KR20210081645A - Preparing method of highly functional peptide derived from natural fermentation extracts - Google Patents

Abstract

Disclosed is a method for revealing that ginseng fruits contain high protein and peptide and increasing the peptide or protein content in fermented ginseng fruits. The present invention provides a method of increasing the peptide or protein content by solvent fractionation of a fermented product obtained by inoculating and fermenting a Bacillus sp. strain in a fermentation medium containing a ginseng fruit extract.

Description

Method for producing high-functional peptides derived from natural fermented products {PREPARING METHOD OF HIGHLY FUNCTIONAL PEPTIDE DERIVED FROM NATURAL FERMENTATION EXTRACTS}

The present invention relates to a method for producing a high-functionality peptide, and more particularly, to a method for producing a high-functionality peptide derived from a natural fermented product.

Fermentation refers to a process in which enzymes possessed by microorganisms change raw materials to produce beneficial ingredients. During the fermentation process, active ingredients are extracted and the content of functional substances increases. In addition, fermentation technology has overlap in meaning with terms such as bioconversion, biosynthesis, and biocatalyst, and refers to a technology for producing a desired product from a precursor by using the enzymatic function of a microorganism. Therefore, fermentation technology that uses biological functions in the cell or enzyme stage is a major process that constitutes the current biological industry with the main purpose of producing useful substances.

If fermented food uses one-dimensional fermentation technology that utilizes naturally occurring microorganisms, fermented cosmetics require more advanced technology. In addition, if the first-generation fermentation simply used the traditional fermentation method, the second-generation fermentation was the development of practical technology for effective substances, and through advanced fermentation production technology that can produce only useful physiologically active substances in large quantities, the foundation of the cosmetic functional material industry was laid. Bioconversion fermentation technology is establishing itself as the third-generation fermentation technology.

Since Koreans use fermented foods such as kimchi and soybean paste a lot, the concept of fermentation is very familiar, and cosmetics using fermented raw materials are also used without objection. Also, the general public has a high preference for fermented cosmetics as it is known that when new materials are developed using fermentation technology, they show completely new effects from existing materials or that many harmful ingredients are changed into safe ingredients, and the technology development is also in other advanced countries. It is not inferior to the level. However, it is known that many fermented materials currently applied to cosmetics use natural fermentation or a fermentation method close to natural aging.

The recent research trend in domestic fermentation technology is known to use natural fermentation or a fermentation method close to natural fermentation. Natural fermentation is a process that uses the power of nature such as water, wind, earth, and seasons while leaving various unprocessed raw materials as they are. It is a method that naturally produces ingredients useful for the skin, although it takes a long time. Its strength is that it can obtain various substances, so that it can obtain effects such as moisturizing, antioxidant, wrinkle improvement, cell activity, and skin texture improvement. However, fermentation of natural fermentation or natural aging method has disadvantages in that it is difficult to obtain a fermentation product of a certain quality for each production batch, and it is difficult to control the quality.

The Korean fermented cosmetics market has been growing at a high rate of 40% in the last three to four years, and is expected to reach about 450 billion won in 2012, 650 billion won in 2013, and 1 trillion won (estimated) in 2014. is looking forward

Fermented products can be largely classified into fermentation filtrates and fermentation lysates. Fermentation filtrates use a supernatant from which microorganisms are removed through a filtration device after fermentation is complete, and fermentation Lysate is a method in which microorganisms are killed by using heat, pH, enzymes (Lysozyme), high pressure, etc. after completion of fermentation.

On the other hand, ginseng (the root of Panax ginseng CAMeyer) is one of the traditional medicines used for various diseases worldwide. The pharmacological component is ginsenoside, which is one of glycosides, and it is known that there are about 30 types of ginsenoside so far.

Recently, as ginseng has been known to have antioxidant effects, anti-wrinkle effects, and anti-aging effects on the skin, ginseng-related beauty products with various functionalities are currently being sold at high prices and the demand for them is gradually increasing. It is 4-6 years long, and it is known that 100-150 g (fresh weight) of fresh ginseng per root can be harvested and continuous cultivation is impossible after 6 years of cultivation.

In the case of ginseng cosmetics, various companies have been producing the product for a long time based on the high preference of domestic consumers for ginseng, and the most representative ginseng cosmetic manufacturers are Amorepacific (trade name: Sulwhasoo) and Korea Tobacco and Ginseng Corporation (trade name). Donginbi) is one of the representatives, and each has built its own product line and is receiving a good response from consumers. These companies used ginseng and red ginseng extract as main ingredients in the past, but recently they are improving the quality by separating and prescribing only the ingredients of saponin or ginsenoside. It also meets the needs.

Ginseng berry, a kind of ginseng by-product, is a raw material that can be easily obtained at a lower price than ginseng, but contains active ingredients with efficacy and high content comparable to that of ginseng itself.

Ginseng fruit began to attract attention when the results of a study at the University of Chicago Medical School in the United States that the unique saponin of ginseng fruit called 'ginsenoside Re' prevents diabetes and obesity was published in the American medical journal Diabetes in 2002. When the research team administered ginseng berries to rats that were manipulated to become diabetic, fasting blood sugar levels were significantly reduced. In addition, it was confirmed that the cholesterol level and body weight of the experimental mice decreased and the energy consumption rate increased.

In particular, the effect of ginseng berry improving wrinkles and enhancing skin elasticity was confirmed. The anti-aging effect was found to be superior to that of ginseng root because ginseng fruit contains abundant vitamin E and anthocyanin, which have excellent antioxidant effects. Ginsenoside Re, the main component of ginseng fruit saponin, has a similar role to phytoestrogens. It is also known to help relieve the symptoms of menopause in women.

In the case of ginseng fruit, because it contains a large amount of active ingredients, it can be used as a cosmetic ingredient, pharmaceutical raw material, and feed additive at home and abroad. The possibility of becoming an industrial material is increasing due to the relaxation of safety standards, a constraint factor. Considering the industrial use value of ginseng by-product, it is necessary to develop various uses and application methods. In particular, it is necessary to expand the utility of ginseng fruit, a by-product of ginseng, through research on functional peptides in the ginseng fruit as well as the well-known ginsenoside series. The need for research is urgent.

Ginsenoside is a major component of ginseng and has been widely used as a functional ingredient in pharmaceuticals, health functional foods, and cosmetics. became engulfed In addition, Jin-Ho Chung et al. (Food and Chemical Toxicology 118 (2018) 645.652) confirmed the toxicity by culturing rat smooth muscle (muscle composing the intestinal wall) cells in Rg3 culture medium, and culture medium with Rg3 concentration of 10 μM or more. induced apoptosis of cardiac smooth muscle cells, and the negative effect on smooth muscle activity was confirmed even at a lower concentration of 1 μM. The mechanism is that Rg3 is 'F-actin', which is responsible for muscle contraction and relaxation of smooth muscle cells. It was confirmed that 'Bmf protein, which was released by breaking down the binding state of the protein and also separating the 'Bmf' protein bound to F-actin, moves to the mitochondria of the cell and interferes with the energy production activity in the mitochondria to induce cell death. Therefore, it was confirmed through the paper that Rg3, which is known to have an anticancer effect, has a fairly strong cytotoxicity, and does not selectively kill only cancer cells but also destroys normal cells.

Of course, there are various ginsenosides other than Rg3 in ginseng, and it cannot be considered that ginseng is harmful to the human body because the toxicity of all of them has not been confirmed. It is true that it is necessary.

In addition to ginsenoside, ginseng or a byproduct of ginseng contains various functional ingredients, and in particular, a lot of research has been done on the immune function of ginseng polysaccharides. Although ginseng polysaccharide extract has been individually certified and used in health functional foods, the reality is that there is a lack of research on ginseng protein or peptide, and it has not been identified as an ingredient that exerts the efficacy of ginseng.

An object of the present invention is to provide a method for isolating peptides having various functions in a fermented ginseng fruit, and a cosmetic composition containing the functional peptide separated by the method as an active ingredient.

In order to solve the above problems, the present invention provides a method for isolating functional peptides in fermented ginseng fruit, including the step of inoculating and fermenting ginseng fruit (Panax Ginseng Berry) with bacteria and then filtering to remove microorganisms.

In addition, the fermentation provides a method, characterized in that it is carried out at 30 to 50 ℃ 2 to 4 days.

In addition, the bacteria provides a method, characterized in that the bacteria of the genus Bacillus ( Bacullus sp.).

In addition, the functional peptide provides a method, characterized in that obtained through solvent fractionation and size exclusion chromatography.

In addition, the functional peptide provides a method, characterized in that it comprises a sequence of (glycine-alanine) n (n is an integer of 2 to 10).

In addition, the functional peptide provides a method, characterized in that it comprises the sequence of glycine-alanine-glycine-alanine-glycine-alanine.

In addition, the functional peptide provides a method, characterized in that it has skin wrinkle improvement and anti-aging functions.

In addition, the functional peptide provides a method characterized in that it has a function of increasing the skin cell regeneration rate by promoting skin cell growth.

In addition, the functional peptide promotes collagen production in human fibroblasts and inhibits the activity of collagen degrading enzyme (MMP-1) to provide a method characterized in that it has a skin wrinkle improvement function.

In addition, the fermented ginseng fruit and the functional peptide provide a method, characterized in that it has a function of promoting the proliferation of human skin keratinocytes.

In order to solve the another problem, the present invention provides a cosmetic composition containing the functional peptide isolated according to the above method as an active ingredient.

The present invention confirmed that the pattern of proteins and peptides in the ginseng fruit was converted through fermentation of the ginseng fruit, and it was also confirmed that the higher the peptide content through the separation and purification, the higher the anti-wrinkle activity. In particular, peptides showing excellent efficacy were finally isolated and the toxicity, efficacy, and physical properties of a single peptide were confirmed.

The functional peptide derived from ginseng fruit provided through the present invention may have various uses as a cosmetic composition.

1 is a schematic diagram showing the manufacturing process of fermented ginseng fruit purified product according to the present invention;
2 is a graph showing the protein quantification results after fractionation of the fermented ginseng fruit of Experimental Example 2 of the present invention;
3 is a photograph of TLC results after column separation and purification of ginseng fruit fermentation product of Experimental Example 3 of the present invention;
4 is a graph showing the protein content confirmation result after column separation and purification of the ginseng fruit fermentation product of Experimental Example 4 of the present invention;
5 is a graph showing the results of confirming the target peptide content for each purification step of the fermented ginseng fruit of Experimental Example 5 of the present invention;
6 is a graph showing the results of high performance liquid chromatography analysis according to the present invention (Gly-Ala-Gly-Ala-Gly-Ala);
7 is a graph showing the results of NMR and FAB-mass analysis of ginseng berry peptide (ApepHexapeptide-01) of Experimental Example 6 according to the present invention (Gly-Ala-Gly-Ala-Gly-Ala);
8 is a graph showing the MMP-1 inhibitory ability measurement results of ginseng fruit extraction and fermentation fractions of Experimental Example 8 of the present invention;
9 is a graph showing the results of the fermented ginseng fruit, fractions, and purified products of Experimental Example 9 of the present invention, the results of promoting intracellular collagen production;
10 and 11 A graph showing the results of measuring the cell proliferation ability of Experimental Example 10 (ginseng fruit purified product) of the present invention in 3T3 cells and HACAT cells;
12 and 13 are graphs showing the optical stability and thermal stability measurement results of Hexapeptide AP-1 of the present invention.

Hereinafter, the present invention will be described in detail through examples. Prior to this, the terms or words used in the present specification and claims should not be construed as being limited to their ordinary or dictionary meanings, and the inventor should properly understand the concept of the term in order to best describe his invention. Based on the principle that can be defined, it should be interpreted as meaning and concept consistent with the technical idea of the present invention. Accordingly, since the configuration of the embodiments described in the present specification is only the most preferred embodiment of the present invention and does not represent all the technical spirit of the present invention, various equivalents and modifications that can be substituted for them at the time of the present application It should be understood that there may be

As a result of repeated research to develop a cosmetic composition effective in preventing skin aging and raw materials with an antioxidant effect in order to effectively prevent or improve skin aging and wrinkles, the present inventors have fermented ginseng fruit extract, or ginseng It has been found that, when a peptide derived from a fermented fruit is used, it is possible to improve the effect of inhibiting the activity of a collagen degrading enzyme (Matrix Metalloproteinase-1), promoting intracellular collagen production, and promoting epidermal cell proliferation, leading to the present invention.

Accordingly, the present invention is a method for providing a cosmetic composition comprising a fermented ginseng fruit or a fermented ginseng fruit as an active ingredient, inoculating and fermenting ginseng fruit (Panax Ginseng Berry) with bacteria and then filtering to remove microorganisms Disclosed is a method for isolating a functional peptide in a fermented ginseng fruit.

In the present invention, the ginseng fruit (Panax ginseng Berry) is a perennial plant belonging to the Araliaceae family, and is widely cultivated for medicinal purposes. The ginseng fruit is opened only in ginseng aged 4 years or older, and various pharmacological actions have been revealed, and it has been developed as a cosmetic and used for skin beauty and anti-aging.

In the present invention, the fermented ginseng fruit refers to a fermented product after drying and extracting ginseng fruit, and the ginseng fruit extract has been used as a drug effective for various diseases, and is rich in ingredients having various effects such as saponins. It is a drug whose efficacy has been confirmed such as anti-cancer, antioxidant, anti-cancer, and liver disease improvement.

The fermented ginseng fruit or fermented ginseng fruit of the present invention is a raw material that acts on the skin to help improve skin wrinkles and alleviate skin aging, and by refining specific effective ingredients, the effect is further enhanced compared to before purification It can be used, and the fermented ginseng fruit and the fermented ginseng fruit can be used together.

In one aspect, the present invention provides a method for isolating a functional peptide in a fermented ginseng fruit, and the method for isolating a functional peptide in a fermented ginseng fruit is a ginseng fruit (Panax Ginseng Berry) inoculated and fermented with bacteria, followed by filtration to remove microorganisms.

Hereinafter, the present invention will be described in detail through the method for producing the fermented ginseng fruit.

In the present invention, the step of inoculating the ginseng fruit extract with microorganisms and fermenting it to prepare a fermented product is a step for increasing the content of functional substances in the ginseng fruit fruit.

The ginseng fruit extract may be prepared using a conventional extraction method known in the art, for example, a solvent extraction method. The extraction solvent used in the preparation of the mixed extract using the solvent extraction method is water, a lower alcohol having 1 to 4 carbon atoms (eg, methanol, ethanol, propanol and butanol) or a mixture thereof, which is a hydrous lower alcohol, propylene glycol, 1, 3-butylene glycol, glycerin, acetone, diethyl ether, ethyl acetate, butyl acetate, dichloromethane, dichloromethane, hexane, and mixtures thereof may be selected from the group consisting of, and selected from water, alcohol, or mixtures thereof When alcohol is used as the extraction solvent, the alcohol is preferably a lower alcohol having 1 to 4 carbon atoms, and the lower alcohol is more preferably selected from methanol or ethanol, and even more preferably ethanol. have.

In addition, when the ginseng fruit extract is extracted with ethanol, for example, 99.5% (v/v) ethanol is added in an amount 5 to 20 times the weight of the ginseng fruit, and the extract can be extracted at room temperature for 18 to 30 hours. , The sugar content of the ginseng fruit extract may be 4 to 10 brix, preferably 6 to 8 birx.

In addition, when the ginseng fruit extract is extracted by hot water extraction, for example, purified water may be added at 5 to 20 times the weight of the ginseng fruit, and extracted at 100 to 140° C. for 4 to 8 hours, the ginseng The sugar content of the fruit extract may be 4 to 10 brix, preferably 6 to 8 birx.

In the present invention, the microorganism may be fermented into a Bacillus sp. strain, a Lactobacillus sp. strain or a mixed strain thereof, and the Bacillus sp. strain is Bacillus methylotropicus ( Bacillus methylotrophicus ) or Bacillus subtilis ( Bacillus subtilis ), the Lactobacillus genus ( Lactobacillus sp.) strain may be Lactobacillus paracasei ( Lactobacillus paracasei ), preferably Bacillus subtilis and Bacillus methyl It may be a mixed strain of Rotropicus.

The fermentation refers to a process in which enzymes possessed by microorganisms change raw materials to create beneficial ingredients, and during the fermentation process, active ingredients are extracted and the content of functional substances increases. In addition, in the present invention, the fermentation is performed ^{by adjusting the culture medium of the microorganism strain to 10 7} to 10 ¹⁰ cfu/ml, and inoculating 0.1 to 10% by weight, preferably 0.5 to 5% by weight of the ginseng fruit extract. and may be carried out at 25 to 45 °C, preferably 30 to 40 °C, for 1 to 5 days, preferably 2 to 4 days.

In addition, the composition of the medium for culturing and fermenting the microorganisms may be glucose 0.2 to 1% by weight, enzyme extract 0.1 to 0.5% by weight, soytone 0.05% by weight, and distilled water (residual amount).

In the present invention, the step of preparing a fermented ginseng fruit by removing the microorganism from the fermented product is a step of removing microorganisms to prevent further fermentation of the fermented ginseng fruit, and the microorganism removal is to be removed through centrifugation and filtration. For example, after separating the microorganisms through a centrifuge in the fermented product, it is possible to remove the microorganisms by filtering with a 0.1 to 0.3 μm filter.

1 is a schematic diagram showing the manufacturing process of fermented ginseng fruit purified product according to the present invention.

1 , the solvent fractionation of the fermented ginseng fruit is a step of extracting peptides by purifying the fermented ginseng fruit. The solvent fractionation is sequentially performed through hexane, dichloromethane, ethyl acetate, butanol and water. can In addition, it may be purified through a column after the solvent fractionation.

In addition, in the present invention, the fermented product of ginseng fruit may be a hexapeptide derived from ginseng fruit, and the hexapeptide is glycine (Glycine, Gly)-alanine (Alanine, Ala)-glycine (Glycine, Gly) -alanine (Alanine, Ala) -glycine (Glycine, Gly) - may be alanine (Alanine, Ala).

In the present invention, the cosmetic composition may contain 1 to 90% by weight of the fermented ginseng fruit or the purified ginseng fruit, preferably 1 to 20% by weight. In addition, the cosmetic composition may include the fermented ginseng fruit and the fermented ginseng fruit, and may contain 1 to 20% by weight, respectively.

It was confirmed that the cosmetic composition according to the present invention has a very excellent effect of inhibiting the activity of collagen-degrading enzymes such as collagenase and MMP-1 (Matrix Metalloproteinase-1), and also has an effect of promoting intracellular collagen production and skin It was also confirmed that the effect of improving the regenerative capacity of cells was very good.

The cosmetic composition according to the present invention may be applied in a gel type, a skin type, a cream type, an ointment type, etc., but is not limited thereto. The above composition can be suitably prepared by a known method by adding an appropriate conventional softening agent, emulsifying agent, thickening agent or other substances known in the art according to its type.

The gel-type composition may be prepared by adding an emollient such as trimethylolpropane, polyethylene glycol, or glycerin, a solvent such as propylene glycol, ethanol, or isostatic alcohol, and purified alcohol.

The skin type composition includes fatty alcohols such as stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol, isocetyl alcohol, butylene glycol, glycerin, allantoin, methyl paraben, EDTA- It can be prepared by adding 2-sodium, xanthan gum, dimethicone, polyethylene glycol-60 hydrogenated castol oil, polysorbate 60 and purified water.

The cream-type composition comprises fatty alcohols such as stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol, isocetyl alcohol, lecithin, phosphatidylcholine, phosphatidylethanolamine, sofatizylserine, sofphatidyl Lipids such as inositol, derivatives thereof, emulsifiers such as glyceryl stearate, sorbitan palmitate, and sorbitan stearate, natural fats or oils such as avocado oil, apricot oil, babassu oil, lucidium oil, camellia oil, It can be prepared by adding a solvent such as propylene glycol and purified water.

The ointment-type composition may be prepared by adding a softening agent, an emulsifying agent, and waxes such as microcrystalline lead, paraffin, ceresin, beeswax, beeswax, and vaseline.

The cosmetic composition of the present invention may further contain one or more skin wrinkle-improving ingredients exhibiting the same or similar function in addition to the active ingredient. The skin wrinkle improvement component may be any one or more selected from the group consisting of vitamin C, retinoic acid, TGF, animal placental-derived protein, betulinic acid, and chlorella extract, but is not limited thereto. In addition, the cosmetic composition of the present invention may further contain excipients including fluorescent substances, fungicides, hydrotropes, humectants, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreens, vitamins, plant extracts, etc. can

In the present invention, the cosmetic composition is a softening lotion, astringent lotion, nourishing lotion, eye cream, serum, nourishing cream, massage cream, cleansing cream, cleansing lotion, cleansing foam, cleansing water, powder, essence, pack, hair tonic, hair treatment It may be formulated as a conditioner, shampoo or rinse.

The cosmetic composition may be formulated by a conventional method. In the formulation of external skin preparations, reference may be made to the contents disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA, and in the formulation of cosmetic compositions, the International cosmetic ingredient dictionary, 6th ed (The cosmetic, Toiletry and Fragrance Association) , Inc, Washington, 1995) may be referred to.

Specifically, the cosmetic composition can be prepared in general emulsified formulations and solubilized formulations. For example, lotion, such as a softening lotion or a nourishing lotion; emulsions such as facial lotions and body lotions; creams such as nourishing creams, moisturizing creams, and eye creams; essence; makeup ointment; spray; gel; pack; sunscreen; makeup base; foundations such as liquid type, solid type or spray type; powder; makeup removers such as cleansing creams, cleansing lotions, and cleansing oils; Alternatively, it may be formulated as a cleaning agent such as a cleansing foam, soap, body wash, but is not limited thereto. In addition, the external preparation for skin may be formulated as an ointment, patch, gel, cream or spray, but is not limited thereto.

In the cosmetic composition, in addition to the essential components in each formulation, other components may be appropriately blended within the range that does not impair the purpose according to the present invention, depending on the type of formulation or purpose of use.

The cosmetic composition may include a conventionally acceptable carrier, for example, oil, water, surfactant, humectant, lower alcohol, thickener, chelating agent, colorant, preservative, fragrance, etc. may be appropriately mixed, but is limited thereto no.

The acceptable carrier may vary depending on the formulation. For example, when formulated into an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or their Mixtures may be used.

When the cosmetic composition is formulated as a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or mixtures thereof may be used as a carrier component, and in the case of a spray, chlorofluoro It may further comprise a propellant such as hydrocarbon, propane, butane or dimethyl ether.

When the cosmetic composition is formulated as a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate, propylene glycol, 1,3-Butylglycol oil may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan may be used.

When the cosmetic composition is formulated as a suspension, a liquid diluent such as water, ethanol or propylene glycol as a carrier component, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester; Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like can be used.

The cosmetic composition may contain fatty substances, organic solvents, solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, and fragrances commonly used in the industry depending on the quality or function of the final product. , surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, commonly used in cosmetics It may additionally contain adjuvants commonly used in cosmetic or dermatological fields, such as any other ingredients. However, the adjuvant and its mixing ratio may be appropriately selected so as not to affect the desirable properties of the cosmetic composition according to the present invention.

The cosmetic composition comprising the fermented ginseng fruit or fermented ginseng fruit prepared according to the present invention as an active ingredient improves the activity of collagen degrading enzyme (Matrix Metalloproteinase-1) by including the fermented ginseng fruit or fermented ginseng fruit. It can inhibit, promote the production of intracellular collagen, and proliferate epidermal cells.

Hereinafter, the present invention will be described in more detail with reference to the preparation examples and experimental examples of the present invention.

Preparation Example 1

After adding 2 kg of purified water to 200 g of dried ginseng fruit, hot water extraction was performed at 120° C. for 6 hours. After extraction, a ginseng fruit extract having a sugar content of 6 to 8 birx was used. After extraction, the Lactobacillus paracasei (purchased from the Korea Cell Line Bank) strain was cultured, and the ginseng fruit extract was inoculated with 1 wt% of a culture solution ^{having a concentration of 1.0×10 9 cfu/ml measured by a spectrophotometer, and 37°C.} was fermented for 3 days, and the medium used for culture was a medium composed of glucose 0.6% by weight, enzyme extract 0.3% by weight, soytone 0.1% by weight, and distilled water. After fermentation, the fermentation broth was centrifuged at 4,000 rpm for 10 minutes to separate lactic acid bacteria and culture broth, and the culture broth was filtered through a 0.2 μm filter to prepare a fermented ginseng fruit.

Preparation 2

A fermented ginseng fruit was prepared in the same manner as in Preparation Example 1, except that Bacillus methylotrophicus (Apep microrganism S001, purchased from Korea Cell Line Bank) was used as a microorganism.

Preparation 3

A fermented ginseng fruit was prepared in the same manner as in Preparation Example 1, except that Bacillus subtilis (Apep microrganism K007, purchased from Korea Cell Line Bank) was used as a microorganism.

Preparation 4

As microorganisms in Preparation Example 1, Bacillus methylotrophicus (Bacillus methylotrophicus, Apep microrganism S001, purchased from Korea Cell Line Bank) and Bacillus subtilis (Bacillus subtilis, Apep microrganism K007, purchased from Korea Cell Line Bank) were 1:1 weight A fermented ginseng fruit was prepared in the same manner except for using a mixed strain mixed by part.

Preparation 5

2 kg of 70% (v/v) ethanol was added to 200 g of dried ginseng fruit and extracted at 25° C. for 24 hours. After extraction, a ginseng fruit extract having a sugar content of 6 to 8 birx was used. After extraction, the Lactobacillus paracasei (purchased from the Korea Cell Line Bank) strain was cultured, and the ginseng fruit extract was inoculated with 1 wt% of a culture solution ^{having a concentration of 1.0×10 9 cfu/ml measured by a spectrophotometer, and 37°C.} was fermented for 3 days, and the medium used for culture was a medium composed of glucose 0.6% by weight, enzyme extract 0.3% by weight, soytone 0.1% by weight, and distilled water. After fermentation, the fermentation broth was centrifuged at 4,000 rpm for 10 minutes to separate lactic acid bacteria and culture broth, and the culture broth was filtered through a 0.2 μm filter to prepare a fermented ginseng fruit.

Preparation 6

A fermented ginseng fruit was prepared in the same manner as in Preparation Example 5, except that Bacillus methylotrophicus (Apep microrganism S001, purchased from Korea Cell Line Bank) was used as a microorganism.

Preparation 7

A fermented ginseng fruit was prepared in the same manner as in Preparation Example 5, except that Bacillus subtilis (Apep microrganism K007, purchased from Korea Cell Line Bank) was used as a microorganism.

Preparation 8

In Preparation Example 5, Bacillus methylotrophicus (Bacillus methylotrophicus, Apep microrganism S001, purchased from Korea Cell Line Bank) and Bacillus subtilis (Bacillus subtilis, Apep microrganism K007, purchased from Korea Cell Line Bank) as microorganisms in Preparation Example 5 were 1:1 weight A fermented ginseng fruit was prepared in the same manner except for using a mixed strain mixed by part.

Preparation 9

To 200 g of dried ginseng fruit, 2 kg of purified water was added, and hot water extraction was performed at 120° C. for 6 hours to prepare a ginseng fruit extract having a sugar content of 6 to 8 birx.

Preparation 10

To 200 g of dried ginseng fruit, 2 kg of 70% (v/v) ethanol was added and extracted at 25° C. for 24 hours to prepare a ginseng fruit extract having a sugar content of 6 to 8 birx.

refinement example

1) n-hexane, dichloromethane, ethyl acetate and butanol were sequentially added to the fermented ginseng fruit prepared in Preparation Example 4, and separated into an n-hexane fraction, a dichloromethane fraction, an ethyl acetate fraction, a butanol fraction and a water fraction. Fractions were prepared.

2) After the separated water fraction was concentrated under reduced pressure, silica column chromatography was performed. At this time, a silica column was used as the column, and the mobile phase solvent was distilled water CH ₂ Cl ₂ : Water 90: 10, 80: 20 by changing 1L in the order fr1(1), fr2(2), fr3( 3), fr4(4), fr5(5), fr6(6-7), fr7(8-10), fr8(11), fr9(12), fr10(13-15).

3) The separated fr.1 was prep. It was purified by HPLC (C18 ODS column, 3ml/min). Solvent A: Water in 0.1% TFA, Solvent B: Acetonitrile (ACN) in 0.1% TFA, 98:2 conditions were carried out, and comp1 was purified from the fermented ginseng fruit.

Experimental Example 1

In order to confirm the skin wrinkle improvement effect of the fermented ginseng fruit prepared in Preparation Examples 1 to 10, an in vitro collagenase activity inhibition experiment was performed according to the following method, and the results are shown in Table 1 shown in

[Test method]

(1) Prepare by dissolving collagen in phosphate buffer (100 ppm). At this time, adenosine is prepared at the same concentration as a positive control.

(2) Prepare a mixture containing 100 mM Tris-cl buffer, 100 mM CaCl ₂ and 0.25 mg/ml collagenase, and add each test substance and positive control to the mixture at a concentration of 5% (v/v) was added to proceed with the reaction.

(3) The reaction is terminated by adding 25 mM citric acid to 400 μl/tube.

(4) Then, 1.5 ㎖/tube of ethyl acetate is added, and after vortexing, the supernatant is taken and transferred to sodium sulfate 150 mg/ep-tube, and centrifuged after vortexing (10,000 rpm, 3 minutes).

(5) Take 300 μl/ep-tube of the supernatant, transfer it to a quartz 96-well plate, and measure the absorbance at 320 nm.

division Collagenase inhibitory ability (%) Control group (untreated group) 100 Preparation Example 1 77 Preparation 2 71 Preparation 3 69 Preparation 4 62 Preparation 5 69 Preparation 6 63 Preparation 7 64 Preparation 8 58 Preparation 9 88 Preparation 10 91 Control (Adenosine) 59

Referring to Table 1, it can be seen that all fermented products (Preparation Examples 1 to 8) have a collagenase inhibitory ability, and it can be confirmed that the collagenase inhibitory ability is superior to that of the case without fermentation (Preparation Examples 9 and 10).

In addition, when using the Lactobacillus sp. strain (Preparation Example 1 and Production Example 5) than when using the Bacillus sp. strain (Preparation Examples 2 to 4 and Preparation Examples 6 to 8) is excellent in the collagenase inhibitory ability, the single strain It can be seen that the collagenase inhibitory ability in the case of using the mixed strain (Preparation Examples 4 and 8) is superior to the case of using (Preparation Examples 1, 2, 3, 5, 6 and 7).

In addition, it can be seen that the collagenase inhibitory ability is superior to that of the case of hot water extraction (Preparation Examples 1 to 4) than the case of ethanol extraction (Preparation Examples 5 to 8).

Experimental Example 2

The protein content of the ginseng fruit extract of Preparation Example 10, the fermented ginseng fruit of Preparation Example 8 and the fractions isolated in Purification Example was measured using the lowry method, and the measurement results are shown in Table 2 and FIG. 2 below. It was.

division Protein quantitative value (mg/ml) Amount of protein in fraction (mg) Total (%) Preparation 9
(ginseng fruit extract) 0.219 49.427 - Preparation 4
(Fermented ginseng fruit) 0.321 72.657 - refinement example
(Hexane, n-Hex) 0.032 0 0.00 refinement example
(dichloromethane, CH2Cl2) 0.046 0 0.00 Purification example (ethyl acetate) 0.099 2.498 3.67 refinement example
(butanol, n-BuOH) 0.302 16.767 24.63 refinement example
(water) 0.759 48.814 71.70 system - 68.079 100

Referring to Table 2 and FIG. 2, it can be seen that the quantitative value of the protein content of the fermented ginseng fruit (Preparation Example 8) is increased by about 147% compared to the ginseng fruit extract (Preparation Example 10), and the ginseng fruit fermentation It can be confirmed that the fraction (purified example) has the highest protein content of the water fraction.

In addition, it can be confirmed that the protein content (mg/ml) of the fermented ginseng fruit fraction (purified example) is 2.36 times or more than that of the ginseng fruit fermented product (Preparation Example 8).

Experimental Example 3

Silica gel thin layer chromatography (TLC) for fr.1 to 10 isolated in the above purification example was performed in a mixed solvent of dichloromethane: 99.5% (v/v) methanol (2:1), and the TLC pattern according to this is shown in FIG. 3 .

Referring to FIG. 3 , as a result of confirming the separated material, it can be confirmed at 210 UV, and when the content is checked at lowry, it can be confirmed that it is a protein, and thus protein purification conditions can be confirmed.

Experimental Example 4

The protein content contained in water (water layer), fr.1, and prep1 separated in the purification example was measured using the lowry method, and the measurement results are shown in Tables 3 and 4 below.

division Protein Quantitation
(peptide content, mg/ml) Total protein (mg) Purification example (water layer) 0.759 26.077 Purification example (Slica column -fr.1) 2.31 48.814 Purification Example (HPLC Prep1) 2.66 143.7848

Referring to Table 3 and FIG. 4 , it was confirmed that the protein content increased during separation and purification through High Pressure Liquid Chromatography (HPLC), but it was confirmed that the protein content did not increase above a certain level.

Experimental Example 6

The content of the target peptide (Hexapeptide AP-1) included in the fermented ginseng fruit according to Preparation Example 4, the water layer of the purification example, fr.1 and prep.1 to 4 was measured using the same HPLC as in the purification example, and measured The results are shown in Table 4 and FIG. 5 below.

HPLC analysis result target peptide Fermented Ginseng Fruit Unknown water floor 2.16 silica gel column 4.21 HPLC prep.1 93.1 prep.2 4.21 prep.3 1.54 prep.4 Unknown

Referring to Tables 4 and 5, in the case of the fermented ginseng fruit (Preparation Example 4) and prep.4 (Purified Example), the target peptide did not appear, but different peptides were present, and prep 1 (Purified Example) In this case, it was confirmed that the content of the target peptide was improved to 93% through HPLC content confirmation, and the result is shown in FIG. 6 .

Experimental Example 7

In order to confirm the comp1 structure obtained after HPLC purification, an experiment was performed according to the following experimental method, and the results are shown in FIG. 7 .

[Test method]

After preparing the sample at 100 ppm, the mass was measured using Mass (FAB-Mass). Then, using 600 MHz NMR (Agilent Technologies (DD2 600 MHz FT NMR)), 1H, 13C, HMBC, HMQC, COSY and DEPT were measured to confirm the structure.

Referring to Figure 7, in the case of Hexapeptide AP-1 ^{, the exact structure was identified through 1} H NMR, ¹³ C NMR, and 2D NMR, and _{CH 3} at δ 1.260 in ¹ H NMR was confirmed to have overlapped peaks, d3.7011 The peaks of CH2 at 3.804 and the peaks of CH at d4.237,4.197 were confirmed. In addition, by checking C * 6, CH * 3, CH2 * 3, CH * 3 through DEPT, the total _{number of CH, CH 2} , and CH ₃ was identified, and each C, CH, CH ₂ , Binding of CH ₃ was confirmed. Through this, when compared with 403, which is the molecular weight confirmed through FAB-Mass, it was confirmed that Gly-Ala was repeatedly attached to the structure. After that, peptide synthesis was carried out, and it was confirmed that a peak appeared at the same time by comparing the synthesized peptide and the peptide separated and purified from the fermented product through HPLC.

Experimental Example 8

Substances that inhibit the production of matrix methaloprotease-1 (MMP-1, collagenase) protect collagen and maintain the mechanical properties of the skin tissue to prevent elasticity and sagging of the skin. By measuring the active ingredients, it can be evaluated that it can be usefully used in the development of cosmetics for wrinkle improvement and elastic skin. Accordingly, the MMP-1 production inhibitory ability test of the fermented ginseng fruit of Preparation Example 4, the ginseng fruit extract of Preparation Example 10 and the fractions of Purified Example was performed in the following manner, and the results are shown in FIG. 8 . Retinol was used as a control.

[Test method]

Human dermal fibroblasts (HDF) cells were cultured in DMEM (Dulbecco's modified Eagle's media, Sigma) in a medium supplemented with 10% fetal bovine serum (Sigma) at 37°C and 5% CO ₂ conditions. . After culturing the cells at a concentration of 2 X 10 ⁵ cells/well in a 24-well plate and confirming cell adhesion, the control group was treated with nothing and only a solvent was added, and retinol was added to the dish at 10 ug/ml for the experimental group and control group. It was treated so that it became a concentration. After adding the test substance to each dish, it was cultured for 2 days. After 2 days, the medium was collected and the inhibition of MMP-1 production was measured using Matrix Metalloproteinase-1 (MMP-1), Human, biotrak, and ELISA kit (GE healthcare RPN2610).

Referring to FIG. 8 , it was confirmed that the amount of MMP-1 produced was decreased in most of the experimental groups, and it was confirmed that there was a difference according to the composition and combination of functional raw materials in the composition even within the experimental group. In particular, it is judged that most of the substances inhibiting MMP-1 expression are distributed in the water layer (purified example) during fractionation after fermentation of ginseng fruit. From these results, when UV that induces MMP-1 production on the skin is applied as a factor, it is more useful to use a mixture of fermented ginseng fruit and fermented ginseng fruit. It is thought to be able to suppress skin aging.

Experimental Example 9

The intracellular collagen-producing ability of the fermented ginseng fruit of Preparation Example 4, the water layer of the purified example, and fr1 and comp1 (Hexapeptide AP-1) was performed in the following manner, and the results are shown in FIG. 9 . As a control, transforming growth factor beta (TGFβ) was used.

[Test method]

Human dermal fibroblst (HDF) cells to be used for the test are medium 106 (cascade biologics, M-106-500) added with 1X LSGS (Low Serum Growth Supplement, cascade biologics S-003-10). 37 ℃, 5% CO ₂ Incubated under conditions. Cells were cultured in a 24-well plate at ^{a concentration of 1X 10 5} cells/well and cell adhesion was confirmed, and TGFβ was treated to a concentration of 1 mg/ml and 10 μg/ml, respectively, as an experimental group and a control group. After adding the test substance to each dish, it was cultured for 2 days. After 2 days, the medium was collected and the amount of collagen produced was measured using the Procollagen Type I C-peptide (hereinafter PIP) EIA kit (takara MK101).

Referring to FIG. 9 , it was confirmed that collagen production increased in all experimental groups and control groups, and the purified (Purified Example water layer, fr1 and comp1 (Hexapeptide AP-1)) was superior to the unrefined (fermented ginseng fruit). It can be confirmed that there is a collagen production effect, and it can be seen that the most purified comp1 (Hexapeptide AP-1) has the highest collagen production effect.

From these results, it is presumed that a large amount of ingredients that promote collagen production in the skin are contained in the ginseng fruit, and using a mixture of fermented ginseng fruit and fermented ginseng fruit can increase collagen production and reduce skin aging. It is believed that it can be suppressed.

Experimental Example 10

In order to confirm the cell proliferation ability of comp1 of the purification example, the experiment was carried out in the following manner, and the results are shown in FIGS. 10 and 11 . Cell proliferation was measured using the MTT assay.

[Test method]

^{3T3 cells and HACAT cells were seeded to a cell number of 4 X 10 4} cells/24well per well in a 24-well plate, and then incubated at 37° C., 5% CO ₂ conditions for 24 hours. It was washed twice with PBS (phosphate buffer saline), and each sample was treated with each concentration (1mg~1ug/mL) and incubated for 24 hours. After incubation, 0.5% of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in DPBS (Dulbecco's Phosphate-Buffered Saline) was mixed with the culture medium at 1:9 (v/v). After addition, incubate in a _{CO 2 incubator for 2 hours.} Then, the generated formazan (frmazan) was dissolved in DMSO (dimethyl sulfoxide) and absorbance was measured at 570 nm using ELISA.

10 and 11, with respect to HACAT cells and 3T3 cells, it was confirmed that one type of peptide (comp1) treated from a low concentration to a high concentration had a cell proliferation ability in a concentration-dependent manner, and there was no significant change in the microscopic observation of the cells. not observed.

Through this, it can be seen that the cosmetic composition of the present invention has no toxicity to skin-related cells, and it appears to have a skin cell proliferation effect. Therefore, it can be predicted that even if the cosmetic composition of the present invention is treated on the skin, it will not have a significant effect.

Experimental Example 11

In order to confirm the thermal and photostability of comp1 of the tablet example, the experiment was conducted in the following manner, and the results are shown in FIGS. 12 and 13 .

[Test method]

Each sample is dissolved in 50 mM Tris-HCl (pH 8.0) buffer so that the sample is 10 mg/mL, aliquoted into a glass vial, and stored at 50° C. for 4 weeks to measure the loss of peptide in the composition due to heat. and stability under sunlight conditions was measured in the same manner.

12 and 13, as a result of measuring the loss of the peptide due to heat and measuring the stability in sunlight conditions, all one peptide (comp1) showed less than 10% peptide loss up to 4 weeks, so that heat and sunlight It was confirmed that it has high stability in the storage period.

Cosmetic composition preparation example

The cream-type composition was prepared in the following way. In the cream type composition, the formulation was constituted by including the above Hexapeptide AP-1 in a weight ratio of 1% of the composition (see Table 5 below).

1) Preparation of [Aqueous B]

Water is heated and stirred at 60 to 70° C., and powdered raw materials are sequentially added sequentially, stirred with a disper mixer until thickened, and dissolved evenly, and then cooled. Prepare in advance for ease of processing.

2) Preparation of [Aqueous A]

After the powdery raw material is sufficiently impregnated with glycerin, water is added and heated to 50 to 60° C. to dissolve evenly with a disper mixer.

3) [Water A] + [Water B] Mix Stirring Preparation

Add pre-prepared aqueous phase B to aqueous phase A and warm while mixing evenly. Prepare to be maintained at 65 to 75 ℃.

4) [Oil phase] Mixed and stirred manufacturing

Each raw material is weighed in a separate beaker and melted by heating and stirring. (Above 80℃) Prepare the melted phase to maintain a temperature of 75℃ or higher.

5) Whole cream preparation

The oil phase is slowly added to the prepared aqueous mixture and stirred vigorously (6,000 rpm) for about 10 minutes to proceed with the emulsification process. At this time, the temperature is to be maintained at 65 to 75 ℃.

After completion, the additive A is prepared and the pH adjustment and neutralization process is performed. At this time, since the viscosity of the contents rises, stir vigorously (6,000 rpm) evenly for 5 minutes or more.

When the content cools below 50°C, add Additive B and mix slowly for about 2 minutes (3,000 to 4,000 rpm). When the temperature is below 40°C, add Additive C and Additive D sequentially and slowly for about 2 minutes (3,000 to 4,000 rpm) After stirring, the contents are collected through a degassing/filtration process.

After completion, the pH adjustment and neutralization process is performed. At this time, since the viscosity of the contents rises, stir vigorously (6,000 rpm) evenly for 5 minutes or more.

When the content cools below 50℃, add a preservative and mix slowly for about 2 minutes (3,000 to 4,000 rpm). When the temperature is below 40℃, add ginseng berry peptide and flavorings and stir slowly (3,000 to 4,000 rpm) for about 2 minutes. The contents are collected through a degassing/filtration process.

The prepared cream was stored at low temperature (4 ℃), high temperature (50 ℃), room temperature (25 ℃), and sunlight conditions to check the stability of the cream. Discoloration, discoloration, phase change, and pH change of the cream for 4 weeks under all conditions Such phenomena were not found, and through this, it was confirmed that the stability of the cream was maintained. In particular, the stability of the ginseng fruit peptide was maintained, and as a result of HPLC analysis, it was confirmed that the content was maintained at 90% or more for 4 weeks under all conditions (see Table 6 below).

division Low temperature (4℃) room temperature (25℃) high temperature (50℃) daylight 1 week later 99 98 98 97 After two weeks 99 95 96 96 after 3 weeks 98 95 95 95 after 4 weeks 98 93 93 94

Preferred embodiments of the present invention described above are disclosed to solve the technical problem, and various modifications, changes, additions, etc. will be possible within the spirit and scope of the present invention by those skilled in the art to which the present invention pertains. , such modifications and changes should be regarded as belonging to the following claims.

Claims (11)

A method of isolating functional peptides from fermented ginseng berries, including the step of inoculating and fermenting ginseng berry (Panax Ginseng Berry) with bacteria and then filtering to remove microorganisms. According to claim 1,
The fermentation method, characterized in that it is carried out at 30 to 50 ℃ 2 to 4 days. According to claim 1,
The bacteria is a method, characterized in that the bacteria of the genus Bacillus ( Bacullus sp.). According to claim 1,
The method, characterized in that the functional peptide is obtained through solvent fractionation and size exclusion chromatography. 5. The method of claim 4,
The functional peptide is (glycine-alanine) n (n is an integer from 2 to 10) The method characterized in that it comprises a sequence. 6. The method of claim 5,
The method, characterized in that the functional peptide comprises the sequence of glycine-alanine-glycine-alanine-glycine-alanine. According to claim 1,
The functional peptide method, characterized in that it has a skin wrinkle improvement and anti-aging function. According to claim 1,
The method characterized in that the functional peptide has a function of increasing the skin cell regeneration rate by promoting skin cell growth. According to claim 1,
The functional peptide promotes collagen production in human fibroblasts, and inhibits the activity of collagen degrading enzyme (MMP-1), characterized in that it has a skin wrinkle improvement function. According to claim 1,
The method, characterized in that the fermented ginseng fruit and the functional peptide have a function of promoting the proliferation of human skin keratinocytes. A cosmetic composition comprising the functional peptide isolated according to any one of claims 1 to 10 as an active ingredient.

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