WO2022124871A1 - Souche de lactobacillus sp. , souche d'eubacterium sp., ou souche de coprococcus sp., vésicules dérivées de ces dernières, et utilisation anti-inflammatoire et antibactérienne correspondante - Google Patents

Souche de lactobacillus sp. , souche d'eubacterium sp., ou souche de coprococcus sp., vésicules dérivées de ces dernières, et utilisation anti-inflammatoire et antibactérienne correspondante Download PDF

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WO2022124871A1
WO2022124871A1 PCT/KR2021/018882 KR2021018882W WO2022124871A1 WO 2022124871 A1 WO2022124871 A1 WO 2022124871A1 KR 2021018882 W KR2021018882 W KR 2021018882W WO 2022124871 A1 WO2022124871 A1 WO 2022124871A1
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Prior art keywords
strain
endoplasmic reticulum
lysate
lactobacillus
culture medium
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PCT/KR2021/018882
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English (en)
Korean (ko)
Inventor
김정현
강기성
Original Assignee
주식회사 바이오뱅크힐링
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Priority claimed from KR1020200173827A external-priority patent/KR102269962B1/ko
Priority claimed from KR1020200173828A external-priority patent/KR102269963B1/ko
Priority claimed from KR1020200173825A external-priority patent/KR102296287B1/ko
Application filed by 주식회사 바이오뱅크힐링 filed Critical 주식회사 바이오뱅크힐링
Publication of WO2022124871A1 publication Critical patent/WO2022124871A1/fr

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    • 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
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • 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
    • 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

  • Microbiome refers to microorganisms that exist in a specific environment and their entire genetic information, and refers to a collection of genomes that refer 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 preventing 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 it is free to move between cells.
  • the endoplasmic reticulum contains membrane lipids, membrane proteins, DNA or RNA, and the like, 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 has been recognized as a medium for information exchange between cells, so methods for using the vesicle as a drug carrier have been developed.
  • accession number KCTC 14264BP Lactobacillus genus ( Lactobacillus sp. ) belonging to Lactobacillus fermentum ( Lactobacillus fermentum ) strain
  • accession number KCTC 14265BP Lactobacillus genus ( Lactobacillus sp. ) belonging to Bacillus plantarum ( Lactobacillus plantarum ) Strain
  • accession number KCTC 14313BP accession number KCTC 14313BP
  • 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 or bacterial infection administered to an individual in need thereof, including the strain, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or a mixture thereof. .
  • 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 use in the preparation of a composition for the prevention or treatment of inflammatory diseases or bacterial infections.
  • accession number KCTC 14264BP Lactobacillus genus ( Lactobacillus sp. ) belonging to Lactobacillus fermentum ( Lactobacillus fermentum ) strain
  • accession number KCTC 14265BP Lactobacillus genus ( Lactobacillus sp. ) belonging to Bacillus plantarum ( Lactobacillus plantarum ) Strain
  • accession number KCTC 14313BP accession number KCTC 14313BP
  • Eubacterium genus Eubacterium sp.
  • the Lactobacillus permentum strain may be a strain deposited with accession number KCTC 14264BP.
  • the Lactobacillus plantarum strain may be a strain deposited with accession number KCTC 14265BP.
  • the Eubacterium rimosum strain may be a strain deposited with accession number KCTC 14313BP.
  • the Coprococcus comes strain may be a strain deposited with accession number KCTC 14374BP.
  • the Lactobacillus permentum strain may be a strain comprising 16S rRNA of SEQ ID NO: 1.
  • the Lactobacillus plantarum strain may be a strain comprising 16S rRNA of SEQ ID NO: 2.
  • the Eubacterium rimosum strain may be a strain comprising 16S rRNA of SEQ ID NO: 3.
  • the Coprococcus cometh strain may be a strain comprising 16S rRNA of SEQ ID NO: 4.
  • 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).
  • Lactobacillus Fermentum Lactobacillus plantarum, Eubacterium rimosum
  • it provides an endoplasmic reticulum derived from a Coprococcus comes strain, a lysate of the strain, a culture solution, an extract of the culture solution, or a mixture thereof.
  • the strain is as described above.
  • vesicle refers to a particle secreted from a cell and released into the extracellular space, and includes an exosome, an ectosome, a microvesicle, and a microparticle. , 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.
  • the endoplasmic reticulum is a membrane-structured endoplasmic reticulum, which is divided into an inside and an outside, and has a plasma membrane lipid, a plasma membrane protein, a nucleic acid, and a cytoplasmic component of the cell, and is larger than the original cell. may be small.
  • the endoplasmic reticulum is the Lactobacillus permentum, Lactobacillus plantarum, Eubacterium rimosum, Or it may be one isolated from the cell lysate of the culture solution of the Coprococcus comes 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.
  • the term "culture medium” can be used interchangeably with “culture supernatant”, “conditioned culture medium” or “conditioned medium”, and Lactobacillus fermentum capable of supplying nutrients to grow and survive in vitro It may refer to the entire medium including the strain obtained by culturing the strain in a medium for a certain period of time, 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 medium 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 bottom 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 medium and taking 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 to the lower layer of the culture 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 the Lactobacillus permentum, Lactobacillus plantarum, Eubacterium rimosum, Or one obtained by culturing the Coprococcus comes strain 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, 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.
  • lysate may refer to a product obtained by crushing 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 fractions obtained by fractionation thereof.
  • Lactobacillus Fermentum Lactobacillus plantarum, Eubacterium rimosum, Or Coprococcus comes strain, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or an extract of the culture medium provides a disease improvement, prevention or treatment use.
  • 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 caused by 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 intestinal health may be a help in intestinal beneficial growth and suppression of harmful bacteria, help in intestinal health by regulating immunity, or 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 ), Helicobactor , Escherichia , Salmonella , Staphylococcus ), Streptococcus ), Haemophilus ( Haemophilus ) ), Klebsiella , Moraxella , Enterobacter , Proteus , Serratia , Pseudomonas , Acinetobacter , Citrobacter ), Stenotrophomonas , Bacteroides , Prevotella ( Prevotella ), Fusobacterium ( Fusobacterium ) It may include an infection caused by bacteria belonging to the genus. More specifically, the bacterial infection is Clostridium difficile infection (CDI), or Clostridium difficile associated disease (CDAD), for example, Clostridium difficile associated diarrhea. diarrhea) may be included.
  • CDI Clostridium difficile infection
  • 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% with respect to 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 the strain, a lysate thereof, a culture medium, or an extract of the culture medium 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 an additional component 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 can also be added to nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonated beverages 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, transdermally delivered patch, drug-containing bandage, lotion, or a combination thereof.
  • the external preparation for skin is a component usually 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-blocking 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-blocking 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 extracts, various herbal medicines, tocopherol acetate, glitylittic acid, tranexamic acid and 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 may be administered directly to a subject by any means, for example, 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 reaction sensitivity, and those skilled in the art
  • the dosage may be appropriately adjusted in consideration of these factors.
  • the number of administration may be once a day or twice or more within the range of clinically acceptable side effects, and may be administered to one or two or more places for the site of administration, 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 a human per kg, or the above dose is converted, for example, by the volume ratio (for example, average value) of the target animal and the organ (heart, etc.) of the human One dose can be administered.
  • novel strain and its derived endoplasmic reticulum there is an effect that can be usefully used for the 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- ⁇ and 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.
  • N negative control
  • P untreated control
  • EV endoplasmic reticulum of Example 2.
  • FIG. 3 is a graph showing the protein expression level of inflammatory cytokines (CCL2) and anti-inflammatory cytokines (IL-10) in cell treatment of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: the endoplasmic reticulum of Example 2.
  • CCL2 inflammatory cytokines
  • IL-10 anti-inflammatory cytokines
  • FIG. 4 is a graph showing the culture rate according to the co-culture of the strain and C. difficile according to one 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
  • BBH005 ER of the Example 1 strain
  • Type strain ER of the Lactobacillus permentum standard strain.
  • CdEV Clostridium difficile ER
  • BBH005 ER of the Example 1 strain
  • Type strain ER of the Lactobacillus Fermentum sub-strain.
  • Figure 9 is a graph showing the amount of nitric oxide production according to 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.
  • FIG. 11 is a graph showing the protein expression levels of inflammatory cytokines (CCL2) and anti-inflammatory cytokines (IL-10) in 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 cytokines
  • IL-10 anti-inflammatory cytokines
  • 12 is a graph showing the culture rate according to the co-culture of the strain and C. difficile according to one embodiment.
  • FIG. 13 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
  • BBH006 ER of Example 1 strain
  • Type strain ER of Lactobacillus plantarum standard strain.
  • CdEV Clostridium difficile ER
  • BBH006 ER of Example 1 strain
  • Type strain ER of Lactobacillus plantarum 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. 19 is a graph showing the protein expression levels of inflammatory cytokines (CCL2) and anti-inflammatory cytokines (IL-10) 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 cytokines
  • IL-10 anti-inflammatory cytokines
  • 20 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
  • BBH009 ER of the Example 1 strain
  • Type strain ER of the Eubacterium rimosum standard strain.
  • CdEV Clostridium difficile ER
  • BBH009 ER of the Example 1 strain
  • Type strain ER of the Eubacterium rimosum 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.
  • 26 is a graph showing the protein expression levels of inflammatory cytokines (CCL2) and anti-inflammatory cytokines (IL-10) in 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 cytokines
  • IL-10 anti-inflammatory cytokines
  • 27 is a graph showing the culture rate according to the co-culture of the strain and C. difficile according to an embodiment.
  • FIG. 28 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 endoplasmic reticulum
  • BBH011 endoplasmic reticulum of strain
  • Type strain endoplasmic reticulum of Coprococcus comes standard strain strain.
  • FIG. 31 is a graph showing the anti-inflammatory activity of the endoplasmic reticulum of the strain according to one embodiment against inflammation induced by Clostridium difficile; CdEV: Clostridium difficile endoplasmic reticulum, BBH011: endoplasmic reticulum of strain Example 1 strain, Type strain: endoplasmic reticulum of Coprococcus comes standard strain strain.
  • Fecal Microbiota Transplantation was performed as follows to isolate and identify strains from the composition.
  • Lactobacillus fermentum BBH 005 having 98% homology was isolated.
  • the selected Lactobacillus fermentum BBH 005 strain was deposited with the Korea Biological Resources Center on August 6, 2020 and was given an accession number KCTC 14264BP, and the Lactobacillus fermentum BBH 005 strain has a 16S rRNA sequence of SEQ ID NO: 1 (complementary DNA).
  • Lactobacillus plantarum BBH 006 having 98% homology was isolated.
  • the selected Lactobacillus plantarum BBH 006 strain was deposited with the Korea Biological Resources Center on August 6, 2020 and was given an accession number KCTC 14265BP, and the Lactobacillus plantarum BBH 006 strain has a 16S rRNA sequence of SEQ ID NO: 2 (complementary DNA).
  • Eubacterium limosum BBH 009 having 98% homology was isolated.
  • the selected Eubacterium limosum BBH 009 strain was deposited with the Korea Center for Biological Resources on September 16, 2020 and was given accession number KCTC 14313BP, and the Eubacterium limosum BBH 009 strain has a 16S rRNA sequence of SEQ ID NO: 3 (complementary DNA).
  • Coprococcus comes BBH 011 having 98% homology was isolated.
  • the selected Coprococcus comes BBH 011 strain was deposited with the Korea Biological Resources Center on November 19, 2020 and was given an accession number KCTC 14374BP, and the Coprococcus comes BBH 011 strain has a 16S rRNA sequence of SEQ ID NO: 4 (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 0.01 to 100 ug/ml and treated, and then incubated at 37° C. for 16 hours.
  • 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 for each strain, respectively. , are shown in FIGS. 17 and 24 .
  • 1, 9, 17 and 24 are graphs showing the production amount of nitric oxide according to 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.
  • the endoplasmic reticulum of the strain according to one embodiment was found to further reduce the amount of NO production of the cells induced by inflammation than the level of the negative control.
  • the inflammatory cytokine inhibitory activity of the endoplasmic reticulum of the strain was measured.
  • the LPS-treated Raw264.7 cells in the same manner as above were treated with the endoplasmic reticulum at a concentration of 1 or 10 ug/ml, and then cultured at 37° C. for 48 hours.
  • the protein expression of TNF- ⁇ , IL-6, CCL2 and IL-10 of 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 each figure. 2 and 3, 10 and 11, 18 and 19, 25 and 26 are shown for each strain.
  • 2, 10, 18 and 25 are graphs showing the protein expression level of inflammatory cytokines (TNF- ⁇ and 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.
  • N negative control
  • P untreated control
  • EV endoplasmic reticulum of Example 2.
  • 3, 11, 19 and 26 are graphs showing the protein expression levels of inflammatory cytokines (CCL2) and anti-inflammatory cytokines (IL-10) in 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 cytokines
  • IL-10 anti-inflammatory cytokines
  • the endoplasmic reticulum of the strain significantly reduced inflammatory cytokines compared to the untreated control group, and significantly increased the anti-inflammatory cytokines compared to the untreated control group.
  • strain according to one embodiment can be usefully used for 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 FIGS. 4, 12 and 27 .
  • 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, 13, 20 and 28 .
  • 5, 13, 20 and 28 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 were 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.
  • N negative control
  • P untreated control
  • EV endoplasmic reticulum of Example 2.
  • Clostridium difficile endoplasmic reticulum (CdEV) was treated in mouse macrophage Raw264.7 cells in an amount of 100gu/ml, and then cultured at 37°C for 4 hours. Thereafter, the standard strain of Lactobacillus permentum (ATCC14931), the standard strain of Lactobacillus plantarum (ATCC14917), the standard strain of Eubacterium rimosum (ATCC8486) or the standard strain of Coprococcus comes (ATCC27758) and After treating the endoplasmic reticulum of the strain of Example 1 in an amount of 0.01 - 10ug/ml, incubated for 16 hours at 37°C, the cell viability was measured in the same manner as in Experimental Example 2, and the results are shown in FIG. 7, 15, 22 and 30 are shown.
  • CdEV Clostridium difficile ER
  • BBH005 ER of the Example 1 strain
  • Type strain ER of the Lactobacillus permentum standard strain.
  • CdEV Clostridium difficile ER
  • BBH006 ER of Example 1 strain
  • Type strain ER of Lactobacillus plantarum standard strain.
  • CdEV Clostridium difficile ER
  • BBH009 ER of the Example 1 strain
  • Type strain ER of the Eubacterium rimosum standard strain.
  • CdEV Clostridium difficile endoplasmic reticulum
  • BBH011 endoplasmic reticulum of strain
  • Type strain endoplasmic reticulum of Coprococcus comes standard strain strain.
  • CdEV Clostridium difficile ER
  • BBH005 ER of the Example 1 strain
  • Type strain ER of the Lactobacillus permentum standard strain.
  • CdEV Clostridium difficile ER
  • BBH006 ER of Example 1 strain
  • Type strain ER of Lactobacillus plantarum standard strain.
  • CdEV Clostridium difficile ER
  • BBH009 ER of the Example 1 strain
  • Type strain ER of the Eubacterium rimosum standard strain.
  • FIG. 31 is a graph showing the anti-inflammatory activity of the endoplasmic reticulum of the strain according to one embodiment against inflammation induced by Clostridium difficile; CdEV: Clostridium difficile endoplasmic reticulum, BBH011: endoplasmic reticulum of strain Example 1 strain, Type strain: endoplasmic reticulum of Coprococcus comes standard strain strain.
  • cytotoxicity was not confirmed in both the endoplasmic reticulum of the strain and the standard strain according to one embodiment.
  • the strain according to one embodiment significantly reduces the amount of cytokines increased by the endoplasmic reticulum of Clostridium difficile compared to the standard strain for pro-inflammatory cytokines TNF, CCL2 and IL-6. , it was found that the anti-inflammatory cytokine IL-10 was significantly increased compared to the standard strain.
  • 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 un nouveau micro-organisme, un lysat associé, et une culture associée, un extrait de la culture, une vésicule associée, et une utilisation anti-inflammatoire et/ou antibactérienne correspondante. La nouvelle souche et la vésicule dérivée de cette dernière selon un aspect de l'invention ont pour effet d'être utiles dans la prévention, l'atténuation ou le traitement d'affections liées à une inflammation ou d'infections bactériennes.
PCT/KR2021/018882 2020-12-11 2021-12-13 Souche de lactobacillus sp. , souche d'eubacterium sp., ou souche de coprococcus sp., vésicules dérivées de ces dernières, et utilisation anti-inflammatoire et antibactérienne correspondante WO2022124871A1 (fr)

Applications Claiming Priority (8)

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KR10-2020-0173828 2020-12-11
KR10-2020-0173826 2020-12-11
KR1020200173826 2020-12-11
KR10-2020-0173825 2020-12-11
KR1020200173827A KR102269962B1 (ko) 2020-12-11 2020-12-11 유박테리움 리모숨 균주, 및 그의 유래의 소포체 및 그의 항염증 및 항균 용도
KR1020200173828A KR102269963B1 (ko) 2020-12-11 2020-12-11 코프로코커스 코메스 균주, 및 그의 유래의 소포체 및 그의 항염증 및 항균 용도
KR10-2020-0173827 2020-12-11
KR1020200173825A KR102296287B1 (ko) 2020-12-11 2020-12-11 락토바실러스 퍼멘텀 균주, 및 그의 유래의 소포체 및 그의 항염증 및 항균 용도

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KR20190060271A (ko) * 2017-11-24 2019-06-03 주식회사 고바이오랩 락토바실러스 퍼멘텀 kbl 375 균주 및 그 용도
KR102021883B1 (ko) * 2018-09-07 2019-09-17 재단법인 전남생물산업진흥원 락토바실러스 퍼멘텀 lm1016 균주 및 이를 포함하는 염증 질환 또는 대사 질환의 예방 또는 치료용 조성물
KR102296287B1 (ko) * 2020-12-11 2021-09-01 주식회사 바이오뱅크힐링 락토바실러스 퍼멘텀 균주, 및 그의 유래의 소포체 및 그의 항염증 및 항균 용도

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180050072A1 (en) * 2016-08-22 2018-02-22 Genmont Biotech Inc. Composition and use of lactobacillus fermentum gmnl-296 to produce composition for improving symptoms of clostridium difficile infection
KR20190060271A (ko) * 2017-11-24 2019-06-03 주식회사 고바이오랩 락토바실러스 퍼멘텀 kbl 375 균주 및 그 용도
KR101937758B1 (ko) * 2018-09-05 2019-01-11 마이크로바이오주식회사 락토바실러스 퍼멘텀 ubc-u32, 및 이의 퇴행성 뇌질환 예방 및 치료 용도
KR102021883B1 (ko) * 2018-09-07 2019-09-17 재단법인 전남생물산업진흥원 락토바실러스 퍼멘텀 lm1016 균주 및 이를 포함하는 염증 질환 또는 대사 질환의 예방 또는 치료용 조성물
KR101981333B1 (ko) * 2018-10-31 2019-05-23 주식회사 메디오젠 항비만 활성을 갖는 인체 유래 락토바실러스 퍼멘툼 mg4231 또는 락토바실러스 퍼멘툼 mg4244 균주 및 이를 포함하는 조성물
KR102296287B1 (ko) * 2020-12-11 2021-09-01 주식회사 바이오뱅크힐링 락토바실러스 퍼멘텀 균주, 및 그의 유래의 소포체 및 그의 항염증 및 항균 용도

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