WO2022239916A1 - Nouvelle souche de bifidobacterium longum et son utilisation - Google Patents

Nouvelle souche de bifidobacterium longum et son utilisation Download PDF

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WO2022239916A1
WO2022239916A1 PCT/KR2021/015958 KR2021015958W WO2022239916A1 WO 2022239916 A1 WO2022239916 A1 WO 2022239916A1 KR 2021015958 W KR2021015958 W KR 2021015958W WO 2022239916 A1 WO2022239916 A1 WO 2022239916A1
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culture
bifidobacterium longum
adipocytes
strain
culture supernatant
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Korean (ko)
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WO2022239916A9 (fr
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김용식
샤밈 라만엠디
강인석
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주식회사 비티진
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Priority to JP2023570106A priority Critical patent/JP2024521057A/ja
Publication of WO2022239916A1 publication Critical patent/WO2022239916A1/fr
Publication of WO2022239916A9 publication Critical patent/WO2022239916A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • 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
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/06—Preparations for care of the skin for countering cellulitis
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0653—Adipocytes; Adipose tissue
    • 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
    • A23V2200/332—Promoters of weight control and weight loss
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/72—Undefined extracts from bacteria
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • 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

Definitions

  • the present invention relates to a novel Bifidobacterium longum strain and its use, and more specifically, to a novel Bifidobacterium longum infantis having anti-obesity activity ( Bifidobacterium longum subsp. Infantis ) YB0411 strain And pharmaceutical compositions, health functional foods, cosmetic compositions, feed compositions and reagent compositions for anti-obesity use containing the culture of the strain.
  • Adipose tissue is a central tissue for maintaining systemic homeostasis, and excessive accumulation of white adipose tissue causes obesity.
  • the conversion process from immature pre-adipocytes to adipocytes is called adipogenesis, and this process is regulated by several factors including hormonal signals and various transcriptional factors.
  • adipogenic hormone stimulation is required to induce expression of early transcription factors such as CCAAT/enhancer-binding protein (C/EBP)- ⁇ and - ⁇ , together with suppression of Wnt/ ⁇ -catenin signaling. do.
  • C/EBP CCAAT/enhancer-binding protein
  • PPAR ⁇ peroxisome prolifertor-activated receptor ⁇
  • C/EBP ⁇ peroxisome prolifertor-activated receptor ⁇
  • FABP4 protein 4
  • Korean Patent Publication No. 10-2019-0118985 discloses adipocyte differentiation inhibitory activity as an anti-obesity effect of Bifidobacterium longum strain. Although it is suggested that the expression of the gene is significantly increased compared to the untreated control, this is related to the brown adiposity of white adipocytes.
  • Patent Document 1 KR10-2019-0118985A
  • An object to be solved by the present invention is to provide a novel Bifidobacterium longum strain that inhibits the maturation of pre-adipocytes into adipocytes.
  • the problem to be solved in the present invention is to provide a pharmaceutical composition for anti-obesity use, health functional food, cosmetic composition, feed composition and reagent composition containing the strain culture.
  • an object to be solved in the present invention is to provide a method of inhibiting maturation into adipocytes by treating the strain culture to isolated pre-adipocytes.
  • the present invention provides a Bifidobacterium longum subsp. Infantis YB0411 strain, accession number KCTC14393BP.
  • the present invention provides a pharmaceutical composition for preventing or treating obesity containing a culture of Bifidobacterium longum subsp .
  • the culture is preferably selected from the group consisting of a culture medium, a culture concentrate, a dried culture medium, a culture medium extract, a culture supernatant, a culture supernatant concentrate, a dried culture supernatant, and a culture supernatant extract.
  • the present invention provides a health functional food for preventing or improving obesity, including a culture of Bifidobacterium longum subsp .
  • the culture is preferably selected from the group consisting of a culture medium, a culture concentrate, a dried culture medium, a culture medium extract, a culture supernatant, a culture supernatant concentrate, a dried culture supernatant, and a culture supernatant extract.
  • the present invention provides a cosmetic composition for slimming comprising a culture of Bifidobacterium longum subsp. Infantis YB0411 strain having accession number KCTC14393BP.
  • the culture is preferably selected from the group consisting of a culture medium, a culture concentrate, a dried culture medium, a culture medium extract, a culture supernatant, a culture supernatant concentrate, a dried culture supernatant, and a culture supernatant extract.
  • the cosmetic composition is preferably a formulation selected from the group consisting of lotion, lotion, cream, essence, foam and pack.
  • the present invention provides a feed composition for the prevention or improvement of obesity (obesity) comprising a culture of Bifidobacterium longum subsp .
  • the culture is preferably selected from the group consisting of a culture medium, a culture concentrate, a dried culture medium, a culture medium extract, a culture supernatant, a culture supernatant concentrate, a dried culture supernatant, and a culture supernatant extract.
  • the present invention is for inhibiting the maturation of pre-adipocytes into adipocytes, including the culture of Bifidobacterium longum subsp.
  • a reagent composition is provided.
  • the culture is preferably selected from the group consisting of a culture medium, a culture concentrate, a dried culture medium, a culture medium extract, a culture supernatant, a culture supernatant concentrate, a dried culture supernatant, and a culture supernatant extract.
  • the present invention is a pre-adipocyte comprising treating a culture of Bifidobacterium longum subsp. It provides a method for inhibiting maturation into adipocytes.
  • Figure 1 is a schematic diagram showing the screening process of Bifidobacterium longum subsp .
  • Figure 2 is the accession number KCTC14393BP of the present invention Bifidobacterium longum infantis ( Bifidobacterium longum subsp. Infantis )
  • PA Pre-adipocytes
  • MDI Methylisobutylxanthine-Dexamethasone-Insulin
  • Rosi Rosiglitazone
  • MRS MRS medium
  • YB B. longum subsp.
  • Infantis YB0411 Each represents a cultural supernatant.
  • PA Pre-adipocytes
  • MDI Methylisobutylxanthine-Dexamethasone-Insulin
  • Rosi Rosiglitazone
  • MRS MRS medium
  • YB B. longum subsp. Infantis YB0411
  • FIG. 5 shows the pre-adipogenesis process (adipogenesis) marker CEBP/ of pre-adipocytes after treating the culture of Bifidobacterium longum subsp.
  • PA Pre-adipocytes
  • MDI Methylisobutylxanthine-Dexamethasone-Insulin
  • Rosi Rosiglitazone
  • MRS MRS medium
  • YB B. longum subsp.
  • Infantis YB0411 Each represents a cultural supernatant.
  • the inventors of the present invention tested the anti-obesity effect from various lactic acid bacteria cultures for the development of a novel anti-obesity treatment, and in the culture of Bifidobacterium longum subsp.
  • the present invention was completed after confirming that there is an activity of inhibiting the maturation of pre-adipocytes into adipocytes.
  • the present invention provides a Bifidobacterium longum subsp. Infantis YB0411 strain with accession number KCTC14393BP.
  • the novel Bifidobacterium longum infantis of the present invention ( Bifidobacterium longum subsp. Infantis ) YB0411 strain was published on November 30, 2020 by the Korea Research Institute of Bioscience and Biotechnology (Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology) ) under the accession number KCTC14393BP.
  • the culture of Bifidobacterium longum subsp. Infantis YB0411 strain, accession number KCTC14393BP of the present invention, can be applied for anti-obesity purposes because it has the effect of inhibiting the maturation process of pre-adipocytes into adipocytes. have.
  • accession number KCTC14393BP is treated with pre-adipocytes, early and late stage markers of adipogenesis, Cebp/ ⁇ , Cebp/ ⁇ , Cebp/ ⁇ , Ppar ⁇ , aP2 and adiponectin mRNA expression significantly decreased.
  • the present invention provides a pharmaceutical composition for preventing or treating obesity, containing a culture of Bifidobacterium longum subsp .
  • the culture is preferably selected from the group consisting of a culture medium, a culture concentrate, a dried culture medium, a culture medium extract, a culture supernatant, a culture supernatant concentrate, a dried culture supernatant, and a culture supernatant extract.
  • the pharmaceutical composition comprising the culture of Bifidobacterium longum subsp. It can be formulated and used in various forms such as oral formulations such as capsules, suspensions, emulsions, syrups, and aerosols, and injections of sterile injection solutions. It can be administered through various routes.
  • compositions may further include carriers, excipients, or diluents, and examples of suitable carriers, excipients, or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. etc. can be mentioned.
  • the pharmaceutical composition of the present invention may further include fillers, anti-agglomerating agents, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, and the like.
  • solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the pharmaceutical composition, for example, starch, calcium carbonate, It is formulated by mixing sucrose, lactose, gelatin, etc.
  • lubricants such as magnesium stearate and talc may be used in addition to simple excipients.
  • liquid preparations for oral use may include suspensions, internal solutions, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, Fragrance, preservatives, etc. may be included.
  • preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, suppositories, and the like.
  • Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • Conventional additives such as solubilizers, tonicity agents, suspending agents, emulsifiers, stabilizers, preservatives and the like may be included in the injection.
  • the culture of YB0411 strain of Bifidobacterium longum subsp. Infantis , accession number KCTC14393BP of the present invention, is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type, severity, drug activity, It may be determined according to factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field.
  • the pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.
  • the effective amount of the culture of Bifidobacterium longum subsp in the pharmaceutical composition of the present invention, the effective amount of the culture of Bifidobacterium longum subsp.
  • 1 to 5,000 mg, preferably 100 to 3,000 mg per kg of body weight may be administered daily or every other day or divided into 1 to 3 times a day.
  • the dosage since it may increase or decrease according to the route of administration, severity of disease, sex, weight, age, etc., the dosage is not limited to the scope of the present invention in any way.
  • composition of the present invention may be administered to a subject through various routes. All modes of administration can be envisaged, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.
  • administration means providing a predetermined substance to a patient by any suitable method, and the route of administration of the pharmaceutical composition of the present invention is oral or parenteral through all common routes as long as it can reach the target tissue. can be administered orally.
  • the composition of the present invention may be administered using any device capable of delivering active ingredients to target cells.
  • subject includes, but is not particularly limited to, for example, human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig. and, preferably, a mammal, more preferably a human.
  • the present invention provides a health functional food for preventing or improving obesity, including a culture of Bifidobacterium longum subsp .
  • the culture is preferably selected from the group consisting of a culture medium, a culture concentrate, a dried culture medium, a culture medium extract, a culture supernatant, a culture supernatant concentrate, a dried culture supernatant, and a culture supernatant extract.
  • the health functional food of the present invention can be used in a variety of ways, such as foods and beverages effective for preventing or improving obesity.
  • the health functional food of the present invention includes, for example, various foods, beverages, gum , tea, There are vitamin complexes, health supplements, etc., and can be used in the form of powder, granules, tablets, capsules or beverages.
  • the culture of Bifidobacterium longum subsp. Infantis YB0411 strain, accession number KCTC14393BP of the present invention can generally be added in an amount of 0.01 to 15% by weight of the total food weight, and the health drink composition is based on 100 ml It may be added at a rate of 0.02 to 10 g, preferably 0.3 to 1 g.
  • the health functional food of the present invention may contain food additives such as natural carbohydrates and various flavors as additional components.
  • Examples of the natural carbohydrate include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • natural flavoring agents such as stevia such as thaumatin, rebaudioside A or glycyrrhizin, and synthetic flavoring agents such as saccharin and aspartame may be used.
  • the proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 ml of the health functional food of the present invention.
  • the health functional food of the present invention is various nutrients, vitamins, minerals, flavors such as synthetic flavors and natural flavors, colorants and enhancers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloids It may contain thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, and the like.
  • the health functional food of the present invention may contain fruit flesh for production of natural fruit juice, fruit juice beverages, and vegetable beverages. These components may be used independently or in combination. The ratio of these additives is generally selected from the range of 0.01 to about 20 parts by weight per 100 parts by weight of delphinidin and pharmaceutically acceptable salts thereof of the present invention.
  • the present invention provides a cosmetic composition for slimming comprising a culture of Bifidobacterium longum subsp. Infantis YB0411 strain having accession number KCTC14393BP.
  • the culture is preferably selected from the group consisting of a culture medium, a culture concentrate, a dried culture medium, a culture medium extract, a culture supernatant, a culture supernatant concentrate, a dried culture supernatant, and a culture supernatant extract.
  • the "slimming” refers to a phenomenon in which the volume of the entire body or a specific region is reduced, and for example, it may be the removal or reduction of fat in the entire body or a specific region.
  • the active ingredient of the cosmetic composition of the present invention may be included in an amount of 0.01 to 50% by weight, preferably 0.1 to 20% by weight, more preferably 1 to 10% by weight, based on the total weight of the composition. Appropriate formulation stability can be secured within this content range, and a desired slimming effect can be expected.
  • composition of the present invention in addition to the active ingredient of the present invention, can be used by further including known natural extracts or other ingredients for the effects of antioxidant, anti-aging, moisturizing, and skin regeneration promotion within the range that does not inhibit the effective activity of the present invention.
  • compositions may include ingredients commonly added to cosmetic compositions, for example, fatty substances, organic solvents, solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, Cosmetics such as fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic actives; It can be molded into a specific dosage form including adjuvants or carriers commonly used in the field of dermatology.
  • ingredients commonly added to cosmetic compositions for example, fatty substances, organic solvents, solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, Cosmetics such as fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preserv
  • the cosmetic composition of the present invention can be prepared in any formulation commonly prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, pack, soap, surfactant- It may be formulated as containing cleansing, oil and spray, etc., but is not limited thereto. More specifically, lotion, lotion, softening lotion, nourishing lotion, cream, nourishing cream, massage cream, essence, eye cream, powder foundation, emulsion foundation, wax foundation, cleansing cream, foam, cleansing foam, cleansing water, pack , Spray, powder, pact, lip gloss, lipstick, shadow, may be prepared in formulations such as shampoo and rinse.
  • the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.
  • lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additionally chlorofluorohydrocarbon, propellants such as propane/butane or dimethyl ether.
  • a solvent, solubilizing agent or emulsifying agent is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, fatty acid esters of glycerol, polyethylene glycol or sorbitan.
  • the formulation of the present invention is a suspension
  • a liquid diluent such as water, ethanol or propylene glycol
  • a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Star cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like may be used.
  • the formulation of the present invention is surfactant-containing cleansing
  • carrier components aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide Ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester may be used.
  • the present invention provides a feed composition for the prevention or improvement of obesity (obesity) comprising a culture of Bifidobacterium longum subsp .
  • the culture is preferably selected from the group consisting of a culture medium, a culture concentrate, a dried culture medium, a culture medium extract, a culture supernatant, a culture supernatant concentrate, a dried culture supernatant, and a culture supernatant extract.
  • the term "feed” may refer to any natural or artificial diet, one meal, etc., or a component of the one meal meal, for or suitable for eating, ingesting, and digesting by an animal.
  • the type of feed is not particularly limited, and feeds commonly used in the art may be used.
  • Non-limiting examples of the feed include vegetable feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal or grain by-products, proteins, inorganic materials, and animal feeds such as oils and fats, mineral oils, fats and oils, unicellular proteins, zooplankton, or food. These may be used alone or in combination of two or more.
  • the present invention is for inhibiting the maturation of pre-adipocytes into adipocytes, including a culture of Bifidobacterium longum subsp.
  • a reagent composition is provided.
  • the culture is preferably selected from the group consisting of a culture medium, a culture concentrate, a dried culture medium, a culture medium extract, a culture supernatant, a culture supernatant concentrate, a dried culture supernatant, and a culture supernatant extract.
  • the reagent composition may be applied as a reagent composition for research, for example.
  • the reagent composition of the present invention can be administered to cells or laboratory animals under normal laboratory conditions, and specific administration methods and safety rules are preferably carried out according to the internal regulations of each laboratory of the experimenter.
  • the present invention is a pre-adipocyte comprising treating a culture of Bifidobacterium longum subsp. It provides a method for inhibiting maturation into adipocytes.
  • the pre-adipocytes are those isolated from a subject by natural or artificial methods.
  • the cells isolated from the subject may be cultured for several days to several weeks under appropriate culture conditions, and in some cases may be cryopreserved.
  • a method of administering the active ingredient of the present invention to the isolated cells is obvious to those skilled in the art, and may be preferably followed as described in the following examples of the present specification.
  • Dexamethasone Isobutyl-1-metylxanthine (IBMX), Insulin, Rosiglitazone (ROSI), Oil Red O dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 4% formaldehyde was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM) and newborn calf serum (NBCS) were purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum was purchased from Atlas Biologicals (Fort Collins, CO, USA). Penicillin-streptomycin solution was manufactured by Hyclone Laboratories, Inc.
  • DMEM Dulbecco's modified Eagle's medium
  • NBCS newborn calf serum
  • Fetal bovine serum was purchased from Atlas Biologicals (Fort Collins, CO, USA). Penicillin-streptomycin solution was manufactured by Hyclo
  • 3T3-L1 mouse embryo fibroblasts were obtained from the Korean Cell Line Bank (KCLB-10092.1), and 10% (v/v) heat-inactivated NCS and 1% (v/v) antibiotic/antimycotic solution (100 U/ml penicillin, In a high-glucose DMEM medium supplemented with 100 U/ml streptomycin), the cells were cultured at 37° C. in a humidified incubator containing 5% CO 2 .
  • 3T3-L1 pre-adipocyte differentiation was induced by replacement with DMEM (MDI) containing 10% fetal bovine serum (FBS), 0.5 mM IBMX, 1 ⁇ M dexamethasone and 10 ⁇ g/ml insulin.
  • the MDI medium was replaced with DMEM supplemented with 10% FBS and 10 ⁇ g/ml insulin. After 5-7 days of induction of differentiation, about 90% of pre-adipocytes were converted into round-shaped mature adipocytes. The insulin-fed medium was changed every 2 days.
  • 3T3-L1 pre-adipocytes were seeded, grown on a 96-well plate at a density of 1 ⁇ 10 4 cells/well, and treated with strain cultures at various concentrations for the indicated times. After incubation was complete, 20 ⁇ l of 5 mg/ml MTT solution was added, and the cells were incubated at 37° C. for 3 hours. The obtained formazan crystals were dissolved in 150 ⁇ l of DMSO, and absorbance was measured at 590 nm using a VictorTM X3 multilabel reader (Perkin Elmer, Waltham, MA, USA).
  • MRS medium was used under absolutely anaerobic conditions, and oxygen present in the medium was removed using N 2 gas for anaerobic conditions, followed by sterilization.
  • 0.1 g of the collected fecal sample was suspended in 10 ml MRS medium, diluted in stages, and 100 ⁇ l each was spread on MRS plate medium or blood agar medium, and cultured at 37 ° C. for 2 days under anaerobic conditions. As a result, the resulting single colony was subcultured, pure isolated, long-term preserved, and used.
  • 3T3-L1 cells were mixed with 10% NBCS and 1% penicillin-streptomycin in DMEM GlutaMax and cultured in a 5% CO 2 incubator at 37 °C.
  • the cell concentration of 3T3-L1 cells reaches 70-80%, they are seeded into 48-well plates.
  • MDI Insulin, Dexamethasone, Isobutyl-1-methylxanthine (IBMX)
  • IBMX Isobutyl-1-methylxanthine
  • RT-PCR reverse transcription-polymerase chain reaction
  • cDNA was synthesized with Maxime RT PreMix kit (Intron Biotechnology, Seoul, Korea), and Veriti 96-Well Thermal Cycler (Applied Biosystems , Singapore).
  • Quantitative real-time PCR was performed using the iQ TM SYBR Green Supermix kit (Bio-Rad, Singapore) on a CFX96 TM Real-Time PCR detection system (Bio-Rad, Singapore).
  • the sequences of the primers used in this study are shown in Table 1.
  • the target gene expression level was normalized to Tbp .
  • PVDF polyvinylidene fluoride
  • the membrane was washed 3 times for 5 minutes each with 1 ⁇ TBST.
  • the membrane was incubated with the specific horseradish peroxidase-conjugated secondary antibody at room temperature for 1.5 hours on a shaker and washed three times for about 5 minutes. All washing steps were performed in 1 ⁇ TBST.
  • Immune response protein signals were detected using a chemiluminescent ECL assay and measured using molecular imaging software (Bio-Rad). Brightness and contrast were slightly adjusted for better visualization of the data, but the same changes were applied across the entire image panel, so that no part was lost from the image.
  • Anti- ⁇ -actin was used as a control.
  • Western blot data were quantified using ImageJ software (National Institutes of Health, USA).
  • 3T3-L1 pre-adipocytes were treated with Rosiglitazone (Rosi), a PPAR ⁇ antagonist, as a positive control, and lactic acid bacteria culture at a concentration of 5 ⁇ l/ml.
  • Rosi Rosiglitazone
  • 3T3-L1 preadipocytes were reacted with ice-cold MetOH at -20 °C for 15 minutes, and then treated with 3% BSA and 0.1% Triton X-100 for 15 minutes to permearbilize.
  • the cells were reacted with 1ug/ml DAPI for 1 minute, washed three times with 1x PBST, mounted with mounting medium containing an anti-fading agent (Dako, CA, USA) and mounted under a confocal microscope (Olympus, Tokyo, USA). Japan).
  • the inventors of the present invention determined an appropriate dose of lactic acid bacteria cultures for use in 3T3-L1 pre-adipocytes and differentiated 3T3-L1 cells. was determined through cell viability assay. As shown in FIG. 2, when 3T3-L1 pre-adipocytes were treated with lactic acid bacteria cultures at concentrations of 1 ⁇ l/ml or 5 ⁇ l/ml for 24, 48, and 72 hours, no remarkable cytotoxicity was observed.
  • post-confluent 3T3-L1 pre-adipocytes were treated with MDI medium in the presence or absence of lactic acid bacteria culture medium at a concentration of 1 ⁇ l / ml or 5 ⁇ l / ml. Differentiation into adipocytes was induced by treatment. Microscopic observation after Oil Red O staining on day 6 showed that the treatment with the lactic acid bacteria culture medium had an inhibitory effect on intracellular triglyceride accumulation in a concentration-dependent manner compared to the control group exposed to the MDI medium alone (FIG. 3).
  • C/EBP ⁇ plays an important role in the early differentiation process into adipocytes, and expression of PPAR ⁇ is very important in the later maturation process.
  • C/EBP ⁇ and C/EBP ⁇ play an important role in regulating the expression of C/EBP ⁇ and PPAR ⁇ in adipocyte differentiation.
  • AdipoQ and aP2 as PPAR ⁇ target genes, are highly expressed during the maturation process of adipocytes.
  • the inventors of the present invention investigated whether lactic acid bacteria cultures affect the expression of adipogenic transcription factors during adipogenesis.
  • the 3T3-L1 culture medium was replaced with MDI medium.
  • the lactic acid bacteria culture of the present invention was treated, the mRNA expression of Cebp ⁇ , Cebp ⁇ , Cebp ⁇ , PPAR ⁇ , adipoQ and aP2 was significantly decreased (FIG. 4).
  • the lactic acid bacteria culture greatly reduced the concentrations of C/EBP ⁇ , C/EBP ⁇ , PPAR ⁇ , and aP2 even at the translation stage (FIG. 5).

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Abstract

La présente invention concerne une nouvelle souche Bifidobacterium longum et son utilisation et, plus particulièrement, une nouvelle souche Bifidobacterium longum subsp. Infantis YB0411 ayant une activité anti-obésité, et une composition pharmaceutique, un aliment fonctionnel de santé, une composition cosmétique, une composition d'aliments pour animaux, une composition de réactifs, ou similaire pour une utilisation anti-obésité, comprenant une culture de la souche. La culture de la souche de Bifidobacterium longum subsp. Infantis YB0411 portant le numéro de dépôt de KCTC14393BP de la présente invention présente un effet inhibiteur sur le processus de maturation des pré-adipocytes en adipocytes. Ainsi, la culture de souche peut être appliquée pour une utilisation dans une composition pharmaceutique anti-obésité, un aliment fonctionnel pour la santé, une composition cosmétique, une composition d'alimentation, une composition de réactif et similaires. Ainsi, la culture de la souche peut être appliquée pour une utilisation dans une composition pharmaceutique anti-obésité, un aliment fonctionnel de santé, une composition cosmétique, une composition d'aliments pour animaux, une composition de réactifs ou similaires.
PCT/KR2021/015958 2021-05-13 2021-11-04 Nouvelle souche de bifidobacterium longum et son utilisation WO2022239916A1 (fr)

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