JPH11255792A - Obtaining of isoflavone glycoside - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an isoflavone glycoside from an inexpensive raw material. SOLUTION: A bean-curd refuse is extracted with water and the resultant water extract solution is then brought into contact with an adsorbent to adsorb an isoflavone glycoside in the extract solution thereto. The resultant isoflavone glycoside is subsequently obtained by using an aqueous solution of an alcohol. In this case, water used for the water extraction is a dilute acid or a dilute alkali solution.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for obtaining isoflavone glycosides by extracting okara with water.

[0002]

2. Description of the Related Art Conventionally, soybeans contain daidzin, glycitin, genistin, acetyldaidin and acetylgenistin as isoflavone compounds or daidzein, glycitein, genistein as their aglycones, and these contain estrogenic action, antibacterial action,
It has been confirmed that it has many pharmacological effects including antioxidant and anticancer effects. On the other hand, recently some soybeans

[0003]

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[0004]

Embedded image The presence of malonyl isoflavone glycosides such as malonyl daidzin and malonyl genistin shown in Table 1 was confirmed, and these were found to be the main components of the isoflavone compounds in soybean. This malonyl isoflavone glycoside is easily soluble in water, has an antioxidant effect itself, is expected to have the above-mentioned pharmacological effects due to its structural similarity, and is also expected to be used as an active ingredient of bath salts. Things.

Accordingly, the present inventors first extract soybeans with water, contact the aqueous extract with an adsorbent to adsorb malonyl isoflavone glycosides, and elute this with an aqueous alcohol solution. And obtained a patent application (Japanese Patent Application Laid-Open No. 8-283283). In addition, soybean hypocotyls used for feeds and the like have a large content of isoflavone compounds, and malonyl isoflavone glycosides can be obtained from this as a raw material.

[0006]

An object of the present invention is to obtain malonyl daidzin, malonyl genistin, daidzin, genistin (hereinafter collectively referred to as isoflavone glycoside) using inexpensive raw materials. It is characterized by using okara, which is a by-product of tofu and soy milk production, as its raw material. The present invention is the first method for obtaining isoflavone glycosides from okara, which is easily rotted, most of which are discarded, and used at most only as feed for livestock.

[0007]

Means for Solving the Problems The present inventors have conducted various studies from the viewpoint of effective use of okara, and surprisingly found that a large amount of isoflavone glycosides existed in okara, and this was extracted with water. The present invention was completed by finding that an isoflavone glycoside can be obtained by adsorbing the extract to an adsorbent and eluting with an alcohol.

Hereinafter, the present invention will be described in detail. Okara as a raw material for obtaining isoflavone glycosides is tofu,
It is a common okara obtained as a by-product of frying and soy milk production, and is a residue generated in the production process of isolated soy protein. Such okara is extracted with water, but the temperature of water used for extraction is not limited. That is, any of room temperature water, warm water and hot water may be used.

The water used for the extraction is made into a dilute acid solution having a pH of 4 to 5 with hydrochloric acid, phosphoric acid, acetic acid, lactic acid, or the like, thereby stabilizing the extracted isoflavone glycoside and, at the same time, extracting the okara after the extraction. Has an acidic effect, which has the effect of suppressing the progress of spoilage by microorganisms. In addition, the extraction rate of the isoflavone glycoside can be improved by making the water used for the extraction into a dilute alkaline solution having a pH of 8 to 10 with sodium hydroxide, potassium hydroxide or the like.

[0010] In order to extract okara using such extracted water, 1 to 10 times (w / w) amount of extracted water is added to okara, and the mixture is sufficiently stirred and mixed. Solid-liquid separation is performed by a separation device, for example, a filter-type filter, a screw decanter, or the like. The obtained extract is used as it is, or a filtrate from which the protein is removed using an ultrafiltration membrane as needed, or a protein is precipitated by adjusting the pH to about 4.0 to 5.0 with hydrochloric acid, and the supernatant is adsorbed. Contact with the agent.

The above extract, filtrate or supernatant is directly contacted with an adsorbent,
After adjusting to about 8.0, there is a method of contacting. In the former case, the amount of adsorption to the adsorbent is increased, and in the latter case, there is an advantage that it is easy to separate daidzin and genistin mixed with malonyl isoflavone glycoside. In any case, the adsorbent used is, for example, a synthetic adsorbent, activated carbon, alumina or the like, and specifically, Diaion HP-2
0 (manufactured by Mitsubishi Chemical), purified Shirasagi activated carbon (manufactured by Takeda Pharmaceutical), activated alumina (manufactured by Wako Pure Chemical Industries), and the like. The contact may be a general method such as a batch method or a column method,
For example, the extraction can be carried out by passing the extract through a column filled with an adsorbent, whereby most of the isoflavone glycosides in the extract are adsorbed by the adsorbent.

Next, the isoflavone glycoside adsorbed on the adsorbent is eluted using an aqueous alcohol solution or an aqueous alkaline alcohol solution. The resulting solution is concentrated under reduced pressure, or concentrated under reduced pressure, and then freeze-dried to obtain a concentrated solution or a dry powder of isoflavone glycoside.

The above-mentioned concentrated liquid or dry powder is daidzin,
It is a mixture of genistin, malonyl daidzin and malonyl genistin.

In order to efficiently obtain malonyl daidzin and malonyl genistin, the following method is preferred. That is, until the extract of okara is brought into contact with the adsorbent, the same as above, but elution is performed sequentially by changing the concentration of the aqueous alcohol solution, and roughly malonyl daidzin,
Malonylgenistin or daidzin and genistin are separated, and these eluates are purified by an ODS column. According to the present invention, malonyl daidzin, malonyl genistin or daidzin and genistin can be easily separated from the extract of okara in this way. Moreover, malonyl daidzin and malonyl genistin can be converted into daidzin and genistin by using these as a raw material by an acid or alkali treatment, and the aglycone daidzein and genistein can be obtained from daidzin and genistin.

For example, a solution in which malonyl daidzin and malonyl genistin are dissolved is adjusted to pH 8 to 13 with ammonia, caustic soda, sodium carbonate, or the like, and left for 0.5 hour or more to be converted into daidzin and genistin. In the alkali treatment, the higher the pH, the higher the conversion. In addition, a solution in which malonyl daidzin and malonyl genistin are dissolved is 70 to 1
By heating at 50 ° C. for 0.5 to 12 hours, these malonyl compounds are converted to daidzin and genistin. The higher the heating temperature and the longer the heating time, the faster the conversion rate.
The conversion rate can be further increased by combining the pH of the solution and the heating temperature. The daidzin and genistin thus converted are adsorbed to the ODS resin, eluted with an aqueous alcohol solution, concentrated under reduced pressure, and lyophilized to obtain purified daidzin and genistin.

The daidzin and genistin obtained as described above can be further converted to daidzein and genistein (isoflavone aglycone) by acid treatment or enzyme treatment. For example, in the case of acid treatment, daidzin and genistin are mixed at a ratio of 4.5 of methanol to 1 of concentrated hydrochloric acid, refluxed at 70 ° C. or more for about 6 hours, and cooled.
Water is added to obtain a precipitate. This is filtered, washed with water, dissolved in hot ethanol, warm water 4 is added to this ethanol solution 6, and the mixture is allowed to cool at room temperature to precipitate isoflavone aglycone. In the acid treatment in this case, isoflavone aglycone can be similarly obtained even when the raw materials are malonyl daidzin and malonyl genistin.

In the case of enzymatic treatment, daidzin and genistin are dispersed in a soybean-derived β-glucosidase solution dissolved in a 1/10 M phosphate buffer (pH 5.0).
For about 6 hours.
After filtration through an ultrafiltration membrane of 000, the filtrate is adsorbed to an ODS resin column, eluted with an aqueous alcohol solution, concentrated under reduced pressure, and lyophilized to obtain purified isoflavone aglycone.

[0018]

According to the present invention, isoflavone glycosides can be easily and efficiently obtained from an aqueous extract of okara, and various isoflavone compounds can be obtained by treating them with acid or alkali.

[0019]

EXAMPLES The present invention will be described in detail with reference to the following Examples. Example 1 After washing 1,000 kg of commercially available US soybean (IOM) with tap water,
It was immersed and swelled in 5 times the amount of tap water for 16 hours. The swollen soybeans are ground while adding tap water, and the obtained go
After heating at 3 ° C. for 3 minutes, the mixture was cooled to 85 ° C. and solid-liquid separated by a screw decanter to obtain 1,540 kg of okara. To this okara, double volume of 0.05N hydrochloric acid solution was added, mixed and stirred, then solid-liquid separated with a screw decanter,
2,800 L of the extract was obtained.

[0020] This extract is used as a synthetic adsorbent DIAION HP-2.
Column filled with 0 (Mitsubishi Chemical) (30 × 100cm, 25L)
The mixture was passed through the column at a flow rate of 70 L / hr to adsorb the isoflavone glycoside, and then washed with 100 L of distilled water. Next, the fraction (A) eluted with 100 L of a 5% aqueous ethanol solution, 200 L of a 20% aqueous ethanol solution and 150 L of a 30% aqueous ethanol solution, and the fraction (B) eluted with 200 L of a 40% aqueous ethanol solution were concentrated under reduced pressure.
6 L of each concentrated solution was obtained. In the categories (A) and (B), malonyl daidzin 28.9 g, 12.3 g, malonyl genistin 7.1 g, 54.1 g, daidzin 11.2 g, 0 g, genistin 7.6 g,
12.5 g, and the category (A) was a malonyl daidzin concentrate and the category (B) was a malonylgenistin concentrate.

<Purification of malonyl daidzin> Next, the above-mentioned malonyl daidzin-containing concentrate (Category A) was treated with 2N-NaOH at pH 8.
0, and the column was filled with a synthetic resin ODS resin packed column (1
(0 × 60 cm, 4 L) at a flow rate of 50 ml / min., And washed with 10 L of distilled water. Next, elution was carried out with 20 L of a 5% aqueous ethanol solution, the fraction containing the target substance was collected, concentrated, and lyophilized to obtain 20.77 g of the former and 6.4 g of the latter using malonyl daidzin and daidzin as sodium salts.

<Purification of Malonylgenistin> The above-mentioned concentrated solution containing malonylgenistin (Category B) was adjusted to pH with 2N-NaOH.
It was adjusted to 8.0, passed through an ODS resin packed column in the same manner as described above, and washed with 10 L of distilled water. Then 1
Elution was carried out with 10 L of a 0% aqueous ethanol solution, the fraction containing the target substance was collected, concentrated, and lyophilized to give malonylgenistin and genistin as sodium salts, the former being 41.80.
g, the latter 6.8 g.

The thus obtained malonyl daidzin, malonyl genistin and daidzin, 1H and 13 of genistin
C-NMR is described in the literature {Agric. Biol. Chem., 55, (9) 2227 (19
91) It was consistent with the numerical value described in ①. The okara used as a raw material was prepared according to the method of Wang et al., J. Agric. Food Chem., 42, 1666 (1.
994)｝ The isoflavone compounds were analyzed. That is, 100 ml of acetonitrile was added to 2.0 g of a freeze-dried powder sample (water content 2%) of the raw material okara (water content 78%).
After adding 20 ml of a 0.1N hydrochloric acid solution and stirring at room temperature for 2 hours, the mixture was filtered with Toyo Filter Paper No. 2 and concentrated to dryness at 30 ° C. or lower by a rotary evaporator. This was dissolved in 10 ml of 80% methanol, filtered through a membrane filter, and 20 μl thereof was subjected to HPLC. The average value of the three analyzes was 12.9 mg of malonyl daidzin, 13.3 mg of malonyl genistin, 4.6 mg of daidzin, 5.3 mg of genistin, 1.8 mg of daidzein and 2.5 mg of genistein per 100 g of raw okara. From the above, the yields of malonyl daidzin, malonyl genistin, daidzin, and genistin in Example 1 were 10%, 20%, 9%, and 8%, respectively. In the normal case, okara rots at room temperature, but rot of okara after this extraction treatment was not observed even after standing at room temperature for 2 days.

Example 2 Okara was extracted and adsorbed and eluted in the same manner as in Example 1 except that okara was extracted and tap water used for extraction was adjusted to pH 8.0 with sodium hydroxide. , Malonyl daidzin 52.1 g, malonyl genistin 75.7 g, daidzin 2
Each concentrate containing 7.9 g and 33.0 g of genistin was obtained.

Claims (2)

[Claims] The present invention is characterized in that isoflavone glycosides are extracted from okara with water, the aqueous extract is brought into contact with an adsorbent to adsorb the isoflavone glycosides in the extract, and then eluted with an alcohol aqueous solution. Method for obtaining isoflavone glycoside. 2. The method for obtaining an isoflavone glycoside according to claim 1, wherein the water used for the water extraction is a dilute acid or dilute alkali solution.