WO2017119795A1 - Novel lactobacillus casei gfc1 strain, fermented ginseng extract using strain, and preparation method thereof - Google Patents

Abstract

The present invention relates to a novel Lactobacillus casei GFC1 strain (deposit number: KFCC18333P), a fermented ginseng extract using the strain, and a preparation method thereof. When the novel Lactobacillus casei GFC1 strain (deposit number: KCTC18333P) provided from the present invention is inoculated into ginseng and cultured, a fermented ginseng extract, of which the content of ginsenoside Rg2 is remarkably increased, can be obtained, by converting ginsenoside Re, contained in ginseng and having low bioavailability, into ginsenoside Rg2 having high bioavailability. In addition, the fermented ginseng extract has excellent skin antioxidation, skin whitening, anti-inflammatory and wrinkle reducing effects, and can control skin troubles, caused by a distribution imbalance between skin flora, by changing the community of the skin flora, thereby having an excellent effect as a cosmetic composition when the fermented ginseng extract is applied to a cosmetic base as an active ingredient.

Description

Novel Lactobacillus casei GFC 1 strain, ginseng fermented extract using the strain and preparation method thereof

The present invention relates to a novel Lactobacillus casei GFC 1 strain, a ginseng fermented extract using the strain and a method for preparing the same.

Panax ginseng CA Meyer is a plant belonging to the genus Ogapi ginseng and has been used in Korea, China and Japan for more than 2,000 years, and has been used for the purpose of preventing disease and extending the lifespan. The effects of ginseng so far known are the effects on the central nervous system, anticarcinogenic effect, anticancer activity, immune function control effect, antidiabetic effect, liver function antifungal effect, cardiovascular disorder improvement, anti-arteriosclerosis, blood pressure control action, menopausal disorder improvement , Effects on osteoporosis, antistress action, anti-fatigue, antioxidant activity, anti-aging.

Ginseng contains more than 40 ginsenosides, which are glycosides, as well as non-saponin-based physiologically active substances, and contains various components useful for the human body such as polyacetylene, phenolic compounds, acid polysaccharides, peptides, and alkaloids.

Ginsenoside, a representative physiologically active ingredient of ginseng, refers to saponin contained in ginseng, and ginseng contains various kinds of ginsenosides. Ginsenoside is distributed evenly in the ground and underground parts of ginseng, especially ginsenoside content and composition is known according to the ginseng root (root), ginseng leaf, ginseng fruit.

Ginseng ginsenosides account for about 5% of ginseng dry weight, and Glucose, Arabinose, Xylose, and Rhamnose in Triterpenoid-type Dammarane skeletons (Rhamnose) is a neutral glycoside combined. Protopanaxadiol (PPD) family saponins for two (3, 12 and 12) depending on the number of -OH attached to the non-sugar portion, protopanaxtriol for three (3, 6, 12) (Protopanaxatriol; PPT) family is called saponin. Glucose, arabinose, xylose, rhamnose and the like are combined with -OH at the positions R1, R2 and R3 of the PPD and PPT. More than 30 species of ginsenosides have been reported from ginseng, but a substantial amount of ginsenosides were detected by extracting and analyzing ginseng, and Rb1, Rb2, Rc, Rd, Re and Rg1 accounted for more than 90% of the total saponin Saponin components are low in content.

Ginsenosides are not absorbed in the human body as they are, but are decomposed by microorganisms such as Bacteriodes, Lactobacillus, Fusobacterium, Eubacterium, and Provetella species present in the human intestine to absorb their metabolites. Ginsenoside Rb1 is sequentially converted to ginsenoside Rd, Compound K (hereinafter abbreviated as CKra) and protopanaxanadiol by intestinal microorganisms. PPD ginsenosides Rb1, Rb2, Rc and Rd are metabolized and absorbed by 20-O-ß-D-glucopyranosyl-20 (S) -protopanaxadiol (CK) in the intestine. PPT ginsenosides Re, Rf, Rg1 are metabolized in the intestine into Rh1, F1, Protopanaxatriol. Rb1, Rb2, Rc, Rd and the like are converted to 20 (R)-/ 20 (S) -ginsenoside Rg3 by intestinal microorganisms, and Rg3 is converted to Rh2 by intestinal microorganisms.

Recently, the active ingredients of ginseng are easily absorbed into the human body, and as a method of reinforcing the components present in ginseng in trace amounts, the method of fermenting ginseng using intestinal microorganisms or lactic acid bacteria or treating enzymes is used. Republic of Korea Patent Publication No. 10-1017378 relates to the production method of fermented ginseng, fermented red ginseng and suitable Bacillus subtilis new strain derived from Cheonggukjang, ginseng using Bacillus genus microorganism isolated from Cheonggukjang And a method for preparing fermented ginseng and fermented red ginseng, characterized by enhancing the content of ginsenoside Rd by fermenting red ginseng, and a technique related to Bacillus substilly new strain derived from Cheonggukjang.

In addition, the Republic of Korea Patent Publication No. 10-1240192 relates to the ginseng fermented extract for improving insulin secretion using Lactobacillus asidophilus K-59 strain, K-59 strain and a method for preparing the same, newly identified and identified Lactobacillus acidophilus K-59 (KFCC11467P) strain was used to convert ginsenosides Rg1, Rb1, Rg1 or Re into ginsenosides Rh1, Rg3, Rh2 or CK using the low bioavailability of ginseng. A method of switching is disclosed.

Therefore, the present inventors have developed a novel Lactobacillus microorganism, that is, Lactobacillus casei GFC 1 strain (Accession No .: KCTC18333P), which has the ability to produce Ginsenoside Rg2, and ginseng When inoculated in and then cultured to prepare a ginseng fermentation extract, the ginsenoside Re containing low bioavailability in ginseng is converted to Ginsenoside Rg2 with high bioavailability, Ginsenoside The present invention was completed by discovering that not only can significantly increase the content of Rg2 but also impart skin antioxidant, whitening, anti-inflammatory, wrinkle improvement and skin trouble control effects.

It is an object of the present invention to provide a novel Lactobacillus casei GFC 1 strain (Accession No .: KCTC18333P).

Another object of the present invention is to inoculate a novel Lactobacillus casei GFC 1 strain (Accession No .: KCTC18333P) into ginseng and incubate the low bioavailability Ginsenoside Re contained in ginseng. By converting into high availability Ginsenoside Rg2, it can not only significantly increase the content of Ginsenoside Rg2, but also impart skin antioxidant, whitening, anti-inflammatory, anti-wrinkle and skin trouble control effects. It is to provide a ginseng fermented extract and a preparation method thereof.

In order to achieve the above object, the present invention is a Lactobacillus casei GFC 1 strain ( trusted ) characterized in that it has a bioconversion capacity from Ginsenoside Re to Ginsenoside Rg2 No .: KCTC18333P).

In addition, the present invention is inoculated in ginseng, Lactobacillus casei GFC 1 strain (accession number: KCTC18333P) having a bioconversion ability from Ginsenoside Re (Ginsenoside) Rg2 in ginseng, cultured It provides a method for producing a ginseng fermented extract, comprising the step of.

In the present invention, the ginseng is characterized in that the geumpungjong.

In the present invention, the ginseng is characterized in that it is selected from the group comprising leaves, fruits, roots and outposts.

In the present invention, the strain is characterized in that the spawn culture in a medium to which ginseng saponin is added.

In the present invention, the culture is characterized in that it is carried out for 3 to 7 days at 27 ~ 37 ℃.

In the present invention, the ginseng fermented extract is characterized in that the content of ginsenoside (Ginsenoside) Rg2 is increased.

In addition, the present invention ginsenoside (Ginsenoside Re) ginseng fermented extracts fermented with Lactobacillus casei GFC 1 strain (accession number: KCTC18333P) having a bioconversion ability from Ginsenoside Rg2 to provide.

In the present invention, the ginseng fermented extract is characterized by having skin antioxidant, whitening, anti-inflammatory, wrinkle improvement and skin trouble control effect.

The present invention also provides a cosmetic composition comprising the ginseng fermented extract.

When inoculating and incubating a novel Lactobacillus casei GFC 1 strain (Accession No .: KCTC18333P) provided from the present invention to ginseng, the low bioavailability of Ginsenoside Re contained in ginseng The ginseng fermentation extract with significantly increased content of Ginsenoside Rg2 can be obtained by converting into high availability Ginsenoside Rg2.

In addition, the ginseng fermented extract is not only excellent in skin antioxidant, whitening, anti-inflammatory and anti-wrinkle effect, but also by changing the population of the skin flora, it can adjust the skin trouble due to the imbalance distribution between the skin flora, When the ginseng fermented extract is applied to the cosmetic base as an active ingredient, the effect as a cosmetic composition is excellent.

1 is a TLC development result showing the saponin conversion pattern of Lactobacillus casei GFC 1 strain (Accession Number: KCTC18333P). (S: standard, C: control, 1: 1day, 3: 3day, 5: 5day, 7: 7day)

Figure 2 is a graph showing the cell viability (Cell viability) results of the ginseng fermented extract obtained from Example 1 of the present invention.

3 is a graph showing the results of DPPH (2,2-Diphenyl-1-picrylhydrazyl) scavenging activity of the ginseng fermentation extract obtained from Example 1 of the present invention.

Figure 4 is a graph showing the results of tyrosinase enzyme inhibitory activity of the ginseng fermented extract obtained from Example 1 of the present invention.

5 is a graph showing the results of tyrosinase enzyme inhibitory activity of the ginseng fermented extract obtained from Comparative Example 1 of the present invention.

6 is a graph showing the results of the elastase inhibitory activity of the ginseng fermented extract obtained from Example 1 of the present invention.

7 is a graph showing the NO (Nitric Oxide) inhibitory activity results of the ginseng fermented extract obtained from Example 1 of the present invention.

8 is a result of confirming through the DNA sequencing the changes in the dermatological bacteria before and after the application of the ginseng fermented extract obtained from Example 1 to the skin.

The inventors of the present invention have attempted to develop a new strain that can not only significantly increase the content of Ginsenoside Rg2, but also has excellent efficacy as a skin cosmetic composition such as skin antioxidant, whitening, anti-inflammatory, wrinkle improvement and skin trouble control effect. .

Therefore, in the present invention, as a result of separating a new strain specifically grown in ginseng to develop a new strain having the ability to produce Ginsenoside Rg2, Lactobacillus casei GFC 1 strain is a living body It was confirmed that the low availability Ginsenoside Re can be converted to the high bioavailability Ginsenoside Rg2 to significantly enhance the content of Ginsenoside Rg2.

The species and genus were identified by 16S rDNA partial sequencing identifying the strain in terms of molecular genetics, and the newly isolated Lactobacillus genus microorganisms were identified as Lactobacillus casei GFC 1 strain (Accession No .: KCTC18333P) and it was deposited on October 21, 2014, Korea Research Institute of Bioscience and Biotechnology, and was given accession number KCTC18333P.

 In addition, the ginseng fermented extract obtained by inoculating and cultivating the strain in ginseng was confirmed that it can give excellent skin antioxidant, whitening, anti-inflammatory, wrinkle improvement and skin trouble control effect and completed the present invention.

Accordingly, the present invention relates to a novel Lactobacillus casei GFC 1 strain (accession number: KCTC18333P) (hereinafter abbreviated as GFC 1) capable of fermenting ginseng.

The GFC 1 strain newly identified by the present invention has a bioconversion ability to bioconvert Ginsenoside Re having low bioavailability contained in Ginseng to Ginsenoside Rg2 having high bioavailability.

As described above, GFC 1 strains hydrolyze sugars of low bioavailability Ginsenoside Re contained in ginseng to bioconvert into high bioavailability Ginsenoside Rg1 and Rg2, and ginsenosides. Ginsenoside Rg1 is further bioconverted to Ginsenoside F1. Preferably, the GFC 1 strain may enhance the content of Ginsenoside Rg2 by bioconverting Ginsenoside Re into Rg2.

In addition, the present invention provides a method for producing a ginseng fermented extract comprising the step of inoculating GFC 1 strain into ginseng, and culturing. "Ginseng fermented extract" described above means a product obtained by inoculating GFC 1 on ginseng and culturing.

According to a preferred embodiment of the present invention, the ginseng used in the production of the ginseng fermentation extract is not particularly limited in variety, parts, but it is preferable to use the ginseng varieties selected from the group containing Geumpung, as a part of ginseng May be selected from the group containing leaves, berries, roots and outposts, but using the fruit of ginseng can significantly increase the content of Ginsenoside Rg2 compared to other parts. More effective. In addition, the form of ginseng may be used in various forms such as ginseng extract, ginseng powder, ginseng concentrate and is not particularly limited.

According to a preferred embodiment of the present invention, the GFC 1 strain may be seed cultured in a medium to which ginseng saponin is added. When culturing the GFC 1 strain, the addition of 1 to 2% by weight of ginseng saponin to the total amount of MRS medium can induce the activity of the GFC 1 strain, so the bioavailability of Ginsenoside Re with low bioavailability is low. The bioconversion activity to this high Ginsenoside Rg2 is more effective. At this time, when the content of ginseng saponin is less than 1% by weight based on the total amount of MRS medium, it is not preferable because sufficient strain cannot be induced, and when it exceeds 2% by weight, sufficient efficacy is not required even if it contains more than 2% by weight. It is uneconomical because it can be represented.

According to a preferred embodiment of the present invention, the culture is preferably performed for 3 to 7 days at 27 ~ 37 ℃ because it can further increase the content of Ginsenoside Rg2. In addition, Yeast extract and Lactose may be additionally added to facilitate the growth of the strain and increase the activity of the enzyme.

The ginseng fermented extract obtained by inoculating GFC 1 strain into ginseng and culturing as described above is subjected to centrifugation and sterilization, and the bactericidal strain is used to prevent destruction of the active ingredient by breeding in the future and stability of the product. desirable.

As described above, the ginseng fermented extract prepared by inoculating and culturing GFC 1 strain into ginseng is a low bioavailability ginsenoside Re contained in ginseng (Ginsenoside Re) high bioavailability ginsenoside ( Conversion to Ginsenoside Rg2 can significantly enhance the content of Ginsenoside Rg2. In addition, the ginseng fermentation extract obtained through the fermentation process has excellent skin antioxidant, whitening, anti-inflammatory, wrinkle improvement and skin trouble control effect compared to ginseng extract that has not undergone the fermentation process, and can be applied as a cosmetic composition effective for skin beauty. .

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention to those skilled in the art. Will be self explanatory.

Isolation and Screening of Strains

Separation of lactic acid bacteria from ginseng was sterilized in ozone water and sealed and aged naturally for 3 months. The ginseng fermented product was diluted to 10 ⁰ ~ 10 ^-5 using sterile distilled water, and then 100 μl of MRS agar was incubated in a 30 ° C. incubator. The strains were selected by visual observation of shape, color, transparency, size, and external structure. The selected strains were streaking in MRS agar medium for four passages to purely isolate single colony. Purely isolated strains were made into 20% Glycerol stock solution and stored in -70 ℃ cryogenic freezer.

Next, in order to select strains with ß-glucosidase activity, Esculin-MRS agar medium containing Esculin was inoculated using Esculin agar method (Atlas, 1993) to observe the color change in the medium. The composition of Esculin-MRS agar medium is shown in Table 1 below.

Table 1 ingredient Amount (g / L) Pancreatic digest of casein 13.0 Sodium chloride 5 Yeast extract 5.0 Heart muscle, solids from infusion 20.0 Esulin 1.0 Ferric ammonium citrate 0.5 Agar 15.0

Esculin is separated into Glucose and Esculetin by ß-glucosidase, and Esculetin reacts with Ferric ammonium citrate to form Black complex around colony. Therefore, the strain forming the black complex around the colony was determined to have a ß-glucosidase activity, and selectively selected the GFC 1 strain having saponin converting ability through the reaction with Ginsenoside Re.

Identification of Isolated Strains by 16S rRNA Gene Sequencing

After extracting DNA from the selected GFC 1 strain, 16S rRNA gene sequencing was commissioned by Genotech Co., Ltd., and confirmed homology with Type strain using NCBI database for systematic analysis. 16S rRNA sequences of type strains were aligned using the BioEdit program (Hall, 1999) and the Clustal X program (Thompson et al., 1997) and tracking the evolution of the strains was carried out by Kimura two-parameter model (Kimura, 1983), and phylogenetic location was determined by the 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 (SEQ ID NO: 1), and the homology of the strain was analyzed using the BLAST program. As a result, the GFC 1 strain was isolated from the 16S rRNA gene with Lactobacillus casei ( Lactobacillus casei ). It was confirmed that it has 99% homology. Thus GFC 1 strain is Lactobacillus casei is found to be a new strain belonging to the (Lactobacillus casei), Lactobacillus casei (Lactobacillus casei) GFC 1 (accession number: KCTC18333P) as named, and it Korea Research Institute of Bioscience and Biotechnology Microbial Resource Center 2014 It was deposited on October 21, 1989 and was given accession number KCTC18333P.

The results of comparing the GFC 1 strain obtained from the 16S rRNA gene sequencing and the conventional Lactobacillus type strain are shown in Tables 2 and 3 below.

1: GFC 1 strain

2: Lactobacillus casei type stain (BL23)

3: Lactobacillus paracasei type stain (JCM1171)

4: Lactobacillus plantarum type stain (ATCC14917)

5: Lactobacillus parabrevis type stain (LMG11984)

TABLE 2 Characteristic One 2 3 4 5 Motility + + + + + escluin hydrolyzation + + - - + Tolerance of 10% red ginseng extract + - - - -

TABLE 3 Characteristic One 2 3 4 5 Ginsenoside Rg2 Generation + - - - - Generating Ginsenoside CK + (+) - - (+) Re, Rd Degradation Activity + (+) - - (+)

* (+): Weak effect

As a result, as shown in Tables 2 and 3, it was confirmed that the GFC 1 strains differed from the existing strains because the enzymes secreted by the strains were different and the environments in which the strains could grow were different. Unlike the existing Lactobacillus type strain, which was unable to produce Rg2, it was able to produce Ginsenoside Rg2, which was progressed by the activity of GFC 1 to decompose Ginsenoside Re. .

EXAMPLE

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1

GFC 1 strain was cultured and inoculated into ginseng and then fermented. In order to more efficiently ferment the strain, the strain was incubated with stirring at 37 ° C. for 3 days through the seed culture. At the time of spawn culture, 1% of ginseng saponin isolated from ginseng root was added to MRS medium to induce strain activity, and a medium was prepared. The grown spawn was inoculated directly into ginseng to produce fermented extract.

Next, 100 ml of water was added to 5 g of the fruit powder of Geumpungjong among the varieties of Panax ginseng CA Meyer and sterilized at 85 ° C. for 60 minutes, and then inoculated with GFC 1 strain. At this time, 5g ~ 10g and Lactose 0.5g ~ 10g Yeast extract was added to make the growth of the strain more smoothly, and increase the enzyme activity. The fermentation culture made through incubation for 7 days at 37 ℃ 8000rpm, centrifuged for 20 minutes to remove the cells, and then through the filter and sterilized ginseng fermented extract was prepared.

Example 2

Ginseng fermented extract was prepared in the same manner as in Example 1, using leaves instead of Geumpung fruit of Panax ginseng CA Meyer .

Example 3

The ginseng fermented extract was prepared in the same manner as in Example 1, using the root instead of the Geumpung fruit of Panax ginseng CA Meyer .

Example 4

The ginseng fermented extract was prepared in the same manner as in Example 1, using root + leaf and fruit (1: 1) instead of Geumpung fruit of Panax ginseng CA Meyer .

<Comparative Examples 1 to 3> Preparation of ginseng extract

Comparative Example 1

100 g of water was added to 5 g of Geumpung fruit powder of Panax ginseng CA Meyer and extracted at 85 ° C. for 3 hours to prepare ginseng extract.

Comparative Example 2

Ginseng extract was prepared in the same manner as in Comparative Example 1 using leaves instead of Geumpung fruit of Panax ginseng CA Meyer .

Comparative Example 3

Ginseng extract was prepared in the same manner as in Comparative Example 1 by using the root instead of the Geumpung fruit of Panax ginseng CA Meyer .

Test Example 1 Ginsenoside Content Analysis (HPLC; High Performance Liquid Chromatography)

5 g of saturated butanol was added to 1 g of ginseng fermentation extract and 1 ginseng extract prepared from Examples 1 to 4 and Comparative Examples 1 to 3, followed by mixing. Then, the supernatant of the butanol mixture was taken and concentrated under reduced pressure. The obtained concentrate was dissolved in Methanol (HPLC grade), filtered through a 0.2 μm Membrane filter and used as a sample for HPLC analysis. HPLC analysis was performed at 203 nm using a C18 column (3.0 × 50 mm, ID 5). The mobile phase was given gradient using Acetonitrile (solvent A) and water (solvent B) and separated at 1.6 ㎖ / min flow rate.

Table 4 Time (min) Mobile phase acetonitrile (solvent A) water (solvent B) 0.00 17 83 6.00 17 83 9.00 23 77 16.5 23.5 76.5 19.00 31 69 29.00 45 55 31.00 47 53 33.00 90 10 34.00 90 10 35.00 17 83 37.00 17 83

The results for the ginsenoside component changes of Examples 1 to 4 and Comparative Examples 1 to 3 are shown in Table 5.

Table 5 Ginseng area Re (mg) Rg1 (mg) Rg2 (mg) Example 1 (Ginseng Fermented Extract) Fruit 5.84 6.32 10.22 Example 2 (Ginseng Fermented Extract) leaf 3.51 2.14 6.21 Example 3 (Ginseng Fermented Extract) Root 0.42 0.08 1.41 Example 4 (Ginseng Fermented Extract) Root + Leaf, Fruit (1: 1) 4.21 0.79 6.93 Comparative Example 1 (Ginseng Extract) Fruit 32.45 3.34 1.08 Comparative Example 2 (Ginseng Extract) leaf 12.11 15.23 0.78 Comparative Example 3 (Ginseng Extract) Root 2.21 1.34 0.1

As a result, as shown in Table 5, the ginsenoside component changes of Examples 1 to 4 and Comparative Examples 1 to 3 were analyzed for each ginseng portion, and the ginseng fermented extracts of Examples 1 to 4 were compared. It was confirmed that the content of ginsenoside (Ginsenoside) Rg2 is significantly higher than the ginseng extract of 1 to 3. This indicates that when ginseng fermented ginseng using the new GFC 1 strain, the content of Ginsenoside Re and Rg1 decreased and the content of Ginsenoside Rg2 increased. It is shown that Ginsenoside Re, Rg1 is converted to Ginsenoside Rg2 due to the GFC 1 strain.

In addition, as a result of comparing the Examples 1 to 4 fermented ginseng by parts, it was confirmed that the highest content of ginsenoside (Ginsenoside) Rg2 when using the fruit of the ginseng site.

Test Example 2 Analysis of Ginsenoside Rg2 Formation Capacity

As a result of ginsenoside analysis of Examples 1 to 4 and Comparative Examples 1 to 3, a mixed solution of 5 g of saturated butanol was added to 1 g of ginseng fermented extract obtained by the method of Example 1, in which the content of Rg2 was significantly improved. Silica gel 60 F254 TLC plate (Merck, Germany) was instilled and then developed using a mixed solvent of Chloroform / methanol / water (65:35:10 v / v / v, lower phase). The developed TLC plate was sprayed with 10% H ₂ SO ₄ and heated to confirm the saponin conversion.

As a result, as can be seen in Figure 1 as time passes, the content of Ginsenoside Re is reduced and the content of Ginsenoside Rg2 is increased, which means that the Ginsenoside Re is It shows the conversion to Ginsenoside Rg2. (S: standard, C: control, 1: 1day, 3: 3day, 5: 5day, 7: 7day)

Test Example 3 Cytotoxicity Test

The cytotoxicity of the ginseng fruit fermented extract obtained by the method of Example 1, in which the content of Ginsenoside Rg2 was significantly improved as a result of Ginsenoside analysis of Examples 1 to 4 and Comparative Examples 1 to 3 Was measured.

In order to determine the cytotoxicity of the extract according to the present invention, specifically, human keratinocyte HaCat (ACTT, CLS 300493, USA) using a hemacytometer (Hemacytometer) 1.5 x 10 ⁴ cells in a 96well plate After counting the same / well each, and incubated for 24 hours at 37 ℃, 5% carbon dioxide conditions. After culturing for 24 hours, the cell culture obtained was removed, and the ginseng fruit fermented extract obtained in Example 1 was prepared at concentrations of 0, 0.125, 0.25, 0.5, 1, 2, and 4%. 4 μl of each concentration was mixed and mixed with DMEM medium, and then treated by each well concentration. This was incubated for 24 hours at 37 ℃, 5% carbon dioxide conditions. Then, 100 μl of 10% WST (high sensitive Water Soluble Tetrazolium salt) per well was added and incubated for 2 hours under the same conditions. After 2 hours, absorbance at 450 nm was measured using an ELISA reader (Thermo, Multiskan EX). In addition, absorbance was measured using distilled water instead of the sample as a control.

The degree of cell activity can be represented by Equation 1 below, and the results are shown in Table 6 and FIG. 2.

[Equation 1]

% Cell activity = (absorbance of experimental group / absorbance of control group) X 100

Table 6 sample Example 1 (Ginseng fruit fermented extract) density(%) 0 0.125 0.25 0.5 One 2 4 Cell viability (%) 100 102.11 99.25 99.57 95.38 96.24 96.73

As a result, as shown in Table 6 and FIG. 2, the use concentration of the ginseng fruit fermentation extract showed no cytotoxicity even at the highest concentration of 4%, confirming that it can be used as a safe material without toxicity.

<Test Example 4> antioxidant efficacy verification test: DPPH radical scavenging activity

As a result of Ginsenoside analysis of Examples 1 to 4 and Comparative Examples 1 to 3, DPPH (2,2-Diphenyl-) of the ginseng fruit fermented extract obtained by the method of Example 1, in which the content of Rg2 was significantly enhanced. 1-picrylhydrazyl) scavenging activity was measured.

Free radicals generally produce excessive amounts of free radicals against external stimuli, destroying the skin's moisturizing layer and drying the skin, and acting as a pathological factor showing an immune response sensitive to external stimuli. Antioxidant activity of the ginseng fruit fermented extract according to the present invention can confirm the free radical scavenging ability through the DPPH experiment.

In order to examine the antioxidant effect on the extract according to the present invention, specifically prepared by diluting the ginseng fermented extract obtained in Example 1 to various concentrations, these samples and 100uM DPPH (1,1-diphenyl-2 -picrylhydrazyl) solution was mixed and reacted at 4 ° C. for 30 minutes, and then placed in a 96well plate to measure DPPH amount at 520 nm. In addition, the free radical scavenging activity (%) of the sample was calculated by using Equation 2 below when the sample was not added. The degree of antioxidant activity by DPPH free radical scavenging assay was expressed by Equation 2 and the results of antioxidant activity are shown in Table 7 and FIG. 3.

[Equation 2]

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

TABLE 7 sample Example 1 (Ginseng fruit fermented extract) density(%) 0 0.0625 0.125 0.25 0.5 One 2 4 free radical scavenging (%) 0 3.55 15.42 22.60 41.83 60.54 86.09 93.22

As a result, as shown in Table 7 and Figure 3, when treated with the concentration of Example 1, the ginseng fruit fermented extract showed a significant antioxidant activity by showing significant DPPH scavenging activity.

<Test Example 5> Whitening efficacy verification test: Tyrosinase inhibitory activity

As a result of Ginsenoside analysis of the Examples 1 to 4 and Comparative Examples 1 to 3, the ginseng fruit fermented extract obtained by the method of Example 1, in which the content of Rg2 was significantly enhanced, was obtained by the method of Comparative Example 1. Tyrosinase inhibitory effect was measured in comparison with the extract.

 Tyrosinase inhibitory activity was measured by colorimetry (Pomerantz et al. 1966) of DOPA chrome produced as a result of the action of tyrosinase. After dispensing 140 μl of 0.1 M sodium phosphate buffer (pH 7.0), 20 μl of the sample was added thereto. Thereafter, 20 µl of 1,500 U / ml of Tyrosinase from Mushroom was added and mixed, followed by reaction at 37 ° C. for 6 minutes. After adding 20 μl of 0.06 mM L-DOPA, the reaction was carried out at 37 ° C. for 5 minutes, the reaction was stopped by standing on ice, and the absorbance was measured at 475 nm. In addition, for comparison of test results, albumin (arbumin) at a concentration of 10 uM as a whitening ingredient was used as a positive control group.

The results for measuring tyrosinase inhibitory activity are shown in Tables 8 and 9 and FIGS. 4 and 5.

Table 8 sample Arbumin control Example 1 (Ginseng fruit fermented extract) Concentration (%) 10 uM 100 0.1 0.2 0.5 One Tyrosinase Activity (%) 31 100 95 83 69 58

Table 9 sample Arbumin control Comparative Example 1 (Ginseng Fruit Extract) Concentration (%) 10 uM 100 0.1 0.2 0.5 One Tyrosinase Activity (%) 32 100 92 91 81 79

As a result, as shown in Tables 8, 9 and 4 and 5, Example 1, which is the ginseng fruit fermentation extract and Comparative Example 1, which was not fermented ginseng fruit extract, had a concentration-dependent tyrosinase activity. It was found that the effect was shown by suppressing, and the efficacy also showed a significant result.

However, the ginseng fruit fermented extract prepared in Example 1 was found to have a superior whitening effect due to a higher tyrosinase inhibition rate compared to the ginseng fruit extract prepared in Comparative Example 1.

<Test Example 6> Wrinkle improvement efficacy verification test: elastase inhibitory activity

As a result of Ginsenoside analysis of Examples 1 to 4 and Comparative Examples 1 to 3, the elastase inhibitory activity of the ginseng fruit fermented extract obtained by the method of Example 1, in which the content of Rg2 was significantly increased. Was measured.

Elastase is an enzyme that breaks down elastin, an important substrate for maintaining skin elasticity in the dermis. Elastase is also a nonspecific hydrolase that can degrade collagen, another important substrate protein. Therefore, an elastase inhibitor has an effect of improving skin wrinkles, and ursolic acid is used as an elastase inhibitor. Elastase is known as an enzyme that causes wrinkles by breaking down the elastin (Elastin), which is an insoluble elastic fiber protein of animal connective tissue, to break the network structure of the skin's dermis. In the dermal tissue of the skin, collagen and elastin related to the elasticity of the skin form a network structure. As the network structure is broken, elastin (Elastin) is decomposed by elasatase (Elastase). As the skin sags and wrinkles, endogenous skin aging occurs. Therefore, it is possible to suppress skin aging by lowering the activity of elastinase, an elastin degrading enzyme which is one of the main causes of skin aging.

The ginseng fruit fermentation extract obtained in Example 1 was diluted to various concentrations, 40 μl of the sample was taken in an E-tube, and N-succinyl- (Ala) 3-p- dissolved in 10 mM Tris-HCl buffer (pH 8.0). Nitroanilide (S4760, Sigma) Add 200 μl of the substrate at 1.6 mM concentration, and add 40 μl of 0.4 U / mL of Elastase from porcine pancreas Type IV (E0258, Sigma) dissolved in 10 mM Tris-HCl buffer (pH8.0). After reacting at 25 ° C. for 5 minutes, absorbance was measured at 410 nm. Elastase inhibitory activity was calculated using Equation 3 below as a control when no sample was added.

Wrinkle improvement efficacy results through the elastase inhibitory activity is shown in Table 10 and FIG.

[Equation 3]

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

Table 10 sample Example 1 (Ginseng fruit fermented extract) density(%) 0 0.0625 0.125 0.25 0.5 One 2 4 Elastase inhibition (%) 0 2.15 5.58 10.80 21.24 29.35 45.21 82.21

As a result, as shown in Table 10 and Figure 6 it was confirmed that the treatment of Example 1, the ginseng fruit fermentation extract according to the concentration showed a significant elastase (Elastase) inhibition rate has a skin wrinkle improvement effect.

<Test Example 7> Anti-inflammatory efficacy verification test: Nitric oxide (NO) inhibitory activity

As a result of Ginsenoside analysis of Examples 1 to 4 and Comparative Examples 1 to 3, Nitric oxide (NO) inhibitory activity of the ginseng fruit fermentation extract obtained by the method of Example 1, in which the content of Rg2 was remarkably improved Measured.

Nitric oxide (NO) is produced when L-arginine produces citrulline in the body, and it is known as a free radical that promotes oxidation of the body when excess production occurs. After activating LPS (Lipopolysaccharide), an inflammation-inducing factor, in RAW 264.7 cells to activate NO production, Nitric oxide expression was measured by Griess analysis. LPS is an important constituent of the outer membrane that surrounds the peptideglycans on the surface of Gram-negative bacteria and is known as an "inflammatory factor." It is known that this LPS is capable of synthesizing NO by inducing activating signals to macrophage through receptors on the surface of macrophage and affecting iNOs expression. This reaction converts monocyte form RAW 264.7 cells into macrophge form.

In order to examine the anti-inflammatory effect on the ginseng fermented extract according to the present invention, the raw 264.7 cells were counted equally by 2.0 x 10 ⁴ cell / well in a 24well plate using a hemacytometer, and then dispensed. Incubated for 24 hours at 37 ℃, 5% carbon dioxide conditions. After culturing for 24 hours, the cell culture solution was removed, and 40 μl of the sample prepared at 0, 0.125, 0.25, 0.5, 1, 2, and 4% of the ginseng fruit fermented extract obtained in Example 1 was taken for each concentration. After mixing with DMEM medium, it was treated for each well concentration. This was incubated for 24 hours at 37 ℃, 5% carbon dioxide conditions.

At this time, in order to express NO, LPS was also treated with 1 µg / ml. Then, 100 ml of the culture solution was taken in a 96 well plate, 50 μl of Griess reagent A and 50 μl of Griess reagent B were added thereto, and then reacted for 10 min. The absorbance was measured at 540 nm using an ELISA plate reader. In addition, sodium nitrite was measured as a standard, and the control group was treated with LPS and the ginseng fermented extract of Example 1, and the concentration of NO ₂ ^{− in} the experimental group was compared. Are shown in Table 11 and FIG.

Table 11 sample Example 1 (Ginseng fruit fermented extract) density(%) 0 0.125 0.25 0.5 One Nitric Oxideinhibition (%) 0 54.4 91.2 111.6 106.4

As a result, when the ginseng fermented extract obtained from the method of Example 1 was treated for each concentration, as shown in Table 11 and FIG. 7, it showed a significant NO (Nitric Oxide) inhibition rate, it was confirmed that the anti-inflammatory effect is very excellent. .

Test Example 8 Changes in Dermatitis

Each sample was prepared by mixing 10 g of ginseng fermented extract of Example 1 with 100 g of sterile water, and treating the sample on the face and hands for 5 hours at 5 ml for 3 hours and then applying the sample before and after DNA was extracted from the face and hands to prepare samples before use (Control) and two days after use, respectively. Next, the 16s rDNA region was amplified by PCR using 27F (SEQ ID NO: 2) and 1492R (SEQ ID NO: 3) universial primer, and then subjected to denaturing gradient gel electrophoresis (DGGE). The DGGE gel band DNA was recovered, eluted and PCR re-amplified and purified, followed by cloning using a Topo-Blunt vector (Thermo Fisher) to secure a single clone, followed by isolation of the insert DNA and sequencing to macrogen. Commissioned. Sequencing results were statistically analyzed using Gelcompar2 Program.

As can be seen in Figure 8, as a result of observing the change in microbial diversity on the face, before the use of Example 1, all the subjects showed a variety of microbial species rich and showed a variety of band patterns, but after using Example 1 The band pattern was simplified and the band pattern between subjects was changed to a very similar form.

This is not simply a change in species and a similar form, but a pattern in which species belonging to the acne-producing microorganism Propionibacterium is decreasing, and a pattern in which a balance of dermatophytes such as Coagulase-negative Staphylococci or Corynebacterium is maintained is maintained. Seemed.

As a result, it was confirmed that the diversity of species was high in the face before using Example 1, and the band pattern similarity between subjects was about 45%, but after the use, the pattern was similar and simplified about 83%. This affected the microbial change by using the ginseng fermentation extract of Example 1, it can be predicted that the change caused by the ginseng fermentation extract of Example 1.

In addition, as a result of confirming the sequence of the DGGE band, the band in which the intensity of the band was increased or further activated was identified as Staphylococcus and Corynebacterium , which are commonly found in the skin. Intensity was confirmed to be reduced, and the species belonging to Propionibacterium was found to be reduced. The use of Example 1 caused a change in the microbial community of the facial skin, which can be expected to have a positive effect on each other, it was predicted that the distribution of the skin flora can be controlled due to the imbalance.

As described above in detail specific parts of the present invention, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Since the ginseng fermented extract of the present invention not only has excellent skin antioxidant, whitening, anti-inflammatory and anti-wrinkle effect, but also changes the cluster of dermatophytes, it can control skin troubles due to the imbalance of the skin flora. When the ginseng fermented extract is applied to the cosmetic base as an active ingredient, the effect as a cosmetic composition is excellent.

[Revision 19.04.2017 under Rule 26]

Claims (10)

Lactobacillus casei GFC 1 strain (Accession Number: KCTC18333P) characterized by having a bioconversion capacity from Ginsenoside Re to Ginsenoside Rg2. Inoculating ginseng and Lactobacillus casei GFC 1 strain (accession number: KCTC18333P) having a bioconversion ability from Ginsenoside Re to Ginsenoside Rg2 (GC) Method of producing ginseng fermented extract, characterized in that. According to claim 2, wherein the ginseng is a method of producing a ginseng fermented extract, characterized in that the Geumpungjong. The method of claim 2, wherein the ginseng is selected from the group consisting of leaves, berries, roots and starches. The method according to claim 2, wherein the strain is cultured in a seed culture in a medium to which ginseng saponin is added. The method of claim 2, wherein the culturing is carried out for 3 to 7 days at 27 ~ 37 ℃ manufacturing method of fermented ginseng extract. The method of claim 2, wherein the ginseng fermented extract is a method for producing a ginseng fermented extract, characterized in that the content of Ginsenoside Rg2 is increased. A ginseng fermented extract fermented with Lactobacillus casei GFC 1 strain (Accession No .: KCTC18333P) having a bioconversion ability from Ginsenoside Re to Ginsenoside Rg2. The ginseng fermented extract according to claim 8, wherein the ginseng fermented extract has skin antioxidant, whitening, anti-inflammatory, anti-wrinkle and skin trouble control effects. Cosmetic composition comprising a ginseng fermented extract of claim 8.

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