KR101817776B1 - Lespedeza cuneata G.Don Fermented extract Using Lactobacillus pentosus-GFC LP and Method for Preparing the Same - Google Patents

Abstract

The present invention provides a Lactobacillus pentosus-GFC LP strain (Entrustment No. KCCM11950P), and the strain is grafted onto a lespedeza cuneata G.Don extract and cultivated to manufacture a sericea lespedeza fermented extract. Accordingly, staphylococcus epidermidis, which is beneficial for skin, is increased, and moreover, the present invention has an excellent effect of improving skin elasticity and reducing wrinkles, thereby bringing an excellent effect as a cosmetic composition when applied to a cosmetic base as an effective component.

Description

[Technical Field] The present invention relates to a fermented extract of Lactobacillus pentosus-GFC LP and a method for preparing the fermented Lactobacillus pentosus-GFC LP,

The present invention relates to a non-fermented fermented extract prepared by inoculating and culturing Lactobacillus pentosus strain into an extract of Lespedeza cuneata G. Don , and a method for producing the fermented extract.

Skin is one of the largest organs in the body, with millions of microbial communities populated, and most microbes live in symbiosis and play a beneficial role in skin. There are many variables in the formation of microorganisms in the skin. Among them, geographical position, internal factor of the host, environmental factors are representative. Human Immunity and External Immunity The function of the immune system is carried out by the microorganisms that are present on the skin, and the human body acquires a variety of immune responses depending on the kinds of microorganisms present in the skin. Such skin immunity is directly or indirectly related to skin aging. Typically, lack of immunity against external factors will affect skin barrier and skin aging. Atopy is a typical phenomenon caused by the collapse of skin immunity and barriers, and atopy is accompanied by symptoms such as skin aging. In order to prevent this balance from being overturned, the skin-borne microorganisms form a defense system to prevent the transition of microorganisms that can cause skin troubles and aging. Previously, it was difficult to analyze the skin microbes, but due to the development of molecular genetic techniques, it is possible to analyze microbial populations that are forming a community on the skin. A variety of analytical methods have shown that many species of the genus such as Streptococcus , Propionibacterium , and Corynebacterium cause skin imbalance, The microbial community of the skin may be applied to anti-aging using Promicrobial or Antimicrobial technology. Probiotics, which collectively refers to microorganisms that have a beneficial effect on human body, and Synbiotics, which include prebiotics that improve the environment and improve the health of the host, . To date, most known as probiotics are known as lactobacilli, and many studies have been conducted in the field where many different microorganisms are present in body parts. In addition to the current market, research on skin microbiology and skin diseases has also begun. Skin is a connection point with the external environment, and it forms various habitats such as pores, sebum, and slit, and it is suitable to study a variety of microorganisms by providing an environment in which a wide range of microorganisms can grow. Probiotics are also reported to inhibit most harmful microflora and activate beneficial microflora by allowing microbial community control. Microbiom approaches microbial communities through changes in microbial communities. Are reported to be related.

On the other hand, Lespedeza cuneata G.Don is a perennial plant of soybean which grows in mountainous areas all over Korea and is widely distributed in Japan, China, Taiwan, India and Australia. There is a characteristic that it grows well at the foot of the mountain, the mountain road, the desolate land, the roadside, the field, The taste is very high and the quality is slightly warm or cool and not poisonous.

It has been reported that it is particularly effective for the lack of oocyte in males and the enhancement of tincture in men, and has been used as an effective medicinal product for the treatment of puberty, premature ejaculation, oil wells, cough, and asthma in the private. Further, it contains physiologically active substances such as pinitol, flavonoid and phenol components, and is excellent in antioxidative action effect, has no toxicity and side effects, and is widely used as a cosmetic composition for preventing aging and the like .

The technology applied to cosmetic materials using the non-aqueous extract includes Korean Patent Registration No. 10-1441291 entitled " Cosmetic Composition for Whitening and Antioxidation Containing Briquette and Unsulfuric Acid Extracts ", Korea Patent Publication No. 2014- 0123374 entitled " Wound-healing functional cosmetic composition containing naked eye extract, " Korean Patent Laid-Open Publication No. 2014-0002827 entitled " And " a composition containing an extract of an insect wing having an antioxidative action ".

Accordingly, the present inventors isolated and identified a strain of Lactobacillus pentosus- GFC LP (accession number: KCCM11950P) which produces a high-activity? -Glucosidase from intestinal lactobacilli, When the fermented extract is prepared by inoculating and cultivating the extract of Lespedeza cuneata G. Doon , the extract of Staphylococcus epidermidis , which is a useful protein, ), As well as the effect of improving wrinkles and skin elasticity can be imparted, and the present invention has been completed.

Korean Patent Registration No. 10-1441291 Korean Patent Laid-Open Publication No. 2014-0123374 Korean Patent Publication No. 2014-0002827 Korean Patent Publication No. 2012-0055476

It is an object of the present invention to provide a fermented Asahi extract and a method for producing the fermented extract, characterized by including a step of inoculating and culturing a novel strain to an extract of Lespedeza cuneata G. Don .

Another object of the present invention is to provide a cosmetic composition for improving wrinkles and skin elasticity, which comprises an aspartame fermented extract as an active ingredient.

In order to achieve the above object, the present invention provides a method for culturing Lactobacillus pentosus- GFC LP ( Lactobacillus pentosus- GFC LP) strain (accession number: KCCM11950P) inoculated with an extract of Lespedeza cuneata G. Don and culturing Wherein the fermented extract is a fermented extract.

In the present invention, the Lactobacillus pentosus- GFC LP strain (accession number: KCCM11950P) has the ability to produce beta -glucosidase.

In the present invention, the culturing is performed at 20 to 37 ° C for 2 to 7 days.

In addition, the present invention provides a fermented ashless extract prepared through the above-described production method.

In the present invention, the non-fermented fermentation extract has an effect of enhancing Staphylococcus epidermidis .

The present invention also provides a cosmetic composition for improving wrinkles and skin elasticity, which comprises the above fermented Asahi extract as an active ingredient.

( Lactobacillus pentosus- GFC LP) strain (accession number: KCCM11950P) provided from the present invention is inoculated into an insecticidal extract ( Lespedeza cuneata G. Don) and cultured, It is possible to increase Staphylococcus epidermidis , which is a skin benefit bacteria, as well as to improve wrinkles and improve skin elasticity. Therefore, when this is applied to a cosmetic base as an effective ingredient, The effect as a composition is excellent.

FIG. 1 is a photograph showing a final selection of one kind of β-glucosidase high-frequency strain (GFC LP) among thirteen strains selected.
Fig. 2 shows a phylogenetic analysis of a strain of Lactobacillus pentosus- GFC LP (accession number: KCCM11950P).
FIG. 3 is a graph showing the cytotoxicity evaluation of the non-sushi extract (before fermentation) and the non-sushi fermentation extract (after fermentation).
4 is a graph showing the activity of suppressing the elastase activity of the non-sushi extract (before fermentation) and the non-sushi fermentation extract (after fermentation).
FIG. 5 is a graph showing the activity of inhibiting MMP-1 synthesis of the non-fermented extract (before fermentation) and the non-fermented fermentation extract (after fermentation).
FIG. 6 is a graph showing the skin elasticity measurement result (unit: AU) of the test product containing the fermented extract of Asuncle.
7 is a graph showing the skin elasticity improvement rate (unit:%) of the test product containing the fermented extract of Asahi fermented extract.
FIG. 8 shows the results of analysis of the changes in the skin supernatant by the denaturing gradient gel electrophoresis (DGGE) of the insecticidal extracts (before fermentation) and the non-fermented fermentation extract (after fermentation) for the subjects 1 to 5.
FIG. 9 shows the result of quantifying and analyzing the image of the skin of the non-sushi extract (before fermentation) and the non-fermented fermentation extract (after fermentation) to the subject 1 by using GelCompar2 and Image lab.
Fig. 10 shows the result of quantifying and analyzing the image of the skin of the non-sushi extract (before fermentation) and the non-sushi fermentation extract (after fermentation) to the subject 2 by using GelCompar2 and Image lab.
Fig. 11 shows the result of quantitative analysis of images of the skin of the non-sushi extract (before fermentation) and the non-fermented fermentation extract (after fermentation) on the subject 3 by using GelCompar2 and an image lab.
Fig. 12 shows the result of quantifying and analyzing the image of the skin of the non-sushi extract (before fermentation) and the non-fermented fermentation extract (after fermentation) to the subject 4 by using GelCompar2 and Image lab.
Fig. 13 shows the result of quantitative analysis of images of the skin-biodegradable bacteria of the non-sushi extract (before fermentation) and non-fermented fermentation extract (after fermentation) on the subject 5 using GelCompar2 and Image lab.

Therefore, in the present invention, Lactobacillus pentosus- GFC LP strain (accession number: KCCM11950P), which is excellent in the ability to produce β-glucosidase, is added to the extract of Lespedeza cuneata G.Don It was confirmed that Staphylococcus epidermidis , which is a skin benefit microorganism, can be augmented and that the effect of improving wrinkles and improving skin elasticity is excellent when the fermented extract is prepared by inoculation and culturing Respectively.

Accordingly, the present invention relates to a method for producing a recombinant Lactobacillus pentosus strain, which comprises inoculating an Lactobacillus pentosus- GFC LP strain (accession number: KCCM11950P) (hereinafter abbreviated as "GFC LP") to an extract of Lespedeza cuneata G. Don , And culturing the fermentation broth of the present invention.

At this time, the GFC LP strain has the ability to produce? -Glucosidase.

Wherein the non-sushi extract is a fraction of non-sushi hot water extract, non-sari organic solvent extract or organic solvent extract. That is, at least one extract selected from the group consisting of hot water extraction at 80 to 100 ° C for 6 to 48 hours, extraction by immersion in an organic solvent at 25 to 80 ° C for 1 to 7 days or fractionation of the organic solvent extract Method is preferable in that the yield of the extract can be most effectively improved.

The step of inoculating and culturing the GFC LP strain into an extract of Lespedeza cuneata G.Don is performed at 20 to 37 ° C for 2 to 7 days. If the incubation temperature and time are out of the above ranges, the culturing conditions of the fermenting bacteria do not match, so that not only β-glucosidase is produced but also the strain does not grow, which is not preferable.

In addition, the present invention relates to a fermented ashless extract prepared through the above-described method for producing fermented fermented extract.

The non-fermented fermentation extract has an effect of enhancing Staphylococcus epidermidis . That is, it has an effect of inducing a change in the skin supernatant which increases the population of Staphylococcus epidermidis , which is a skin beneficial microorganism, and thus it is possible to realize fundamental skin health improvement as well as skin beauty. Further, it is effective for wrinkle improvement and skin elasticity improvement, so that the effect as a cosmetic composition is excellent.

Accordingly, the present invention relates to a cosmetic composition for improving wrinkles and improving skin elasticity comprising the above fermented Asahi extract as an active ingredient.

The cosmetic composition for improving wrinkles and skin elasticity according to the present invention can be used for ointment for external use, cream, softener, softener, pack, essence, hair tonic, shampoo, , Skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutritional cream, eye cream, moisturizing cream, hand cream, foundation, nutrition essence, sunscreen, soap, cleansing foam, cleansing lotion, cleansing cream , Body lotions, and body cleansers, but are not limited thereto. The composition of each of these formulations may contain various kinds of bases and additives necessary for formulation of the formulation, and the kinds and amounts of these ingredients can be easily selected by those skilled in the art.

When the formulation of the present invention is a paste, cream or gel, animal fibers, plant fibers, wax, paraffin, starch, tracant, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide are used as carrier components .

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

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

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

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component is selected from aliphatic alcohol sulfates, aliphatic alcohol ether sulfates, sulfosuccinic acid monoesters, isethionates, imidazolinium derivatives, methyltaurate, sarcosinates, fatty acid amides Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolenic derivatives or ethoxylated glycerol fatty acid esters.

In the cosmetic composition for improvement of wrinkles and skin elasticity according to the present invention, the non-fermented fermentation extract may contain 0.01 to 99% by weight, preferably 5 to 20% by weight, based on the total weight of the composition But is not limited thereto, including an effective amount that can provide a desired skin whitening or wrinkle reducing effect.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not to be construed as limiting the scope of the present invention. It will be self-evident.

Example 1 Isolation and Selection of Strain

Fermentation strain isolation focused on isolating lactic acid bacteria with high β-glucosidase activity, which is known to convert isoflavone glycosides in plant material to non-glycosides. First, ginseng samples were diluted to 10 ^-2 , 10 ^-3 and 10 ^-4 with decanter, and then MRS medium (Proteose peptone No. 3. 10 g, Beef extract 10 g, Yeast extract 5.0 g) containing 0.002% of bromophenol blue , Dextrose 20g, Polysorbate 80 1.0g, Ammonium citrate 2.0g, Sodium acetate 5.0g, Magnesium sulfate 0.1g, Manganese sulfate 0.05g, Dipotassium phosphate 2.0g, Agar 15.0g / l, Difco, USA) A colony forming a yellow circle was isolated from about 100 candidate members of lactic acid bacteria. A strain producing β-glucosidase having a function of degrading glycosides existing in plants among the firstly isolated lactic acid bacteria was cultured in Esculin agar medium (Esculin 3.0 g, Ferric citrate 0.2 g, Proteose peptone No. 3. 10 g, Beef extract 10.0 g Yeast extract 5.0 g Dextrose 20 g Polysorbate 80 1.0 g Ammonium citrate 2.0 g Sodium acetate 5.0 g Magnesium sulfate 0.1 g Manganese sulfate 0.05 g Dipotassium phosphate 2 g Agar 15.0 g). Esculin is a glycoside derived from a simple monosaccharide. When carbohydrates and non-carbohydrates are linked by an acetal linkage, aglycone is produced. Acetal is hydrolyzed by acids of lactic acid bacteria. Esculetin produced after digestion is screened by black complex formed by ferric ion. Among the previously selected candidate lactic acid bacteria, 13 strains producing β-glucosidase were selected by Esculin agar method and were selected according to whether they produced Black complex by Esculin.

The isolated strains were stored in a 30% glycerol stock solution at -70 ° C in a Deep freezer. In order to select a strain having an excellent activity of β-glucosidase, a lactic acid bacterium having β-glucosidase production was cultured in an MRS broth medium. The supernatant obtained by centrifuging was dissolved in a crude enzyme solution And the activity was measured using ρ-nitrophenyl-β-D-glucopyranoside (ρNPG) as a substrate. One unit of β-glucosidase was defined as the amount required to produce 1 μmol of ρ-nitrophenol (ρNP) in ρ-nitrophenyl-β-D-glucopyranoside (p-NPG) , And 13 strains of selected bacteria were finally selected as one type of β-glucosidase high-frequency strain (GFC LP) (Fig. 1).

<Example 2> Sequence analysis of 16s rRNA of the selected strain

The identification of GFC LP, a β-glucosidase high-titer strain, was performed by 16S rRNA sequence analysis. (5 'AGA GTT TGA TCM TGG CTC AG 3') and SEQ ID NO: 2: 1492R (5 'TAC GGY TAC CTT GTT ACG ACTT 3') from the gDNA obtained by performing chromosomal DNA extraction PCR was carried out to analyze the nucleotide sequence. Based on the results obtained by performing 16s rRNA sequencing, the homology test was performed through the BLAST search provided by NCBI. It was identified with the base sequence isolated strains exhibited a Lactobacillus pento suspension (Lactobacillus pentosus) and 99% of similarity belongs to the Lactobacillus (Lactobacillus) genus (genus). Based on the results, Lactobacillus pentosus- GFC LP was named as Lactobacillus pentosus- GFC LP and deposited on Nov. 29, 2016 at the Korean Microorganism Conservation Center (KCCM), which is an international microorganism patent depository, and the deposit number KCCM11950P . MEGA 6.0 (Megasoftware, USA) program was used to create a schematic diagram by neighbor-joining method (Figure 2).

Example 3: Preparation of non-sushi fermented extract

The non-sushi was purchased at the Seoul Yakunyeong market, which was completely dried, and prepared in a size of about 30cm. 70% EtOH was soaked 10 times by weight of the non-sludge, and then extracted at room temperature for 7 days. The extracts were filtered with filter paper Whatman No.2 and then evaporated under reduced pressure with a rotary vacuum evaporator (400 series, Eyela, Tokyo, Japan). Powders that did not become a powder by evaporation under reduced pressure were lyophilized to powder. The weight of the final aspartame extract powder was measured, and the final yield was 8.2% by weight. At this time, the prepared syrup extract (before fermentation) was used as a comparative sphere.

Next, 0.1% by weight of the above non-aqueous extract and 1.0% by weight of glucose were added to the liquid medium, sterilized at 121 ° C for 15 minutes, then inactivated GFC LP was inoculated and cultured at 37 ° C for 5 days under optimal fermentation conditions To thereby prepare a final fermentation extract (after fermentation).

In order to maximize the active ingredient, the prepared insecticidal extract (before fermentation) and non-fermented fermentation extract (after fermentation) were respectively centrifuged to remove pellets such as cells, followed by filtration at 0.22um to obtain a supernatant. The fractions were fractionated with ethyl acetate to increase.

<Example 4> Anti-wrinkle improvement effect test

Various anti-aging-related in-vitro activity studies were carried out using the non-sushi extracts and non-sushi fermentation extracts prepared in Example 3 above.

(1) Cytotoxicity test

The cell toxicity test was carried out on HS-68 fibroblasts in order to confirm the irritation of the cells using the non-sushi extracts prepared from Example 3 and the non-sushi fermentation extracts.

B16-F10 melanoma cells (ATCC ^ⓡ CRL 6475 ™) is H. Masai sat meter (hemacytometer) in 96 well plate (Corning 3590) 5 * 10 ^ 3 / culture as well and hFF (Human Foreskin Fibroblast, CCD- 986Sk) in (ATCC ^ⓡ CRL-1947 ™) was seeded with ³ * 10 ^{^} 3 / well and cultured for 24 hours at 37 ℃, CO ₂ conditions. The cell culture medium was removed and a sample diluted with DMEM medium (Dulbecco's Modified Eagle Medium, Gibco BRL, USA) containing 10% fetal bovine serum (FBS) 100 쨉 l per well was added to the wells and cultured at 37 째 C and 5% CO ₂ for 24 hours. To measure the survival rate of cells, WST-1 (high sensitive Water Soluble Tetrazolium salt) reaction solution was diluted 1/10-fold in DMEM medium, treated with 100 쨉 l per well and allowed to react for 2 hours. Then, ELISA absorption analyzer , Thermo scientific). Cell viability was calculated using the following equation (1) as compared with the untreated group (untreated group).

[Equation 1]

Cell survival rate (%) = (absorbance of sample treated group / absorbance of no treated group) * 100

As a result, as shown in FIG. 3, it was confirmed that the non-sushi extract and the non-sushi fermented extract had no irritation to HS-68 fibroblasts with a survival rate of 90% or more at a maximum concentration of 400 ㎍ / ㎖, Respectively.

(2) Elastase inhibitory activity

A sample diluted with various concentrations (400, 200, 100, 50, 25, and 12.5 ppm) of the non-sushi extract and the non-sushi fermentation extract prepared in Example 3 was dissolved in 1.6 mM N 2 N 2 dissolved in 10 mM Tris-HCl buffer (pH 8.0) (Ala) 3-p-nitroanilide (S4760, Sigma) was added to the reaction mixture, followed by the addition of 10 mM Tris-HCl buffer (pH 8.0) (E0258, Sigma) of 0.4 U / mL of the enzyme dissolved in 50 μL of the enzyme was reacted at 25 ° C. for 5 minutes, and the absorbance was measured at 410 nm in a spectroscopic analyzer. Elastase inhibitory activity was calculated using the following equation (2) as a control group when no sample was added.

The results of wrinkle-reducing efficacy through the inhibition of elastase activity are shown in Fig.

&Quot; (2) &quot;

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

As a result, as shown in FIG. 4, when the fermented extract of Asahi fermented product (after fermentation) was treated by concentration, it showed significant Elastase inhibition rate and it was confirmed that it has an effect of improving skin wrinkles. In addition, it was confirmed that the IC ₅₀ value of the fermented Asahi fermented extract (after fermentation) was 316.41 / / ㎖, which was greatly increased compared to the fermentation of Asurii extract (before fermentation) at a maximum of about 42.73% at 200 / / ㎖.

(3) MMP-1 synthesis inhibitory activity

The inhibitory activity of MMP-1 on the inhibition of MMP-1 synthesis was evaluated by using an ELISA kit based on the test method of the phytophthora due to the use of the non-sushi extract and the non-sushi fermentation extract prepared in Example 3 above. Fibroblasts were plated on a 24-well plate at a density of 1.5 × 10 ⁴ cells / well and cultured at 37 ° C and 5% CO ₂ for 24 hours. The medium was discarded and further incubated for 6 hours with serum-free media. After the medium was discarded, DPBS was added. UV irradiation (15 ~ 22 mJ / cm ² ) was carried out with DPBS and DPBS was removed. At this time, Retinoic acid was used as a control group. After 48 hours, the culture solution was taken and the amount of collagenase (MMP-1) was measured by ELISA. As described in the ELISA kit, 100 μL of RD1-52 buffer was taken and added to each well of an ELISA kit. 100 μL of each sample and standard was added and left in an orbital shaker for 2 hours. At this time, the sample was diluted 1/10 with RD5-19 buffer. After washing 4 times with Wash buffer, 200 μL of Humman Pro-MMP-1 solution was added and left in orbital shaker for 2 hours. After washing 4 times with Wash Buffer, 200 μL of coloring reagent was added and reacted at room temperature for 20 minutes. 50 [mu] L of 2N sulfuric acid was added and immediately measured with a 450 nm ELISA reader.

As a result, as shown in FIG. 5, it was confirmed that the non-fermented fermentation extract (after fermentation) exhibited an activity of inhibiting MMP-1 synthesis in proportion to the concentration, thus improving wrinkles of the skin. In addition, the maximum inhibition rate of 81.36% when treated with 50 μg / ml of the fermented non-fermented extract (after fermentation), and 78% with the maximum of 100 μg / ㎖ of the fermented non-fermented extract (before fermentation) were confirmed. In other words, it was confirmed that the non-sludge fermentation extract (after fermentation) showed an excellent inhibitory effect on MMP-1 synthesis even at a low concentration compared to the non-sludge extract (before fermentation).

The GFC LP strain having the ability to produce beta -glucosidase converts the glycoside in the fermentation extract to the non-glycoside, thereby inhibiting the elastase (Elastase) and MMP-1 It was confirmed that the effect of wrinkle improvement was improved by improving the synthesis inhibitory ability.

<Example 5> Skin elasticity improvement effect test

Various skin elasticity improving effects were tested using the non-sushi fermentation extract prepared in Example 3 above.

First, a cream containing the non-fermented fermented extract was prepared. Cream contains 1% by weight of fermented Asahi extract, 0.5% by weight of 1,2-hexanediol, 0.5% by weight of Caprylyl glycol, 30% by weight of Carbomer, 8% by weight of Gycerin, 6% by weight of Caprylic / Capric Triglyceride, 2% by weight Butyrospermum Parkii Butter, 1% by weight Cetearyl olivate, 1% by weight Sorbitan olivate, 0.5% by weight C14-22 Alcohols, 0.5% by weight C12-20 Alkyl Glucoside, 1.5% by weight Glyceryl stearate, Polyglyceryl-10 Stearate 1 By weight, 4% by weight of Dicaprylyl carbonate, 0.45% by weight of Aiginne and the balance of water.

(1) Subjects

The subjects were adult women aged 40 to 55 years. The test product was used on the facial area according to the method of use, and the skin elasticity improvement effect was evaluated. The evaluation was carried out in accordance with the SOP of the Center, and the matters not specified in the Food and Drug Safety Guidelines were referred to the references. 20 adult women aged 40 to 55 years were selected for the subjects who satisfied the selection criteria and did not meet the exclusion criteria, and the test site was performed on the subject's face (eye area).

(2) Test method

The subjects were kept clean and dry in order to make the measurement conditions the same, and the skin was stabilized at the place where the temperature and humidity (22 ± 2 ℃, RH 40 ~ 60%) were maintained for at least 30 minutes. Skin elasticity was measured using a cutometer (MPA 580, Courage and Khazaka Electronic Co., Germany). The cutometer measures the extent to which the skin is inhaled into the probe by applying a negative pressure to the surface of the skin using an optical system that can pass through the skin. At this time, the used light measured the degree of elasticity by the ratio of the intensity of light that passes through the skin through infrared rays and the intensity of light that does not pass through the skin. At this time, the measurement coefficient of elasticity evaluation is an arbitrary unit (AU). Before the product was used (0 weeks, start date of the test), subjects were selected according to the exclusion criterion, and the skin condition of the subject was checked and then a cutometer was performed. The effect of the test product distributed 2 weeks after using the product was evaluated by using a skin irritation evaluation and a cutometer together with the subjects who used the product for 2 weeks according to the use method. The effect of the test product distributed 4 weeks after using the product was evaluated by using the skin irritation evaluation, the cutter and the questionnaire evaluation in the subjects who used for 4 weeks according to the use method.

(3) Assessment of skin irritation

It is recommended that the test product be free of adverse reactions such as erythema, edema, scaling, itching, stinging, burning, tightness, prickling, The presence or absence of the reaction was observed closely and the grade was displayed according to severity when the skin adverse reaction appeared and the test findings were prepared. If you can not attend the exam anymore even though it is not the day you visit, you have to write the "Attendance Disclaimer" attached with your signature.

(4) Analysis of results

Skin elasticity was measured using a cutometer (MPA 580, Courage and Khazaka Electronic Co., Germany) at a distance of 3 cm from the eye margin. Measurements were made before the product was used (0 weeks), 2 weeks after using the product, 4 weeks after using the product, and the measurement area was measured three times for each subject and the average value was used. R 2 (maximum roughness) and R 5 (arithmetic average roughness) were selected as the variables. The higher the value, the better the skin elasticity. At this time, the measurement coefficient is an arbitrary unit (AU). Statistical analysis was performed by Paired t-test method to compare the difference between before and after use of the product. Statistical analysis was performed using SPSS 18.0 software. All statistical results were considered statistically significant at 5% (p <0.05).

(5) Test results

The skin elasticity measurement results (unit: A.U.) are shown in Table 1 and FIG. 6, and the statistical analysis results are shown in Table 2.

R2 R5 Before use
(0 weeks) use
after 2 weeks use
After 4 weeks Before use
(0 weeks) use
after 2 weeks use
After 4 weeks Average 0.67403 0.69822 0.71349 0.39528 0.40114 0.40622 Standard Deviation 0.04193 0.03112 0.03226 0.04018 0.03547 0.03486

Statistical analysis results R2 R5 p-value After 2 weeks of product use 0.001 (p < 0.05) 0.041 (p < 0.05) After 4 weeks of use 0.000 (p < 0.05) 0.002 (p < 0.05)

As a result, R2 was 0.69822AU after 2 weeks of product usage and 0.71349AU after 4 weeks of product use at 0.67403AU before using the test product (0 weeks), and R5 was 0.3 weeks And 0.40622AU after 4 weeks of using the product, respectively. Compared with before use (0 weeks), both R2 and R5 showed statistically significant improvement in skin elasticity after 2 weeks and 4 weeks of use (p <0.05).

The skin elasticity change amount (unit: A.U.) is shown in Table 3, and the skin elasticity improvement ratio (%) is shown in Table 4 and FIG.

R2 R5 After 2 weeks of use -
Before use (0 weeks) After 4 weeks of use -
Before use (0 weeks) After 2 weeks of use -
Before use (0 weeks) After 4 weeks of use -
Before use (0 weeks) Average 0.02419 0.03946 0.00585 0.01094 Standard Deviation 0.02670 0.02980 0.01193 0.01378

R2 R5 use
after 2 weeks use
After 4 weeks use
after 2 weeks use
After 4 weeks Average 3.76534 6.05045 1.64058 2.96966 Standard Deviation 4.38132 4.89518 3.12418 3.71705

As a result, the cream containing non-fermented fermented extract was 3.76534% after 2 weeks of use of the product, 6.05045% after 4 weeks of using the product, 1.64058% after 2 weeks of using the product, After 4 weeks, the skin elasticity improvement rate was 2.96966%.

As a reference, during the course of the study, no skin adverse events were observed on the subjects' power test products as a result of the questionnaire survey. No skin adverse events were observed in the visual evaluation by the investigator after use of the test product.

&Lt; Example 6 >

The diabetic extract (before fermentation) and the non-fermented fermentation extract (after fermentation) prepared in Example 3 were subjected to a variety of skin topical change tests.

(1) Subjects

(1) Skin Microbiome Sample Collection

In order to select the skin with relatively little change from the external factors, the experiment was performed on the forehead, which is a region with a large amount of sebum among the skin of the face, and the jaw which is a region of the U zone with less sebum. Control samples were collected from the subjects' skin before using the fermented extracts of Asahi fermented extracts. Skin samples were collected by swab test method using sterilized water and gauze based on references. All work was carried out in a clean bench.

(2) Genomic DNA extraction and DNA amplification of skin microorganisms

Samples collected from the skin were centrifuged at 3,600 rpm for 10 minutes, and DNA was extracted from the pellet using FastDNA SPIN KIT (MP Biomedical, France). For amplification of the 16S ribosomal DNA gene of the extracted DNA, SEQ ID NO: 3: 5'-CCTACGGGAGGCAGCAG-3 'with attached 5'-CGCCCGGGGCGCGCCGGGGGCGGCGGGGGCGGGGGGGGGGCACGGGGGG-3'5'-ATTACCGCGGCTGCTGG-3') was used. 1 μl of DNA template (20 μg / ml), 1.0 μM forward primer and 1.0 μM reverse primer were added to 25 μl of GoTaq ^ⓡ Colorless Master Mix (Promega, USA) and the remaining volume was added with distilled water. Mu] l. PCR was performed using a Touch down PCR method, which amplifies the PCR product at a temperature of 0.5 ° C per cycle from 64 ° C to 50 ° C using C1000-Dual (Bio-Rad, USA) After treatment, it was stored at 4 ° C. The obtained PCR product was used for DGGE (Denaturing Gradient Gel Electrophoresis) analysis.

(3) Denaturing Gradient Gel Electrophoresis (DGGE)

For DGGE analysis, D-code system (Bio-rad, USA) was used. The polyacrylamide gel concentration used was 8% and a 40% polyacrylamide bis-solution 29: 1 (3.3% C) (Bio-Rad, USA) was made to have a concentration gradient of between 40% and 60% vertically. The denaturants used were 7M urea and 40% (w / v) Formamide (Sigma, USA). D-code system was filled with approximately 7 L of TAE buffer (20 mM Tris, 10 mM acetic acid, 0.5 mM EDTA, pH 8.0) and incubated with 2 × loading dye (0.05% bromophenol blue, 0.05% xylene cyanol, 70% The same amount of the product was mixed and electrophoresis was performed at 50 V for 800 minutes. The electrophoretic polyacrylamide gels were analyzed for specific bands after 15 minutes of staining in a solution of Red safe (Intron, Korea) (Fig. 8). Band intensity of DGGE results were histogram and quantified using GelCompar2 and Image lab Respectively.

(4) Analysis of the results of skin flora change

Gel Compar II software (ApplideMaths) was used for the analysis of DGGE results, and it was confirmed visually that Staphylococcus epidermidis , which is a skin benefit protein, was increased in five subjects' face (forehead and jaw) And Image Lab program.

As a result of analysis using Image lab program, Staphylococcus epidermidis , which is known as a beneficial bacteria among the skin lyophilized bacteria, was increased in the sample treated area (jaw, forehead) before and after treatment with the non-fermented fermented extract Respectively. As can be seen from Figs. 9 to 13, the result of subject 1 was 34.12%, jaw 104.62%, subject 2 had 67.80%, jaw 99.18%, subject 3 had 164.68%, 110.23%, subject 4 had forehead 112.20% %, Subject 5 results 34.36% forehead and 31.46% chin.

Further, the subject 1-5 in the case of the forehead bisuri fermentation extract-treated (control) as compared to, bisuri fermentation extract treatment after (treatment group) with a mean 82.63% to about 34.12 ~ 164.68% Stability Philo nose kusu epi deolmi disk (Staphylococcus epidermidis ) band density of the jaws was increased, and the average value of the jaws was increased by about 83.06% (31.43 ~ 110.23%) after the fermented extract treatment. Therefore, it was confirmed that all five subjects induced the increase of Staphylococcus epidermidis in the forehead and the jaw treated area during the treatment of the non-sushi fermentation extract.

Similarity of the DGGE band was also investigated using the Gel comparII program. As a result, the sample of the subject 1 showed 20 bands on the DGGE gel and the similarity before and after the treatment of the non-fermented fermented extract was 100%. The jaw samples showed 20 bands on DGGE gel, and the similarity was 90% before and after the treatment of non-fermented fermented extract. Sample 2 of the forehead sample showed 21 bands on DGGE gel and the similarity before and after the treatment of the non-fermented fermented extract was 95.23%. The jaw samples showed 26 bands on DGGE gel and the similarities before and after the treatment of non - fermented fermented extracts were 76.92%. The subject 's forehead sample showed 16 bands on DGGE gel, and the similarity was 87.5% before and after treatment with the non - fermented fermented extract. The jaw samples showed 33 bands on DGGE gel, and the similarities before and after the treatment of the non-fermented fermented extract were 90.90%. Subjects' 4 forehead samples showed 13 bands on DGGE gel, and the similarities before and after treatment with non-fermented fermented extract were 92.30%. The jaw samples showed 24 bands on DGGE gel, and the similarities were 91.66% before and after the treatment of non - fermented fermented extract. Sample 5 of the forehead sample showed 30 bands on DGGE gel, and similarity before and after the treatment of non-fermented fermented extract was 100%. The jaw samples showed 31 bands on DGGE gel, and the similarities before and after the treatment of non-fermented fermented extract were 83.87%.

DGGE bands and similarity of 5 samples showed that more band was observed in the jaw with an average of 20 in the forehead and 26.8 in the jaw, and similarity was 95% in the forehead and 86.37% in the jaw. Thus, it was confirmed that the non-sushi fermentation extract increased Staphylococcus epidermidis , which is a useful protein, without greatly impairing the growth of the fungi in the skin.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Korea Microorganism Conservation Center (KCCM) KCCM11950P 20161129

<110> GFC Co., Ltd.          Seoulcosmetics. Co., Ltd. <120> Lespedeza cuneata G. Don Fermented extract Using Lactobacillus          pentosus GFC-LP and Method for Preparing the Same <130> P17077 <160> 5 <170> KoPatentin 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Universal primer 27F <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Universal primer 1492R <400> 2 tacggytacc ttgttacgac tt 22 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> GC clamp <400> 3 cgcccggggc gcgccccggg cggggcgggg gcacgggggg 40 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Primer 341F <400> 4 cctacgggag gcagcag 17 <210> 5 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Primer 518R <400> 5 attaccgcgg ctgctgg 17

Claims (6)

The method of the present invention includes a step of inoculating a Lactobacillus pentosus- GFC LP strain (accession number: KCCM11950P) into an extract of Lespedeza cuneata G.Don and culturing, (EN) The present invention relates to a method for producing fermented Asahi extract having the ability to increase Staphylococcus epidermidis .
2. The fermented extract according to claim 1, wherein the Lactobacillus pentosus- GFC LP strain (accession number: KCCM11950P) has the ability to produce beta -glucosidase &Lt; / RTI &gt;
The method according to claim 1, wherein the culturing is performed at 20 to 37 ° C for 2 to 7 days.
A non-fermented fermented extract having the ability to enhance Staphylococcus epidermidis , which is a skin benefit bacterium, produced by the method of any one of claims 1 to 3.


delete A cosmetic composition for improving wrinkles and skin elasticity, which comprises the fermented extract of Asahi fermentation of claim 4 as an active ingredient.

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