JP2005224162A - Method for producing isoflavone aglycone - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To efficiently produce an isoflavone aglycone-containing composition from a composition containing ≥3.0 wt.% of an isoflavone glycoside. <P>SOLUTION: The isoflavone aglycone-containing composition is industrially produced more efficiently by making enzyme(s), especially the genus Rhizopus- and/or Aspergillus-derived one(s), on the composition containing ≥3.0 wt.% of the isoflavone glycoside, thus producing the objective isoflavone aglycone regarded as being physiologically useful. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for producing an isoflavone aglycone, wherein an isoflavone glycoside is converted into an isoflavone aglycone by an enzyme, and the isoflavone aglycone is recovered as a substance insoluble in water.

  Isoflavones are represented by the following formula (I) and are present in naturally occurring legumes, roses, irises, mulberries, and amaraceae, particularly legumes.

  The isoflavone compound has an isoflavone represented by the above formula (I) as a basic skeleton, and is typically represented by the following formula (II).

[式中、R1は、グリコシド基、ヒドロキシル基、またはアルコキシル基を表し、R2は、水素原子、ヒドロキシル基、またはアルコキシル基を表し、そしてR3は、水素原子、ヒドロキシル基、またはアルコキシル基を表す]

[Wherein R1 represents a glycoside group, a hydroxyl group, or an alkoxyl group, R2 represents a hydrogen atom, a hydroxyl group, or an alkoxyl group, and R3 represents a hydrogen atom, a hydroxyl group, or an alkoxyl group]

  Among isoflavone compounds, aglyconized daidzein, genistein, etc. are said to be more effective for physiological actions such as atherosclerosis suppression than the glycosides daidin, genistin and the like. Izumi et al. Have found that soy isoflavone aglycone is absorbed into the body faster than glycosides in humans [J. Nutr. Vol. 130, p1695-1699 (2000) (Non-patent Document 1)] Murota et al. Examined absorption and migration of isoflavones using human intestinal Caco-2 cells, and showed that the isoflavone aglycones daidzein and genistein are more effectively absorbed than glycosides. [J. Nutr. Vol. 132, p1956-1961 (2002) (non-patent document 2)]. Yamakoshi et al. Have found an inhibitory effect of isoflavone aglycone alone on the formation of atherosclerosis in cholesterol-ingested rabbits [J. Nutr. Vol. 130, p1887-1893 (2000) (Non-patent Document 3)].

  For these reasons, there is interest in the production method of isoflavone aglycone, but many methods have been devised for the production method of aglycone. For example, a method for producing an aglycone by hydrolyzing isoflavone glycosides produced during the production process of soy sauce (Japanese Patent Laid-Open No. 5-170756 (Patent Document 1)), inoculating cereals (soy beans etc.) with microorganisms such as koji molds Then, isoflavone glycosides present in a large amount in beans are converted to aglycone, and this aglycone is recovered (Japanese Patent Laid-Open No. 9-23822 (Patent Document 2)) [Patent Publication 3383718 (Patent Document 3)] [Patent Publication 33403795 (Patent Document 4)] [Japanese Patent Laid-Open No. 2000-281673 (Patent Document 5)].

  In addition, through research on clover as feed for livestock, especially sheep and cattle, it has long been known that enzymes are involved in the conversion to aglycone [Food Add. Contam. Vol. 2, p73-106 (1985 (Non-Patent Document 4)] [Aust. J. Agric. Res. Vol. 15, p223-230 (1964) (Non-Patent Document 5)], and many proposals have been made for a method for producing an isoflavone aglycone by an enzyme. Yes.

  A method for hydrolyzing isoflavone glycosides and producing and recovering aglycone by using β-glucosidase, which is one of the enzymes of soybean itself (Japanese Patent Laid-Open No. 1-258669 (Patent Document 6)), plant protein There is known a method of aglyconization using β-glucosidase or esterase after the extraction of Japanese quince (Japanese Patent Publication No. 9-503781 (Patent Document 7)). In addition, there is a report on a method for producing isoflavone aglycone by converting an isoflavone glycoside in soybean hypocotyl into aglycone by an enzyme derived from the genus Aspergillus [Japanese Patent Laid-Open No. 11-89589 (Patent Document 8)].

  However, Japanese Patent Application Laid-Open No. 1-258669 has the disadvantages that the isoflavone content of the exudate is low, and because the soybean's own enzyme is used, the conversion efficiency to aglycone is low and the apparatus becomes large-scale. Yes, in Japanese National Publication No. 9-503781, since a commercially available enzyme is added as an auxiliary agent, the conversion rate to aglycone is improved, but the content of isoflavone in the exudate is low and the apparatus becomes large-scale There is.

  In addition, in JP-A-11-89589, an enzyme dispersed in a small amount of water is directly added to soybean hypocotyls to cause it to act directly, but in the exemplified method, in order to deactivate trypsin inhibitor, 150 Pretreatment was performed at 0 ° C., and the hypocotyl had to be powdered in order to increase the conversion rate to aglycone. Considering pretreatment, powdering effort, filtration device for removing hypocotyl residue, etc., it is considered unsuitable for industrialization.

Japanese Patent Laid-Open No. 5-170756 Japanese Unexamined Patent Publication No. 9-23822 Patent publication 3383718 Patent Publication No. 3340395 JP 2000-281673 A Japanese Unexamined Patent Publication No. 1-258669 Japanese National Patent Publication No. 9-503781 Japanese Patent Laid-Open No. 11-89589 J.Nutr.Vol.130, p1695-1699 (2000) J.Nutr.Vol.132, p1956-1961 (2002) J.Nutr.Vol.130, p1887-1893 (2000) Food Add.Contam.Vol.2, p73-106 (1985) Aust.J.Agric.Res.Vol.15, p223-230 (1964)

  An object of the present invention is to industrially produce a more efficient isoflavone aglycone-containing composition.

  The present inventor contains 3.0% or more of isoflavone glycosides using as a raw material an isoflavone glycoside concentrate extracted from plants, in particular, a composition containing concentrated isoflavone glycosides extracted from soybeans or soybean hypocotyls. It has been found that an effective isoflavone aglycone can be produced by allowing an enzyme, particularly a glucosidase enzyme derived from Rhizopus or Aspergillus to act on the composition.

That is, the present invention
(1) A method for producing an isoflavone aglycone comprising treating a composition containing 3.0% or more of isoflavone glycosides with a glucosidase enzyme and recovering insoluble components after the enzyme treatment,
(2) The method for producing an isoflavone aglycone according to (1), wherein the glucosidase enzyme is derived from the genus Rhizopus and / or the genus Aspergillus.

  In the present invention, the glucosidase enzyme is an enzyme having an ability to convert an isoflavone glycoside into an aglycone, and the enzyme may be prepared from a culture solution of a microorganism or the like, but can also be selected from commercially available enzymes. is there. In particular, an enzyme derived from the genus Rhizopus or an enzyme derived from the genus Aspergillus is preferable. The ratio of enzyme addition in the following description is the ratio when a commercially available enzyme is used.

  In the present invention, the “composition” refers to a product extracted from a plant or extracted and purified from a plant, for example, a product extracted from soybean or soybean hypocotyl with an extraction solvent, or a product obtained by further preparing an extract with a synthetic adsorption resin or the like. .

  In this composition, isoflavone glycoside is concentrated to 3.0% or more in the solid component, and this is made into a 5 to 50% suspension, preferably a 10 to 25% suspension, to which glucosidase enzyme is added. It is added at a rate of 1 to 20, preferably 4 to 10, with respect to the isoflavone-containing solid content weight of 200, and stirred or shaken at room temperature to 60 ° C, preferably 40 to 60 ° C for 6 to 24 hours. After completion of the enzyme reaction, insoluble components increase with the production of isoflavone aglycone, and these insoluble components are recovered by centrifugation or filtration.

  FIG. 1 shows an example of a manufacturing process scheme. In each step 1) to 4) shown in FIG. 1, samples were collected and dried, and the isoflavone composition was analyzed using HPLC (High Performance Liquid Chromatography) (Table 2).

  Here, what was extracted using soybean hypocotyl as a raw material was subjected to resin treatment with synthetic adsorption resin Sepa beads SP825 (Mitsubishi Chemical), and a concentrated preparation was obtained as a starting material. An enzyme was allowed to act on this, and the enzyme used was Rhizopus-derived Uniase 2K (manufactured by Yakult Pharmaceutical Co., Ltd.). At the time of starting material 1), the total isoflavone content was 25.6%, and the proportion of aglycone in the total isoflavone was 0.02, and at the time of 2) after the enzymatic reaction, the total isoflavone content was 16.8% and the proportion of aglycone was 0.62. In the product obtained by centrifuging the product at the time of 2) and collecting the precipitate, the total isoflavone content increased to 30.3% and the aglycone ratio increased to 0.78. As a control, the supernatant was adsorbed on Sepabead SP825, and a recovered product 4) was obtained. The total isoflavone content was 11.5% and the aglycone ratio was 0.31.

  Therefore, when the amount of aglycone 3) obtained by the production method of the present invention is compared with the amount of aglycone 4) obtained by preparing from the supernatant, 30.3 × 0.78 = 23.634 and 11.5 × 0.31 = 3.565. Therefore, the amount of aglycone 3) obtained by the production method of the present invention is about 6.63 times the amount of aglycone 4) obtained from the supernatant.

  That is, a high content isoflavone aglycone composition can be efficiently obtained by combining (I) an aglycone production step with an enzyme and (II) a recovery step of the produced precipitate.

  By the method disclosed in the present invention, an isoflavone aglycone that is efficiently and physiologically useful can be produced from an isoflavone glycoside concentrate extracted from a plant or extracted and purified from a plant.

  Hereinafter, the present invention will be specifically described with reference to examples.

  Many commercially available industrial enzymes are crudely purified products, and often have glucosidase activity as an impurity, that is, activity to convert isoflavone glycosides to aglycone. The conversion ability of glycosides to aglycone was examined for these commercially available enzymes.

  5.0 g of 40.0% isoflavone glycoside preparation prepared from soybean hypocotyl was suspended in 50 ml of ion exchange water, 0.5 g of each enzyme shown in Table 1 was added thereto, and the mixture was shaken at 50 ° C. for 18 hours. This was dried and analyzed by HPLC. Table 1 shows the ratio (conversion rate) of aglycone in the total isoflavones. In this study, Uniase 2K (conversion rate 0.64) and pectinase G Amano (conversion rate 0.82) showed relatively high conversion rates (Table 1).

  A synthetic adsorbent resin SP825 adsorbate derived from soybean hypocotyl was obtained, and this was used as a raw material for enzyme treatment, and an aglycon was prepared according to the scheme of FIG. This raw material contained 25.6% as total isoflavones, and the ratio of aglycone in the total isoflavones was 0.02 (Table 2- (1)). 10.0 g of the raw material was suspended in 50 ml of ion-exchanged water, 0.50 g of Rhizopus-derived Uniase 2K (manufactured by Yakult) was added, and the mixture was shaken at 50 ° C. for 18 hours for enzyme treatment. After the reaction, a precipitate was formed, which was collected by centrifugation at 5000 r.p.m. The dried precipitate was 2.8 g, the total isoflavone content increased to 30.3%, and the aglycone ratio increased to 0.78 (Table 2- (3)).

  When the starting material was enzyme-treated, the total isoflavone content was 16.8%, the aglycone ratio was 0.62 (Table 2- (2)), and the dried supernatant obtained by resin treatment of the supernatant after centrifugation as a control was used. The total isoflavone content was 11.5%, and the proportion of aglycone was 0.31 (Table 2- (4)).

  Thus, as described above, when the amount of aglycon (3) obtained by the production method of the present invention and the amount of aglycon (4) obtained by preparing from the supernatant were compared, the production method of the present invention The amount of aglycon (3) obtained by the above is approximately 6.63 times the amount of aglycon (4) obtained from the supernatant.

  An ethanol extract derived from soybean hypocotyl was obtained using water-containing 70% ethanol, and this was used as a raw material for enzyme treatment. This raw material contained 4.6% as total isoflavones, and the proportion of aglycone in all isoflavones was 0.03. 500 g of the raw material was suspended in 2500 ml of ion-exchanged water, 12.5 g of Aspergillus pruberlentus-derived pectinase G (manufactured by Amano Enzyme) and 12.5 g were added, and the mixture was shaken at 50 ° C. for 18 hours for enzyme treatment. After the reaction, a precipitate was formed, which was collected using a Buchner filter. The dried precipitate weighed 56.8 g, the total isoflavone content rose to 15.23% and the aglycone proportion increased to 0.94.

  That is, the proportion of aglycone in the total isoflavones is about 31 times that of the raw material by the production method of the present invention.

[Comparative Example 1]
Attempts were made to prepare isoflavone aglycones according to JP-A-11-89589. The soybean hypocotyl was pretreated in an oven at 150 ° C. for 30 minutes, crushed, and sieved with 35 mesh, and then the enzyme treatment was performed. This soybean hypocotyl contained 1.8% as total isoflavones, and the proportion of aglycone in all isoflavones was 0.01. 20 g of soybean hypocotyl, Aspergillus pruberlentus-derived pectinase G (manufactured by Amano Enzyme) dispersed in 10 ml of water, 0.5 g was added, mixed well, and subjected to an enzyme reaction at 50 ° C. for 18 hours. Dried in 3 hours. The total isoflavone content of the dried soybean hypocotyl after the reaction was 0.96%, and the proportion of aglycone in the total isoflavone was 0.78.

  10 g of the dried soybean hypocotyl after the enzyme treatment was extracted twice with 50 ml of water-containing 70% ethanol. The extracted solid was 2.5 g, the total isoflavone content was 2.71%, and the proportion of aglycone in the total isoflavone was 0.75. The method of carrying out an enzymatic reaction by a method according to JP-A-11-89589 and performing ethanol extraction following this enzymatic reaction is the reverse of the preparation method of Example 3, but the total isoflavone content is In Example 3, it was 15.23%, but it was as low as 2.71%, and the conversion rate to aglycone was also 0.75, compared to 0.94 in Example 3.

  Therefore, the amount of aglycone is 15.23 × 0.94 = 14.32 in Example 3, and 2.71 × 0.75 = 2.03 in Comparative Example 1, and the amount of aglycon in Comparative Example is only about 0.14 times the amount of aglycon in Example 3.

The figure which shows the scheme of the manufacturing process of isoflavone aglycone.

Claims (2)

  A method for producing an isoflavone aglycone, comprising treating a composition containing 3.0% or more of an isoflavone glycoside with a glucosidase enzyme and recovering an insoluble component after the enzyme treatment.   The method for producing an isoflavone aglycone according to claim 1, wherein the glucosidase enzyme is derived from Rhizopus and / or Aspergillus.

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