WO2020091179A9 - Composition for preventing or treating secondary osteoporosis comprising probiotics as active ingredient - Google Patents

Composition for preventing or treating secondary osteoporosis comprising probiotics as active ingredient Download PDF

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Publication number
WO2020091179A9
WO2020091179A9 PCT/KR2019/007317 KR2019007317W WO2020091179A9 WO 2020091179 A9 WO2020091179 A9 WO 2020091179A9 KR 2019007317 W KR2019007317 W KR 2019007317W WO 2020091179 A9 WO2020091179 A9 WO 2020091179A9
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Prior art keywords
osteoporosis
present
strain
composition
culture
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PCT/KR2019/007317
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French (fr)
Korean (ko)
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WO2020091179A2 (en
WO2020091179A3 (en
Inventor
이인옥
박나현
김병국
이철상
김세헌
최인석
Original Assignee
주식회사 종근당바이오
고려대학교 산학협력단
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Publication of WO2020091179A2 publication Critical patent/WO2020091179A2/en
Publication of WO2020091179A3 publication Critical patent/WO2020091179A3/en
Publication of WO2020091179A9 publication Critical patent/WO2020091179A9/en

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    • 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
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • 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
    • 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
    • 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
    • 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
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/306—Foods, ingredients or supplements having a functional effect on health having an effect on bone mass, e.g. osteoporosis prevention
    • 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
    • A23V2400/143—Fermentum
    • 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

  • composition for the prevention or treatment of secondary osteoporosis containing probiotics as an active ingredient
  • the present invention was accomplished by PET117051-3 under the support of the Ministry of Food, Agriculture, Forestry and Food of the Republic of Korea.
  • the present invention relates to a composition for preventing or treating secondary osteoporosis containing probiotics as an active ingredient.
  • Osteoporosis is primary osteoporosis (primary, primary) in which bone mass decreases due to normal physiological changes such as age and menopause, and secondary osteoporosis (secondary, secondary) caused by exposure to diseases or drugs regardless of age.
  • primary osteoporosis primary, primary
  • secondary osteoporosis secondary, secondary
  • Glucocorticoid has anti-inflammatory and immunosuppressive properties, so it is commonly used in the treatment of various diseases such as rheumatoid arthritis, asthma, atopic dermatitis, and arthritis. It is a drug. Long-term administration of GC effectively suppresses these diseases, but in most patients, it inhibits the normal activity of osteoblasts and osteoclasts, leading to osteoporosis.
  • Statistical findings in patients receiving GC, 12% of bone density within one year It has been reported that the bone density is gradually decreased by 2 to 5% every year due to a rapid decrease in bone loss and thereafter.
  • the purpose of the present invention is to provide the Lactobacillus plantarum A41 strain with accession number KCTC13686BP.
  • Another purpose of the present invention is the above lactobacillus
  • Another object of the present invention is the above Lactobacillus plantarum (LactoZ?
  • Another object of the present invention is the above Lactobacillus plantarum (LactoZ?
  • the present invention is the accession number KCTC13686BP
  • Lactobacillus plantarum (LactoZ? i plantarum) A41
  • the strain (accession number: KCTC13686BP) was newly isolated.
  • the present inventors described the Lactobacillus plantarum) A41 strain (accession number:
  • KCTC13686BP was used to prepare fermented milk. 2020/091179 1»(:1 ⁇ 1 ⁇ 2019/007317
  • the fermented milk was administered with the above fermented milk. It was confirmed that the expression level of calcium absorption-related proteins expressed in the intestine of the experimental animal model was increased, the calcium concentration in the blood was increased, and the expression level of genes related to the inhibition of osteoclast differentiation and promotion of osteoblast differentiation was confirmed.
  • osteoporosis refers to a reduction in bone mass and microstructure.
  • Bone strength refers to bone mass (quantity)
  • Bone mass is mainly expressed by bone density (BMD), and bone quality is composed of structure, bone replacement rate, mineralization, micro-damage accumulation, etc.
  • BMD bone density
  • osteoporosis is diagnosed by measuring bone density.
  • Osteoporosis is classified into primary and secondary osteoporosis.
  • Primary osteoporosis or primary osteoporosis is classified as type 1 osteoporosis due to menopause and type 2 osteoporosis due to aging for convenience, but since it is combined and progressed at about the same time, it should be accurately classified.
  • Secondary osteoporosis refers to an increase in the risk of fracture due to changes in bone microstructure and decrease in bone mass due to underlying diseases or drugs. Endocrine diseases (thyroidism, diabetes, hyperparathyroidism, etc.) as a cause of secondary osteoporosis.
  • Gastrointestinal diseases gastric resection line, chronic liver disease, malabsorption disorder syndrome, inflammatory growth disease, alcohol addiction, etc.), bone marrow disease, connective tissue disease (ankylosing spondylitis, rheumatoid arthritis, bone dysplasia, etc.), and drugs.
  • the most common causes are hypogonadism and administration of glucocorticoids.
  • the osteoporosis is secondary osteoporosis
  • glucocorticoid drug is, but is not limited to, cortisol, prednisone, prednisolone, methylprednisolone, cortisone, hydroxycortisone, triamcinolone, betamethasone, or dexamethasone.
  • the Lactobacillus plantarum (1 ⁇ 3 ⁇ 4 ⁇ 0& ⁇ 3 ⁇ 4 ⁇ 7/ 1 time 11111) Show 41 strain is "Korea Research Institute of Bioscience and Biotechnology Microbial Resource Center (Korean Collection for Type Culture, KCTC) It has been deposited under the "accession number"KCTC13686BP".
  • [28]-It is part of the normal flora of the human mouth, stomach, and feminine organs, and is generally considered safe for use in food (probiotics and fermented foods).
  • L. plantarum is a member of the genus Lactobacillus, is generally found in many fermented foods and substances of anaerobic plants, and is also present in saliva.
  • the present invention is the accession number
  • It provides a pharmaceutical composition for the prevention or treatment of osteoporosis comprising the KCTC13686BP, Lac plantarum A41 strain, or a culture thereof as an active ingredient.
  • the present invention is the accession number KCTC13686BP
  • Lactobacillus plantarum plantarum Provides a food composition for preventing or improving osteoporosis comprising the A41 strain, or a culture thereof, as an active ingredient.
  • the composition is an active ingredient, Lactobacillus fermentum, Lactobacillus casei,
  • Lactobacillus acidophilus Lactobacillus acidophilus
  • Lactococcus lactis Lactococcus lactis), Enterococcus faecium, Enterococcus fecalis, Streptococcus thermophilus ( « Sireptococci thermophilus), Bifidobacterium bifidum, and Bifidobacterium bifidum. It further comprises one or more bacteria selected from the group consisting of Bifidobacterium lactis, or a culture thereof.
  • the Lactobacillus Fermentum may be the Lactobacillus Fermentum SRK414 strain (accession number KCTC13687BP).
  • KCTC has been deposited as a deposit number for'KCTC13687BP'.
  • [39]-It is part of the normal flora of the human mouth, stomach, and feminine organs, and is generally considered safe for use in food (probiotics and fermented foods).
  • L. fermentum is a member of the genus Lactobacillus, and the species belonging to this genus have various uses.
  • the above strain, or a culture thereof, which is an active ingredient of the compositions of the present invention is a culture containing cells in addition to the cells isolated and/or purified from the above strains, extracts of cells, and cultures 2020/091179 1»(:1 ⁇ 1 ⁇ 2019/007317 Supernatant, these concentrates, concentrates, dry matters, as well as diluents, dilutions, etc., if necessary, including culture solutions and all conditions obtained by processing the cultures do.
  • the medium for cultivating the cells usually contains milk proteins such as skim milk, whey and casein, sugars, yeast extract, etc.
  • milk proteins such as skim milk, whey and casein, sugars, yeast extract, etc.
  • various general aerobic or anaerobic methods can be appropriately used.
  • a neutralization culture method in which, for example, 35 45 O C is set as the culture temperature, and during culture, an alkali such as sodium hydroxide is used to maintain the pH of the medium from neutral to acidic, for example, about 5-6.
  • an alkali such as sodium hydroxide
  • any culture method such as batch culture can be used, and after culture, the culture or its supernatant can be concentrated, dried, or diluted if necessary.
  • centrifugal separation or membrane separation may be used to separate a fluid [45] in addition, centrifugal separation or membrane separation.
  • the cells can be separated and recovered in a concentrated state.
  • the cells can be subjected to ultrasonic treatment or enzyme treatment to extract the components in the cells, or to dry the culture or its supernatant, the cells or their extracts. It can be used as an active ingredient in a cosmetic composition.
  • composition of the present invention is prepared as a food composition, the above
  • lactic acid bacteria In addition to lactic acid bacteria, it may contain ingredients that are normally added in food manufacturing.
  • the additive ingredients include, for example, proteins, carbohydrates, fats, nutrients, flavoring agents and flavoring agents.
  • the carbohydrates include monosaccharides (for example, monosaccharides). Glucose, fructose, etc.), disaccharides (e.g. maltose, sucrose, oligosaccharide, etc.), and
  • Polysaccharides for example, regular sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • Natural flavoring agents taumatin, stevia extracts (for example, rebaudioside A, glysir) as flavoring agents) Hijin, etc.
  • synthetic flavoring agents sacharin, aspartame, etc.
  • the food composition of the present invention is manufactured as a drink link agent, citric acid, liquid fructose, sugar, glucose, acetic acid, apple acid, fruit juice, jujube extract, licorice extract, etc. Can be included.
  • the food composition of the present invention is food, functional food,
  • Food compositions of this type are in accordance with conventional methods known in the art. It can be manufactured in various forms.
  • the lactic acid bacteria itself can be prepared and consumed in the form of tea, juice, and drink, or it can be ingested by granulating, encapsulating and powdering.
  • beverages including alcoholic beverages
  • fruits and processed foods thereof eg:
  • Canned fruit, bottled, jam, marmalade, etc. fish, meat and processed foods (eg ham, sausage corn beef, etc.), bread and noodles (eg udon, buckwheat noodles, ramen, spaghetti, macaroni) 2020/091179 1» (:1/10 ⁇ 019/007317, etc.), fruit juice, various drinks, cookies, syrup, dairy products (eg yogurt, fermented milk, butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, retort It can be manufactured by adding the lactic acid bacteria of the present invention such as food, frozen food, and various seasonings (eg, miso, soy sauce, sauce, etc.).
  • lactic acid bacteria of the present invention such as food, frozen food, and various seasonings (eg, miso, soy sauce, sauce, etc.).
  • the lactic acid bacteria of the present invention in the form of a food additive, it can be prepared and used in the form of powder or concentrate.
  • the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is normally used in the formulation. It is used as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline
  • the pharmaceutical composition may additionally contain lubricants, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents, preservatives, and the like.
  • composition of the present invention can be administered orally or parenterally,
  • composition of the present invention can be formulated in various oral or parenteral administration forms as follows, but is not limited thereto.
  • Formulations for oral administration include, for example, tablets, pills, hard/soft capsules, liquids, suspensions, emulsifiers, syrups, granules, elixirs, etc. These formulations include fillers commonly used in addition to the above active ingredients, One or more diluents or excipients such as extenders, wetting agents, disintegrants, lubricants, binders, surfactants, etc. can be used. Disintegrants include agar, starch, alginic acid or its sodium salt, and anhydrous calcium monohydrogen phosphate. Silica, talc, stearic acid or its magnesium salt or calcium salt, polyethylene glycol, etc. can be used as lubricants, and as binders, magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose,
  • Hydroxypropyl cellulose can be used.
  • lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine can be used as diluents, and in some cases, commonly known boiling mixtures, absorbents, colorants, flavors It can be used together with sweeteners and sweeteners.
  • composition may be sterilized or contain supplements such as preservatives, stabilizers, hydrating agents or emulsification accelerators, salts for regulating osmotic pressure, buffers, and other therapeutically useful substances, and may be used in conventional mixing, granulation or coating methods. It can be formulated according to.
  • the appropriate dosage of the pharmaceutical composition of the present invention is the formulation method, administration method, patient 2020/091179 1»(:1 ⁇ 1 ⁇ 2019/007317 It can be prescribed differently depending on factors such as age, weight, sex, morbidity, food, administration time, route of administration, excretion rate, and response sensitivity.
  • composition of the present invention is commonly used in the technical field to which the present invention belongs.
  • the dosage form is oil or water-based. It may be in the form of a solution, suspension, syrup or emulsion in a medium, or in the form of extracts, powders, powders, granules, tablets or capsules, and may additionally contain dispersants or stabilizers.
  • the present invention is the accession number KCTC13686BP
  • plantarum Provides a method for preventing, improving or treating osteoporosis, comprising administering to a subject a composition containing strain A41 or a culture thereof as an active ingredient.
  • Osteoporosis the disease target of the treatment method or improvement method of the present invention, is the same as defined in relation to the disease target of the pharmaceutical composition.
  • the subject is a mammal or human.
  • A41 strain or its culture is a method of using the same active ingredient as a composition containing as an active ingredient, so the description of duplicate contents is omitted to avoid excessive complexity in this specification.
  • the present invention provides the Lactobacillus plantarum (LactoZwd/to plantarum) A41 strain with accession number KCTC13686BP.
  • the present invention provides a pharmaceutical composition and food composition comprising the Lactose plantarum A41 strain, or a culture thereof, with accession number KCTC13686BP as an active ingredient.
  • strains and their cultures of the present invention can be usefully used as food compositions and pharmaceutical compositions for the purpose of preventing, ameliorating, or treating osteoporosis, especially secondary osteoporosis.
  • 1 is a diagram showing bone mineM density, mg/cm 3 for each experimental group.
  • FIG. 2 is a diagram showing the bone ratio (percent bone volume, %) for each experimental group.
  • FIG. 3 shows the mRNA expression level of TRPV6, an intestinal calcium-receiving protein for each experimental group. 2020/091179 1»(:1 ⁇ 1 ⁇ 2019/007317 is shown.
  • Figure 5 shows the mRNA expression level of PMCA1, an intestinal calcium-receiving protein for each experimental group.
  • Fig. 6 is a diagram showing the blood calcium concentration for each experimental group.
  • Figure 7 is a diagram showing the expression level of RANKL mRNA for each experimental group.
  • Fig. 8 is a diagram showing the expression level of OPG mRNA for each experimental group.
  • Fig. 9 is a diagram showing the expression level of BSP mRNA for each experimental group.
  • liquid/liquid (volume/volume) is %.
  • Example 1 Preparation of probiotic strain and fermentation milk
  • probiotic strains used in this experiment are feces and traditions in infants and young children.
  • skim milk Each of the probiotic strains isolated from the above was inoculated in 13 ⁇ 4 medium and cultured for 48 hours at 37°(:). Fermented milk after freeze-drying after fermentation was freeze-dried and suspended at a concentration of 1 0 10 0 ⁇ /1111 to feed the experimental animals.
  • the composition of the skim milk medium used in the present invention and a list of probiotic strains are shown in Tables 1 and 2.
  • composition of the skim milk medium of the present invention is composition of the skim milk medium of the present invention
  • the secondary osteoporosis animal model was produced as follows: 8-week-old Wistar female rats (Samtako, South Korea) weighing 175-185 g were supplied with sufficient pellet-type feed and water, and adapted to individual cages for 1 week.
  • the secondary osteoporosis model was established by Lucinda et al. (PHYTOTHERAPY RESEARCH 27(4), (2012), 515-520) and Sousa et al. (Archives of Oral Biology 77 (2017) 55-61), a type of glucocorticoid by intramuscular injection
  • Dexamethasone (DEX) was administered once a week at a concentration of 7 mg/kg BW for 5 weeks.
  • the experimental group was classified into 9 groups as shown in Table 3. 7 experimental animals were assigned to each group, and dexamethasone (DEX) was administered according to the method in 2.1 above for all groups except the normal group. It was administered for 5 weeks at a concentration of 7 mg/kg (BW) once.
  • DEX dexamethasone
  • Bone mineral density and percent bone volume are the most basic checks when measuring bone strength.
  • Micro Ct imaging Analyzer Sky Scan 1176
  • NRecon a program to reconstruct it
  • fermented milk fermented with fermentum SRK414, L. plantarum B719, and L. casei 393 recovered the reduction in bone density and bone ratio, which are symptoms of secondary osteoporosis caused by the administration of dexamethasone.
  • the present inventors are done to find out the amount of mRNA expression of the intestinal calcium-receiving protein according to the administration of the fermented milk of the present invention! -1(11 analyzes were conducted, and the relationship with blood calcium concentration was confirmed.
  • Example 5 Analysis of indicators related to bone metabolism according to administration of fermentation oil of the present invention
  • RANKL Receptor Activator of Nuclear Factor Kappa B Ligand
  • RANK Receptor Activator of Nuclear Factor Kappa B
  • OPG Ostoprotegerin
  • the differentiation of osteoclasts can be inhibited by interfering with the binding of RANK and RANKL.
  • the present inventors determined the amount of mRNA expression of RANKL, OPG, and BSP, which are bone metabolism-related markers, to confirm the effect of the administration of the fermentation oil of the present invention on bone metabolism.
  • the primers for qRT-PCT analysis are shown in Table 5. The degree of total transcription was (from the 3-actin transcription level)

Abstract

The present invention provides a Lactobacillus plantarum A41 strain with accession number KCTC13686BP, and pharmaceutical and food compositions comprising, as an active ingredient, the strain or a culture thereof. The strain according to the present invention and the culture thereof have an excellent effect in preventing, alleviating, or treating osteoporosis, particularly secondary osteoporosis, and thus can be usefully employed in food and pharmaceutical compositions for said purpose.

Description

2020/091179 1»（：1^1{2019/007317 명세서 2020/091179 1»（：1^1{2019/007317 specification
발명의 명칭 :프로바이오틱스를유효성분으로포함하는이차성 골다공증의 예방또는치료용조성물 기술분야 Title of Invention: Composition for the prevention or treatment of secondary osteoporosis containing probiotics as an active ingredient
[1] 본발명은대한민국농림축산식품부의지원하에서과제번히 PET117051-3에 의해이루어진것으로서,상기과제의연구관리전문기관은 [1] The present invention was accomplished by PET117051-3 under the support of the Ministry of Food, Agriculture, Forestry and Food of the Republic of Korea.
농림식품기술기획평가원,연구사업명은 "고부가가치식품기술개발사업 ", 연구과제명은 "원발성및이차성골다공증개선효과를갖는기능성 Agriculture, Forestry and Food Technology Planning and Evaluation Institute, the name of the research project is "High Value-added Food Technology Development Project", and the name of the research project is "Functionality that has the effect of improving primary and secondary osteoporosis.
프로바이오틱스제품개발”,주관기관은（주）종근당바이오,연구기간은 Probiotics product development”, the leading agency is Chong Kun Dang Bio, the research period
2017.06.15-2019.12.31이다. It is 2017.06.15-2019.12.31.
[2] 본특허출원은 2018년 W월 30일에대한민국특허청에제출된대한민국 [2] This patent application was filed with the Korean Intellectual Property Office on W/30, 2018.
특허출원제 10-2018-0131060호에대하여우선권을주장하며,상기특허출원의 개시사항은본명세서에참조로서삽입된다. Priority is claimed for patent application No. 10-2018-0131060, and the disclosures of the patent application are incorporated by reference in this specification.
[3] 본발명은프로바이오틱스를유효성분으로포함하는이차성골다공증의 예방 또는치료용조성물에관한것이다. [3] The present invention relates to a composition for preventing or treating secondary osteoporosis containing probiotics as an active ingredient.
[4] [4]
배경기술 Background
[5] 골다공증（Osteoporosis）은연령과폐경등정상생리적변화에기인하여골량이 감소하는일차성골다공증（원발성, primary）과,연령과관계없이질환이나 약물에노줄되어초래되는이차성골다공증（속발성 , secondary）,크게두가지로 분류된다.흔히골다공증은노년기의질병이라고생각하기쉽지만,이차성 골다공증의경우노령이아닌경우에도발병할수있고,이경우향후환자의 삶의질에미치는영향이매우크기때문에이차성골다공증의치료및예방 방법을개발하는것은매우중요하다. [5] Osteoporosis is primary osteoporosis (primary, primary) in which bone mass decreases due to normal physiological changes such as age and menopause, and secondary osteoporosis (secondary, secondary) caused by exposure to diseases or drugs regardless of age. Although it is easy to think that osteoporosis is a disease of old age, secondary osteoporosis can develop even if it is not old age, and in this case, the effect on the quality of life of the patient in the future is very large, so the treatment of secondary osteoporosis And it is very important to develop preventive measures.
[6] 이차성골다공증의원인들은매우다양하다.여러전신질환과약물등이 [6] The causes of secondary osteoporosis are very diverse. Various systemic diseases and drugs
원인으로알려져있으나,이중글루코코르티코이드복용으로인한골다공증이 가장흔하다.글루코코르티코이드（Glucocorticoid: GC）는항염증과면역억제 기능이 있어류마티스관절염,천식,아토피피부염,관절염등여러질병의 치료에보편적으로사용되는약제이다. GC의장기간투여는이러한질병을 효과적으로억제하지만,대부분환자에게서조골세포와파골세포의정상적인 활성을저해하여골다공증을초래한다.통계적으로살펴보면, GC를투여하는 환자의경우 1년이내골밀도의 12%가급격히감소되고그이후로는뼈의 손실이서서히진행되어 2~5%정도의골밀도가매년점차적으로감소됨이 보고된바있다.위와같은 GC투여에의한이차성골다공증을예방하기 위해서는 GC의투여를중단하는것이가장이상적인방법이기는하나,해당 질병의치료를위해서는소량이라도복용이지속되어야한다는점에서문제가 2020/091179 1»（：1^1{2019/007317 된다. Although known as the cause, osteoporosis caused by the use of double glucocorticoids is the most common. Glucocorticoid (GC) has anti-inflammatory and immunosuppressive properties, so it is commonly used in the treatment of various diseases such as rheumatoid arthritis, asthma, atopic dermatitis, and arthritis. It is a drug. Long-term administration of GC effectively suppresses these diseases, but in most patients, it inhibits the normal activity of osteoblasts and osteoclasts, leading to osteoporosis. Statistical findings, in patients receiving GC, 12% of bone density within one year It has been reported that the bone density is gradually decreased by 2 to 5% every year due to a rapid decrease in bone loss and thereafter. To prevent secondary osteoporosis caused by the above GC administration, it is necessary to stop the administration of GC. Although this is the most ideal method, the problem is that even a small amount must be sustained for the treatment of the disease. 2020/091179 1»（：1^1{2019/007317 becomes.
[7] GC의장기투여로인한골다공증을예방할수있는식품을함께섭취하는것도 좋은대안이될수있지만,시중에서판매중인제품들은대부분폐경기여성,및 노인성골다공증과같은’원발성골다공증’을개선하기위한건강기능식품이며, 이차성골다공증을예방할수있는제품은아직까지개발된바없다.따라서 이차성골다공증의예방,개선또는치료용도의식품또는의약품의개발이 절실히필요한실정이다. [7] Consumption of foods that can prevent osteoporosis caused by long-term administration of GC can be a good alternative, but products on the market are mostly health functional foods to improve'primary osteoporosis' such as menopausal women and senile osteoporosis. And, a product that can prevent secondary osteoporosis has not yet been developed. Therefore, there is an urgent need to develop foods or drugs for the prevention, improvement or treatment of secondary osteoporosis.
[8] [8]
발명의상세한설명 Detailed description of the invention
기술적과제 Technical task
[9] 본발명자들은향후골다공증의 예방,개선또는치료용도의식품및 [9] In the future, the present inventors will provide food and beverages for the prevention, improvement or treatment of osteoporosis.
의약품으로응용이가능한프로바이오틱스균주및이를이용한발효유를 개발하고자하였다.그결과,프로바이오틱스균주를사용하여발효유를제조한 후이를글루코코르티코이드-유도이차성골다공증실험동물에게경구 투여하면골밀도및골비율의증가되고,칼슘관련흡수및대사에관련된 유전자의발현이증가됨을규명함으로써,본발명을완성하게되었다. It was attempted to develop a probiotic strain that could be applied as a pharmaceutical and a fermented milk using the same. As a result, when a fermented milk was prepared using a probiotic strain and administered orally to a glucocorticoid-induced secondary osteoporosis test animal, the bone density and bone ratio increased. The present invention was completed by finding that the expression of genes related to calcium-related absorption and metabolism was increased.
[1이 따라서,본발명의목적은수탁번호 KCTC13686BP인락토바실러스플란타럼 ( Lactobacillus plantarum) A41균주를제공하는것이다. [1] Accordingly, the purpose of the present invention is to provide the Lactobacillus plantarum A41 strain with accession number KCTC13686BP.
[11] 본발명의다른목적은상기락토바실러
Figure imgf000004_0001
[11] Another purpose of the present invention is the above lactobacillus
Figure imgf000004_0001
A41균주를유효성분으로포함하는골다공증의 예방또는치료용약제학적 조성물을제공하는것이다. It is to provide a pharmaceutical composition for the prevention or treatment of osteoporosis comprising strain A41 as an active ingredient.
[12] 본발명의또다른목적은상기락토바실러스플란타럼 (LactoZ?예
Figure imgf000004_0002
[12] Another object of the present invention is the above Lactobacillus plantarum (LactoZ?
Figure imgf000004_0002
) A41균주를유효성분으로포함하는골다공증의예방또는개선용 ) For the prevention or improvement of osteoporosis containing the A41 strain as an active ingredient
식품조성물을제공하는것이다. It is to provide food composition.
[13] 본발명의또다른목적은상기락토바실러스플란타럼 (LactoZ?예
Figure imgf000004_0003
[13] Another object of the present invention is the above Lactobacillus plantarum (LactoZ?
Figure imgf000004_0003
) A41균주를유효성분으로포함하는조성물을대상체에투여하는단계를 포함하는골다공증의예방,개선또는치료방법을제공하는것이다. ) It is to provide a method for preventing, improving or treating osteoporosis comprising administering a composition containing the A41 strain as an active ingredient to a subject.
[14] 본발명의다른목적및이점은하기의발명의상세한설명,청구범위및도면에 의해보다명확하게된다. [14] Other objectives and advantages of the present invention will become more apparent by the detailed description, claims, and drawings of the present invention.
[15] [15]
과제해결수단 Problem solving means
[16] 본발명의일양태에따르면,본발명은수탁번호 KCTC13686BP인 [16] According to one aspect of the present invention, the present invention is the accession number KCTC13686BP
Figure imgf000004_0004
plantarum) A41균주를제공한다.
Figure imgf000004_0004
plantarum) A41 strain.
[17] 본발명자들은락토바실러스플란타럼 (LactoZ?예 i plantarum) A41 [17] The present inventors are Lactobacillus plantarum (LactoZ? i plantarum) A41
균주 (수탁번호 : KCTC13686BP)를신규분리하였다.또한,본발명자들은상기 락토바실러스
Figure imgf000004_0005
plantarum) A41균주 (수탁번호 : The strain (accession number: KCTC13686BP) was newly isolated. In addition, the present inventors described the Lactobacillus
Figure imgf000004_0005
plantarum) A41 strain (accession number:
KCTC13686BP)를이용하여발효유를제조하였다.상기균주에의해제조된 2020/091179 1»（：1^1{2019/007317 발효유는이차성골다공증을유도한실험동물모델에투여한결과,골밀도및 골비율이증가되는효과가있음을확인하였다.나아가,상기 발효유를투여한 실험동물모델의장내에서발현되는칼슘흡수관련단백질의 발현량이 증가되고,혈중칼슘농도가증가되며 ,파골세포분화억제 및조골세포분화 촉진과관련된유전자의 발현량을조절함을확인하였다. KCTC13686BP) was used to prepare fermented milk. 2020/091179 1»（：1^1{2019/007317 As a result of administration of fermented milk to an experimental animal model that induces secondary osteoporosis, it was confirmed that it has the effect of increasing bone density and bone ratio. Furthermore, the fermented milk was administered with the above fermented milk. It was confirmed that the expression level of calcium absorption-related proteins expressed in the intestine of the experimental animal model was increased, the calcium concentration in the blood was increased, and the expression level of genes related to the inhibition of osteoclast differentiation and promotion of osteoblast differentiation was confirmed.
[18] 위결과로부터，본발명의락토바실러스플란타럼 (LactoZwd// plantarum) A41 균주 (수탁번호 : KCTC13686BP)를이용하여 제조된발효유는이차성 [18] From the above results, the fermented milk prepared using the Lactobacillus plantarum (LactoZwd// plantarum) A41 strain (accession number: KCTC13686BP) of the present invention is secondary
골다공증의 예방,개선,또는치료에유용하게사용될수있음을확인하였다. It has been confirmed that it can be usefully used in the prevention, improvement, or treatment of osteoporosis.
[19] [19]
[20] 본명세서에서용어 "골다공증 (osteoporosis)”은”골량의감소와미세구조의 [20] In the present specification, the term "osteoporosis" refers to a reduction in bone mass and microstructure.
이상을특징으로하는전신적인골격계질환으로,결과적으로뼈가약해져서 부러지기 쉬운상태가되는질환”을말한다.골강도는골량 (quantity)과 It is a systemic skeletal disease characterized by the above, and as a result, the bone becomes weak and becomes brittle.” Bone strength refers to bone mass (quantity) and
골질 (quality)에 의해결정된다.골량은주로골밀도 (BMD)에의해표현되고 골질은구조,골교체율,무기질화,미세손상축적등으로구성된다.현재는 골밀도를측정하여골다공증을진단하고있다. Bone mass is mainly expressed by bone density (BMD), and bone quality is composed of structure, bone replacement rate, mineralization, micro-damage accumulation, etc. Currently, osteoporosis is diagnosed by measuring bone density.
[21] 골다공증은일차성과이차성골다공증으로분류된다.일차성골다공증또는 원발성 골다공증은폐경으로인한제 1형 골다공증과노화로인한제 2형 골다공증으로편의상분류하지만거의같은시기에 병합되어 진행되므로 정확히분류하기 어려운것으로알려져 있다.이차성골다공증은기저질환이나 약물에 의해뼈의 미세구조가변하고골량이감소하여 골절위험도가증가한 것을말한다.이차성골다공증의 원인으로내분비질환 (갑상선중독증,당뇨병, 부갑상선기능항진증등),위장관질환 (위절제줄,만성 간질환,흡수장애증후군, 염증성장질환,알코올중독등),골수질환,결체조직질환 (강직성척추염, 류마티스관절염,골형성부전증등),약물등이 있다.남성에서 가장흔한원인은 성선기능저하증,글루코코르티코이드투여등이다. [21] Osteoporosis is classified into primary and secondary osteoporosis. Primary osteoporosis or primary osteoporosis is classified as type 1 osteoporosis due to menopause and type 2 osteoporosis due to aging for convenience, but since it is combined and progressed at about the same time, it should be accurately classified. Secondary osteoporosis refers to an increase in the risk of fracture due to changes in bone microstructure and decrease in bone mass due to underlying diseases or drugs. Endocrine diseases (thyroidism, diabetes, hyperparathyroidism, etc.) as a cause of secondary osteoporosis. Gastrointestinal diseases (gastric resection line, chronic liver disease, malabsorption disorder syndrome, inflammatory growth disease, alcohol addiction, etc.), bone marrow disease, connective tissue disease (ankylosing spondylitis, rheumatoid arthritis, bone dysplasia, etc.), and drugs. The most common causes are hypogonadism and administration of glucocorticoids.
[22] 본발명의 일구현예에서,상기골다공증은이차성골다공증이고,보다 [22] In one embodiment of the present invention, the osteoporosis is secondary osteoporosis,
구체적으로는글루코코르티코이드계 약물투여에의한이차성골다공증이다. 상기글루코코르티코이드계 약물은코르티솔,프레드니손,프레드니솔론, 메틸프레드니솔론,코르티손,하이드록시코르티손,트리암시놀론,베타메타손, 또는덱사메타손이나,이에 한정되는것은아니다 Specifically, it is secondary osteoporosis caused by administration of a glucocorticoid drug. The glucocorticoid drug is, but is not limited to, cortisol, prednisone, prednisolone, methylprednisolone, cortisone, hydroxycortisone, triamcinolone, betamethasone, or dexamethasone.
[23] [23]
[24] 본발명에있어서,상기락토바실러스플란타럼(1^¾^0&<¾^7/ 1 때11111)쇼41 균주는 "한국생명공학연구원미생물자원센터 (Korean Collection for Type Culture, KCTC)”에수탁번호” KCTC13686BP”로기탁된것이다. [24] In the present invention, the Lactobacillus plantarum (1^¾^0&<¾ ^7/ 1 time 11111) Show 41 strain is "Korea Research Institute of Bioscience and Biotechnology Microbial Resource Center (Korean Collection for Type Culture, KCTC) It has been deposited under the "accession number"KCTC13686BP".
[25] 상기락토바실러스플란타럼 (LactoZ¾?cz7/i«· plantarum) A41균주의균체성상은 다음과같다: [25] The cell properties of the Lactobacillus plantarum (LactoZ¾?cz7/i«· plantarum) A41 strain are as follows:
[26] -서열번호 1의 16s rRNA서열을포함함 [26]-Contains the 16s rRNA sequence of SEQ ID NO: 1
[27] -그람양성 막대모양또는구형간균의 형태임 2020/091179 1»（：1^1{2019/007317 [27] -In the form of a gram-positive rod or spheroid 2020/091179 1»（：1^1{2019/007317
[28] -인간의 입,위장,여성성기관의정상세균총의 일부이며,일반적으로음식에 사용하는것에 대해 안전하다고여겨진다 (probiotics와발효음식 ). L. plantarum은 Lactobacillus속구성원이며,일반적으로많은발효식품과혐기성식물의 물질에서 발견되며타액에도존재하기도한다. [28]-It is part of the normal flora of the human mouth, stomach, and feminine organs, and is generally considered safe for use in food (probiotics and fermented foods). L. plantarum is a member of the genus Lactobacillus, is generally found in many fermented foods and substances of anaerobic plants, and is also present in saliva.
[29] [29]
[3이 따라서，본발명의다른일양태에따르면,본발명은수탁번호 [3] Accordingly, according to another aspect of the present invention, the present invention is the accession number
KCTC13686BP인락토바실러스플란타럼 (Lac plantarum) A41균주, 또는이의 배양물을유효성분으로포함하는골다공증예방또는치료용 약제학적조성물을제공한다. It provides a pharmaceutical composition for the prevention or treatment of osteoporosis comprising the KCTC13686BP, Lac plantarum A41 strain, or a culture thereof as an active ingredient.
[31] 본발명의또다른일양태에 따르면,본발명은수탁번호 KCTC13686BP인 [31] According to another aspect of the present invention, the present invention is the accession number KCTC13686BP
락토바실러스플란타럼 plantarum) A41균주,또는이의 배양물을 유효성분으로포함하는골다공증예방또는개선용식품조성물을제공한다. Lactobacillus plantarum plantarum) Provides a food composition for preventing or improving osteoporosis comprising the A41 strain, or a culture thereof, as an active ingredient.
[32] [32]
[33] 본발명의 일구현예에서,상기조성물은유효성분으로락토바실러스퍼멘텀 ( Lactobacillus fermentum),락토바실러스카제이 {Lactobacillus casei), [33] In one embodiment of the present invention, the composition is an active ingredient, Lactobacillus fermentum, Lactobacillus casei,
락토바실러스애시도필러스 (Lactobacillus acidophilus),락토바실러스 Lactobacillus acidophilus, Lactobacillus acidophilus
불가리쿠스 (Lactobacillus delbrueckii ssp bulgaricus),락토코커스락티스 ( Bulgaricus (Lactobacillus delbrueckii ssp bulgaricus), Lactobacillus lactis (
Lactococcus lactis),엔테로코커스페시움 (Enterococcus faecium),엔테로코커스 페칼리 :느 (Enterococcus fecalis),스트렙토코커스써모필러스 («Sireptococci thermophilus),비피도박테리움비피덤 (Bifidobacterium bifidum),및비피도 박테리움락티스 (Bifidobacterium lactis)로이루어진군으로부터선택되는 1종 이상의균,또는이의 배양물을추가적으로포함한다. Lactococcus lactis), Enterococcus faecium, Enterococcus fecalis, Streptococcus thermophilus ( « Sireptococci thermophilus), Bifidobacterium bifidum, and Bifidobacterium bifidum. It further comprises one or more bacteria selected from the group consisting of Bifidobacterium lactis, or a culture thereof.
[34] 본발명의구체적인구현예에서 ,상기 락토바실러스퍼멘텀은락토바실러스 퍼멘텀 SRK414균주 (수탁번호 KCTC13687BP)일수있다. [34] In a specific embodiment of the present invention, the Lactobacillus Fermentum may be the Lactobacillus Fermentum SRK414 strain (accession number KCTC13687BP).
[35] 본발명에 있어서,상기락토바실러스퍼멘텀 SRK414균주는 [35] In the present invention, the lactobacillus fermentum SRK414 strain
"한국생명공학연구원미생물자원센터 (Korean Collection for Type Culture, "Korea Research Institute of Bioscience and Biotechnology Microorganism Resource Center (Korean Collection for Type Culture,
KCTC)’’에수탁번호’’KCTC13687BP”로기탁된것이다. KCTC) has been deposited as a deposit number for'KCTC13687BP'.
[36] 상기락토바실러스퍼멘텀 SRK414균주의균체성상은다음과같다: [36] The cell properties of the Lactobacillus fermentum SRK414 strain are as follows:
[37] -서열번호 2의 16s rRNA서열을포함함 [37]-Contains the 16s rRNA sequence of SEQ ID NO: 2
[38] -그람양성 막대모양또는구형간균의 형태임 [38] -In the form of a gram-positive rod or spheroid bacilli
[39] -인간의 입,위장,여성성기관의정상세균총의 일부이며,일반적으로음식에 사용하는것에 대해 안전하다고여겨진다 (probiotics와발효음식). L. fermentum 은 Lactobacillus속구성원이며,이속에속하는종들은다양한활용이 [39]-It is part of the normal flora of the human mouth, stomach, and feminine organs, and is generally considered safe for use in food (probiotics and fermented foods). L. fermentum is a member of the genus Lactobacillus, and the species belonging to this genus have various uses.
가능하다고한다.이러한활용은식품과사료발효를포함한다. L. fermentum중 몇균주들은항생제 및화학요법제에 대해자연내성을갖는것으로밝혀져 있다. These uses include fermentation of food and feed. Several strains of L. fermentum have been shown to have natural resistance to antibiotics and chemotherapeutic agents.
[4이 [4 this
[41] 본발명의조성물들의유효성분인상기균주,또는이의 배양물은상기균주를 단리 및/또는정제한균체외에균체를포함하는배양물,균체의추출물,배양물 2020/091179 1»（：1^1{2019/007317 상층액,이들의농축액,농축물,건조물,또한필요에 따라서 희석액,희석물 등이며 ,배양액 ,배양물을처리하여 얻어지는모든상태의 것을포함한다. [41] The above strain, or a culture thereof, which is an active ingredient of the compositions of the present invention, is a culture containing cells in addition to the cells isolated and/or purified from the above strains, extracts of cells, and cultures 2020/091179 1»（：1^1{2019/007317 Supernatant, these concentrates, concentrates, dry matters, as well as diluents, dilutions, etc., if necessary, including culture solutions and all conditions obtained by processing the cultures do.
[42] 상기균체의 배양법,추출법,분리법,농축법,건조법,희석법등은특별히 [42] The culture method, extraction method, separation method, concentration method, drying method, dilution method, etc.
한정되지 않는다. Not limited.
[43] 균체를배양하기 위한배지로는통상적으로탈지유,훼이 ,카제인등의우유 단백질,당류,효모엑기스등을포함하고있으며,배양방법으로는일반적인 각종호기적또는혐기적인방법을적당히사용할수있다. [43] The medium for cultivating the cells usually contains milk proteins such as skim milk, whey and casein, sugars, yeast extract, etc. As a culture method, various general aerobic or anaerobic methods can be appropriately used.
[44] 배양온도로는예를들어 35 45OC를설정하고,배양중에는수산화나트륨등의 알칼리를사용하여 배지의 pH를중성으로부터산성,예를들어 pH가 5~6정도가 되도록유지하는중화배양법을사용할수도있다.이와같은중화배양법외에 회분배양법등의 임의의 배양방법을사용할수있으며,배양한후에는필요에 따라서 배양물이나그상층액을농축,건조,희석등을할수도있다. [44] A neutralization culture method in which, for example, 35 45 O C is set as the culture temperature, and during culture, an alkali such as sodium hydroxide is used to maintain the pH of the medium from neutral to acidic, for example, about 5-6. In addition to such neutralization culture, any culture method such as batch culture can be used, and after culture, the culture or its supernatant can be concentrated, dried, or diluted if necessary.
[45] 또한원심분리법이나막분리법을사용하여 배양물의상층액과균체를 [45] In addition, centrifugal separation or membrane separation may be used to
분리하여균체를농축한상태로회수할수도있다.그리고균체에초음파처리나 효소처리등을행하여균체내의성분을추출하거나,배양물이나그상층액, 균체나그추출물등을건조할수도있다.이들은본발명의상기조성물의유효 성분으로서사용할수있다. The cells can be separated and recovered in a concentrated state. In addition, the cells can be subjected to ultrasonic treatment or enzyme treatment to extract the components in the cells, or to dry the culture or its supernatant, the cells or their extracts. It can be used as an active ingredient in a cosmetic composition.
[46] 본발명의조성물이식품조성물로제조되는경우,유효성분으로서상기 [46] When the composition of the present invention is prepared as a food composition, the above
유산균뿐만아니라,식품제조시에통상적으로첨가되는성분을포함할수 있다.상기 첨가성분은예컨대단백질,탄수화물,지방,영양소,조미제및 향미제를포함한다.상기탄수화물로는모노사카라이드（예를들어,포도당,과당 등）,디사카라이드（예를들어 말토스,수크로스,올리고당등）및 In addition to lactic acid bacteria, it may contain ingredients that are normally added in food manufacturing. The additive ingredients include, for example, proteins, carbohydrates, fats, nutrients, flavoring agents and flavoring agents. The carbohydrates include monosaccharides (for example, monosaccharides). Glucose, fructose, etc.), disaccharides (e.g. maltose, sucrose, oligosaccharide, etc.), and
폴리사카라이드（예를들어 덱스트린,사이클로덱스트린등과같은통상적인당 및자일리톨,소르비톨,에리트리톨등의 당알콜이다.향미제로서천연향미제 （타우마틴,스테비아추출물（예를들어 레바우디오시드 A,글리시르히진등））및 합성 향미제（사카린,아스파르탐등）를사용할수있다. Polysaccharides (for example, regular sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavoring agents (taumatin, stevia extracts (for example, rebaudioside A, glysir) as flavoring agents) Hijin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.
[47] 예컨대,본발명의식품조성물이드링크제로제조되는경우에는본발명의 유효성분인상기균주이외에구연산,액상과당,설탕,포도당,초산,사과산, 과즙,대추추출액또는감초추출액등을추가로포함시킬수있다. [47] For example, when the food composition of the present invention is manufactured as a drink link agent, citric acid, liquid fructose, sugar, glucose, acetic acid, apple acid, fruit juice, jujube extract, licorice extract, etc. Can be included.
[48] 본발명의식품조성물은식품,기능성식품（functional food）, [48] The food composition of the present invention is food, functional food,
영양보조제（nutritional supplement）,건강식품（heal比 i food）및식품첨가제（food additives）등의모든천연소재의가공형태를포함한다.상기유형의식품 조성물은당업계에공지된통상적인방법에 따라다양한형태로제조될수있다. Includes processing forms of all natural materials such as nutritional supplements, health foods and food additives. Food compositions of this type are in accordance with conventional methods known in the art. It can be manufactured in various forms.
[49] 예를들면,건강식품으로는상기유산균자체를차,주스및드링크의 형태로 제조하여 음용하도록하거나,과립화,캡슐화및분말화하여 섭취할수있다. 또한,식품으로는음료（알콜성음료포함）,과실및그의 가공식품（예: [49] For example, as a health food, the lactic acid bacteria itself can be prepared and consumed in the form of tea, juice, and drink, or it can be ingested by granulating, encapsulating and powdering. In addition, as food, beverages (including alcoholic beverages), fruits and processed foods thereof (eg:
과일통조림,병조림,잼,마아말레이드등）,어류,육류및그가공식품（예:햄, 소시지콘비이프등）,빵류및면류（예:우동,메밀국수,라면,스파게티,마카로니 2020/091179 1»（：1/10公019/007317 등）,과즙,각종드링크,쿠키,엿,유제품（예:요거트,발효유,버터,치즈등）, 식용식물유지,마아가린,식물성단백질,레토르트식품,냉동식품,각종 조미료（예:된장,간장,소스등）등본발명의유산균을첨가하여제조될수있다. 또한,본발명의유산균을식품첨가제의형태로사용하기위해서는분말또는 농축액형태로제조하여사용할수있다. 본발명의조성물이약제학적조성물로제조되는경우,본발명의약제학적 조성물은약제학적으로허용되는담체를포함한다.본발명의약제학적 조성물에포함되는약제학적으로허용되는담체는제제시에통상적으로 이용되는것으로서,락토스,덱스트로스,수크로스,솔비톨,만니톨,전분, 아카시아고무,인산칼슘,알기네이트,젤라틴,규산칼슘,미세결정성 Canned fruit, bottled, jam, marmalade, etc.), fish, meat and processed foods (eg ham, sausage corn beef, etc.), bread and noodles (eg udon, buckwheat noodles, ramen, spaghetti, macaroni) 2020/091179 1» (：1/10公019/007317, etc.), fruit juice, various drinks, cookies, syrup, dairy products (eg yogurt, fermented milk, butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, retort It can be manufactured by adding the lactic acid bacteria of the present invention such as food, frozen food, and various seasonings (eg, miso, soy sauce, sauce, etc.). In addition, in order to use the lactic acid bacteria of the present invention in the form of a food additive, it can be prepared and used in the form of powder or concentrate. When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is normally used in the formulation. It is used as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline
셀룰로오스,폴리비닐피롤리돈,셀룰로오스,물,시럽,메틸셀룰로오스, 메틸히드록시벤조에이트,프로필히드록시벤조에이트,활석,스테아르산 마그네슘및미네랄오일등을포함하나,이에제한되는것은아니다.본발명의 약제학적조성물은상기성분들이외에윤활제 ,습윤제 ,감미제 ,향미제 ,유화제 , 현탁제,보존제등을추가로포함할수있다. Cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, including, but not limited to. In addition to the above ingredients, the pharmaceutical composition may additionally contain lubricants, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents, preservatives, and the like.
본발명의약제학적조성물은경구또는비경구투여할수있으며 , The pharmaceutical composition of the present invention can be administered orally or parenterally,
바람직하게는경구투여방식으로적용된다.본발명의약제학적조성물은 하기의다양한경구또는비경구투여형태로제형화할수있으나,이에 한정되는것은아니다. It is preferably applied by oral administration. The pharmaceutical composition of the present invention can be formulated in various oral or parenteral administration forms as follows, but is not limited thereto.
[53] 경구투여용제형으로는예를들면정제,환제,경/연질캅셀제,액제,현탁제, 유화제,시럽제.과립제,엘릭시르제등이있는데,이들제형은상기유효성분 이외에통상적으로사용되는충진제 ,증량제 ,습윤제，붕해제 ,활택제 ,결합제 , 계면활성제등의희석제또는부형제를 1종이상사용할수있다.붕해제로는 한천,전분,알긴산또는이의나트륨염,무수인산일수소칼슘염등이사용될수 있고,활택제로는실리카,탈크,스테아르산또는이의마그네슘염또는칼슘염 , 폴리에틸렌글리콜등이사용될수있으며,결합제로는마그네슘알루미늄 실리케이트,전분페이스트,젤라틴,트라가칸스,메틸셀룰로오스, [53] Formulations for oral administration include, for example, tablets, pills, hard/soft capsules, liquids, suspensions, emulsifiers, syrups, granules, elixirs, etc. These formulations include fillers commonly used in addition to the above active ingredients, One or more diluents or excipients such as extenders, wetting agents, disintegrants, lubricants, binders, surfactants, etc. can be used. Disintegrants include agar, starch, alginic acid or its sodium salt, and anhydrous calcium monohydrogen phosphate. Silica, talc, stearic acid or its magnesium salt or calcium salt, polyethylene glycol, etc. can be used as lubricants, and as binders, magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose,
나트륨카복시메틸셀룰로오스,폴리비닐피롤리딘,저치환도 Sodium carboxymethyl cellulose, polyvinylpyrrolidine, low substitution degree
하이드록시프로필셀룰로오스등이사용될수있다.이외에도락토즈, 덱스트로오스,수크로오스,만니톨,소르비톨,셀룰로오스.글리신등을 희석제로사용할수있으며,경우에따라서는일반적으로알려진비등혼합물, 흡수제,착색제,향미제,감미제등을함께사용할수있다. Hydroxypropyl cellulose can be used. In addition, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine can be used as diluents, and in some cases, commonly known boiling mixtures, absorbents, colorants, flavors It can be used together with sweeteners and sweeteners.
상기조성물은멸균되거나또는방부제,안정화제,수화제또는유화촉진제, 삼투압조절을위한염,완충제등의보조제및기타치료적으로유용한물질을 함유할수있으며,통상적인방법인혼합,과립화또는코팅방법에따라 제제화할수있다. The above composition may be sterilized or contain supplements such as preservatives, stabilizers, hydrating agents or emulsification accelerators, salts for regulating osmotic pressure, buffers, and other therapeutically useful substances, and may be used in conventional mixing, granulation or coating methods. It can be formulated according to.
[551 본발명의약제학적조성물의적합한투여량은제제화방법 ,투여방식 ,환자의 2020/091179 1»（：1^1{2019/007317 연령,체중,성,병적상태,음식,투여시간,투여경로,배설속도및반응 감응성과같은요인들에의해다양하게처방될수있다. [551 The appropriate dosage of the pharmaceutical composition of the present invention is the formulation method, administration method, patient 2020/091179 1»（：1^1{2019/007317 It can be prescribed differently depending on factors such as age, weight, sex, morbidity, food, administration time, route of administration, excretion rate, and response sensitivity.
[56] 본발명의약제학적조성물은당해발명이속하는기술분야에서통상의 [56] The pharmaceutical composition of the present invention is commonly used in the technical field to which the present invention belongs.
지식을가진자가용이하게실시할수있는방법에따라,약제학적으로허용되는 담체및/또는부형제를이용하여제제화함으로써단위용량형태로제조되거나 또는다용량용기내에내입시켜제조될수있다.이때제형은오일또는수성 매질중의용액,현탁액,시럽제또는유화액형태이거나엑스제,산제,분말제, 과립제,정제또는캅셀제형태일수도있으며,분산제또는안정화제를 추가적으로포함할수있다. According to a method that can be easily implemented by a knowledgeable person, it can be prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient, or it can be prepared by incorporating it into a multi-dose container. In this case, the dosage form is oil or water-based. It may be in the form of a solution, suspension, syrup or emulsion in a medium, or in the form of extracts, powders, powders, granules, tablets or capsules, and may additionally contain dispersants or stabilizers.
[57] 본발명의일양태에따르면,본발명은수탁번호 KCTC13686BP인 [57] According to one aspect of the present invention, the present invention is the accession number KCTC13686BP
Figure imgf000009_0001
plantarum) A41균주,또는이의배양물을 유효성분으로포함하는조성물을대상체에투여하는단계를포함하는, 골다공증의 예방,개선또는치료방법을제공한다.
Figure imgf000009_0001
plantarum) Provides a method for preventing, improving or treating osteoporosis, comprising administering to a subject a composition containing strain A41 or a culture thereof as an active ingredient.
[58] 본발명의치료방법,또는개선방법의대상질병인골다공증은상기약제학적 조성물의치료대상질환과관련하여정의한것과같다. [58] Osteoporosis, the disease target of the treatment method or improvement method of the present invention, is the same as defined in relation to the disease target of the pharmaceutical composition.
[59] 본발명의일구현예에서 ,상기대상체는포유동물또는인간이다. [59] In one embodiment of the present invention, the subject is a mammal or human.
[6이 본발명의골다공증의치료방법또는개선방법은상술한수탁번호 [6 The accession number mentioned above for the treatment method or improvement method for osteoporosis of this invention
Figure imgf000009_0002
plantarum) A41균주, 또는이의배양물을유효성분으로포함하는조성물과동일한유효성분을 사용하는방법이므로,중복되는내용에대해서는본명세서의과도한복잡성을 피하기위하여그기재를생략한다.
Figure imgf000009_0002
plantarum) A41 strain or its culture is a method of using the same active ingredient as a composition containing as an active ingredient, so the description of duplicate contents is omitted to avoid excessive complexity in this specification.
[61] [61]
발명의효과 Effects of the Invention
[62] 본발명의특징및이점을요약하면다음과같다: [62] The features and advantages of the present invention are summarized as follows:
[63] (a)본발명은수탁번호 KCTC13686BP인락토바실러스플란타럼 (LactoZwd/to plantarum) A41균주를제공한다. [63] (a) The present invention provides the Lactobacillus plantarum (LactoZwd/to plantarum) A41 strain with accession number KCTC13686BP.
[64] (b)본발명은수탁번호 KCTC13686BP인락토바실러스플란타럼 (Lactose plantarum) A41균주,또는이의배양물을유효성분으로포함하는약제학적 조성물및식품조성물을제공한다. [64] (b) The present invention provides a pharmaceutical composition and food composition comprising the Lactose plantarum A41 strain, or a culture thereof, with accession number KCTC13686BP as an active ingredient.
[65] 본발명의균주및그배양물은골다공증,특히이차성골다공증을예방하거나 개선,또는치료하기위한목적의식품조성물및약제학적조성물로유용하게 사용될수있다. [65] The strains and their cultures of the present invention can be usefully used as food compositions and pharmaceutical compositions for the purpose of preventing, ameliorating, or treating osteoporosis, especially secondary osteoporosis.
[66] [66]
도면의간단한설명 Brief description of the drawing
[67] 도 1은실험그룹별골밀도 (bone mineM density, mg/cm3)를나타낸도이다. 1 is a diagram showing bone mineM density, mg/cm 3 for each experimental group.
[68] 도 2는실험그룹별골비율 (percent bone volume, %)을나타낸도이다. 2 is a diagram showing the bone ratio (percent bone volume, %) for each experimental group.
[69] 도 3은실험그룹별장내칼슘수용단백질인 TRPV6의 mRNA발현량을 2020/091179 1»（：1^1{2019/007317 나타낸도이다.[69] Figure 3 shows the mRNA expression level of TRPV6, an intestinal calcium-receiving protein for each experimental group. 2020/091179 1»（：1^1{2019/007317 is shown.
이
Figure imgf000010_0001
발현량을 나타낸도이다.this
Figure imgf000010_0001
It is a diagram showing the expression level.
1] 도 5는실험그룹별장내칼슘수용단백질인 PMCA1의 mRNA발현량을 1] Figure 5 shows the mRNA expression level of PMCA1, an intestinal calcium-receiving protein for each experimental group.
나타낸도이다. It is a diagram shown.
2] 도 6은실험그룹별혈중칼슘농도를나타낸도이다. 2] Fig. 6 is a diagram showing the blood calcium concentration for each experimental group.
3] 도 7은실험그룹별 RANKL mRNA의발현량을나타낸도이다. 3] Figure 7 is a diagram showing the expression level of RANKL mRNA for each experimental group.
4] 도 8은실험그룹별 OPG mRNA의발현량을나타낸도이다. 4] Fig. 8 is a diagram showing the expression level of OPG mRNA for each experimental group.
5] 도 9는실험그룹별 BSP mRNA의발현량을나타낸도이다. 5] Fig. 9 is a diagram showing the expression level of BSP mRNA for each experimental group.
6] 6]
발명의실시를위한형태 Modes for the implementation of the invention
7] 이하,실시예를통하여본발명을더욱상세히설명하고자한다.이들실시예는 오로지본발명을보다구체적으로설명하기위한것으로,본발명의요지에 따라본발명의범위가이들실시예에의해제한되지않는다는것은당업계에서 통상의지식을가진자에있어서자명할것이다. 7] Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention more specifically, and the scope of the present invention is limited by these examples according to the gist of the present invention. It will be obvious to those of ordinary skill in the art that it does not.
8] 8]
9] 실시예 9] Examples
[8이 [8 this
[81] 본명세서전체에걸쳐,특정물질의농도를나타내기위하여사용되는
Figure imgf000010_0002
는 별도의언급이없는경우,고체/고체는（중량/중량） %,고체/액체는（중량/부피）[81] Throughout this specification, it is used to indicate the concentration of a specific substance.
Figure imgf000010_0002
Unless otherwise stated, solid/solid (weight/weight) %, solid/liquid (weight/volume)
%，그리고액체/액체는（부피/부피） %이다. %, and liquid/liquid (volume/volume) is %.
[82] [82]
[83] 실시예 1:프로바이오틱스균주및발효유의제조 [83] Example 1: Preparation of probiotic strain and fermentation milk
[84] 본실험에사용된프로바이오틱스균주들은영유아의분변과전통 [84] The probiotic strains used in this experiment are feces and traditions in infants and young children.
발효식품으로부터분리되었다（표 2） .본발명의발효유를제조하기위하여,탈지 우유知난!!! 1¾배지에상기에서분리한프로바이오틱스균주를각각접종하여 37ᄋ（：에서 48시간동안배양하였다.발효가완료된발효유는동결건조후분말화 하여 1 010 0^/1111농도로현탁하여실험동물에게급여하였다.본 발명에사용된탈지우유배지의조성과,프로바이오틱스균주목록은표 1및표 2에나타내었다. Separated from fermented food (Table 2). To manufacture the fermented milk of the present invention, skim milk Each of the probiotic strains isolated from the above was inoculated in 1¾ medium and cultured for 48 hours at 37°(:). Fermented milk after freeze-drying after fermentation was freeze-dried and suspended at a concentration of 1 0 10 0^/1111 to feed the experimental animals. The composition of the skim milk medium used in the present invention and a list of probiotic strains are shown in Tables 1 and 2.
[85] 2020/091179 1»(：1^1{2019/007317 [85] 2020/091179 1»(：1^1{2019/007317
[86] [표 1] [86] [Table 1]
본발명의탈지우유 (Skim milk)배지의조성 Composition of the skim milk medium of the present invention
Figure imgf000011_0001
Figure imgf000011_0001
[87] [표 2] [87] [Table 2]
본발명의프로바이오틱스균주목록 List of probiotic strains of the present invention
Figure imgf000011_0002
Figure imgf000011_0002
[88] [88]
[89] 실시예 2:이차성골다공증동물모델의제작및실험군설정 [89] Example 2: Preparation of secondary osteoporosis animal model and setting of experimental group
[9이 2.1이차성골다공증동물모델의제작 [9 Yi 2.1 Production of secondary osteoporosis animal model
[91] 이차성골다공증동물모델은다음과같이제작하였다.체중 175-185 g내외의 8주령 Wistar계암컷랫드 (Samtako, South Korea)를펠렛형사료와물을충분히 공급하여 1주일간개별케이지에적응시킨후실험에사용하였다.이차성 골다공증모델의수립은 Lucinda et al.(PHYTOTHERAPY RESEARCH 27(4), (2012), 515-520)와 Sousa et al. (Archives of Oral Biology 77 (2017) 55-61)에 의거하여근육내주사법으로글루코코르티코이드의일종인 [91] The secondary osteoporosis animal model was produced as follows: 8-week-old Wistar female rats (Samtako, South Korea) weighing 175-185 g were supplied with sufficient pellet-type feed and water, and adapted to individual cages for 1 week. The secondary osteoporosis model was established by Lucinda et al. (PHYTOTHERAPY RESEARCH 27(4), (2012), 515-520) and Sousa et al. (Archives of Oral Biology 77 (2017) 55-61), a type of glucocorticoid by intramuscular injection
덱사메타손 (Dexamethasone, DEX)을 7 mg/kg BW의농도로주 1회씩 5주간 투여하였다. Dexamethasone (DEX) was administered once a week at a concentration of 7 mg/kg BW for 5 weeks.
[92] [92]
[93] 2.2실험군의설정및발효유의급여 [93] 2.2 Establishment of experimental groups and benefits of fermentation
[94] 본발명의프로바이오틱스균주및발효유의이차성골다공증에대한 [94] Probiotic strain and fermentation-induced secondary osteoporosis of the present invention
치료효과를확인하기위하여,실험군을하기표 3과같이총 9개의그룹으로 분류하였다.각그룹별로실험동물을 7마리씩배정하였고, Normal그룹을 제외한모든그룹은상기 2.1의방법대로덱사메타손 (DEX)을주 1회 7 mg/kg(BW)의농도로 5주간투여하였다. To confirm the treatment effect, the experimental group was classified into 9 groups as shown in Table 3. 7 experimental animals were assigned to each group, and dexamethasone (DEX) was administered according to the method in 2.1 above for all groups except the normal group. It was administered for 5 weeks at a concentration of 7 mg/kg (BW) once.
[95] 모든발효유는 lxl01Q CFU/ml의농도로 PBS(Phosphate buffer saline)에희석하여 2020/091179 1»(：1^1{2019/007317 위관영양법 (0¾1 으로경구투여하였다.한편,본실험의목적이 프로바이오틱스를이용한우유발효물의항골다공증효과를확인하기 위함이기때문에대조군으로는발효를진행하지않은 3 111 111뾰투여군 (SMP)을 사용하였다. [95] All fermented milk was diluted in PBS (Phosphate buffer saline) at a concentration of lxl0 1Q CFU/ml. 2020/091179 1»(：1^1{2019/007317 It was administered orally by gavage (0¾1). On the other hand, fermentation proceeds as a control because the purpose of this experiment is to confirm the anti-osteoporosis effect of milk fermentation using probiotics. Untreated 3 111 111 puncture group (SMP) was used.
[96] [표 3] [96] [Table 3]
본발명의실험군설정 Experimental group setting of the present invention
Figure imgf000012_0001
Figure imgf000012_0001
[97] [97]
[98] 실시예 3:본발명의발효유투여에따른골의형태학적분석 [98] Example 3: Morphological Analysis of Bone Following Administration of Fermentation Oil of the Invention
[99] 골밀도 (Bone mineral density)와골비율 (Percent bone volume)은뼈의강도를 측정할때가장기본적으로확인하는지표이다.본발명의프로바이오틱스균주 및발효유의골밀도개선효과를측정하기위하여, Micro Ct영상분석기 (Sky Scan 1176)와이를재구성하는프로그램 (NRecon),그리고이를분석하는 [99] Bone mineral density and percent bone volume are the most basic checks when measuring bone strength. To measure the effect of improving the bone density of the probiotic strains and fermented milk of the present invention, Micro Ct imaging Analyzer (Sky Scan 1176), a program to reconstruct it (NRecon), and
프로그램 (CT An)을이용하였다. The program (CT An) was used.
[10이 실험결과,덱사메타손을투여한 DEX그룹에서는골밀도및골비율이 Normal 그룹대비유의적으로감소한것으로보아이차성골다공증이유도됨을확인할 수있었다.이차성골다공증약물 (alendrcmate)을덱사메타손과함께투여한 Drug 그룹에서는덱사메타손투여로인해감소된골밀도와골비율이회복된것으로 나타났다.본발명의프로바이오틱스균주로발효시킨발효유를투여한 A41 그룹, SRK414그룹, B719그룹,및 393그룹도 drug그룹과유사한정도의골밀도 및골비율회복효과를나타내었다. (도 1및도 2) [10 As a result of this experiment, it was confirmed that secondary osteoporosis was induced in the DEX group administered with dexamethasone, as the bone density and bone ratio decreased significantly compared to the normal group. The drug group administered a secondary osteoporosis drug (alendrcmate) together with dexamethasone In Figure 2, the reduced bone density and bone ratio were recovered due to the administration of dexamethasone. The A41 group, SRK414 group, B719 group, and 393 group administered with fermented milk fermented with the probiotic strain of the present invention also had similar bone density and bone density to the drug group. The ratio recovery effect was shown. (Figure 1 and Figure 2)
[101] 상기결과로부터,본발명의프로바이오틱스균주인 L. plantarum A41, L. [101] From the above results, L. plantarum A41, L. plantarum A41, the probiotic strain of the present invention.
fermentum SRK414, L. plantarum B719, L. casei 393으로발효시킨발효유가 덱사메타손의투여로유발된이차성골다공증의증상인골밀도,골비율감소를 회복시킴을확인하였다. It was confirmed that fermented milk fermented with fermentum SRK414, L. plantarum B719, and L. casei 393 recovered the reduction in bone density and bone ratio, which are symptoms of secondary osteoporosis caused by the administration of dexamethasone.
[102] 2020/091179 1»(：1^1{2019/007317 [102] 2020/091179 1»(：1^1{2019/007317
[103] [103]
[104] 실시예 4.본발명의발효유투여에의한칼슘흡수관련지표분석 [104] Example 4 Analysis of Indicators Related to Calcium Absorption by Administration of Fermented Oil of the Invention
[105] 혈중칼슘농도가부족할경우,뼈에있는칼슘이혈중으로유리되어골소실이 증가하게된다.따라서장내에서칼슘흡수를촉진시킴으로써혈중칼슘농도를 증가시키는것은골소실을낮출수있는중요한예방책이될수있다.칼슘은 장에서칼슘을이동시
Figure imgf000013_0001
[105] If the blood calcium concentration is insufficient, bone loss increases as calcium in the bones is released into the blood. Therefore, increasing the calcium concentration in the blood by promoting calcium absorption in the intestine is an important preventive measure that can reduce bone loss. Calcium transfers calcium from the intestine
Figure imgf000013_0001
등에의해소장에서혈관으로유입되는데,이들의발현량이증가한다면칼슘 흡수가원활히진행된다고볼수있다.따라서본발명자들은본발명의발효유 투여에따른장내칼슘수용단백질의 mRNA발현량을알아보기위해서 다요! - 1（11분석을실시하였으며,혈중칼슘농도와의관련성을확인하였다. It is introduced into the blood vessels from the small intestine by the back, and if their expression increases, it can be said that calcium absorption proceeds smoothly. Therefore, the present inventors are done to find out the amount of mRNA expression of the intestinal calcium-receiving protein according to the administration of the fermented milk of the present invention! -1（11 analyzes were conducted, and the relationship with blood calcium concentration was confirmed.
[106] [106]
[107] 4.1장내칼슘수용체유전자발현확인 [107] 4.1 Confirmation of expression of intestinal calcium receptor gene
[108] 발효유가
Figure imgf000013_0002
발현량에 미치는효과를알아보기위해서다요! -：的¾분석을진행하였다.구체적으로,실험 기간종료후각실험동물의회장을적출하여균질화（110111（¾£11뇨£（1）과정을거친 후 1요뇨01（111 1]'（¾11句을사용하여조직에포함되어있는 RNA를추출하였다. 정제된 RNA는역전사효소 01^0（（11）프라이머와함께 cDNA합성에 [108] fermented milk prices
Figure imgf000013_0002
To see the effect on the expression level! -: 的¾ analysis was conducted. Specifically, after the end of the experiment period, the halls of each experimental animal were excised and homogenized (110111（ ¾£11 urine £ (1)) and then 1 urinary urine 01 (111 1)' (¾11句RNA contained in the tissue was extracted using. Purified RNA is used for cDNA synthesis with reverse transcriptase 01^0 ((11) primer.
사용되었으며 , 1＞幻1조건은 95ᄋ（：에서 3분이후, 94ᄋ（：에서 초 60ᄋ（:에서 It was used, and the condition 1＞幻1 was 95ᄋ（： at 3 minutes later, 94ᄋ（： at seconds 60 ᄋ（: at
30초로,총 44주기반복하였다. ¾요1'-1）（그분석을위한프라이머는표 4에 나타내었다.총전사의정도는（3 - 전사레벨로부터 In 30 seconds, a total of 44 weeks were restored. ¾ required 1'-1) (Primers for the analysis are shown in Table 4. The degree of total warriors is (3-from warrior level)
정규화 010]111 뇨 1011）하여그래프로나타내었다（도 3내지도 5）. Normalized 010]111 urine 1011) and displayed as a graph (Figs. 3 to 5).
[109] [109]
[110] [표 4] [110] [Table 4]
Figure imgf000013_0004
Figure imgf000013_0004
[111] 도 3내지 5에서나타낸바와같이,본발명의발효유투여그룹중에서쇼41 그룹및 3요 414그룹이다른그룹에비해상대적으로높은장내칼슘수용 단백질（
Figure imgf000013_0003
발현량을나타내었다（도 3내지도 5）. [111] As shown in Figs. 3 to 5, in the fermented milk administration group of the present invention, the show 41 group and the 3 yo 414 group are relatively high in the intestinal calcium-receiving protein (
Figure imgf000013_0003
The expression level was shown (Figures 3 to 5).
[112] [112]
[113] 4.2혈중칼슘농도조절효과 [113] 4.2 Effect of controlling calcium concentration in blood
[114] 본발명의발효유투여가혈중칼슘농도에미치는영향을확인하기위하여, 2020/091179 1»(：1^1{2019/007317 혈중칼슘농도를확인하였다.구체적으로,동물실험기간종류후 [114] To confirm the effect of the administration of fermented milk of the present invention on blood calcium concentration, 2020/091179 1»(：1^1{2019/007317 Blood calcium concentration was confirmed. Specifically, after the animal experiment period
심장천자 (heart puncture)법으로혈액을채취하였으며원심분리 (2500 g, 15분, 4 )하여수집한혈청을분석전까지 -70 에보관하였다.혈청내에있는 칼슘 (Ca)농도는 Calcium activity assay kit ((주)엠비엘, Korea)를사용하여 분석하였다.결과는도 6에나타내었다. Blood was collected by the heart puncture method, and the collected serum was centrifuged (2500 g, 15 minutes, 4) and stored at -70 until analysis. Calcium activity assay kit (calcium activity assay kit) MBL Co., Ltd., Korea) was used to analyze the results. The results are shown in FIG.
[115] 도 6에나타낸바와같이,장내칼슘수용단백질의발현량과마찬가지로본 발명의발효유투여그룹중에서 A41그룹및 SRK414그룹이다른그룹에비해 상대적으로높은혈중칼슘농도를나타냄을확인하였다.따라서본발명의 L. plantarum A41,및 L. fermentum SRK414균주에의해발효된발효유는,발효유가 투여된동물의소장내칼슘흡수를촉진시킴으로써높은혈중칼슘농도 유지하여골소실을효과적으로예방할수있다고사료된다. [115] As shown in Fig. 6, it was confirmed that among the fermented milk administration group of the present invention, the A41 group and SRK414 group showed a relatively higher blood calcium concentration than the other groups, similar to the expression amount of the intestinal calcium-receiving protein. Therefore, the present invention Fermented milk fermented by L. plantarum A41 and L. fermentum SRK414 strains of L. plantarum A41 and L. fermentum SRK414 are believed to be able to effectively prevent bone loss by maintaining high blood calcium concentration by promoting calcium absorption in the small intestine of animals to which fermented milk was administered.
[116] [116]
[117] [117]
[118] 실시예 5.본발명의발효유투여에따른골대사관련지표분석 [118] Example 5. Analysis of indicators related to bone metabolism according to administration of fermentation oil of the present invention
[119] 조골세포또는활성화된면역세포에서 RANKL(Receptor Activator of Nuclear Factor Kappa B Ligand)이발현되면파골전구세포세포막의 RANK(Receptor Activator of Nuclear Factor Kappa B)와결합하여파골세포의분화가 [119] When RANKL (Receptor Activator of Nuclear Factor Kappa B Ligand) is expressed in osteoblasts or activated immune cells, it binds to RANK (Receptor Activator of Nuclear Factor Kappa B) in the osteoclast cell membrane, thereby preventing the differentiation of osteoclasts.
이루어지는데,이경우뼈흡수가촉진되어골다공증이유발될수있다.반면 OPG(Osteoprotegerin)는 RANKL대신 RANK에수용되는인자로, OPG가 In this case, bone resorption may be promoted, leading to osteoporosis. On the other hand, OPG (Osteoprotegerin) is a factor that is accepted by RANK instead of RANKL, and OPG is
RANK와 RANKL의결합을방해함으로써파골세포의분화를억제시킬수있다. The differentiation of osteoclasts can be inhibited by interfering with the binding of RANK and RANKL.
[120] 본발명자들은본발명의발효유투여가골대사에미치는영향을확인하기 위하여 ,골대사관련마커인 RANKL, OPG,및 BSP의 mRNA발현양을 [120] The present inventors determined the amount of mRNA expression of RANKL, OPG, and BSP, which are bone metabolism-related markers, to confirm the effect of the administration of the fermentation oil of the present invention on bone metabolism.
qRT-PCR을통해분석하였다.구체적으로,실험동물의넙다리뼈와골피질을 균질화 (homogenization)한후 TRIzol reagent(Thermofisher, USA)를이용해종 RNA를추출하였다.정제된총 RNA는 cDNA합성을위해사용하였다. PCR은 Biorad RT-PCR을사용하였으며 95 OC에서 3분간처리한후, 94 OC에서 10초, 60 OC에서 30초를총 44주기반복하였다. qRT-PCT분석을위한프라이머는표 5에 나타내었다.총전사의정도는 (3-actin전사레벨로부터 Analysis was carried out by qRT-PCR. Specifically, after homogenization of the femur and bone cortex of experimental animals, RNA was extracted using TRIzol reagent (Thermofisher, USA). The purified total RNA was used for cDNA synthesis. Used. For PCR, Biorad RT-PCR was used, and after 3 minutes at 95 O C, 10 seconds at 94 O C and 30 seconds at 60 OC were repeated for a total of 44 weeks. The primers for qRT-PCT analysis are shown in Table 5. The degree of total transcription was (from the 3-actin transcription level)
정규화 (normalization)하여그래프에나타내었다 (도 7내지 9). Normalized and shown in the graph (Figs. 7 to 9).
[121] [표 5] [121] [Table 5]
Figure imgf000014_0001
2020/091179 1»（：1^1{2019/007317
Figure imgf000014_0001
2020/091179 1»（：1^1{2019/007317
[122] 도 7에나타낸바와같이,쇼41그룹,및 311X414그룹에서
Figure imgf000015_0001
그룹에비해
Figure imgf000015_0002
미하게감소하여쇼41과 311X414발효유가 파골세포의분화를감소시키는것을확인하였다. [122] As shown in Fig. 7, in the show 41 group and the 311X414 group
Figure imgf000015_0001
Compared to the group
Figure imgf000015_0002
It was confirmed that the fermented milk of Sho41 and 311X414 reduced the differentiation of osteoclasts by a slight decrease.
[123] 또한,도 8에나타낸바와같이,
Figure imgf000015_0003
제외한 모든발효유투여그룹에서모두유의미하게증가하여파골세포분화의억제를 확인하였다. [123] Also, as shown in Fig. 8,
Figure imgf000015_0003
All except the fermented milk administration group significantly increased, confirming the inhibition of osteoclast differentiation.
[124] 마지막으로,도 9에나타낸바와같이,궁극적인조골세포분화표지인자인 6815여01祀 8 1애] 11）발현량역시쇼41, 811X414그리고 6기 9그룹에서 상대적으로높은발현량을나타내어조골세포분화가촉진되어골다공증의 치료효과가뛰어남을확인할수있었다. [124] Finally, as shown in Fig. 9, the ultimate osteoblast differentiation marker, 681 5 female 01 祀 8 1 ae] 11) The expression level was also relatively high in the 9 groups of show 41, 811X414 and 6th stage. It was confirmed that osteoblast differentiation was promoted and the treatment effect of osteoporosis was excellent.
[125] 상기결과를바탕으로본발명의 I」. ]31&1^표1'11111쇼41균주,및 I」. 6]1116111；11111 811X414균주로발효시킨발효유를동물모델에투여하였을때체내조골세포가 0?0생성을촉진시키고 RANKL의생성을억제시켜파골세포의분화를 억제하고,조골세포의분화를촉진함으로써이차성골다공증에대한예방 효과를나타내는것을확인하였다. [125] Based on the above results, I of the present invention". ]3 1&1^Table 1'11111 Show 41 strain, and I". 6] 1116111; 11111 When fermented milk fermented with strain 811X414 is administered to an animal model, osteoblasts in the body promote 0 to 0 production, inhibit the production of RANKL, inhibit the differentiation of osteoclasts, and promote the differentiation of osteoblasts. It was confirmed that it exhibits a preventive effect on secondary osteoporosis.
[126] [126]
[127] [수탁번히 [127] [Confidentially
[128] [기탁기관명]한국생명공학연구원 [128] [Name of deposit institution] Korea Research Institute of Bioscience and Biotechnology
[129] [수탁
Figure imgf000015_0004
[129] [Consignment
Figure imgf000015_0004
[130] [수탁일자] 20181025 [130] [Consignment Date] 20181025
[131] [131]
[132] [기탁기관명]한국생명공학연구원 [132] [Name of deposit institution] Korea Research Institute of Bioscience and Biotechnology
[133] [수탁
Figure imgf000015_0005
[133] [Consignment
Figure imgf000015_0005
[134] [수탁일자] 20181025 [134] [Consignment Date] 20181025
[135] [135]
\¥0 2020/091179 1»（：1^1{2019/007317\¥0 2020/091179 1»（：1^1{2019/007317
[1 36] 특허奪차상 며營·還· 기학의 국제적 승'?]에; #¾ [1 36] To the international victory of patent 奪cha award and 營·還· kihak'?];#¾
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정정용지 （규칙 제 91조） / \¥02020/091179 1»（：1^1{2019/007317Correction sheet (Article 91 of the Rules) / \¥02020/091179 1»（：1^1{2019/007317
[137] [137]
，허奪聲公피.寒卷 지탁의 국#적 安안에 德意 ，Heo 奪聲公Pi .寒卷 Ji-Tak’s national character security
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¾기탁에 대한 수탁증 ¾ trust certificate for deposit
Figure imgf000017_0001
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정정용지 （규칙 제 91조） / Correction sheet (Article 91 of the Rules) /

Claims

2020/091179 1»（：1/10公019/007317 청구범위 2020/091179 1»（：1/10公019/007317 Claims
［청구항 1］
Figure imgf000018_0001
[Claim 1]
Figure imgf000018_0001
plantarum) A41균주. plantarum) A41 strain.
［청구항 2］ 수탁번호 KCTC13686BP인락토바실러
Figure imgf000018_0002
[Claim 2] Accession No. KCTC13686BP Inlactoba Sealer
Figure imgf000018_0002
plantarum) A41균주,또는이의배양물을유효성분으로포함하는 골다공증예방또는치료용약제학적조성물. plantarum) A pharmaceutical composition for preventing or treating osteoporosis, comprising the A41 strain, or a culture thereof as an active ingredient.
［청구항 3］ 제 2항에 있어서,상기골다공증은이차성골다공증인,약제학적조성물. ［청구항 4］ 제 2항에 있어서,상기조성물은락토바실러
Figure imgf000018_0003
[Claim 3] The pharmaceutical composition according to claim 2, wherein the osteoporosis is secondary osteoporosis. [Claim 4] According to claim 2, the composition is lactobacilli
Figure imgf000018_0003
fermentum)，락토바실러스카제이 (Lactobacillus casei),락토바실러스 애시도필러ᅀ (Jxictobadllus acidophilus),락토바실러스불가리쿠스 ( Lactobacillus delbrueckii ssp bulgaricus),
Figure imgf000018_0004
fermentum), Lactobacillus casei, Lactobacillus acidophilus (Jxictobadllus acidophilus), Lactobacillus delbrueckii ssp bulgaricus,
Figure imgf000018_0004
lactis),엔테로코커스 ^ll ^-(Enterococcus faecium),엔테로코커스 페칼리 :느 (Enterococcus fecalis),스트렙토코커스써모필러스: (Streptococcus thermophilus),비피도박테리움비피덤 (Bifidobacterium bifidum),및비피도
Figure imgf000018_0005
lactis)로이루어진군으로부터선택되는 1종이상의균,또는이의배양물을추가적으로포함하는것인,약제학적 조성물. lactis), Enterococcus ^ll ^-(Enterococcus faecium), Enterococcus fecalis, Streptococcus thermophilus: (Streptococcus thermophilus), Bifidobacterium bifidum, and Bifido
Figure imgf000018_0005
lactis), one or more bacteria selected from the group consisting of, or a culture thereof, additionally comprising a pharmaceutical composition.
［청구항 5］ 수탁번호 KCTC13686BP인락토바실러
Figure imgf000018_0006
[Claim 5] Accession No. KCTC13686BP Inlactoba Sealer
Figure imgf000018_0006
plantarum) A41균주,또는이의배양물을유효성분으로포함하는 골다공증예방또는개선용식품조성물. plantarum) A food composition for preventing or improving osteoporosis, comprising the A41 strain or a culture thereof as an active ingredient.
［청구항 6］ 제 5항에 있어서,상기골다공증은이차성골다공증인,식품조성물. [Claim 6] The food composition according to claim 5, wherein the osteoporosis is secondary osteoporosis.
［청구항 7］ 제 5항에 있어서,상기식품조성물은발효유인,조성물. [Claim 7] The composition according to claim 5, wherein the food composition is a fermentation oil.
［청구항 8］ 제 5항에 있어서,상기조성물은락토바실러스
Figure imgf000018_0007
[Claim 8] According to claim 5, the composition is lactobacilli
Figure imgf000018_0007
fermentum)，락토바실러스카제이 (Lactobacillus casei),락토바실러스 애시도필러ᅀ (Jxictobadllus acidophilus),락토바실러스불가리쿠스 ( Lactobacillus delbrueckii ssp bulgaricus),
Figure imgf000018_0008
fermentum), Lactobacillus casei, Lactobacillus acidophilus (Jxictobadllus acidophilus), Lactobacillus delbrueckii ssp bulgaricus,
Figure imgf000018_0008
lactis),엔테로코커스 ^ll ^-(Enterococcus faecium),엔테로코커스 페칼리 :느 (Enterococcus fecalis),스트렙토코커스써모필러스: (Streptococcus thermophilus),비피도박테리움비피덤 (Bifidobacterium bifidum),및비피도
Figure imgf000018_0009
lactis)로이루어진군으로부터선택되는 1종이상의균,또는이의배양물을추가적으로포함하는것인, 식품조성물. lactis), Enterococcus ^ll ^-(Enterococcus faecium), Enterococcus fecalis, Streptococcus thermophilus: (Streptococcus thermophilus), Bifidobacterium bifidum, and Bifido
Figure imgf000018_0009
lactis), one or more bacteria selected from the group consisting of, or a food composition further containing a culture thereof.
［청구항 9］ 수탁번호 KCTC13686BP인락토바실러
Figure imgf000018_0010
[Claim 9] Accession No. KCTC13686BP Inlactoba Sealer
Figure imgf000018_0010
plantarum) A41균주,또는이의배양물을유효성분으로포함하는 조성물을대상체에투여하는단계를포함하는,골다공증의 예방,개선 또는치료방법 . plantarum) A41 strain, or a method for preventing, improving, or treating osteoporosis, comprising administering to a subject a composition containing the active ingredient thereof.
［청구항 ］ 제 9항에 있어서,상기골다공증은이차성골다공증인,방법. [Claim] The method according to claim 9, wherein the osteoporosis is secondary osteoporosis.
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