WO2023287025A1 - Souche de cutibacterium avidum, milieu de culture dérivé de celle-ci, et son utilisation antibactérienne - Google Patents

Souche de cutibacterium avidum, milieu de culture dérivé de celle-ci, et son utilisation antibactérienne Download PDF

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Publication number
WO2023287025A1
WO2023287025A1 PCT/KR2022/008319 KR2022008319W WO2023287025A1 WO 2023287025 A1 WO2023287025 A1 WO 2023287025A1 KR 2022008319 W KR2022008319 W KR 2022008319W WO 2023287025 A1 WO2023287025 A1 WO 2023287025A1
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Prior art keywords
strain
culture medium
avidum
lysate
mixture
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PCT/KR2022/008319
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English (en)
Korean (ko)
Inventor
이주혁
오병섭
강세원
김지선
박승환
이정숙
Original Assignee
한국생명공학연구원
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Priority claimed from KR1020220070353A external-priority patent/KR20230010577A/ko
Application filed by 한국생명공학연구원 filed Critical 한국생명공학연구원
Publication of WO2023287025A1 publication Critical patent/WO2023287025A1/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
    • 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
    • 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
    • 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 microbiome refers to microorganisms existing in a specific environment and their entire genetic information, and refers to the collection of genomes that represent 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 largest number of microorganisms in the body exists in the intestine as an optimal environment for microorganisms to consume nutrients and form a systematic community.
  • Intestinal microbes supply nutrients that cannot be produced by the host's own 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 In recent years, the imbalance of the intestinal microbiota has deteriorated due to Western eating habits and indiscriminate use of antibiotics, leading to deterioration of intestinal health. there is.
  • One aspect is to provide a Cutibacterium avidum strain belonging to the genus Cutibacterium ( Cutibacterium sp. ) deposited under accession number KCTC 14558BP.
  • Another aspect is to provide a lysate or culture medium derived from the strain.
  • Another aspect is to provide a pharmaceutical composition for preventing or treating bacterial infection comprising the strain, a lysate of the strain, a culture thereof, 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, a lysate of the strain, a culture medium, or a mixture thereof as an active ingredient.
  • Another aspect is to provide a cosmetic composition
  • a cosmetic composition comprising the strain, a lysate of the strain, a culture medium, or a mixture thereof as an active ingredient.
  • Another aspect is to provide a method for preventing or treating a bacterial infection or inflammatory disease by administering the strain, a lysate, a culture medium, or a mixture thereof of the strain to a subject in need thereof.
  • Another aspect is to provide a strain, a lysate of the strain, a culture medium, or a mixture thereof for use in preparing a composition for preventing or treating bacterial infections or inflammatory diseases.
  • Cutibacterium avidum strain belonging to the genus Cutibacterium ( Cutibacterium sp. ) deposited under accession number KCTC 14558BP.
  • the strain may be a strain containing 16s rRNA of SEQ ID NO: 1.
  • Another aspect provides a lysate or culture medium derived from the strain.
  • Another aspect provides a pharmaceutical composition for preventing or treating bacterial infections comprising the strain, a lysate of the strain, a culture thereof, or a mixture thereof as an active ingredient.
  • the bacterial infection may be Clostridium difficile infection (CDI).
  • CDI Clostridium difficile infection
  • inflammatory bowel disease comprising the strain, a lysate of the strain, a culture thereof, or a mixture thereof as an active ingredient; irritable bowel syndrome; enteritis; Crohn's disease; Behcet's disease; Or it may be to provide a pharmaceutical composition for preventing or treating ulcerative colitis (ulcerative colitis).
  • IBD inflammatory bowel diseases
  • Another aspect provides a health functional food for preventing or improving bacterial infection comprising the strain, a lysate of the strain, a culture medium, or a mixture thereof as an active ingredient.
  • the bacterial infection may be Clostridium difficile infection (CDI).
  • CDI Clostridium difficile infection
  • a health functional food for improving intestinal health by inhibiting Clostridium difficile or inhibiting inflammation comprising the strain, a lysate, a culture medium, or a mixture thereof of the strain as an active ingredient may be providing
  • inflammatory bowel disease comprising the strain, a lysate of the strain, a culture thereof, or a mixture thereof as an active ingredient; irritable bowel syndrome; enteritis; Crohn's disease; Behcet's disease; Or it may be to provide a health functional food for preventing or improving ulcerative colitis (ulcerative colitis).
  • IBD inflammatory bowel diseases
  • Another aspect provides a cosmetic composition
  • a cosmetic composition comprising the strain, a lysate of the strain, a culture medium, or a mixture thereof as an active ingredient.
  • Another aspect provides a method for preventing or treating a bacterial infection or inflammatory disease by administering the strain, a lysate of the strain, a culture medium, or a mixture thereof to a subject in need thereof.
  • Another aspect provides a strain, a lysate of the strain, a culture medium, or a mixture thereof for use in preparing a composition for preventing or treating bacterial infections or inflammatory diseases.
  • the strain may be derived from a human body, in particular from a human feces sample, but is not limited thereto.
  • the strain may be a live strain or an attenuated strain (killed strain).
  • attenuated means modified to reduce the pathogenicity of the strain. Attenuation can be done for the purpose of reducing toxicity and other side effects when the strain is administered to a subject.
  • Attenuated strains can be prepared through various methods known in the art. For example, attenuation can be achieved by deleting or disrupting virulence factors that allow the strain to survive in the host cell. The deletion and destruction are well known in the art, and may be performed by methods such as homologous recombination, chemical mutagenesis, irradiation mutagenesis, or transposon mutagenesis.
  • the strain may have antibacterial activity.
  • the strain may inhibit the growth of bacteria (eg , C. difficile ).
  • 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) it could be
  • Cutibacterium genus ( Cutibacterium sp .) Belonging to Cutibacterium avidum ( Cutibacterium avidum ) Provides a lysate, a culture medium, an extract of the culture medium, or a mixture thereof.
  • the strain is as described above.
  • lysate may mean a product obtained by disrupting the cell wall of the strain itself by chemical or physical force.
  • the term "culture medium” may be used interchangeably with “culture supernatant”, “conditioned culture medium” or “conditioned medium”, and may supply nutrients so that Bailonella seminalis can grow and survive in vitro. It may mean the entire medium including the strain, its metabolites, extra nutrients, etc. obtained by culturing the strain for a certain period of time in a medium that can be used.
  • the culture solution may mean a culture solution obtained by removing the cells from the cell culture solution obtained by culturing the strain.
  • the liquid from which the cells are removed from the culture solution is also called “supernatant", and the culture solution is left still for a certain period of time to take only the liquid of the upper layer except for the part sunk in the lower layer, remove the cells through filtration, or centrifuge the culture solution to It can be obtained by removing the precipitate and taking only the upper liquid.
  • the "cell” refers to the strain itself of the present invention, and includes the strain itself isolated and selected from a fecal sample or the strain isolated from the culture medium by culturing the strain. The cells can be obtained by centrifuging the culture solution and taking the part that has settled in the lower layer, or can be obtained by removing the upper liquid with the lower part of the culture medium that has settled for a certain period of time.
  • the culture solution may include a culture solution itself obtained by culturing the strain, a concentrate thereof, or a lyophilized product or a culture supernatant obtained by removing the strain from the culture solution, a concentrate thereof, or a lyophilisate.
  • the culture medium is obtained by culturing Cutibacterium avidum in an appropriate medium (eg, Reinforced Clostridial Medium medium) at any temperature above 10 ° C or below 40 ° C for a certain period of time, for example, 4 to 50 hours can
  • the culture supernatant of the strain may be obtained by centrifuging or filtering the strain culture medium to remove the strain.
  • the concentrate may be obtained by concentrating the supernatant obtained after filtering the strain culture medium itself, or the culture medium using a centrifugal separation or filter.
  • the culture medium and culture conditions for culturing the Cutibacterium avidum can be appropriately selected or modified by those skilled in the art.
  • culture medium extract refers to an extract obtained from the culture medium or a concentrate thereof, and may include an extract, a diluent or concentrate of the extract, a dried product obtained by drying the extract, a purified product obtained by purifying the same, or a fraction obtained by fractionating the same. there is.
  • Another aspect provides a disease improvement, prevention or treatment use of a Cutibacterium avidum strain, a lysate derived from the strain, a culture medium, or an extract of the culture medium.
  • the term "treat” may mean that inflammation is cured in a shorter time compared to natural healing.
  • the treatment may include amelioration or alleviation of inflammation or amelioration or alleviation of a bacterial infection.
  • the treatment may refer to healing or recovery of symptoms caused by inflammation or bacterial infection.
  • Uses of the strain may include preventing, ameliorating, or treating inflammatory diseases, or preventing, ameliorating, or treating bacterial infections (antibacterial activity), or preventing or improving intestinal health.
  • the inflammatory diseases include inflammatory bowel diseases (IBD); irritable bowel syndrome; Behcet's disease; enteritis, Crohn's disease; ulcerative colitis; It may be any one selected from the group consisting of.
  • IBD inflammatory bowel diseases
  • Behcet's disease enteritis, Crohn's disease
  • ulcerative colitis It may be any one selected from the group consisting of.
  • the improvement of intestinal health may be helpful for proliferation of beneficial bacteria in the intestine and suppression of harmful bacteria, help for intestinal health by regulating immunity, or help for smooth bowel movement.
  • antimicrobial agent refers to inhibiting, reducing or preventing the growth of bacteria, inhibiting or reducing the ability of bacteria to cause an infection in a subject, or inhibiting or reducing the ability of bacteria to proliferate or remain infectious in the environment Refers to a substance that can be reduced.
  • examples of the bacterial infections may include infections caused by gram-positive bacteria or gram-negative bacteria.
  • the bacterial infection is Clostridium , Helicobacter, Helicobactor , Escherichia , Salmonella , Staphylococcus , Streptococcus , Haemophilus ), Klebsiella , Moraxella , Enterobacter , Proteus , Serratia , Pseudomonas , Acinetobacter , Citrobacter ), Stenotrophomonas , Bacteroides , Prevotella , and Fusobacterium .
  • the bacterial infection is Clostridium difficile infection (CDI), or Clostridium difficile associated disease (CDAD: Clostridium difficile associated disease), for example, Clostridium difficile associated diarrhea (Clostridium difficile associated disease) diarrhea) may be included.
  • compositions for preventing or treating a bacterial infection comprising a cutibacterium avidum strain, a lysate of the strain, a culture medium, an extract of the culture medium, or a mixture thereof as an active ingredient.
  • the composition is 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%, 0.00001% to 5%, 0.05% to 60%, 0.05% to 40%, 0.05% to 30%, 0.05% to 20%, 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 medium, or an extract of a culture medium thereof.
  • the term "included as an active ingredient” means that the strain of the present specification, a lysate of the strain, a culture medium, or an extract of the culture medium is added to the extent that the above-mentioned effects can be exhibited, and drug delivery and stabilization It means that various components are added as subcomponents for the purpose of formulation in various forms.
  • the composition can be a pharmaceutical composition.
  • the pharmaceutical composition may additionally include a pharmaceutically acceptable diluent or carrier.
  • the diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, 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 calcium carboxymethylcellulose, sodium starch glycolate, 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 for oral or parenteral administration.
  • Oral dosage forms may be granules, powders, solutions, tablets, capsules, dry syrups, or combinations thereof.
  • Parenteral dosage forms may be injections.
  • the composition may be a health functional food composition.
  • the health functional food composition may be used alone or in combination with the strain or its culture medium or other food or food component, 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 (prevention, 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.
  • beverage compositions may contain various flavoring agents or natural carbohydrates as additional components, like conventional beverages.
  • 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 thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used.
  • the health food composition may also contain nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonated beverages. carbonation agent used, or a combination thereof.
  • the health functional food composition may also contain natural fruit juice, fruit juice beverages, fruit flesh for preparing vegetable beverages, or a combination thereof.
  • the composition may be a cosmetic composition.
  • the cosmetic composition may have, for example, softening lotion, nutrient lotion, massage cream, nutrient cream, essence, pack, gel, ampoule, or skin-adhesive cosmetic formulation.
  • ingredients included in the cosmetic composition may include ingredients commonly used in cosmetic compositions other than the composition as active ingredients, for example, conventional adjuvants and carriers such as stabilizers, solubilizers, vitamins, pigments and flavors. can include
  • composition may be a composition for external application for skin.
  • the external skin preparation may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal delivery patch, drug-containing bandage, lotion, or a combination thereof.
  • the external skin preparation is a component usually used in external preparations for skin such as cosmetics or pharmaceuticals, for example, water-based components, oil-based components, powder components, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, and fragrances. , colorants, various skin nutrients, or combinations thereof and may be suitably blended as needed.
  • the external skin preparations include metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, bellapamil, licorice extract, glabridin, and calin's fruit hot water extracts, various herbal medicines, tocopherol acetate, glycylitnic acid, tranexamic acid and its derivatives or salts, vitamin C, magnesium ascorbate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose , saccharides such as trehalose, etc. can also be blended appropriately.
  • metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, bellapamil, licorice extract, glabridin, and calin's fruit hot water extracts, various
  • Another aspect also provides a method for preventing, ameliorating, or treating a condition in a subject comprising treating or administering to a subject in need thereof an effective amount of the above composition.
  • the condition of the subject may be a condition related to inflammation.
  • the administration may be administered by a method known in the art.
  • the administration is directly administered to a subject by any means, such as, for example, intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. It can be.
  • the administration may be administered 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 an improvement effect of a condition related to inflammation.
  • the administration of the composition according to one embodiment is 0.00001 mg to 1,000 mg per day per subject, 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 variously prescribed depending on factors such as administration method, patient's age, weight, sex, medical condition, food, administration time, administration route, excretion rate and reaction sensitivity, and those skilled in the art take these factors into consideration.
  • the number of administrations can be once a day or twice or more within the range of clinically acceptable side effects, and the administration site can be administered to one or more than two sites, daily or every 2 to 5 days, total
  • the number of administration days may be administered from 1 day to 30 days per treatment. If necessary, the same treatment can be repeated after a titration period.
  • the same dosage per kg as for humans is used, or the above dosage is converted by the volume ratio (eg, average value) of the organ (heart, etc.) between the target animal and the human.
  • a single dose can be administered.
  • Figure 1 is a diagram showing the phylogenetic tree analysis of C. avidum KGMB02810 strain isolated from feces of healthy people according to one embodiment.
  • Figure 2 is a diagram showing the identification of C. avidum KGMB02810 strain isolated from feces of a healthy person according to one embodiment.
  • FIG. 3 is a graph measuring the concentration-dependent antibacterial activity of the culture medium (CaCFS) of C. avidum strains according to one embodiment; CaCFS: C. avidum cell free supernatant
  • Figure 4 is a photograph showing the antibacterial activity of live bacteria of C. avidum strain; CaCFS: C. avidum cell free supernatant
  • Figure 5 is a graph comparing the antibacterial activity against C. difficile of cultures of other strains (KGMB09337, KCTC 5339) of C. avidum species according to one embodiment.
  • Figure 6 is a graph showing the effect of the culture medium (CaCFS) of C. avidum strains on beneficial microorganisms in the intestine according to one embodiment.
  • Figure 7 is a graph measuring the degree of antibacterial activity according to the incubation time of the culture solution (CaCFS) of C. avidum strains according to one embodiment.
  • Figure 8 is a graph measuring the degree of antibacterial activity according to the culture temperature of the culture solution (CaCFS) of C. avidum strains according to one embodiment.
  • Figure 9 is a graph measuring the antibacterial activity after proteolytic enzyme treatment in order to identify the active ingredient of the C. avidum strain culture medium (CaCFS) according to one embodiment.
  • C. avidum strain culture medium (CaCFS) according to one embodiment.
  • Figure 11 is a graph measuring the antibacterial activity after treatment with ethyl acetate in order to identify the active ingredient of the C. avidum strain culture medium (CaCFS) according to one embodiment.
  • CaCFS C. avidum strain culture medium
  • Figure 12 is a graph showing the survival rates of a group orally administered with a C. avidum strain (KGMB02810) and a control group according to one embodiment; NC: control group, CD: negative control group, Ca: experimental group
  • FIG. 13 is a graph showing changes in body weight of a group orally administered with a C. avidum strain (KGMB02810) according to one embodiment and a control group; NC: control group, CD: negative control group, Ca: experimental group
  • FIG. 14 is a graph showing the amount of C. difficile in feces of a group orally administered with a C. avidum strain (KGMB02810) according to one embodiment and a control group; NC: control group, CD: negative control group, Ca: experimental group
  • TcdA and TcdB which are toxins of C. difficile, in feces of a group orally administered with a C. avidum strain (KGMB02810) and a control group according to one embodiment; NC: control group, CD: negative control group, Ca: experimental group
  • Figure 16 is a graph showing the effect of reducing inflammatory cytokines in a CDI mouse model of C. avidum strain culture medium (CaCFS) according to one embodiment; NC: control group, CD: negative control group, Ca: experimental group
  • FIG. 17 is a photograph of intestinal tissue in a CDI mouse model of a culture medium (CaCFS) of a C. avidum strain according to one embodiment; NC: control group, CD: negative control group, Ca: experimental group
  • Figure 18 is a photograph showing the effect of preventing long-length shrinkage in a CDI mouse model of a culture solution (CaCFS) of a C. avidum strain according to one embodiment; NC: control group, CD: negative control group, Van: positive control group, Ca: experimental group
  • NC control group
  • CD negative control group
  • Ca experimental group
  • 20 is a hierarchical heat map comparing the correlation between intestinal microbes at the family level using metagenome data of the fecal intestinal microbiome of a CDI mouse model according to an embodiment and the number of beneficial bacteria in the intestine
  • NC control group
  • CD negative control group
  • Ca experimental group
  • 21 is a graph showing changes in C. difficile after co-cultivation of C. difficile and CaPPT by Gram staining, SEM, and TEM in order to investigate the mechanism of action of the antibacterial activity of the culture medium (CaCFS) of the C. avidum strain according to one embodiment. It is a photograph observed with; RCMPPT: negative control, CaPPT: experimental group (protein in CaCFS), Vancomycin: positive control
  • FIG. 22 is a photograph confirming leakage of cellular contents of C. difficile after co-cultivation of C. difficile and CaPPT in order to investigate the mechanism of action of the antibacterial activity of the culture medium (CaCFS) of the C. avidum strain according to one embodiment. to be; RCMPPT: negative control group, CaPPT: experimental group (protein in CaCFS)
  • Figure 23 is a graph measuring the amount of leaked nucleic acid in the supernatant after co-cultivation of C. difficile and CaPPT in order to investigate the mechanism of action of the antibacterial activity of the culture medium (CaCFS) of the C. avidum strain according to one embodiment.
  • CaCFS culture medium
  • CaPPT experimental group (protein in CaCFS)
  • intestinal microorganisms were isolated using an obligate anaerobic microorganism isolation technique in the intestine.
  • C. avidum KGMB02810 strain, C. avidum KGMB09337 strain, and C. avidum ATCC 25577T strain share 1,983 genes through whole-genome sequencing, and strain identification through each strain-specific gene identification could.
  • Figure 1 is a diagram showing the phylogenetic tree analysis of C. avidum KGMB02810 strain isolated from feces of healthy people according to one embodiment.
  • Figure 2 is a diagram showing the identification of C. avidum KGMB02810 strain isolated from feces of a healthy person according to one embodiment.
  • CFS supernatant
  • 10 ⁇ l of heat-treated C. avidum and live C. avidum were applied to a plate coated with 100 ⁇ l of C. difficile with an OD of 0.5. After dropping it on top, it was incubated at 37 ° C. for 10 hours, and the results are shown in FIG. 4 .
  • FIG. 3 is a graph measuring the concentration-dependent antibacterial activity of the culture medium (CaCFS) of C. avidum strains according to one embodiment; CaCFS: C. avidum cell free supernatant
  • Figure 4 is a photograph showing the antibacterial activity of live bacteria of C. avidum strain; CaCFS: C. avidum cell free supernatant
  • C. avidum culture medium (CaCFS) had a concentration-dependent antibacterial activity against C. difficile.
  • the antibacterial activity against C. difficile was not shown in heat-treated C. avidum, and when the generation of a clear zone was observed in live C. avidum, live C. avidum also showed antibacterial activity against C. difficile. confirmed to have.
  • Figure 5 is a graph showing the antibacterial activity against C. difficile of cultures of other strains (KGMB09337, KCTC 5339) of C. avidum species according to one embodiment.
  • Figure 6 is a graph showing the effect of the culture medium (CaCFS) of C. avidum strains on beneficial microorganisms in the intestine according to one embodiment.
  • C. avidum was prepared at regular intervals (24, 26, 30, 34, 38, 42, 46 hours), and then An antibacterial test was conducted in the manner described in Example 2 above.
  • an antibacterial test was conducted after giving CaCFS heat between 30 and 90 ° C for a certain period of time (0 to 80 minutes), and the results are shown in FIGS. 7 and 8 .
  • Figure 7 is a graph measuring the degree of antibacterial activity according to the incubation time of the culture solution (CaCFS) of C. avidum strains according to one embodiment.
  • Figure 8 is a graph measuring the degree of antibacterial activity according to the culture temperature of the culture solution (CaCFS) of C. avidum strains according to one embodiment.
  • the heat-sensitive antibacterial substance is a protein
  • several enzymes Phosphatase, Catalase, a-amylase, Proteinase K, Pepsin, Papain, Chymotrypsin, Trypsin
  • ammonium sulfate protein precipitation was used to obtain proteins in CaCFS.
  • 40% (w/v) Ammonium sulfate was added to 1 L of CaCFS, stirred at 4 °C for 1 hour, and then centrifuged.
  • CaCFS genus proteins were measured for their antibacterial activity in the manner described in Example 2. through liquid culture.
  • CaCFS was extracted with ethyl acetate. Ethyl acetate was added in the same amount as 500ml CaCFS, stirred at room temperature for 30 minutes, and left as it was to separate the aqueous layer and the organic layer.
  • Figure 9 is a graph measuring the antibacterial activity after proteolytic enzyme treatment in order to identify the active ingredient of the C. avidum strain culture medium (CaCFS) according to one embodiment.
  • C. avidum strain culture medium (CaCFS) according to one embodiment.
  • Figure 11 is a graph measuring the antibacterial activity after treatment with ethyl acetate in order to identify the active ingredient of the C. avidum strain culture medium (CaCFS) according to one embodiment.
  • CaCFS C. avidum strain culture medium
  • the antibacterial activity of CaCFS was reduced by more than 40% by the proteolytic enzymes Pepsin and Trypsin.
  • FIG. 10 it was confirmed that CaPPT has antibacterial activity similar to that of CaCFS.
  • FIG. 11 the organic matter in the CaCFS fraction did not show antibacterial activity.
  • antibiotic drinking water Keratin 400mg, Vancomycin 45mg, Colistin 28.3mg, Gentamycin 35mg, Metronidazole 215mg in 1L DW
  • CD group negative control group
  • Ca group experimental group
  • Ca group received 1 X 10 9 CFU per mouse.
  • IL-6 and TNF-alpha As inflammatory cytokine indicators for C. difficile infection, the amount of IL-6 and TNF-alpha was measured using an ELISA kit after obtaining mouse serum. Analysis was performed, and the results are shown in FIGS. 16 and 17.
  • mice were sacrificed and the length of the large intestine was measured, and the results are shown in FIG. 18 .
  • Figure 12 is a graph showing the survival rates of a group orally administered with a C. avidum strain (KGMB02810) and a control group according to one embodiment; NC: control group, CD: negative control group, Ca: experimental group
  • FIG. 13 is a graph showing changes in body weight of a group orally administered with a C. avidum strain (KGMB02810) according to one embodiment and a control group; NC: control group, CD: negative control group, Ca: experimental group
  • FIG. 14 is a graph showing the amount of C. difficile in feces of a group orally administered with a C. avidum strain (KGMB02810) according to one embodiment and a control group; NC: control group, CD: negative control group, Ca: experimental group
  • TcdA and TcdB which are toxins of C. difficile, in feces of a group orally administered with a C. avidum strain (KGMB02810) and a control group according to one embodiment; NC: control group, CD: negative control group, Ca: experimental group
  • Figure 16 is a graph showing the effect of reducing inflammatory cytokines in a CDI mouse model of C. avidum strain culture medium (CaCFS) according to one embodiment; NC: control group, CD: negative control group, Ca: experimental group
  • FIG. 17 is a photograph of intestinal tissue in a CDI mouse model of a culture medium (CaCFS) of a C. avidum strain according to one embodiment; NC: control group, CD: negative control group, Ca: experimental group
  • Figure 18 is a photograph showing the effect of preventing long-length shrinkage in a CDI mouse model of a culture solution (CaCFS) of a C. avidum strain according to one embodiment; NC: control group, CD: negative control group, Van: positive control group, Ca: experimental group
  • the survival rate of the group orally administered with C. avidum was significantly higher than that of the C. difficile control group (CD group) by 70%.
  • the body weight decreased less than that of the negative control group (CD group), but increased the same or more than that of the C. difficile non-inoculated group (NC group) from the 5th day.
  • the amount of C. difficile inside the feces was reduced 100 times in the group administered with C. avidum KGMB02810 compared to the negative control group (CD group).
  • the amount of C. difficile toxin in the feces of the experimental group (Ca group) was reduced by more than 3 times compared to that of the negative control group (CD group).
  • NGS next-generation sequencing
  • NC control group
  • CD negative control group
  • Ca experimental group
  • 20 is a hierarchical heat map comparing the correlation between intestinal microbes at the family level using metagenome data of the fecal intestinal microbiome of a CDI mouse model according to an embodiment and the number of beneficial bacteria in the intestine
  • NC control group
  • CD negative control group
  • Ca experimental group
  • CD group C. difficile control group
  • NC group C. difficile non-inoculated group
  • the positive clusters (Lactobacillaceae, Ruminococcaceae, Lachnospiraceae, Muribaculaceae) and negative clusters of the C. difficile non-inoculated group (NC group) and the group orally administered with C. avidum KGMB02810 (Ca group) are similar, It was confirmed that it was clearly distinguished from the C. difficile control group (CD group). Compared to the C. difficile non-inoculated group (NC group), the abundance of the C. difficile control group (CD group) decreased noticeably, but the abundance of the group orally administered with C. avidum KGMB02810 (Ca group) recovered. Confirmed.
  • 21 is a graph showing changes in C. difficile after co-cultivation of C. difficile and CaPPT by Gram staining, SEM, and TEM in order to investigate the mechanism of action of the antibacterial activity of the culture medium (CaCFS) of the C. avidum strain according to one embodiment. It is a photograph observed with; RCMPPT: negative control, CaPPT: experimental group (protein in CaCFS), Vancomycin: positive control
  • FIG. 22 is a photograph confirming leakage of cellular contents of C. difficile after co-cultivation of C. difficile and CaPPT in order to investigate the mechanism of action of the antibacterial activity of the culture medium (CaCFS) of the C. avidum strain according to one embodiment. to be; RCMPPT: negative control group, CaPPT: experimental group (protein in CaCFS)
  • Figure 23 is a graph measuring the amount of leaked nucleic acid in the supernatant after co-cultivation of C. difficile and CaPPT in order to investigate the mechanism of action of the antibacterial activity of the culture medium (CaCFS) of the C. avidum strain according to one embodiment.
  • CaCFS culture medium
  • CaPPT experimental group (protein in CaCFS)
  • the antibacterial active protein against C. difficile produced by the C. avidum KGMB02810 strain has a mechanism of action that destroys the cell wall of C. difficile.

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Abstract

La présente invention concerne une souche déposée, un lysat de celle-ci, un milieu de culture, un extrait du milieu de culture, et leurs utilisations antibactériennes. La souche et le milieu de culture dérivés de celle-ci, selon un aspect de la présente invention, peuvent être utilisés efficacement dans la prévention, l'atténuation ou le traitement des infections bactériennes.
PCT/KR2022/008319 2021-07-12 2022-06-13 Souche de cutibacterium avidum, milieu de culture dérivé de celle-ci, et son utilisation antibactérienne WO2023287025A1 (fr)

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KR10-2021-0090936 2021-07-12
KR20210090936 2021-07-12
KR1020220070353A KR20230010577A (ko) 2021-07-12 2022-06-09 큐티박테리움 아비덤 균주, 그의 유래의 배양액 및 그의 항균 용도
KR10-2022-0070353 2022-06-09

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Citations (4)

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US20070196341A1 (en) * 2004-04-13 2007-08-23 Meiji Dairies Corporation Preventive and/or remedy for inflammatory bowel diseases
KR20100126285A (ko) * 2008-02-29 2010-12-01 메이지 데어리즈 코포레이션 항알레르기제
KR101925135B1 (ko) * 2018-08-23 2018-12-04 주식회사 지놈앤컴퍼니 신규의 큐티박테리움 아비덤 균주, 및 이러한 균주 또는 이의 배양물을 포함하는 아토피 피부염 예방 또는 치료용 조성물
KR20200065004A (ko) * 2017-09-12 2020-06-08 소파 에스피에이 클로스트리듐 디피실리 감염을 치료하기 위한 새로운 용도

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US20070196341A1 (en) * 2004-04-13 2007-08-23 Meiji Dairies Corporation Preventive and/or remedy for inflammatory bowel diseases
KR20100126285A (ko) * 2008-02-29 2010-12-01 메이지 데어리즈 코포레이션 항알레르기제
KR20200065004A (ko) * 2017-09-12 2020-06-08 소파 에스피에이 클로스트리듐 디피실리 감염을 치료하기 위한 새로운 용도
KR101925135B1 (ko) * 2018-08-23 2018-12-04 주식회사 지놈앤컴퍼니 신규의 큐티박테리움 아비덤 균주, 및 이러한 균주 또는 이의 배양물을 포함하는 아토피 피부염 예방 또는 치료용 조성물

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