WO2021194011A1 - Probiotics for prevention or treatment of oral disease and food composition comprising same for prevention or treatment of oral disease - Google Patents
Probiotics for prevention or treatment of oral disease and food composition comprising same for prevention or treatment of oral disease Download PDFInfo
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- WO2021194011A1 WO2021194011A1 PCT/KR2020/007649 KR2020007649W WO2021194011A1 WO 2021194011 A1 WO2021194011 A1 WO 2021194011A1 KR 2020007649 W KR2020007649 W KR 2020007649W WO 2021194011 A1 WO2021194011 A1 WO 2021194011A1
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- strain
- oral
- composition
- smfm2016
- alveolar bone
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- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000007183 wilkins-chalgren anaerobe broth Substances 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
Images
Classifications
-
- 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
-
- 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
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/312—Foods, ingredients or supplements having a functional effect on health having an effect on dental health
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
Definitions
- Lactobacillus curvatus Lactobacillus curvatus
- SMFM2016-NK strain accession number: KCTC14138BP
- oral disease prevention or improvement food composition comprising the strain, oral disease prevention or treatment pharmaceutical composition, probiotics, and feed to the composition.
- Oral disease refers to all diseases that can occur in the oral cavity, including teeth.
- Common oral diseases include dental caries, that is, periodontal diseases such as tooth decay, periodontitis, and gingivitis. This can be.
- Periodontal disease includes inflammatory diseases that damage soft tissues such as periodontal ligaments and gingiva surrounding teeth and hard tissues such as alveolar bone. plays a role In alveolar bone, bone formation by osteoblasts and bone resorption by osteoclasts are metabolized. When bone resorption exceeds bone formation due to various causes, the alveolar bone lowers, exposing the tooth roots, and eventually supporting the teeth. cannot be performed. This loss of alveolar bone can also be caused by gingivitis and periodontitis.
- Gingivitis is an inflammation limited to the soft tissues of the gums and is a disease with relatively light and recoverable symptoms. It causes inflammation and bone loss of the periodontal ligament.
- Such oral disease or periodontal disease is not only a disease that can occur most often in the adult population, regardless of domestic or foreign, but also a disease of public health interest that requires considerable cost in terms of personal medical care and social management system.
- the number of periodontal disease patients in Korea between 2012 and 2016 increased from 7.07 million in 2012 to 11.07 million in 2016, an increase of about 56.6% in four years, which corresponds to an average annual growth rate of 12%. Therefore, various surgical treatment methods exist and have been developed for the treatment of such periodontal disease, but for individuals, surgical treatment is not only psychologically reluctant but also carries a huge cost burden, There is a problem that continuous treatment at an appropriate time is difficult because there are cases where it is performed. Therefore, for oral diseases including periodontal disease, it can be said that not only surgical treatment, but also establishment of personal oral hygiene and prevention through this is more important.
- Lactobacillus curvatus SMFM2016-NK strain decreases the number of osteoclasts, increases the number of osteoblasts, decreases the volume of cancellous bone loss, increases the volume of alveolar bone, reduces the number of inflammatory cells, and harmful effects in the oral cavity
- the present invention was completed by confirming that it exhibits excellent antibacterial activity against bacteria.
- probiotics reached about $33 billion (about KRW 36 trillion) in 2015, and is expected to increase by 7% every year to reach about $ 47 billion (about KRW 53 trillion) by 2020.
- probiotics are highly likely to be used not only as health functional foods using them, but also as quasi-drugs and medicines for oral care and oral disease management, the significance of the present invention is also very large.
- the present invention aims to solve the above problems and other problems related thereto.
- An exemplary object of the present invention is to provide a newly isolated and identified Lactobacillus curvatus SMFM2016-NK strain (Accession No.: KCTC14138BP) from kimchi.
- Another exemplary object of the present invention is to provide a food composition for preventing or improving oral diseases, including the Lactobacillus curbatus SMFM2016-NK strain, its culture, concentrate, dry matter or lysate.
- Another exemplary object of the present invention is to provide a pharmaceutical composition for preventing or treating oral diseases, including the strain, its culture, concentrate, dry matter or lysate.
- Another exemplary object of the present invention is to provide a probiotic comprising the above strain, a culture, concentrate, dried product or lysate thereof.
- Another exemplary object of the present invention is to provide a quasi-drug composition comprising the strain, its culture, concentrate, dried product or lysate.
- Lactobacillus curvatus (Lactobacillus curvatus) SMFM2016-NK strain.
- the strain was isolated from kimchi and was deposited at the Korea Institute of Biotechnology and Biotechnology Biological Resources Center (KCTC) on February 17, 2020, and was given an accession number KCTC 14138BP.
- the strain includes the genomic characteristics of Table 7, FIGS. 5 and 6 and the 16s rDNA sequence shown in SEQ ID NO: 1, newly isolated and identified by the present inventors who confirmed that there is no identical strain as a result of genomic analysis is a strain This corresponds to a probiotic strain, is harmless to the human body, and can be used without side effects.
- the strain can exhibit the effect of reducing the number of osteoclasts, increasing the number of osteoblasts, decreasing the volume of cancellous bone loss, increasing the volume of alveolar bone, and reducing the number of inflammatory cells in oral teeth or periodontal tissue, etc.
- Intermedia Prevotella intermedia
- Fusobacterium nucleatum Fusobacterium nucleatum
- Aggregatibacter actinomycetemcomitans Aggregatibacter actinomycetemcomitans
- It may be one that exhibits antibacterial activity against harmful bacteria in the oral cavity.
- Lactobacillus curbatus SMFM2016-NK strain (Accession No.: KCTC14138BP), its culture, concentrate, dry matter or lysate, including oral disease prevention or improvement food A composition is provided.
- Lactobacillus ( Lactobacillus ) refers to a microorganism of the genus Gram-positive bacillus of aerobic or facultative anaerobic nature widely distributed in nature, and the description of the Lactobacillus curvatus SMFM2016-NK strain is as described above.
- the term "culture” refers to a medium containing by-products and strains generated through the ingestion of nutrients and metabolism by culturing the strain in a medium, and the culture of the Lactobacillus curvatus SMFM2016-NK strain. It could mean water.
- the food composition of the present invention may include a culture of the above strain as well as a concentrate, a dried product, or a lysate thereof.
- oral disease refers to all diseases that can occur in the oral cavity, including teeth, specifically, a decrease in the number of osteoclasts in the oral tissue, an increase in the number of osteoblasts, a decrease in the volume of cancellous bone loss, and alveolar bone. Includes all diseases that can be prevented, ameliorated and/or treated due to an increase in volume, a decrease in the number of inflammatory cells, and an antibacterial effect on harmful oral bacteria.
- the oral disease is alveolar bone breakage, alveolar bone osteoporosis, alveolar bone osteomalacia, alveolar bone osteopenia, dental caries, gingivitis ) and periodontitis (periodontitis) may be one or more diseases selected from the group consisting of, but is not particularly limited thereto.
- the food composition of the present invention includes the Lactobacillus curbatus SMFM2016-NK strain, its culture, concentrate, dry matter or lysate, the number of osteoclasts in the oral cavity or periodontal tissue, etc. decreases, the number of osteoblasts increases, cancellous bone Loss volume reduction, alveolar bone volume increase, and inflammatory cell count reduction effects can be equally represented, and Prevotella intermedia , Fusobacterium nucleatum ) or Aggregatibacter actinomycetemcomy Antibacterial activity against harmful bacteria in the oral cavity, such as Aggregatibacter actinomycetemcomitans, may also be exhibited in the same manner.
- the strain decreases the number of osteoclasts, increases the number of osteoblasts, decreases the volume of cancellous bone loss, increases the volume of alveolar bone and reduces the number of inflammatory cells, and the specific use thereof as long as it can exhibit antibacterial activity against harmful bacteria in the oral cavity , may be included in any amount (effective amount) depending on the formulation and purpose of mixing, and a typical effective amount will be determined within the range of 0.001 wt % to 50.0 wt % based on the total weight of the composition.
- the "effective amount” refers to the amount of the active ingredient contained in the composition of the present invention that can exhibit the intended functional and pharmacological effects according to the composition of the present invention to a mammal, preferably a human, to be applied during the administration period by a medical professional or the like. say Such effective amounts can be determined empirically within the ordinary ability of one of ordinary skill in the art.
- Subjects to which the composition of the present invention can be applied are mammals and humans, particularly preferably humans.
- the food may include a health functional (sexual) food.
- the term "health functional food” refers to a food manufactured and processed using raw materials or ingredients having useful functions in the human body.
- the “functionality” refers to obtaining useful effects for health purposes such as regulating nutrients or physiological actions with respect to the structure and function of the human body.
- the health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art.
- the formulation of the health functional food is also recognized as a health functional food, it can be prepared without limitation.
- the food composition of the present invention can be prepared in various forms, and unlike general drugs, there is no side effect that may occur when taking the drug for a long time using food as a raw material.
- the food composition of the present invention can be prepared in any form, and specifically, health functional food preparations such as tablets, capsules, pills, granules, liquids, powders, pieces, pastes, syrups, gels, jellies, bars, etc.; It may be one or more formulations selected from the group consisting of beverages, gums and candies, but is not particularly limited thereto.
- the food composition of the present invention may contain food additives in addition to the active ingredient.
- Food additives can be generally understood as substances that are mixed or infiltrated with food in manufacturing, processing, or preserving food, and their safety must be ensured as they are consumed daily and for a long period of time with food.
- the food additives are divided into sweeteners, flavoring agents, preservatives, emulsifiers, acidulants, thickeners, etc. in terms of function, and are not particularly limited as long as the food composition of the present invention meets the intended purpose.
- the food composition of the present invention may contain physiologically active substances or minerals known in the art for the purpose of functional and nutritional supplementation and guaranteed stability as a food additive in addition to the food additives.
- the physiologically active substances or minerals are not particularly limited as long as the food composition of the present invention meets the intended purpose.
- the food additives as described above may be included in an effective amount to achieve the purpose of the addition according to the type of product, and with respect to other food additives that may be included in the food composition of the present invention, the food additives of each country Or you can refer to the Food Additives Ordinance.
- the term “improvement” refers to the effect of alleviating the symptoms of oral disease or periodontal disease by applying the composition comprising the Lactobacillus curbatus SMFM2016-NK strain of the present invention
- prevention means any action that suppresses or delays oral disease or periodontal disease-related symptoms by administration of the composition of the present invention.
- Lactobacillus curvatus Lactobacillus curvatus
- SMFM2016-NK strain Accession No.: KCTC14138BP
- Lactobacillus, Lactobacillus curvatus SMFM2016-NK strain, culture and oral disease are the same as described above.
- the pharmaceutical composition of the present invention includes the Lactobacillus curbatus SMFM2016-NK strain, its culture, concentrate, dry matter or lysate, the number of osteoclasts in the oral cavity or periodontal tissue, etc. decreases, the number of osteoblasts increases, bone loss volume reduction, and equally it represents an increase in bone volume and inflammatory cell count reduction, freebo telra intermediate (Prevotella intermedia), Pew jobak Te Leeum New Clegg Atum (Fusobacterium nucleatum) or Agde Leganes tee bakteo solution Tino My setem Antibacterial activity against harmful bacteria in the oral cavity, such as Aggregatibacter actinomycetemcomitans, may also be exhibited in the same manner.
- the pharmaceutical composition of the present invention is a pharmaceutically acceptable carrier, excipient or diluent commonly used in the preparation of pharmaceutical compositions in addition to the Lactobacillus curbatus SMFM2016-NK strain, its culture, concentrate, dried product or lysate.
- the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used, but is not particularly limited thereto.
- the pharmaceutical composition of the present invention is selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents and freeze-drying agents according to a conventional method, respectively. It may have any one dosage form, and the dosage form may be in various forms, either oral or parenteral. In the case of formulation, it may be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants, but is not limited thereto. Specifically, tablets, pills, powders, granules, capsules, etc.
- the solid preparation may be used in the solid preparation for oral administration, and the solid preparation includes at least one or more excipients such as starch, calcium carbonate, sucrose or lactose, Gelatin or the like may be used.
- excipients such as starch, calcium carbonate, sucrose or lactose, Gelatin or the like
- lubricants such as magnesium stearate and talc may be used in addition to simple excipients, but the present invention is not limited thereto.
- liquid formulations for oral administration suspensions, internal solutions, emulsions, syrups, etc. may be used, and various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc.
- a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a freeze-dried formulation, or a suppository may be used as a formulation for parenteral administration.
- the non-aqueous solvent and suspending agent may include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
- the optimal amount and dosing interval of individual administration of the pharmaceutical composition of the present invention will be determined by the nature and extent of oral disease or periodontal disease, dosage form, route and site, and the age and health condition of the subject, and the doctor will ultimately use it. It will be appreciated by those skilled in the art that appropriate dosing will be determined. Such dosing may be repeated as often as appropriate, and the dosage and frequency may be altered or reduced according to normal clinical practice.
- the administration route of the composition of the present invention may be administered through any general route as long as the object of the present invention can be achieved.
- the pharmaceutical composition of the present invention may be administered intraperitoneally, intravenously, subcutaneously, intradermally, orally, but is not limited thereto.
- treatment refers to any action in which the symptoms of oral disease or periodontal disease are improved or beneficially changed by administration of the composition of the present invention.
- Lactobacillus curvatus SMFM2016-NK strain (Accession No.: KCTC14138BP), its culture, concentrate, dry matter or lysate, including probiotics.
- the term Lactobacillus, Lactobacillus curvatus SMFM2016-NK strain and culture are as described above.
- the term "probiotics” refers to living microorganisms that provide beneficial effects in the body.
- the microflora in the oral cavity is improved through the interaction of microorganisms and furthermore, oral disease or periodontal disease is prevented, improved and treated Microorganisms that can achieve the purpose of
- the strain used as the probiotic for the purpose of the present invention is the Lactobacillus curbatus SMFM2016-NK strain, and may further include other probiotic strains as long as they are effective in preventing, improving and treating oral diseases or periodontal diseases.
- the probiotics containing the Lactobacillus curbatus SMFM2016-NK strain of the present invention are characterized in that they are used for preventing and improving oral diseases, and the strain reduces the number of osteoclasts in teeth or periodontal tissues in the oral cavity, and the number of osteoblasts. increase, may indicate bone loss volume reduction, increased bone volume and inflammatory cell count reduction, freebo telra intermediate (Prevotella intermedia), Pew jobak Te Leeum New Clegg Atum (Fusobacterium nucleatum) or Agde Leganes tee bakteo solution Tino My setem It may exhibit antibacterial activity against harmful bacteria in the oral cavity, such as Aggregatibacter actinomycetemcomitans.
- the probiotics of the present invention may be used in the form of Lactobacillus curbatus SMFM2016-NK strain, its culture, concentrate, dried product or crushed product, including powder, fermented milk, granules, capsules, pills, gums, films, etc. It is not particularly limited.
- Another aspect of the present invention for achieving the above object provides a quasi-drug composition
- a quasi-drug composition comprising the Lactobacillus curvatus SMFM2016-NK strain (Accession No.: KCTC14138BP), its culture, concentrate, dry matter or lysate.
- Lactobacillus, Lactobacillus curvatus SMFM2016-NK strain, culture and oral disease are the same as described above.
- the quasi-drug composition of the present invention includes the Lactobacillus curbatus SMFM2016-NK strain, its culture, concentrate, dry matter, or lysate, the number of osteoclasts in the oral cavity or periodontal tissue, etc. decreases, the number of osteoblasts increases, cancellous bone Loss volume reduction, alveolar bone volume increase, and inflammatory cell count reduction effects can be equally represented, and Prevotella intermedia , Fusobacterium nucleatum ) or Aggregatibacter actinomycetemcomy Antibacterial activity against harmful bacteria in the oral cavity, such as Aggregatibacter actinomycetemcomitans, may also be exhibited in the same manner.
- the quasi-drug composition of the present invention may include a quasi-drug for oral use.
- the quasi-drug composition of the present invention may include ingredients commonly used in the composition for oral quasi-drugs, such as abrasives, wetting agents, binders, It may include a foaming agent, a sweetener, a preservative, an active ingredient, a flavoring agent, a colorant, a solvent, a brightener, a solubilizer, or a pH adjuster.
- the quasi-drug composition of the present invention may be prepared in any formulation conventionally prepared in the art, and specifically, toothpaste, mouthwash, gum, candy, oral spray, oral ointment, oral varnish, It may be one or more formulations selected from the group consisting of oral film and gum massage cream, but is not particularly limited thereto.
- the quasi-drug composition of the present invention when it is a toothpaste formulation, it may include a wetting agent, abrasive agent, binder, foaming agent, flavoring agent, sweetener, coloring agent, preservative, active ingredient, solvent, pH adjusting agent, and the like.
- the Lactobacillus curvatus SMFM2016-NK strain (Accession No.: KCTC14138BP) of the present invention is a probiotic strain newly isolated and identified from kimchi, and decreases the number of osteoclasts, increases the number of osteoblasts, decreases the volume of cancellous bone loss, and the volume of alveolar bone. It can be usefully used as a composition for prevention, improvement, and treatment of oral diseases because it has an effect of increasing, reducing the number of inflammatory cells, and exhibiting excellent antibacterial activity against harmful bacteria in the oral cavity.
- H&E histopathological hematozylin-eosin
- Figure 4 shows the cell morphological SEM image of the Lactobacillus curbatus SMFM2016-NK strain of the present invention.
- FIG. 6 shows the analysis results of the unweighted pair group method with arithmetic mean (UPGMA) tree based on the average sequence similarity (ANI %) of the Lactobacillus curbatus SMFM2016-NK strain of the present invention and other Lactobacillus sp. strains of the present invention.
- UGMA unweighted pair group method with arithmetic mean
- ANI average sequence similarity
- Example 1 Lactobacillus curvatus Isolation and identification of SMFM2016-NK strain
- kimchi collected in Seoul was placed in a sterile filter bag, 100 mL of 0.1% buffered peptone water (BPW) was added, and then homogenized for 1 minute using a pummeler (Bag mixer 400; Interscience Co., Paris, France). 100 ⁇ L of the homogenized solution was taken and spread on MRS agar (Becton, Dickinson and Company, Sparks, MD, USA), and incubated at 35° C. for 24 hours. The isolated strain was inoculated in MRS broth (BD, USA) medium, then cultured at 35°C for 24 hours, and a glycerol stock containing 20% glycerol was prepared and stored at -80°C before use.
- BPW buffered peptone water
- the isolated strain was identified through 16s rRNA sequencing. After pure separation of the isolates, the identification was requested to Bionics (Seoul) on MRS agar plate. 16s rRNA sequencing was performed using universal primers 518F (5'-CCAGCAGCCGCGGTAATACG-3') and 800R (5'-TACCAGGGTATCTAATCC-3'). The sequence information was as shown in the attached SEQ ID NO: 1.
- Example 3 Oral disease animal model
- Antibiotics (1 mg/mL sulfamethoxazole and 200 ⁇ g/mL trimethoprim in water) were put into a drinking water bottle and ingested for 3 days from the day following the end of the acclimatization period of the experimental animals (Sprague-Dawley rat, male, 6-week-old).
- Zoletil (1 mg/mL) and Lumpun were mixed in a 4:1 (v/v) ratio and administered intraperitoneally to experimental animals using a syringe at a dose of 1 mL/kg.
- the lower right first molar was ligated to the boundary between the gum and periodontal area using sterile nylon. After ligation, sterile nylon was pushed to be buried in the gums, and oral administration was performed while maintaining the ligation for 28 days (FIG. 1).
- lactic acid bacteria glycerol stock solution 100 ⁇ L was inoculated into a 50 mL tube containing 45 mL of MRS broth (Becton, Dickinson and Company), and incubated at 35 °C for 18-24 hours.
- SD periodontitis-induced Sprague-Dawley
- Example 4 Micro-CT analysis and cancellous bone loss volume analysis
- micro-CT was measured by setting a predetermined periodontitis-induced site, and the volume of loss of trabecular tissue was measured.
- the periodontitis-induced group showed a statistically significant loss of cancellous bone volume compared to the normal group, confirming normal periodontitis induction, and periodontitis in the L. curvatus SMFM2016-NK treatment group. It was confirmed that the volume of cancellous bone loss was reduced by 11.6% compared to the induced group (FIG. 2).
- Histopathological examination was performed after H&E staining using specimens of experimental animals measured by Micro-CT. Histomorphological changes were scored based on the following criteria, and inflammatory cell numbers, osteoblast cell numbers, osteoclast cell numbers, and alveolar process volumes were examined. Items were measured (Table 3).
- the histomorphological change score was significantly reduced in the L. curvatus SMFM2016-NK treatment group after periodontitis induction compared to the periodontitis-inducing group, which was the L. curvatus of the present invention. This indicates that inflammatory cell infiltration in the gingival and periodontal ligaments was reduced, and alveolar bone resorption and cementum destruction were significantly reduced when the SMFM2016-NK strain was treated (FIG. 3).
- curvatus SMFM2016-NK treatment group after periodontitis induction Periodontitis+ L. curvatus SMFM2016-NK+ statistically significant, compared to normal group by pairwise t-test ( p ⁇ 0.05)* statistically significant, compared to negative control group by pairwise t-test ( p ⁇ 0.05)
- Example 6 Lactobacillus curvatus Characterization of SMFM2016-NK strain
- the strain of the present invention was cultured in MRS broth (BD, USA) at 35° C. for 12 hours.
- the bacteria were subcultured in MRS broth containing slide glass (0.5x0.5 cm) so that the bacteria grew attached to the slide glass surface.
- a 1.8% glutaldehyde solution was dropped on a piece of glass slide and washed with sterile distilled water. After dropping a 2% osmium tetroxide (Sigma, St. Louis, MO, USA) solution, the reaction was carried out in the dark at room temperature.
- HMDS hexamethyldisilazane
- FE-SEM field emission scanning electrochemical microscopy
- DNA link (Seoul) was requested to analyze the genome.
- a 20 kb library was constructed using the Pacific Biosciences system (Pacific Biosciences, Menlo Park, CA, USA) sequencing platform, and de novo assembly was performed based on the HGAP.3 protocol.
- the strain was identified as a strain consisting of a total of 4 contigs, and as a result of confirming each contig using the BLAST program based on the NCBI GenBank database, one chromosome (2,139,352 bp, GC content 41.97) %) and the genome with three plasmids.
- the chromosome was confirmed to be most similar to L. curvatus.
- the genetic characteristics (location of gene, coding region, function of gene) of the chromosome of the corresponding strain were analyzed, and structural characteristics ( The number of genes, their location) was confirmed with the following DNA plot. Specifically, it was confirmed that the L. curvatus SMFM2016-NK chromosome consists of 2,307 coding sequences (CDS), 18 rRNA, 67 tRNA, and 8,500 transcripts (FIG. 5).
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Abstract
The present invention relates to a Lactobacillus curvatus SMFM2016-NK strain (accession number: KCTC14138BP), and a food composition for prevention or treatment of oral disease, a pharmaceutical composition for prevention or treatment of oral disease, probiotics, and a feedstuff composition, each comprising the strain. The Lactobacillus curvatus SMFM2016-NK strain (accession number: KCTC14138BP) of the present invention, which is a probiotic strain newly isolated and identified from kimchi, exhibits the effect of decreasing a number of osteoclasts, increasing a number of osteoblasts, decreasing a loss volume of the spongy bone, increasing a volume of the alveolar bone, and decreasing a number of inflammatory cells, and excellent antibacterial activity against harmful bacteria in the mouth and, as such, can be advantageously used in a composition for prevention, alleviation, and treatment of oral disease.
Description
본 발명은 락토바실러스 커바투스(Lactobacillus curvatus) SMFM2016-NK 균주(기탁번호: KCTC14138BP), 상기 균주를 포함하는 구강질환 예방 또는 개선용 식품 조성물, 구강질환 예방 또는 치료용 약학적 조성물, 프로바이오틱스, 및 사료 조성물에 관한 것이다.The present invention Lactobacillus curvatus ( Lactobacillus curvatus ) SMFM2016-NK strain (accession number: KCTC14138BP), oral disease prevention or improvement food composition comprising the strain, oral disease prevention or treatment pharmaceutical composition, probiotics, and feed to the composition.
구강질환은 치아를 포함한 구강 내에서 발생할 수 있는 모든 질환을 일컫는 것으로, 흔히 발생하는 구강질환으로는 대표적으로 치아 우식증(dental caries), 즉 충치와 치주염(periodontitis), 치은염(gingivitis) 등의 치주질환이 있을 수 있다. 치주질환은 치아 주변을 둘러싸고 있는 치주 인대, 치은 등 연조직과 치조골 등 경조직을 손상시키는 염증성 질환 등을 포함하며, 특히 치조골(alveolar bone)은 악골의 기저골에 단단히 부착되어 있는 골조직으로 치아를 단단히 지지하는 역할을 한다. 치조골에서는 조골세포에 의한 골형성과 파골세포에 의한 골흡수의 대사가 이루어지는데, 다양한 원인에 의해 골흡수가 골형성을 초과하게 되는 경우 치조골이 낮아져 치아 뿌리가 노출되게 되고 결국 치아를 지지하는 역할을 수행할 수 없게 된다. 이러한 치조골의 소실은 치은염 및 치주염에 의해서도 발생할 수 있다. 치은염은 잇몸 연조직에 국한된 염증으로 비교적 증상이 가볍고 회복이 가능한 질환에 해당되나, 치주염은 치은염이 악화된 경우로 염증이 잇몸과 치조골 주변까지 진행되어 치주낭(periodontal pocket)을 형성하고, 깊어진 치주낭으로 인해 치주인대의 염증과 골소실을 유발하게 된다.Oral disease refers to all diseases that can occur in the oral cavity, including teeth. Common oral diseases include dental caries, that is, periodontal diseases such as tooth decay, periodontitis, and gingivitis. This can be. Periodontal disease includes inflammatory diseases that damage soft tissues such as periodontal ligaments and gingiva surrounding teeth and hard tissues such as alveolar bone. plays a role In alveolar bone, bone formation by osteoblasts and bone resorption by osteoclasts are metabolized. When bone resorption exceeds bone formation due to various causes, the alveolar bone lowers, exposing the tooth roots, and eventually supporting the teeth. cannot be performed. This loss of alveolar bone can also be caused by gingivitis and periodontitis. Gingivitis is an inflammation limited to the soft tissues of the gums and is a disease with relatively light and recoverable symptoms. It causes inflammation and bone loss of the periodontal ligament.
이러한 구강질환 또는 치주질환은 국내외를 불문하고 성인 인구에 대해 가장 흔하게 발생할 수 있는 질환에 해당할 뿐만 아니라, 개인 의료 및 사회적인 관리 체계 면에서도 상당한 비용이 요구되는 공중 보건의 관심질환에 해당한다. 국민건강보험공단에 따르면 2012-2016년 국내 치주질환 환자의 수는 지난 2012년 707만명에서 2016년 1107만명으로 4년 새 약 56.6% 증가하였고, 이는 연평균 무려 12%의 증가율에 해당하는 수치이다. 따라서 이러한 치주질환의 치료를 위해 다양한 외과적인 치료 방법이 존재하고 개발되었으나, 개인에게 있어 외과적인 치료는 심리적으로 꺼려질 뿐 아니라 막대한 비용의 부담도 있으며, 구강질환의 특성상 어느 정도 질환이 진행된 이후에야 행해지는 경우가 있어 적절한 시기에 지속적인 치료가 어렵다는 문제가 있다. 이에, 치주질환을 비롯한 구강질환에 있어서만큼은 외과적 치료뿐 아니라 개인의 평소 구강 위생 확립과 이를 통한 예방이 보다 더 중요하다고 할 수 있다.Such oral disease or periodontal disease is not only a disease that can occur most often in the adult population, regardless of domestic or foreign, but also a disease of public health interest that requires considerable cost in terms of personal medical care and social management system. According to the National Health Insurance Corporation, the number of periodontal disease patients in Korea between 2012 and 2016 increased from 7.07 million in 2012 to 11.07 million in 2016, an increase of about 56.6% in four years, which corresponds to an average annual growth rate of 12%. Therefore, various surgical treatment methods exist and have been developed for the treatment of such periodontal disease, but for individuals, surgical treatment is not only psychologically reluctant but also carries a huge cost burden, There is a problem that continuous treatment at an appropriate time is difficult because there are cases where it is performed. Therefore, for oral diseases including periodontal disease, it can be said that not only surgical treatment, but also establishment of personal oral hygiene and prevention through this is more important.
따라서, 이러한 구강질환 또는 치주질환을 평소에 예방하고 그 증상을 개선시키며, 나아가 치료 효과까지 달성할 수 있는 물질에 대한 개발이 절실히 필요한 상황이다. 이러한 배경 하에 본 발명의 발명자들은 최근 체내에서 유익한 효과를 제공하는 미생물, 즉 구강질환의 예방, 개선 및 치료에 효과적인 프로바이오틱스(probiotics)를 새롭게 분리, 동정하고자 예의 노력한 결과, 김치로부터 새롭게 분리, 동정된 프로바이오틱스 균주, 락토바실러스 커바투스(Lactobacillus curvatus) SMFM2016-NK 균주(기탁번호: KCTC14138BP)가 파골세포 수 감소, 조골세포 수 증가, 해면골 소실 부피 감소, 치조골 부피 증가, 염증세포 수 감소 효과 및 구강 내 유해세균에 대한 우수한 항균 활성을 나타냄을 확인하여 본 발명을 완성하였다.Therefore, there is an urgent need to develop a material that can prevent such oral disease or periodontal disease on a regular basis, improve its symptoms, and even achieve a therapeutic effect. Under this background, the inventors of the present invention recently made an effort to newly isolate and identify microorganisms that provide beneficial effects in the body, that is, probiotics effective for the prevention, improvement and treatment of oral diseases. Probiotic strain, Lactobacillus curvatus SMFM2016-NK strain (Accession No.: KCTC14138BP) decreases the number of osteoclasts, increases the number of osteoblasts, decreases the volume of cancellous bone loss, increases the volume of alveolar bone, reduces the number of inflammatory cells, and harmful effects in the oral cavity The present invention was completed by confirming that it exhibits excellent antibacterial activity against bacteria.
프로바이오틱스의 시장 규모는 2015년 기준 약 330억불(한화 약 36조원)에 달하였으며, 매년 7%씩 증가하여 2020년 약 470억불(한화 53조원)에 이를 것으로 예상된다. 또한 프로바이오틱스는 이를 활용한 건강기능식품뿐 아니라 구강케어 및 구강질환 관리를 위한 의약외품, 의약품으로도 그 활용 가능성이 높게 전망되는 점에서, 본 발명의 의의 또한 매우 크다고 볼 수 있을 것이다.The market size of probiotics reached about $33 billion (about KRW 36 trillion) in 2015, and is expected to increase by 7% every year to reach about $ 47 billion (about KRW 53 trillion) by 2020. In addition, since probiotics are highly likely to be used not only as health functional foods using them, but also as quasi-drugs and medicines for oral care and oral disease management, the significance of the present invention is also very large.
본 발명은 전술한 문제 및 이와 연관된 다른 문제를 해결하는 것을 목적으로 한다.SUMMARY OF THE INVENTION The present invention aims to solve the above problems and other problems related thereto.
본 발명의 일 예시적 목적은 김치로부터 새롭게 분리, 동정된 락토바실러스 커바투스(Lactobacillus curvatus) SMFM2016-NK 균주(기탁번호: KCTC14138BP)를 제공하는 것이다.An exemplary object of the present invention is to provide a newly isolated and identified Lactobacillus curvatus SMFM2016-NK strain (Accession No.: KCTC14138BP) from kimchi.
본 발명의 다른 예시적 목적은 상기 락토바실러스 커바투스 SMFM2016-NK 균주, 이의 배양물, 농축물, 건조물 또는 파쇄물을 포함하는 구강질환 예방 또는 개선용 식품 조성물을 제공하는 것이다.Another exemplary object of the present invention is to provide a food composition for preventing or improving oral diseases, including the Lactobacillus curbatus SMFM2016-NK strain, its culture, concentrate, dry matter or lysate.
본 발명의 또 다른 예시적 목적은 상기 균주, 이의 배양물, 농축물, 건조물 또는 파쇄물을 포함하는 구강질환 예방 또는 치료용 약학적 조성물 제공하는 것이다.Another exemplary object of the present invention is to provide a pharmaceutical composition for preventing or treating oral diseases, including the strain, its culture, concentrate, dry matter or lysate.
본 발명의 또 다른 예시적 목적은 상기 균주, 이의 배양물, 농축물, 건조물 또는 파쇄물을 포함하는 프로바이오틱스를 제공하는 것이다.Another exemplary object of the present invention is to provide a probiotic comprising the above strain, a culture, concentrate, dried product or lysate thereof.
본 발명의 또 다른 예시적 목적은 상기 균주, 이의 배양물, 농축물, 건조물 또는 파쇄물을 포함하는 의약외품 조성물을 제공하는 것이다.Another exemplary object of the present invention is to provide a quasi-drug composition comprising the strain, its culture, concentrate, dried product or lysate.
본 명세서에 개시된 발명의 기술적 사상에 따라 이루고자 하는 기술적 과제는 이상에서 언급한 문제점을 해결하기 위한 과제로 제한되지 않으며, 언급되지 않은 또 다른 과제는 아래의 기재로부터 통상의 기술자에게 명확하게 이해될 수 있을 것이다.The technical problem to be achieved according to the technical idea of the invention disclosed in this specification is not limited to the problem for solving the above-mentioned problems, and another problem not mentioned can be clearly understood by those skilled in the art from the description below. There will be.
이를 구체적으로 설명하면 다음과 같다. 한편, 본 출원에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 출원에서 개시된 다양한 요소들의 모든 조합이 본 출원의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 출원의 범주가 제한된다고 볼 수 없다.This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below.
상기 목적을 달성하기 위한 본 발명의 하나의 양태는 락토바실러스 커바투스(Lactobacillus curvatus) SMFM2016-NK 균주를 제공한다. 상기 균주는 김치로부터 분리된 것으로 2020년 2월 17일자로 한국생명공학연구원 생물자원센터(KCTC)에 기탁되어 기탁번호 KCTC 14138BP를 부여받았다.One aspect of the present invention for achieving the above object provides a Lactobacillus curvatus (Lactobacillus curvatus) SMFM2016-NK strain. The strain was isolated from kimchi and was deposited at the Korea Institute of Biotechnology and Biotechnology Biological Resources Center (KCTC) on February 17, 2020, and was given an accession number KCTC 14138BP.
또한, 상기 균주는 표 7, 도 5 및 도 6의 유전체 특성 및 서열번호 1로 표시되는 16s rDNA 서열을 포함하는 것으로, 유전체 분석 결과 동일한 균주가 없는 것을 확인한 본 발명자들에 의해 새롭게 분리, 동정된 균주이다. 이는 프로바이오틱스 균주에 해당하며, 인체에 무해하며, 부작용 없이 사용될 수 있다.In addition, the strain includes the genomic characteristics of Table 7, FIGS. 5 and 6 and the 16s rDNA sequence shown in SEQ ID NO: 1, newly isolated and identified by the present inventors who confirmed that there is no identical strain as a result of genomic analysis is a strain This corresponds to a probiotic strain, is harmless to the human body, and can be used without side effects.
본 발명의 실시예에 따르면, 상기 균주는 구강 내 치아 또는 치주 조직 등에 있어서 파골세포 수 감소, 조골세포 수 증가, 해면골 소실 부피 감소, 치조골 부피 증가 및 염증세포 수 감소 효과를 나타낼 수 있으며, 프리보텔라 인터미디어(Prevotella intermedia), 퓨조박테리움 뉴클레아툼(Fusobacterium nucleatum) 또는 아그레가티박터 액티노마이세템코미탄스(Aggregatibacter actinomycetemcomitans) 등 구강 내 유해세균에 대한 항균 활성을 나타내는 것일 수 있다.According to an embodiment of the present invention, the strain can exhibit the effect of reducing the number of osteoclasts, increasing the number of osteoblasts, decreasing the volume of cancellous bone loss, increasing the volume of alveolar bone, and reducing the number of inflammatory cells in oral teeth or periodontal tissue, etc. Intermedia ( Prevotella intermedia ), Fusobacterium nucleatum ( Fusobacterium nucleatum ) or Aggregatibacter actinomycetemcomitans ( Aggregatibacter actinomycetemcomitans ) It may be one that exhibits antibacterial activity against harmful bacteria in the oral cavity.
상기 목적을 달성하기 위한 본 발명의 다른 하나의 양태는 락토바실러스 커바투스 SMFM2016-NK 균주(기탁번호: KCTC14138BP), 이의 배양물, 농축물, 건조물 또는 파쇄물을 포함하는, 구강질환 예방 또는 개선용 식품 조성물을 제공한다.Another aspect of the present invention for achieving the above object is Lactobacillus curbatus SMFM2016-NK strain (Accession No.: KCTC14138BP), its culture, concentrate, dry matter or lysate, including oral disease prevention or improvement food A composition is provided.
본 발명에서 용어 "락토바실러스(Lactobacillus)"는 자연계에 널리 분포하는 호기성, 또는 통성 혐기성의 그람양성 간균 속 미생물을 의미하며, 상기 락토바실러스 커바투스 SMFM2016-NK 균주에 대한 설명은 전술한 바와 같다.In the present invention, the term "Lactobacillus ( Lactobacillus )" refers to a microorganism of the genus Gram-positive bacillus of aerobic or facultative anaerobic nature widely distributed in nature, and the description of the Lactobacillus curvatus SMFM2016-NK strain is as described above.
본 발명에서 용어 "배양물"은 균주를 배지에 배양하여 균주가 영양분을 섭취하고 물질대사를 통해 생겨난 부산물과 균주 등이 포함된 배지를 의미하는 것으로, 상기 락토바실러스 커바투스 SMFM2016-NK 균주의 배양물을 의미할 수 있다. 또한 본 발명의 식품 조성물은 상기 균주의 배양물 뿐 아니라 이의 농축물, 건조물 또는 파쇄물을 포함하는 것일 수 있다.In the present invention, the term "culture" refers to a medium containing by-products and strains generated through the ingestion of nutrients and metabolism by culturing the strain in a medium, and the culture of the Lactobacillus curvatus SMFM2016-NK strain. It could mean water. In addition, the food composition of the present invention may include a culture of the above strain as well as a concentrate, a dried product, or a lysate thereof.
또한, 본 발명에서 용어 "구강질환"은 치아를 포함하여 구강 내에서 발생할 수 있는 모든 질환을 의미하는 것으로, 구체적으로 구강 조직 내에서 파골세포 수 감소, 조골세포 수 증가, 해면골 소실 부피 감소, 치조골 부피 증가, 염증세포 수 감소 및 구강 유해세균에 대한 항균 효과로 인해 예방, 개선 및/또는 치료될 수 있는 모든 질환을 포함한다. 보다 구체적으로, 상기 구강질환은 치조골 파손(alveolar bone breakage), 치조골다공증(alveolar bone osteoporosis), 치조골연화증(alveolar bone osteomalacia), 치조골감소증(alveolar bone osteopenia), 치아 우식증(dental caries), 치은염(gingivitis) 및 치주염(periodontitis)으로 구성된 군으로부터 선택되는 하나 이상의 질환일 수 있으며, 이에 특별히 제한되는 것은 아니다.In addition, in the present invention, the term "oral disease" refers to all diseases that can occur in the oral cavity, including teeth, specifically, a decrease in the number of osteoclasts in the oral tissue, an increase in the number of osteoblasts, a decrease in the volume of cancellous bone loss, and alveolar bone. Includes all diseases that can be prevented, ameliorated and/or treated due to an increase in volume, a decrease in the number of inflammatory cells, and an antibacterial effect on harmful oral bacteria. More specifically, the oral disease is alveolar bone breakage, alveolar bone osteoporosis, alveolar bone osteomalacia, alveolar bone osteopenia, dental caries, gingivitis ) and periodontitis (periodontitis) may be one or more diseases selected from the group consisting of, but is not particularly limited thereto.
본 발명의 식품 조성물은 상기 락토바실러스 커바투스 SMFM2016-NK 균주, 이의 배양물, 농축물, 건조물 또는 파쇄물을 포함하는 것이므로, 구강 내 치아 또는 치주 조직 등에 있어서 파골세포 수 감소, 조골세포 수 증가, 해면골 소실 부피 감소, 치조골 부피 증가 및 염증세포 수 감소 효과를 동일하게 나타낼 수 있으며, 프리보텔라 인터미디어(Prevotella intermedia), 퓨조박테리움 뉴클레아툼(Fusobacterium nucleatum) 또는 아그레가티박터 액티노마이세템코미탄스(Aggregatibacter actinomycetemcomitans) 등 구강 내 유해세균에 대한 항균 활성 또한 동일하게 나타낼 수 있다.Since the food composition of the present invention includes the Lactobacillus curbatus SMFM2016-NK strain, its culture, concentrate, dry matter or lysate, the number of osteoclasts in the oral cavity or periodontal tissue, etc. decreases, the number of osteoblasts increases, cancellous bone Loss volume reduction, alveolar bone volume increase, and inflammatory cell count reduction effects can be equally represented, and Prevotella intermedia , Fusobacterium nucleatum ) or Aggregatibacter actinomycetemcomy Antibacterial activity against harmful bacteria in the oral cavity, such as Aggregatibacter actinomycetemcomitans, may also be exhibited in the same manner.
본 발명의 조성물에서 상기 균주는 파골세포 수 감소, 조골세포 수 증가, 해면골 소실 부피 감소, 치조골 부피 증가 및 염증세포 수 감소 효과 및 구강 내 유해세균에 대한 항균 활성을 나타낼 수 있는 한, 그 구체적인 용도, 제형 및 배합 목적 등에 따라 임의의 양(유효량)으로 포함될 수 있는데, 통상적인 유효량은 조성물 전체 중량을 기준으로 할 때 0.001 중량 % 내지 50.0 중량 % 범위 내에서 결정될 것이다. 상기 "유효량"이란 의료 전문가 등에 의한 투여 기간 동안 적용 대상인 포유동물, 바람직하게는 사람에게 본 발명 조성물에 따라 의도한 기능적·약리학적 효과를 나타낼 수 있는, 본 발명의 조성물에 포함되는 유효성분의 양을 말한다. 이러한 유효량은 당업자의 통상의 능력 범위 내에서 실험적으로 결정될 수 있다. 본 발명의 조성물이 적용될 수 있는 대상은 포유동물 및 사람이며, 특히 사람인 경우가 바람직하다.In the composition of the present invention, the strain decreases the number of osteoclasts, increases the number of osteoblasts, decreases the volume of cancellous bone loss, increases the volume of alveolar bone and reduces the number of inflammatory cells, and the specific use thereof as long as it can exhibit antibacterial activity against harmful bacteria in the oral cavity , may be included in any amount (effective amount) depending on the formulation and purpose of mixing, and a typical effective amount will be determined within the range of 0.001 wt % to 50.0 wt % based on the total weight of the composition. The "effective amount" refers to the amount of the active ingredient contained in the composition of the present invention that can exhibit the intended functional and pharmacological effects according to the composition of the present invention to a mammal, preferably a human, to be applied during the administration period by a medical professional or the like. say Such effective amounts can be determined empirically within the ordinary ability of one of ordinary skill in the art. Subjects to which the composition of the present invention can be applied are mammals and humans, particularly preferably humans.
본 발명의 식품 조성물에 있어서, 상기 식품에는 건강기능(성)식품이 포함될 수 있다. 본 발명에서 용어 "건강기능식품"이란, 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 말한다. 상기 "기능성" 은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻는 것을 의미한다. 본 발명의 건강기능식품은 당업계에서 통상적으로 사용되는 방법에 의하여 제조 가능하며, 상기 제조시에는 당업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. 또한 상기 건강기능식품의 제형 또한 건강기능식품으로 인정되는 제형이면 제한없이 제조될 수 있다. 본 발명의 식품 조성물은 다양한 형태의 제형으로 제조될 수 있으며, 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있다. 본 발명의 식품 조성물은 어떠한 형태로도 제조될 수 있으며, 구체적으로는 정제, 캡슐, 환, 과립, 액상, 분말, 편상, 페이스트상, 시럽, 겔, 젤리, 바 등의 건강기능식품 제제류, 음료류, 껌류 및 캔디류로 구성된 군으로부터 선택되는 하나 이상의 제형일 수 있으나, 이에 특별히 제한되는 것은 아니다.In the food composition of the present invention, the food may include a health functional (sexual) food. In the present invention, the term "health functional food" refers to a food manufactured and processed using raw materials or ingredients having useful functions in the human body. The "functionality" refers to obtaining useful effects for health purposes such as regulating nutrients or physiological actions with respect to the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, if the formulation of the health functional food is also recognized as a health functional food, it can be prepared without limitation. The food composition of the present invention can be prepared in various forms, and unlike general drugs, there is no side effect that may occur when taking the drug for a long time using food as a raw material. The food composition of the present invention can be prepared in any form, and specifically, health functional food preparations such as tablets, capsules, pills, granules, liquids, powders, pieces, pastes, syrups, gels, jellies, bars, etc.; It may be one or more formulations selected from the group consisting of beverages, gums and candies, but is not particularly limited thereto.
또한, 본 발명의 식품 조성물에는 그 유효성분 이외에 식품첨가물이 포함될 수 있다. 식품첨가물은 일반적으로 식품을 제조, 가공 또는 보존함에 있어 식품에 첨가되어 혼합되거나 침윤되는 물질로서 이해될 수 있으며, 식품과 함께 매일 그리고 장기간 섭취되므로 그 안전성이 보장되어야 한다. 상기 식품첨가물은 기능 면에 있어서 감미제, 풍미제, 보존제, 유화제, 산미료, 점증제 등으로 구분되며, 본 발명의 식품 조성물이 달성하고자 하는 목적에 부합하는 한 특별히 제한되지 않는다. 또한, 본 발명의 식품 조성물은 상기 식품첨가물 이외에 기능성과 영양 보충의 목적으로 당업계에 공지되고 식품첨가물로서 안정성이 보장된 생리활성 물질 또는 미네랄류를 포함할 수 있다. 상기 생리활성 물질 또는 미네랄류는 본 발명의 식품 조성물이 달성하고자 하는 목적에 부합하는 한 특별히 제한되지 않는다.In addition, the food composition of the present invention may contain food additives in addition to the active ingredient. Food additives can be generally understood as substances that are mixed or infiltrated with food in manufacturing, processing, or preserving food, and their safety must be ensured as they are consumed daily and for a long period of time with food. The food additives are divided into sweeteners, flavoring agents, preservatives, emulsifiers, acidulants, thickeners, etc. in terms of function, and are not particularly limited as long as the food composition of the present invention meets the intended purpose. In addition, the food composition of the present invention may contain physiologically active substances or minerals known in the art for the purpose of functional and nutritional supplementation and guaranteed stability as a food additive in addition to the food additives. The physiologically active substances or minerals are not particularly limited as long as the food composition of the present invention meets the intended purpose.
본 발명의 식품 조성물에는 전술한 바의 식품첨가물이 제품 유형에 따라 그 첨가 목적을 달성할 수 있는 유효량으로 포함될 수 있으며, 본 발명의 식품 조성물에 포함될 수 있는 기타의 식품첨가물과 관련하여서는 각국 식품공전이나 식품첨가물 공전을 참조할 수 있다.In the food composition of the present invention, the food additives as described above may be included in an effective amount to achieve the purpose of the addition according to the type of product, and with respect to other food additives that may be included in the food composition of the present invention, the food additives of each country Or you can refer to the Food Additives Ordinance.
또한, 본 발명에서 용어 "개선"은 본 발명의 락토바실러스 커바투스 SMFM2016-NK 균주를 포함하는 조성물을 적용하여, 구강질환 또는 치주질환의 증상을 완화시키는 효과를 나타내는 것을 의미하며, 용어 "예방"은 본 발명 조성물의 투여로 구강질환 또는 치주질환 관련 증상을 억제 또는 지연시키는 모든 행위를 의미한다.In addition, in the present invention, the term "improvement" refers to the effect of alleviating the symptoms of oral disease or periodontal disease by applying the composition comprising the Lactobacillus curbatus SMFM2016-NK strain of the present invention, and the term "prevention" means any action that suppresses or delays oral disease or periodontal disease-related symptoms by administration of the composition of the present invention.
상기 목적을 달성하기 위한 본 발명의 또 다른 하나의 양태는 락토바실러스 커바투스(Lactobacillus curvatus) SMFM2016-NK 균주(기탁번호: KCTC14138BP), 이의 배양물, 농축물, 건조물 또는 파쇄물을 포함하는, 구강질환 예방 또는 치료용 약학적 조성물을 제공한다. 상기 용어 락토바실러스, 락토바실러스 커바투스 SMFM2016-NK 균주, 배양물 및 구강질환은 전술한 바와 같다.Another aspect of the present invention for achieving the above object is Lactobacillus curvatus ( Lactobacillus curvatus ) SMFM2016-NK strain (Accession No.: KCTC14138BP), its culture, concentrate, oral disease, including dry matter or lysate A pharmaceutical composition for prevention or treatment is provided. The term Lactobacillus, Lactobacillus curvatus SMFM2016-NK strain, culture and oral disease are the same as described above.
본 발명의 약학적 조성물은 상기 락토바실러스 커바투스 SMFM2016-NK 균주, 이의 배양물, 농축물, 건조물 또는 파쇄물을 포함하는 것이므로, 구강 내 치아 또는 치주 조직 등에 있어서 파골세포 수 감소, 조골세포 수 증가, 해면골 소실 부피 감소, 치조골 부피 증가 및 염증세포 수 감소 효과를 동일하게 나타낼 수 있으며, 프리보텔라 인터미디어(Prevotella intermedia), 퓨조박테리움 뉴클레아툼(Fusobacterium nucleatum) 또는 아그레가티박터 액티노마이세템코미탄스(Aggregatibacter actinomycetemcomitans) 등 구강 내 유해세균에 대한 항균 활성 또한 동일하게 나타낼 수 있다.Since the pharmaceutical composition of the present invention includes the Lactobacillus curbatus SMFM2016-NK strain, its culture, concentrate, dry matter or lysate, the number of osteoclasts in the oral cavity or periodontal tissue, etc. decreases, the number of osteoblasts increases, bone loss volume reduction, and equally it represents an increase in bone volume and inflammatory cell count reduction, freebo telra intermediate (Prevotella intermedia), Pew jobak Te Leeum New Clegg Atum (Fusobacterium nucleatum) or Agde Leganes tee bakteo solution Tino My setem Antibacterial activity against harmful bacteria in the oral cavity, such as Aggregatibacter actinomycetemcomitans, may also be exhibited in the same manner.
본 발명의 약학적 조성물은 상기 락토바실러스 커바투스 SMFM2016-NK 균주, 이의 배양물, 농축물, 건조물 또는 파쇄물 이외에 약학적 조성물의 제조에 통상적으로 사용하는 약학적으로 허용가능한 담체, 부형제 또는 희석제를 추가로 포함할 수 있다. 상기 담체, 부형제 및 희석제의 구체적인 예로는 락토오스, 덱스트로즈, 수크로오스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 또는 광물유 등이 사용될 수 있으며, 이에 특별히 제한되지 않는다.The pharmaceutical composition of the present invention is a pharmaceutically acceptable carrier, excipient or diluent commonly used in the preparation of pharmaceutical compositions in addition to the Lactobacillus curbatus SMFM2016-NK strain, its culture, concentrate, dried product or lysate. can be included as Specific examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used, but is not particularly limited thereto.
또한, 본 발명의 약학적 조성물은 각각 통상의 방법에 따라 정제, 환제, 산제, 과립제, 캡슐제, 현탁제, 내용액제, 유제, 시럽제, 멸균된 수용액, 비수성용제 및 동결 건조제로 이루어진 군으로부터 선택되는 어느 하나의 제형을 가질 수 있으며, 상기 제형은 경구 또는 비경구의 여러 가지 형태일 수 있다. 제제화할 경우에는 통상적으로 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있으며, 이에 제한되지 않는다. 구체적으로, 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 사용될 수 있으며, 상기 고형제제는 적어도 하나 이상의 부형제 예컨대 전분, 탄산칼슘, 수크로오스(sucrose) 또는 락토오스(lactose), 젤라틴 등이 사용될 수 있다. 또한, 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제 등이 사용될 수 있으며, 이에 제한되지 않는다. 또한, 경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 사용될 수 있으며, 통상적으로 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예컨대 습윤제, 감미제, 방향제, 보존제 등이 사용될 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제 또는 좌제 등이 사용될 수 있다. 상기 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테로 등이 사용될 수 있으며, 이에 제한되지 않는다.In addition, the pharmaceutical composition of the present invention is selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents and freeze-drying agents according to a conventional method, respectively. It may have any one dosage form, and the dosage form may be in various forms, either oral or parenteral. In the case of formulation, it may be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants, but is not limited thereto. Specifically, tablets, pills, powders, granules, capsules, etc. may be used in the solid preparation for oral administration, and the solid preparation includes at least one or more excipients such as starch, calcium carbonate, sucrose or lactose, Gelatin or the like may be used. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients, but the present invention is not limited thereto. In addition, as liquid formulations for oral administration, suspensions, internal solutions, emulsions, syrups, etc. may be used, and various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to commonly used simple diluents such as water and liquid paraffin this can be used A sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a freeze-dried formulation, or a suppository may be used as a formulation for parenteral administration. The non-aqueous solvent and suspending agent may include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
본 발명 약학적 조성물의 개별적인 투약의 최적량 및 투약 간격은 구강질환 또는 치주질환의 성질 및 정도, 투여 제형, 경로 및 부위, 그리고 적용 대상의 나이와 건강상태에 의해 결정될 것이고, 의사가 궁극적으로 사용될 적절한 투약을 결정할 것이라는 것은 당해 분야의 당업자가 알 수 있을 것이다. 이러한 투약은 적절할 정도로 자주 반복될 수 있고, 보통의 임상 진료에 따라서 투여량 및 빈도를 변경하거나 또는 감소시킬 수 있다. 또한, 본 발명 조성물의 투여 경로는 본 발명의 목적을 달성할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다. 예컨대 본 발명의 약학적 조성물은 복강내 투여, 정맥내 투여, 피하 투여, 피내 투여, 경구 투여될 수 있으나, 이에 제한되지는 않는다.The optimal amount and dosing interval of individual administration of the pharmaceutical composition of the present invention will be determined by the nature and extent of oral disease or periodontal disease, dosage form, route and site, and the age and health condition of the subject, and the doctor will ultimately use it. It will be appreciated by those skilled in the art that appropriate dosing will be determined. Such dosing may be repeated as often as appropriate, and the dosage and frequency may be altered or reduced according to normal clinical practice. In addition, the administration route of the composition of the present invention may be administered through any general route as long as the object of the present invention can be achieved. For example, the pharmaceutical composition of the present invention may be administered intraperitoneally, intravenously, subcutaneously, intradermally, orally, but is not limited thereto.
또한, 본 발명에서 용어 "치료"는 본 발명 조성물의 투여로 구강질환 또는 치주질환의 증상이 호전되거나 이롭게 변경되는 모든 행위를 의미한다.In addition, in the present invention, the term "treatment" refers to any action in which the symptoms of oral disease or periodontal disease are improved or beneficially changed by administration of the composition of the present invention.
상기 목적을 달성하기 위한 본 발명의 또 다른 하나의 양태는 락토바실러스 커바투스 SMFM2016-NK 균주(기탁번호: KCTC14138BP), 이의 배양물, 농축물, 건조물 또는 파쇄물을 포함하는, 프로바이오틱스를 제공한다. 상기 용어 락토바실러스, 락토바실러스 커바투스 SMFM2016-NK 균주 및 배양물은 전술한 바와 같다.Another aspect of the present invention for achieving the above object is Lactobacillus curvatus SMFM2016-NK strain (Accession No.: KCTC14138BP), its culture, concentrate, dry matter or lysate, including probiotics. The term Lactobacillus, Lactobacillus curvatus SMFM2016-NK strain and culture are as described above.
본 발명에서 용어 "프로바이오틱스"는 체내에서 유익한 효과를 제공하는 살아있는 미생물을 의미하는 것으로, 특히 본 발명에서는 미생물의 상호작용을 통해 구강 내 균총을 개선하고 나아가 구강질환 또는 치주질환을 예방, 개선 및 치료하는 목적을 달성할 수 있는 미생물을 의미한다. 본 발명 목적상 상기 프로바이오틱스로 사용되는 균주는 락토바실러스 커바투스 SMFM2016-NK 균주이며, 구강질환 또는 치주질환의 예방, 개선 및 치료에 효과를 나타내는 한 다른 프로바이오틱스 균주를 추가로 더 포함할 수 있다.In the present invention, the term "probiotics" refers to living microorganisms that provide beneficial effects in the body. In particular, in the present invention, the microflora in the oral cavity is improved through the interaction of microorganisms and furthermore, oral disease or periodontal disease is prevented, improved and treated Microorganisms that can achieve the purpose of The strain used as the probiotic for the purpose of the present invention is the Lactobacillus curbatus SMFM2016-NK strain, and may further include other probiotic strains as long as they are effective in preventing, improving and treating oral diseases or periodontal diseases.
본 발명의 락토바실러스 커바투스 SMFM2016-NK 균주를 포함하는 프로바이오틱스는 구강질환 예방 및 개선용으로 사용되는 것을 특징으로 하는 것으로, 상기 균주는 구강 내 치아 또는 치주 조직 등에 있어서 파골세포 수 감소, 조골세포 수 증가, 해면골 소실 부피 감소, 치조골 부피 증가 및 염증세포 수 감소 효과를 나타낼 수 있고, 프리보텔라 인터미디어(Prevotella intermedia), 퓨조박테리움 뉴클레아툼(Fusobacterium nucleatum) 또는 아그레가티박터 액티노마이세템코미탄스(Aggregatibacter actinomycetemcomitans) 등 구강 내 유해세균에 대한 항균 활성을 나타낼 수 있다.The probiotics containing the Lactobacillus curbatus SMFM2016-NK strain of the present invention are characterized in that they are used for preventing and improving oral diseases, and the strain reduces the number of osteoclasts in teeth or periodontal tissues in the oral cavity, and the number of osteoblasts. increase, may indicate bone loss volume reduction, increased bone volume and inflammatory cell count reduction, freebo telra intermediate (Prevotella intermedia), Pew jobak Te Leeum New Clegg Atum (Fusobacterium nucleatum) or Agde Leganes tee bakteo solution Tino My setem It may exhibit antibacterial activity against harmful bacteria in the oral cavity, such as Aggregatibacter actinomycetemcomitans.
본 발명의 프로바이오틱스는 락토바실러스 커바투스 SMFM2016-NK 균주, 이의 배양물, 농축물, 건조물 또는 파쇄물을 포함하는, 분말, 발효유, 과립, 캡슐, 환, 껌, 필름 등의 형태로 사용될 수 있으나, 이에 특별히 제한되는 것은 아니다.The probiotics of the present invention may be used in the form of Lactobacillus curbatus SMFM2016-NK strain, its culture, concentrate, dried product or crushed product, including powder, fermented milk, granules, capsules, pills, gums, films, etc. It is not particularly limited.
상기 목적을 달성하기 위한 본 발명의 또 다른 하나의 양태는 락토바실러스 커바투스 SMFM2016-NK 균주(기탁번호: KCTC14138BP), 이의 배양물, 농축물, 건조물 또는 파쇄물을 포함하는, 의약외품 조성물을 제공한다. 상기 용어 락토바실러스, 락토바실러스 커바투스 SMFM2016-NK 균주, 배양물 및 구강질환은 전술한 바와 같다.Another aspect of the present invention for achieving the above object provides a quasi-drug composition comprising the Lactobacillus curvatus SMFM2016-NK strain (Accession No.: KCTC14138BP), its culture, concentrate, dry matter or lysate. The term Lactobacillus, Lactobacillus curvatus SMFM2016-NK strain, culture and oral disease are the same as described above.
본 발명의 의약외품 조성물은 상기 락토바실러스 커바투스 SMFM2016-NK 균주, 이의 배양물, 농축물, 건조물 또는 파쇄물을 포함하는 것이므로, 구강 내 치아 또는 치주 조직 등에 있어서 파골세포 수 감소, 조골세포 수 증가, 해면골 소실 부피 감소, 치조골 부피 증가 및 염증세포 수 감소 효과를 동일하게 나타낼 수 있으며, 프리보텔라 인터미디어(Prevotella intermedia), 퓨조박테리움 뉴클레아툼(Fusobacterium nucleatum) 또는 아그레가티박터 액티노마이세템코미탄스(Aggregatibacter actinomycetemcomitans) 등 구강 내 유해세균에 대한 항균 활성 또한 동일하게 나타낼 수 있다.Since the quasi-drug composition of the present invention includes the Lactobacillus curbatus SMFM2016-NK strain, its culture, concentrate, dry matter, or lysate, the number of osteoclasts in the oral cavity or periodontal tissue, etc. decreases, the number of osteoblasts increases, cancellous bone Loss volume reduction, alveolar bone volume increase, and inflammatory cell count reduction effects can be equally represented, and Prevotella intermedia , Fusobacterium nucleatum ) or Aggregatibacter actinomycetemcomy Antibacterial activity against harmful bacteria in the oral cavity, such as Aggregatibacter actinomycetemcomitans, may also be exhibited in the same manner.
구체적으로, 본 발명의 의약외품 조성물은 구강용 의약외품을 포함하는 것일 수 있다. 본 발명의 의약외품 조성물은 상기 락토바실러스 커바투스 SMFM2016-NK 균주, 이의 배양물, 농축물, 건조물 또는 파쇄물 이외에 구강용 의약외품 조성물에 통상적으로 사용되는 성분들을 포함할 수 있으며, 예컨대 연마제, 습윤제, 결합제, 기포제, 감미제, 방부제, 약효성분, 향미제, 색소, 용제, 증백제, 가용화제 또는 pH 조정제를 포함할 수 있다.Specifically, the quasi-drug composition of the present invention may include a quasi-drug for oral use. In addition to the Lactobacillus curbatus SMFM2016-NK strain, its culture, concentrate, dried or crushed product, the quasi-drug composition of the present invention may include ingredients commonly used in the composition for oral quasi-drugs, such as abrasives, wetting agents, binders, It may include a foaming agent, a sweetener, a preservative, an active ingredient, a flavoring agent, a colorant, a solvent, a brightener, a solubilizer, or a pH adjuster.
또한, 본 발명의 의약외품 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 구체적으로는 치약, 구강청결제, 껌, 캔디류, 구강스프레이, 구강용 연고제, 구강용 바니쉬(varnish), 구강필름 및 잇몸 마사지 크림으로 구성된 군으로부터 선택되는 하나 이상의 제형일 수 있으나, 이에 특별히 제한되는 것은 아니다. 하나의 예로서, 본 발명의 의약외품 조성물이 치약의 제형일 경우, 습윤제, 연마제, 결합제, 기포제, 향미제, 감미제, 착색제, 보존제, 약효성분, 용제, pH 조절제 등을 포함할 수 있다.In addition, the quasi-drug composition of the present invention may be prepared in any formulation conventionally prepared in the art, and specifically, toothpaste, mouthwash, gum, candy, oral spray, oral ointment, oral varnish, It may be one or more formulations selected from the group consisting of oral film and gum massage cream, but is not particularly limited thereto. As an example, when the quasi-drug composition of the present invention is a toothpaste formulation, it may include a wetting agent, abrasive agent, binder, foaming agent, flavoring agent, sweetener, coloring agent, preservative, active ingredient, solvent, pH adjusting agent, and the like.
본 발명의 락토바실러스 커바투스(Lactobacillus curvatus) SMFM2016-NK 균주(기탁번호: KCTC14138BP)는 김치로부터 새롭게 분리, 동정된 프로바이오틱스 균주로서, 파골세포 수 감소, 조골세포 수 증가, 해면골 소실 부피 감소, 치조골 부피 증가, 염증세포 수 감소 효과 및 구강 내 유해세균에 대한 우수한 항균 활성을 나타내어 구강질환의 예방 및 개선, 치료를 위한 조성물로서 유용하게 활용될 수 있다. The Lactobacillus curvatus SMFM2016-NK strain (Accession No.: KCTC14138BP) of the present invention is a probiotic strain newly isolated and identified from kimchi, and decreases the number of osteoclasts, increases the number of osteoblasts, decreases the volume of cancellous bone loss, and the volume of alveolar bone. It can be usefully used as a composition for prevention, improvement, and treatment of oral diseases because it has an effect of increasing, reducing the number of inflammatory cells, and exhibiting excellent antibacterial activity against harmful bacteria in the oral cavity.
다만, 본 명세서에 개시된 기술의 일 실시예에 따른 효과는 이상에서 언급한 것들로 제한되지 않으며, 언급하지 않은 또 다른 효과들은 아래의 기재로부터 통상의 기술자에게 명확하게 이해될 수 있을 것이다.However, the effects according to an embodiment of the technology disclosed in the present specification are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.
본 명세서에서 인용되는 도면을 보다 충분히 이해하기 위하여 각 도면의 간단한 설명이 제공된다.In order to more fully understand the drawings cited herein, a brief description of each drawing is provided.
도 1은 치주염 동물모델을 사용한 본 발명 프로바이오틱스 균주의 효능 분석 실험의 진행 일정을 나타낸 것이다.1 shows the progress schedule of the efficacy analysis experiment of the present invention probiotic strain using an animal model of periodontitis.
도 2는 Micro-CT 분석을 통해 본 발명 프로바이오틱스 균주 처리 시 해면골 조직의 소실 부피를 측정한 결과를 나타낸 것이다.2 shows the results of measuring the volume of cancellous bone tissue lost during the treatment of the present invention probiotic strain through Micro-CT analysis.
도 3은 본 발명의 프로바이오틱스 균주 처리 시 조직형태학적 변화, 염증세포 수, 치조골 부피, 파골세포 및 조골세포의 수를 분석하고자 조직병리학적 hematozylin-eosin (H&E) 분석 결과를 나타낸 것이다.3 shows the results of histopathological hematozylin-eosin (H&E) analysis to analyze the histomorphological changes, the number of inflammatory cells, the volume of alveolar bone, the number of osteoclasts and osteoblasts during treatment with the probiotic strain of the present invention.
도 4는 본 발명의 락토바실러스 커바투스 SMFM2016-NK 균주의 세포형태학적 SEM 이미지를 나타낸 것이다.Figure 4 shows the cell morphological SEM image of the Lactobacillus curbatus SMFM2016-NK strain of the present invention.
도 5는 본 발명의 락토바실러스 커바투스 SMFM2016-NK 균주의 유전체 특징 분석 결과를 나타낸 것이다.5 shows the results of analysis of the genome characteristics of the Lactobacillus curbatus SMFM2016-NK strain of the present invention.
도 6은 본 발명의 본 발명의 락토바실러스 커바투스 SMFM2016-NK 균주와 다른 락토바실러스 속 균주의 평균 염기서열 유사도(ANI %) 기준 unweighted pair group method with arithmetic mean (UPGMA) tree 분석결과를 나타낸 것이다.6 shows the analysis results of the unweighted pair group method with arithmetic mean (UPGMA) tree based on the average sequence similarity (ANI %) of the Lactobacillus curbatus SMFM2016-NK strain of the present invention and other Lactobacillus sp. strains of the present invention.
이하, 본 발명을 하기 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예만으로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through the following examples. However, these Examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited only to these Examples.
실시예 1: Example 1:
Lactobacillus curvatusLactobacillus curvatus
SMFM2016-NK 균주의 분리 및 동정 Isolation and identification of SMFM2016-NK strain
1-1: 젖산균주의 분리1-1: Isolation of lactic acid bacteria
서울 지역에서 수집된 김치 50 g을 멸균 필터백에 담고 0.1% buffered peptone water (BPW) 100 mL를 넣은 후 pummeler (Bag mixer 400; Interscience Co., Paris, France)를 이용하여 1분 간 균질화하였다. 균질화된 용액으로부터 100 μL를 취하여 MRS agar (Becton, Dickinson and Company, Sparks, MD, USA)에 도말하고, 35℃에서 24시간 동안 배양하였다. 분리된 균주는 MRS broth (BD, USA) 배지에 접종한 후, 35℃에서 24시간 동안 배양하였고 glycerol 20%를 첨가한 glycerol stock을 만들어 -80 ℃에 보관하며 사용하였다.50 g of kimchi collected in Seoul was placed in a sterile filter bag, 100 mL of 0.1% buffered peptone water (BPW) was added, and then homogenized for 1 minute using a pummeler (Bag mixer 400; Interscience Co., Paris, France). 100 μL of the homogenized solution was taken and spread on MRS agar (Becton, Dickinson and Company, Sparks, MD, USA), and incubated at 35° C. for 24 hours. The isolated strain was inoculated in MRS broth (BD, USA) medium, then cultured at 35°C for 24 hours, and a glycerol stock containing 20% glycerol was prepared and stored at -80°C before use.
1-2: 젖산균주의 동정1-2: Identification of lactic acid bacteria
분리된 균주는 16s rRNA 염기서열 분석을 통해 동정하였다. 분리균주를 순수 분리한 후 MRS agar plate 상태로 바이오닉스(서울)에 동정을 의뢰하였다. Universial primer 518F (5'-CCAGCAGCCGCGGTAATACG-3')와 800R (5'-TACCAGGGTATCTAATCC-3')을 사용하여 16s rRNA 염기서열 분석을 수행하였고, 그 결과 해당균주는 1,504 bp의 염기서열로 확인되었으며, 염기서열 정보는 첨부된 서열번호 1과 같았다.The isolated strain was identified through 16s rRNA sequencing. After pure separation of the isolates, the identification was requested to Bionics (Seoul) on MRS agar plate. 16s rRNA sequencing was performed using universal primers 518F (5'-CCAGCAGCCGCGGTAATACG-3') and 800R (5'-TACCAGGGTATCTAATCC-3'). The sequence information was as shown in the attached SEQ ID NO: 1.
해당 염기서열정보를 GeneBank database에 등록된 정보를 대상으로 BLAST 프로그램을 이용하여 상동성을 확인한 결과, Lactobacillus curvatus와 99% 상동성이 나타났고, 따라서 해당 균주를 L. curvatus로 최종 동정하였다.As a result of confirming the homology of the corresponding base sequence information with the information registered in the GeneBank database using the BLAST program, 99% homology with Lactobacillus curvatus was found, and thus the strain was finally identified as L. curvatus.
실시예 2: 구강 유해세균에 대한 우수한 항균 효과 확인Example 2: Confirmation of excellent antibacterial effect against oral harmful bacteria
각 프로바이오틱스 후보 균주의 항균효과를 확인하기 위해 한국구강미생물자원은행(KCOM)으로부터 분양받은 구강 유해세균(Prevotella intermedia KCOM1933, Fusobacterium nucleatum KCOM1250, Aggregatibacter actinomycetemcomitans KCOM1299)에 대해 Dual agar assay를 진행하였다.To confirm the antibacterial effect of each probiotic candidate strain, a dual agar assay was performed on oral harmful bacteria (Prevotella intermedia KCOM1933, Fusobacterium nucleatum KCOM1250, Aggregatibacter actinomycetemcomitans KCOM1299) received from the Korea Oral Microorganism Resources Bank (KCOM).
10 mL MRS broth(Becton, Dickinson and Company)에 35 ℃, 24시간 배양된 프로바이오틱스 후보 균주의 배양액(OD550nm=1.0)을 MRS agar(Becton, Dickinson and Company)에 10 μL 접종한 후 35 ℃, 24시간 배양하였다. 10 mL Wilkins Chalgren Anaerobe broth (Oxoid, Basingstoke, Hampshire, UK)에 90% N2, 5% CO2, 5% H2 조건에서 배양된 구강 유해세균을 Brain Heart Infusion agar (Becton, Dickinson and Company)에 접종한 후 각 프로바이오틱스 후보 균주가 접종된 플레이트에 10 mL 분주하였다. 플레이트를 90% N2, 5% CO2, 5% H2 조건에서 35 ℃, 24-48시간 동안 배양한 후 억제대를 측정하였다.In 10 mL MRS broth (Becton, Dickinson and Company), 35 ℃, 24 hours after inoculating 10 μL of the culture solution (OD550nm=1.0) of the probiotic candidate strain in MRS agar (Becton, Dickinson and Company) at 35 ℃, 24 hours cultured. In 10 mL Wilkins Chalgren Anaerobe broth (Oxoid, Basingstoke, Hampshire, UK), 90% N 2 , 5% CO 2 , 5% H 2 Oral harmful bacteria cultured in Brain Heart Infusion agar (Becton, Dickinson and Company) After inoculation, 10 mL of each probiotic candidate strain was dispensed to the inoculated plate. The plate was incubated in 90% N 2 , 5% CO 2 , 5% H 2 conditions at 35° C. for 24-48 hours, and then the inhibition zone was measured.
측정 결과, L. curvatus SMFM2016-NK 균주가 다른 프로바이오틱스 후보 균주들에 비해 구강 유해세균 3종에 대해 평균적으로 억제대가 넓은 것으로 확인되었고, 이에 따라 구강 유해세균에 대한 항균력이 우수하다고 판단하였다(표 1).As a result of the measurement, it was confirmed that the L. curvatus SMFM2016-NK strain had a wider inhibition zone on average for 3 types of oral harmful bacteria compared to other probiotic candidate strains, and thus it was judged that the antibacterial activity against harmful oral bacteria was excellent (Table 1). ).
| 분리원separatist | 균주strain | 구강 유해세균oral harmful bacteria | 전체 평균overall average | ||
| P. intermedia KCOM1933 P. intermedia KCOM1933 | F. nucleatum KCOM1250 F. nucleatum KCOM1250 | A.actinomycetemcomitans KCOM1299 A.actinomycetemcomitans KCOM1299 | |||
| 김치Kimchi | L. curvatus SMFM2016-NK L. curvatus SMFM2016-NK | 3.6±1.13.6±1.1 | 2.5±0.72.5±0.7 | 3.9±1.03.9±1.0 | 3.3±0.73.3±0.7 |
실시예 3: 구강질환 동물모델Example 3: Oral disease animal model
3-1: 치주염 유발3-1: Cause periodontitis
실험동물(Sprague-Dawley rat, male, 6-week-old)의 순화 기간 종료 다음 날부터 3일간 항생제(1 mg/mL sulfamethoxazole and 200 μg/mL trimethoprim in water)를 음수병에 넣어 섭취시켰다. 치주염 유발 당일에 Zoletil (1 mg/mL)과 럼푼을 4:1(v/v)비율로 섞어 1 mL/kg용량으로 주사기를 이용하여 실험동물에게 복강투여하였다. 우측 아랫쪽 첫번째 어금니를 sterile nylon을 이용하여 잇몸과 치주부위 경계선면을 결찰하였다. 결찰 후 sterile nylon은 잇몸에 묻히도록 밀어 넣었고, 28일 동안 결찰을 유지하면서 경구투여를 진행하였다(도 1).Antibiotics (1 mg/mL sulfamethoxazole and 200 μg/mL trimethoprim in water) were put into a drinking water bottle and ingested for 3 days from the day following the end of the acclimatization period of the experimental animals (Sprague-Dawley rat, male, 6-week-old). On the day of induction of periodontitis, Zoletil (1 mg/mL) and Lumpun were mixed in a 4:1 (v/v) ratio and administered intraperitoneally to experimental animals using a syringe at a dose of 1 mL/kg. The lower right first molar was ligated to the boundary between the gum and periodontal area using sterile nylon. After ligation, sterile nylon was pushed to be buried in the gums, and oral administration was performed while maintaining the ligation for 28 days (FIG. 1).
3-2: 균액 준비 및 시험3-2: Preparation and test of bacterial solution
젖산균의 글리세롤 보존액 100 μL를 MRS broth (Becton, Dickinson and Company) 45 mL이 들어가 있는 50 mL tube에 접종하고, 35 ℃에서 18-24시간 동안 정치배양 시켰다. 배양된 균액을 1912 xg, 4 ℃, 15분 조건으로 2번 원심분리 진행하였다. 원심분리 후 pellet에 PBS 45 mL을 추가하여 균질화시킨 프로바이오틱스 9-10 log CFU/mL을 치주염 유발 Sprague-Dawley (SD) SPF 랫드(수컷 6주령, n=8/group)(Orient Bio, Korea)에 10 mL/kg/day를 4주간 경구투여하였다. 또한 이 중 9 mL을 별도로 취하여 1912 xg, 4 ℃, 15분간 원심분리한 후 0.2% CMC 농도의 PBS 용액 9 mL에 현탁하여 각 동물의 치주염 유발부위에 0.5 mL/day를 4주간 분주하였다.100 μL of lactic acid bacteria glycerol stock solution was inoculated into a 50 mL tube containing 45 mL of MRS broth (Becton, Dickinson and Company), and incubated at 35 °C for 18-24 hours. The cultured bacterial solution was centrifuged twice under the conditions of 1912 x g, 4 °C, and 15 minutes. After centrifugation, 9-10 log CFU/mL of probiotics homogenized by adding 45 mL of PBS to the pellet was added to periodontitis-induced Sprague-Dawley (SD) SPF rats (6 weeks old male, n=8/group) (Orient Bio, Korea). 10 mL/kg/day was orally administered for 4 weeks. In addition, 9 mL of this was separately taken, centrifuged at 1912 x g, 4 °C, for 15 minutes, suspended in 9 mL of 0.2% CMC concentration PBS solution, and 0.5 mL/day was dispensed to the periodontitis-inducing site of each animal for 4 weeks.
실시예 4: Micro-CT 분석 및 해면골 소실 부피 분석Example 4: Micro-CT analysis and cancellous bone loss volume analysis
각 실험동물의 하악골을 분리한 후 10% 중성 완충 포르말린용액에 고정한 다음, 그 중 각 군당 4 마리를 이용하여 Micro-CT 분석을 진행하였다. Micro-CT는 치주염 유발 일정 부위를 설정하여 측정하였으며, 섬유주 조직(trabecular tissue)의 소실 부피를 측정하였다. Micro-CT 분석을 통해 해면골 소실 부피를 측정한 결과, 치주염 유발군은 정상군과 비교하여 해면골 부피가 통계학적으로 유의하게 소실되어 정상적인 치주염 유발이 확인되었고, L. curvatus SMFM2016-NK 처리군에서 치주염 유발군 대비 해면골 소실 부피가 11.6% 감소한 것으로 확인되었다(도 2).After separating the mandibles of each experimental animal, they were fixed in 10% neutral buffered formalin solution, and then micro-CT analysis was performed using 4 animals in each group. Micro-CT was measured by setting a predetermined periodontitis-induced site, and the volume of loss of trabecular tissue was measured. As a result of measuring the cancellous bone loss volume through micro-CT analysis, the periodontitis-induced group showed a statistically significant loss of cancellous bone volume compared to the normal group, confirming normal periodontitis induction, and periodontitis in the L. curvatus SMFM2016-NK treatment group. It was confirmed that the volume of cancellous bone loss was reduced by 11.6% compared to the induced group (FIG. 2).
| 그룹group | 해면골 소실 부피(mm3)Cancellous bone loss volume (mm 3 ) | 치주염 유발군 대비 감소율Decrease rate compared to periodontitis-inducing group |
| 정상군normal group | 0.417±0.0200.417±0.020 | N/AN/A |
| 치주염 유발군Periodontitis causative group | 0.776±0.088*0.776±0.088* | N/AN/A |
| 치주염 유발 후 L. curvatus SMFM2016-NK 처리군 L. curvatus SMFM2016-NK treatment group after periodontitis induction | 0.686±0.0910.686±0.091 | - 11.6%- 11.6% |
| 정상군; phosphate buffered saline(PBS), 치주염 유발군; 치주염+PBS, 치주염 유발 후 L. curvatus SMFM2016-NK 처리군; 치주염+L. curvatus SMFM2016-NK* statistically significant, compared to normal group by pairwise t-test (p<0.05)normal group; phosphate buffered saline (PBS), periodontitis-inducing group; Periodontitis+PBS, L. curvatus SMFM2016-NK treatment group after periodontitis induction; Periodontitis+ L. curvatus SMFM2016-NK* statistically significant, compared to normal group by pairwise t-test ( p <0.05) | ||
(mean±SE, n=4)(mean±SE, n=4)
실시예 5: 조직병리학적 검사Example 5: Histopathological examination
조직병리학적 검사는 Micro-CT를 측정한 실험동물의 검체를 이용하여 H&E 염색 후 검사를 실시하였다. 조직형태학적인 변화는 아래의 기준으로 score를 측정하였고, 염증세포 수(inflammatory cell numbers), 조골세포 수(osteoblast cell numbers), 파골세포 수(osteoclast cell numbers) 및 치조골 부피(alveolar process volumes)를 검사항목으로 측정하였다(표 3).Histopathological examination was performed after H&E staining using specimens of experimental animals measured by Micro-CT. Histomorphological changes were scored based on the following criteria, and inflammatory cell numbers, osteoblast cell numbers, osteoclast cell numbers, and alveolar process volumes were examined. Items were measured (Table 3).
| 조직형태학적 scorehistomorphological score | 평가 기준Evaluation standard |
| 00 | 존재하지 않거나 불연속적인 세포 침윤(염증성 세포 침윤이 거의 없고, 치은 부위에 제한됨), 정상적인 치조골과 백악질Non-existent or discontinuous cell infiltration (little inflammatory cell infiltration, limited to the gingival region), normal alveolar bone and cementum |
| 1One | 중도의 세포 침윤(치은 이상의 범위에서 염증성 세포 침윤이 발생), 경미한 치조골과 백악질의 흡수Moderate cell infiltration (inflammatory cell infiltration occurs beyond the gingiva), mild resorption of alveolar bone and cementum |
| 22 | 중증도의 세포 침윤(치은 및 치주 인대에서의 염증성 세포 침윤이 발생), 치조골 분해와 백악질의 부분적 파괴Moderate cell infiltration (inflammatory cell infiltration of the gingival and periodontal ligaments occurs), alveolar bone degradation and partial destruction of cementum |
| 33 | 중증도의 세포 침윤, 완전한 치조골 흡수와 심각한 백악질의 파괴Moderate cell infiltration, complete alveolar bone resorption and severe cementum destruction |
5-1: 조직형태학적 변화 분석5-1: Histomorphological change analysis
조직형태학적 변화 score를 측정하여 비교한결과, 치주염 유발 후 L. curvatus SMFM2016-NK 처리군에서는 치주염 유발군과 비교하여 조직형태학적 변화 score가 현저히 감소된 것으로 분석되었고, 이는 본 발명의 L. curvatus SMFM2016-NK 균주 처리시 치은 및 치주 인대에서 염증성 세포 침윤이 감소하고, 치조골 흡수 및 백악질 파괴가 현저히 감소하였음을 나타내는 것이다(도 3).As a result of measuring and comparing the histomorphological change score, it was analyzed that the histomorphological change score was significantly reduced in the L. curvatus SMFM2016-NK treatment group after periodontitis induction compared to the periodontitis-inducing group, which was the L. curvatus of the present invention. This indicates that inflammatory cell infiltration in the gingival and periodontal ligaments was reduced, and alveolar bone resorption and cementum destruction were significantly reduced when the SMFM2016-NK strain was treated (FIG. 3).
| 그룹group | 조직형태학적 변화histomorphological changes |
| 정상군normal group | 0.50±0.290.50±0.29 |
| 치주염 유발군Periodontitis causative group | 2.75±0.25+2.75±0.25+ |
| 치주염 유발 후 L. curvatus SMFM2016-NK 처리군 L. curvatus SMFM2016-NK treatment group after periodontitis induction | 1.75±0.25*1.75±0.25* |
| 정상군; phosphate buffered saline (PBS), 치주염 유발군; 치주염+PBS, 치주염 유발 후 L. curvatus SMFM2016-NK 처리군; 치주염+L. curvatus SMFM2016-NK+ statistically significant, compared to normal group by pairwise t-test (p<0.05)* statistically significant, compared to negative control group by pairwise t-test (p<0.05)normal group; phosphate buffered saline (PBS), periodontitis-inducing group; Periodontitis+PBS, L. curvatus SMFM2016-NK treatment group after periodontitis induction; Periodontitis+ L. curvatus SMFM2016-NK+ statistically significant, compared to normal group by pairwise t-test ( p <0.05)* statistically significant, compared to negative control group by pairwise t-test ( p <0.05) | |
(mean±SE, n=4)(mean±SE, n=4)
5-2: 염증세포 수 변화 분석5-2: Analysis of changes in the number of inflammatory cells
조직병리학적 검사 결과 L. curvatus SMFM2016-NK 처리군에서는 치주염 유발군과 비교하여 치은 조직의 염증세포 수가 현저히 감소한 것을 확인하였고(도 3), 이는 본 발명의 L. curvatus SMFM2016-NK 균주 처리가 치주 인대, 치은 등 연조직과 치조골 등 경조직을 손상시키는 염증성 질환에 대해 우수한 효과가 있음을 나타내는 것이다.As a result of histopathological examination , it was confirmed that the number of inflammatory cells in the gingival tissue was significantly reduced in the L. curvatus SMFM2016-NK treatment group compared to the periodontitis-induced group (FIG. 3), which indicates that the L. curvatus SMFM2016-NK strain treatment of the present invention was significantly reduced. This indicates that it has an excellent effect on inflammatory diseases that damage soft tissues such as ligaments and gingiva and hard tissues such as alveolar bone.
| 그룹group | 염증세포수(cells/mm2 of gingival tissues)Number of inflammatory cells (cells/mm 2 of gingival tissues) |
| 정상군normal group | 49.00±14.3949.00±14.39 |
| 치주염 유발군Periodontitis causative group | 238.75±42.66+238.75±42.66+ |
| 치주염 유발 후 L. curvatus SMFM2016-NK 처리군 L. curvatus SMFM2016-NK treatment group after periodontitis induction | 152.50±19.40*152.50±19.40* |
| 정상군; phosphate buffered saline (PBS), 치주염 유발군; 치주염+PBS, 치주염 유발 후 L. curvatus SMFM2016-NK 처리군; 치주염+L. curvatus SMFM2016-NK+ statistically significant, compared to normal group by pairwise t-test (p<0.05)* statistically significant, compared to negative control group by pairwise t-test (p<0.05)normal group; phosphate buffered saline (PBS), periodontitis-inducing group; Periodontitis+PBS, L. curvatus SMFM2016-NK treatment group after periodontitis induction; Periodontitis+ L. curvatus SMFM2016-NK+ statistically significant, compared to normal group by pairwise t-test ( p <0.05)* statistically significant, compared to negative control group by pairwise t-test ( p <0.05) | |
(mean±SE, n=4)(mean±SE, n=4)
5-3: 치조골 부피, 파골세포 수 및 조골세포 수 변화 분석5-3: Analysis of changes in alveolar bone volume, number of osteoclasts, and number of osteoblasts
또한, 조직병리학적 검사 결과 L. curvatus SMFM2016-NK 처리군에서는 치주염 유발군과 비교하여 치조골 부피가 증가하고, 치은 조직의 파골세포 수는 2배 이상 감소한 반면 조골세포 수는 2배 이상 증가한 것을 확인하였다(도 3). 이는 본 발명의 L. curvatus SMFM2016-NK 균주 처리가 치조골에서 조골세포에 의한 골형성을 촉진하는 한편 파골세포에 의한 골흡수를 저해시킴으로써 치조골의 부피를 증가시키고, 이를 통해 치조골 파손, 치조골다공증, 치조골연화증 및 치조골감소증에 대해 예방, 개선 또는 치료 효과를 나타낼 수 있음을 나타내는 것이다.In addition, as a result of histopathological examination, it was confirmed that in the L. curvatus SMFM2016-NK treatment group, the alveolar bone volume increased and the number of osteoclasts in the gingival tissue decreased by more than 2 times, while the number of osteoblasts increased more than 2 times compared to the periodontitis-induced group. (FIG. 3). This is because the treatment of the L. curvatus SMFM2016-NK strain of the present invention promotes bone formation by osteoblasts in alveolar bone while inhibiting bone resorption by osteoclasts to increase the volume of alveolar bone, thereby causing alveolar bone damage, alveolar osteoporosis, and alveolar bone. It indicates that it can exhibit a preventive, ameliorating or therapeutic effect for osteomalacia and alveolar osteopenia.
| 그룹group | 치조골 부피 (%)Alveolar bone volume (%) | 파골세포수(cells/mm2 of alveolar gingival tissues)Number of osteoclasts (cells/mm 2 of alveolar gingival tissues) | 조골세포수 (cells/mm2 of alveolar gingival tissues)Osteoblast count (cells/mm 2 of alveolar gingival tissues) |
| 정상군normal group | 67.80±3.6667.80±3.66 | 8.00±1.638.00±1.63 | 66.00±2.5866.00±2.58 |
| 치주염 유발군Periodontitis causative group | 35.51±2.75+35.51±2.75+ | 34.00±4.76+34.00±4.76+ | 21.00±3.42+21.00±3.42+ |
| 치주염 유발 후L. curvatus SMFM2016-NK 처리군 L. curvatus SMFM2016-NK treatment group after periodontitis induction | 49.78±3.0*49.78±3.0* | 13.00±1.91*13.00±1.91* | 46.00±5.29*46.00±5.29* |
| 정상군; phosphate buffered saline (PBS), 치주염 유발군; 치주염+PBS, 치주염 유발 후 L. curvatus SMFM2016-NK 처리군; 치주염+L. curvatus SMFM2016-NK+ statistically significant, compared to normal group by pairwise t-test (p<0.05)* statistically significant, compared to negative control group by pairwise t-test (p<0.05)normal group; phosphate buffered saline (PBS), periodontitis-inducing group; Periodontitis+PBS, L. curvatus SMFM2016-NK treatment group after periodontitis induction; Periodontitis+ L. curvatus SMFM2016-NK+ statistically significant, compared to normal group by pairwise t-test ( p <0.05)* statistically significant, compared to negative control group by pairwise t-test ( p <0.05) | |||
(mean±SE, n=4)(mean±SE, n=4)
실시예 6: Example 6:
Lactobacillus curvatusLactobacillus curvatus
SMFM2016-NK 균주의 특성 분석 Characterization of SMFM2016-NK strain
6-1: 6-1:
Lactobacillus curvatusLactobacillus curvatus
SMFM2016-NK 균주의 주사전자 현미경 이미지 Scanning electron microscope image of SMFM2016-NK strain
본 발명의 균주를 MRS broth (BD, USA)에 35℃에서 12시간 배양하였다. 슬라이드 글라스(0.5x0.5 cm)를 넣은 MRS broth에 균을 계대 배양하여 세균이 슬라이드 글라스 표면에 붙어 자라도록 하였다. 슬라이드 글라스 조각에 1.8% glutaldehyde solution을 떨어뜨리고 멸균증류수로 세척하였다. 2% osmium tetroxide(Sigma, St. Louis, MO, USA) 용액을 떨어뜨린 후 암실에 실온 반응하였다. 멸균증류수, 에탄올 25%, 50%, 75%, 90%, 100%, hexamethyldisilazane (HMDS; Sigma Aldrich)을 이용하여 세척하고 건조시킨 후 주사전자현미경(FE-SEM; field emission scanning electrochemical microscopy)(Jeol Ltd., Tokyo, Japan)을 통해 관찰하였다. 주사전자 현미경 관찰결과, 본 발명의 균주는 그람 양성의 간균으로 확인되었다(도 4).The strain of the present invention was cultured in MRS broth (BD, USA) at 35° C. for 12 hours. The bacteria were subcultured in MRS broth containing slide glass (0.5x0.5 cm) so that the bacteria grew attached to the slide glass surface. A 1.8% glutaldehyde solution was dropped on a piece of glass slide and washed with sterile distilled water. After dropping a 2% osmium tetroxide (Sigma, St. Louis, MO, USA) solution, the reaction was carried out in the dark at room temperature. After washing and drying using sterile distilled water, ethanol 25%, 50%, 75%, 90%, 100%, hexamethyldisilazane (HMDS; Sigma Aldrich), scanning electron microscope (FE-SEM; field emission scanning electrochemical microscopy) (Jeol) Ltd., Tokyo, Japan). As a result of scanning electron microscopy, the strain of the present invention was confirmed as a Gram-positive bacillus (FIG. 4).
6-2: 6-2:
Lactobacillus curvatusLactobacillus curvatus
SMFM2016-NK 균주의 유전체 분석 Genome analysis of SMFM2016-NK strain
본 발명 균주의 유전체 분석을 위해, 디엔에이링크(서울)에 유전체 분석을 의뢰하였다. Pacific Biosciences system (Pacific Biosciences, Menlo Park, CA, USA) 시퀀싱 플랫폼을 이용하여 20 kb의 라이브러리를 구축하였고, HGAP.3 프로토콜을 바탕으로 de novo assembly를 진행하였다. 그 결과, 해당균주는 총 4개의 콘티그(contigs)로 구성된 균주로 확인되었고, 각 콘티그에 대해 NCBI GenBank database를 바탕으로 한 BLAST 프로그램을 이용하여 확인한 결과, 1개의 염색체(2,139,352 bp, GC content 41.97%)와 3개의 플라스미드를 가진 유전체로 분석되었다. 염색체는 L. curvatus와 가장 유사한 것으로 확인되었다.For the genome analysis of the strain of the present invention, DNA link (Seoul) was requested to analyze the genome. A 20 kb library was constructed using the Pacific Biosciences system (Pacific Biosciences, Menlo Park, CA, USA) sequencing platform, and de novo assembly was performed based on the HGAP.3 protocol. As a result, the strain was identified as a strain consisting of a total of 4 contigs, and as a result of confirming each contig using the BLAST program based on the NCBI GenBank database, one chromosome (2,139,352 bp, GC content 41.97) %) and the genome with three plasmids. The chromosome was confirmed to be most similar to L. curvatus.
| 내용Contents | Lactobacillus curvatus SMFM2016-NK Lactobacillus curvatus SMFM2016-NK | |
| 시퀀싱 플랫폼(Sequencing platform)Sequencing platform | Pacific BiosciencesTM Pacific Biosciences ™ | |
| 라이브러리 사이즈(Library size)Library size | 20 kb20 kb | |
| 프로토콜(Assembly protocol)Assembly protocol | HGAP.3HGAP.3 | |
| 콘티그 수(The number of contigs)The number of contigs | 44 | |
| 유전체 사이즈(Genome size)Genome size | Contig 1Contig 1 | 2,139,352 bp (GC content 41.97%)2,139,352 bp (GC content 41.97%) |
| Contig 2Contig 2 | 41,082 bp (GC content 39.16%)41,082 bp (GC content 39.16%) | |
| Contig 3Contig 3 | 17,282 bp (GC content 37.23%)17,282 bp (GC content 37.23%) | |
| Contig 4Contig 4 | 10,972 bp (GC content 41.67%)10,972 bp (GC content 41.67%) |
염기서열의 유전체 정보를 바탕으로 해당 균주 염색체의 유전적 특성(location of gene, coding region, function of gene)을 분석하였고, open reading frame (ORF), coding sequence (CDS) 정보를 바탕으로 구조적 특징(유전자 수, 위치)을 아래와 같은 DNA plot으로 확인되었다. 구체적으로, L. curvatus SMFM2016-NK 염색체는 2,307 coding sequences(CDS), 18 rRNA, 67 tRNA, 8,500 transcripts로 구성되어 있음이 확인되었다(도 5).Based on the genomic information of the base sequence, the genetic characteristics (location of gene, coding region, function of gene) of the chromosome of the corresponding strain were analyzed, and structural characteristics ( The number of genes, their location) was confirmed with the following DNA plot. Specifically, it was confirmed that the L. curvatus SMFM2016-NK chromosome consists of 2,307 coding sequences (CDS), 18 rRNA, 67 tRNA, and 8,500 transcripts (FIG. 5).
또한, NCBI GenBank database에 등록된 11개의 다른 L. curvatus 균주들과 특징(유전체 크기, GC 비율, tRNA 수, plasmid 수)을 분석한 결과, 평균염기서열 유사도(ANI; average nucleotide identity, %)는 98.21% - 99.4%로 L. curvatus DSM20019와 가장 유사한 것으로 확인되었다(도 6).In addition, as a result of analyzing the characteristics (genome size, GC ratio, tRNA number, plasmid number) with 11 other L. curvatus strains registered in the NCBI GenBank database, the average nucleotide identity (ANI; average nucleotide identity, %) was 98.21% - 99.4% was confirmed to be the most similar to L. curvatus DSM20019 (FIG. 6).
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.
From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.
Claims (16)
- 락토바실러스 커바투스(Lactobacillus curvatus) SMFM2016-NK 균주(기탁번호: KCTC14138BP). Lactobacillus curvatus ( Lactobacillus curvatus ) SMFM2016-NK strain (Accession No.: KCTC14138BP).
- 제1항에 있어서, 상기 균주는 서열번호 1로 표시되는 16s rDNA 서열을 포함하는 것을 특징으로 하는, 균주.The strain according to claim 1, wherein the strain comprises a 16s rDNA sequence represented by SEQ ID NO: 1.
- 락토바실러스 커바투스(Lactobacillus curvatus) SMFM2016-NK 균주(기탁번호: KCTC14138BP), 이의 배양물, 농축물, 건조물 또는 파쇄물을 유효성분으로 포함하는, 구강질환 예방 또는 개선용 식품 조성물. Lactobacillus curvatus ( Lactobacillus curvatus ) SMFM2016-NK strain (Accession No.: KCTC14138BP), its culture, concentrate, dry matter or lysate comprising as an active ingredient, a food composition for preventing or improving oral disease.
- 제3항에 있어서, 상기 구강질환은 치조골 파손(alveolar bone breakage), 치조골다공증(alveolar bone osteoporosis), 치조골연화증(alveolar bone osteomalacia), 치조골감소증(alveolar bone osteopenia), 치아 우식증(dental caries), 치은염(gingivitis) 및 치주염(periodontitis)으로 구성된 군으로부터 선택되는 하나 이상의 질환인 것인, 식품 조성물.According to claim 3, wherein the oral disease is alveolar bone breakage, alveolar bone osteoporosis, alveolar bone osteomalacia, alveolar bone osteopenia, dental caries, gingivitis (gingivitis) and periodontitis (periodontitis) of one or more diseases selected from the group consisting of, the food composition.
- 제3항에 있어서, 상기 균주는 파골세포 수 감소, 조골세포 수 증가, 해면골 소실 부피 감소, 치조골 부피 증가, 염증세포 수 감소 효과 및 구강 내 유해세균에 대한 항균 활성을 나타내는 것을 특징으로 하는, 식품 조성물. The food according to claim 3, wherein the strain reduces the number of osteoclasts, increases the number of osteoblasts, decreases the volume of cancellous bone loss, increases the volume of alveolar bone, reduces the number of inflammatory cells, and exhibits antibacterial activity against harmful bacteria in the oral cavity, food composition.
- 제5항에 있어서, 상기 유해세균은 프리보텔라 인터미디어(Prevotella intermedia), 퓨조박테리움 뉴클레아툼(Fusobacterium nucleatum) 또는 아그레가티박터 액티노마이세템코미탄스(Aggregatibacter actinomycetemcomitans)인, 식품 조성물.The food composition of claim 5, wherein the harmful bacteria are Prevotella intermedia , Fusobacterium nucleatum , or Aggregatibacter actinomycetemcomitans.
- 제3항에 있어서, 상기 조성물은 정제, 캡슐, 환, 과립, 액상, 분말, 편상, 페이스트상, 시럽, 겔, 젤리, 바 등의 건강기능식품 제제류, 음료류, 껌류 및 캔디류로 구성된 군으로부터 선택되는 하나 이상의 제형인 것인, 식품 조성물.The composition according to claim 3, wherein the composition is selected from the group consisting of health functional food preparations such as tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies and bars, beverages, gums and candies. One or more formulations selected, the food composition.
- 락토바실러스 커바투스(Lactobacillus curvatus) SMFM2016-NK 균주(기탁번호: KCTC14138BP), 이의 배양물, 농축물, 건조물 또는 파쇄물을 유효성분으로 포함하는, 구강질환 예방 또는 치료용 약학적 조성물. Lactobacillus curvatus ( Lactobacillus curvatus ) SMFM2016-NK strain (Accession No.: KCTC14138BP), its culture, concentrate, dry matter or lysate comprising as an active ingredient, oral disease prevention or treatment pharmaceutical composition.
- 제8항에 있어서, 상기 구강질환은 치조골 파손(alveolar bone breakage), 치조골다공증(alveolar bone osteoporosis), 치조골연화증(alveolar bone osteomalacia), 치조골감소증(alveolar bone osteopenia), 치아 우식증(dental caries), 치은염(gingivitis) 및 치주염(periodontitis)으로 구성된 군으로부터 선택되는 하나 이상의 질환인 것인, 약학적 조성물.The method of claim 8, wherein the oral disease is alveolar bone breakage, alveolar bone osteoporosis, alveolar bone osteomalacia, alveolar bone osteopenia, dental caries, gingivitis (gingivitis) and one or more diseases selected from the group consisting of periodontitis (periodontitis), the pharmaceutical composition.
- 제8항에 있어서, 상기 균주는 파골세포 수 감소, 조골세포 수 증가, 해면골 소실 부피 감소, 치조골 부피 증가, 염증세포 수 감소 효과 및 구강 내 유해세균에 대한 항균 활성을 나타내는 것을 특징으로 하는, 약학적 조성물.According to claim 8, wherein the strain reduces the number of osteoclasts, increases the number of osteoblasts, decreases the volume of cancellous bone loss, increases the volume of alveolar bone, reduces the number of inflammatory cells, and exhibits antibacterial activity against harmful bacteria in the oral cavity, pharmaceutical enemy composition.
- 제10항에 있어서, 상기 유해세균은 프리보텔라 인터미디어(Prevotella intermedia), 퓨조박테리움 뉴클레아툼(Fusobacterium nucleatum) 또는 아그레가티박터 액티노마이세템코미탄스(Aggregatibacter actinomycetemcomitans)인, 약학적 조성물.The pharmaceutical composition of claim 10, wherein the harmful bacteria are Prevotella intermedia , Fusobacterium nucleatum , or Aggregatibacter actinomycetemcomitans. .
- 락토바실러스 커바투스(Lactobacillus curvatus) SMFM2016-NK 균주(기탁번호: KCTC14138BP), 이의 배양물, 농축물, 건조물 또는 파쇄물을 유효성분으로 포함하는, 프로바이오틱스. Lactobacillus curvatus ( Lactobacillus curvatus ) SMFM2016-NK strain (Accession No.: KCTC14138BP), its culture, concentrate, dry matter or lysate comprising the as an active ingredient, probiotics.
- 제12항에 있어서, 상기 프로바이오틱스는 구강질환 예방 및 개선용으로 사용되는 것을 특징으로 하는, 프로바이오틱스.The probiotics according to claim 12, wherein the probiotics are used for preventing and improving oral diseases.
- 락토바실러스 커바투스(Lactobacillus curvatus) SMFM2016-NK 균주(기탁번호: KCTC14138BP), 이의 배양물, 농축물, 건조물 또는 파쇄물을 유효성분으로 포함하는, 구강질환 예방 또는 개선용 의약외품 조성물. Lactobacillus curvatus ( Lactobacillus curvatus ) SMFM2016-NK strain (Accession No.: KCTC14138BP), its culture, concentrate, dry matter or lysate comprising as an active ingredient, a quasi-drug composition for preventing or improving oral diseases.
- 제14항에 있어서, 상기 균주는 파골세포 수 감소, 조골세포 수 증가, 해면골 소실 부피 감소, 치조골 부피 증가, 염증세포 수 감소 효과 및 구강 내 유해세균에 대한 항균 활성을 나타내는 것을 특징으로 하는, 의약외품 조성물.The quasi-drug of claim 14, wherein the strain reduces the number of osteoclasts, increases the number of osteoblasts, decreases the volume of cancellous bone loss, increases the volume of alveolar bone, reduces the number of inflammatory cells, and exhibits antibacterial activity against harmful bacteria in the oral cavity, quasi-drugs composition.
- 제14항에 있어서, 상기 의약외품 조성물은 치약, 구강청결제, 껌, 캔디류, 구강스프레이, 구강용 연고제, 구강용 바니쉬(varnish), 구강필름 및 잇몸 마사지 크림으로 구성된 군으로부터 선택되는 하나 이상의 제형인 것인, 의약외품 조성물.15. The method of claim 14, wherein the quasi-drug composition is one or more formulations selected from the group consisting of toothpaste, mouthwash, gum, candy, oral spray, oral ointment, oral varnish, oral film, and gum massage cream. Phosphorus, a quasi-drug composition.
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| KR102573674B1 (en) * | 2021-12-28 | 2023-09-01 | 한국식품연구원 | Composition for preventing, improving or treating inflammatory disease comprising the Latilactobacillus curvatus WiKim0140 |
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