WO2020091180A9 - 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

Info

Publication number
WO2020091180A9
WO2020091180A9 PCT/KR2019/007320 KR2019007320W WO2020091180A9 WO 2020091180 A9 WO2020091180 A9 WO 2020091180A9 KR 2019007320 W KR2019007320 W KR 2019007320W WO 2020091180 A9 WO2020091180 A9 WO 2020091180A9
Authority
WO
WIPO (PCT)
Prior art keywords
lactobacillus
osteoporosis
present
strain
culture
Prior art date
Application number
PCT/KR2019/007320
Other languages
French (fr)
Korean (ko)
Other versions
WO2020091180A2 (en
WO2020091180A3 (en
Inventor
이인옥
박나현
김병국
이철상
김세헌
최인석
Original Assignee
주식회사 종근당바이오
고려대학교 산학협력단
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
Application filed by 주식회사 종근당바이오, 고려대학교 산학협력단 filed Critical 주식회사 종근당바이오
Publication of WO2020091180A2 publication Critical patent/WO2020091180A2/en
Publication of WO2020091180A3 publication Critical patent/WO2020091180A3/en
Publication of WO2020091180A9 publication Critical patent/WO2020091180A9/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • 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
    • A23V2400/169—Plantarum
    • 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
    • C12R2001/25—Lactobacillus plantarum

Definitions

  • the present invention was made by the project number IPET117051-3 under the support of the Ministry of Agriculture, Food and Rural Affairs of the Republic of Korea.
  • the research and management professional institution for the project is the Agency for Food and Technology Planning and Evaluation, the name of the research project is "High Value Added Food Technology Development Project”, and the name of the research project is " “Development of functional probiotics products with the effect of improving primary and secondary osteoporosis", the main institution is Chong Kun Dang Bio, the research period is from June 15, 2017 to December 31, 2019.
  • the present invention relates to a composition for preventing or treating secondary osteoporosis comprising 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. It is classified into two categories. It is easy to think that osteoporosis is a disease of old age, but secondary osteoporosis can develop even when not in old age, and in this case, the effect on the quality of life of patients in the future is very large, so that the treatment and prevention methods for secondary osteoporosis are developed. Is very important.
  • Glucocorticoid has anti-inflammatory and immunosuppressive properties and is a commonly used drug for the treatment of various diseases such as rheumatoid arthritis, asthma, atopic dermatitis, and arthritis. Long-term administration of GC effectively inhibits these diseases, but in most patients, it inhibits the normal activity of osteoblasts and osteoclasts, resulting in osteoporosis.
  • the present inventors have attempted to develop a probiotic strain that can be applied as food and pharmaceuticals for preventing, improving or treating osteoporosis in the future, and fermented milk using the same. As a result, it was found that when fermented milk was prepared using a probiotic strain and then administered orally to glucocorticoid-induced secondary osteoporosis experimental animals, bone mineral density and bone ratio were increased, and expression of genes related to calcium-related absorption and metabolism was increased. The present invention has been completed.
  • an object of the present invention is to provide a Lactobacillus fermentum SRK414 strain with accession number KCTC13687BP.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating osteoporosis comprising the Lactobacillus fermentum SRK414 strain, or a culture thereof as an active ingredient.
  • Another object of the present invention is to provide a food composition for preventing or improving osteoporosis comprising the Lactobacillus fermentum SRK414 strain, or a culture thereof as an active ingredient.
  • Another object of the present invention is to provide a method for preventing, improving, or treating osteoporosis comprising administering to a subject a composition comprising the Lactobacillus fermentum SRK414 strain of accession number KCTC13687BP as an active ingredient.
  • the present invention provides a Lactobacillus fermentum ( Lactobacillus fermentum ) SRK414 strain of accession number KCTC13687BP.
  • the present inventors newly isolated Lactobacillus fermentum SRK414 strain (accession number: KCTC13687BP).
  • the present inventors prepared a fermented milk using the Lactobacillus fermentum SRK414 strain (accession number: KCTC13687BP).
  • KCTC13687BP accession number: KCTC13687BP
  • the expression level of calcium absorption-related proteins expressed in the intestine of the experimental animal model administered with the fermented milk is increased, the calcium concentration in the blood is increased, and the expression level of genes related to the inhibition of osteoclast differentiation and promotion of osteoblast differentiation was confirmed.
  • the fermented milk prepared using the Lactobacillus fermentum SRK414 strain (accession number: KCTC13687BP) of the present invention can be usefully used in the prevention, improvement, or treatment of secondary osteoporosis.
  • osteoporosis refers to a "systemic skeletal system disease characterized by a decrease in bone mass and abnormalities in microstructures, and as a result, a disease in which the bone becomes weak and becomes brittle".
  • Bone strength is determined by bone quantity and quality. Bone mass is mainly expressed by bone density (BMD), and bone quality is composed of structure, bone replacement rate, mineralization, and accumulation of micro-damages.
  • 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 it is known that it is difficult to accurately classify it because it is combined and progressed at about the same time.
  • Secondary osteoporosis refers to an increase in the risk of fracture due to changes in the microstructure of bones and decreases in bone mass due to underlying diseases or drugs.
  • the causes of secondary osteoporosis include endocrine diseases (thyroidism, diabetes, hyperparathyroidism, etc.), gastrointestinal diseases (gastric resection lines, chronic liver disease, malabsorption syndrome, inflammatory bowel disease, alcoholism, etc.), bone marrow disease, connective tissue disease ( Ankylosing spondylitis, rheumatoid arthritis, bone insufficiency, etc.), and drugs.
  • endocrine diseases thyroidism, diabetes, hyperparathyroidism, etc.
  • gastrointestinal diseases gastric resection lines, chronic liver disease, malabsorption syndrome, inflammatory bowel disease, alcoholism, etc.
  • bone marrow disease connective tissue disease ( Ankylosing spondylitis, rheumatoid arthritis, bone insufficiency, etc.)
  • drugs The most common causes in men are hypogonadism and glucocorticoid administration.
  • the osteoporosis is secondary osteoporosis, more specifically secondary osteoporosis caused by administration of a glucocorticoid drug.
  • the glucocorticoid-based drug is cortisol, prednisone, prednisolone, methylprednisolone, cortisone, hydroxycortisone, triamcinolone, betamethasone, or dexamethasone, but is not limited thereto.
  • Lactobacillus fermentum SRK414 strain is deposited under the accession number "KCTC13687BP" in the "Korean Collection for Type Culture (KCTC)".
  • the cell properties of the Lactobacillus fermentum SRK414 strain are as follows:
  • L. fermentum is a member of the genus Lactobacillus , and the species belonging to this genus are said to have various uses. These uses include food and feed fermentation. L. fermentum Several strains of these have been found to have natural resistance to antibiotics and chemotherapeutic agents.
  • the present invention provides a pharmaceutical composition for preventing or treating osteoporosis comprising the Lactobacillus fermentum SRK414 strain, or a culture thereof, of accession number KCTC13687BP, as an active ingredient.
  • the present invention provides a food composition for preventing or improving osteoporosis comprising the Lactobacillus fermentum SRK414 strain of accession number KCTC13687BP, or a culture thereof as an active ingredient.
  • the composition comprises as an active ingredient Lactobacillus Planta column (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei), Lactobacillus ash FIG pillar's (Lactobacillus acidophilus), Lactobacillus Bulgaria kusu (Lactobacillus delbrueckii ssp bulgaricus ), Lactococcus lactis , Enterococcus faecium ), Enterococcus fecalis , Streptococcus thermophilus , Bifidobacterium Bifidobacterium bifidum ), and Bifidobacterium lactis lactis ) and one or more fungi selected from the group consisting of, or a culture thereof.
  • Lactobacillus Planta column Lactobacillus plantarum
  • Lactobacillus casei Lactobacillus casei
  • Lactobacillus ash FIG pillar's Lacto
  • the Lactobacillus plantarum may be a Lactobacillus plantarum A41 strain (accession number KCTC13686BP).
  • Lactobacillus plantarum A41 strain is deposited with the accession number "KCTC13686BP" to the "Korea Research Institute of Bioscience and Biotechnology Microbial Resource Center (Korean Collection for Type Culture, KCTC)".
  • Lactobacillus plantarum A41 strain The microbial properties of the Lactobacillus plantarum A41 strain are as follows:
  • L. plantarum is a member of the genus Lactobacillus , is generally found in many fermented foods and anaerobic plant materials, and is also present in saliva.
  • the 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 strain, extracts of cells, supernatant of cultures, concentrates, concentrates thereof, It is a dried product, and, if necessary, a dilution liquid, a dilution product, etc., and includes a culture solution and any condition obtained by treating the culture product.
  • the culture method, extraction method, separation method, concentration method, drying method, dilution method, etc. of the cells are not particularly limited.
  • the medium for culturing the cells typically contains milk proteins such as skim milk, whey, and casein, sugars, yeast extract, and the like, and as a culture method, various general aerobic or anaerobic methods can be appropriately used.
  • the culture temperature for example, 35 ⁇ 45°C is set, and during the 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. May be.
  • an alkali such as sodium hydroxide
  • any culture method such as a batch culture method may be used. After the culture, the culture product or its supernatant may be concentrated, dried, or diluted if necessary.
  • centrifugation or membrane separation may be used to separate the supernatant and the cells of the culture, and the cells may be recovered in a concentrated state. Then, the cells may be subjected to ultrasonic treatment, enzyme treatment, or the like to extract components within the cells, or to dry the culture product, its supernatant, the cells or its extract, and the like. These can be used as an active ingredient of the composition of the present invention.
  • composition of the present invention when prepared as a food composition, it may include not only the lactic acid bacteria as an active ingredient, but also ingredients that are commonly added during food production.
  • the additive ingredients include, for example, proteins, carbohydrates, fats, nutrients, flavoring agents and flavoring agents.
  • the carbohydrates include monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, oligosaccharides, etc.), and polysaccharides (e.g., dextrin, cyclodextrin, etc.) Sugar and sugar alcohols such as xylitol, sorbitol, erythritol, etc.
  • flavoring agents natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartic acid) Tom, etc.) can be used.
  • the food composition of the present invention when prepared as a drink, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, jujube extract, licorice extract, etc. can be additionally included in addition to the above strain, which is an active ingredient of the present invention. have.
  • the food composition of the present invention includes processed forms of all natural materials such as food, functional food, nutritional supplement, health food and food additives.
  • Food compositions of this type may be prepared in various forms according to conventional methods known in the art.
  • the lactic acid bacteria itself may be prepared in the form of tea, juice, and drinks to be consumed, or granulated, encapsulated, and powdered to be consumed.
  • foods include beverages (including alcoholic beverages), fruits and processed foods thereof (e.g., canned fruit, bottled, jam, marmalade, etc.), fish, meat and processed foods thereof (e.g. ham, sausage corn beef, etc.) ), bread and noodles (e.g. udon, soba, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, syrup, dairy products (e.g.
  • yogurt, fermented milk, butter, cheese, etc. edible vegetable oil, margarine, Vegetable protein, retort food, frozen food, various seasonings (eg, miso, soy sauce, sauce, etc.) can be prepared by adding the lactic acid bacteria of the present invention.
  • lactic acid bacteria of the present invention in the form of a food additive, it may be prepared and used in the form of a powder or a concentrate.
  • the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used at the time of formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, etc. It does not become.
  • the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components.
  • the pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably applied by way of oral administration.
  • the pharmaceutical composition of the present invention may be formulated in the following various oral or parenteral dosage forms, but is not limited thereto.
  • Formulations for oral administration include, for example, tablets, pills, hard/soft capsules, solutions, suspensions, emulsifiers, and syrups. There are granules, elixirs, and the like, and these formulations may include one or more diluents or excipients such as fillers, extenders, wetting agents, disintegrants, lubricants, binders, surfactants, etc., which are commonly used in addition to the active ingredients. Agar, starch, alginic acid or its sodium salt, anhydrous monohydrogen phosphate calcium salt, etc.
  • the disintegrant may be used as the disintegrant, and as the lubricant, silica, talc, stearic acid or its magnesium salt or calcium salt, polyethylene glycol, etc. may be used.
  • the binder magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine, low-substituted hydroxypropylcellulose, and the like may be used.
  • lactose, dextrose, sucrose, mannitol, sorbitol, and cellulose. Glycine or the like may be used as a diluent, and in some cases, commonly known boiling mixtures, absorbents, colorants, flavors, sweeteners, and the like may be used together.
  • the composition may be sterilized or contain adjuvants such as preservatives, stabilizers, hydrating agents or emulsification accelerators, salts for regulating osmotic pressure, buffers, and other therapeutically useful substances, which are conventional methods of mixing, granulating or coating. It can be formulated according to.
  • the appropriate dosage of the pharmaceutical composition of the present invention is prescribed in various ways depending on factors such as the formulation method, the mode of administration, the patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity. Can be.
  • the pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the technical field to which the present invention belongs. Or it can be made by incorporating it into a multi-dose container.
  • the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.
  • the present invention provides a method for preventing, improving, or treating osteoporosis comprising administering to a subject a composition comprising the Lactobacillus fermentum SRK414 strain of accession number KCTC13687BP as an active ingredient. to provide.
  • Osteoporosis which is the target disease of the treatment method or improvement method of the present invention, is the same as defined in relation to the disease to be treated by the pharmaceutical composition.
  • the subject is a mammal or human.
  • the method for treating or improving osteoporosis of the present invention is a method of using the same active ingredient as a composition containing the above-described accession number KCTC13687BP, Lactobacillus fermentum SRK414 strain, or a culture thereof as an active ingredient, and thus overlapping Description of the contents will be omitted to avoid excessive complexity of the present specification.
  • the present invention provides a Lactobacillus fermentum ( Lactobacillus fermentum ) SRK414 strain of accession number KCTC13687BP.
  • the present invention provides a pharmaceutical composition and a food composition comprising the accession number KCTC13687BP Lactobacillus fermentum SRK414 strain, or a culture thereof as an active ingredient.
  • strains and cultures thereof of the present invention can be usefully used as food compositions and pharmaceutical compositions for the purpose of preventing, improving, or treating osteoporosis, particularly secondary osteoporosis.
  • FIG. 1 is a diagram showing bone mineral density (mg/cm 3 ) for each experimental group.
  • Figure 2 is a diagram showing the bone ratio (percent bone volume, %) for each experimental group.
  • FIG. 3 is a diagram showing the mRNA expression level of TRPV6, an intestinal calcium receptor protein for each experimental group.
  • FIG. 4 is a diagram showing the mRNA expression level of Calbindin-D9k, an intestinal calcium receptor protein for each experimental group.
  • FIG. 5 is a diagram showing the mRNA expression level of PMCA1, an intestinal calcium receptor protein, for each experimental group.
  • FIG. 6 is a diagram showing blood calcium concentration for each experimental group.
  • FIG. 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.
  • % used to indicate the concentration of a specific substance is (weight/weight)% for solids/solids, (weight/volume)% for solids/liquids, and Liquid/liquid is (vol/vol) %.
  • Example One Probiotics Preparation of strains and fermented milk
  • the probiotic strains used in this experiment were isolated from infant feces and traditional fermented foods (Table 2).
  • skim milk (skim milk medium) was inoculated with the separated probiotic strain for 48 hours at 37° C.
  • the fermented milk was freeze-dried, powdered, and 1 ⁇ in PBS. It was suspended at a concentration of 10 10 CFU/ml and fed to the experimental animals
  • the composition of the skim milk medium used in the present invention and the list of probiotic strains are shown in Tables 1 and 2.
  • composition of the Skim milk medium of the present invention Serial number Raw material name Capacity (g/L)
  • One Skim milk powder 100 2 Glucose 20 3 Yeast extract 3 4 Bacto peptone 3
  • the secondary osteoporosis animal model was constructed as follows. Eight-week-old Wistar female rats (Samtako, South Korea) weighing 175 to 185 g were fed enough pellet-type feed and water, and were adapted to individual cages for one week, and then used for the experiment. Secondary osteoporosis model was established by intramuscular injection based on Lucinda et al. (PHYTOTHERAPY RESEARCH 27(4), (2012), 515-520) and Sousa et al. (Archives of Oral Biology 77 (2017) 55-61). As a result, dexamethasone (DEX), a type of glucocorticoid, was administered once a week at a concentration of 7 mg/kg BW for 5 weeks.
  • DEX dexamethasone
  • the experimental group was classified into a total of 9 groups as shown in Table 3 below. 7 experimental animals were assigned to each group, and all groups except the normal group were administered dexamethasone (DEX) once a week at a concentration of 7 mg/kg (BW) for 5 weeks according to the method of 2.1.
  • DEX dexamethasone
  • Experimental group setting of the present invention Serial number Experimental group density Abbreviation One Normal-PBS group - Normal 2 Dexamethasone administration group 7 mg/kg Dex 3 Skim milk administration group SMP 4 Fermented milk administration group ( L. plantarum A41) 1 ⁇ 10 10 CFU/ml A41 5 Fermented milk administration group ( L. consumermtum SKR414) SKR414 6 Fermented milk administration group ( L. plantarum B719) B719 7 Fermented milk administration group ( L. casei 393) 393 8 Fermented milk administration group ( L. plantarum KSK411) KSK411 9 Secondary osteoporosis drug (alendronate) administration group 0.16 mg/kg Drug
  • Example 3 Morphological analysis of bone according to the administration of fermented milk of the present invention
  • Bone mineral density and percent bone volume are the most basic indicators to check when measuring bone strength.
  • a Micro Ct image analyzer Sky Scan 1176
  • NRecon a program for reconstructing the same
  • CTAn a program for analyzing the same
  • the fermented milk fermented with L. plantarum A41, L. fermentum SRK414, L. plantarum B719, and L. casei 393, which are probiotic strains of the present invention decreases bone density and bone ratio, which are symptoms of secondary osteoporosis induced by administration of dexamethasone. It was confirmed that it recovered.
  • Example 4 Analysis of indicators related to calcium absorption by administration of fermented milk of the present invention
  • RNA contained in the tissue was extracted using TRIzol (Invitrogne). Purified RNA was used for cDNA synthesis with reverse transcriptase oligo (dT) primer, and PCR conditions were repeated at 95°C for 3 minutes, 94°C for 10 seconds and 60°C for 30 seconds, for a total of 44 cycles. Primers for qRT-PCT analysis are shown in Table 4. The degree of total transcription was normalized from the ⁇ -actin transcription level and expressed as a graph (FIGS. 3 to 5 ).
  • the A41 group and SRK414 group among the fermented milk administration group of the present invention showed a relatively high expression level of intestinal calcium-receptive proteins (TRPV6, Calbindin-D9k, PMCA1) compared to other groups (Fig. 3 to 5).
  • the blood calcium concentration was confirmed. Specifically, blood was collected by a heart puncture method after the animal experiment period, and the collected serum was stored at -70°C until analysis by centrifugation (2500 g, 15 minutes, 4°C). Calcium (Ca) concentration in serum was analyzed using a calcium activity assay kit (MBL, Korea). The results are shown in FIG. 6.
  • the fermented milk fermented by the L. plantarum A41 and L. fermentum SRK414 strains of the present invention can effectively prevent bone loss by maintaining a high blood calcium concentration by promoting calcium absorption in the small intestine of animals to which the fermented milk was administered. .
  • Example 5 Analysis of indicators related to bone metabolism according to the administration of fermented milk of the present invention
  • osteoclasts When RANKL (Receptor Activator of Nuclear Factor Kappa B Ligand) is expressed in osteoblasts or activated immune cells, osteoclasts are differentiated by binding with RANK (Receptor Activator of Nuclear Factor Kappa B) in the osteoclast cell membrane. Bone resorption is promoted, which can lead to osteoporosis.
  • OPG Ostoprotegerin
  • OPG is a factor that is accepted by RANK instead of RANKL, and OPG can inhibit the differentiation of osteoclasts by interfering with the binding of RANK and RANKL.
  • the present inventors analyzed the mRNA expression levels of RANKL, OPG, and BSP, which are bone metabolism related markers, through qRT-PCR in order to confirm the effect of the administration of fermented milk of the present invention on bone metabolism. Specifically, after homogenization of the femur and bone cortex of experimental animals, total RNA was extracted using a TRIzol reagent (Thermofisher, USA). Purified total RNA was used for cDNA synthesis. For PCR, Biorad RT-PCR was used, and after treatment at 95° C. for 3 minutes, 10 seconds at 94° C. and 30 seconds at 60° C. were repeated for a total of 44 cycles. Primers for qRT-PCT analysis are shown in Table 5. The degree of total transcription was normalized from the ⁇ -actin transcription level and shown in the graph (FIGS. 7 to 9 ).
  • OPG mRNA expression level was also significantly increased in all fermented milk administration groups except for 393 and KSK, confirming the inhibition of osteoclast differentiation.
  • BSP bone sialoprotein

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Mycology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Rheumatology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Polymers & Plastics (AREA)
  • Food Science & Technology (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Nutrition Science (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)

Abstract

The present invention provides a Lactobacillus fermentum SRK414 strain with accession number KCTC13687BP, 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

프로바이오틱스를 유효 성분으로 포함하는 이차성 골다공증의 예방 또는 치료용 조성물Composition for preventing or treating secondary osteoporosis comprising probiotics as an active ingredient
본 발명은 대한민국 농림축산식품부의 지원 하에서 과제번호 IPET117051-3에 의해 이루어진 것으로서, 상기 과제의 연구관리 전문기관은 농림식품기술기획평가원, 연구사업명은 "고부가가치식품기술개발사업", 연구과제명은 "원발성 및 이차성 골다공증 개선 효과를 갖는 기능성 프로바이오틱스 제품 개발", 주관기관은 ㈜종근당바이오, 연구기간은 2017.06.15-2019.12.31 이다. The present invention was made by the project number IPET117051-3 under the support of the Ministry of Agriculture, Food and Rural Affairs of the Republic of Korea. The research and management professional institution for the project is the Agency for Food and Technology Planning and Evaluation, the name of the research project is "High Value Added Food Technology Development Project", and the name of the research project is " "Development of functional probiotics products with the effect of improving primary and secondary osteoporosis", the main institution is Chong Kun Dang Bio, the research period is from June 15, 2017 to December 31, 2019.
본 특허출원은 2018년 10월 30일에 대한민국 특허청에 제출된 대한민국 특허출원 제10-2018-0131027호에 대하여 우선권을 주장하며, 상기 특허출원의 개시사항은 본 명세서에 참조로서 삽입된다.This patent application claims priority to the Korean Patent Application No. 10-2018-0131027 filed with the Korean Intellectual Property Office on October 30, 2018, and the disclosure of the patent application is incorporated herein by reference.
본 발명은 프로바이오틱스를 유효 성분으로 포함하는 이차성 골다공증의 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing or treating secondary osteoporosis comprising probiotics as an active ingredient.
골다공증(Osteoporosis)은 연령과 폐경 등 정상 생리적 변화에 기인하여 골량이 감소하는 일차성 골다공증(원발성, primary)과, 연령과 관계없이 질환이나 약물에 노출되어 초래되는 이차성 골다공증(속발성, secondary), 크게 두 가지로 분류된다. 흔히 골다공증은 노년기의 질병이라고 생각하기 쉽지만, 이차성 골다공증의 경우 노령이 아닌 경우에도 발병할 수 있고, 이 경우 향후 환자의 삶의 질에 미치는 영향이 매우 크기 때문에 이차성 골다공증의 치료 및 예방 방법을 개발하는 것은 매우 중요하다. 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. It is classified into two categories. It is easy to think that osteoporosis is a disease of old age, but secondary osteoporosis can develop even when not in old age, and in this case, the effect on the quality of life of patients in the future is very large, so that the treatment and prevention methods for secondary osteoporosis are developed. Is very important.
이차성 골다공증의 원인들은 매우 다양하다. 여러 전신 질환과 약물 등이 원인으로 알려져 있으나, 이 중 글루코코르티코이드 복용으로 인한 골다공증이 가장 흔하다. 글루코코르티코이드(Glucocorticoid: GC)는 항염증과 면역억제 기능이 있어 류마티스 관절염, 천식, 아토피 피부염, 관절염 등 여러 질병의 치료에 보편적으로 사용되는 약제이다. GC의 장기간 투여는 이러한 질병을 효과적으로 억제하지만, 대부분 환자에게서 조골세포와 파골세포의 정상적인 활성을 저해하여 골다공증을 초래한다. 통계적으로 살펴보면, GC를 투여하는 환자의 경우 1년 이내 골밀도의 12%가 급격히 감소되고 그 이후로는 뼈의 손실이 서서히 진행되어 2~5% 정도의 골밀도가 매년 점차적으로 감소됨이 보고된 바 있다. 위와 같은 GC 투여에 의한 이차성 골다공증을 예방하기 위해서는 GC의 투여를 중단하는 것이 가장 이상적인 방법이기는 하나, 해당 질병의 치료를 위해서는 소량이라도 복용이 지속되어야 한다는 점에서 문제가 된다. The causes of secondary osteoporosis are very diverse. Various systemic diseases and drugs are known to be the cause, but among them, osteoporosis caused by taking glucocorticoids is the most common. Glucocorticoid (GC) has anti-inflammatory and immunosuppressive properties and is a commonly used drug for the treatment of various diseases such as rheumatoid arthritis, asthma, atopic dermatitis, and arthritis. Long-term administration of GC effectively inhibits these diseases, but in most patients, it inhibits the normal activity of osteoblasts and osteoclasts, resulting in osteoporosis. Statistically, it has been reported that in the case of patients receiving GC, 12% of bone density decreases rapidly within one year, and bone loss gradually progresses thereafter, resulting in a gradual decrease in bone density of 2-5% every year. . In order to prevent secondary osteoporosis caused by the GC administration as described above, stopping the administration of GC is the most ideal method, but it is a problem in that even a small dose must be continued for the treatment of the disease.
GC의 장기 투여로 인한 골다공증을 예방할 수 있는 식품을 함께 섭취하는 것도 좋은 대안이 될 수 있지만, 시중에서 판매중인 제품들은 대부분 폐경기 여성, 및 노인성 골다공증과 같은 '원발성 골다공증'을 개선하기 위한 건강기능식품이며, 이차성 골다공증을 예방할 수 있는 제품은 아직까지 개발된 바 없다. 따라서 이차성 골다공증의 예방, 개선 또는 치료용도의 식품 또는 의약품의 개발이 절실히 필요한 실정이다. Consuming 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 been developed yet. Therefore, there is an urgent need to develop foods or drugs for the prevention, improvement or treatment of secondary osteoporosis.
본 발명자들은 향후 골다공증의 예방, 개선 또는 치료용도의 식품 및 의약품으로 응용이 가능한 프로바이오틱스 균주 및 이를 이용한 발효유를 개발하고자 하였다. 그 결과, 프로바이오틱스 균주를 사용하여 발효유를 제조한 후 이를 글루코코르티코이드-유도 이차성 골다공증 실험동물에게 경구 투여하면 골밀도 및 골비율의 증가되고, 칼슘 관련 흡수 및 대사에 관련된 유전자의 발현이 증가됨을 규명함으로써, 본 발명을 완성하게 되었다. The present inventors have attempted to develop a probiotic strain that can be applied as food and pharmaceuticals for preventing, improving or treating osteoporosis in the future, and fermented milk using the same. As a result, it was found that when fermented milk was prepared using a probiotic strain and then administered orally to glucocorticoid-induced secondary osteoporosis experimental animals, bone mineral density and bone ratio were increased, and expression of genes related to calcium-related absorption and metabolism was increased. The present invention has been completed.
따라서, 본 발명의 목적은 수탁번호 KCTC13687BP 인 락토바실러스 퍼멘텀(Lactobacillus fermentum) SRK414 균주를 제공하는 것이다. Accordingly, an object of the present invention is to provide a Lactobacillus fermentum SRK414 strain with accession number KCTC13687BP.
본 발명의 다른 목적은 상기 락토바실러스 퍼멘텀(Lactobacillus fermentum) SRK414 균주, 또는 이의 배양물을 유효성분으로 포함하는 골다공증의 예방 또는 치료용 약제학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating osteoporosis comprising the Lactobacillus fermentum SRK414 strain, or a culture thereof as an active ingredient.
본 발명의 또 다른 목적은 상기 락토바실러스 퍼멘텀(Lactobacillus fermentum) SRK414 균주, 또는 이의 배양물을 유효성분으로 포함하는 골다공증의 예방 또는 개선용 식품조성물을 제공하는 것이다.Another object of the present invention is to provide a food composition for preventing or improving osteoporosis comprising the Lactobacillus fermentum SRK414 strain, or a culture thereof as an active ingredient.
본 발명의 또 다른 목적은 수탁번호 KCTC13687BP 인 락토바실러스 퍼멘텀(Lactobacillus fermentum) SRK414 균주를 유효성분으로 포함하는 조성물을 대상체에 투여하는 단계를 포함하는 골다공증의 예방, 개선 또는 치료방법을 제공하는 것이다.Another object of the present invention is to provide a method for preventing, improving, or treating osteoporosis comprising administering to a subject a composition comprising the Lactobacillus fermentum SRK414 strain of accession number KCTC13687BP as an active ingredient.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.
본 발명의 일 양태에 따르면, 본 발명은 수탁번호 KCTC13687BP 인 락토바실러스 퍼멘텀(Lactobacillus fermentum) SRK414 균주를 제공한다.According to one aspect of the present invention, the present invention provides a Lactobacillus fermentum ( Lactobacillus fermentum ) SRK414 strain of accession number KCTC13687BP.
본 발명자들은 락토바실러스 퍼멘텀(Lactobacillus fermentum) SRK414 균주 (수탁번호 : KCTC13687BP)를 신규 분리하였다. 또한, 본 발명자들은 상기 락토바실러스 퍼멘텀(Lactobacillus fermentum) SRK414 균주 (수탁번호: KCTC13687BP)를 이용하여 발효유를 제조하였다. 상기 균주에 의해 제조된 발효유는 이차성 골다공증을 유도한 실험동물모델에 투여한 결과, 골밀도 및 골비율이 증가되는 효과가 있음을 확인하였다. 나아가, 상기 발효유를 투여한 실험동물모델의 장내에서 발현되는 칼슘 흡수 관련 단백질의 발현량이 증가되고, 혈중 칼슘농도가 증가되며, 파골세포 분화 억제 및 조골세포 분화 촉진과 관련된 유전자의 발현량을 조절함을 확인하였다. The present inventors newly isolated Lactobacillus fermentum SRK414 strain (accession number: KCTC13687BP). In addition, the present inventors prepared a fermented milk using the Lactobacillus fermentum SRK414 strain (accession number: KCTC13687BP). As a result of administration of the fermented milk produced by the strain to an experimental animal model that induced secondary osteoporosis, it was confirmed that it has an effect of increasing bone density and bone ratio. Furthermore, the expression level of calcium absorption-related proteins expressed in the intestine of the experimental animal model administered with the fermented milk is increased, the calcium concentration in the blood is increased, and the expression level of genes related to the inhibition of osteoclast differentiation and promotion of osteoblast differentiation Was confirmed.
위 결과로부터, 본 발명의 락토바실러스 퍼멘텀(Lactobacillus fermentum) SRK414 균주 (수탁번호 : KCTC13687BP)를 이용하여 제조된 발효유는 이차성 골다공증의 예방, 개선, 또는 치료에 유용하게 사용될 수 있음을 확인하였다. From the above results, it was confirmed that the fermented milk prepared using the Lactobacillus fermentum SRK414 strain (accession number: KCTC13687BP) of the present invention can be usefully used in the prevention, improvement, or treatment of secondary osteoporosis.
본 명세서에서 용어 "골다공증(osteoporosis)"은 "골량의 감소와 미세구조의 이상을 특징으로 하는 전신적인 골격계 질환으로, 결과적으로 뼈가 약해져서 부러지기 쉬운 상태가 되는 질환"을 말한다. 골강도는 골량(quantity)과 골질(quality)에 의해 결정된다. 골량은 주로 골밀도 (BMD)에 의해 표현되고 골질은 구조, 골교체율, 무기질화, 미세 손상 축적 등으로 구성된다. 현재는 골밀도를 측정하여 골다공증을 진단하고 있다.In the present specification, the term "osteoporosis" refers to a "systemic skeletal system disease characterized by a decrease in bone mass and abnormalities in microstructures, and as a result, a disease in which the bone becomes weak and becomes brittle". Bone strength is determined by bone quantity and quality. Bone mass is mainly expressed by bone density (BMD), and bone quality is composed of structure, bone replacement rate, mineralization, and accumulation of micro-damages. Currently, osteoporosis is diagnosed by measuring bone density.
골다공증은 일차성과 이차성 골다공증으로 분류된다. 일차성 골다공증 또는 원발성 골다공증은 폐경으로 인한 제1형 골다공증과 노화로 인한 제2형 골다공증으로 편의상 분류하지만 거의 같은 시기에 병합되어 진행되므로 정확히 분류하기 어려운 것으로 알려져 있다. 이차성 골다공증은 기저질환이나 약물에 의해 뼈의 미세구조가 변하고 골량이 감소하여 골절위험도가 증가한 것을 말한다. 이차성 골다공증의 원인으로 내분비질환(갑상선중독증, 당뇨병, 부갑상선기능항진증 등), 위장관질환(위절제줄, 만성 간질환, 흡수장애증후군, 염증성 장질환, 알코올 중독 등), 골수질환, 결체 조직질환(강직성 척추염, 류마티스 관절염, 골형성부전증 등), 약물 등이 있다. 남성에서 가장 흔한 원인은 성선기능 저하증, 글루코코르티코이드 투여 등이다.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 it is known that it is difficult to accurately classify it because it is combined and progressed at about the same time. Secondary osteoporosis refers to an increase in the risk of fracture due to changes in the microstructure of bones and decreases in bone mass due to underlying diseases or drugs. The causes of secondary osteoporosis include endocrine diseases (thyroidism, diabetes, hyperparathyroidism, etc.), gastrointestinal diseases (gastric resection lines, chronic liver disease, malabsorption syndrome, inflammatory bowel disease, alcoholism, etc.), bone marrow disease, connective tissue disease ( Ankylosing spondylitis, rheumatoid arthritis, bone insufficiency, etc.), and drugs. The most common causes in men are hypogonadism and glucocorticoid administration.
본 발명의 일구현예에서, 상기 골다공증은 이차성 골다공증이고, 보다 구체적으로는 글루코코르티코이드계 약물 투여에 의한 이차성 골다공증이다. 상기 글루코코르티코이드계 약물은 코르티솔, 프레드니손, 프레드니솔론, 메틸프레드니솔론, 코르티손, 하이드록시코르티손, 트리암시놀론, 베타메타손, 또는 덱사메타손이나, 이에 한정되는 것은 아니다.In one embodiment of the present invention, the osteoporosis is secondary osteoporosis, more specifically secondary osteoporosis caused by administration of a glucocorticoid drug. The glucocorticoid-based drug is cortisol, prednisone, prednisolone, methylprednisolone, cortisone, hydroxycortisone, triamcinolone, betamethasone, or dexamethasone, but is not limited thereto.
본 발명에 있어서, 상기 락토바실러스 퍼멘텀 SRK414 균주는 "한국생명공학연구원 미생물자원센터(Korean Collection for Type Culture, KCTC)"에 수탁번호 "KCTC13687BP"로 기탁된 것이다. In the present invention, the Lactobacillus fermentum SRK414 strain is deposited under the accession number "KCTC13687BP" in the "Korean Collection for Type Culture (KCTC)".
상기 락토바실러스 퍼멘텀 SRK414 균주의 균체 성상은 다음과 같다:The cell properties of the Lactobacillus fermentum SRK414 strain are as follows:
- 서열번호 2의 16s rRNA 서열을 포함함 -Contains the 16s rRNA sequence of SEQ ID NO: 2
- 그람 양성 막대모양 또는 구형 간균의 형태임-Gram-positive rod-shaped or spherical bacillus form
- 인간의 입, 위장, 여성 성기관의 정상 세균총의 일부이며, 일반적으로 음식에 사용하는 것에 대해 안전하다고 여겨진다 (probiotics와 발효음식). L. fermentum은 Lactobacillus 속 구성원이며, 이 속에 속하는 종들은 다양한 활용이 가능하다고 한다. 이러한 활용은 식품과 사료 발효를 포함한다. L. fermentum 중 몇 균주들은 항생제 및 화학요법제에 대해 자연내성을 갖는 것으로 밝혀져 있다. -It is part of the normal flora of the human mouth, stomach and female sexual 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 are said to have various uses. These uses include food and feed fermentation. L. fermentum Several strains of these have been found to have natural resistance to antibiotics and chemotherapeutic agents.
따라서, 본 발명의 다른 일 양태에 따르면, 본 발명은 수탁번호 KCTC13687BP인 락토바실러스 퍼멘텀 SRK414 균주, 또는 이의 배양물을 유효성분으로 포함하는 골다공증 예방 또는 치료용 약제학적 조성물을 제공한다. Therefore, according to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating osteoporosis comprising the Lactobacillus fermentum SRK414 strain, or a culture thereof, of accession number KCTC13687BP, as an active ingredient.
본 발명의 또 다른 일 양태에 따르면, 본 발명은 수탁번호 KCTC13687BP인 락토바실러스 퍼멘텀 SRK414 균주, 또는 이의 배양물을 유효성분으로 포함하는 골다공증 예방 또는 개선용 식품조성물을 제공한다.According to another aspect of the present invention, the present invention provides a food composition for preventing or improving osteoporosis comprising the Lactobacillus fermentum SRK414 strain of accession number KCTC13687BP, or a culture thereof as an active ingredient.
본 발명의 일구현예에서, 상기 조성물은 유효성분으로 락토바실러스 플란타럼(Lactobacillus plantarum), 락토바실러스 카제이(Lactobacillus casei),락토바실러스 애시도필러스(Lactobacillus acidophilus), 락토바실러스 불가리쿠스(Lactobacillus delbrueckii ssp bulgaricus), 락토코커스 락티스(Lactococcus lactis), 엔테로코커스 페시움(Enterococcus faecium), 엔테로코커스 페칼리스(Enterococcus fecalis), 스트렙토코커스 써모필러스(Streptococcus thermophilus), 비피도박테리움 비피덤(Bifidobacterium bifidum), 및 비피도 박테리움 락티스(Bifidobacterium lactis)로 이루어진 군으로부터 선택되는 1종 이상의 균, 또는 이의 배양물을 추가적으로 포함한다. In one embodiment, the composition comprises as an active ingredient Lactobacillus Planta column (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei), Lactobacillus ash FIG pillar's (Lactobacillus acidophilus), Lactobacillus Bulgaria kusu (Lactobacillus delbrueckii ssp bulgaricus ), Lactococcus lactis , Enterococcus faecium ), Enterococcus fecalis , Streptococcus thermophilus , Bifidobacterium Bifidobacterium bifidum ), and Bifidobacterium lactis lactis ) and one or more fungi selected from the group consisting of, or a culture thereof.
본 발명의 구체적인 구현예에서, 상기 락토바실러스 플란타럼은 락토바실러스 플란타럼(Lactobacillus plantarum) A41 균주(수탁번호 KCTC13686BP)일 수 있다. In a specific embodiment of the present invention, the Lactobacillus plantarum may be a Lactobacillus plantarum A41 strain (accession number KCTC13686BP).
본 발명에 있어서, 상기 락토바실러스 플란타럼(Lactobacillus plantarum) A41 균주는 "한국생명공학연구원 미생물자원센터(Korean Collection for Type Culture, KCTC)"에 수탁번호 "KCTC13686BP"로 기탁된 것이다. In the present invention, the Lactobacillus plantarum A41 strain is deposited with the accession number "KCTC13686BP" to the "Korea Research Institute of Bioscience and Biotechnology Microbial Resource Center (Korean Collection for Type Culture, KCTC)".
상기 락토바실러스 플란타럼(Lactobacillus plantarum) A41 균주의 균체 성상은 다음과 같다:The microbial properties of the Lactobacillus plantarum A41 strain are as follows:
- 서열번호 1의 16s rRNA 서열을 포함함-Contains the 16s rRNA sequence of SEQ ID NO: 1
- 그람 양성 막대모양 또는 구형 간균의 형태임-Gram-positive rod-shaped or spherical bacillus form
- 인간의 입, 위장, 여성 성기관의 정상 세균총의 일부이며, 일반적으로 음식에 사용하는 것에 대해 안전하다고 여겨진다 (probiotics와 발효음식). L. plantarum은 Lactobacillus 속 구성원이며, 일반적으로 많은 발효 식품과 혐기성 식물의 물질에서 발견되며 타액에도 존재 하기도 한다.-It is part of the normal flora of the human mouth, stomach and female sexual 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 anaerobic plant materials, and is also present in saliva.
본 발명의 조성물들의 유효성분인 상기 균주, 또는 이의 배양물은 상기 균주를 단리 및/또는 정제한 균체 외에 균체를 포함하는 배양물, 균체의 추출물, 배양물 상층액, 이들의 농축액, 농축물, 건조물, 또한 필요에 따라서 희석액, 희석물 등이며, 배양액, 배양물을 처리하여 얻어지는 모든 상태의 것을 포함한다. The 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 strain, extracts of cells, supernatant of cultures, concentrates, concentrates thereof, It is a dried product, and, if necessary, a dilution liquid, a dilution product, etc., and includes a culture solution and any condition obtained by treating the culture product.
상기 균체의 배양법, 추출법, 분리법, 농축법, 건조법, 희석법 등은 특별히 한정되지 않는다. The culture method, extraction method, separation method, concentration method, drying method, dilution method, etc. of the cells are not particularly limited.
균체를 배양하기 위한 배지로는 통상적으로 탈지유, 훼이, 카제인 등의 우유 단백질, 당류, 효모 엑기스 등을 포함하고 있으며, 배양 방법으로는 일반적인 각종 호기적 또는 혐기적인 방법을 적당히 사용할 수 있다. The medium for culturing the cells typically contains milk proteins such as skim milk, whey, and casein, sugars, yeast extract, and the like, and as a culture method, various general aerobic or anaerobic methods can be appropriately used.
배양 온도로는 예를 들어 35~45℃를 설정하고, 배양 중에는 수산화나트륨 등의 알칼리를 사용하여 배지의 pH를 중성으로부터 산성, 예를 들어 pH가 5~6정도가 되도록 유지하는 중화배양법을 사용할 수도 있다. 이와 같은 중화배양법 외에 회분배양법 등의 임의의 배양 방법을 사용할 수 있으며, 배양한 후에는 필요에 따라서 배양물이나 그 상층액을 농축, 건조, 희석 등을 할 수도 있다. As the culture temperature, for example, 35~45℃ is set, and during the 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. May be. In addition to the neutralization culture method, any culture method such as a batch culture method may be used. After the culture, the culture product or its supernatant may be concentrated, dried, or diluted if necessary.
또한 원심분리법이나 막분리법을 사용하여 배양물의 상층액과 균체를 분리하여 균체를 농축한 상태로 회수할 수도 있다. 그리고 균체에 초음파 처리나 효소 처리 등을 행하여 균체 내의 성분을 추출하거나, 배양물이나 그 상층액, 균체나 그 추출물 등을 건조할 수도 있다. 이들은 본 발명의 상기 조성물의 유효 성분으로서 사용할 수 있다.In addition, centrifugation or membrane separation may be used to separate the supernatant and the cells of the culture, and the cells may be recovered in a concentrated state. Then, the cells may be subjected to ultrasonic treatment, enzyme treatment, or the like to extract components within the cells, or to dry the culture product, its supernatant, the cells or its extract, and the like. These can be used as an active ingredient of the composition of the present invention.
본 발명의 조성물이 식품 조성물로 제조되는 경우, 유효성분으로서 상기 유산균 뿐만 아니라, 식품 제조 시에 통상적으로 첨가되는 성분을 포함할 수 있다. 상기 첨가성분은 예컨대 단백질, 탄수화물, 지방, 영양소, 조미제 및 향미제를 포함한다. 상기 탄수화물로는 모노사카라이드(예를 들어, 포도당, 과당 등), 디사카라이드(예를 들어 말토스, 수크로스, 올리고당 등) 및 폴리사카라이드(예를 들어 덱스트린, 사이클로덱스트린 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 향미제로서 천연 향미제 (타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등)) 및 합성 향미제(사카린, 아스파르탐 등)를 사용할 수 있다.When the composition of the present invention is prepared as a food composition, it may include not only the lactic acid bacteria as an active ingredient, but also ingredients that are commonly added during food production. The additive ingredients include, for example, proteins, carbohydrates, fats, nutrients, flavoring agents and flavoring agents. The carbohydrates include monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, oligosaccharides, etc.), and polysaccharides (e.g., dextrin, cyclodextrin, etc.) Sugar and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents, natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartic acid) Tom, etc.) can be used.
예컨대, 본 발명의 식품 조성물이 드링크제로 제조되는 경우에는 본 발명의 유효성분인 상기 균주 이외에 구연산, 액상과당, 설탕, 포도당, 초산, 사과산, 과즙, 대추 추출액 또는 감초 추출액 등을 추가로 포함시킬 수 있다.For example, when the food composition of the present invention is prepared as a drink, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, jujube extract, licorice extract, etc. can be additionally included in addition to the above strain, which is an active ingredient of the present invention. have.
본 발명의 식품조성물은 식품, 기능성 식품(functional food), 영양보조제(nutritional supplement), 건강식품(health food) 및 식품 첨가제(food additives) 등의 모든 천연소재의 가공 형태를 포함한다. 상기 유형의 식품 조성물은 당업계에 공지된 통상적인 방법에 따라 다양한 형태로 제조될 수 있다.The food composition of the present invention includes processed forms of all natural materials such as food, functional food, nutritional supplement, health food and food additives. Food compositions of this type may be prepared in various forms according to conventional methods known in the art.
예를 들면, 건강식품으로는 상기 유산균 자체를 차, 주스 및 드링크의 형태로 제조하여 음용하도록 하거나, 과립화, 캡슐화 및 분말화하여 섭취할 수 있다. 또한, 식품으로는 음료(알콜성 음료 포함), 과실 및 그의 가공식품(예: 과일통조림, 병조림, 잼, 마아말레이드 등), 어류, 육류 및 그 가공식품(예: 햄, 소시지 콘비이프 등), 빵류 및 면류(예: 우동, 메밀국수, 라면, 스파게티, 마카로니 등), 과즙, 각종 드링크, 쿠키, 엿, 유제품(예: 요거트, 발효유, 버터, 치즈 등), 식용식물유지, 마아가린, 식물성 단백질, 레토르트 식품, 냉동식품, 각종 조미료(예: 된장, 간장, 소스 등) 등 본 발명의 유산균을 첨가하여 제조될 수 있다. 또한, 본 발명의 유산균을 식품 첨가제의 형태로 사용하기 위해서는 분말 또는 농축액 형태로 제조하여 사용할 수 있다.For example, as a health food, the lactic acid bacteria itself may be prepared in the form of tea, juice, and drinks to be consumed, or granulated, encapsulated, and powdered to be consumed. In addition, foods include beverages (including alcoholic beverages), fruits and processed foods thereof (e.g., canned fruit, bottled, jam, marmalade, etc.), fish, meat and processed foods thereof (e.g. ham, sausage corn beef, etc.) ), bread and noodles (e.g. udon, soba, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, syrup, dairy products (e.g. yogurt, fermented milk, butter, cheese, etc.), edible vegetable oil, margarine, Vegetable protein, retort food, frozen food, various seasonings (eg, miso, soy sauce, sauce, etc.) can be prepared by adding the lactic acid bacteria of the present invention. In addition, in order to use the lactic acid bacteria of the present invention in the form of a food additive, it may be prepared and used in the form of a powder or a concentrate.
본 발명의 조성물이 약제학적 조성물로 제조되는 경우, 본 발명의 약제학적 조성물은 약제학적으로 허용되는 담체를 포함한다. 본 발명의 약제학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로오스, 폴리비닐피롤리돈, 셀룰로오스, 물, 시럽, 메틸 셀룰로오스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 제한되는 것은 아니다. 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.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. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used at the time of formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, etc. It does not become. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components.
본 발명의 약제학적 조성물은 경구 또는 비경구 투여할 수 있으며, 바람직하게는 경구 투여 방식으로 적용된다. 본 발명의 약제학적 조성물은 하기의 다양한 경구 또는 비경구 투여 형태로 제형화할 수 있으나, 이에 한정되는 것은 아니다.The pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably applied by way of oral administration. The pharmaceutical composition of the present invention may be formulated in the following various oral or parenteral dosage forms, but is not limited thereto.
경구 투여용 제형으로는 예를 들면 정제, 환제, 경/연질 캅셀제, 액제, 현탁제, 유화제, 시럽제. 과립제, 엘릭시르제 등이 있는데, 이들 제형은 상기 유효성분 이외에 통상적으로 사용되는 충진제, 증량제, 습윤제, 붕해제, 활택제, 결합제, 계면활성제 등의 희석제 또는 부형제를 1종 이상 사용할 수 있다. 붕해제로는 한천, 전분, 알긴산 또는 이의 나트륨염, 무수인산일수소 칼슘염 등이 사용될 수 있고, 활택제로는 실리카, 탈크, 스테아르산 또는 이의 마그네슘염 또는 칼슘염, 폴리에틸렌 글리콜 등이 사용될 수 있으며, 결합제로는 마그네슘 알루미늄 실리케이트, 전분 페이스트, 젤라틴, 트라가칸스, 메틸셀룰로오스, 나트륨카복시메틸셀룰로오스, 폴리비닐피롤리딘, 저치환도 하이드록시프로필셀룰로오스 등이 사용될 수 있다. 이외에도 락토즈, 덱스트로오스, 수크로오스, 만니톨, 소르비톨, 셀룰로오스. 글리신 등을 희석제로 사용할 수 있으며, 경우에 따라서는 일반적으로 알려진 비등 혼합물, 흡수제, 착색제, 향미제, 감미제 등을 함께 사용할 수 있다.Formulations for oral administration include, for example, tablets, pills, hard/soft capsules, solutions, suspensions, emulsifiers, and syrups. There are granules, elixirs, and the like, and these formulations may include one or more diluents or excipients such as fillers, extenders, wetting agents, disintegrants, lubricants, binders, surfactants, etc., which are commonly used in addition to the active ingredients. Agar, starch, alginic acid or its sodium salt, anhydrous monohydrogen phosphate calcium salt, etc. may be used as the disintegrant, and as the lubricant, silica, talc, stearic acid or its magnesium salt or calcium salt, polyethylene glycol, etc. may be used. As the binder, magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine, low-substituted hydroxypropylcellulose, and the like may be used. In addition, lactose, dextrose, sucrose, mannitol, sorbitol, and cellulose. Glycine or the like may be used as a diluent, and in some cases, commonly known boiling mixtures, absorbents, colorants, flavors, sweeteners, and the like may be used together.
상기 조성물은 멸균되거나 또는 방부제, 안정화제, 수화제 또는 유화 촉진제, 삼투압 조절을 위한 염, 완충제 등의 보조제 및 기타 치료적으로 유용한 물질을 함유할 수 있으며, 통상적인 방법인 혼합, 과립화 또는 코팅 방법에 따라 제제화할 수 있다. The composition may be sterilized or contain adjuvants such as preservatives, stabilizers, hydrating agents or emulsification accelerators, salts for regulating osmotic pressure, buffers, and other therapeutically useful substances, which are conventional methods of mixing, granulating or coating. It can be formulated according to.
본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. The appropriate dosage of the pharmaceutical composition of the present invention is prescribed in various ways depending on factors such as the formulation method, the mode of administration, the patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity. Can be.
본 발명의 약제학적 조성물은 당해 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액, 시럽제 또는 유화액 형태이거나 엑스제, 산제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the technical field to which the present invention belongs. Or it can be made by incorporating it into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.
본 발명의 일 양태에 따르면, 본 발명은 수탁번호 KCTC13687BP 인 락토바실러스 퍼멘텀(Lactobacillus fermentum) SRK414 균주를 유효성분으로 포함하는 조성물을 대상체에 투여하는 단계를 포함하는 골다공증의 예방, 개선 또는 치료방법을 제공한다.According to an aspect of the present invention, the present invention provides a method for preventing, improving, or treating osteoporosis comprising administering to a subject a composition comprising the Lactobacillus fermentum SRK414 strain of accession number KCTC13687BP as an active ingredient. to provide.
본 발명의 치료방법, 또는 개선방법의 대상 질병인 골다공증은 상기 약제학적 조성물의 치료 대상 질환과 관련하여 정의한 것과 같다. Osteoporosis, which is the target disease of the treatment method or improvement method of the present invention, is the same as defined in relation to the disease to be treated by the pharmaceutical composition.
본 발명의 일 구현예에서, 상기 대상체는 포유동물 또는 인간이다. In one embodiment of the present invention, the subject is a mammal or human.
본 발명의 골다공증의 치료방법 또는 개선방법은 상술한 수탁번호 KCTC13687BP 인 락토바실러스 퍼멘텀(Lactobacillus fermentum) SRK414 균주, 또는 이의 배양물을 유효성분으로 포함하는 조성물과 동일한 유효성분을 사용하는 방법이므로, 중복되는 내용에 대해서는 본 명세서의 과도한 복잡성을 피하기 위하여 그 기재를 생략한다. The method for treating or improving osteoporosis of the present invention is a method of using the same active ingredient as a composition containing the above-described accession number KCTC13687BP, Lactobacillus fermentum SRK414 strain, or a culture thereof as an active ingredient, and thus overlapping Description of the contents will be omitted to avoid excessive complexity of the present specification.
본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:
(a) 본 발명은 수탁번호 KCTC13687BP인 락토바실러스 퍼멘텀(Lactobacillus fermentum) SRK414 균주를 제공한다. (a) The present invention provides a Lactobacillus fermentum ( Lactobacillus fermentum ) SRK414 strain of accession number KCTC13687BP.
(b) 본 발명은 수탁번호 KCTC13687BP인 락토바실러스 퍼멘텀(Lactobacillus fermentum) SRK414 균주, 또는 이의 배양물을 유효성분으로 포함하는 약제학적 조성물 및 식품조성물을 제공한다.(b) The present invention provides a pharmaceutical composition and a food composition comprising the accession number KCTC13687BP Lactobacillus fermentum SRK414 strain, or a culture thereof as an active ingredient.
본 발명의 균주 및 이의 배양물은 골다공증, 특히 이차성 골다공증을 예방하거나 개선, 또는 치료하기 위한 목적의 식품조성물 및 약제학적 조성물로 유용하게 사용될 수 있다. The strains and cultures thereof of the present invention can be usefully used as food compositions and pharmaceutical compositions for the purpose of preventing, improving, or treating osteoporosis, particularly secondary osteoporosis.
도 1은 실험 그룹 별 골밀도(bone mineral density, mg/cm3)를 나타낸 도이다. 1 is a diagram showing bone mineral density (mg/cm 3 ) for each experimental group.
도 2는 실험 그룹 별 골비율(percent bone volume, %)을 나타낸 도이다. Figure 2 is a diagram showing the bone ratio (percent bone volume, %) for each experimental group.
도 3은 실험 그룹 별 장내 칼슘 수용 단백질인 TRPV6의 mRNA 발현량을 나타낸 도이다. 3 is a diagram showing the mRNA expression level of TRPV6, an intestinal calcium receptor protein for each experimental group.
도 4는 실험 그룹 별 장내 칼슘 수용 단백질인 Calbindin-D9k의 mRNA 발현량을 나타낸 도이다. 4 is a diagram showing the mRNA expression level of Calbindin-D9k, an intestinal calcium receptor protein for each experimental group.
도 5는 실험 그룹 별 장내 칼슘 수용 단백질인 PMCA1의 mRNA 발현량을 나타낸 도이다. 5 is a diagram showing the mRNA expression level of PMCA1, an intestinal calcium receptor protein, for each experimental group.
도 6은 실험 그룹 별 혈중 칼슘 농도를 나타낸 도이다. 6 is a diagram showing blood calcium concentration for each experimental group.
도 7은 실험 그룹 별 RANKL mRNA의 발현량을 나타낸 도이다. 7 is a diagram showing the expression level of RANKL mRNA for each experimental group.
도 8은 실험 그룹 별 OPG mRNA의 발현량을 나타낸 도이다.8 is a diagram showing the expression level of OPG mRNA for each experimental group.
도 9는 실험 그룹 별 BSP mRNA의 발현량을 나타낸 도이다. 9 is a diagram showing the expression level of BSP mRNA for each experimental group.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
실시예Example
본 명세서 전체에 걸쳐, 특정 물질의 농도를 나타내기 위하여 사용되는 "%"는 별도의 언급이 없는 경우, 고체/고체는 (중량/중량) %, 고체/액체는 (중량/부피) %, 그리고 액체/액체는 (부피/부피) %이다.Throughout the present specification, "%" used to indicate the concentration of a specific substance is (weight/weight)% for solids/solids, (weight/volume)% for solids/liquids, and Liquid/liquid is (vol/vol) %.
실시예Example 1: One: 프로바이오틱스Probiotics 균주 및 발효유의 제조 Preparation of strains and fermented milk
본 실험에 사용된 프로바이오틱스 균주들은 영유아의 분변과 전통 발효식품으로부터 분리되었다(표 2). 본 발명의 발효유를 제조하기 위하여, 탈지 우유(skim milk 배지에 상기에서 분리한 프로바이오틱스 균주를 각각 접종하여 37℃에서 48시간동안 배양하였다. 발효가 완료된 발효유는 동결건조 후 분말화 하여 PBS에 1×1010 CFU/ml 농도로 현탁하여 실험동물에게 급여하였다. 본 발명에 사용된 탈지 우유 배지의 조성과, 프로바이오틱스 균주 목록은 표 1 및 표 2에 나타내었다. The probiotic strains used in this experiment were isolated from infant feces and traditional fermented foods (Table 2). In order to prepare the fermented milk of the present invention, skim milk (skim milk medium) was inoculated with the separated probiotic strain for 48 hours at 37° C. The fermented milk was freeze-dried, powdered, and 1× in PBS. It was suspended at a concentration of 10 10 CFU/ml and fed to the experimental animals The composition of the skim milk medium used in the present invention and the list of probiotic strains are shown in Tables 1 and 2.
본 발명의 탈지 우유 (Skim milk) 배지의 조성Composition of the Skim milk medium of the present invention
연번Serial number 원료명Raw material name 용량 (g/L)Capacity (g/L)
1One Skim milk powder Skim milk powder 100100
22 Glucose Glucose 2020
33 Yeast extractYeast extract 33
44 Bacto peptoneBacto peptone 33
본 발명의 프로바이오틱스 균주 목록List of probiotic strains of the present invention
연번Serial number 균주명Strain name 약어Abbreviation
1One Lactobacillus plantarum A41 Lactobacillus plantarum A41 A41A41
22 Lactobacillus fermentum SRK414 Lactobacillus fermentum SRK414 SRK414SRK414
33 Lactobacillus plantarum B719 Lactobacillus plantarum B719 B719B719
44 Lactobacillus casei 393 Lactobacillus casei 393 393393
55 Lactobacillus plantarum KSK411 Lactobacillus plantarum KSK411 KSK411KSK411
실시예Example 2: 이차성 골다공증 동물모델의 제작 및 2: Preparation of secondary osteoporosis animal model and 실험군Experimental group 설정 Settings
2.1 이차성 골다공증 동물모델의 제작2.1 Construction of secondary osteoporosis animal model
이차성 골다공증 동물 모델은 다음과 같이 제작하였다. 체중 175~185 g 내외의 8주령 Wistar계 암컷 랫드(Samtako, South Korea)를 펠렛형 사료와 물을 충분히 공급하여 1주일 간 개별 케이지에 적응시킨 후 실험에 사용하였다. 이차성 골다공증 모델의 수립은 Lucinda et al.(PHYTOTHERAPY RESEARCH 27(4), (2012), 515-520) 와 Sousa et al.(Archives of Oral Biology 77 (2017) 55-61)에 의거하여 근육내 주사법으로 글루코코르티코이드의 일종인 덱사메타손(Dexamethasone, DEX)을 7 mg/kg BW의 농도로 주 1회씩 5주간 투여하였다. The secondary osteoporosis animal model was constructed as follows. Eight-week-old Wistar female rats (Samtako, South Korea) weighing 175 to 185 g were fed enough pellet-type feed and water, and were adapted to individual cages for one week, and then used for the experiment. Secondary osteoporosis model was established by intramuscular injection based on Lucinda et al. (PHYTOTHERAPY RESEARCH 27(4), (2012), 515-520) and Sousa et al. (Archives of Oral Biology 77 (2017) 55-61). As a result, dexamethasone (DEX), a type of glucocorticoid, was administered once a week at a concentration of 7 mg/kg BW for 5 weeks.
2.2 2.2 실험군의Experimental group 설정 및 발효유의 급여 Setting and salary of fermented milk
본 발명의 프로바이오틱스 균주 및 발효유의 이차성 골다공증에 대한 치료효과를 확인하기 위하여, 실험군을 하기 표 3과 같이 총 9개의 그룹으로 분류하였다. 각 그룹별로 실험동물을 7마리씩 배정하였고, Normal 그룹을 제외한 모든 그룹은 상기 2.1의 방법대로 덱사메타손(DEX)을 주 1회 7 mg/kg(BW)의 농도로 5주간 투여하였다. In order to confirm the therapeutic effect of the probiotic strain and fermented milk of the present invention on secondary osteoporosis, the experimental group was classified into a total of 9 groups as shown in Table 3 below. 7 experimental animals were assigned to each group, and all groups except the normal group were administered dexamethasone (DEX) once a week at a concentration of 7 mg/kg (BW) for 5 weeks according to the method of 2.1.
모든 발효유는 1×1010 CFU/ml의 농도로 PBS(Phosphate buffer saline)에 희석하여 위관 영양법(oral gavage)으로 경구 투여하였다. 한편, 본 실험의 목적이 프로바이오틱스를 이용한 우유 발효물의 항 골다공증 효과를 확인하기 위함이기 때문에 대조군으로는 발효를 진행하지 않은 Skim milk 투여군(SMP)을 사용하였다.All fermented milk was diluted in PBS (Phosphate buffer saline) at a concentration of 1×10 10 CFU/ml and administered orally by gavage. On the other hand, since the purpose of this experiment was to confirm the anti-osteoporosis effect of fermented milk using probiotics, the Skim milk administration group (SMP) without fermentation was used as a control group.
본 발명의 실험군 설정Experimental group setting of the present invention
연번Serial number 실험그룹Experimental group 농도density 약어Abbreviation
1One Normal-PBS투여군Normal-PBS group -- Normal Normal
22 Dexamethasone 투여군 Dexamethasone administration group 7 mg/kg7 mg/kg DexDex
33 Skim milk 투여군Skim milk administration group SMPSMP
44 발효유 투여군 (L. plantarum A41)Fermented milk administration group ( L. plantarum A41) 1×1010 CFU/ml1×10 10 CFU/ml A41A41
55 발효유 투여군 (L. fermemtum SKR414)Fermented milk administration group ( L. fermemtum SKR414) SKR414SKR414
66 발효유 투여군 (L. plantarum B719)Fermented milk administration group ( L. plantarum B719) B719 B719
77 발효유 투여군 (L. casei 393)Fermented milk administration group ( L. casei 393) 393393
88 발효유 투여군 (L. plantarum KSK411)Fermented milk administration group ( L. plantarum KSK411) KSK411KSK411
99 이차성 골다공증 약물(alendronate) 투여군Secondary osteoporosis drug (alendronate) administration group 0.16 mg/kg0.16 mg/kg DrugDrug
실시예Example 3: 본 발명의 발효유 투여에 따른 골의 형태학적 분석 3: Morphological analysis of bone according to the administration of fermented milk of the present invention
골밀도(Bone mineral density)와 골비율(Percent bone volume)은 뼈의 강도를 측정할 때 가장 기본적으로 확인하는 지표이다. 본 발명의 프로바이오틱스 균주 및 발효유의 골밀도 개선효과를 측정하기 위하여, Micro Ct 영상분석기(Sky Scan 1176)와 이를 재구성하는 프로그램(NRecon), 그리고 이를 분석하는 프로그램(CTAn)을 이용하였다.Bone mineral density and percent bone volume are the most basic indicators to check when measuring bone strength. In order to measure the effect of improving the bone density of the probiotic strain and fermented milk of the present invention, a Micro Ct image analyzer (Sky Scan 1176), a program for reconstructing the same (NRecon), and a program for analyzing the same (CTAn) were used.
실험 결과, 덱사메타손을 투여한 DEX 그룹에서는 골밀도 및 골비율이 Normal 그룹 대비 유의적으로 감소한 것으로 보아 이차성 골다공증이 유도됨을 확인할 수 있었다. 이차성 골다공증 약물(alendronate)을 덱사메타손과 함께 투여한 Drug 그룹에서는 덱사메타손 투여로 인해 감소된 골밀도와 골비율이 회복된 것으로 나타났다. 본 발명의 프로바이오틱스 균주로 발효시킨 발효유를 투여한 A41 그룹, SRK414 그룹, B719 그룹, 및 393 그룹도 drug 그룹과 유사한 정도의 골밀도 및 골비율 회복 효과를 나타내었다. (도 1 및 도 2)As a result of the experiment, it was confirmed that secondary osteoporosis was induced in the DEX group to which dexamethasone was administered, as the bone density and bone ratio decreased significantly compared to the normal group. In the Drug group administered with a secondary osteoporosis drug (alendronate) along with dexamethasone, the decreased bone density and bone ratio were recovered due to the administration of dexamethasone. The A41 group, SRK414 group, B719 group, and 393 group to which fermented milk fermented with the probiotic strain of the present invention was administered also showed similar degree of bone mineral density and bone ratio recovery effect to the drug group. (Fig. 1 and Fig. 2)
상기 결과로부터, 본 발명의 프로바이오틱스 균주인 L. plantarum A41, L. fermentum SRK414, L. plantarum B719, L. casei 393 으로 발효시킨 발효유가 덱사메타손의 투여로 유발된 이차성 골다공증의 증상인 골밀도, 골비율 감소를 회복시킴을 확인하였다.From the above results, the fermented milk fermented with L. plantarum A41, L. fermentum SRK414, L. plantarum B719, and L. casei 393, which are probiotic strains of the present invention, decreases bone density and bone ratio, which are symptoms of secondary osteoporosis induced by administration of dexamethasone. It was confirmed that it recovered.
실시예Example 4. 본 발명의 발효유 투여에 의한 칼슘 흡수 관련 지표 분석 4. Analysis of indicators related to calcium absorption by administration of fermented milk of the present invention
혈중 칼슘농도가 부족할 경우, 뼈에 있는 칼슘이 혈중으로 유리되어 골소실이 증가하게 된다. 따라서 장내에서 칼슘 흡수를 촉진시킴으로써 혈중 칼슘 농도를 증가시키는 것은 골소실을 낮출 수 있는 중요한 예방책이 될 수 있다. 칼슘은 장에서 칼슘을 이동시키는 역할을 하는 단백질인 TRPV6, Calbindin-D9k, PMCA1 등에 의해 소장에서 혈관으로 유입되는데, 이들의 발현량이 증가한다면 칼슘 흡수가 원활히 진행된다고 볼 수 있다. 따라서 본 발명자들은 본 발명의 발효유 투여에 따른 장내 칼슘 수용 단백질의 mRNA 발현량을 알아보기 위해서 qRT-PCR 분석을 실시하였으며, 혈중 칼슘 농도와의 관련성을 확인하였다. When the calcium concentration in the blood is insufficient, calcium in the bone is released into the blood and bone loss increases. Therefore, increasing the blood calcium concentration by promoting calcium absorption in the intestine can be an important preventive measure that can lower bone loss. Calcium is introduced into the blood vessels from the small intestine by proteins TRPV6, Calbindin-D9k, and PMCA1, which are proteins that move calcium in the intestine. If their expression levels increase, calcium absorption can be seen to proceed smoothly. Therefore, the present inventors performed qRT-PCR analysis to determine the mRNA expression level of the calcium-receiving protein in the intestine according to the administration of fermented milk of the present invention, and confirmed the relationship with the blood calcium concentration.
4.1 장내 칼슘 수용체 유전자 발현 확인4.1 Intestinal calcium receptor gene expression confirmation
발효유가 장내 칼슘 수용 단백질(TRPV6, Calbindin-D9k, PMCA1)의 발현량에 미치는 효과를 알아보기 위해서 qRT-PCR 분석을 진행하였다. 구체적으로, 실험 기간 종료 후 각 실험동물의 회장을 적출하여 균질화(homogenized)과정을 거친 후 TRIzol(Invitrogne)을 사용하여 조직에 포함되어 있는 RNA를 추출하였다. 정제된 RNA는 역전사효소 oligo(dT) 프라이머와 함께 cDNA합성에 사용되었으며, PCR 조건은 95 ℃에서 3분 이후, 94 ℃에서 10초 60 ℃에서 30초로, 총 44주기 반복하였다. qRT-PCT 분석을 위한 프라이머는 표 4에 나타내었다. 총 전사의 정도는 β-actin 전사 레벨로부터 정규화(normalization)하여 그래프로 나타내었다(도 3 내지 도 5). QRT-PCR analysis was performed to determine the effect of fermented milk on the expression levels of intestinal calcium receptor proteins (TRPV6, Calbindin-D9k, PMCA1). Specifically, after the end of the experiment period, the ileum of each experimental animal was excised, subjected to a homogenization process, and RNA contained in the tissue was extracted using TRIzol (Invitrogne). Purified RNA was used for cDNA synthesis with reverse transcriptase oligo (dT) primer, and PCR conditions were repeated at 95°C for 3 minutes, 94°C for 10 seconds and 60°C for 30 seconds, for a total of 44 cycles. Primers for qRT-PCT analysis are shown in Table 4. The degree of total transcription was normalized from the β-actin transcription level and expressed as a graph (FIGS. 3 to 5 ).
유전자명Gene name Forward primer (5' to 3')Forward primer (5' to 3') Reverse primer (5' to 3')Reverse primer (5' to 3')
TRPV6TRPV6 GAT GGC ACG ACC CTT TGGGAT GGC ACG ACC CTT TGG CTT CGG GAG GTA CTT CGA GAC ACTT CGG GAG GTA CTT CGA GAC A
Calbindin-D9kCalbindin-D9k CGC TAA GAA ATC TCC CGA AGCGC TAA GAA ATC TCC CGA AG CTC CAT CAC CGT TCT TAT CCACTC CAT CAC CGT TCT TAT CCA
PMCA1PMCA1 ACT CTG GGG CCA GCT TAT TTACT CTG GGG CCA GCT TAT TT GTT CGG CAT GGT CAA TCT CTGTT CGG CAT GGT CAA TCT CT
도 3 내지 5에서 나타낸 바와 같이, 본 발명의 발효유 투여 그룹 중에서 A41 그룹 및 SRK414 그룹이 다른 그룹에 비해 상대적으로 높은 장내 칼슘 수용 단백질(TRPV6, Calbindin-D9k, PMCA1)의 발현량을 나타내었다(도 3 내지 도 5).As shown in Figs. 3 to 5, the A41 group and SRK414 group among the fermented milk administration group of the present invention showed a relatively high expression level of intestinal calcium-receptive proteins (TRPV6, Calbindin-D9k, PMCA1) compared to other groups (Fig. 3 to 5).
4.2 혈중 칼슘 농도 조절효과4.2 Blood calcium concentration control effect
본 발명의 발효유 투여가 혈중 칼슘 농도에 미치는 영향을 확인하기 위하여, 혈중 칼슘 농도를 확인하였다. 구체적으로, 동물실험 기간 종류 후 심장천자(heart puncture)법으로 혈액을 채취하였으며 원심분리(2500 g, 15분, 4℃)하여 수집한 혈청을 분석 전까지 -70 ℃에 보관하였다. 혈청 내에 있는 칼슘(Ca) 농도는 Calcium activity assay kit (㈜엠비엘, Korea)를 사용하여 분석하였다. 결과는 도 6에 나타내었다.In order to confirm the effect of the administration of fermented milk of the present invention on the blood calcium concentration, the blood calcium concentration was confirmed. Specifically, blood was collected by a heart puncture method after the animal experiment period, and the collected serum was stored at -70°C until analysis by centrifugation (2500 g, 15 minutes, 4°C). Calcium (Ca) concentration in serum was analyzed using a calcium activity assay kit (MBL, Korea). The results are shown in FIG. 6.
도 6에 나타낸 바와 같이, 장내 칼슘 수용 단백질의 발현량과 마찬가지로 본 발명의 발효유 투여 그룹 중에서 A41 그룹 및 SRK414 그룹이 다른 그룹에 비해 상대적으로 높은 혈중 칼슘 농도를 나타냄을 확인하였다. 따라서 본 발명의 L. plantarum A41, 및 L. fermentum SRK414 균주에 의해 발효된 발효유는, 발효유가 투여된 동물의 소장 내 칼슘 흡수를 촉진시킴으로써 높은 혈중 칼슘 농도 유지하여 골소실을 효과적으로 예방할 수 있다고 사료된다.As shown in FIG. 6, it was confirmed that the A41 group and SRK414 group among the fermented milk administration group of the present invention exhibited relatively high blood calcium concentration compared to the other groups, similar to the expression level of the intestinal calcium-receiving protein. Therefore, it is believed that the fermented milk fermented by the L. plantarum A41 and L. fermentum SRK414 strains of the present invention can effectively prevent bone loss by maintaining a high blood calcium concentration by promoting calcium absorption in the small intestine of animals to which the fermented milk was administered. .
실시예Example 5. 본 발명의 발효유 투여에 따른 골 대사 관련 지표 분석 5. Analysis of indicators related to bone metabolism according to the administration of fermented milk of the present invention
조골세포 또는 활성화된 면역세포에서 RANKL(Receptor Activator of Nuclear Factor Kappa B Ligand)이 발현되면 파골전구세포 세포막의 RANK(Receptor Activator of Nuclear Factor Kappa B)와 결합하여 파골세포의 분화가 이루어지는데, 이 경우 뼈 흡수가 촉진되어 골다공증이 유발될 수 있다. 반면 OPG(Osteoprotegerin)는 RANKL 대신 RANK에 수용되는 인자로, OPG가 RANK와 RANKL의 결합을 방해함으로써 파골세포의 분화를 억제시킬 수 있다. When RANKL (Receptor Activator of Nuclear Factor Kappa B Ligand) is expressed in osteoblasts or activated immune cells, osteoclasts are differentiated by binding with RANK (Receptor Activator of Nuclear Factor Kappa B) in the osteoclast cell membrane. Bone resorption is promoted, which can lead to osteoporosis. On the other hand, OPG (Osteoprotegerin) is a factor that is accepted by RANK instead of RANKL, and OPG can inhibit the differentiation of osteoclasts by interfering with the binding of RANK and RANKL.
본 발명자들은 본 발명의 발효유 투여가 골 대사에 미치는 영향을 확인하기 위하여, 골대사 관련 마커인 RANKL, OPG, 및 BSP의 mRNA 발현양을 qRT-PCR을 통해 분석하였다. 구체적으로, 실험동물의 넙다리뼈와 골피질을 균질화(homogenization)한 후 TRIzol reagent(Thermofisher, USA)를 이용해 총 RNA를 추출하였다. 정제된 총 RNA는 cDNA합성을 위해 사용하였다. PCR은 Biorad RT-PCR을 사용하였으며 95 ℃에서 3분간 처리한 후, 94 ℃에서 10초, 60 ℃에서 30초를 총 44주기 반복하였다. qRT-PCT 분석을 위한 프라이머는 표 5에 나타내었다. 총 전사의 정도는 β-actin 전사 레벨로부터 정규화(normalization)하여 그래프에 나타내었다(도 7 내지 9).The present inventors analyzed the mRNA expression levels of RANKL, OPG, and BSP, which are bone metabolism related markers, through qRT-PCR in order to confirm the effect of the administration of fermented milk of the present invention on bone metabolism. Specifically, after homogenization of the femur and bone cortex of experimental animals, total RNA was extracted using a TRIzol reagent (Thermofisher, USA). Purified total RNA was used for cDNA synthesis. For PCR, Biorad RT-PCR was used, and after treatment at 95° C. for 3 minutes, 10 seconds at 94° C. and 30 seconds at 60° C. were repeated for a total of 44 cycles. Primers for qRT-PCT analysis are shown in Table 5. The degree of total transcription was normalized from the β-actin transcription level and shown in the graph (FIGS. 7 to 9 ).
유전자명Gene name Forward primer (5' to 3')Forward primer (5' to 3') Reverse primer (5' to 3')Reverse primer (5' to 3')
TRPV6TRPV6 CCC ATC GGG TTC CCA TAA AGT C CCC ATC GGG TTC CCA TAA AGT C GCC TGA AGC AAA TGT TGG CGT AGCC TGA AGC AAA TGT TGG CGT A
Calbindin-D9kCalbindin-D9k ACG GTT TGC AAA AGA TGT CCACG GTT TGC AAA AGA TGT CC GTG AGC TGC AGT TGG TGT GTGTG AGC TGC AGT TGG TGT GT
PMCA1PMCA1 AGA AAG AGC AGC ACG GTT GAG TAGA AAG AGC AGC ACG GTT GAG T GAC CCT CGT AGC CTT CAT AGC CGAC CCT CGT AGC CTT CAT AGC C
도 7에 나타낸 바와 같이, A41 그룹, 및 SRK414 그룹에서 Dex 그룹에 비해 RANKL mRNA의 발현량이 유의미하게 감소하여 A41과 SRK414 발효유가 파골세포의 분화를 감소시키는 것을 확인하였다. As shown in FIG. 7, it was confirmed that the expression level of RANKL mRNA was significantly reduced in the A41 group and the SRK414 group compared to the Dex group, so that A41 and SRK414 fermented milk reduced the differentiation of osteoclasts.
또한, 도 8에 나타낸 바와 같이, OPG mRNA 발현량도 393과 KSK을 제외한 모든발효유 투여 그룹에서 모두 유의미하게 증가하여 파골세포 분화의 억제를 확인하였다. In addition, as shown in FIG. 8, OPG mRNA expression level was also significantly increased in all fermented milk administration groups except for 393 and KSK, confirming the inhibition of osteoclast differentiation.
마지막으로, 도 9에 나타낸 바와 같이, 궁극적인 조골세포 분화 표지인자인 BSP(Bone sialoprotein) 발현량 역시 A41, SRK414 그리고 B719 그룹에서 상대적으로 높은 발현량을 나타내어 조골세포 분화가 촉진되어 골다공증의 치료효과가 뛰어남을 확인할 수 있었다.Finally, as shown in FIG. 9, the expression level of bone sialoprotein (BSP), which is the ultimate osteoblast differentiation marker, is also relatively high in the A41, SRK414, and B719 groups, thereby promoting osteoblast differentiation to treat osteoporosis. Was able to confirm excellence.
상기 결과를 바탕으로 본 발명의 L. plantarum A41 균주, 및 L. fermentum SRK414 균주로 발효시킨 발효유를 동물모델에 투여하였을 때 체내 조골세포가 OPG 생성을 촉진시키고 RANKL의 생성을 억제시켜 파골세포의 분화를 억제하고, 조골세포의 분화를 촉진함으로써 이차성 골다공증에 대한 예방 효과를 나타내는 것을 확인하였다.Based on the above results, when fermented milk fermented with L. plantarum A41 strain and L. fermentum SRK414 strain of the present invention was administered to an animal model, osteoblasts in the body promote OPG production and inhibit RANKL production, thereby differentiation of osteoclasts. It was confirmed that it exhibited a preventive effect against secondary osteoporosis by inhibiting and promoting the differentiation of osteoblasts.
[수탁번호][Accession number]
[기탁기관명] 한국생명공학연구원[Name of deposit institution] Korea Research Institute of Bioscience and Biotechnology
[수탁번호] KCTC13686BP[Accession number] KCTC13686BP
[수탁일자] 20181025[Consignment Date] 20181025
[기탁기관명] 한국생명공학연구원[Name of deposit institution] Korea Research Institute of Bioscience and Biotechnology
[수탁번호] KCTC13687BP[Accession number] KCTC13687BP
[수탁일자] 20181025[Consignment Date] 20181025
[규칙 제91조에 의한 정정 20.12.2019]　
Figure WO-DOC-TABLE-135
 [Amendment 20.12.2019 under Rule 91]
Figure WO-DOC-TABLE-135
[규칙 제91조에 의한 정정 20.12.2019]　
Figure WO-DOC-TABLE-136
 [Amendment 20.12.2019 under Rule 91]
Figure WO-DOC-TABLE-136

Claims (10)

  1. 수탁번호 KCTC13687BP인 락토바실러스 퍼멘텀(Lactobacillus fermentum) SRK414 균주.Accession number KCTC13687BP Lactobacillus fermentum ( Lactobacillus fermentum ) SRK414 strain.
  2. 수탁번호 KCTC13687BP인 락토바실러스 퍼멘텀(Lactobacillus fermentum) SRK414 균주, 또는 이의 배양물을 유효성분으로 포함하는 골다공증 예방 또는 치료용 약제학적 조성물.Accession number KCTC13687BP Lactobacillus fermentum ( Lactobacillus fermentum ) SRK414 strain, or a pharmaceutical composition for the prevention or treatment of osteoporosis comprising a culture thereof as an active ingredient.
  3. 제2항에 있어서, 상기 골다공증은 이차성 골다공증인, 약제학적 조성물.The pharmaceutical composition according to claim 2, wherein the osteoporosis is secondary osteoporosis.
  4. 제2항에 있어서, 상기 조성물은 락토바실러스 플란타럼(Lactobacillus plantarum), 락토바실러스 카제이(Lactobacillus casei),락토바실러스 애시도필러스(Lactobacillus acidophilus), 락토바실러스 불가리쿠스(Lactobacillus delbrueckii ssp bulgaricus), 락토코커스 락티스(Lactococcus lactis), 엔테로코커스 페시움(Enterococcus faecium), 엔테로코커스 페칼리스(Enterococcus fecalis), 스트렙토코커스 써모필러스(Streptococcus thermophilus), 비피도박테리움 비피덤(Bifidobacterium bifidum), 및 비피도 박테리움 락티스(Bifidobacterium lactis)로 이루어진 군으로부터 선택되는 1종 이상의 균, 또는 이의 배양물을 추가적으로 포함하는 것인, 약제학적 조성물.According to claim 2, wherein the composition is Lactobacillus plantarum ( Lactobacillus plantarum ), Lactobacillus casei ( Lactobacillus casei ), Lactobacillus acidophilus ( Lactobacillus acidophilus ), Lactobacillus bulgaricus ( Lactobacillus delbrueckii ssp bulgaricus ), Lactococcus lactis ), Enterococcus faecium ), Enterococcus fecalis , Streptococcus thermophilus , Bifidobacterium Bifidobacterium bifidum ), and Bifidobacterium lactis ( Bifidobacterium lactis ), which additionally comprises one or more bacteria selected from the group consisting of, or a culture thereof.
  5. 수탁번호 KCTC13687BP인 락토바실러스 퍼멘텀(Lactobacillus fermentum) SRK414 균주, 또는 이의 배양물을 유효성분으로 포함하는 골다공증 예방 또는 개선용 식품조성물.Accession number KCTC13687BP Lactobacillus fermentum ( Lactobacillus fermentum ) SRK414 strain, or a food composition for preventing or improving osteoporosis comprising a culture thereof as an active ingredient.
  6. 제5항에 있어서, 상기 골다공증은 이차성 골다공증인, 식품조성물.The food composition according to claim 5, wherein the osteoporosis is secondary osteoporosis.
  7. 제5항에 있어서, 상기 식품조성물은 발효유인, 조성물.The composition of claim 5, wherein the food composition is fermented milk.
  8. 제5항에 있어서, 락토바실러스 플란타럼(Lactobacillus plantarum), 락토바실러스 카제이(Lactobacillus casei),락토바실러스 애시도필러스(Lactobacillus acidophilus), 락토바실러스 불가리쿠스(Lactobacillus delbrueckii ssp bulgaricus), 락토코커스 락티스(Lactococcus lactis), 엔테로코커스 페시움(Enterococcus faecium), 엔테로코커스 페칼리스(Enterococcus fecalis), 스트렙토코커스 써모필러스(Streptococcus thermophilus), 비피도박테리움 비피덤(Bifidobacterium bifidum), 및 비피도 박테리움 락티스(Bifidobacterium lactis)로 이루어진 군으로부터 선택되는 1종 이상의 균, 또는 이의 배양물을 추가적으로 포함하는 것인, 식품조성물.According to claim 5, Lactobacillus plantarum ( Lactobacillus plantarum ), Lactobacillus casei ( Lactobacillus casei ), Lactobacillus acidophilus ( Lactobacillus acidophilus ), Lactobacillus bulgaricus ( Lactobacillus delbrueckii) ssp bulgaricus ), Lactococcus lactis ), Enterococcus faecium , Enterococcus fecalis ), Streptococcus thermophilus , Bifidobacterium bifidum , and at least one fungus selected from the group consisting of Bifidobacterium lactis , or a culture thereof The food composition that further comprises.
  9. 수탁번호 KCTC13687BP인 락토바실러스 퍼멘텀(Lactobacillus fermentum) SRK414 균주, 또는 이의 배양물을 유효성분으로 포함하는 조성물을 대상체에 투여하는 단계를 포함하는, 골다공증의 예방, 개선 또는 치료방법.Accession number KCTC13687BP Lactobacillus fermentum ( Lactobacillus fermentum ) SRK414 strain, comprising the step of administering to a subject a composition containing a culture thereof as an active ingredient, prevention, improvement or treatment method of osteoporosis.
  10. 제9항에 있어서, 상기 골다공증은 이차성 골다공증인, 방법.The method of claim 9, wherein the osteoporosis is secondary osteoporosis.
PCT/KR2019/007320 2018-10-30 2019-06-18 Composition for preventing or treating secondary osteoporosis comprising probiotics as active ingredient WO2020091180A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020180131027A KR20200049999A (en) 2018-10-30 2018-10-30 Composition for Preventing or Treating Secondary Osteoporosis Comprising Probiotics
KR10-2018-0131027 2018-10-30

Publications (3)

Publication Number Publication Date
WO2020091180A2 WO2020091180A2 (en) 2020-05-07
WO2020091180A3 WO2020091180A3 (en) 2020-07-23
WO2020091180A9 true WO2020091180A9 (en) 2021-02-18

Family

ID=70462259

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2019/007320 WO2020091180A2 (en) 2018-10-30 2019-06-18 Composition for preventing or treating secondary osteoporosis comprising probiotics as active ingredient

Country Status (2)

Country Link
KR (1) KR20200049999A (en)
WO (1) WO2020091180A2 (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111419882B (en) * 2020-05-26 2022-07-29 北京科拓恒通生物技术股份有限公司 Bifidobacterium lactis for preventing and treating osteoporosis and application thereof
CN111718873B (en) * 2020-06-19 2022-09-06 江南大学 Lactobacillus fermentum with effect of relieving osteoporosis and application thereof
CN116058469B (en) * 2023-03-06 2023-06-27 天津创源生物技术有限公司 Preparation method and application of fermented lactobacillus mucilaginosus IOB802 fermented metapowder

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102009037089A1 (en) * 2009-08-11 2011-03-03 Heller, Knut J., Prof. Dr. Composition with strains of Lactobacillus fermentum
KR101279852B1 (en) * 2011-06-29 2013-07-09 주식회사 쎌바이오텍 Composition for preventing or treating osteoporosis comprising multi-species pobiotic mixture
KR101349139B1 (en) * 2012-02-27 2014-01-10 한국식품연구원 Method for Preparing Vitamin K2 and Fermented Milk Using Lactobacillus fermentum LC272
KR101771692B1 (en) * 2015-09-03 2017-08-29 호서대학교 산학협력단 Composition for Preventing, Treating or Improving Menopausal Syndrome comprising Sparassis crispa Wulf.: Fr. and Lactic Acid Bacteria as Active Ingredient

Also Published As

Publication number Publication date
WO2020091180A2 (en) 2020-05-07
KR20200049999A (en) 2020-05-11
WO2020091180A3 (en) 2020-07-23

Similar Documents

Publication Publication Date Title
WO2020091179A2 (en) Composition for preventing or treating secondary osteoporosis comprising probiotics as active ingredient
JP5801802B2 (en) Lipid metabolism improver
WO2017082611A1 (en) Strain having ability to inhibit obesity and pharmaceutical composition containing same
DK2478910T3 (en) ANTI-ADIPOSITY AGENT, ANTI-ADIPOSITA NUTRITION OR DRINK, GLUCOSE TOLERANCE EFFECTIVE AGENT, AND NUTRITION OR DRINK FOR IMPROVING GLUCOSE TOLERANCE
EP2615163B1 (en) Composition comprising probiotics having aryl hydrocarbon receptor activating potency for use as anti-inflammatory agents
KR101981333B1 (en) Lactobacillus fermentum MG4231 or MG4244 having antiobesity activity derived from human and composition comprising the same
WO2020091180A9 (en) Composition for preventing or treating secondary osteoporosis comprising probiotics as active ingredient
WO2019199094A1 (en) Novel bifidobacterium longum or lactobacillus rhamnosus strain having effect of preventing or treating obesity, and use thereof
KR20090122454A (en) Agent for reducing visceral fat
WO2020091166A1 (en) Novel lactic acid bacteria and use thereof
EP3166619B1 (en) Use of lactobacillus paracasei for promoting recovery of the intestinal microbiota diversity after dysbiosis
EP2634269A1 (en) New strain of lactic acid bacteria and its combinations producing probiotic preparations
KR101985792B1 (en) Lactobacillus fermentum MG4231 or MG4244 having antiobesity activity derived from human and composition comprising the same
US20170028000A1 (en) Use of lactobacillus rhamnosus for promoting recovery of the intestinal microbiota diversity after dysbiosis
US20100203026A1 (en) Probiotics for inducing satiety and/or satiation
CN114786692A (en) Novel probiotic compositions for modulating gut immunity
CN111886332A (en) Novel bacterium belonging to the genus Bifidobacterium and composition containing the same
KR101402926B1 (en) Pharmaceutical composition and functional food for prevention or treatment of bone disease comprising the lactic acid bacteria ferment of hwangryunhaedoktang
WO2020226467A1 (en) Enterococcus lactis wikim0107 strain having effects of regulating immune functions and alleviating inflammatory bowel disease and use thereof
WO2020226468A1 (en) Pediococcus inopinatus ｗikim0108 strain having effect of regulating immune function and alleviating inflammatory bowel disease, and use thereof
WO2020045971A1 (en) Human-derived lactobacillus fermentum mg4231 or lactobacillus fermentum mg4244 strain having anti-obesity activity, and composition comprising same
EP2545930A1 (en) Agent for controlling the increase and decrease of lactobacillus bifidus in colon
EP2746398A1 (en) Process for producing conjugated linolenic acid from linolenic acid employing Bifidobacterium breve, Bifidobacterium bifidum, or Lactobacillus oris strains.
KR101611833B1 (en) Use of lactobacillus acidophilus cbt la1 in the prevention or treatment of obesity and obesity-related metabolic syndrome and composition comprising the same
CN117460427A (en) Composition for improving joint function

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: 19879988

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19879988

Country of ref document: EP

Kind code of ref document: A2