobacillus plantarum subsp. It showed 99.93% identity to the nucleotide sequence of the 16S rRNA gene of Lactobacillus (JCM1149) and 99.73% identity to the nucleotide sequence of the 16S rRNA gene of Lactobacillus paraplantarum (DSM10667). However, no microorganism with a 16S rRNA gene that completely matches the base sequence of the isolate E 16S rRNA gene was found.
  • isolate E formed circular colonies. As shown in FIG. 5B, isolate E was positive for Gram staining. Tables 9 and 10 show the results of physiological and biochemical property tests and fermentability tests of isolate E. Isolate E was a non-motile Gram-positive bacillus, did not form spores, was negative in catalase and oxidase reactions, and fermented glucose. These properties were consistent with those of the genus Lactobacillus, for which the possibility of attribution was shown as a result of 16S rDNA partial nucleotide sequence analysis.
  • isolate E fermented galactose, fructose, ⁇ -methyl-D-mannoside and melezitose, etc., but did not ferment glycerol, D-xylose, etc.
  • Isolate E showed no arginine dihydrolase activity and grew at 15°C. These properties were attributed to L. cerevisiae, whose attribution was suggested as a result of 16S rDNA partial nucleotide sequence analysis.
  • pentosus and L Among L. plantarum, L. plantarum shows no fermentation of glycerol and D-xylose. Unlike L. pentosus, L. It matched the properties of plantarum. Therefore, isolate E was found to be a novel isolate belonging to Lactobacillus plantarum. The isolate E was internationally deposited as NITE BP-03202.
  • periodontal disease-causing bacteria Pg, Tf and Td
  • the causative bacteria of periodontal disease were suspended in GAM broth, and McFarland No. 0.5 (approximately 1 ⁇ 10 8 to 2 ⁇ 10 8 cfu/mL). This was further diluted 10-fold to obtain a periodontal disease-causing bacterial solution 11 (about 1 ⁇ 10 7 to 2 ⁇ 10 7 cfu/mL).
  • 100 ⁇ L of periodontal disease bacterium liquid 11 was dropped on an agar medium 12 for measurement, and was evenly applied with a Conlarge stick.
  • the bacterial cell preparation of Lactobacillus plantarum is obtained by culturing any of NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201 and NITE BP-03202 in MRS Broth at a temperature of 30 ° C. Then, the culture supernatant (cell culture) obtained by sedimenting the cells by centrifugation was used. P. g. fungus, T. f. fungi and T. d. The results of antibacterial activity against fungi are shown in Tables 12, 13 and 14, respectively. A medium for lactic acid bacteria (MRS Broth) was used as a negative control.
  • Sensidisc “MEPM” manufactured by Nippon Becton Dickinson
  • Sensidisc “LVFX” manufactured by Nippon Becton Dickinson
  • ATCC-11454 is a nisin-A producing lactic acid bacterium (Lactococcus subsp. lactis). The nisin A-producing strain was cultured in the same manner as Lactobacillus plantarum, and the culture supernatant was prepared.
  • NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201 and NITE BP-03202 are P. g. fungus, T. f. fungi and T. d.
  • Both bacterial cell cultures of Lactobacillus plantarum are P. g. fungus, T. f. fungi and T. d. It was shown that the growth of bacteria can be suppressed.
  • the culture supernatant of the nisin A-producing strain was T. cerevisiae. d. Although it inhibited the growth of P. g. fungi and T. f. It was not possible to suppress the growth of bacteria.
  • Experiment 7 was performed in the same manner as Experiment 6, except that Goldoni bacteria were used as bacteria for verifying antibacterial activity.
  • Gordonii was pre-cultured according to the medium and culture conditions described in Table 15.
  • Gordonii bacteria were suspended in GAM broth and added to McFarland No. 0.5 (approximately 1 ⁇ 10 8 to 2 ⁇ 10 8 cfu/mL). This was further diluted 10-fold to obtain Gordonii fungus solution 11 (about 1 ⁇ 10 7 to 2 ⁇ 10 7 cfu/mL). 100 ⁇ L of the Gordonii fungus solution 11 was dropped on the agar medium 12 for measurement, and was evenly applied with a Conlarge stick.
  • Experiment 8 was carried out in the same manner as Experiment 6, except that B. salivarius was used as the bacterium for verifying the antibacterial activity.
  • B. salivarius was pre-cultured according to the medium and culture conditions described in Table 17.
  • Salivarius was suspended in GAM broth and plated with McFarland No. 0.5 (approximately 1 ⁇ 10 8 to 2 ⁇ 10 8 cfu/mL). This was further diluted 10-fold to obtain Salivarius bacterial solution 11 (approximately 1 ⁇ 10 7 to 2 ⁇ 10 7 cfu/mL). 100 ⁇ L of the bacterial solution 11 of Salivarius salivarius was dropped on the agar medium 12 for measurement, and was evenly applied with a Conlarge stick.
  • NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201 and NITE BP-03202 caused growth inhibition circles to form in the growth medium of Salivarius. It was shown that the bacterial cell culture of Lactobacillus plantarum has little effect on oral flora. On the other hand, tetracycline also suppressed the growth of oral flora.
  • Bacteria that cause periodontal disease, bacteria that promote plaque formation, or fungal fluids of oral bacteria 12. Agar medium for measurement, 13. Lactobacillus plantarum bacterial cell preparation, 14. Growth inhibition circle.

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Abstract

Provided is an oral composition that includes Lactobacillus plantarum cells or cell cultures or an extract of these. The Lactobacillus plantarum is ideally at least one type selected from NITE-BP-03198, NITE-BP-03199, NITE-BP-03200, NITE-BP-03201, and NITE-BP-03202. Also provided is a prophylactic or therapeutic method for oral cavity diseases, that includes the application of Lactobacillus plantarum cells or cell cultures or an extract of these to the oral cavity of animals.

Description

口腔用組成物、および口腔内疾患の予防または治療方法Oral composition and method for prevention or treatment of oral disease
　本発明は、口腔用組成物、および口腔内疾患の予防または治療方法に関する。 The present invention relates to an oral composition and a method for preventing or treating oral diseases.
　歯周病は、歯周病菌等の病原微生物によって引き起こされ、人類の多くが罹患する感染症である。歯周病を抑制するために、病原菌に対する抗菌活性を有する成分を含む組成物が開発されている。国際公開第２０１１／００７５８４号（特許文献１）には、ラクトバチルス・ラムノーサスＫＯ３株、ラクトバチルス・カゼイＹＵ３株およびラクトバチルス・パラカゼイＹＵ４株から選ばれる１種以上の細菌の菌体もしくは菌体培養物等を有効成分する口腔内疾患の予防または治療剤が開示されている。国際公開第２０１２／１０８５１８号（特許文献２）には、ラクトバチルス・ラムノーサスＫＯ１株およびラクトバチルス・ラムノーサスＫＯ３株から産生されるＫｏｇ１またはＫｏｇ３を含む口腔内疾患の予防または治療用組成物が開示されている。 　Periodontal disease is caused by pathogenic microorganisms such as periodontal disease bacteria, and is an infectious disease that affects many humans. Compositions containing ingredients with antibacterial activity against pathogenic bacteria have been developed to control periodontal disease. In International Publication No. 2011/007584 (Patent Document 1), one or more bacterial cells or bacterial cell cultures selected from Lactobacillus rhamnosus KO3 strain, Lactobacillus casei YU3 strain and Lactobacillus paracasei YU4 strain A prophylactic or therapeutic agent for oral diseases is disclosed, which contains a substance, etc. as an active ingredient. International Publication No. 2012/108518 (Patent Document 2) discloses a composition for preventing or treating oral diseases containing Kog1 or Kog3 produced from Lactobacillus rhamnosus KO1 strain and Lactobacillus rhamnosus KO3 strain. ing.
　中国特許出願公開第１０８６８５７１６号明細書（特許文献３）には、ナイシンを含む練り歯磨きが開示されている。中国特許出願公開第１０５３２６６５６号明細書（特許文献４）には、ナイシンおよびポリリジンを含む口腔用抗菌組成物が開示されている。独国特許出願公開第１０２０１１１１６３２５号明細書（特許文献５）には、ラクトバチルス・クリスパタス、ラクトバチルス・ガセリおよびラクトバチルス・アシドフィルスに属する細菌の菌体またはその一部を含む口腔ケア用の組成物が開示されている。 Chinese Patent Application Publication No. 108685716 (Patent Document 3) discloses a toothpaste containing nisin. Chinese Patent Application Publication No. 105326656 (Patent Document 4) discloses an oral antibacterial composition comprising nisin and polylysine. German Patent Application Publication No. 102011116325 (Patent Document 5) discloses a composition for oral care comprising cells of bacteria belonging to Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus acidophilus or parts thereof. is disclosed.
国際公開第２０１１／００７５８４号WO2011/007584 国際公開第２０１２／１０８５１８号WO2012/108518 中国特許出願公開第１０８６８５７１６号明細書Chinese Patent Application Publication No. 108685716 中国特許出願公開第１０５３２６６５６号明細書Chinese Patent Application Publication No. 105326656 独国特許出願公開第１０２０１１１１６３２５号明細書DE 102011116325 A1
　本発明は、口腔内疾患を抑制し得る新規組成物を提供することを目的とする。 An object of the present invention is to provide a novel composition capable of suppressing oral diseases.
　本発明は、以下に例示する項目に関する。
［１］　ラクトバチルス・プランタラムの菌体もしくは菌体培養物またはこれらの抽出物を含む口腔用組成物。
［２］　食品である、［１］に記載の組成物。
［３］　前記食品は発酵食品である、［２］に記載の組成物。
［４］　洗口剤または歯磨剤である、［１］に記載の組成物。
［５］　フッ素化合物をさらに含む、［１］または［４］に記載の組成物。
［６］　前記ラクトバチルス・プランタラムは、ＮＩＴＥ－ＢＰ－０３１９８、ＮＩＴＥ－ＢＰ－０３１９９、ＮＩＴＥ－ＢＰ－０３２００、ＮＩＴＥ－ＢＰ－０３２０１およびＮＩＴＥ－ＢＰ－０３２０２から選択される少なくとも１つである、［１］～［５］のいずれかに記載の組成物。
［７］　口腔内疾患の予防または治療用組成物である、［１］～［６］のいずれかに記載の組成物。
［８］　前記口腔内疾患は歯周病である、［７］に記載の組成物。
［９］　ラクトバチルス・プランタラムの菌体もしくは菌体培養物またはこれらの抽出物を動物の口腔に適用することを含む、口腔内疾患の予防または治療方法。
［１０］　前記口腔内疾患は歯周病である、［９］に記載の方法。
［１１］　ラクトバチルス・プランタラムの菌体もしくは菌体培養物またはこれらの抽出物を含む、歯周病の原因菌に対する増殖抑制剤または殺菌剤。
［１２］　前記歯周病の原因菌は、ポルフィロモナス・ジンジバリス、タンネレラ・フォーサイシアおよびトレポネーマ・デンティコーラから選択される少なくとも１つを含む、［１１］に記載の増殖抑制剤または殺菌剤。
［１３］　ラクトバチルス・プランタラムの菌体もしくは菌体培養物またはこれらの抽出物を歯周病の原因菌に接触させることを含む、歯周病菌の増殖抑制または殺菌方法。 The present invention relates to items exemplified below.
[1] An oral composition containing Lactobacillus plantarum cells, cell cultures, or extracts thereof.
[2] The composition according to [1], which is a food.
[3] The composition according to [2], wherein the food is a fermented food.
[4] The composition of [1], which is a mouthwash or dentifrice.
[5] The composition according to [1] or [4], further comprising a fluorine compound.
[6] The Lactobacillus plantarum is at least one selected from NITE-BP-03198, NITE-BP-03199, NITE-BP-03200, NITE-BP-03201 and NITE-BP-03202, The composition according to any one of [1] to [5].
[7] The composition according to any one of [1] to [6], which is a composition for preventing or treating oral diseases.
[8] The composition of [7], wherein the oral disease is periodontal disease.
[9] A method for preventing or treating oral diseases, which comprises applying Lactobacillus plantarum cells, cell cultures, or extracts thereof to the oral cavity of an animal.
[10] The method of [9], wherein the oral disease is periodontal disease.
[11] A growth inhibitor or bactericidal agent for periodontal disease-causing bacteria, containing Lactobacillus plantarum cells, cell cultures, or extracts thereof.
[12] The growth inhibitor or fungicide according to [11], wherein the periodontal disease-causing bacteria include at least one selected from Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola.
[13] A method for inhibiting the growth of or sterilizing periodontal disease bacteria, which comprises contacting Lactobacillus plantarum cells, cell cultures, or extracts thereof with periodontal disease-causing bacteria.
　本発明によれば、口腔内疾患を抑制し得る組成物を提供することができる。 According to the present invention, it is possible to provide a composition capable of suppressing oral diseases.
実験１において、ＮＩＴＥ　ＢＰ－０３１９８の（Ａ）コロニーの形状および（Ｂ）グラム染色結果を示す図である。FIG. 2 shows (A) colony shape and (B) Gram staining results of NITE BP-03198 in Experiment 1. FIG. 実験２において、ＮＩＴＥ　ＢＰ－０３１９９の（Ａ）コロニーの形状および（Ｂ）グラム染色結果を示す図である。Fig. 2 shows (A) colony shape and (B) Gram staining results of NITE BP-03199 in Experiment 2. 実験３において、ＮＩＴＥ　ＢＰ－０３２００の（Ａ）コロニーの形状および（Ｂ）グラム染色結果を示す図である。FIG. 10 shows (A) colony shape and (B) Gram staining results of NITE BP-03200 in Experiment 3. FIG. 実験４において、ＮＩＴＥ　ＢＰ－０３２０１の（Ａ）コロニーの形状および（Ｂ）グラム染色結果を示す図である。FIG. 10 shows (A) colony shape and (B) Gram staining results of NITE BP-03201 in Experiment 4. FIG. 実験５において、ＮＩＴＥ　ＢＰ－０３２０２の（Ａ）コロニーの形状および（Ｂ）グラム染色結果を示す図である。FIG. 10 shows (A) colony shape and (B) Gram staining results of NITE BP-03202 in Experiment 5. FIG. 実験６～８において、細菌に対する抗菌活性評価試験を説明する図である。FIG. 10 is a diagram illustrating an antibacterial activity evaluation test against bacteria in Experiments 6 to 8. FIG.
　以下、本発明を実施するための形態について詳細に説明する。なお、本発明は、以下の実施形態に限定されるものではない。本明細書において「Ａ～Ｂ」という形式の表記は、範囲の上限下限（すなわちＡ以上Ｂ以下）を意味し、Ａにおいて単位の記載がなく、Ｂにおいてのみ単位が記載されている場合、Ａの単位とＢの単位とは同じである。 Hereinafter, the embodiments for carrying out the present invention will be described in detail. In addition, this invention is not limited to the following embodiment. In this specification, the notation of the form "A to B" means the upper and lower limits of the range (that is, from A to B). and the unit of B are the same.
　［口腔用組成物］
　本発明の一実施形態に係る口腔用組成物は、ラクトバチルス・プランタラム（Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｐｌａｎｔａｒｕｍ）の菌体もしくは菌体培養物またはこれらの抽出物を含む。本発明に係る組成物は、歯周病菌に対する抗菌活性を有する。 [Composition for oral cavity]
An oral composition according to one embodiment of the present invention comprises cells or cell cultures of Lactobacillus plantarum or extracts thereof. The composition according to the present invention has antibacterial activity against periodontal bacteria.
　ラクトバチルス・プランタラムは、好ましくはＮＩＴＥ－ＢＰ－０３１９８、ＮＩＴＥ－ＢＰ－０３１９９、ＮＩＴＥ－ＢＰ－０３２００、ＮＩＴＥ－ＢＰ－０３２０１およびＮＩＴＥ－ＢＰ－０３２０２から選択される少なくとも１つである。ＮＩＴＥ－ＢＰ－０３１９８、ＮＩＴＥ－ＢＰ－０３１９９、ＮＩＴＥ－ＢＰ－０３２００、ＮＩＴＥ－ＢＰ－０３２０１およびＮＩＴＥ－ＢＰ－０３２０２は、それぞれ受託番号ＮＩＴＥ　ＢＰ－０３１９８（原寄託日：２０２０年４月９日）、受託番号ＮＩＴＥ　ＢＰ－０３１９９（原寄託日：２０２０年４月９日）、受託番号ＮＩＴＥ　ＢＰ－０３２００（原寄託日：２０２０年４月９日）、受託番号ＮＩＴＥ　ＢＰ－０３２０１（原寄託日：２０２０年４月９日）、受託番号ＮＩＴＥ　ＢＰ－０３２０２（原寄託日：２０２０年４月９日）として、独立行政法人製品評価技術基盤機構特許微生物寄託センター（ＮＰＭＤ、住所：〒２９２－０８１８　千葉県木更津市かずさ鎌足２－５－８　１２２号室）にブダペスト条約に基づいて国際寄託されている細菌である。上記細菌は、いずれも乳酸菌の一種であるラクトバチルス・プランタラム（Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｐｌａｎｔａｒｕｍ）に属する細菌である。上記細菌の菌学的性質については、後述する表１～表１０および図１～図５に示す。 Lactobacillus plantarum is preferably at least one selected from NITE-BP-03198, NITE-BP-03199, NITE-BP-03200, NITE-BP-03201 and NITE-BP-03202. NITE-BP-03198, NITE-BP-03199, NITE-BP-03200, NITE-BP-03201 and NITE-BP-03202 are respectively accession number NITE BP-03198 (original deposit date: April 9, 2020) , Accession No. NITE BP-03199 (Original Deposit Date: April 9, 2020), Accession No. NITE BP-03200 (Original Deposit Date: April 9, 2020), Accession No. NITE BP-03201 (Original Deposit Date: April 9, 2020), under the accession number NITE BP-03202 (original deposit date: April 9, 2020), National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (NPMD, Address: 292-0818 Chiba It is a bacterium that has been internationally deposited under the Budapest Treaty at Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Prefecture. All of the above bacteria belong to Lactobacillus plantarum, which is a kind of lactic acid bacteria. The mycological properties of the bacteria are shown in Tables 1 to 10 and FIGS. 1 to 5, which will be described later.
　ＮＩＴＥ－ＢＰ－０３１９８は発酵食品中、ＮＩＴＥ－ＢＰ－０３１９９、ＮＩＴＥ－ＢＰ－０３２００、ＮＩＴＥ－ＢＰ－０３２０１およびＮＩＴＥ－ＢＰ－０３２０２は、自然環境中に存在するため、人への安全性も高いと考えられる。上記乳酸菌は、単離された細菌であってよい。 Since NITE-BP-03198 exists in fermented foods, and NITE-BP-03199, NITE-BP-03200, NITE-BP-03201 and NITE-BP-03202 exist in the natural environment, they are highly safe to humans. it is conceivable that. The lactic acid bacteria may be isolated bacteria.
　本発明の一実施形態に係る口腔用組成物は、ＮＩＴＥ－ＢＰ－０３１９８、ＮＩＴＥ－ＢＰ－０３１９９、ＮＩＴＥ－ＢＰ－０３２００、ＮＩＴＥ－ＢＰ－０３２０１およびＮＩＴＥ－ＢＰ－０３２０２から選択される少なくとも１種の細菌の菌体もしくは菌体培養物またはこれらの抽出物を含む。菌体は、発酵食品中または環境中から単離されたものであってもよく、培養されたものであってもよい。菌体は死菌であっても生菌であってもよい。菌体は培養液、緩衝液等に存在しているものであってもよく、これらを濃縮して液体を除いたものまたはその凍結乾燥物であってもよく、凍結ストックであってもよい。 The oral composition according to one embodiment of the present invention is at least one selected from NITE-BP-03198, NITE-BP-03199, NITE-BP-03200, NITE-BP-03201 and NITE-BP-03202 bacteria or bacterial cell cultures or extracts thereof. The fungus may be isolated from the fermented food or the environment, or may be cultured. The cells may be dead cells or live cells. The cells may exist in a culture medium, buffer solution, or the like, or may be concentrated to remove the liquid, a lyophilized product thereof, or a frozen stock.
　菌体培養物は、細菌の分泌物、代謝物等を含んでよい。菌体培養物には、細菌が産生するペプチド、タンパク質、糖、酵素、有機酸およびこれらを含む培地（液体培地および固形培地）が含まれ得る。菌体培養物は、細菌を培養した後の上清であってもよい。培養上清は、例えば細菌を培養した液体培地から遠心分離、ろ過操作等で細菌を除いて得ることができる。 The bacterial cell culture may contain bacterial secretions, metabolites, and the like. Cell cultures can contain peptides, proteins, sugars, enzymes, organic acids produced by bacteria, and media containing these (liquid media and solid media). The cell culture may be the supernatant after culturing bacteria. The culture supernatant can be obtained, for example, by removing bacteria from a liquid medium in which bacteria have been cultured, by centrifugation, filtration, or the like.
　ＮＩＴＥ－ＢＰ－０３１９８、ＮＩＴＥ－ＢＰ－０３１９９、ＮＩＴＥ－ＢＰ－０３２００、ＮＩＴＥ－ＢＰ－０３２０１およびＮＩＴＥ－ＢＰ－０３２０２は、乳酸菌の通常の培養方法に従って培養することができる。代表的な培養方法としては、ＭＲＳ（ｄｅ　Ｍａｎ，Ｒｏｇｏｓａ　ａｎｄ　Ｓｈａｒｐｅ）液体培地またはＭＲＳ寒天培地を用いて温度３０℃で培養する方法が挙げられる。 NITE-BP-03198, NITE-BP-03199, NITE-BP-03200, NITE-BP-03201 and NITE-BP-03202 can be cultured according to a normal culture method for lactic acid bacteria. A typical culture method includes a method of culturing at a temperature of 30° C. using MRS (de Mann, Rogosa and Sharpe) liquid medium or MRS agar medium.
　菌体または菌体培養物の抽出物は、上記乳酸菌の菌体または菌体培養物が有する歯周病菌に対する抗菌活性を失わないように調製される。抽出物は、例えば細菌の菌体または菌体培養物を超音波破砕、ビーズ摩砕、凍結融解、化学的溶解等の処理をすることにより得ることができる。抽出物は、菌体または菌体培養物を塩析、限外ろ過、イオン交換クロマトグラフィーまたは有機溶媒を用いた液相抽出等を行って得てもよい。これらの処理は適宜組合わせて行なうことができる。抽出物は、細菌の菌体の断片、核酸、ペプチド、タンパク質、糖および酵素を含み得る。本明細書において、細菌の菌体もしくは菌体培養物またはこれらの抽出物を「菌体調製物」とも記す。 The bacterial cell or bacterial cell culture extract is prepared so as not to lose the antibacterial activity against periodontal disease bacteria possessed by the lactic acid bacteria cell or bacterial cell culture. Extracts can be obtained, for example, by subjecting bacterial cells or cell cultures to ultrasonic disruption, bead grinding, freeze-thawing, chemical lysis, or the like. The extract may be obtained by salting-out, ultrafiltration, ion-exchange chromatography, liquid-phase extraction using an organic solvent, or the like, on the cells or cell culture. These treatments can be performed in combination as appropriate. The extract may contain bacterial cell fragments, nucleic acids, peptides, proteins, sugars and enzymes. In the present specification, bacterial cells, bacterial cell cultures, or extracts thereof are also referred to as "microbial cell preparations."
　口腔用組成物は、口腔から体内に摂取される経口組成物であってよい。口腔用組成物は、食品であってよく、ヒト以外の動物の食品または飼料であってよい。口腔用組成物は、口腔内で使用され、使用後は口腔内から排出される組成物であってもよい。口腔用組成物は、口腔外で使用される組成物であってもよい。口腔外で使用される組成物は、例えば、義歯洗浄剤であってもよい。口腔用組成物は、例えば口腔ケア用品であってよい。 The oral composition may be an oral composition that is taken into the body through the oral cavity. The oral composition may be a food product and may be a non-human animal food or feed. The oral composition may be a composition that is used in the oral cavity and is discharged from the oral cavity after use. The oral composition may be a composition for extra-oral use. The composition for extraoral use may be, for example, a denture cleanser. The oral composition can be, for example, an oral care product.
　口腔用組成物は、好ましくは経口組成物は、本発明の効果を損なわない範囲で添加成分を配合することができる。経口摂取に適した添加成分としては、水等の溶媒、糖質、タンパク質、脂質、ビタミン類、ミネラル類、生体必須微量金属（硫酸マンガン、硫酸亜鉛、塩化マグネシウム、炭酸カリウム等）、香料、食品衛生上または薬学的に許容可能な担体、食品添加物等が挙げられる。添加成分は、１種または２種以上の組み合わせを適宜選択して、必要に応じて配合することができる。 The oral composition, preferably the oral composition, can contain additive components within a range that does not impair the effects of the present invention. Additives suitable for oral intake include solvents such as water, carbohydrates, proteins, lipids, vitamins, minerals, trace metals essential for living organisms (manganese sulfate, zinc sulfate, magnesium chloride, potassium carbonate, etc.), fragrances, and foods. sanitary or pharmaceutically acceptable carriers, food additives and the like. Additive components can be added as needed by appropriately selecting one or a combination of two or more.
　糖質としては、糖類、加工澱粉（デキストリン、可溶性澱粉、ブリティッシュスターチ、酸化澱粉、澱粉エステル、澱粉エーテル等）、食物繊維等が挙げられる。 Carbohydrates include saccharides, processed starch (dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like.
　タンパク質としては、全脂粉乳、脱脂粉乳、部分脱脂粉乳、カゼイン、ホエイ粉、ホエイタンパク質、ホエイタンパク質濃縮物、ホエイタンパク質分離物、α－カゼイン、β－カゼイン、κ－カゼイン、β－ラクトグロブリン、α－ラクトアルブミン、ラクトフェリン、大豆タンパク質、鶏卵タンパク質、および肉タンパク質等の動植物性タンパク質、ならびにこれらの加水分解物、ならびに、バター、乳性ミネラル、クリーム、ホエイ、非タンパク態窒素、シアル酸、リン脂質、および乳糖等の各種乳由来成分等が挙げられる。 Proteins include whole milk powder, skimmed milk powder, partially skimmed milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein, β-lactoglobulin, Animal and plant proteins such as α-lactalbumin, lactoferrin, soy protein, chicken egg protein, and meat protein, and their hydrolysates, as well as butter, milk minerals, cream, whey, non-protein nitrogen, sialic acid, and phosphorus Examples include lipids and various milk-derived components such as lactose.
　脂質としては、ラード、魚油等、これらの分別油、これらの水素添加油、およびこれらのエステル交換油等の動物性油脂、ならびに、パーム油、サフラワー油、コーン油、ナタネ油、ヤシ油、これらの分別油、これらの水素添加油、およびこれらのエステル交換油等の植物性油脂等が挙げられる。 Lipids include animal oils such as lard, fish oil, fractionated oils thereof, hydrogenated oils thereof, transesterified oils thereof, palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, Vegetable oils and fats such as these fractionated oils, these hydrogenated oils, and these interesterified oils are included.
　ビタミン類としては、ビタミンＡ、カロチン類、ビタミンＢ群、ビタミンＣ、ビタミンＤ群、ビタミンＥ、ビタミンＫ群、ビタミンＰ、ビタミンＱ、ナイアシン、ニコチン酸、パントテン酸、ビオチン、イノシトール、コリン、葉酸等が挙げられる。 Vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline, and folic acid. etc.
　ミネラル類としては、カルシウム、カリウム、マグネシウム、ナトリウム、銅、鉄、マンガン、亜鉛、セレン等が挙げられる。 　Minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, and selenium.
　口腔用組成物（好ましくは経口組成物）は、溶液、懸濁液、乳濁液、粉末、ペースト、半固体成形物、固体成形物等、経口摂取可能な形態であればよい。食品（飲料を含む）としては、例えば、牛乳、植物性ミルク、乳飲料、清涼飲料、発酵乳、乳酸菌飲料、乳性飲料、調製粉乳、液体ミルク、流動食、病者用食品、冷凍食品、発酵食品、加工食品、菓子類、調味料その他の市販食品等が挙げられる。より具体的には、ヨーグルト、チーズ、アイスクリーム、氷菓、チョコレート、タブレット（錠菓）、グミ、キャンディー、ゼリー、ガム、パン、ビスケット、クラッカー、ピッツァクラスト等が挙げられる。口腔用組成物は、サプリメント、健康食品、機能性食品、特別用途食品、保健機能食品、特定保健用食品、栄養機能食品、機能性表示食品、医薬用部外品、化粧品等であってもよく、錠剤、カプセル剤、散剤、顆粒剤、ゼリー剤等であってもよい。 The oral composition (preferably oral composition) may be in an orally ingestible form such as solution, suspension, emulsion, powder, paste, semi-solid molding, or solid molding. Foods (including beverages) include, for example, milk, vegetable milk, milk beverages, soft drinks, fermented milk, lactic acid beverages, lactic beverages, infant formula, liquid milk, liquid diets, foods for the sick, frozen foods, Examples include fermented foods, processed foods, confectionery, seasonings and other commercially available foods. More specific examples include yoghurt, cheese, ice cream, ice cream, chocolate, tablets, gummies, candies, jellies, gums, breads, biscuits, crackers, pizza crusts, and the like. The oral composition may be supplements, health foods, functional foods, foods for special dietary uses, foods with health claims, foods for specified health uses, foods with nutrient function claims, foods with function claims, quasi-drugs, cosmetics, and the like. , tablets, capsules, powders, granules, jelly and the like.
　口腔用組成物は、好ましくは口腔ケア用品は、本発明の効果を損なわない範囲で、添加成分を配合することができる。添加成分としては、例えばフッ素化合物、薬用成分、研磨剤、粘結剤、粘稠剤、界面活性剤、矯味剤、防腐剤、香料、着色剤、ｐＨ調整剤、溶剤、可溶化剤、基剤、洗浄剤、吸着剤等が挙げられる。添加成分は、組成物の剤形に応じて適宜選択し得る。添加成分は、１種または２種以上の組み合わせを適宜選択して、必要に応じて配合することができる。 The oral composition, preferably the oral care product, can contain additive components within a range that does not impair the effects of the present invention. Examples of additive components include fluorine compounds, medicinal ingredients, abrasives, binders, thickening agents, surfactants, corrigents, preservatives, fragrances, coloring agents, pH adjusters, solvents, solubilizers, and bases. , detergents, adsorbents, and the like. The additive component can be appropriately selected according to the dosage form of the composition. Additive components can be added as needed by appropriately selecting one or a combination of two or more.
　フッ素化合物としては、フッ化ナトリウム、フッ化カリウム、フッ化アンモニウム、フッ化スズ、アミンフッ化物、モノフルオロリン酸ナトリウム、モノフルオロリン酸カリウム、フッ化ケイ素ナトリウム、フッ化ケイ素カルシウム等が挙げられ、好ましくはフッ化ナトリウムまたはモノフルオロリン酸ナトリウムである。フッ素化合物は、う蝕を抑制することができる。 Examples of fluorine compounds include sodium fluoride, potassium fluoride, ammonium fluoride, tin fluoride, amine fluoride, sodium monofluorophosphate, potassium monofluorophosphate, sodium silicon fluoride, and calcium silicon fluoride. Sodium fluoride or sodium monofluorophosphate is preferred. Fluorine compounds can inhibit caries.
　フッ素化合物は、組成物中の濃度が例えばフッ素濃度として１００１ｐｐｍ～３０００ｐｐｍ、好ましくは１００１ｐｐｍ～２０００ｐｐｍ、より好ましくは１００１ｐｐｍ～１５００ｐｐｍとなる量で使用される。 The fluorine compound is used in an amount such that the fluorine concentration in the composition is, for example, 1001 ppm to 3000 ppm, preferably 1001 ppm to 2000 ppm, more preferably 1001 ppm to 1500 ppm.
　薬用成分としては、殺菌剤、抗炎症剤、血行促進剤、歯石沈着抑制剤、ステイン除去剤、知覚過敏抑制剤、ビタミン剤、生薬エキス、歯垢分解酵素等を、上記口腔用組成物に配合することができる。これらの薬効成分は、医薬品等に使用しうるものであれば特に限定されない。 As medicinal ingredients, bactericides, anti-inflammatory agents, blood circulation promoters, tartar deposition inhibitors, stain removers, hypersensitivity inhibitors, vitamins, crude drug extracts, plaque-degrading enzymes, etc. are added to the oral composition. can do. These medicinal ingredients are not particularly limited as long as they can be used for pharmaceuticals and the like.
　殺菌剤としては、塩化セチルピリジニウム、塩化ベンザルコニウム、塩化ベンゼトニウム、塩酸クロルヘキシジン、およびグルコン酸クロルヘキシジン等のカチオン性殺菌剤；ドデシルジアミノエチルグリシン等の両性殺菌剤；トリクロサン、およびイソプロピルメチルフェノール等の非イオン性殺菌剤；ならびに、ヒノキチオール等が挙げられる。 Bactericides include cationic fungicides such as cetylpyridinium chloride, benzalkonium chloride, benzethonium chloride, chlorhexidine hydrochloride, and chlorhexidine gluconate; amphoteric fungicides such as dodecyldiaminoethylglycine; ionic fungicides; and hinokitiol and the like.
　抗炎症剤としては、β－グリチルレチン酸、グリチルレチン酸、グリチルリチン酸、グリチルリチン酸二アンモニウム、グリチルリチン酸二ナトリウム、グリチルリチン酸三ナトリウム、グリチルリチン酸二カリウム、グリチルリチン酸モノアンモニウム、ε－アミノカプロン酸、アズレンスルホン酸ナトリウム水和物、アラントイン、アラントインクロルヒドトキシアルミニウム、アルクロキサ、アラントインジヒドロキシアルミニウム、アルジオキサ、エピジヒドロコレステリン、ジヒドロコレステロール、リゾチーム塩酸塩等が挙げられる。 Anti-inflammatory agents include β-glycyrrhetinic acid, glycyrrhetinic acid, glycyrrhizic acid, diammonium glycyrrhizinate, disodium glycyrrhizinate, trisodium glycyrrhizinate, dipotassium glycyrrhizinate, monoammonium glycyrrhizinate, ε-aminocaproic acid, azulene sulfonic acid. Sodium hydrate, allantoin, allantoin chlorohydroxyaluminum, alcloxa, allantoin dihydroxyaluminum, aldioxa, epidihydrocholesterin, dihydrocholesterol, lysozyme hydrochloride and the like.
　血行促進剤としては、塩化ナトリウム等が挙げられる。 Examples of blood circulation promoting agents include sodium chloride.
　歯石沈着抑制剤としては、ゼオライト、リン酸水素二ナトリウム、ピロリン酸二水素二ナトリウム、ピロリン酸ナトリウム、無水ピロリン酸ナトリウム、ピロリン酸四ナトリウム（無水）、リン酸一水素二ナトリウム、リン酸水素ナトリウム水和物、リン酸水素二ナトリウム（結晶）、リン酸三ナトリウム、ポリリン酸ナトリウム等が挙げられる。 As a tartar deposition inhibitor, zeolite, disodium hydrogen phosphate, disodium dihydrogen pyrophosphate, sodium pyrophosphate, anhydrous sodium pyrophosphate, tetrasodium pyrophosphate (anhydrous), disodium monohydrogen phosphate, sodium hydrogen phosphate hydrate, disodium hydrogen phosphate (crystal), trisodium phosphate, sodium polyphosphate and the like.
　ステイン除去剤としては、マクロゴール（マクロゴール２００、マクロゴール３００、マクロゴール４００、マクロゴール６００、マクロゴール１０００、マクロゴール１５００、マクロゴール１５４０、マクロゴール４０００、マクロゴール６０００、マクロゴール２００００など）、ポリリン酸ナトリウム、ポリビニルピロリドン等が挙げられる。 As a stain remover, macrogol (Macrogol 200, Macrogol 300, Macrogol 400, Macrogol 600, Macrogol 1000, Macrogol 1500, Macrogol 1540, Macrogol 4000, Macrogol 6000, Macrogol 20000, etc.) , sodium polyphosphate, polyvinylpyrrolidone, and the like.
　知覚過敏抑制剤としては、硝酸カリウム、乳酸アルミニウム等が挙げられる。 Examples of antihypersensitivity agents include potassium nitrate and aluminum lactate.
　ビタミン剤としては、アスコルビン酸、Ｌ－アスコルビン酸、アスコルビン酸ナトリウム、Ｌ－アスコルビン酸ナトリウム、ピリドキシン塩酸塩、酢酸ＤＬ－α－トコフェロール、トコフェロール酢酸エステル、ニコチン酸ｄl－α－トコフェロール、トコフェロールニコチン酸エステル等が挙げられる。 Vitamin preparations include ascorbic acid, L-ascorbic acid, sodium ascorbate, sodium L-ascorbate, pyridoxine hydrochloride, DL-α-tocopherol acetate, tocopherol acetate, dl-α-tocopherol nicotinate, tocopherol nicotinate etc.
　研磨剤としては、無水ケイ酸、シリカ（結晶性シリカ又は非晶性シリカ）、シリカゲル、およびアルミノシリケート等のシリカ系研磨剤、ゼオライト、リン酸水素カルシウム無水和物、リン酸水素カルシウム２水和物、ピロリン酸カルシウム、炭酸カルシウム、水酸化アルミニウム、アルミナ、炭酸マグネシウム、第３リン酸マグネシウム、ケイ酸ジルコニウム、第３リン酸カルシウム、ハイドロキシアパタイト、第４リン酸カルシウム、ならびに、合成樹脂系研磨剤等が挙げられる。 Examples of abrasives include silicic anhydride, silica (crystalline silica or amorphous silica), silica gel, silica-based abrasives such as aluminosilicate, zeolite, calcium hydrogen phosphate anhydrate, and calcium hydrogen phosphate dihydrate. substances, calcium pyrophosphate, calcium carbonate, aluminum hydroxide, alumina, magnesium carbonate, magnesium phosphate tertiary, zirconium silicate, calcium phosphate tertiary, hydroxyapatite, calcium tetraphosphate, and synthetic resin abrasives.
　粘結剤としては、プルラン、ゼラチン、メチルセルロース、ヒドロキシエチルセルロース、ヒドロキシプロピルセルロース、カルボキシメチルセルロースナトリウム、カラギーナン、アルギン酸ナトリウム、キサンタンガム、ポリアクリル酸ナトリウム、アラビアガム、グアーガム、ローカストビーンガム、ポリビニルアルコール、ポリビニルピロリドン、およびカルボキシビニルポリマー等の有機系粘結剤、ならびに、増粘性無水ケイ酸、およびベントナイト等の無機系粘結剤等が挙げられる。 Binders include pullulan, gelatin, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, sodium carboxymethylcellulose, carrageenan, sodium alginate, xanthan gum, sodium polyacrylate, gum arabic, guar gum, locust bean gum, polyvinyl alcohol, polyvinylpyrrolidone, and organic binders such as carboxyvinyl polymer, and inorganic binders such as thickening silicic anhydride and bentonite.
　粘稠剤としては、多価アルコール（さらに具体的には、ソルビット、グリセリン、濃グリセリン、エチレングリコール、プロピレングリコール、１,３－ブチレングリコール、プロパンジオール(１，３－プロパンジオール)、ポリエチレングリコール、ポリプロピレングリコール、キシリトール、マルチトール、ラクチトール等）、トレハロース、ヒアルロン酸ナトリウム、加水分解コラーゲン等が挙げられる。 Examples of thickening agents include polyhydric alcohols (more specifically, sorbitol, glycerin, concentrated glycerin, ethylene glycol, propylene glycol, 1,3-butylene glycol, propanediol (1,3-propanediol), polyethylene glycol, polypropylene glycol, xylitol, maltitol, lactitol, etc.), trehalose, sodium hyaluronate, hydrolyzed collagen, and the like.
　界面活性剤としては、アニオン界面活性剤では、Ｎ－アシルアミノ酸塩、α－オレフィンスルホン酸塩、Ｎ－アシルスルホン酸塩、アルキル硫酸塩（例えば、ラウリル硫酸ナトリウムなど）、グリセリン脂肪酸エステルの硫酸塩等が挙げられる。
　ノニオン界面活性剤としては、ポリオキシエチレンアルキルエーテル、ポリオキシエチレン－ポリオキシプロピレンブロック共重合体、ポリオキシエチレン硬化ヒマシ油、グリセリンエステルのポリオキシエチレンエーテル、ショ糖脂肪酸エステル、アルキロールアミド、グリセリン脂肪酸エステル、アルキルグリコシド等が挙げられる。
　両性界面活性剤としては、アルキルベタイン系界面活性剤、アミンオキサイド系界面活性剤、イミダゾリニウムベタイン系界面活性剤が挙げられる。これらの具体例としては、２－アルキル－Ｎ－カルボキシメチル－Ｎ－ヒドロキシエチルイミダゾリニウムベタイン、ラウリルジメチルアミノ酢酸ベタイン、ヤシ油アルキルベタイン（ヤシ油アルキルジメチルアミノ酢酸ベタイン）、ステアリルジメチルアミノ酢酸ベタイン、ステアリルジメチルベタインナトリウム、ヤシ油脂肪酸アミドアルキルベタイン、パーム油脂肪酸アミドプロピルベタイン、ラウリン酸アミドプロピルベタイン、リシノレイン酸アミドプロピルベタイン、ステアリルジヒドロキシエチルベタイン等が挙げられる。 Surfactants include anionic surfactants such as N-acyl amino acid salts, α-olefin sulfonates, N-acyl sulfonates, alkyl sulfates (eg, sodium lauryl sulfate, etc.), sulfates of glycerin fatty acid esters. etc.
Nonionic surfactants include polyoxyethylene alkyl ether, polyoxyethylene-polyoxypropylene block copolymer, polyoxyethylene hydrogenated castor oil, polyoxyethylene ether of glycerin ester, sucrose fatty acid ester, alkylolamide, glycerin. fatty acid esters, alkyl glycosides, and the like;
Amphoteric surfactants include alkyl betaine surfactants, amine oxide surfactants, and imidazolinium betaine surfactants. Specific examples of these include 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine, lauryldimethylaminoacetic acid betaine, coconut oil alkylbetaine (coconut alkyldimethylaminoacetic acid betaine), stearyldimethylaminoacetic acid betaine. .
　矯味剤としては、例えば、Ｌ－グルタミン酸ナトリウム、サッカリン、サッカリンナトリウム、グリチルリチン酸二ナトリウム、グリチルリチン酸三ナトリウム、ショ糖、ブドウ糖、果糖、乳糖、ハチミツ、アスパルテーム、ステビア、スクラロース、キシリトール、イノシトール、Ｄ－ソルビトール、Ｄ－マンニトール、アラビトール、ラフィノース、ラクチュロース、ラクチトール、エリスリトール、還元パラチノース、パラチノース、パラチニット、アセスルファムＫ、マルトース、マルトシルトレハロース、マルチトール、ネオヘスペリジンジヒドロカルコン、ペリラルチン、ｐ－メトキシシンナミックアルデヒド、ソーマチン等が挙げられる。 Examples of flavoring agents include sodium L-glutamate, saccharin, sodium saccharin, disodium glycyrrhizinate, trisodium glycyrrhizinate, sucrose, glucose, fructose, lactose, honey, aspartame, stevia, sucralose, xylitol, inositol, and D-sorbitol. , D-mannitol, arabitol, raffinose, lactulose, lactitol, erythritol, reduced palatinose, palatinose, palatinit, acesulfame K, maltose, maltosyltrehalose, maltitol, neohesperidin dihydrochalcone, perillartine, p-methoxycinnamic aldehyde, thaumatin, etc. is mentioned.
　防腐剤としては、例えば、グリシン、安息香酸ナトリウム、メチルパラベン、エチルパラベン、ブチルパラベン、イソプロピルパラベン、プロピルパラベン、イソブチルパラベン、およびベンジルパラベン等のパラオキシ安息香酸エステル、フェノキシエタノール、およびエタノール等のアルコール類、ソルビン酸、安息香酸、デヒドロ酢酸、プロピオン酸およびこれらの塩、エチレンジアミン四酢酸塩、塩化ベンザルコニウム、塩化ベンゼトニウム、塩化セチルピリジニウム、ならびに、塩酸アルキルジアミノエチルグリシン等が挙げられる。 Examples of antiseptics include glycine, sodium benzoate, paraoxybenzoic acid esters such as methylparaben, ethylparaben, butylparaben, isopropylparaben, propylparaben, isobutylparaben, and benzylparaben; alcohols such as phenoxyethanol and ethanol; acid, benzoic acid, dehydroacetic acid, propionic acid and salts thereof, ethylenediaminetetraacetate, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, and alkyldiaminoethylglycine hydrochloride.
　香料としては、例えば、Ｌ－メントール、ペパーミント、スペアミント、フルーツ香料、ハッカ油等が挙げられる。香料は、唾液分泌を刺激するという利点も有する。 Flavors include, for example, L-menthol, peppermint, spearmint, fruit flavors, and peppermint oil. Perfumes also have the advantage of stimulating salivation.
　着色剤としては、ベニバナ赤色素、クチナシ黄色素、クチナシ青色素、シソ色素、紅麹色素、赤キャベツ色素、ニンジン色素、ハイビスカス色素、カカオ色素、スピルリナ青色素、およびクマリンド色素等の天然色素、赤色３号、赤色１０４号、赤色１０５号、赤色１０６号、黄色４号、黄色５号、緑色３号、および青色１号等の法定色素、リボフラビン、銅クロロフィリンナトリウム、ならびに、二酸化チタン等が挙げられる。 Coloring agents include safflower red pigment, gardenia yellow pigment, gardenia blue pigment, perilla pigment, monascus pigment, red cabbage pigment, carrot pigment, hibiscus pigment, cacao pigment, spirulina blue pigment, and natural pigment such as coumarind pigment, red Legal pigments such as No. 3, Red No. 104, Red No. 105, Red No. 106, Yellow No. 4, Yellow No. 5, Green No. 3, and Blue No. 1, riboflavin, sodium copper chlorophyllin, and titanium dioxide. .
　ｐＨ調整剤としては、ギ酸、乳酸、酢酸、塩酸、硫酸、硝酸、クエン酸、リン酸、リンゴ酸、グルコン酸、マレイン酸、コハク酸、グルタミン酸、ピロリン酸、酒石酸、酢酸水酸化ナトリウム、水酸化カリウム、酢酸ナトリウム、炭酸ナトリウム、クエン酸ナトリウム、クエン酸水素ナトリウム、リン酸、リン酸ナトリウム、リン酸一水素ナトリウム、およびリン酸二水素ナトリウムリン酸二水素カリウム等の酸、アルカリ、および緩衝剤等が挙げられる。 pH adjusters include formic acid, lactic acid, acetic acid, hydrochloric acid, sulfuric acid, nitric acid, citric acid, phosphoric acid, malic acid, gluconic acid, maleic acid, succinic acid, glutamic acid, pyrophosphoric acid, tartaric acid, sodium acetate hydroxide, hydroxide Acids, alkalis, and buffers such as potassium, sodium acetate, sodium carbonate, sodium citrate, sodium hydrogen citrate, phosphoric acid, sodium phosphate, sodium monohydrogen phosphate, and sodium dihydrogen phosphate potassium dihydrogen phosphate etc.
　溶剤としては、水、ならびに、エタノール、およびプロパノールなどの低級アルコール等が挙げられる。 Examples of solvents include water and lower alcohols such as ethanol and propanol.
　可溶化剤は、水への上記添加剤または薬効成分の溶解を促進させるために添加してもよい。そのような可溶化剤の例として、プロピレングリコール、ジプロピレングリコール、ブチレングリコール、およびポリエチレングリコール等の多価アルコール類等を挙げることができる。 A solubilizer may be added to promote the dissolution of the above additives or medicinal ingredients in water. Examples of such solubilizers include polyhydric alcohols such as propylene glycol, dipropylene glycol, butylene glycol, and polyethylene glycol.
　基剤としては、炭酸水素ナトリウム等が挙げられる。 Examples of bases include sodium hydrogen carbonate.
　洗浄剤としては、ポリリン酸ナトリウムなどを挙げることができる。 Examples of cleaning agents include sodium polyphosphate.
　吸着剤としては、β－シクロデキストリンなどを挙げることができる。 Examples of adsorbents include β-cyclodextrin.
　口腔用組成物は、例えば洗口剤（マウスウォッシュ）、歯磨剤（練歯磨、液体歯磨、歯磨粉等）、口中清涼剤、ガム剤、トローチ剤、バッカル錠、歯肉付着性テープ製剤、口腔内用ゲル製剤、口腔内用軟膏、口腔内用スプレー剤、口腔内用パスタ剤、口腔内用ペースト剤、口腔用丸剤、口腔内用錠剤、口腔用散剤、口腔内用粉剤、口腔内用液剤、口腔内用懸濁剤、口腔内用乳剤、口腔内用顆粒剤、口腔内用カプセル剤、義歯洗浄剤が挙げられる。口腔用組成物は、好ましくは洗口剤または歯磨剤である。口腔用組成物は、医薬部外品、衛生用品または化粧品であってもよい。 Oral compositions include, for example, mouthwashes, dentifrices (toothpaste, liquid toothpaste, toothpaste, etc.), mouth fresheners, gums, lozenges, buccal tablets, gingival adhesive tape preparations, intraoral oral gel, oral ointment, oral spray, oral paste, oral paste, oral pill, oral tablet, oral powder, oral powder, oral liquid , intraoral suspensions, intraoral emulsions, intraoral granules, intraoral capsules, and denture cleaners. The oral composition is preferably a mouthwash or dentifrice. Oral compositions may be quasi-drugs, hygiene products or cosmetics.
　口腔用組成物の製造方法は特に限定されない。口腔用組成物は、一般的な経口組成物および口腔ケア用品等が製造される任意の製造方法によって製造されてよい。口腔用組成物の製造方法は、例えば一般的な経口組成物および口腔ケア用等の製造方法のいずれかの工程において、上記乳酸菌の菌体調製物を添加する工程を含んでよい。また、口腔用組成物の製造方法は、例えば、製造された経口組成物および口腔ケア用品等に、上記乳酸菌の菌体調製物を添加する工程を含んでよい。口腔用組成物は、上記乳酸菌の菌体調製物そのものであってもよい。口腔用組成物は、上記乳酸菌の菌体調製物を経口組成物および口腔ケア用品等に付着、吸収または混合させやすくするための液体、展着剤等を含んでよい。 The manufacturing method of the oral composition is not particularly limited. The oral composition may be manufactured by any manufacturing method by which common oral compositions, oral care products, and the like are manufactured. A method for producing an oral composition may include a step of adding the cell preparation of the lactic acid bacterium to any step of a general oral composition, an oral care composition, or the like. In addition, the method for producing an oral composition may include, for example, a step of adding the cell preparation of lactic acid bacteria to the oral composition, oral care product and the like produced. The composition for oral cavity may be the bacterial cell preparation itself of the above-mentioned lactic acid bacteria. The oral composition may contain liquids, spreading agents, and the like to facilitate the attachment, absorption, or mixing of the lactic acid bacteria cell preparation with the oral composition, oral care products, and the like.
　口腔用組成物に含まれる上記乳酸菌の菌体調製物の量は、歯周病菌に対する抗菌活性を示す程度であれば特に限定されない。口腔用組成物中の上記乳酸菌の菌体調製物の合計量は、例えば１×１０３～１×１０１２ｃｆｕ（コロニー形成単位）／ｇであってもよく、１×１０４～１×１０１２ｃｆｕ／ｇであってもよく、１×１０５～１×１０１２ｃｆｕ／ｇであってもよい。上記コロニー形成単位は、寒天平板培養法によって求めることができる。口腔用組成物中の上記乳酸菌の菌体調製物の合計量（含有割合）は、口腔用組成物重量に対して０．０００１質量％～３０質量％、好ましくは０．００１質量％～１０質量％であってよい。口腔用組成物中の上記乳酸菌の菌体調製物の合計の濃度は、０．１～１００,０００ｐｐｍであってもよく、１～１００,０００ｐｐｍであってもよく、１０～１００,０００ｐｐｍであってもよい。当該合計量は、天秤等で秤量可能である。 The amount of the lactic acid bacteria cell preparation contained in the oral cavity composition is not particularly limited as long as it exhibits an antibacterial activity against periodontal disease bacteria. The total amount of the cell preparation of lactic acid bacteria in the oral composition may be, for example, 1× 10 3 to 1×10 12 cfu (colony forming units)/g. 12 cfu/g, or 1×10 5 to 1×10 12 cfu/g. The colony-forming unit can be determined by an agar plate culture method. The total amount (content ratio) of the bacterial cell preparation of lactic acid bacteria in the oral composition is 0.0001% by mass to 30% by mass, preferably 0.001% by mass to 10% by mass relative to the weight of the oral composition. %. The total concentration of the lactic acid bacteria cell preparation in the oral composition may be 0.1 to 100,000 ppm, may be 1 to 100,000 ppm, or may be 10 to 100,000 ppm. may The total amount can be weighed with a balance or the like.
　口腔用組成物は、動物へ適用し得る。動物は、ヒトまたはヒト以外の動物であってよい。動物は、哺乳類を含む。哺乳類は、齧歯目、ウサギ目、食肉目、鯨偶蹄目、奇蹄目、霊長目を含んでよい。より具体的には上記哺乳類は、マウス、ラット、ハムスター、モルモット、ハリネズミ、フェレット、ウサギ、イヌ、ネコ、ウシ、ブタ、ヤギ、ウマ、ヒツジ、サル、オランウータン、チンパンジー等が挙げられる。動物は、ペット、家畜または実験動物であってもよい。 The oral composition can be applied to animals. The animal may be a human or non-human animal. Animals include mammals. Mammals may include Rodentia, Lagomorpha, Carnivora, Cetatodactyla, Perissodactyla, Primate. More specifically, the above mammals include mice, rats, hamsters, guinea pigs, hedgehogs, ferrets, rabbits, dogs, cats, cows, pigs, goats, horses, sheep, monkeys, orangutans, chimpanzees and the like. Animals may be pets, farm animals or laboratory animals.
　本発明の一実施形態は、口腔用組成物の製造におけるラクトバチルス・プランタラムの菌体もしくは菌体培養物またはこれらの抽出物の使用である。 One embodiment of the present invention is the use of Lactobacillus plantarum cells or cell cultures or extracts thereof in the production of oral compositions.
　本発明に係る口腔用組成物は、口腔内疾患の予防または治療用組成物であってもよい。本明細書において、治療には、症状の緩和、症状の好転および完治を含む。予防または治療用組成物は、医薬品または動物用医薬品、医薬部外品、化粧品等であってよい。 The oral composition according to the present invention may be a preventive or therapeutic composition for oral diseases. As used herein, treatment includes alleviation of symptoms, amelioration of symptoms, and complete cure. The prophylactic or therapeutic composition may be pharmaceuticals or veterinary drugs, quasi-drugs, cosmetics, and the like.
　ラクトバチルス・プランタラムは、口腔内疾患の原因菌に対して抗菌活性を示すため、ラクトバチルス・プランタラムを用いて口腔内疾患を予防または治療することができる。口腔内疾患としては、歯肉炎、および歯周炎等の歯周病；う蝕；口腔カンジダ症；粘膜炎；舌炎；口唇炎；ならびに、口角炎等が挙げられる。歯周病の原因菌としては、ポルフィロモナス・ジンジバリス（Ｐｏｒｐｈｙｒｏｍｏｎａｓ　ｇｉｎｇｉｖａｌｉｓ；以下、「Ｐ．ｇ．菌」ともいう。）、タンネレラ・フォーサイシア（Ｔａｎｎｅｒｅｌｌａ　ｆｏｒｓｙｔｈｉａ；以下、Ｔ．ｆ．菌」ともいう。）、トレポネーマ・デンティコーラ（Ｔｒｅｐｏｎｅｍａ　ｄｅｎｔｉｃｏｌａ；以下、「Ｔ．ｄ．菌」ともいう。）、プレボテラ・インターメディア（Ｐｒｅｖｏｔｅｌｌａ　ｉｎｔｅｒｍｅｄｉａ）、アクチノバチルス・アクチノミセテムコミタンス（Ａｃｔｉｎｏｂａｃｉｌｌｕｓ　ａｃｔｉｎｏｍｙｃｅｔｅｍｃｏｍｉｔａｎｓ）、フソバクテリウム・ヌクレアタム（Ｆｕｓｏｂａｃｔｅｒｉｕｍ　ｎｕｃｌｅａｔｕｍ）等が挙げられる。う蝕の原因菌としては、ストレプトコッカス・ミュータンス（Ｓｔｒｅｐｔｏｃｏｃｃｕｓ　ｍｕｔａｎｓ）、ストレプトコッカス・ソブリナス（Ｓｔｒｅｐｔｏｃｏｃｃｕｓ　ｓｏｂｒｉｎｕｓ）等が挙げられる。カンジダ菌としては、カンジダ・アルビカンス（Ｃａｎｄｉｄａ　ａｌｂｉｃａｎｓ）、カンジダ・グラブラータ（Ｃａｎｄｉｄａ　ｇｌａｂｒａｔａ）、カンジダ・トロピカリス（Ｃａｎｄｉｄａ　ｔｒｏｐｉｃａｌｉｓ）等が挙げられる。 Because Lactobacillus plantarum exhibits antimicrobial activity against causative bacteria of oral diseases, Lactobacillus plantarum can be used to prevent or treat oral diseases. Oral diseases include periodontal diseases such as gingivitis and periodontitis; dental caries; oral candidiasis; mucositis; glossitis; Bacteria that cause periodontal disease include Porphyromonas gingivalis (hereinafter also referred to as "Pg bacteria") and Tannerella forsythia (hereinafter also referred to as Tf bacteria). ), Treponema denticola (hereinafter also referred to as "T.d. bacteria"), Prevotella intermedia, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum nucleatum) and the like. Caries-causing bacteria include Streptococcus mutans, Streptococcus sobrinus, and the like. Candida fungi include Candida albicans, Candida glabrata, Candida tropicalis, and the like.
　歯周病の原因菌のうち、Ｐ．ｇ．菌、Ｔ．ｆ．菌およびＴ．ｄ．菌は、歯周病を重症化させる菌として知られている。ラクトバチルス・プランタラムの菌体もしくは菌体培養物またはこれらの抽出物は、悪性度が高い上記３つの歯周病菌の全てに対して抗菌活性を有する。従って、ラクトバチルス・プランタラムの菌体調製物を含む組成物は、口腔内疾患の予防または治療に効果的である。ラクトバチルス・プランタラムの菌体調製物を含む組成物は、抗生物質（ミノサイクリン等）および化学殺菌剤（塩化セチルピリジニウム（ＣＰＣ）、イソプロピルメチルフェノール（ＩＰＭＰ）等）より口腔常在菌への影響が少ない。このため、ラクトバチルス・プランタラムの菌体調製物を含む組成物は、口腔微生物叢のバランスを崩しにくい。ラクトバチルス・プランタラムの菌体調製物は、歯垢形成を促進する細菌の増殖も抑制することができる。歯垢形成を促進する細菌としては、例えば、ストレプトコッカス・ゴルドニ（Ｓｔｒｅｐｔｏｃｏｃｃｕｓ　ｇｏｒｄｏｎｉｉ；以下、「ゴルドニ菌」ともいう。）が挙げられる。 Among the causative bacteria of periodontal disease, P. g. fungus, T. f. fungi and T. d. Bacteria are known to aggravate periodontal disease. Cells or cell cultures of Lactobacillus plantarum, or extracts thereof, have antibacterial activity against all of the above three highly malignant periodontal bacteria. Therefore, compositions containing a bacterial cell preparation of Lactobacillus plantarum are effective in preventing or treating oral diseases. Compositions containing bacterial cell preparations of Lactobacillus plantarum are more effective than antibiotics (minocycline, etc.) and chemical fungicides (cetylpyridinium chloride (CPC), isopropylmethylphenol (IPMP), etc.) on oral flora. Less is. For this reason, compositions containing a cell preparation of Lactobacillus plantarum are less likely to unbalance the oral microflora. Cell preparations of Lactobacillus plantarum can also inhibit the growth of bacteria that promote plaque formation. Bacteria that promote plaque formation include, for example, Streptococcus gordonii (hereinafter also referred to as “Gordonii”).
　予防または治療用組成物は、口腔内疾患の原因菌に対する他の抗菌薬および抗生物質を含んでもよく、含まなくてもよい。予防または治療用組成物の剤型および製造方法は、上記組成物の剤型および製造方法と同じであってよい。予防または治療用組成物におけるラクトバチルス・プランタラムの菌体調製物の含有量は、歯周病菌に対する抗菌活性を示す程度であれば特に限定されず、上記口腔用組成物における含有量と同じ範囲であってよい。 The preventive or therapeutic composition may or may not contain other antibacterial agents and antibiotics against causative bacteria of oral diseases. The dosage form and manufacturing method of the preventive or therapeutic composition may be the same as the dosage form and manufacturing method of the composition described above. The content of the bacterial cell preparation of Lactobacillus plantarum in the preventive or therapeutic composition is not particularly limited as long as it exhibits antibacterial activity against periodontal disease bacteria, and is the same range as the content in the oral composition. can be
　本発明の一実施形態は、口腔内疾患の予防または治療用組成物の製造におけるラクトバチルス・プランタラムの菌体もしくは菌体培養物またはこれらの抽出物の使用である。 One embodiment of the present invention is the use of Lactobacillus plantarum cells or cell cultures or extracts thereof in the manufacture of compositions for the prevention or treatment of oral diseases.
　［口腔内疾患の予防または治療方法］
　本発明の一実施形態に係る口腔内疾患の予防または治療方法は、ラクトバチルス・プランタラムの菌体もしくは菌体培養物またはこれらの抽出物を動物の口腔に適用することを含む。動物は、ヒトまたはヒト以外の動物であってよい。口腔内疾患としては、歯肉炎、および歯周炎等の歯周病；う蝕；口腔カンジダ症；粘膜炎；舌炎；口唇炎；ならびに、口角炎等が挙げられる。ラクトバチルス・プランタラムの菌体調製物は、経口摂取されてもよく、口腔内で使用後、口腔外に排出されてもよい。ラクトバチルス・プランタラムの菌体もしくは菌体培養物またはこれらの抽出物は、上記口腔内組成物として供給されてもよい。 [Method for preventing or treating oral diseases]
A method for preventing or treating oral diseases according to one embodiment of the present invention includes applying Lactobacillus plantarum cells or cell cultures or extracts thereof to the oral cavity of an animal. The animal may be a human or non-human animal. Oral diseases include periodontal diseases such as gingivitis and periodontitis; dental caries; oral candidiasis; mucositis; glossitis; The bacterial cell preparation of Lactobacillus plantarum may be orally ingested, or may be excreted out of the oral cavity after being used in the oral cavity. Cells or cell cultures of Lactobacillus plantarum or extracts thereof may be supplied as the intraoral composition.
　本発明の一実施形態は、口腔内疾患の予防または治療のためのラクトバチルス・プランタラムの菌体もしくは菌体培養物またはこれらの抽出物の使用である。 One embodiment of the present invention is the use of Lactobacillus plantarum cells or cell cultures or extracts thereof for the prevention or treatment of oral diseases.
　［歯周病の原因菌に対する増殖抑制剤または殺菌剤、および歯周病の原因菌の増殖抑制方法または殺菌方法］
　本発明の一実施形態に係る歯周病の原因菌に対する増殖抑制剤または殺菌剤は、ラクトバチルス・プランタラムの菌体もしくは菌体培養物またはこれらの抽出物を含む。歯周病の原因菌は、例えばポルフィロモナス・ジンジバリス、タンネレラ・フォーサイシアおよびトレポネーマ・デンティコーラから選択される少なくとも１つを含む。ラクトバチルス・プランタラムの菌体もしくは菌体培養物またはこれらの抽出物は、歯周病の原因菌を増殖抑制または殺菌することができ、特に上記３つの原因菌の全てに対する抗菌活性を有する。増殖抑制剤または殺菌剤は、上記口腔用組成物と同じ形態であってよい。増殖抑制剤または殺菌剤は、口腔内疾患の原因菌に対する他の抗菌薬および抗生物質を含んでもよく、含まなくてもよい。 [Growth inhibitor or bactericidal agent for periodontal disease-causing bacteria, and growth inhibitory method or sterilization method for periodontal disease-causing bacteria]
A growth inhibitor or fungicide for periodontal disease-causing bacteria according to one embodiment of the present invention contains Lactobacillus plantarum cells, cell cultures, or extracts thereof. Periodontal disease-causing bacteria include, for example, at least one selected from Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. Lactobacillus plantarum cells, cell cultures, or extracts thereof can inhibit the growth of or kill periodontal disease-causing bacteria, and particularly have antibacterial activity against all of the above three causative bacteria. The antiproliferative agent or bactericidal agent may be in the same form as the oral composition. Antiproliferative agents or antiseptics may or may not include other antibacterial agents and antibiotics against causative agents of oral disease.
　増殖抑制剤または殺菌剤に含まれる上記乳酸菌の菌体調製物の含有量は特に限定されないが、例えば１×１０３～１×１０１２ｃｆｕ／ｍＬであってもよく、１×１０５～１×１０１２ｃｆｕ／ｍＬであってもよく、１×１０７～１×１０１２ｃｆｕ／ｍＬであってもよい。上記コロニー形成単位は、寒天平板培養法によって求めることができる。増殖抑制剤または殺菌剤に含まれる上記乳酸菌の菌体調製物の合計の濃度は、特に限定されず、０．１～１００，０００ｐｐｍであってもよく、１～１００，０００ｐｐｍであってもよく、１０～１００，０００ｐｐｍであってもよい。当該合計の濃度は、天秤等で秤量可能である。 The content of the lactic acid bacteria cell preparation contained in the growth inhibitor or bactericide is not particularly limited, but may be, for example, 1×10 3 to 1×10 12 cfu/mL, or 1×10 5 to 1×10 cfu/mL. It may be x10 12 cfu/mL, or may be from 1 x 10 7 to 1 x 10 12 cfu/mL. The colony-forming unit can be determined by an agar plate culture method. The total concentration of the lactic acid bacteria cell preparation contained in the growth inhibitor or disinfectant is not particularly limited, and may be 0.1 to 100,000 ppm, or 1 to 100,000 ppm. , from 10 to 100,000 ppm. The total concentration can be weighed with a balance or the like.
　本発明の一実施形態に係る歯周病の原因菌の増殖抑制または殺菌方法は、ラクトバチルス・プランタラムの菌体もしくは菌体培養物またはこれらの抽出物を歯周病の原因菌に接触させることを含む。ラクトバチルス・プランタラムの菌体調製物と歯周病の原因菌とを接触させる方法は特に限定されず、例えば歯周病の原因菌が存在する環境にラクトバチルス・プランタラムの菌体調製物を添加してもよく、歯周病の原因菌が存在する物体をラクトバチルス・プランタラムの菌体調製物を含む液体に浸漬してもよく、歯周病の原因菌が感染した宿主にラクトバチルス・プランタラムの菌体調製物を含む組成物を経口投与してもよい。ラクトバチルス・プランタラムの菌体調製物は、歯周病の原因菌の増殖抑制剤または殺菌剤の形態で供給されてもよい。 A method for inhibiting or sterilizing the growth of periodontal disease-causing bacteria according to one embodiment of the present invention comprises contacting Lactobacillus plantarum bacteria or bacterial cell cultures or extracts thereof with periodontal disease-causing bacteria. Including. The method of contacting the bacterial cell preparation of Lactobacillus plantarum with the causative bacteria of periodontal disease is not particularly limited. may be added, an object in which periodontal disease-causing bacteria are present may be immersed in a liquid containing a bacterial cell preparation of Lactobacillus plantarum, and a host infected with periodontal disease-causing bacteria may be lacto Compositions containing the bacterial cell preparation of Bacillus plantarum may be administered orally. The bacterial cell preparation of Lactobacillus plantarum may be supplied in the form of a periodontal disease-causing bacteria growth inhibitor or bactericide.
　本発明の一実施形態は、歯周病の原因菌の増殖抑制剤または殺菌剤の製造におけるラクトバチルス・プランタラムの菌体もしくは菌体培養物またはこれらの抽出物の使用である。本発明の一実施形態は、歯周病の原因菌の増殖抑制または殺菌のためのラクトバチルス・プランタラムの菌体もしくは菌体培養物またはこれらの抽出物の使用である。 One embodiment of the present invention is the use of Lactobacillus plantarum cells or cell cultures or extracts thereof in the production of growth inhibitors or fungicides for periodontal disease-causing bacteria. One embodiment of the present invention is the use of Lactobacillus plantarum cells or cell cultures or extracts thereof for growth inhibition or sterilization of periodontal disease-causing bacteria.
　以下、実施例を挙げて本発明をより詳細に説明するが、本発明はこれらの実施例に限定されるものではない。 The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.
　［実験１：ＮＩＴＥ　ＢＰ－０３１９８の分離および同定］
　分離源（イカ塩辛）を滅菌水と共に摩砕した。摩砕液を適宜希釈して１／２ＭＲＳ液体培地に添加し、集積培養した。集積培養液を炭酸カルシウム含有のＭＲＳ寒天培地に塗抹し、ハローを形成した微生物を分離した。以下、この分離株を分離株Ａという。過酸化水素に分離株Ａの培養液を懸濁したところ、気泡が発生しなかった。分離株Ａは、カタラーゼ活性を有しないことから、乳酸菌であることが確認された。 [Experiment 1: Separation and identification of NITE BP-03198]
The isolate (squid salted fish) was ground with sterile water. The homogenate was appropriately diluted and added to 1/2 MRS liquid medium for enrichment culture. The enriched culture was smeared on a calcium carbonate-containing MRS agar medium to isolate microorganisms that formed halos. Hereinafter, this isolate will be referred to as isolate A. Suspension of isolate A cultures in hydrogen peroxide did not generate bubbles. Isolate A was confirmed to be a lactic acid bacterium because it did not have catalase activity.
　１６Ｓ　ｒＲＮＡ遺伝子解析、形態観察および生理・生化学的性状試験によって、分離株Ａを同定した。
　（１）１６Ｓ　ｒＲＮＡ遺伝子解析
　分離株ＡからゲノムＤＮＡを抽出し、得られたゲノムＤＮＡを鋳型として、クローニング用フォワードプライマー９Ｆおよびクローニング用リバースプライマー１５１０Ｒ（中川恭好他：遺伝子解析法　１６Ｓ　ｒＲＮＡ遺伝子の塩基配列決定法、日本放線菌学会編、放線菌の分類と同定、８８－１１７ｐｐ．日本学会事務センター、２００１）を用いて１６Ｓ　ｒＲＮＡ遺伝子のＰＣＲ増幅を行った。ＰＣＲ増幅はＴｋｓ　Ｇｆｌｅｘ　ＤＮＡ　ポリメラーゼ（タカラバイオ社製）を用いて行い、ＰＣＲ後の増幅産物を精製した。 Isolate A was identified by 16S rRNA gene analysis, morphological observation, and physiological and biochemical characterization tests.
(1) 16S rRNA gene analysis Genomic DNA was extracted from the isolate A, and the resulting genomic DNA was used as a template for cloning forward primer 9F and cloning reverse primer 1510R (Yoshiyoshi Nakagawa et al.: Genetic analysis method of 16S rRNA gene. PCR amplification of the 16S rRNA gene was performed using a nucleotide sequencing method, Actinomycetes Society of Japan, Classification and Identification of Actinomycetes, 88-117 pp. Japan Society Office, 2001). PCR amplification was performed using Tks Gflex DNA polymerase (manufactured by Takara Bio Inc.), and the amplified product after PCR was purified.
　精製したＰＣＲ後の増幅産物を用いてサイクルシーケンス反応を行った。サイクルシーケンス反応は、ＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　ｖ３．１　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｋｉｔを用いて行った。得られた反応液を精製し、精製液をＤＮＡシーケンス解析（３１３０ｘｌ　ＤＮＡ　Ａｎａｌｙｚｅｒ）に供して、分離株Ａから抽出した鋳型ＤＮＡの１６Ｓ　ｒＲＮＡ遺伝子の塩基配列を決定した。シークエンス解析用プライマーとしては、９Ｆ、５１５Ｆ、１０９９Ｆ、５３６Ｒ、９２６Ｒ、１５１０Ｒ（中川恭好他：遺伝子解析法　１６Ｓ　ｒＲＮＡ遺伝子の塩基配列決定法、日本放線菌学会編、放線菌の分類と同定、８８－１１７ｐｐ．日本学会事務センター、２００１）を用いた。 A cycle sequencing reaction was performed using the purified amplified product after PCR. The cycle sequencing reaction was performed using BigDye Terminator v3.1 Cycle Sequencing Kit. The resulting reaction solution was purified, and the purified solution was subjected to DNA sequence analysis (3130xl DNA Analyzer) to determine the base sequence of the 16S rRNA gene of the template DNA extracted from isolate A. Sequence analysis primers include 9F, 515F, 1099F, 536R, 926R, and 1510R (Yoshiyoshi Nakagawa et al.: Gene analysis method 16S rRNA gene sequencing method, Actinomycetes Society of Japan, Classification and Identification of Actinomycetes, 88 -117 pp. Japan Society Administrative Center, 2001) was used.
　分離株Ａの１６Ｓ　ｒＲＮＡ遺伝子の塩基配列を微生物同定システム「ＥＮＫＩ」（テクノスルガ・ラボ社製）を用いて、微生物同定データベースＤＢ－ＢＡ１５．０（テクノスルガ・ラボ社製）、国際塩基配列データベース（ＤＤＢＪ／ＥＮＡ（ＥＭＢＬ）／ＧｅｎＢａｎｋ）に対してＢＬＡＳＴ相同性検索を行った。分離株Ａの１６Ｓ　ｒＲＮＡ遺伝子の塩基配列は、Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｐｅｎｔｏｓｕｓ（ＪＣＭ１５５８）の１６Ｓ　ｒＲＮＡ遺伝子の塩基配列に対して同一性９９．８７％、Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｐｌａｎｔａｒｕｍ　ｓｕｂｓｐ．ｐｌａｎｔａｒｕｍ（ＪＣＭ１１４９）の１６Ｓ　ｒＲＮＡ遺伝子の塩基配列に対して同一性９９．８７％、Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｐａｒａｐｌａｎｔａｒｕｍ（ＤＳＭ１０６６７）の１６Ｓ　ｒＲＮＡ遺伝子の塩基配列に対して同一性９９．７３％を示した。しかし、分離株Ａの１６Ｓ　ｒＲＮＡ遺伝子の塩基配列と完全に一致する１６Ｓ　ｒＲＮＡ遺伝子を持つ微生物は存在しなかった。 Using the microorganism identification system "ENKI" (manufactured by Techno Suruga Lab), the base sequence of the 16S rRNA gene of isolate A was identified as a microorganism identification database DB-BA15.0 (manufactured by Techno Suruga Lab), an international base sequence database A BLAST homology search was performed against (DDBJ/ENA(EMBL)/GenBank). The nucleotide sequence of the 16S rRNA gene of isolate A has 99.87% identity to the nucleotide sequence of the 16S rRNA gene of Lactobacillus pentosus (JCM1558), Lactobacillus plantarum subsp. It showed 99.87% identity to the nucleotide sequence of the 16S rRNA gene of Lactobacillus (JCM1149) and 99.73% identity to the nucleotide sequence of the 16S rRNA gene of Lactobacillus paraplantarum (DSM10667). However, there was no microorganism with a 16S rRNA gene that completely matched the base sequence of the 16S rRNA gene of isolate A.
　（２）形態観察および生理・生化学的性状試験
　ＭＲＳ寒天培地に分離菌Ａを塗布し、温度３０℃で４８時間好気培養し、以下の方法で細胞形態、グラム染色性、運動性およびコロニー形態を観察した。細胞形態は、光学顕微鏡ＢＸ５０Ｆ４（オリンパス社製）によって観察した。グラム染色ではフェイバーＧ「ニッスイ」（日水製薬社製）を用いた。コロニー形態は、実体顕微鏡ＳＭＺ８００Ｎ（ニコン社製）によって観察した。Ｂａｒｒｏｗ＆Ｆｅｌｔｈａｍ（Ｃｏｗａｎ　ａｎｄ　Ｓｔｅｅｌ’ｓ　Ｍａｎｕａｌ　ｆｏｒ　ｔｈｅ　Ｉｄｅｎｔｉｆｉｃａｔｉｏｎ　ｏｆ　Ｍｅｄｉｃａｌ　Ｂａｃｔｅｒｉａ，３ｒｄ　ｅｄ．Ｃａｍｂｒｉｄｇｅ：Ｃａｍｂｒｉｄｇｅ　Ｕｎｉｖｅｒｓｉｔｙ　Ｐｒｅｓｓ；１９９３．）に記載の方法に基づき、カタラーゼ反応、オキシダーゼ反応、ブドウ糖からの酸／ガス産生およびブドウ糖の酸化／発酵（Ｏ／Ｆ）について試験を行った。ＡＰＩ５０ＣＨＢキット（ｂｉｏＭｅｒｉｅｕｘ社製、フランス）を用いて、細菌の生理・生化学的性状反応を調べた。 (2) Morphological Observation and Physiological/Biochemical Property Test The isolate A was applied to an MRS agar medium and aerobically cultured at a temperature of 30° C. for 48 hours. Morphology was observed. Cell morphology was observed with an optical microscope BX50F4 (manufactured by Olympus). For Gram staining, Favor G "Nissui" (manufactured by Nissui Pharmaceutical Co., Ltd.) was used. Colony morphology was observed with a stereoscopic microscope SMZ800N (manufactured by Nikon Corporation). Based on the method described in Barrow & Feltham (Cowan and Steel's Manual for the Identification of Medical Bacteria, 3rd ed. Cambridge: Cambridge University Press; 1993.), catalase reaction, oxidase reaction, production gas from glucose and acid / Testing was done for oxidation/fermentation (O/F). The API50CHB kit (manufactured by bioMerieux, France) was used to examine the physiological and biochemical properties of the bacteria.
　図１の（Ａ）に示すように、分離株Ａは円形のコロニーを形成した。図１の（Ｂ）に示すように、分離株Ａはグラム染色性が陽性であった。分離株Ａの生理・生化学的性状試験および発酵性試験の結果を表１および表２に示す。分離株Ａは運動性を示さないグラム陽性の桿菌で、芽胞を形成せず、カタラーゼ反応およびオキシダーゼ反応は陰性を示し、グルコースを発酵した。これらの性状は、１６Ｓ　ｒＤＮＡ部分塩基配列解析の結果、帰属の可能性が示されたＬａｃｔｏｂａｃｉｌｌｕｓ属の性状と一致した。ＡＰＩキットを用いて行った発酵性試験の結果、分離株Ａはガラクトース、フラクトースおよびメレチトース等を発酵し、グリセロール、Ｄ－キシロース等を発酵しなかった。分離株Ａは、アルギニンジヒドロラーゼ活性を示さず、１５℃で生育した。これらの性状は、１６Ｓ　ｒＤＮＡ部分塩基配列解析の結果、帰属が示唆されたＬ．ｐｅｎｔｏｓｕｓおよびＬ．ｐｌａｎｔａｒｕｍのうち、グリセロールおよびＤ－キシロースの発酵を示さない点がＬ．ｐｅｎｔｏｓｕｓと異なり、Ｌ．ｐｌａｎｔａｒｕｍの性状と一致した。従って、分離株ＡはＬａｃｔｏｂａｃｉｌｌｕｓ　ｐｌａｎｔａｒｕｍに属する新規分離株であることがわかった。分離株ＡをＮＩＴＥ　ＢＰ－０３１９８として国際寄託した。 As shown in Figure 1 (A), isolate A formed circular colonies. As shown in FIG. 1(B), isolate A was positive for Gram staining. Tables 1 and 2 show the results of physiological/biochemical property tests and fermentability tests of isolate A. Isolate A was a non-motile Gram-positive bacillus, did not form spores, was negative in catalase and oxidase reactions, and fermented glucose. These properties were consistent with those of the genus Lactobacillus, for which the possibility of attribution was shown as a result of 16S rDNA partial nucleotide sequence analysis. As a result of a fermentability test conducted using an API kit, isolate A fermented galactose, fructose, melezitose, etc., but did not ferment glycerol, D-xylose, etc. Isolate A showed no arginine dihydrolase activity and grew at 15°C. These properties were attributed to L. cerevisiae, whose attribution was suggested as a result of 16S rDNA partial nucleotide sequence analysis. pentosus and L. Among L. plantarum, L. plantarum shows no fermentation of glycerol and D-xylose. Unlike L. pentosus, L. It matched the properties of plantarum. Therefore, isolate A was found to be a novel isolate belonging to Lactobacillus plantarum. The isolate A was internationally deposited as NITE BP-03198.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　［実験２：ＮＩＴＥ　ＢＰ－０３１９９の分離および同定］
　分離源として、レンブを用いた以外は、実験１と同じ方法により、分離株Ｂを分離した。分離株Ｂの同定を実験１と同じ方法によって行なった。 [Experiment 2: Separation and identification of NITE BP-03199]
Isolate B was isolated by the same method as Experiment 1, except that Lembu was used as the isolation source. Identification of isolate B was performed by the same method as in Experiment 1.
　分離株Ｂの１６Ｓ　ｒＲＮＡ遺伝子の塩基配列は、Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｐｅｎｔｏｓｕｓ（ＪＣＭ１５５８）の１６Ｓ　ｒＲＮＡ遺伝子の塩基配列に対して同一性１００．０％、Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｐｌａｎｔａｒｕｍ　ｓｕｂｓｐ．ｐｌａｎｔａｒｕｍ（ＪＣＭ１１４９）の１６Ｓ　ｒＲＮＡ遺伝子の塩基配列に対して同一性１００．０％、Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｐａｒａｐｌａｎｔａｒｕｍ（ＤＳＭ１０６６７）の１６Ｓ　ｒＲＮＡ遺伝子の塩基配列に対して同一性９９．８０％を示した。 The base sequence of the 16S rRNA gene of isolate B has 100.0% identity to the base sequence of the 16S rRNA gene of Lactobacillus pentosus (JCM1558), Lactobacillus plantarum subsp. It showed 100.0% identity to the nucleotide sequence of the 16S rRNA gene of Lactobacillus (JCM1149) and 99.80% identity to the nucleotide sequence of the 16S rRNA gene of Lactobacillus paraplantarum (DSM10667).
　図２の（Ａ）に示すように、分離株Ｂは円形のコロニーを形成した。図２の（Ｂ）に示すように、分離株Ｂはグラム染色性が陽性であった。分離株Ｂの生理・生化学的性状試験および発酵性試験の結果を表３および表４に示す。分離株Ｂは運動性を示さないグラム陽性の桿菌で、芽胞を形成せず、カタラーゼ反応およびオキシダーゼ反応は陰性を示し、グルコースを発酵した。これらの性状は、１６Ｓ　ｒＤＮＡ部分塩基配列解析の結果、帰属の可能性が示されたＬａｃｔｏｂａｃｉｌｌｕｓ属の性状と一致した。ＡＰＩキットを用いて行った発酵性試験の結果、分離株Ｂはガラクトース、フラクトース、α－メチル－Ｄ－マンノシドおよびメレチトース等を発酵し、グリセロール、Ｄ－キシロース等を発酵しなかった。分離株Ｂは、アルギニンジヒドロラーゼ活性を示さず、１５℃で生育した。これらの性状は、１６Ｓ　ｒＤＮＡ部分塩基配列解析の結果、帰属が示唆されたＬ．ｐｅｎｔｏｓｕｓおよびＬ．ｐｌａｎｔａｒｕｍのうち、グリセロールおよびＤ－キシロースの発酵を示さない点がＬ．ｐｅｎｔｏｓｕｓと異なり、Ｌ．ｐｌａｎｔａｒｕｍの性状と一致した。従って、分離株ＢはＬａｃｔｏｂａｃｉｌｌｕｓ　ｐｌａｎｔａｒｕｍに属する新規分離株であることがわかった。分離株ＢをＮＩＴＥ　ＢＰ－０３１９９として国際寄託した。 As shown in Figure 2 (A), isolate B formed circular colonies. As shown in FIG. 2B, isolate B was positive for Gram staining. Tables 3 and 4 show the results of physiological and biochemical property tests and fermentability tests of isolate B. Isolate B was a non-motile Gram-positive bacillus, did not form spores, was negative in catalase and oxidase reactions, and fermented glucose. These properties were consistent with those of the genus Lactobacillus, for which the possibility of attribution was shown as a result of 16S rDNA partial nucleotide sequence analysis. As a result of a fermentability test conducted using an API kit, isolate B fermented galactose, fructose, α-methyl-D-mannoside and melezitose, etc., but did not ferment glycerol, D-xylose, etc. Isolate B showed no arginine dihydrolase activity and grew at 15°C. These properties were attributed to L. cerevisiae, whose attribution was suggested as a result of 16S rDNA partial nucleotide sequence analysis. pentosus and L. Among L. plantarum, L. plantarum shows no fermentation of glycerol and D-xylose. Unlike L. pentosus, L. It matched the properties of plantarum. Therefore, isolate B was found to be a novel isolate belonging to Lactobacillus plantarum. Isolate B was internationally deposited as NITE BP-03199.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　［実験３：ＮＩＴＥ　ＢＰ－０３２００の分離および同定］
　分離源として、アダンを用いた以外は、実験１と同じ方法により、分離株Ｃを分離した。分離株Ｃの同定を実験１と同じ方法によって行なった。 [Experiment 3: Separation and identification of NITE BP-03200]
Isolate C was isolated by the same method as in Experiment 1, except that pandanus was used as the isolation source. Identification of isolate C was performed by the same method as in Experiment 1.
　分離株Ｃの１６Ｓ　ｒＲＮＡ遺伝子の塩基配列は、Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｐｅｎｔｏｓｕｓ（ＪＣＭ１５５８）の１６Ｓ　ｒＲＮＡ遺伝子の塩基配列に対して同一性９９．８７％、Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｐｌａｎｔａｒｕｍ　ｓｕｂｓｐ．ｐｌａｎｔａｒｕｍ（ＪＣＭ１１４９）の１６Ｓ　ｒＲＮＡ遺伝子の塩基配列に対して同一性９９．８７％、Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｐａｒａｐｌａｎｔａｒｕｍ（ＤＳＭ１０６６７）の１６Ｓ　ｒＲＮＡ遺伝子の塩基配列に対して同一性９９．６６％を示した。しかし、分離株Ｃの１６Ｓ　ｒＲＮＡ遺伝子の塩基配列と完全に一致する１６Ｓ　ｒＲＮＡ遺伝子を持つ微生物は存在しなかった。 The base sequence of the 16S rRNA gene of isolate C has 99.87% identity to the base sequence of the 16S rRNA gene of Lactobacillus pentosus (JCM1558), Lactobacillus plantarum subsp. It showed 99.87% identity to the nucleotide sequence of the 16S rRNA gene of Lactobacillus (JCM1149) and 99.66% identity to the nucleotide sequence of the 16S rRNA gene of Lactobacillus paraplantarum (DSM10667). However, there was no microorganism with a 16S rRNA gene that completely matched the base sequence of the isolate C 16S rRNA gene.
　図３の（Ａ）に示すように、分離株Ｃは円形のコロニーを形成した。図３の（Ｂ）に示すように、分離株Ｃはグラム染色性が陽性であった。分離株Ｃの生理・生化学的性状試験および発酵性試験の結果を表５および表６に示す。分離株Ｃは運動性を示さないグラム陽性の桿菌で、芽胞を形成せず、カタラーゼ反応およびオキシダーゼ反応は陰性を示し、グルコースを発酵した。これらの性状は、１６Ｓ　ｒＤＮＡ部分塩基配列解析の結果、帰属の可能性が示されたＬａｃｔｏｂａｃｉｌｌｕｓ属の性状と一致した。ＡＰＩキットを用いて行った発酵性試験の結果、分離株Ｃはガラクトース、フラクトース、α－メチル－Ｄ－マンノシドおよびメレチトース等を発酵し、グリセロール、Ｄ－キシロース等を発酵しなかった。分離株Ｃは、アルギニンジヒドロラーゼ活性を示さず、１５℃で生育した。これらの性状は、１６Ｓ　ｒＤＮＡ部分塩基配列解析の結果、近縁と示されたＬ．ｐｅｎｔｏｓｕｓおよびＬ．ｐｌａｎｔａｒｕｍのうち、グリセロールおよびＤ－キシロースの発酵を示さない点がＬ．ｐｅｎｔｏｓｕｓと異なり、Ｌ．ｐｌａｎｔａｒｕｍの性状と一致した。従って、分離株ＣはＬａｃｔｏｂａｃｉｌｌｕｓ　ｐｌａｎｔａｒｕｍに属する新規分離株であることがわかった。分離株ＣをＮＩＴＥ　ＢＰ－０３２００として国際寄託した。 As shown in Figure 3 (A), isolate C formed circular colonies. As shown in FIG. 3B, isolate C was positive for Gram staining. Tables 5 and 6 show the results of physiological and biochemical property tests and fermentability tests of isolate C. Isolate C was a non-motile Gram-positive bacillus, did not form spores, was negative in catalase and oxidase reactions, and fermented glucose. These properties were consistent with those of the genus Lactobacillus, for which the possibility of attribution was shown as a result of 16S rDNA partial nucleotide sequence analysis. As a result of a fermentability test conducted using an API kit, isolate C fermented galactose, fructose, α-methyl-D-mannoside and melezitose, etc., but did not ferment glycerol, D-xylose, etc. Isolate C showed no arginine dihydrolase activity and grew at 15°C. These properties were shown to be closely related as a result of 16S rDNA partial nucleotide sequence analysis. pentosus and L. Among L. plantarum, L. plantarum shows no fermentation of glycerol and D-xylose. Unlike L. pentosus, L. It matched the properties of plantarum. Therefore, isolate C was found to be a novel isolate belonging to Lactobacillus plantarum. Isolate C was internationally deposited as NITE BP-03200.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　［実験４：ＮＩＴＥ　ＢＰ－０３２０１の分離および同定］
　分離源として、ギランイヌビワを用いた以外は、実験１と同じ方法により分離株Ｄを分離した。分離株Ｄの同定を実験１と同じ方法によって行なった。 [Experiment 4: Separation and identification of NITE BP-03201]
Isolate D was isolated by the same method as in Experiment 1, except that G. japonica was used as the isolation source. Identification of isolate D was performed by the same method as in Experiment 1.
　分離株Ｄの１６Ｓ　ｒＲＮＡ遺伝子の塩基配列は、Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｐｅｎｔｏｓｕｓ（ＪＣＭ１５５８）の１６Ｓ　ｒＲＮＡ遺伝子の塩基配列に対して同一性９９．９３％、Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｐｌａｎｔａｒｕｍ　ｓｕｂｓｐ．ｐｌａｎｔａｒｕｍ（ＪＣＭ１１４９）の１６Ｓ　ｒＲＮＡ遺伝子の塩基配列に対して同一性９９．９３％、Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｐａｒａｐｌａｎｔａｒｕｍ（ＤＳＭ１０６６７）の１６Ｓ　ｒＲＮＡ遺伝子の塩基配列に対して同一性９９．７３％を示した。しかし、分離株Ｄの１６Ｓ　ｒＲＮＡ遺伝子の塩基配列と完全に一致する１６Ｓ　ｒＲＮＡ遺伝子を持つ微生物は存在しなかった。 The base sequence of the 16S rRNA gene of isolate D has 99.93% identity to the base sequence of the 16S rRNA gene of Lactobacillus pentosus (JCM1558), Lactobacillus plantarum subsp. It showed 99.93% identity to the nucleotide sequence of the 16S rRNA gene of Lactobacillus (JCM1149) and 99.73% identity to the nucleotide sequence of the 16S rRNA gene of Lactobacillus paraplantarum (DSM10667). However, no microorganism with a 16S rRNA gene that completely matches the base sequence of the 16S rRNA gene of isolate D was found.
　図４の（Ａ）に示すように、分離株Ｄは円形のコロニーを形成した。図４の（Ｂ）に示すように、分離株Ｄはグラム染色性が陽性であった。分離株Ｄの生理・生化学的性状試験および発酵性試験の結果を表５および表６に示す。分離株Ｄは運動性を示さないグラム陽性の桿菌で、芽胞を形成せず、カタラーゼ反応およびオキシダーゼ反応は陰性を示し、グルコースを発酵した。これらの性状は、１６Ｓ　ｒＤＮＡ部分塩基配列解析の結果、帰属の可能性が示されたＬａｃｔｏｂａｃｉｌｌｕｓ属の性状と一致した。ＡＰＩキットを用いて行った発酵性試験の結果、分離株Ｄはガラクトース、フラクトースおよびメレチトース等を発酵し、グリセロール、Ｄ－キシロース等を発酵しなかった。分離株Ｄは、アルギニンジヒドロラーゼ活性を示さず、１５℃で生育した。これらの性状は、１６Ｓ　ｒＤＮＡ部分塩基配列解析の結果、帰属が示唆されたＬ．ｐｅｎｔｏｓｕｓおよびＬ．ｐｌａｎｔａｒｕｍのうち、グリセロールおよびＤ－キシロースの発酵を示さない点がＬ．ｐｅｎｔｏｓｕｓと異なり、Ｌ．ｐｌａｎｔａｒｕｍの性状と一致した。従って、分離株ＤはＬａｃｔｏｂａｃｉｌｌｕｓ　ｐｌａｎｔａｒｕｍに属する新規分離株であることがわかった。分離株ＤをＮＩＴＥ　ＢＰ－０３２０１として国際寄託した。 As shown in Figure 4 (A), isolate D formed circular colonies. As shown in FIG. 4B, isolate D was positive for Gram staining. Tables 5 and 6 show the results of physiological/biochemical property tests and fermentability tests of isolate D. Isolate D was a non-motile Gram-positive bacillus, did not form spores, was negative in catalase and oxidase reactions, and fermented glucose. These properties were consistent with those of the genus Lactobacillus, for which the possibility of attribution was shown as a result of 16S rDNA partial nucleotide sequence analysis. As a result of a fermentability test conducted using an API kit, isolate D fermented galactose, fructose and melezitose, etc., but did not ferment glycerol, D-xylose, etc. Isolate D showed no arginine dihydrolase activity and grew at 15°C. These properties were attributed to L. cerevisiae, whose attribution was suggested as a result of 16S rDNA partial nucleotide sequence analysis. pentosus and L. Among L. plantarum, L. plantarum shows no fermentation of glycerol and D-xylose. Unlike L. pentosus, L. It matched the properties of plantarum. Therefore, isolate D was found to be a novel isolate belonging to Lactobacillus plantarum. Isolate D was internationally deposited as NITE BP-03201.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　［実験５：ＮＩＴＥ　ＢＰ－０３２０２の分離および同定］
　分離源として、マツボックリを用いた以外は、実験１と同じ方法により分離株Ｅを分離した。分離株Ｅの同定を実験１と同じ方法によって行なった。 [Experiment 5: Separation and identification of NITE BP-03202]
Isolate E was isolated by the same method as in Experiment 1, except that Pinus chinensis was used as the isolation source. Identification of isolate E was performed by the same method as in Experiment 1.
　分離株Ｅの１６Ｓ　ｒＲＮＡ遺伝子の塩基配列は、Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｐｅｎｔｏｓｕｓ（ＪＣＭ１５５８）の１６Ｓ　ｒＲＮＡ遺伝子の塩基配列に対して同一性９９．９３％、Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｐｌａｎｔａｒｕｍ　ｓｕｂｓｐ．ｐｌａｎｔａｒｕｍ（ＪＣＭ１１４９）の１６Ｓ　ｒＲＮＡ遺伝子の塩基配列に対して同一性９９．９３％、Ｌａｃｔｏｂａｃｉｌｌｕｓ　ｐａｒａｐｌａｎｔａｒｕｍ（ＤＳＭ１０６６７）の１６Ｓ　ｒＲＮＡ遺伝子の塩基配列に対して同一性９９．７３％を示した。しかし、分離株Ｅの１６Ｓ　ｒＲＮＡ遺伝子の塩基配列と完全に一致する１６Ｓ　ｒＲＮＡ遺伝子を持つ微生物は存在しなかった。 The nucleotide sequence of the 16S rRNA gene of isolate E has 99.93% identity to the nucleotide sequence of the 16S rRNA gene of Lactobacillus pentosus (JCM1558), Lactobacillus plantarum subsp. It showed 99.93% identity to the nucleotide sequence of the 16S rRNA gene of Lactobacillus (JCM1149) and 99.73% identity to the nucleotide sequence of the 16S rRNA gene of Lactobacillus paraplantarum (DSM10667). However, no microorganism with a 16S rRNA gene that completely matches the base sequence of the isolate E 16S rRNA gene was found.
　図５の（Ａ）に示すように、分離株Ｅは円形のコロニーを形成した。図５の（Ｂ）に示すように、分離株Ｅはグラム染色性が陽性であった。分離株Ｅの生理・生化学的性状試験および発酵性試験の結果を表９および表１０に示す。分離株Ｅは運動性を示さないグラム陽性の桿菌で、芽胞を形成せず、カタラーゼ反応およびオキシダーゼ反応は陰性を示し、グルコースを発酵した。これらの性状は、１６Ｓ　ｒＤＮＡ部分塩基配列解析の結果、帰属の可能性が示されたＬａｃｔｏｂａｃｉｌｌｕｓ属の性状と一致した。ＡＰＩキットを用いて行った発酵性試験の結果、分離株Ｅはガラクトース、フラクトース、α－メチル－Ｄ－マンノシドおよびメレチトース等を発酵し、グリセロール、Ｄ－キシロース等を発酵しなかった。分離株Ｅは、アルギニンジヒドロラーゼ活性を示さず、１５℃で生育した。これらの性状は、１６Ｓ　ｒＤＮＡ部分塩基配列解析の結果、帰属が示唆されたＬ．ｐｅｎｔｏｓｕｓおよびＬ．ｐｌａｎｔａｒｕｍのうち、グリセロールおよびＤ－キシロースの発酵を示さない点がＬ．ｐｅｎｔｏｓｕｓと異なり、Ｌ．ｐｌａｎｔａｒｕｍの性状と一致した。従って、分離株ＥはＬａｃｔｏｂａｃｉｌｌｕｓ　ｐｌａｎｔａｒｕｍに属する新規分離株であることがわかった。分離株ＥをＮＩＴＥ　ＢＰ－０３２０２として国際寄託した。 As shown in Figure 5 (A), isolate E formed circular colonies. As shown in FIG. 5B, isolate E was positive for Gram staining. Tables 9 and 10 show the results of physiological and biochemical property tests and fermentability tests of isolate E. Isolate E was a non-motile Gram-positive bacillus, did not form spores, was negative in catalase and oxidase reactions, and fermented glucose. These properties were consistent with those of the genus Lactobacillus, for which the possibility of attribution was shown as a result of 16S rDNA partial nucleotide sequence analysis. As a result of a fermentability test conducted using an API kit, isolate E fermented galactose, fructose, α-methyl-D-mannoside and melezitose, etc., but did not ferment glycerol, D-xylose, etc. Isolate E showed no arginine dihydrolase activity and grew at 15°C. These properties were attributed to L. cerevisiae, whose attribution was suggested as a result of 16S rDNA partial nucleotide sequence analysis. pentosus and L. Among L. plantarum, L. plantarum shows no fermentation of glycerol and D-xylose. Unlike L. pentosus, L. It matched the properties of plantarum. Therefore, isolate E was found to be a novel isolate belonging to Lactobacillus plantarum. The isolate E was internationally deposited as NITE BP-03202.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
　［実験６：歯周病の原因菌に対するラクトバチルス・プランタラムの抗菌活性評価試験］
　ＮＩＴＥ　ＢＰ－０３１９８、ＮＩＴＥ　ＢＰ－０３１９９、ＮＩＴＥ　ＢＰ－０３２００、ＮＩＴＥ　ＢＰ－０３２０１およびＮＩＴＥ　ＢＰ－０３２０２の菌体調製物が、歯周病の原因菌の増殖を抑制するかを検証した。 [Experiment 6: Antibacterial activity evaluation test of Lactobacillus plantarum against bacteria causing periodontal disease]
It was verified whether the cell preparations of NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201 and NITE BP-03202 inhibit the growth of periodontal disease-causing bacteria.
　図６を参照して実験方法を説明する。表１１に記載の培地および培養条件に従って、歯周病の原因菌（Ｐ．ｇ．菌、Ｔ．ｆ．菌およびＴ．ｄ．菌）を前培養した。歯周病の原因菌をＧＡＭ　ｂｒｏｔｈに懸濁し、ＭｃＦａｒｌａｎｄ　Ｎｏ．０．５（約１×１０８～２×１０８ｃｆｕ／ｍＬ）となるように調整した。これをさらに１０倍希釈して歯周病の原因菌の菌液１１（約１×１０７～２×１０７ｃｆｕ／ｍＬ）とした。１００μＬの歯周病菌の菌液１１を測定用寒天培地１２に滴下し、コンラージ棒で均一に塗布した。寒天培地の表面が乾いたら、１０μＬのラクトバチルス・プランタラムの菌体調製物１３を滴下し、３５℃で嫌気性環境下で培養した。Ｐ．ｇ．菌は５日間、Ｔ．ｆ．菌は７日間、Ｔ．ｄ．菌は４日間培養した。ラクトバチルス・プランタラムの菌体調製物が歯周病の原因菌に対する抗菌活性を有するとき、生育阻止円１４が形成される。ラクトバチルス・プランタラムの菌体調製物は、ＮＩＴＥ　ＢＰ－０３１９８、ＮＩＴＥ　ＢＰ－０３１９９、ＮＩＴＥ　ＢＰ－０３２００、ＮＩＴＥ　ＢＰ－０３２０１およびＮＩＴＥ　ＢＰ－０３２０２のいずれかの乳酸菌をＭＲＳ　Ｂｒｏｔｈで温度３０℃で培養し、遠心分離によって菌体を沈殿させた培養上清（菌体培養物）を用いた。Ｐ．ｇ．菌、Ｔ．ｆ．菌およびＴ．ｄ．菌に対する抗菌活性の結果を表１２、１３および１４にそれぞれ示す。ネガティブコントロールとして乳酸菌用培地（ＭＲＳ　Ｂｒｏｔｈ）を用いた。ポジティブコントロールとして、メロペネム１０μｇ／６ｍｍφを含むセンシ・ディスク「ＭＥＰＭ」（日本ベクトン・ディッキンソン社製）またはキノロン系抗菌剤レボフロキサシン１０μｇ／６ｍｍφを含むセンシディスク「ＬＶＦＸ」（日本ベクトン・ディッキンソン社製）を用いた。ＡＴＣＣ－１１４５４は、ナイシンＡを産生する乳酸菌（Ｌａｃｔｏｃｏｃｃｕｓ　ｓｕｂｓｐ．ｌａｃｔｉｓ）である。ナイシンＡ産生株は、ラクトバチルス・プランタラムと同じ方法で培養し、培養上清を調整した。 The experimental method will be described with reference to FIG. According to the medium and culture conditions described in Table 11, periodontal disease-causing bacteria (Pg, Tf and Td) were precultured. The causative bacteria of periodontal disease were suspended in GAM broth, and McFarland No. 0.5 (approximately 1×10 8 to 2×10 8 cfu/mL). This was further diluted 10-fold to obtain a periodontal disease-causing bacterial solution 11 (about 1×10 7 to 2×10 7 cfu/mL). 100 μL of periodontal disease bacterium liquid 11 was dropped on an agar medium 12 for measurement, and was evenly applied with a Conlarge stick. After the surface of the agar medium was dry, 10 μL of the cell preparation 13 of Lactobacillus plantarum was dropped and cultured at 35° C. in an anaerobic environment. P. g. The fungus was killed for 5 days, T. f. The fungus was killed for 7 days, T. d. The fungus was cultured for 4 days. Growth inhibition circles 14 are formed when the bacterial cell preparation of Lactobacillus plantarum has antibacterial activity against periodontal disease-causing bacteria. The bacterial cell preparation of Lactobacillus plantarum is obtained by culturing any of NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201 and NITE BP-03202 in MRS Broth at a temperature of 30 ° C. Then, the culture supernatant (cell culture) obtained by sedimenting the cells by centrifugation was used. P. g. fungus, T. f. fungi and T. d. The results of antibacterial activity against fungi are shown in Tables 12, 13 and 14, respectively. A medium for lactic acid bacteria (MRS Broth) was used as a negative control. As a positive control, use Sensidisc "MEPM" (manufactured by Nippon Becton Dickinson) containing meropenem 10 μg/6 mmφ or Sensidisc "LVFX" (manufactured by Nippon Becton Dickinson) containing 10 μg/6 mmφ quinolone antibacterial agent levofloxacin. board. ATCC-11454 is a nisin-A producing lactic acid bacterium (Lactococcus subsp. lactis). The nisin A-producing strain was cultured in the same manner as Lactobacillus plantarum, and the culture supernatant was prepared.
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000014
Figure JPOXMLDOC01-appb-T000014
　ＮＩＴＥ　ＢＰ－０３１９８、ＮＩＴＥ　ＢＰ－０３１９９、ＮＩＴＥ　ＢＰ－０３２００、ＮＩＴＥ　ＢＰ－０３２０１およびＮＩＴＥ　ＢＰ－０３２０２の菌体培養物はいずれも、Ｐ．ｇ．菌、Ｔ．ｆ．菌およびＴ．ｄ．菌の生育培地の全てに生育阻止円を形成させた。ラクトバチルス・プランタラムの菌体培養物はいずれも、Ｐ．ｇ．菌、Ｔ．ｆ．菌およびＴ．ｄ．菌の増殖を抑制できることが示された。一方、ナイシンＡ産生株の培養上清は、Ｔ．ｄ．菌の増殖を抑制したが、Ｐ．ｇ．菌およびＴ．ｆ．菌の増殖を抑制することができなかった。 All of the cell cultures of NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201 and NITE BP-03202 are P. g. fungus, T. f. fungi and T. d. A circle of growth inhibition was formed in all of the fungal growth medium. Both bacterial cell cultures of Lactobacillus plantarum are P. g. fungus, T. f. fungi and T. d. It was shown that the growth of bacteria can be suppressed. On the other hand, the culture supernatant of the nisin A-producing strain was T. cerevisiae. d. Although it inhibited the growth of P. g. fungi and T. f. It was not possible to suppress the growth of bacteria.
　［実験７：歯垢形成を促進する菌に対するラクトバチルス・プランタラムの抗菌活性評価試験］
　ＮＩＴＥ　ＢＰ－０３１９８、ＮＩＴＥ　ＢＰ－０３１９９、ＮＩＴＥ　ＢＰ－０３２００、ＮＩＴＥ　ＢＰ－０３２０１およびＮＩＴＥ　ＢＰ－０３２０２の菌体調製物が、歯垢形成を促進する菌の増殖を抑制するかを検証した。 [Experiment 7: Antibacterial activity evaluation test of Lactobacillus plantarum against bacteria that promotes plaque formation]
It was verified whether bacterial cell preparations of NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201 and NITE BP-03202 inhibit the growth of bacteria that promote plaque formation.
　実験７は、抗菌活性を検証する細菌として、ゴルドニ菌を用いたこと以外は、実験６と同様の方法で行った。表１５に記載の培地および培養条件に従って、ゴルドニ菌を前培養した。ゴルドニ菌をＧＡＭ　ｂｒｏｔｈに懸濁し、ＭｃＦａｒｌａｎｄ　Ｎｏ．０．５（約１×１０８～２×１０８ｃｆｕ／ｍＬ）となるように調整した。これをさらに１０倍希釈してゴルドニ菌の菌液１１（約１×１０７～２×１０７ｃｆｕ／ｍＬ）とした。１００μＬのゴルドニ菌の菌液１１を測定用寒天培地１２に滴下し、コンラージ棒で均一に塗布した。寒天培地の表面が乾いたら、１０μＬのラクトバチルス・プランタラムの菌体調製物１３を滴下し、３５℃で２４時間培養した。ゴルドニ菌に対する抗菌活性の結果を表１６に示す。ネガティブコントロールとして乳酸菌用培地（ＭＲＳ　Ｂｒｏｔｈ）を用いた。ポジティブコントロールとして、テトラサイクリン３０μｇ／６ｍｍφを含むセンシ・ディスク「ＴＣ」（日本ベクトン・ディッキンソン社製）を用いた。ＡＴＣＣ－１１４５４は、ナイシンＡを産生する乳酸菌（Ｌａｃｔｏｃｏｃｃｕｓ　ｓｕｂｓｐ．ｌａｃｔｉｓ）である。 Experiment 7 was performed in the same manner as Experiment 6, except that Goldoni bacteria were used as bacteria for verifying antibacterial activity. Gordonii was pre-cultured according to the medium and culture conditions described in Table 15. Gordonii bacteria were suspended in GAM broth and added to McFarland No. 0.5 (approximately 1×10 8 to 2×10 8 cfu/mL). This was further diluted 10-fold to obtain Gordonii fungus solution 11 (about 1×10 7 to 2×10 7 cfu/mL). 100 μL of the Gordonii fungus solution 11 was dropped on the agar medium 12 for measurement, and was evenly applied with a Conlarge stick. After the surface of the agar medium was dry, 10 µL of the cell preparation 13 of Lactobacillus plantarum was dropped and cultured at 35°C for 24 hours. Table 16 shows the results of the antibacterial activity against Gordonii. A medium for lactic acid bacteria (MRS Broth) was used as a negative control. As a positive control, Sensidisc "TC" (manufactured by Nippon Becton Dickinson Co., Ltd.) containing 30 μg/6 mmφ of tetracycline was used. ATCC-11454 is a nisin-A producing lactic acid bacterium (Lactococcus subsp. lactis).
Figure JPOXMLDOC01-appb-T000015
Figure JPOXMLDOC01-appb-T000015
Figure JPOXMLDOC01-appb-T000016
Figure JPOXMLDOC01-appb-T000016
　ＮＩＴＥ　ＢＰ－０３１９８、ＮＩＴＥ　ＢＰ－０３１９９、ＮＩＴＥ　ＢＰ－０３２００、ＮＩＴＥ　ＢＰ－０３２０１およびＮＩＴＥ　ＢＰ－０３２０２の菌体培養物はいずれも、ゴルドニ菌の生育培地に生育阻止円を形成させた。ラクトバチルス・プランタラムの菌体培養物は、ラクトバチルス・プランタラムの増殖を抑制できることが示された。 All of the cell cultures of NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201 and NITE BP-03202 formed growth inhibition circles in the growth medium of Gordonii fungus. It was shown that the cell culture of Lactobacillus plantarum can suppress the growth of Lactobacillus plantarum.
　［実験８：口腔常在菌に対するラクトバチルス・プランタラムの抗菌活性評価試験］
　ＮＩＴＥ　ＢＰ－０３１９８、ＮＩＴＥ　ＢＰ－０３１９９、ＮＩＴＥ　ＢＰ－０３２００、ＮＩＴＥ　ＢＰ－０３２０１およびＮＩＴＥ　ＢＰ－０３２０２の菌体調製物が、口腔常在菌の増殖を抑制するかを検証した。 [Experiment 8: Antibacterial activity evaluation test of Lactobacillus plantarum against oral bacteria]
It was verified whether the cell preparations of NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201 and NITE BP-03202 suppress the growth of oral flora.
　実験８は、抗菌活性を検証する細菌として、サリバリウス菌を用いたこと以外は、実験６と同様の方法で行った。表１７に記載の培地および培養条件に従って、サリバリウス菌を前培養した。サリバリウス菌をＧＡＭ　ｂｒｏｔｈに懸濁し、ＭｃＦａｒｌａｎｄ　Ｎｏ．０．５（約１×１０８～２×１０８ｃｆｕ／ｍＬ）となるように調整した。これをさらに１０倍希釈してサリバリウス菌の菌液１１（約１×１０７～２×１０７ｃｆｕ／ｍＬ）とした。１００μＬのサリバリウス菌の菌液１１を測定用寒天培地１２に滴下し、コンラージ棒で均一に塗布した。寒天培地の表面が乾いたら、１０μＬのラクトバチルス・プランタラムの菌体調製物１３を滴下し、３５℃で２４時間培養した。サリバリウス菌に対する抗菌活性の結果を表１８に示す。ネガティブコントロールとして乳酸菌用培地（ＭＲＳ　Ｂｒｏｔｈ）を用いた。ポジティブコントロールとして、テトラサイクリン３０μｇ／６ｍｍφを含むセンシ・ディスク「ＴＣ」（日本ベクトン・ディッキンソン社製）を用いた。ＡＴＣＣ－１１４５４は、ナイシンＡを産生する乳酸菌（Ｌａｃｔｏｃｏｃｃｕｓ　ｓｕｂｓｐ．ｌａｃｔｉｓ）である。 Experiment 8 was carried out in the same manner as Experiment 6, except that B. salivarius was used as the bacterium for verifying the antibacterial activity. B. salivarius was pre-cultured according to the medium and culture conditions described in Table 17. Salivarius was suspended in GAM broth and plated with McFarland No. 0.5 (approximately 1×10 8 to 2×10 8 cfu/mL). This was further diluted 10-fold to obtain Salivarius bacterial solution 11 (approximately 1×10 7 to 2×10 7 cfu/mL). 100 μL of the bacterial solution 11 of Salivarius salivarius was dropped on the agar medium 12 for measurement, and was evenly applied with a Conlarge stick. After the surface of the agar medium was dry, 10 µL of the cell preparation 13 of Lactobacillus plantarum was dropped and cultured at 35°C for 24 hours. Table 18 shows the results of antibacterial activity against S. salivarius. A medium for lactic acid bacteria (MRS Broth) was used as a negative control. As a positive control, Sensidisc "TC" (manufactured by Nippon Becton Dickinson Co., Ltd.) containing 30 μg/6 mmφ of tetracycline was used. ATCC-11454 is a nisin-A producing lactic acid bacterium (Lactococcus subsp. lactis).
Figure JPOXMLDOC01-appb-T000017
Figure JPOXMLDOC01-appb-T000017
Figure JPOXMLDOC01-appb-T000018
Figure JPOXMLDOC01-appb-T000018
　ＮＩＴＥ　ＢＰ－０３１９８、ＮＩＴＥ　ＢＰ－０３１９９、ＮＩＴＥ　ＢＰ－０３２００、ＮＩＴＥ　ＢＰ－０３２０１およびＮＩＴＥ　ＢＰ－０３２０２の菌体培養物はいずれも、サリバリウス菌の生育培地に生育阻止円を形成させなかった。ラクトバチルス・プランタラムの菌体培養物は、口腔常在菌への影響が少ないことが示された。一方、テトラサイクリンは、口腔常在菌の増殖も抑制した。 None of the cell cultures of NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201 and NITE BP-03202 caused growth inhibition circles to form in the growth medium of Salivarius. It was shown that the bacterial cell culture of Lactobacillus plantarum has little effect on oral flora. On the other hand, tetracycline also suppressed the growth of oral flora.
　１１　歯周病の原因菌、歯垢形成を促進する菌又は口腔常在菌の菌液、１２　測定用寒天培地、１３　ラクトバチルス・プランタラムの菌体調製物、１４　生育阻止円。 11. Bacteria that cause periodontal disease, bacteria that promote plaque formation, or fungal fluids of oral bacteria, 12. Agar medium for measurement, 13. Lactobacillus plantarum bacterial cell preparation, 14. Growth inhibition circle.

Claims (13)

  1. 　ラクトバチルス・プランタラムの菌体もしくは菌体培養物またはこれらの抽出物を含む口腔用組成物。 An oral composition containing Lactobacillus plantarum cells or cell cultures or extracts thereof.
  2. 　食品である、請求項１に記載の組成物。 The composition according to claim 1, which is a food.
  3. 　前記食品は発酵食品である、請求項２に記載の組成物。 The composition according to claim 2, wherein the food is a fermented food.
  4. 　洗口剤または歯磨剤である、請求項１に記載の組成物。 The composition according to claim 1, which is a mouthwash or dentifrice.
  5. 　フッ素化合物をさらに含む、請求項１または４に記載の組成物。 The composition according to claim 1 or 4, further comprising a fluorine compound.
  6. 　前記ラクトバチルス・プランタラムは、ＮＩＴＥ－ＢＰ－０３１９８、ＮＩＴＥ－ＢＰ－０３１９９、ＮＩＴＥ－ＢＰ－０３２００、ＮＩＴＥ－ＢＰ－０３２０１およびＮＩＴＥ－ＢＰ－０３２０２から選択される少なくとも１つである、請求項１または２に記載の組成物。 Claim 1, wherein the Lactobacillus plantarum is at least one selected from NITE-BP-03198, NITE-BP-03199, NITE-BP-03200, NITE-BP-03201 and NITE-BP-03202. Or the composition according to 2.
  7. 　口腔内疾患の予防または治療用組成物である、請求項１～４のいずれか１項に記載の組成物。 The composition according to any one of claims 1 to 4, which is a composition for preventing or treating oral diseases.
  8. 　前記口腔内疾患は歯周病である、請求項７に記載の組成物。 The composition according to claim 7, wherein the oral disease is periodontal disease.
  9. 　ラクトバチルス・プランタラムの菌体もしくは菌体培養物またはこれらの抽出物を動物の口腔に適用することを含む、口腔内疾患の予防または治療方法。 A method for preventing or treating oral diseases, which comprises applying Lactobacillus plantarum cells or cell cultures or extracts thereof to the oral cavity of animals.
  10. 　前記口腔内疾患は歯周病である、請求項９に記載の方法。 The method according to claim 9, wherein the oral disease is periodontal disease.
  11. 　ラクトバチルス・プランタラムの菌体もしくは菌体培養物またはこれらの抽出物を含む、歯周病の原因菌に対する増殖抑制剤または殺菌剤。 A growth inhibitor or bactericidal agent for periodontal disease-causing bacteria containing Lactobacillus plantarum cells or cell cultures or extracts thereof.
  12. 　前記歯周病の原因菌は、ポルフィロモナス・ジンジバリス、タンネレラ・フォーサイシアおよびトレポネーマ・デンティコーラから選択される少なくとも１つを含む、請求項１１に記載の増殖抑制剤または殺菌剤。 The growth inhibitor or bactericide according to claim 11, wherein the periodontal disease-causing bacteria include at least one selected from Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola.
  13. 　ラクトバチルス・プランタラムの菌体もしくは菌体培養物またはこれらの抽出物を歯周病の原因菌に接触させることを含む、歯周病菌の増殖抑制または殺菌方法。 A method for inhibiting the growth of or sterilizing periodontal disease bacteria, comprising contacting Lactobacillus plantarum cells or cell cultures or extracts thereof with periodontal disease-causing bacteria.
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