The present invention relates to an aglucone isoflavone enriched vegetable flour and an aglucone isoflavone enriched vegetable grit, and methods for providing such by converting isoflavone glucosides in a vegetable material to aglucone isoflavones and processing the vegetable material into a vegetable flour or grit.
Isoflavones occur in a variety of leguminous plants, including vegetable protein materials such as soybeans. These compounds include daidzin, 6xe2x80x3-OAc daidzin, 6xe2x80x3-OMal daidzin, daidzein, genistin, 6xe2x80x3-OAc genistin, 6xe2x80x3-OMal genistin, genistein, glycitin, 6xe2x80x3-OAc-glycitin, 6xe2x80x3-OMal glycitin, glycitein, biochanin A, formononentin, and coumestrol. Typically these compounds are associated with the inherent, bitter flavor of soybeans.
The isoflavones in vegetable protein materials include isoflavone glucosides (glucones) and aglucone isoflavones. Isoflavone glucosides have a glucose molecule attached to an isoflavone moiety. Additional moieties may be attached to the glucose molecule of an isoflavone glucoside, for example, 6xe2x80x3-OAc genistin contains an acetate group attached to the six position of the glucose molecule of genistin. Aglucone isoflavones consist solely of an isoflavone moiety.
Soy contains three xe2x80x9cfamiliesxe2x80x9d of isoflavone compounds having corresponding glucoside and aglucone members: the genistein family, the daidzein family, and the glycitein family. The genistein family includes the glucoside genistin, the conjugates 6xe2x80x3-OMal genistin (6xe2x80x3-malonate ester of genistin) and 6xe2x80x3-OAc genistin (6xe2x80x3-acetate ester of genistin); and the aglucone genistein. The daidzein family includes the glucoside daidzin, the conjugates 6xe2x80x3-OMal daidzin, and 6xe2x80x3-OAc daidzin; and the aglucone daidzein. The glycitein family includes the glucoside glycitin, the conjugate 6xe2x80x3-OMal glycitin, and the aglucone glycitein.
In the production of commercial products the focus has been to remove these materials. For example, in a conventional process for the production of a soy protein isolate or concentrate isoflavones in the protein are usually removed by exhaustive washing. The washes are typically discarded.
It has recently been recognized that the isoflavones contained in vegetable materials such as soybeans, clover, alfalfa, peanuts, and nearly all types of legumes have medicinal value. While all the isoflavones are of interest in medical evaluation, the aglucones are the specific isoflavones of most interest. Genistein and daidzein may significantly reduce cardiovascular risk factors. xe2x80x9cPlant and Mammalian Estrogen Effects on Plasma Lipids of Female Monkeysxe2x80x9d, Circulation, vol. 90, p. 1259 (October 1994). Genistein and daidzein are also thought to reduce the symptoms of conditions caused by reduced or altered levels of endogenous estrogen in women, such as menopause or premenstrual syndrome, as disclosed in U.S. Pat. No. 5,499,691. It has recently been recognized that aglucone isoflavones may inhibit the growth of human cancer cells, such as breast cancer cells and prostate cancer cells, as described in the following articles: xe2x80x9cGenistein Inhibition of the Growth of Human Breast Cancer Cells, Independence from Estrogen Receptors and the Multi-Drug Resistance Genexe2x80x9d by Peterson and Barnes, Biochemical and Biophysical Research, Communications, Vol. 179, No. 1, pp. 661-667, Aug. 30, 1991; xe2x80x9cGenistein and Biochanin A Inhibit the Growth of Human Prostrate Cancer Cells but not Epidermal Growth Factor Receptor Tyrosine Autophosphorylationxe2x80x9d by Peterson and Barnes, The Prostate, Vol. 22, pp. 335-345 (1993); and xe2x80x9cSoybeans Inhibit Mammary Tumors in Models of Breast Cancerxe2x80x9d by Barnes, et al., Mutagens and Carcinogens in the Diet, pp. 239-253 (1990).
As noted above, the aglucone isoflavones include daidzein, genistein, and glycitein. These aglucones have the following general formula: 
wherein, R1, R2, R3 and R4 may be selected from the group consisting of H, OH and OCH3. Genistein has the formula above where R1xe2x95x90OH, R2xe2x95x90H, R3xe2x95x90OH, and R4xe2x95x90OH, daidzein has the formula above where R1xe2x95x90OH, R2xe2x95x90H, R3xe2x95x90H, and R4xe2x95x90OH, and glycitein has the formula above where R1xe2x95x90OH, R2xe2x95x90OCH3, R3xe2x95x90H, and R4xe2x95x90OH.
It is therefore to the aglucones and enrichment of a vegetable flour and a vegetable grit with these compounds to which the present invention is directed. The present invention is also directed to methods of making an aglucone enriched vegetable flour and an aglucone isoflavone enriched vegetable grit from a vegetable material containing isoflavone glucosides.
Processes are known in the art for converting isoflavone glucosides to aglucone isoflavones, such as described in Japanese Patent Application 258,669 to Obata, et al. These processes achieve only a moderate extent of conversion of the glucosides to aglucones, and require a substantial period of time to effect this moderate extent conversion. In addition, known processes such as described in the ""669 application are directed to removing the isoflavones from vegetable materials, and do not provide aglucone isoflavone enriched vegetable flour or grit products.
A process is also known for producing an aglucone isoflavone enriched vegetable protein fiber from a vegetable protein material, as described in U.S. Pat. No. 5,320,949 to Shen. The process is a process in which isoflavone glucosides in a vegetable fiber material are converted to aglucone isoflavones with a beta glucosidase enzyme.
An aglucone isoflavone enriched vegetable flour or grit and processes for producing the same are desirable, since production of commercial vegetable flours and grits is relatively inexpensive. An aglucone isoflavone enriched vegetable flour or grit, therefore, would be an inexpensive source of aglucone isoflavone enriched products.
It is therefore an object of the present invention to provide an aglucone isoflavone enriched vegetable flour and a process for producing the same from a vegetable material containing isoflavone glucosides.
It is further object of the present invention to provide an aglucone isoflavone enriched vegetable grit, and a process for producing the same from a vegetable material containing isoflavone glucosides.
In one aspect, the present invention is a process for producing an aglucone isoflavone enriched vegetable flour or grit from a vegetable material containing isoflavone glucosides. An aqueous slurry is formed of the vegetable material. An enzyme is contacted with the isoflavone glucosides in the slurry at a temperature and a pH for a time period sufficient to convert the isoflavone glucosides to aglucone isoflavones. The vegetable material is then comminuted to form an aglucone isoflavone enriched vegetable flour or grit.
In one embodiment, the enzyme is contacted with the isoflavone glucosides in the slurry at a temperature of about 5xc2x0 C. to about 75xc2x0 C. and a pH of about 3 to about 9. Preferably the enzyme is contacted with the isoflavone glucosides in the slurry for a period of about 1 to about 24 hours, most preferably for about 1 to about 3 hours, to convert at least a majority of the isoflavone glucosides to aglucone isoflavones.
In another embodiment of the invention, the enzyme is a supplemental enzyme which is added to the slurry in an amount effective to convert the isoflavone glucosides to aglucone isoflavones. Preferably, the supplemental enzyme is a saccharidase enzyme capable of cleaving 1,4-glucoside bonds. Most preferably, the supplemental enzyme converts substantially all of the isoflavone glucosides to aglucone isoflavones in about 1 hour to about 3 hours.
In a further aspect, the invention is a process for producing an aglucone isoflavone enriched vegetable flour or grit from a vegetable material containing isoflavone glucosides, where the vegetable material is initially comminuted to form a vegetable flour or grit. An aqueous slurry is formed of the vegetable flour or grit, and an enzyme is contacted with the isoflavone glucosides in the slurry at a temperature and a pH for a time period sufficient to convert the isoflavone glucosides to aglucone isoflavones. Preferably the enzyme is a supplemental enzyme contacted with the isoflavone glucosides in the slurry at about 5xc2x0 C. to about 75xc2x0 C. and at a pH of about 3 to about 9 for about 1 hour to about 24 hours. Most preferably the supplemental enzyme is a saccharidase enzyme capable of cleaving 1,4-glucoside bonds which is contacted with the isoflavone glucosides for a period of about 1 to 3 hours to convert substantially all of the isoflavone glucosides to aglucone isoflavones.
In still another aspect, the invention is a process for producing an aglucone isoflavone enriched soy flour or grit A soy material containing isoflavone glucosides is provided from a group comprising soy cake, soy meal, soy chips, soy flakes, or combinations thereof. The isoflavone glucosides of the soy material are contacted with an enzyme at a temperature and a pH for a time period sufficient to convert the isoflavone glucosides to aglucone isoflavones. The soy material is comminuted to form an aglucone isoflavone enriched soy flour or grit.
The starting material of the process for producing the aglucone isoflavone enriched vegetable flour or grit of the present invention is any vegetable material or plant material containing isoflavone glucosides. The preferred starting material is a soybean material, however, a wide array of vegetable and plant materials may be used, including clover, alfalfa, peanuts, and nearly all types of legumes.
The vegetable or plant starting material is preferably processed into a finely divided form to render the isoflavone glucosides in the material more accessible to the various reagents and enzymes as described below. If the vegetable or plant starting material is in a state such that the isoflavone compounds in the plant material are readily accessible to external reagents or reactants, such as vegetable materials having small leafy portions, it may not be necessary to subject the plant material to such processing. The vegetable or plant starting material may be processed into a finely divided form by comminuting the material according to conventional methods for comminuting vegetable or plant materials such as grinding, shearing, or crushing.
Preferably the vegetable or plant starting material is comminuted into a flour or a grit. xe2x80x9cFlourxe2x80x9d products, as that term is used herein, refers to a comminuted form of a defatted or fat containing vegetable or plant material formed of particles having a size such that the particles can pass through a No. 100 mesh (U.S. Standard) screen and which contains vegetable protein and vegetable fiber. xe2x80x9cGritxe2x80x9d products, as used herein, refers to a comminuted form of a defatted vegetable or fat containing vegetable or plant material formed of particles having a size such that the particles can pass through No. 10 to No. 20 mesh (U.S. Standard) screens (coarse grit), No. 20 to No. 40 mesh screens (medium grit), or No. 40 up to No. 100 mesh screens (fine grit) which contains vegetable protein and vegetable fiber. A vegetable flour or a vegetable grit is defined herein as containing at least 40% and less than 60% vegetable protein, by dry weight, from 2% to 4% crude fiber, by dry weight, and from 30% to 35% carbohydrates, by dry weight.
If the vegetable material to be used as a starting material is a soybean material, the material may be soybeans or a soybean derivative. Preferably the soybean starting material is either soybean cake, soybean chips, soybean meal, soybean flakes, or a mixture of these materials. The soybean cake, chips, meal, or flakes may be formed from soybeans according to conventional procedures in the art, where soybean cake and soybean chips are formed by extraction of part of the oil in soybeans by pressure or solvents, soybean flakes are formed by cracking, heating, and flaking soybeans and reducing the oil content of the soybeans by solvent extraction, and soybean meal is formed by grinding soybean cake, chips, or flakes. The soy cake, chips, flakes, meal, or mixture of the materials may be comminuted into a soy flour or a soy grit, as described above, to render the isoflavone glucosides in the soy material more accessible to the reagents and reactants described below.
An aqueous slurry is formed of the vegetable or plant starting material by mixing the material with a sufficient amount of water. The amount of water is not critical, so long as a relatively uniform mixture or dispersion is formed and the slurry is not so viscous that mixing or agitation of the slurry is inhibited. Preferably the slurry is formed so that the vegetable or plant material is present in a concentration of about 10% to about 30% by weight, and most preferably, about 15% to about 20% by weight.
The isoflavone glucosides in the vegetable or plant material are converted to aglucone isoflavones by contacting the isoflavone glucosides with an enzyme in the slurry. The conversion produces an aglucone isoflavone enriched vegetable or plant material by converting at least a majority, and preferably substantially all of the isoflavone glucosides to aglucone isoflavones.
The conversion has been found to be dependent on the concentration of enzymes present in the slurry, and their characteristics. The enzymes required to effect the conversion are enzymes capable of cleaving the glucosidic linkage between the isoflavone moiety and the glucose molecule of the isoflavone glucosides. In a preferred embodiment, the enzymes are saccharidase or gluco-amylase enzymes capable of cleaving 1,4-glucoside bonds.
The concentration of enzymes required to convert the isoflavone glucosides to aglucone isoflavones is dependent on a variety of factors including the types of enzymes present in the slurry, distribution of enzyme concentrations, activities of the enzymes, and the pH and temperature of the slurry during the conversion. The enzymes may be inherently present in the slurry either from the vegetable or plant material or from microbial growth in the slurry. Such inherently present enzymes are referred to herein as xe2x80x9cresidualxe2x80x9d enzymes, and enzymes that are added to the slurry are referred to herein as xe2x80x9csupplementalxe2x80x9d enzymes.
Sufficient enzyme should be present in the slurry to convert at least a majority, and preferably substantially all, of the isoflavone glucosides to aglucone isoflavones. Generally, if the residual enzymes in the slurry are insufficient to effect the conversion, supplemental enzymes should be added to the slurry. In a preferred embodiment, supplemental enzymes are added to the slurry regardless whether sufficient residual enzymes are present in the slurry since addition of supplemental enzymes dramatically decreases the time necessary to effect the conversion of the isoflavone glucosides to aglucone isoflavones. If supplemental enzymes are added, the supplemental enzymes should be added so that the total concentration of enzyme present is about 0.1% to about 10% by weight of the vegetable or plant material on a dry basis.
Supplemental enzymes are selected based on optimum activity at selected pH and temperature conditions, and cost effectiveness. The supplemental enzymes are enzymes capable of cleaving the bond between the isoflavone moiety and the glucose molecule of the isoflavone glucosides, such as saccharidase and gluco-amylase enzymes capable of cleaving 1,4-glucoside bonds. Preferred supplemental enzymes are commercially available alpha-and beta-glucosidase enzymes, beta-galactosidase enzymes, gluco-amylase enzymes, and pectinase enzymes. Particularly preferred are enzymes such as Biopectinase 100L (which is preferably utilized at a pH range of from about 3 to about 6), Biopectinase 300L (optimum pH range from about 3 to about 6), Biopectinase OK 70L (optimum pH range from about 3 to about 6), Biolactase 30,000 (optimum pH range from about 3 to about 6) Neutral Lactase (optimum pH range from about 6 to about 8), all of which are available from Quest International, 1833 57th Street, Post Office Box 3917, Sarasota, Fla. 34243. Also especially preferred are Lactase F (which is preferably utilized at a pH range of from about 4 to about 6), and Lactase 50,000 (optimum pH range from about 4 to about 6), both available from Amano International Enzyme Co., Inc., Post Office Box 1000, Troy, Va. 22974. Other particularly preferred supplemental enzymes include G-Zyme G990 (optimum pH from about 4 to about 6) and Enzeco Fungal Lactase Concentrate (optimum pH from about 4 to about 6):available from Enzyme Development Corporation, 2 Penn Plaza, Suite 2439, New York, N.Y. 10121; Lactozyme 3000L (which preferably is utilized at a pH range from about 6 to about 8), and Alpha-Gal 600L (which preferably is utilized at a pH range of from about 4 to about 6.5), available from Novo Nordisk Bioindustrials, Inc., 33 Turner Road, Danbury, Conn. 06813; Maxilact L2000 (which is preferably utilized at a pH range of from about 4 to about 6), available from Gist Brocades Food Ingredients, Inc., King of Prussia, Pa., 19406; and Neutral Lactase (which is preferably utilized at a pH range of from about 6 to about 8), available from Pfizer Food Science Group, 205 East 42nd Street, New York, N.Y. 10017.
The pH range for enzymatic conversion of the isoflavone glucosides to aglucone isoflavones is from about 3 to about 9. The pH that is utilized depends primarily upon the type of enzyme used, and should be selected accordingly. The residual enzyme is active within a pH range of about 7 to about 9, although it is believed that the pH of the extract is lowered during the course of the conversion. The supplemental enzymes are active within an optimum pH range specified by the manufacturer of the enzyme, as shown above for several specific enzymes. Typically the supplemental enzymes are active either in a neutral pH range from about 6 to about 8, or in an acidic pH range from about 3 to about 6.
The pH of the slurry may be adjusted to a desired value for conducting the isoflavone conversion. The pH may be reduced or increased using conventional acidic and basic reagents, respectively, such as hydrochloric acid, acetic acid, sulfuric acid, sodium hydroxide, and potassium hydroxide.
The temperature range for the isoflavone glucoside conversion is from about 5xc2x0 C. to about 75xc2x0 C. The temperature significantly affects the activity of the enzymes, and therefore, the rate of conversion. The supplemental enzymes may be active above 70xc2x0 C., for example Alpha-Gal 600L is active at 75xc2x0 C., however, it is preferred to conduct the conversion at lower temperatures to avoid enzyme deactivation. In preferred embodiment, the conversion is effected between about 35xc2x0 C. and about 45xc2x0 C.
The time required for the isoflavone glucoside conversion step depends upon enzyme-related factors, particularly concentration and the temperature and pH of the system. In most instances it is possible to achieve substantially complete conversion within 24 hours, however, it is preferred that supplemental enzyme be added to dramatically decrease the time required for the conversion. The selected supplemental enzyme, enzyme concentration, pH and temperature preferably cause substantially complete conversion within 3 hours, and most preferably within 1 hour.
The very high degrees of conversion with this process are such that at least a majority, and preferably substantially all, the isoflavone glucosides present in the slurry are converted to aglucone form. The term xe2x80x9ca majorityxe2x80x9d refers to an extent of conversion of isoflavone glucosides to aglucone isoflavones of at least about 50%. The term xe2x80x9csubstantially allxe2x80x9d refers to an extent of conversion of isoflavone glucosides to aglucone isoflavones of at least about 80%, and most preferably at least about 90%.
Upon completion of the conversion of isoflavone glucosides to aglucone isoflavones, the aglucone enriched vegetable or plant material may be comminuted to produce an aglucone enriched vegetable or plant flour or grit if the material was not comminuted before formation of the slurry, or if the material had not been sufficiently comminuted to form a flour or a grit. Preferably the aglucone isoflavone enriched vegetable or plant material is separated from the slurry and dried prior to being comminuted. If the vegetable or plant material had been sufficiently comminuted to form a flour or a grit prior to the conversion, the material is separated from the slurry and dried.
The vegetable or plant material is separated from the slurry by dewatering the vegetable or plant material. xe2x80x9cDewateringxe2x80x9d as used herein is defined as removing water from the vegetable or plant material slurry without removing substantial amounts of the protein, aglucone isoflavone, fiber, and carbohydrate components of the vegetable or plant material. Dewatering includes evaporation, flash vaporization under reduced pressure, and spray drying.
The aglucone isoflavone rich vegetable flour or grit of the invention contains substantially increased amounts of aglucone isoflavones relative to conventional vegetable flours or grits. Conventional commodity vegetable flours and grits typically contain from 5 to 75 parts per million (xe2x80x9cppmxe2x80x9d) genistein (2-17% genistein relative to all genistein family isoflavones by weight normalized to only the genistein component of the genistein family isoflavones, where xe2x80x9cgenistein family isoflavonesxe2x80x9d is herein specifically defined as genistein, genistin, 6xe2x80x3-O-Mal genistin, and 6xe2x80x3-O-Ac genistin); and from 2 to 125 ppm daidzein (2-15% daidzein relative to all daidzein family isoflavones by weight normalized to only the daidzein component of the daidzein familiy isoflavones, where xe2x80x9cdaidzein family isoflavonesxe2x80x9d is herein specifically defined as daidzein, daidzin, 6xe2x80x3-O-Mal daidzin, and 6xe2x80x3-O-Ac daidzin). Specialty flours and grits, such as flours and grits derived from high sucrose soybeans, may contain up to 200 ppm of genistein and 225 ppm of daidzein. The vegetable flours and grits of the present invention, however, contain at least 400 ppm of genistein and 400 ppm of daidzein; more preferably at least 500 ppm genistein and 500 ppm of daidzein; and most preferably at least 700 ppm of genistein and 700 ppm of daidzein. The vegetable flours and grits of the present invention also contain at least 50% genistein relative to all genistein family isoflavones by weight normalized to only the genistein component of the isoflavones; and at least 50% daidzein relative to all daidzein family isoflavones by weight normalized to only the daidzein component of the isoflavones, and more preferably contain at least 80% genistein relative to all genistein family isoflavones and at least 80% daidzein relative to all daidzein family isoflavones, and most preferably at least 90% genistein relative to all genistein family isoflavones and at least 90% daidzein relative to all daidzein family isoflavones.
The present invention is illustrated in more detail by the following examples using a soybean material as the vegetable material containing isoflavone glucosides. The soybean material is in the form of finely ground soybean flakes (soy flour) which have been extracted with a solvent to remove oil from the flakes.
As noted above, soy materials include the genistein, daidzein, and glycitein xe2x80x9cfamiliesxe2x80x9d of isoflavones having corresponding glucoside, conjugate, and aglucone members, where the genistein family contains the conjugates 6xe2x80x3-OMal genistin, and 6xe2x80x3-OAc genistin, the glucoside genistin, and the aglucone genistein; the daidzein family contains the conjugates 6xe2x80x3-OMal daidzin, 6xe2x80x3-OAc daidzin, the glucoside daidzin, and the aglucone daidzein; and the glycitein family contains the conjugate 6xe2x80x3-OMal glycitin, the glucoside glycitin, and the aglucone glycitein.
In the following examples the relative concentrations of the isoflavones are measured as a percentage of a family of isoflavones. For example, in the genistein family: % genistin+% 6xe2x80x3-OMal genistin+% 6xe2x80x3-OAc genistin+% genistein=100%. The extent of conversion of gluco sides to aglucones can be determined by comparing the relative percentages of the glucoside to the aglucone compounds in a respective isoflavone family.