KR101139508B1 - Fermented ginseng materials comprising bio-conversion compound K and manufacturing method, functional rice wine using the same - Google Patents

Abstract

The present invention relates to a ginseng fermentation product and a method for preparing the same, and a functional makgeolli using biocondensation of ginseng saponin to compound K to enhance the absorption of the saponin in the body, and more specifically, fermented ginseng using the probiotic Hwangkuk bacterium and The present invention relates to a ginseng fermentation product containing saponin metabolite bioconverted into compound K, which is a glycoside component of ginseng saponin decomposed during fermentation, and a method for preparing the same, and functional makgeolli using the same.

The ginseng fermentation product of the present invention is a saponin metabolite comprising a compound K (20-O-β-D-glucopyranosyl-20 (S) -protopanaxadiol) which is bioconverted with saponins of ginseng as a fermentation strain of H. pylori ( Aspergillus oryzae KCTC6292). It contains as an active ingredient.

Ginseng, Bioconversion, Compound K, Fermentation, Ginseng Saponins (Ginsengsides), Metabolites

Description

Fermented ginseng materials containing bio-converted compound 및 and method for preparing the same, and functional ginseng makgeolli using the same {Fermented ginseng materials comprising bio-conversion compound K and manufacturing method, functional rice wine using the same}

The present invention relates to a ginseng fermentation product and a method for preparing the same, and a functional makgeolli using biocondensation of ginseng saponin to compound K to enhance the absorption of the saponin in the body, and more specifically, fermented ginseng using the probiotic Hwangkuk bacterium and The present invention relates to a ginseng fermentation product containing saponin metabolite bioconverted into compound K, which is a glycoside component of ginseng saponin decomposed during fermentation, and a method for preparing the same, and functional makgeolli using the same.

Ginseng has been used as a traditional drug in Korea and China since ancient times (Kang BS. Et al., Boncho-Hak, 6th Ed., Seoul: Young-Lim Press, pp400-401, 1991). The Panax genus is one of the most important genus in Chinese medicine. It is one of the genus of about 120 plants distributed in East Asia and North America.

Recent scientific research shows that ginseng contains various bioactive substances such as saponins, polyacetylenes, polyphenolic compounds, organic acids, amino acids, peptides, polysaccharides, vitamins, and the like. The pharmacological action has been reported to be central nerve suppression, protein synthesis, immune function regulation, insulin-like action, detoxification, anti-cancer and anti-cancer activity.

Saponins are glycosides composed of glycosides and aglycones and are widely distributed in the plant family. Ginseng saponins are characterized by the fact that the non-sugar portion is a dammarane-based triterpene. It has been classified into protopanaxdiol and protopanaxtriol systems. To date, 38 ginsenoside chemical structures have been identified.

However, ginsenosides are linked to the polymer component and are not absorbed by the body without being degraded by the unique microorganisms that inhabit the human intestines after ingestion, and thus are ineffective. Therefore, it must be decomposed by the microorganisms living in the intestine to be absorbed by the human body in the form of small molecules.

Among these ginsenosides, ginsenosides Rb1, Rb2, Rc and Rd are metabolized by enterobacteriaceae and are bioconverted to compound K (compound K, IH-901), which has the greatest anticancer effect. Compound K (20-O-beta-D-glucopyranosyl-20 (S) -protopanaxadiol) is a metabolite of protopanaxadiol ginsenoside, which contains a variety of immunological pharmacological effects (Wakabayashi C). , 1998, Hasegawa H, 1995, Lee SJ, 1999), anti-allergic effects (Bae EA, Choo MK, 2003, Kim DH, 2003, Lee JH, 2006). Several studies have shown that some ginsenosides exhibit glucocorticoid-like activity, which directly or indirectly activates the glucocorticoid receptor (GR) (Lee YJ, 1997, Lee YN, 1996). Other studies have suggested a novel treatment for inflammatory diseases by harmonizing control between homeostasis and immune action by interactions between toll like receptor 4 (TLR4) and GR (Ogawa S, 2005).

Human excreta were sampled and tested for the hydrolytic ability of intestinal microorganisms to ginsenoside Rb1. 21% of the samples showed no degradability, and 70% of those containing microorganisms also degraded ginsenosides. It was confirmed that there was a difference in their ability to do so.

In addition, the composition of the intestinal microorganism is susceptible to the constitution, eating habits, taking antibiotics, and stress, so the ability to break down glycosides shows a remarkable individual difference. Therefore, there is a significant difference in the absorption of glycosides, which is known as one cause of individual differences in drug efficacy (Hasegawa H. et al., Planta Medica, 64, pp 436-440, 1997).

To date, studies have shown that ginsenosides in orally administered saponins are converted into metabolites that are metabolized by intestinal microorganisms, especially Prevotella. It turns out that oris is involved in this. These metabolic pathways depend on the intestinal microorganisms. Bifidobacterium K-103 converts ginsenoside Rc to compound K via ginsenoside Rd and Bifidobacterium K-506 to ginsenoside Mb to compound K.

Kim Jae-baek, a professor of Wonkwang University's pharmacy team, has also developed a yeast ginseng with significantly improved pharmacological components by fermenting 4 years old ginseng for 1 month at an appropriate temperature, pH and moisture. In addition, the world's first lactic acid bacterium fermented ginseng was developed by the team of Ilhwa Central Research Institute and Professor Kim Dong-hyun of Kyung Hee University's College of Pharmacy, confirming the cancer metastasis inhibitory ability and health promoting action of the metabolite of Ginsenoside IH-901 (compound K).

Therefore, the present inventors convert the glycoside form of ginseng into a glycoside form using a microbial probiotic that produces various kinds of degrading enzymes, thereby increasing the content of specific components, improving the intestinal effect, increasing digestive absorption rate, and increasing bioactivity or newly biosynthesizing. Expected metabolites. In particular, the hydrolysis and reducing ability of the fungus has been well known for a long time has been studied by the conversion of microorganisms in the field of steroids, alkaloids, antibiotics, the present invention was also expected to bioconversion of ginseng saponin component by Bacillus bacteria.

The present invention is a ginseng fermented product containing compound K which has excellent physiological activity, and particularly has an effect of anticancer and immunomodulation by bioconversion of the saponin component of ginseng to be absorbable in the body by using a probiotic, Hwang Kuk bacteria, and Its purpose is to provide a method of manufacturing the same, and a functional makgeolli using the same.

The ginseng fermentation product of the present invention for achieving the above object is a compound K (20-O-β-D-glucopyranosyl-20 (S) -protopanaxadiol, which is a bio-converted saponin of ginseng using as a fermentation strain Hwang Kyun ( Aspergillus oryzae KCTC6292) It is characterized by containing a saponin metabolite comprising a) as an active ingredient.

And the production method of the ginseng fermentation product of the present invention for achieving the above object is the addition step of adding ginseng to the medium; Aspergillus in the medium oryzae An inoculation step of inoculating KCTC6292; It characterized by comprising a culture step of culturing the sulfur bacteria and the saponin bioconversion into a saponin metabolite containing compound K (20-O-β-D-glucopyranosyl-20 (S) -protopanaxadiol).

The addition step is characterized in that 10 parts by weight of the ginseng is added to 100 parts by weight of the medium prepared by mixing potato starch, dextrose in distilled water.

The culturing step is characterized by incubating for 5 to 8 days by stirring at 120rpm at 30 ℃ while injecting air at 30 vvm.

And saponin metabolite of the present invention for achieving the above object is a Hwang Kuk ( Aspergillus oryzae It is characterized in that it comprises a compound K (20-O-β-D-glucopyranosyl-20 (S) -protopanaxadiol) obtained by separating and purifying from the ginseng fermentation cultured with KCTC6292) as a fermentation strain.

And functional ginseng Makgeolli of the present invention for achieving the above object is characterized by containing the saponin metabolite as an active ingredient.

As described above, according to the present invention, ginseng is fermented using commercially available probiotics, Hwang-gyun, and in this process, glycoside components of ginseng saponins are decomposed and converted into saponin metabolites, so that saponins contained in ginseng are easily absorbed by the body. To be possible. Therefore, by increasing the absorption of saponin in the body, it is possible to eliminate the difference in absorption and efficacy due to the presence or absence of intestinal microorganisms or the difference in hydrolytic capacity.

In addition, the present invention can provide an effective bioconversion method to compound K which is a saponin component excellent in physiological activity, it is possible to give high value of ginseng and diversify the ginseng product.

And ginseng Makgeolli containing saponin metabolite including Compound K has high physiological activity such as anticancer and immunomodulation in addition to the advantages of existing Makgeolli.

Hereinafter, a ginseng fermentation product containing a saponin metabolite bioconverted to compound K according to a preferred embodiment of the present invention will be described in detail with reference to the accompanying drawings.

The ginseng fermentation product according to an embodiment of the present invention is a sulfur bacteria ( Aspergillus) oryzae A saponin metabolite containing compound K (20-O-β-D-glucopyranosyl-20 (S) -protopanaxadiol) obtained by bioconversion of ginseng saponin using KCTC6292) as a fermentation strain is contained as an active ingredient.

The ginseng used as a raw material in the present invention is Panax ginseng, Panax quinquefolia, Panax notoginseng, Panax notifinseng, Panax japonica, Panax trifolia, Himalaya ginseng, Panax pseudoginseng, Vietnam Panax vietnamensis, such as Panax plants. The ginseng may be used in the form of ginseng washed with water, dried dried ginseng, steamed and dried red ginseng, rice ginseng after harvesting. In addition, the ginseng may be used in the form of powder or extract, of course.

The compound K (20-O-beta-D-glucopyranosyl-20 (S) -protopanaxadiol) is a saponin metabolite in which protopanaxadiol ginsenoside is bioconverted by metabolism of a fermentation strain. The ginseng fermentation product of the present invention may include other saponin metabolites in addition to the compound K.

In order to prepare the ginseng fermented product, an addition step of adding ginseng to the medium, an inoculation step of inoculating the medium with Aspergillus oryzae KCTC6292, and the saponin by culturing the bacterium, the compound K (20-O-β- Culture step of bioconversion to saponin metabolite including D-glucopyranosyl-20 (S) -protopanaxadiol). The medium used is a mixture of potato starch and dextrose in distilled water. The amount of ginseng added to the medium is 5 to 15 parts by weight of ginseng based on 100 parts by weight of the medium. Preferably 10 parts by weight is added. The culturing step is incubated at 30 ° C 120 rpm for 5 to 8 days.

As another example of the present invention, the ginseng may be added to the medium after ultra high pressure treatment in the above-described addition step. The ultra-high pressure treatment is performed by cutting the ginseng through a forming mold to correspond to the internal space of the vacuum chamber of the ultra-high pressure processor, and then applied for 30 to 60 minutes at a pressure of 600 to 1200 MPa in the vacuum chamber. By such ultra-high pressure treatment, the structure of saponin can be easily decomposed to increase the yield of saponin metabolites. In addition, it is possible to sterilize ginseng as a raw material through ultra-high pressure treatment to prevent harmful bacteria from being incorporated into the batch through ginseng.

The ultrahigh pressure processor has a vacuum chamber of a predetermined size that can accommodate an object to be subjected to a high pressure treatment, and injects ginseng into the vacuum chamber. At this time, after cutting the cut ginseng in a vacuum pack, it is preferable to fix the shape to be all or part of the inner shape of the vacuum chamber by using a molding mold corresponding to the shape of the vacuum chamber. For example, when the vacuum chamber is formed in a cylindrical shape, the ginseng formed by the forming mold has a cylindrical or semi-cylindrical or fan-shaped cross section having an outer diameter corresponding to the inner diameter of the vacuum chamber and overlapping each other to form a single cylindrical shape. It may be formed in the shape of a fan-shaped pillar having.

After molding into a specific shape by the mold, it is charged into the vacuum chamber and then the cover of the vacuum chamber is closed to seal the inside. Then, the compressed medium is supplied to the vacuum chamber to apply a pressure of 600 to 1200 MPa for 30 to 60 minutes. Pressure and processing time in the ultrahigh pressure treatment may be appropriately adjusted within the pressure and time range according to the dose of ginseng added. When the pressure is less than 600MPa during the ultrahigh pressure treatment, the ultrahigh pressure treatment efficiency decreases, such as a decrease in sterilization effect, and when the pressure exceeds 1200MPa, the ultrahigh pressure treatment efficiency does not increase significantly compared to the consumption of power for increasing the pressure. It is desirable to be within the scope. In addition, if the time is also shorter than the time range, sufficient ultra-high pressure treatment effect cannot be obtained, and if the time is exceeded, there is almost no synergistic effect. The ultra high pressure ginseng is added to the medium after grinding finely with a blender.

On the other hand, the functional ginseng Makgeolli of the present invention ( Aspergillus oryzae A saponin metabolite comprising compound K (20-O-β-D-glucopyranosyl-20 (S) -protopanaxadiol) obtained by separating and purifying from ginseng fermentation cultured using KCTC6292) as a fermentation strain is contained as an active ingredient.

Saponin metabolites can be added to any of the conventional processes for producing makgeolli. As an example of the manufacturing method of the functional ginseng makgeolli of the present invention by adding a saccharifying agent such as malt or koji to the cooked rice, the starch is saccharified with sugar to prepare a saccharified solution, and then fermented by adding a base liquor to the saccharified liquid. It is prepared by injecting it into a packaging container and packing it. After the preparation step, saponin metabolites are added. In addition, ginseng fermentation itself containing saponin metabolites instead of saponin metabolites can be added to the rice wine. Saponin metabolite or ginseng fermentation may be added in an amount of 0.1 to 5% by weight based on the whole makgeolli.

Hereinafter, the present invention will be described through experimental examples. However, the following experimental example is for describing the present invention in detail, and the scope of the present invention is not limited to the following experimental example.

Experimental Example 1: Bioconversion of Fresh Ginseng by Hwang Kyun

Six years old ginseng was purchased from the ginseng market in Bujeon Market, Busan, Korea, and was used as a raw material. A. oryzae KCTC6292, used as a probiotic, was distributed from Korea Research Institute of Bioscience and Biotechnology (KRIBB). After addition of 10 parts of fresh ginseng by weight relative to the medium 100 parts by weight After a culture by inoculating a seed culture a hwanggukgyun 10 parts by weight of A. oryzae It was confirmed that ginseng is converted by KCTC6292. The medium used was Potato Dextrose Broth (Difco, USA), which mixed 4 g of potato starch and 20 g of dextrose in 1 L of distilled water. Fresh ginseng fermented product was prepared.

In order to identify and examine a new bioconversion substance, saponin metabolites, BuOH extracts from fermented ginseng were concentrated and deposited on silica TLC plates and developed with C / M / W (65/35/10, downstairs), followed by anisaldehyde sulfuric acid. As a result of the color development by spraying, it was possible to observe the change of the component expected to be a new conversion material near the Rf value 0.9 of the fresh ginseng fermentation products (AO1, AO2) as shown in FIG.

In Figure 1, AO ^* shows the results of the culture of Rhodibacterial bacillus without added ginseng, AO1 is a ginseng fermented product, AO2 is a ginseng fermented product, G1 is a sterilized ginseng juice, G2 is not a sterilized ginseng juice.

As a result of re-establishing the presence of sterilization of the fresh ginseng and only the bacillus bacterium, no clear conversion material could be identified in the culture medium in which the ginseng was cultured without adding the fresh ginseng or in the sterilized or not sterilized ginseng juice. It is thought that the newly converted material is derived from fresh ginseng that was converted by the Kukki bacteria, not of the Kukki bacteria culture itself.

Experimental Example 2: Analysis of Bioconverters

In order to identify the converted material, a sample (fermented ginseng fermentation product) was placed at a position 5 mm from the bottom of the silica gel TLC plate (60F254, Merck, Germany), and the diameter of each droplet was minimized. The spot generated by the side sample was 0.5 cm or more apart so that the sample was not disturbed. The developing solvent of the sample was developed using the lower layer of chloroform / methanol / water (4/2/1, v / v / v). After development, it was sprayed with anisaldehyde sulfuric acid and warmed at 110 ° C. for about 10 minutes to develop color.

According to the concentration of the fresh ginseng fermented fraction to the separated water 1 mg / ml was injected by 5 ㎕, the mobile phase from 0 to 5 minutes, _{30% CH 3 CN, 5 ~} 40 minutes, _{30-80% CH 3 CN, 40 ~} 50 The flow rate was 0.5 ml / min at 80% CH ₃ CN, and YMC-Pack C18 column (ø6.0x150 mm, YMC Co., Japan) was used as the stationary phase. Compound K, Rh2, Rb1, and Rg3 were used as standards.

As shown in FIG. 2, Rb1, a protopanaxatriol ginsenoside of fresh ginseng, was identified at 24 minutes, and Rg3 was removed at 33 and 36 minutes by degrading one glucose from Rb1. In addition, in the case of Rh2 in which two glucoses were removed and removed in Rb1, and one glucose was removed in Rg3, it was confirmed at 43.5 minutes. On the other hand, in the case of compound K, which was decomposed by two glucoses, such as Rh2, as protopanaxadiol ginsenoside, it was confirmed at 43.0 minutes similar to Rh2.

In FIG. 3, 10 μl (50 μg), 5 μl (25 μg), and 1 μl (5 μg) of 5 mg / ml concentration of Compound K were injected to compare the content of Compound K, corresponding to compound K in a concentration-dependent manner. It can be seen that the area of the peak increases.

Experimental Example 3: Bioconversion of Fresh Ginseng According to Amount and Culture Period of Fresh Ginseng

In order to examine the production of the metabolite of Rf value 0.9 shown in FIG. 1, the experiment was conducted in the same manner as in Experimental Example 1, but the amount of cultured ginseng and the culture period were varied. The amount of H. erythrocytes was fixed, and the amount of ginseng was 5 parts by weight, 10 parts by weight and 20 parts by weight, respectively.

Figure 4 shows the change depending on the culture period according to the amount of added ginseng. The first picture is a picture of 5 parts by weight of fresh ginseng, the second picture is a picture of 10 parts by weight of fresh ginseng, and the third picture is a picture of 20 parts by weight of fresh ginseng.

As a result of shaking culture for 10 days, the conversion material began to be produced from the 3rd day and showed the maximum conversion at 5-8 days after the incubation. In particular, it was confirmed by TLC that the maximum conversion was made in the fresh ginseng added 10 parts by weight. To confirm the conversion, ginsenosides Rb1, Rg3, Rh2 and compound K, which are the major components of fresh ginseng or ginseng, were analyzed by HPLC, and the converted material was consistent with 43 minutes of the same retention time (RT) as compound K. It was confirmed that the compound K was contained in the metabolite converted by the bioconversion, which is a saponin component of fresh ginseng, by Hwang-gyun bacteria.

In order to mass-produce the converted material, 3 kg of fresh ginseng was pulverized with a mixer, sterilized with 30 L of potato dextrose broth medium, and 10 parts by weight of the spawned cultivated Rheum bacteria were inoculated at 30 ° C. and 120 ° C. in a 50 L fermenter (Biotron, Korea). Incubated at rpm for 5 days. The fermentor used was Biotron's Pilot 50L fermentor, and the pH of the initial culture was 4.91, but the pH was increased to pH 6.6 on the second day of culture, pH 6.96 on the fourth day of culture, and 7.67 on the fifth day of incubation (FIG. 5). During the incubation period, air was fixed at 30 vvm, and antifoaming agents (LS-303, Dow-corning, Korea) were added twice on the 1st and 4th days to prevent foaming by saponin. 2, 4, and 5 days of fermentation were recovered, MeOH extracted, and the recovered samples were compared by HPLC to compare the amount of converted compound K in the fermentation by date. As shown in FIG. It started to be confirmed that the maximum production on the fifth day

<Experimental Example 4: Isolation and purification of the conversion material>

The culture was centrifuged at 5,000 rpm in order to separate the new conversion material including compound K from the fermentation, and the precipitate was extracted with 30 L of MeOH for 2 days. Stepwise elution method to increase the ratio of chloroform and methanol in silica gel column filled with chloroform by extracting MeOH extract of fresh ginseng suspended in distilled water with ethylacetate (EtOAc) three times (chloroform ： methanol = 50 ： 1 → 10: 1, v / v) to separate WG1, WG2, and WG3. Among them, WG2 containing saponin metabolites was selected and subjected to secondary silica gel column chromatography, where chloroform / MeOH / water ( 65/35/10, downstair) was used as a developing solvent to separate the fractions of WG2-1 and WG2-2. The WG2-2 fraction was finally separated from the conversion material WG2-2-2 by RP-18 column chromatography using 80% MeOH as a developing solvent.

<Experimental Example 5: Identification of the transition material WG2-2-2>

WG2-2-2 isolated in Experimental Example 4 was estimated as a ginsenoside compound through HPLC, TLC analysis, UV absorption and color reaction. HPLC and TLC analysis of standard K of the compound K showed that WG2-2-2 was estimated to be the same as that of compound K. To confirm this, LR-MS was performed to compare the fragmented molecular ion peaks. 6 is shown.

Referring to FIG. 6, the molecular weight of the conventionally known compound K was 621, and thus the molecular ion peaks segmented at 135, 207, 341, and 425 could be confirmed, and the segmentation pattern of WG2-2-2 was the same as that of the compound K. It was confirmed that WG2-2-2 is a metabolite bioconverted to compound K.

<Experimental Example 6: Biological Activity Measurement of conversion material WG2-2-2>

In order to measure the physiological activity of WG2-2-2 isolated in Experimental Example 4, cytotoxicity against cancer cells known as physiological activity of ginseng was examined. To determine the cell toxicity on PC-3 cells, 1x104 cells / 250 μL were dispensed on a 48-well plate and the samples were collected at 12 μg / ml, 50 μg / ml, 25 μg / ml, 12.5 after 12 hours. Treated to μg / ml. After incubation for 48 hours at 37 ℃, 5% CO ₂ incubator, the supernatant was removed and the Triazolyl Blue Tetrazolium Bromide was added to 0.5 mg / ml, and left for 4 hours at 37 ℃. After confirming that the Fomazan crystals were formed in purple, DMSO was added to completely dissolve the crystals, and then, 100 μl was dispensed into the 96-well and measured by an ELISA reader having an absorbance of 540 nm.

Referring to FIG. 7, as a result of measuring cytotoxicity of PC-3 prostate cancer cells of the intermediate fraction, the finally isolated conversion material WG2-2-2 showed a strong cytotoxicity in a concentration-dependent manner against PC-3 cells. In addition, WG2-2-2, which was identified as Compound K, had stronger cytotoxic activity compared to WG2-2-1, and the results showed that the non-glycoside ginsenoside was more biologically active than the glycoside ginsenoside. -2-1 is believed to be a saponin metabolite, an intermediate metabolite that is converted to WG2-2-2 (Compound K).

In the above, the present invention has been described with reference to one embodiment, which is merely exemplary, and it will be understood by those skilled in the art that various modifications and equivalent embodiments are possible.

Therefore, the true scope of protection of the present invention should be defined only by the appended claims.

1 is a photograph of the culture solution of Experimental Example 1 by TLC analysis,

2 and 3 are graphs showing the culture of Experimental Example 2 analyzed by HPLC and quantified Compound K,

Figure 4 is a photograph of the culture solution of Experimental Example 3 with different contents and culture period of fresh ginseng by TLC,

5 is a graph analyzing the amount of converted compound K during the culture of fresh ginseng when cultured in large quantities in Experimental Example 3,

6 is a graph showing the LR-mass spectrum of the conversion material WG2-2-2 in Experimental Example 5,

7 is a graph showing cytotoxicity of PC-3 cancer cells of the converting material WG2-2-2.

Claims (6)

delete delete delete delete delete Saponin metabolite containing compound K (20-O-β-D-glucopyranosyl-20 (S) -protopanaxadiol) obtained by isolating and purifying from ginseng ferment cultured with Aspergillus oryzae KCTC6292 as a fermentation strain After the formation process of The ginseng fermentation product is an addition step of adding 10 parts by weight of ground ginseng pulverized by a mixer after ultra high pressure treatment with respect to 100 parts by weight of a medium prepared by mixing 1 g of distilled water with 4 g of potato starch and 20 g of dextrose; The inoculation step of inoculating the Kukki bacteria in the medium and: the culture step of culturing the Kukgi bacteria to bioconvert the saponin to a saponin metabolite containing the compound K; In the addition step, the ultra-high pressure treatment is performed by cutting the ginseng, packing it in a pack, and then charging it in a vacuum chamber and applying a pressure of 600 to 1200 MPa for 30 to 60 minutes. The culturing step of the functional ginseng makgeolli characterized in that the culture for 5 to 8 days by stirring at 120rpm at 30 ℃ while injecting air at 30 vvm.

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