WO2023282356A1 - 口腔用組成物、および口腔内疾患の予防または治療方法 - Google Patents

口腔用組成物、および口腔内疾患の予防または治療方法 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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English (en)
French (fr)
Japanese (ja)
Inventor
幹雄 青木
和樹 味方
貴子 清水
敏裕 甲斐
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Sumitomo Chemical Co Ltd
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Sumitomo Chemical Co Ltd
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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 OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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; 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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