KR101987639B1 - Novel strain of Fusidium coccineum spp., and composition for improving skin beauty comprising a culture solution of the strain - Google Patents

Abstract

The present invention relates to a novel foshiidium cocinum strain, a composition for improving skin beauty comprising the culture medium of the strain, and a method for producing the strain culture. According to a composition for improving skin beauty according to one aspect of the present invention, the culture medium of Fushidium coccinium can be used for skin barrier enhancement, skin moisturization, skin cell regeneration, skin vitality enhancement, antioxidant, antiinflammation or antiatopia. According to another aspect of the present invention, there is provided a method for producing a fusidium coccinium culture, which comprises culturing the cultured Fusidium coccinium strain in a cosmetic, food, pharmaceutical composition, .

Description

[TECHNICAL FIELD] The present invention relates to novel fusidium coccineum spp., And a composition for improving the skin beauty comprising the fusidium coccineum strain and a culture of the strain,

A novel foshium coccinium strain and a culture solution of the strain.

Fushimi Stadium in (Fusidium sp.) Fungi and microorganisms, the steroidal husidin San antibiotics (fusidic acid) are co-Fushimi Stadium Stadium Cine (Fusidium coccineum strains. Fucidin acid is effective against Gram-positive bacteria, especially staphylococcal infections, and is also used as underwear and external medicine. Fusidic acid in the drug is used in the form of sodium fusidate.

Chinese Patent Publication No. 101812498 relates to a method for fermenting fucidin acid, and discloses a method for producing fucindinic acid through a fermentation process using F. coccineum strain.

However, much research has not been conducted on the F. coccineum strain itself, and there have been no studies using Fushidium coccinum strain for skin cosmetic improvement.

Chinese Patent Publication No. 101812498.

One aspect is Fusidium coccineum strain (accession number: KCCM12013P).

Another aspect is Fusidium < RTI ID = 0.0 > coccineum ) culture medium as an active ingredient.

Another aspect provides a method of producing a fusidium coccinium culture.

One aspect is Fusidium coccineum strain (accession number: KCCM12013P).

The " Fusidium " coccineum "is a kind of fungus which may be isolated from a plant. The plant is not limited in its kind but may be, for example, a ginkgo leaf. The strain is obtained by inoculating a plant sample with potato agar medium The strain may be a strain isolated by a method of purely isolating and culturing colonies. The strain may be one which grows to form a pale yellow colonies. The strain may be grown in a potato agar medium.

Another aspect is Fusidium < RTI ID = 0.0 > coccineum ) culture medium as an active ingredient.

Such skin aesthetic enhancement may be skin barrier enhancement, skin moisturization, skin cell regeneration, increased skin vitality, antioxidant, anti-inflammatory, or anti-atopic. The culture medium of the Fusidium coccinum strain is superior to the stem cell culture solution in that it has a skin barrier strengthening effect, a skin moisturizing effect, a skin cell regeneration effect, a skin vitality increasing effect, an antioxidative effect, an antiinflammatory effect or an antiatopic effect, A composition comprising a culture of a nociceptic strain may be used as a composition for skin barrier strengthening, skin moisturizing, skin cell regeneration, skin vitality enhancement, antioxidant, anti-inflammatory, or anti-atopic composition.

The term "skin barrier enhancement" may refer to any action that is located at the outermost position of the skin, thereby enhancing the function of the skin barrier to prevent moisture and nutritional loss.

The term "skin moisturizing" may mean any action that maintains skin moisture or prevents moisture loss.

The term "skin cell regeneration" may mean any action that, when a portion of the skin is lost, replenishes that part or promotes proliferation of the skin cells.

The term "increased skin vitality" may refer to any action that increases the activity of mitochondria in the cells of the skin, thereby increasing the vitality or vitality of the skin.

The term "antioxidant" may mean any action that inhibits oxidation. The antioxidative action can prevent, ameliorate, or inhibit skin aging.

The term "anti-inflammatory" can be used interchangeably with "inflammation inhibition or improvement ", and may mean all actions in which the immune response is alleviated and NO production is suppressed.

The term "anti-atopy" can be used interchangeably with " inhibiting, improving or alleviating atopy ", and includes all actions that inhibit, ameliorate, or alleviate the symptoms of atopic dermatitis (pruritus) It can mean.

The term "improvement" may mean any action that at least reduces the degree of symptomatology, such as a condition associated with relief or treatment of a condition.

The strain may be isolated from food, soil, sea or the like.

In a composition according to one embodiment, the strain may be a Fusidium coccineum strain (accession number: KCCM12013P).

In the composition according to one embodiment, the culture may be one wherein the cells are removed from the culture medium obtained by culturing the strain.

In a composition according to one embodiment, the culture may be, but is not limited to, include fusidic acid.

The culture solution may be used in an amount of 0.001 to 80 wt%, for example, 0.01 to 60 wt%, 0.01 to 40 wt%, 0.01 to 30 wt%, 0.01 to 20 wt% %, 0.01% to 10%, 0.01% to 5%, 0.05% to 60%, 0.05% to 40%, 0.05% to 30%, 0.05% to 20% From 0.05% to 10%, from 0.05% to 5%, from 0.1% to 60%, from 0.1% to 40%, from 0.1% to 30%, from 0.1% to 20% % To 10 wt%, 0.1 wt% to 5 wt%, 0.1 wt% to 3 wt%, and 0.1 wt% to 2 wt%.

The composition according to one embodiment may be one that increases the expression of filaggrin, and more specifically, the expression of pilar green to increase the skin barrier.

The composition according to one embodiment may increase the expression of ceramide synthase 3, b-glucocerebrosidase, or AQP3 (Aquaporin 3), and more specifically, 3, b-glucocerebrosidase, or AQP3, thereby exhibiting a skin moisturizing effect.

The composition according to one embodiment may be one that increases intracellular mitochondrial activity, specifically, increases mitochondrial activity in fibroblasts, thereby exhibiting skin cell regeneration or skin vitality enhancement.

The composition according to one embodiment may inhibit the expression of IL-1a, IL-6, or TSLP, and specifically inhibits the expression of IL-1a, IL-6, or TSLP to produce anti- .

The composition according to one embodiment may contain the above culture medium as an effective amount or as an active ingredient. The effective amount may be appropriately selected depending on the individual. The severity of the disease or condition, the age, weight, health, sex, sensitivity of the individual to the extract, time of administration, route and rate of excretion, duration of administration, factors including other compositions combined or co- May be determined according to factors well known in the art, physiology or medical field.

The composition for improving skin care according to one embodiment may further comprise a cosmetically, pharmaceutically or pharmaceutically acceptable excipient or carrier. The carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low substituted hydroxy cellulose, or a combination thereof. The disintegrant may be sodium starch glycolate, calcium monohydrogen phosphate anhydrous, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or combinations thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The composition may be formulated into a parenteral dosage form. The parenteral dosage form may be an injection or an external preparation for skin. The external skin preparation may be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a drug-containing bandage, a lotion, or a combination thereof.

The external preparation for skin is usually used as a component used in external skin preparations such as cosmetics or medicines such as an aqueous component, an oily component, a powder component, an alcohol, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, an antiseptic, , Coloring agents, various skin nutrients, and the like can be appropriately blended as needed.

The external preparation for skin may be a metal blocker such as sodium edetate, sodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate or gluconic acid, caffeine, tannin, bellapamil, licorice extract, glabridine, Vitamin C, ascorbic acid magnesium phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, fructose, fructose and other herbal medicines, various herbal medicines, tocopherol acetate, glycyrrhizic acid, Sugars such as trehalose and the like can also be appropriately compounded.

The composition according to one embodiment may be a cosmetic composition. In this case, the culture solution can be used as a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream, a nutrition cream, a moisturizing cream, a hand cream, a foundation, , A soap, a cleansing foam, a cleansing lotion, a cleansing cream, a body lotion, a body cleanser, a suspension, a gel, a powder, a paste, a mask pack or a sheet or an aerosol composition. Compositions of such formulations may be prepared according to methods conventional in the art. The cosmetic composition may further contain preservatives, stabilizers, surfactants, solubilizers, moisturizers, emollients, ultraviolet absorbers, preservatives, bactericides, antioxidants, pH regulators, organic and inorganic pigments, fragrances, have. The amount of the additional component such as the above-mentioned moisturizing agent and the like can be easily selected by those skilled in the art within a range not to impair the purpose and effect of the present invention. The amount of the additional component is 0.001 to 5% by weight, % ≪ / RTI >

The composition according to one embodiment may be a food composition. In this case, it can be formulated into a conventional health functional food formulation known in the art. The food composition may be prepared by conventional formulations such as powders, granules, tablets, pills, capsules, suspensions, emulsions, syrups, And may be manufactured in the form of any health food such as confectionery, pizza, ramen, other noodles, gums, jellies, dairy products including ice cream, various soups, drinks, tea, drinks, alcoholic beverages and vitamin complexes. A pharmaceutically acceptable carrier or additive may be used for the formulation of the health food, and any carrier or additive known to be usable in the art for the preparation of the formulation to be prepared may be used. As the above-mentioned additives, there can be used various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, Carbonating agents, and the like. In addition, it may contain natural fruit juice, fruit juice beverage and flesh for the production of vegetable beverages. These additive components may be used independently or in combination, and the proportion of the additive may be 0.001 to 5 wt%, specifically 0.01 to 3 wt%, based on the total weight of the composition.

The content of the culture medium in the food composition may be suitably determined according to the intended use (prevention or improvement). Generally, it may contain 0.01 to 15% by weight of the total food weight, and when it is prepared as a beverage, it may contain 0.02 to 10 g, specifically 0.3 to 1 g, based on 100 mL.

The beverage may further contain ingredients other than the above extract, and may further contain various flavors or natural carbohydrates commonly used in beverages. Examples of the natural carbohydrate include conventional sugars such as monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; and sugars such as xylitol, sorbitol, erythritol, Of sugar alcohols may be contained. In addition, as a flavoring agent, a natural flavoring agent (e.g., tau Martin, stevia extract, etc.) and a synthetic flavoring agent (e.g., saccharin, aspartame, etc.) may be contained. The ratio of the natural carbohydrate may be generally about 1 to 20 g, specifically about 5 to 12 g per 100 mL of beverage.

The composition according to one embodiment may be a pharmaceutical composition. Specifically, the pharmaceutical composition may be a pharmaceutical composition for preventing or treating a skin disease. The skin disease may be a disease caused by skin barrier function impairment, skin aging, skin wounds, skin scarring or skin inflammation, or atopic dermatitis. The term " prevention " includes inhibiting the occurrence of a disease. The term " treatment " includes inhibiting, alleviating, or eliminating the development of the disease.

Damage to the skin barrier function may mean all the changes that appear on the skin due to the degradation or damage of the skin barrier function. For example, it may include, but is not limited to, increased wrinkles, dryness, dermatitis, atopic dermatitis, allergic dermatitis, acne and the like. The pharmaceutical composition according to one embodiment of the present invention can enhance the expression of pillar green to enhance the skin barrier function, thereby preventing or treating damage to the skin barrier function.

The aging of the skin can mean all types and intangible changes that appear on the skin as the age goes on. For example, it may include, but is not limited to, increased skin wrinkles, drying, decreased wound healing ability, decreased skin immune function, and the like. The pharmaceutical composition according to one embodiment can prevent or treat skin aging by the effects of strengthening skin barrier, maintaining skin moisture, preventing loss of skin moisture, regenerating skin cells, and antioxidation.

The skin wound or skin scar may mean any disease that can be improved or remedied by maintaining the continuity of skin cells regenerated, differentiated, proliferating, interfering with the synthesis of scarring collagen and promoting collagen degradation. But are not limited to, scars such as bruises, abrasions, lacerations, scars such as hypertrophic scars, keloid scars, and the like. The pharmaceutical composition according to one embodiment can prevent or treat skin wounds or skin scars due to the effects of skin moisture retention, prevention of skin moisture loss, skin cell regeneration, and anti-inflammation.

The skin inflammatory disease may mean all diseases caused by immune stimulation. But are not limited to, for example, atopic dermatitis, eczema, seborrheic dermatitis, psoriasis, acne, and the like. The pharmaceutical composition according to one embodiment of the present invention can prevent or treat skin inflammation diseases by enhancing skin barrier, maintaining skin moisture, preventing skin moisture loss, anti-inflammation, and anti-atopy.

Another aspect provides a method of improving skin beauty of an individual comprising administering the composition to a subject. The composition is the same as described above.

Specifically, the method may include a method of enhancing a skin barrier function of an individual, a method of efficiently maintaining skin moisture or preventing moisture loss of an individual, a method of regenerating skin cells of an individual, a method of increasing skin vitality of an individual, A method of improving the antioxidant activity in an individual, a method of improving or treating inflammation of an individual, a method of improving, treating or preventing atopic dermatitis of an individual.

The terms "administering "," introducing "and" transplanting "are used interchangeably and refer to a method of delivering a composition according to one embodiment ≪ / RTI > may refer to the arrangement of the composition according to the invention. May be administered by any suitable route that delivers at least a portion of the culture broth or culture medium components of the composition according to one embodiment to the desired location within the living organism.

Administration can be by any method known in the art. Administration may be by any means directly administered to a subject, such as by intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration, . The administration can be systemically or locally administered.

The subject may be a mammal, such as a person, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat. The subject may be an individual in need of skin aesthetic improvement, such as skin barrier enhancement, skin moisturization, skin cell regeneration, increased skin vitality, antioxidant, anti-inflammatory, or anti-atopic effect.

The administration may be carried out in a culture medium of the fusidium oxycinum strain in an amount of 0.1 mg to 1,000 mg, for example, 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg From 1 mg to 500 mg, from 1 mg to 100 mg, from 1 mg to 50 mg, from 1 mg to 25 mg, from 5 mg to 1000 mg, from 5 mg to 500 mg, from 5 mg to 100 mg, from 5 mg to 50 mg , 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg. However, the dose may be variously prescribed depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate and responsiveness of the patient, These factors can be taken into account to appropriately adjust the dosage. The number of doses may be at least once per day or within the range of clinically acceptable side effects and may be administered at one or two or more doses at the site of administration and may be administered daily or every two to five days The number of days of administration can be administered from one day to 30 days at one time of treatment. If necessary, the same treatment may be repeated after the appropriate time. For an animal other than a human, the dosage may be the same as that of a human per kg, or the dosage may be converted into a dose in terms of a volume ratio (for example, an average value) One dose may be administered.

Another aspect is Fusidium < RTI ID = 0.0 > coccineum ); Centrifuging the cell culture solution obtained after culturing to remove the cells and recovering the supernatant; And filtering the recovered supernatant to remove the remaining microbial cells. The present invention also provides a method for producing a fusidium coccinium culture.

The above-mentioned F. coccineum strain is the same as described above.

In a method according to one embodiment, the strain may be a strain of Fusidium coccineum (accession number: KCCM12013P).

The culturing can be carried out at 20 to 35 DEG C, 25 to 30 DEG C for 12 to 120 hours, 24 to 100 hours, 48 to 80 hours.

The fusidium coccinum culture prepared by the above method can enhance the expression of pillar green to enhance the skin barrier and increase the expression of ceramide synthase 3, b-glucocerebrosidase or AQP3, And can exhibit an effect of increasing skin cell regeneration or skin vitality by increasing intracellular mitochondrial activity, exhibiting an antioxidative effect, inhibiting the expression of IL-la, IL-6 or TSLP, Can exhibit an atopy effect. Skin barrier enhancement, skin moisturization, skin cell regeneration, skin vitality increase, antioxidant, antiinflammation or anti-atopy effect of the culture medium of Fushidium coccinium are superior to those of stem cell culture. Therefore, the culture medium of the Fusidium coccinium strain produced by the above method can be used for various purposes such as cosmetics, foods, pharmaceutical compositions and the like.

According to a novel aspect of the present invention, a novel F. coccineum strain can be used for improving skin beauty by applying various activities of the strain.

According to another aspect of the present invention, there is provided a composition for improving skin beauty, comprising the F. coccineum culture broth as a skin barrier enhancement, skin moisturizing, skin cell regeneration, skin vitality enhancement, antioxidant, anti- Can be used.

According to another aspect of the present invention, there is provided a method for producing a fusidium coccinium culture, which comprises culturing the cultured Fusidium coccinium strain in a cosmetic, food, pharmaceutical composition, .

Figure 1 is a graph showing mRNA relative levels of filaggrin. (-): Negative control group. ** p <0.01 vs (-) control, * p <0.05 vs (-) control.
Figure 2 is a graph showing mRNA relative levels of bGC and CerS3. (-): Negative control group.
FIG. 3 is a graph showing the mRNA relative level of the moisturizing factor AQP3. (-): Negative control group.
FIG. 4 is a graph showing mRNA relative levels of cytokine IL-1 a as a result of confirming anti-inflammatory effects. (-): Negative control group.
FIG. 5 is a graph showing the relative level of mRNA of IL-6, a cytokine, as a result of confirming the anti-inflammatory effect. (-): Negative control group.
FIG. 6 is a graph showing mRNA relative levels of TSLP as a result of confirming the anti-atopic effect. (-): Negative control group. (+): Stimulation induction group.
FIG. 7 is a graph showing relative cell viability (% of untreated group) as a result of confirming cell regeneration and vitality effect. Control: Negative control group.
8 is a graph showing antioxidant activity. (-): negative control group, negative control group; AA: positive control group AA treatment group; Fusidium culture medium: treated with 1% (v / v) of fusidium cochineum culture; Stem cell culture: Stem cell culture 1% (v / v) treated group.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One: Fushidium Coincidence ( F. coccineum ) Isolation and Identification of Strain

Dried ginkgo leaves collected from Chungbuk and Jecheon were added to 100 ml of sterilized distilled water and stirred for 1 hour to isolate the microorganisms. The stirred sample was taken and 30 mycelial colonies cultured in potato agar medium (manufactured by Beckton Dickinson, USA) were selected. The selected strains were consigned to Macrogen Co., Ltd., and the Fungi ITS part was selectively analyzed and the homology with the reference strains was analyzed through NCBI Blast. As a result, it was confirmed that the fungus of white mycelia separated by CXHB-8 had a homology of 98% with Fusidium coccineum . Specifically, the previously known Fushidium cochineum standard strain grows to form a white colonies, but the isolated strain forms a pale yellow colon. In addition, while the conventional Fushidium cochinum strain is not suited to nutritional conditions, it can be confirmed that the isolated strain does not grow or grow outside the potato agar medium. Thus, the isolated strain was named Fushidium coccumenum CX-3FU, deposited at the Korean Microorganism Conservation Center on Apr. 14, 2017, and assigned the deposit number KCCM12013P.

Example  2: Fushidium Coincidence ( F. coccineum ) Preparation of culture broth

A culture solution of F. coccineum isolated in Example 1 was prepared.

Specifically, it was confirmed that the isolated fusidium cochineum CX-3FU showed optimal growth environment in the potato agar medium. 5 g of potato agar medium was mixed with 100 ml of sterilized distilled water and sterilized at 121 ° C for 15 minutes. After sterilization, the mixture was cooled to 25 DEG C and 1 g of the separated CX-3FU cells was inoculated. The inoculated medium was incubated at 28 ° C with stirring at 100 rpm and cultured for 7 days. After 7 days, the cells were sterilized by passing through a 0.2 μm filter and used as a culture medium of fusidium coccinium.

Comparative Example  1: Preparation of stem cell culture liquid

Human stem cell culture was prepared. Human-derived stem cells were purchased from ATCC (Rockville, MD, USA) and cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS, ATCC, Rockville, MD, USA) and 1% Antibiotic / Antimycotic solution using the AA, ATCC, Rockville, MD, USA), a DMEM (HyClone, Logan, UT, USA was added) 37, 5%, and cultured in an incubator of CO ₂ conditions. The culture was centrifuged to remove the cells and only the upper layer culture was used.

Experimental Method 1: mRNA  Expression studies (RT- PCR )

HaCaT, a human keratinocyte cell line, was inoculated to a 6-well plate at 5 × 10 ⁵ cells / well and cultured for 24 hours using a medium supplemented with 10% FBS and 1% AA. Then, Fosmidium cochineum culture or stem cell culture solution, Poly I: C, and IL-4 were further cultured in a medium to which no FBS was added for 24 hours. To extract total RNA, 1 mL of RNA iso (Takara Bio Inc., Otsu, Japan) was added to each well to dissolve the cells and 200 μL of chloroform (Sigma-Aldrich, St. Louis, MO) And centrifuged for 10 minutes. The supernatant was transferred to a new tube and the same amount of isopropanol (Sigma-Aldrich, St. Louis, MO) was added and the RNA was isolated by centrifugation at 14,000 rpm for 10 minutes. Centrifuged at 7,500 rpm using 99% ethanol (Sigma-Aldrich, St. Louis, MO), washed twice, dried and dissolved in diethyl pyrocarbonate (DEPC) -water (Ambion, Austin, TX, USA) . RNA was quantitated using Nanodrop 2000 (Thermo Fisher Scientific, Waltham, Mass., USA) and 2 μg of total RNA was heated with DEPC at 70 ° C for 5 minutes, then transferred to a reverse transcription primer (ELPIS-Biotech, Daejeon, Korea) To a final volume of 20 μL. The reaction was carried out at 42 ° C for 55 minutes and at 70 ° C for 15 minutes, and cDNA was synthesized and used for PCR. To amplify genes from the resulting cDNA, add 2 μL of 5-fold diluted cDNA to the StepOne Plus RT-PCR system (10 μL) with 1 μL of primer, 7 μL of DEPC, and 10 μL of SYBR Green Master Mix (Life Technologies, Grand Island, Applied Biosystems, Foster City, CA, USA). The reaction was carried out at 50 ° C for 2 minutes and at 95 ° C for 10 minutes, followed by 40 cycles of 95 ° C for 10 seconds and 60 ° C for 1 minute. Table 1 shows the primer sequences of the genes to be amplified.

gene order
number primer  designation primer  direction primer  order Filaggrin One Filaggrin-F Forward 5'-AGTGCACTCAGGGGGCTCACA-3 ' 2 Filaggrin-R Reverse 5'-CCGGCTTGGCCGTAATGTGT-3 ' Claudin 1 3 Claudin 1-F Forward 5'-GCTCTAGAATTCTCACACGTAGTCTTTCCCGCT-3 ' 4 Claudin 1-R Reverse 5'-GCTCTAGAATTCTCACACGTAGTCTTTCCCGCT-3 ' Claudin 4 5 Claudin 4-F Forward 5'-ACTTTGATAACTGCTCCTCTGAC-3 ' 6 Claudin 4-R Reverse 5'-TTCGTGTCCAGCAGAGTACC-3 ' α-Sphingomyelinase 7 Alpha-Sphingomyelinase-F Forward 5'-ACTTTGATAACTGCTCCTCTGAC-3 ' 8 Alpha-Sphingomyelinase-R Reverse 5'-TTCGTGTCCAGCAGAGTACC-3 ' Ceramide synthase 3 9 Ceramide synthase 3-F Forward 5'-ACATTCCACAAGGCAACCATTG-3 ' 10 Ceramide synthase 3-R Reverse 5'-CTCTTGATTCCGCCGACTCC-3 ' b-Glucocerebrosidase 11 b-Glucocerebrosidase-F Forward 5'-ACCTACACTCTCTGGGGACC-3 ' 12 b-Glucocerebrosidase-R Reverse 5'-TTCAGCCCACTTCCCAGACC-3 ' Hyaluronan synthase 3 13 Hyaluronan synthase 3-F Forward 5'-CTTAAGGGTTGCTTGCTTGC-3 ' 14 Hyaluronan synthase 3-R Reverse 5'-GTTCGTGGGAGATGAAGGAA-3 ' Aquaporin 3 15 Aquaporin 3-F Forward 5'-AGACAGCCCCTTCAGGATTT-3 ' 16 Aquaporin 3-R Reverse 5'-TCCCTTGCCCTGAATATCTG-3 ' IL-6 17 IL-6-F Forward 5'-TACCCCCAGGAGAAGATTCC-3 ' 18 IL-6-R Reverse 5'-TTTTCTGCCAGTGCCTCTTT-3 ' TNF-a 19 TNF-alpha-F Forward 5'-GCTATCTGGTGCCCAGGCTAT-3 ' 20 TNF-alpha-R Reverse 5'-CGACGCCACAATCCTTGTAAT-3 ' TSLP 21 TSLP-F Forward 5'-GCTATCTGGTGCCCAGGCTAT-3 ' 22 TSLP-R Reverse 5'-CGACGCCACAATCCTTGTAAT-3 ' TARC 23 TARC-F Forward 5'-CTTCTCTGCAGCACATCC-3 ' 24 TARC-R Reverse 5'-AAGACCTCTCAAGGCTTTG-3 ' β-Actin
25 Beta-Actin-F Forward 5'-GGCCATCTCTTGCTCGAAGT-3 ' 26 Beta-Actin-R Reverse 5'-GACACCTTCAACACCCCAGC-3 '

Experiment method 3: Data analysis and statistical processing

Group-to-group statistics for cell analysis were Student's t-test. (P <0.05), ** P <0.01, and (+) # P <0.05 and P <0.01 compared to the control group, respectively.

Experimental Method 4: Cell proliferation Acceleration ability  evaluation

In order to compare the cell proliferation promoting activities of the examples and the comparative examples, experiments were carried out with Hs68 fibroblasts, a human fibroblast, using MTT (3- (4,5-dimethylthiazol-2yl) -2,5-diphenyltetrazolium bromide) reagent .

Human fibroblast Hs68 fibroblasts were divided into 96-well plates at a concentration of 2 × 10 ⁴ cells / well and cultured in an incubator at 37 ° C. and 5% CO ₂ for 24 hours. Thereafter, the cell culture medium was replaced with a medium containing 10 μg / m 2 of each of the samples of the examples and the comparative examples, followed by further culturing for 24 hours. After the incubation, the medium of each well was removed, and the DPBS washing procedure was performed once. Then, 0.5 mg / mL MTT reagent was added and reacted at 37 ° C for 4 hours. The formazan crystal formed in the reaction was dissolved in DMSO (dimethyl sulfoxide) after removing the MTT reagent, and the absorbance was measured at 590 nm using a spectrophotometer. Cell viability was expressed as a percentage of the mean absorbance value of the control group added without sample.

Experimental Example  1: Confirm skin barrier strengthening effect

Experiments were conducted to confirm the skin barrier strengthening effect of F. coccineum culture.

Specifically, in accordance with Experimental Method 1, human horny cells were treated with 1% or 5% (v / v) of the Fushymicocinum culture of the Example, or 1% or 5% (v / / v), and then the relative level of mRNA of pilar green was determined.

Figure 1 is a graph showing mRNA relative levels of filaggrin. (-): Negative control group. ** p <0.01 vs (-) control, * p <0.05 vs (-) control.

As shown in FIG. 1, when the expression ratio of filaggrin, which is an important factor in constituting the skin barrier, was analyzed by RT-PCR, it was found that when the Fosmidomocinum culture medium was treated, it was about 8 times that of the Fosmidomocineum culture 1% treatment) and about 10 times (treated with 5% Fushidium cochineum culture). In addition, it was confirmed that the expression rate was more than 8 times that of the stem cell culture solution of the comparative example. Therefore, it was found that the Fusidium cochineum culture enhances the skin barrier enhancement because it increases the expression of filaggreen which promotes the differentiation of keratinocytes and thereby helps to improve the skin barrier.

Experimental Example  2: Confirm skin moisturizing effect

Experiments were conducted to confirm skin moisturizing effect of F. coccineum culture.

2-1. Moisturizing factor CerS3  And bGC's  Confirmation of increased expression

(Ceramide synthase 3: CerS3) and b-glucocerebrosidase (bGC) were increased by the culture medium of Fosmidium cocinum.

Specifically, in accordance with Experimental Method 1, human horny cells were treated with 1% or 5% (v / v) of the Fushymicocinum culture of the Example, or 1% or 5% (v / / v), the relative levels of mRNA of bGC and CerS3 were determined.

Figure 2 is a graph showing mRNA relative levels of bGC and CerS3. (-): Negative control group.

As shown in Fig. 2, when RT-PCR was performed to measure the expression ratios of bGC and CerS3 genes, which are important factors for skin moisturization, it was found that when Fusiodium kosineum culture medium was treated, And 1% treatment of coccinium cultures). However, as the concentration of the treatment increased, the expression rate of the moisturizing factor tended to decrease. In addition, it was confirmed that the expression ratio of the stem cell culture fluid of the comparative example was about twice as good.

2-2. Moisturizing factor AQP3  Confirmation of increased expression

Experiments were conducted to determine if the expression of Aquaporin 3 (AQP3), which is a moisturizing factor, is increased by the fucoidiumcocineum culture.

Specifically, in accordance with Experimental Method 1, human horny cells were treated with 1% or 5% (v / v) of the Fushymicocinum culture of the Example, or 1% or 5% (v / / v), the relative level of mRNA of AQP3 was confirmed. 1 μM of retinol was used as a positive control.

FIG. 3 is a graph showing the mRNA relative level of the moisturizing factor AQP3. (-): Negative control group.

As shown in FIG. 3, in order to measure the expression ratio of AQP3 gene, which is an important factor in skin moisturization, by RT-PCR, it was found that when Fusidium coccinium cultures were treated, about 1.5 times 1% treatment of the culture medium). In the case of AQP3, the expression level was decreased when treated at a high concentration, and thus the optimal expression level was confirmed to be 1% of the Fusidium cochineum culture.

Experimental Example  3: Anti-inflammatory and Anti-atopic  Check the effect

Experiments were carried out to confirm the anti-inflammatory and anti-atopic effects of F. coccineum culture.

3-1. Identification of inhibitory effects of inflammatory factors IL-1a and IL-6

Experiments were performed to confirm the inhibition of the expression of IL-1α and IL-6 genes, which are known to be important cytokines as inflammatory factors, by the culture medium of fosmidium coccineum.

Specifically, in accordance with Experimental Method 1, human horny cells were treated with 1% or 5% (v / v) of the Fushymicocinum culture of the Example, or 1% or 5% (v / / v), the relative levels of IL-1 a and IL-6 mRNA were determined. As a positive control, 1 μM of Dexamethasone (Dex) was used.

FIG. 4 is a graph showing mRNA relative levels of cytokine IL-1 a as a result of confirming anti-inflammatory effects. (-): Negative control group.

FIG. 5 is a graph showing the relative level of mRNA of IL-6, a cytokine, as a result of confirming the anti-inflammatory effect. (-): Negative control group.

As shown in FIG. 4 and FIG. 5, it was confirmed that the fusidium cochineal culture supernatant was about twice as high as that of the positive control group, dexamethasone, for the inhibition of IL-1 a and IL-6. On the other hand, it was confirmed that IL-6 had a higher inhibition rate at a concentration of 1% than the concentration of 5% concentration of Fosmidium cochineum. Therefore, it was found that the fusidium cochineum culture medium had an excellent anti-inflammatory effect.

3-2. Itching factor TSLP's  Confirmation of inhibition

Atopic dermatitis occurs when pruritus occurs, in which TSLP (Thymic Stromal Lymphopoietin) factor plays an important role. RT-PCR was performed to determine the inhibition rate of TSLP by the fusidium oxycinum culture.

Specifically, in accordance with Experimental Method 1, human horny cells were treated with 1% or 5% (v / v) of the Fushymicocinum culture of the Example, or 1% or 5% (v / / v), the relative level of mRNA of TSLP was confirmed. As a positive control, 1 μM of Dexamethasone (Dex) was used.

FIG. 6 is a graph showing mRNA relative levels of TSLP as a result of confirming the anti-atopic effect. (-): Negative control group. (+): Stimulation induction group.

As shown in FIG. 6, in the case of Fosmidomocinum culture solution, the inhibitory effect of TSLP expression was confirmed to be similar to that of the positive control dexamethasone. Therefore, it was found that the fusidium cochineum culture medium had an excellent anti-atopic effect.

Experimental Example  4: Confirm cell proliferation effect

Experiments were carried out to measure the regeneration and vitality of the cells by the Fosmidium cochineum culture.

Specifically, in accordance with Experimental Method 4, fibroblasts were treated with 1% or 5% (v / v) of the fusidium coccinium culture solution of the Example, or 1% or 5% (v / v) ), And relative cell viability was confirmed.

FIG. 7 is a graph showing relative cell viability (% of untreated group) as a result of confirming cell regeneration and vitality effect. Control: Negative control group.

As shown in FIG. 7, the Fusidium cochineum culture solution showed a cell proliferation effect of about 20% or more as compared with the untreated group, and the effect thereof was confirmed to increase in a concentration-dependent manner.

Experimental Example  5: Confirmation of antioxidant effect

Experiments were carried out to confirm the antioxidant effect of Fosmidium cochineum.

Specifically, the antioxidant test was carried out by measuring antioxidative activity using a DPPH solution which was quantitatively discolored by an antioxidant having a radical scavenging activity. First, 0.1 mM DPPH / 80% MeOH was prepared. Next, 0.05 ml of sample and ascorbic acid were added to 2.95 ml of DPPH solution. The mixture was vortexed and left in a dark room at room temperature for 30 minutes. UV was measured at 517 nm.

8 is a graph showing antioxidant activity. (-): negative control group, negative control group; AA: positive control group AA treatment group; Fusidium culture medium: treated with 1% (v / v) of fusidium cochineum culture; Stem cell culture: Stem cell culture 1% (v / v) treated group.

As shown in FIG. 8, the Fusidium cochineum culture showed an antioxidative power of 14% as compared to the untreated group, and the antioxidative capacity of the stem cell culture solution was found to be lower than that of the untreated group.

Korea Microorganism Conservation Center (overseas) KCCM12013 20170414

<110> Cosmax Co., Ltd. <120> Novel strain of Fusidium coccineum spp., And composition for          improving skin beauty comprising a culture solution of the strain <130> PN117190 <160> 26 <170> KoPatentin 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Filaggrin-F primer <400> 1 agtgcactca gggggctcac a 21 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Filaggrin-R primer <400> 2 ccggcttggc cgtaatgtgt 20 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Claudin 1-F primer <400> 3 gctctagaat tctcacacgt agtctttccc gct 33 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Claudin 1-R primer <400> 4 gctctagaat tctcacacgt agtctttccc gct 33 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Claudin 4-F primer <400> 5 actttgataa ctgctcctct gac 23 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Claudin 4-R primer <400> 6 ttcgtgtcca gcagagtacc 20 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Alpha-Sphingomyelinase-F primer <400> 7 actttgataa ctgctcctct gac 23 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Alpha-Sphingomyelinase-R primer <400> 8 ttcgtgtcca gcagagtacc 20 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Ceramide synthase 3-F primer <400> 9 acattccaca aggcaaccat tg 22 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ceramide synthase 3-R primer <400> 10 ctcttgattc cgccgactcc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> b-Glucocerebrosidase-F primer <400> 11 acctacactc tctggggacc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> b-Glucocerebrosidase-R primer <400> 12 ttcagcccac ttcccagacc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hyaluronan synthase 3-F primer <400> 13 cttaagggtt gcttgcttgc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hyaluronan synthase 3-R primer <400> 14 gttcgtggga gatgaaggaa 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Aquaporin 3-F primer <400> 15 agacagcccc ttcaggattt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Aquaporin 3-R primer <400> 16 tcccttgccc tgaatatctg 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-6-F primer <400> 17 tacccccagg agaagattcc 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> IL-6-R primer <400> 18 ttttctgcca gtgcctcttt 20 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha-F primer <400> 19 gctatctggt gcccaggcta t 21 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha-R primer <400> 20 cgacgccaca atccttgtaa t 21 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TSLP-F primer <400> 21 gctatctggt gcccaggcta t 21 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TSLP-R primer <400> 22 cgacgccaca atccttgtaa t 21 <210> 23 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TARC-F primer <400> 23 cttctctgca gcacatcc 18 <210> 24 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TARC-R primer <400> 24 aagacctctc aaggctttg 19 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta-Actin-F primer <400> 25 ggccatctct tgctcgaagt 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta-Actin-R primer <400> 26 gacaccttca acaccccagc 20

Claims (15)

It is isolated from gingko leaves and forms a pale yellow colony and is capable of expressing at least one of filaggrin, ceramide synthase 3, b-glucocerebrosidase and AQP3, and IL-1a, IL-6 and TSLP Fusidium coccineum strain (accession number: KCCM12013P) having the ability to inhibit expression of any one of the above. The present invention relates to a composition for improving skin beauty comprising a culture solution of Fusidium coccineum strain (Accession No .: KCCM12013P) as an active ingredient. The skin cosmetic composition can be used for skin barrier enhancement, skin moisturization, skin cell regeneration, Antioxidant, anti-inflammatory, or anti-atopic. delete The composition for improving skin care according to claim 2, wherein the culture solution is obtained by culturing a strain, and the cells are removed from the culture medium. The composition for improving skin care according to claim 2, wherein the culture liquid contains fusidic acid. The composition for improving skin care according to claim 2, wherein the culture liquid is contained in an amount of 0.01 to 10% by weight based on the total weight of the composition. The composition according to claim 2, wherein the expression of filaggrin is increased. The composition according to claim 2, wherein the expression of ceramide synthase 3, b-glucocerebrosidase, or AQP3 is increased. The composition according to claim 2, wherein the expression of IL-1a, IL-6, or TSLP is inhibited. The composition according to claim 2, wherein the composition is a cosmetic composition. The composition of claim 2, wherein the composition is a food composition. The composition according to claim 2, wherein the composition is a pharmaceutical composition. Culturing the Fusidium coccineum strain (accession number: KCCM12013P) of claim 1 in a potato agar medium;
Centrifuging the cell culture solution obtained after culturing to remove the cells and recovering the supernatant; And
And filtering the recovered supernatant to remove the remaining microbial cells. delete 14. The method according to claim 13, wherein said culturing is carried out at 25 to 30 DEG C for 12 to 120 hours.

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