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WO2023239024A1 - Nouveau microorganisme de la famille des iamiaceae et ses utilisations - Google Patents

Nouveau microorganisme de la famille des iamiaceae et ses utilisations Download PDF

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Publication number
WO2023239024A1
WO2023239024A1 PCT/KR2023/003949 KR2023003949W WO2023239024A1 WO 2023239024 A1 WO2023239024 A1 WO 2023239024A1 KR 2023003949 W KR2023003949 W KR 2023003949W WO 2023239024 A1 WO2023239024 A1 WO 2023239024A1
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Prior art keywords
strain
skin
hominis
culture medium
dermatobacter
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English (en)
Korean (ko)
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조형우
이동걸
강승현
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Cosmax Inc
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Cosmax Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • 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 skin is a point of contact with the external environment and is also a collection site for various microorganisms (fungi, bacteria, viruses, and small larvae). Depending on the selection of physical and chemical functions, microorganisms adapt to specialized niches and establish habitats.
  • the surface of the skin is cold, shows slightly acidic properties, and is maintained in a dry state.
  • the epidermis forms the skin barrier and plays an important role in blocking the penetration of microorganisms and toxins and maintaining moisture.
  • the uppermost layer of the epidermis is composed of the stratum corneum.
  • the epidermis has a form called a 'brick and mortar structure'.
  • the skin tissue goes through a continuous self-recovery process, and the scales that have completed the differentiation process constantly repeat the process of being shed from the skin tissue. It has been revealed that microorganisms influence these skin changes.
  • Probiotics are a general term for microorganisms that have beneficial effects on the human body. They refer to microorganisms that benefit our bodies. Most probiotics known to date are known as lactic acid bacteria. Probiotics have been reported to produce effective effects through various beneficial effects on the human body, but research on the relationship between skin flora and skin is insufficient.
  • the skin barrier is composed of dead keratinocytes and intercellular lipids, and plays a key role in skin health as a skin protective film that protects the skin from external stimuli and prevents moisture from evaporating from the skin. In other words, it prevents excessive release of moisture from the body and prevents harmful substances such as chemicals or microorganisms from entering our body.
  • the keratinocyte envelope which constitutes the surface of dead keratinocytes, plays an important role in the stability of intercellular lipids. Keratinocytes differentiate and create a skin barrier through the keratinization process. The skin barrier function can be destroyed by aging or external factors, and damage to the skin barrier can reduce skin moisture and cause wrinkles.
  • the present inventors observed changes in the skin due to changes in skin flora and further studied whether changes in skin flora could lead to potential improvement in the skin environment. Iamiaceae strains were isolated and identified from and confirmed to be useful for skin-related conditions, and the present invention was completed.
  • One aspect is to provide a new Dermatobacter hominis strain belonging to the Iamiaceae family.
  • Another aspect is to provide a lysate, culture medium, or extract of the culture medium of the strain.
  • compositions comprising a Dermatobacter hominis strain, its lysate, a culture, an extract of the culture, or a mixture thereof.
  • One aspect provides a novel Dermatobacter hominis strain belonging to the Iamiaceae family.
  • Strains belonging to the Iamiaceae family may have one or more of the following mycological properties;
  • Strains belonging to the Iamiaceae family may have one or more of the following mycological properties;
  • DNA G+C content is 72.2 mol%
  • MK-9(H8) tetrahydrogenated menaquinone with nine units is the isoprenoid quinone detected
  • the main polar lipids are diphosphatidylglycerol (DPG), three unidentified phosphoglycolipid (PGL), unidentified aminophosphoglycolipid (APGL), and unidentified aminophospholipid ( at least one selected from the group consisting of unidentified aminophospholipid (APL); and
  • the phylogenetic characteristics of the strain belonging to the Iamiaceae family show a clear difference from other genera to which other strains belong, and the chemical taxonomic characteristics also show a clear difference, resulting in the new Dermatobacter . I sympathized with him in my heart.
  • the Dermatobacter strain may include Dermatobacter hominis .
  • the Dermatobacter hominis may have one or more of the following mycological properties;
  • the length of the cell is 0.8 ⁇ 1.4 ⁇ m and the diameter is 0.4 ⁇ 0.7 ⁇ m;
  • R2A Reasoner's 2A
  • TSA Teryptic Soy Agar
  • NA Nutrient Agar
  • LBA Lysogeny Broth Agar
  • Nitrate is not reduced to nitrite
  • Esterase (C4) Esterase lipase (C8), leucine arylamidase, crystalline arylamidase, acid phosphatase, and naphthol-AS -BI-phosphohydrolase activity. Any one or more activities selected from the group consisting of;
  • the main fatty acids are C 16:0 , C 18:0, C 18:1 ⁇ 9c or C 20:1 ⁇ 9c;
  • the Dermatobacter hominis may include 16s rRNA containing the base sequence represented by SEQ ID NO: 1. Additionally, the Dermatobacter hominis may have been deposited under the deposit number KACC 22415.
  • Another aspect provides lysate, culture fluid, or extract of the culture fluid of the strain.
  • culture medium may be used interchangeably with “culture supernatant,” “conditioned culture medium,” or “conditioned medium,” and refers to a medium that can supply nutrients to enable the strain to grow and survive in vitro. It may refer to the entire medium containing the strain, its metabolites, extra nutrients, etc. obtained by culturing the strain for a certain period of time. Additionally, the culture medium may refer to a culture medium obtained by removing the bacterial cells from the bacterial culture medium obtained by culturing the strain.
  • the liquid from which the bacteria have been removed from the culture medium is also called “supernatant", and the culture medium is left alone for a certain period of time and only the liquid in the upper layer excluding the part that has settled in the lower layer is taken, the bacteria are removed through filtration, or the culture medium is centrifuged and the lower layer is removed. It can be obtained by removing the precipitation and taking only the upper liquid.
  • the "bacteria” refers to the strain itself of the present invention, and includes the strain itself isolated and selected from a skin sample, etc., or a strain isolated from the culture medium by culturing the strain. The bacterial cells can be obtained by centrifuging the culture medium and taking the part that has sunk to the lower layer. Alternatively, since they sink to the lower layer of the culture medium by gravity, they can be obtained by leaving them still for a certain period of time and then removing the upper liquid.
  • the culture medium is the culture medium itself, its concentrate, or freeze-dried product obtained by cultivating a strain belonging to the Iamiaceae family, or the culture supernatant obtained by removing the strain from the culture medium, its concentrate, or freeze-dried product. may include.
  • the culture medium may be obtained by culturing the strain in a medium (e.g., 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.
  • a medium e.g., R2A medium or TSA medium
  • 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 strain culture medium itself, or the supernatant obtained after filtering the culture medium using centrifugation or a filter.
  • the culture medium and culture conditions for cultivating the KERA-3 strain belonging to the Iamiaceae family can be appropriately selected or modified by those skilled in the art.
  • lysate may refer to a product obtained by breaking the cell wall of the strain itself using chemical or physical force.
  • culture extract refers to an extract from the culture medium or its concentrate, and may include extracts, diluted or concentrated extracts, dried products obtained by drying the extracts, crude or purified products thereof, and fractions thereof. You can.
  • Another aspect provides the use of the strain, a lysate of the strain, a culture medium, or an extract of the culture medium.
  • a composition comprising the above strain, its lysate, culture medium, or extract of the culture medium is provided.
  • Uses of the strain may include improving skin condition, improving skin beauty, preventing, improving or treating skin diseases, etc.
  • the skin condition improvement or skin beauty improvement may be suppressing or improving skin aging, anti-wrinkle, skin whitening, strengthening the skin barrier, or skin moisturizing.
  • skin aging refers to both tangible and intangible changes that occur in the skin as one ages, such as thinning of the epidermis, number of cells or blood vessels in the dermis, DNA damage repair ability, cell replacement cycle, It refers to a decrease in wound recovery, skin barrier function, epidermal moisture retention, sweat secretion, sebum secretion, vitamin D production, physical damage defense, chemical removal ability, immune response, sensory function, and temperature regulation.
  • the strain or its culture medium may be used to improve skin aging caused by exogenous factors or endogenous factors.
  • the exogenous factors refer to various external factors, such as ultraviolet rays (light), and the endogenous factors are also referred to as chronological factors and mainly refer to factors that occur due to the passage of time.
  • the skin aging is not only a premature aging phenomenon induced by external stimuli such as ultraviolet rays, pollution, cigarette smoke, chemicals, etc., but also a natural aging phenomenon that occurs as the proliferation of skin cells decreases with age. It is a concept that includes wrinkles, loss of elasticity, skin sagging, and dryness.
  • wrinkles include stimulation caused by changes in internal and external factors that change the components that make up skin tissue, causing wrinkles.
  • the aging may be photoaging.
  • photoaging is a phenomenon caused by external environmental factors, the most representative factor being ultraviolet rays. Ultraviolet rays cause damage to biological components such as activation of proteolytic enzymes, chain cutting of matrix proteins, and abnormal cross-linking, and repetition of this mechanism causes skin aging that is evident in appearance.
  • wrinkle refers to a state in which the skin loses its elasticity and becomes loose, for example, the skin may be folded.
  • prevention or improvement of skin wrinkles may refer to any action that prevents or improves wrinkles by suppressing the expression of factors related to wrinkles, or increases the total amount of collagen.
  • skin barrier strengthening may refer to any action that improves the function of the skin barrier, which is located on the outermost layer of the skin and prevents moisture and nutrition loss.
  • skin moisturizing may refer to any action that maintains skin moisture or prevents moisture loss.
  • the “skin disease” may be a disease caused by damage to the skin barrier function, skin aging, skin wound, skin scar, or skin inflammation.
  • prevention includes suppressing the occurrence of a disease.
  • treatment includes inhibiting, alleviating, or eliminating the development of a disease.
  • the damage to the skin barrier function may mean any change that appears in the skin due to decreased or damaged skin barrier function. For example, it may include increased skin wrinkles, dryness, dermatitis, atopic dermatitis, allergic dermatitis, acne, etc.
  • the strain may be used together with another strain belonging to Iamiaceae that has a skin-improving effect to exhibit a synergistic effect.
  • Strains belonging to the Aiaemia family include, for example, Desertimonas Genus (e.g., Desertimonas flava ) , Ilumatobacter Genera (e.g., Ilumatobacter coccineus ), Ilumatobacter fluminis ( Ilumatobacter fluminis ), Ilumatobacter nonamiensis ( Ilumatobacter nonamiensis ), etc. may be included.
  • the composition is 0.001% to 80% by weight, for example, 0.01% to 60% by weight, 0.01% to 40% by weight, 0.01% to 30% by weight, 0.01% to 20% by weight, based on the total weight of the composition.
  • % 0.01% to 10% by weight, 0.01% 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
  • It may include % to 10% by weight, or 0.1% to 5% by weight of the strain, its lysate, culture medium, or extract of the culture medium.
  • the effect of improving skin condition for example, inhibiting skin aging, whitening skin, strengthening the skin barrier, inhibiting skin wrinkles, or moisturizing the skin
  • the effect is not sufficiently effective.
  • the term "included as an active ingredient” means that the strain of the present specification, its lysate, culture medium, or extract of the culture medium is added to the extent that it can exhibit the effects mentioned above, drug delivery, stabilization, etc. This means that it is formulated into various forms by adding various ingredients as sub-ingredients.
  • the composition may be a cosmetic composition.
  • the cosmetic composition may have, for example, an softening lotion, nourishing lotion, massage cream, nourishing cream, essence, pack, gel, ampoule, or skin-adhesive type cosmetic formulation.
  • Ingredients included in the cosmetic composition may include ingredients commonly used in cosmetic compositions in addition to the composition as an active ingredient, for example, conventional auxiliaries and carriers such as stabilizers, solubilizers, vitamins, pigments, and fragrances. may include.
  • composition may be a composition for external skin application.
  • the external skin agent may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal delivery patch, drug-containing bandage, lotion, or a combination thereof.
  • the skin external preparations include ingredients commonly used in external skin preparations such as cosmetics and medicines, such as aqueous ingredients, oil-based ingredients, powder ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, and fragrances. , colorants, various skin nutrients, or a combination thereof may be appropriately mixed according to need.
  • the skin external preparations include metal sequestrants such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belafamil, licorice extract, glablidin, and various herbal medicines. , drugs such as tocopherol acetate, glythylitic acid, tranexamic acid and its derivatives or salts, and sugars such as vitamin C, magnesium ascorbate phosphate, ascorbate glucoside, arbutin, kojic acid, glucose, fructose, and trehalose. It can be mixed easily.
  • metal sequestrants such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belafamil, licorice extract, glablidin, and various herbal medicines.
  • drugs such as tocopherol acetate,
  • the composition may 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, and the lubricant may be magnesium stearate, talc, or a combination thereof.
  • the carrier may be an excipient, disintegrant, binder, 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 anhydride, 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 syrup, or combinations thereof.
  • Parenteral dosage forms may be injectable.
  • the composition may be a health functional food composition.
  • the health functional food composition can be used alone or with other foods or food ingredients, such as the strain or its culture medium, and can be used appropriately according to conventional methods.
  • the mixing amount of the active ingredient can 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 materials.
  • beverage compositions may contain various flavoring agents or natural carbohydrates as additional ingredients like ordinary 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.
  • a sweetener natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame can be used.
  • the health food composition also contains 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. It may contain the carbonating agent used, or a combination thereof.
  • the health functional food composition may also contain pulp for the production of natural fruit juice, fruit juice beverage, vegetable beverage, or a combination thereof.
  • Another aspect provides a method of preventing, ameliorating, or treating a condition of a subject comprising treating or administering an effective amount of the composition described above to the subject in need thereof.
  • the condition of the subject may be a skin-related condition or an inflammation-related condition.
  • administering As used herein, the terms “administering,” “introducing,” and “implanting” are used interchangeably and are used interchangeably to introduce a composition into an individual by a method or route that results in at least partial localization to the desired site according to one embodiment. It may refer to the arrangement of the composition according to one embodiment of.
  • Administration can be done by methods known in the art. Administration may be administered directly to the subject by any means, such as, for example, intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. You can. 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 an individual in need of skin beauty improvement, for example, skin moisturizing, skin barrier strengthening, skin inflammation inhibition, and skin wrinkle improvement effects.
  • the administration of the composition according to one embodiment is 0.1 mg to 1,000 mg, for example, 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to 1 mg per day.
  • the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, gender, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and those skilled in the art will Taking these factors into consideration, the dosage can be adjusted appropriately.
  • the frequency of administration can be once a day or two or more times within the range of clinically acceptable side effects, and can be administered at one or two or more locations, daily or at intervals of 2 to 5 days.
  • the number of days of administration can be from 1 to 30 days per treatment. If necessary, the same treatment can be repeated after an appropriate period.
  • the dosage per kg is the same as for humans, or the above dosage is converted into, for example, the volume ratio (e.g., average value) of the organs (heart, etc.) between the target animal and human.
  • One dose can be administered.
  • the new Aemiaceae family strain has phylogenetically and chemically distinct characteristics compared to strains of other or other genera of the same family. Additionally, the strain or its culture medium can be usefully used to prevent, improve, or treat skin-related conditions.
  • Figure 1 is a diagram showing the systematic classification results of KERA-3.
  • Figure 2 shows the results of confirming the morphological characteristics of KERA-3.
  • Figure 3 shows the results showing the effect of KERA-3 strain on the expression of filaggrin in human keratinocytes.
  • Figure 4 shows the results showing the effect of KERA-3 strain on the expression of HAS-3 in human keratinocytes.
  • Figure 5 shows the results showing the effect of KERA-3 strain on the expression of ABCA12 in human keratinocytes.
  • Figure 6 shows the results showing the effect of KERA-3 strain on the expression of ELOVL4 in human keratinocytes.
  • Figure 7 shows the results showing the effect of KER-3 strain on the expression of CERS3 in human keratinocytes.
  • a sample human epidermal keratinocyte obtained by washing the skin of a healthy adult with sterile distilled water was inoculated into R2A (Reasoner's 2A) medium (Becton Dickinson, Cockeysville, MD) diluted 1/10. After inoculation, the colonies were cultured in an incubator at 30°C for 240 hours, and then 100 colonies formed were pure, isolated and cultured, and re-cultured in an incubator at 30°C for 240 hours. The 16s rRNA gene sequence was identified for the colonies whose culture was completed. The primers used at this time were designed to react and amplify only bacteria (SEQ ID NOs. 2 and 3).
  • PCR amplification was performed in 30 cycles of 95°C for 1 minute, 55°C for 1 minute, and 75°C for 1 minute and 30 seconds, and was finally treated at 72°C for 8 minutes and stored at 4°C.
  • the DNA sequences of the isolated and cultured species were determined using ABI-3730XL (ABI, USA).
  • the base sequence of the 16s rRNA region determined among the isolated and cultured microbial colonies was compared and analyzed with other strains registered with the BLAST program provided on the US National Center for Biotechnology Information (NCBI) website. Only novel species with 97% or less homology were selected and used, and among them, novel microorganisms with 94% or less homology (hereinafter referred to as “KERA-3”) were selected.
  • KERA-3 has a 16s rRNA sequence of SEQ ID NO: 1 (complementary DNA).
  • KERA-3 was Actinomarinicola tropica SCSIO 58843, Aquihabitans daechonnesis CH22-21, Iamia majanohamensis F12, Illumatobacter It was confirmed that it had 90.94%, 90.39%, 89.56%, and 89.20% homology with Ilumatobacter fluminis YM22-133, respectively.
  • Figure 1 is a diagram showing the systematic classification results of KERA-3.
  • KERA-3 was confirmed to be a new genus belonging to the Iamiaceae family that has never been reported so far.
  • KERA-3 The morphological characteristics of KERA-3 were analyzed as follows.
  • the cell morphology of KERA-3 was cultured in R2A at 25°C for 5 days and then observed at 1000x magnification using a microscope (Olympus microscope, GX71).
  • the motility of the strain was evaluated by inoculating fresh cultured KERA-3 cells into 0.3% agar slant medium.
  • KERA-3 was inoculated on R2A (Becton Dickinson, Cockeysville, MD) solid medium, and a membrane filter was attached to the colonies formed after culturing for 5 days.
  • the membrane filter to which the colony was attached was separated and fixed by treatment in 2.5% glutaraldehyde solution for 2 hours, and then washed by soaking in PBS for 5 minutes each. Afterwards, it was treated with 2% osmium tetroxide solution for 1 hour and dehydrated using 40, 50, 60, 70, 80, 90, and 100% ethanol.
  • KERA-3 had the following morphological characteristics.
  • the length of the cell is 0.8 ⁇ 1.4 ⁇ m and the diameter is 0.4 ⁇ 0.7 ⁇ m.
  • the colony shape is oval and white.
  • the optimal growth temperature was set from 4°C to 50°C to confirm the optimal culture temperature, and the optimal salt concentration was determined by adding 0% to 10% (w/v) of NaCl to the medium at 25°C. It was confirmed by culturing for 10 days. pH was checked using the following buffer system; Sodium acetate/acetic acid (pH ⁇ 6), Tris/HCl (pH 6 to 9), and glycine/sodium hydroxide (pH>9).
  • R2A Reasoner's 2A
  • TSA Teryptic Soy Agar
  • NA Nutrient Agar
  • LBA Lysogeny Broth Agar
  • API-ZYM test control Alkaline phosphatase - Esterase (C4) + Esterase Lipase (C8) + Lipase (C14) - Leucine arylamidase + Valine arylamidase - Cystine arylamidase - Trypsin - a-chymotrypsin - Acid phosphatase - Naphtol-AS-BI-phosphohydrolase + ⁇ -galactosidase - ⁇ -galactosidase - ⁇ -glucuronidase - ⁇ -glucosidase - ⁇ -glucosidase - N -acetyl - ⁇ -glucosaminidase - a - mannosidase - a-fucosidase - Use of carbon source Glucose, + Sucrose, + Pyruvate, + Soluble starch, + Xylose, -
  • minimal salt medium proteose peotone 0.05%, Casamino acids 0.05%, Dipotassium phosphate, Magnesium sulfate
  • carbon source Glucose, Sucrose, Pyruvate, Soluble starch, Xylose, Galactose, Mannose, Mannitol, Lactose, Fructose
  • Aerobic growth was confirmed by culturing under aerobic culture conditions and anaerobic culture conditions (using an anaerobic pack).
  • EPI-7 had the following culture and physiological characteristics.
  • R2A Reasoner's 2A
  • TSA Teryptic Soy Agar
  • NA Nutrient Agar
  • LBA Lysogeny Broth Agar
  • the optimal growth medium is R2A medium, which grows at 18°C to 37°C (optimum 25°C), but does not grow at temperatures outside this;
  • Nitrate is not reduced to nitrite
  • the DNA G+C content of KERA-3 was confirmed by whole genome sequencing using Illumina Miseq equipment.
  • the Cellular Fatty Acid composition, G+C composition, and Quinone analysis test methods and conditions are as follows.
  • the cellular fatty acid composition of the isolated strains was determined according to Miller's method. After transferring about 40 mg of cultured cells to a tube, 1 ml of a solution containing 15% NaOH in 50% methanol was added, heated at 100°C for 30 minutes, and cooled to room temperature. 2 ml of methanolic-HCl (a mixed solution of 325 ml of 6.0N HCl and 275 ml of methanol) was added thereto, heated at 80°C for 10 minutes, and then rapidly cooled. Afterwards, 1.25 ml of hexane/methyl-tert-butylether (1:1, v/v) was added and mixed well for 10 minutes.
  • methanolic-HCl a mixed solution of 325 ml of 6.0N HCl and 275 ml of methanol
  • HPLC YOUNG LIN YL9100 (YL9111 Binary pump)
  • Polar lipid analysis was performed after culturing the strain in R2A medium at 25°C for 96 hours. First, polar lipids from the strains were purified using Minnikin D.E. Extraction was performed according to the method described in et al (1984) (J Microbiol Methods 2, 233-241). Afterwards, polar lipids were identified using two-dimensional TLC ( Komakata K et al., 1987, Methods Microbiol 19, 161-206).
  • KERA-3 has the following biochemical characteristics
  • DNA G+C content is 72.2 mol%
  • MK-9(H8) tetrahydrogenated menaquinone with nine units is the isoprenoid quinone detected
  • the main polar lipids are diphosphatidylglycerol (DPG), three unidentified phosphoglycolipids (PGL), unidentified aminophosphoglycolipid (APGL), and unidentified aminophospholipids ( at least one selected from the group consisting of unidentified aminophospholipid (APL); and
  • DPG DPG, PI, PIM N.D. N.D. DPG, PI, PC, PIM Major fatty acids C16:0, C18:1w9c ISO-C16:0(17.9), Iso-C17:1 ⁇ 9c(14.7); ISO-C17:0(11.3) C16: 0, , iso-C16:0 16:1 ⁇ 5c, 16:0, 17:1 ⁇ 8c,18:1 ⁇ 9c C17:0, C17:1w8c iso-C16:0, iso-C14:0, iso-C15:0, ai-C15: 0,C17: 1 ⁇ 6c. 17:1 ⁇ 8c, 17:0
  • DPG diphosphatidylglycerol
  • PI phosphatidylinositol
  • PC phospatidylcholine
  • PIM phosphatidylinositolmannoside
  • unidentified phospholipids PGL: unidentified unidentified phosphoglycolipid
  • APGL unidentified aminophosphoglycolipid
  • APL unidentified aminophospholipid
  • KERA-3 has the same characteristics as those in Table 3 above, and its main biochemical characteristics are different from the relative strain, and the combination of these chemical classification results is the result of the phylogenetic classification of the strain. Together, it proves that KERA-3 is a new species.
  • KERA-3 isolated from human epidermal keratinocyte is a new genus of microorganism belonging to the Iamiaceae family, and it was named Dermatobacter according to the systematic nomenclature.
  • the genus was named Hominis .
  • the Dermatobacter Hominis strain belonging to the Iemia family was deposited in the National Institute of Agricultural Science's Microbial Bank (Korean Agricultural Culture Collection, KACC) on February 15, 2022, with deposit number KACC 22415. was granted.
  • filaggrin a factor related to skin barrier strengthening and moisturizing activity following treatment of KERA-3 in human keratinocyte cell line (HaCaT)
  • HAS3 hyaluronic acid synthase 3
  • ABCA12 ATP-binding cassette sub-family A member 12
  • ELOVL4 Elongation of very long chain fatty acids protein 4
  • CERS3 Ceramide synthase 3
  • HaCaT cells a human keratinocyte cell line, and human-derived Epidermal Keratinocytes were cultured in DMEM medium (Dulbecco's modified Eagle's Medium, Gibco 1210-0038) containing 10% fetal bovin serum. All cultures were performed in a 37°C CO 2 incubator. As a positive control, the cultured cell lines were treated with retinol at a dose of 1mM. Additionally, KERA-3 cultures were treated at concentrations of 1.0% and 10.0%. Afterwards, HaCaT cells were cultured for an additional 24 hours, then recovered, and RNA was isolated by adding 1 ml of Trizol (RNA iso, DAKARA, Japan).
  • Trizol RNA iso, DAKARA, Japan
  • Figure 3 shows the results showing the effect of KERA-3 strain on the expression of filaggrin in human keratinocytes.
  • Figure 4 shows the results showing the effect of KERA-3 strain on the expression of HAS-3 in human keratinocytes.
  • Figure 5 shows the results showing the effect of KERA-3 strain on the expression of ABCA12 in human keratinocytes.
  • Figure 6 shows the results showing the effect of KERA-3 strain on the expression of ELOVL4 in human keratinocytes.
  • Figure 7 shows the results showing the effect of KER-3 strain on the expression of CERS3 in human keratinocytes.
  • KERA-3 culture medium (Example 1) compared to the untreated control and KERA-3 culture medium (Comparative Example 1) of filaggrin, HAS3, ABCA12, ELOVL4 and CERS3 It was confirmed that expression was significantly high.
  • the novel strain belonging to the Aemia family can be used to improve skin conditions such as inhibiting or improving skin aging, anti-wrinkle, strengthening the skin barrier, or moisturizing the skin.

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Abstract

La présente invention concerne un micro-organisme appartenant à la famille des Iamiaceae, présentant des caractéristiques phylogénétiquement et chimiotaxonomiquement différentes par comparaison avec des souches du même genre ou d'un genre différent. En outre, la souche ou une solution de culture de celle-ci peut être utilisée efficacement pour prévenir, améliorer ou traiter des affections liées à la peau.
PCT/KR2023/003949 2022-06-09 2023-03-24 Nouveau microorganisme de la famille des iamiaceae et ses utilisations Ceased WO2023239024A1 (fr)

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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE Nucleotide 24 May 2021 (2021-05-24), ANONYMOUS : "Acidimicrobiales bacterium strain kera_5 16S ribosomal RNA gene, partial sequence", XP093114640, retrieved from NCBI Database accession no. MZ234082.1 *
FERNÁNDEZ-NATAL I., SÁEZ-NIETO J.A., MEDINA-PASCUAL M.J., ALBERSMEIER A., VALDEZATE S., GUERRA-LASO J.M., RODRÍGUEZ H., MARRODÁN T: "Dermabacter hominis: a usually daptomycin-resistant gram-positive organism infrequently isolated from human clinical samples", NEW MICROBES AND NEW INFECTIONS, ELSEVIER LTD, GB, vol. 1, no. 3, 1 December 2013 (2013-12-01), GB , pages 35 - 40, XP093114635, ISSN: 2052-2975, DOI: 10.1002/2052-2975.31 *
FUNKE G, ET AL.: "CHARACTERISTICS OF CDC FROUP 3 AND GROUP 5 CORYNEFORM BACTERIA ISOLATED FROM CLINICAL SPECIMENS AND ASSIGNMENT TO THE GENUS DERMABACTER", JOURNAL OF CLINICAL MICROBIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 32, no. 05, 1 May 1994 (1994-05-01), US , pages 1223 - 1228, XP002948527, ISSN: 0095-1137 *
KIM YOUNG IN, PARK KYOUNG UN, PARK IL JOONG, PARK SEO-JIN, LEE WEE GYO: "A Case of Misidentification of Dermabacter hominis as Listeria grayi", KOREAN JOURNAL OF CLINICAL MICROBIOLOGY, vol. 14, no. 2, 1 January 2011 (2011-01-01), pages 79 - 82, XP093114639, ISSN: 1229-0025, DOI: 10.5145/KJCM.2011.14.2.79 *
LEE HYE-JIN, CHO CHI-HYUN, KWON MIN-JUNG, NAM MYUNG-HYUN, LEE KAP-NO, LEE CHANG-KYU: "A Patient with Fatal Septicemia Caused by a Rare Pathogen Dermabacter Hominis", INFECTION & CHEMOTHERAPY, vol. 43, no. 1, 1 January 2011 (2011-01-01), pages 86 - 88, XP093114633, ISSN: 2093-2340, DOI: 10.3947/ic.2011.43.1.86 *

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