KR101860312B1 - Cosmetic composition including extract of fermented ginseng using lactobacillus casei gfc1 and manufacturing method for the same - Google Patents

Abstract

An embodiment of the present invention relates to a cosmetic composition comprising a fermented ginseng extract using Lactobacillus casei GFC1 (KCTC18333P) strain having excellent SNARE protein activity inhibitory activity. Accordingly, the present invention includes a fermented ginseng extract using Lactobacillus casei GFC1 (KCTC18333P) strain having excellent SNARE protein activity inhibitory activity, thereby increasing the content of active ingredients and polysaccharides, Provided is a cosmetic composition excellent in the effect of minimizing toxicity and skin irritation, excellent in anti-inflammatory and antioxidative activity effects, inhibiting the activity of collagenolytic enzyme, inhibiting the activity of snare protein and improving skin wrinkles and a method for producing the same can do.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cosmetic composition comprising a fermented ginseng extract using Lactobacillus casei GFC1 strain having excellent snake protein activity inhibitory effect and a method for producing the same,

The present invention relates to a cosmetic composition comprising a fermented ginseng extract using Lactobacillus casei GFC1 strain having excellent snare protein activity inhibitory effect and a method for producing the same.

The most closely related to wrinkles is skin cell senescence. In general, the cause of skin aging can be classified into natural aging caused by aging and internal aging caused by external environment. Natural aging is difficult to control because it involves many genetic factors. Therefore, much research has been conducted to control environmental factors such as ultraviolet rays, oxidation, and drying.

Environmental factors mainly collagen protein, elastin protein contained in the dermis, such as destruction or denaturation causes wrinkles. In particular, ultraviolet rays of the sun are typical environmental factors for oxidizing skin and producing active oxygen. Such oxidative power and active oxygen can cause protein breakdown in skin cells, oxidation of sugar, genetic modification, and the like, which can cause skin aging and wrinkles.

On the other hand, the fact that the SNARE protein (SNAP receptor protein) is involved in the development of skin wrinkles is known from recent studies. Snare protein is a membrane protein involved in transport follicles and target membrane fusions. It is thought to promote wrinkles by promoting secretion of neurons that form wrinkles. In the past, a technique has been described in which a snare protein is limited and separated by using a Vortolium neurotoxin to block neurotransmission and paralyze muscle cells infiltrated with neurotoxins to improve wrinkles. However, the Vortolium neurotoxin causes tolerance to the human body. As the frequency of use increases, there is a disadvantage that the effect of the neurotoxin is reduced compared to the usage amount. Therefore, a high cost and high purification equipment for purifying the neurotoxin is required.

In recent years, studies on raw materials that are significantly lower than those of conventional peptides and protein materials have been actively conducted while controlling the activity of snare proteins to a useful level by using natural substances. In addition, the side effects caused by neurotoxins or synthetic substances are minimized, and there is a growing demand for cosmetic compositions that can be used without any limitations on the content of the ingredients, such as skin irritation and cytotoxicity.

One of the objects of the present invention is to provide a method for preparing a fermented ginseng extract comprising a fermented ginseng extract using Lactobacillus casei GFC1 (KCTC18333P) strain having excellent SNARE protein activity inhibitory activity, thereby increasing the content of active ingredients and polysaccharides, A cosmetic composition having excellent cytotoxicity and skin irritation, excellent antiinflammatory and antioxidative activity effects, inhibiting activity of collagenolytic enzyme, inhibiting the activity of snare protein and improving skin wrinkles, and a method for producing the same .

An embodiment of the present invention relates to a cosmetic composition comprising a fermented ginseng extract using Lactobacillus casei GFC1 (KCTC18333P) strain having excellent SNARE protein activity inhibitory activity.

The fermented ginseng extract may be fermented by inoculating the lactobacillus casei strain GFC1 into a composition for fermentation comprising ginseng fruit pulverized product and lactose.

Wherein the composition for fermentation further comprises 7 to 10% by weight of ginseng fruit pulverized product, 4.5 to 6% by weight of garlic pulverized product, 5 to 6% by weight of garlic pulverized product, The pulverized product may contain 1.5 wt% to 2 wt% of lactose, 0.5 wt% to 10 wt% of lactose, and 72 wt% to 86 wt% of solvent.

The cosmetic composition may be formulated into any one of lotion, essence, foundation, powder, lotion, cream, pack, gel, ointment, patch, spray, mask sheet and detergent.

The cosmetic composition may further have an effect of inhibiting elastase activity, and may be one for improving wrinkles.

In another embodiment of the present invention, a fermentation composition comprising ginseng is inoculated with a strain Lactobacillus casei GFC1 (KCTC18333P), which has excellent SNARE protein activity inhibitory activity, Culturing the fermented product for 1 to 7 days to prepare a fermented product, and then centrifuging the fermented product to prepare a fermented ginseng extract from which cells have been removed.

The method of manufacturing the cosmetic composition may further include sterilizing the fermentation composition at 80 ° C to 90 ° C for 30 minutes to 90 minutes before the lactobacillus casei GFC1 strain is inoculated into the composition for fermentation.

The preparation of the fermented ginseng extract may include centrifuging the fermented product at 6000 rpm to 10000 rpm for 15 minutes to 25 minutes to remove the precipitate, and then filtering the supernatant of the fermented product from which the precipitate has been removed.

The method for producing the cosmetic composition according to the present invention is characterized in that 7 to 10% by weight of the ginseng fruit pulverized product, 4.5 to 6% by weight of the garlic pulverized product, 1.5 to 2% by weight of the omega crushed product, May range from 0.5 wt% to 10 wt%, and the solvent may be from 72 wt% to 86 wt%.

The method of manufacturing the cosmetic composition is a method of preparing a pharmaceutical composition prepared by mixing a fermented ginseng extract with an excipient into any one of a lotion, essence, foundation, powder, lotion, cream, pack, gel, ointment, patch, spray, mask sheet and detergent And < / RTI >

The present invention includes a fermented ginseng extract using Lactobacillus casei GFC1 (KCTC18333P) strain having excellent SNARE protein activity inhibitory activity, thereby increasing the content of active ingredients and polysaccharides, The present invention can provide a cosmetic composition having excellent effects of minimizing irritation, excellent anti-inflammatory and antioxidant activity, inhibiting activity of collagenase, inhibiting the activity of snare protein and improving skin wrinkles, and a method for producing the same .

1 is a graph showing the results of cytotoxicity tests on the extracts of Example 1 and Comparative Example 1 of the present invention.

An embodiment of the present invention relates to a cosmetic composition comprising a fermented ginseng extract using Lactobacillus casei GFC1 (KCTC18333P) strain having excellent SNARE protein activity inhibitory activity. Accordingly, it is an object of the present invention to provide a pharmaceutical composition containing an active ingredient and a polysaccharide derived from ginseng, increasing the content of the active ingredient and polysaccharide, minimizing cytotoxicity and skin irritation, exhibiting excellent antiinflammatory and antioxidant activity, The present invention can provide a cosmetic composition excellent in the effect of inhibiting the activity of the skin and improving the wrinkles of the skin and a method for producing the same.

In order to enhance the efficacy of ginseng and to identify microorganisms which can be used as a cosmetic composition, the ginseng pulverization solution is suspended in sterilized saline, diluted, laid on NA agar medium, cultured at 30 DEG C for 24 hours, (colony) was subcultured to isolate a strain having excellent SNARE protein activity.

The strain thus isolated has been identified as a novel strain belonging to Lactobacillus casei. The present inventors named the selected strain as Lactobacillus casei GFC1 (KCTC18333P) It was deposited on Oct. 21, and was granted accession number KCTC18333P.

These Lactobacillus casei GFC1 strains were confirmed to be 99% or more homologous to Lactobacillus casei by phylogenetic analysis and morphological characterization. Phylogenetic characterization was performed by 16S rDNA sequence analysis and PCR. Gram staining, endospore staining, and Scanning Electron Microscope (SEM) imaging were performed to analyze the morphological characteristics.

The Lactobacillus casei strain GFC1 identified by the above-described assay methods is excellent in the SNARE protein activity inhibiting activity and the erasastase activity inhibiting activity, and the active ingredient derived from ginseng when used for fermentation of ginseng The efficacy of increasing the polysaccharide content is excellent.

The present invention uses the fermented ginseng extract using the strain Lactobacillus casei GFC1 (KCTC18333P) as a cosmetic composition to increase the content of active ingredients and polysaccharides derived from ginseng to minimize cytotoxicity and skin irritation Can provide a cosmetic composition which is excellent in anti-inflammatory and antioxidant activity, inhibits activity of collagenase, inhibits the activity of snare protein, and has an effect of improving skin wrinkles.

The cosmetic composition of the present invention comprising the Lactobacillus casei strain GFC1 is more safe than conventional neurotoxins and synthetic medicines having snare protein activity inhibiting activity or elastase activity inhibiting activity, Low cost of production, high productivity, and excellent economical efficiency.

In addition, the cosmetic composition of the present invention can be particularly superior in industrial utility when used as a cosmetic composition for improving wrinkles through the above-mentioned effects.

The term "fermented ginseng extract" in the present specification means a fermentation product prepared by inoculating a fermentation composition containing ginseng and inoculating the strain Lactobacillus casei GFC1 (KCTC18333P) and / or an extract isolated from the fermentation product . ≪ / RTI > Thus, the cosmetic composition not only increases the content of the active ingredient and the polysaccharide derived from ginseng, but also can be converted into a form that is easily absorbed into the skin when it acts on the skin.

As used herein, the term "extract" includes not only extracts obtained by using extraction solutions, but also extracts obtained by ordinary purification processes. For example, a variety of additional purification methods, such as separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatographies (made for separation by size, charge, hydrophobicity or affinity) The fraction of the extract obtained through the above step is also included in the extract of the present invention. In addition, the extract can be prepared in powder form by an additional process such as vacuum distillation and freeze-drying or spray drying.

The ginseng (Panax ginseng C.A. Meyer) contained in the composition for fermentation is a plant belonging to the genus Aralma and Ginseng, and is used as a typical medicinal plant in Korea, China and Japan. By using ginseng in the composition for fermentation, the fermented ginseng extract contains saponin and ginsenoside complex carbohydrate (polysaccharide), which are major active ingredients of ginseng. In such a case, the cosmetic composition can improve skin elasticity, anti-inflammation and antioxidant effect.

Specifically, the fermented ginseng extract of the present invention can be obtained by inoculating and fermenting the above-mentioned lactobacillus casei GFC1 (KCTC18333P) strain into a fermentation composition containing ginseng to obtain Rb1, Rb2, Rc, Rd , Re, Rg1 and other minor amounts of saponin derivative components. In addition, the content of ginsenoside Rg2 can be significantly increased by converting ginsenoside Re with low bioavailability into ginsenoside Rg2 having high bioavailability. In such a case, the cosmetic composition contains a high content of ginsenoside in a stabilized form, and it is possible to prevent a phenomenon such as separation of formulation or discoloration occurring at high temperature after formulation.

The composition for fermentation is ginseng, and one or more of roots, leaves and fruits of ginseng can be used. For example, the composition for fermentation may contain ginseng fruit as ginseng. In this case, the fermented ginseng extract may be further increased in polysaccharide and saponin content by fermentation using Lactobacillus casei strain GFC1.

In the composition for fermentation, the powder of the ginseng fruit may be contained in an amount of 7 to 10% by weight. In this case, the fermented ginseng composition can further improve skin elasticity, antiinflammation and antioxidant effect.

The composition for fermentation may further contain lactose in addition to ginseng. In this case, the fermented ginseng extract promotes the growth of the Lactobacillus casei strain GFC1 to improve the fermentation degree more properly, and it can increase the activity of the enzyme produced by the Lactobacillus casei strain GFC1. In this case, the cosmetic composition not only increases the content of polysaccharide and saponin of ginseng by improving the fermentation degree, but also promotes the growth of the strain to inhibit the activity of collagenase, inhibits the activity of snare protein, The improvement effect can be realized at a higher level.

The lactose in the fermentation composition may be contained in an amount of 0.5 to 10% by weight. In this case, the fermented ginseng composition may be more effective in improving the activity of the lactobacillus casei GFC1 strain.

The composition for fermentation may further comprise a garlic pulverized product, an excipient pulverized product and a solvent. In such a case, the fermented ginseng extract further contains ingredients useful for skin derived from garlic and anagaki, minimizes cytotoxicity and skin irritation, and can further enhance anti-inflammatory and antioxidant activity effects.

Garlic which contains the above-mentioned fermented composition Ali, a kind of carbohydrate (disk rotoseu) and amino acids in perennial bulbous plants belonging to the genus liliales Liliaceae Allium taxonomically plant (alliin; s-allylcysteine-s -oxide), C 6 H ₁₁ O ₃ NS, cyclo-alliin, allyl-L-cysteine, allicin, scorodose, vitamin B and the like. The composition for fermentation may contain antioxidative substances derived from garlic by further containing garlic powder, and these components may act on skin cells when used as a cosmetic composition to improve antimicrobial, antioxidant and anti-aging effects . By using the garlic as a fermentation composition, the active ingredient of garlic, which is irritating and easily deteriorated, can be converted into non-magnetic, and the fermented composition can further improve the antibacterial and antioxidative effect.

The garlic pulverized product in the fermentation composition may be contained in an amount of 4.5 wt% to 6 wt%. In this case, the fermented ginseng composition can further improve the antimicrobial and antioxidant efficacy.

The plant belonging to the genus Glycine include the lignan glycosides syringaresinol diglycoside, acanthide D, syringoside, eleutheroside B and eleutheroside E, falcarindol, methyl-hexacosanoate, coniferin, pimaradiene diterpene and sumogaside, antioxidant And eleutheroside B having activity. The composition for fermentation may further contain antioxidants derived from an omega by additionally containing an omega crushed product. The antioxidants may be used as a cosmetic composition to improve antioxidative and antioxidative properties. In addition, the roots of acanthoic acid (acanthoic acid), which has not been reported in other species of Acanthopanax senticosus, are contained in the root of the acanthopanax that can further enhance antioxidant and antiinflammatory activity.

The ocher-crushed product of the fermentation composition may be contained in an amount of 1.5 to 2% by weight. In this case, the fermented ginseng composition can further improve the antioxidant and anti-inflammatory effects.

As the solvent, a conventional extraction solvent may be used. Examples of the solvent include water, acetone, ethyl acetate, butyl acetate, diethyl ether, benzene, chloroform, hexane, and alcohols having 1 to 10 carbon atoms. These may be used alone or in combination of two or more. In one embodiment, the alcohols having 1 to 10 carbon atoms include lower monohydric alcohols such as methanol, ethanol, butanol, pentanol, and hexanol; alcohols such as 1,2-pentanediol, 1,5-pentanediol, hexanediol, , Intermediate dihydric alcohols such as octanediol and decanediol, lower trivalent alcohols such as propylene glycol, 1,3-butylene glycol and glycerin, and the like can be used. These may be used alone or in combination of two or more.

The solvent in the composition for fermentation may be contained in an amount of 72% by weight to 86% by weight. In this case, the fermented ginseng composition can increase the extraction efficiency of the useful ingredient while minimizing cytotoxicity and skin irritation, and can further improve the growth of the strain.

0.1 to 5: 0.1 to 5, more specifically 1: 0.1 to 1: 0.1 to 1, more preferably 1: 0.6 to 1: 1 by weight of the ginseng fruit pulverized product: garlic pulverized product: : 0.2 weight ratio. Within the above range, synergistic action occurs when each active ingredient contained in ginseng, garlic, and ginger is fermented by the strain, so that it has excellent anti-inflammatory and antioxidative effects without causing cytotoxicity, The formulation stability of the cosmetic composition can be further improved.

The fermented ginseng extract may be contained in an amount of 0.01 to 50% by weight based on the total weight of the whole cosmetic composition. Within the above range, synergistic action occurs when the respective active ingredients contained in ginseng, garlic, and Ogapi are fermented by the strain, thereby exhibiting excellent anti-inflammatory and antioxidative effects without causing cytotoxicity, , The formulation stability of the cosmetic composition can be further improved.

The cosmetic composition of the present invention may further contain, in addition to the above fermented ginseng extract, an excipient for formulation into various cosmetics so as to be contained in an amount of 50% by weight to 99.99% by weight.

Such excipients may include ingredients that may be formulated in conventional cosmetic compositions. Specifically, the excipient may be at least one of a solubilizer, a surfactant, a moisturizer, a thickener, a pH adjuster, an antiseptic, an antioxidant, a sequestering agent, a bactericide, an antiinflammatory agent, an antimicrobial agent, a solubilizing agent, .

The dissolution aid may be selected from, for example, diisopropyl oxalate, diisopropyl sebacate, tea tree oil, ethoxydiglycol, ethoxydiglycolic behenate, ethoxydiglycol, sostearate, ethoxydiglycol Olite, and the like.

The surfactant includes, for example, anionic surfactants such as ammonium laurylsulfosuccinate, ammonium lauryl sulfate and the like and nonionic surfactants such as polyoxyethylene alkyl ether, polyoxyethylene fatty acid ester and the like; Cationic surfactants such as tertiary aliphatic amine salts, alkyltrimethylammonium chloride and the like and amphoteric surfactants such as betaine and amide betaine; And the like.

The humectant may be, for example, sodium hyaluronate, glycerin, propylene glycol, 1,3-butylene glycol, dipropylene glycol, sorbitol and the like.

The thickening agent may be, for example, a water-soluble polymer compound such as xanthan gum, methyl cellulose, hydroxyethyl cellulose, carrageenan, carboxymethyl cellulose, hydroxymethyl cellulose and the like.

The pH adjuster may be, for example, citric acid, sodium hydroxide, triethanolamine, sodium citrate, phosphoric acid, sodium phosphate, lactic acid and the like.

The preservative may be, for example, benzoic acid, paraoxybenzoic acid ester, a mixture of methylchloroisothiazolinone, phenoxyethanol, DMDMantantoin, and the like.

The antioxidant may be, for example, dibutylhydroxytoluene, ascorbic acid or the like.

The metal ion sequestering agent may be, for example, disodium ethylenediamine tetraacetate, tetra sodium ethylenediamine tetraacetate, or the like.

The bactericide may be, for example, chlorhexidine gluconate, quaternary ammonium salt, pyrrothonol amine, zinc pyrithione suspension, urethropropyl butylcarbamate, salicylic acid and the like.

The anti-inflammatory component may be, for example, monoammonium glycyrrhizinate, dipotassium glycyrrhizinate, allantoin, and mixtures thereof.

The antimicrobial component may be, for example, phenoxyethanol, chlorhexidine, chlorhexidine gluconate, benzoic acid and its salts, benzyl alcohol, salicylic acid, trichlorocarban, zinc pyrithione suspensions and mixtures thereof.

The solubilizing agent may be, for example, Pigment 60-Hydrogenated castor oil or the like.

The above-mentioned flavoring agents, pigments and dyes may be those conventionally used in the field of cosmetic compositions of the present invention.

The cosmetic composition of the present invention can be prepared into any formulation conventionally produced in the art. Specifically, the cosmetic composition may be formulated into any one of lotion, essence, foundation, powder, lotion, cream, pack, gel, ointment, patch, spray, mask sheet and detergent.

In another embodiment of the present invention, a composition for fermentation comprising ginseng is inoculated with a Lactobacillus casei GFC1 (KCTC18333P) strain having excellent SNARE protein cleavage activity, To 7 days, to prepare a fermented product, and then centrifuging the fermented product to prepare a fermented ginseng extract from which cells have been removed. The cosmetic composition prepared by this method can further increase the content of active ingredients and polysaccharides derived from ginseng using the above strain, minimize cytotoxicity and skin irritation, exhibit excellent anti-inflammatory and antioxidative activity effects, The activity of the snare protein is inhibited and the effect of improving skin wrinkles is excellent.

When the fermentation product has a fermentation temperature of less than 27 ° C or more than 37 ° C, it is difficult to sufficiently achieve the effect of inhibiting the activity of the snare protein and the activity of inhibiting the activity of the collagenase (Elastase).

If the fermentation period of the fermented product is less than 3 days, the fermentation by the strain is difficult to proceed sufficiently, and if it exceeds 7 days, the stability of the active ingredient and the fermentation efficiency may be lowered.

In one embodiment, the fermented ginseng extract is prepared by pulverizing ginseng with a solvent to a size of 5 mesh to 50 mesh to prepare a composition for fermentation, directly inoculating Lactobacillus casei GFC1 to prepare a fermented product, And then centrifuging the fermentation product to remove the cells.

In another embodiment, the fermented ginseng extract is prepared by adding 30 to 500 parts by weight of a solvent for extraction to 100 parts by weight of a composition for fermentation comprising ginseng, extracting the mixture for 3 hours to 10 days, , And inoculating the strain to produce a fermented product, followed by centrifuging the fermented product to remove the bacterial cells. In this case, the solids of the composition for fermentation can be easily removed, the disappearance of the active ingredient can be minimized, and the fractionation time of the fermented product can be shortened, which is advantageous for mass production and production of highly concentrated products.

The Lactobacillus casei GFC1 (KCTC18333P) strain may be used for inoculation after seed culture with agitation for 3 to 5 days at 35 DEG C to 37 DEG C in the MRS medium during the inoculation.

In one embodiment, the seed culture of Lactobacillus casei GFC1 can be seeded in a medium prepared by adding 5 to 20 parts by weight of ginseng, garlic, and Ogaki to 100 parts by weight of the MRS medium.

The composition for fermentation is ginseng, and one or more of roots, leaves and fruits of ginseng can be used. For example, the composition for fermentation may contain ginseng fruit as ginseng. The composition for fermentation may further contain lactose in addition to ginseng. In addition, the composition for fermentation may further comprise a garlic pulverized product, an excipient pulverized product and a solvent.

The method for producing the cosmetic composition according to the present invention is characterized in that 7 to 10% by weight of the ginseng fruit pulverized product, 4.5 to 6% by weight of the garlic pulverized product, 1.5 to 2% by weight of the omega crushed product, May range from 0.5 wt% to 10 wt%, and the solvent may be from 72 wt% to 86 wt%.

The effect of the content and the weight ratio of each component is as described above.

The method of manufacturing the cosmetic composition may further include sterilizing the fermentation composition at 80 ° C to 90 ° C for 30 minutes to 90 minutes before the lactobacillus casei GFC1 strain is inoculated into the composition for fermentation. In this case, the activity of the Lactobacillus casei strain GFC1 is suppressed, the activity of the snare protein is inhibited, and the effect of improving the skin wrinkle can be further improved, and the proliferation of harmful bacteria can be prevented, The preservability can be improved.

The preparation of the fermented ginseng extract may include centrifuging the fermented product at 6000 rpm to 10000 rpm for 15 minutes to 25 minutes to remove the precipitate, and then filtering the supernatant of the fermented product from which the precipitate has been removed. In this case, the composition stability of the cosmetic composition can be improved, the activity of the elastase can be inhibited while effectively preventing the precipitation, and the content of the fermented ginseng extract inhibiting the activity of the snare protein can be maintained at an effective content. Further, by removing the cells, further fermentation can be prevented, and the balance of ingredients can be maintained during formulation.

The method of manufacturing the cosmetic composition may further include concentrating the fermented ginseng extract prepared as described above. The concentration can be carried out using an evaporation concentration or a reduced pressure concentration method. Specifically, it may be added to a rotary vacuum evaporator (Eyela, Japan) of the fermented ginseng extract to be concentrated.

The method of manufacturing the cosmetic composition is a method of preparing a pharmaceutical composition prepared by mixing a fermented ginseng extract with an excipient into any one of a lotion, essence, foundation, powder, lotion, cream, pack, gel, ointment, patch, spray, mask sheet and detergent And < / RTI > The specific contents and components of the excipient are the same as those described above. The method of formulating the cosmetic composition is not particularly limited.

Example

Hereinafter, the structure and operation of the present invention will be described in more detail with reference to preferred embodiments of the present invention. It is to be understood, however, that the same is by way of illustration and example only and is not to be construed in a limiting sense. The contents not described in this specification can be sufficiently technically inferred by those skilled in the art, and a description thereof will be omitted.

Manufacturing example  One

(1) Isolation and selection of strains

The ginseng was sterilized by ozonated water, sealed, and aged for 3 months. The strain was separated from the aged ginseng. The ginseng-aged material was diluted with sterilized distilled water to a concentration of 1 to 10 ^-5 times, and then plated in an MRS agar medium in an amount of 100 μl each and cultured in an incubator at 30 ° C. The strains were selected by visual observation of shape, color, transparency, size, and external structure. Strainings of the selected strains were streaked on MRS agar medium and cultured for 4 times to isolate single colonies. The pure isolates were prepared in 20% Glycerol stock solution and stored at -70 ° C in a cryogenic freezer.

(2) Identification of isolated strains by 16S rRNA gene sequencing

DNA was extracted from the selected strains, 16S rRNA gene sequencing was commissioned by Genentech Co., Ltd., and homology with Type strain was confirmed using NCBI database for phylogenetic analysis. Type strain 16S rRNA sequences were aligned using the BioEdit program (Hall, 1999) and the Clustal X program (Thompson et al., 1997) and the Kimura two-parameter model (Kimura, 1983), and the phylogenetic location was determined by Neighbor-joining method (Saitou and Nei, 1987) of the MEGA 3 program (Kumar et al., 2004).

DNA was extracted from the strain and subjected to 16S rRNA analysis. The homology of the strain was analyzed using the BLAST program. As a result, the GFC 1 strain showed 99% homology with the 16S rRNA gene with Lactobacillus casei . To: (KCTC18333P accession number) naming and microbial resource centers them, Korea Research Institute of Bioscience and Biotechnology to the GFC 1 strain is Lactobacillus casei is found to be a new strain belonging to the (Lactobacillus casei), Lactobacillus casei (Lactobacillus casei) GFC 1 It was purchased on October 21, 2014, and granted accession number KCTC18333P.

Example 1

The MFC medium was inoculated with GFC 1, cultured at 37 ° C for 3 days with stirring, and seeded. The grown seedlings were directly inoculated with the ginseng fruit to produce a fermented product as follows.

100 g of water was added to 10 g of ginseng fruit and sterilized at 85 캜 for 60 minutes, and then the seed culture of the GFC 1 was inoculated. At this time, 0.5 g to 10 g of lactose was added to smooth the growth of the strain and increase the enzyme activity, and the fermented product was fermented by culturing at 37 ° C for 7 days. The fermented product was centrifuged at 8000 rpm for 20 minutes to remove cells, followed by filtration and sterilization to prepare a fermented extract.

Example 2

To increase the active substance-producing activity of the strain, 1 to 2 g of ginseng powder was added to the MRS medium, and the mixture was cultured in the medium at 37 DEG C for 3 days with stirring, followed by seed culture. The grown seedlings were directly inoculated into a mixture of ginseng fruit powder, garlic powder, and oatmeal millet as follows to prepare a fermented product.

The ginseng fruit was prepared by finely dividing the fruit portion of ginseng into 50 mesh pieces. The garlic was prepared by cutting the garlic into a size of 50 meshes and the stem was cut into 50 mesh size pieces.

Next, 100 ml of water was added to 10 g of the ginseng fruit pulverized product, 6 g of the garlic pulverized product, and 2 g of the finely ground gruel, and the mixture was sterilized at 85 ° C for 60 minutes, followed by inoculation with the strain GFC 1. At this time, 0.5 g to 10 g of lactose was added to smooth the growth of the strain and increase the enzyme activity, and the fermented product was fermented by culturing at 37 ° C for 7 days. The fermented product was centrifuged at 8000 rpm for 20 minutes to remove cells, followed by filtration and sterilization to prepare a fermented ginseng extract.

Comparative Example 1

The extract was prepared in the same manner as in Example 1, except that 100 ml of water was added to 10 g of ginseng fruit, sterilized at 85 캜 for 60 minutes, and then the strain was not inoculated.

Comparative Example 2

The extract was prepared in the same manner as in Example 2, except that the ginseng fruit pulverized product, the garlic pulverized product and the excipient pulverized product were mixed at a weight ratio of 1: 0.3: 0.3 and then the strain was not inoculated.

Comparative Example 3

Ginseng fruits pulverized 10g, garlic pulverized 6g and Acanthopanax was added to 100ml of water to the grinding water 2g sterilized at 85 ℃ for 60 minutes, and, as in Example 2 except that the inoculated Lactobacillus casei general strain instead of Lactobacillus casei GFC1 strain The extract was prepared in the same manner.

Experimental Example

(1) Measurement of polysaccharide content

Each of the samples of Examples 1 to 2 and Comparative Examples 1 to 3 was immersed in ethanol in an amount of 80% or more and precipitated for 4 to 24 hours. The precipitates were separated by centrifugation for 20 minutes. The separated precipitate was dissolved by adding 5% distilled water, and the ethanol was distilled by heating at 105. The filtrate was filtered through an ultrafiltration membrane, and the filtrate was separated by freeze drying in a freeze dryer (Eyela FDU 540, Japan) And the water-soluble polysaccharide content was converted into the water-soluble polysaccharide content. The results are shown in Table 1 below.

division Polysaccharide content (mg / g) Example 1 29.9 ± 0.58 Example 2 47.2 ± 0.28 Comparative Example 1 7.01 + - 0.33 Comparative Example 2 11.1 ± 0.38 Comparative Example 3 30.0 ± 0.31

Referring to the results shown in Table 1, it was confirmed that the extracts of Examples 1 and 2 had a polysaccharide content 4.2 times higher than that of Comparative Examples 1 and 2, respectively, containing the non-fermented extract. In addition, Example 2 fermented using Lactobacillus casei GFC1 strain had a better polysaccharide content than Comparative Example 3 using the same strain ( Lactobacillus casei ).

(2) Cytotoxicity test

The degree of cytotoxicity according to the concentration was evaluated by diluting the cells of Example 1 and Comparative Example 1 with concentrations of 0, 3.125, 6.25, 12.5, 25, 50, 100 (占 퐂 / ml).

RAW 264.7 cells, a type of macrophage, were counted in a DMEM medium containing 1% penicillin / scrap tromycin and 10% FBS (fetal bovineserum) in the same number of 1.0 × 10 ⁴ cells / well on a 24-well plate , Followed by incubation at 37 ° C and 5% carbon dioxide for 24 hours.

The cells cultured for 24 hours were mixed with the culture medium of each of the concentrations of Example 1 and Comparative Example 1, and 1 ml of each was added to each well. After 24 hours of incubation at 37 ° C and 5% CO _{2 in} an incubator, well of the supernatant was separately prepared and the WST-1 assay solution (ez-cytox) was added to each well. After incubation for 2 hours in an incubator, the cell viability was determined by measuring the absorbance at 450 nm with an ELISA reader. Respectively.

1 is a graph showing the results of cytotoxicity tests on the extracts of Example 1 and Comparative Example 1. Fig. 1, the extract of Example 1 was prepared by the same method even at a high concentration of 100 μg / ml. However, it was found that the extract of Example 1 was less cytotoxic than the extract of Comparative Example 1 inoculated with Lactobacillus casei GFC1.

(3) Inflammatory Inducing Factor Inhibition Test

In order to measure the anti-inflammatory activity of the extracts prepared in Examples 1 to 2 and Comparative Examples 1 to 3, nitric oxide (NO) was diluted to a concentration of 50 (占 퐂 / ml) Was measured.

RAW 264.7 cells, a type of macrophage, were counted by the same number of 1.0 x 104 cells / well on a 24-well plate using DMEM medium containing 1% penicillin / scrap tromycin and 10% FBS (fetal bovineserum) , And cultured at 37 ° C and 5% carbon dioxide for 24 hours.

The cells of the above Example and Comparative Example were mixed with the culture medium at a concentration of 50 (/ / ml), and 1 ml of the extract was added to each well, followed by incubation in an incubator under the conditions of 37 ° C and 5% CO ₂ for 24 hours . At this time, LPS (lipo poly saccharide), which is an inflammatory inducer for expressing NO, was treated at a concentration of 1 μg / ml and allowed to react at 37 ° C and 5% carbon dioxide for 24 hours. Next, take the supernatant of each well separately and take 100 ml of the culture solution into each well using Nitric Oxide detection kit. Add 50 ㎕ of Griess reagent A and 50 ㎕ of Griess reagent B to each well. Min, and the absorbance was measured at 540 nm using an ELISA plate reader. The results are shown in Table 2 below.

Griess Reagent A: N-1-Naphthylethylenediamine (NEDHC)

Griess Reagent B: Sulfanilamide

division NO production concentration (μM) Positive control group 15 ± 0.15 Negative control group 55 ± 0.21 Example 1 27 ± 0.24 Example 2 12 ± 0.36 Comparative Example 1 58 ± 0.71 Comparative Example 2 60 ± 0.32 Comparative Example 3 41 ± 0.68

Referring to the results of Table 2, it was found that the extracts of Examples 1 and 2 were significantly superior in inhibiting nitric oxide (NO) production than Comparative Examples 1 to 3.

(4) Free radical removal ability test

Free radicals are active oxygen, and the oxygen that we breathe is thousands of times higher in the process of making energy and reducing it to water. It is produced by the metabolic processes of cells in plants and animals, or by stress, light stimulation, and bacterial infiltration. When excessive amount of active oxygen is present in the body, normal cells become indiscriminate attacks, diseases and aging. In addition, it acts as a pathological factor showing an immune response sensitive to external stimuli. Whether the extract of the present invention exhibited antioxidant activity and whether it had an inflammation-alleviating effect was confirmed as a free radical scavenging ability of DPPH. The extracts prepared in Examples 1 to 2 and Comparative Examples 1 and 2 were each administered at a concentration of 50 (㎍ / ml) And the degree of DPPH inhibition was evaluated.

The above Examples 1 to 2 and Comparative Examples 1 to 3 were diluted to a concentration of 50 μg / ml and then mixed with 250 μl of 0.1 M DPPH (1,1-diphenyl-2-picrylhydrazyl) Lt; / RTI > At the end of the reaction, each sample was placed in a 96-well plate and absorbance was measured at 520 nm using an ELISA reader.

The free radical scavenging activity (%) of the sample was calculated according to the following equation 1 using the case where the above examples and comparative examples were not used as a control group, and the results are shown in Table 3 below. At this time, ascorbic acid was used as a positive control.

[Equation 1]

Free radical scavenging ability (%) = [100- (absorbance of the group treated with the sample / absorbance of the group not treated with the sample X 100)]

division Free radical scavenging ability (%) Positive control group 66.74 + - 0.98 Example 1 60.9 ± 0.24 Example 2 82.1 ± 0.68 Comparative Example 1 55.3 ± 0.13 Comparative Example 2 51.4 ± 0.44 Comparative Example 3 78.9 ± 0.55

Referring to Table 2, the extracts of Examples 1 and 2 were prepared by the same method, but the DPPH scavenging ability was superior to that of Comparative Examples 1 and 2 in which the strain was not inoculated. In addition, Example 2 fermented using Lactobacillus casei GFC1 strain showed better free radical scavenging ability (antioxidative ability) than Comparative Example 3 using the same strain ( Lactobacillus casei ).

(5) Wrinkle prevention efficacy verification test

The extracts prepared in Examples 1 to 2 and Comparative Examples 1 to 3 were each diluted to a concentration of 50 (占 퐂 / ml) to evaluate the degree of inhibition of Elastase.

The above Examples 1 and 2 and Comparative Examples 1 and 2 were diluted to a concentration of 50 μg / ml and then diluted with N-succinyl- (Ala) 3-p-nitroanilide (10 μl) dissolved in 10 mM Tris- S4760, Sigma). After incubation at 25 ° C for 5 minutes, the absorbance at 410 nm was measured in a 96-well plate by mixing 1.6 mM substrate and 0.4 U / mL of Elastase from porcine pancreas Type IV (E0258, Sigma).

The results are shown in Table 4 below. The results are shown in Table 4 below. At this time, Oleanolic acid was used as a positive control.

[Formula 2]

Elastase inhibitory activity (%) = 100- (absorbance of the group treated with the sample / absorbance of the group not treated with the sample X 100)

division Elastase inhibitory activity (%) Positive control group 88.30 + - 0.50 Negative control group 6.1 ± 0.50 Example 1 44 ± 0.43 Example 2 79.3 ± 0.64 Comparative Example 1 28 ± 0.55 Comparative Example 2 50.0 + - 0.33 Comparative Example 3 69.1 ± 0.35

Referring to Table 4, it can be seen that the extracts of Examples 1 and 2 are far superior in the effect of inhibiting the elastase from the extracts of Comparative Examples 1 and 2 and are effective in improving wrinkles.

(6) Inhibition of Snare membrane fusion effect

In order to search for membrane fusion inhibitors, membrane fusion inhibitory effects of the extracts prepared in Examples 1 to 2 and Comparative Examples 1 to 3 were tested. The SNARE protein was obtained by over-expressing the protein in the transformed E. coli and then selectively expressing the expressed protein using glutathione agarose beads. In order to prepare liposomes, which are fine coating particles marked with the following fluorescent substance, 1-palmitoyl-1,2-diolureo-sn-glycero-3-phosphatidylcholine (POPC, 62 mol%), -sn-glycero-3-phosphatidylserine (DOPS, 35 mol%), 1,2-diolureo-sn- glycero-3-phosphocerine-N- (7-nitro- 10 mM liposome (v-vesicle) was prepared by mixing the above compound (5 mg / ml) and Rhodamin-PE (1.5 mol%) as a fluorescent substance. Next, in order to prepare a liposome without fluorescent substance labeling, DOPS and POPC were mixed in a molar concentration of 35: 65 to prepare a 50 mM liposome (t-vesicle). To prepare a complex of purified SNAP-25 and syntactic 1a, the mixture was mixed at a molar concentration of 1: 1 and reacted at room temperature for 1 hour. Then, the mixture was mixed with the liposome not labeled with the fluorescent substance to a molar ratio of 100: -2 was combined with a liposome containing a fluorescent substance in a molar ratio of 50: 1. Subsequently, the two kinds of liposomes were dialyzed and mixed at a ratio of 1: 9 (v-vesicle: t-vesicle)

Fluorescence intensity was measured using SpectraMax M2, manufacturer: Molecular Device).

division Membrane fusion inhibition (%) Positive control group
(Myricetin) 50.60 0.35 Negative control group 2.2 ± 0.31 Example 1 28.1 ± 0.22 Example 2 49.3 ± 0.35 Comparative Example 1 16.1 ± 0.29 Comparative Example 2 20.0 + - 0.84 Comparative Example 3 27.3 + - 0.54

(7) Formulation stability evaluation

The extracts of Examples 1 and 2 and Comparative Examples 1 and 2 were applied to the ingredients listed in Table 6 below to prepare lotions using conventional methods.

Ingredients Content (unit:% by weight) Purified water 88.97 1,3-butylene glycol 2.0 glycerin 1.0 Carbomer 15.0 Triethanolamine 0.2 Stearic acid 3.0 Beads wax 0.5 Squalene 5.0 Glyceryl stearate 2.0 Cetyl alcohol 1.0 antiseptic 0.01 extract 5.0 Sum 100

In order to measure the stability of the cosmetic material containing the extracts of Examples 1 to 2 and Comparative Examples 1 and 2 to the degree of separation and discoloration, a thermostat was kept constant at 45 ° C, 25 ° C and 4 ° C, Each of the Examples 1 to 2 and Comparative Examples 1 and 2 was put in a container for 5 weeks and stored for 4 weeks. The degree of separation and discoloration were compared and the results are shown in Table 7 below. The degree of separation and discoloration of the product was classified into the following six grades and evaluated.

* Separation and discoloration evaluation criteria

4: Severely separated (discolored), 5: Severely separated (discolored), 2: slightly separated (discolored), 3: slightly separated (discolored)

Evaluation items Degree of separation and discoloration Example Comparative Example One 2 One 2 45 ° C One One One 2 25 ℃ 0 0 0 0 4 ℃ 0 0 0 0 daylight One One One One

Referring to the results of Table 7, it was found that the cosmetic compositions of Examples 1 and 2 hardly suffered discoloration and separation phenomenon, and that the formulation stability was excellent.

It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (10)

A cosmetic composition comprising a fermented ginseng extract using a strain of Lactobacillus casei GFC1 (KCTC18333P) having SNARE protein activity inhibitory activity.
The method according to claim 1,
Wherein the fermented ginseng extract is fermented by inoculating the lactobacillus casei strain GFC1 into a composition for fermentation comprising ginseng fruit pulverized product and lactose.
3. The method of claim 2,
Wherein the composition for fermentation further comprises 7 to 10% by weight of ginseng fruit pulverized product, 4.5 to 6% by weight of garlic pulverized product, 5 to 6% by weight of garlic pulverized product, Wherein the pulverized product comprises 1.5 wt% to 2 wt% lactose, 0.5 wt% to 10 wt% lactose, and 72 wt% to 86 wt% solvent.
The method according to claim 1,
Wherein the cosmetic composition is formulated into any one of a lotion, an essence, a foundation, a powder, a lotion, a cream, a pack, a gel, an ointment, a patch, a spray, a mask sheet and a detergent.
The method according to claim 1,
Wherein the cosmetic composition is more effective for inhibiting the activity of elastase, and is for improving wrinkles.
Lactobacillus casei GFC1 (KCTC18333P) strain having the SNARE protein activity inhibitory effect was inoculated into the fermentation composition containing ginseng and cultured at 27 ° C to 37 ° C for 3 to 7 days Preparing a fermented product, and then centrifuging the fermented product to prepare a fermented ginseng extract from which cells have been removed.
The method according to claim 6,
Further comprising sterilizing the composition for fermentation at 80 캜 to 90 캜 for 30 minutes to 90 minutes before the lactobacillus casei GFC1 strain is inoculated into the composition for fermentation.
The method according to claim 6,
The preparation of the fermented ginseng extract comprises centrifuging the fermented product at 6000 rpm to 10000 rpm for 15 minutes to 25 minutes to remove the precipitate, and then filtering the supernatant of the fermented product from which the precipitate has been removed. Way.
The method according to claim 6,
Wherein the composition for fermentation comprises ginseng powdered ginseng powder, and further comprises garlic pulverized product, omega crushed product and lactose,
The whole fermentation composition contains 7 wt% to 10 wt% of ginseng fruit pulp, 4.5 wt% to 6 wt% of pulverized garlic, 1.5 wt% to 2 wt% of omega crushed pulp, 0.5 wt% to 10 wt% By weight, and the solvent is from 72% by weight to 86% by weight.
The method according to claim 6,
The cosmetic composition may be prepared by mixing a fermented ginseng extract with an excipient into one of a lotion, essence, foundation, powder, lotion, cream, pack, gel, ointment, patch, spray, mask sheet and detergent ≪ / RTI >

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