KR100413025B1 - Aglucon isoflavone-enriched vegetable protein whey, whey protein material, aglucon isoflavone material, high zenistein content, high diad zein content and vegetable protein whey - Google Patents

Abstract

The present invention relates to a method for producing them from vegetable protein whey, which is rich in agroglutin isoflavones, vegetable protein whey, whey protein material, high genistein content, high dyedzein material and aglucon isoflavone material. to provide. The isoflavone complex in the vegetable protein whey is converted to isoflavone glucoside by treating the whey for a time sufficient to effect the conversion reaction at the desired temperature and pH. Isoflavone glucoside is converted to aglucon isoflavones by an enzymatic reaction that produces a vegetable protein whey rich in aglucon isoflavones. Aglucon isoflavone whey protein material is recovered from vegetable protein whey rich in aglucon isoflavones. Materials with high genistein content, high dyedzein content and aglucon isoflavone materials are prepared from alcohol extracts of aglucon isoflavone whey protein material.

Description

Aglucon isoflavone fortified vegetable protein whey, whey protein material, aglucon isoflavone material, high genistein content and high dyedzein material and method for preparing them from vegetable protein whey

The present invention prepares them from aglucon isoflavone fortified vegetable protein whey, aglucon isoflavone fortified whey protein material, aglucon isoflavone material, high genistein content, high dyedzein material and vegetable protein whey. It is about how to.

Isoflavones are produced in many legumes, including vegetable protein materials such as soybeans. These compounds include dydazine, 6 "-OAc didazine, 6" -OMal dydazine, dydzein, zenithine, 6 "-OAc zenithine, 6" -OMal zenithine, zenithine, glycidin, 6 " -OAc-glycithin, 6 "-OMal glycidine, glycidine, biocanin A, formonectin and cumestrol. Typically, these compounds are associated with the intrinsic bitter taste of soybeans.

In the manufacture of conventional products such as vegetable protein isolates and concentrates, the removal of these substances has been the focus. For example, in the conventional method of preparing soy protein isolates that extract soy flakes with an aqueous alkaline medium, most of the isoflavones are solubilized together with the soy protein in the extract, the protein precipitates from the extract by acidification of the extract, It separates to form a isolate, leaving a whey containing large amounts of solubilized isoflavones. Residual isoflavones remaining in the acid precipitated protein isolate are usually removed by complete washing of the isolate. Whey and washings are usually discarded. Isoflavones in vegetable protein whey include isoflavone glucoside (glucon), isoflavone complexes, and aglucon isoflavones. Isoflavone glucoside has a glucose molecule attached to the isoflavone portion of the compound. The isoflavone complex comprises an additional moiety bound to the glucose molecule, for example, a 6 "-OAc zenithine containing an acetate group bonded at the 6 position of the glucose molecule. Aglucon isoflavones bind to the bound glucose molecule. It consists of parts of isoflavones that do not have.

Soy whey includes "systems" of isoflavone compounds corresponding to glucosides, complexes and aglucones, ie, genisteine, dyedzeine and glycineine. Genistein systems include glucoside zenithine: complex 6 "-OMal zenithine (6" -malonate ester of zenithine) and 6 "-OAc zenithine (6" -acetate ester of zenithine); And aglucon genistein. Dyezein series includes glucoside didazine; Complex 6 "-OMal didazine and 6" -OAc didazine and aglucon diedzein. Glycysteine classes include glucoside glycidine, complex 6 "-OMal glycidine and aglucon glycine.

All isoflavones are of high interest in medical evaluation, and aglucon is the specific isoflavone that is of most interest. Genistein and dyedzein can greatly reduce cardiovascular risk factors. References ["Plant and Mammalian Estrogen Effects on Plasma Lipids of Female Monkeys", Circulation, vol. 90, page 1259 (October 1994)]. Genistein and dyedzein are believed to mitigate the signs of conditions caused by a decrease or change in endogenous estrogen levels in women, such as menopause or premenstrual signs. In addition, recently confirmed, aglucon isoflavones can inhibit the growth of human cancer cells such as breast cancer cells and prostate cancer cells. Peterson and Barnes, Biochemical and Biophysical Research, Communication, Vol. . 179, No. 1, pp. 661-667 (August 30, 1991) entitled "Genistein Inhibition of the Growth of Human Breast Cancer Cells, Independence from Estrogen Receptors and the Multi-Drug Resistance Gene"; Peterson and Barnes, The Prostate, Vol. 22, pp. 335-345 (1993), entitled "Genistein and Biochanin A Inhibit the Growth of Human Prostate Cancer Cells but not Epidermal Growth Factor Receptor Tyrosine Autophosphorylation"; Barnes et al., Mutagens and Carcinogens in the Diet, pp. 239-253 (1990), entitled "Soybeans Inhibit Mammary Tumors in Models of Breast Cancer."

As mentioned above, aglucon isoflavones include dydzein, genistein, glycidine and the like. The structural formula of the aglucon isoflavone is represented by the following formula (1).

[Formula 1]

Where

R ₁ , R ₂ , R ₃ and R ₄ may be selected from the group consisting of H, OH and OCH ₃ . Zenysteine is a compound of Formula 1 wherein R ₁ is OH, R ₂ is H, R ₃ is OH, R ₄ is OH, Dyzedine is R ₁ is OH, R ₂ is H, R ₃ Is H, R ₄ is OH, and glycine is a compound of Formula 1 wherein R ₁ is OH, R ₂ is OCH ₃ , R ₃ is H, and R ₄ is OH. .

Therefore, the present invention relates to the agglucon and vegetable protein whey of the present invention and to the fortified protein of the whey protein including the compound, and in particular, a material having a high content of genistein, a high content of dyzein and an aglucon isoflavone material. The present invention also relates to an aglucon-enriched vegetable protein whey, an aglucon-enriched vegetable whey protein material, a high genistein content, a high dyedzein content, and a method for producing an aglucon isoflavone material.

Methods for converting plant protein isoflavone complexes to aglucone isoflavones are known and described in currently pending US Application No. 08 / 477,102, filed June 7, 1995, assigned to the assignee of the present application. It is. It is well known to convert isoflavone glucoside to aglucon isoflavone. A method for converting isoflavone glucoside to aglucon isoflavone in vegetable protein whey is described in the assignee's own pending PCT patent application No. PCT / US94 / 10699.

Other methods known in the art for converting isoflavone glucoside to aglucon isoflavones are described, for example, in Japanese Patent Application No. 258,669 (Obata et al.). This method does not provide a method for converting the isoflavone complex to aglucone isoflavone or provides a material with high zesteine content, a high dyedzein material or an aglucon isoflavone material. In addition, these methods can only obtain moderate conversions in converting glucosides to aglucon, and it takes considerable time to obtain such intermediate conversions. Thus, this method is undesirable for large scale commercial operations.

It is a first object of the present invention to provide an aglucon isoflavone fortified vegetable protein whey and a method for preparing them from vegetable protein whey.

A second object of the present invention is to provide an aglucon isoflavone whey protein material and a method for preparing them from vegetable protein whey.

It is a third object of the present invention to provide a material having a high content of genistein and a method for producing them from vegetable protein whey,

A fourth object of the present invention is to provide a material having a high content of dyedzein and a method for producing them from vegetable protein whey.

A fifth object of the present invention is to provide an aglucon isoflavone material and a method for preparing them from vegetable protein whey.

These and other objects will be described in detail by the detailed description of the invention described below.

The present invention relates to a method for producing aglucon isoflavone fortified vegetable protein whey from vegetable protein whey comprising an isoflavone complex. This method involves treating the vegetable protein whey comprising the isoflavone complex at a predetermined temperature and pH for a time sufficient to convert the isoflavone complex to isoflavone glucoside. The enzyme contacts the isoflavone glucoside in most vegetable protein whey with isoflavone glucoside in the vegetable protein whey at a predetermined pH and temperature for a predetermined time sufficient to be converted to aglucone isoflavone.

In one embodiment of the invention, the vegetable protein whey is treated at a pH of about 6 to about 13.7 at a temperature of about 2 ° C to about 121 ° C to convert the isoflavone complex to isoflavone glucoside.

In one embodiment of the invention, the isoflavone glucoside is contacted with an enzyme in the vegetable protein whey at a pH of about 3 to about 9 at a temperature of about 5 ° C to about 75 ° C to convert the isoflavone glucoside to aglucon isoflavone. Switch.

The isoflavone complex can be converted to isoflavone glucoside and to high conversion in converting isoflavone glucoside to aglulon isoflavone. In one embodiment, at least 80% of the isoflavone complexes are converted to isoflavone glucosides and at least 80% of the isoflavone glucosides are converted to aglucon isoflavones.

A first aspect of the present invention is directed to a method for producing an aglucon isoflavone whey protein material from vegetable protein whey comprising an aglucon isoflavone whey protein material and an isoflavone complex. Aglucon isoflavone whey protein material, including protein and aglucon isoflavones, is recovered from the aglucon fortified vegetable protein whey. In one embodiment of the invention, the aglucon isoflavone whey protein material is recovered by one of ultrafiltration, thermal coagulation and dehydration.

A second aspect of the invention relates to a method for producing a high genistein content and a high genistein content from a vegetable protein whey comprising an isoflavone complex. Agglucon isoflavone whey protein derived from vegetable protein whey was extracted with an aqueous alcohol extract to prepare aglucon isoflavone fortified extract. The extract is contacted with the adsorbent material for a time sufficient to separate the high genistein content from the extract.

A third aspect of the invention relates to a method for producing a high dyedzein material and a high dyedzein material from a vegetable protein whey comprising an isoflavone complex. Agglucon isoflavone whey protein derived from vegetable protein whey was extracted with an aqueous alcohol extract to prepare aglucon isoflavone fortified extract. The extract is contacted with the adsorbent material for a time sufficient to separate a material with a high content of dydzein from the extract.

A fourth aspect of the present invention is directed to a method for producing an aglucon isoflavone material from a vegetable protein whey comprising an aglucon isoflavone material and an isoflavone complex. Agglucon isoflavone whey protein material derived from vegetable protein whey was extracted with an aqueous alcohol extract to prepare aglucon isoflavone fortified extract. The extractant was concentrated to about 15% to about 30% of the initial volume and water was added to the extract to precipitate the aglucon isoflavone material.

In one embodiment of the present invention, the high genistein content is separated from the aglucon isoflavone material. The aglucon isoflavone material is solvated in an aqueous solution of alcohol, and the adsorbent material is brought into contact with the aqueous solution of alcohol for a time sufficient to separate the high genistein content.

In one embodiment of the present invention, the high content of the diyzedine is separated from the aglucon isoflavone material. The aglucon isoflavone material is solvated in an aqueous solution of alcohol and the adsorbent material is brought into contact with the aqueous solution of alcohol for a time sufficient to separate the material with a high content of dydzein.

The starting material in this method is the vegetable protein whey, wherein the vegetable protein whey is defined as soluble protein, isoflavones and other water soluble compounds remaining after removal of the vegetable protein curd from the vegetable protein extract. In a preferred embodiment, the starting material is soy whey, because the method is characterized by agglomerate isoflavone fortified whey and aglucon isoflavone whey protein material, a high genistein content, a diyzedine content from soybean material. This is because it is particularly suitable for producing high materials and aglucon isoflavone materials.

Vegetable protein whey starting materials include isoflavone complexes, isoflavone glucosides and aglucon isoflavones. Soy whey, for example, isoflavone-glucoside-geninistin, dydzin and glycidine; Isoflavone complexes-6 "-malonate esters of zenithine, dydazine and glycidine, and 6" -acetate esters of zenithine and dydazine; Aglucon isoflavone-genysteine, dydzein and glycidane. Isoflavones in soy whey starting materials are predominantly isoflavone complexes.

Vegetable protein whey starting materials are usually obtained as a by-product of conventional methods for preparing vegetable protein separations. Vegetable protein sources, such as soy flakes degreased by solvent extraction, are treated with an aqueous extractant having a pH above the isoelectric point of the protein in the vegetable protein source, other compounds solubilized from the vegetable protein source by other extracts, Isoflavones and proteins can be prepared. The extract is separated from the vegetable material not dissolved in the extract. The pH of the extract containing the solubilized protein and isoflavone is adjusted to the isoelectric point of the protein, in the case of soy protein, at about pH 4.4 to 4.6 to precipitate the protein from the extract. The precipitated protein is separated to produce a vegetable protein isolate, leaving the vegetable protein whey starting material. Most isoflavones are solubilized in whey. In order to maximize recovery of isoflavones in the whey, further washing of the precipitated protein may be desirable, with each wash being added to the whey.

The vegetable protein whey starting material may be spray dried on the vegetable protein whey slurried in water. To facilitate handling, vegetable protein whey can be spray dried to recover whey protein (protein is still dissolved in whey after precipitation of protein isolates), isoflavones, and other compounds as a solid material. Spray dried material may be added to the water to reconstitute the vegetable protein whey starting material. In a preferred embodiment, the slurry comprises about 2-10 g of spray-dried material in 100 g of water so that the viscosity of the whey is not too high, while the desired aglucon isoflavone fortified whey, aglucon isoflavone whey Sufficient isoflavones are provided to prepare protein materials, high genistein content, high dyedzein material and aglucon isoflavone material.

During the first conversion step or manipulation, the isoflavone complex in the vegetable protein whey starting material is converted to isoflavone glucoside to prepare the isoflavone glucoside enriched vegetable protein whey. It can be seen that the conversion depends on the pH and temperature of the whey.

The pH range for converting the isoflavone complex to isoflavone glucoside is from about 6 to about 13.5. The pH of the vegetable protein whey should be adjusted to the desired pH if necessary. Soy protein whey typically has a pH of about 4.4 to 4.6, which must be adjusted with base or basic reagents in the desired pH range. The pH can be adjusted with any suitable base, caustic or basic agent, such as sodium hydroxide, potassium hydroxide and calcium hydroxide, which increases the pH of the system. It has been found that under relatively strong basic conditions, the conversion reaction proceeds more easily, preferably at pH 9-11. The pH should be maintained below pH 12, because isoflavone glucosides—geninistin, dydazine and glycidine, especially dydazine—are prone to denaturation above pH 12. The reaction proceeds less easily under low pH conditions, for example at about pH 6, while the reaction proceeds under high temperature and / or elevated pressure.

The temperature range required for the conversion of the isoflavone complex to isoflavone glucoside is from about 2 ° C to about 121 ° C. The temperature at which the conversion reaction is easily carried out depends on the pH of the whey. The inventors have found that when the pH is relatively high, the conversion reaction is easily performed even at low temperatures. For example, at a whey pH of about 11, the conversion reaction is performed easily and efficiently within the temperature range of about 5 ° C to about 50 ° C. At a whey pH of about 9, the conversion reaction takes place efficiently within the temperature range of about 45 ° C to about 73 ° C. If the whey pH is relatively low, the conversion reaction takes place at high temperatures. For example, at a whey pH of 6, a conversion reaction occurs in the temperature range of about 80 ° C to about 121 ° C. In a preferred embodiment, the conversion reaction occurs at about 35 ° C. at a whey pH of about 11. In another preferred embodiment, the conversion reaction is conducted at a temperature of about 73 ° C. at a whey pH of about 9.

The time required for the nearly complete conversion of the isoflavone complex to isoflavone glucoside depends on the pH and temperature of the vegetable protein whey. The time is 15 minutes to 24 hours. The conversion reaction occurs rapidly at higher pH and higher temperatures. At a pH of about 9-10, the conversion reaction is nearly complete at 73 ° C. for about 4 to about 6 hours. At a pH of about 10-11, the conversion reaction is nearly complete at 35 ° C. for about 30 minutes −1 hour. In a preferred embodiment, the isoflavone complex is converted to isoflavone glucoside at a pH of about 11 and a temperature of about 35 ° C. for about 45 minutes.

The first conversion step is very efficient at converting from about 80% to about 100% of the isoflavone complexes to isoflavone glucosides. In particular at least 95% conversion is observed. Such high conversion rates are particularly useful for large commercial operations.

In the second conversion step or operation, the isoflavone glucoside prepared in the first step, as well as the isoflavone glucoside already remaining in the whey, is converted to the aglucon isoflavone by enzymatic reaction. The conversion reaction produces aglucon isoflavone fortified vegetable protein whey from isoflavone glucoside fortified whey.

It was found that the second conversion step depends on the concentration of the enzyme present in the whey and its properties. The enzyme required to carry out the conversion reaction is an enzyme capable of breaking down the glucoside bond between the isoflavone moiety and the glucose molecule of the isoflavone glucoside. In a preferred embodiment, the enzyme is capable of breaking 1,4-glucoside bond Is a sugar enzyme. The concentration of enzyme required to convert isoflavone glucoside to aglucone isoflavone can be determined by various factors including the type of enzyme present in the whey, distribution of enzyme concentration, activity of enzyme, concentration of isoflavone glucoside, and during the conversion reaction. It depends on the pH and temperature of the whey.

Enzymes can be naturally present in the vegetable protein whey, from the growth of microorganisms in the whey, or added as a supplement to the whey. Enzymes that exist naturally or from microbial growth in whey are called "residue" enzymes, and enzymes added to whey are called "supplement" enzymes.

Sufficient enzymes must be present in the whey to convert at least most, preferably almost all isoflavone glucosides, into aglucon isoflavones. In general, residual enzymes in whey are insufficient to conduct a conversion reaction and a supplemental enzyme must be added to the whey. As mentioned above, various factors are determined as to whether the enzyme is present at an appropriate concentration to perform the conversion reaction.

When supplementary enzyme is added, the supplementary enzyme should be added such that the total concentration of enzyme present is from about 0.1% to about 10% by weight of whey solids, based on dry weight. In a preferred embodiment, the supplemental enzyme should be added to the whey irrespective of whether there is enough residual enzyme present in the whey, which greatly reduces the time required for the addition of the supplemental enzyme to almost completely convert the glucoside to aglucon. .

Supplementary enzymes are selected based on optimal activity in terms of predetermined pH and temperature conditions and cost effectiveness. Supplementary enzymes are enzymes capable of breaking down the bond between the isoflavone moiety and the glucos moiety of the isoflavone glucoside, for example a sugar enzyme capable of breaking the 1,4-glucoside bond. Preferred supplemental enzymes include commercially available α- and β-glucosidase enzymes, β-galactosidase enzymes, gluco-amylase enzymes and pectinase enzymes. Examples of particularly preferred enzymes include 100 L of Biopectinase (preferably used at a pH of about 3 to about 6), 300 L of Biopectinase (optimal pH of about 3 to about 6), and 70 L of Biopectinase OK (optimal pH of about 3 to about 6). ), Biolactase 30,000 (optimum pH is about 3 to about 6), Neutral Lactase (optimum pH is about 6 to about 8), and all of these enzymes are 34243 Sarasota Post Office Box 3917, 57, Florida, USA. Available from Quest International on Avenue 1833. Particularly preferred enzymes are Lactase F (optimal pH is about 4 to about 6) and Lactase 50,000 (optimal pH) available from Amano International Enzyme Company, Inc., 22974 Troy Post Office Box 1000, Virginia, USA. Is about 4 to about 6). Other particularly preferred supplemental enzymes include G-Zyme G990 (optimal pH is about 4 to about 6) and Enzeco Fungal Lactase available from Enzyme Development Coporation, 10121 New York Sweet 2439 Penn Plaza 2, New York, USA. Concentrate (optimal pH is about 4 to about 6); Novo Nordisk Bioindustry, Inc., 06813 Danbury Turner Road, Connecticut, USA, Lactozyme 3000L (optimal pH is about 6 to about 8) and Alpha-Gal 600L (optimal pH is about 4 to About 6.5); Neutral Lactase (optimal pH is about 6 to about 8) sold by Pfizer Food Science Group, 205 East 42nd Street, New York, NY 10017; Maxilact L2000 (optimal pH of about 4 to about 6), available from Gist Brocade Foods Inc., Inc., 19406 King of Prussia, Pennsylvania, USA.

Once a sufficient concentration of enzyme is present from the residual enzyme, supplemental enzyme, or both, the enzyme may be at a predetermined pH for a time sufficient to convert at least most, preferably almost all, isoflavone glucosides to aglucon isoflavones. And isoflavone glucoside in the whey at temperature. If necessary, the pH of the isoflavone glucoside enriched whey should be adjusted within the pH range at which the enzyme will react with isoflavone glucoside actively. The pH range where the mixed residual enzyme and the supplemental enzyme will react with isoflavone glucoside is from about 3 to about 9.

The inventors have found that while the pH of whey is known to decrease during the reaction, residual enzymes in whey are active within a pH range of about 7 to about 9. Supplementary enzymes have activity within the optimal pH range specified by the enzyme manufacturer as described above for the various specific enzymes. Typically, supplemental enzymes are active within a neutral pH range of about 6 to about 8, or an acidic pH range of about 4 to about 6. In addition, the acidic enzyme was shown to have activity at a pH of about 3.

The pH of the whey can in most cases be adjusted to decrease from the relatively high or basic pH of the first step by the addition of one or more suitable acetic acid, sulfuric acid, phosphoric acid, hydrochloric acid or any suitable agent. The formulations used may be edible acidic formulations or acids.

The temperature range for converting isoflavone glucoside to aglucon isoflavone is from about 5 ° C to about 75 ° C. Temperature greatly affects the activity of the enzyme, which in turn affects the conversion rate. Supplementary enzymes can be active at 72.5 ° C. or higher, for example Alpha-Gal 600L is active at 75 ° C., but it is preferred to carry out the conversion reaction at low temperature to avoid enzyme deactivation. In a preferred embodiment, the conversion reaction is preferably performed at about 35 ° C to about 45 ° C.

The enzymatic reaction is preferably carried out at the same temperature as the first conversion step so that there is no need to change the temperature of the whey after the first conversion step. Most preferably both the second conversion step and the first conversion step are carried out at 35 ° C.

It is also desirable to maintain a constant temperature during the conversion reaction of isoflavone glucoside to aglucon isoflavone. In some cases, however, it may be desirable to raise, lower or change the temperature during the reaction.

The time taken for the second conversion step depends on the factors associated with the enzyme, the specific concentration and the temperature and pH of the whey. In most cases, a complete conversion reaction can be performed within 24 hours, but it is desirable to add supplemental enzymes to greatly increase the reaction rate. The selected supplemental enzyme, enzyme concentration, pH and temperature are preferably within 2 hours, most preferably Preferably performs a nearly complete conversion reaction within 1 hour.

The degree of conversion of isoflavone glucoside to aglucon isoflavone in the second conversion step is about 80% to 100%. Typically, at least 95% conversion of isoflavone glucoside to aglucon isoflavones can be obtained.

After conversion of isoflavone glucoside to aglucon isoflavone, the aglucon isoflavone fortified whey can be used as desired without drying or removing the protein in whey or recovering the aglucon isoflavone whey protein material Agglucon isoflavones in the protein material can be concentrated. As used herein, aglucon isoflavone whey protein material is defined as a material comprising proteins, aglucon isoflavones and residual vegetable compounds that can be precipitated and separated from vegetable protein whey. Aglucon isoflavone fortified protein material can be recovered by conventional procedures such as ultrafiltration, thermal coagulation and dehydration. The resulting aglucon isoflavone whey protein material can be dehydrated and dried by conventional methods.

Aglucon isoflavone whey protein material can be recovered from whey by cooling the whey. Aglucon isoflavone whey protein material is insoluble in the cooled whey and can be separated as a precipitate from the whey by centrifuging the cooled whey. It is desirable to cool the whey to about 4 ° C. to precipitate the protein material.

In a preferred embodiment, the whey is concentrated to facilitate recovery of the aglucon isoflavone whey protein material. Increasing the ratio of solid: liquid by concentrating whey has been found to increase the capture of aglucon isoflavone whey protein material from whey. Whey can be concentrated by heating, placing the whey under reduced pressure, or both. The whey is preferably in a solid: liquid ratio of about 1: 3 to about 1: 6, most preferably about 1: 3.

Materials with high genistein content and high dyedzein content can be prepared from the recovered aglucon isoflavone whey protein material. As used herein, a material having a high genistein content is defined as a vegetable material comprising at least 40% genistein, most preferably at least 90% genistein, with residual vegetable material, wherein the residual vegetable material has a high genistein content. When high material is recovered from soy whey, it is a residual soy material. Materials with a high content of dydzein include at least 40% dyedzein together with residual vegetable materials.

In order to produce materials with high zesteine content and high dyedzein content, the aglucon isoflavone whey protein material was initially washed to remove undesirable salts and sugars and then dried. To wash the aglucon isoflavone whey protein material, the material was diluted with water, preferably with about 1% solids to about 6% solids, most preferably about 2% solids. However, the wash water can be of any temperature, but is preferably about 25 ° C to about 75 ° C, most preferably about 60 ° C. After washing the aglucon isoflavone whey protein material, the material is separated from the wash water and dried. In a preferred embodiment, the material is centrifuged and the supernatant is tilted away from the material to separate the material.

Aglucon isoflavone whey protein material was extracted with an aqueous alcohol extract to remove aglucon isoflavones from whey protein and an aglucon isoflavone fortified extract was prepared. Low molecular weight alcohols such as methanol, in particular ethanol, are preferred as alcohol components of the extractant. Aglucon isoflavones have been found to be soluble at almost all alcohol concentrations of the extractant. Aglucon isoflavones are particularly soluble when the extractant comprises about 30% alcohol to about 90% alcohol, most preferably about 60% alcohol to about 80% alcohol. If aqueous alcohol is the preferred solvent, water, acetonitrile, methylene chloride, acetone and ethyl acetate and other solvents including mixtures of these solvents can be used to extract the aglucon isoflavones from the whey protein material.

Extraction is carried out using a small amount of extractant. It is preferable that the weight ratio of extractant: aglucon isoflavone whey protein material does not exceed 11: 1. In one embodiment, the whey protein material may be extracted using a reverse extraction method with a weight ratio of extractant: material of about 6: 1 to about 8: 1. In another embodiment, the whey protein material may be extracted in two fractions of extractant whose weight ratio of extractant: material does not exceed 11: 1.

Although extraction can be performed at any pH, the extractant preferably has a pH at the isoelectric point of the protein in the aglucon isoflavone whey protein material that minimizes the solubility of the protein in the extractant. The extractant preferably has a pH of about 3 to about 6 and most preferably about 4.5 when the whey protein is soy whey protein.

Extraction can be carried out at any temperature below the boiling point of the extractant, preferably at a temperature of about 25 ° C to about 70 ° C. In order to reduce the time it takes to extract aglucon isoflavones from the aglucon isoflavone whey protein material, the extraction is preferably carried out at a temperature above room temperature and most preferably about 60 ° C.

After extraction, the extract is contacted with an adsorbent material for a time sufficient to separate the high genistein content and the high dyed zein material from the extract, thereby increasing the high genistein content and the diyzein content from the aglucon isoflavone fortified extract. High material can be separated. In a preferred embodiment, materials with high Genistein content and high Dyzedine content are separated from the extract by reverse phase high performance liquid chromatography ("HPLC"). Other isoflavones and impurities in the extracts are separated by eluting the extracts in a manner specific to the compound through particles of adsorbents which are separably bound to zenithine, dydzein, other isoflavones and impurities. Genistein and Dyzedine are separated from each other.

Aglucon isoflavone enriched extract is initially filtered to remove insoluble matters that may block the HPLC column. HPLC columns are prepared by filling a conventional HPLC column with particulate sorbents that will releasably bind genistein, dydzein, other isoflavones and impurities in a compound-specific manner. The adsorbent may be any reverse phase HPLC filler material, but the preferred filler may be selected according to criteria taking into account load capacity, separation efficiency and cost. One preferred filler is Kromasil C18 16 μm 100 μs beads commercially available from Eka Nobel, Nobel Industries, Sweden.

The filtered extract is passed through the column until all binding sites of the packed HPLC column are completely saturated with isoflavones, which is detected by the appearance of isoflavones in the eluted eluate from the column. The HPLC column is then separated by eluting with a polar eluent. In a preferred embodiment, the eluent is an aqueous alcohol. The alcohol content of the aqueous alcohol eluent is from about 30 to about 90% alcohol, preferably about 50%, to provide both good separation and good solubility of the isoflavones. The alcohol is preferably methanol or ethanol, and ethanol is preferable when a product having a high content of Zenysteine or a high content of Dyzeze is used for food or pharmaceutical use.

The high zenistein content and the high dyedzein material are collected from the column eluate. The eluate fraction comprising dyedzein is eluted in the first column, followed by the eluting glycine fraction and eluting the more polar genistein fraction. Dyzedine and Genistein fractions are collected when they are eluted from the column. If necessary, the Genistein fractions are also collected.

The alcohol in the fraction is evaporated off, and then the material with high content of Zenysteine and high content of Dyzezein can be recovered by conventional separation methods such as centrifugation or filtration. The material with high recovered genistein content contains at least 40%, preferably at least 90%, of genistein with residual vegetable material, when the genistein is recovered from soy whey, the residual vegetable material becomes a residual soybean material. High materials having a recovered dyedzein content typically contain at least 40% dyedzein along with residual vegetable material comprising a significant amount of glycidane.

In another embodiment of the invention, the aglucon isoflavone material is prepared from an aglucon isoflavone fortified extract. As used herein, aglucon isoflavone material is defined as a material comprising at least 10% genistein and at least 5% dyed, as well as other isoflavones and residual vegetable compounds.

After extracting the aglucon isoflavone whey protein material, the aglucon isoflavone fortified extract is concentrated to facilitate precipitation of the aglucon isoflavone from the extract. The extract can be heated and the extract can be concentrated under reduced pressure or by both. In a preferred embodiment, the extract is concentrated to about 15% to about 30% of the initial volume.

Aglucon isoflavone material is precipitated from the extract by adding water to the extract. In a preferred embodiment, about 6 to about 8 parts of water is added per part of the concentrated extract. When water is added to the extract, some aglucon isoflavone material precipitates.

In order to maximize recovery of the aglucon isoflavone material from the extract, the extract and water are thoroughly mixed and cooled. The extract and water are mixed for a predetermined time, preferably from about 30 minutes to about 1 hour. In a preferred embodiment, the extract and water are mixed at a temperature of about 50 ° C to about 75 ° C, preferably about 70 ° C. After complete mixing of water and extract, the mixture is cooled to precipitate the aglucon isoflavone material. The extract / water mixture is preferably cooled to a temperature of about 5 ° C. to about 20 ° C., most preferably about 10 ° C., for a time sufficient to precipitate almost all aglucon isoflavone material. Precipitated aglucon isoflavones can be separated from the extract / water mixture by conventional methods such as centrifugation or filtration.

The separated aglucon isoflavone material can then be washed with water. In a preferred embodiment, the aglucon isoflavone material is washed for about 5 minutes at a temperature of about 70 ° C. with a weight ratio of wash water: material of about 0.8: 1 to about 2: 1. The aglucon isoflavone material is separated from the wash water by conventional methods such as filtration or centrifugation and dried. The recovered aglucon isoflavone material typically comprises at least 20% genistein and at least 10% dyzezein, and the residual content of the material is formed from residual vegetable materials, including other aglucon isoflavones. The residual vegetable material becomes the soybean material when the aglucon isoflavone material is separated from the soy whey.

The recovered aglucon isoflavone material is further refined to produce a high genistein content comprising at least 40% genistein, preferably at least 90% genistein and a high dyedzein content comprising at least 40% dyedzein. Can be manufactured. The aglucon isoflavone material may be solvated with an aqueous alcohol solvent. Low molecular weight alcohols are preferred as the alcohol component of the solvent, and ethanol is most preferred for food and pharmaceutical use because of its low toxicity. Preferably, the alcohol component of the solvent is from about 30% to about 90%, wherein about 80% of the alcohol component is most preferred to provide good solvation of the aglucon isoflavone material.

The aqueous alcohol solution comprising the solvated aglucon isoflavone material may be contacted with the adsorbent material for a time sufficient to separate the high zenistein content and the high dyedzein content from the aqueous alcohol solution. In a preferred embodiment, the high zenisteine content and the high dyedzein content were separated from the aqueous alcohol solution by reverse phase HPLC. The HPLC column was prepared as described above, the aqueous solution of alcohol containing the aglucon isoflavone material was charged to the column, and the high genistein content and the high dyedzein material were eluted from the column in the manner described above. A material with a high content of genistein contains at least 40% of genistein, preferably at least 90% of genistein, together with residual vegetable material, and when the genistein is recovered from soy whey, the residual vegetable material becomes a residual soybean material. Materials with a high content of dydzein include at least 40% dyedzein together with residual vegetable materials.

Experiment

The present invention is more specifically illustrated by the following examples which use soy whey as vegetable protein whey. The following examples are illustrative and are not intended to limit the scope of the invention in any form.

As noted above, soy whey comprises the Genistein, Dyzezein and Glycsteine “system” of isoflavones having the corresponding glucosides, complexes, and aglucone members, wherein the genistein system is complex 6 ”-OMal. Zenithine, 6 "-OAc zenithine, glucoside zenithine, and aglucon genistein, and the dyedzein series is a complex 6" -OMal dydazine, a 6 "-OAc dydazine, a glucoside dydazine and agloo Cone Dyzezein; Glycinein systems include the complex 6 "-OMal glycidine, glucoside glycidine and aglucon glycinein. In the table below, the relative concentrations of isoflavones are measured as a percentage of the isoflavone system. For example, in the case of genistein, genistin (%) + 6 "-OMal genistin (%) + 6" -OAc genistin (%) + genistein (%) = 100%. The complex is glucoside, not glucoside. The conversion to glucon can be determined by comparing the percentage of the form of each compound in the isoflavone system.

Example 1

In the first experiment, the conversion of isoflavone complexes to isoflavone glucosides was investigated. Conversion was determined by decreasing the quantitative percentages of the malonate and acetate esters of the isoflavones relative to the corresponding quantitative percentage increase of the glucosides of the same isoflavones.

The effect under various different pH conditions on the first stage conversion from isoflavone complex to isoflavone glucoside was measured at two different temperatures. Spray dried soy whey was slurried in water to form a 2% solids soy whey suspension. Soy whey was divided into two groups for four samples. Samples in each group were adjusted to pH 6.0, 7.0, 9.0 and 11.0, respectively. A group of samples was incubated for 24 hours, one group of samples was incubated at 45 ° C. and the other group of samples at 72.5 ° C. Periodic analysis was performed for each sample at 0, 2, 4, 6, 8 and 24 hours to determine the isoflavone content of the sample. Tables 1a-1b below show the changes and distribution of isoflavones throughout the course of the experiment.

TABLE 1a

TABLE 1b

As can be seen from the decrease in the relative concentrations of the 6 "-OMal and 6" -OAc isoflavone complex compounds and the corresponding increase in the concentrations of glucoside zenithine, dydazine and glycidine, the primary conversion step is a higher pH. That is, they complete most rapidly at more basic pH conditions and high temperature conditions. Complete conversion of isoflavone complexes to isoflavone glucosides occurred in samples at pH 9 and pH 11 at both 45 ° C and 72.5 ° C. The conversion reaction also proceeds close to completion in samples of pH 6 and 7 at 72.5 ° C.

Example 2

In a second experiment, the conversion of isoflavone glucoside to aglucon isoflavone was investigated. The second conversion step was investigated using the isoflavone glucoside enriched whey produced by the first conversion step. The conversion was determined by the quantitative decrease in the percentage of glucosides in the isoflavones, which correlated with the corresponding quantitative increase in the percentage of agluconone in the same isoflavones.

The pH of soy whey was adjusted to 11.0 and incubated at 35 ° C. for 30 minutes to convert soy whey to whey rich in isoflavone glucoside. One sample of glucoside-enriched whey was incubated at 45 ° C. for 24 hours to determine the conversion of isoflavone glucoside to aglucon isoflavone by residual enzymes in the whey. Other samples of glucoside-enriched whey were biopectinase 100L, Biopectinase 300L, Biopectinase OK70L, Lactase F, Alpha-Gal 600L, G-Zyme G990, Quest Biolactase 30,000, Novo Lactozyme 3000L, Maxilact L2000, Enzeco Fungal Lactase, Pfizer Neutral Lt Inoculation with commercially available supplemental enzymes such as Neutral Lactase. Samples inoculated with Alpha-Gal 600L, C-Zyme G990, Biopectinase 100L, Biopectinase 300L, Biopectinase OK70L, Lactase F and Enzeco Fungal Lactase were adjusted to pH 4.5 before inoculation. Samples inoculated with Novo Lactozyme 3000L, Maxilact L2000, Pfizer Neutral Lactase and Quest Biolactase 30,000 and Quest Neutral Lactase were adjusted to pH 4.5 and 7.0 prior to inoculation. Lactase F samples were incubated at 35 ° C. and supplemental enzyme samples were incubated at 50 ° C. except that Biopectinase 300L and Biopectinase OK70L samples were incubated at 40 ° C. Subsequent intervals were taken and measured for isoflavone content. Tables 2a to 2f below show the changes and distribution of isoflavones throughout the course of the experiment.

TABLE 2a

TABLE 2b

TABLE 2c

Table 2d

TABLE 2e

TABLE 2f

The conversion of isoflavone glucoside to substantially complete aglucon isoflavones is achieved, as can be seen in the conversion of zenithine, dydazine and glycidine to genistein, dydzein and glycidine, respectively. The supplemental enzyme significantly increased the conversion rate, and nearly complete conversion was performed within 1 hour using the specific supplemental enzyme. Supplementary enzymes that have been shown to be most effective at pH 4.5 are Biopectinase 100L, Biopectinase 300L, Lactase F, Alpha-Gal 600L, G-Zyme G990, Quest Biolactase 30,000 and Enzeco Fungal Lactase. The most efficient supplemental enzymes at pH 7 are Quest Biolactase 30,000 and Quest Neutral Lactase.

Example 3

In another experiment, aglucon isoflavone whey protein material was recovered from aglucon isoflavone fortified soy whey. 163 g (concentration ratio-1) of a first sample of 1,000 g weight of aglucon isoflavone fortified soy whey, comprising 30 mg genistein, 37 mg dyedzein and 7 mg glycidane. : 6.1). The concentrated whey is heated to coagulate the protein material in the whey and centrifuged to further concentrate the whey protein material. 21 g of whey protein material comprising 25 mg of Genistein, 32 mg of Dyzedine and 6 mg of Glycithin is separated from the whey. The recovered whey protein material comprises 82% Genistein, 88% Dyzedine and 77% Glycysteine in the mixed whey and whey protein material.

A second sample of aglucone isoflavone fortified soy whey, weighing 400 g and containing 12 mg of Genistein, 15 mg of Dyzedine and 3 mg of Glycsteine, was heated to concentrate the whey protein without whey concentration. The material was solidified. The coagulated whey protein material and whey are centrifuged to further concentrate the whey protein material. 8.7 g of whey protein material was recovered, including 5 mg of Genistein, 7 mg of Dyzedine, and 1 mg of Glycsteine. The recovered whey protein material comprises 44% Genistein, 47% Dyzedine and 34% Glycinein in the mixed whey protein material and whey.

Comparing the whey protein materials of the first and second samples, it was shown that concentrating the aglucon isoflavone fortified whey prior to separating the whey protein material increased the capture of aglucon isoflavones in the whey protein material.

Example 4

In another experiment, the aglucon isoflavone whey protein material was extracted with an aqueous alcohol extract and the aglucon isoflavone material was precipitated from the extract to recover the aglucon isoflavone material.

Convert the isoflavone complex and isoflavone glucoside in the whey to aglucon isoflavones and recover the aglucon isoflavone whey protein material from the whey to yield 86% protein dry weight, 4.7 g genistein, 2.2 g die 821 g of aglucon isoflavone whey protein material was provided comprising zein and 0.36 g of glycine. Aglucon isoflavone whey protein material was extracted with 6,360 g of 80:20 wt% ethanol / water solution (7.7: 1 solution / aglucon isoflavone whey protein material) at 60 ° C. for 45 minutes. After extraction, the resulting slurry was cooled to 25 ° C. and Whatman No. Filter with 4 filter paper. A wet cake weighing 1,584 g comprising 798 g of solid, 0.8 g of Zensteine, 0.4 g of Dyzedine and 0.02 g of Glycsteine, is 23 g of Solid, 3.9 g of Zenysteine and 1.8 g of Dydein And 3,397 g of clear extract containing 0.34 g of glycinein.

The cake was extracted twice at 25 ° C. for 5 minutes with 2,000 g of 80:20 wt% ethanol / water solution (2.3: 1 solution / initial aglucon isoflavone whey protein material). After two extractions, the resulting slurry was returned to Whatman No. Filter on 4 filter paper. A wet cake comprising 794 g solids, 0.3 g Genistein, 0.1 g Dyzedine and 0.01 g Glycsteine and weighing 1,542 g was 4.0 g solids, 0.5 g Genistein, 0.3 g Dydzein And a second extract comprising 0.01 g of glycine and weighing 2,042 g. The extracts were combined, which initially contained 94% genistein and 95% dyedzein in the aglucon isoflavone whey protein material.

The extract was then concentrated in Buch evaporator at 70 ° C. under vacuum to 1528 g (20% of the initial combined extract volume). 6,000 g of deionized water was added to the concentrated extract (4: 1 water / extract) A white isoflavone precipitate formed when water was added. The precipitate slurry was heated to 70 ° C. for 45 minutes. The slurry was then refrigerated at 4 ° C. for 24 hours to allow the isoflavone precipitate to form and settle. 7,300 g of the supernatant was decanted from the precipitate and the remaining slurry was centrifuged to recover the precipitate. The recovered precipitate was washed with 600 g of deionized water at 70 ° C. for 15 minutes. The precipitate was recovered by centrifugation and dried at 50 ° C. under vacuum.

A dried aglucon isoflavone material weighing 7.3 g and comprising 49% Genistein, 19% Dyzezein and 4% Glycsteine was obtained.

Example 5

In another experiment, high zenistein and high dyedzein materials were separated from the aglucon isoflavone material by reverse phase HPLC. 2 g of aglucone isoflavone material comprising 55% Zenysteine, 21% Dyzezein and 4% Glycsteine on a dry weight basis in 1 liter of 50:50 weight% methanol / water solution Added. The solution of Whatman No. After filtration with 5 filter paper, it was filtered with 0.45μ filter paper.

The solution was poured into a 25 cm long, 2 "diameter HPLC column filled with Kromasil filler (Kromasil C18 16 μm, 100 μs beads). 64 mL / min mobile phase consisting of 50:50 wt% methanol / water solution The appearance of dydzein, glycidane and genistein from the column effluent was detected by UV absorption, and dyedzein was collected in the first fraction and genistein was collected in the second fraction. Diedzein and Genistein fractions were evaporated to remove alcohols, which resulted in the precipitation of materials with high Genistein content and high Dyzein content in each fraction. Recovered by centrifugation and dried in a vacuum oven The recovered high genistein content comprises about 95% genistein and the recovered dyedzein content This high material contains about 45% of Dyzedine.

In the experiments described above, 6 "-OMal-Genistine, 6" -OAc-Genistine, 6 "-OMal-Didazine, 6" -OAc-Didazine, 6 "-OMal-Glycithin and Glycinein All percentages for are theoretical values The percentages for enzyme concentration are calculated from conventional enzyme formulations (g) for 100 g whey solids or 100 g whey in each sample.

The following describes a method for quantifying isoflavones in soybean products. 0.75 g of the sample (spray dried or pulverized powder) was mixed with 50 ml of 80/20 methanol / water solvent to extract isoflavones from the soybean product. The mixture was stirred for 2 h at orbital stirrer. After 2 hours, the remaining undissolved material was removed from Whatman No. It was removed by filtration with 42 filter paper. 5 ml of filtrate was diluted with 4 ml of water and 1 ml of methanol,

The extracted isoflavones were separated by HPLC (high performance liquid chromatography) using Hewlett Packard C18 Hypersil reverse phase column. Isoflavones were injected into the column and eluted with a solvent gradient starting with 88% methanol, 10% water and 2% glacial acetic acid. All isoflavone-zenithine, 6 "-O-acetylgenistine, 6" -O-malonylgenistin, genistein, dydazine, 6 "-O-acetylidazine, 6" -O- at flow rate 0.4 ml / min Malonylidene, dydzein, glycidine, 6 "-O-malonylglycitin and glycidine were all dissolved. The peak was detected by UV absorption at 260 nm. By HPLC-weight spectroscopy Peak analysis was performed.

Quantitative analysis was performed using pure standards (Geninistine, Genistein, Dydazine and Dyzedine) commercially available from Indopine Chemical Company, Somerville, NJ. Reaction factors (integrated area / concentration) were calculated for each of the compounds and used to quantify unknown samples. For complex forms where pure standards were not available, the response factor was assumed to be the parent molecule, but the molecular weight difference was corrected. The response factor for glycidine was estimated to be that of genistin, correcting for molecular weight differences.

The method of the present invention provides for the quantification of individual isoflavones, conveniently, it is possible to calculate total genistein, total dydzein and total glycidine, which is when all complex forms are converted to their respective noncomplex forms. And aggregate weight of these compounds. In addition, these total amounts can also be measured directly by the method using acid hydrolysis which converts a non-complex form.

The above descriptions are merely preferred embodiments of the present invention. Various modifications and variations can be made without departing from the spirit and various features of the invention as set forth in the claims below, which are to be interpreted by principles of patent law, including equivalent theory.

Claims (61)

(a) treating vegetable protein whey at pH 6-13.7 and temperature 2 ° C. to 121 ° C. for 15 minutes to 24 hours to convert the isoflavone complex to isoflavone glucoside; (b) between the isoflavone portion of the isoflavone glucoside in the vegetable protein whey and the glucose molecule at pH 3-9 and temperature 5 ° C.-75 ° C. within 24 hours to convert isoflavone glucoside to aglucone isoflavone. A method for producing an aglucon isoflavone fortified vegetable protein whey from a vegetable protein whey comprising an isoflavone complex comprising contacting an enzyme capable of cleaving a glucoside bond. The method of claim 1, wherein the vegetable protein whey is treated at a temperature of 45 ℃ to 73 ℃ and pH 9 to 10. The method of claim 2, wherein the time for converting the isoflavone complex to isoflavone glucoside is between 4 hours and 6 hours. The method of claim 1, wherein the vegetable protein whey is treated at a temperature of 5 ℃ to 50 ℃ and pH 10-11. The method of claim 4, wherein the time for converting the isoflavone complex to isoflavone glucoside is 0.5 hour to 1 hour. The method of claim 1, wherein at least 80% of the isoflavone complexes are converted to isoflavone glucosides. The method of claim 6, wherein at least 90% of the isoflavone complexes are converted to isoflavone glucosides. The method of claim 1, wherein the enzyme is contacted with isoflavone glucoside in vegetable protein whey at a temperature between 35 ° C. and 45 ° C. 3. The method of claim 1, wherein contacting the isoflavone glucoside with the enzyme in the vegetable protein whey comprises adding a supplement enzyme to the vegetable protein whey so that the total enzyme concentration present is 0.1% to 10% of the whey solids, based on dry content. To percent by weight. The method of claim 9, wherein said supplemental enzyme comprises a saccharidase enzyme capable of breaking 1,4-glucoside bonds. The method of claim 10, wherein the supplemental enzyme is from the group comprising α-glucosidase enzyme, β-glucosidase enzyme, β-galactosidase enzyme, gluco-amylase enzyme, pectinase enzyme and combinations thereof. Which method is chosen. The method of claim 1, wherein at least 80% of the isoflavone glucoside is converted to aglucon isoflavones. 13. The method of claim 12, wherein at least 90% of the isoflavone glucoside is converted to aglulon isoflavones. The method of claim 1, wherein the vegetable protein whey comprises soy whey. The method of claim 1, further comprising recovering the aglucon isoflavone whey protein material containing the protein and aglucon isoflavone from the aglucon isoflavone fortified vegetable protein whey. The method of claim 15, wherein the aglucon isoflavone whey protein material is recovered by at least one of ultrafiltration, thermal coagulation and dehydration. 16. The method of claim 15, wherein the vegetable protein whey is cooled and the aglucon isoflavone whey protein material is separated from the cooled whey to recover the aglucon isoflavone whey protein material. The method of claim 15, wherein the aglucon isoflavone whey protein material is recovered from a concentrated vegetable protein whey. The method of claim 15, (a) extracting the aglucon isoflavone whey protein material with an aqueous alcohol extract to produce an aglucon isoflavone fortified extract. (b) contacting the extract with an adsorbent material to separate material containing at least 40% genistein from the extract. 20. The method of claim 19, wherein said aqueous alcohol extractant comprises 30% to 90% alcohol. The method of claim 19, wherein the aqueous alcohol extractant has a pH of the isoelectric point of the protein in the aglucon isoflavone whey protein material. 22. The method of claim 21, wherein the pH of the aqueous alcohol extractant is between 3 and 6. 20. The method of claim 19, wherein the aglucon isoflavone whey protein material is extracted with an extractant wherein the weight ratio of extractant: whey protein material does not exceed 11: 1. 20. The aqueous composition of claim 19, wherein the aglucon isoflavone whey protein material is combined with two fractions of the combined weight of two fractions of the aqueous alcohol extractant: the weight ratio of whey protein material does not exceed a total weight ratio of 11: 1. Extraction with an alcoholic extractant. 20. The method of claim 19, wherein the adsorbent material is particulate. 20. The method of claim 19, wherein contacting the extract with an adsorbent material further comprises separably binding genistein in the extract with the adsorbent material. The method of claim 19, wherein the extract is eluted through an adsorbent material using an eluent to separate material containing at least 40% genistein from the extract. The method of claim 27, wherein the material comprises at least 90% genistein. The method of claim 15, (a) extracting the aglucon isoflavone whey protein material with an aqueous alcohol extract to produce an aglucon isoflavone fortified extract, (b) contacting said extract with an adsorbent material to separate a material containing at least 40% dyedzein from said extract. 30. The method of claim 29, wherein the aqueous alcohol extract comprises 30% to 90% alcohol. 30. The method of claim 29, wherein the aqueous alcohol extractant has a pH of the isoelectric point of the protein in the aglucon isoflavone whey protein material. 32. The method of claim 31 wherein the pH of the aqueous alcohol extractant is between 3 and 6. 30. The method of claim 29, wherein the aglucon isoflavone whey protein material is extracted with an extractant wherein the weight ratio of extractant: whey protein material does not exceed 11: 1. 30. The method of claim 29, wherein the aglucon isoflavone whey protein material is extracted from two fractions of aqueous alcohol in which the weight ratio of two fractions of the aqueous alcohol extractant: the weight ratio of whey protein material does not exceed a total weight ratio of 11: 1. Zero extraction. The method of claim 29, wherein the adsorbent material is particulate. 30. The method of claim 29, wherein contacting the extract with an adsorbent material further comprises separably binding dyedzein in the extract with the adsorbent material. 30. The method of claim 29, wherein said extract is eluted through an adsorbent material using an eluent to separate said dyedzein-containing material from said extract. The method of claim 15, (a) extracting the aglucon isoflavone whey protein material with an aqueous alcohol extract to produce an aglucon isoflavone fortified extract, (b) further comprising contacting said extract with an adsorbent material to separate glycidine-containing material from said extract. The method of claim 15, (a) extracting the aglucon isoflavone whey protein material with an aqueous alcohol extract to produce an aglucon isoflavone fortified extract, (b) concentrating the aglucon isoflavone fortified extract to 15% to 30% of its initial volume, (c) adding water to the extract to further precipitate the aglucon isoflavone material. 40. The method of claim 39, wherein the aqueous alcohol extract comprises 30% to 90% alcohol. 40. The method of claim 39, wherein the aglucon isoflavone whey protein material is extracted with an extractant wherein the weight ratio of extractant to whey protein material does not exceed 11: 1. 40. The aqueous formulation of claim 2, wherein the aglucon isoflavone whey protein material is formulated in a formulated weight ratio of two fractions of the aqueous alcohol extractant: the weight ratio of whey protein material does not exceed a total weight ratio of 11: 1. Extraction with an alcoholic extractant. The method of claim 38, wherein the aqueous alcohol extractant has a pH of the isoelectric point of the protein in the aglucon isoflavone whey protein material. 44. The method of claim 43, wherein the pH of the aqueous alcohol extractant is between 3 and 6. 40. The method of claim 39, wherein water is added to the extract in a ratio of water: extract weight ratio of 6: 1 to 8: 1. 40. The method of claim 39, further comprising washing the precipitated aglucon isoflavone material with water in a ratio of water: agglucon isoflavone material in a ratio of 0.8: 1 to 2: 1. 40. The method of claim 39, further comprising cooling the extract and water to maximize precipitation of the aglucon isoflavone material. The method of claim 39, (a) solvating the aglucon isoflavone material in an aqueous alcohol solution, (b) contacting said aqueous solution of said alcohol comprising said solvated aglucon isoflavone material with an adsorbent material to separate said material containing at least 40% genistein from said aqueous solution of alcohol. The method of claim 39, (a) solvating the aglucon isoflavone material in an aqueous alcohol solution, (b) further comprising contacting said aqueous solution of said alcohol comprising said solvated aglucon isoflavone material with an adsorbent material to separate said material containing at least 40% dyedzein from said aqueous solution of alcohol. . Aglucon isoflavone fortified vegetable protein whey prepared by the method of claim 1. Aglucon isoflavone whey protein material prepared by the method of claim 15. A material containing genistein prepared by the method of claim 19. A material containing diyzedine prepared by the method of claim 29. A material containing genistein prepared by the method of claim 48. A material containing dyedzein prepared by the method of claim 49. 40. Aglucon isoflavone material prepared by the method of claim 39. Aglucon isoflavone whey protein material, including whey protein and aglucon isoflavone. An aglucon isoflavone material comprising soybean material, at least 10% genistein and at least 5% dyedzein. A material containing Genistein, comprising soybean material and at least 40% Genistein. 60. The material of claim 59, wherein the material comprises genistein comprising at least 90% genistein. A material containing dyedzein comprising soybean material and at least 40% dyedzein.