KR20190071883A - Cosmetic composition for prevention of skin-wrinkle containing low-molecular weight peptide through microbial fermentation of bilberry fruit - Google Patents

Abstract

The present invention relates to a cosmetic composition for skin protection and wrinkle reduction containing a low molecular peptide extract through purification after bilberry fruit fermentation using microorganisms. More particularly, the present invention relates to a cosmetic composition for protecting skin and reducing wrinkles, containing a microbial extract obtained by the fermentation of bilberry fruit, which is fermented with a fermenting microorganism (Saccharomyces cerevisiae), and the microorganism is purified to increase active ingredients by lowering the molecular weight to a peptide of a certain size or less.

Description

TECHNICAL FIELD The present invention relates to a cosmetic composition for prevention of skin wrinkles containing low-molecular peptides derived from microbial fermentation of fruit of bilberry,

The present invention relates to a skin protecting and wrinkle-improving cosmetic composition containing a low-molecular peptide extract through purification after the fermentation of a bilberry fruit using microorganisms, and more particularly to a cosmetic composition for improving the skin protection and wrinkle of cosmetic compositions comprising a fermenting microorganism ( Saccharomyces The present invention relates to a cosmetic composition for protecting skin and improving wrinkles containing a microbial extract through fermentation with a microbial fermentation method comprising fermenting the microorganism with a peptide of a certain size or less and increasing an effective ingredient.

Bilberry ( Vaccinium Myrtillus ) is a deciduous broad-leaved shrub distributed mainly in Europe, northern Asia, Greenland, Western Canada, and the western United States. The height is about 15 ~ 50cm, the stem is yellow and the young branch is green. Leaves are ovate to elliptical, 2 ~ 3cm long, 1 ~ 1.5cm wide, rarely keratinized on the back side and vein of the leaf, and the edge has a teeth angle. From late summer to autumn, the leaves are red. The flowers bloom in May and June come out one by one from the leaves, face downwards, and just like shape, white to light red. The fruit ripened from July to September is round and dark blue, with a maximum diameter of about 2cm.

Bilberry fruit is sometimes called European blueberry or wild blueberry. Bilberry began to gain more attention after the British Air Force pilots who consumed Bilberry jam as much as the thickness of the bread during World War II reported good night vision and helped in the battle with Germany. In fact, some say that Germany has created false rumors to hide the fact that the UK is using radar technology that it does not have, but it actually prevents bilberries from attacking reactive oxygen species, especially in retinal maculae, It has been shown that it has a good role to alleviate visual impairment such as vision, cataract, diabetes, and glaucoma, and even restore vision to its original state.

The main ingredient in Bilberry that helps to improve eye health is anthocyanins (or anthocyanosides). Anthocyanin is an antioxidant that plants produce to protect themselves from ultraviolet rays and viruses. Anthocyanin protects blood vessels by antioxidant action and increases blood circulation in microvessels, which helps blood and nutrition in the eyes.

Since aging is caused by the oxidation of cells, the notion that antioxidants, which prevent oxidation, can prevent aging and its diseases has long been widely recognized in academia. In the US Department of Agriculture, Bilberry has been reported to have the highest antioxidant properties of the 20 fresh fruits and berries, with the highest oxygen radical elimination. In addition to anthocyanins, vitamin C, vitamin E, and resveratrol (antioxidants in red wine) play a role in the antioxidant capacity of bilberry. The fruit contains 3 to 4 times anthocyanin and flavonoid compound compared with blueberry. It is used in various diets as a healthy fruit together with blueberry. It is useful for improving blood circulation, sight recovery and diarrhea, and is commercialized as a functional food. Leaves have long been used for diabetes, retinopathy due to sugar beet, and high blood pressure.

Korean Patent Laid-Open No. 10-2013-0085012 discloses a process for producing a Chinese cabbage such as ginseng, licorice, rice, barley, soybean, cabbage, lettuce, spinach, grape, apple, blueberry, strawberry, raspberry, , A plant selected from the group consisting of berry, rosehip, lemongrass, cape chamomile, mung bean, red bean, plum, plum, asai, gugija, omija, coriander, angelica, golden bamboo shoot, sage, mung bean, And a fermented product thereof as an active ingredient.

Thus, the present inventors have sought to find a method of obtaining a peptide component useful for skin as much as possible at a higher yield than the above-mentioned prior art, and strive to apply it to cosmetics. As a result, it has been found that the method of producing a bilberry fruit fermented microorganism extract as a peptide can produce a peptide content as a skin-useful component at a high concentration, and shows excellent skin protection and wrinkle-reducing effect when applied to cosmetics I got it.

Accordingly, an object of the present invention is to provide a cosmetic composition for protecting skin and improving wrinkles containing a low-molecular peptide through fermentation of microbial fruit of bilberry fruit.

The objects and advantages of the present invention will become more apparent from the detailed description and the claims that follow.

The present invention relates to a skin containing a low-molecular peptide through fermentation of microbial fruit of a bilberry fruit, characterized in that it contains a peptide obtained by extracting the fermenting microorganism after fermentation of the fruit of Bilberry with Saccharomyces cerevisiae (KCTC 7904) Protecting and wrinkle-improving cosmetic composition.

Preferably, the cosmetic composition is obtained by filtering the fermented microorganism obtained by fermenting the fruit of Bilberry with Saccharomyces cerevisiae (KCTC 7904), purifying to contain only 100,000 daltons to 500,000 daltons of the peptide, and concentrating Followed by drying.

Preferably, the cosmetic composition has a flexible lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder formulation.

Preferably, the cosmetic composition has an effect of improving wrinkles.

Preferably, the cosmetic composition has an antioxidative effect.

Preferably, the cosmetic composition has an effect of improving skin elasticity.

The cosmetic composition according to the present invention contains a low molecular peptide through microbial fermentation of the fruit of bilberry and is free of skin irritation and has excellent solubility, affinity and stability for various base materials, additives and organic solvents of cosmetics, The improvement effect was excellent.

The present invention provides a cosmetic composition containing a extract of a fermented microbial extract of Bilberry fruit, which is obtained by fermenting a bilberry fruit with a microorganism, extracting and filtering the fermented microorganism, purifying the fermented microorganism with a peptide having a predetermined size or less, and concentrating and cooling.

According to the present invention, the extract of B. berry fruit fermented microorganism has increased peptide content as an active ingredient by peptide purification treatment, and it is possible to provide uniform quality by concentrating and drying the active ingredient after peptide purification treatment, , Wrinkle improving effect (Experimental Example 4), inflammatory reaction inhibitory effect (Experimental Example 5) and antioxidative effect (Experimental Example 6).

Therefore, the cosmetic composition of the present invention containing a low-molecular peptide through microbial fermentation of a bilberry fruit having various functions can be effectively used because it has a high content of peptide as an active ingredient and has an anti-aging effect on the skin.

Unless defined otherwise, all technical terms used in the present invention have the following definitions and are consistent with the meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In addition, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention. The contents of all publications referred to herein are incorporated herein by reference. The term " drug " is used herein to refer to a reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, Level, value, number, frequency, percentage, dimension, size, quantity, weight or length of a sample,

Throughout this specification, the words " comprising " and " comprising ", unless the context clearly requires otherwise, include the steps or components, or groups of steps or elements, And that they are not excluded.

The present invention relates to a process for producing a microbial fermentation product comprising the steps of extracting and filtering this microorganism after fermentation with a fermenting microorganism using a bilberry fruit and then adding a small amount of a peptide comprising a peptide obtained by a purification process of a certain size or smaller, ≪ / RTI >

More specifically, the fruit of the berry is fermented by using a fermenting microorganism selected from the group consisting of yeast, lactic acid bacteria, Bacillus subtilis, and a mixture thereof, and the microorganism is extracted and filtered, and the resulting product is filtered using ultrafiltration It is characterized in that only peptides within the range of 10,000 to 500,000 Daltons are purified and used. In this case, the active ingredient may contain the extract of the fermented Bilberry fruit which has an increased peptide content. If the ultrafiltration is less than 100,000 daltons, the stability of the cosmetic composition is poor and the economical efficiency at the time of purification is low. If it exceeds 500,000 daltons, the effect of increasing the peptide content effective on the skin is insufficient, The reaction inhibition effect and the antioxidant effect were poor.

The cosmetic composition of the present invention as described above contains a low-molecular peptide through microbial fermentation of the fruit of a bilberry obtained by fermenting the fruit of a bilberry, extracting, filtering and purifying the microorganism.

The cosmetic composition of the present invention comprising the above components is suitable as a cosmetic composition for protecting skin and improving wrinkles.

The present inventors have found that, in order to find a component of a peptide having various effects, particularly, a method which can be obtained at the highest possible yield through a filtration process, a method of fermenting a microorganism using a bilberry fruit, And the filtration process, it was confirmed that the content of the active ingredient was increased, and that it was applied to a cosmetic composition to have a skin wrinkle reducing effect, an inflammation reaction inhibiting effect, and an antioxidative effect, thereby completing the present invention.

The extract of the fruit juice fermented with the extract of the present invention contains the fruit of the present invention as a fermenting microorganism bacterium selected from the group consisting of yeast, lactic acid bacteria, bacteria of the genus Bacillus, and a mixture thereof, at a pH of 5 to 7 And fermenting it for 1 to 7 days. Preferably Saccharomyces cerevisisae, and most preferably Saccharomyces cerevisiae KCTC 7904 can be used for fermentation.

The bilberry berries used in the present invention are commercially available bilberry berries, which can be sufficiently washed and then milled and finely adjusted using about 300 mesh. The crushed bilberry fruit is added to the culture medium of Saccharomyces sp. (Saccharomyces cerevisiae KCTC 7904) at a concentration of 10 to 100 g / L. Here, 1 to 4% of glucose is used as a carbon source, preferably 1 to 2% is added, and 0.1 to 1% of peptone is added and preferably 0.2 to 0.5% is further added. The fermentation can be carried out by incubating the fermentation broth in a 5 L fermenter at a temperature of 20 to 40 ° C for 1 to 7 days while maintaining the pH at 5 to 7.

The subsequent extraction process may be carried out by extracting the cultured microorganism with an extraction solvent selected from the group consisting of purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane, chloroform, ethyl ether, butylene glycol, hexane, Extraction with a solvent, more preferably ethanol, 70% ethanol aqueous solution or water, and most preferably 70% ethanol aqueous solution.

After extraction, it can be filtered through a filter paper such as Whatman # 2 filter paper. Ultrafiltration was then carried out to purify the extract to include the extract of a fermented microbial extract containing a peptide having a size within the range of 10 to 500,000 Da. When the purification process of the ultrafilteration is completed, in the present invention, it is transferred to a thickener, concentrated under reduced pressure, and lyophilized. The concentration of the supernatant was concentrated using a rotary vacuum concentrator. Concentration conditions were as follows: 700 ml of extract was added to a 1,000 ml round-bottomed flask, and the mixture was concentrated under reduced pressure for 2 hours while rotating at a temperature of from 650 to 1200 rpm at a temperature of from 650 to 750 mmHg at a temperature of from 650 to 750 mmHg.

The lyophilized concentrate was subjected to preliminary freezing to lower the temperature to -40 ° C, maintain the temperature at -40 ° C for 2 to 4 hours, reduce the temperature to -80 ° C by freezing the trap, adjust the vacuum to 10 to 30 mTorr And then raised to -40 ° C over 120 minutes. Thereafter, the temperature is maintained at -20 ° C for 240 minutes, at 0 ° C for 40 minutes, at 15 ° C for 340 minutes, at 30 ° C for 130 minutes, and at 1300 minutes until completely dried.

For reference, the extract of the fruit juice fermentation of Bilberry fruit is an extract obtained by ultra-high pressure treatment of a microorganism obtained by fermenting the fruit of Bilberry fruit, a powder obtained by vacuum-concentrating the extract, lyophilized, or a combination of the powder with an extraction solvent.

The extract of the fruit juice fermented with the bilberry fruit is not limited to a specific range, but is preferably contained in an amount of 0.0001 to 30% by weight based on the total weight of the cosmetic composition. When the content of the extract is less than 0.0001% by weight, the skin protection effect is not exhibited. When the content of the extract is more than 30% by weight, the effect of increasing the content of the extract is insignificant, and there is a problem in safety and stability of the cosmetic composition.

The ingredients contained in the cosmetic composition of the present invention include, in addition to the above-mentioned effective ingredients, the ingredients conventionally used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, And may include a carrier.

The cosmetic composition of the present invention may be prepared in any form conventionally produced in the art and may be in the form of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, Oils, powder foundations, emulsion foundations, wax foundations and sprays, but are not limited thereto.

More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in the case of spray, additionally chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan.

When the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide ether Alkylamido betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester.

When the cosmetic composition of the present invention is a soap, a surfactant-containing cleansing agent, or a surfactant-free cleansing agent, it may be applied to the skin and then wiped off or removed or washed with water. The surfactant-containing cleansing formulation is a cleansing foam, a cleansing water, a cleansing towel, and a cleansing pack. The surfactant-free cleansing formulation may be a cleansing cream, , Cleansing lotion, cleansing water and cleansing gel, but is not limited thereto.

The cosmetic composition of the present invention can be applied to cosmetics for skin protection (antioxidant, inflammatory reaction improvement) and wrinkle reduction (collagen production effect).

The present invention also provides a cosmetic method for obtaining a skin protection and wrinkle-improving effect by using a cosmetic composition containing a low-molecular peptide through microbial fermentation of the above-mentioned bilberry fruit.

The cosmetic process of the present invention refers to all cosmetic processes using the cosmetic composition of the present invention. That is, all methods known in the art using a cosmetic composition belong to the cosmetic method of the present invention.

The cosmetic composition of the present invention may be used alone or in combination, or may be used by overlapping with other cosmetic compositions other than the present invention. In addition, the cosmetic composition having excellent skin aging-preventing effect according to the present invention can be used according to a conventional method of use, and the number of times of use can be varied depending on the skin condition or taste of the user.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, and it is to be understood by those skilled in the art that the present invention is not limited thereto It will be obvious.

Example  One : Bilberry  Through microbial fermentation Low molecule Of peptide  Produce

Bilberry fruit was selected as a sample, thoroughly washed, and finely pulverized so as to pass through a 300-mesh sieve. This pulverized product was added to a culture medium of Saccharomyces cerevisiae (Saccharomyces cerevisiae KCTC 7904) at 10 to 100 g / L. 2% (W / V) of glucose was added as a carbon source and 0.5% (w / v) of peptone was further added thereto. The cultures were incubated for 5 days at 37 ° C and pH 6 using a 5 L fermenter. After culturing the microorganisms, the microorganisms in the lower layer were purified using a centrifuge on the bilberry fruit fermented product, the temperature of the microorganisms was raised to 50 ° C, treated for 24 hours under ultrahigh pressure, and cooled to room temperature.

After that, ethanol was added to the ultrahigh-pressure treated bilberry fruit microorganism so as to be a final 70% (v / v) ethanol aqueous solution, refluxed three times for 5 hours, cooled, and filtered through a Whatman # 2 filter paper And filtration process).

When the filtration was completed, the peptide having a size within the range of 10 to 500,000 Daltons was purified using ultrafiltration to purify the low molecular weight peptide (purification process).

The purified extract having an increased peptide content was used in a rotary vacuum concentrator at 650 to 750 Hg. In this example, the extract was concentrated under reduced pressure by treatment at 700 Hg, 50 ° C., and 1200 rpm for 2 hours, followed by lyophilization ). The lyophilized product was dissolved in 50% glycerin to a concentration of 2% to prepare a bilberry fruit fermented microorganism extract (Example 1).

Comparative Example  One : Bilberry fruit  Solvent extract preparation

Comparative Example 1 was a general organic solvent extract of Bilberry fruit. The Bilberry fruit was selected as a sample, washed thoroughly, and then pulverized and passed through a 300-mesh sieve to finely crush the product. Ethanol was added to make a final concentration of 70% (v / v) ethanol aqueous solution, refluxed three times for 5 hours, cooled down, and filtered through a filter of whatman # 2. The filtered extract was concentrated under reduced pressure and lyophilized. The powder was dissolved in 50% glycerin to 2% (W / V) to prepare a bilberry fruit extract (Comparative Example 1).

Comparative Example  2 : Bilberry fruit  Fermented extract preparation

Bilberry fruit was stirred at a speed of 400 rpm in a constant temperature bath maintained at 37 ℃ to allow the air to mix well in the reaction solution and to allow the fermentation reaction to occur using oxygen in the air. After the fermentation process for 72 hours, the reaction solution was collected, and pure fermentation supernatant was collected through ultrafast centrifugation (3600 rpm) and ultrafiltration (pore size = 10 μm). The resulting fermentation supernatant was concentrated to a solid, and the concentrated solid was dissolved in 50% glycerin to a concentration of 2% (W / V) to prepare a bilberry fruit fermented extract (Comparative Example 2).

Example  2 : Bilberry  Through microbial fermentation Low molecule Peptides  Manufacture of nutritional cream containing

The nutritional cream containing the low molecular peptide (Example 1) through microbial fermentation of the bilberry fruit of the present invention was prepared. A step of warming the purified water portion to 80 DEG C to dissolve it, a step of emulsifying by adding an oily portion heated to 80 DEG C, a step of adding and mixing a thickener dissolved by heating at 60 DEG C, Mixing and cooling the mixture to 30 DEG C, followed by defoaming and filtration, and aging at 50 DEG C, to prepare a nutritional cream as shown in Table 1.

Comparative Examples 3 and 4 having the compositions shown in the following Table 1 were also prepared.

Classification Raw material name Example 2 Comparative Example 3 Comparative Example 4 Water component Purified water 59.02 59.02 59.02 Dipropylene glycol 5 5 5 glycerin 6 6 6 Xanthan gum 0.1 0.1 0.1 1,2-hexanediol 2 2 2 Low molecular weight peptide through fermentation of microbial fruit of bilberry fruit (Example 1) 2 - - Bilberry fruit extract (Comparative Example 1) - 2 - Bilberry fruit fermented extract (Comparative Example 1) - - 2 Arginine 0.24 0.24 0.24 Oil component Squalane 8 8 8 Cetyl alcohol 2.2 2.2 2.2 Cyclopentasiloxane 2 2 2 Stearic acid 1.5 1.5 1.5 Glyceryl monostearate One One One Microcrystalline wax 0.6 0.6 0.6 PIG-100 0.3 0.3 0.3 Tocopheryl acetate One One One Sheer butter 2 2 2 Dimethicone 0.5 0.5 0.5 Polyoxyethylene sorbitan monostearate 0.8 0.8 0.8 Incrementer Purified water 2.4 2.4 2.4 Carbomer 0.24 0.24 0.24 Beauty ingredient Sodium ascorbate 0.1 0.1 0.1 Phospholipids One One One Marchia extract 2 2 2 Sum 100.0 100.00 100.00

Example  3: Bilberry  Through microbial fermentation Low molecule Peptides  Containing Facial  Manufacture of essences

Facial essences were prepared at the composition ratios shown in Table 2 below. The manufacturing method includes a step of adding and dissolving a purified water portion in purified water at 60 ° C, a step of adding and dissolving the moisturizing elastic component dissolved at 40 ° C, a step of cooling to 30 ° C to dissolve the functional cosmetic ingredient, And then mixed with a low molecular peptide through microbial fermentation of the bilberry fruit prepared in Example 1 to prepare facial essence.

Classification Raw material name Example 3 Comparative Example 3 Comparative Example 4 Purified water
part Purified water 56.18 56.18 56.18 glycerin 7 7 7 Dipotassium glycyrrinate 0.05 0.05 0.05 Disodium iodide 0.02 0.02 0.02 Hydroxyethyl cellulose 0.05 0.05 0.05 Dipropylene glycol 3 3 3 1,2-hexanediol 7 7 7 Moisturizing
Elastic component Squalane 3 3 3 Caprylic / capric triglyceride One One One Sorbitan stearate 0.88 0.88 0.88 Sorbitan laurate 0.12 0.12 0.12 beauty
ingredient Sodium hyaruronate 0.5 0.5 0.5 Collagen One One One Hibiscus flower extract 0.2 0.2 0.2 Low molecular weight peptide through fermentation of microbial fruit of bilberry fruit (Example 1) 20.0 - - Bilberry fruit extract (Comparative Example 1) - 20.0 - Bilberry fruit fermented extract (Comparative Example 1) - 20.0 Sum 100.00 100.00 100.00

Experimental Example  1: Skin irritation experiment

The skin irritation of the nutrition cream containing the low molecular peptide (Example 2) through microbial fermentation of the bilberry fruit according to the present invention was examined.

A predetermined amount (10 m / 5 cm) of the nutritional cream prepared in Example 2, Comparative Examples 3 and 4 was applied to the first half of 30 healthy males and females (15 males and 15 females) 30 minutes after shielding, long wavelength ultraviolet rays were irradiated at a dose of 10.2 J / cm ² using an ultraviolet light irradiator, and skin conditions (erythema, edema, papules, etc.) were observed after 24 hours. As a result, no adverse effects such as erythema were observed on the skin, so that the nutritional cream had no phototoxicity and skin protection effect was confirmed as shown in Table 3.

Erythema No stimulation Example 2 - 28 Comparative Example 3 11 20 Comparative Example 4 5 23

Experimental Example  2: skin elasticity improvement experiment

The degree of skin elasticity improvement of the nutrition cream containing the low molecular peptide (Example 2) through microbial fermentation of the fruit of bilberry according to the present invention was tested.

(Average age 36.3 years) at a temperature of 24-26 DEG C and a relative humidity of 75% were subjected to the cosmetic composition of Example 2 on the left side and the cosmetic composition of Comparative Examples 3 to 4 for 12 weeks After application, skin elasticity was measured using a skin elasticity meter (Cutometer SEM 575, C + K Electronic Co., Germany). The results are shown in Table 4 below as R8 values of Cutometer SEM 575, where R8 values represent viscoelasticity of the skin.

R8 Example 2 0.40 Comparative Example 3 0.13 Comparative Example 4 0.02

Experimental Example  3: Skin stability test

The skin safety of the nutrition cream (Example 2) and the facial essence (Example 3) containing a low molecular peptide through microbial fermentation of the bilberry fruit according to the present invention was confirmed.

The nutritional cream and facial essence prepared in Examples 2 and 3 and Comparative Examples 3 to 6 were applied to the forehead of 30 healthy male and female subjects (15 males and 15 females) to perform a human skin patch test. Specifically, the entire portion of the test subjects was thoroughly wiped with 70% ethanol, and then the pin chambers containing 20 nutritional creams prepared in Example 2 were clogged with adhesive tape for 24 hours in a usual cosmetic base. After 24 hours, the adhesive tape and the pin chamber were removed, and the sample remaining on the skin with the gauze was gently wiped off and the skin condition was observed and again after 24 hours.

sample After 24 hours (n = 30) After 48 hours (n = 30) Remarks Example 2 0/30 0/30 No stimulation Example 3 0/30 0/30 No stimulation Comparative Example 3 4/30 1/30 Irritation Comparative Example 4 3/30 3/30 Irritation Comparative Example 5 5/30 1/30 Irritation Comparative Example 6 7/30 2/30 Irritation

As shown in Table 5, it was confirmed that the nutrient cream containing the low molecular peptide through the microbial fermentation of the fruit of the present invention did not show a positive reaction in the human skin test to check the skin irritation, . However, when the comparative examples 3 to 6 were tested, it was confirmed that all of them were irritated. Therefore, it was confirmed that the low molecular peptide through microbial fermentation of the bilberry fruit of the present invention helps skin irritation.

Experimental Example  4 : Bilberry  Through microbial fermentation Low molecule Of peptide  Experiment to confirm collagen production

The effect of collagen production was measured using the low molecular peptide obtained through fermentation of the microbial fermentation of the bilberry fruit obtained in Example 1 above.

One of the causes of skin wrinkles is the lack of collagen in the skin. Collagen is a major protein that constitutes the skin dermis and plays a role in maintaining the skin structure and elasticity. It is known that collagen is decreased in production with increasing age, and is decomposed to induce depression of the skin dermis layer to generate skin wrinkles. Fibroblasts present in the dermis are responsible for the synthesis of collagen, a major protein in the skin. Therefore, by measuring the degree of increase of collagen production using fibroblast, the collagen aggregation performance of the extract can be improved and the wrinkle improvement effect can be evaluated.

The degree of collagen synthesis was assayed at the cellular level through fibroblast culture. Human fibroblast-derived HDFn cell line (C-004-5C: purchased from Cascade Biologics) was used as the fibroblast. Fibroblasts were inoculated into 48 well plates at 1 × 10 ⁶ cells / well and cultured in DMEM medium containing 10% serum for 24 hours at 37 ° C and 5% CO ₂ . The medium was replaced with serum-free DMEM medium, and the extracts were added at different concentrations and cultured for 24 hours. After culturing, each supernatant was taken and centrifuged at 13,000 rpm for 5 minutes to collect only the supernatant. The amount of procollagen type I C-peptide (PIP) was measured using a kit (Takara, Japan) The amount was measured. The amount of PIP was converted to ng / ml, and the collagen biosynthesis rate was calculated according to the following formula (1).

FIG. 1 is a graph showing the synthesis rate of procollagen type I C-peptide (PIP) according to the concentration of low molecular peptide through microbial fermentation of the fruit of Bilberry according to the present invention. Respectively.

Experimental Example  5: Bilberry  Through microbial fermentation Low molecule Of peptide  Measurement of inflammation reaction inhibition effect by concentration

In the same manner as in Experimental Example 4, a sample of a control group and an experimental group of a low molecular peptide through microbial fermentation of a bilberry fruit was used. Nitric oxide (NO) is known to play a role in blood pressure regulation, neurotransmission, platelet aggregation inhibition, and immune function. It is synthesized by nitric oxide synthase from L-arginine in various tissues and cells. NOS is largely divided into cNOS and iNOS, and iNOS is independent of intracellular calcium concentration, and LPS, IFN-r, IL-1 and TNF in various cells such as macrophages, vascular smooth muscle cells, endothelial cells, hepatocytes and myocardial cells -a < / RTI > to produce a large amount of NO over a long period of time. However, when NO is generated more than necessary, it causes vasodilation due to shock, tissue damage caused by inflammatory reaction, damage of nerve tissue, and so on, and is harmful to living body. In the macrophage, NO produced by L-arginine eliminates tumor cells and fungi, and thus acts as an important substance in the function of macrophages involved in the immune response. In addition, NO acts as a major vasodilator. In this case, acetylcholine or bradykinin is used as a receptor. At this time, NO diffused to the surrounding smooth muscle cells increases cGMP and eventually relaxes the muscle cells by the increased cGMP and causes vasodilation. Based on this fact, development of an inflammatory drug that inhibits the production of NO by iNOS is under way, and the inhibitory effect of inflammation can be judged by measuring the NO production ability of the extract.

As an experimental method, RAW 264.7 cells, which are mouse macrophage cells, were treated with artificially induced inflammation-inducing substance LPS to evaluate whether they inhibited inflammation. Cells were seeded at 2 × 10 ⁵ cells per well in a 96-well plate and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. Overnight, and then exchanged for new medium. LPS (1 ug / ml) and extract were added at different concentrations and cultured for 24 hours. After the culture, the supernatant of the culture medium was taken and centrifuged at 13,000 rpm for 3 minutes to collect only the supernatant. The supernatant was collected and reacted with Griess reagent reagent 1: 1, and absorbance was measured at 570 nm. 2 shows the inhibitory effect of Nitric Oxide on the concentration of the extract of the fermented microorganism of Bilberry fruit. It was confirmed that the extract of the present invention was more effective than the extract of the present invention and the fermented extract.

Experimental Example  6: Of bilberry fruit  Through microbial fermentation Low molecule Of peptide  Depending on concentration DPPH On the offensive  Antioxidant effect

In the same manner as in Experimental Example 4, a sample of a control group and an experimental group of a low molecular peptide through microbial fermentation of a bilberry fruit was used.

Skin aging processes by active oxygen include lipid peroxidation, DNA oxidation, melanogenesis, cleavage of connective tissue and abnormal cross-linking. These reactions are almost always radical reactions and involve autoxidation processes. Antioxidants are responsible for blocking the autoxidation reaction in vivo. As the ability to give electrons, the greater the reducing power, the stronger the antioxidant.

1,1-diphenyl-2 picrylhydrazyl radical (DPPH) is a reagent for measuring the reducing power of antioxidants. DPPH is present as a radical in the stabilized structure of the nitrogen center in the compound. The maximum absorption at 517 nm is shown, and absorption reduces at 517 nm. The magnitude of the reducing power of the low molecular peptide through microbial fermentation of the fruit of the present invention is represented by the scavenging activity (SC50), and SC50 represents the concentration required to reduce the concentration of DPPH by 50%.

FIG. 3 is a graph showing the antioxidative effect of DPPH-cleaving ability of a low-molecular peptide through microbial fermentation of the fruit of a berry of the present invention produced by the present invention. More specifically, the concentration of a low molecular peptide through microbial fermentation of a bilberry fruit is The antioxidative effect of DPPH eradication ability was increased in proportion to the concentration when tested at 0.2, 0.4, 0.8, 1.7, 3.3, 6.7 (%, V / V) Thus, it can be confirmed that the extract of the present invention containing a low molecular peptide through microbial fermentation of the fruit of Bilberry has more excellent effects than the conventional extract and the fermented extract.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but on the contrary, ≪ RTI ID = 0.0 > and / or < / RTI >

Claims (5)

The present invention relates to a skin protection agent containing a low molecular peptide through fermentation of a microbial fruit of a bilberry fruit, which comprises a peptide obtained by extracting the fermenting microorganism after fermentation using a saccharomyces cerevisiae (KCTC 7904) A cosmetic composition for improving wrinkles. The cosmetic composition according to claim 1, wherein the cosmetic composition is obtained by filtering the fermented microorganism obtained by fermenting the fruit of Bilberry with the use of Saccharomyces cerevisiae (KCTC 7904), and purifying it to contain only peptides of 100,000 to 500,000 daltons, The present invention relates to a cosmetic composition for protecting skin and improving wrinkles containing low molecular peptides by fermentation of microbial fruit of bilberry fruit. The cosmetic composition according to claim 1, characterized in that the cosmetic composition has a flexible lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, A cosmetic composition for protecting skin and improving wrinkles containing low molecular weight peptide through fermentation of microorganism of fruit. The cosmetic composition for skin protection and wrinkle improvement according to claim 1, wherein the cosmetic composition has an antioxidative effect, and contains a low molecular peptide through microbial fermentation of a bilberry fruit. The cosmetic composition for skin protection and wrinkle improvement according to claim 1, wherein the cosmetic composition has an effect of improving skin elasticity, the composition comprising a low molecular peptide through microbial fermentation of a bilberry fruit.