KR20200018047A - Aspergillus oryzae GBE174 and method for preparing fermentated extract of ginseng berry using the same - Google Patents

Abstract

The present invention relates to a novel Aspergillus oryzae GBE174 strain (accession number: KCTC 13526BP), a fermented extract of ginseng berries using the strain, and a method for manufacturing the same. When the Aspergillus oryzae GBE174 strain (accession number: KCTC 13526BP) provided in the present invention is inoculated into an extract of ginseng berries and cultured, ginsenoside Re contained in the ginseng berry extract can be completely decomposed to be converted into ginsenosides Rg1 and F1, thus it is possible to simply obtain a fermented extract of ginseng berries with a significantly increased content of minor ginsenoside F1.

Description

Novel Aspergillus duck GBE174 strain and method for preparing ginseng fruit fermented extract using same {Aspergillus oryzae GBE174 and method for preparing fermentated extract of ginseng berry using the same}

The present invention relates to a novel Aspergillus oryzae GBE174 strain (KCTC 13526BP), ginsenoside bioconversion method contained in the ginseng fruit fermentation extract using the strain.

Ginseng is a perennial seminegative plant belonging to the genus Araliaceae, and its medicinal effect is widely used in oriental medicine. Ginseng begins to bloom in the third year or more, and fruit begins to grow in May, but farmers use the root of ginseng, so when the ginseng fruit begins to grow to promote root growth, only a part is left and the rest is discarded. . Ginseng fruit has more than 2 times more saponin content than ginseng root, and it has been reported to have ginsenoside content and composition different from ginseng root, and ginsenoside ingredient with superior pharmacological action is better than root. It is known to be more than 30 times.

To date, about 40 species of ginsenosides have been identified and isolated from ginseng, red ginseng, and ginseng fruit, including protopanaxadiol and ginsenosides containing ginsenosides Rb1, Rb2, Rc, and Rd. Re, Rg1 and the like can be divided into protopanaxatriol system. These ginsenosides are not absorbed in the human body as it is, but are decomposed by the intestinal microorganisms present in the human intestine after ingestion, and the metabolites are absorbed and have physiological activity. The typical ginsenoside Rb1, Rb2, Rc, and the like are metabolized to CK (compound K), and ginsenoside Re and Rg1, which are protoparanaxtriols, are metabolized to ginsenosides Rh1 and F1 to show various physiological activities. Major ginsenosides containing relatively high amounts in ginseng and ginseng fruit include Rb1, Rb2, Rc, and Re, and minor ginsenosides containing Rg3, Rh2, F1, and Compound K. These minor minor ginsenosides have been reported to be easily absorbed into the human body and have pharmacological effects such as antitumor, radical scavenging, neurotoxicity inhibition, and anti-inflammatory activity. Methods for converting to active ginsenosides, such as the treatment of is studied. (Korean Registered Patent Publication Nos. 10-1017378, 10-1240192, and 10-1718465)

More specifically, Korean Patent No. 10-2016-0014096 discloses ginsenosides comprising a process of mixing ginseng with one or more enzymes selected from the group consisting of cellulase, hemicellulase, lactase, betaglucosidase and naringinase. And to a method for processing ginseng fortified with aglycone and processed ginseng, wherein the cellulase, hemicellulase, lactase, betaglucosidase or naringinase enzyme derived from Aspergillus oryzae is ginseng. A process of bioconversion to ginsenoside F1 by mixing and treating the same is disclosed. Reacting to obtain a bioconversion reaction solution containing ginsenoside F1. It is disclosed about a ginsenoside F1 produced by the production method and the method of ginsenoside F1 Gene Using.

In addition, although studies to convert to active ginsenoside Rh1 or F1 using ginsenoside Re, one of the major ginsenosides, most of the methods for preparing Rh1 using enzymes or lactic acid bacteria, Although studies have been carried out to produce F1 using enzymes obtained from Penicillium aculeatum and Aspergillus sp ., It is necessary to purify the enzyme by reaction with compound (Re) or by strain culture. There is a hassle.

Accordingly, the present inventors have developed a strain having the activity of producing ginsenoside F1, Aspergillus oryzae GBE174 (Strain Accession No .: KCTC 13526BP), and when inoculated directly into ginseng fruit extract, It is confirmed that the ginseng fruit fermentation extract with increased content of ginsenoside F1 can be obtained more simply by completely decomposing ginsenoside Re included in ginseng fruit extract and converting it to ginsenoside Rg1 and F1. The invention has been completed.

It is an object of the present invention to provide a novel Aspergillus duck GBE174 strain (Strain Accession Number: KCTC 13526BP), the strain.

Another object of the present invention is to inoculate and cultivate the novel Aspergillus duck GBE174 strain (Bacterial Accession No .: KCTC 13526BP) in ginseng fruit extract to completely decompose the ginsenoside Re contained in the ginseng fruit extract ginsenoside It is to provide a ginseng fruit fermentation extract and a method of producing a ginsenoside F1 markedly enhanced by conversion to Rg1 and F1.

The present invention is a Aspergillus oryzae having a bioconversion ability to convert Ginsenoside Re contained in Ginseng berry extract to Ginsenoside Rg1 and Ginsenoside F1. ) GBE174 strain (Accession Number: KCTC 13526BP).

The strain produces the ginsenosides Rg1 and F1 in a weight ratio of 2: 1, and 70 to 75% of the ginsenosides Re is bioconverted to the ginsenosides Rg1 and F1. It is also characterized by including the 18S rRNA sequence of SEQ ID NO: 1.

In another aspect, the present invention is inoculated with Ginseng fruit extract Aspergillus oryzae GBE174 strain (strain accession number: KCTC 13526BP), and culturing; Ginsenoside (Gginsenoside) Provides a ginsenoside bioconversion manufacturing method comprising the step of converting the ginsenosides Rg1 and F1. In the converting step, the ginsenosides Rg1 and F1 are converted to a weight ratio of 2: 1.

In another aspect, the present invention provides a ginseng fruit fermented extract fermented by the method.

When inoculated with Aspergillus duck material GBE174 (Bacterial Accession No .: KCTC 13526BP) provided from the present invention in ginseng fruit extract, the ginsenoside Re included in the ginseng fruit extract is completely decomposed to ginsenoside Rg1 and By converting to F1, the ginseng fruit fermentation extract with a markedly enhanced content of minor ginsenoside F1 can be obtained.

1 is a TLC development result showing the ginsenoside conversion pattern of the various strains cultured using ginseng fruit extract. (C: control (prior ginseng fruit extract), S: standard (ginsenoside Re, Rg1, F1 and C_K), 1-7: each microbial culture, 8: Aspergillus duck GBE174 culture)
Figure 2 is a TLC result of the ginseng fruit fermented extract cultured for 7 days using Aspergillus duck GBE174 strain (strain accession number: KCTC 13526BP). (C: control (prior ginseng fruit extract), S: standard (ginsenoside Re, Rg1, F1 and C_K), GBE174: culture of Aspergillus duck GBE174)
Figure 3 is an HPLC result of ginseng fruit fermentation extract using Aspergillus duck GBE174 strain (strain accession number: KCTC 13526BP).
Figure 4 shows the difference in the content of ginsenosides Re, Rg1 and F1 contained in ginseng fruit fermentation extract using Aspergillus duck GBE174 strain (strain accession number: KCTC 13526BP).

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1 Isolation and Screening of Strains

Separation of Aspergillus strains from ginseng fruit extract was obtained by inoculating mixed microorganisms obtained from various fermented foods into a culture solution containing ginseng fruit extract and incubating for 20 days and then decomposing ginsenoside Re included in ginseng fruit extract through TLC. Microbial groups were selected. Selected microbial groups were plated on MRS agar and PDA medium, and each plate was incubated at 25 ° C. Strains grown on the plate were visually selected and observed, such as shape, color, transparency, and size. The selected strains were purely separated by passage 4-5 times in MRS agar and PDA medium. Purely isolated strains were cultured in ginseng fruit extract, respectively, to identify ginsenoside Re converting ginsenoside Re to F1, a fungal strain (GBE174), which was made of 20% Glycerol stock solution and stored at -80 ° C ultra low temperature freezer.

Example 2 Identification of Isolated Strains by 18S rRNA Gene Sequencing

The selected fungal strain (GBE174) was commissioned by Macrogen Co., Ltd. for 18S rRNA sequencing by streaking on PDA medium, and determined the phylogenetic location by confirming homology with Type strain using NCBI database for systematic analysis. .

As a result of 18S rRNA analysis (SEQ ID NO: 1) from the strain and analysis of the homology of the strain using the BLAST program, the strain was 99% homologous to the 18S rRNA gene with Aspergillus oryzae . It was confirmed to have. Therefore GBE174 strain Aspergillus duck is found to be a new strain belonging to the material (Aspergillus oryzae), Aspergillus duck material (Aspergillus oryzae) GBE174 (strain accession number: KCTC 13526BP) as named, and it Korea Research Institute of Bioscience and Biotechnology Microbial Resources It was entrusted to the center (KCTC) on May 10, 2018 and was given accession number KCTC 13526BP (see FIGS. 5 and 6).

<Example 3>

A portion of the GBE174 strain grown on a PDA (potato dextrose agar) plate was taken and directly inoculated in M9 medium containing 5% (w / v) ginseng fruit extract to proceed with fermentation. The medium containing the ginseng fruit extract inoculated with GBE174 was incubated at 200 rpm in a 24 ° C. incubator for 5 days. It was confirmed by TLC that ginsenosides Re was converted to ginsenosides Rg1 and F1 from the second day after inoculation. On the final 5th day, Re was almost disappeared and the contents of Rg1 and F1 were significantly increased.

After the culture was completed, the fermentation broth was centrifuged at 8000 rpm for 10 minutes to remove the cells, and then the fermented ginseng fruit extract was prepared through a filter.

<Example 4>

The fermented ginseng fruit extract prepared in Example 2 was lyophilized and concentrated, and the obtained concentrate was dissolved in 50% Methanol, filtered through a 0.2 μm membrane filter, and used as a sample for HPLC analysis. HPLC analysis was detected at UV 203 nm using a ZORBAX Eclipse Plus C18 column (5 μm, 4.6 × 150 mm). The mobile phase was separated at a flow rate of 1.0 ml / min using Acetonitrile (solvent A) and water (solvent B), and the mobile phase conditions are shown in Table 1 below.

Example 5

In order to measure the content of ginsenoside Re, Rg1 and F1 contained in ginseng fruit extract and ginseng fruit fermentation extract, standard compounds were dissolved at concentrations of 0.0625, 0.125, 0.25, 0.5 and 1 mg / ml, and HPLC was performed. Using the area corresponding to each concentration, the standard curve was drawn and the ginsenoside content of the sample before and after the culture was measured. As a result, it was confirmed that the content of ginsenoside Re disappeared completely after incubation, the content of ginsenoside Rg1 increased by about 5 times from 5.2 mg / g to 42.5 mg / g, and the content of ginsenoside F1 was 0.4. It was confirmed that the increase was more than about 50-fold from 2 mg / g to 21.6 mg / g (see Table 2).

The above description is merely illustrative of the present invention, and those skilled in the art to which the present invention pertains will be capable of various modifications without departing from the essential characteristics of the present invention. The examples are not intended to limit the invention but to explain, and the spirit and scope of the invention are not limited by these embodiments.

The scope of protection of the present invention should be interpreted by the following claims, and all the technologies within the scope equivalent thereto should be construed as being included in the scope of the present invention.

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Claims (7)

Aspergillus oryzae GBE174 strain with bioconverting ability to convert Gginsenoside Re from Ginseng berry extract to Ginsenoside Rg1 and Ginsenoside F1 (Accession Number: KCTC 13526BP)
The strain of claim 1, wherein the ginsenosides Rg1 and F1 are produced in a weight ratio of 2: 1.
According to claim 1, wherein the strain is characterized in that 70 to 75% by weight of the ginsenoside Re is bioconverted to ginsenoside Rg1 and F1
According to claim 1, wherein said strain is characterized in that it comprises a 18S rRNA sequence of SEQ ID NO: 1
Inoculating Ginseng Berry extract with Aspergillus oryzae GBE174 strain (Strain Accession Number: KCTC 13526BP) and culturing;
Ginsenoside (Gginsenoside) Reconversion of ginsenosides Rg1 and F1; ginsenoside bioconversion production method comprising the
The method according to claim 5, wherein the ginsenosides Rg1 and F1 are converted to a weight ratio of 2: 1 in the converting step.
Ginseng fruit fermented extract fermented by the manufacturing method according to claim 5

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