KR20180114783A - Composition for improving skin beauty comprising extract of fermented soybean fermented with Aspergillus cristatus strain - Google Patents

Abstract

The present invention relates to a composition for improving skin beauty comprising an extract of fermented soybean fermented with an Aspergillus cristatus strain. According to one aspect of the composition for improving the skin beauty, the extract of fermented soybean fermented with the Aspergillus cristatus strain has skin moisturizing, skin barrier enhancing, whitening or skin regenerating properties, thereby being useful for improving the skin beauty.

Description

TECHNICAL FIELD The present invention relates to a composition for improving skin beauty comprising an extract of a fermented soybean fermented by an Aspergillus cristatus strain,

And an extract of fermented soybean fermented by an Aspergillus cristatus strain.

Aspergillus cristatus ) is a microorganism dominated by Fuzhuan brick tea, a post fermented tea in China, and it is called a golden flower fungus because the Chinese form spores like the gold flower. . A. cristatus has been used for a long time in the production of brick tea as a safe strain because it does not produce carcinogenic mycotoxins. In addition, Yongyi Ge et al. (2016) reported that the presence and expression of mycotoxin gene was not observed in A. cristatus isolated from the blastocyst .

Soybean is one of cosmetic raw materials, and it helps skin to be enriched and helps to improve blood circulation, and it gives the skin vitality. In one room, it has the effect of enhancing the immunity of the human body, and it is known that the blood pressure is lowered, blood is removed, blood is removed from the blood, and prevention and improvement of tumor are prevented. Therefore, beans have been used as raw materials for cosmetics.

However, there is no study on fermented soybeans fermented using A. cristatus .

 Youngyi Ge et al., "Comparative genomic and transcriptomic analyzes of the Fuzhan brick tea-fermentation fungus Aspergillus cristatus ", BMC Genomics (2016) 17: 428.

One aspect is that the beans are produced by Aspergillus The present invention also provides a composition for improving skin beauty comprising an extract of fermented soybean fermented with a cristatus strain as an effective ingredient.

Another aspect is that the beans are produced by Aspergillus cristatus ) to produce fermented soybeans; And contacting the fermented soybeans with a C1-C6 alcohol, water, or a mixture thereof.

One aspect is that the beans are produced by Aspergillus The present invention also provides a composition for improving skin beauty comprising an extract of fermented soybean fermented with a cristatus strain as an effective ingredient.

The skin cosmetic improvement may be skin moisturizing, barrier strengthening, wrinkle improvement, whitening, or skin regeneration. Extracts of fermented soybeans fermented with A. cristatus strain of soybeans showed better skin moisturizing effect and barrier enhancement than those of soybean extract and A. cristatus cellulase, A wrinkle improving effect, a whitening effect, or a skin regeneration effect, the composition containing the fermented soybean extract can be used as a composition for skin moisturizing, barrier strengthening, wrinkle improvement, whitening, or skin regeneration.

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

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 "improvement of skin wrinkles" may mean all actions that inhibit the expression of factors related to wrinkles to prevent or improve wrinkles, or inhibit collagenase activity and thereby increase the total amount of collagen.

The term "skin whitening" may mean any action that inhibits the synthesis of melanin to inhibit or prevent skin deposition.

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

The term "improvement" may mean all actions that at least reduce the degree of relief or the parameters associated with the treatment, for example, symptoms.

The term " Aspergillus & cristatus "is a kind of yeast fungus that forms spores like the golden flower and is also called golden flower fungus. The above A. cristatus can be separated from food, soil, And may be, for example, isolated from Fuzhuan brick tea, a post fermented tea of China, for example.

In a composition according to one embodiment, the strain may be an Aspergillus cristatus Cosmax-GF strain (accession number: KCCM11820P). The Aspergillus cristatus Cosmax-GF strain is a novel strain isolated from a zygotic body manufactured in China, specifically, diluting a zygotic body with a NaCl solution; Culturing the diluted zygotes in potato agar medium supplemented with chloramphenicol; And a step of selecting strains which form gold colonies in the medium. The NaCl solution may contain from 0.50% w / w to 0.99% w / w, from 0.60% w / w to 0.95% w / w, from 0.70% w / w to 0.90% w / ), 0.80% (w / w) to 0.90 (w / w), 0.84% (w / w) to 0.86% (w / w), such as 0.85% (w / w) NaCl solution. The culturing can be carried out at a temperature of 20 to 40 DEG C, 25 to 40 DEG C, 25 to 35 DEG C, 27 to 35 DEG C, and 28 to 32 DEG C. The culture may be performed for 10 to 100 hours, 20 to 90 hours, 30 to 80 hours, 40 to 70 hours, 48 to 65 hours, and 48 to 60 hours.

The term "soybean" is a plant of dicotyledonous roots and legumes, which is also called "soybeans ". The soybean can be used without limitation, and for example, various varieties such as black bean, black bean, brown bean, stain bean, and black bean can be used, but the present invention is not limited thereto. The soybeans may be those obtained by a conventionally available method, for example, those sold in the market. The soybean may be sterilized soybean, and the sterilization method may be carried out by a conventional method in the art.

In a composition according to one embodiment, the fermentation may be a solid fermentation. The term "solid fermentation" may mean fermenting microorganisms in a solid state raw material having low water content. The solid fermentation method may be advantageous in that it can be easily recovered without contamination of germs compared with the conventional liquid fermentation method or semi-solid fermentation method.

In the composition according to one embodiment, the fermented soybeans are soybean Aspergillus way may be one which loose Christa tooth (A. cristatus), specifically Aspergillus Christa tooth (A. cristatus) inoculated with the spore suspension on the solid fermentation .

In a composition according to one embodiment, the culture substrate can be converted to a functional material by extracellular enzyme of A. cristatus through the fermentation. Examples of the extracellular enzymes include, but are not limited to, alkaline phosphatase, esterase (C4), esterase lipase (C8), acid phosphatase, Beta-glucosaminidase, alpha-galactosidase, beta-galactosidase, beta-glucosidase, N-acetyl-beta-glucosaminidase ), Alpha-mannosidase, and the like, but are not limited thereto. Therefore, it is important to maintain the phase of conversion from mycelium to spore, which is the highest point of biosynthesis of the enzyme in the life cycle of Aspergillus cristatus . Therefore, in order to maximize the expression phase of extracellular enzyme of A. cristatus to maximize the non-glycosylated component of the soybean, the fermentation temperature, time, and humidity may be suitably selected .

The fermented soybeans may be fermented at 25 to 40 ° C, 25 to 35 ° C, 28 to 35 ° C, 30 to 35 ° C, 31 to 34 ° C, 32 to 34 ° C and 33 ° C. When the fermentation temperature is higher than 35 ° C, the time required for the strain to form spores is increased, so that the enzyme activity and expression level can be relatively lowered. On the contrary, when the temperature is lower than 30 ° C, the enzyme reaction rate is slowed, Can be lowered.

The fermented soybeans may be cultured for 10 to 100 hours, 20 to 100 hours, 20 to 90 hours, 30 to 90 hours, 30 to 80 hours, 40 to 80 hours, 40 to 70 hours, 48 to 65 hours, To 65 hours, 60 to 65 hours. The fermentation time can be appropriately selected in accordance with the fermentation temperature so as to terminate until spores are formed in consideration of the growth of hyphae.

Wherein the fermented soybeans have a moisture content of 10 to 99.9%, a moisture content of 20 to 99.9%, a moisture content of 30 to 99.9%, a moisture content of 40 to 99.9%, a moisture content of 50 to 99.9%, a moisture content of 50 to 90%, a moisture content of 60 to 90% Humidity, 75 to 85% humidity, and 80 to 85% humidity. If the humidity is less than 75%, the time for spore formation of the strain becomes faster so that the enzyme activity and expression amount can be lowered. On the contrary, when the humidity is higher than 85%, the moisture content is high, The growth of the strain A. cristatus may be slowed and the rate of contamination by other microorganisms may be increased.

For example, the fermented soybeans may be fermented at 30 to 35 DEG C for 48 to 65 hours at 75 to 85% humidity.

The fermented soybeans may be the entire fermented soybean, a part thereof, or a material derived therefrom. The fermented soybeans used for the extraction may be the whole, a part thereof, or a material derived therefrom, which may be pulverized or cut or suitably dried. The fermented soybeans may be dried to have a water content of less than 20%, less than 15%, such as less than 12%.

The extract may be extracted with a hydrophilic solvent, for example, alcohol, water, or a combination thereof. The alcohol may be a compound having at least one-OH group of C1 to C10. The alcohol may be a C1 to C6 alcohol, or a C3 to C6 polyhydric alcohol. The alcohol may be methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, isobutanol, tert-butanol, n-pentanol, n-hexanol or mixtures thereof. The solvent may be, for example, a mixture of water and an alcohol, that is, an aqueous solution of an alcohol. The alcohol concentration of the alcohol aqueous solution may be 1 to 100% (w / w), such as 1 to 99.5% (w / w), 10 to 100% (w / w), 20 to 100% (w / (W / w), 40 to 100% (w / w), 50 to 100% (w / w), 60 to 100% (W / w), 60 to 90% (w / w), 60 to 80% (w / w), 65 to 75% have. The aqueous alcohol solution may be methanol, ethanol, or an aqueous butanol solution. In a composition according to one embodiment, the extract may be an ethanol extract.

The extract may be extracted by a conventional method in the art such as warm extraction, pressure extraction, ultrasonic extraction, hot water extraction, reflux cooling extraction, subcritical extraction, or supercritical extraction.

The extraction solvent may be selected from the group consisting of 5 to 15 (volume / weight) times, such as 5 to 13 (volume / weight), 5 to 11 (volume / , 8 to 13 (volume / weight) times, 8 to 11 (volume / weight) times, or 9 to 11 (volume / weight) times. For example, 0.5 to 1.5 L of the extraction solvent may be added to 100 g of the whole fermented soybean, a part thereof, or a material derived therefrom.

The extraction may be carried out at a temperature of 4 ° C to 70 ° C, for example 4 ° C to 50 ° C, 4 ° C to 40 ° C, 4 ° C to 30 ° C, 10 ° C to 70 ° C, 15 ° C to 70 ° C, 10 C to 50 C, 10 C to 50 C, 4 to 40 C, 4 to 30 C, 10 to 40 C, 10 to 35 C, or 10 to 30 C or room temperature .

The extraction time can be appropriately selected according to the selected temperature and extraction method. For example, the extraction time may be from 1 hour to 3 days, from 1 hour to 2 days, from 1 hour to 1 day, from 5 hours to 3 days, from 5 hours to 2 days, from 5 hours to 1 day, from 10 hours to 3 days, from 15 Hour to 2 days, 15 hours to 36 hours, 18 hours to 30 hours, 1 day to 3 days, 1 day to 2 days, or 24 hours.

For example, the fermented soybean powder can be extracted by ultrasonic extraction by immersing the fermented soybean powder in the extraction solvent for 2 to 4 hours, or by immersing in a solvent for 24 to 72 hours at room temperature. The extraction may be one or more times, for example, one to five times, one to four times, one to three times, two to five times, or two to four times, each extraction being performed in the same way, It can be performed in other ways.

The above extraction can be performed by separating the plant residue and the extract by a known method such as filtration. The extraction can also include removing the solvent from the resulting extract by known methods such as concentration under reduced pressure. The extraction may also comprise preparing the dried extract by drying, such as lyophilization, of the resulting extract.

The extract may contain from 0.001% to 80%, such as from 0.01% to 60%, from 0.01% to 40%, from 0.01% to 30%, from 0.01% to 20% %, 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%, or 0.1 wt% to 5 wt%.

The composition according to one embodiment of the present invention may contain, as an active ingredient, a fraction of the fermented soybean extract instead of the fermented soybean extract. The composition according to one embodiment may further comprise a fraction of the extract of the fermented soybean. The term "fraction" refers to a substance in which the extract of the fermented soybean is divided into its components, that is, a fractionated substance. The fraction may be obtained by solvent fractionation. The solvent fractionation may be a step of mixing the fermented soybean extract with a solvent and separating the substance present in the solvent. The fraction may be a methylene chloride fraction, an ethyl acetate fraction, a butanol fraction, or a water fraction obtained by suspending the fermented soybean extract in water and sequentially fractionating the fraction with methylene chloride, ethyl acetate, butanol, and water.

Specifically, the methylene chloride fraction is prepared by mixing the fermented soybean extract with water, mixing the mixture with methylene chloride, allowing the mixture to stand for a predetermined time, separating the methylene chloride layer, separating the separated methylene chloride layer , ≪ / RTI > Separation of the fractions may include removing methylene chloride from the methylene chloride layer. Further, the ethyl acetate fraction was obtained by mixing the remaining water after the methylene chloride fraction was separated with ethyl acetate, leaving it for a predetermined time, separating the ethyl acetate layer, and separating the fraction from the separated ethyl acetate layer Lt; / RTI > Separation of the fractions can include removal of the ethyl acetate from the ethyl acetate layer. In addition, the butanol fraction may be obtained by mixing water remaining after the ethyl acetate fraction is separated again with butanol, leaving the butanol layer separated for a predetermined time, and separating the fraction from the separated butanol layer. Separation of the fractions may include removing butanol from the butanol layer. Conditions for such fractionation, such as temperature conditions, pressure conditions, time, amount or concentration of solvent used, stirring, etc., may be as described for the extracts used to prepare the above fermented soybean extract. The fractionation may be repeated one or more times, for example, 1 to 5 times.

Separation of the fractions may be accomplished by known methods such as filtration. The fractionation may also include removing the solvent from the fraction obtained by known methods such as concentration under reduced pressure. The fractionation may also include concentration and / or drying of the obtained fractions. The concentration may be reduced pressure concentrated. The drying may include vacuum drying, boiling drying, spray drying, room temperature drying or freeze drying.

The composition according to one embodiment may be one that increases the expression of HAS3, or AQP3, and more specifically, that increases the expression of HAS3, or AQP3, to exhibit skin moisturizing effects.

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 be one that inhibits the expression of MMP-1, specifically, to suppress the expression of MMP-1 to improve skin wrinkles.

The composition according to one embodiment may be one that inhibits tyrosinase activity, specifically, exhibits a skin whitening effect by inhibiting melissin activity.

The composition according to one embodiment may be one which promotes proliferation of fibroblasts, specifically, one that exhibits a skin regeneration effect by promoting proliferation of fibroblasts.

The composition according to one embodiment may comprise the extract 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 extract 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 moisturizer 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 extract 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 pigmentation, skin wounds, skin scars, or skin inflammation. 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, improving skin wrinkles, maintaining skin moisture, preventing loss of skin moisture, and regenerating skin.

The skin pigmentation disorder may mean all diseases caused by skin pigmentation due to increased production of melanin. Examples include, but are not limited to, spots, freckles, spots, daylight pigmentation, pigmentation after drug use, post-inflammatory pigmentation, and pregnancy pigmentation. The pharmaceutical composition according to one embodiment can prevent or treat skin pigmentation diseases by inhibiting tyrosinase activity.

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, and skin regeneration.

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 inflammatory diseases by the effects of maintaining skin moisture, preventing loss of skin moisture, strengthening skin barrier, and regenerating skin.

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 effectively maintaining skin moisture or preventing moisture loss of an individual, a method of enhancing a skin barrier function of an individual, a method of improving skin wrinkles of an individual, a method of improving skin tone of an individual, Or to treat or prevent diseases caused by pigment increase, or to improve or enhance skin regeneration 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 of delivery of at least a portion of the extract or extract components of the composition according to one embodiment to the desired location in the living individual.

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 requiring an improvement in skin care, for example, skin moisturizing, barrier strengthening, wrinkle improvement, whitening, or skin regeneration.

The administration may be carried out in an amount of 0.1 mg to 1,000 mg, such as 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 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 that the beans are produced by Aspergillus cristatus ) to produce fermented soybeans; And contacting the fermented soybeans with a C1-C6 alcohol, water, or a mixture thereof.

The above-mentioned extracts of soybean, A. cristatus strain, fermentation, fermented soybean, and fermented soybean are the same as described above.

In a method according to one embodiment, the strain may be an Aspergillus cristatus Cosmax-GF strain (accession number: KCCM11820P).

In a method according to one embodiment, the fermentation may be a solid fermentation.

In a method according to one embodiment, the fermentation can be performed at 30-35 < 0 > C for 48-65 hours at 75-85% humidity conditions.

The extract of the fermented soybean produced by the above method can increase the expression of HAS3 or AQP3 to exhibit skin moisturizing effect, increase the expression of filaggrin to enhance the skin barrier, inhibit the expression of MMP-1 Skin wrinkles can be improved, tyrosinase activity can be suppressed, skin whitening effect can be exhibited, and proliferation of fibroblasts can be promoted, and skin regeneration effect can be exhibited. The skin moisturizing, barrier strengthening, wrinkle improvement, whitening, or skin regeneration effects of such fermented soybean extracts are superior to those of non-fermented soybean extracts or A. cristatus cell lysate. Therefore, the fermented soybean extract prepared by the above method can be used for various purposes such as cosmetics, foods, pharmaceutical compositions, and the like.

According to the composition for improving skin beauty according to one aspect, the extract of fermented soybean fermented by A. cristatus strain can be used for skin moisturizing, barrier strengthening, wrinkle improvement, whitening, skin regeneration have.

According to another aspect of the present invention, it is possible to produce an extract of fermented soybeans having an effect of improving the skin's beauty, so that the extract can be applied to various fields such as cosmetics, foods, and pharmaceutical compositions.

1 is a photograph showing fermented soybeans solid-cultured with A. cristatus Cosmax-GF (KCCM11820P).
2 is a graph showing the mRNA relative level of the moisturizing factor HAS3. (-): keratinocytes not treated with negative controls; Retinol: a group treated with retinol, which is a positive control; Comparative Example 1: soybean extract treatment group; Comparative Example 2: Aspergillus cristatus cell lysate treatment group; And Example 2: fermented soybean treated group.
3 is a graph showing the mRNA relative level of the moisturizing factor AQP3. (-): keratinocytes not treated with negative controls; Retinol: a group treated with retinol, which is a positive control; Comparative Example 1: soybean extract treatment group; Comparative Example 2: Aspergillus cristatus cell lysate treatment group; And Example 2: fermented soybean treated group.
FIG. 4 is a graph showing the mRNA relative level of filaggrin. (-): keratinocytes not treated with negative controls; Comparative Example 1: soybean extract treatment group; Comparative Example 2: Aspergillus cristatus cell lysate treatment group; And Example 2: fermented soybean treated group.
FIG. 5 is a graph showing the relative concentration (%) of total MMP-1 as a result of confirming the effect of suppressing the expression of MMP-1 as a wrinkle factor. (-) is fibroblasts not treated with TNF-α, and the second to sixth bars are fibroblasts treated with TNF-α to induce an aging reaction. Retinol: a group treated with retinol, which is a positive control; Comparative Example 1: soybean extract treatment group; Comparative Example 2: Aspergillus cristatus cell lysate treatment group; And Example 2: fermented soybean treated group.
6 is a graph showing the intracellular tyrosinase activity (%) as a result of confirming the melanin production inhibitory effect. Con is a pigmented cell not treated with α-MSH, and the second to sixth bars are pigment cells that induce melanogenesis by treating α-MSH. Kojic acid: a positive control group treated with kojic acid; Comparative Example 1: soybean extract treatment group; Comparative Example 2: Aspergillus cristatus cell lysate treatment group; And Example 2: fermented soybean treated group.
FIG. 7 shows fluorescence microscopic observation of the degree of proliferation of cells after 24 hours after scraping of cells as a result of confirming the skin cell regeneration effect. Con: negative control; Comparative Example 1: soybean extract treatment group; Example 2: Fermented soybean 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 1. Aspergillus cristatus ( A. cristatus ) Isolation and Identification of Strain

Ten grams of a bicomponent manufactured in China was appropriately serially diluted with a 0.85% (w / w) NaCl solution and applied to a Potato Dextrose Agar (PDA, Himedia, India, M1942) supplemented with chloramphenicol And then cultured at 30 DEG C for 60 hours. Among the dominant strains, strains forming gold colonies were selected and identified. Identification was made by Charles River Laboratories Korea, Inc. and analyzed the Internal Transcribed Spacer (ITS2) part. As a result, 99.9% homology with Aspergillus cristatus was obtained and used in the present invention. The isolated strain was named A. cristatus Cosmax-GF, deposited at the Korean Microorganism Conservation Center on March 22, 2016, and assigned the deposit number KCCM11820P.

Example 2. Preparation of Fermented Soybeans and Extracts

Soybean was fermented with A. cristatus isolated in Example 1 to prepare fermented soybeans, and fermented soybean extract was prepared.

Specifically, the beans used for the solid fermentation were prepared by washing after purchase in the market. The washed soybeans were sterilized at 121 DEG C for 30 minutes. Then, A. cristatus of Example 1, which had been grown on a potato agar medium, was prepared as a spore suspension, and the suspension was added to the sterilized soybeans and incubated at 33 ° C. for 60 to 65 hours at 75 to 85% Lt; / RTI > The solid fermented soybean was dried and pulverized at 60 ° C until the moisture content was less than 12%.

Powdered fermented soybeans (100 g) were placed in an extraction vessel, and immersed in 1 L of a 70 wt% ethanol aqueous solution as an extraction solvent and left at room temperature for 24 hours to obtain an extract. The supernatant, which was centrifuged at 3000 rpm for 10 minutes, was filtered through a 0.45 μm membrane to prepare a filtrate. The filtrate was concentrated under reduced pressure using a rotary evaporator to obtain 9 to 11 g of concentrated extract. 1 is a photograph showing fermented soybeans solid-cultured with A. cristatus Cosmax-GF (KCCM11820P).

Comparative Example 1. Preparation of soybean extract

100 g of pulverized soybeans were placed in an extraction vessel and immersed in 1 L of a 70 wt% ethanol aqueous solution as an extraction solvent and left at room temperature for 24 hours to obtain an extract. The supernatant, which was centrifuged at 3000 rpm for 10 minutes, was filtered through a 0.45 μm membrane to prepare a filtrate. The filtrate was concentrated under reduced pressure using a rotary evaporator to obtain 9 to 10 g of concentrated extract.

Comparative Example 2 Aspergillus cristatus ( A. cristatus ) Preparation of cell lysate

A. cristatus isolated in Example 1 was inoculated on a plate cultured in a potato agar medium (PDA) at 33 ° C. for 4 days in a buffer (Tris 5 mmol / L, pH 7.5) 15 ml Was added thereto and scraped with a cell scraper to obtain a suspension of A. cristatus cells. The cell suspensions obtained from the cultured plates were centrifuged at 4,000 rpm at 4 ° C for 10 minutes to recover the cells. The recovered cells were washed twice by suspending and centrifuging in buffer twice. To 6.5 g of the washed cells, 15 ml of 4 ° C buffer (Tris 50 mmol / L, pH 7.5) and 2 g of 0.5 mm glass beads (Sigma) were added and vortexed for 3 minutes. The sample was left on ice for 2 minutes, cooled, and then vortexed for 3 minutes. The cells were further physically disrupted by repeating this twice. The homogenate was centrifuged at 6,500 rpm at 4 ° C for 15 minutes and filtered through 0.45 μm membrane.

Experimental Example 1. Confirmation of Increasing Effect of Moisturizing Factor on Fermented Soybean

Experiments were conducted to confirm the effect of increasing the moisturizing function of fermented soybeans.

First, human keratinocytes were inoculated in a 6-well cell culture dish at a density of ⁵ × 10 ⁵ , and cultured in a 5% CO ₂ incubator at 37 ° C. for 24 hours. Then, the samples of Comparative Example 1, Comparative Example 2 and Example 2 were diluted in the culture medium to a concentration of 50 μg / ml, and treated with the corresponding wells for 24 hours. Then, RNA was isolated from the cells using RNA iso (DAKARA, Japan) solution. The amount of each RNA was quantified using Nanodrop 2000 (Thermo, USA) and then 55 minutes at 42 ° C and 15 minutes at 70 ° C (Reverse Transcriptase Mix, ELPIS biotech, Korea). The synthesized cDNA was amplified by real-time PCR using a mixture of a target protein HAS3 (Hyaluronic acid synthase-3), a template of AQP3 (Aquaporins 3) and a cyanine dye CyBR Green supermix (Applied Biosystems, USA) time PCR machine (Step One Plus, Applied Biosystems, USA) to evaluate the expression level of HAS3 and AQP3 genes. The primer sequences used in the real-time PCR were shown in Table 1 below. The real-time PCR reaction was carried out at 94 ° C for 5 minutes, after activation of the polymerase, for 40 cycles at 95 ° C for 30 seconds, 54 ° C for 1 minute and 72 ° C for 1 minute Respectively. The expression level of the gene was finally analyzed by correction for the β-actin gene.

gene SEQ ID NO: Name of the primer Primer direction Primer sequence HAS3 One HAS3-F Forward 5'-CTTAAGGGTTGCTTGCTTGC-3 ' 2 HAS3-R Reverse 5'-GTTCGTGGGAGATGAAGGAA-3 ' AQP3 3 AQP3-F Forward 5'-AGACAGCCCCTTCAGGATTT-3 ' 4 AQP3-R Reverse 5'-TCCCTTGCCCTGAATATCTG-3 ' β-Actin 5 Beta-Actin-F Forward 5'-GGCCATCTCTTGCTCGAAGT-3 ' 6 Beta-Actin-R Reverse 5'-GAGACCTTCAACACCCCAGC-3 '

2 is a graph showing the mRNA relative level of the moisturizing factor HAS3. (-): keratinocytes not treated with negative controls; Retinol: a group treated with retinol, which is a positive control; Comparative Example 1: soybean extract treatment group; Comparative Example 2: Aspergillus cristatus cell lysate treatment group; And Example 2: fermented soybean treated group.

3 is a graph showing the mRNA relative level of the moisturizing factor AQP3. (-): keratinocytes not treated with negative controls; Retinol: a group treated with retinol, which is a positive control; Comparative Example 1: soybean extract treatment group; Comparative Example 2: Aspergillus cristatus cell lysate treatment group; And Example 2: fermented soybean treated group.

As shown in Figs. 2 and 3, the expression of HAS3, which is a moisturizing factor, in the fermented soybean-treated group of Example 2 was greatly increased as compared with Comparative Examples 1 and 2. In addition, the fermented soybean-treated group of Example 2 also markedly increased the expression of AQP3 and was found to be equivalent to that of the positive control group. Therefore, it was found that the fermented soybean had an excellent effect of increasing the moisturizing function.

Experimental Example 2 Confirmation of Skin Barrier Strength of Fermented Soybeans

Experiments were conducted to confirm the skin barrier strengthening effect of fermented soybeans.

First, human keratinocytes were inoculated in a 6-well cell culture dish at a density of ⁴ × 10 ⁵ , and cultured in a 5% CO ₂ incubator at 37 ° C. for 24 hours. Thereafter, the samples of Comparative Example 1, Comparative Example 2 and Example 2 were diluted in a culture medium to a concentration of 50 μg / ml, treated in the respective wells, and then cultured for an additional 24 hours. Then, RNA was isolated from the cells using RNA iso (DAKARA, Japan) solution. The amount of each RNA was quantified using Nanodrop 2000 (Thermo, USA) and then 55 minutes at 42 ° C and 15 minutes at 70 ° C (Reverse Transcriptase Mix, ELPIS biotech, Korea). The synthesized cDNA was amplified using a real-time PCR machine (Step One Plus, Applied Biosystems, USA) using a mixture of a filaggrin template and a cyanine dye (SYBR Green supermix, Applied Biosystems, USA) USA), and the degree of expression of the fila green gene was finally evaluated by performing a real-time PCR reaction. In the real-time PCR, real-time PCR was carried out at 95 ° C for 30 seconds, 54 ° C for 1 minute, and 72 ° C for 1 minute after activation of the polymerase at 94 ° C for 5 minutes. . The expression level of the gene was finally analyzed by correction for the β-actin gene.

gene SEQ ID NO: primer  designation primer  direction Primer sequence Filaggrin 7 Filaggrin-F Forward 5'-GGCACTGAAAGGCAAAAAGG-3 ' 8 Filaggrin-R Reverse 5'-AAACCCGGATTCACCATAATCA-3 '

FIG. 4 is a graph showing the mRNA relative level of filaggrin. (-): keratinocytes not treated with negative controls; Comparative Example 1: soybean extract treatment group; Comparative Example 2: Aspergillus cristatus cell lysate treatment group; And Example 2: fermented soybean treated group.

As shown in Fig. 4, the expression of filaggrin in the fermented soybean-treated group of Example 2 was much higher than that of Comparative Examples 1 and 2. Therefore, it was found that the fermented soybean had a very excellent skin barrier strengthening effect.

Experimental Example 3. Confirmation of Inhibitory Effect of Fermented Soybean on MMP-1 Production

Experiments were conducted to confirm wrinkle improvement and wrinkle suppression effect of fermented soybeans.

First, human fibroblasts were inoculated on a 48-well plate at a density of ⁴ × 10 ⁵ , and then cultured in a 5% CO ₂ incubator at 37 ° C. for 24 hours. Thereafter, the aging reaction was induced by using 10 ng / ml of TNF-α, and 20 μg / ml of retinol as a positive control and 100 μg / ml of the samples of Comparative Example 1, Comparative Example 2, And treated with the respective wells, followed by incubation for an additional 48 hours. Subsequently, the culture medium was recovered and the amount of MMP-1 released into the medium was assayed by using the MMP-1 kit (Human Total MMP-1 DY901), antigen-antibody reaction, blocking, , And the absorbance was measured at 450 nm. The absorbance value was corrected by cell number and the total MMP-1 amount by concentration was calculated for each condition.

FIG. 5 is a graph showing the relative concentration (%) of total MMP-1 as a result of confirming the effect of suppressing the expression of MMP-1 as a wrinkle factor. (-) is fibroblasts not treated with TNF-α, and the second to sixth bars are fibroblasts treated with TNF-α to induce an aging reaction. Retinol: a group treated with retinol, which is a positive control; Comparative Example 1: soybean extract treatment group; Comparative Example 2: Aspergillus cristatus cell lysate treatment group; And Example 2: fermented soybean treated group.

As shown in FIG. 5, when the aging reaction was induced by TNF-α, the production amount of MMP-1, which is a pleat factor in fibroblasts, was increased. However, the fermented soybean- It was confirmed that the amount of MMP-1 produced was significantly lower than that of the Aspergillus cristatus cell disruption treated group of Comparative Example 2. [ Thus, it was found that the fermented soybean of Example 2 had an excellent wrinkle suppressing effect.

Experimental Example 4. Determination of whitening effect of fermented soybeans

As an experiment for confirming the whitening effect of the fermented soybeans, an experiment was conducted to confirm the inhibitory effect of tyrosinase activity in cells.

First, pigment cells (B16F10) were inoculated on a 12-well plate and cultured for 24 hours. Subsequently, the medium was replaced and each sample was treated with 100 ng / ml of α-melanocyte stimulating hormone (α-MSH) 10 nM and cultured for 48 hours. After washing with phosphate buffered saline (PBS), the cells were recovered by centrifugation at 5,000 rpm for 5 minutes. The recovered cells were dissolved in 0.1 M sodium phosphate buffer (pH 7.0) containing 1% Triton x 100 and sonicated at 4 ° C for 1 hour to break the cells. The supernatant was recovered by centrifugation at 13,000 rpm for 20 minutes. 40 μl of 5 mM L-DOPA and 0.1 M PBS (pH 6.8) was added to 40 μg of the same protein through protein quantification for each sample, and reacted at 37 ° C. in a 96-well plate. Then, the absorbance at 475 nm was measured using an ELISA microplate reader to confirm the activity of the tyrosinase enzyme.

6 is a graph showing the intracellular tyrosinase activity (%) as a result of confirming the melanin production inhibitory effect. Con is a pigmented cell not treated with α-MSH, and the second to sixth bars are pigment cells that induce melanogenesis by treating α-MSH. Kojic acid: a positive control group treated with kojic acid; Comparative Example 1: soybean extract treatment group; Comparative Example 2: Aspergillus cristatus cell lysate treatment group; And Example 2: fermented soybean treated group.

As shown in FIG. 6, it was confirmed that the tyrosinase activity in the fermented soybean treated group of Example 2 was lowered to a higher level than that of Comparative Examples 1 and 2 and the positive control group. Thus, it was found that the fermented soybean of Example 2 had a very excellent whitening effect.

Experimental Example 5. Confirmation of skin cell regeneration effect of fermented soybean

Experiments were conducted to confirm the skin cell regeneration effect of fermented soybeans.

First, human fibroblasts were inoculated in a 6-well cell culture dish at a density of ⁴ x 10 ⁵ , and then cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. After the center of the cultured cells was scraped off using a cell scraper, the samples of Comparative Example 1 or 2 were added and cultured for 24 hours, and the degree of cell proliferation was photographed using a fluorescence microscope.

FIG. 7 shows fluorescence microscopic observation of the degree of proliferation of cells after 24 hours after scraping of cells as a result of confirming the skin cell regeneration effect. Con: negative control; Comparative Example 1: soybean extract treatment group; Example 2: Fermented soybean treated group.

As shown in Fig. 7, it was confirmed that the fermented soybean of Example 2 had much better cell proliferation than Comparative Example 1. Therefore, it was found that the fermented soybean of Example 2 had excellent skin cell regeneration effect.

Korea Microorganism Conservation Center (overseas) KCCM11820 20160322

<110> Cosmax Co., Ltd. <120> Composition for improving skin          fermented soybean fermented with Aspergillus cristatus strain <130> PN117163 <160> 8 <170> KoPatentin 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HAS3-F primer <400> 1 cttaagggtt gcttgcttgc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HAS3-R primer <400> 2 gttcgtggga gatgaaggaa 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AQP3-F primer <400> 3 agacagcccc ttcaggattt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AQP3-R primer <400> 4 tcccttgccc tgaatatctg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta-Actin-F primer <400> 5 ggccatctct tgctcgaagt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta-Actin-R primer <400> 6 gagaccttca acaccccagc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Filaggrin-F primer <400> 7 ggcactgaaa ggcaaaaagg 20 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Filaggrin-R primer <400> 8 aaacccggat tcaccataat ca 22

Claims (19)

Aspergillus The present invention relates to a composition for improving skin beauty comprising an extract of fermented soybean fermented by a cristatus strain as an active ingredient. The composition according to claim 1, wherein the improvement in skin care is skin moisturizing, barrier strengthening, wrinkle improvement, whitening, or skin regeneration. The composition of claim 1, wherein the strain is an Aspergillus cristatus Cosmax-GF strain (accession number: KCCM11820P). The composition of claim 1, wherein the fermentation is a solid fermentation. The composition for improving skin care according to claim 1, wherein the fermented soybean is fermented at 30 to 35 DEG C for 48 to 65 hours at 75 to 85% humidity. The composition of claim 1, wherein the extract is an extract of C1-C6 alcohol, water, or a mixture thereof. The composition of claim 1, wherein the extract is an ethanol extract. The composition for improving skin care according to claim 1, wherein the extract is an extract obtained by immersing the fermented soybeans in an extraction solvent at room temperature. The composition for improving skin care according to claim 1, wherein the extract is contained in an amount of 0.001 to 10% by weight based on the total weight of the composition. The composition according to claim 1, wherein the expression of HAS3 or AQP3 is increased. The composition according to claim 1, wherein the expression of filaggrin is increased. The composition according to claim 1, wherein the expression of MMP-1 is inhibited. The composition according to claim 1, wherein the tyrosinase activity is inhibited. The composition of claim 1, further comprising a cosmetically, pharmaceutically or pharmaceutically acceptable excipient or carrier. The composition of claim 1, wherein the composition is a cosmetic, a food, or a pharmaceutical composition. Aspergillus cristatus ) to produce fermented soybeans; And
Contacting said fermented soybeans with an alcohol, water, or a mixture thereof of C1-C6. The method according to claim 16, wherein the strain is an Aspergillus cristatus Cosmax-GF strain (accession number: KCCM11820P). 17. The method of claim 16, wherein the fermentation is a solid fermentation. 17. The method of claim 16, wherein the fermentation is performed at 75-85% humidity conditions at 30-35 &lt; 0 &gt; C for 48-65 hours.

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