WO2021242056A1 - Souche de l'espèce bifidobacterium et vésicule extracellulaire dérivée de cette dernière, et leurs utilisations anti-inflammatoires et antibactériennes - Google Patents

Souche de l'espèce bifidobacterium et vésicule extracellulaire dérivée de cette dernière, et leurs utilisations anti-inflammatoires et antibactériennes Download PDF

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WO2021242056A1
WO2021242056A1 PCT/KR2021/006702 KR2021006702W WO2021242056A1 WO 2021242056 A1 WO2021242056 A1 WO 2021242056A1 KR 2021006702 W KR2021006702 W KR 2021006702W WO 2021242056 A1 WO2021242056 A1 WO 2021242056A1
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strain
endoplasmic reticulum
lysate
culture medium
mixture
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PCT/KR2021/006702
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English (en)
Korean (ko)
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김정현
강기성
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주식회사 바이오뱅크힐링
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Priority claimed from KR1020200173823A external-priority patent/KR102296285B1/ko
Priority claimed from KR1020200189817A external-priority patent/KR102271909B1/ko
Application filed by 주식회사 바이오뱅크힐링 filed Critical 주식회사 바이오뱅크힐링
Publication of WO2021242056A1 publication Critical patent/WO2021242056A1/fr

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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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

Definitions

  • Microbiome refers to microorganisms that exist in a specific environment and their entire genetic information, and refers to a collection of genomes, which refers to the entire genetic information of a single organism. Therefore, the human microbiome refers to microorganisms living inside and outside the human body and their entire genetic information.
  • the human body lives in a symbiotic relationship with many microorganisms, and in particular, the intestine contains the most microorganisms as an optimal environment for the microorganisms to take in nutrients and form a systematic community.
  • Intestinal microbes supply nutrients that cannot be produced by the host’s enzymes alone and are closely related to the host’s metabolism and immune system, while helping to prevent various diseases such as irritable bowel syndrome, obesity, atopy, depression, rheumatoid arthritis, autism spectrum disorder, and dementia. It has been reported to be associated with
  • the endoplasmic reticulum is a nano-sized material of about 20 to 200 nm produced and discharged by cells, and is free to move between cells.
  • the endoplasmic reticulum contains membrane lipids, membrane proteins, DNA or RNA, etc., and these genetic materials act as a complex to deliver toxic factors between cells and regulate inflammation and immune response. From unicellular organisms to multicellular organisms, information exchange between cells is an essential process of life phenomena, and recently, the endoplasmic reticulum is recognized as a medium for information exchange between cells, so methods for using the vesicle as a drug carrier have been developed.
  • Bifidobacterium genus Bifidobacterium sp.
  • Bifidobacterium breve Bifidobacterium breve
  • KCTC14221BP Bifidobacterium sp.
  • Another aspect is to provide an endoplasmic reticulum derived from the strain, a lysate of the strain, or a culture solution.
  • Another aspect is to provide a pharmaceutical composition for preventing or treating inflammatory diseases comprising the strain, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or a mixture thereof as an active ingredient.
  • Another aspect is to provide a health functional food for the prevention or improvement of inflammatory diseases comprising the strain, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or a mixture thereof as an active ingredient.
  • Another aspect is to provide a health functional food for improving intestinal health comprising the strain, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or a mixture thereof as an active ingredient.
  • Another aspect is to provide a pharmaceutical composition for preventing or treating a bacterial infection comprising the strain, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or a mixture thereof as an active ingredient.
  • Another aspect is to provide a health functional food for preventing or improving bacterial infection comprising the strain, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or a mixture thereof as an active ingredient.
  • Another aspect is to provide a cosmetic composition
  • a cosmetic composition comprising the strain, the endoplasmic reticulum derived from the strain, a lysate of the strain, a culture solution, or a mixture thereof.
  • Another aspect is to provide an antibacterial composition for external application for skin comprising the strain, the endoplasmic reticulum derived from the strain, a lysate of the strain, a culture solution, or a mixture thereof.
  • Another aspect is to provide a method for preventing or treating an inflammatory disease by administering the strain, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or a mixture thereof to an individual in need thereof.
  • Another aspect is to provide a method for preventing or treating a bacterial infection by administering the strain, the endoplasmic reticulum derived from the strain, a lysate of the strain, a culture medium, or a mixture thereof to an individual in need thereof.
  • Another aspect is to provide the use of the strain, the endoplasmic reticulum derived from the strain, a lysate of the strain, a culture solution, or a mixture thereof for the preparation of a medicament for the prevention or treatment of inflammatory diseases.
  • Another aspect is to provide the use of the strain, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or a mixture thereof for the manufacture of a medicament for the prevention or treatment of bacterial infections.
  • One aspect provides a strain of Bifidobacterium breve belonging to the genus Bifidobacterium sp .
  • the Bifidobacterium breve strain may be a strain deposited with accession number KCTC14195BP.
  • the Bifidobacterium breve strain may be a strain comprising 16s rRNA of SEQ ID NO: 1.
  • One aspect provides a strain of Bifidobacterium animalis subsp. lactis belonging to the genus Bifidobacterium sp .
  • the Bifidobacterium animalis lactis strain may be a strain deposited with accession number KCTC14221BP.
  • the Bifidobacterium animalis lactis strain may be a strain comprising 16s rRNA of SEQ ID NO: 2.
  • the strain may have anti-inflammatory and/or antibacterial activity.
  • the strain inhibits the production of nitric oxide in the inflamed cells, inhibits the expression of inflammatory cytokines (eg, TNF- ⁇ or IL-6), or inhibits the proliferation of bacteria (eg, C. difficile ) may be inhibiting
  • the strain reduces inflammatory factors induced by C. difficile, for example, pro-inflammatory cytokines (eg, TNF, or CCL2), or increases anti-inflammatory cytokines (IL-10).
  • Another aspect provides an endoplasmic reticulum derived from the Bifidobacterium breve strain, a lysate of the strain, a culture solution, an extract of the culture solution, or a mixture thereof.
  • Another aspect provides an endoplasmic reticulum derived from Bifidobacterium animalis subsp. lactis, a lysate of the strain, a culture solution, an extract of the culture solution, or a mixture thereof.
  • the strain is as described above.
  • the term “vesicle” refers to particles secreted from cells and released into the extracellular space, and includes exosomes, ectosomes, microvesicles, and microparticles. , exosome-like vesicles, and the like.
  • the extracellular ER can reflect the state of the secreting cell of origin (donor cell), exhibit various biological activities depending on which cell it is secreted from, and play an important role in cell-to-cell interactions by transferring genetic material and proteins between cells can do.
  • the cell-derived substances including the endoplasmic reticulum cause disease or stimulate immune cells to fight disease, and have the effect of helping to break down and absorb substances that humans cannot digest through the metabolic process of microorganisms. have.
  • the endoplasmic reticulum is a membrane-structured endoplasmic reticulum, which is divided into inside and outside, and has plasma membrane lipid and plasma membrane protein, nucleic acid, and cytoplasmic components of the cell, and is larger than the original cell. may be small.
  • the endoplasmic reticulum may be isolated from the cell lysate of the culture solution of the Bifidobacterium breve strain.
  • the endoplasmic reticulum may be isolated from the cell lysate of the culture solution of the Bifidobacterium animalis lactis strain.
  • the extracellular vesicles may have a diameter of 10 nm to 400 nm.
  • it may be 10 nm to 400 nm, 10 nm to 350 nm, 10 nm to 300 nm, or 10 nm to 250 nm.
  • culture medium may be used interchangeably with “culture supernatant”, “conditioned culture medium” or “conditioned medium”, and Bifidobacterium breve or Bifidobacterium animalis lactis is It may mean the entire medium including the strain obtained by culturing the strain for a certain period of time in a medium capable of supplying nutrients to grow and survive, its metabolites, and extra nutrients.
  • the culture solution may refer to a culture solution obtained by culturing the strain in which the cells are removed from the culture solution.
  • the liquid from which the cells have been removed from the culture solution is also called a “supernatant”, and the culture solution is left for a certain period of time to take only the liquid from the upper layer except for the part that has sunk to the lower layer, remove the cells through filtration, or centrifuge the culture solution to the lower part It can be obtained by removing the precipitation of and taking only the liquid at the top.
  • the "cell” of the present invention refers to the strain itself of the present invention, and includes the strain isolated and selected from a skin sample, or the strain isolated from the culture solution by culturing the strain.
  • the cells can be obtained by centrifuging the culture solution to take the part that has sunk to the lower layer, or by removing the liquid from the upper layer after leaving it still for a certain period of time because it sinks into the lower layer of the culturing solution by gravity.
  • the culture medium may include the culture medium itself obtained by culturing the strain, its concentrate, or a lyophilisate or a culture supernatant obtained by removing the strain from the culture medium, its concentrate or lyophilisate.
  • the culture medium is prepared by culturing Bifidobacterium breve or Bifidobacterium animalis lactis in an appropriate medium (eg, R2A medium or TSA medium) at any temperature above 10°C or below 40°C for a certain period of time, for example, , may be obtained by culturing for 4 to 50 hours.
  • an appropriate medium eg, R2A medium or TSA medium
  • the culture supernatant of the strain may be obtained by centrifuging or filtering the strain culture to remove the strain.
  • the concentrate may be obtained by concentrating the supernatant obtained after filtering the strain culture solution itself, or the culture solution using centrifugation or a filter.
  • the culture medium and culture conditions for culturing the Bifidobacterium breve or Bifidobacterium animalis lactis may be appropriately selected or modified by those of ordinary skill in the art.
  • lysate may refer to a product obtained by disrupting the cell wall of the strain itself by chemical or physical force.
  • culture solution extract means extraction from the culture solution or its concentrate, and may include an extract, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or these prepared or purified products, and a fraction obtained by fractionating it.
  • Another aspect provides a disease improvement, prevention or treatment use of Bifidobacterium breve, the endoplasmic reticulum derived from the strain, a lysate of the strain, a culture solution, or an extract of the culture solution.
  • Another aspect provides the use of Bifidobacterium animalis lactis, an endoplasmic reticulum derived from the strain, a lysate of the strain, a culture solution, or an extract of the culture solution for improving, preventing or treating diseases.
  • the term “treat” may mean that inflammation or bacterial infection is cured in a shorter time compared to natural healing.
  • the treatment may include amelioration and/or alleviation of inflammation or bacterial infection.
  • the treatment may refer to healing and/or recovery of symptoms resulting from inflammation or bacterial infection.
  • the use of the strain may include preventing, ameliorating, or treating an inflammatory disease (anti-inflammatory activity), preventing, ameliorating, or treating a bacterial infection (antibacterial activity), or preventing or improving intestinal health.
  • an inflammatory disease anti-inflammatory activity
  • preventing, ameliorating, or treating a bacterial infection antibacterial activity
  • preventing or improving intestinal health may include preventing, ameliorating, or treating an inflammatory disease (anti-inflammatory activity), preventing, ameliorating, or treating a bacterial infection (antibacterial activity), or preventing or improving intestinal health.
  • the inflammatory disease may include inflammation of the digestive system (gastrointestinal tract, etc.), intraocular inflammation, oral inflammation, respiratory system inflammation including lung, skin inflammation, cardiovascular inflammation, brain inflammation, and ear inflammation. have.
  • the inflammatory disease is inflammatory bowel disease (IBD); irritable bowel syndrome; Behcet's disease; enteritis, Crohn's disease; ulcerative colitis; vasculitis; mucositis; stomatitis; peri-implantitis; periodontitis; pulpitis; gingivitis; Pneumonia; dermatitis; atopic dermatitis; contact dermatitis; CREST syndrome; dermatitis herpetiformis; dermatomyositis; systemic scleroderma; erythema nodosum; Henoch-Schonlein purpura; Hidradenitis suppurativa; lichen planus; Majeed syndrome; Schnitzler syndrome; psoriasis; eczema; acne; mouth ulcers; uveitis; pharyngitis; tonsillitis; otitis, including otitis media; arthritis (psoriatic arthritis); synovitis; men
  • the improvement of the intestinal health may be a help in intestinal beneficial growth and suppression of harmful bacteria, a help in intestinal health by regulating immunity, or a help in smooth bowel movements.
  • antibacterial agent refers to (i) inhibiting, reducing or preventing the growth of bacteria; (ii) inhibit or reduce the ability of the bacteria to cause infection in a subject; or (iii) a substance capable of inhibiting or reducing the ability of a bacterium to multiply or maintain infectivity in the environment.
  • the bacterial infection may include an infection caused by gram-positive bacteria or gram-negative bacteria.
  • the bacterial infection is Clostridium (Clostridium), Helicobacter (Helicobactor), Escherichia (Escherichia), Salmonella (Salmonella), Staphylococcus (Staphylococcus), Streptococcus (Streptococcus), by a brush loose (Haemophilus ), Klebsiella (Klebsiella), morak Cellar (Moraxella), Enterobacter bakteo (Enterobacter), Proteus (Proteus), Serratia marcescens (Serratia), Pseudomonas (Pseudomonas), ahsine Sat bakteo (Acinetobacter), bakteo (Citrobacter a sheet ), Stenotrophomonas ( Stenotrophomonas ), Bacteroides ), Prevotella ( Prevotella ( Pre
  • the composition comprises 0.00001 wt% to 80 wt%, for example, 0.00001 wt% to 60 wt%, 0.00001 wt% to 40 wt%, 0.00001 wt% to 30 wt%, 0.00001 wt% to 20 wt%, based on the total weight of the composition %, 0.00001% to 10% by weight, 0.00001% to 5% by weight, 0.05% to 60% by weight, 0.05% to 40% by weight, 0.05% to 30% by weight, 0.05% to 20% by weight, 0.05% to 10% by weight, 0.05% to 5% by weight, 0.1% to 60% by weight, 0.1% to 40% by weight, 0.1% to 30% by weight, 0.1% to 20% by weight, 0.1% by weight % to 10% by weight, or 0.1% to 5% by weight of a strain, a lysate thereof, a culture solution, or an extract of a culture solution thereof.
  • the term, "included as an active ingredient” means that the strain of the present specification, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or the extract of the culture medium is added to the extent that it can exhibit the above-mentioned effects,
  • the term, “included as an active ingredient” means that the strain of the present specification, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or the extract of the culture medium is added to the extent that it can exhibit the above-mentioned effects.
  • drug delivery and stabilization it is meant to include formulations in various forms by adding various components as sub-components.
  • the composition may be a pharmaceutical composition.
  • the pharmaceutical composition may further include a pharmaceutically acceptable diluent or carrier.
  • the diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and the lubricant may be magnesium stearate, talc, or a combination thereof.
  • the carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof.
  • the excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof.
  • the disintegrant may be carboxymethyl cellulose calcium, sodium starch glycolate, anhydrous calcium monohydrogen phosphate, or a combination thereof.
  • the binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof.
  • the lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.
  • the pharmaceutical composition may be formulated as an oral or parenteral dosage form.
  • Oral dosage forms may be granules, powders, solutions, tablets, capsules, dry syrups, or a combination thereof.
  • the parenteral dosage form may be an injection.
  • the composition may be a health functional food composition.
  • the health functional food composition may be used alone or in combination with other foods or food ingredients of the strain or its culture, and may be appropriately used according to a conventional method.
  • the mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prophylactic, health or therapeutic treatment).
  • the composition of the present specification may be added in an amount of 15 parts by weight or less based on the raw material.
  • the beverage composition may contain various flavoring agents or natural carbohydrates as additional components, like a conventional beverage.
  • the natural carbohydrates 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.
  • sweetener natural sweeteners such as taumartin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used.
  • the health food composition may also be added to nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonated beverages the carbonation agent used, or a combination thereof.
  • the health functional food composition may also contain natural fruit juice, fruit juice beverage, fruit flesh for the production of vegetable beverage, or a combination thereof.
  • the composition may be a cosmetic composition.
  • the cosmetic composition may have a cosmetic formulation of, for example, a softening lotion, a nourishing lotion, a massage cream, a nourishing cream, an essence, a pack, a gel, an ampoule, or a skin adhesion type.
  • Components included in the cosmetic composition may include components commonly used in cosmetic compositions in addition to the composition as an active ingredient, for example, conventional adjuvants and carriers such as stabilizers, solubilizers, vitamins, pigments and fragrances. may include.
  • composition may be a composition for external application to the skin.
  • the external preparation for skin may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, drug-containing bandage, lotion, or a combination thereof.
  • the external preparation for skin is a component normally used in external preparations for skin such as cosmetics or pharmaceuticals, for example, an aqueous component, an oily component, a powder component, alcohol, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, a preservative, an antioxidant, a surfactant, a fragrance , colorant, various skin nutrients, or a combination thereof may be appropriately formulated as needed.
  • the external preparation for skin includes metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belapamil, licorice extract, glablidine, and kaline.
  • metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belapamil, licorice extract, glablidine, and kaline.
  • Fruit hot water extract, various herbal medicines, tocopherol acetate, glycyrrhizic acid, tranexamic acid and its derivatives or salts thereof, vitamin C, magnesium ascorbate phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, Sugars, such as trehalose, etc. can be mix
  • Another aspect also provides a method of preventing, ameliorating, or treating a condition in a subject comprising treating or administering to the subject in need thereof an effective amount of the composition described above.
  • the subject's condition may be a condition associated with inflammation or a condition associated with a bacterial infection.
  • Administration may be administered by methods known in the art. Administration can be administered directly to a subject by any means, for example, by routes such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. can The administration may be systemically or locally.
  • the subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat.
  • the subject may be a subject in need of improvement of a condition associated with inflammation or a condition associated with bacterial infection.
  • the administration is 0.00001 mg to 1,000 mg per subject per day, for example, 0.00001 mg to 500 mg, 0.00001 mg to 100 mg, 0.00001 mg to 50 mg, 0.00001 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg may be administered.
  • the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity, and those skilled in the art
  • the dosage may be appropriately adjusted in consideration of these factors.
  • the number of administration can be once a day or twice or more within the range of clinically acceptable side effects, and it can be administered to one or two or more places for the administration site, and total daily or at intervals of 2 to 5 days.
  • the number of days of administration may range from 1 to 30 days per treatment. If necessary, the same treatment can be repeated after a titration period.
  • the dose is the same as that of humans per kg, or the above dose is converted, for example, by the volume ratio (for example, the average value) of the target animal and the organ (heart, etc.) One dose can be administered.
  • novel strain and its derived endoplasmic reticulum there is an effect that can be usefully used for prevention, improvement, or treatment of inflammation-related conditions, or bacterial infections.
  • N negative control
  • P untreated control
  • EV endoplasmic reticulum of Example 2.
  • Figure 2 is a graph showing the protein expression level of inflammatory cytokines (TNF- ⁇ ) in the cell treatment of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.
  • N negative control
  • P untreated control
  • EV endoplasmic reticulum of Example 2.
  • Figure 3 is a graph showing the protein expression level of the inflammatory cytokine (IL-6) in the cell treatment of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.
  • IL-6 inflammatory cytokine
  • FIG. 4 is a graph showing the culture rate according to the co-culture of the strain and C. difficile according to an embodiment.
  • FIG. 5 is a graph showing the culture rate of C. difficile according to the treatment with C. difficile of the endoplasmic reticulum of the strain according to one embodiment.
  • N negative control
  • P untreated control
  • EV endoplasmic reticulum of Example 2.
  • CdEV Clostridium difficile ER
  • BBH001 ER of Example 1 strain
  • Type strain ER of Bifidobacterium breve standard strain.
  • CdEV Clostridium difficile ER
  • BBH001 ER of Example 1 strain
  • Type strain ER of Bifidobacterium breve standard strain.
  • N negative control
  • P untreated control
  • EV endoplasmic reticulum of Example 2.
  • N negative control
  • P untreated control
  • EV endoplasmic reticulum of Example 2.
  • FIG. 11 is a graph showing the protein expression level of the inflammatory cytokine (IL-6) in the cell treatment of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.
  • IL-6 inflammatory cytokine
  • FIG. 12 is a graph showing the protein expression level of the inflammatory cytokine (CCL2) in the cell treatment of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.
  • CCL2 inflammatory cytokine
  • 13 is a graph showing the culture rate of C. difficile in the supernatant of the strain according to one embodiment.
  • N negative control
  • P untreated control
  • EV endoplasmic reticulum of Example 2.
  • CdEV Clostridium difficile ER
  • BBH003 ER of the Example 1 strain
  • Type strain ER of the Bifidobacterium animalis standard strain.
  • CdEV Clostridium difficile ER
  • BBH003 ER of the Example 1 strain
  • Type strain ER of the Bifidobacterium animalis standard strain.
  • Fecal Microbiota Transplantation was performed as follows to isolate and identify the Bifidobacterium breve strain from the composition.
  • Bifidobacterium breve BBH 001 having 99% homology was isolated.
  • the selected Bifidobacterium breve BBH 001 strain was deposited with the Korea Center for Biological Resources on May 25, 2020 and was given accession number KCTC14195BP, and the Bifidobacterium breve BBH 001 strain has a 16s rRNA sequence of SEQ ID NO: 1 (complementary DNA).
  • serial dilution Serial dilution
  • BHI medium Brain Heart Infusion-supplemented, Eco cell
  • PCR amplification was performed on the cultured colonies, and the nucleotide sequence of the 16S rRNA region determined among the isolated and cultured microbial colonies was transferred to another registered BLAST program provided on the website of the National Center for Biotechnology Information (NCBI). The strains were compared and analyzed. As a result of comparative analysis, Bifidobacterium animalis BBH 003 having 99% homology was isolated.
  • the selected Bifidobacterium animalis BBH 003 strain was deposited with the Korea Biological Resources Center on June 23, 2020 and was given an accession number KCTC14214BP, and the Bifidobacterium animalis BBH 003 strain has a 16s rRNA sequence of SEQ ID NO: 2 (complementary DNA).
  • the endoplasmic reticulum of the strain isolated in the above example was isolated.
  • the isolated strain was cultured in BHIs broth (Brain Heart Infusion-supplemented, Eco cell) at 30° C. anaerobic conditions for 3 days. Thereafter, the culture medium was centrifuged at 3000 RPM for 20 minutes and then centrifuged again at 3000 RPM for 40 minutes to remove bacterial debris.
  • BHIs broth Brain Heart Infusion-supplemented, Eco cell
  • Mouse macrophage Raw264.7 cells were treated with RPMI1640 culture medium containing 20% fetal bovine serum (FBS) and 1% antibiotics (100 U/mL penicillin and 100 ⁇ g/mL streptomycin) in the presence of 5% CO 2 37 Incubated at °C. Thereafter, the Raw 264.7 cells were aliquoted at a concentration of 5 ⁇ 10 4 cells/well in a 48-well plate by 250 ⁇ L, and incubated at 37° C. and 24 hours in a CO 2 incubator.
  • FBS fetal bovine serum
  • antibiotics 100 U/mL penicillin and 100 ⁇ g/mL streptomycin
  • the well supernatant was discarded and a medium supplemented with lipopolysaccharide (LPS) 10ug/ml was dispensed to induce inflammation, followed by further incubation for 4 hours.
  • the supernatant containing LPS was discarded and the endoplasmic reticulum was added to the medium at a concentration of 1 or 10 ug/ml for treatment, and then cultured at 37° C. for 16 hours. Thereafter, 50 ⁇ L of the well supernatant and 50 ⁇ L of Griess reagent were mixed and reacted at room temperature for 10 minutes, and then absorbance was measured at 570 nm with a plate reader to measure the amount of nitric oxide produced, and the results are shown in FIGS. 1 and 9 .
  • N negative control
  • P untreated control
  • EV endoplasmic reticulum of Example 2.
  • the ER of the strain As shown in FIGS. 1 and 9 , it was found that the ER of the strain according to one embodiment reduced the amount of NO production of the inflammation-induced cells to the level of the negative control.
  • the inflammatory cytokine inhibitory activity of the endoplasmic reticulum of the strain was measured. Specifically, the Raw264.7 cells treated with LPS in the same way as above The endoplasmic reticulum was treated at a concentration of 0.1, 1 or 10 ug/ml and then incubated at 37° C. for 48 hours. Then, the protein expression of TNF- ⁇ , IL-6 and / or CCL2 in the cells was measured for absorbance at 540 nm using an ELISA kit (eBioscience, USA) according to the manufacturer's instructions, and the results are shown in Fig. 2, 3 and 10 to 12 are shown.
  • Figure 2 is a graph showing the protein expression level of inflammatory cytokines (TNF- ⁇ ) in the cell treatment of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.
  • N negative control
  • P untreated control
  • EV endoplasmic reticulum of Example 2.
  • Figure 3 is a graph showing the protein expression level of the inflammatory cytokine (IL-6) in the cell treatment of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.
  • IL-6 inflammatory cytokine
  • N negative control
  • P untreated control
  • EV endoplasmic reticulum of Example 2.
  • FIG. 11 is a graph showing the protein expression level of the inflammatory cytokine (IL-6) in the cell treatment of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.
  • IL-6 inflammatory cytokine
  • FIG. 12 is a graph showing the protein expression level of the inflammatory cytokine (CCL2) in the cell treatment of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.
  • CCL2 inflammatory cytokine
  • strain according to one embodiment can be usefully used for the prevention, improvement, or treatment of inflammatory diseases, particularly inflammatory bowel disease or irritable bowel syndrome.
  • Example 1 The antibacterial activity of the strain isolated in Example 1. was analyzed.
  • the strain of Example 1 and C.difficile in 5ml BHI broth (Brain Heart Infusion, Eco cell) It was pre-cultured and adjusted to OD (600 nm) 0.5 using a spectrophotometer. After that, the strain culture medium was inoculated in 30ml BHI broth at a rate of 10% and then cultured anaerobically at 37°C for 48 hours. The cultured strain was centrifuged at 4000RPM for 30 minutes to separate the pellet and the supernatant, and then the pellet was washed 3 times with PBS and then resuspended.
  • C. difficile and the strain of the above example were inoculated into BHI broth at the same ratio in the same amount, and then co-cultured in BHI broth medium under anaerobic conditions for 2 days. Thereafter, the culture rate of C. difficile was measured by the method of colony forming unit calculation, and the results are shown in FIG. 4 .
  • the culture supernatant of the strain was centrifuged at 4000 for 30 minutes to separate the endoplasmic reticulum.
  • the separated supernatant was concentrated using centrifuge tubes (Amicon centrifuge tubes), and inoculated to 3 X 10 6 cfu/ml C. difficile at a ratio of 90%, anaerobic conditions for 1 day in BHI broth medium cultured in Thereafter, the culture rate of C. difficile was measured by the method of colony forming unit calculation, and the results are shown in FIGS. 5 and 13 .
  • FIG. 4 is a graph showing the culture rate according to the co-culture of the strain and C. difficile according to an embodiment.
  • 5 and 13 are graphs showing the culture rate of C. difficile according to the treatment with C. difficile of the endoplasmic reticulum of the strain according to one embodiment.
  • the strain and its derived endoplasmic reticulum according to one embodiment was found to significantly reduce the culture rate of C. difficile.
  • the strain and/or the endoplasmic reticulum derived therefrom according to one embodiment have antibacterial activity against bacteria, for example, Gram-negative bacteria.
  • these results show that the strain and/or the endoplasmic reticulum derived therefrom according to one embodiment are Clostridium difficile infection (CDI), or the prevention, improvement, or treatment of irritable bowel syndrome resulting therefrom.
  • CDI Clostridium difficile infection
  • the PreMix WST-1 Cell Proliferation Assay System was used for the cytotoxicity test of the endoplasmic reticulum of the strain isolated in Example 2.
  • 6 and 14 are graphs showing the cytotoxicity results of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.
  • Clostridium difficile endoplasmic reticulum (CdEV) in mouse macrophage Raw264.7 cells was treated in an amount of 100gu/ml, and then cultured at 37°C for 4 hours.
  • the standard strain of Bifidobacterium breve (ATCC15700) and the endoplasmic reticulum of the strain of Example 1 were treated in an amount of 0.1ug/ml, and cultured at 37°C for 16 hours, cell viability and oxidation Nitrogen production was measured in the same manner as in Experimental Examples 1 and 3, and the results are shown in FIG. 7 .
  • mice macrophage Raw264.7 cells were treated with Clostridium difficile endoplasmic reticulum (CdEV) in an amount of 100gu/ml, and then cultured at 37°C for 4 hours.
  • CdEV Clostridium difficile endoplasmic reticulum
  • ATCC25527 the standard strain of Bifidobacterium animalis
  • endoplasmic reticulum of the strain of Example 1 were treated with an amount of 1ug/ml or 10ug/ml, and then cultured at 37°C for 16 hours, cells
  • the survival rate was measured in the same manner as in Experimental Example 3, and the results are shown in FIG. 15 .
  • CdEV Clostridium difficile ER
  • BBH001 ER of Example 1 strain
  • Type strain ER of Bifidobacterium breve standard strain.
  • CdEV Clostridium difficile ER
  • BBH001 ER of Example 1 strain
  • Type strain ER of Bifidobacterium breve standard strain.
  • CdEV Clostridium difficile ER
  • BBH003 ER of the Example 1 strain
  • Type strain ER of the Bifidobacterium animalis standard strain.
  • CdEV Clostridium difficile ER
  • BBH003 ER of the Example 1 strain
  • Type strain ER of the Bifidobacterium animalis standard strain.
  • the endoplasmic reticulum of the strain significantly reduced the amount of cytokines increased by the endoplasmic reticulum of Clostridium difficile compared to the standard strain for pro-inflammatory cytokines TNF and CCL2 It was found that the amount of anti-inflammatory cytokine IL-10 was significantly increased.
  • strain and/or endoplasmic reticulum derived therefrom has significant anti-inflammatory activity, Clostridium difficile infection (CDI), or prevention, improvement, or irritable bowel syndrome resulting therefrom It means that it can be usefully used for treatment.
  • CDI Clostridium difficile infection

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Abstract

La présente invention concerne : une nouvelle souche de Bifidobacterium breve ou Bifidobacterium animalis subsp. Lactis appartenant au genre Bifidobacterium ; un lysat associé ; un milieu de culture ; un extrait du milieu de culture ; une vésicule extracellulaire ; et leurs utilisations anti-inflammatoires et/ou antibactériennes. Une nouvelle souche et une vésicule extracellulaire dérivée de cette dernière, selon un aspect, peuvent être efficacement utilisées dans la prévention, le soulagement ou le traitement d'états associés à une inflammation ou d'infections bactériennes.
PCT/KR2021/006702 2020-05-28 2021-05-28 Souche de l'espèce bifidobacterium et vésicule extracellulaire dérivée de cette dernière, et leurs utilisations anti-inflammatoires et antibactériennes WO2021242056A1 (fr)

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KR20200064592 2020-05-28
KR10-2020-0064592 2020-05-28
KR20200076997 2020-06-24
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KR1020200173823A KR102296285B1 (ko) 2020-05-28 2020-12-11 비피도박테리움 속 균주, 및 그의 유래의 소포체 및 그의 항염증 및 항균 용도
KR10-2020-0173823 2020-12-11
KR10-2020-0189817 2020-12-31
KR1020200189817A KR102271909B1 (ko) 2020-06-24 2020-12-31 비피도박테리움 아니말리스 균주, 및 그의 유래의 소포체 및 그의 항염증 및 항균 용도

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