WO2021060649A1 - Souche de staphylococcus intermedius st-11 et son utilisation pour améliorer l'état de la peau - Google Patents

Souche de staphylococcus intermedius st-11 et son utilisation pour améliorer l'état de la peau Download PDF

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WO2021060649A1
WO2021060649A1 PCT/KR2020/006988 KR2020006988W WO2021060649A1 WO 2021060649 A1 WO2021060649 A1 WO 2021060649A1 KR 2020006988 W KR2020006988 W KR 2020006988W WO 2021060649 A1 WO2021060649 A1 WO 2021060649A1
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skin
strain
staphylococcus
culture solution
composition
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English (en)
Korean (ko)
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이동걸
김민지
김미선
강승현
박명삼
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코스맥스 주식회사
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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
    • 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
    • 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
    • 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
    • C12R2001/44—Staphylococcus

Definitions

  • the skin ecosystem provides a variety of habitats for microorganisms, and a wide range of microorganisms live there.
  • Humans which are hosts, have a symbiotic relationship with them, and are known to have many positive effects on the host.
  • the skin consists of various types of habitats such as depressions and specialized crevices, and helps a wide distribution of microorganisms to grow. Basically, the skin forms a physical film and helps to defend against potential hazards and toxic substances from the outside.
  • the skin becomes a point of connection with the external environment and is also a collection of various microorganisms (fungi, bacteria, viruses and small larvae). In accordance with the choice of physical and chemical functions, microorganisms prepare habitats by adapting to specialized niches.
  • the skin is cold, acidic, and remains dry. Structurally, the epidermis forms the skin barrier, blocks the penetration of microorganisms and toxins, and plays an important role in maintaining moisture.
  • the uppermost layer of the epidermis is composed of the stratum corneum.
  • the epidermis has a shape called'brick and mortar structure', and the skin tissue goes through a continuous self-healing process, and the scales that have gone through the end of the differentiation process are constantly being removed from the skin tissue.
  • Probiotics is a generic term for microorganisms that have a beneficial effect on the human body, and refers to microorganisms that provide benefits to our body. Most of the probiotics known to date are known as lactobacilli. Probiotics have been reported to produce effective effects through various beneficial actions on the human body, but studies on the relationship between skin flora and skin are insufficient.
  • One aspect is to provide a Staphylococcus intermedius ST-11 (Staphylococcus intermedius ST-11) strain belonging to the genus Staphylococcus sp.
  • Staphylococcus intermedius ST-11 Staphylococcus intermedius ST-11
  • Another aspect is to provide a lysate of the strain, a culture medium, or an extract of the culture medium.
  • Another aspect is to provide a composition
  • a composition comprising the strain, a lysate thereof, a culture medium, and an extract of the culture medium.
  • Another aspect is to provide a method for improving, preventing, or treating a skin condition comprising administering the strain, its lysate, a culture medium, and an extract of the culture medium to an individual.
  • One aspect provides a Staphylococcus intermedius ST-11 strain.
  • the Staphylococcus intermedius ST-11 strain may be a strain deposited with accession number KCCM12556P.
  • the Staphylococcus intermedius ST-11 strain may be a strain containing 16s rRNA of SEQ ID NO: 1.
  • the strain may be one having an effect of improving skin beauty, for example, inhibiting skin aging, whitening skin, strengthening skin barrier, inhibiting skin wrinkles, or moisturizing skin.
  • the strain may be one that increases the expression of hyaluronic acid synthase 3 (HAS3: Hyaluronan synthase 3) or filaggrin.
  • HAS3 Hyaluronan synthase 3
  • filaggrin filaggrin
  • Another aspect provides a lysate of the strain, a culture medium, or an extract of the culture medium.
  • the strain is as described above.
  • culture medium may be used interchangeably with “culture supernatant”, “conditional culture medium” or “conditioned medium”, and Staphylococcus intermedius ST-11 strain can grow and survive in vitro. It may mean a whole medium including the strain, its metabolites, and extra nutrients obtained by culturing the strain in a medium capable of supplying nutrients for a certain period of time.
  • the culture solution may mean a culture solution in which cells are removed from the cell culture solution obtained by culturing the strain.
  • the liquid from which the cells have been removed from the culture solution is also referred to as the "supernatant solution", and the culture solution is left for a certain period of time to take only the liquid in the upper layer excluding the submerged part, or remove the cells through filtration, or It can be obtained by removing the precipitation of and taking only the upper liquid.
  • the "bacterial body” refers to the strain itself of the present invention, and includes the strain itself or separated from the culture solution by culturing the strain selected by separating from skin samples and the like.
  • the cells can be obtained by taking the part that has settled in the lower layer by centrifuging the culture solution, or by allowing it to stand still for a certain period of time and then removing the upper liquid because it sinks into the lower layer of the culture solution by gravity.
  • the culture solution may include a culture solution itself obtained by culturing a strain, a concentrate thereof, or a culture supernatant obtained by removing a strain from a lyophilisate or a culture solution, a concentrate thereof, or a lyophilisate.
  • the culture medium is Staphylococcus intermedius ST-11 strain in a medium (for example, R2A medium or TSA medium) at any temperature of more than 10 °C or less than 40 °C for a certain period of time, for example, 4 to 50 hours. It may be obtained by culturing.
  • a medium for example, R2A medium or TSA medium
  • the culture supernatant of the strain may be obtained by centrifuging or filtering the strain culture solution to remove the strain.
  • the concentrate may be obtained by concentrating the obtained supernatant after filtering the strain culture solution itself or the culture solution using a centrifuge or filter.
  • the culture medium and culture conditions for culturing the Staphylococcus intermedius ST-11 strain may be appropriately selected or modified by a person of ordinary skill in the art.
  • lysate may refer to a product obtained by crushing the cell wall of the strain itself by chemical or physical force.
  • culture solution extract refers to extraction from the culture solution or its concentrate, and includes an extract, a dilution or concentrate of the extract, a dried product obtained by drying the extract, or these preparations or purified products, and a fraction obtained by fractionating them. I can.
  • Another aspect provides the use of the strain, a lysate of the strain, a culture solution, or an extract of the culture solution. Specifically, it provides a composition comprising an extract of Staphylococcus intermedius ST-11 strain, a lysate, a culture solution, or a culture solution thereof.
  • the use of the strain may include improving skin conditions, improving skin beauty, preventing, improving or treating skin diseases, preventing, improving, or treating inflammatory diseases of the skin.
  • the skin condition improvement or skin beauty improvement may be suppressing or improving skin aging, anti-wrinkle, skin whitening, skin barrier strengthening, or skin moisturizing.
  • skin aging refers to the morphological and intangible changes that appear in the skin as we age, for example, the phenomenon of thinning the thickness of the epidermis, the number of cells or blood vessels in the dermis, the ability to repair DNA damage, the cell replacement cycle, It refers to the reduction of wound recovery, skin barrier function, epidermal moisture retention, sweat secretion, sebum secretion, vitamin D production, physical damage protection, chemical removal ability, immune response, sensory function, and body temperature regulation.
  • the strain or a culture solution thereof may be for improving skin aging caused by an exogenous factor or an endogenous factor.
  • the exogenous factor refers to various external factors, such as ultraviolet (light), and the endogenous factor is also referred to as a chronological factor, and refers mainly to a factor caused by the passage of time.
  • the skin aging is not only a symptom of premature aging induced by external stimuli caused by ultraviolet rays, pollution, tobacco smoke, chemical substances, etc., but also a natural aging phenomenon that occurs as the proliferation of skin cells decreases with age. It includes, and includes all of wrinkles, elasticity reduction, sagging and dryness of the skin.
  • wrinkles include that irritation caused by changes in internal and external factors causes wrinkles by changing components constituting the skin tissue.
  • the aging may be photoaging.
  • photoaging is a phenomenon caused by external environmental factors, and the most representative factor is ultraviolet rays. Ultraviolet rays cause damage to biological components such as activation of proteases, chain cleavage of matrix proteins, and abnormal cross-linking, and repetition of this mechanism causes apparent skin aging.
  • wrinkle refers to a state in which the elasticity of the skin is lost and loosened, and for example, the skin may be folded.
  • Pigmentation refers to a condition in which the amount of pigment in the body is abnormal or where the pigment appears, and may be, for example, spots and freckles.
  • skin wrinkle prevention or improvement may mean any action of preventing or improving wrinkles by inhibiting the expression of factors related to wrinkles, or increasing the total amount of collagen.
  • skin whitening may mean not only brightening skin tone by inhibiting the synthesis of melanin pigment, but also improving skin hyperpigmentation such as spots and freckles caused by ultraviolet rays, hormones, or genetics.
  • skin barrier strengthening may refer to any action that enhances the function of the skin barrier that is located on the outermost side of the skin and prevents loss of moisture and nutrition.
  • the “skin moisturizing” may mean any action of maintaining skin moisture or preventing moisture loss.
  • anti-inflammatory may be used interchangeably with “inhibitory or ameliorating inflammation”, and may mean any action in which the immune response is alleviated and NO production is suppressed.
  • the “skin disease” may be a disease caused by impaired skin barrier function, skin aging, skin wounds, skin scars, or skin inflammation.
  • prevention includes controlling the occurrence of disease.
  • treatment includes inhibiting, alleviating, or eliminating the development of a disease.
  • the damage to the skin barrier function may mean all changes that appear on the skin due to a decrease or damage to the function of the skin barrier. For example, it may include increased skin wrinkles, dryness, dermatitis, atopic dermatitis, allergic dermatitis, acne, and the like.
  • the skin inflammatory disease may be any one selected from the group consisting of skin wounds, dermatitis, atopic dermatitis, pruritus, eczema skin disease, dry eczema, erythema, urticaria, psoriasis, weak rash, and acne.
  • the strain may be used together with a strain belonging to the genus Staphylococcus other strains having a skin improvement effect to exhibit a synergistic effect.
  • strains belonging to the genus Staphylococcus are S. argenteus , S. aureus , S. schweitzeri , S. simiae, S. auricularis, S. carnosus , S. condimenti , S. debuckii , S. massiliensis , S. piscifermentans , S. simulans, S. capitis , S. caprae , S. epidermidis , S. saccharolyticus, S. devriesei , S.
  • strains belonging to the genus Staphylococcus include Staphylococcus capitis ST-1, Staphylococcus lentus ST-2, Staphylococcus lentus ST-2, and Staphylococcus cornii.
  • Staphylococcus cohnii ST-3 Staphylococcus gallinarum ST-4, Staphylococcus saprophyticus ST-5, Staphylococcus saprophyticus ST-5, Staphylococcus epi Staphylococcus epidermidis ST-6, Staphylococcus sciuri ST-7, Staphylococcus haemolyticus ST-8, Staphylococcus haemolyticus ST-8, Staphylococcus simul Staphylococcus simulans ST-9, Staphylococcus xylosus ST-10, Staphylococcus intermedius ST-11, Staphylococcus intermedius ST-11, and Staphylococcus Reneri ST-12 ( Staphylococcus warneri ST-12), or Staphylococcus schleiferi ST-13 (Staphylococcus schleiferi ST-13).
  • the composition is from 0.001% to 80% by weight based on the total weight of the composition, for example, from 0.01% to 60% by weight, from 0.01% to 40% by weight, from 0.01% to 30% by weight, from 0.01% to 20% by weight %, 0.01% to 10%, 0.01% 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 the strain, a lysate thereof, a culture solution, or an extract of a culture solution thereof.
  • the term "included as an active ingredient” means that the strain of the present specification, its lysate, culture medium, or extract of the culture medium thereof is added to the extent that the above-mentioned effect can be exhibited, and various It means that the ingredients are added as an auxiliary ingredient to be formulated in various forms.
  • the composition may be a cosmetic composition.
  • the cosmetic composition may be, for example, a softening lotion, a nutritional lotion, a massage cream, a nutritional cream, an essence, a pack, a gel, an ampoule, or a cosmetic formulation of a skin adhesion type.
  • ingredients included in the cosmetic composition may include ingredients commonly used in cosmetic compositions in addition to the composition as an active ingredient.
  • conventional adjuvants and carriers such as stabilizers, solubilizers, vitamins, pigments and fragrances It may include.
  • composition may be a composition for external application for skin.
  • the external preparation for skin may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal delivery patch, drug-containing bandage, lotion, or a combination thereof.
  • the above skin external preparations are ingredients commonly used in external preparations for skin such as cosmetics and pharmaceuticals, such as aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances. , Colorants, various skin nutrients, or a combination thereof and may be appropriately formulated according to need.
  • the external preparations for the skin include metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, bellapamil, licorice extract, glavidine, and caline.
  • Hot water extract of fruit, various herbal medicines, drugs such as tocopherol acetate, glytilithic acid, tranexamic acid and derivatives or salts thereof, vitamin C, ascorbic acid magnesium phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, Sugars such as trehalose can also be appropriately blended.
  • 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 as a lubricant, 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 calcium carboxymethylcellulose, 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.
  • the oral dosage form may be a granule, a powder, a liquid, a tablet, a capsule, a dry syrup, 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, and may be appropriately used according to a conventional method.
  • the mixing amount of the active ingredient may be appropriately determined according to 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.
  • the beverage composition may contain various flavoring agents or natural carbohydrates as an additional component, like ordinary beverages.
  • the natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • sweetener natural sweeteners such as taumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame can be used.
  • the health food composition may also be used in nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonated beverages. Carbonating agents used, or combinations thereof.
  • the health functional food composition may also contain natural fruit juice, fruit juice beverage, pulp for the production of vegetable beverages, or a combination thereof.
  • Another aspect also provides a method of preventing, ameliorating, or treating a condition in an individual comprising the step of treating or administering to an individual in need thereof an effective amount of the above composition.
  • the condition of the individual may be a condition related to skin or a condition related to inflammation.
  • administering is used interchangeably and one embodiment into a subject by a method or route that results in at least partial localization of a composition to a desired site according to one embodiment. It may mean the arrangement of the composition according to the example.
  • Administration can be administered by a method known in the art. Administration may be administered directly to a subject by any means, for example by routes such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. I can. The administration can be administered systemically or locally.
  • the subject may be a mammal, for example a human, cow, horse, pig, dog, sheep, goat, or cat.
  • the individual may be an individual in need of an effect of improving skin beauty, for example, moisturizing the skin, strengthening the skin barrier, inhibiting skin inflammation, and improving skin wrinkles.
  • the administration is 0.1 mg to 1,000 mg per subject per day, 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 the composition according to an embodiment.
  • 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 according to factors such as formulation method, administration mode, patient's age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate, and response sensitivity. In consideration of these factors, the dosage can be appropriately adjusted.
  • the number of administration can be once a day or two or more times within the range of clinically tolerable side effects, and the administration site 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 administered from 1 to 30 days per treatment. If necessary, the same treatment can be repeated after an appropriate time period.
  • 1 is a graph showing the effect of the strain on the expression of COL1A1 according to an embodiment.
  • Figure 2 is a graph showing the effect of the strain on the expression of HAS3 according to an embodiment.
  • 3 is a graph showing the effect of the strain on the expression of pilagrin according to an embodiment.
  • a sample human epidermal keratinocyte obtained by washing the skin of a healthy woman with sterile distilled water was inoculated into R2A medium (Becton Dickinson, Cockeysville, MD). After culturing in a 28°C incubator for 48 hours after inoculation, 100 colonies formed were separated and cultured with pure water, and cultivated in a 28°C incubator for 48 hours. The cultured colonies were subjected to 16s rRNA gene sequence identification. The primers used at this time were designed to amplify in response to only bacteria (SEQ ID NOs: 2 and 3).
  • PCR amplification was carried out in 30 cycles of 95°C for 1 minute, 55°C for 1 minute, 75°C for 1 minute and 30 seconds, and finally treated at 72°C for 8 minutes and then stored at 4°C.
  • DNA sequences of the isolated and cultured species were determined using ABI-3730XL (ABI, USA).
  • the nucleotide sequence of the 16S rRNA site determined among the isolated and cultured microbial colonies was compared with other strains registered with the BLAST program provided on the website of the National Center for Biotechnology Information (NCBI), when compared with 98% homology.
  • NCBI National Center for Biotechnology Information
  • ST-11 novel microorganism Staphylococcus intermedius ST-11 strain
  • ST-11 novel microorganism Staphylococcus intermedius ST-11 strain
  • the selected ST-11 strain was deposited with the Korean Microbial Conservation Center on June 11, 2019, and was given the accession number KCCM12556P, and the ST-11 strain has a 16s rRNA sequence of SEQ ID NO: 1 (complementary DNA).
  • Type Strain KCCM 40149 Staphylococcus intermedius strain (sold in Korea Microbial Conservation Center)
  • the human fibroblast cell line Human dermal fibroblast, Hs68
  • Hs68 human dermal fibroblast, Hs68
  • DPBS phosphatidylcholine
  • a real-time PCR machine (Step One Plus, Applied Biosystems, USA) by adding cybergreen (SYBR Green supermix, Applied Biosystems, USA) together with primers and cDNA to the target gene COL1A1 using the synthesized cDNA as a template.
  • Real-time polymerase chain reaction was performed at.
  • the expression level of the gene was finally analyzed through correction for the ⁇ -actin gene, and the results are shown in FIG. 1.
  • strain culture solution The effect of the strain culture solution on skin barrier strengthening and skin moisturizing activity was analyzed.
  • HaCaT cells a human keratinocyte
  • DMEM medium Dulbecco'smodified Eagle's Medium, Gibco 1210-00308
  • the cultured cell line was treated with the culture solution (1% (w/w)) of the ST-11 strain and further cultured for 24 hours.
  • the expression level of HAS3 and filaggrin was measured in the same manner as in the above experimental example, except that primers for HAS3 (hyaluronic acid synthase) and filaggrin, which are factors related to skin barrier strengthening and moisturizing, were used. I did. The results are shown in FIGS. 2 and 3, respectively.

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Abstract

La présente invention concerne un nouveau micro-organisme, un lysat de celui-ci, un bouillon de culture de celui-ci, ou un extrait du bouillon de culture de celui-ci, et son utilisation pour améliorer un état de la peau.
PCT/KR2020/006988 2019-09-27 2020-05-29 Souche de staphylococcus intermedius st-11 et son utilisation pour améliorer l'état de la peau WO2021060649A1 (fr)

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KR20170003478A (ko) * 2015-06-30 2017-01-09 (주)아모레퍼시픽 피부 개선용 균주 및 이를 포함하는 피부 개선용 키트
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KR101980071B1 (ko) * 2017-04-28 2019-05-20 코스맥스 주식회사 스트렙토코커스 써모필러스 균주 및 이의 배양액을 포함하는 조성물
KR102123022B1 (ko) 2018-12-31 2020-06-16 코스맥스 주식회사 피부에 효능이 있는 고농도 프로바이오틱스를 포함하는 조성물

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EP2361632A1 (fr) * 2010-02-19 2011-08-31 Protectimmun GmbH Bactérie environnementale spécifique pour la protection contre le traitement des maladies allergiques, inflammatoires chroniques et/ou auto-immunes
KR20170003478A (ko) * 2015-06-30 2017-01-09 (주)아모레퍼시픽 피부 개선용 균주 및 이를 포함하는 피부 개선용 키트
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