Drug – bio-affecting and body treating compositions – Plant material or plant extract of undetermined constitution... – Containing or obtained from leguminosae
Reexamination Certificate
2001-01-25
2002-09-03
Gitomer, Ralph (Department: 1627)
Drug, bio-affecting and body treating compositions
Plant material or plant extract of undetermined constitution...
Containing or obtained from leguminosae
C426S046000, C530S378000
Reexamination Certificate
active
06444239
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a process for obtaining a composition containing soybean isoflavone aglycones mainly comprising genistein.
2. Description of the Related Art
Isoflavone compounds, such as malonyldaidzin, malonylglycitin, malonylgenistin, daidzin, glycitin, genistin, daidzein, glycitein, and genistein, are known to have estrogenic activity, antioxidative activity, antibacterial activity, antilipemia activity, anticholesterol activity, and the like. In recent years, cancer cell differentiation and induction activity, oncogene inhibitory activity, and prophylactic activity on cancers have been confirmed. Thus, the usefulness of these isoflavone compounds has been attracting attention. Many researches have revealed that the pharmacological effects, such a cancer prophylactic effect, of isoflavone compounds are primarily attributed not to the glycosides themselves but their aglycones, such as daidzein or genistein. Of the soybean isoflavone aglycones genistein has recently been proved particularly excellent in physiological activities, including antiosteoporosis activity, antiarteriosclerotic activity, and anticancer activities in the breast, the stomach and the prostate (see M. Numoto,
Cancer Research
, vol. 53, p. 5815 (1993) and S. Barnes,
Biochem. Biophys. Res. Commun.,
vol. 179, p. 661 (1991).
Methods of obtaining isoflavone compounds include the methods described in Japanese Patent Laid-Open Nos. 62-126186 and 11-89589, for example. Since 95% or more of isoflavone compounds in soybeans are present in the form of glycosides, however, the isoflavone compounds obtained by the former method mainly comprise glycosides with little amount of aglycones. Further, the isoflavone aglycones obtained by the latter method, which uses soybean hypocotyl tissue as a raw material, mainly comprise daidzein with the genistein content being about ⅙ of daidzein.
The inventors of the present invention previously applied for a patent based on their findings that soy sauce cake and soy sauce oil, which are by-produced in soy sauce production, contain isofalvone compounds substantially comprising aglycones such as daidzein and genistein and that these aglycones can be obtained efficiently by organic solvent extraction (see Japanese Patent Laid-Open No. 5-170756). The problems associated with this method, however, are that the yield of aglycones is low, the raw materials are uncommon, and a special extraction operation is needed.
SUMMARY OF THE INVENTION
The present inventors have conducted extensive investigations on production of isoflavone aglycones from soybeans and found as a result that a composition containing isoflavone aglycones the majority of which is genistein can easily be obtained by allowing an enzyme preparation having protease activity and &bgr;-glucosidase activity to thoroughly act on a soybean raw material that is easily available in the market and recovering water-insoluble matter.
Having been completed based on the above finding, the present invention provides a process for producing an isoflavone aglycone-containing composition comprising allowing a protease and &bgr;-glucosidase to act on a soy protein raw material, an extract of a soy protein raw material or a by-product of a soy protein raw material to water-solubilize the protein of soybean origin and to convert isoflavone glycosides to the corresponding aglycones, separating water-soluble components from the enzymatic reaction mixture, and recovering water-insoluble matter. The majority of the aglycones present in the resulting composition is genistein.
According to the present invention, a composition containing isoflavone aglycones mainly comprising genistein can be obtained from an easily commercially available raw material through an easy and simple operation.
DETAILED DESCRIPTION OF THE INVENTION
The raw material used in the present invention is a soy protein raw material, an extract of a soy protein raw material or a by-product of a soy protein raw material. The soy protein raw material includes whole soybeans, dehulled soybeans, and defatted soybeans. The extract of a soy protein raw material includes a soy protein isolate, a soy protein concentrate, soybean milk, and a soybean extract containing isoflavone glycosides. The by-product of a soy protein raw material includes soybean molasses, soybean whey, and soybean curd waste. To help a protease enzyme act, it is preferred that these raw materials be previously heat treated to denature the protein and deactivate the trypsin inhibitor. The heat treatment is preferably carried out at 80 to 200° C. for about 10 to 60 minutes.
The enzymes which can be used in the present invention, i.e., a protease and glucosidase, can be crude enzymes as produced by culturing microorganisms or commercially available crude enzymes, such as protease preparations. The crude enzymes may be used as concentrated or purified by ultrafiltration or column chromatography. While not limiting, the amounts of the enzymes (protease and &bgr;-glucosidase) preferably range from 0.1 to 10 parts by weight in terms of the solid content per 100 parts by weight of the soybean raw material.
The enzymes (protease and &bgr;-glucosidase) are preferably of acid species whose optimum pH is in a range of from 2.0 to 6.0 and of thermostable species whose optimum temperature is 50° C. or higher.
Any commercially available crude enzymes can be used as long as they have protease activity and &bgr;-glucosidase activity. Enzyme preparations of the genus Penicilliuni or Aspergillus origin are preferred. Analysis of &bgr;-glucosidase activity on various commercially available protease preparations by the method of Ebata, el al. (
Nippon Nogeikagaku Kaishi
, vol. 46, p. 323 (1972)) revealed that those of Penicillium or Aspergillits origin, especially those having an optimum pH in an acidic region (acid protease preparations) exhibit high activity. Specific examples of preferred commercial enzyme preparations having both protease activity (PA) and &bgr;-glucosidase activity (GA) are Molsin F (PA: 38,000 U/g; GA: 260 U/g; available from Kikkoman Corp.), Sumizyme FP (PA: 50,000 U/g; GA: 172 U/g; available from Shin-nihon Kagaku Kogyo K.K.), Protease M Amano (PA: 5,500 U/g; GA: 132 U/g; available from Amano Pharmaceutical Co., Ltd.), Miso Koso Amano Al (PA: 3,000 U/g; GA: 28 U/g; available from Amano Pharmaceutical Co., Ltd.), AO Protease (PA: 24,000 U/g; GA: 20 U/g; available from Kikkoman Corp.), and IP Koso (PA: 34,000 U/g; GA: 4 U/g; available from Kikkoman Corp.). These enzyme preparations are sold as enzymes for food so that their safety as food has been established.
Of these enzyme preparations those having a protease activity of 5,000 U/g or more (as determined by the method described in P. E. Wilcox,
Methods Enzymol., vol.
19, pp. 64-80 (1970)) and a &bgr;-glucosidase activity of 100 U/g or more (as determined by the method described in J. Ebata et al.,
Nippon Nogeikagaku Kaishi,
pp. 323-329 (1972)) are preferred.
The manner of carrying out the enzymatic reaction is not particularly restricted. For example, the soybean raw material is added to a previously prepared aqueous dispersion of the enzyme preparation followed by stirring, or the aqueous enzyme dispersion is sprayed directly on the soybean raw material, or the enzyme preparation is added to a mixture of the soybean raw material and water, followed by stirring, or enzymes (protease and &bgr;-glucosidase) are brought into contact with the soybean raw material to impregnate. While the amount of water added is not particularly limited, too much water requires of necessity larger equipment to increase the cost and to reduce the productivity. A preferred amount of water is about 50 to 10,000 parts by weight per 100 parts by weight of the soybean raw material.
The enzyme reaction conditions, while not particularly limited unless abrupt deactivation of the enzymes or putrefaction by bacteria occurs, are preferably 40 to 70° C. and 1 to 24 hours. Since isoflavone glycosides are sparingl
Izumi Toru
Kikuchi Mamoru
Manaka Tatuo
Obata Akio
Saito Makoto
Gitomer Ralph
Kikkoman Corporation
Young & Thompson
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