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WO2022039572A1 - Culture mycélienne d'une nouvelle souche de porostereum sp. (kctc18837p) et composition cosmétique la comprenant pour améliorer l'état de la peau - Google Patents

Culture mycélienne d'une nouvelle souche de porostereum sp. (kctc18837p) et composition cosmétique la comprenant pour améliorer l'état de la peau Download PDF

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WO2022039572A1
WO2022039572A1 PCT/KR2021/011159 KR2021011159W WO2022039572A1 WO 2022039572 A1 WO2022039572 A1 WO 2022039572A1 KR 2021011159 W KR2021011159 W KR 2021011159W WO 2022039572 A1 WO2022039572 A1 WO 2022039572A1
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porostereum
kctc18837p
culture
skin
mycelium
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Korean (ko)
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김병천
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Mycel Lab Co Ltd
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Mycel Lab Co Ltd
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    • 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
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • 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
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • 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
    • 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
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • 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
    • A61Q19/08—Anti-ageing preparations

Definitions

  • the present invention relates to a Porostereum sp. (KCTC18837P) mycelium culture and a cosmetic composition for improving skin condition comprising the same.
  • KCTC18837P Porostereum sp.
  • Mushrooms are rich in carbohydrates, proteins, lipids, minerals, vitamins, etc., and contain a large amount of physiologically active substances including beta-glucan and extracellular polysaccharides.
  • a significant part of the ingredients and physiological activity mechanisms of mushrooms have been scientifically proven, so research such as discovering new mushrooms, developing a mass propagation method, and searching for functionality is needed.
  • Mushrooms can be divided into mycellium and fruit body.
  • Mycelium contains various useful substances like the fruiting body, and fruiting body induction takes a long time for cultivation and technology development, whereas the liquid culture method of mycelium uses hygienic and automated equipment to stably and mass-produce in a short time. It has the advantage that it can be cultivated.
  • the genus Porostereum belongs to the genus Basidiomycota, Agaricomycetes, Polyporales, Phanerochaetaceae, and Porostereum. . (National Arboretum National Standard Mushroom List)
  • Korean Patent No. 1401997 describes that Porostereum spadiceum (KUC8602) has the ability to decolorize a liquid dye, but it is specifically related to the genus Porostereum sp. KCTC18837P The species is different and there is a difference from the use of the present invention in that it is used for dyeing wastewater treatment.
  • It is an object of the present invention to provide a cosmetic composition for improving skin condition comprising a new strain Porostereum sp. (KCTC18837P) or a mycelium culture thereof as an active ingredient.
  • the skin condition improvement may be at least one selected from the group consisting of antioxidant, wrinkle improvement, skin moisturizing, skin whitening and anti-aging.
  • the wrinkle improvement may be to promote collagen synthesis.
  • the skin moisturizing may be to increase the filaggrin expression of the skin.
  • the skin moisturizing may be to inhibit melanin production.
  • the anti-aging may be to increase the cell viability or increase the cell regeneration rate.
  • An object of the present invention is based on the total amount of the liquid medium, starch 0.2 ⁇ 2% (w / v), sucrose 0.2 ⁇ 2% (w / v), glucose 0.2 ⁇ 2% (w / v), soybean powder 1.5% ( w/v), FeSO 4 0.001% (w/v), KH 2 PO 4 0.001% (w/v), MgSO 4 0.003% (w/v), vitamin B7 0.003% (w/v), vitamin B6 0.002 % (w/v), in a liquid medium with a pH of 5-6, the air supply is 0.02-1vvm, the stirring speed is 25-100rpm, and the culture temperature is 18-27°C for 5-7 days.
  • An object of the present invention is to provide a cosmetic composition for improving skin condition comprising a new strain Porostereum sp. (KCTC18837P) or a mycelium culture thereof as an active ingredient.
  • the present invention provides a new strain Porostereum sp. (KCTC18837P) or a mycelium culture thereof.
  • the new strain Porostereum sp. (KCTC18837P) mycelium culture is characterized in that it contains beta-glucan, extracellular polysaccharide, 5-hydroxy-6,7-dimethoxyphthalide.
  • the present invention also provides a cosmetic composition for improving skin condition comprising a new strain Porostereum sp. (KCTC18837P) or a mycelium culture thereof as an active ingredient.
  • the skin condition improvement may be antioxidant, wrinkle improvement, skin moisturizing, skin whitening, skin cell survival increase, or skin regeneration.
  • the wrinkle improvement may be to promote collagen synthesis.
  • the skin moisturizing may be to increase the filaggrin expression of the skin.
  • the skin moisturizing may be to inhibit melanin production.
  • the present invention is to provide optimal culture conditions for the new strain Porostereum sp. (KCTC18837P).
  • the liquid medium conditions of the new strain Porostereum sp. (KCTC18837P) are based on the total amount of the broth: starch 0.2 to 2 % (w/v), sucrose 0.2 to 2 % (w/v), glucose 0.2 to 2 % (w/v). v), soybean powder 1.5% (w/v), FeSO 4 0.001% (w/v), KH 2 PO 4 0.001% (w/v), MgSO 4 0.003% (w/v), vitamin B7 0.003% ( w/v), vitamin B6 0.002% (w/v), and may be a liquid medium having a pH of 5-6.
  • the new strain Porostereum sp. (KCTC18837P) may be cultured for 5-7 days at an air supply amount of 0.02 ⁇ 1vvm, agitation speed of 25 ⁇ 100rpm, and a culture temperature of 18 ⁇ 27°C.
  • the present invention provides a cosmetic composition for improving skin condition containing the culture medium produced under the above conditions and a dried product thereof as an active ingredient.
  • the present invention is Porostereum sp. (KCTC18837P) It relates to a cosmetic composition for improving skin condition comprising a mycelium culture as an active ingredient.
  • the Porostereum sp. (KCTC18837P) Mycelium culture can produce complex polysaccharides and beta-glucan as well as 5-hydroxy-6,7-dimethoxyphthalide, vitamins, aluminum, magnesium, calcium, and the like.
  • the 5-hydroxy-6,7-dimethoxyphthalide is known to have a therapeutic effect on atopic dermatitis.
  • the aluminum, magnesium and calcium are known to be effective in strengthening the skin barrier as inorganic elements, promoting skin regeneration, and cosmetically removing wastes such as sebum and acne and removing blemishes.
  • the vitamins are known to have various effects such as antioxidant, anti-inflammatory, and skin improvement, and are preferably vitamins B, C, D and E.
  • the Porostereum sp. (KCTC18837P) Mycelium culture is Porostereum sp. (KCTC18837P) It can be obtained by liquid culture of mycelium.
  • the culture may be a culture medium containing both the mycelium and the culture medium, or the mycelium and the culture medium separated from the culture medium, respectively.
  • it is a culture medium in which the mycelium and the culture medium are separated.
  • the culture is a liquid culture medium, a culture medium powder obtained by drying the culture medium, water, C1-C4 lower alcohol, acetone, n-hexane, dichloromethane and ethyl acetate
  • One or more solvents selected from the group consisting of It may be an extract extracted by adding it, but is not limited thereto.
  • the dried culture solution may be powdered using a conventional drying method of the culture solution. Preferably, it is freeze-dried.
  • the C1-C4 lower alcohol may be methanol, ethanol, propanol, isopropanol, butanol, or the like.
  • the extraction method of the extract may be selected from any one of hot water extraction method, cold extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method, compression method, and the like.
  • the desired extract may be further subjected to a conventional fractionation process, and may be purified using a conventional purification method.
  • the components of the liquid medium during the liquid culture may affect the growth of the strain and the production of active ingredients. Therefore, the present invention Porostereum sp. (KCTC18837P) It is necessary to establish the conditions for the components and content of the liquid medium optimized for strain culture.
  • the liquid medium is a carbon source as maltose, glucose, lactose, starch, dextrin, sucrose, fructose, galactose, mannose ( Mannose) and at least one selected from sugar (oligosaccharide) may be used.
  • it contains starch, sucrose and glucose, more preferably starch 0.2 to 2% (w/v), sucrose 0.2 to 2% (w/v) and glucose 0.2 to 2% based on the liquid medium. (w/v), most preferably starch 1% (w/v), sucrose 0.5% (w/v) and glucose 0.5% (w/v) based on the liquid medium.
  • the liquid medium is a nitrogen source, soy flour (soy flour), yeast extract (yeast extract), L- glutamic acid (L-glutamic acid), soy peptone (soy peptone), malt extract (malt extract), ammonium (ammonium), nitric acid
  • It may include one or more selected from the group consisting of calcium (cacium nitrate), potassium nitrate (potassium nitrate), sodium nitrate (sodium nitrate), and the like, but is not limited thereto.
  • it contains 0.5 to 3% (w/v) of soybean powder based on the liquid medium.
  • the liquid medium may include one or more selected from the group consisting of KH 2 PO 4 , ZnSO 4 , MgSO 4 , CuSO 4 , FeSO 4 and CaCl 2 as trace elements, but is not limited thereto.
  • it contains FeSO 4 , KH 2 PO 4 , MgSO 4 , and more preferably 0.001 to 0.3% (w/v) FeSO 4 , 0.001 to 0.3% (w/v) KH 2 PO based on the liquid medium. 4 , 0.001 to 0.3% (w/v) MgSO 4 .
  • it contains 0.001% (w/v) FeSO 4 , 0.001% (w/v) KH 2 PO 4 , and 0.003% (w/v) MgSO 4 , based on the liquid medium.
  • the liquid medium may contain vitamins.
  • vitamins B, C and E but not limited thereto. More preferably, 0.001 to 0.01% (w/v) of each vitamin is included based on the liquid medium.
  • 0.005% (w/v) of vitamin B is included based on the liquid medium. Most preferably, it contains 0.003% (w/v) of biotin (B7) and 0.002% (w/v) of pyridoxine (B6).
  • the liquid medium may have a pH of 4.5 to 6.5. Preferably it is pH 5.5.
  • the pH can affect the shape and activity of the protein by changing the charge of the amine group or carboxyl group of amino acids, which are units of enzyme proteins important for cell metabolism.
  • changes in pH in the external environment may affect the ionization of microbial nutrients, thereby affecting microbial intake.
  • the pH of the liquid medium is less than 5, aerobic and acidic algae or archaea easily propagate, and when the pH exceeds 7, alkaline actinomyces and mold fungi breed. It is not preferable because it is easy and the mycelium does not grow.
  • the carbon source is starch 1% (w/v), sucrose 0.5% (w/v), glucose 0.5% (w/v), and the nitrogen source is soybean powder 1.5% (w/v)
  • Minerals are 0.001%(w/v) FeSO 4 , 0.001%(w/v) KH 2 PO 4 , 0.003%(w/v) MgSO 4
  • Vitamins are Biotin (B7) 0.003%(w/v), Pyridoxine (B6) 0.002% (w/v), most preferably having a pH of 5.5.
  • the culture may use an agitated bioreactor, and the growth of the strain may be affected depending on the air supply amount, agitation speed, culture temperature and culture time, so that the Porostereum sp. It is important to establish optimal culture conditions for the strain.
  • the air supply amount may be 0.02 ⁇ 1vvm, preferably 0.1 ⁇ 0.5vvm, more preferably 0.2vvm.
  • the stirring speed may be 25-100 rpm, preferably 50-100 rpm, more preferably 60 rpm.
  • the incubation temperature may be 18 ⁇ 27 °C, most preferably 23 °C.
  • the temperature is less than 20 °C, the mycelium activity is slowed and the incubation period is prolonged, resulting in lower productivity and a sharp increase in production cost. It is lowered to, and if it exceeds 28 °C, the mycelium is undesirable to die.
  • the culture may have a pH of 4 to 7, preferably a pH of 5 to 6, and most preferably a pH of 5.5.
  • the incubation time may be 4 to 9 days, preferably 5 to 7 days, and more preferably 6 days. If the incubation time is less than 4 days, the amount of mycelium is small and the content of the active ingredient is low. If the culture time is more than 7 days, the content of the active ingredient no longer increases or decreases gradually, and if it exceeds 9 days, the content of the active ingredient is decreased. It is undesirable because it decreases rapidly.
  • the Porostereum sp. (KCTC18837P)
  • the mycelium and the culture medium should be separated for concentration.
  • a filtering process must be performed, and the filtrate obtained by filtration with a filter press may have a sugar content of 0.5 to 1 brix, a viscosity of 2 to 10 Cp, and a moisture content of 95 to 98%.
  • the filtrate may be subjected to a concentration process to reduce the volume.
  • Reverse osmosis (RO: reverse osmosis) can be used as a concentration method that prevents the reduction of active ingredients due to heat and has an excellent processing speed.
  • the concentrate concentrated 12 times by performing RO may have a sugar content of 5 to 10 brix, a viscosity of 30 to 100 Cp, and a moisture content of 10 to 20%, and can be used as a cosmetic material.
  • the concentrate can be powdered through freeze-drying, and powdered products can also be used as cosmetic materials.
  • the Porostereum sp. (KCTC18837P) Mycelium culture not only contains extracellular polysaccharides and beta-glucan, but also can produce 5-hydroxy-6,7-dimethoxyphthalide, vitamins, magnesium, calcium, and the like.
  • the Porostereum sp. (KCTC18837P) Mycelium culture can suppress cell damage caused by aging and oxidation of free radicals, improve wrinkles, increase skin moisture, and increase skin whitening.
  • Porostereum sp. (KCTC18837P) Mycelium culture can increase the survival rate of skin cells and promote skin cell regeneration.
  • the cosmetic composition is the Porostereum sp. (KCTC18837P) Adjuvants commonly used in the field of mycelium culture and cosmetics, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, blockers, pigments, odorants or dyes may contain.
  • Adjuvants commonly used in the field of mycelium culture and cosmetics such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, blockers, pigments, odorants or dyes may contain.
  • the Porostereum sp. (KCTC18837P) Mycelium culture may contain 0.001 to 50% by weight, preferably 0.001 to 30% by weight, based on the total weight of the cosmetic composition.
  • the amount of the adjuvant is an amount commonly used in the art, for example, 0.001 to 50% by weight, preferably 0.001 to 70% by weight, based on the total weight of the cosmetic composition.
  • the auxiliary agent and its ratio may be selected so as not to adversely affect the desirable properties of the cosmetic composition according to the present invention.
  • the cosmetic composition is Porostereum sp. (KCTC18837P)
  • a known compound or plant extract known to have an effect on improving skin condition may be further included.
  • compounds or plant extracts known to have an effect on improving the skin condition include mercaptosuccinic acid, mercaptodextran, teprenone, dihydroxy-isoquinoline, indomethacin, vitamin K, lemon extract, cucumber extract, mulberry extract , licorice extract, rosemary extract, acerola extract, ginkgo extract, carob extract, geranium extract, and the like, but are not limited thereto.
  • the cosmetic composition may be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil , powder foundation, emulsion foundation, wax foundation, and spray.
  • a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil , powder foundation, emulsion foundation, wax foundation, and spray Preferably, it may be prepared in the form of a softening lotion, a nourishing lotion, a massage cream, a nourishing cream, a pack, a gel, a skin adhesion type, etc., but is not limited thereto.
  • the cosmetic may consist of one or more formulations selected from the group consisting of lotions, skin softeners, skin toners, astringents, creams, foundations, essences, packs, soaps, cleansing foams, and body cleansers, but is not limited thereto.
  • the present invention provides a cosmetic comprising the cosmetic composition.
  • the present invention is Porostereum sp. (KCTC18837P) It relates to a cosmetic composition for skin improvement comprising a mycelium culture as an active ingredient, and an unused natural resource, Porostereum sp. (KCTC18837P) A culture method for effective industrialization of mycelium and a method for preparing a composition were established. Also, Porostereum sp.
  • KCTC18837P It was confirmed that the mycelium culture contains a lot of active ingredients such as inorganic ingredients, amino acids, and vitamins, in addition to extracellular polysaccharide, beta-glucan, which is effective for skin improvement and beauty, and these cultures inhibit skin antioxidant It was confirmed that the effect of improving wrinkles, enhancing skin moisture, promoting skin whitening, enhancing skin cell survival rate, and promoting skin cell regeneration were excellent.
  • the mycelium culture is expected to be usefully used in the development of cosmetics for skin improvement.
  • KCTC18837P is a copy of the depository certificate of the strain.
  • FIG. 2 is Porostereum sp. (KCTC18837P) is a phylogenetic diagram showing the taxonomic position of the strain.
  • the present inventors are rich in beta-glucan, extracellular polysaccharide, vitamins and minerals in the process of conducting research on industrially useful mushrooms and fungi, Porostereum sp. (KCTC18837P) strain was identified and optimal culture conditions were established, and it was deposited at the National Institute of Biotechnology and Biotechnology (KCTC) (FIG. 1).
  • the active ingredient produced by the culture method according to the present invention is Porostereum sp.
  • KCTC18837P is an extracellular polysaccharide and beta-glucan produced by Porostereum sp.
  • KCTC18837P mycelium culture or the Porostereum sp.
  • the dried product and concentrate of the mycelium culture can be widely used as a cosmetic material to protect and improve skin condition.
  • the production of the active ingredient for improving skin condition of the present invention can shorten the culture period and increase the content of the active ingredient by optimizing the composition, oxygen supply, temperature, and stirring speed of the culture medium for Porostereum sp. (KCTC18837P) mycelia.
  • the present invention relates to a mycelium culture of Porostereum sp. (KCTC18837P) comprising extracellular polysaccharide and beta-glucan and the extracellular polysaccharide and beta-glucan produced by Porostereum sp. (KCTC18837P) and the Porostereum sp. (KCTC18837P) It provides a method for preparing a mycelium culture or a dried product and an extract thereof.
  • composition of the present invention comprises the extracellular polysaccharide and beta-glucan produced by Porostereum sp. (KCTC18837P) and Porostereum sp. (KCTC18837P) containing the extracellular polysaccharide and beta-glucan Mycelium culture or dried product and extract thereof contains as an active ingredient.
  • Example 1 Establishment of optimal culture conditions of Porostereum sp. (KCTC18837P) strain
  • Example 1.1 Composition of nutrient medium for culture of Porostereum sp. (KCTC18837P) strain
  • the biological incubator used in this experiment was a 100l biological incubator installed in CL Bio (Yeongdong-gun, Chungcheongbuk-do). For each experiment, 30 liters of liquid medium containing nutrient medium was sterilized at 123 ° C and 1.25 bar for 20 minutes under high pressure, cooled to 23 ° C, inoculated with 600 ml of Porostereum sp. (KCTC18837P) strain, the temperature was 23 ⁇ 1 ° C, and air supply Silver was cultured for 5 days while maintaining 0.2 vvm, 20% dissolved oxygen, and pH 5.5.
  • CL Bio Yeongdong-gun, Chungcheongbuk-do
  • KCTC18837P Porostereum sp.
  • soybean powder, soy peptone, yeast extract, ammonium, calcium nitrate, and sodium nitrate were added to each 1% (w/v).
  • 600ml of Porostereum sp. (KCTC18837P) strain was inoculated, the temperature was 23 ⁇ 1°C, the air supply was 0.2 vvm, the dissolved oxygen was 20%, and the pH was 5.5. Table 2 shows.
  • culture water 30 liters carbon source Starch 1%, sucrose 0.5%, glucose 0.5% (W/V) nitrogen source composition ratio 1% (W/V) Soybean Powder Soipeeptone yeast extract ammonium calcium nitrate sodium nitrate mycelium weight (g/100ml) 46.74g 37.45g 44.91g 15.65g 18.43g 17.25g
  • Example 11.3 Mineral crystals for culture of Porostereum sp. (KCTC18837P)
  • KCTC18837P The effect of 6 types of minerals most commonly used in culturing microorganisms on the growth of Porostereum sp. (KCTC18837P) mycelia was tested. Starch 1%, sucrose 0.5%, glucose 0.5% (W/V), soybean powder 1% (W/V) based on the liquid medium added, after, KH 2 PO 4 , MgSO 4 , CaCl 2 , FeSO 4 , ZnSO 4 and CuSO 4 were added to be 0.005% (w/v), respectively, and 600 ml of Porostereum sp. (KCTC18837P) strain was inoculated. The temperature was 23 ⁇ 1°C, the air supply was 0.2 vvm, and the dissolved oxygen was 20%. , cultured for 5 days while maintaining pH 5.5, the mycelium was harvested and the weight was measured and shown in Table 3.
  • Table 4 shows the results of experiments on the effects of the six most commonly used vitamins in culturing microorganisms on the growth of Porostereum sp. (KCTC18837P) mycelium. Other experimental conditions were the same as in Examples 1.1.1 to 4.
  • the increase in mycelium was the largest in vitamin B6 or vitamin B7 conditions among vitamins A, vitamin B1, vitamin B2, vitamin B6, vitamin B7, and vitamin C.
  • a preferred embodiment for liquid culture of Porostereum sp. (KCTC18837P) mycelium in step (a) is 1% starch (w/v), 0.5% sucrose (w/v), 0.5% glucose as a carbon source (w/v), nitrogen source is soybean powder 1% (w/v), minerals are MgSO 4 0.003% (w/v), KH 2 PO 4 0.001% (w/v), FeSO 4 0.001% (w/v) ), vitamins are biotin (B7) 0.003% (w / v), pyridoxine (B6) 0.002% (w / v) weight %.
  • KCTC18837P KCTC18837P strain into the culture medium containing biotin (B7) 0.003 % (w/v) and pyridoxine (B6) 0.002 % (w/v) for vitamins, and maintain the air volume at 0.2 vvm and incubated for 5 days at 23°C.
  • Porostereum sp. (KCTC18837P) mycelium weight increased the most at an air supply of 0.1 to 0.5 vvm, and then Porostereum sp. (KCTC18837P) culture was performed at a stirring speed of 60 to 70 rpm.
  • the increase in the mycelium weight of Porostereum sp. was the largest at 22 ⁇ 24°C in culture temperature. Most preferably, it was 23°C.
  • the pH is 6 at this time.
  • the amount of mycelium was measured after culturing the composition with sodium hydroxide and glacial acetic acid so that each pH was 4-7. The results are shown in Table 8 below.
  • the optimal pH of the mycelium of Porostereum sp. was 5-6, and most preferably pH 5.5.
  • the optimum pH for liquid culture of general mushroom mycelium is known to be 4 to 6, but in the case of Porostereum sp. (KCTC18837P) mycelium, at pH 4, the weight of Porostereum sp. wrote Therefore, by establishing the optimal culture pH of Porostereum sp. (KCTC18837P), rapid mass culture of Porostereum sp. (KCTC18837P) became possible.
  • Example 2.5 Determination of culture period of Porostereum sp. (KCTC18837P) strain
  • the incubation period is preferably 5-7 days, and most preferably 6 days.
  • the Porostereum sp. (KCTC18837P) mycelium culture was completed, filtered, concentrated, and dried.
  • the culture medium itself has a large volume and is easy to be contaminated with various germs, so it has many problems during commercialization and distribution. Therefore, in order to reduce the volume, it is necessary to go through a concentration or drying process, and when high temperature heat is applied in this process, the protein and vitamin components contained in the culture medium are reduced, so a non-heating method must be used.
  • a non-heating concentration method with high efficiency there is a method using reverse osmosis (RO).
  • RO reverse osmosis
  • the mycelium in the culture medium and the culture medium must be separated, and for this purpose, a filter press process must be performed.
  • the standard of the filter cloth installed on the filter press is 0.5 ⁇ 2.0 ⁇ m pore size, and in the case of Porostereum sp. (KCTC18837P) mycelium culture, 1.0 ⁇ m pore size is preferable.
  • the filtrate may have a sugar content of 0.5 to 1 brix, a viscosity of 2 to 10 Cp, and a moisture content of 95 to 98%.
  • the 12-fold concentrate may have a sugar content of 5 to 10 brix, a viscosity of 30 to 100 Cp, and a moisture content of 10 to 20%.
  • the concentrate may be powdered through freeze-drying.
  • the freeze-dried powder is usually obtained in an amount of 1.2 to 1.8% of the weight of the culture medium. Preferably, it is 1.5% by weight of the weight of the culture medium, and the moisture content may be 0.01 to 0.03%.
  • KCTC18837P Porostereum sp. (KCTC18837P) mycelium culture or the dried culture material obtained above as a cosmetic material, after extraction with a solvent, extracellular polysaccharide or beta-glucan, which is an active ingredient of the culture according to the present invention, is isolated And it provides a manufacturing method.
  • the dry powder was stirred by adding 100 ml of distilled water per 2 g of powder, followed by centrifugation (10,000 rpm, 20 minutes), and then ethanol corresponding to three times the amount was added to the supernatant and refrigerated conditions (2-8 ° C) After allowing to stand for 16 hours, centrifugation (10,000 rpm, 20 minutes) of the above-mentioned sediment is taken and freeze-dried to extract extracellular polysaccharide (EPS).
  • EPS extracellular polysaccharide
  • the dry powder was suspended by adding 50 ml of distilled water per 10 g of the powder, and then the pH was adjusted to 10.0 using 20% sodium carbonate at room temperature, and then left for about 10 hours to sufficiently soften the powder sample.
  • ⁇ -amylase which is stable at high temperature, is added, stirred in a constant temperature water bath at 60° C. for 2 hours, subjected to enzyme treatment, and then cooled to room temperature.
  • This solution was adjusted to pH 5 and then stirred with amyloglucosidase in a constant temperature water bath at 60° C. for 2 hours. Then, after adjusting the pH to 4.5 in order to inactivate the enzyme, it is stirred in a constant temperature water bath at 95° C. for 30 minutes and then cooled.
  • the cooled extract is centrifuged (30 min ⁇ 8000 rpm) to take only the supernatant, and then concentrated under reduced pressure at a temperature of 40°C. A 4-fold amount of ethanol was added to this concentrate, left for about 4 hours, and the precipitate obtained by centrifugation was again dissolved in water, the pH was adjusted to 4.5, and the enzyme was re-treated with amyloglucosidase at 58°C for 2 hours. .
  • the enzyme After reprocessing, the enzyme is inactivated at 95°C for 30 minutes, cooled, centrifuged, and the supernatant is taken, and then reprecipitated with ethanol to extract crude beta-glucan ( ⁇ -glucan).
  • the extract of the mycelium culture of the present invention is not limited to the above method, but according to a conventional method known in the art, hot water extraction, room temperature extraction, warming extraction, ultrasonic extraction, supercritical extraction, etc. It can be prepared by methods such as extraction.
  • the cultured Porostereum sp. (KCTC18837P) mycelium cultured according to the preferred embodiment not only contains complex polysaccharides and beta-glucan, but also contains 5-hydroxy-6,7-dimethoxyphthalide, vitamins, magnesium, Calcium and the like can be produced. Component analysis was conducted at the Korea Functional Food Research Institute, and the results are shown in Table 10.
  • beta glucan 34.23 (%/g) extracellular polysaccharide 43.11 (%/g) 5-hydroxy-6,7-dimethoxyphthalide 29.51 ( ⁇ g/g) vitamin 1.34 (%/g) magnesium 0.52 (%/g) calcium 0.74 (%/g)
  • Porostereum sp. (KCTC18837P) mycelium culture of the present invention contains a significant amount of active ingredients reported to be effective for skin care.
  • Example 5 Skin protection efficacy test
  • the Porostereum sp. (KCTC18837P) mycelium culture solution (hereinafter referred to as Po-D) was tested for efficacy in the Department of Bioindustry, Department of Life and Environment, Daegu University and the Beauty Research Institute, Gyeongbuk Health University.
  • Example 5.1.1 Measurement of DPPH (1,1-diphenyl-2-picryhydrazyl) free radical scavenging activity
  • the DPPH radical scavenging activity method was used. 2 ⁇ l of Po-D by concentration (0 mg/mL, 0.5 mg/mL, 1 mg/mL, 5 mg/mL) was put in a 96 well plate, and 198 ⁇ l of 100 ⁇ M DPP solution was added and mixed well. After standing at room temperature for 30 minutes, the absorbance at 540 nm was measured to calculate the extinction rate compared to the control, and the results are shown in Table 11.
  • the DPPH free radical scavenging activity gradually increased as the amount of Po-D added increased, and when the Po-D concentration was 5 mg/mL, the DPPH radical scavenging rate of about 79% was confirmed to have an excellent antioxidant effect.
  • Example 5.1.2 Measurement of free radical scavenging activity of ABTS(2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate)
  • Antioxidant activity was measured using another method of measuring antioxidant activity, ABTS free radical scavenging activity. After mixing 2.45 mM potassium sulfate in 7.4 mM ABTS solution, it was reacted for about 24 hours, and it was diluted with phosphate buffered saline so that the absorbance at 732 nm was 0.700 ⁇ 0.030. Po-D was added at each concentration (0 mg/mL, 0.5 mg/mL, 1 mg/mL, 5 mg/mL) to 990 ⁇ l of the diluted solution, reacted for 10 minutes, and absorbance at 732 nm was measured for control. (Things not added) The elimination ratio was calculated, and the results are shown in Table 12 below.
  • the ABTS free radical scavenging activity gradually increased as the amount of Po-D was increased, and when the Po-D concentration was 1 mg/mL, the ABTS radical scavenging rate was about 78%, confirming the excellent antioxidant effect.
  • the collagen synthesis promoting effect was measured using human fibroblasts.
  • Human fibroblasts were dispensed in a 24 well plate at a concentration of 1 ⁇ 10 5 cells/well, treated with Po-D by concentration (0.5 mg/ml, 1 mg/ml, 5 mg/ml), and then cultured for 48 hours.
  • ⁇ -MSH ⁇ -melanocyte stimulating hormone
  • a cell suspension was prepared, 20 ⁇ l of cell suspension and 10 ⁇ l of antibody-POD conjugate solution were dispensed in a 96 well plate, and incubated for 3 hours.
  • TMBZ tetramethoxybenzil
  • Keratinocytes were dispensed in a 24 well plate at a concentration of 1 ⁇ 10 4 cells/well and incubated for 24 hours, treated with Po-D at different concentrations (30 ⁇ g/ml, 100 ⁇ g/ml, 300 ⁇ g/ml), and then After culturing for 24 hours, the cells were lysed by treatment with a protein extraction solution, centrifuged to recover the supernatant, and the protein concentration was measured. After diluting with the same amount of protein, NuPAGE LDS sample buffer was mixed and boiled at 100°C for 5 minutes. Then, it was analyzed by western blot, and the results are shown in Table 14.
  • B16 melanoma cells were treated at a concentration of each concentration (0.5 mg/ml, 1 mg/ml, 5 mg/ml,) in a 96-well plate at a concentration of 1 ⁇ 10 5 cells/well, and then cultured for 48 hours. and treated with 50 nM of ⁇ -MSH ( ⁇ -melanocyte stimulating hormone) and incubated for 48 hours. Cells were recovered with Trypsin-EDTA and then centrifuged to remove the supernatant. Add protein extraction buffer and lyse cells at low temperature for 40 minutes. The supernatant is recovered by centrifugation, and then reacted with a protein assay kit for 15 minutes. The absorbance at 540 nm was compared with a negative control group (not added) and a positive control group (treated with 50 ⁇ g/ml of arbutin, a tyrosinase inhibitor), and the results are shown in Table 15.
  • B16 melanoma cells were treated with Po-D at different concentrations to conduct cell culture experiments, and the results are shown in Table 16.
  • Po-D dose (mg/ml) B16 melanoma cells (% of control) 0 100 0.5 115 2.5 117 5 119 10 121
  • the survival rate was increased when Po-D was added, and it was confirmed that Po-D had an anti-aging effect on skin cells as it increased by about 21% compared to that without addition when 10 ⁇ g/ml treatment was performed.
  • MTS [3-(4,5-dimethyl-thiazol-2-yl)-5-carboxy-methoxyphenyl-2 -(4-sulfophenyl)-2H-tetrazolium] was treated for 1 hour, and then absorbance was measured at 490 nm with a microplate reader (Perkin Elmer 1420 Multilabel Counter VICTORTM, USA). Cell viability was expressed as the absorbance of the sample treated group compared to the control group. The results are shown in Table 17.
  • Po-D dose ( ⁇ g/ml) Cell proliferation rate (%) control 100.0 ⁇ 2.3 50 ⁇ g/ml 103.2 ⁇ 2.5 100 ⁇ g/ml 114.1 ⁇ 1.3 200 ⁇ g/ml 127.7 ⁇ 1.8 300 ⁇ g/ml 131.2 ⁇ 2.2

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Abstract

La présente invention concerne une composition cosmétique comprenant une culture mycélienne de Porostereum sp. (KCTC18837P) en tant que principe actif pour améliorer un état de la peau. La culture mycélienne de Porostereum sp. (KCTC18837P) s'est avérée générer récemment un ingrédient 5-hydroxy-6,7-diméthoxyphtalide en plus des polysaccharides extracellulaires et du bêta-glucane efficaces pour améliorer l'état et la beauté de la peau et contenir des composants efficaces tels que des composants inorganiques, des acides aminés, des vitamines, etc., et identifiés comme ayant d'excellents effets d'antioxydation, de réduction des rides, d'hydratation de la peau, de blanchiment de la peau, d'augmentation de la viabilité des cellules de la peau et de régénération des cellules de la peau. Ainsi, la culture mycélienne de Porostereum sp. (KCTC18837P) devrait trouver des applications avantageuses dans le développement d'un produit cosmétique destiné à améliorer un état de la peau.
PCT/KR2021/011159 2020-08-20 2021-08-20 Culture mycélienne d'une nouvelle souche de porostereum sp. (kctc18837p) et composition cosmétique la comprenant pour améliorer l'état de la peau Ceased WO2022039572A1 (fr)

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