KR20040064490A - Method for extracting isoflavone from soy bean - Google Patents

Abstract

The present invention relates to a method for extracting isoflavones from soybean, specifically, the present invention is to separate the liquid component generated as a by-product from the steamed soybean during the process of making meju from steamed soybean, and the separated liquid component Is purified by column chromatography to extract isoflavones. In addition, the present invention is to separate the solid component suspended in the upper layer in the process of fermenting the meju to prepare the miso, add the alcohol or an aqueous alcohol solution to the separated solid component, stirred and filtered, and then the filtrate by column chromatography Purification is to extract isoflavones. The present invention makes it possible to extract isoflavones in a simple process and to improve productivity by extracting isoflavones from by-products generated during the preparation of meju and miso.

Description

How to extract isoflavones from beans {METHOD FOR EXTRACTING ISOFLAVONE FROM SOY BEAN}

The present invention relates to a method for extracting isoflavones from soybeans.

Isoflavones are a type of vegetable compound found in soybeans. This isoflavone is not only structurally similar to estrogen, which is a type of female hormone, but also has a similar biological action, and is called phytoestrogens. Specifically, isoflavones are structurally similar to estrogen hormones and biologically similar to each other, and when administered into the body, the isoflavone antagonizes the estrogen hormone and regulates the action of the estrogen hormone. Thus, isoflavones are known to be effective in treating various cancers associated with estrogen hormones, menopausal syndrome, hormonal diseases including cardiovascular disease and osteoporosis.

Such isoflavones exist in the form of glycosides in which sugar residues are bound to flavonoid compounds and in the form of nonglycosides consisting only of flavonoid compounds. Glycoside isoflavones include didzin, genistin, glycidin, malonyl-daidzin, malonyl-glycitin, malonyl- There are malonyl-genistin, acetyl-daidzin, acetyl-glycitin or acetyl-genistin. Nonglycoside isoflavones include dydzein, genistein or glycidane, which are present mainly in fermented foods. In fermented foods, sugar residues are degraded from glycoside-formed isoflavones by bacteria involved in fermentation to form non-glycosided isoflavones. This isoflavone is known to be contained in a lot of beans among other plants, in particular, is known to be contained in a lot of embryos. Therefore, isoflavones are extracted from fermented foods or soybeans.

Conventional methods for extracting isoflavones from soybeans include organic acids and organic solvents, adsorption resins, and β-glucosidase enzymes.

The method using the organic acid and the organic solvent is a method of layer separation of the extract from the soybean using an organic acid selected from glacial acetic acid or lactic acid and an organic solvent selected from acetone, ethyl acetate, and isolating the isoflavone non-glycoside contained in the separated organic solvent to be.

In the method using the adsorption resin, soybean embryos are extracted with an aqueous alkali solution at room temperature to remove the fiber, and the protein is removed from the fiber-free extract, and then passed through the adsorption resin to adsorb isoflavone and saponin, and polarity such as methanol or ethanol. The solvent is flowed and the isoflavone and saponin are dried under reduced pressure in a desorption far infrared furnace to obtain a mixture of isoflavone and saponin. The saponins and isoflavones are separated from each other by an extraction method using acetone, and then dried under reduced pressure in a far infrared furnace to obtain isoflavones.

The method using β-glucosidase ferments soybean embryos using the fermentation strain Bacillus Licheniformis CK-14, and mainly glycosides in soybean embryos using β-glucosidase produced therefrom. It is a method of converting the isoflavone component present in the form into a nonglycoside form having excellent absorption in vivo. Another method of using β-glucosidase is to extract a vegetable protein containing glycoform isoflavones with an aqueous extractant having a pH above the isoelectric point of the protein to generate an aqueous extract containing protein and isoflavone glycosides, and then extract A method for producing isoflavone nonglycoside extracts by reacting isoflavone glycosides with β-glucosidase under conditions of time, temperature and pH sufficient to convert most of the isoflavone glycosides into isoflavone nonglycosides.

However, the conventional method for extracting isoflavones from soybeans has a problem in that the extraction process is complicated and the productivity is lowered by directly extracting isoflavones from the beans.

The present invention is designed to solve the above problems, an object of the present invention is to extract isoflavones by a simple extraction process, by extracting isoflavones from the by-products generated in the process of manufacturing meju and miso using soybeans It is to improve productivity.

1 shows column chromatography of a liquid component in the course of extracting isoflavones from the liquid component according to the first aspect of the invention,

FIG. 2 shows the column chromatography of the filtrate in the process of extracting isoflavones by putting solid components in 80% aqueous methanol solution according to the second aspect of the present invention and stirring at 25 ° C.

Figure 3 shows a column chromatograph of the filtrate in the process of extracting isoflavones by putting the solid component in 40% ethanol aqueous solution according to the second aspect of the present invention and stirred at 25 ℃,

4 shows column chromatography of the filtrate in the process of extracting isoflavones by adding solid components to 60% aqueous ethanol solution according to the second aspect of the present invention and stirring at 25 ° C.

FIG. 5 shows that the filtrate was column chromatographed in the process of extracting isoflavones by adding solid components to 60% aqueous ethanol solution according to the second aspect of the present invention and stirring at 50 ° C.

FIG. 6 shows that the filtrate was column chromatographed in the process of extracting isoflavones by adding solid components to an aqueous 80% ethanol solution and stirring at 25 ° C. according to the second aspect of the present invention.

7 shows column chromatography of isoflavones extracted from a liquid component according to the first aspect of the present invention using an analytical column,

FIG. 8 shows column chromatography of isoflavones extracted from doenjang residue by adding solid components to an aqueous 60% ethanol solution and stirring at 50 ° C. according to a second aspect of the present invention.

In order to achieve the above object, the present invention is to separate the liquid components generated from the steamed soybeans in the process of preparing meju with steamed soybeans, and to extract the isoflavones by purification of the separated liquid components by column chromatography will be.

In addition, the present invention is to separate the solid component suspended in the upper layer in the process of fermenting the meju to prepare the miso, add the alcohol or an aqueous alcohol solution to the separated solid component, stirred and filtered, and then the filtrate by column chromatography Purification is to extract isoflavones.

Hereinafter, the present invention will be described in detail.

The first aspect of the invention

1) adding water to the beans and steaming, and then filtering to separate the solid and liquid components (step 1);

2) purifying the liquid component by column chromatography (step 2).

Separation of the solid component and the liquid component in step 1 is generally performed in the process of preparing meju.

Specifically, the first step in the process of preparing meju is to add water to the beans and to steam at a temperature of 100 ° C. The beans can then be soaked in water for a period of time before the beans are boiled in water. The second step is to formulate meju using steamed beans. In general, the liquid component generated during the steaming of soybean is a by-product generated during the steaming process, and it is necessary to remove a certain amount of the liquid component because it exists in excess in making the formulation of Meju. The method for removing the liquid component can be removed by filtration of the steamed state, or by centrifugation. In the present invention, isoflavones are extracted from the resulting liquid components, and the productivity of extracting isoflavones from beans is improved by using the liquid components produced as by-products in the process of preparing meju.

Purifying the liquid component by column chromatography in step 2 may be performed with various kinds of columns and mobile phase conditions. Preferably, the mobile phase causes mobile phase A and mobile phase B to have a concentration gradient of mobile phase A / mobile phase B = 100/0 to 70/30. The mobile phase A comprises water: acetonitrile: acetic acid = 94.9: 5.0: 0.1 (volume). %) And the mobile phase B is water: acetonitrile: acetic acid = 5.0: 94.9: 0.1 (vol%). The mobile phase was kept at 1 ml / min.

Isoflavones purified by column chromatography may be detected using light having a wavelength capable of detecting flavonoid compounds. Preferably at 260 nm.

Further, the second aspect of the present invention

1) drying the meju prepared from soybeans, adding water and salt to the dried meju and fermenting the meju (step 1);

2) separating the solid component suspended in the upper portion in the fermentation process (step 2);

3) adding an alcohol or an aqueous solution of alcohol to the solid component, stirring and filtering (step 3); and

4) purification of the filtrate by column chromatography (step 4).

Fermenting the meju in step 1 is a part of preparing the miso. Specifically, the meju is prepared by the method described in the first aspect of the present invention, and the meju is dried for a certain period of time, and then fermented with water and salt in the meju for a certain period of time. Solid components are produced. This solid component is produced as a by-product in the process of fermentation of meju by bacteria. Generally, the solid component is removed and the remaining fermented meju is stirred to prepare miso. In the present invention, isoflavones are extracted from the resulting solid components, and the productivity of extracting isoflavones from soybeans is improved by using the solid components produced as by-products in the process of making miso.

Step 2 is to separate the suspended solid component produced in step 1.

Step 3 is a step of adding an alcohol or an aqueous alcohol solution to the separated solid component, stirring, and then filtering.

The alcohol is preferably a C ₁ ~ C ₄ alcohol, most preferably methanol or ethanol can be used. The aqueous alcohol solution contains 20 to 80% (volume / volume) of alcohol. By using a highly polar solvent such as methanol or ethanol, highly polar isoflavones can be efficiently extracted.

It is preferable that an alcohol or an aqueous alcohol solution is added to the solid component, and stirring is performed at 25 to 50 ° C.

In addition, the filtrate prepared through the above step contains a lot of impurities, so that the filtrate can be prepared by further concentrating the filtrate, followed by centrifugation and refiltration.

In step 4, the filtrate obtained from step 3 is purified by column chromatography. Column chromatography purification can be performed with various types of columns and mobile phase conditions. Preferably, the mobile phase causes the mobile phase A and the mobile phase B to achieve a concentration gradient of mobile phase A / mobile phase B = 100/0 to 70/30. The mobile phase A is water: acetonitrile: acetic acid = 94.9: 5.0: 0.1 (volume: volume: volume%), and the mobile phase B is water: acetonitrile: acetic acid = 5.0: 94.9: 0.1 (volume: volume: volume%) to be. The mobile phase was kept at 1 ml / min.

Isoflavones purified by column chromatography may be detected using light having a wavelength capable of detecting flavonoid compounds. Preferably at 260 nm.

Hereinafter, an Example demonstrates this invention.

However, the present invention is not limited by this embodiment.

Example 1 Extracting Isoflavones from Soybean According to the First Aspect of the Present Invention

Soybeans are soaked in water for 6 hours, soaked soybean is added 1.5 times the weight of soybeans, and steamed for 2 hours at a temperature of 100 ° C. or higher. The boiled solution is maintained at 70 ° C. for 1 hour, centrifuged at 60,000 rpm for 1 hour to remove solid components and to separate liquid components.

1 μl of the liquid component was injected into a preparative column filled with 15 μm particles (Licrospher100RP-C _18, Merck, USA) and eluted. The mobile phase solvent was mobile phase A (water: acetonitrile: acetic acid = 94.9: 5.0: 0.1 (volume: volume: volume (%))) and mobile phase B (water: acetonitrile: acetic acid = 5.0: 94.9: 0.1 (volume: volume) Volume (%)) was eluted with a mobile phase A / mobile phase B = 100/0 to 70/30 concentration gradient and the elution rate of the mobile phase solvent was maintained at 1 mL / min.

In addition, the wavelength for detecting isoflavones was 260 nm. In order to determine the position of the isoflavones, the same experiment was conducted with a standard sample containing Genistin, Dyzedein and Genistein.

The column chromatography was shown in FIG. 1 . As shown in FIG. 1 , it can be seen that the liquid component according to the present invention contains a glycoside isoflavone including digazine, glycidine and malonyl-geninistin and a nonglycoside isoflavone comprising genistein. .

In addition, the same experiment with US soybeans yielded the same results as those with Korean soybeans.

Example 2 A method for extracting isoflavones from soybeans according to a second aspect of the invention

Soybeans are soaked in water for 6 hours, soaked soybean is added 1.5 times the weight of soybeans, and steamed for 2 hours at a temperature of 100 ° C. or higher. Steamed beans are filtered to separate the solid components and meju is prepared. After the prepared meju is dried in a conventional manner, water and salt are added to the dried meju in a ratio of 1: 4: 0.8 with respect to meso beans and fermented.

In the fermentation process, the solid component suspended in the upper layer is separated.

200 g of 80% aqueous methanol solution was added to 50 g of the separated solid component, and stirred at 3,000 rpm for 3 hours at 25 ° C. The stirred solution was filtered through a filter paper, and then concentrated. The concentrate was placed in a centrifuge (Optima LE-80K), centrifuged at 60,000 rpm for 1 hour at 4 ° C., and then filtered through a 0.45 μm filter paper.

1 μl of the filtrate was injected into a preparative column (Licrospher 100RP-C _18, Merck, USA) filled with 15 μm particles and eluted. The mobile phase solvent was mobile phase A (water: acetonitrile: acetic acid = 94.9: 5.0: 0.1 (volume: volume: volume (%))) and mobile phase B (water: acetonitrile: acetic acid = 5.0: 94.9: 0.1 (volume: volume) Volume (%)) was eluted with a concentration gradient of mobile phase A / mobile phase B = 100/0 to 70/30, and the elution rate of the mobile phase solvent was maintained at 1 ml / min. The wavelength was 260 nm.

The column chromatography was shown in FIG. 2 .

As shown in FIG. 2 , it contains a glycoside isoflavone comprising a diglycoside isoflavone including diedzein and genistein and malonyl-genistine, dydazine, glycidine, genistin and acetyl-geninistin. Able to know. In addition, it can be seen that the content of genistein, which is a nonglycoside isoflavone, among them is high. Such high content of non-glycoside isoflavones is thought to be high content of non-glycoside isoflavones by bacteria involved in fermenting soybean paste by decomposing glycoside isoflavones contained in soybeans.

Example 3 Method 2 Extracting Isoflavones from Soybean According to a Second Aspect According to the Present Invention

200 g of 40% ethanol aqueous solution was added to 50 g of the solid component separated in Example 2, and isoflavones were extracted using the same method as in Example 2 except that the solution was stirred at 25 ° C.

The column chromatography was shown in FIG. 3 .

As shown in FIG. 3 , nonglycoside isoflavones including didzein, glycidine and genistein and malonyl-zenithine, dydazine, glycidine, zenithine, malonyl-dydazine and acetyl-zenithine It can be seen that the glycoside isoflavone containing a. In addition, it can be seen that the content of genistein, which is a nonglycoside isoflavone, among them is high.

Example 4 A method for extracting isoflavones from soybeans according to a second aspect of the present invention

200 g of an aqueous 60% ethanol solution was added to 50 g of the solid component separated in Example 2, and isoflavones were extracted using the same method as Example 2 except for stirring at 25 ° C.

The column chromatography was shown in FIG. 4 .

As shown in FIG. 4 , non-glycoside isoflavones including didzein, glycidine and genistein and malonyl-zenithine, dydazine, glycidine, genistin, malonyl-didezin and acetyl-zenithine It can be seen that the glycoside isoflavone containing a. In addition, it can be seen that the content of genistein, which is a nonglycoside isoflavone, among them is high.

Example 5 Method 4 Extracting Isoflavones from Soybean According to a Second Aspect According to the Present Invention

200 g of 60% ethanol aqueous solution was added to 50 g of the solid component separated in Example 2, and isoflavone was extracted using the same method as Example 2 except for stirring at 50 ° C.

The column chromatography was shown in FIG. 5 .

As shown in FIG. 5 , it can be seen that a glycoside isoflavone including didazine and genistein is contained, and glycoside isoflavone including dydazine, glycidin and genistin.

Example 6 A method for extracting isoflavones from soybeans according to a second aspect of the present invention 5

200 g of 80% ethanol aqueous solution was added to 50 g of the solid component separated in Example 2, and isoflavone was extracted in the same manner as in Example 2 except that the solution was stirred at 25 ° C.

The column chromatography was shown in FIG. 6 .

As shown in FIG. 6 , a nonglycoside isoflavone comprising diedzein, glycidine and genistein and a glycoside isoflavone comprising malonyl-zenithine, acetyl-genistine, dydazine, glycidine and genistin It can be seen that it contains.

Experimental Example 1 Analysis of Isoflavones Extracted According to the First Aspect of the Present Invention

Using isoflavone extracted from the liquid component of Example 1 as a sample, it was analyzed with an analytical column (0.46 cm x 25 cm, packed with 5 μm particles).

The analysis conditions are as follows.

In the mobile phase, the mobile phase A and the mobile phase B had a concentration gradient of mobile phase A / mobile phase B = 100/0 to 70/30. The mobile phase A is water: acetonitrile: acetic acid = 94.9: 5.0: 0.1 (volume: volume: volume (%)) and the mobile phase B is water: acetonitrile: acetic acid = 5.0: 94.9: 0.1 (volume: volume: volume) (%))to be. The mobile phase was kept at 1 ml / min and the isoflavone detection wavelength was 260 nm. The sample was injected with 1 μl of isoflavone prepared in Example 1.

In addition, in order to find out the components of the isoflavone extracted from the liquid component according to the present invention, the same experiment was repeated using a mixture of Genistin and Dyzedine as a standard sample.

The results of analyzing the isoflavones extracted from the soybean extract using the analytical column are shown in FIG. 7 . As shown in FIG. 7 , it can be seen that the isoflavone extracted from the soybean extract according to the present invention contains glycoside isoflavone including didazine, glycidine, genistin and malonyl-geninistin.

Experimental Example 2 Analysis of Isoflavones Extracted According to the Second Aspect of the Present Invention

Using isoflavone extracted from Example 5 as a sample, it was analyzed with an analytical column (0.46 cm x 25 cm, filled with 5 μm particles).

The analysis conditions are as follows.

In the mobile phase, the mobile phase A and the mobile phase B had a concentration gradient of mobile phase A / mobile phase B = 100/0 to 70/30. The mobile phase A is water: acetonitrile: acetic acid = 94.9: 5.0: 0.1 (vol%) and the mobile phase B is water: acetonitrile: acetic acid = 5.0: 94.9: 0.1 (vol%). The mobile phase was kept at 1 ml / min and the isoflavone detection wavelength was 260 nm. The sample was injected with 1 μl of the extract prepared in Examples 2-6.

The isoflavones extracted from the solid components using the analytical column are shown in FIG. 8 . As shown in FIG. 8 , isoflavones extracted from the solid component according to the present invention include nonglycoside isoflavones including genistein, dydzein, and glycidin, malonyl-geninistin, genistin, malonyl-dydazine, Glycoside isoflavones, including glycidine, dydazine and acetyl zenithine. In particular, it can be seen that the content of non-glycoside isoflavones is relatively higher than isoflavones extracted according to the first aspect of the present invention.

The method for extracting isoflavones from the soybean of the present invention by the above configuration has the following effects.

First, the liquid component, which is a by-product generated in the process of preparing meju, is purified by column chromatography, or the solid component, which is a by-product generated in the process of making doenjang, is added with a solvent to separate the filtrate and purified by column chromatography. Isoflavones can be extracted by a simple process.

Second, it is possible to improve the productivity of extracting isoflavones from soybeans by extracting isoflavones from by-products generated in the process of preparing meju and miso using soybeans without extracting isoflavones directly from soybeans.

Claims (6)

1) adding water to the beans and steaming, and then filtering to separate the solid and liquid components (step 1); 2) A method for extracting isoflavones from soybean, characterized in that the liquid component is purified by column chromatography (step 2). 1) drying the meju prepared from soybeans, adding water and salt to the dried meju and fermenting the meju (step 1); 2) separating the solid component suspended in the upper portion in the fermentation process (step 2); 3) adding an alcohol or an aqueous solution of alcohol to the solid component, stirring and filtering (step 3); and 4) A method for extracting isoflavones from soybean, characterized in that the filtrate is purified by column chromatography (step 4). 3. The method of claim 2, wherein the alcohol is a C ₁ to C ₄ alcohol. 4. The method of claim 3 wherein the alcohol is methanol or ethanol. The method of claim 2 wherein the aqueous solution contains 20-80% (volume / volume) of alcohol. The method of claim 2 wherein said agitation is carried out at 25-50 ° C.