WO2020175869A2 - 신규한 락토바실러스 사케이 cvl-001 균주 또는 이의 배양액을 포함하는 골 질환 또는 대사성 질환 개선, 예방 또는 치료용 조성물 - Google Patents

신규한 락토바실러스 사케이 cvl-001 균주 또는 이의 배양액을 포함하는 골 질환 또는 대사성 질환 개선, 예방 또는 치료용 조성물 Download PDF

Info

Publication number
WO2020175869A2
WO2020175869A2 PCT/KR2020/002612 KR2020002612W WO2020175869A2 WO 2020175869 A2 WO2020175869 A2 WO 2020175869A2 KR 2020002612 W KR2020002612 W KR 2020002612W WO 2020175869 A2 WO2020175869 A2 WO 2020175869A2
Authority
WO
WIPO (PCT)
Prior art keywords
strain
cvl
lactobacillus
culture
culture solution
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/KR2020/002612
Other languages
English (en)
French (fr)
Other versions
WO2020175869A3 (ko
Inventor
박종환
최주희
정도헌
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Industry Foundation of Chonnam National University
Original Assignee
Industry Foundation of Chonnam National University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020190022671A external-priority patent/KR102242668B1/ko
Priority claimed from KR1020200012763A external-priority patent/KR102276576B1/ko
Application filed by Industry Foundation of Chonnam National University filed Critical Industry Foundation of Chonnam National University
Priority to JP2021549784A priority Critical patent/JP2022522151A/ja
Priority to US17/434,080 priority patent/US20220135935A1/en
Priority to CN202080016504.6A priority patent/CN113840908A/zh
Priority to EP20762101.2A priority patent/EP3932416A4/en
Priority to SG11202109231WA priority patent/SG11202109231WA/en
Publication of WO2020175869A2 publication Critical patent/WO2020175869A2/ko
Publication of WO2020175869A3 publication Critical patent/WO2020175869A3/ko
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

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; 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 OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/02—Separating microorganisms from their culture media
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/179—Sakei
    • 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
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A61K2035/115—Probiotics
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • C12N2500/14—Calcium; Ca chelators; Calcitonin
    • 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
    • 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
    • C12R2001/245—Lactobacillus casei

Definitions

  • Composition for improving, preventing or treating bone diseases or metabolic diseases including cultures thereof
  • the present invention relates to the new Lactobacillus scikei CVL-001 strain and its separation method. More specifically, the novel Han Lactobacillus sakei CVL-001 strain identified by separating from kimchi. will be.
  • the present invention uses Lactobacillus SAK CVL-001 strain or a culture solution thereof.
  • compositions for improving, preventing or treating bone diseases or metabolic diseases including, and in more detail, a new company deposited under the accession number KCTC13816BP.
  • composition containing the Lactobacillus sakei CVL-001 strain or its culture solution for improving bone disease or metabolic disease It is about the improvement, prevention or treatment effect of the composition containing the Lactobacillus sakei CVL-001 strain or its culture solution for improving bone disease or metabolic disease.
  • Lactobacillus which is abundantly present in traditional fermented foods such as kimchi, coexists in the digestive system of the human body and plays a role in decomposing fiber and complex proteins to make it an important nutrient component. It inhibits the propagation of harmful bacteria such as Chlostridium sp., improves diarrhea and constipation, plays a role in vitamin synthesis, lowering blood cholesterol, etc.
  • lactic acid bacteria have the property of being able to bind strongly to the intestinal mucosa and epithelial cells. It helps a lot in the intestinal function.
  • lactic acid bacteria are known to promote the proliferation of macrophages to enhance the cognition and sterilization ability of harmful bacteria in the intestine of macrophages, and to promote the secretion of immune-related substances, thereby exhibiting an immune enhancing effect.
  • lactic acid bacteria are known to promote the proliferation of macrophages to enhance the cognition and sterilization ability of harmful bacteria in the intestine of macrophages, and to promote the secretion of immune-related substances, thereby exhibiting an immune enhancing effect.
  • Microorganisms of the genus Lactobacillus are lactic acid bacillus that undergo homogeneous or heterogeneous fermentation
  • GM is made up of a huge amount of bacteria that contain 150 times more genes than the human genome, which is obtained at birth and is a distinct reality. Regardless of this, it can clearly be regarded as a multicellular organism that resonates with the human genome and communicates and influences its host in various ways.
  • composition of GM is driven by a number of environmental factors such as food and antibiotic therapy.
  • Molecules produced by intestinal microflora can be beneficial and harmful, and are known to affect the endocrine cells, gut nervous system, intestinal permeability and immune system.
  • the disturbed microbial composition is Crohn's disease, ulcerative colitis. , Rheumatoid arthritis, multiple sclerosis, diabetes, food allergy, eczema and asthma, as well as obesity and metabolic syndrome, have been implicated in a variety of inflammatory conditions both inside and outside the intestine.
  • Cortical bone ( ⁇ )1 15011 the body constitutes about 80% of the bone in the body, and several studies have shown that cortical bone is a major determinant of bone strength and thus a major determinant of fracture sensitivity. Due to loss 2010, May 15; 375(9727):1729-36).
  • Skeletal is formed by bone-forming osteoblasts ([ « and bone resorption osteoclasts (00 ⁇ ))
  • Macrophage colony stimulating factor not only increases proliferation and survival of 00 ⁇ progenitor cells, but also nuclear factor at 00 ⁇ -
  • the purpose of the present invention is Lactobacillus deposited under accession number KCTC13816BP
  • Another purpose of the present invention is Lactobacillus deposited under accession number KCTC13816BP
  • Another object of the present invention is to provide a method for isolating the Lactobacillus company CVL-001 strain deposited under accession number KCTC13816BP, including a culture step of culturing kimchi extract.
  • Another purpose of this invention is to improve, prevent or treat bone diseases or metabolic diseases.
  • Another object of the present invention is Lactobacillus Corporation, which is deposited under the accession number KCTC13816BP, which has the activity of improving, preventing or treating bone diseases or metabolic diseases.
  • Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of bone diseases or metabolic diseases, including the culture solution of the Lactobacillus sakei CVL-001 strain deposited under accession number KCTC13816BP.
  • Another object of the present invention is Lactobacillus deposited under accession number KCTC13816BP 2020/175869 1»(:1/10 ⁇ 020/002612 Sakei CVL-001 is to provide a food composition for improving bone disease or metabolic disease, including the culture medium.
  • Another object of the present invention is to improve, prevent and treat bone diseases or metabolic diseases of the Lactobacillus sakei CVL-001 strain deposited under the accession number KCTC13816BP.
  • the present invention is a novel Lactobacillus sakei CVL-001 strain, its isolation method, Bone disease or metabolic disease improvement, prevention or treatment comprising Lactobacillus Sake CVL-001 strain or a culture medium thereof It is about dragon composition.
  • the present inventors isolated the strain by culturing the kimchi extract, and selected the Gram-positive and catalase-negative strain from this, and isolated the Lactobacillus SAK CVL-001 strain, and the strain or its culture medium inhibited the differentiation of osteoclasts. It was confirmed that it is effective in treating bone diseases, and
  • One aspect of the present invention is the Lactobacillus SK CVL-001 strain deposited under the accession number KCTC13816BP.
  • the strain may have a 16S rRNA sequence represented by SEQ ID NO: 1.
  • Another aspect of the present invention is Lactobacillus deposited under accession number KCTC13816BP
  • the strain may have a 16S rRNA sequence represented by SEQ ID NO: 1.
  • Another aspect of the present invention comprises a culture step of culturing the kimchi extract
  • the kimchi extract may be a kimchi solution that is filtered by grinding the kimchi.
  • it may be a kimchi solution diluted by adding water to the kimchi solution, but is not limited thereto.
  • the culture step may be a first culture step of culturing the kimchi extract; and a second culture step of culturing the strain isolated in the first culture step.
  • the first culture step may be performed in a medium to which 0.5 to 2% of calcium carbonate or 0.004 to 0.006% of bromocresol purple is added for the differentiation of lactic acid bacteria, for example, calcium carbonate is added. It may be done on a badge, but is not limited to this.
  • the first cultivation step above may be performed at 20 to 32 O C for 36 to 60 hours, for example, it may be performed at 30 O C for 48 hours, but is not limited thereto. Isolate the strain that formed Steps 2020/175869 PCT/KR2020/002612 are applicable.
  • the second culturing step may be a step of selecting and culturing lactic acid bacteria out of the separated strains. Specifically, a step of selecting and culturing gram-positive and catalase-negative ones among the separated strains as lactic acid bacteria.
  • the second culture step above may be performed for 20 to 32 to 12 to 36 hours, for example, at 30 O C for 24 hours, but is not limited thereto.
  • Another aspect of the present invention is to improve bone disease, prevent or have therapeutic activity.
  • Lactobacillus company CVL-001 strain deposited under the accession number KCTC13816BP.
  • the strain above may be isolated from kimchi, but is not limited thereto.
  • Another aspect of the present invention is to improve bone disease, prevent or have therapeutic activity.
  • Another aspect of the present invention is a pharmaceutical composition for the prevention or treatment of bone diseases comprising the Lactobacillus sakei CVL-001 strain deposited under the accession number KCTC13816BP or a culture solution thereof.
  • the above bone diseases are osteoporosis, bone metastatic cancer,
  • osteomalacia rickets
  • fibrotic bone disease amorphous bone disease
  • metabolic bone disease amorphous bone disease
  • periodontal disease for example, osteoporosis, but is not limited thereto.
  • the culture solution is, Lactobacillus SAK CVL-001 strain is cultured, and the strain is
  • It may be a freeze-dried product.
  • the culture solution is Lactobacillus SAK CVL-001 strain for 12 to 30 hours,
  • BME Basal Media Eagle
  • MEM Minimum Essential Media Eagle
  • Alpha MEM is an amino acid, 2020/175869 1»(:1 ⁇ 1 ⁇ 2020/002612 Vitamins, etc. are additionally added, which is useful for DNA transformation of animal cells.
  • the above composition contains a culture solution of 50 to 400, 50 to 350, 50 to 300, 50 to 250, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 150 to 400, 150 to 350, 150 to 300, 150 to 250, 180 to 400, 180 to 350 or 180 to 300 ⁇ , for example, 180 to 250 may include a concentration of 111/1111, but is not limited thereto.
  • the culture medium may be 1 ⁇ 6.5 to 8.5, 6.5 to 8.0, 6.5 to 7.5, 7.0 to 8.5 or 7.0 to 8.0, for example, ! ⁇ 7.0 to 7.5, but is not limited thereto.
  • Lactobacillus deposited under the accession number He(:1381661 5 ) It is a pharmaceutical composition for preventing or treating metabolic diseases containing a culture medium.
  • the metabolic diseases include obesity, diabetes, hypertension, hyperlipidemia, cardiovascular disease and
  • It may be one or more selected from the group consisting of hyperinsulinemia.
  • It may be a freeze-dried product.
  • the culture medium may be 1 ⁇ 6.5 to 8.5, 6.5 to 8.0, 6.5 to 7.5, 7.0 to 8.5 or 7.0 to 8.0, for example, ! ⁇ 7.0 to 7.5, but is not limited thereto.
  • the pharmaceutical composition of the present invention is Lactobacillus sakei (: ⁇ 1-001 strain culture solution)
  • composition containing a pharmaceutically effective amount and/or a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate ,Gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose,
  • the pharmaceutical composition of the present invention is a lubricant, humectant, sweetener, flavoring agent in addition to the above ingredients. , Emulsifiers, suspending agents, preservatives, etc. may additionally be included.
  • the pharmaceutical composition according to the present invention can be administered to mammals, including humans, by various routes.
  • the mode of administration can be any commonly used method, for example, oral, skin, intravenous, muscle, subcutaneous, etc. Can be administered by route,
  • it can be administered orally.
  • the appropriate dosage of the pharmaceutical composition of the present invention depends on factors such as formulation method, administration method, patient's age, weight, sex, morbidity, food, administration time, route of administration, excretion rate and response sensitivity. It varies, and usually a trained doctor can easily determine and prescribe a dosage that is effective for the desired treatment or prevention.
  • the pharmaceutical composition of the present invention is formulated in a unit dosage form using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily implemented by a person with ordinary knowledge in the technical field to which the present invention belongs.
  • the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet, capsule or gel (e.g., hydrogel).
  • It may additionally contain stabilizers.
  • Lactobacillus deposited under the accession number He(:1381661 5 ) It is a food composition for improving bone disease comprising a culture medium.
  • the bone disease may be selected from the group consisting of osteoporosis, bone metastases, osteomalacia, rickets, fibrotic osteopathy, amorphous bone disease, metabolic bone disease, and periodontal disease.
  • it may be osteoporosis, but limited to this. It doesn't work.
  • It may be a freeze-dried product.
  • 12 to 30 hours, 12 to 24 hours, 18 to 36 hours, 18 to 30 hours or 18 to 24 hours, for example, may be obtained by culturing for 22 to 26 hours, but limited to this It doesn't work.
  • the above composition contains culture solutions of 20 to 400, 20 to 350, 20 to 300, 20 to 250, 50 to 400, 50 to 350, 50 to 300, 50 to 250, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 150 to 400, 150 to 350, 150 to 300, 150 to 250, 180 to 400, 180 to 350 or 180 to 300 ⁇ /sen, for example, at a concentration of 180 to 250 111/1111 May include, but is not limited to.
  • the culture medium may be 1 ⁇ 6.5 to 8.5, 6.5 to 8.0, 6.5 to 7.5, 7.0 to 8.5 or 7.0 to 8.0, for example, ! ⁇ 7.0 to 7.5, but is not limited thereto.
  • This improvement of bone disease may be achieved through inhibition of osteoclast differentiation, but is not limited thereto.
  • accession number (Lactobacillus deposited under (:1381661 5 ) It is a health functional food composition for improving metabolic diseases containing culture medium.
  • the metabolic diseases are obesity, diabetes, hypertension, hyperlipidemia, cardiovascular disease and
  • It may be one or more selected from the group consisting of hyperinsulinemia.
  • It may be a freeze-dried product.
  • the culture medium may be 1 ⁇ 6.5 to 8.5, 6.5 to 8.0, 6.5 to 7.5, 7.0 to 8.5 or 7.0 to 8.0, for example, ! ⁇ 7.0 to 7.5, but is not limited thereto.
  • the food composition of the present invention is preferably 15% by weight or less based on the raw material. It may be added in an amount of 10% by weight or less.
  • the drink contains various flavors or natural carbohydrates as additional ingredients.
  • the natural carbohydrates described above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame. Can be used.
  • the ratio of the natural carbohydrate can be appropriately determined by the choice of the person skilled in the art.
  • the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, fectic acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, It may contain preservatives, glycerin, alcohol, carbonic acid used in carbonated beverages, etc.
  • the food composition of the present invention may contain pulp for the manufacture of natural fruit juice, fruit juice beverages and vegetable beverages. These ingredients independently They can be used either individually or in combination. The proportion of these additives can also be appropriately selected by the person skilled in the art.
  • the present invention is a new Korean lactobacillus sealer
  • the above strain is a lactic acid bacteria that can be separated from kimchi, it can be effectively used as a probiotic or food additive.
  • compositions for improving, preventing or treating bone diseases or metabolic diseases including its culture solution
  • the Lactobacillus sakei strain isolated from kimchi and its culture media inhibits osteoclast differentiation, improves osteoporosis disease, inhibits adipose cell differentiation and increases weight. Since it exhibits an inhibitory effect, it can be effectively used for improving, preventing or treating osteoporosis or obesity diseases.
  • Lactobacillus sakei 001 [Shun (111 « ⁇ ri ⁇ ))
  • Figure 2 is a lactobacillus Induction of osteoclast differentiation of nutrient solution
  • Figure 4 is a lactobacillus in a mouse model induced osteoporosis
  • Figure 5 is a graph showing the inhibitory effect on the differentiation of adipocytes of Lactobacillus SAK CVL-001 culture solution.
  • Figure 6 is a graph showing the weight-suppression effect of the Lactobacillus sakei CVL-001 strain in the high-fat diet experimental group over time.
  • Figure 7 is a graph showing the effect of reducing the weight of epididymis fat by the Lactobacillus sakei CVL-001 strain in the high-fat diet experimental group according to the experimental group.
  • Figure 8 is a Lactobacillus sakei CVL-001 strain in the high fat diet experimental group
  • Figure 9 is a Lactobacillus sakei CVL-001 strain in the high fat diet experimental group
  • Fig. W is the Lactobacillus sakei CVL-001 strain in the high fat diet experimental group
  • 11 is a graph showing the blood glucose lowering effect in the glucose load test by the Lactobacillus sakei CVL-001 strain in the high fat diet experimental group according to the experimental group.
  • FIG. 13 is a graph showing the blood glucose lowering effect in the insulin load test by the Lactobacillus sakei CVL-001 strain in the high-fat diet experimental group according to the experimental group. Best mode for carrying out the invention
  • the present invention is Lactobacillus sakei, which has the activity of improving, preventing or treating bone diseases or metabolic diseases, deposited under accession number KCTC13816BP.
  • solid/solid is (weight/weight)%
  • solid/liquid is (weight/volume)%
  • liquid/liquid is (volume/volume)%.
  • a stock was made and stored at -70 O C and used.
  • Example 2 Identification of lactic acid strains
  • 16S rRNA base sequence analysis was performed using a universal primer set of 1492R (5, -GGTTACCTTGTTACGACTT-3,).
  • Lactobacillus sakei LZm Lactobacillus sakei LZ2157 and 99% It was finally identified as Lactobacillus Sakei LZ217, indicating the province of costume.
  • the isolated strain was named Lactobacillus Sarkei CVL-0()1, and was deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center, 823, Sinjeong-dong, Jeongeup-si, Jeollabuk-do, Korea.
  • Lactobacillus SAK CVL-0()1 was incubated for 24 hours, and after centrifugation, the supernatant was separated and adjusted to pH 7.4.
  • Penicillin-Streptomycin hereinafter PS
  • macrophage colony stimulating factor hereinafter referred to as M-CSF
  • PS Penicillin-Streptomycin
  • M-CSF macrophage colony stimulating factor
  • RANKL Receptor activator of nuclear factor kappa-B ligand
  • Nuclei were stained by adding a chromogenic substrate to phosphatase (Tartrate resistance acid phosphatase; hereinafter, TRAP), and cells with three or more nuclei multinucleated were observed and imaged.
  • a chromogenic substrate to phosphatase (Tartrate resistance acid phosphatase; hereinafter, TRAP)
  • TRAP phosphatase
  • the TRAP-positive cells are stimulated with inflammatory factors such as RANKL and TNF-oc, the cells are fused and differentiated into multikaryotype TRAP-positive cells.
  • Example 5 Evaluation of inhibition of protein expression inducing osteoclast differentiation of Lactobacillus SAK CVL-001 strain culture
  • Lactobacillus SK CVL-001 strain culture solution (100 ul/ml) was treated for 2 hours. Then, RANKL was treated with 100 ng/ml and reacted for 0, 5, 15 and 30 minutes. Made it.
  • the protein was extracted using a protein lysis buffer, and then quantified, and 30 ug of the protein was separated on an SDS-PAGE gel.
  • primary and secondary antibodies p-jnk (Cell Signaling Technology, #925 IS), p-p38 (Cell Signaling Technology, 921 IS), p-ERK (santa cruz, sc- 7383), Ikbalpha (Cell Signaling Technology, 9242S), p-p65 (Cell Signaling Technology, 303 IS) were sequentially processed and expressed as a kit (BIO-RAD, ECL solution detection kit) and immediately designated as a machine (Chemidoc). .
  • RANKL binds to RANK, and through TRA, MAPK and NF-KB proteins
  • Lactobacillus SAK culture solution inhibited the differentiation into osteoclasts by inhibiting phosphorylation of JNK, P38, and ERK.
  • Example 6 Evaluation of inhibition of expression of osteoclast differentiation-inducing gene of Lactobacillus saK culture solution
  • Lactobacillus SAK culture solution was treated on mouse macrophages to determine gene expression for the osteoclast differentiation mechanism-related genes TRAP, DC-STAMP, Cathepsin K, and NFATcl through real-time PCR.
  • Bone marrow-derived macrophages (hereinafter BMDMs) were cultured in 12 wells at a density of lxlO 5 /well for 24 hours. After treating the medium containing only 1% PS without FBS for 1 hour, 100 ul/ml of the Lactobacillus SAK culture solution was treated for 2 hours. After that, RANKL was treated and reacted for 24 hours.
  • RNA was isolated using TRIZol solution, and cDNA was synthesized using RT premix based on RNA quantification.
  • the synthesized cDNA was amplified through real-time feeding using a primer.
  • the primers used were Mouse TRAP, DC-STAMP, Cathepsin K, and NFATcl.
  • the relative amount was compared with the control gene GAPDH.
  • TRAP which activates NFATcl, a transcription factor important for differentiation into osteoclasts, is involved in osteoclastogenesis.
  • Cathepsine K increases the expression of DC-STAMP.
  • Example 7 Induction of an osteoporosis animal model through ovariectomy of a mouse
  • Mouse 0576 ⁇ 6 female (7 weeks old) was used and reared in a plastic cage in an environment set at 22 ⁇ 2o (: and 50+10% relative humidity, 12-hour light and dark cycle). Prior to ovariectomy of mice, the same environment In about 1 week, it was adapted to the breeding conditions.
  • Lactobacillus Sake was administered orally after pre-culture and main culture, calculated as 1x10 ⁇ 1 10 8 and 1 0 9 0 118/1111.
  • Example 8 Lactobacillus sakei Lactobacillus and Bone Mineral Density Evaluation in Osteoporosis-induced Mice for Administration of Its Culture Solution
  • Example 9 Lactobacillus sakei isolated from kimchi (: ⁇ -001 Experiment on the inhibitory effect of adipocyte differentiation using culture solution (year 0)
  • Adipocyte differentiation is induced when the media components are replaced with FBS rather than Bovine calf serum (BCS).
  • 3T3-L1 cells were inoculated with a medium to which 10% FBS and 1% PS were added at a density of 5 X 10 5 cells/well in a 12 well plate. After 2 days at the point where the cells grew at a density of 100%, the culture solution (12.5, 25.0 and 50.0%) was treated for 2 hours on day 0, and the adipocyte differentiation inducer MDI was treated.
  • the culture medium was prepared by culturing L. mkei at a concentration of 1 X 10 8 cfu/ml in DMEM (High glucose) + FBS (10%) + PS (1%) medium for 24 hours, and the culture solution was diluted. Was carried out using DMEM medium.
  • differentiated adipocytes When differentiated adipocytes were observed, they were replaced with 4% formalin for staining and the cells were fixed for 10 minutes. After washing twice with D.W, oil red O staining reagent and D.W were mixed at a ratio of 6:4, and the fat cells were stained for 30 minutes. After washing with D.W and observation under a microscope, oil red O was extracted by treatment with 100% isopropanol, and the degree of differentiation was quantitatively measured by absorbance at 5 W nm.
  • Lactobacillus SAK CVL-001 culture solution is used to promote differentiation into adipocytes.
  • Example 10 Preparation of an obese mouse animal model for confirming the effect of improving obesity (in vivo)
  • [183] was prepared in the live cells to handle the mouse. Specifically, the solid medium (MRS agar) and incubated at 30 O C for 24 hours to collect a single colony (single colony) ago in the liquid medium (MRS broth) The cultivation was carried out. The pre-culture was carried out in 5 ml medium (media) at 150 rpm 30 O C for 24 hours, and then diluted in W-fold and the main culture was carried out for 3 hours under the same conditions.
  • the absorbance is 0.D value 0.6 at 600 ran.
  • the concentration was adjusted in PBS using a spectrophotometer. This means that lactic acid bacteria exist as much as 0.89xl0 9 CFU/ml under this condition. 200 ul per mouse individual (lxl0 8 CFU/mouse, lxl0 9 CFU/) mouse) to be administered, centrifugation was performed at 3,000 RPM for 15 minutes to prepare a viable medium (media).
  • the MRS medium containing Lactobacillus sakei was cultured for 24 hours at 150 rpm and 30 O C for 24 hours. After that, centrifugation was performed at 3,000 RPM for 15 minutes to separate the supernatant. And the pH was adjusted to 7.4. After filtering with 0.45 um membrane filter paper, it was stored at 4 O C.
  • Example 11 Lactobacillus sakei isolated from kimchi (: ⁇ -001 strain and its
  • the weight of epididymis fat in group 05 fed with high-fat diet was significantly increased from the standard diet group (31), and the weight of epididymis fat in group 05 fed with high-fat diet was 10 9
  • the weight gain was significantly increased in the group 3 fed the high-fat diet compared to the group fed the standard diet (31).
  • the weight was significantly lower than that in 03.
  • the weight was induced by the high fat diet. It turned out to be unable to suppress weight gain.
  • mice were tested for blood sugar after fasting for 12 hours from 9 p.m. the day before the experiment to 9 p.m. the next day.
  • Mouse animal models fed a high fat diet are known to maintain very high fasting blood sugar.
  • Conditions for each experiment group are: The procedure was carried out under the same conditions as in 2-3 above, and the groups to which the strain was administered and the group to which the culture solution was administered are shown in Tables 8 and 9, respectively.
  • the blood sugar level was reduced, so it was similar to the standard diet group ( 31).
  • the present invention relates to the new Lactobacillus scikei CVL-001 strain and its separation method. More specifically, the novel Han Lactobacillus sakei CVL-001 strain identified by separating from kimchi. will be.
  • the present invention uses Lactobacillus Skay CVL-001 strain or a culture solution thereof
  • composition for improving, preventing, or treating bone diseases containing, in more detail, the new Lactobacillus SAK CVL-001 strain deposited under the accession number KCTC13816BP or a composition containing a culture solution thereof to improve, prevent, or It is about the therapeutic effect.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Polymers & Plastics (AREA)
  • Nutrition Science (AREA)
  • Food Science & Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Rheumatology (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Diabetes (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Hematology (AREA)
  • Obesity (AREA)
  • Pediatric Medicine (AREA)

Abstract

본 발명은 신규한 락토바실러스 사케이 CVL-001 균주 및 이의 분리 방법에 관한 것으로서, 상기 균주는 김치로부터 분리할 수 있는 유산균이므로, 이를 효과적으로 생균제 또는 식품 첨가제로서 이용할 수 있다. 또한, 본 발명은 락토바실러스 사케이 CVL-001 균주(Lactobacillus sakei) 또는 이의 배양액을 포함하는 골 질환 또는 대사성 질환 개선, 예방 또는 치료용 조성물에 관한 것으로서, 김치로부터 분리한 락토바실러스 사케이 균주와 그의 배양액이 파골세포 분화 억제, 골다공증 질환 개선, 지방세포 분화 억제 및 체중 증가 억제 효과를 나타내므로, 이를 효과적으로 골다공증 또는 비만 질환의 개선, 예방 또는 치료에 이용할 수 있다.

Description

명세서
발명의명칭:신규한락토바실러
Figure imgf000003_0001
이의배양액을포함하는골질환또는대사성질환개선，예방또는 치료용조성물
기술분야
[1] 본발명은과학기술정보통신부의지원하에서과제번호 2018R1A2B3004143에 의해이루어진것으로서,상기과제의연구관리전문기관은한국연구재단, 연구사업명은”중견연구자지원사업”,연구과제명은”병원체감염에대한 Nod2 매개세포/조직특이적방어시스템연구”,주관기관은전남대학교산학협력단, 연구기간은 2018.03.01 ~ 2022.02.28이다.
[2] 본특허출원은 2019년 2월 26일에대한민국특허청에제출된대한민국
특허출원제 10-2019-0022670호및제 10-2019-00226기호,및 2020년 2월 3일에 대한민국특허청에제출된대한민국특허출원제 10-2020-0012763호에대하여 우선권을주장하며,상기특허출원의개시사항은본명세서에참조로서 삽입된다.
[3] 본발명은신규한락토바실러스사케이 (Lactobacillus scikei) CVL-001균주및 이의분리방법에관한것으로서,더욱상세하게는김치로부터분리하여동정한 신규한락토바실러스사케이 CVL-001균주에관한것이다.
[4] 또한본발명은락토바실러스사케이 CVL-001균주또는이의배양액을
포함하는골질환또는대사성질환개선,예방또는치료용조성물에관한 것으로서,더욱상세하게는수탁번호 KCTC13816BP로기탁된신규한
락토바실러스사케이 CVL-001균주또는이의배양액을포함하는조성물의골 질환또는대사성질환개선,예방또는치료효과에관한것이다.
배경기술
[5] 김치와같은전통발효식품에풍부하게존재하는유산균은인체의소화계에 공생하면서섬유질및복합단백질을분해하여중요한영양성분으로만드는 역할을담당하며,장내 pH를산성으로유지시켜대장균이나크로스트리디움 ( Chlostridium sp.)과같은유해균의번식을억제하고설사와변비를개선할뿐만 아니라,비타민합성,혈중콜레스테롤저하등의역할을한다.특히유산균은 장의점막과상피세포에강하게결합할수있는특성이있어정장작용에많은 도움을준다.
[6] 또한,유산균은대식세포 (macrophage)의증식을족진하여대식세포의장내 유해세균에대한인지능력,살균능력을강화시키고,면역관련물질의분비를 촉진하여면역증강효과를나타내는것으로알려져있다 (Gabriela peridgon et al. J of food Protection 53: 404-411, 1990: Katsumasa sato et al” Microbiol Immunol., 32(7): 689-698, 1998). 2020/175869 1»（：1^1{2020/002612
[7] 락토바실러스속미생물은동형또는이형발효를하는유산간균으로서 ,
유제품이나채소의발효과정에서흔히볼수있는균이다.최근락토바실러스속 미생물을생균제,식품첨가제등으로개발하고자하는연구가활발히진행되고 있다.
[8] 최근에 ,건강및질병모두를위해장내미생물 아 111 (^(^;이하
Figure imgf000004_0001
중요성이집중적으로연구되어왔다. GM은총괄적으로인간게놈보다 150배더 많은유전자를함유하는엄청난양의박테리아로구성된다.이는출생시 얻어지며,구별되는실재
Figure imgf000004_0002
임에도불구하고,분명히인간게놈과 함께공진화하고,다양한방식으로이의숙주와소통하고영향을미치는다세포 생물로간주될수있다.
[9] GM의구성은음식및항생체치료와같은다수의환경적요인에의해
조절된다.장내미생물에의해생산된분자들은유익할수도있고해로울수도 있으며,장내의내분비세포,장신경계,장투과성및면역시스템에영향을 미치는것으로알려져있다.동요된미생물구성은크론병,궤양성대장염, 류마티스성관절염,다발성경화증,당뇨,음식알레르기,습진및천식뿐만 아니라비만및대사증후군을포함하여장내외에서다양한염증조건에 연루되는것으로상정되어왔다.
[1이 갱년기의여성에게서는여러폐경증후들이나타나는데,특히에스트로겐의 감소로인해뼈에서칼슘이빠져나가뼈의질량이감소하고구멍이많아지므로 골손실의증가등으로골다공증질환의발병률이높아지게되었다.폐경기이후 후기변화는증상이나타나기까지다소시간이걸릴수있으므로문제를쉽게 자각하지못하는경우가많다.보건복지부국민건강통계치에서 30세이상에서 골다공증유병률이남성은 1%,여성은 9%로여성유병률이남성대비 9배나높게 나타난것으로보고되었다.
[11] 골다공증에의해야기되는골절은주된건강문제를구성하며건강관리
시스템상에막대한경제적부담을준다.골다공증으로인한골절의위험은 서양에서높으며 (여성에서약 50%및남성에서약 20%),골절은현저한치사율 및이환율과연관된다.피질골 (¥]1노신15011句은신체에서뼈의약 80%를 구성하며,여러연구들은피질골이뼈강도의주요결정인자이며,이에따라 골절민감성의주요결정인자임을보여주었다 . 65세이후에뼈손실은 해면질골이아니라주로피질골에서의손실에기인한다
Figure imgf000004_0003
2010, May 15; 375(9727):1729-36).
[12] 골격은뼈형성골아세포 (〔« 및뼈흡수파골세포 (00本)에의해
리모델링된다.대식세포콜로니자극인자 (MCSF)는 00^전구세포의증식및 생존을증가시킬뿐만아니라 00^에서핵인자-
Figure imgf000004_0004
발현을상향조절한다.이는 RANK리간드 (RANKL)가바인딩하고
Figure imgf000004_0005
형성을 이 11는시그널링캐스케이드를개시하도록한다. 1 ^의영향은 1 볘 ^에 대한유인리셉터인오스테오프로테게린 (0모(3)에의해저해될수있다. [13] 또한,최근빠른경제성장과식생활의서구화와함께유전적,환경적요인 등으로인한비만및당뇨등의대사성질환이증가하고있는추세이다.이를 예방및치료할수있는물질들에대한연구들이진행되고있는가운데최근 다양한기능성식품개발이요구되고있다.
[14] 비만은전세계적으로유병률이늘어나고있으며,비만으로인한대사관련 합병증으로인해비만치료에대한관심이고조되고있다.현재사용되고있는 비만치료제는포만감을유도하여식욕을억제시키거나지방의흡수를 저하시켜체중감소를유도한다.포만감은노르에피네프린혹은세로토닌의 신경접합부에서이러한신경전달물질의농도를증가시킴으로써유발되기도 하고,세로토닌수용체나아드레날린성수용제를자극하여유발되기도한다.
[15] 그러나비만치료를위해개발된약물들은효과에비해부작용이심각하므로, 항비만효과는뛰어나면서도부작용이적은소재의새로운작용기전을가진 물질의개발이절실히요구되고있다.
발명의상세한설명
기술적과제
[16] 이에본발명자들은전국에서수집된김치시료에서분리및동정을통해
신규한락토바실러스사케이 (Lactobacillus sakei)균주를분리하고,김치로부터 분리한락토바실러스사케이균주와그의배양액이파골세포분화억제, 골다공증질환개선,지방세포분화억제및체중증가억제효과를나타냄을 확인하였다.
[17] 이에,본발명의목적은수탁번호 KCTC13816BP로기탁된락토바실러스
사케이 CVL-001균주를제공하는것이다.
[18] 본발명의다른목적은수탁번호 KCTC13816BP로기탁된락토바실러스
사케이 CVL-001균주의배양액을제공하는것이다.
[19] 본발명의또다른목적은김치추출물을배양하는배양단계를포함하는, 수탁번호 KCTC13816BP로기탁된락토바실러스사케이 CVL-001균주의분리 방법을제공하는것이다.
[2이 본발명의또다른목적은골질환또는대사성질환개선,예방또는치료
활성을가지는수탁번호 KCTC13816BP로기탁된락토바실러스사케이
CVL-001균주를제공하는것이다.
[21] 본발명의또다른다른목적은골질환또는대사성질환개선,예방또는치료 활성을가지는수탁번호 KCTC13816BP로기탁된락토바실러스사케이
CVL-001균주의배양액을제공하는것이다.
[22] 본발명의또다른목적은수탁번호 KCTC13816BP로기탁된락토바실러스 사케이 CVL-001균주의배양액을포함하는골질환또는대사성질환예방또는 치료용약학적조성물을제공하는것이다.
[23] 본발명의또다른목적은수탁번호 KCTC13816BP로기탁된락토바실러스 2020/175869 1»（：1/10公020/002612 사케이 CVL-001균주의 배양액을포함하는골질환또는대사성질환개선용 식품조성물을제공하는것이다.
본발명의또다른목적은수탁번호 KCTC13816BP로기탁된락토바실러스 사케이 CVL-001균주의골질환또는대사성질환개선,예방및치료용도에 관한것이다.
과제해결수단
[25] 본발명은신규한락토바실러스사케이 (Lactobacillus sakei) CVL-001균주,이의 분리 방법 ,락토바실러스사케이 CVL-001균주또는이의 배양액을포함하는골 질환또는대사성 질환개선,예방또는치료용조성물에관한것이다.
본발명자들은김치추출물을배양하여균주를분리하고,이로부터그람 양성이고카탈라제음성인균주를선별하여 락토바실러스사케이 CVL-001 균주를분리하였고,상기균주또는그의 배양액이파골세포의분화를 억제시키고골질환을치료하는효과가있음을확인하였으며,이를
고지방식이로비만유도된마우스에 대하여 경구투여한결과,체중증가가 억제되고혈당수치가감소하는것을관찰하였다.
[27] 이하본발명을더욱자세히설명하고자한다.
[28] 본발명의 일양태는수탁번호 KCTC13816BP로기탁된락토바실러스사케이 CVL-001균주이다.
[29] 상기균주는서열번호 1로표시되는 16S rRNA서열을가지는것일수있다.
[3이 본발명의다른양태는수탁번호 KCTC13816BP로기탁된락토바실러스
사케이 CVL-001균주의 배양액이다.
[31] 상기균주는서열번호 1로표시되는 16S rRNA서열을가지는것일수있다.
본발명의또다른양태는김치추출물을배양하는배양단계를포함하는,
] ] 수탁번호 KCTC13816BP로기탁된락토바실러스사케이 CVL-001균주의분리 45
22 33324611
방법이다.
[33] 상기김치추출물은김치를마쇄하여거른김치마쇄액인것일수있고,예를 들어,상기 김치마쇄액에물을첨가하여 희석한김치액인것일수있으나,이에 한정되는것은아니다.
상기 배양단계는김치추출물을배양하는제 1배양단계 ;및상기제 1배양 단계에서분리한균주를배양하는제 2배양단계인것일수있다.
상기제 1배양단계는유산균감별을위해 0.5내지 2%의 탄산칼슘또는 0.004 내지 0.006%의브로모크레졸퍼늘 (bromocresol purple)이 첨가된배지에서 수행되는것일수있고,예를들어 ,탄산칼슘이 첨가된배지에서수행되는것일 수있으나,이에 한정되는것은아니다.
[36] 상기제 1배양단계는 20내지 32OC에서 36내지 60시간동안수행되는것일수 있고,예를들어 , 30OC에서 48시간동안수행되는것일수있으나,이에 한정되는 것은아니다.이후투명환을형성한균주를분리하여 이에 대하여제 2배양 2020/175869 PCT/KR2020/002612 단계를적용할수있다.
[37] 상기제 2배양단계는분리된균주중유산균을선별하여배양하는단계인것일 수있다.구체적으로,상기분리된균주중그람양성이고카탈라제음성인것을 유산균으로선별하여배양하는단계인것일수있다.
[38] 상기제 2배양단계는 20내지 32 에서 12내지 36시간동안수행되는것일수 있고,예를들어 , 30OC에서 24시간동안수행되는것일수있으나,이에한정되는 것은아니다.
[39] 본발명의또다른양태는골질환개선,예방또는치료활성을가지는
수탁번호 KCTC13816BP로기탁된락토바실러스사케이 CVL-001균주이다. 상기균주는김치로부터분리된것일수있으나,이에한정되는것은아니다.
[4이 본발명의또다른양태는골질환개선,예방또는치료활성을가지는
수탁번호 KCTC13816BP로기탁된락토바실러스사케이 CVL-001균주의 배양액이다.
[41] 본발명의또다른양태는수탁번호 KCTC13816BP로기탁된락토바실러스 사케이 CVL-001균주또는이의배양액을포함하는골질환예방또는치료용 약학적조성물이다.
[42] 상기골질환은골다공증 (osteoporosis),뼈전이암 (bone metastatic cancer),
골연화증 (osteomalacia),구루병,섬유성골염,무형성골질환,대사성골질환및 치주질환으로이루어진군으로부터선택되는것일수있고,예를들어, 골다공증일수있으나,이에한정되는것은아니다.
[43] 상기배양액은,락토바실러스사케이 CVL-001균주를배양하고,균주를
제거한후수득된배양상층액 ,이의농축물,이의분획물또는이의
동결건조물인것일수있다.
[44] 상기배양액은락토바실러스사케이 CVL-001균주를 12시간내지 30시간,
12시간내지 24시간, 18시간내지 30시간또는 18시간내지 24시간,예를들어 ,
22시간내지 26시간동안배양하여수득한것일수있으나,이에한정되는것은 아니다.배양시간은 24시간을기점으로하여그이상배양하는것이배양시간 의존적으로파골세포분화억제활성에대하여현저한상승효과를나타내는 것은아니다.
[45] 상기락토바실러스사케이 CVL-001균주의배양을위해서는 Alpha-MEM
배지를이용할수있다.
[46] 구체적으로, BME(Basal Media Eagle)배지는세포성장에필요한필수영양분 및무기염류와비타민류에대한연구의결과로얻어진배지로현재널리 사용되는 MEM과 DMEM의기본조성이된배지이며 , MEM(Minimum Essential Media Eagle)배지는 BME에비해서아미노산의농도가더높고 C02/NaHC03의 완충작용을위해 Earle's salts를포함하고있다.세포종류에따라서
비필수아미노산이더첨가된배지와 Earle's salts대신 Hank's salts가포함된 배지를사용하기도한다. Alpha MEM(MEM, Alpha modificiation)은아미노산, 2020/175869 1»（：1^1{2020/002612 비타민등이추가로첨가되어 있어동물세포의 DNA형질전환에유용하다.
[47] 상기조성물은배양액을 50내지 400, 50내지 350, 50내지 300, 50내지 250, 100 내지 400, 100내지 350, 100내지 300, 100내지 250, 150내지 400, 150내지 350, 150내지 300, 150내지 250, 180내지 400, 180내지 350또는 180내지 300山 , 예를들어 , 180내지 250 111/1111의농도로포함하는것일수있으나,이에 한정되는 것은아니다.
[48] 상기 배양액은 1^ 6.5내지 8.5, 6.5내지 8.0, 6.5내지 7.5, 7.0내지 8.5또는 7.0 내지 8.0,예를들어, !^ 7.0내지 7.5인것일수있으나,이에 한정되는것은 아니다.
[49] 화될
Figure imgf000008_0003
[5이 상기골질환예방및치료는파골세포분화억제를통해달성되는것일수
있으나,이에 한정되는것은아니다.
[51] 본발명의또다른양태는수탁번호 （그（：13816615로기탁된락토바실러스
Figure imgf000008_0001
배양액을포함하는대사성 질환예방또는 치료용약학적조성물이다.
[52] 상기 대사성 질환은비만,당뇨,고혈압,고지혈증,심혈관질환및
고인슐린혈증으로이루어진군으로부터선택되는 1종이상인것일수있다.
Figure imgf000008_0004
22시간내지 26시간동안배양하여수득한것일수있으나,이에 한정되는것은 아니다.배양시간은 24시간을기점으로하여그이상배양하는것이 배양시간 의존적으로체중증가억제또는혈당수치감소효과에 대하여 현저한
상승효과를나타내는것은아니
[55] 상기 배양액은,락토바실러스 양하고,균주를
제거한후수득된배양상층액 ,
Figure imgf000008_0002
또는이의
동결건조물인것일수있다.
[56] 상기 배양액은 1^ 6.5내지 8.5, 6.5내지 8.0, 6.5내지 7.5, 7.0내지 8.5또는 7.0 내지 8.0,예를들어, !^ 7.0내지 7.5인것일수있으나,이에 한정되는것은 아니다.
[57] 화될
Figure imgf000008_0005
2020/175869 1»（：1^1{2020/002612
[58] 본발명의 약학적조성물은락토바실러스사케이（：\1-001균주배양액의
약학적유효량및/또는약학적으로허용되는담체를포함하는약학적조성물로 이용될수있다.
[59] 본명세서에서용어 "약학적유효량”은상술한락토바실러
Figure imgf000009_0001
균주배양액의효능또는활성을달성하는데충분한양을의미한다.
[6이 본발명의 약학적조성물에포함되는약학적으로허용되는담체는제제시에 통상적으로이용되는것으로서 ,락토스,덱스트로스,수크로스,솔비톨,만니톨, 전분,아카시아고무,인산칼슘,알기네이트,젤라틴,규산칼슘,미세결정성 셀룰로스,폴리비닐피롤리돈,셀룰로스,물,시럽,메틸셀룰로스,
메틸히드록시벤조에이트,프로필히드록시벤조에이트,활석,스테아르산 마그네슘및미네랄오일등을포함하나,이에 한정되는것은아니다.본발명의 약학적조성물은상기성분들이외에윤활제,습윤제,감미제,향미제,유화제, 현탁제,보존제등을추가로포함할수있다.
[61] 본발명에 따른약학적조성물은인간을포함하는포유동물에다양한경로로 투여될수있다.투여 방식은통상적으로사용되는모든방식일수있으며, 예컨대,경구,피부,정맥,근육,피하등의경로로투여될수있으며,
바람직하게는경구로투여될수있다.
[62] 본발명의 약학적조성물의 적합한투여량은제제화방법 ,투여방식 ,환자의 연령,체중,성별,병적상태,음식,투여시간,투여 경로,배설속도및반응 감응성과같은요인들에 의해다양하며,보통으로숙련된의사는소망하는치료 또는예방에효과적인투여량을용이하게결정 및처방할수있다.
[63] 본발명의 약학적조성물은당해발명이속하는기술분야에서통상의지식을 가진자가용이하게실시할수있는방법에 따라,약학적으로허용되는담체 및/또는부형제를이용하여 제제화함으로써단위용량형태로제조되거나또는 다용량용기내에내입시켜 제조될수있다.이때제형은오일또는수성 매질중의용액,현탁액또는유화액형태이거나엑스제,분말제,과립제,정제, 캅셀제또는젤（예컨대,하이드로젤）형태일수도있으며,분산제또는
안정화제를추가적으로포함할수있다.
[64] 본발명의또다른양태는수탁번호 （그（：13816615로기탁된락토바실러스
Figure imgf000009_0002
배양액을포함하는골질환개선용식품 조성물이다.
[65] 상기골질환은골다공증,뼈전이암,골연화증,구루병,섬유성 골염,무형성 골질환,대사성 골질환및치주질환으로이루어진군으로부터선택되는것일수 있고,예를들어,골다공증일수있으나,이에 한정되는것은아니다.
[66] 양하고,균주를
Figure imgf000009_0003
또는이의
동결건조물인것일수있다.
Figure imgf000009_0004
2020/175869 1»（：1^1{2020/002612
12시간내지 30시간, 12시간내지 24시간, 18시간내지 36시간, 18시간내지 30시간또는 18시간내지 24시간,예를들어 , 22시간내지 26시간동안배양하여 수득한것일수있으나,이에 한정되는것은아니다.
[68] 상기조성물은배양액을 20내지 400, 20내지 350, 20내지 300, 20내지 250, 50 내지 400, 50내지 350, 50내지 300, 50내지 250, 100내지 400, 100내지 350, 100 내지 300, 100내지 250, 150내지 400, 150내지 350, 150내지 300, 150내지 250, 180내지 400, 180내지 350또는 180내지 300山/센,예를들어 , 180내지 250 111/1111의농도로포함하는것일수있으나,이에 한정되는것은아니다.
[69] 상기 배양액은 1^ 6.5내지 8.5, 6.5내지 8.0, 6.5내지 7.5, 7.0내지 8.5또는 7.0 내지 8.0,예를들어, !^ 7.0내지 7.5인것일수있으나,이에 한정되는것은 아니다.
이 상기골질환개선은파골세포분화억제를통해달성되는것일수있으나,이에 한정되는것은아니다.
1] 본발명의또다른양태는수탁번호 （그（：13816615로기탁된락토바실러스
Figure imgf000010_0001
배양액을포함하는대사성 질환개선용 건강기능식품조성물이다.
2] 상기 대사성 질환은비만,당뇨,고혈압,고지혈증,심혈관질환및
고인슐린혈증으로이루어진군으로부터선택되는 1종이상인것일수있다.
Figure imgf000010_0003
30시간또는 18시간내지 24시간,예를들어 , 22시간내지 26시간동안배양하여 수득한것일수있으나,이에 한정되는것은아니다.
5] 양하고,균주를
Figure imgf000010_0002
또는이의
동결건조물인것일수있다.
[76] 상기 배양액은 1^ 6.5내지 8.5, 6.5내지 8.0, 6.5내지 7.5, 7.0내지 8.5또는 7.0 내지 8.0,예를들어, !^ 7.0내지 7.5인것일수있으나,이에 한정되는것은 아니다.
7] 본발명의식품조성물을식품첨가물로사용할경우,상기식품조성물을
그대로첨가하거나다른식품또는식품성분과함께사용될수있고,통상적인 방법에 따라적절하게사용될수있다.일반적으로,식품또는음료의제조시에 본발명의식품조성물은원료에 대하여 15중량%이하,바람직하게는 10중량% 이하의 양으로첨가될수있다.
8] 상기식품의종류에는특별한제한은없다.상기물질을첨가할수있는식품의 2020/175869 1»（：1^1{2020/002612 예로는육류,소세지,빵,초콜릿,캔디류,스넥류,과자류,피자,라면,기타면류, 껌류,아이스크림류를포함한낙농제품,각종스프,음료수,차,드링크제 ,알콜 음료및비타민복합제등이 있으며 ,통상적인의미에서의식품을모두 포함한다.
9] 상기음료는여러가지향미제또는천연탄수화물등을추가성분으로서
함유할수있다.상술한천연탄수화물은포도당,과당과같은모노사카라이드, 말토스,슈크로스와같은디사카라이드,및덱스트린,사이클로덱스트린과같은 천연감미제나,사카린,아스파르탐과같은합성감미제등을사용할수있다. 상기천연탄수화물의비율은당업자의선택에의해적절하게결정될수있다.
[8이 상기외에본발명의식품조성물은여러가지영양제 ,비타민,전해질,풍미제 , 착색제,펙트산및그의염,알긴산및그의염,유기산,보호성콜로이드증점제,
Figure imgf000011_0001
방부제,글리세린,알콜,탄산음료에사용되는탄산화제 등을함유할수있다.그밖에본발명의식품조성물은천연과일쥬스,과일쥬스 음료및야채음료의제조를위한과육을함유할수있다.이러한성분은 독립적으로또는조합하여사용할수있다.이러한첨가제의비율또한당업자에 의해적절히선택될수있다.
발명의효과
[81] 본발명은신규한락토바실러
Figure imgf000011_0002
관한것으로서,상기균주는김치로부터분리할수있는유산균이므로,이를 효과적으로생균제또는식품첨가제로서이용할수있다.
[82] 또한,
Figure imgf000011_0003
이의배양액을포함하는골질환또는대사성질환개선,예방또는치료용 조성물에관한것으로서,김치로부터분리한락토바실러스사케이균주와그의 배양액이파골세포분화억제 ,골다공증질환개선,지방세포분화억제및체중 증가억제효과를나타내므로,이를효과적으로골다공증또는비만질환의 개선,예방또는치료에이용할수있다.
도면의간단한설명
[83] 도 는락토바실러스사케이에001（[셌 （ 111 «決리·）균주배양액의
파골세포분화억제효과를나타내는사진이다.
[84] 도내는락토바실러스사케이
Figure imgf000011_0004
균주배양액의파골세포분화억제 효과를나타내는그래프이다.
[85] 도 2는락토바실러
Figure imgf000011_0005
양액의파골세포분화유도
단백질의인산화억제효과를나타내는웨스턴블로팅결과사진이다.
[86] 도 3은락토바실러스사케이 균주배양액의파골세포분화유도
유전자의발현억제효과를나타내는그래프이다.
[87] 도 4는골다공증을유도한마우스모델에서락토바실러
Figure imgf000011_0006
균주및이의배양액으로인한골밀도증가효과를나타낸그래프이다. [88] 도 5는락토바실러스사케이 CVL-001배양액의지방세포분화에대한억제 효과를나타내는그래프이다.
[89] 도 6은고지방식이실험군에서의락토바실러스사케이 CVL-001균주에의한 체중억제효과를시간경과에따라나타낸그래프이다.
[9이 도 7은고지방식이실험군에서의락토바실러스사케이 CVL-001균주에의한 부고환지방무게감소효과를실험군에따라나타낸그래프이다.
[91] 도 8은고지방식이실험군에서의락토바실러스사케이 CVL-001균주
배양액에의한체중억제효과를시간경과에따라나타낸그래프이다.
[92] 도 9는고지방식이실험군에서의락토바실러스사케이 CVL-001균주
배양액에의한부고환지방무게감소효과를실험군에따라나타낸그래프이다.
[93] 도 W은고지방식이실험군에서의락토바실러스사케이 CVL-001균주
배양액에의한당부하검사에서의혈당강하효과를실험군에따라나타난 그래프이다.
[94] 도 11은고지방식이실험군에서의락토바실러스사케이 CVL-001균주에의한 당부하검사에서의혈당강하효과를실험군에따라나타난그래프이다.
[95] 도 12는고지방식이실험군에서의락토바실러스사케이 CVL-001균주
배양액에의한인슐린부하검사에서의혈당강하효과를실험군에따라나타낸 그래프이다.
[96] 도 13은고지방식이실험군에서의락토바실러스사케이 CVL-001균주에의한 인슐린부하검사에서의혈당강하효과를실험군에따라나타낸그래프이다. 발명의실시를위한최선의형태
[97] 본발명은수탁번호 KCTC13816BP로기탁된,골질환또는대사성질환개선, 예방또는치료활성을가지는락토바실러스사케이（Lactobacillus sakei）
CVL-001균주에관한것이다.
발명의실시를위한형태
[98] 이하,본발명을하기의실시예에의하여더욱상세히설명한다.그러나이들 실시예는본발명을예시하기위한것일뿐이며,본발명의범위가이들 실시예에의하여한정되는것은아니다.
[99] 본명세서전체에걸쳐,특정물질의농도를나타내기위하여사용되는
Figure imgf000012_0001
별도의언급이없는경우,고체/고체는（중량/중량）%,고체/액체는（중량/부피）%, 그리고액체/액체는（부피/부피）%이다.
0이
[101] 실시예 1.균주의분리
[102] 전국에서담금직후의배추김치를수집하였으며，수집된김치는 6±0.5 에서 0주부터 5주까지저장하면서유산균분리를위한김치시료로사용하였다.상기 김치 500 g을핸드블렌더（hand blender, Hanil Co, Korea）로마쇄하고,이를 멸균거즈로거른김치액에멸균수를추가하여 101배내지 107배의순차적인 배율로희석하였다.최종적으로는 107배로희석된김치액도말배지를 분리하였다.
[103] 희석한김치액을탄산칼슘 (CaC03)이 2%첨가된 MRS배지 (Difco Co.,
France)배지에도말하고, 30OC에서 48시간동안배양한다음,투명환을형성한 균주를분리하였다.상기분리된균주에대해그람염색 (Gram strain kit, BD Co., USA)과카탈라제테스트 (Biomerieux Co., France)를수행하여 ,그람양성이고, 카탈라제음성인집락을유산균으로잠정적으로확인하였다.
[104] 상기분리된유산균주들은 MRS액체배지에접종한후, 30OC에서 24시간동안 배양하여글리세롤 (glycerol)을 25%(Wv)가되게첨가하여글리세롤
스탁 (stock)을만들어 -70OC에서보관하며사용하였다.
[105]
[106] 실시예 2:유산균주의동정
[107] 최종선정된유산균락토바실러스사케이 CYL-OOKLactobacillus scikei)은 16S rRNA염기서열분석을통하여동정을수행하였다.분리균주는순수분리한후 MRS플레이트 (plate)상태로냉장택배를이용하여솔젠트 (대전)에의뢰하였고, !YFW-AGAGTTTGATCCTGGCTCAG-J)와
1492R(5，-GGTTACCTTGTTACGACTT-3，)의유니버설프라이머세트 (universal primer set)를사용하여 16S rRNA염기서열분석을수행하였다.
[108] 상기분리균주락토바실러스사케이 CVL-001£] 16S rRNA코딩염기서열로 총 l,439bp의염기서열을결정하였으며,그결과를표 1에나타내었다.
[109] [표 1]
Figure imgf000014_0001
[110] 상기수행한염기서열을기초로염기서열의상동성검사는등록된
정보 (GeneBank database)를대상으로블라스트프로그램 (Blast program, http://www.ncbi.nlm.nhi.gov) °1]의해실행하였다.그결과락토바실러스사케이 LZm (Lactobacillus sakei LZ217)와 99%의상동성을나타내어 ,락토바실러스 사케이 LZ217로최종동정하였다.
[111] 상기한결과,즉,형태학적특성,당대사능및 16S rRNA염기서열분석결과, 만니톨 (mannitol)생성능및김치액내생존율 (우점율)이우수한락토바실러스 사케이 CVL-001균주는락토바실러스사케이 LZ217으로동정되었다.
[112] 그결과분리균주를락토바실러스사케이 CVL-0()1로명명하였고,대한민국 전라북도정읍시신정동 823한국생명공학연구원생물자원센터에기탁하였다.
[113]
[114] 실시예 3:락토바실러스사케이 CVL-001균주배양액의제조
[115] 세포에처리하기위한배양액을제조하기위하여 alpha-MEM배지에
락토바실러스사케이 CVL-0()1를 24시간동안배양하고원심분리후상층액을 분리하여 pH 7.4로맞추었다.
[116] [117] 실시예 4:락토바실러스사케이 CVL-001균주배양액의파골세포분화에대한 억제평가
[118] 마우스대식세포를 12웰플레이트 (well plate)에 2xl05/웰의밀도로 24시간동안 배양하였으며소태아혈청 (fetal bovine serum;이하 FBS), 1%
페니실린-스트렙토마이신 (Penicillin-Streptomycin;이하 PS),대식세포콜로니 자극인자 (macrophage colony stimulating factor;이하 M-CSF)가첨가된배지로 교체해준후배양액 (50, 100, 200 ul/ml)을 2시간동안처리하였다.이후
RANKL(Receptor activator of nuclear factor kappa-B ligand)을 100 ng/ml처리하여 24시간동안반응시키고,위와같은방법으로 6일동안분화시켰다.
[119] 이후파골세포의세포화학적표지효소인타르타르산염저항성산성
포스파테이즈 (Tartrate resistance acid phosphatase;이하 TRAP)에발색성기질을 첨가하여핵을염색하였고핵이 3개이상으로다핵화된세포를관찰하고 이미지화하였다.
[ 12이 대식세포에 RANKL을처리하면 RANK에결합하며 TRAP양성세포로
분화하게된다. TRAP양성세포에 RANKL, TNF-oc와같은염증인자로자극하면 세포끼리융합되어다핵형 TRAP양성세포로분화된다.
[121] 도 la, lb및표 2에서확인할수있듯이,대식세포에 RANKL을처리했을때 파골세포로의분화가증가하였으며배양액처리에의해서파골세포의수가 현저히농도의존적으로감소했음을확인했다.이것으로보아락토바실러스 사케이 CVL-001배양액이파골세포로의분화를억제시킴을확인할수있었다.
[122] [표 2]
Figure imgf000015_0001
[123]
[124] 실시예 5:락토바실러스사케이 CVL-001균주배양액의파골세포분화유도 단백질발현에대한억제평가
[125] 마우스대식세포에 RANKL및배양액을처리했을때 MAPK(m加 gen-activated protein kinase)및 N '- KB신호기전을확인하고자하였다.마우스대식세포를 12 웰플레이트에 4xl05/웰의밀도로 24시간동안배양하였다. FBS, 1% PS,
M-CSF가첨가된배지로교체해준후락토바실러스사케이 CVL-001균주 배양액 (100 ul/ml)을 2시간동안처리해주었다.이후 RANKL을 100 ng/ml 처리하여 0, 5, 15및 30분동안반응시켰다.
[126] 배지를제거하고단백질용해버퍼 (Protein lysis buffer)를이용하여단백질을 추출한후정량하여, 30 ug의단백질을 SDS-PAGE겔 (gel)에서분리하였다. 단백질을멤브레인 (membrane)으로옮긴후 1차, 2차항체 (p-jnk(Cell Signaling Technology, #925 IS), p-p38(Cell Signaling Technology, 921 IS), p-ERK(santa cruz, sc-7383), Ikbalpha(Cell Signaling Technology, 9242S), p-p65(Cell Signaling Technology, 303 IS))를순차적으로처리하여키트 (BIO-RAD, ECL solution detection kit)로발현되면기계 (Chemidoc)로즉정하였다.
[127] RANKL은 RANK에결합하여 TRA的를통해서 MAPK및 NF-KB단백질
발현을증가시키고여러가지전사인자들의증가를유도해파골세포의분화를 촉진시키게된다.따라서이들기전에대해락토바실러스사케이배양액이 파골세포분화를억제시키는지확인하기위해 MAPK단백질인 ERK, JNK, p38의인산화 (phosphorylation)를웨스턴블랏 (western blotting)을통해서 확인하였다.
[128] 그결과 RANKL의처리에의하여 5분및 15분째에 MAPK단백질의인산화가 증가했고배양액처리에의해서 JNK, P38및 ERK의인산화가감소했음을 확인하였다.또한 IkB알파 (alpha)의분해는억제되었으며 p65의인산화는 억제되었다.
[129] 도 2에서확인할수있듯이,락토바실러스사케이배양액은 JNK, P38및 ERK의 인산화를억제함으로써파골세포로의분화를억제하는것으로확인되었다.
[B이
[131] 실시예 6:락토바실러스사케이배양액의파골세포분화유도유전자발현에 대한억제평가
[132] 락토바실러스사케이배양액을마우스대식세포에처리하여파골세포분화 기전관련유전자인 TRAP, DC-STAMP, Cathepsin K, NFATcl에대한유전자 발현 (gene expression)을실시간 (Real-time) PCR을통해확인하였다.골수-유래 대식세포 (bone marrow-derived macrophages;이하 BMDMs)를 12웰늘레이트에 lxlO5/웰의밀도로 24시간동안배양하였다. 1시간동안 FBS가없는 1% PS만 첨가된배지를처리해준후락토바실러스사케이배양액 100 ul/ml을 2시간동안 처리해주었다.이후 RANKL을처리하여 24시간동안반응시켰다.위와같은 방법으로 3일동안분화시킨후트리졸 (TRIzol)용액을이용하여세포내 RNA를 분리하고 RNA정량값을토대로 RT프리믹스 (premix)를이용하여 cDNA를 합성하였다.합성된 cDNA는프라이머를이용하여실시간모이을통해 증폭시켰다.
[133] 사용된프라이머는 Mouse TRAP, DC-STAMP, Cathepsin K, NFATcl이고
대조군 (Control)유전자인 GAPDH와비교하여상대적인양을비교하였다.
[134] 대식세포에 RANKL을처리하면 RANK에결합하여 TRAP양성인다핵화된 파골세포로분화가된다.또한파골세포로분화하는데중요한전사인자인 NFATcl을활성화시키고파골세포분화기전 (osteoclastogenesis)에관여하는 TRAP, Cathepsine K, DC-STAMP의발현을증가시킨다.
[135] 도 3및표 3에서확인할수있듯이, RANKL처리에의하여증가된 TRAP, 2020/175869 1»（：1^1{2020/002612
DC-STAMP, X, 쇼1^ 1의발현이 락토바실러스사케이 배양액처리에 의해서감소했음을확인했다.이것으로보아전사인자인 NFATcl의 활성을 감소시키고그로인해파골세포분화기전관련유전자인 요쇼!5, DC-STAMP,
X의발현을감소하여파골세포로의분화를억제시킴을확인할수 있었다.
[136] [표 3]
Figure imgf000017_0003
[137]
[138] 실시예 7:마우스의 난소적출수술을통한골다공증동물모델유도
[139] 마우스 0576^6암컷（7주령）을사용하여 22±2ᄋ（:및상대습도 50+10%, 12시간 명암주기로설정된환경에서플라스틱 케이지에사육하였다.마우스의 난소절제 이전에동일환경에서 약 1주정도사육조건에 적응시켰다.
[140] 졸레틸（2：01 1）과럼펀（1111고]31111）을근육주사하여마취한후난소수술부위를 제모하고소독하였다. 1 0111가량의피부를절개하고다른장기에손상이 가해지지 않도록주의하여자궁을따라난소를확인하여봉합용실로난소를 결찰한뒤 양측의 난소를모두절제하였다.난소절제후각장기를복강내로 재위치시킨뒤봉합용실로봉합하였다. 일이후부터 8주동안치료물질을 투여하였다.
[141] 유산균투여군제조:락토바실러스사케이는전배양및본배양시킨뒤 1x10^ 1 108및 1 09 0 118/1111로계산하여경구투여하였다.
[142] 배양액투여군제조:락토바실러스사케이는전배양및본배양시킨뒤 1x10^
108및 1^（^。태 로계산하였다.그리고 MRS배지에 배 희석하여 24시간 동안 30ᄋ（:로배양하였다.그다음원심분리기를이용하여균을가라앉힌뒤 배양액인상층액을수집하여
Figure imgf000017_0001
7.4로맞추어 200 경구투여하였다.
[143]
[144] 실험군은아래와같이설정하였다.
[145] 0576^6마우스; 80마리
[146] ①난소비절제 대조군（此 ）, 10마리, MRS배지（대조군）투여
[147] ②난소비절제 대조군, 마리,배양액
[148] ③난소비절제 대조군, 마리,유산균
[149] ④난소절제실험군（0¥ ）, 10마리, MR
Figure imgf000017_0002
[15이 ⑤난소절제실험군, W마리,배양액（1/4희석）투여
[151] ⑥난소절제실험군, W마리,배양액（1/2희석）투여
[152] ⑦난소절제실험군, W마리,배양액（원액）투여
[153] ⑧난소절제실험군, W마리 ,유산균 lxlO7 cells/ml투여
[154] ⑨난소절제실험군, W마리 ,유산균 lxlO8 cells/ml투여
[155] ⑩난소절제실험군, W마리 ,유산균 lxlO9 cells/ml투여
[156]
[157] 실시예 8:락토바실러스사케이유산균및그의 배양액투여에 대한골다공증 유도마우스의골밀도평가
[158] 골밀도측정은이전의단순방사선사진촬영법이나컴퓨터 단층촬영법에 비해고해상도영상자료를제공하는마이크로 CT（microcomp uted tomography）# 이용하였다.
[159] 실험종료후마우스다리를포르말린에고정시킨뒤
대구경북첨단의료산업진흥재단실험동물센터에골밀도분석을의뢰하였다.
[16이 난소절제된마우스는에스트로겐결핍으로인해골밀도가감소하는것으로 알려져 있다.따라서난소절제된마우스동물모델은골다공증질환연구에 많이 이용되고있다.
[161] 도 4및표 4에서확인할수있듯이,난소절제된마우스그룹（G4）은 Sham군에 비해유의적으로골밀도가감소하는것으로관찰되었다.한편,락토바실러스 사케이균주와그의 배양액은농도의존적으로골밀도를증가시키는것으로 나타났다.특히 배양액원액을투여한그룹（G7）과높은농도의균을투여한 그룹（G10）에서난소를절제하지 않은 Sham군의골밀도만큼회복시키는것으로 확인되었다.
[162] [표 4]
Figure imgf000018_0002
[163]
[164] 실시예 9:김치로부터분리된락토바실러스사케이（：^-001배양액을이용한 지방세포분화억제효과에 대한실험（ 년 0）
[165] 9-1.락토바실러스사케이（：^-001배양액제조
[166]
Figure imgf000018_0001
[167]
[168] 9-2.미분화된지방전구세포주 313-1그을이용한지방분화 [169] ATCC( American type culture collection, USA)에서구입한 3T3-L1 지방전구세포 (preadipocyte cell)를 DMEM배지에 10%우아혈청 (Bovine calf serum; BCS)및 1% PS를첨가하고계대배양하여유지하였다.플레이트 (plate)에 접종 (seeding)한후밀도가 100%가되었을때를 - 2 day로지정하고, 2일동안더 배양하면서배양된플레이트내의모든세포가 G1세포주기정지 (arrest)상태가 되도록하였다.
[170] 0 day부터 IBMX (3-isobutyl- 1-methylxanthine),덱사메타손 (Dexamethasone), 인슐린 (Insulin, MDI)을첨가하여분화를시작하였다 . 2 day에는인슐린만포함된 배지로교체하였다. 4 day부터는 10%소태아혈청 (Fetal bovine serum; FBS)이 들어간배지로 2일간격으로교체하여분화될때까지배양하였다.
[171]
[172] 9-3.락토바실러스사케이 CVL-001배양액의지방세포분화에대한억제평가
(Oil red O staining)
[173] 지방세포분화는배지구성성분을우아혈청 (Bovine calf serum; BCS)이아닌 FBS로교체했을때부터유도된다. 3T3-L1세포는 12웰플레이트 (well plate)에 5 X 105 cells/well의밀도로 10% FBS, 1% PS가첨가된배지와함께접종하였다. 100%의밀도로세포가자란지점에서 2일후, 0 day에배양액 (12.5, 25.0및 50.0 %)을 2시간동안처리하고지방세포분화유도물질 MDI를처리하였다.
[174] 배양액은 1 X 108 cfu/ml농도의 L. mkei를 DMEM (High glucose) + FBS (10%) + PS (1%)배지 (media)에 24시간동안배양하여제조하였고,배양액희석은 DMEM 배지를이용하여수행하였다.
[175] 2 day에는,배지를교체하면서배양액 (12.5, 25.0및 50.0 %)을 2시간동안
처리한후인슐린을처리하였다 . 4 day에는,배지를교체하면서배양액 (12.5, 25.0 및 50.0 %)을처리하였다. 2일간격으로분화될때까지동일한과정을
반복하였다.
[176] 분화된지방세포가관찰되면염색을위해 4%포르말린으로교체하여 10분 동안세포를고정시켰다. D.W로 2번세척하고 Oil red O염색시약과 D.W를 6:4로혼합하여넣은후 30분동안지방세포를염색하였다. D.W로세척후 현미경으로관찰한뒤 , 100%이소프로판올 (isopropanol)을처리하여 Oil red O을 추출하였고 5W nm의흡광도에서측정하여분화정도를정량적으로
분석하였다.
[177] 도 5및표 5에서확인할수있듯이, 3T3-L1세포성장배지에 MDI를첨가하면 지방세포로분화하게된다.본연구결과에서는 3T3-L1세포에 MDI를처리했을 때지방세포로의분화가증가하였으며배양액처리에의해서농도의존적으로 지방세포의수가현저히감소했음을확인하였다. [178] [표 5]
Figure imgf000020_0001
[179] 이로부터락토바실러스사케이 CVL-001배양액은지방세포로의분화를
억제함을확인할수있었다.
[18이
[181] 실시예 10:비만개선효과의확인을위한비만마우스동물모델의제조 (in vivo)
[182] 10-1.락토바실러스사케이 CVL-001생균제조
[ 183] 마우스에처리하기위한생균을제조하였다.구체적으로,고체배지 (MRS agar)에 24시간동안 30OC에서배양한후단일콜로니 (single colony)를채취하여 액체배지 (MRS broth)에서전배양을진행하였다.전배양은 5 ml배지 (media)에 150 rpm 30OC조건에서 24시간동안수행하였으며,이후 W배로희석하여동일한 조건에서 3시간동안본배양을진행하였다.
[184] 균수를맞추기위해흡광도 600 ran에서 0.D값 0.6이나오도록
분광광도계 (spectrophotometer)를이용하여 PBS에농도를맞췄다.이는유산균이 이조건에서 0.89xl09 CFU/ml만큼존재함을의미한다.마우스개체마다 200 ul씩 (lxl08CFU/mouse, lxl09CFU/mouse)투여할것을고려하여 3,000 RPM에 15분조건으로원심분리를실시하여생균배지 (media)를제조하였다.
[185]
[186] 10-2.락토바실러스사케이 CVL-001배양액제조
[187] 본배양이완료된락토바실러스사케이가들어있는 MRS배지를 W배희석하여 150 rpm 30OC조건에서 24시간동안배양하였다.이후 3,000 RPM에 15분 조건으로원심분리를실시하여상층액을분리하고 pH 7.4로조절하였다. 0.45 um막여과지 (membrane filter paper)로여과한후 4OC에서보관하였다.
[188]
[189] 10-3.락토바실러스사케이 CVL-001균주및배양액에대한항비만실험 (in vivo)
[19이 7주령의 C57BL/6수컷마우스에생균 (1일단회 200 ul/개체),배양액 (1일단회
200 ul/개체)및고지방식이 (High Fat diet)를투여하였다.이후 2주간격으로 체중을측정하였다. 11주째에혈당검사를실시하였고, 14주째에부검하여 혈액을채취하고장기의무게를측정하였다.
[191]
[192] 균주이용실험에이용된그룹의정보는다음과같다.
[193] G1:대조군 (Control Group,표준식이 (Normal diet)급이) PBS (phosphate buffered saline)경구투여 (n=6) 2020/175869 1»(：1^1{2020/002612
[194] 02:대조군 I」. «決리· 109경구투여知=6）
[195] 03:고지방군（ 沙므 （¾0때,고지방식이급이） ?68경구투여여=8）
[196] 04:고지방군 I «決리· 108경구투여知=8）
[197] 05:고지방군 I «決리· 109경구투여知=8）
[198]
[199] 배양액이용실험에이용된그룹의정보는다음과같다.
[200] （31:대조군（표준식이급이） MRS경구투여여=6）
[201] 02:대조군배양액（원액）경구투여知=6）
[202] 03:고지방군（고지방식이급이） MRS경구투여知=8）
[203] 04:고지방군배양액（1/2희석）경구투여知=8）
[204] 05:고지방군배양액（원액）경구투여知=8）
[205]
[206] 실시예 11:김치로부터분리된락토바실러스사케이（：^-001균주와그의
배양액을이용한효과에대한실험（111 0）
[207] 11-1.락토바실러스사케이（：^-001균주또는배양액에의한체중변화의
확인（ 1 ¥ 0）
[208] 60%의지방으로구성된고지방식이를마우스에경구투여시키면표준식이를 먹인마우스에비해체중이증가하는것으로알려져 있다.따라서고지방식이로 유도된비만마우스동물모델은비만질환연구에많이이용되고있다.
[209] 도 6에서확인할수있듯이,고지방식이를먹인그룹必3）에서표준식이를먹인 그룹（（31）에비해유의적으로체중이증가되는것으로관찰되었다.특히 고지방식이와함께락토바실러스사케이 0/ᄂ001균주를 108과 109의수로먹인 그룹 4및 05）에서 03보다유의적으로체중이감소되어있는것으로나타났다.
[21이 [표 6]
Figure imgf000021_0002
[211] 도 7및표 6에서확인할수있듯이,부고환지방무게또한고지방식이를먹인 03이표준식이그룹（31보다유의적으로증가하였고,고지방식이와함께균주 10 9을먹인그룹 05의부고환지방의무게가유의적으로감소되어 있는것을 확인하였다.도 8에서확인할수있듯이,고지방식이를먹인그룹 3）에서 표준식이를먹인그룹（（31）에비해유의적으로체중이증가되는것이
관찰되었다.
Figure imgf000021_0001
원액 5）을먹인그룹에서 03보다유의적으로체중이감소되어있는것으로 나타났다.한편배양액의 1/2를투여한그룹 4）에서는고지방식이로유도된 체중증가를억제시키지못하는것으로나타났다.
[212] [표 7]
Figure imgf000022_0002
[213] 도 9및표 7에서확인할수있듯이,부고환지방무게또한고지방식이를먹인 G3이표준식이그룹이보다유의적으로증가되었고,고지방식이와함께배양액 원액을먹인그룹 G5의부고환지방의무게가유의적으로감소되어 있는것을 확인하였다.
[214]
[215] 11-2.락토바실러스사케이 CVL-001균주와배양액투여에대한비만유도 마우스의공복시혈당평가 (Fasting Blood Sugar Test)
[216] 마우스는실험전날오후 9시부터다음날 9시까지 12시간동안공복유지후 혈당검사를수행하였다.고지방식이를먹인마우스동물모델은공복시혈당이 매우높게유지되는것으로알려져 있다.실험군별조건은상기 2-3과같은 조건으로진행하였으며,균주를투여한군과배양액을투여한군을각각표 8및 9로나타내었다.
[217] [표 8]
Figure imgf000022_0003
[218] 표 8에서확인할수있듯이,본실시예에서고지방식이를먹인 03그룹은
표준식이를먹인그룹 (31에비해공복시혈당수치가상당히높게유지되어 있는것으로관찰되었다.특히고지방식이와함께락토바실러스사케이
Figure imgf000022_0001
농도를각각다르게투여한 04와 05그룹에서 03보다
혈당수치가감소되어표준식이먹이그룹 (31과비슷한수준인것으로
관찰되었다. [219] [표 9]
Figure imgf000023_0001
[22이 표 9에서확인할수있듯이,본실시예에서고지방식이를먹인 G3그룹은 표준식이를먹인그룹이에비해공복시혈당수치가상당히높게유지되어 있는것으로관찰되었다.특히고지방식이와함께락토바실러스사케이 CVL-001배양액원액을투여한 G5그룹에서 G3보다혈당수치가감소되는 경향을보여표준식이를먹인그룹 G1과비슷한수준인것으로관찰되었다.
[221]
[222] 11-3.락토바실러스사케이 CVL-001균주와배양액투여에대한비만유도 마우스의당부하검사 (Glucose Tolerance Test)
[223] 실험군별조건은상기 W-3과같은조건으로진행하되,마우스는실험전날 오후 9시부터다음날 9시까지 12시간동안공복유지후다음검사를 수행하였다. 0.2 um여과장치 (filteration)로멸균시킨 10%글루코스 (Glucose)를 함유하는 PBS용액을준비하여각개체마다 2 mg(glucose/g. volume(ul)= body weight(g) x 20)씩복강투여 (27_gauge sterile needle)하였다.마우스를케이지에 다시넣고혈당수치를 0, 30, 60, 90및 120분간격으로측정하였다.혈당검사는 꼬리에서채혈하여수행하여표 10,표 11,도 W및도 11로나타내었다.
[224] [표 1이
[225] 표 및도 에서확인할수있듯이,고지방식이를먹인그룹 3)에서 표준식이를먹인그룹 ((31)에비해유의적으로혈당이높게유지되는것이 관찰되었다.
Figure imgf000024_0001
원액 5)을먹인그룹에서 03보다 30분에서유의적으로혈당이감소되어있는 것으로나타났다.한편배양액의 1/2를투여한그룹必4)에서는고지방식이로 유도된혈당증가를억제시키지못하는것으로나타났다.
[226] [표 11]
Figure imgf000024_0002
[227] 표 11및도 11에서확인할수있듯이,고지방식이를먹인그룹 (G3)에서
표준식이를먹인그룹 (G1)에비해유의적으로혈당이높게유지되는것이 관찰되었다.특히고지방식이와함께락토바실러스사케이 CVL-001균주를 108 과 109의수로먹인그룹 (G4및 G5)에서 G3보다혈당이감소되어있는것으로 나타났지만유의적인차이는보이지않았다.
[228]
[229] 11-4.락토바실러스사케이 CVL-001균주와배양액투여에대한비만유도
마우스의인슐린부하검사 (Insulin tolerance Test)
[23이 실험군별조건은상기 W-3과같은조건으로진행하되,마우스는실험당일날 오전 9시부터오후 1시까지 4시간동안공복유지후다음검사를수행하였다. 2 mg/ml인슐린 (HC1을희석하여 pH 3으로맞춘 D.W용액)을준비하여각 개체마다 1 U(0.03846 mg/kg. Volume(ul)= Body weight(g) x 20)씩복강
투여 (27-gauge sterile needle)하였다.마우스를케이지에다시넣고혈당수치를 0, 30, 60, 90및 120분간격으로측정하였다.혈당검사는꼬리에서채혈하여 수행하였다. [231] [표 12]
Figure imgf000025_0002
[232] 표 12및도 12에서확인할수있듯이,고지방식이를먹인그룹 3）에서
표준식이를먹인그룹（（31）에비해유의적으로혈당이높게유지되는것이 관찰되었다.
Figure imgf000025_0001
원액 5）을먹인그룹에서 03보다 90분및 120분에서유의적으로혈당이 감소되어 있는것으로나타났다.한편배양액의 1/2를투여한그룹必4）에서는 03 보다 120분에서유의적으로혈당이감소되어 있는것으로나타났다.
[233] [표 13]
Figure imgf000025_0003
[234] 표 13및도 13에서확인할수있듯이,고지방식이를먹인그룹 3）에서
표준식이를먹인그룹（（31）에비해유의적으로혈당이높게유지되는것이 관찰되었다.특히고지방식이와함께락토바실러스사케이 균주를 108 과 109£]수로먹인그룹 4및 05）에서 03보다혈당이 0분을제외한모든시간 2020/175869 PCT/KR2020/002612 대에서유의적으로감소되어 있는것으로나타났다.
산업상이용가능성
[235] 본발명은신규한락토바실러스사케이 (Lactobacillus scikei) CVL-001균주및 이의분리방법에관한것으로서,더욱상세하게는김치로부터분리하여동정한 신규한락토바실러스사케이 CVL-001균주에관한것이다.
[236] 또한,본발명은락토바실러스사케이 CVL-001균주또는이의배양액을
포함하는골질환개선,예방또는치료용조성물에관한것으로서,더욱 상세하게는수탁번호 KCTC13816BP로기탁된신규한락토바실러스사케이 CVL-001균주또는이의배양액을포함하는조성물의골질환개선,예방또는 치료효과에관한것이다.
2020/175869 1»(：1/10公020/002612
[237]
1
국제용 양식
원기탁에 관한수령
규정 7. 1에 따른 발행 수신: 전남대학교산학협력단
전남내학교
77, 용봉로， 북구, 광주 （우） 61186
대한민국
Figure imgf000027_0001
11)1八 1 1^/4 00：] 的ä 17 ) 단일 페이지

Claims

2020/175869 1»（：1/10公020/002612 청구범위
[청구항 1] 수탁번호 KCTC13816BP로기탁된락토바실러스사케이 (Lactobacillus sakei) CVL-001균주.
[청구항 2] 제 1항에 있어서,상기균주는서열번호 1로표시되는 16S rRNA서열을 가지는것인,락토바실러스사케이 CVL-001균주.
[청구항 3] 수탁번호 KCTC13816BP로기탁된락토바실러스사케이 (Lactobacillus sakei) CVL-001균주의배양액.
[청구항 4] 제 3항에 있어서,상기균주는서열번호 1로표시되는 16S rRNA서열을 가지는것인,락토바실러스사케이 CVL-001균주의배양액.
[청구항 5] 김치추출물을배양하는배양단계를포함하는,수탁번호 KCTC13816BP 로기탁된락토바실러스사케이 (Lactobacillus sakei) CVL-001균주의분리 방법.
[청구항 6] 제 5항에 있어서,상기김치추출물은김치를마쇄하여거른김치
마쇄액인것인,락토바실러스사케이 CVL-001균주의분리방법 .
[청구항 7] 제 5항에 있어서 ,상기배양단계는김치추출물을배양하는제 1배양 단계 ;및상기제 1배양단계에서분리한균주를배양하는제 2배양 단계인것인,락토바실러스사케이 CVL-001균주의분리방법.
[청구항 8] 제 7항에 있어서 ,상기제 1배양단계는탄산칼슘이첨가된배지에서
수행되는것인,락토바실러스사케이 CVL-001균주의분리방법.
[청구항 9] 제 7항에 있어서 ,상기제 1배양단계는 20내지 32OC에서 36내지 60시간 동안수행되는것인,락토바실러스사케이 CVL-001균주의분리방법.
[청구항 10] 제 7항에 있어서,상기제 2배양단계는분리된균주중그람양성인것을 선별하여배양하는단계인것인,락토바실러스사케이 CVL-001균주의 분리방법.
[청구항 11] 제 7항에 있어서,상기제 2배양단계는분리된균주중카탈라제음성인 것을선별하여배양하는단계인것인,락토바실러스사케이 CVL-001 균주의분리방법.
[청구항 12] 제 7항에 있어서 ,상기제 2배양단계는 20내지 32 에서 12내지 36시간 동안수행되는것인,락토바실러스사케이 CVL-001균주의분리방법.
[청구항 13] 수탁번호 KCTC13816BP로기탁된락토바실러스사케이 (Lactobacillus sakei) CVL-001균주또는이의배양액을포함하는골질환또는대사성 질환예방또는치료용약학적조성물.
[청구항 14] 제 D항에 있어서,상기골질환은골다공증 (osteoporosis),뼈전이암 (bone metastatic cancer),골연화증 (osteomalacia),구루병,섬유성골염,무형성 골질환,대사성골질환및치주질환으로이루어진군으로부터선택되는 것인,약학적조성물.
[청구항 15] 제 13항에 있어서,상기대사성질환은비만,당뇨,고혈압,고지혈증, 2020/175869 PCT/KR2020/002612 심혈관질환및고인슐린혈증으로이루어진군으로부터선택되는 1종 이상인것인,약학적조성물.
［청구항 16］ 제 13항에 있어서 ,상기배양액은,락토바실러스사케이 CVL-001균주를 배양하고,균주를제거한후수득된배양상층액 ,이의농축물,이의 분획물또는이의동결건조물인것인,약학적조성물.
［청구항 17］ 제 13항에 있어서 ,상기약학적조성물은락토바실러스사케이 CVL-001 균주를 5 X 105내지 5 X 1012 CFU/ml의농도로포함하는것인,약학적 조성물.
［청구항 18］ 제 13항에 있어서,상기배양액은락토바실러스사케이 CVL-001균주를
12시간내지 30시간동안배양하여수득한것인,약학적조성물.
［청구항 19］ 제 13항에 있어서 ,상기조성물은배양액을 50내지 400 ul/ml의농도로
포함하는것인,약학적조성물.
［청구항 2이 제 13항에 있어서 ,상기배양액은 pH 6.5내지 8.5인것인,약학적조성물.
［청구항 21］ 수탁번호 KCTC13816BP로기탁된락토바실러스사케이 CVL-001(
Lactobacillus sakei)균주또는이의배양액을포함하는골질환또는 대사성질환개선용식품조성물.
［청구항 22］ 제 21항에 있어서 ,상기골질환은골다공증 (osteoporosis),뼈전이암 (bone metastatic cancer),골연화증 (osteomalacia),구루병,섬유성골염,무형성 골질환,대사성골질환및치주질환으로이루어진군으로부터선택되는 것인,식품조성물.
［청구항 23］ 제 21항에 있어서,상기대사성질환은비만,당뇨,고혈압,고지혈증,
심혈관질환및고인슐린혈증으로이루어진군으로부터선택되는 1종 이상인것인,식품조성물.
［청구항 24］ 제 21항에 있어서 ,상기배양액은,락토바실러스사케이 CVL-001균주를 배양하고,균주를제거한후수득된배양상층액 ,이의농축물,이의 분획물또는이의동결건조물인것인,식품조성물.
［청구항 25］ 제 21항에 있어서 ,상기배양액은락토바실러스사케이 CVL-001균주를
12시간내지 30시간동안배양하여수득한것인,식품조성물.
［청구항 26］ 제 21항에 있어서 ,상기식품조성물은락토바실러스사케이 CVL-001 균주를 5 X 105내지 5 X 1012 CFU/ml의농도로포함하는것인,식품조성물.
［청구항 27］ 제 21항에 있어서 ,상기조성물은배양액을 50내지 400 ul/ml의농도로
포함하는것인,식품조성물.
［청구항 28］ 제 21항에 있어서 ,상기배양액은 pH 6.5내지 8.5인것인,식품조성물.
PCT/KR2020/002612 2019-02-26 2020-02-24 신규한 락토바실러스 사케이 cvl-001 균주 또는 이의 배양액을 포함하는 골 질환 또는 대사성 질환 개선, 예방 또는 치료용 조성물 Ceased WO2020175869A2 (ko)

Priority Applications (5)

Application Number Priority Date Filing Date Title
JP2021549784A JP2022522151A (ja) 2019-02-26 2020-02-24 新規なラクトバチルスサケイｃｖｌ－００１菌株又はその培養液を含む骨疾患又は代謝性疾患改善、予防又は治療用組成物
US17/434,080 US20220135935A1 (en) 2019-02-26 2020-02-24 Composition comprising lactobacillus sakei cvl-001 or culture liquid thereof for alleviating, preventing, or treating bone diseases or metabolic diseases
CN202080016504.6A CN113840908A (zh) 2019-02-26 2020-02-24 含新型沙克乳酸杆菌cvl-001菌株或其培养液的用于改善、预防或治疗骨病或代谢性疾病的组合物
EP20762101.2A EP3932416A4 (en) 2019-02-26 2020-02-24 COMPOSITION FOR IMPROVING, PREVENTING OR TREATING BONE DISEASES OR METABOLIC DISEASES, COMPRISING A NEW STRAIN LACTOBACILLUS SAKEI CVL-001 AND CULTURE MEDIUM THEREOF
SG11202109231WA SG11202109231WA (en) 2019-02-26 2020-02-24 Composition comprising lactobacillus sakei cvl-001 or culture liquid thereof for alleviating, preventing, or treating bone diseases or metabolic diseases

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
KR10-2019-0022671 2019-02-26
KR1020190022671A KR102242668B1 (ko) 2019-02-26 2019-02-26 락토바실러스 사케이 cvl-001 균주 또는 이의 배양액을 포함하는 골 질환 개선, 예방 또는 치료용 조성물
KR10-2019-0022670 2019-02-26
KR20190022670 2019-02-26
KR1020200012763A KR102276576B1 (ko) 2020-02-03 2020-02-03 락토바실러스 사케이 cvl-001 균주 또는 이의 배양액을 포함하는 대사성 질환 예방 또는 치료용 조성물
KR10-2020-0012763 2020-02-03

Publications (2)

Publication Number Publication Date
WO2020175869A2 true WO2020175869A2 (ko) 2020-09-03
WO2020175869A3 WO2020175869A3 (ko) 2020-10-22

Family

ID=72239777

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2020/002612 Ceased WO2020175869A2 (ko) 2019-02-26 2020-02-24 신규한 락토바실러스 사케이 cvl-001 균주 또는 이의 배양액을 포함하는 골 질환 또는 대사성 질환 개선, 예방 또는 치료용 조성물

Country Status (6)

Country Link
US (1) US20220135935A1 (ko)
EP (1) EP3932416A4 (ko)
JP (1) JP2022522151A (ko)
CN (1) CN113840908A (ko)
SG (1) SG11202109231WA (ko)
WO (1) WO2020175869A2 (ko)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022223005A1 (zh) * 2019-06-25 2022-10-27 生合生物科技股份有限公司 使用植物乳杆菌twk10来促进骨愈合的方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114525233B (zh) * 2022-04-22 2022-07-22 中国疾病预防控制中心传染病预防控制所 清酒乳杆菌和水苏糖组合物在制备便秘治疗药物中的应用
KR102752054B1 (ko) * 2024-06-25 2025-01-10 전남대학교 산학협력단 락토바실러스 사케이 cvl-001 균주를 포함하는 점막염 예방, 치료 또는 개선용 조성물

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20190022671A (ko) 2016-06-21 2019-03-06 코닝 인코포레이티드 드로우 바닥부에서의 장력 조절을 갖는 유리 제조 장치를 위한 다중-상승 구동 장치
KR20190022670A (ko) 2016-06-23 2019-03-06 아레보 에이비 제올라이트와 염기성 l-아미노산을 함유하는 비료 조성물
KR20200012763A (ko) 2018-07-27 2020-02-05 가부시키가이샤 코쿠사이 엘렉트릭 기판 처리 장치, 반도체 장치의 제조 방법, 및 기록매체

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4580542B2 (ja) * 2000-05-17 2010-11-17 株式會社バイオニア 肥満又は糖尿病治療用微生物及びその微生物を含む医薬組成物
TWI346554B (en) * 2008-04-30 2011-08-11 Genmont Biotech Inc Lactobacillus isolates having anti-inflammatory activities and uses of the same
KR101005747B1 (ko) * 2008-06-24 2011-01-06 목포대학교산학협력단 김치로부터 분리된 유산균 및 그의 용도
KR20120100608A (ko) * 2011-03-04 2012-09-12 주식회사 알엔에이 면역관련 및 염증성 질환의 예방 및 치료 활성을 갖는 신규한 락토바실러스 사케이(Ｌａｃｔｏｂａｃｉｌｌｕｓ ｓａｋｅｉ) Ｋ101
KR101575867B1 (ko) * 2013-12-18 2015-12-09 한국식품연구원 유리 칼슘 함량이 향상된 곡물 조성물 제조방법
KR101740583B1 (ko) * 2014-02-17 2017-05-29 경희대학교 산학협력단 비만 억제 효능을 갖는 신규 유산균 및 이의 용도
KR20160057855A (ko) * 2014-11-14 2016-05-24 대상에프앤에프 주식회사 김치에서 분리한 항진균 활성을 갖는 유산균 및 이를 포함하는 조성물
KR101708173B1 (ko) * 2015-03-06 2017-02-21 경희대학교 산학협력단 혈당 조절 효능을 갖는 신규 유산균 및 이의 용도
CN105176856A (zh) * 2015-05-19 2015-12-23 浙江工商大学 一种清酒乳杆菌lz217筛选和培养方法及其应用
KR101736033B1 (ko) * 2016-05-09 2017-05-17 한국식품연구원 항비만 활성을 가지는 락토바실러스 사케아이
JP6684966B2 (ja) * 2016-09-30 2020-04-22 シージェイ チェイルジェダン コーポレイション 新規なラクトバチルス・サケイ及びそれを含む組成物

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20190022671A (ko) 2016-06-21 2019-03-06 코닝 인코포레이티드 드로우 바닥부에서의 장력 조절을 갖는 유리 제조 장치를 위한 다중-상승 구동 장치
KR20190022670A (ko) 2016-06-23 2019-03-06 아레보 에이비 제올라이트와 염기성 l-아미노산을 함유하는 비료 조성물
KR20200012763A (ko) 2018-07-27 2020-02-05 가부시키가이샤 코쿠사이 엘렉트릭 기판 처리 장치, 반도체 장치의 제조 방법, 및 기록매체

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GABRIELA PERIDGON ET AL., J OF FOOD PROTECTION, vol. 53, 1990, pages 404 - 411
KATSUMASA SATO ET AL., MICROBIOL IMMUNOL., vol. 32, no. 7, 1998, pages 689 - 698
LANCET, vol. 375, no. 9727, 2010, pages 1729 - 36
See also references of EP3932416A4

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022223005A1 (zh) * 2019-06-25 2022-10-27 生合生物科技股份有限公司 使用植物乳杆菌twk10来促进骨愈合的方法

Also Published As

Publication number Publication date
EP3932416A2 (en) 2022-01-05
CN113840908A (zh) 2021-12-24
EP3932416A4 (en) 2022-12-28
US20220135935A1 (en) 2022-05-05
JP2022522151A (ja) 2022-04-14
SG11202109231WA (en) 2021-09-29
WO2020175869A3 (ko) 2020-10-22

Similar Documents

Publication Publication Date Title
KR101494279B1 (ko) 지방세포 분화 유도에 관여하는 유전자의 발현을 억제함으로써 지방세포 분화 억제 효능을 갖는 락토바실러스 플란타룸 케이와이1032 및 이를 유효성분으로 함유하는 제품
US11273189B2 (en) Lactobacillus plantarum TCI378 and its uses in losing fat and improving gastrointestinal functions
TW200944215A (en) Lactobacillus isolates having anti-inflammatory activities and uses of the same
KR102242668B1 (ko) 락토바실러스 사케이 cvl-001 균주 또는 이의 배양액을 포함하는 골 질환 개선, 예방 또는 치료용 조성물
TW201043151A (en) Agent for preventing or improving obesity
WO2020175869A2 (ko) 신규한 락토바실러스 사케이 cvl-001 균주 또는 이의 배양액을 포함하는 골 질환 또는 대사성 질환 개선, 예방 또는 치료용 조성물
KR102323783B1 (ko) 클로스트리디움 디피실레(Clostridium difficile) 성장 억제효과를 갖는 클로스트리디움 신덴스 균주
JP2021527414A (ja) ロイコノストック属菌株を含む腸機能改善用組成物
KR102486028B1 (ko) 락토바실러스 사케이 cvl001 균주 배양액 유래 세포외소포체를 포함하는 골 질환 예방, 치료 또는 개선용 조성물
KR102495246B1 (ko) 체지방 감소 활성을 갖는 락토바실러스 헬베티쿠스 bcc-lh-04 및 이를 포함하는 조성물
KR20220026201A (ko) 락토바실러스 퍼멘텀을 포함하는 갱년기 예방 또는 치료용 조성물
KR102190239B1 (ko) 신규한 락토바실러스 플란타룸 Lb41 균주 및 이를 포함하는 비만의 예방 및 치료용 약학적 조성물
KR101266328B1 (ko) 지방세포 분화 유도에 관여하는 유전자의 발현을 억제함으로써 지방세포 분화 억제 효능을 갖는 락토바실러스 가세리 에이치와이7021 및 이를 유효성분으로 함유하는 제품
KR102916706B1 (ko) 파골세포분화 억제 효과를 갖는 비피도박테리움 락티스 kl101 균주 및 그의 골질환 예방 및 치료 용도
KR100865075B1 (ko) 장내 정착성이 우수한 신규 락토바실러스속 ｓｍ1 균주 및이들이 생산하는 생균제
KR20190102498A (ko) 질염 원인균에 대한 증식억제활성을 갖는 락토바실러스 속 균주 조합 및 이를 유효성분으로 함유하는 제품
KR20180040894A (ko) 신규한 락토바실러스 플란타룸 Lb41 균주 및 이를 포함하는 비만의 예방 및 치료용 약학적 조성물
KR20190122187A (ko) 신규한 락토바실러스 플란타룸 Lb41 균주 및 이를 포함하는 비만의 예방 및 치료용 약학적 조성물
KR101809616B1 (ko) 항비만효과를 가지는 케피어 유래 프로바이오틱 균주
KR102144838B1 (ko) 락토바실러스 쿠르바투스 WiKim38 균주 또는 이의 배양액을 포함하는 골 질환 개선, 예방 또는 치료용 조성물
KR20240086744A (ko) 엔테로코커스 패시움 lcm001 균주를 포함하는 생리전증후군 완화 또는 치료용 약학적 조성물
RU2782361C1 (ru) Композиция для облегчения, предупреждения или лечения заболеваний костей или метаболических заболеваний, содержащая новый штамм lactobacillus sakei cvl-001 и ее культуральную среду
KR102916582B1 (ko) 파골세포분화 억제 효과를 갖는 레빌락토바실러스 브레비스 kl251 균주 및 그의 골질환 예방 및 치료 용도
JP7408070B2 (ja) ビフィドバクテリウム・ビフィダムを含む免疫力増強または改善用組成物
KR101535077B1 (ko) 인슐린 저항성 개선 효능을 가지는 비피도박테리움 락티스 hy8101 및 이를 유효성분으로 함유하는 제품

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20762101

Country of ref document: EP

Kind code of ref document: A2

ENP Entry into the national phase

Ref document number: 2021549784

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020762101

Country of ref document: EP

Effective date: 20210927