KR20130099277A - A cosmetic composition comprising hydrolysates of ecklonia cava - Google Patents

Abstract

PURPOSE: A hydrolysate of ecklonia cava is provided to have excellent elastase inhabitation activity and typosinase inhabitation activity, thereby having excellent whitening and anti-wrinkling effect. CONSTITUTION: A cosmetic composition comprises hydrolysates that Ecklonia cava is hydrolyzed by a biocatalyst including at least one selected from Microbulbifer sp., the strain therein, the culture medium thereof, and the concentrate of the culture medium. The strain in the Microbulbifer sp. is Microbulbifer sp. SC092. The cosmetic composition has whitening and anti-wrinkling effect.

Description

Cosmetic composition containing E. coli hydrolyzate as active ingredient {A COSMETIC COMPOSITION COMPRISING HYDROLYSATES OF ECKLONIA CAVA}

The present invention relates to a cosmetic composition comprising a natural substance having a skin care function as an active ingredient.

In the past, cosmetics, which were previously thought to be used by women, are used not only as a beauty expression tool but also as household goods for men and women of all ages, and as the industry develops, the demand for high-quality and diversification of cosmetics increases. As a result, there is a growing interest in 'functional' products that expect a particular effect or efficacy.

The functional cosmetics currently recognized in Korea include whitening products that help whitening the skin, wrinkle improvement products that help improve the wrinkles of the skin, and sunscreen products that help to burn the skin finely or protect the skin from ultraviolet rays. . In general, such functional cosmetics are interpreted as cosmetics that affect the structure or function of the body as well as cosmetic effects, and prevent and treat. In particular, recently, as the sales proportion of functional cosmetics in the total sales of cosmetics significantly increased, researches to find new efficacy ingredients on the human body and use them effectively on the skin have been actively conducted.

In particular, due to the recent increase in the elderly population due to the aging of the population and the improvement of the quality of life according to the economic development, in addition to the wrinkles and hyperpigmentation of the skin, which are naturally accompanied by aging, it also suppresses blemishes, swelling and blemishes. Or there is an increasing demand for products that can be controlled. In particular, due to the increase in the social activity opportunities of the elderly population, the interest in beauty of the elderly population is increasing, while the factors of stimulation and aging for skin such as ultraviolet rays are increasing, and functional cosmetics related to whitening and wrinkle prevention Demand and usage for the is increasing significantly.

In relation to the raw materials of such functional cosmetics, recent researches related to functional materials have been focused on natural materials derived from natural products mainly in conjunction with environmentally friendly fever, and in particular, researches on using algae which are more resource-rich than land foods. It's going on.

KR 1108886 B KR 0762181 B KR 0733503 B KR 0359996 B

The present invention is to improve the problems of the prior art as described above, an object of the present invention to provide a cosmetic composition comprising a natural substance as an active ingredient.

In order to achieve the above object, the present invention provides a cosmetic composition comprising the Ecklonia caum hydrolyzate as an active ingredient.

More specifically, the present invention is a hydrolyzate hydrolyzed by the biocatalyst comprising at least one selected from the group consisting of microbulbifer sp., Microbulbifer sp., The culture medium of the strain and the concentrated solution of the culture solution It provides a cosmetic composition comprising the as an active ingredient.

The strain of the genus MicroBulper may preferably be a microbulbifer sp. SC092 strain.

The cosmetic composition may be a cosmetic composition for improving wrinkles. In addition, the cosmetic composition may be a cosmetic composition for whitening.

The inventors of the present invention, in the course of studying the functionality of Ecklonia cava in natural algae resources, in the case of the hydrolyzate reacted to the Ecklonia cava strain of Microbulbifer sp. The tyrosinase inhibitory activity and the elastase inhibitory activity experiment confirmed that the whitening function and the anti-wrinkle function, which are functions related to the composition, were remarkably improved, thereby completing the present invention.

In the present invention, the cosmetic composition is a composition used in the human body to clean and beautify the human body to add charm and brighten the appearance, or to maintain or promote the health of skin and hair. Detergents and cosmetics (cosmetics) The concept includes.

In the present invention, the active ingredient means a component of the cosmetic composition applied to the skin condition to provide a prophylactic, improvement or supplementary effect.

In the present invention, the hydrolyzate means a hydrolyzate hydrolyzed by a microorganism or a biocatalyst including a microorganism or a microbial culture medium, and the Ecklonia hydrolyzate means a substance in which the Ecklonia cava is hydrolyzed by the microorganism or the biocatalyst. . The biocatalyst includes at least one selected from the group consisting of microorganisms, culture medium of the microorganisms, and concentrated solution of the culture solution.

In the present invention, the biocatalyst may include a microorganism that acts as a catalyst in various chemical reactions or biochemical reactions or a substance secreted by the microorganisms. For example, the biocatalyst is a protein that catalyzes a chemical reaction or a biochemical reaction. It may be to include an enzyme (enzyme).

In the present invention, the culture medium means a culture medium obtained by culturing microorganisms, and may be a culture medium containing the microorganisms or a cell-free culture medium in which the cells are removed.

The culture medium means that the nutrients required by the culture in order to culture the tissues of microorganisms or animals or animals as a main component, or for the special purpose further comprises an additional material in addition to the main component. The culture medium may be a conventional medium for culturing microorganisms or tissues of animals and plants. The concentrate of the culture solution means that the culture solution of the microorganism is concentrated by a conventional method.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a cosmetic composition comprising an Ecklonia caum hydrolyzate as an active ingredient.

The Ecklonia cava belongs to the brown seaweed of the algae plant kelp seaweed seaweed family, and refers to the perennial brown algae in which vegetative cells grow for more than one year. The Ecklonia inhabited by forming a sea forest in the deep of the lunch. In Korea, it is widely distributed on the south coast and Jeju island, and has been used for food since ancient times.

The hydrolyzate may be a hydrolyzate hydrolyzed the Ecklonia cava with microbulbifer sp. Or a biocatalyst.

The strain of the genus MicroBulper may preferably be a microbulbifer sp. SC092 strain. The microbulbifer sp. SC092 strain is a microbulbifer sp. It may be a strain having excellent resolution of alginic acid (alginic acid) and agar (agar), that is, the resolution of agar.

The biocatalyst may be at least one selected from the group consisting of the microbulb bee strain, the culture medium of the strain, and the concentrated solution of the culture solution.

The hydrolyzate may be reacted with the Ecklonia cava SC092 strain ( Microbulbifer sp. SC092) at 30 ℃ to 40 ℃ or 33 ℃ to 39 ℃ or 35 ℃ to 37 ℃.

The cosmetic composition may be a cosmetic composition for improving wrinkles. In addition, the cosmetic composition may be a cosmetic composition for whitening.

In the conventional researches, studies on the functionality of existing seaweeds or solvent extracts of seaweeds have been carried out, but researches to improve the functionality by reacting with specific microorganisms, specifically, by reacting microorganisms, biocatalysts or enzymes with Ecklonia spp. For example, research on improving functionalities such as anti-whitening, anti-wrinkle or UV-blocking related to cosmetics or improving functionalities such as antioxidant activity, anti-diabetic activity or alcohol degrading activity and hangover elimination activity has not been conducted. In order to improve the functionality, researches related to the reaction of specific microorganisms, specifically, microbephericus strains, have not been conducted. The invention which is revealed that the prior art It may be having a significant effect which is not obtained.

In general, tyrosinase is a polyphenol oxidase widely distributed in the flora and fauna and catalyzes two reactions in the body. One function is tyrosinase oxidase, which oxidizes monophenol to o-diphenol, and the other function as dopa oxidase, which oxidizes o-diphenol to o-quinone.

Tyrosinase plays an important role in the biosynthesis of melanin polymers in the body due to its function as an oxidase, and particularly plays a crucial role in producing melanin in melanocytes. The melanin appears on the skin as lesions such as blemishes, freckles, melanin hyperpigmentation and the like. Therefore, when the tyrosinase inhibitory activity is excellent, it is expected to inhibit melanin production and excellent whitening effect.

As described above, when it is possible to inhibit tyrosinase, it is possible to derive a whitening effect by inhibiting melanin production, and as a result of confirming through the experiment the Ecklonia hydrolyzate which is an active ingredient of the cosmetic composition of the present invention, Tyro In view of excellent synthase inhibitory activity, and since the substance derived from a natural substance is an active ingredient, the side effects are not a problem and are safe, the cosmetic composition of the present invention can be applied to skin for which whitening activity is required. In this aspect, the cosmetic composition of the present invention may be a cosmetic composition for whitening.

In addition, the Ecklonia hydrolyzate, which is an active ingredient of the cosmetic composition of the present invention, has been confirmed through experiments, and has excellent elastase inhibitory activity, and since a substance derived from a natural substance is an active ingredient, side effects are not a problem and are safe. In that regard, the cosmetic composition of the present invention can be applied to the skin for the purpose of preventing wrinkles. In this aspect, the cosmetic composition of the present invention may be a cosmetic composition for improving wrinkles, cosmetic composition for preventing or improving wrinkles or cosmetic composition for anti-wrinkle.

The cosmetic composition may include 0.0001% to 50% by weight, preferably 0.001% to 30% by weight, of the Ecklonia hydrolyzate on the skin. That is, the content of the Ecklonia hydrolyzate should be more than the minimum value in order to achieve the skin-related functionality, specifically, the whitening effect, the anti-wrinkle effect or the moisturizing effect. In consideration of this, it can be freely adjusted within the above range. At this time, the content of the active ingredient and the content of the additional component may be appropriately adjusted within the above range according to the content of the components contained in the formulation or cosmetic composition.

The cosmetic composition of the present invention may be provided for use in skin external ointments, basic cosmetics, makeup cosmetics, body cosmetics or shaving cosmetics, and the like, and examples of the basic cosmetics include essences, lotions, creams, gels, lotions, packs, massages, and the like. There may be creams, latexes, and the like, and examples of the makeup cosmetics may include foundation, lipstick, eyeshadow, eyeliner, mascara, eyebrow pencil, makeup base, and the like, and body cosmetics include soap, liquid cleaner, and bathing agent. , Sun screen cream, sun oil, and the like, and shaving cosmetics include aftershave and shaving cream.

The cosmetic composition of the present invention, in addition to containing the Ecklonia hydrolyzate as an active ingredient, may further include substances such as functional substances having a whitening effect, wrinkle improvement effect or moisturizing effect, or excipients or diluents for improving physical properties. .

For example, the cosmetic composition may be additionally added to the perfume, pigments, fungicides, antioxidants, preservatives, moisturizers, thickeners, inorganic salts or synthetic polymer materials to improve physical properties. In addition to the blending components that may be added, it may be a fat or oil component, an emollient agent, a surfactant, an organic pigment, an inorganic pigment, an organic powder, an ultraviolet absorber, a pH adjuster, an alcohol, a blood circulation accelerator, a cooling agent, a limiting agent, or purified water.

In addition, the cosmetic composition may further comprise one or more selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingolipids and seaweed extract.

The compounding component which may be added other than the said active ingredient is not limited to this, Any said component can be mix | blended within the range which does not impair the objective and effect of this invention.

In addition, the cosmetic composition may take the form of a solution, an emulsion, a viscous mixture, and the like, and any formulation commonly prepared in the art, for example, emulsion, cream, lotion, pack, foundation, lotion, beauty liquid, hair cosmetic, etc. It may be a formulation.

More specifically, the cosmetic composition of the present invention, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisturizing cream, hand cream, foundation, essence, nutrition Formulations of essences, packs, soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions and body cleansers.

For example, when the formulation of the present invention is a paste, cream or gel, the carrier component is animal fiber, vegetable fiber, wax, paraffin, starch, trakant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide. This can be used.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as the carrier component, and especially when the formulation of the present invention is a spray, additionally chlorofluoro Propellants such as rohydrocarbon, propane / butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solvating agent, or emulsifying agent is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, Fatty acid esters of 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or 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, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-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 sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolenic derivatives or ethoxylated glycerol fatty acid esters.

As described above, the E. coli hydrolyzate reacted with the microbulbifer sp. SC092 strain compared to the conventional Eckloniasis not only has improved anti-whitening or anti-wrinkle function, but also antioxidant activity, anti-diabetic activity and hangover. Resolution can also be improved.

In this aspect, the present invention may be a composition for improving the functionality that can improve the functionality of the Ecklonia.

The active ingredient of the functional improving composition may be a microbulbifer sp. SC092 strain or a biocatalyst of the genus Microbulbifer.

The biocatalyst may be at least one selected from the group consisting of the microbulbifer sp. SC092 strain ( Microbulbifer sp. SC092), the culture medium of the strain and the concentrated solution of the culture solution.

SC092 strain of the genus Microbulbifer ( Microbulbifer sp. SC092) or functional improvement composition comprising the biocatalyst as an active ingredient in response to the Eckloniasis antioxidant activity, anti-inflammatory, anti-diabetic, alcohol-degrading or whitening function activity Can be improved.

In addition, the present invention may relate to a method for preparing an Ecklonia cava hydrolyzate having anti-wrinkle activity and whitening activity.

The preparation method of the present invention is selected from the group consisting of the microbulbifer sp. SC092 strain ( Microbulbifer sp. SC092) or the microbulbifer sp. SC092 strain ( Microbulbifer sp. It relates to a method for producing an Ecklonia hydrolyzate having anti-wrinkle activity and whitening activity comprising the step of reacting a biocatalyst which may be at least one species.

The reaction step may be to react the Ecklonia cava SC092 strain ( Microbulbifer sp. SC092) or the biocatalyst at 30 ℃ to 40 ℃ or 33 ℃ to 39 ℃ or 35 ℃ to 37 ℃.

Ecklonia cava hydrolyzate of the present invention, specifically, E. coli is a microbulbifer sp. The Ecklonia hydrolyzate reacted with SC092 showed excellent antioxidative activity through DPPH radical scavenging activity and SOD activity, and also showed anti-diabetic effect, anti-diabetic effect through α-glucosidase inhibitory effect, anti-inflammatory effect and ADH and ALDH. As a result of measuring activity, alcohol decomposability was excellent, and it was confirmed through tyrosinase inhibitory effect and elastase inhibitory effect. As a result, the whitening function and anti-wrinkle function were also excellent. Since safety is also excellent, it can be used as a natural material for securing various functionalities, and in particular, the industrial effect in terms of the material of the cosmetic composition will be excellent.

1 is a table showing a phylogenetic relationship with other bacteria based on the nucleotide sequence of 16S rDNA of SC092 strain isolated from Busan Gijang area according to an embodiment of the present invention.
Figure 2 relates to the glycolytic activity of the SC092 strain according to an embodiment of the present invention, the vertical axis is the relative activity (%) of sugar resolution based on the resolution for agar (agar), the horizontal axis is the type of substrate it means. In FIG. 2, FU is a case where 1% fucoidan is reacted with a substrate, LN is a case where 1% laminarin is reacted with a substrate, and AA is a case where 1% alginic acid is reacted with a substrate. In the case of ST, 1% starch is reacted with a substrate, CT is 1% chitin with a substrate, and AG is 1% agar with a substrate.
Figure 3 shows the inhibitory activity against alpha-glucosidase of α-glucosidase by SC092 strain of Ecklonia cava according to an embodiment of the present invention. In FIG. 3, Acarbose means that 0.5 mg / ml of acarbose was treated as a positive control.
Figure 4 shows the nitrite scavenging effect (Nitrite scavenging effect) according to the pH of the hydrolyzate by the SC092 strain of Ecklonia cava according to an embodiment of the present invention. 4 is treated with 0.5 mg / ml of the hydrolyzate, and Vit C means that 0.5 mg / ml of vitamin C (ascorbic acid) was treated as a positive control.
Figure 5 shows the tyrosinase inhibitory effect (Tyrosinase inhibitory effect) of the hydrolyzate by SC092 strain of Ecklonia cava according to an embodiment of the present invention. In FIG. 5, Kojic acid means that Kojic acid is treated with 0.1 mg / ml as a positive control group.
Figure 6 shows the elastase inhibitory effect (Elastase inhibitory effect) of the hydrolyzate by SC092 strain of Ecklonia cava according to an embodiment of the present invention. In Figure 6, the hydrolyzate means that Vit C is treated with 1 mg / ml of vitamin C (ascorbic aci) as a positive control.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the scope of the present invention is not limited by the following examples.

< Example  1> Isolation of strain

1-1. Isolation of strain

To isolate strains that degrade the indigestible complex polysaccharides of algae from the sea, samples were taken from marine water near the Gijang coast near Busan, Korea, and cultured in marine broth (MB, Difco) medium containing 1.5% agar. After incubation, it was selected to pit or clear zone around colony on agar plate surface, and this isolated strain was named SC092 and identified by GenBank and 16S rDNA sequencing by PCR product direct sequencing. Based on the alignment result of NCBI database, it was analyzed by RDF program (http://rdp.cme.msu.edu). The analysis results are shown in FIG. 1.

As shown in FIG. 1, the SC092 strain showed high similarity with Microbulbifer sp. In particular, Microbulbifer sp. 16S rDNA sequence analysis was similar to that of EU1357141480 , finally identified as Microbulbifer sp., Microbulbifer sp. Named SC092.

1-2. Coenzyme Preparation of Strains

Microbulbifer sp. Coenzyme production of SC092 was determined by Microbulbifer sp. The strain was pre-cultivated under optimum conditions, then added to 0.1% in 250 ml of MB medium containing 0.3% agar, and then cultured at 37 ° C. for 72 hours (200 rpm). The culture solution was centrifuged at 6,000 x g for 30 minutes to remove the cells and the supernatant was used as a coenzyme solution.

The crude enzyme solution was saturated with 30% ammonium sulfate and then precipitated at 4 ° C. for 24 hours. The precipitate was centrifuged at 8,000 × g for 30 minutes, the supernatant was again saturated with 70% ammonium sulfate and precipitated at 4 ° C. for 24 hours, followed by centrifugation at 10,000 × g for 30 minutes, and the precipitate was washed with TE buffer (25 mM Tris -HCl, 1 mM EDTA, pH 8.0) and dialyzed (cut-off value, 12,000-14,000 MW).

The content of the coenzyme was performed by measuring the absorbance at 595 nm using BSA (Bovine Serum Albumin) as a standard protein.

The substrate specificity of the coenzyme prepared above was reacted with the coenzyme solution by adding 1% (w / v) of fucoidan, laminarin, alginic acid, starch, chitin and agar, respectively, which are non-digestible polysaccharides present in seaweed in TE buffer. The resulting monosaccharides were quantified to determine the specificity of the substrate for the enzyme. The measurement results are shown in FIG. 2.

As shown in FIG. 2, when the coenzyme is compared to the relative activity of agar, the resolution of fucoidan, laminarin, starch, and chitin substrates is relatively weak, but alginic acid has a high activity of 146.2%. From the above results, the crude, specifically, Microbulbifer sp. The SC092 strain was found to have excellent enzymatic activity against agar and alginic acid.

Regarding the sugar concentration of the Ecklonia cava product by the hydrolase, 0.5% (w / v) Ecklonia cava powder was added to 8 ml of the crude enzyme solution having a protein content of 1.7 mg / ml, and 37 ° C water. Reacting for 48 hours in the bath, reducing sugar, the reaction product of SC092 by the DNS (3,5-Dinitrosalicylic acid) method, the amount of reducing sugar that produces 1 μmol galactose per minute 1 As a result of unit measurement, a sugar concentration of 2.5 mg / ml was obtained.

< Example  2> Moth Hydrolyzate  Functional check

Microbulbifer sp. Prepared in Example 1. When the Ecklonia cava was hydrolyzed by the coenzyme using the SC092 strain, the functionality of the Ecklonia hydrolyzate was confirmed.

The Ecklonia cava ( E. cava Kjellman) used in the above example was purchased from Jeju seaweed food in 2011, washed thoroughly, dried naturally in the shade for a week, and then pulverized with a blender (HMF-1000A, Hanil Electronics, Korea) and then 150 um sieve. Was used as a sample for enzyme extraction.

2-1. Antioxidant measurement

The antioxidant activity of the Ecklonia cava hydrolyzate was confirmed by measuring DPPH radical scavenging activity and SOD activity.

Free radicals are well known as a major cause of biological damage, which is one of the causes of aging and disease, and produces free radicals in vivo. Free radicals in the human body are substances that can promote aging of a living body by reacting with lipids or proteins, and research on natural substances that can remove such free radicals is being actively conducted. In particular, DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging method is an antioxidant measure using the electron donating ability of antioxidants is commonly used to evaluate the free radical scavenging activity of natural antioxidants.

The DPPH radical scavenging activity was measured by the hydrogen donating effect on DPPH (1,1-Diphenyl-2-picrylhydrazyl). Specifically, the DPPH solution for measuring DPPH radical scavenging ability is dissolved DPPH 1.5 × 10 ^-4 M in 100 ml ethanol and then mixed with 100 ml of distilled water, Whatman filter paper No. It filtered by 2 and prepared. The DPPH radical scavenging ability was measured in a 96 well plate by mixing the coenzyme solution or vitamin C (ascorbic acid) with the prepared DPPH solution in a ratio of 1: 4, and reacting at 37 ° C. for 30 minutes, followed by ELISA reader (Molecular Device, VersaMax Microplate Reader, USA) was used to measure the absorbance at 520 nm and then calculate by the following formula 1. In the following formula 1, the control absorbance means absorbance measured after no sample was added.

[Equation 1]

Superoxide dismutase (SOD) is an antioxidant enzyme that converts oxygen radicals that are harmful to cells into hydrogen peroxide and converts them into harmless water and oxygen molecules by catalase.

The SOD activity (superoxide dismutase assay) was measured using a SOD assay kit (Dojindo Molecular Technologies, Rockville, USA), and the absorbance was measured using the ELISA reader. Specifically, the measuring method is as follows. First, the samples were diluted by concentration, 20 μl were dispensed into 96 well plates, and 200 μl of the WST working solution was added and mixed. Thereafter, 20 μl of enzyme working solution was added thereto, followed by incubation at 37 ° C. for 20 minutes, and absorbance was measured at 450 nm. SOD activity was calculated by the following Equation 2 using the measured absorbance. . In the following formula 2, A is the absorbance of the sample addition sphere, B is the absorbance of the sample addition sphere.

[Equation 2]

The results of measuring the DPPH radical scavenging activity and the SOD activity are shown in Table 1 below.

Concentration (mg / ml) Vit C concentration (mg / ml) 0.1 0.5 One 2.5 0.1 0.5 DPPH 10.4 ± 1.5 40.1 ± 1.2 61.6 ± 0.7 84.1 ± 0.7 96.7 ± 0.7 - SOD 19.0 ± 2.0 65.4 ± 2.6 80.7 ± 1.7 89.6 ± 2.1 - 97.4 ± 2.0

As shown in Table 1, DPPH radical scavenging ability showed an antioxidant activity of 84.1% at a concentration of 2.5 mg / ml, which is similar to the antioxidant activity (96.3%) using Vit C as a positive control at a concentration of 0.1 mg / ml. It was confirmed to have a degree of excellent antioxidant activity.

In addition, as shown in Table 2, SOD activity of the Ecklonia cava products increased concentration-dependently, it was confirmed that the SOD activity of 89.6% at 2.5 mg / ml concentration. In relation to the SOD activity, it was confirmed to have a good antioxidant activity similar to the antioxidant activity (97.4%) using a positive control Vit C at a concentration of 0.5 mg / ml.

From the above results, Microbulbifer sp. Hydrolysates using the SC092 strain were expected to be highly useful as antioxidants.

2-2. Anti-diabetic  Active measurement

α-glucosidase finally converts the sugars degraded by α-amylase into monosaccharides. Therefore, the inhibition of the α-glucosidase activity is used as a therapeutic effect for glycemic-related diseases such as type 2 diabetes by limiting the sugar concentration after meals by delaying the glycolysis and absorption process.

In this aspect, Microbulbifer sp. Antidiabetic activity of the hydrolyzate using the SC092 strain was confirmed through the inhibitory effect of α-glucosidase activity.

More specifically, the substrate solution for confirming the α-glucosidase activity inhibitory effect is dissolved in ρ-nitrophenol-α-D-glucopyranoside (PNPG, Sigma, USA) in 50mM sodium succinate buffer (pH 4.2) 1 mg / ml It was prepared at a concentration of. The activity inhibitory effect was measured by mixing the prepared substrate solution 0.2 ml and enzyme solution (Sigma, USA) 0.075 unit / ml, 0.1 ml of the prepared crude enzyme solution was reacted for 30 minutes at 37 ℃, 1 N NaOH 0.1 It was developed by adding ml. The control was added 0.1 ml TE buffer (pH 8.0) in place of the crude enzyme solution. In addition, acarbose was used instead of the coenzyme solution as a positive control.

In the α-glucosidase enzyme activity measurement, the absorbance of the generated ρ-nitrophenol (PNP) was measured by a spectrophotometer at 400 nm, and the inhibition rate was calculated by the following Equation 3 using the measured absorbance. The results are shown in FIG.

[Equation 3]

As shown in FIG. 3, Microbulbifer sp. The α-glucosidase inhibitory activity of the hydrolyzate using SC092 strain was increased in a concentration-dependent manner, and the inhibitory activity was found to be 58.7% at the concentration of 2.5 mg / ml, and 0.5 mg of the a-glucosidase inhibitor of acarbose used as a positive control. / ml showed an inhibitory effect of 40.4%, the microbulbifer sp. Hydrolysates using the SC092 strain are expected to play an important role in the treatment and prevention of diabetes.

2-3. Anti-inflammatory effect measurement

Microbulbifer sp. The anti-inflammatory effect of the hydrolyzate using the SC092 strain was confirmed by Microbulbifer sp. The nitrite scavenging ability of the hydrolyzate using the SC092 strain was measured.

The nitrite scavenging ability was adjusted to pH 1.2, pH 3.0 and pH 6.0 by using 0.1 N HCl and 0.2 M citric acid buffer solution in the mixed solution of the crude enzyme solution 0.5 mg / ml and nitrite solution. The pH-adjusted mixed solution was reacted at 37 ° C. for 1 hour, and then, Griess reagent was added and left at room temperature for 15 minutes, and then absorbance was measured at 520 nm. From the measured absorbance, nitrite scavenging ability was calculated according to the following formula (4). In Formula 4, A is the absorbance after the reaction for 1 hour by adding the extraction sample to 1 mM NaNO ₂ solution, B is: the absorbance of the NaNO ₂ solution, C means the absorbance of the extraction sample itself. The results measured according to Formula 4 are shown in FIG. 4.

[Equation 4]

As shown in FIG. 4, Microbulbifer sp. The nitrite scavenging ability of the hydrolyzate using the SC092 strain showed 56.3% scavenging ability according to pH, pH 1.2, 40.8% scavenging activity for pH 3.0, and 12.6% scavenging activity for pH 6.0. The lower the pH, the higher the scavenging activity. On the other hand, when treated with a positive control Vit C at a concentration of 0.5 mg / ml, it was confirmed that 46.0% at pH 1.2. Therefore, from the above results, Microbulbifer sp. The hydrolyzate using the SC092 strain showed better activity than Vit C, a positive control, and the microbulbifer sp. The hydrolyzate using SC092 strain is considered to be of great industrial value because it can be directly used in foods as a safe anti-inflammatory material of natural materials.

2-4. Hangover  Measure

Most of the alcohol absorbed into the body is absorbed by the stomach and small intestine, moved to the liver and then metabolized and broken down. Chronic alcohol intake can lead to impaired absorption of nutrients and impaired liver function. Alcohol metabolism in liver tissue can first be regulated by factors affecting ADH activity. Most alcohols in the body are rapidly decomposed into acetaldehyde by the hepatic ADH enzyme and then oxidized by the ALDH enzyme. The actual hangover symptoms after drinking alcohol are due to the toxic action of acetaldehyde, so in relation to hangover ability, ADH enzyme and ALDH enzyme, more specifically, can be confirmed through ALDH enzyme activity.

Therefore, in relation to hangover ability, ADH activity and ALDH activity were measured.

Specifically, the measurement of ADH activity was confirmed by the following method. First, the reaction solution composition was added to 1.4 ml of distilled water, 0.75 ml of 1.0 M Tris-HCl buffer (pH 8.8), 20 mM NAD ⁺ 0.3 ml, 0.3 ml of 0.2 M EtOH and 0.1 ml of the coenzyme solution, followed by a 25 ° C constant temperature water bath. The reaction solution was added to a cuvette for 10 minutes and the reaction solution was adjusted to 3 ml in a cuvette, preincubated at 25 ° C. for 5 minutes, and then absorbance was measured at 340 nm for 5 minutes. . In the experiment control was added TE buffer (pH 8.0) in place of the crude enzyme solution. The activity of the ADH was expressed as the ratio of the maximum absorbance at the end of the reaction to the maximum absorbance of the control, specifically calculated by the following formula 5, the results are shown in Table 2 below. In Formula 5, A means the maximum absorbance of the control, B means: the maximum absorbance of the experiment.

[Equation 5]

In addition, ALDH activity measurement was confirmed by the following method. First, the composition of the reaction solution was distilled water 2.1 ml, 1.0 M Tris-HCl buffer (pH 8.0) 0.3 ml, 20 mM NAD ⁺ 0.1 ml, 0.1 M acetaldehyde 0.1 ml, 3.0 M KCl 0.1 ml, 0.33 M ^2- mercapto-ethanol After mixing 0.1 ml and 0.1 ml of the coenzyme solution, react for 10 minutes in a 25 ° C. constant temperature water bath, add 0.1 ml of ALDH (1 unit / ml), adjust the reaction solution to 3 ml, and preincubation at 25 ° C. for 5 minutes. After that, it was carried out by measuring the change in absorbance at 340 nm for 5 minutes. In the experiment control was added TE buffer (pH 8.0) in place of the crude enzyme solution. The activity of ADH was expressed as the ratio of the maximum absorbance at the end of the reaction to the maximum absorbance of the control, specifically calculated by the following formula 6, the results are shown in Table 2 below. In Formula 6, A means the maximum absorbance of the control, B means: the maximum absorbance of the experimental.

[Equation 6]

Concentration (mg / ml) 0.1 0.5 One 2.5 ADH 94.7 ± 0.6 104.7 ± 1.1 112.3 ± 0.7 123.3 ± 1.0 ALDH 134.8 ± 2.7 153.3 ± 2.3 170.4 ± 2.0 215.2 ± 1.7

As shown in Table 2, the maximum absorbance showed a significant difference in all the experimental groups, when the control group absorbance value was set to 100, the microbulbifer sp. The ADH activity of the hydrolyzate using the SC092 strain increased. Specifically, Microbulbifer sp. When the hydrolyzate using the SC092 strain was treated at a concentration of 2.5 mg / ml, the rate of promoting ADH activity was found to be 123.3%. From the above results, Microbulbifer sp. Hydrolysates using the SC092 strains were found to increase the rate of ADH activity increase as the concentration was increased, through which Microbulbifer sp. Hydrolysates using the SC092 strain were estimated to be available for hangover relief.

In addition, as shown in Table 2, Microbulbifer sp. The ALDH activity of the hydrolyzate using the SC092 strain was found to be able to promote ALDH activity in a concentration-dependent manner. Specifically, Microbulbifer sp. When the hydrolyzate using the SC092 strain was treated with the hydrolyzate at a concentration of 2.5 mg / ml, the rate of ALDH activity was found to be 215.2%.

From the above results, Microbulbifer sp. The hydrolyzate using the SC092 strain enables the promotion of ADH activity and the promotion of ALDH activity, and in particular, promotes ALDH activity and has been evaluated as having hangover function.

2-5. Tyrosinase ( tyrosinase ) Through the inhibitory effect Whitening activity  Measure

Tyrosinase is a polyphenol oxidase that is widely distributed in the flora and fauna, and is involved in the conversion of L-tyrosine from L-tyrosine, L-3,4-dihydroxyphenylphenyline (DOPA), to L-dopaquinone during the initial stage of melanin synthesis. It is an enzyme that is involved in the production of melanin pigment, which is finally blackish brown, and plays a crucial role in producing melanin in melanocytes. The melanin appears on the skin as lesions such as blemishes, freckles, melanin hyperpigmentation and the like. In particular, the skin is exposed to ultraviolet rays and the action of tyrosinase causes melanin to be synthesized in the melanosome, which promotes skin aging, and causes browning of foods and the deposition of dark brown pigments on the skin. .

Therefore, tyrosinase activity inhibitors are widely applied to medicines, cosmetics, etc. and have a low side effect on the human body, and the tyrosinase activity inhibition effect was measured in relation to the whitening activity.

Specifically, the inhibitory effect of tyrosinase activity was obtained by adding mushroom tyrosinase (1,000 unit / ml) and distilled water to a mixture of 2 mM L-tyrosine solution and the coenzyme solution as a substrate in 50 mM sodium phosphate buffer (pH 6.5). After reacting for 30 minutes in a 25 ℃ constant temperature water bath, the absorbance (SAbs) was measured at 490 nm. In the experiment, the comparison value (Blank absorbance) was added 20 μl of 50 mM sodium phosphate buffer (pH 6.5) instead of the coenzyme solution, and the absorbance (BAbs) was measured. In addition, the control (Control absorbance) was added 10 μl of TE buffer (pH 8.0) in place of the crude enzyme solution, and the absorbance (CAbs) was measured.

Tyrosinase inhibitory activity was calculated by the following equation (7), the results are shown in FIG.

[Equation 7]

As shown in FIG. 5, Microbulbifer sp. Hydrolysates using the SC092 strain were found to have tyrosinase inhibitory activity in a concentration dependent manner. Specifically, kojic acid as a positive control showed tyrosinase inhibitory activity of 57.4% at 0.1 mg / ml, and the microbulbifer sp. Hydrolysates using the SC092 strain showed 42.2% tyrosinase inhibitory activity at a concentration of 2.5 mg / ml.

From the above results, Microbulbifer sp. The tyrosinase inhibitory activity of the hydrolyzate using the SC092 strain was almost similar to that of kojic acid, which is a conventional tyrosinase inhibitory activity, and the microbulbifer sp. The tyrosinase inhibitory activity of the hydrolyzate using the SC092 strain has excellent whitening activity and is a natural material, so Microbulbifer sp. Hydrolysates using SC092 strains are highly valued as cosmetic and food materials.

2-6. Elastase  Through inhibitory effect Anti-wrinkle activity  Measure

Human skin can be classified into epidermis, dermis, and subcutaneous fat. Fibroblasts differentiated from mesenchynal cells are located in the dermis to produce and shape the necessary substances. The important proteins in the skin are elastin and collagen, and the skin has a balance between enzymes that make major proteins such as elastin and collagen and enzymes that break down them, and have elasticity and structure as normal skin. It is expected to be possible.

Of these proteins, elastin is only 2% of the total protein in the dermis, but plays an important role in the elasticity and physiological function of human skin and is produced in fibroblasts. The skin is exfoliated with age because the elastin breaks down and builds up on the epidermis.

When the activity of elastase, an enzyme that degrades elastin, increases, it causes wrinkles and loss of elasticity of the skin. Thus, the skin is inhibited through an inhibitory activity against elastase, which is one of the causes of wrinkles of the skin. It was confirmed that the effect of giving elasticity to and improving wrinkles. Therefore, anti-wrinkle activity was measured through the elastase inhibitory activity.

Specifically, the effect of inhibiting elastase activity was dissolved in 0.5 mM N-succinyl-Ala-Ala-Ala-ρ-nitroniline as a substrate in 50 mM Tris-HCl buffer (pH 8.2), and then the mixture and the elastase of the coenzyme solution. The first (1 unit / ml) enzyme solution was added and reacted in a 25 ° C. constant temperature water bath for 10 minutes, and the absorbance was measured at 415 nm. The measured absorbance was calculated by the following Equation 8, and the results are shown in FIG. In the following Equation 8, S is the absorbance of the crude enzyme solution, C is the absorbance of the non-added sphere.

[Equation 8]

As shown in FIG. 6, Microbulbifer sp. Hydrolysates using the SC092 strain were found to have elastase inhibitory activity in a concentration dependent manner. Specifically, the positive control vitamin C (Vit C) showed an elastase inhibitory activity of 72.2% at a concentration of 1 mg / ml, Microbulbifer sp. Hydrolysates using the SC092 strain showed elastase inhibitory activity of 73.1% at a concentration of 2.5 mg / ml.

From the above results, Microbulbifer sp. The elastase inhibitory activity of the hydrolyzate using the SC092 strain was almost similar to that of vitamin C known to have the existing elastase inhibitory activity, so that Microbulbifer sp. The elastase inhibitory activity of the hydrolyzate using the SC092 strain has excellent anti-wrinkle activity and is a natural material, so Microbulbifer sp. Hydrolysates using SC092 strains are expected to have high value as beauty and food materials to prevent the action of elastase to give skin elasticity and reduce wrinkles.

Claims (5)

Ecklonia cava ( Ecklonia cava ) microbulbifer sp. ( Microbulbifer sp.) Or microbulbifer sp. ( Microbulbifer sp.), The living medium comprising at least one selected from the group consisting of the culture medium and the concentrate of the culture medium Cosmetic composition comprising a hydrolyzed by a catalyst as an active ingredient. The method of claim 1,
The microbulbifer sp. Strain ( Microbulbifer sp.) Is a microbulbifer sp. SC092 strain ( Microbulbifer sp. SC092) cosmetic composition. The method of claim 1,
The cosmetic composition is a cosmetic composition for wrinkle improvement cosmetic composition. The method of claim 1,
The cosmetic composition is a cosmetic composition is a cosmetic composition for whitening. Wherein the Ecklonia cava micro beolbi flops in SC092 isolates (Microbulbifer sp. SC092) or the micro beolbi flops in SC092 isolates (Microbulbifer sp. SC092), production can be more than one member selected from the group consisting of the culture medium and the concentrate of the culture solution of the strain Reacting the catalyst at 35 ° C. to 37 ° C.
Method for preparing Ecklonia cava hydrolyzate having anti-wrinkle activity and whitening activity.

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