MXPA97006806A - Extract of vegetable protein and protein material enriched with aglucon isoflavone, and high content genistein and daidzein materials and procedure to produce myself - Google Patents

Abstract

a material protein and vegetable protein enriched isoflavone aglycone is provided, as wellas also or material high genistein content and material of high daidzein, isoflavone conjugates in a plant material are converted to isoflavone glucosides treating the plant material at a temperature and a pH for a time sufficient to effect the conversion period, the isoflavone glucosides are converted to isoflavone aglucosa by enzymatic reaction, the plant material is extracted with an agent aqueous extraction having a pH above about the isoelectric point of the protein in the vegetable material to extract protein and the isoflavones either entities or after converting the isoflavone conjugates to isoflavone glucosides or to convert the isoflavone glucosides isoflavone aglycone, it occurs a protein material enriched with isofalvone of aglucone precipitand or protein and isoflavones agulcona from the extract, a material high genistein content material or a high daidzein can be produced from the protein extract enriched isoflavone aglycone or protein material enriched isoflavone aglycone or the protein material enriched with aglucone isoflavone by separating the high-content genistein material or the high daidzein content of the extract or protein material.

Description

EXTRACT OF VEGETABLE PROTEIN AND PROTEIN MATERIAL ENRICHED WITH ISOFLAVONE OF ATLUCONfl- AND HIGH-CONTENT GENISTEIN AND DAIDZEIN MATERIALS AND PROCEDURE FOR PRODUCE THEMSELVES FIELD OF THE INVENTION The present invention relates to an extract of vegetable protein and protein material enriched with aglucone isoflavone, and methods for providing such by performing a two-step procedure for converting the isoflavone conjugates into a vegetable protein material for aglucone isoflavones. and to a high genistein content material and a high daidzein content material and methods for providing such from a protein material enriched with aglucone isoflavone.

BACKGROUND OF THE INVENTION Isoflavones exist in several legume plants, including plant protein materials such as soy. These compounds include daidzin, 6"-OAc daidzin, 6" -drial daidzin, daidzein, genistin, 6"-OAc genistin, 6" -Ol1 to genistin, genistein, glycitin. 6"-0Ac glycitin, 6" -OMal glycitin, glycitein, biochanin A, forrnononentin, and cistrol. Typically, these compounds they are associated with the inherent bitter taste of the soy. Isoflavones in plant protein materials include isoflavone glucosides (glucones), isoflavone conjugates and aglucone isoflavones. The isoflavone glycosides have a glucose molecule bound to a portion of isoflavone. The .isoflavone conjugates have additional portions attached to the glucose molecule of an isoflavone glucoside, for example, 6"-0Ac genistin contains an acetate group attached to the six position of the genistin glucose molecule.The aglucone isoflavones They consist of only one portion of isoflavone, soy contains three "families" of isoflavone compounds that have corresponding glycoside, conjugate and aglucone members: the genistein family, the daidzein family and the glycitein family. genistein includes the genistin glycoside, the conjugates 6"0Mal genistin (ester of 6" - genistin nalonate) and 6"-0Ac genistin (6" ester-genistin acetate), and genlisine aglucona. daidzein includes the daidzin glucoside, the conjugates 6"-0Mal daidzin and 6" -0Ac daidzin, and the aglycone daidzein.The glycitein family includes the glycitide glycitin, the conjugate 6"-0Mal glycitin; and aglucona glycitein. In the production of commercial products, such as isolates and vegetable protein concentrates, the objective has been to remove these materials. For example, in a procedure conventionally to produce a soy protein isolate or concentrate in which soy flakes are extracted with an aqueous alkaline medium, much of the isoflavones are solubilized in the extract together with the soy protein. The rotein is precipitated from the extract by acidification of the extract and separated to form an isolate or a concentrate, leaving a serum that retains a large part of the solubilized isoflavones. The residual isoflavones remaining in the precipitated acid protein are often removed by thorough washing. Typically, serum and washes are discarded.

It has recently been recognized that isoflavones contained in plant proteins such as soybean have medicinal value. Although all isoflavones are of interest in medical evaluation, agglucones are the specific isoflavones of most interest. Genistein and daidzein can significantly reduce cardiovascular risk factors. "Plant and Mamp-aJan Estrogen Effects on Plasma Lipids of Fe ale Mon eys", Circulation, vol. 90, p. 1259 (Oct 1994). It is also thought that genistein and daidzein reduce the symptoms of conditions caused by reduced or altered levels of endogenous estrogen in women, such as menopause or premenstrual syndrome. In addition, it has recently been recognized that aglucone isoflavones can inhibit the growth of human cancer cells, such as breast cancer cells and prostate cancer cells, as described in US Pat. following articles: "Genistem Inhibition of the Growth of Human Breast Cancer Cells, Tndependence from Estrogen Receptors and the Multi-Drug Resistance Gene," by Peterson and Barnee, Biochemistry and Biophysical Research Communications. Vol. 179, No. 1, pp. 661-667, flgo. 30, 1991; "Gemstein and Biochamn A Inhibit the Growth of Human Prostate Cancer Cells but not Epidermal Growth Factor Receptor Tyrosme Autophosphorylation", by Peterson and Barnes, The Prostate, Vol. 22, pp. 335-345 (1993); and "Soybeans Tnhibit Mammary Turnors m Models of Breast Cancer", by Barnes and others, Mutagens and C rcinogens in the Diet, pp. 239-253 (1990). As mentioned above, the isoflavones of aglucone include daidzein, genistein and glycitein. These agluconas have the following general formula: wherein Ri, 2) R3 and RA can be selected from the group consisting of H, OH and OCH3. Genistein has the above formula, wherein R? = OH, R2-H, R3 = 0H and R < ¡= OH, daidzein has the above formula wherein R? = OH, R2 = H, R3 = H and R-i = OH, and the glycitema has the above formula wherein R1 = OH. R2 = 0CH3, R3 ^ H and A = OH. Therefore, the present invention is directed to the aglucones and to the enrichment of a vegetable protein extract and a vegetable protein material with these compounds, and also to a material of high gemsteic content and a material of high daidzein content. The present invention is also directed to methods for obtaining a vegetable protein extract enriched with aglucone, a plant protein material enriched with aglucone isoflavone, a material of high gemstein content and a material of high daidzein content. A general procedure for converting plant protein isoflavone conjugates to aglucone isoflavones is known and is provided in the currently pending application of E.U.A. Ser. No. 08 / 477,102, filed on June 7, 1995, recognized by the agent of the present application. Processes for converting glycosides into agjucone isoflavones are also known. A method for converting the isoflavone glycosides to aglucone isoflavones to produce a plant protein extract enriched with aglucone isoflavone and a plant protein isolate enriched with aglucone isoflavone is provided in the currently pending PCT patent application No.

P0 / US94 / 10697, recognized by the agent of the present invention. Other methods for converting isoflavone glycosides to aglucone isoflavones, as described in Japanese Patent Application No. 258,669 by Obata et al., Are also known in the art. Said methods do not provide for the conversion of the isoflavone conjugates to aglucone isotlavones, nor do they provide a high-content material of gemstema or a material of high daidzein content derived from a plant protein isolate enriched with aglucone. Furthermore, these procedures achieve only a moderate degree of conversion of the glucosides into aglucones, and require a substantial period to effect this conversion of moderate degree. Therefore, such procedures are not convenient for large-scale commercial operations. It is therefore an object of the present invention to provide an extract of vegetable protein enriched with aglucone isoflavone and a process for producing the same. Another objective of the present invention is to provide a vegetable protein material enriched with aglucone isoflavone, and a process for producing the same. It is still another object of the present invention to provide a high gemstein material and a high daidzein content material and processes for produce them from a plant protein material enriched with aglucone isoflavone.

BRIEF DESCRIPTION OF THE INVENTION The invention is an extract enriched with isoflavone of aglucone and a process for producing the same from a plant material containing isoflavone and protein conjugates. The method comprises extracting the plant material containing the isoflavone conjugates with an aqueous extraction agent having a pH above about the isoelectric point of the protein in the plant material. The aqueous extract is treated at a temperature and pH for a sufficient period to convert the isoflavone conjugates to isoflavone glycosides. An enzyme is contacted with the isoflavone glycosides in the aqueous extract at a temperature and a pH for a sufficient period to convert the isoflavone glycosides into aglucone isoflavones, producing the extract enriched with aglucone isoflavone. In one embodiment of the invention, the extraction is carried out at a pH of from about 6 to about 10. Preferably, the weight ratio of the extraction agent to the vegetable protein material is from about 8: 1 to about 16. :1. In another embodiment of the invention, the conjugates of isoflavone are converted to isoflavone glycosides by treating the aqueous extract at a temperature of about 2 ° C to about 121 ° C and a pH value of about 6 to about 13.5. Preferably, the conversion is carried out at a pH of about 11 and at a temperature of about 5 ° C to about 50 ° C or, alternatively, at a pH of about 9 and at a temperature of about 45 ° C to about 75 ° C. . In yet another embodiment of the invention, the isoflavone glycosides are converted to aglucone isoflavones by contacting the isoflavone glycosides with an enzyme in the aqueous extract at a temperature between about 5 ° C and about 75 ° C. pH value of between about 3 and about 9. Preferably, the enzyme is a saccharose enzyme capable of cutting the 1,4-glucosidic bonds. In another embodiment of the invention, the pH of the aglucone isoflavone-enriched extract is adjusted to approximately the isoelectric point of the protein in the extract to precipitate a protein material containing protein and aglucone isoflavones. High conversion rates of isoflavone conjugates are made in isoflavone glycosides, and isoflavone glycosides in aglucone isoflavones. In an embodiment, most and preferably substantially all isoflavone conjugates are converted to isoflavones of aglucona In another aspect, the invention is a protein material enriched with aglucone isoflavone, and a process for producing the same from a protein material enriched with isoflavone glucoside derived from a plant material containing isoflavone and protein conjugates. The plant material is extracted with an aqueous extraction agent having a pH above about the isoelectric point of the protein in the plant material. The aqueous extract is treated at a temperature and pH for a period sufficient to convert the isoflavone conjugates to isoflavone glycosides. A protein material containing the isoflavone glycosides is separated from the extract, and the isoflavone glycosides in the protein material are contacted with an enzyme at a pH and a temperature for a sufficient period to convert the isoflavone glycosides into isoflavones. of agl? cona. In still another aspect, the invention is an extract enriched with aglucone isoflavone, and a process for producing the same from a plant material enriched with isoflavone glucoside derived from a plant material containing isoflavone and protein conjugates. An aqueous suspension is formed of the plant material, and the suspension is treated at a pH and at a temperature for a sufficient period to convert the isoflavone conjugates to isoflavone glycosides. The enriched plant material with isoflavone glycoside is then extracted with an aqueous extraction agent having a pH above about the isoelectric point of the protein in the plant material. The isoflavone glycosides in the extract are contacted with an enzyme at a temperature and pH for a period sufficient to convert the isoflavone glycosides to the aglucone oflavones. In a preferred embodiment, the isoflavone glycosides in the extract are contacted with an enzyme by adding an effective amount of an enzyme complementary to the extract, wherein the complementary enzyme is preferably a saccharide enzyme capable of cutting the 1,4-linkages. cosmetics. In another embodiment, a protein material enriched with aglucone isoflavone is formed from the aglucone isoflavone-enriched extract by adjusting the pH of the extract to approximately the isoelectric point of the protein to precipitate a protein material containing protein and isoflavone from aglucone. . In still another aspect, the invention is a protein material enriched with aglucone isoflavone, and a process for producing the same from a protein material derived from a plant material containing isoflavone and protein conjugates. The plant material is extracted with an aqueous extraction agent having a pH above about the isoelectric point of the protein.

A protein material containing the isoflavone conjugates is separated from the extract by adjusting the pH of the extract to approximately the isoelectric point of the protein. An aqueous suspension is formed of the protein material, and the aqueous suspension is treated at a pH and at a temperature for a sufficient period to convert the isoflavone conjugates into isoflavone glycosides. The isoflavone glycosides in the suspension are contacted with an enzyme at a pH and at a temperature for a sufficient period to convert the isoflavone glycosides to aglucone isoflavones. In a preferred embodiment, the isoflavone glycosides in the suspension are contacted with an enzyme by adding an effective amount of an enzyme complementary to the suspension, wherein the complementary enzyme is preferably an saccharide enzyme capable of cutting the bonds 1,4-gl? Cosídicos. In yet another aspect, the invention is a material of high genietein content and a method for recovering same from a plant protein material enriched with aglucone isoflavone. A vegetable protein material enriched with aglucone isoflavone is supplied and extracted with an aqueous extraction alcohol to produce an extract enriched with aglucone isoflavone. The extract is contacted with an adsorbent material for a sufficient period to separate a high-content material. genistein from the extract. In a final aspect, the invention is a material of high daidzein content and a process for producing same from a plant protein material enriched with aglucone isoflavone. A plant protein material enriched with aglucone isoflavone is supplied and extracted with an aqueous extraction alcohol to produce an extract enriched with aglucone isoflavone. The extract is contacted with an adsorbent material for a period sufficient to remove a material of high daidzein content from the extract.

DESCRIPTION OF THE PREFERRED MODALITIES The starting material of the method of the preferred embodiment is any vegetable protein material or plant material containing isoflavone conjugates and a vegetable protein. In a preferred embodiment, the starting material is a soy material, since the process is particularly suitable for producing extracts and protein materials enriched with aglucone isoflavone from the soy material. The term "soy material" as used in the present invention refers to soy or any type of soy derivative. The most preferred starting material are soy flakes from which the oil has been removed by solvent extraction in accordance with conventional procedures in the art. The current procedure is generally applied to a wide range of plant protein materials in addition to soy or soy materials. Depending on the type of plant material containing the isoflavone conjugates, it may be necessary in some cases to process the plant material in a finely divided form. This may be convenient for making the isoflavone compounds contained in the plant material accessible to the different reatives, as described in greater detail below. The material can be milled, crushed or otherwise processed by conventional methods known in the art. If the plant material is in such a state that the isoflavone compounds in the plant material are readily accessible to external reagents, such as small foliate portions of certain plants, it may not be necessary to sut the plant material to such processing. In First step or operation, the vegetable protein and the isoflavone compounds including isoflavone conjugates are extracted from the vegetable protein material. The flakes are extracted with an aqueous extraction agent having a pH above about the isoelectric point of the plant material, preferably at a pH of about 5.0 to about 10.0, and more preferably at a pH of about 6.7 to about 9.7. If necessary, alkaline reagents can be used such as sodium hydroxide, potassium hydroxide and calcium hydroxide to raise the pH of the aqueous extraction agent. The isoflavone compounds and the desired vegetable proteins are soluable in the aqueous extract. It is preferred, in order to maximize the recovery of these compounds in the aqueous extract, that the weight ratio of the soy flakes or other vegetable protein material to the extraction agent be controlled at specific levels to solubilize as much as possible the isoflavones in the plant material. The extraction of proteins and isoflavones can be carried out by conventional extraction methods which include countercurrent extraction of the vegetable protein material, preferably at a weight ratio of the aqueous extraction agent to the vegetable protein material, of about 8%. : 1 to approximately 16: 1. After extracting the plant protein material, the extraction agent provides an aqueous extract of protein and isoflavones. Alternatively, a two-step extraction process may be used, wherein preferably the weight ratio of the extractant to the vegetable protein material in an initial extraction is about 10: 1, and the weight ratio of the extractant to the Plant protein material in a second extraction is about 6: 1, or less, so that the combined weight ratio of the extraction agent Regarding the vegetable protein material in both extractions, it does not exceed a total weight ratio of the extraction agent to the vegetable protein material of approximately 16: 1. Other extraction methods may also be used in which the weight ratio of the extractant to the vegetable protein material is preferably 16: 1 or less. In the first step? Isoflavone conversion operation, the isoflavone conjugates in the aqueous extract are converted into isoflavone glucids to produce an extract enriched with isoflavone glycosides. It has been found that the conversion depends on the pH and the temperature of the aqueous extract. The pH scale for converting the isoflavone conjugates into isoflavone glycosides is from about 6 to about 13.5. The pH of the aqueous extract should be adjusted to the desired pH, if necessary, with an appropriate base, caustic agent, or basic reagent if the pH is to rise or, if the pH is to be reduced, with an appropriate acid or acid reagent. It has been found that the conversion of the isoflavone conjugates to isoflavone glycosides is base-catalyzed and thus, it is more preferred to use a high pH to achieve rapid conversion. The most preferred pH for converting the isoflavone conjugates to isoflavone glucosides is a pH of about a to about 11. The temperature scale for converting the Isoflavone conjugates in isoflavone glycosides is from about 2 ° C to about 121 ° C. The temperature scale at which the conversion occurs rapidly depends on the pH of the aqueous extract. The inventors have found that conversion occurs easily at lower temperatures when the pH is relatively high. For example, at a pH of about 11, the conversion occurs rapidly and efficiently at a temperature range of about 5 ° C to about 50 ° C. At a pH of about 9, the conversion occurs efficiently within a temperature range of about 45 ° C to about 75 ° C. When the pH of the aqueous extract is relatively low, the conversion occurs at higher temperatures. For example, at a pH of about 6, the conversion occurs within a temperature range of about 80 ° C to about 121 ° C. In a preferred embodiment, the conversion is carried out at about 35 ° C and at a pH of about 11. In another preferred embodiment, the conversion is carried out at a temperature of about 73 ° C and a pH of about 9. The period that is required to convert the isoflavone conjugates into isoflavone glycosides in the first step depends mainly on the pH and the temperature scale used. Said conversion times typically vary from about 15 minutes to several hours or more. The conversion occurs more rapidly at a higher pH and at a higher temperature. At a pH of approximately 9, the Conversion concludes substantially in about 4 hours to about 6 hours at 73 ° C. In a more preferred embodiment, the isoflavone conjugates are converted to isoflavone glycosides in about 30 minutes to about 1 hour, preferably about 45 minutes, to a pH of about 11 and at a temperature of about 35 ° C. The first conversion step is preferably carried out in an aqueous system. Other compatible aqueous components, such as low molecular weight alcohol and other water-soluble solvents, may also be present in the system. The first conversion step of the isoflavone is remarkably efficient, with at least the majority and preferably substantially all of the isoflavone conjugates in ieoflavone glycosides becoming. Typically from about 80% to about 100% of the isoflavone conjugates are converted to isoflavone glycosides. By using the preferred reaction parameters described above, it is possible to achieve 95% conversion or rne. This high conversion speeds are particularly attractive for large-scale commercial operations. In a second step or operation of conversion of the io flavone, the isoflavone glucosides produced in the first conversion step, as well as the isoflavone glycosides previously resident in the aqueous extract, are converted into aglucone isoflavones by reaction enzymatic The conversion produces an extract enriched with aglucone isoflavone from the extract enriched with isoflavone glucoside. It has been found that the second conversion step depends on the concentration of the enzymes present in the extract, and their characteristics. The enzymes that are required to effect the conversion are enzymes capable of cutting the glycosidic bond between the isoflavone portion and the glucose molecule of isoflavone glucosides. In a preferred embodiment, the enzymes are sacapdases, esterases or glyco-archaea capable of cutting the 1, 4-gl? Cosid? C links. The concentration of the enzymes required to convert isoflavone glycosides to isoflavone agglomerates depends on several factors including the type of enzymes present in the aqueous extract, the distribution of enzyme concentrations, the activities of the enzymes and the pH and temperature of the extract during the conversion. The enzymes may be inherently present in the extract either from the plant protein material or from the microbial growth in the extract. Said inherently present enzymes are referred to herein as "residual" enzymes, and the enzymes that are added to the extract are referred to herein as "complementary" enzymes. Sufficient enzyme must be present in the extract to convert at least the majority of, and preferably substantially all, the isoflavone glycosides in aglucone isoflavones. In general, if the residual enzymes in the extract are insufficient to effect the conversion, enzymes complementary to the extract should be added. In a preferred embodiment, enzymes complementary to the extract are added regardless of whether sufficient residual enzymes are present in the extract, since the addition of the complementary enzymes dramatically decreases the time necessary to substantially carry out the complete conversion of the glucosides to aglucone. If complementary enzymes are added, the complementary enzymes should be added in such a way that the total concentration of the enzyme present is from about 0.1% to about 10% by weight of the vegetable protein material on a dry weight basis. Lae complementary enzymes are selected based on optimal activity at selected pH and temperature conditions, and cost effectiveness. Complementary enzymes are enzymes capable of cutting the bond between the isoflavone portion and the glucose molecule of the isoflavone glycosides, such as saccharose enzymes, esterases and gluco-amylases capable of cutting the 1,4-glucoeidic bonds. Preferred complementary enzymes are alpha- and beta-glucosidase enzymes, beta-galactosidase enzymes, gl-co-anilase enzymes, and commercially available pectinase enzymes. Particularly preferred are enzymes such as Biopectinase 1001, (which is preferably used on a scale from H about 3 to about 5), Biopectinase 3001 (optimal pH scale from about 3 to about 6), Biopectinase OK 701 (optimal pH scale from about 3 to about 6), Biolactase 30,000 (optimal scale pH from about 3 to about 6), Neutral Lactase (optimum pH scale from about 6 to about 8), all of which are available from Quest International, 1833 57th Street, Post Office Box 3917, Sarasota, Florida 34243. Also especially preferred are Lactase F (which is preferably used at a pH scale of about 6), and Lactase 50,000 (optimum pH scale of about 4 to about 6), both available from Amano International Enzyme Co., Inc., Post Office Box 1000, Troy, Virginia 22974. Other particularly preferred complementary enzymes include G-Zyrne G990 (optimum pH from about 4 to about 6) and Enzeco Fungal Lactase Concentrate (pH). thymus of around 4 to about 6) available from Enzyme Development Corporation, 2 Penn Plaza, Suite 2439, New York, New York 10121; Lactozyrne 3000L (which is preferably used at a pH scale of about 6 to about 8) and Alpha-Gal 600L (which is preferably used at a pH scale of about 4 to about 6.5), available from Novo Nordisk Bioindustrials, Inc., 33 Turner Road, Danbury, Connecticut 06813; Maxilact 1,2000 (which is preferably used at a pH scale of about 4 to about 6), available from Gist Brocades Food Ingredients, Inc., King of Prussia, Pennsylvania, 19406; and Neutral Lactase (which is preferably used at a pH scale of about 6 to about 8), available from Pfizer Food Science Group, 205 East 42nd Street, New York, New York 10017. The pH scale for converting the isoflavone glycosides in aglucone isoflavones ee from about 3 to about 9. The pH used depends mainly on the type of enzyme used, and should be selected co or corresponds. The residual enzyme is active within a pH range of about 7 to about 9, although it is thought that the pH of the extract decreases during the course of the conversion. The complementary enzymes are active within an optimal pH range specified by the enzyme manufacturer, as shown above for several specific enzymes. Typically, the complementary enzymes are active on a neutral pH scale of from about 6 to about 8, or on an acid pH scale of about 3 to about 6. The pH can be adjusted to a desired value to drive the second step of isoflavone conversion. In most cases, the pH is reduced from the relatively high or basic pH of the first conversion step of the isoflavone, by the addition of one or more appropriate acids such as acetic acid, sulfuric acid, phosphoric acid, hydrochloric acid or any other appropriate reagent.

The temperature scale for the second conversion step of the isoflavone is from about 5 ° C to about 7-5 ° C. The temperature significantly affects the activity of the enzymes and, therefore, the speed of conversion. The complementary enzymes can be active above 70 ° C, for example Alpha-Gal 600L is active at 75 ° C; however, it is preferred to perform the conversion at lower temperatures to avoid inactivation of the enzyme. In a preferred embodiment, the conversion takes place between about 35 ° C and about 45 ° C. The time required for the second conversion step of isoflavone depends on factors related to the enzyme, particularly the concentration, and the temperature and pH of seven a. In most cases, it is possible to achieve a substantially complete conversion within 24 hours; however, it is preferred to add the complementary enzyme to dramatically increase the rate of the reaction. The selected complementary enzyme, the concentration of the enzyme, the pH and the temperature, preferably cause the substantially complete conversion within 2 hours, and more preferably within 1 hour. Very high degrees of conversion with this process are such that at least most, and preferably all subetanially, isoflavone glycosides present in the extract are converted to the agglomerate form. The term "the majority" refers to a degree of conversion of isoflavone glycosides in aglucone isoflavones of at least about 50%. The term "substantially all" refers to a degree of conversion of isoflavone glycosides to agglomerate isoflavones of at least about 80%, and preferably at least about 90%. Such high conversion rates on a reliable basis are remarkable, and are convenient for commercial applications. A protein material enriched with egoflavone of aglucone can be recovered from the extract enriched with isoflavone aglucone. After concluding the second conversion step of the isoflavone, the pH is adjusted by the addition of acid, if necessary, to about the isoelectric point for the vegetable protein, for the soy protein generally between about 4.0 to about 5.0 and preferably between about 4.4 to about 4.6. The protein is precipitated from the extract adjusted in its pH in the form of a rennet. A significant portion of the aglucone isoflavone is captured in the rennet. After the precipitation, the rennet or the precipitated protein are separated from the extract to form a protein material enriched with aglucone isoflavones. Preferably, the protein material enriched with aglucone isoflavone is separated from the extract by centrifugation or filtration. In the most preferred embodiment, the washing of the separated protein material is totally avoided or minimized to substantially reduce the removal of agglomerate isoflavones from the protein material. Therefore, the washing of the protein material with water can be completely avoided, or limited to a single wash with water during which the weight ratio of the water to the protein material is between about 2: 1 to about 6: 1. The lack of washing of the precipitated rennet provides a protein material enriched with the desired levels of isoflavones, even though a more extensive wash can be carried out with a lower recovery of the isoflavones. The separated protein material can be dehydrated by centrifugation or concentration or a combination of the rnisrnae, and dried in a conventional manner. The preferred embodiment is not intended to be limited by a particular dehydration means, although it is preferred to use conventional dehydration and drying techniques such as centrifugation and spray drying to form a dry protein material. The method of the preferred embodiment described above utilizes both the first and the second conversion steps of the isoflavone immediately after obtaining an extract. The present invention also includes a process in which a plant material containing isoflavone and protein conjugates is extracted with an aqueous extraction agent having a pH above about the isoelectric point of the protein; the first step of ? Isoflavone conversion is carried out on the extract; A protein material containing the isoflavone glycosides is separated from the extract; and the second conversion step of the isoflavone is carried out on the protein material. The steps in this procedure can be carried out in the same general manner as described above. The present invention further includes a process in which an aqueous suspension of a plant material containing isoflavone and protein conjugates is formed, the first step of conveying the isoflavone is carried out on the aqueous suspension; the plant material is extracted with an aqueous extraction agent having a pH above about the isoelectric point of the protein; and the second conversion step of the isoflavone is carried out on the isoflavone glycosides in the extract. The aqueous suspension of the plant material preferably contains up to 20% of the vegetable material by weight. The steps of this procedure may be carried out in the same general manner as described above. In addition, a protein material containing the isoflavones of aglycone can be separated from the extract after the second conversion step of the isoflavone in the manner described above. The present invention also includes a process in which a plant protein material containing isoflavone and protein conjugates is extracted with a aqueous extract having a pH above about the isoelectric point of the protein; a protein material containing the isoflavone conjugates is separated from the extract; an aqueous suspension is formed of the protein material; and the first and second conversion steps of the isoflavone are carried out on the aqueous suspension of the protein material. The aqueous suspension of the protein material preferably contains up to 30% by weight of the protein material. The steps of this procedure can be carried out in the same general manner as described above. It is contemplated that the first and second conversion steps of the isoflavone may be carried out on an aqueous suspension of the plant material containing isoflavone and protein conjugates, on an extract of said plant material, and on a protein material separated from said extract. . The present invention includes a combination of any of the above steps for forming an extract or protein material enpquecidoe with aglucone isoflavone. A high gemstein material and a high daidzein material can be produced from the recovered protein material enriched with aglucone isoflavone. As used in the present inventionA material of high gemstema content is defined as a plant material containing at least 40% genistein, and more preferably at least 90% genietein, together with residual plant material, which is residual soy material if the high gemstein material is recovered from a soy material. A high daidzein material contains at least 40% daidzein together with residual plant material, which is soy material if the high daidzein material is recovered from a soy material. The protein material enriched with isoflavone agglomerate can be washed and filtered initially to remove salts and undesirable sugars. The protein material enriched with aglucone isoflavone is mixed with water, where water is present in up to a ratio of 6: 1 relative to the protein material. The water must be cold to minimize the solubility of the aglucone isoflavones in the water, and preferably has a temperature of about 5 ° C to about 30 ° C. The protein material is mixed in the water for about 15 minutes to about 30 minutes, and then the protein material is filtered from the water using any conventional filtration means, preferably by filtering the mixture through conventional filter paper. If desired, washing and filtering can be avoided to minimize any potential loss of the aglucone isoflavone in the water bath. The protein material enriched with aglucone isoflavone can then be extracted with an aqueous alcohol of extraction to remove the isoflavones of aglucone from the protein material and produce an isoflavone extract of agglomerate. Low molecular weight alcohols such as methanol are preferred, and particularly ethanol as the alcohol component of the extractant. It has been found that aglucone isoflavones are soluble at almost all alcohol concentrations of the extraction agent. The aglucone isoflavones are particularly soluble when the extractant contains between about 30% alcohol and about 90% alcohol, and more preferably the extractant contains between about 60% alcohol and about 80% alcohol. Although the aqueous alcohol is the preferred solvent, other solvents including water, acetonitop, inethylene chloride, acetone and ethyl acetate can be used to effect the extraction of the aglucone isoflavones from the protein material. The extraction is carried out using a minimal amount of the extraction agent. It is preferred that the weight ratio of the extraction agent to the protein material enriched with aglucone isoflavone does not exceed 11: 1. The extraction can be carried out by any conventional extraction method, including countercurrent extraction, or a double extraction, where the weight ratio of the combined extracts to the protein material does not exceed 11: 1. In a preferred embodiment, the protein material it is initially extracted with 80% ethanol, where the weight ratio of the extraction agent to the protein material is approximately 6: 1. The extraction agent is separated from protein material by a conventional separation means, such as a centrifuge or a pressure filter, and the extract is collected. The protein material is extracted again with 80% ethanol, where the weight ratio of the extraction agent to the protein material is approximately 4: 1. Again, the extraction agent is collected and added to the collected initial extract. The protein material is then washed with a water jet, where the weight ratio of the water to the protein material is about 4: 1, and the water is added to the extracts collected. Although the extraction can be carried out at any pH, it is preferred that the extraction agent have a pH of about 7 to about 10. The formation of protein gel within the preferred pH range and, if the Protein will be recovered as well as the aglucone isoflavone extract, the formation of inconvenient by-product amino acids within the protein material within the preferred pH scale is avoided. The extraction can be carried out at any temperature up to the boiling point of the extraction agent, and is preferably carried out between about 25 ° C and about 70 ° C. To carry out the Maximum removal of the aglucone isoflavones from the protein material, it is preferred that the extraction be carried out at a temperature of about 50 ° C to about 70 ° C, preferably at about 60 ° C. After the extraction, a material of high content of genistein and a material of high content of daidzein can be separated from the isoflavone extract of aglucone by contacting the extract with an adsorbent material for a sufficient time to separate the materials from high. content of daidzein and high content of genistein from the extract. In a preferred embodiment, materials of high daidzein content and high genistein content are separated from the extract by reverse phase high performance liquid chromatography (HPLC). Genistein and daidzein are separated from other isoflavones and impurities in the extract by eluting the extract through particles of an adsorbent material that binds in free form genistein, daidzein, other isoflavones and impurities in a specific manner of the compound, allowing so that each of the compounds be separated. The aglucone isoflavone extract is initially filtered to remove the ineffective material that can clog a CLAR column. The extract can be filtered by any conventional filtration method. More preferably, the extract is filtered in a conventional ultrafiltration process that also removes the protein residual that may be in the extract. A column of CLAR is prepared by packing a conventional commercially available CLAR column with a particulate adsorbent material that will free-form bind the genistema, daidzein, other isoflavones, and impurities in a specific manner of the compound. The adsorbent material can be any reversed phase HPLC packing material; however, a preferred packaging material can be chosen by the criteria of load capacity, separation efficiency and cost. One of these preferred packaging materials are the 16 μ-lOOfl Kro asil C18 spheres available from Fka Nobel, Nobel Industries, Sweden. The filtered extract is passed through the packed HPLC column until all the binding sites in the column are fully saturated with isoflavones, which are detected by the appearance of isoflavones in the effluent of the column. The HPLC column can then be eluted with a polar eluent to effect separation. In a preferred embodiment, the eluent is an aqueous alcohol. The aqueous eluent alcohol may have an alcohol content of between about 30% to about 90% alcohol, and preferably has an alcohol content of about 50% alcohol to provide good separation and good solubility of the isoflavones. The alcohol is preferably methanol or ethanol, and ethanol is preferred when the materials of the high content of daidzein and high genistein content will be used in drug or food applications. Materials with a high content of genistein and high content of daidzein are collected from the effluent of the column. First a fraction of the effluent containing daidzein is eluted from the column, followed by a fraction of glycitein, which is followed by the more polar fraction of the genistema. The daidzein and genistein fractions are collected as they elute from the column. The glycitein fraction can also be collected, if desired. The alcohol in the fractions can be removed by evaporation, after which materials of high content of genistein and high content of daidzein, and a material of high content of glycitein, can be recovered by conventional separation methods such as centrifugation or filtration. The recovered high genistein material contains at least 40% genistein, and preferably at least 90% genistein, together with residual plant material, which is residual soy material if the genistein is recovered from a serum of soy. The recovered material with a high daidzein content contains at least 40% daidzein, together with residual plant material.

EXPERIMENTS The present invention is described in detail by the following examples using a soybean material or the plant material. The examples are intended to be illustrative, and should not be construed as limiting or otherwise restricting the invention in any way. As noted above, the soy material includes the "families" of genistein ieoflavones, daidzein and glycitein having corresponding glycoside, conjugate and aglycone members, wherein the genistein family contains the conjugates 6 '-OMal genistin and 6"~ 0Ac genistin, the genistin glycoside, and aglucona genistein, the daidzein family contains the conjugates 6" -0Mal daidzin and 6"-OAc daidzin, the daidzin glucoside, and the dalzezein aglycone, and the glycitein family contains the conjugate 6"-0Mal glycitin, the glucoside glycitin, and the aglycone glycitein. In the following tables, the relative concentrations of the isoflavones are measured in a percentage of an isoflavone family. For example, in the genistein family:% of genistin +% of 6"-OMal genistin +% of 6" -OAc genistin +% of genistein = 100%. The degree of conversion of the conjugates into glycosides, and of glucosides in aglucones can be determined by comparing the percentages of each type of compound in a family of isoflavone.

EXAMPLE 1 In a first experiment, the conversion of the isoflavone conjugates into isoflavone glucosidoe in a soy extract is examined. The degree of conversion is determined by the quantitative decrease in the percentage of rnalonate and acetate esters of a family of the isoflavone coupled with a corresponding quantitative increase of the percentage of the glucoside from the same isoflavone family. A soy extract is prepared by putting it in suspension 400g of degreased soy flakes finely ground with 4000g of water. The pH is adjusted to 9.7 with sodium hydroxide and the suspension is heated at 38 ° C for 15 minutes with stirring. The suspension is then centrifuged and the extract collected as a supernatant. The conversion of the isoflavone conjugates into isoflavone glycosides is examined at different pH conditions and different temperatures. The 600g samples of the extract are adjusted to a pH of 6, 7, 9 and 11 with hydrochloric acid or sodium hydroxide. For each pH, the 600 g sample is divided into two 300 g samples, and these samples are incubated at 45 ° C and 72.5 ° C for 24 hours. Periodic analysis is performed on each sample at 0, 2, 4 and 24 hours to determine the isoflavone content of the samples. Table 1 below shows the change and distribution of isoflavones during the cure of the experiment.

TABLE 1 SAMPLE 6ENISTINE 6EMSTINE 8ENISTII-A SENISTEINFI DAIDZIN DAIDZIN DAIDZIN DAHZEIN 6LICITIN GLICITIKA 6LICITEI S'-Oflal d "-OAc 6" -OHal B "-OAc 6" -OHal PERCENTAGES pH, 45"C t = 0 41 47 0 13 35 48 1 16 40 37 23 t = 10 lin 42 49 0 9 39 49 1 11 41 37 22 t = 2 hrs 29 51 0 19 27 49 1 22 37 36 27 t = 4 hrs 25 50 0 25 22 49 1 28 36 35 29 t - • 5 hrs 23 50 0 27 19 48 1 31 35 35 30 t = 2 * hrs; 15 43 1 40 12 42 0 45 30 32 37 pH7, 45 ° C t = fl 44 47 0 13 35 49 1 16 40 37 23 i - 10 lin 43 48 0 9 40 48 1 11 42 36 22 t = 2 hrs 38 48 0 13 35 48 1 16 40 37 23 t = 4 hrs 37 47 0 16 32 47 1 28 41 36 23 t = 6 hrs 38 46 0 18 31 46 1 22 41 35 24 t = 24 hrs 18 42 0 39 13 41 0 46 31 34 35 pH9, 45 ° C ts 0 44 47 0 13 35 48 1 16 40 37 23 t: 10 lin 46 46 0 8 43 46 1 10 45 33 23 t - 2 hrs 51 41 0 8 49 40 1 10 49 30 21 ts hrs 57 36 0 7 54 35 1 10 52 27 21 t = 6 hrs 60 33 0 7 58 31 0 10 54 25 21 t - 8 hrs 58 26 0 15 55 25 0 20 50 23 27 pHll, 45'C t = 0 44 47 0 13 35 40 1 16 40 37 23 t = 10 li No. 73 20 0 8 71 19 0 10 62 19 19 t = 2 hrβ. 92 0 0 7 91 0 0 9 82 0 1 t = 4 hrs. 93 0 0 7 90 0 0 10 82 0 1 t = 6 hrs. 93 0 0 7 90 0 0 10 81 0 1 t s 24 hrs. 95 0 0 5 87 0 0 13 78 0 2 TABLE 1 (CONTINUED! FLUESTRA GENISTINE 6ENISTINE 6ENISTINE GENISTEINE DAIDZIN DAIDZIN DAIDZIN DAIDZEIN GLICITIN GLICITIN 6ÜC 6"-OBal 6'-OAc 6" -Ol.al B "-OAc d'-OHal PERCENTAGES pH6, 72.5'C t = 0 44 47 0 13 35 48 1 15 40 37 t = 10 lin. 42 48 0 9 40 48 1 12 40 35 t s 2 hrs. 50 41 0 9 47 40 1 12 47 29 t = 4 hrs. 50 34 0 9 53 34 1 12 52 23 t = 6 hrs. 61 30 0 9 50 29 2 12 53 23 t = 24 hrs. 84 7 0 9 88 6 2 12 66 5 pH7, 72.5 ° C t - 0 44 47 0 13 35 48 1 16 40 37 - 10 lin. 45 40 0 9 41 47 1 11 43 36 t = 2 hrs. 54 21 0 8 50 38 1 10 47 30 t: 4 hrs. 61 11 0 8 58 30 1 10 52 24 t = 6 hrs. 67 6 0 4 63 25 1 10 56 20 t = 24 hrs. 90 0 0 5 85 4 1 9 68 4 pH9, 72.5ßC t = 0 44 47 8 13 35 48 1 16 40 37 i - 10 lin. 53 40 0 7 50 39 1 10 47 31 t - 2 hrs. 73 21 0 6 70 20 0 9 58 22 t = 4 hrs. 83 11 0 6 80 10 0 9 67 14 t = 6 hrs. 80 6 0 5 85 6 0 9 73 8 t - 24 hrs. 96 0 0 4 91 0 0 9 80 0 pHll, 72.5 ° C t = 0 44 47 0 13 35 48 1 15 40 37 t = 10 lin. 89 3 0 8 87 3 0 9 79 3 t s 2 hrs. 94 0 0 6 90 0 0 10 81 0 t = 4 hrs. 94 0 0 6 87 0 0 13 75 3 t = 6 hrs. 94 0 0 5 86 0 0 14 74 3 t = 24 hrs. 95 0 0 3 78 0 2 20 70 4 As indicated by the relative concentration dimensions of the conjugated compounds of isoflavone 6". OMal and 6'P OAc, and the corresponding increases in concentration of the glycosides genistin, daidzein and glycitin, the first step of conversion is faster and more complete. The most complete conversion of the isoflavone conjugates into isoflavone glycosides occurs in the pH 9 and pH 11 samples at 45 ° C and 72.5 ° C, however, the daidzein and the glycid they were degraded in 11 samples at pH 11 at 45 ° C and 72.5 ° C, however, daidzin and glycitin were degraded at pH 11, 72.5 ° C. Conversion also proceeds to term in pH 6 and 7 samples. 72.5 ° C. Substantial conversion of isoflavone glycosides to aglucone isoflavones by residual enzyme in the extract occurs at intervals of pH 6 and 7 at 45 ° C, although the conversion of isoflavone conjugates to gluc isoflavone is not particularly effective under these conditions.

EXAMPLE 2 In a second experiment, the conversion of the isoflavone glycosides into aglucone isoflavone was examined. The degree of conversion is determined by the quantitative decrease in the percentage of the glycoside of an isoflavone family coupled with a quantitative increase corresponding to the percentage of the aglucone of the isoflavone family. An extract enriched with isoflavone glycoside is produced from soy flakes by adjusting the pH of a soybean extract to about 1.1 at a temperature of about 35 ° C for about 1 hour. In the first samples, the conversion of the isoflavone glycosides to iso (-) aglucone lavonas is carried out using the residual enzyme present in an extract enriched with isoflavone glycoside, adjusting the pH of the samples to pH 7.0 and pH 9.0 and maintaining the samples at 45 ° C for 24 hours The conversion of the isoflavone glycosides into aglucone isoflavones is carried out using complementary enzymes by dosing the samples of an extract enriched with isoflavone glycoside with the following commercially available complementary enzymes: Biolactase 30,000, Lactase Neutral Quest, Lactase 50,000, Biopectinase 1001 and Alpha Gal 600. The amount of enzyme added to each sample is indicated in Table 2. Each sample is adjusted to a pH at which the complementary enzyme is active, either 4.0, 4.5 or 7.0 Lae rnotetras are incubated at temperatures ranging from 35 ° C to 75 ° C. Samples are taken at selected times and are measured for isoflavone TABLE 2 SAMPLE GENISTINE 6ENISTINE GENISTINE 6ENISTEINE DAIDZIN DAIDZIN DAIDZIN DAIDZEIN 6LICITIN GLICITIN GLICIT 5"-nal 6 * -OAc 6'-Or1al B" -OAc 5"-OHal PERCENTAGES Residual enzyme; pH7.0; 45'C t - 0 94 1 1 5 93 1 0 6 75 2 23 t = 3 hrs. 94 1 1 5 93 1 0 6 75 2 22 t - 6 hrs. 84 1 1 14 06 1 1 13 73 3 24 t = 24 hrs. 29 1 1 69 42 2 2 54 45 3 53 Residual enzyme; pH9.0; 45 * C t = 0 94 1 1 5 93 1 0 6 75 2 2 t - 3 hrs. 93 1 1 5 94 1 0 6 74 2 24 t = 6 hrs. 93 1 1 5 94 1 0 6 74 2 24 t = 24 hrs. fl 1 0 99 1 1 4 93 18 5 77 Lactase 50,000; pH4.0; 50 ° C; 0.2 g / 100 of the extract t = 0 100 0 0 0 100 0 0 0 100 0 0 t s 1 hr. 6 0 0 94 30 0 0 70 40 19 41 lyolactase 30,000; pH4.5; 35'C; 0.05 g / 100 g of the extract t = 0 93 4 0 4 93 3 0 5 100 0 0 t - 1 hr. 25 4 0 0 5 3 0 80 40 0 60 t = 2 hrs. 10 4 0 85 4 3 0 92 26 0 74 t = 3 hrs. 5 4 0 91 0 3 0 97 19 0 81 Alpha 691500; pH4.5; 75'C; 10 g / 1009 of the extract t = 0 91 0 0 9 89 0 0 11 78 0 22 t - 24 hrs. 1 0 0 99 0 0 2 98 0 0 100 Liopectinase 100 L; pH4.0; 5B "C; 0.29 / IOO 9 of the extract t s 0 100 0 0 0 100 0 0 0 100 0 0 t s l hr 67 0 0 33 50 0 0 42 07 13 0 Ques Neutral Lactase; pH7.0; 35'C; 0.05 g / lOH 9 of the extract t s 0 93 4 0 4 93 3 0 5 100 0 0 t = 1 hr. 65 4 0 30 66 3 0 31 77 0 2 t = 2 hrs. 50 4 0 46 51 3 0 46 67 0 33 t = 3 hrs. 36 4 0 59 37 3 0 59 50 0 4 t s 24 hrs. 1 4 0 95 0 3 0 97 0 0 10 As shown by the conversion of genistma, daidzin and glycite in gemstein, daidzein and glycitein, respectively, the conversion is achieved subs. Complete entity of the isoflavone glycosides in aglucone isoflavone. The selected complementary enzymes considerably increase the rate of conversion compared to the conversion by the residual enzyme in the extract, carrying out the substantially complete conversion within 1 hour at an effective concentration, temperature and pH.

EXAMPLE 3 In another experiment, a protein material enriched with isoflavone from aglucone is recovered from an extract enriched with isoflavone from aglucone, and conventional protein material is recovered from a conventional extract. The content of isoflavone in the protein materials recovered from each extract is determined at a pH of separation of 4.0, 4.5 and 5.0. An extract of soy enriched with isoflavone from agl? Cona is prepared 1) by extracting defatted soy flakes with an aqueous alkaline solution; 2) adjusting the pH of the extract to 11 and keeping the extract at 35 ° C for one hour to produce an extract enriched with isoflavone glycoside; and 3) adding 0.1% Lactase 50,000 to the extract (ftrnano International Enzyme Co.) by weight of the solids in the extract enriched with isoflavone glucoside, which is then treated at 50 ° C and pH 4.5 for 1 hour to produce the extract enriched with isoflavone aglucone. A conventional soy extract is also prepared, wherein the conventional extract is prepared by extracting defatted soy flakes with an alkaline aqueous solution. A sample containing lOg of solids are obtained from each extract, and the samples of each extract are adjusted to pH 4.5. A protein material is separated from each sample by centrifuging the sample and decanting the supernatant serum from the protein material. The isoflavone content of the protein material separated from each sample is then determined. Table 3 below shows the total isoflavone content in milligrams per sample and the percentage of each type of isoflavone from a family of isoflavone present in the protein material of each of the samples.

TABLE 3 GENISTINE GENISTINE GENISINE GENISINE DIAZONE DAIDZINE DAIDZINE GENEINE DAIDZEIN GLICITINE GLICITINE GLICIT 6"-OMal 5'-OAc 6'-OMal 6 * -OAc 6'-OI1al HG / FLUESTRA Protein material enriched with aglucone isoflavone; Separation pH: 4.5. Protein 14 00 00 97 00 00 00 58 04 00 05 Conventional protein material; Separation pH: 4.5. Protein 16 43 00 15 07 23 00 12 00 04 04 PERCENTAGE Protein material enriched with aglucone isoflavone; Separation pH: 4.5. Protein 13 0 0 87 0 0 0 100 49 0 51 flaterial of conventional protein; Separation pH: 4.5.

Protein 21 58 0 21 16 55 0 28 0 52 48 By comparing the isoflavone content of the protein material from the extract enriched with aglucone isoflavone, and the protein material from the conventional extract, it can be seen that the protein material from the extract enriched with isoflavone from aglycone contains significantly higher than aglucone isoflavones, particularly gemstein and daidzein, that the protein material from the conventional extract. The source material from the conventional extract contains substantial amounts of isoflavone conjugates missing from the protein material enriched with isoflavone from aglucone due to conversion of the isoflavone conjugates to aglucone isoflavones in the extract enriched with isoflavone from aglucone. In the previous examples, all the percentages indicated for 6"-0Mal genistin, 6" -00c genistin, 6"-orial daidzin, 6" -0Rc daidzin, glycitin, 6"-0Hal glycite and glycitein are calculated values. or the concentration of enzymes are calculated from grams of commercial enzyme preparation per lOOg of solid in each sample, followed by a method to quantify isoflavones in soybean product.Isoflavones are extracted from soy products mixing sample G.75g (dry powder by spraying or finely ground powder) with 500 ml of rnetanol / water solvent at 80/20 The mixture is stirred for 2 hours at room temperature with an orbital shaker.

After 2 hours, the remaining undissolved materials are removed by filtration through Uhat an No. 42 filter paper. Five ml of filtrate are diluted with 4 ml of water and 1 ml of rnetanol. The extracted isoflavones are separated by CLflR (High performance liquid chromatography) using a Hypereil C18 reverse phase column from Hewlett Pacl ^ ard. The isoflavones are injected into the column and eluted with a solvent gradient starting with 88% methanol, 10% water and 2% glacial acetic acid, and ending with 98% methanol and 2% glacial acetic acid. fl a flow rate of 0.4 ml / rnin, all isoflavones - genietin, 6"~ 0-acetylgenistin, 6" ~ 0-malonylgenistin, genistein, daidzin, 6"-0-acetyldaidzin, 6" -O-malonyldaidzin, Daidzin, glycitin and its derivatives and glycitein - are clearly resolved. The maximum detection is by absorbance of UV light at 260 nm. The identification of the maximum values was done by means of CLAR-spectrometer of rnasa. The antifi cation is achieved using pure standards (genistin, genistein, daidzin and daidzein) purchased from Indofine Chemical Company, Somrnerville, NJ. The response factors (integrated area / concentration) are calculated for each of the above compounds and used to quantify the unknown samples. For conjugated forms for which there are no pure patterns, it is assumed that the response factors they are those of the original molecule, but corrected for molecular weight difference. It is assumed that the response factor for glycine is that for the corrected genistme for the molecular weight difference. This method provides the quantities of each individual isoflavone. By convection, total genistein, total daidzein, and total glycitema can be calculated, and represent the aggregate weight of these compounds if all conjugated forms are converted to their respective unconjugated forms. These totals can also be measured directly by a method using acid hydrolysis to convert the conjugated forms. The embodiments of the invention in which an exclusive quality or privilege is claimed, are defined as follows.

Claims (67)

NOVELTY OF THE INVENTION CLAIMS

1. - A process for producing an aglucone isoflavone-enriched extract from a plant material comprising: extracting a plant material containing isoflavone and protein conjugates with an aqueous extraction agent having a pH above about the isoelectric point of said protein in said plant material; treating said aqueous extract at a temperature of about 2 ° C to about 1.21 ° C and a pH of about 6 to about 13.5 for a period sufficient to convert said isoflavone conjugates into glycosides of ieoflavone; and contacting an enzyme capable of cleaving the glycoside linkages with said isoflavone glycosides in said aqueous extract at a temperature of from about 5 ° C to about 75 ° C and a pH of from about 3 to about 9 during a sufficient period to convert said isoflavone glycosides to aglucone isoflavones.

2. The process as set forth in claim 1, wherein the extraction is carried out at a pH of about 6 to about 10.

3. The process of claim 1, wherein said aqueous extract is treated at a pH value of about 9 and a temperature of about 45 ° C to about 75 ° C to convert said isoflavone conjugates to isoflavone glycosides.

4. The process of claim 1, wherein said aqueous extract is treated, at a pH value of about 11 and at a temperature of about 5 ° C to about 50 ° C, to convert said isoflavone conjugates into glycosides. of isoflavone.

5. The method of claim 1, wherein contacting an enzyme with said glycosides of ieoflavone comprises adding an effective amount of an enzyme complementary to said aqueous extract containing isoflavone glycosides.

6. The method of claim 5, wherein said complementary enzyme is selected from the group consisting of alpha-galactosidase enzymes, beta-galactosidase enzymes, gluco-amylase enzymes, pectinase enzymes and combinations thereof.

7. The method of claim 5, wherein said complementary enzyme is added in such a way that the total concentration of enzyme present in said aqueous extract is from about 0.1% to about 10% by weight of said plant material, over a dry weight basis.

8. The process as set forth in claim 1, wherein said plant material comprises a soy material.

9.- The procedure co or is exposed in the claim 1, wherein a majority of said isoflavone conjugates and said isoflavone glycosides are converted to agl? cona isoflavones.

10. The method as set forth in claim 1, wherein substantially all said isoflavone conjugates and said isoflavone glycosides are converted to aglucone isoflavones.

11. The process as set forth in claim 1, further comprising adjusting the pH of said extract enriched with isoflavone from agglomerate to approximately the isoelectric point of said protein to precipitate a protein material containing protein and said isoflavones from agl? cona.

12. The method as set forth in claim 11, wherein said protein material is washed away.

13. The method as set forth in claim 1, wherein said protein material is washed with water in an amount by weight that is about 6 times the weight of said precipitated protein material.

14. The aglucone isoflavone enriched extract produced by the method of claim 1.

15. The protein material enriched with aglucone ieoflavone produced by the method of claim 11.

16. - A process for producing a protein material enriched with aglucone isoflavone from a plant material comprising: extracting a plant material containing isoflavone and protein conjugates with an aqueous extraction agent having a pH above about the point isoelectric of said protein in said plant material; treating said aqueous extract at a temperature of from about 2 ° C to about 121 ° C and a pH of about 6 to about 13.5 for a sufficient period to convert said ieoflavone conjugates into isoflavone glycosides; separating protein material containing said isoflavone glycosides from said aqueous extract; and contacting said isoflavone glycosides in said protein material with an enzyme capable of cutting glycosidic bonds at a temperature of about 5 ° C to about 75 ° C and a pH of about 3 to about 9 over a period of time. sufficient to convert said isoflavone glycosides to aglucone isoflavones.

17. The process as set forth in claim 16, wherein said extraction is carried out at a pH of from about 6 to about 10.

18. The process as set forth in claim 16, wherein said aqueous extract is treated. at a pH of about 9 to about 11 and at a temperature of about 5 ° C to about 75 ° C to convert said isoflavone conjugates into said glycosides of isofl vona

19. The process as set forth in claim 16, wherein separating a protein material containing said isoflavone glycosides from said aqueous extrudate further comprises adjusting the pH of said aqueous extract to approximately the isoelectric point of said pro-eine material for precipitating said protein material from said extract.

20. The method as set forth in claim 16, wherein contacting said isoflavone glycosides in said protein material with an enzyme comprises adding an effective amount of an enzyme complementary to said protein material.

21. The process as set forth in claim 20, wherein said complementary enzyme is selected from the group consisting of alpha-galactosidase enzymes, beta-galactoeidase enzymes, glucoalase enzymes, pectin enzymes and combinations thereof.

22. The method as set forth in claim 20, wherein said complementary enzyme is added to said protein material at a concentration of from about 0.1% to about 10% by weight of said protein material, on a weight basis. dry.

23. The method as set forth in claim 15, wherein said plant material is a soy material.

24. The method of claim 16, wherein a majority of said isoflavone conjugates are converted to aglucone isoflavones.

25. The method of claim 16, wherein substantially all of said isoflavone conjugates are converted to agl? Cona isoflavone.

26. The protein material enriched with aglucone isoflavone produced by the process of claim 16.

27.- A process for producing an extract enriched with isoflavone aglucone from a plant material, comprising: forming an aqueous suspension of? n plant material containing protein and isoflavone conjugates; treating said aqueous suspension of said plant material at a temperature of from about 2 ° C to about 121 ° C and an H of from about 6 to about 13.5 dur-before a sufficient period to convert said isoflavone conjugates into isoflavone glycosides; extracting said plant material with an aqueous extraction agent having a pH above about the isoelectric point of said protein in said plant material; and contacting said isoflavone glycosides in said aqueous extract with an enzyme capable of cutting the glycosidic linkages at a temperature of about 5 ° C to about 75 ° C and a pH of about 3 to about 9 over a period of time. sufficient to convert said isoflavone glycosides to aglucone isoflavone.

28. The process as set forth in claim 27, wherein said aqueous suspension contains up to 20% by weight of said plant material.

29. The method as set forth in claim 27, wherein said extraction is carried out at a pH of from about 6 to about 10.

The method as set forth in claim 27, wherein contacting said Isoflavone glycosides in said extract with an enzyme comprises adding an effective amount of an enzyme complementary to said extract.

31. The method as set forth in claim 30, wherein said complementary enzyme is selected from the group consisting of alpha-galactosidase enzymes, beta-galactosidase enzymes, glucoamylase enzymes, pectinase enzymes, and combinations thereof.

32. The process as set forth in claim 30, wherein said complementary enzyme is added to said extract in a concentration of about 0.1% to about 10% by weight of said plant material, on a dry weight basis.

33. The method as set forth in claim 27, wherein said plant material comprises a soy material.

34.- The procedure of claim 27, wherein a majority of said isoflavone conjugates are converted into said aglucone isoflavones.

35. The method of claim 27, wherein substantially all of said isoflavone conjugates are converted to aglucone isoflavone.

36. The aglucone isoflavone-enriched extract produced by the process of claim 27.

37. The method as set forth in claim 27, further comprising adjusting the pH of said enriched isoflavone extract from aglucone to approximately the isoelectric point of said protein to precipitate a protein material containing protein and said aglucone isoflavones.

38.- The protein material enriched with aglucone isoflavone produced by the method of claim 37.

39.- A process for producing protein material enriched with aglucone isoflavone from a plant material comprising: extracting a plant material containing isoflavone and protein conjugates with an aqueous extraction agent having a pH above about the isoelectric point of said protein in said plant material; separate a protein material containing said Isoflavone conjugates from said extract; forming an aqueous suspension of said protein material; treating said aqueous suspension at a temperature of about 2 ° C to about 121 ° C and a pH of about 6 to about 13.5 for a sufficient period to convert said isoflavone conjugates into isoflavone glycosides; and contacting said isoflavone glycosides in said aqueous suspension with an enzyme capable of cutting the glycosidic bonds at a temperature of about 5 ° C to about 75 ° C and a pH of about 3 to about 9 for a sufficient period to converting said isoflavone glycosides into aglucone isoflavone.

40.- The method as set forth in claim 39, wherein the extraction is carried out at a pH of about 6 to about 10.

41.- The method as set forth in claim 39, wherein said protein material is separated from said extract by adjusting the pH of said extr-act to approximately the isoelectric point of said protein to precipitate said protein material from said extract.

42. The method as set forth in claim 39, wherein said aqueous suspeneion contains up to about 30% by weight of said protein material.

43.- The procedure co or is exposed in the claim 39, wherein said aqueous suspension is treated at a pH of about 9 to about 11 and a temperature of about 5 ° C to about 75 ° C to convert said isoflavone conjugates into isoflavone glycosides.

44. The method as set forth in claim 39, wherein contacting said isoflavone glycosides in said aqueous suspension with an enzyme comprises adding an effective amount of an enzyme complementary to said suspension.

45. The method as set forth in claim 44, wherein said complementary enzyme is selected from the group consisting of enzyme alpha-galactosidases, beta-galactosidase enzymes, glucoamylase enzymes, pectinase enzymes, and combinations thereof.

46. The method as set forth in claim 44, wherein in said complementary enzyme is added to said aqueous suspension in a concentration of about 0.1% to about 10% by weight of said plant material, on a dry weight basis .

47. The method as set forth in claim 39, wherein said plant material is comprised of a soy material.

48. The process as set forth in claim 39, wherein a majority of said isoflavone conjugates are converted to aglucone isoflavones.

49. - The method as set forth in claim 39, wherein substantially all said isoflavone conjugates are converted to isoflavones, aglucona.

50.- The protein material enriched with aglucone isoflavone formed by the method of claim 39.

51.- The process for recovering a material of high genistein content from a vegetable protein material enriched with aglucone, comprising: providing a plant protein material enriched with aglucone isoflavone; extracting said plant protein material enriched with aglucone isoflavone with an extraction agent to produce an extract enriched with aglucone ieoflavone; and contacting said extract with an adsorbent material for a time sufficient to separate a material of high genistein content from said extract.

52. The method as set forth in claim 51, wherein said extraction agent is an aqueous alcohol containing between about 30% alcohol and about 90% alcohol

53. The process as set forth in claim 51, wherein said extraction agent has a pH value of about the isoelectric point of said protein in said plant protein material enriched with aglucone isoflavone.

54. - The method as set forth in claim 51, wherein said plant protein material enriched with aglucone isoflavone is extracted with said extraction agent in a weight ratio of the extraction agent with the material not exceeding about 11: 1

55. The method as set forth in claim 51, wherein said extract is eluted through said adsorbent material with eluent to contact said extract with said adsorbent material to separate a material of high genistein content from of said extract by differentially binding in a free form said genistein in said extract to said adsorbent material.

56. The method as set forth in claim 51, wherein said high genistema containing material contains at least 40% genistein.

57. The method as set forth in claim 56, wherein said high genistein content material contains at least 90% genistein.

58.- The method as set forth in claim 51, further comprising removing the residual vegetable protein material from said extract.

59.- A material with a high content of genistein produced by the process of claim 51. 60.- The process for recovering a material with a high content of daidzein from a protein material. plant enriched with aglucona, comprising: providing plant protein material enriched with aglucone isoflavone; extracting said plant protein material enriched with aglucone isoflavone with an extraction agent to produce an extract enriched with aglucone isoflavone; and contacting said extract with an adsorbent material for a sufficient time to separate a material of high daidzein content from said extract. 61.- The method as set forth in claim 60, wherein said extraction agent is an aqueous alcohol containing between about 30% and about 90% alcohol. 62. The method as set forth in claim 60, wherein said extraction agent has a pH value of approximately the isoelectric point of said protein in said plant protein material enriched with isoflavone aglycaene. 63. The method as set forth in claim 60, wherein said plant protein material enriched with aglucone isoflavone is extracted with said extraction agent where a weight ratio of the extraction agent with the material does not exceed about 11: 1 64. The process as set forth in claim 60, wherein said extract is eluted through said adsorbent material with an eluent to contact said extract with said adsorbent material for separating a material of high daidzein content from said extract uniting said daidzein to said adsorbent material in a free-flowing manner. 65.- The process as set forth in claim 60, wherein said high daidzein material contains at least 40% daidzein. 66. The method as set forth in claim 60, further comprising removing the residual vegetable protein material from said extract. 67.- A material of high daidzein content produced by the method of claim

60. SUMMARY OF THE INVENTION A protein material and a plant protein extract enriched with aglucone isoflavone, as well as a high gemstein content material and a high daidzein content material are provided; the isoflavone conjugates in a plant material are converted to isoflavone glycosides by treating the plant material at a temperature and pH for a sufficient period to effect the conversion; the isoflavone glycosides are converted to aglucose ieoflavone by enzymatic reaction; the plant material is extracted with an aqueous extraction agent having a pH above about the isoelectric point of the protein in the plant material to extract the protein and the isoflavones, either before or after converting the isoflavone conjugates into glycosides of isoflavone, or of converting the glycosides of isoflavone into aglucone isoflavone; a protein material enriched with aglucone isoflavone is produced by precipitating the protein and lae isoflavone from aglucone from the extract; high genistein content material or a high daidzein material can be produced from the protein extract enriched with aglucone ieoflavone or the protein material enriched with aglucone isoflavone separating the high content of genistein or high content of daidzein from the extract or the rotein material. P97 / 911 MG / blrn * lss * elt * fac *