KR20130076723A - Skin care composition - Google Patents

Abstract

PURPOSE: A cosmetic composition which improves skin conditions, containing gamma-aminobutyric acid (GABA) which is generated from a novel lactic acid bacterium with a buck wheat or sea tangle extract is provided to promote collagen synthesis, to alleviate skin inflammation, and to develop and manufacture a functional cosmetic product and a pharmaceutical composition related to skin care. CONSTITUTION: A cosmetic composition for improving skin conditions contains a sea tangle extract, a buck wheat extract, and a lactic acid bacterium-cultured material which is cultured in a medium containing an ingredient selected from the group consisting of the extracts, as active ingredients. The extracts are a hot water extract or an organic solvent extract. The lactic acid bacterium-cultured material contains GABA generated from the lactic acid bacterium. A method for manufacturing the lactic acid bacterium-cultured material comprises the steps of: culturing the lactic acid bacterium in the medium; and collecting the culture.

Description

Skin improvement composition {Skin care Composition}

The present invention relates to a composition for improving skin, and more particularly, the present invention relates to a cosmetic composition for skin improvement comprising a culture obtained by culturing lactic acid bacteria in a medium containing extracts such as kelp and buckwheat as an active ingredient, the cosmetics Functional cosmetics comprising the composition, a method for producing the culture, a culture prepared by the above method, a pharmaceutical composition for treating inflammatory diseases and the quasi-drug composition comprising the culture.

In general, the wrinkles of the skin can be said to be the form and structure of the skin that is locally formed and settled on the site that is chronically deformed by muscle movement. Examining the wrinkle formation mechanism along with the change can be said to be important for understanding the actual wrinkle formation mechanism. In this regard, as skin internal structure changes due to photoaging, epidermis, thickening of the dermis, accumulation of abnormal elastic material, or three-dimensional structure change of elastic fibers have been reported. In addition, for collagen, a skeleton that is a major constituent of skin structure, microfiberization of collagen fibers, weakening of fibrous form, and the like have been found.

Collagen is a major matrix protein produced by skin fibroblasts. It is an important protein in the epilepsy of cells and accounts for 30% of the total weight of biological proteins. It has a solid triple helix structure. Its main functions are known as mechanical firmness of skin, resistance of connective tissue and adhesion of tissue, support of cell adhesion, induction of cell division and differentiation (when organic growth or wound healing). Such collagen is reduced by light aging due to aging and UV irradiation, and in general, as the aging progresses, the thickness of the skin becomes thin and this phenomenon is known to be closely related to the decrease in elasticity of the skin and the formation of wrinkles.

Various active ingredients that promote the collagen synthesis and improve wrinkles, for example, retinoic acid, transforming growth factor (TGF), animal placental proteins (Japanese Patent Publication No. 1996-231370), betulinic acid (betulinic acid) (Japanese Patent Publication No. 1996-208424), Chlorella extract (Japanese Patent Publication No. 194-040523, Japanese Patent Publication No. 1998-036283) and the like are known, but the active ingredients are irritated upon application of skin. There are limitations on the amount of use due to safety problems such as redness or redness, or there is a problem that the effect of improving skin function can not be expected by substantially promoting the collagen synthesis of the skin due to its insignificant effect. Therefore, there is an urgent need for the development of components that are safe for the living body, stable in active ingredients, and above all, have anti-wrinkle activity, which is more effective than conventional collagen synthesis promoting substances. Recently, gamma aminobutyric acid (γ-aminobutyric acid, Attention is focused on the use of GABA.

GABA is an amino acid neurotransmitter that produces inhibitory postsynaptic potentials, and is one of the most commonly used neurotransmitters in the central nervous system of mammals along with glutamic acid and glycine, a nonproteinaceous amino acid that is widely present in flora and fauna including common grains In animals, it exists in the brain. The GABA has been found to have an action of inhibiting nervous excitement, and has an effect of normalizing sedation, antistress and blood pressure, kidney function. Since such GABA can directly change the activity of the cell to change the activity of the cell in a short time, research has been actively conducted to use GABA as an active ingredient of the cosmetic composition. For example, Korean Patent No. 783556 discloses a cosmetic composition of a granulated pack formulation comprising GABA and starch derivatives, and Korean Patent Publication No. 2007-6960 includes a mung bean lactic acid bacteria culture comprising GABA. It discloses about the cosmetic composition to make. However, since the cosmetic composition described above contains a small amount of GABA, there was a disadvantage in that it does not sufficiently reflect the functionality of the GABA.

Under this background, the present inventors search for a component that can assist with the functionality of GABA produced from lactic acid bacteria, and as the component can be an extract of kelp, buckwheat, etc., by culturing the lactic acid bacteria in a medium containing the component The obtained culture may exhibit collagen synthesis promoting effect, skin inflammation relieving effect, etc. due to GABA produced from a component selected from the group consisting of the kelp extract, buckwheat extract, and combinations thereof and lactic acid bacteria, thereby exhibiting skin improvement effect. The present invention was completed by confirming that it can be used as a functional cosmetic composition and a pharmaceutical composition for treating inflammatory diseases.

One object of the present invention to provide a cosmetic composition for skin improvement comprising the lactic acid bacteria culture as an active ingredient.

Another object of the present invention to provide a functional cosmetic comprising the cosmetic composition for skin improvement.

Still another object of the present invention is to provide a method for preparing the lactic acid bacteria culture.

Another object of the present invention is to provide a lactic acid bacteria culture prepared by the above method.

Still another object of the present invention is to provide a pharmaceutical composition for treating an inflammatory disease comprising the culture of lactic acid bacteria as an active ingredient.

Still another object of the present invention is to provide a quasi-drug composition comprising the lactic acid bacteria culture as an active ingredient.

As one embodiment for achieving the above object, the present invention is a cosmetic for skin improvement comprising a lactic acid bacteria cultured in a medium containing a component selected from the group consisting of kelp extract, buckwheat extract and combinations thereof as an active ingredient To provide a composition.

The term "extract" of the present invention refers to an extract obtained by extracting kelp, buckwheat, or the like with hot water or an organic solvent. The organic solvent is not particularly limited thereto, and methanol, nucleic acid, chloroform, methyl acetate, butanol, and the like may be used. Can be. For the purposes of the present invention, the extract may be used in the fermentation broth of lactic acid bacteria to promote collagen synthesis or to alleviate skin inflammation with GABA produced from lactic acid bacteria, but is not particularly limited thereto.

The term "GABA (γ-aminobutyric acid)" of the present invention is widely present in flora and fauna including general grains, and indicates an excitement inhibitory effect, sedative effect, antistress effect, blood pressure normalizing effect, normalizing kidney function, and the like. , Means an amino acid neurotransmitter that causes a potential after inhibitory synapses. In the present invention, the GABA is produced in lactic acid bacteria cultured in a medium containing an extract such as kelp, buckwheat, and can be used to exhibit collagen synthesis or to reduce the skin inflammation with the extract, but is not particularly limited thereto. Does not.

As used herein, the term "lactic acid bacteria" refers to a bacterium that breaks down sugars such as glucose to produce lactic acid, and may generally be Lactobacillus sp. Or Bacillus sp. Lactobacillus brevis, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus helveticus Delbrueki subspecies Bulgaricus (Lactobacillus delbrueckii subsp bulgaricus) and the like, and most preferably may be Lactobacillus brevis AR1 (KACC91688P), but is not particularly limited thereto. Lactobacillus brevis AR1 (KACC91688P) is a lactic acid bacterium newly identified by the present inventors, and deposited on November 23, 2011, at the National Institute of Agricultural Science, National Genetics Institute, 34, Seoho-ro, Gwon-gu, Suwon-si, Gyeonggi-do.

The term "culture" of the present invention means a culture obtained by culturing a specific microorganism in a medium. For the purpose of the present invention, the culture may be obtained by inoculating the lactic acid bacteria in a culture medium containing an extract obtained by extracting kelp, buckwheat, etc. with hot water or an organic solvent, GABA produced from the cultured lactic acid bacteria It may include, but may exhibit a skin improvement effect such as collagen synthesis promoting effect, skin inflammation alleviating effect by the extract and GABA contained in the culture, but is not particularly limited thereto.

As used herein, the term "skin improvement" broadly means a process of treating, alleviating, or alleviating damage to the skin caused by an intrinsic or exogenous factor of the skin, or an effect thereof. For the purpose of the present invention, the skin improvement may be interpreted as showing an effect of promoting collagen synthesis in skin cells, alleviating inflammation occurring in the skin, but is not particularly limited thereto.

According to one embodiment of the present invention, lactic acid bacteria exhibiting GABA (γ-aminobutyric acid) production ability from kimchi is isolated (FIG. 1), and the separated lactic acid bacteria are various grains (buckwheat, oats, black sesame seeds, black beans, yulpi). , Brown rice or barley) or lactic acid bacteria cultures were obtained by inoculating in a medium containing an extract of kelp, and the result of analyzing the effect of the lactic acid bacteria cultures showed a collagen synthesis promoting effect in a cell line similar to skin cells (FIG. 2). And FIG. 3), which shows cell safety in a concentration-dependent manner (FIG. 4), including GABA produced from lactic acid bacteria (FIG. 6), and showing an alleviation effect on skin inflammation (FIG. 7).

The lactic acid bacteria culture is preferably included in 0.0001 to 50% by weight relative to the total weight of the cosmetic composition. If the content of the lactic acid bacteria culture is less than 0.0001% by weight relative to the total weight of the cosmetic composition, it is difficult to expect a substantial skin improvement effect, if more than 50% by weight may cause problems such as unstable formulation due to salt.

On the other hand, the cosmetic composition according to the invention may further comprise an additive. The kind of the additive is not particularly limited, but may be at least one of, for example, oil, water, a surfactant, a moisturizer, a lower alcohol, a bactericide, an antioxidant, a thickener, a chelating agent, a pigment, a preservative and a perfume. These additives may be used in combination in appropriate amounts as necessary.

According to another aspect, the present invention provides a functional cosmetic comprising the cosmetic composition for skin improvement.

The functional cosmetics of the present invention exhibit skin improvement effects such as collagen synthesis promoting effect and skin inflammation relieving effect, and the formulation thereof is not particularly limited, but for example, solution, emulsion, suspension, paste, cream, lotion, gel , Powders, sprays, surfactant-containing cleansing, oils, soaps, liquid cleansers, baths, foundations, makeup bases, essences, lotions, foams, packs, softening water, sunscreen creams, sun oils, etc. Preferably, it may be prepared in the form of external skin ointment, softening water, nourishing cream, nourishing cream, massage cream, essence, pack, emulsion or oil gel.

For example, the skin external ointment may be prepared to contain 50 to 97% by weight of petrolatum and 0.1 to 5% by weight of polyoxyethylene oleyl-ether phosphate in addition to the lactic acid bacteria fermentation product of the present invention. For example, softener may be prepared to contain 1 to 10% by weight of polyhydric alcohols such as propylene glycol or glycerin and 0.05 to 2% by weight of surfactants such as polyethylene oleyl ether or polyoxyethylene hydrogenated castor oil, The lotion and nourishing cream may be prepared to contain 5 to 20% by weight of oils such as squalane, petrolatum or octyldodecanol and 3 to 15% by weight of wax components such as cetanol, stearyl alcohol or beeswax in addition to the herbal fermented product. , Essence can be prepared to contain 5 to 30% by weight of polyhydric alcohols such as glycerin or propylene glycol.

The massage cream may be prepared to contain 30 to 70% by weight of oils such as liquid paraffin, petrolatum or isononylisononanoate in addition to the lactic acid bacteria fermentation product of the present invention, and the pack may contain 5 to 20% by weight of polyvinyl alcohol. It can be prepared as a peel off pack, and can also be prepared as a wash off pack containing from 5 to 30% by weight of pigments such as kaolin, talc, zinc oxide or titanium dioxide in a general emulsified cosmetic. have.

According to another aspect, the present invention comprises the steps of: (i) culturing the lactic acid bacteria in a medium comprising a component selected from the group consisting of kelp extract, buckwheat extract and combinations thereof; And (ii) provides a method for producing a culture of lactic acid bacteria showing a skin improvement effect, comprising the step of obtaining a culture and lactic acid bacteria culture prepared by the above method.

In the above production method, the extract, lactic acid bacteria, culture and the like are the same as described above, the lactic acid bacteria culture may include GABA (γ-aminobutyric acid) generated from lactic acid bacteria, collagen synthesis promoting effect, skin It may also have a skin-improving effect, such as inflammation relief.

According to another aspect, the present invention provides a composition for treating inflammatory diseases or quasi-drugs comprising a lactic acid bacteria cultured in a medium comprising a component selected from the group consisting of kelp extract, buckwheat extract and combinations thereof as an active ingredient. to provide.

The composition of the present invention may be utilized to prevent or treat an inflammatory response by administering to a subject who is likely to develop or develops an inflammatory disease on the skin. In this case, the inflammatory disease is not particularly limited thereto, but rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, spondyloarthropathy, inflammatory bowel disease, chronic heart failure, diabetes, systemic lupus erythematosus, scleroderma, sarcoidosis, Crohn's disease, multiple myositis / Dermatitis, multiple myelopathy, bone dysplasia syndrome, acute myeloid leukemia, Parkinson's disease, AIDS dementia, Alzheimer's disease, depressive disorder, necrotic pyoderma, septic shock, Behcet's syndrome, Wegener's granulomatosis, Sjogren's syndrome, chronic Obstructive pulmonary disease, periodontal disease, cachexia, central nervous system injury, viral respiratory disease, obesity, cirrhosis, cardiac myxoma, silicosis or AIDS, plasmacytosis, hyperimmunoglobulinemia, anemia, nephritis, Catzl's disease , Vasculitis, systemic lupus erythematosus, ulcerative colitis, acute pancreatitis, combustible idiopathic atrophy or systemic combustible Atrophic deafness, senile deafness or noise-induced hearing loss due to vocal atrophy, vasculitis and Kawasaki disease, Ménière's disease, drug inner ear disorder, viral otitis, purulent otitis, temporal bone fracture or auditory nerve tumor Lupus erythematosus, Atopic dermatitis, Diabetic retinopathy, Retinitis, Macular degeneration, Coronary artery disease, Myocardial infarction, Unstable angina, Vascular stenosis, Giant cell arteritis, Ankylosing spondylitis, Osteoporosis, Diabetic nephropathy, Autoimmune pancreatitis, Chronic pelvic inflammation Diseases, endometritis, tumor metastasis, transplant rejection and chronic prostatitis, allergic rhinitis, COPD, irritable lung disease, irritable pneumonia, interstitial lung disease (ILD), anaphylaxis or hypersensitivity reactions, drug allergies, allergic acupuncture, spondyloarthritis , Scleroderma; Psoriasis and dermatitis, eczema, allergic contact dermatitis, gallbladder, vasculitis, chronic arthritis, psoriatic arthritis and deformable arthritis, polychondritis, chronic active hepatitis, myasthenia gravis, Steven-Johnson syndrome, idiopathic sprue, autoimmune thyroiditis, basset disease, Endocrine eye diseases, Graves' disease, multiple sclerosis, primary biliary combustible diabetes mellitus (true diabetes mellitus type I), dry keratoconjunctivitis and spring keratoconjunctivitis, interstitial pulmonary fibrosis, and glomerulonephritis, including bacterial and fungal diphtheria and pertussis Upper respiratory tract infections including acute and chronic infections, acute and chronic bronchitis, sinusitis and common cold, acute and chronic gastroenteritis and colitis, acute and chronic cystitis and urethritis, acute and chronic dermatitis, acute and chronic conjunctivitis, acute and chronic Meningitis, peritonitis, peritonitis, synovialitis, pleurisy and tendinitis, uremic peritonitis, acute and bay NO-related conditions including cholecystitis, acute and chronic vaginitis, acute and chronic uveitis, drug reactions, wounds in insects, burns (heat, chemicals and electrical burns) and sunburn, arteriosclerosis, stroke Tissue damage due to ischemia and reperfusion, multiple sclerosis, chronic inflammatory demyelinating polyneuropathy, glomerulonephritis, nephritis and the like.

As used herein, the term "individual" means any animal, including humans, who may or may have an inflammatory disease.

At this time, the route of administration of the composition may be administered through any general route as long as it can reach the target tissue. The composition of the present invention may be administered as skin coating, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration, pulmonary administration, rectal administration, but is not limited thereto. Does not. In addition, the composition may be administered by any device capable of transferring the active agent to the target cell.

The composition of the present invention may further comprise a pharmaceutically acceptable diluent, excipient or carrier. The composition comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablet pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose ), Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The composition is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers and suppositories. It may have a formulation.

The term "pharmaceutically effective amount" of the present invention means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, meaning that the composition of the present invention can be administered in a pharmaceutically effective amount. The effective dose level is determined by the type and severity of the subject, the age, sex, activity of the drug, sensitivity to the drug, time of administration, route and rate of excretion, duration of treatment, Can be determined according to the element. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. It is important to take into account all of the above factors and administer an amount that will achieve the maximum effect in the least amount without side effects.

In addition, the composition of the present invention may be used alone or in combination with methods using surgery, hormonal therapy, drug treatment and biological response modifiers for the treatment of inflammatory diseases appearing on the skin.

In the composition, the extract, lactic acid bacteria, cultures and the like are the same as described above, the lactic acid bacteria cultures are produced from GABA (lactic acid bacteria) with a component selected from the group consisting of kelp extract, buckwheat extract and combinations thereof γ-aminobutyric acid) and can be used as a pharmaceutical composition because it shows remarkable safety for cells (FIG. 4).

Cosmetic composition showing the skin improvement effect of the present invention, including GABA produced from a novel lactic acid bacteria with buckwheat or kelp extract, showing the skin improvement effect such as collagen synthesis promoting effect, skin inflammation alleviating effect, as well as functional cosmetics It may be widely used in the development and manufacture of skin-related pharmaceutical compositions.

Figure 1 is a photograph showing the TLC test results for GABA production of kimchi lactic acid bacteria.
Figure 2 is a graph showing the collagen synthesis promoting effect of the culture of lactic acid bacteria containing grains or kelp extract.
Figure 3 is a graph showing the concentration-dependent collagen production ability of lactic acid bacteria culture containing buckwheat or kelp extract.
Figure 4 is a graph showing the concentration-dependent cell safety of lactic acid bacteria culture containing buckwheat or kelp extract.
Figure 5 is a graph showing the growth curve over time of AR1 strain cultured in a medium containing buckwheat or kelp extract.
Figure 6 is a graph showing the content of GABA produced over time in AR1 strains cultured in a medium containing buckwheat or kelp extract.
Figure 7 is a micrograph showing the effect of lactic acid bacteria culture containing buckwheat or kelp extract on the inflammation caused by skin irritation.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not limited by these embodiments.

Example  1: Preparation of Grain and Kelp Extract

Wash various grains (buckwheat, oats, black sesame seeds, black beans, yulpi, brown rice, barley, perilla or wheat) or kelp with water, grind them in a mixer, and add 5 times the volume of water to 100 g of grain or kelp, respectively. Extraction was carried out for 12 hours at a temperature of 60 ℃. After the extraction was completed, the supernatant was obtained by centrifugation (6000 rpm, 15 minutes), and the obtained supernatant was lyophilized to prepare each grain or kelp extract.

Example  2: GABA  Isolation of Producing Lactobacillus

Kimchi contains various lactic acid bacteria and produces various physiologically active substances and has superiority in preventing adult diseases, anticancer effect, digestive and tonic effects. In order to separate the lactic acid bacteria that synthesize GABA from kimchi, 0.1 ml of dilute solution of various cabbage kimchi diluted in 1 ml of sterile water was added to MRS medium (Pancreatic digest of gelatin 10g / l, Beef extract 8g / l, Dextrose 20g /). L, Dipotassium phosphate 2g / l, Polysorbate80 1g / l, Sodium acetate 5g / l, Ammonium citrate 2g / l, Magnesium sulfate 0.2g / l, Manganese sulfate 0.05g / l) and cultured to form colonies. Among the colonies, various types of colonies were selected, the selected colonies were inoculated in a liquid MRS medium to which 5% MSG (monosodium glutamic acid) was added, and then cultured at 37 ° C. for 48 hours to obtain a culture. It was. The obtained culture was centrifuged to recover the supernatant, and distilled water was added to the recovered supernatant to dilute to 50 times the volume. The diluted supernatant was added to TLC and developed as a solvent (butanol: acetic acid: water = 4: 1). After the development in 1), 0.2% ninhydrin was added to confirm whether the color development was confirmed by GABA synthesis (Fig. 1).

Figure 1 is a photograph showing the TLC test results for the GABA production of kimchi lactic acid bacteria, lane 1 represents the GABA standard used as a control, lane 2 represents the kimchi-derived lactic acid bacteria showing the GABA production capacity. As shown in Fig. 1, lactic acid bacteria showing GABA production ability were detected from one type of sample.

The 16s rDNA partial base sequence (SEQ ID NO: 1) was analyzed to identify the detected lactic acid bacteria, and the lactic acid bacteria were 100% identical to 16s rDNA of Lactobacillus brevis IMAU80121. It was named and deposited as KACC91688P on November 23, 2011, at the National Institute of Agricultural Science, National Agricultural Science Institute, 34, Seoho-ro, Gwon-gu, Suwon-si, Gyeonggi-do.

Example  3: Preparation of Lactic Acid Bacteria Culture Containing Grain or Kelp Extract

Sucrose-yeast extract medium (4% sucrose, 1% yeast extract, 4% MSG) was added to 500 g of the grain or kelp extract prepared in Example 1, mixed, and sterilized by heating at 80 ° C. for 1 hour. For buckwheat and kelp mixed extracts, sucrose-yeast extract medium (4% sucrose, 1% yeast extract, 4% MSG) was added to 500 g of 250 g of buckwheat extract and 250 g of kelp extract, followed by heating at 80 ° C. for 1 hour. Treated and used. In this case, the MSG contained in the medium was used as a substrate of GAD (glutamate decarboxylase), which is an enzyme synthesizing GABA, and the GAD used was derived from the AR1 strain identified in Example 2.

AR1 strains identified in Example 2 were precultured in liquid MRS medium for 2 days, inoculated at 1% (v / v) concentration in each of the sterilized sucrose-yeast extract medium and stirred at 120 rpm. Incubation at 30 ° C. for 68 hours yielded each lactic acid bacteria culture. Each of the obtained lactic acid bacteria culture was centrifuged (8000 rpm, 10 minutes) to recover each supernatant, and each of the recovered supernatants was filtered through a microfilter (pore size: 3 μm), and then each of these cells And a supernatant in which the insoluble component was filtered was used as a sample.

Example  4: Validation of Lactic Acid Bacteria Culture Containing Grain or Kelp Extract

Example  4-1: Collagen Synthesis Promoting Effect

The collagen synthesis promoting effect of each sample obtained in Example 3 was confirmed using the Sircol Assay method. The circol assay method is a method for measuring collagen by quantifying the conjugate of collagen and dye solution using a dye that is specifically bound to collagen, and the cells used for collagen synthesis are fibroblast-derived NIH 3T3 cell line (KCLB, Korea) was used.

Specifically, the NIH 3T3 cell line was incubated at 37 ° C. and 5% CO ₂ conditions in DMEM medium (FBS 10%), dispensed in 24-well plates at a concentration of 4 × 10 ⁴ / well, respectively, and 12 hours under the same conditions. Incubated for 2 hours. Then, each sample obtained in Example 3 was treated at a concentration of 0.1% (v / v), and further incubated for 24 hours under the same conditions. At this time, the control group was used NIH 3T3 cell line treated with a sample derived from lactic acid bacteria culture cultured without containing any grain or kelp extract.

After the incubation was completed, each culture was centrifuged at 12,000 xg to obtain a supernatant, and the supernatant was reacted by applying to a assay assay kit (Sircol assay, Biocolor, UK). Absorbance (OD ₅₄₀ ) was measured to quantify the synthesized collagen (FIG. 2).

Figure 2 is a graph showing the collagen synthesis promoting effect of the culture of lactic acid bacteria containing grains or kelp extract. As shown in Figure 2, lactic acid bacteria culture containing buckwheat, oats, black sesame, black beans, yulpi, brown rice and barley extract promotes collagen synthesis to a higher level than the control, especially lactic acid bacteria culture containing buckwheat or kelp extract Water was found to promote the synthesis of collagen at the highest level (300μg / ml or more).

In order to confirm the collagen production ability according to the concentration of the lactic acid bacteria culture containing the identified buckwheat or kelp extract, genistein (0.1, 1.0 or 10 μM) known to have excellent collagen synthesis effect while culturing the NIH 3T3 cell line in a 24-well plate , Lactic acid bacteria culture containing the buckwheat extract (0.01, 0.1 or 0.5%), lactic acid bacteria culture containing the kelp extract (0.01, 0.1 or 0.5%) or lactic acid bacteria culture containing the buckwheat and kelp mixture extract (0.01 , 0.1 or 0.5%), and incubated for 24 hours, followed by the above-described assay method (Fig. 3).

Figure 3 is a graph showing the concentration-dependent collagen production ability of lactic acid bacteria culture containing buckwheat or kelp extract. As shown in FIG. 3, when 0.5% of lactic acid bacteria culture containing kelp extract was treated, collagen synthesis was more accelerated than 10 μM treatment of genistein used as a control, and the lactic acid bacteria culture containing buckwheat extract was also similar to genistein. It was confirmed that collagen synthesis is promoted to the extent.

In addition, in the culture of lactic acid bacteria containing buckwheat and kelp mixed extracts showed twice the collagen synthesis ability at the same concentration than the respective extracts of buckwheat and kelp, it was confirmed that the collagen synthesis capacity is doubled when the two substances are mixed. .

Example  4-2: Cell Safety Test

The lactic acid bacteria culture comprising the buckwheat or kelp extract identified in Example 4-1 was to determine whether or not affect the cell activity.

Specifically, lactic acid bacteria culture comprising buckwheat or kelp extract in the culture solution in the culture solution in the NIH 3T3 cell line cultured in 24-well plate in the same manner as in Example 4-1 to 1, 2, 5 or 10%, respectively After treatment, the cells were incubated for 24 hours under the same conditions, and then reacted with an EZ-cytox cell viability assay kit (Daeillab service, Korea). The absorbance of the reactants (OD ₄₅₀ ) was measured to measure the activity of the NIH 3T3 cell line. (FIG. 4).

Figure 4 is a graph showing the concentration-dependent cell safety of lactic acid bacteria culture containing buckwheat or kelp extract. As shown in Figure 4, as the concentration of the lactic acid bacteria culture containing the buckwheat or kelp extract contained in the culture medium showed a tendency to decrease slightly, even when included in the maximum 10% showed more than 85% of the cell activity It was confirmed.

Therefore, lactic acid bacteria cultures containing buckwheat or kelp extract shows a significant safety for the cells, it can be seen that it can be used as a cosmetic composition and a pharmaceutical composition.

Example  4-3: GABA Generation  analysis

When culturing the AR1 strain in a medium containing the buckwheat or kelp extract confirmed in Example 4-1, it was intended to determine whether GABA can be produced.

Specifically, sucrose-yeast extract medium was added to 500 g of buckwheat or kelp extract prepared by the method of Example 1, mixed, and sterilized by heating at 80 ° C. for 1 hour, and pre-cultured in an MRS medium for 2 days. AR1 strains were inoculated in a sucrose-yeast extract medium containing buckwheat or kelp extract at a concentration of 1% (v / v) and incubated at 30 ° C. for 68 hours while stirring at 120 rpm for each lactic acid bacteria culture every 4 hours. Samples were obtained. Then, the absorbance (OD ₆₀₀ ) of the obtained sample was measured to compare the growth type of the AR1 strain (FIG. 5).

Figure 5 is a graph showing the growth curve over time of AR1 strain cultured in a medium containing buckwheat or kelp extract. As shown in Figure 5, AR1 strain was confirmed that can be more effectively grown in the medium containing buckwheat extract than the medium containing the kelp extract.

On the other hand, the content of GABA contained in the obtained sample was analyzed using HPLC. That is, by using the AccQ-Tag using PITC (phenylisothiocyanate) fluorescently label the GABA contained in each sample, the labeled sample was filtered with a microfilter (pore size: 0.45㎛), and then Nova-Pak C18 Fluorescent label under appropriate elution conditions (developing solvent: AccQ-Tag Eluent A: 69% acetonitrile = 98: 2 (v / v), flow rate: 1 ml / min) by HPLC using HPLC column (150 x 3.9 mm) The amount of GABA was analyzed (FIG. 6). Figure 6 is a graph showing the content of GABA produced over time in AR1 strains cultured in a medium containing buckwheat or kelp extract. As shown in FIG. 6, GABA was started to be produced 45 hours after the start of the stop phase of the AR1 strain in both the buckwheat or kelp extract medium, and after about 68 hours, the GABA was about 180 mM (GABA conversion rate 84%). Was produced, it was confirmed that exhibited similar levels of GABA production ability in the medium containing buckwheat or kelp extract.

These results demonstrate that the lactic acid bacteria of the present invention have an excellent effect on the growth and GABA production in the medium containing buckwheat or kelp extract.

Example  4-4: Dermatitis  Mitigating effect

Known method (Sheu et al., The whether or not the lactic acid bacteria culture obtained by culturing the lactic acid bacteria in the medium containing the buckwheat or kelp extract confirmed in Example 4-1 exhibits an inflammation-inducing effect caused by skin irritation Journal of Investigative Dermatology, 118, pp94-101, 2002).

Specifically, TPA (12-O-tetradecanoylphorbol-13-acetate) 0.05% (wt / vol), known as a skin irritant, is found in and around both ears of CD-1 mice (male & female, 4-6 weeks, Hyochang science, Korea). 10 μl of acetone) solution, and clofibrate, a PPAR α agonist, known to exhibit an anti-inflammatory effect as a control after 45 minutes and 2 hours after the inflammatory reaction occurred in the treated ear. Lactobacillus cultures derived from a medium containing 1 mM, 5% buckwheat extract and lactic acid bacteria cultures derived from a medium containing 5% kelp extract were each treated in an amount of 2 μl / cm 2. The site of inflammation was then removed, fixed with 4% formaldehyde, subjected to H & E staining, and observed under a microscope at 100x magnification (FIG. 7).

Figure 7 is a micrograph showing the effect of the culture of lactic acid bacteria containing buckwheat or kelp extract on inflammation caused by skin irritation, A represents a TPA-treated tissue, B sequentially TPA and clofibrate Represents the treated tissue, C represents a tissue treated sequentially with the lactic acid bacteria culture containing TPA and kelp extract, D denotes a tissue treated sequentially with a lactic acid bacteria culture containing TPA and buckwheat extract. As shown in FIG. 7, it was confirmed that the tissue was thickened in the tissue (A) treated only with TPA, and that inflammation was induced in the tissue (B) sequentially treated with TPA and clofibrate. In the tissue (C) sequentially treated with lactic acid bacteria culture containing TPA and kelp extract was confirmed that the inflammation induced by the level similar to the tissue (B) treated with clofibrate is alleviated, including TPA and buckwheat extract In the tissue (D) treated sequentially with the lactic acid bacteria culture, but relatively lower than the lactic acid bacteria culture (C) containing the kelp extract, it was confirmed that the induced inflammation is alleviated.

Through the results of Examples 4-1 to 4-4, the culture obtained by culturing the lactic acid bacteria in a medium containing a component selected from the group consisting of kelp extract, buckwheat extract and combinations thereof is produced by GABA Was added, indicating safety to the cells, showing a collagen synthesis promoting effect or skin inflammation alleviating effect, it can be seen that the culture can be used as an active ingredient of functional cosmetics and inflammation treatment compositions.

Agricultural Genetic Resource Center KACC91688P 20111123

<110> Dong-eui University Industry-Academic Cooperation Foundation <120> Skin care Composition <130> PA121137 / KR <150> KR10-2011-0145133 <151> 2011-12-28 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1400 <212> DNA <213> Artificial Sequence <220> <223> 16s rDNA <400> 1 cgctggcggc atgcctaata catgcaagtc gaacgagctt ccgttgaatg acgtgcttgc 60 actgatttca acaatgaagc gagtggcgaa ctggtgagta acacgtggga aatctgccca 120 gaagcagggg ataacacttg gaaacaggtg ctaataccgt ataacaacaa aatccgcatg 180 gattttgttt gaaaggtggc ttcggctatc acttctggat gatcccgcgg cgtattagtt 240 agttggtgag gtaaaggccc accaagacga tgatacgtag ccgacctgag agggtaatcg 300 gccacattgg gactgagaca cggcccaaac tcctacggga ggcagcagta gggaatcttc 360 cacaatggac gaaagtctga tggagcaatg ccgcgtgagt gaagaagggt ttcggctcgt 420 aaaactctgt tgttaaagaa gaacaccttt gagagtaact gttcaagggt tgacggtatt 480 taaccagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta ggtggcaagc 540 gttgtccgga tttattgggc gtaaagcgag cgcaggcggt tttttaagtc tgatgtgaaa 600 gccttcggct taaccggaga agtgcatcgg aaactgggag acttgagtgc agaagaggac 660 agtggaactc catgtgtagc ggtggaatgc gtagatatat ggaagaacac cagtggcgaa 720 ggcggctgtc tagtctgtaa ctgacgctga ggctcgaaag catgggtagc gaacaggatt 780 agataccctg gtagtccatg ccgtaaacga tgagtgctaa gtgttggagg gtttccgccc 840 ttcagtgctg cagctaacgc attaagcact ccgcctgggg agtacgaccg caaggttgaa 900 actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagct 960 acgcgaagaa ccttaccagg tcttgacatc ttctgccaat cttagagata agacgttccc 1020 ttcggggaca gaatgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg 1080 gttaagtccc gcaacgagcg caacccttat tatcagttgc cagcattcag ttgggcactc 1140 tggtgagact gccggtgaca aaccggagga aggtggggat gacgtcaaat catcatgccc 1200 cttatgacct gggctacaca cgtgctacaa tggacggtac aacgagtcgc gaagtcgtga 1260 ggctaagcta atctcttaaa gccgttctca gttcggattg taggctgcaa ctcgcctaca 1320 tgaagttgga atcgctagta atcgccgatc agcatgccgc gttgaatact ttcccggggc 1380 ttgtacacac cgcccgtcac 1400

Claims (15)

A cosmetic composition for skin improvement comprising lactic acid bacteria cultured in a medium comprising a component selected from the group consisting of kelp extract, buckwheat extract and combinations thereof as an active ingredient.
The method of claim 1,
The extract is a hot water extract or an organic solvent extract.
The method of claim 1,
The lactic acid bacteria culture composition comprising GABA (γ-aminobutyric acid) generated from lactic acid bacteria.
The method of claim 1,
The lactic acid bacteria is Lactobacillus brevis AR1 (KACC91688P) composition.
The method of claim 1,
A composition that exhibits collagen synthesis promoting effect or skin inflammation relieving effect.
The method of claim 1,
The composition further comprises one or more selected from the group consisting of water, surfactants, moisturizers, lower alcohols, chelating agents, fungicides, antioxidants, preservatives, pigments and flavorings.
A functional cosmetic for skin improvement comprising the composition of any one of claims 1 to 6.
The method of claim 7, wherein
A lotion, a gel, a powder, a spray, a surfactant-containing cleansing oil, a soap, a liquid cleansing agent, a bath agent, a foundation, a makeup base, an essence, a lotion, a foam, Sunscreen cream or sun oil. &Lt; RTI ID = 0.0 &gt; 18. &lt; / RTI &gt;
(Iii) culturing the lactic acid bacteria in a medium comprising a component selected from the group consisting of kelp extract, buckwheat extract, and combinations thereof; And
(Ii) A method of producing a lactic acid bacteria culture having a skin improvement effect, comprising the step of obtaining a culture.
10. The method of claim 9,
The lactic acid bacteria is Lactobacillus brevis AR1 (KACC91688P) method.
10. The method of claim 9,
The lactic acid bacteria culture method comprising GABA (γ-aminobutyric acid) generated from lactic acid bacteria.
A lactic acid bacterium culture prepared by the method of any one of claims 9 to 11 and exhibiting skin improvement effect.
Pharmaceutical composition for the treatment of inflammation comprising lactic acid bacteria cultured in a medium comprising a component selected from the group consisting of kelp extract, buckwheat extract and combinations thereof as an active ingredient.
The method of claim 13,
Wherein said composition further comprises a pharmaceutically acceptable diluent, excipient or carrier.
A quasi-drug composition comprising a lactic acid bacteria cultured in a medium containing a component selected from the group consisting of kelp extract, buckwheat extract, and combinations thereof as an active ingredient.

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