KR20210065855A - Method for preparing compound K using red ginseng, enzymes and natural products - Google Patents

Abstract

The present invention relates to a method for manufacturing compound K, including a step of mixing a red ginseng extract, an enzyme, and a natural product extract. According to the present invention, it is confirmed that the amount of compound K produced according to the method of the present invention is significantly increased compared to when the enzyme is used alone, thereby being able to be usefully used in the manufacture of health functional foods related to red ginseng.

Description

Method for preparing compound K using red ginseng, enzymes and natural products {Method for preparing compound K using red ginseng, enzymes and natural products}

The present invention relates to a method for producing a compound K, including mixing red ginseng, an enzyme and a natural product extract.

Ginsenoside is a saponin contained in red ginseng. It contains Rg1, Rb1, and Rg3, so it is effective in enhancing immunity, stabilizing nerves, and inhibiting cancer prevention. However, natural red ginseng saponin has a problem of poor absorption.

In order for the main component of saponin to have an effect, it must be converted into 'compound K'. This compound K is a substance produced by removing sugar from saponin, an active ingredient of red ginseng, by intestinal microbes, and is not converted to compound K. Unsatisfactory ginseng saponins are excreted from the body. However, according to the Ministry of Food and Drug Safety data, which measured ginsenosides (saponin absorption rate of ginseng) by age and sex, it was confirmed that 25% of the test subjects did not fully absorb ginsenosides into the blood due to enzyme inactivation of intestinal microorganisms. . In addition, in natural red ginseng products, it takes about 10 hours or more for ginsenosides to be converted into compound K in the body and absorbed, and in the process, many parts are not absorbed and are discharged outside the body.

In this respect, the compound K manufacturing technology that has already completed external processing and converts the active ingredient of red ginseng into a form that can be absorbed regardless of an individual's metabolic function is very meaningful, but research on it is still insufficient.

Republic of Korea Patent Publication No. 10-2020-0121196

The inventors of the present invention completed the present invention by confirming that when red ginseng is treated with an enzyme and a natural product extract, the compound K yield is significantly increased compared to an enzyme alone treatment or a natural product alone treatment.

Accordingly, the present invention provides a method for producing a compound K comprising mixing a red ginseng extract, an enzyme and a natural product extract.

The present invention provides a method for preparing compound K comprising mixing a red ginseng extract, an enzyme and a natural product extract, wherein the enzyme is cellulose (Cellulase), beta-glucosidase (β-glucosidase) and hemicellulose (hemicellulose) ) is at least one selected from the group consisting of, and the natural product is at least one selected from the group consisting of shiitake mushrooms, almonds and pumpkin seeds.

In one embodiment of the present invention, the red ginseng extract may be an ethanol extract.

In one embodiment of the present invention, the ethanol may be an aqueous solution of 60 to 80% (v/v) ethanol.

In one embodiment of the present invention, the red ginseng extract may be extracted under 70 to 90 ℃ conditions.

In one embodiment of the present invention, the red ginseng extract may be extracted for 30 to 120 minutes.

In one embodiment of the present invention, the natural product extract may be included in an amount of 30 to 40% (v/v) with respect to the total volume of the red ginseng extract, the enzyme and the natural product extract.

In one embodiment of the present invention, the natural product extract may be a supernatant of the natural product extract.

In one embodiment of the present invention, the supernatant may be prepared by a method comprising the following steps.

a) grinding the natural product;

b) adding a solvent so that the pulverized product of step a) has a concentration of 0.01 to 1 g/ml;

c) immersing the resultant of step b) at 1 to 10° C. for 12 to 36 hours; and

d) obtaining a supernatant from the result of step c).

In one embodiment of the present invention, the solvent in step b) may be distilled water or a buffer solution.

In one embodiment of the present invention, the enzyme may include cellulose (Cellulase), beta-glucosidase (β-glucosidase) and hemicellulose (hemicellulose).

In one embodiment of the present invention, the natural product may include pumpkin seeds.

The present invention relates to a method for producing compound K, including a step of mixing a red ginseng extract, an enzyme and a natural product extract, and the method of the present invention confirms that the amount of compound K is significantly increased compared to when the enzyme is used alone, It is expected to be usefully used in the manufacture of health functional foods related to red ginseng.

1 shows the results of thin film chromatography to confirm the amount of compound K produced by each experimental condition.
2 is a graph showing the amount of compound K generated by experimental conditions.

The inventor of the present invention has completed the present invention by confirming that the yield of compound K is significantly increased compared to the enzyme alone treatment or the natural product alone treatment when the red ginseng extract is treated with the enzyme and the natural product extract.

Accordingly, the present invention provides a method for preparing compound K comprising mixing a red ginseng extract, an enzyme and a natural product extract, wherein the enzyme is cellulase, beta-glucosidase, and hemicellulose ( hemicellulose), and the natural product is at least one selected from the group consisting of shiitake mushrooms, almonds and pumpkin seeds.

In the present invention, compound K (Compound K) is a final metabolite and physiologically active substance of ginsenoside and is represented by the following formula (1). Compound K is known to be difficult to manufacture in large quantities because it is quite difficult to manufacture.

[Formula 1]

In the present invention, the red ginseng is a concept including both unprocessed red ginseng itself or those obtained by physically or chemically processing red ginseng.

In the present invention, an extract is a substance obtained by extracting a specific substance. For example, the extract refers to a result such as a liquid component obtained by immersing red ginseng or natural product in a solvent and then extracting it for a certain period of time at low temperature, room temperature or warming state, and a solid obtained by removing the solvent from the liquid component However, the present invention is not limited thereto, and may include an extract, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or all of these crude products or purified products. The solvent may be one or more solvents selected from the group consisting of water, C ₁ to C ₄ alcohols, hexane, and mixtures thereof, but is not limited thereto.

In the present invention, the red ginseng extract may be an ethanol extract.

In the present invention, the ethanol may be 60 to 80% (v/v) aqueous ethanol solution, preferably 70% (v/v) ethanol aqueous solution.

In the present invention, the red ginseng extract may be extracted under 70 to 90 ℃ conditions, preferably at 72, 75 or 85 ℃, more preferably 72 ℃ conditions may be extracted.

In the present invention, the red ginseng extract may be extracted for 30 to 120 minutes, preferably for 60 minutes.

In the present invention, the enzyme may be one or more enzymes selected from the group consisting of cellulase, beta-glucosidase, and hemicellulose, preferably cellulase, It may be a beta-glucosidase (β-glucosidase) and hemicellulase (hemicellulase) mixed enzyme. There is no limitation on the formulation of the enzyme, but it may preferably be a solid, powder or liquid enzyme.

In the present invention, the natural product may be at least one selected from the group consisting of shiitake mushrooms, almonds and pumpkin seeds, and preferably pumpkin seeds. The shiitake mushrooms, almonds and pumpkin seeds may be raw materials or a physical or chemical processing thereof.

In the present invention, the natural product extract may be included in an amount of 30 to 40% (v/v), preferably 35% (v/v), based on the total volume of the red ginseng extract, enzyme and natural product extract.

In the present invention, the natural product extract may be a supernatant of the natural product extract.

In the present invention, the supernatant may be prepared by a method comprising the following steps:

a) grinding the natural product;

b) adding a solvent so that the pulverized product of step a) has a concentration of 0.01 to 1 g/ml;

c) immersing the resultant of step b) at 1 to 10° C. for 12 to 36 hours; and

d) obtaining a supernatant from the result of step c).

Preferably, the concentration of the pulverized product in step b) may be 0.1 g/ml, and the immersion in step c) may be performed at 4° C. for 24 hours.

In the present invention, the solvent in step b) may be distilled water or a buffer solution. There is no limitation on the pH of the solvent, but as an example, it may be pH 4 to 10, preferably pH 5 to 9, more preferably pH 6 to 8, and most preferably pH 7.

In addition, the buffer solution can be used without limitation as long as it does not impair the activity of the enzyme used in the present invention (proper active pH is 4 to 4.5), and a known buffer solution is appropriately used by those skilled in the art. It is selectable. For example, the buffer may be a citrate buffer, a succinate buffer, a maleate buffer, or an acetate buffer, preferably Tris -HCl buffer solution may be used.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

<Example>

<Example 1: Materials and Preparation>

Red ginseng purchased from the Geumsan Wholesale Market was used, and the red ginseng was ground into a mesh of a certain size using a Stan mixer. Almonds, a natural product, were purchased from Nexfood Co., Ltd. (USA), shiitake mushrooms were purchased from a local mart (Korea), and pumpkin seeds were purchased from Fruit Village Co., Ltd. (China). Edible enzymes Sumizyme AC and Pluszyme 2000 were purchased from Vision Biochem. For the thin film chromatography plate, Merck's aluminum product was used. All other reagents and instruments used in the present invention are commercially available.

<Example 2: Preparation of compound K>

For the preparation of red ginseng extract, 50 g of red ginseng was autoclaved at a temperature of 121° C. and 15 lb psi for 15 minutes and then immersed in 1 L of 70% ethanol. The immersed red rice ginseng was extracted at 85° C. for about 1 hour using a Lihibi cooler. Ethanol was removed from the extracted solution using a rotary evaporator, and the remaining concentrate was used as the red ginseng extract of the present invention.

For the preparation of the natural extract of the present invention, 20 g of shiitake, almond or pumpkin seeds in 20 mM Tris-HCl (pH 7.0) were physically crushed into a mesh of a certain size using a Stan mixer and immersed at 4° C. for 24 hours, The supernatant was collected by centrifugation at 13,000 rpm for 10 minutes, and impurities were removed using a 3 um micro filter and used in the reaction.

The enzyme solution was dissolved in distilled water in advance so as to be v/v % of the final reaction solution (the sum of red ginseng extract, enzyme and natural product extract) and homogenized, and then added to the red ginseng extract just before the reaction.

Specifically, the reaction solution was adjusted to pH between 4 and 5 using citric acid to optimize the enzymatic reaction. The enzyme solution, Sumizyme AC 7% v/v (based on the total volume of the above red ginseng extract, enzyme and natural product extract, the same as below) and natural product extract (shiitake, almond or pumpkin seed extract) 35% v/v was mixed and vortexed to homogenize, and the natural product When the extract was not used, the same amount of distilled water was used for mixing. The prepared mixture was added to the red ginseng extract and reacted for 72 hours at a temperature of 50° C. in a shaker incubator. Upon completion of the reaction, the reaction solution was immediately bathed at 95° C. for 10 minutes to inactivate the enzyme. After centrifugation at 4,000 rpm for 5 minutes, the supernatant was taken and impurities were filtered through a 3 um micro filter.

<Example 3: Analysis of Compound K Content>

For TLC analysis, 1000 ul of the reaction solution after the reaction was completed was mixed with 400 ul of water-saturated butanol, vortexed, and then centrifuged at 13,000 rpm for 1 minute to confirm layer separation. The supernatant was taken and dripped on a TLC plate by 20 ul. . For the TLC developing solution, a developing solvent mixed with chloroform:methanol:water in a ratio of 65:35:10 was used. About 100 ml of the developing solution was put into the developing tank, a thin layer was placed, sealed, and developed to a certain height. After the thin layer was taken out of the developing tank and dried, 10% sulfuric acid was applied, and then dried at 110° C. to check the presence or absence of compound K.

Ginsenoside analysis was commissioned by Geumsan International Ginseng Herbal Research Institute, and the sample was added 1:1 to hexane and methanol, extracted with shaking for 3 hours, and when the layers were separated, concentrated under reduced pressure and completely dissolved in 70% methanol, followed by a 0.45 uM filter was filtered. Agilent's equipment was used for HPLC equipment, and a C18 reversed-phase column was used for the column, and 10 ul of the sample solution was injected into distilled water and acetonitrile mobile phase at a rate of 1 ml / min, and detection at a wavelength of 203 nm was performed.

As a result, when Sumizyme AC and natural product (shiitake, almond, pumpkin seed) extract are mixed and used, significantly higher compound K can be produced compared to other groups, and in particular, when pumpkin seed extract is used as a natural extract, the highest compound K can be produced. It was confirmed (see Table 1, Figures 1 and 2 below).

ingredient Compound K (mg/g) Red Ginseng Extract 0.004 Red Ginseng Extract + Sumizyme AC 7% 0.168 Red Ginseng Extract + Raw Shiitake Extract + Sumizyme AC 7% 0.298 Red Ginseng Extract + Dried Shiitake Extract + Sumizyme AC 7% 0.269 Red Ginseng Extract + Almond Extract + Sumizyme AC 7% 0.299 Red Ginseng Extract + Pumpkin Seed Extract + Sumizyme AC 7% 0.440

Claims (11)

In the method for producing a compound K comprising mixing a red ginseng extract, an enzyme and a natural product extract, the enzyme is cellulase (Cellulase), beta-glucosidase (β-glucosidase) and hemicellulase (hemicellulase) consisting of It is at least one selected from the group, and the natural product is at least one selected from the group consisting of shiitake mushrooms, almonds and pumpkin seeds.
The method of claim 1, wherein the red ginseng extract is an ethanol extract.
The method of claim 2, wherein the ethanol is an aqueous ethanol solution of 60 to 80% (v/v).
The method of claim 1, wherein the red ginseng extract is extracted under 70 to 90°C conditions.
The method of claim 1, wherein the red ginseng extract is extracted for 30 to 120 minutes.
The method of claim 1, wherein the natural extract is included in an amount of 30 to 40% (v/v) based on the total volume of the red ginseng extract, the enzyme and the natural extract.
The method of claim 1, wherein the natural product extract is a supernatant of the natural product extract.
The method of claim 7, wherein the supernatant is prepared by a method comprising the steps of:
a) grinding the natural product;
b) adding a solvent so that the pulverized product of step a) has a concentration of 0.01 to 1 g/ml;
c) immersing the resultant of step b) at 1 to 10° C. for 12 to 36 hours; and
d) obtaining a supernatant from the result of step c).
The method of claim 8, wherein the solvent in step b) is distilled water or a buffer solution.
According to claim 1, wherein the enzyme is cellulase (Cellulase), beta- glucosidase (β-glucosidase) and hemicellulose (hemicellulose) will include, the method for producing compound K.
The method of claim 1, wherein the natural product includes pumpkin seeds.

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