阅读说明：本技术 包含银耳培养液提取物的组合物 (Composition containing tremella fuciformis culture solution extract ) 是由 廉贤淑 吴元辅 金地慧 李慧子 朴尽五 李知愿 金哲镐 吴百禄 金民秀 许仙宴 于 2020-09-29 设计创作，主要内容包括：本发明涉及一种包含银耳培养液提取物的组合物。适用本发明的银耳培养液提取物的细胞毒性较低,而且具有保湿因子生成量增加、胶原蛋白和胶原蛋白纤维生成量增加以及细胞内活性氧抑制效果,可以作为源自于天然物质的保湿、抗皱纹以及抗氧化功能性原材料进行使用,因此可以广泛地在用于改善皮肤状态的美容以及食品领域使用。(The invention relates to a composition containing a tremella fuciformis culture solution extract. The tremella fuciformis culture solution extract to which the present invention is applied has low cytotoxicity, has effects of increasing production of moisturizing factors, increasing production of collagen and collagen fibers, and inhibiting intracellular active oxygen, can be used as a functional raw material derived from natural substances for moisturizing, anti-wrinkle, and anti-oxidation, and thus can be widely used in the fields of beauty and food for improving skin conditions.)

1. A cosmetic composition characterized by:

an extract of Tremella fuciformis (Tremella fuciformis) culture solution is contained as an active ingredient.

2. The cosmetic composition of claim 1, wherein:

the tremella culture solution is a mycelium culture solution.

3. The cosmetic composition of claim 2, wherein:

the tremella fuciformis culture solution contains polysaccharide derived from the mycelium culture solution,

the content of mannose (mannose) in the polysaccharide body is 20 to 60% by weight.

4. The cosmetic composition of claim 1, wherein:

the tremella fuciformis culture solution is cultured for 6 to 48 hours.

5. The cosmetic composition of claim 1, wherein:

the tremella culture solution is cultured at a temperature of 15-35 ℃.

6. The cosmetic composition of claim 1, wherein:

the above extract is polysaccharide derived from Tremella culture fluid.

7. The cosmetic composition of claim 1, wherein:

the extract is obtained by extracting with water and C₁To C₄Or a mixed solvent thereof.

8. The cosmetic composition of claim 1, wherein:

the cosmetic composition has more than 1 selected from skin moistening, wrinkle resisting and antioxidant effects.

9. A food composition characterized by:

an extract of Tremella fuciformis (Tremella fuciformis) culture solution is contained as an active ingredient.

10. The food composition of claim 9, wherein:

the food composition has more than 1 kind of effects selected from skin moisturizing, wrinkle resisting and antioxidant effects.

Technical Field

The present invention relates to a composition comprising an extract of a white fungus culture solution, and more particularly, to a cosmetic composition and a food composition that can improve the skin condition by moisturizing the skin, anti-wrinkle, or anti-oxidation.

This application is a priority claim application to korean patent application No. 10-2019-0138381, filed on 11/01/2019, and all contents disclosed in the specification and drawings of the corresponding application are applicable to this application by reference.

Background

Tremella fuciformis (Tremella fuciformis) belongs to the Tremellales (Tremellales) Tremellaceae (Tremellaceae) of Heterobasidiomycetes (Heterobasidiomycetes), is a wood-rotting edible fungus growing on dead wood, dead branches and trunks of broad-leaved trees from spring to autumn, and is widely distributed in Korea, Japan, China, Europe, America and the like all over the world (Oh et al, 2006). Commonly referred to as tremella or snow ear, etc., and also referred to as Rokikurage (Ko, 2012) in japan. Furthermore, it has been known since ancient times in china that frequent consumption of Auricularia can activate the immune system and prevent cancer and aging, as well as prevent hypertension and arteriosclerosis (Chen and Cai 2008). In studies relating to the physiological activity of Tremella, it has been found that acidic polysaccharides isolated from Tremella may exhibit approximately 37 to 64% of antitumor activity against sarcomas cell lines, sarcomas 180 cells in animal models (Ukai et al, 1972). Further, it has been reported that tremella extract has effects of improving body fat and inhibiting cholesterol increase, and also has long-term and short-term anti-stress effects (Cheng et al, 2002; Chenng, 1996; Ko et al, 2009).

In addition, raw materials derived from various mushrooms such as phellinus linteus, ganoderma lucidum, shiitake mushroom, etc. are widely used in various industrial fields. As a prior art related thereto, a fine dust adsorption activity of polysaccharide derived from Tremella fuciformis was disclosed in Korean registered patent No. 10-1924342, and a skin barrier-strengthening effect of Tremella fuciformis extract was disclosed in Korean published patent No. 10-2018-0124443. However, most of these mushroom-derived substances are fruiting body extracts, and thus are produced only in small amounts and have a problem of high production cost.

Documents of the prior art

Patent document

(patent document 0001) Korean registered patent publication No. 10-1924342

(patent document 0002) Korea publication No. 10-2018-0124443

Disclosure of Invention

Therefore, the present inventors have manufactured an extract of a tremella fuciformis culture solution in order to solve the problems of the prior art as described above, and completed the present invention by confirming the moisturizing, anti-wrinkle and anti-oxidant activities thereof.

Accordingly, an object of the present invention is to provide a composition comprising an extract of Tremella fuciformis (Tremella fuciformis) culture solution as an active ingredient.

In order to achieve the above-mentioned object, the cosmetic composition of the present invention is suitable as an active ingredient containing an extract of Tremella fuciformis (Tremella fuciformis) culture solution.

Wherein the Tremella culture fluid may be mycelium culture fluid.

In this case, the tremella fuciformis culture solution may include a polysaccharide derived from the mycelium culture solution, and the content of mannose (mannose) in the polysaccharide may be 20 to 60% by weight.

In addition, the tremella fuciformis culture solution can be cultured for 6 to 48 hours.

Furthermore, the tremella fuciformis culture solution can be cultured at a temperature of 15-35 ℃.

In addition, the above extract may be a polysaccharide derived from a white fungus culture solution.

The extract can be obtained by using water and C₁To C₄Or a mixed solvent thereof.

In addition, the cosmetic composition can exhibit more than 1 kind of effects selected from skin moisturizing, anti-wrinkle and anti-oxidation.

Further, the food composition to which the present invention is applied contains Tremella fuciformis (Tremella fuciformis) culture solution extract as an active ingredient.

Wherein the food composition can exhibit more than 1 kind of effects selected from skin moisturizing, anti-wrinkle and anti-oxidation.

The tremella fuciformis culture solution extract to which the present invention is applied has low cytotoxicity, has effects of increasing production of moisturizing factors, increasing production of collagen and collagen fibers, and inhibiting intracellular active oxygen, can be used as a functional raw material derived from natural substances for moisturizing, anti-wrinkle, and anti-oxidation, and thus can be widely used in the fields of beauty and food for improving skin conditions.

Drawings

FIG. 1 is a schematic view showing the result of sugar analysis of a Tremella mycelium culture fluid extract to which the present invention is applied.

FIG. 2 is a graph showing the results of molecular weight analysis of polysaccharides derived from a Tremella mycelium culture fluid extract to which the present invention is applied.

Fig. 3 is a schematic diagram showing the results of confirming the production amount of Hyaluronic Acid (HA), which is a moisturizing factor, to which the tremella mycelium culture fluid extract of the present invention is applied.

Fig. 4 is a graph showing the results of confirming the production amount of aquaporin 3 (AQP 3), which is a moisturizing factor, in a tremella mycelium culture fluid extract to which the present invention is applied.

FIG. 5 is a graph showing the results of quantifying the amount of type I procollagen peptide produced when a Tremella mycelium culture extract to which the present invention is applied is treated.

FIG. 6 is a schematic view showing the fluorescence microscopic observation result of collagen fibril formation when the extract of Tremella mycelium culture medium to which the present invention is applied is treated.

FIG. 7 is a schematic view showing the measurement results of active oxygen inhibitory activity when a Tremella mycelium culture fluid extract to which the present invention is applied is treated.

FIG. 8 is a schematic diagram showing the results of confirmation of intracellular reactive oxygen species when a Tremella mycelial culture fluid extract to which the present invention is applied is treated.

Detailed Description

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings in order that those having ordinary skill in the art to which the present invention pertains may easily practice the present invention. However, the present invention can be realized in various forms, and is not limited to the embodiments and drawings described herein.

The cosmetic composition for improving skin conditions to which the present invention is applied contains a Tremella fuciformis (Tremella fuciformis) culture extract as an active ingredient.

The tremella fuciformis culture solution extract applicable to the invention has low cytotoxicity, has the effects of increasing the production of moisturizing factors, increasing the production of collagen and collagen fibers and inhibiting active oxygen in cells, and can be used as a cosmetic composition with skin moisturizing, anti-wrinkle or anti-oxidation functions and derived from natural substances.

Tremella fuciformis is divided into mycelium and fruiting body. The mycelium is a white fluffy or string-shaped main body part of the mushroom, which is equivalent to the root, stem and leaf parts of common plants, and the mushroom lives parasitized or floating in a hypha state in most of the life. The fruit body is a mechanism for the propagation of mushrooms, and corresponds to a flower of a general plant, and after sufficient nutrients are accumulated in hyphae, the hyphae instantaneously grow into a fruit body visible to the naked eye when the climate conditions are satisfied. The sugars and amino acids constituting The mycelia and fruit bodies of mushrooms differ from each other (Lee et al, 1999, "Characteristics of polysaccharides isolated from The fruit body and culture treated mycelia of fungi IY001," The Korea Journal of Mycology 27.6(1999): 424) 429).

However, the above-mentioned tremella culture solution is preferably a mycelium culture solution of tremella in view of effects, but is not limited thereto.

In this case, the tremella fuciformis culture solution may include a polysaccharide derived from the mycelium culture solution, and the content of mannose (mannose) in the polysaccharide may be preferably 20 to 60 wt%, and more preferably 30 to 50 wt%.

In addition, the tremella fuciformis culture solution can be cultured for 6 to 48 hours, and preferably can be cultured for 12 to 36 hours, but is not limited thereto.

When the culture time is less than 6 hours, the content of the active ingredient and the yield of the active ingredient may be low due to insufficient culture of the mycelia, and when the culture time exceeds 48 hours, the culture efficiency of the mycelia may be decreased or the mycelia may be deformed.

The tremella fuciformis culture solution may be cultured at a temperature of 15 to 35 ℃, preferably at a temperature of 20 to 30 ℃, but is not limited thereto.

When the culture temperature is less than 15 ℃, the content of the active ingredient and the yield of the active ingredient may be low due to insufficient culture of the mycelia, and when the culture temperature exceeds 35 ℃, the culture efficiency of the mycelia may be decreased or the mycelia may be deformed.

Further, the extract may be a polysaccharide derived from a culture solution of tremella, and the polysaccharide is a highly viscous polysaccharide, so that the procollagen synthesis performance is improved, the skin moisturizing ability is improved by the characteristics similar to those of gelatin, and skin wrinkles are effectively improved, and the anti-inflammatory effect, the anti-aging effect, the whitening effect, the collagen synthesis promoting effect, and the like are very excellent. In addition, since a stable state can be maintained even when pH changes due to external harmful substances, it has an effect of relieving skin irritation.

In addition, the extract can be used with water or C for improving skin condition₁To C₄Or a mixed solvent thereof. The lower alcohol may be methanol, ethanol, propanol, butanol, and although the mixed solvent is not particularly limited, it may be preferably 20 to 80 vol% methanol, ethanol, propanol, or butanol aqueous solution, and more preferably 60 to 80 vol% ethanol aqueous solution.

Further, the food composition for improving skin conditions to which another aspect of the present invention is applied contains a Tremella fuciformis (Tremella fuciformis) culture solution extract as an active ingredient.

The Tremella culture fluid extract has low cytotoxicity, and has effects of increasing production of moisturizing factor, increasing production of collagen and collagen fiber, and inhibiting intracellular active oxygen, and can be used as natural functional food composition for moisturizing skin, resisting wrinkle or resisting oxidation.

Next, the present invention will be described in more detail with reference to specific examples. The following examples are merely illustrative of the present invention and the present invention is not limited thereto.

Example 1 preparation of Tremella mycelium culture fluid extract

Extracting the culture solution of the tremella mycelium by using ethanol to prepare the tremella mycelium culture solution extract. Specifically, mycelia were separated from Tremella fuciformis and cultured for 24 hours under conditions of 300mL of Mushroom Complete Medium (MCM) at 25 ℃ and 180 rpm. The supernatant was obtained by centrifuging the cultured mycelia. The separated supernatant and ethanol were mixed (separated supernatant: ethanol 1:3 (volume ratio)), and then culture was performed overnight (overnight) while stirring at 4 ℃. The cultured mixture was centrifuged (12,000rpm), thereby obtaining a precipitate, i.e., a polysaccharide derived from a culture broth of Tremella mycelia. The obtained polysaccharide derived from the cultured tremella mycelium was diluted with purified water (polysaccharide: purified water 1:99 (volume ratio)) and filtered to produce a cultured tremella mycelium.

Test example 1 sugar analysis of extract derived from Tremella mycelium culture fluid

1-1 analysis of polysaccharide content of extract derived from Tremella mycelium culture fluid

1-1-1 hydrolysis of neutral sugars

100. mu.g of polysaccharide derived from a Tremella mycelial culture broth and 400. mu.l of 2M Trifluoroacetic acid (TFA) were put into a Microcentrifuge tube (Microcentifuge tube), and hydrolysis was performed at 100 ℃ for 4 hours. Cooling was performed at room temperature and drying treatment was performed using a vacuum centrifugal concentrator, and then the polysaccharide derived from the tremella mycelium culture solution was dissolved in 2ml of triple distilled water and filtered using a 02.Um filter.

1-1-2 hydrolysis of Glucuronic acid (Glucuronic acid)

100. mu.g of polysaccharide derived from a Tremella mycelial culture broth and 400. mu.l of 2M Trifluoroacetic acid (TFA) were put into a Microcentrifuge tube (Microcentifuge tube), and hydrolysis was performed at 100 ℃ for 6 hours. The sample was cooled at room temperature and dried by a vacuum centrifugal concentrator, and then dissolved in 1ml of triple distilled water and filtered by a 02.Um filter.

1-1-3 sugar analysis by High Performance Anion Exchange Chromatography (HPAEC, High-Performance Anion-Exchange Chromatography)

The sugars hydrolyzed in the above-mentioned test examples 1-1-1 and 1-1-2 were analyzed by High Performance Anion Exchange Chromatography (HPAEC). Specific conditions for High Performance Anion Exchange Chromatography (HPAEC) analysis are shown in table 1 below, and sugar analysis results are shown in fig. 1 and table 2.

[ TABLE 1 ]

[ TABLE 2 ]

As shown in table 2, it was confirmed that the content of Glucose (Glucose) in the polysaccharide derived from the culture broth of tremella mycelia was smaller than that of the polysaccharide derived from the sporophores of tremella. On the contrary, it was confirmed that the content of mannose (mannose) in the polysaccharide derived from the culture solution of tremella mycelia was about 8 times higher than that of the polysaccharide derived from the culture solution of tremella sporogenes. Mannose (mannose) to which α -mannan (α -mannan) belongs is a polysaccharide that induces skin immunity and intestinal immunity by binding to a dendritic cell-associated C-type lectin-2 receptor (Dectin-2 receptor) present in a cell membrane.

1-2 analysis of polysaccharide molecular weight of extract derived from Tremella mycelium culture fluid

A Size Exclusion Chromatography (SEC) column (TSK-gel-GMPWXL, 30 cm. times.7.8 mm, 13. mu. mu.M) was utilized in a high performance liquid chromatography-multiangle light scattering (MALS) system (Wyatt Technology, Santa Babara, Calif.)m, Tosoh) the average molecular weight of the polysaccharide derived from the culture broth of tremella mycelial is analyzed. An analyte sample was prepared by dissolving a polysaccharide derived from a culture solution of Tremella mycelia in a phosphate buffer (pH 7.3-7.5) at a concentration of 3 mg/mL. The flow rate of a high performance liquid chromatography sample is 0.5mL/min, and the sample injection volume isThe analysis was carried out under conditions of mobile phase phosphate buffered saline (pH 7.3-7.5) and DAWN HELEOS II as detector, Optilab rEX. The value of dn/dc (specific reactive index increment) at the time of molecular weight calculation was determined to be 0.15mL/g on the premise of reference. ASTRA 6 software (software) (Wyatt Technology) was used in the calculation of the average molecular weight of polysaccharides derived from the culture broth of Tremella mycelia. The high performance liquid chromatography-multiangle light scattering (MALS) results for the polysaccharides derived from the tremella mycelial broth are shown in fig. 2.

As can be confirmed from FIG. 2, the average molecular weight of the polysaccharide derived from the cultured Tremella mycelia was 1.79X 10⁶(±0.721％)。

Test example 2 culture of skin cells

2-1 culture of skin keratinocytes

Human dermal epidermal keratinocytes (HaCaT), which are skin keratinocytes, are commercially available from Cell Line Service GmbH. (Germany). Dulbecco's Modified Eagle's Medium (DMEM) containing 1% penicillin-streptomycin and 10% Fetal Bovine Serum (FBS) was used at 37 ℃ with 5% CO₂The culture was performed in a thermostat, and subculture was performed at intervals of 2 to 3 days.

2-2 culturing of dermal fibroblasts

Normal Human Dermal Fibroblasts (NHDF) as Dermal Fibroblasts were purchased from Lonza (Lonza Walkersville, Inc) and utilized a mixture containing 0.1% Human fibroblast growth factor-B (hFGF-B), insulin, GA-1000, and 2% fetal bovine bloodClear Fibroblast Basal Medium (FBM) at 37 deg.C, 5% CO₂The culture was performed in a thermostat, and subculture was performed at intervals of 3 to 5 days.

Test example 3 evaluation of cytotoxicity of Tremella fuciformis mycelium culture fluid extract

The Ez-cytox test (assay) is a representative cytotoxicity assessment method for measuring cell activity by using the principle that a water-soluble tetrazolium salt (WST) reacts with Dehydrogenase (Dehydrogenase) of active cells to produce vermillicular water-soluble formazan (formazan).

3-1 evaluation of cytotoxicity against skin keratinocytes

To confirm the toxicity in cells, human dermal epidermal keratinocytes (HaCaT) were treated at 5X 10⁴cells/mL concentration into 96-well plates and at 37 ℃, 5% CO₂The culture was carried out under the conditions for 18 hours. The cultured cells were changed to serum-free medium and then treated with the extract of the tremella mycelium culture solution produced in example 1 at various concentrations (0.1, 0.5, 1.0, 2.0, 4.0 v/v%). Next, EZ-cytox was added to each well and 5% CO at 37 deg.C₂The reaction was carried out under the conditions for 30 minutes. The absorbance of each well at 450nm was measured using a microplate reader. The average absorbance value of each sample group was calculated and the cell activity was evaluated by comparing the above absorbance values with those of the control group. The results of cytotoxicity evaluation of the extracts of the culture broth of Tremella fuciformis for skin keratinocytes are shown in Table 3 below.

[ TABLE 3 ]

As shown in table 3, it was confirmed that the extract of the tremella mycelium culture solution had low cytotoxicity to skin keratinocytes.

3-2 evaluation of cytotoxicity to dermal fibroblasts

Normal Human Dermal Fibroblasts (NHDF), Normal Human Dermal Fibroblasts (1X 10) as Dermal Fibroblasts⁴cells/mL were aliquoted into 96-well plates. Cytotoxicity of the extract of Tremella mycelium culture broth of Normal Human Dermal Fibroblasts (NHDF) was evaluated by EZ-cytox test (assay) of test example 2-1 described above. The results of cytotoxicity evaluation of the extracts of the culture broth of Tremella mycelia of dermal fibroblasts are shown in the following Table 4.

[ TABLE 4 ]

As shown in table 4, it was confirmed that the extract of the tremella mycelium culture solution had low cytotoxicity to skin fibroblasts.

Test example 4 confirmation of moisturizing Effect of Tremella fuciformis mycelium culture fluid extract

4-1 confirmation of Effect of increasing production amount of Hyaluronic Acid (HA), a moisturizing factor of an extract of Tremella mycelial Medium

Human skin epidermal keratinocytes (HaCaT) at a ratio of 1.0X 10⁵cells/mL concentration were aliquoted into 24-well plates and incubated at 37 ℃ with 5% CO₂The culture was carried out under the conditions for 18 hours. The cultured cells were cultured in the medium replaced with serum-free Darber Modified Eagle's Medium (DMEM), and then treated with the extract of the Tremella mycelial culture solution prepared in example 1 at concentrations of 0.1, 0.5, and 1.0 v/v%, respectively, for 24 hours. The hyaluronic acid production amount of the culture extract of each test group was then measured. As a control group, Retinoic Acid (RA) was treated at a concentration of 10 μ M. The measurement of the amount of hyaluronic acid produced was performed using an HA-ELISA kit (Cusabio Biotechnology Co., Ltd.), and the test was performed according to the method provided by the manufacturing company. The measurement results of the amount of hyaluronic acid produced by the treatment with the extract of Tremella mycelia culture are shown in FIG. 3.

As can be confirmed from fig. 3, the extract of the tremella mycelium culture solution can increase the production of hyaluronic acid, which is a moisturizing factor, in a concentration-dependent manner. Further, it was confirmed that the culture solution of Tremella mycelia can produce hyaluronic acid at a level similar to that of the control group, i.e., retinoic acid.

4-2 confirmation of Effect of increasing production of aquaporin 3 (AQP 3), a moisturizing factor of Tremella mycelial Medium extract

Human skin epidermal keratinocytes (HaCaT) at a ratio of 1.0X 10⁵cells/mL concentration were aliquoted into 24-well plates and incubated at 37 ℃ with 5% CO₂The culture was carried out under the conditions for 18 hours. The cultured cells were cultured in the medium replaced with serum-free Darber Modified Eagle's Medium (DMEM), and then treated with the extract of the Tremella mycelial culture solution prepared in example 1 at concentrations of 0.1, 0.5, and 1.0 v/v%, respectively, for 24 hours. Next, proteins were extracted after lysing the cells of each test group and the amount of aquaporin 3 produced in the culture extract was measured. As a control group, Retinoic Acid (RA) was treated at a concentration of 10 μ M. The measurement of the amount of aquaporin 3 produced was performed using AQP3-ELISA kit (Cusabio Biotechnology Co., Ltd.), and the test was performed according to the method provided by the manufacturer. The results of measuring the amount of aquaporin 3 produced by the treatment with the extract of Tremella mycelium culture are shown in FIG. 4.

It was confirmed from FIG. 4 that the extract of Tremella mycelium culture solution can increase the production of aquaporin 3, which is a moisturizing factor, concentration-dependently.

From the above test results, it was confirmed that the extract of the cultured Tremella mycelia solution produced in example 1 can increase the production of hyaluronic acid and aquaporin 3. The above results were obtained presumably because the extract of the Tremella mycelium culture broth contained a high concentration of mannose. This confirmed that the extract of the tremella mycelium culture solution was effectively used as a natural moisturizing material.

Test example 5 confirmation of anti-wrinkle Activity of Tremella fuciformis mycelium culture fluid extract

5-1 confirmation of Procollagen type I peptide (PIP) production Effect of Tremella mycelial Medium extract

Normal Human Dermal Fibroblasts (NHDF) as dermal fibroblasts were cultured at 2X 10⁴cells/mL were aliquoted into 24-well plates and cultured under cell culture conditions for 24 hours. After completion of the culture, the culture medium was replaced with a serum-free Fibroblast Basal Medium (FBM), and then the extract of the Tremella mycelium culture solution produced in example 1 was treated at concentrations of 0.1, 0.5, and 1.0 v/v%, respectively, to perform the culture for 24 hours. The supernatants of each group were extracted after the completion of the culture, and the amount of free procollagen (procollagen) in the medium was determined. As a control group, ascorbic acid (ascorbic acid) was used in place of the extract and treated at a concentration of 10. mu.g/ml. For the measurement of the amount of Procollagen, Procollagen type I peptide (PIP) EIA kit (Takara Biomedical Co.) was used, and the amount of collagen was measured by measuring the absorbance at a wavelength of 450nm according to the manufacturer's manual. FIG. 5 shows the results of quantifying the amount of type I procollagen peptide produced by the treatment with the extract of Tremella mycelium culture.

It can be confirmed from FIG. 5 that the extract of the Tremella mycelium culture solution can increase collagen synthesis concentration-dependently.

5-2 confirmation of collagen fibril formation Effect of Tremella mycelium culture fluid extract

Normal Human Dermal Fibroblasts (NHDF) as dermal fibroblasts were cultured at 2X 10⁴cells/mL were aliquoted into 24-well plates and cultured under cell culture conditions for 24 hours. After completion of the culture, the culture medium was replaced with a serum-free Fibroblast Basal Medium (FBM), and then the extract of the Tremella mycelium culture solution produced in example 1 was treated at concentrations of 0.1, 0.5, and 1.0 v/v%, respectively, to perform the culture for 24 hours. After completion of the culture, the cells were washed with a hydroxyethylpiperazine ethanethiosulfonic acid buffer Solution (HEPES-BSS, HEPES Buffered Saline Solution), followed by immunofluorescenceThe staining method stains collagen fibers in cells. The degree of fluorescence expression of the stained cells was measured by fluorescence microscopy. The results of confirming collagen fibril formation when the extract of the tremella mycelium culture solution was treated are shown in FIG. 6.

It was confirmed from FIG. 6 that the extract of Tremella mycelium culture solution can increase the production of collagen fibers in skin fibroblasts in a concentration-dependent manner.

From the above test results, it was confirmed that the extract of the cultured Tremella fuciformis solution produced in example 1 can increase the synthesis of collagen and the production of collagen fibers, and thus can be effectively used as an anti-wrinkle material.

Test example 6 confirmation of antioxidant Effect of Tremella fuciformis mycelium culture fluid extract

6-1. measurement of inhibitory Effect of Reactive Oxygen Species (ROS) in intracellular

For measuring the change of active oxygen in cells, human epidermal keratinocytes (HaCaT) were cultured at a temperature of 1.0X 10⁵The cell/well was inoculated into a 24-well plate and cultured for 24 hours, and then the extract of the Tremella mycelium culture solution prepared in example 1 was pretreated for 24 hours. After washing the pretreated cells with Phosphate Buffered Saline (PBS), the assay was performed using the Intracellular ROS assay kit (Green Fluorescence) according to the manufacturer's manual. The results of measuring the inhibitory effect on intracellular reactive oxygen species are shown in FIG. 7.

It can be confirmed from FIG. 7 that the extract of the Tremella mycelium culture fluid can increase the radical scavenging activity concentration-dependently. In particular, it was confirmed that about 40% or more of the free radicals were removed from the test group treated with the extract of the cultured Tremella mycelia solution at a concentration of 2.0 v/v%.

6-2 measurement of inhibitory Effect of intracellular Reactive Oxygen Species (ROS) by imaging

For visualizing intracellular Reactive Oxygen Species (ROS) changes in skinHuman skin keratinocyte epidermal keratinocyte (HaCaT) of skin keratinocyte at 1.0 × 10⁵cells/well were inoculated into 24-well plates at different concentrations (0.1, 0.5, 1.0, 2.0 v/v%) and then incubated for 24 hours, followed by pretreatment with the Tremella mycelium culture fluid extract prepared in example 1 at different concentrations. After the cultured cells were washed with Phosphate Buffered Saline (PBS), H was used₂O₂The treatment was carried out at a concentration of 300. mu.M, and additional culture was carried out for 3 hours. As a dye for measuring intracellular reactive oxygen species, dichlorofluorescein diacetate (DCF-DA) was added at a concentration of 50. mu.M and incubated for 1 hour. After the cultured cells were washed with Phosphate Buffered Saline (PBS), the degree of fluorescence expression of the washed cells was measured using a fluorescence microscope. The results of intracellular reactive oxygen species measurement of the extract of Tremella mycelium culture fluid are shown in FIG. 8.

As can be seen from FIG. 8, the extract of the Tremella mycelium culture fluid can inhibit the production of intracellular reactive oxygen species in a concentration-dependent manner. In particular, it was confirmed that the activity of inhibiting intracellular reactive oxygen species was excellent in the test groups treated with the extracts of the cultured Tremella fuciformis fluid at concentrations of 0.5, 1.0, and 2.0 v/v%.

The above test results confirmed that the extract of the tremella mycelium culture solution produced in example 1 has a very significant effect of inhibiting intracellular active oxygen, and therefore can be effectively used as an antioxidant material.

In summary, the present inventors produced an extract of a culture solution of Tremella fuciformis berk, and confirmed that the extract has low cytotoxicity, increased production of moisturizing factors, increased production of collagen and collagen fibers, and an effect of inhibiting intracellular reactive oxygen species. This indicates that the extract of the tremella fuciformis culture solution of the present invention can be used as a natural moisturizing, anti-wrinkle and anti-oxidation functional raw material, and the extract of the tremella fuciformis culture solution of the present invention can be widely applied to the fields of beauty and food for improving skin conditions.

Next, the present invention will be described in more detail with reference to production examples. The manufacturing example is merely an illustration of the present invention, and the scope of the present invention should not be construed as being limited by the manufacturing example.

Production example 1 production of cosmetic composition for improving skin Condition

1-1 preparation of smoothing toner (skin lotion)

0.5% by weight of the extract of Tremella culture fluid

BETA-1, 3-glucan 1.0% by weight

2.0% by weight of butanediol

2.0% by weight of propylene glycol

Carboxyvinyl Polymer 0.1% by weight

0.2% by weight of polyethylene glycol-12-anyalkylphenol polyglycol ether

Polysorbate 800.4% by weight

Ethanol 10.0% by weight

Triethanolamine 0.1% by weight

Proper amount of antiseptic, pigment and spice

Adding purified water to 100 wt%

1-2 preparation of nutrient water (milk skin lotion)

0.5% by weight of the extract of Tremella culture fluid

BETA-1, 3-glucan 1.0% by weight

Beeswax 4.0% by weight

Polysorbate 601.5% by weight

Sorbitan sesquioleate 1.5% by weight

Liquid paraffin 0.5 wt%

Caprylic/capric triglyceride 5.0% by weight

3.0% by weight of Glycerol

Butanediol 3.0 wt.%

Propylene glycol 3.0% by weight

Carboxyvinyl Polymer 0.1% by weight

Triethanolamine 0.2% by weight

Proper amount of antiseptic, pigment and spice

Adding purified water to 100 wt%

1-3 preparation of nourishing cream

1.0% by weight of the extract of Tremella culture fluid

BETA-1, 3-glucan 5.0% by weight

Beeswax 10.0 wt%

Polysorbate 601.5% by weight

Polyethylene glycol 60 hardened Castor oil 2.0% by weight

Sorbitan sesquioleate 0.5% by weight

Liquid paraffin 10.0 wt%

Squalane 5.0% by weight

Caprylic/capric triglyceride 5.0% by weight

Glycerol 5.0% by weight

Butanediol 3.0 wt.%

Propylene glycol 3.0% by weight

Triethanolamine 0.2% by weight

Proper amount of antiseptic, pigment and spice

Adding purified water to 100 wt%

Production example 2 production of food preparation for improving skin Condition

2-1 preparation of health food

Tremella culture fluid extract 100mg

Proper amount of vitamin mixture

Vitamin A acetate 70g

Vitamin E1.0 mg

Vitamin B10.13mg

Vitamin B20.15mg

Vitamin B60.5mg

Vitamin B120.2g

Vitamin C10 mg

Biotin 10g

Nicotinamide 1.7mg

Folic acid 50g

Calcium pantothenate 0.5mg

Proper amount of inorganic substance mixture

Ferrous sulfate 1.75mg

Zinc oxide 0.82mg

Magnesium carbonate 25.3mg

Potassium dihydrogen phosphate 15mg

Dipotassium hydrogen phosphate 55mg

Potassium citrate 90mg

Calcium carbonate 100mg

24.8mg of magnesium chloride

The composition ratio of the vitamin and mineral mixture is mixed according to a preferred embodiment relatively suitable for the ingredients of the health food, and the above mixing ratio may be arbitrarily modified, and the above ingredients may be mixed according to a general health food production method to produce granules, and used for the production of the health food composition according to a general method.

2-2 preparation of health drink

Tremella culture fluid extract 100mg

Vitamin C15 g

Vitamin E (powder) 100g

Iron lactate 19.75g

3.5g of zinc oxide

Nicotinamide 3.5g

Vitamin A0.2 g

Vitamin B10.25g

Vitamin B20.3g

Adding water for quantitative determination

The above ingredients were mixed according to a general method for producing a health drink, and then stirred and heated at 85 ℃ for about 1 hour, and the resulting solution was filtered, filled into a 2L container subjected to sterilization treatment, and hermetically sterilized, and then stored under refrigeration, and used for producing the health drink composition of the present invention.

The above composition ratio is mixed in accordance with a preferred embodiment which is relatively suitable for popular beverage ingredients, but may be arbitrarily modified depending on the consumption class, country of consumption, local use, and national preference.

The foregoing is merely an illustration of the present invention, and it will be understood by those having ordinary skill in the art that the present invention may be embodied in various forms without departing from the essential characteristics thereof. Accordingly, the disclosed examples and test examples are illustrative only and not intended to be limiting. The scope of the invention is defined by the appended claims rather than the foregoing description, and all differences within the scope equivalent to the above-described embodiments are intended to be construed as being included in the scope of the present invention.