KR102327435B1 - Culture broth of Porostereum sp.(KCTC18837P) and Cosmetic composition comprising the same for improving skin condition - Google Patents

Abstract

The present invention relates to a cosmetic composition comprising a mycelial culture of Porostereum sp. (KCTC18837P) as an active ingredient for improving a skin condition. The mycelial culture of Porostereum sp. (KCTC18837P) was found to newly generate a 5-hydroxy-6,7-dimethoxyphthalide ingredient in addition to extracellular polysaccharides and beta-glucan effective for improving a skin condition and skin beauty and to contain effective components such as inorganic components, amino acids, vitamins, and so on, and identified to have excellent effects of antioxidation, wrinkle reduction, skin moisturization, skin whitening, an increase of skin cell viability, and skin cell regeneration. Thus, the mycelial culture of Porostereum sp. (KCTC18837P) is expected to find advantageous applications in developing a cosmetic product for improving a skin condition.

Description

The new strain Porostereum sp. (KCTC18837P) mycelium culture and a cosmetic composition for improving skin condition comprising the same {Culture broth of Porostereum sp. (KCTC18837P) and Cosmetic composition comprising the same for improving skin condition}

The present invention relates to a Porostereum sp. (KCTC18837P) mycelium culture and a cosmetic composition for improving skin condition comprising the same.

As interest in skin beauty increases, various functional cosmetic compositions based on natural materials that replace existing chemical materials have been disclosed.

Among the natural resources discovered so far, the available resources with widely known utilization have decreased in scarcity due to excessive competition.

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. In addition, as a significant part of the ingredients and physiological activity mechanisms of mushrooms are scientifically proven, 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. The mycelium contains various useful substances like the fruiting body. Induction of the fruiting body takes a long time to cultivate and technology development takes a long time, whereas the liquid culture method of the mycelium uses a hygienic and automated facility to stably and mass-produce a large amount in a short time. It has the advantage that it can be cultivated.

Therefore, most mushrooms recently made it possible to culture mycelium containing various physiologically active substances through bioconversion using nutrients contained in liquid medium. In addition, since mushroom mycelium has different culture characteristics depending on the medium composition, culture conditions, or mushroom mycelium type, suitable culture conditions for each mushroom must be found in order to improve the liquid culture efficiency of the mycelium. Depending on the nutrient composition and culture conditions, various metabolites having anticancer and antioxidant activities can be obtained (Lee Wi-Young, et al., 2008).

The genus Porostereum belongs to the genus Basidiomycota, Agaricomycetes, Polyporales, Phanerochaetaceae, and Porostereum. . (National Arboretum National Standard Mushroom List)

Twenty-one species of the genus Porostereum have been registered in the International Database (Index Fungroum) so far, and Porsterum crassum and Porostereum spadiceum belong to this genus. Muhammad Hamayun et al. confirmed that Porostereum spadiceum AGH786 could be used as a fungal biofertilizer in soybeans under salt stress (Muhammad H. et al., 2017). However, industrialization research on the genus Orifolia genus strain is still incomplete.

Regarding the genus Porostereum, 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.

Korean Patent No. 1401997, composition for removing chromaticity from dyeing wastewater and method for removing chromaticity from dyeing wastewater, registered on May 26, 2014

Wi-Young Lee, Jin-Kwon Ahn, Gang-Hyeon Ga, Eung-Jun Park, Liquid culture of mushroom mycelium and production of useful substances, 2008, Publication No.: 11-1400377-000243-01, National Academy of Forest Sciences. Muhammad Hamayun1, Anwar Hussain, Sumera A. Khan, Ho-Youn Kim, Abdul L. Khan, Muhammad Waqas, Muhammad Irshad, Amjad Iqbal, Gauhar Rehman, Samin Jan and In-Jung Lee, Gibberellins Producing Endophytic Fungus AGH786 Rescuberellins Producing Endophytic Fungus Poroster spa of Salt Affected Soybean, Frontiers in Microbiology, April 2017, (8); article 686

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. In addition, 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) Including, the new strain Porostereum sp, characterized in that it was produced by culturing for 5-7 days at a pH of 5-6 in a liquid medium with an air supply of 0.02-1vvm, agitation speed of 25-100rpm, and a culture temperature of 18-27℃ (KCTC18837P) or to provide a cosmetic composition for improving skin condition comprising a mycelium culture thereof as an active ingredient.

In order to achieve the above object, 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. In addition, 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 of 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), and 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) It may be a liquid medium having a pH of 5 to 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℃.

In addition, the present invention provides a cosmetic composition for improving skin condition containing a culture solution produced under the above conditions and a dried product thereof as an active ingredient.

Hereinafter, the present invention will be described in detail. The present invention is Poostereum sp. (KCTC18837P) It relates to a cosmetic composition for improving skin condition comprising a mycelium culture 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 have an effect of 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 was obtained from Porostereum sp. (KCTC18837P) Can be obtained by liquid culture of mycelium.

The culture may be a culture medium containing both the mycelium and the culture medium, or may be a culture medium separated from the mycelium and the culture medium. Preferably, 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 dried by drying the culture medium, water, C1-C4 lower alcohol, acetone, n-hexane, one or more solvents selected from the group consisting of dichloromethane and ethyl acetate 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, buntanol, 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. In addition, 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 (Galactose), mannose ( Mannose) and at least any one or more selected from sugar (Oligosaccharide) may be used. Preferably, 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), and 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. Preferably, 0.5 to 3% (w/v) of soybean powder is included based on the liquid medium. Most preferably, it contains 1% (w/v) 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.} Preferably, 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 . Most preferably, 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. Preferably, vitamins B, C and E, but not limited thereto. More preferably, each vitamin is contained in an amount of 0.001 to 0.01% (w/v) based on the liquid medium. Preferably, it contains 0.005% (w/v) of vitamin B 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. In addition, changes in pH in the external environment may affect the ionization of microbial nutrients, thereby affecting microbial intake. In addition, when the pH of the liquid medium is less than 5, aerobic and acidic algae or archaea are easy to propagate, and when the pH is more than 7, alkaline actinomyces and mold fungi breed. It is not preferable because it is easy and the mycelium does not grow.

In the liquid medium, based on the liquid medium, 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 culture temperature may be 18 ~ 27 ℃, most preferably 23 ℃. When the temperature is less than 20 ℃, the activity of the mycelium 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 ℃, 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 gradually decreases, 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) For the mycelium culture, the mycelium and the culture medium should be separated for concentration. For this, 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 in order 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.

In addition, the 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.

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. However, in any case, the adjuvant 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) In addition to the strain culture, it may further include a known compound or plant extract known to have an effect of improving skin condition. Examples of 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 (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. Preferably, it may be prepared in the form of a softening lotion, a nutrient lotion, a massage cream, a nutrient 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 Poostereum 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, vitamins, in addition to extracellular polysaccharides, beta-glucan, which are effective for skin improvement and beauty, and these cultures inhibit skin antioxidants 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 was excellent.

Through this, the Porostereum sp of the present invention. (KCTC18837P) Mycelium culture is expected to be usefully used in the development of cosmetics for skin improvement.

1 shows Porostereum sp. (KCTC18837P) is a copy of the depository certificate of the strain.
Figure 2 is a 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 was deposited at the National Institute of Biotechnology and Biotechnology Biological Resources Center (KCTC) (FIG. 1).

Hereinafter, an embodiment of the present invention will be described in detail. The present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided so that this disclosure will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art. In addition, the terms used in this specification are terms used to properly express the preferred embodiment of the present invention, which may vary according to the intention of the user or operator, or customs in the field to which the present invention belongs.

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. (KCTC18837P) The dried product and concentrate of the mycelium culture can be widely used as a cosmetic material to protect and improve the 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) containing 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.

The 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 It contains active ingredients.

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, Chungbuk). For each experiment, 30 liters of liquid medium containing nutrient medium was autoclaved at 123 ° C and 1.25 bar for 20 minutes, 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.

Example 1.1.1 Porostereum sp. (KCTC18837P) Determination of carbon source for culturing

The effects of the six most commonly used carbon sources in culturing this microorganism on the growth of Porostereum sp. (KCTC18837P) mycelia were tested. Based on the liquid medium, maltose, glucose, lactose, starch, sucrose, and fructose were added to each 2% (w/v). . Thereafter, 600ml of Porostereum sp. (KCTC18837P) strain was inoculated, and the temperature was 23±1℃, the air supply was 0.2 vvm, the dissolved oxygen was 20%, and the pH was 5.5. Table 1 shows.

As shown in Table 1 below, when starch, glucose, and sucrose were used as carbon sources, the average weight of mycelium increased by 18.6% more than when fructose, lactose, and maltose were used as carbon sources. Therefore, starch, glucose, or sucrose was used alone or in combination as a carbon source of Porostereum sp. (KCTC18837P), and 1% (w/v) starch based on the liquid medium in consideration of the mycelium yield under the above alone or mixed conditions. , sucrose 0.5% (w/v) and glucose 0.5% (w/v) were used in subsequent experiments.

culture water 30 liters composition ratio 2% (W/V) carbon source sucrose glucose starch fructose lactose maltose mycelium weight
(g/100ml) 20.31g 20.36g 21.30g 18.62g 16.23g 17.41g

Example 1.1.2 Porostereum sp. (KCTC18837P) Determination of nitrogen source for culturing

The effects of the six most frequently used nitrogen sources in culturing microorganisms on the growth of Porostereum sp. (KCTC18837P) mycelia were tested. Based on the liquid medium, soybean powder, bovine peptone, yeast extract, ammonium, calcium nitrate, and sodium nitrate were added to each 1% (w/v). Thereafter, 600ml of Porostereum sp. (KCTC18837P) strain was inoculated, and the temperature was 23±1℃, the air supply was 0.2 vvm, the dissolved oxygen was 20%, and the pH was 5.5. Table 2 shows.

As shown in Table 2 below, when soybean powder was used as a nitrogen source, the mycelium weight of Porostereum sp. (KCTC18837P) increased the most, and about 3 times more mycelium was obtained than when ammonium was used.

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 1.1.3 Mineral crystals for culture of Porostereum sp. (KCTC18837P)

The effects of 6 types of minerals most commonly used in culturing microorganisms on the growth of Porostereum sp. (KCTC18837P) mycelia were tested. Based on a liquid medium containing 1% starch, 0.5% sucrose, 0.5% glucose (W/V), and 1% (W/V) soybean powder, 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℃, 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.

As shown in Table 3 below, KH ₂ PO ₄ , MgSO ₄ , CaCl ₂ , FeSO ₄ , ZnSO ₄ and CuSO ₄ Among Porostereum sp. (KCTC18837P), the increase in mycelium weight was greatest in KH ₂ PO ₄ , MgSO ₄ , and FeSO ₄ conditions, followed by MgSO ₄ 0.003, KH ₂ PO ₄ 0.001%, FeSO ₄ 0.001% (W/V). It was used by adding it to the medium.

culture water 30 liters carbon source Starch 1%, Sucrose 0.5%, Glucose 0.5% (W/V) nitrogen source Soybean powder 1% (W/V) mineral
composition ratio 0.005% (W/V) KH ₂ PO ₄ MgSO ₄ CaCl ₂ FeSO ₄ ZnSO ₄ CuSO ₄ mycelium weight
(g/100ml) 47.65g 48.44g 45.32g 47.11g 46.33g 46.07g

Example 1.1.4 Porostereum sp. (KCTC18837P) Vitamin crystals for culture

Table 5 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.

As shown in Table 5 below, the mycelium increase was the largest in the vitamin B6 or vitamin B7 condition among vitamins A, vitamin B1, vitamin B2, vitamin B6, vitamin B7, and vitamin C.

culture water 30 liters carbon source 2% (W/V) nitrogen source 0.03% (W/V) mineral MgSO ₄ 0.003, KH ₂ PO ₄ 0.001%, FeSO ₄ 0.001% (W/V) vitamin
composition ratio 0.005% (W/V) vitamin A vitamin B1 vitamin B2 vitamin B6 vitamin B7 vitamin C mycelium weight
(g/100ml) 48.44g 48.74g 48.52g 49.03g 49.11g 47.88g

As a result of the experiment, 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 %.

Example 2. Setting of environmental conditions for culture of Porostereum sp. (KCTC18837P) strain

In order to confirm the optimal culture conditions of the Porostereum sp. (KCTC18837P) mycelium of the present invention, starch 1% (w / v), sucrose 0.5% (w / v), glucose 0.5% (w / v), soybean powder, 1% (w / v), MgSO 4 0.003% (w / v), KH2PO 4 0.001% (w / v), FeSO 4 0.001% (w / v ), inoculate 600ml of Porostereum sp. (KCTC18837P) strain into a 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.

Example 2.1. Determination of the agitation rate of the incubator

In mushroom mycelium culture, the rotational speed of the stirrer during culturing of mushroom mycelium is a very important factor in terms of the metabolic activity of the mycelium. In order to investigate the effect of the rotational speed of the stirrer on the mycelium growth, the results of measuring the mycelium amount after culturing while maintaining the agitation speed of the incubator differently are shown in Table 5.

Stirring speed (rpm) Mycelium weight (g/ℓ) 25 5.7 50 7.9 60 8.8 70 8.5 80 7.4 100 5.9

As a result of the experiment, the weight of Porostereum sp. (KCTC18837P) mycelium increased the most at a stirring speed of 60-70 rpm. Accordingly, the stirring speed of Porostereum sp. (KCTC18837P) culture was set to 60-70 rpm.

Example 2.2. Determination of the air supply of the incubator

In order to investigate the effect of the air supply on the growth of Porostereum sp. (KCTC18837P) mycelium, the results of measuring the amount of mycelium after culturing while maintaining the air supply in the incubator differently are shown in Table 6 below.

Air supply (vvm) Mycelium weight (g/ℓ) 0.05 8.5 0.1 8.7 0.2 8.8 0.5 8.6 One 8.4 2 7.9

As a result of the experiment, 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.

Example 2.3. Determination of the incubation temperature of the incubator

In order to investigate the effect of the culture temperature on the growth of mycelium, the results of measuring the mycelium amount after culturing while maintaining the temperature of the incubator differently are shown in Table 7 below.

Temperature (℃) Mycelium weight (g/ℓ) 18 6.5 19 6.8 20 7.6 22 8.5 23 8.8 24 8.7 25 8.4 26 6.2 27 3.3 28 1.2

As a result of the experiment, the increase in the mycelium weight of Porostereum sp. (KCTC18837P) was the greatest at 22~24℃ in culture temperature. Most preferably, it was 23°C.

Example 2.4. Determination of culture pH in the incubator

When the medium is prepared as in the preferred embodiment, the pH is 6 at this time. In order to investigate the effect of pH on mycelial growth, the amount of mycelium was measured after culturing with sodium hydroxide and glacial acetic acid so that each pH was 4-7. The results are shown in Table 8 below.

pH Mycelium weight (g/ℓ) 4 3.2 4.5 6.1 5 8.6 5.5 8.8 6 8.5 6.5 5.3 7 2.8

As a result of the experiment, the optimal pH of the mycelium of Porostereum sp. (KCTC18837P) 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

In order to investigate the effect of the incubation period on the mycelium growth, After setting the medium and environmental conditions as in the preferred embodiment, and adjusting the pH to 5.5, the amount of mycelium according to the incubation period was measured. The results are shown in Table 9 below.

incubation period Mycelium weight (g/ℓ) 3 3.7 4 6.1 5 8.6 6 8.8 7 8.7 8 7.9 9 7.2

As a result of the experiment, as shown in Table 9, it was confirmed that the mycelium amount of Porostereum sp. (KCTC18837P) increased rapidly from the 4th day, reached the peak on the 6th day, and then gradually decreased from the 7th day. Therefore, the incubation period is preferably 5-7 days, and most preferably 6 days.

Example 3. Preparation of concentrates and lyophilisates and extraction of active ingredients

The Porostereum sp. (KCTC18837P) mycelium culture was completed, filtered, concentrated, and dried.

Example 3.1 filter press

The culture medium itself is too bulky and easily 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. As a non-heating concentration method with high efficiency, there is a method using reverse osmosis (RO). In order to use the reverse osmosis machine, 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 mounted on the filter press is 0.5 ~ 2.0 ㎛ pore size, and in the case of Porostereum sp. (KCTC18837P) mycelium culture, 1.0 ㎛ 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%.

Example 3.2 Reverse Osmosis (RO)

Reverse osmosis can remove a significant portion of the pure water component contained in the culture solution, and the culture solution can be concentrated up to 12 times without applying heat. Before freeze-drying, the volume of the object should be reduced as much as possible. This is because a large-capacity freeze dryer is uncommon, the usage fee is too high, and it takes a long time to dry. 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%.

Example 3.3 Lyophilization

The concentrate may be powdered through freeze-drying. The lyophilized 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%.

Example 3.4 Extraction of active ingredients

When it is difficult to directly use the 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 a method for preparing it.

The dry powder was stirred by adding 100 ml of distilled water per 2 g of the powder, centrifuged (10,000 rpm, 20 minutes), and then ethanol corresponding to 3 times the amount was added to the supernatant and refrigerated conditions (2-8 ° C). After standing for 16 hours, centrifugation (10,000 rpm, 20 minutes) of the above-mentioned material, the precipitate is taken and freeze-dried to extract extracellular polysaccharide (EPS).

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. To this softened sample solution, α-amylase, which is stable at high temperature, is added, stirred in a constant temperature water bath at 60° C. for 2 hours, enzymatically treated, 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), only the supernatant is taken, and then concentrated under reduced pressure at a temperature of 40°C. After adding 4 times the amount of ethanol to this concentrate and leaving it for about 4 hours, the precipitate obtained by centrifugation is again dissolved in water, the pH is adjusted to 4.5, and the enzyme is re-treated with amyloglucosidase at 58°C for 2 hours. .

After reprocessing, the enzyme is inactivated at 95° C. for 30 minutes, then cooled and centrifuged to take the supernatant and reprecipitate with ethanol to extract crude beta-glucan (β-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.

Example 4. Analysis of main component content of Porostereum sp. (KCTC18837P) mycelium

Porostereum sp. (KCTC18837P) mycelium culture 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.

ingredient content beta glucan 34.23 (%/g) extracellular polysaccharide 43.11 (%/g) 5-hydroxy-6,7-dimethoxyphthalide 29.51 (μg/g) vitamin 1.34 (%/g) magnesium 0.52 (%/g) calcium 0.74 (%/g)

Through this, it was found that the 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. Antioxidant effect experiment

Example 5.1.1. Measurement of DPPH (1,1-diphenyl-2-picryhydrazyl) free radical scavenging activity

To confirm the antioxidant effect of Po-D, the DPPH radical scavenging activity method was used. 2 μl of Po-D by concentration (0 mg/mL, 0.5 mg/mL, 1 mg/mL, 5 mg/mL) was put into a 96 well plate, and 198 μl of 100 μM 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.

Po-D dose (mg/ml) DPPH radical scavenging activity (%) 0 0 0.5 26 One 38 5 79

As a result of the experiment, the DPPH free radical scavenging activity gradually increased as the amount of Po-D was increased, and when the Po-D concentration was 5 mg/mL, the DPPH radical scavenging rate was about 79%, confirming that it had an excellent antioxidant effect.

Example 5.1.2. ABTS(2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) free radical scavenging activity measurement

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 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 μl of the diluted solution, reacted for 10 minutes, and absorbance at 732 nm was measured for control. The elimination ratio was calculated compared to (not added), and the results are shown in Table 12 below.

Po-D dose (mg/ml) ABTS radical scavenging activity (%) 0 0 0.5 48 One 78 5 84

As a result of the experiment, 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.

Example 5.2 Wrinkle improvement effect test (collagen synthesis promoting effect)

To confirm the anti-wrinkle effect of Po-D, 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. , α-MSH (α-melanocyte stimulating hormone) was treated with 50 nM and then incubated for 72 hours. After recovering the cells by centrifugation, a cell suspension was prepared, 20 μl of cell suspension and 10 μl of antibody-POD conjugate solution were dispensed in a 96 well plate, and incubated for 3 hours. After washing 3 times with 400 μl of PBS, 100 μl of TMBZ (tetramethoxybenzil) was added, incubated for 15 minutes at room temperature, and then 1 N H2SO4 was added to measure the absorbance at 540 nm, a negative control (not added), It was compared with a positive control group (TGF-β 10ng/ml treatment, which is a tyrosinase inhibitor). The results are shown in Table 13.

Po-D dose (mg/ml) Ttpe 1 procollagen (ng/ml) Control 2,900 10 TGF-β (ng/ml) 4,720 0.5 3,850 One 4,030 5 5,210

As a result of the experiment, as the treatment concentration of Po-D increased, collagen synthesis increased, and when treated with 0.5, 1, and 5 mg/mL, collagen synthesis was increased by 32.7%, 37.9%, and 79.3%, respectively, so that Po-D was effective for collagen synthesis. It was confirmed that

Example 5.3 Skin moisturizing effect test (increase in filaggrin expression)

To confirm the skin moisturizing effect of Po-D, an experiment to increase filaggrin expression was conducted. Keratinocytes were dispensed in a 24 well plate at a concentration of 1×10 ⁴ cells/well, cultured for 24 hours, and Po-D was treated at each concentration (30 μg/ml, 100 μg/ml, 300 μg/ml). 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, the 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.

Po-D dose (㎍/ml) Filaggrin protein expression 0 0.78 30 0.93 100 1.02 300 1.27

As a result of the experiment, it was confirmed that as the concentration of Po-D increased, it promoted the expression of filaggrin protein, a natural moisturizing factor, and thus had a moisturizing effect on the skin.

Example 5.4 Skin whitening effect (inhibition of melanin production) test

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 α-MSH (α-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 (not added) and a positive control (treated with 50 μg/ml of arbutin, a tyrosinase inhibitor), and the results are shown in Table 15.

Po-D dose (mg/ml) B16 cell viability (% of control) Control 34 0 92 0.05 arbutin (mg/ml) 61 0.5 56 One 48 5 44

As a result of the experiment, as the treatment concentration of Po-D increased, melanin production was inhibited, and at a dose of 0.5 mg/mL, melanin synthesis was inhibited better than that of the control group, confirming that Po-D is a very effective substance for skin whitening.

Example 5.5 Skin cell viability increase experiment

In order to confirm the anti-aging effect of Po-D on skin cells, B16 melanoma cells were treated with Po-D at different concentrations and cell culture experiments were performed, 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

As a result of the experiment, 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 μg/ml treatment was performed.

Example 5.6 Skin cell regeneration effect test

Cell experiments were performed to confirm the effect of Po-D skin cell regeneration. To confirm the skin regeneration effect, human dermal fibroblast cells (CCD986sk) were purchased from the Korea Cell Line Bank and cultured in DMEM (Gibco) medium containing 15% calf serum (FBS; Gibco). The cells were aliquoted in a 96-well plate at 4000 cells/well, and 24 hours later, Po-D was treated in a cell culture medium containing 10% FBS for each concentration. MTS [3-(4,5-dimethyl-thiazol-2-yl)-5-carboxy-methoxyphenyl-2 After treatment with -(4-sulfophenyl)-2H-tetrazolium] reagent for 1 hour, absorbance was measured at 490 nm with a microplate reader (Perkin Elmer 1420 Multilabel Counter VICTOR™, 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 (㎍/ml) Cell proliferation rate (%) control 100.0±2.3 50 μg/ml 103.2±2.5 100 μg/ml 114.1±1.3 200 μg/ml 127.7±1.8 300 μg/ml 131.2±2.2

As a result of the experiment, the cell regeneration effect increased as the concentration of Po-D increased in a concentration-dependent manner, and more significant results were shown in the group administered 200 μg/ml or more, confirming that Po-D is an effective substance for promoting skin cell regeneration.

Various modifications and applications of the present invention described above are possible by those skilled in the art to which the present invention pertains, and the scope of the technical idea according to the present invention should be defined by the following claims.

Claims (8)

In the cosmetic composition for improving skin condition comprising the new strain Porostereum sp. (KCTC18837P) or a mycelium culture thereof as an active ingredient,
The new strain Porostereum sp. (KCTC18837P) mycelium culture is a cosmetic composition for improving skin condition, characterized in that it contains beta-glucan, extracellular polysaccharide, 5-hydroxy-6,7-dimethoxyphthalide. delete According to claim 1,
The skin condition improvement is a new strain Porostereum sp. (KCTC18837P), characterized in that at least one selected from the group consisting of antioxidant, wrinkle improvement, skin moisturizing, skin whitening and anti-aging Skin comprising a mycelium culture or its active ingredient A cosmetic composition for improving condition. 4. The method of claim 3,
The wrinkle improvement is a cosmetic composition for improving skin condition comprising a new strain Porostereum sp. (KCTC18837P) or a mycelium culture thereof as an active ingredient, characterized in that it promotes collagen synthesis. 4. The method of claim 3,
The skin moisturizing cosmetic composition for improving skin condition comprising a new strain Porostereum sp. (KCTC18837P) or a mycelium culture thereof as an active ingredient, characterized in that it increases the filaggrin expression of the skin. 4. The method of claim 3,
The skin whitening is a cosmetic composition for improving skin condition comprising a new strain Porostereum sp. (KCTC18837P) or a mycelium culture thereof as an active ingredient, characterized in that it inhibits melanin synthesis. 4. The method of claim 3,
The anti-aging cosmetic composition for improving skin condition comprising a new strain Porostereum sp. (KCTC18837P) or a mycelium culture thereof as an active ingredient, characterized in that the anti-aging increases the cell viability or cell regeneration rate. According to claim 1,
The new strain Porostereum sp. (KCTC18837P) or its mycelium culture 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) Including, the new strain Porostereum sp, characterized in that it was produced by culturing for 5-7 days at a pH of 5-6 in a liquid medium with an air supply of 0.02-1vvm, agitation speed of 25-100rpm, and a culture temperature of 18-27℃ .(KCTC18837P) or a cosmetic composition for improving skin condition comprising a mycelium culture thereof as an active ingredient.

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