MXPA98000653A - Serum of vegetable protein enriched with aglucone isoflavone, serum protein material, aglucone isoflavone material, genistein high content and material with high content of daidzein, and procedure to produce them from a vege protein serum - Google Patents

Abstract

The present invention relates to a plant protein serum enriched with aglucone isoflavone, whey protein material, high genistein content, high daidzein content and aglucone isoflavone material, as well as a process for producing the same from serum of vegetable protein, isoflavone conjugates in a vegetable protein serum are converted to isoflavone glycosides treating the serum at a temperature and pH for a sufficient time to effect the conversion, the isoflavone glycosides are converted to isoflavone glycosides by enzymatic reaction to produce a vegetable protein serum enriched with aglucone isoflavone, whey protein material is recovered with aglucone isoflavone from the vegetable protein whey enriched with aglucone isoflavone, a material with high content of genistein, a material with high content of daidzein and an isoflavone material of aglucone are produced from an alcohol extract of the whey protein material with agluco isoflavone

Description

SERUM OF VEGETABLE PROTEIN ENRICHED WITH AGLUCONE ISOFLAVONE, SERUM PROTEIN MATERIAL, AGLUCONIDE ISOFLAVONE MATERIAL, GENISTEIN HIGH CONTENT AND MATERIAL WITH HIGH CONTENT OF DAIDZEIN, AND PROCEDURE TO PRODUCE THEM FROM A VEGETABLE PROTEIN SERUM BACKGROUND OF THE INVENTION The present invention relates to a plant protein whey enriched with aglucone isoflavone, a whey protein material enriched with aglucone isoflavone, an isoflavone material of aglucone, a material with a high content of genistein, and a material with high content of daidzein, and to the processes for producing them from a whey of vegetable protein. Isoflavones exist in several legume plants, including plant protein materials such as soy. These compounds include daidzin, 6"-0Ac daidzin, 6" -0Mal daidzin, daidzein, genistin, 6"-0Ac genistin, 6" -0Mal genistin, genistein, glycitin, 6"-0Ac glycitin, 6" -0Mal glycitin, glycitein , biochanin A, formononentin, and cu estrol. Typically, these compounds are associated with the inherent bitter taste of the soy beans. 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 conventional process for the production of a soy protein isolate in which soy flakes are extracted with an aqueous alkaline medium, many of the isoflavones are solubilized in the extract together with the soy protein. The protein is precipitated from the extract by acidification of the extract and separated to form an isolate, leaving a serum that retains much of the solubilized isoflavones. The residual isoflavones left in the protein isolate precipitated in acid are usually removed by thorough washing of the isolated. Serum and washes are typically discarded. Isoflavones in the whey of vegetable protein include isoflavone glucosides (glucone), isoflavone conjugates, and "Aglucone isoflavones. Isoflavone glycosides have a glucose molecule bound to the isoflavone portion of the compound. The isoflavone conjugates have additional portions attached to the glucose molecule, for example, 6"-OAC genistin contains an acetate group attached at the 6-position of the glucose molecule. The isoflavones of aglucone consist of a portion of isoflavone without a molecule of glucose bound. Soymilk contains three "families" of isoflavone compounds that have corresponding glycoside, conjugate and aglucone members: the genistein family, the daidzein family, and the glycitein family. The family of genistein includes the genistin glycoside; the conjugates 6 '' - 0Mal genistin (ester 6 '' - genistin malonate) and 6 '' - OAc genistin (ester '' - genistin acetate); and aglucona genistein. The daidzein family includes the daidzin glucoside; the conjugates 6 '' - 0Mal daidzin, and 6"-OAc daidzin; and aglucona daidzeína. The glycitein family includes the glycite glycitide, the conjugate 6"'- 0Mal glycitin, and the glycletin aglucone.Although all isoflavones are of interest in medical evaluation, agglucones are the specific isoflavones of most interest.Geistemis and daidzein can significantly reduce cardiovascular risk factors. "Plant and Mammalian Estrogen Effects on Plasma Lipids of Female Monkeys, 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 in menopause or premenstrual syndrome.In addition, it has recently been recognized that aglucone isoflavones contained in plant material such as soybean, can inhibit the growth of human cancer cells, such as breast cancer cells and prostate cancer cells, as described in the following articles: "Genistein Inhibition of the Growth of Human Breast Cancer Cells, Independence from Estrogen Receptors and the Multi-Drug Resistance Gene," by Peterson and Barnes, Biochemical and Biophysical Research Communications. Vol. 179, No. 1, p. 661-667, August 30, 1991; "Genistein and Biochanin A Inhibit the Growth of Human Prostate Cancer Cells but not Epidermal Growth Factor Receptor Tyrosine Autophosphorylation", by Peterson and Barnes, The Prostate, Vol. 22, p. 335-345 (1993); and "Soybeans Inhibit Mammary Tumors in Models of Breast Cancer," by Barnes and others, utagens and Carcinogens in the Diet, p. 239-253 (1990). As mentioned above, aglucone isoflavones include daidzein, genistein and glycitein. These agluconas have the following general formula: wherein R1, R2, R3 and R4 can be selected from the group consisting of H, OH and 0CH3. Genistein has the above formula, where R1 = 0H, R2 = H, R3 = 0H and R4 = 0H, daidzein has the above formula wherein R1 = 0H, R2 = H, R3 = H and R4 = 0H, and glycitein has the above formula wherein R1 = 0H, R2 = 0CH3, R3 = H and R4 = 0H. Therefore, the present invention is directed to the aglucones and to the enrichment of a whey of vegetable protein and a whey protein material with these compounds, and particularly to a material of high genistein content, a material of high daidzein content. , and an isoflavone material of aglucone. The present invention is also directed to methods for producing a plant protein serum enriched with aglucone, a plant serum protein material enriched with aglucone, a material of high genistein content, a material of high daidzein content, and a material of aglucone isoflavone. 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. Series No. 08 / 477,102, filed on June 7, 1995, recognized by the agent of the present application. Methods for converting isoflavone glycosides to aglucone isoflavones are also known. A process for converting isoflavone glycosides to aglucone isoflavones in a plant protein serum is provided in the pending application No. PCT / US / 94/10699, recognized by the attorney of the present request. Other methods for converting isoflavone glycosides to aglucone isoflavones are also known in the art, as described in Japanese Patent Application 258,669 by Obata et al. Said methods do not provide for the conversion of isoflavone conjugates to aglucone isoflavones, nor do they provide a material with a high content of genistein, a material with a high content of daidzein, or an isoflavone material of 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 a plant protein serum enriched with aglucone isoflavone, and a process for producing the same from vegetable protein whey. Another objective of the present invention is to provide an isoflavone aglucone whey protein material, and a process for producing the same from a whey of vegetable protein. Another objective of the present invention is to provide a material with a high content of genistein and a process for producing same from a whey of vegetable protein. A further objective of the present invention is to provide a material of high daidzein content and a process for manufacturing same from a whey of vegetable protein. Another objective of the present invention is to provide an aglucone isoflavone material and a process for manufacturing same from a whey of vegetable protein. These and other objects are specifically achieved as described in the detailed description of the invention, as indicated below.

BRIEF DESCRIPTION OF THE INVENTION This invention is a whey of vegetable protein enriched with aglucone isoflavone and a process for producing a whey of vegetable protein enriched with isoflavone from aglucone from a whey of vegetable protein containing isoflavone conjugates. The method comprises treating a plant protein whey containing isoflavone conjugates at a temperature and at a pH for a period sufficient to convert the isoflavone conjugates to isoflavone glycosides. An enzyme is contacted with the isoflavone glycosides in the vegetable protein whey at a temperature and at a pH for a sufficient period to convert at least most of the isoflavone glycosides to aglucone isoflavones. In an embodiment of the invention, the isoflavone conjugates are converted to isoflavone glycosides by treating the vegetable protein whey at a temperature between about 2 ° C and 121 ° C and at a pH value of about 6 to 13.7. In another embodiment of the invention, the isoflavone glycosides are converted to aglucone isoflavones by contacting the isoflavone glycosides with an enzyme in the whey of vegetable protein at a temperature between about 5 ° C and about 75 ° C and at a pH value between about 3 and about 9. High conversion rates of the isoflavone conjugates are made in isoflavone glycosides, and of the isoflavone glycosides in aglucone isoflavones. In one embodiment, at least 80% of the isoflavone conjugates are converted to isoflavone glycosides, and at least 80% of the isoflavone glycosides are converted to aglucone isoflavones. In another aspect, this invention is an aglucone isoflavone serum protein material and a process for producing an aglucone isoflavone whey protein material from a whey of vegetable protein containing isoflavone conjugates. An aglucone isoflavone whey protein material containing aglucone protein and isoflavones is recovered from a whey of vegetable protein enriched with aglucone. In one embodiment of the invention, the aglucone isoflavone serum protein material is recovered by means of at least some of ultrafiltration, heat coagulation and dehydration. In yet another aspect, this invention is a material of high genistein content and a process for producing a material of high genistein content from a whey of vegetable protein containing isoflavone conjugates. An aglucone isoflavone whey protein plant material derived from a plant protein whey is extracted with an aqueous alcohol extractant to produce an extract enriched with aglucone isoflavone. The extract is contacted with an adsorbent material for a period sufficient to separate a material of high genistein content from the extract. In another aspect, this invention is a high daidzein content material and a process for producing a high daidzein material from a plant protein whey containing conjugates of aglucone isoflavone. An aglucone isoflavone whey protein material derived from a vegetable protein whey is extracted with an aqueous alcohol extractant to produce an extract enriched with aglucone isoflavone. The extract is contacted with an adsorbent material for a sufficient time to remove a material of high daidzein content from the extract. In another aspect, this invention is an aglucone isoflavone material, and a method for producing an isoflavone material from aglucone from a plant protein serum comprising isoflavone conjugates. An isoflavone whey protein material is extracted from aglucone derived from a vegetable protein, with an aqueous alcohol extracting agent to produce an extract enriched with aglucone isoflavone. The extract is concentrated approximately between 15% and 30% of its original volume, and adding water to the extract precipitates an aglucone isoflavone material from the extract. In one embodiment, a high genistein material is separated from the isoflavone material of aglucone. The aglucone isoflavone material is solvated in an aqueous solution of alcohol and the aqueous alcohol solution is contacted with an adsorbent material for a sufficient time to remove a material of high genistein content. In another embodiment, a material of high daidzein content is separated from the isoflavone material of aglucone. The isoflavone material of aglucone is solvated in an aqueous solution of alcohol, and the aqueous alcohol solution is contacted with an adsorbent material for a sufficient time to remove a material with high daidzein content.

DESCRIPTION OF THE PREFERRED MODALITIES The starting material of the process is a vegetable protein whey; This plant protein serum is defined as an aqueous solution of soluble proteins, isoflavones and other water soluble compounds that remain after the removal of vegetable protein curdled with a vegetable protein extract. In a preferred embodiment, the starting material is a soybean serum, since the process is particularly suitable for the production of serum enriched with aglucone isoflavone, aglucone isoflavone serum protein material, high content material genistein, high daidzein content material, and aglucone isoflavone material, from soy materials. The starting material of vegetable protein whey contains conjugates of isoflavone, isoflavone glycosides and aglucone isoflavones. For example, soy whey contains: isoflavone glycosides -genistine, daidzin, and glycitin; isoflavone-6-ester esters-genistin, daidzin, and glycitin malonate and 6-esters-genistin and daidzin acetate; and aglucone isoflavones -genistein, daidzein, and glycitein. The isoflavones in a soybean starting material are predominantly isoflavone conjugates. The vegetable protein whey starting material can typically be obtained as a byproduct of a conventional production process of vegetable protein isolation. Extraction can be done to a source of vegetable protein, such as soy flakes from which oil has been removed by solvent extraction, with an aqueous extractant having a pH above the isoelectric point of the protein in the vegetable protein source , to produce an extract containing protein, isoflavones and other compounds solubilized from the vegetable protein source by the extract. The extract is separated from the vegetable material not soluble in the extract. The pH of the resulting extract containing the solubilized proteins and isoflavones is then adjusted to approximately the isoelectric point of the protein, approximately pH 4.4-4.6 for soy protein, to precipitate the protein from the extract. The precipitated protein is separated to produce a vegetable protein isolate, leaving the whey protein starting material. The isoflavones, for the most part, remain solubilized in the serum. To maximize the recovery of isoflavone in the serum, an additional wash of the precipitated protein may be convenient, with each wash being added to the serum. The vegetable protein whey starting material may be spray-dried vegetable protein whey suspended in water. For ease of handling, the vegetable protein whey can be spray dried to recover whey protein (protein still soluble in the whey after precipitating the protein isolate), isoflavones and other compounds as a solid material. The spray-dried material can be added in water to reconstitute a whey vegetable protein starting material. In a preferred embodiment, a suspension contains approximately 2-10 g of spray-dried material for each 100 g of water to ensure that the whey is not too viscous, while providing sufficient isoflavones to produce the serum enriched with desired aglucone isoflavone, aglucone isoflavone whey protein material, high genistein content material, high daidzein content material and aglucone isoflavone material. In a first step of conversion or operation, the isoflavone conjugates in the vegetable protein whey starting material are converted to isoflavone glycosides to produce a vegetable protein whey enriched with isoflavone glucoside. It has been found that the conversion depends on the pH and the temperature of the serum. The pH scale for the conversion of the isoflavone conjugates to isoflavone glycosides is from about 6 to about 13.5. The pH of the vegetable protein serum should be adjusted to the desired pH if necessary. Soy protein whey typically has a pH value of about 4.4 to 4.6, and must be adjusted with a base or a basic reagent to the desired pH range. The pH can be adjusted with any suitable base, caustic reagent, or basic reagent that increases the pH of the system, including sodium hydroxide, potassium hydroxide and calcium hydroxide. It has been found that the conversion proceeds more easily under relatively strong basic conditions, preferably pH 9-11. The pH must be maintained below pH 12 since the isoflavone glycosides genistin, daidzin and glycine, particularly daidinine, tend to degrade at pH values of 12 and above. The reaction proceeds less rapidly at lower pH conditions, for example about pH 6, however, the reaction will proceed at higher temperatures and / or under increased pressure.

The temperature scale for the conversion of the isoflavone conjugates to 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 serum. The inventors have found that conversion occurs easily at lower temperatures when the pH is relatively high. For example, at a serum pH of about 11, conversion occurs rapidly and efficiently at a temperature range of about 5 ° C to about 50 ° C. At a serum pH of about 9, the conversion occurs efficiently at a temperature range of about 45 ° C to about 73 ° C. When the pH of the serum is relatively low, the conversion occurs at higher temperatures. For example, at a serum pH of about 6, the conversion occurs at a temperature range of about 80 ° C to about 121 ° C. In a preferred embodiment, the conversion is effected at about 35 ° C at a pH of serum of about 11. In another preferred embodiment, the conversion is effected at about 73 ° C at a serum pH of about 9. The period required for substantially complete conversion of the isoflavone conjugates to isoflavone glycosides to occur depends on the pH and The temperature of the vegetable protein whey The periods vary from 15 minutes to 24 hours Conversion occurs more rapidly at a higher pH and at a higher temperature At a pH of about 9-10, the conversion is substantially complete at about 4 a 6 hours at 73 ° C. At a pH of approximately 10 to 11, the conversion is substantially complete in an approximate time of 30 minutes to 1 hour, to 35 ° C. In a very preferred, the isoflavone conjugates are converted to isoflavone glycosides in about 45 minutes at a pH value of about 11 and at a temperature of about 35 ° C. The first conversion step is remarkably efficient, converting from about 80% to about 100% of the isoflavone conjugates into isoflavone glycosides. Typically, conversion rates of at least 95% were observed. These high conversion speeds are especially attractive for use in large-scale commercial operations. In a second step of conversion or operation, the isoflavone glycosides, the isoflavone glycosides produced in the first step, as well as the isoflavone glycosides previously resident in the serum, are converted to aglucone isoflavones by an enzymatic reaction. The conversion produces a whey of vegetable protein enriched with isoflavone from aglucone from the serum enriched with isoflavone glucoside. It has been found that the second conversion step depends on the concentration of the enzymes present in the serum and its characteristics. The enzymes required to effect the conversion are enzymes capable of breaking the glycosidic bond between the isoflavone portion and the glucose molecule of the isoflavone glycosides. In a preferred embodiment, the enzymes are saccharidase enzymes capable of breaking 1,4-glucosidic bonds. The concentration of enzymes required to convert isoflavone glycosides to aglucone isoflavones depends on a variety of factors including the types of enzymes present in the serum, distribution of enzyme concentrations, activities of enzymes, concentration of isoflavone glycosides , and the pH and temperature of the serum during the conversion. In the vegetable protein serum, the enzymes may be present naturally, or from the microbial growth in the extract, or they may be added to the serum as a complement. Enzymes that are naturally present or by microbial growth in serum are referred to herein as "residual" enzymes, and the enzymes that are added to the serum are referred to herein as "complementary" enzymes. Enzyme must be present in the serum enough to convert at least the most part, and preferably substantially all, the isoflavone glycosides in aglucone isoflavones. Generally, if the residual enzymes in the serum are insufficient to effect the conversion, sufficient complementary enzyme should be added to the serum. As indicated above, a variety of factors determine whether the enzymes are present in adequate concentration to effect the conversion. 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 whey solids on a dry weight basis. In a preferred embodiment, complementary enzymes are added to the serum, regardless of whether sufficient residual enzymes are present in the serum, since the addition of complementary enzymes dramatically decreases the time necessary to effect substantially the complete conversion of the glucosides into aglucones. Complementary enzymes are selected based on optimal activity at a selected pH and temperature conditions, and cost effectiveness. Complementary enzymes are enzymes capable of breaking the bond between the isoflavone portion and the glucose portion of the isoflavone glycosides, such as saccharide enzymes capable of breaking 1,4-glucosidic bonds. Preferred complementary enzymes are the alpha- and beta-glucosidase enzymes, beta-galactosidase enzyme, glucoamylase enzyme and pectinase enzyme, commercially available. Enzymes such as Biopectinase 100L (which is preferably used at a pH scale of about 3 to 6), Biopectinase 300L (optimum pH scale of about 3 to 6), Biopectinase OK 70L (optimum pH scale of about 3) are particularly preferred. to 6), Biolactase 30,000 (optimal pH scale from about 3 to about 6), and Neutral Lactase (optimal pH scale from about 6 to about 8), all of which are available from Quest International, 1833 57 th Street, Post Office Box 3917, Sarasota, Florida 34243. Lactase F (optimal pH scale from about 4 to about 6) and lactase 50,000 (optimum pH scale from about 4 to about 6) are also particularly preferred which are available from Amano International Ezy e Co., Inc., Post Office Box 1000, Troy, Virginia 22974. Other particularly preferred complementary enzymes include G-Zimey G990 (optimum pH of about 4 to about nte 6) and Enzeco fungal lactase concentrate (optimum pH scale from about 4 to about 6) available from Enzyme Development Corporation, 2 Penn Plaza, Suite 2439, New York, New York 10121; Lactozyme 3000L (optimum pH scale from around 6 to approximately 8) and Alpha-Gal 600L (optimum pH from around 4 to approximately 6.5) available from Novo Nordisk Bioindust riais, Inc., 33 Turner Road, Danbury, Connecticut 06813; Neutral Lactase (optimal pH scale from about 6 to about 8) available from Pfizer Food Science Group, 205 East 42nd Street, New York, New York 10017; and Maxilact L2000 (optimum pH scale of about 4 to about 6) available from Gist Brocades Food Ingredients, Inc., King of Prussia, Pennsylvania 19406.

After sufficient concentrations of enzymes are present, either residual enzymes, complementary enzymes, or both, the enzymes are brought into contact with the isoflavone glycosides in the serum at a pH and temperature and for a sufficient time to convert at minus the majority, and preferably substantially all, of the isoflavone glycosides in aglucone isoflavones. If necessary, the pH of the serum enriched with isoflavone glycoside should be adjusted to be within a pH range at which the enzymes react actively with the isoflavone glycosides. The pH scale over which the residual enzymes and the complementary enzymes combined react with the isoflavone glycosides is from about 3 to about 9. The inventors have found that the residual enzyme in the serum is active within a pH range of about 7 to about 9, although it is thought that the pH of the serum is reduced during the course of the reaction. 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 acidic pH scale of about 4 to about 6. It has also been shown that the acidic enzymes are active at a pH of around 3.

The pH of the serum can be adjusted, and in most cases it is reduced, from the relatively high or basic pH of the first step, by the addition of one or more suitable acids such as acetic acid, sulfuric acid, phosphoric acid, acid hydrochloric, or any other suitable reagent. Preferably, the reagent used will be an acid or acid reagent that is used in food products. The temperature scale for converting the isoflavone glycosides to aglucone isoflavones is from about 5 ° C to about 75 ° C. The temperature significantly affects the activity of the enzymes and, therefore, the speed of the conversion. Complementary enzymes can be active above 72.5 ° C, for example, Alpha-Gal 600L is active at 75 ° C; however, it is preferred to perform the conversion at lower temperatures to avoid deactivation of the enzyme. In a preferred embodiment, the conversion takes place between about 35 ° C and about 45 ° C. Preferably, the reaction of the enzyme is carried out at the same temperature as the first conversion step, thus eliminating the need to modify the temperature of the serum after the first conversion step. More preferably, the second conversion step and the first conversion step are both carried out at 35 ° C. It is also preferred that a constant temperature be maintained during the conversion of the isoflavone glycosides to aglucone isoflavones. However, in some cases it may be convenient to raise, decrease, or otherwise vary the temperature during the course of the reaction. The period required for the second conversion step depends on factors related to the enzyme, particularly the concentration, and the temperature and pH of the serum. In most cases, it is possible to achieve complete conversion within 24 hours; however, it is preferred that the complementary enzyme be added to dramatically increase the speed of the reaction. The selected complementary enzyme, the concentration of the enzyme, the pH and the temperature, preferably cause a substantially complete conversion within 2 hours, and more preferably within 1 hour. The degree of conversion of the isoflavone glycosides to aglucone isoflavones in the second conversion step is remarkable, typically at least about 80% up to 100%. The conversion of at least 95% of the isoflavone glycosides into aglucone isoflavones is commonly achieved. After the conversion of isoflavone glycosides into aglucone isoflavones, the aglucone isoflavone enriched serum can be used as desired without drying or removing the protein in the serum or, alternatively, an aglucone isoflavone whey protein material can be recovered to concentrate the aglucone isoflavones in the protein material. The protein material of the aglucone isoflavone serum, as used in the present invention, is defined as a material containing protein, aglucone isoflavones and residual plant compounds that can be precipitated and separated from a whey of vegetable protein. The protein material enriched with aglucone isoflavones can be recovered by conventional methods such as ultrafiltration, thermal coagulation and dehydration. The resulting aglucone isoflavone whey protein material can be dehydrated and dried by conventional means. The protein material of the isoflavone serum of aglucone can also be recovered from the serum by cooling the latter. The protein material of the isoflavone serum of aglucone is insoluble in the cooled serum, and can be separated as a precipitate from the serum by centrifuging the cooled serum. Preferably, the serum is cooled to about 4 ° C to precipitate the protein material. In a preferred embodiment, the serum is concentrated to increase the recovery of the protein material from the isoflavone serum of aglucone. It has been found that the increase in the ratio of solids / liquids in the serum, by concentrating it, increases the capture of the protein material of the isoflavone serum of serum aglucone. The serum can be concentrated by heating, placing it under reduced pressure, or by both methods. Preferably, the whey is concentrated at a solids / liquid ratio of from about 1: 3 to about 1: 6, more preferably about 1: 3. A material of high genistein content and a high daidzein material can be produced from the recovered aglucon isoflavone whey protein material. As used in the present invention, a material of high genistein content is defined as a plant material containing at least 40% genistein, and more preferably at least 90% genistein, together with residual plant material, which is residual soy material if the material with a high content of genistein is recovered from a soy serum. A material with a high daidzein content contains at least 40% daidzein together with residual plant material. To produce high-content genistein and high-daidzein materials, the protein material of aglucone isoflavone is initially washed to remove undesirable salts and sugars, and then dried. To wash the protein material from the aglucone isoflavone serum, the material is diluted with water, preferably to between about 1% solids to about 6% solids, and more preferably up to about 2% solids. The washing water can be at any temperature; however, it is preferred that the water be between about 25 ° C and about 75 ° C, and more preferably about 60 ° C. After washing the protein material from the isoflavone serum of aglucone, the material is separated from the wash and dried. In a preferred embodiment, the material is separated by centrifuging, and the supernatant thereof is decanted. The protein material of the aglucone isoflavone serum can then be extracted with an aqueous alcohol extractor to remove the aglucone isoflavones from the whey protein and produce an extract enriched with aglucone isoflavone. Low molecular weight alcohols such as methanol, and particularly ethanol, are preferred as the alcohol component of the extractant. It has been found that aglucone isoflavones are soluble at almost all alcohol concentrations of the extractant. The aglucone isoflavones are particularly soluble when the extractant contains between about 30% alcohol and about 90% alcohol, more preferably between about 60% alcohol and about 80% alcohol. When the aqueous alcohol is the preferred solvent, other solvents including water, acetonitrile, methylene chloride, acetone and ethyl acetate, and mixtures of these solvents, can be used to effect the extraction of the aglucone isoflavones from the protein material. of the serum. The extraction is carried out using a minimal amount of extractant. It is preferred that the weight ratio of the extractive agent of the aglucone isoflavone whey protein material does not exceed 11: 1. In one embodiment, the material can be extracted using a countercurrent extraction method, wherein the weight ratio of the extractant to the material is between about 6: 1 to about 8: 1. In another embodiment, the material can be extracted with two portions of the extractant, wherein the combined weight ratio of the extractant to the material does not exceed 11: 1. Although the extraction can be carried out at any pH, it is preferred that the extractant has a pH near the isoelectric point of the protein in the whey protein material of aglucone isoflavone to minimize the solubility of the protein in the agent extractor. Preferably, the extractant has a pH value between about 3 and about 6, and more preferably about 4.5 when the whey protein is a soy whey protein. The extraction can be carried out at any temperature up to the boiling point of the extractant, and preferably it is carried out between about 25 ° C and about 70 ° C. To reduce the time for extracting the aglucone isoflavones from the protein material of the aglucone isoflavone serum, it is preferred to perform the extraction at an elevated temperature above room temperature, more preferably at about 60 ° C. After extraction, a high-genistein material and a high-daidzein material can be separated from the extract enriched with aglucone isoflavone by contacting the extract with an adsorbent material for a sufficient time to separate the materials from the extract from the extract. high content of genistein and high daidzein content. In a preferred embodiment, materials of high genistein content and high daidzein 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 the particles of an adsorbent material releasably linking genistein, daidzein, other isoflavones and impurities in a specific manner in the compound, thus allowing each of the compounds to be separated. The extract enriched with aglucone ieoflavone is initially filtered to remove insoluble material that could block a CLAR column. A CLAR column is prepared by packing a commercially available conventional CLAR column with a particulate adsorbent material that will releasably bind genistein, daidzein, other isoflavones and impurities in a specific manner in the compound. The adsorbent material can be any packaging material for reverse phase HPLC; however, a preferred packaging material can be chosen by the criteria of load capacity, separation efficiency and cost. Said preferred packaging material is 16μm-100ft Kromaeil C18 beads, available from Eka Nobel, Nobel Industries, Sweden. The filtered extract is passed through the packed HPLC column until all the binding sites in the column are completely saturated with isoflavones, which is detected by the appearance of isoflavones in the effluent of the column. The HPLC column can be eluted with a polar eluent to effect the preparation. In a preferred embodiment, the eluent is an aqueous alcohol. The aqueous alcohol eluent can 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, wherein ethanol is preferred when the high-genistein or high-daidzein product materials are to 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. A fraction of the effluent containing the daidzein is eluted first from the column, followed by a fraction of glycitein, which is followed by the more polar fraction of genistein. The fractions of daidzein and genistein are collected as they are eluted from the column. The glycitein fraction can also be collected, if desired.

The alcohol in the fraction can be removed by evaporation, after which high-content genistein and high-daidzein materials, as well as a high glycitein content material, 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 reeidual soy material and the geniethein is recovered from a soy serum. The recovered material of high daidzein content contains at least 40% daidzein, together with residual plant material, which typically includes a significant amount of glycitein. In another embodiment of the invention, an aglucone isoflavone material is produced from the extract enriched with aglucone isoflavone. As used in the present invention, an aglucone isoflavone material is defined as a material containing at least 10% genistein and at least 5% daidzein, as well as other isoflavones and reeiduale plant compounds. After extraction of the protein material from the aglucone ieoflavone, the extract enriched with aglucone ieoflavone can be concentrated to facilitate the precipitation of the isoflavone from the extract. The extract can be concentrated by heating it, placing the extract under reduced pressure, or by both methods. In a preferred embodiment, the extract is concentrated to between about 15% and about 30% of its original volume. An isoflavone material of aglucone is precipitated from the extract by adding water to the latter. In a preferred embodiment, between about 6 to about 8 parts of water are added by the concentrated extract. After adding water to the extract, a certain amount of the aglucone isoflavone material is precipitated. To maximize the recovery of the aglucone isoflavone material from the extract, the extract and water are thoroughly mixed, and then cooled. The extract and water are mixed together for a period, preferably between about 30 minutes to about 1 hour. In a preferred embodiment, the extract and water are mixed at a temperature between about 50 ° C and about 75 ° C, more preferably around 70 ° C. After the water and extract are completely mixed, the mixture is cooled to precipitate the isoflavone material from aglucone. Preferably, the extract / water mixture is cooled to between about 5 ° C and about 20 ° C, and more preferably at about 10 ° C, for a period sufficient to subetanially precipitate all of the aglucone isoflavone material. The precipitated aglucone isoflavone can be separated after the extract / water mixture in a conventional manner, such as by centrifugation or filtration.The separated aglucone isoflavone material can be washed after with water In a preferred embodiment, the aglucone ieoflavone material It is washed with water at a temperature of about 70 ° C for about 5 minutes, where the ratio of washing water to the material is between about 0.8: 1 and about 2: 1. The isoflavone material of aglucone It is separated from the washing water by conventional means such as filtration or centrifugation, and then dried.The isoflavone material of The recovered glucone typically contains at least 20% of genistein and at least 10% of daidzein, the remaining content of the material of remediated vegetale materials being formed, including another example of aglucone. The vegetal materials residualee with materiae of eoya and the material of ieoflavone of aglucone is isolated from soy serum. The recovered aglucone isoflavone material can be further purified to produce a high genistein content material containing at least 40% genistein, and preferably at least 90% genistein, and a high daidzein material containing at least 40% daidzein. The aglucone isoflavone material can be solvated in an aqueous alcohol solvent. Low molecular weight alcohols are preferred as the alcohol component of the solvent, wherein ethanol is most preferred for drug and food applications due to its low toxicity. The alcohol content of the solvent is preferably between about 30% and about 90%, wherein an alcohol content of about 80% is more preferred to provide good solvation of the aglucone ieoflavone material. The aqueous solution of alcohol containing the solvated isoflavone isoflavone material can be contacted with an adsorbent material for a time sufficient to separate the high-content genistein and high-daidzein materials from the aqueous alcohol solution. In a preferred embodiment, high content genistein and high daidzein contents are separated from the aqueous alcohol solution by reverse phase HPLC. A column of HPLC is prepared as described above, and the aqueous solution of alcohol containing the isoflavone material of aglucone is loaded onto the column, and a material of high content of genistein and a material of high content of daidzein is eluted from the column in the manner described above. The high genietein material contains at least 40% genistein, preferably at least 90% geniethein, together with reeidual plant material, which is residual soy material and genistein is recovered from a soy serum. The high daidzein material contains at least 40% daidzein, along with residual plant material.

Experiments The present invention is described in more detail by the following examples using an eoya serum as the vegetable protein eyer. The examples are intended to be illustrative, and should not be construed as limiting or otherwise restricting in any way the scope of the invention. As previously observed, soy whey includes the "families" of genistein isoflavone, daidzein and glycitein having corresponding conjugated, glucoside and aglucone members, wherein the genistein family contains the conjugates 6"-0Mal genistin and 6"-0Ac genistin, the glycoside genistin and the aglucona genistein; the family of daidzein contains the conjugates 6"-0tal daidzin and 6" -0Ac daidzin, the glucoside daidzin and the aglucona daidzein; and the glycitein family contains the conjugate 6"-0Mal glycitein, the glycitide glycitin and the aglycone glycitein.In the following tables, the relative concentrations of isoflavones are measured as a percentage of a family of isoflavones. the genistein family:% genistin +% 6"-0Mal genistin +% 6" -0Ac genistin +% genistein = 100%. The degree of conversion of the conjugates to glycosides, and from the glucosides to aglucones, can be determined by comparing the percentages of each type of compound in a family of isoflavones.

EXAMPLE 1 In a first experiment, the conversion of the isoflavone conjugates into isoflavone glycosides is examined. The degree of conversion is determined by the quantitative decrease in the percentage of malonate and lactate esters of a family of isoflavones coupled with a corresponding quantitative increase in the percentage of the glucoside of the same isoflavone family. The effect of the different pH condition on the first stage of conjugation of the ieoflavone conjugate in isoflavone glucides is measured at different temperatures. Eoya whey with aspersion is suspended in water to form a suspension of 2% by weight of eoya-whey solid. The soybean ee eepara in doe grupoe de cuatro mueetrae. The samples from each group are adjusted to a pH of 6.0, 7.0, 9.0 and 11.0, respectively. The groups of mueetrae are incubated for 24 hours with one group of samples being incubated at 45 ° C, and the other group of samples being incubated at 72.5 ° C. Periodic analysis is carried out in each unit at 0, 2, 4, 6, 8 and 24 hours to determine the yeoflavone content of the samples. Table 1 below shows the change and distribution of the isoflavones during the cure of the experiment.

TABLE I 6"-0HaL 6" -0Ac 6"-0MaL 6" -0Ac 6"-0Mal Sample Genistin Genistin Genistein Genistein Daidzin Daidzin Daidzin Daidzein Glycline Glicitin Glicitein PERCENTAGES H e, 45 * C t = 0 16 65 0 19 15 65 2 18 32 33 36 t = 2 hrs 15 62 0 23 15 61 2 21 34 37 28 t = 4 hrs 13 61 0 26 13 60 2 25 30 34 36 t = 6 hrs 11 61 0 28 11 60 2 27 30 33 37 t: 8 hrs 11 60 0 29 10 60 2 28 31 33 36 t = 24 hrs 24 49 2 25 16 52 0 32 30 27 43 pH 7, 45 * C t: 0 16 65 0 19 15 65 2 18 32 35 30 t = 2 hrs 22 59 0 19 20 50 2 19 42 25 33 t = 4 hrs 22 57 0 21 21 57 1 20 35 32 34 ? - 6 hrs 21 57 0 20 20 58 0 21 40 30 30 t = 8 hrs 22 56 0 21 21 57 0 22 37 31 33 t = 24 hrs 17 49 0 15 15 49 0 36 26 28 46 6"-0MaL 6" -0Ac 6"-0MaL 6" -0Ac 6"-0Male Sample Genistin Genistin Genistein Genistein Daidzin Daidzin Daidzin Daidzein Glycline Glicitin Glicitein PERCENTAGES H 9, 45'C t = 0 16 65 0 19 15 65 2 18 32 32 30 t = 2 hrs 50 34 0 16 50 34 0 15 50 20 30: hrs 57 27 0 15 57 27 0 16 49 17 34 t = 6 hrs 62 23 0 15 62 23 0 15 54 13 34 t: 8 hrs 67 19 0 14 67 18 0 15 57 10 34 t = 24 hrs 70 17 0 13 63 17 0 20 50 10 39 DH 11, 45'C t: 0 16 65 0 19 15 65 2 18 32 33 36 t = 2 hrs 85 0 0 15 82 0 0 18 62 0 38 1 = 4 hrs 85 0 0 15 81 0 0 19 63 0 37 t = 6 hrs 86 0 0 14 79 0 0 21 61 0 39 t = 8 hrs 87 0 0 13 77 0 0 23 60 0 40 t = 24 hrs 90 0 0 10 53 0 0 47 46 0 54 PH 6.72.5'C t: 0 16 65 0 19 15 65 2 18 32 33 36 t = 2 hrs 33 48 0 19 33 48 2 17 39 27 34 t = 4 hrs 43 39 0 17 42 39 2 17 46 21 33 t = 6 hrs 51 33 0 17 49 32 3 17 50 19 31 t = 8 hrs 56 28 0 16 54 27 3 16 57 14 29 t = 24 hrs 80 5 0 15 77 5 3 16 66 0 34 PH 7, 72.5 ' C t = 0 16 65 19 15 65 2 18 32 33 36 1 = 2 hrs 41 43 17 41 39 2 17 47 25 29 t = 4 hrs 52 32 15 50 32 2 16 49 18 33 t-6 hrs 58 27 15 56 26 2 16 5. 15 35 t = 8 hrs 64 21 15 62 20 2 16 55 12 32 t = 24 hrs 59 4 38 61 3 0 36 50 0 50 pH 9, 72.5'C t = 0 16 65 0 19 15 65 2 18 32 33 36 t = 2 hrs 83 4 0 13 82 4 0 14 64 0 36 t = 4 hrs 88 2 0 11 84 2 0 15 65 0 35 t = 6 hrs 90 0 0 10 85 0 0 15 65 0 35 t = 8 hrs 91 0 0 9 85 0 0 15 65 0 35 t - 24 hrs 100 0 0 0 85 0 0 15 100 0 0 6"-0MaL 6" -0Ac 6"-0MaL 6" -0Ac 6"-0Male Sample Genistin Genistin Genistein Genistein Daidzin Daidzin Daidzin Daidzein Glycline Glicitin Glicitein PERCENTAGES pH 11, 72.5'C t: 0 16 65 0 19 15 65 2 18 32 33 36 t: 2 hrs 86 0 0 14 76 0 0 24 57 0 43 t: 4 hrs 87 0 0 13 72 0 0 28 54 0 46 t: 6 hrs 87 0 0 13 67 0 0 33 51 0 49 t: 8 hrs 88 0 0 12 61 0 0 39 48 0 52 t: 24 hrs 78 0 0 22 24 0 0 76 31 0 69 As indicated by decreasing the relative concentration of the conjugated compounds of isoflavone 6"-0Mal and 6" -0Ac and the corresponding concentration increases of the glycosides genistin, daidzin and glycitin, the first conversion step is faster and more complete at higher pH and higher pH and temperature conditions. The substantially complete conversion of the isoflavone conjugates into isoflavone glycosides occurs in the mueetrae of pH 9 and 11 at both 45 ° C and 72.5 ° C. Converetion also occurs at term in samples of pH 6 and 7 at 72.5 ° C.

EXAMPLE 2 In a second experiment, the conversion of the isoflavone glycoeidoe into aglucone isoflavone is examined. Serum enriched with isoflavone glycoside produced by the first conversion step is used to examine the second conversion step. The degree of conversion is determined by the quantitative reduction of the percentage of the glycoside of an isoflavone family coupled with a corresponding quantitative increase of the percentage of the aglucone from the same isoflavone family. Soy serum is converted into serum enriched with isoflavone glycoside by adjusting the serum pH to 11.0 and incubating for 30 minutes at 35 ° C. A sample of the glycoside-enriched serum is incubated at 45 ° C for 24 hours to measure the conversion of the isoflavone glycosides to aglucone isoflavones by residual enzymes in the serum. Other samples of the glycoside-enriched serum are inoculated with the following complementary enzymes and are commercially available: Biopectinase 100L, Biopectinase 300L, Biopectinase 0K70L, Lactase F, Alpha-Gal 600L, G-Zyma G990, Quest Biolactase 30,000, Novo Lactozy e 3000L, Maxilact L2000, Enzeco Fungal Lactase, Pfizer Neutral Lactase and Quest Neutral Lactase. Samples inoculated with Alpha-Gal 600L, G-Zyme G990, Biopectinase 100L, Biopectinase 300L, Biopectinase 0K70L, Lactase F and Enzeco Fungal Lactase were adjusted to pH 4.5 before inoculation. The mueetrae inoculated with Novo Lactozyme 3000L, Maxilact L2000, Pfizer Neutral Lactase, Quest Biolactase 30,000 and Quest Neutral Lactase are adjusted to a pH of 4.5 and 7.0 before inoculation. The enzymes of complementary enzymes are incubated at 50 ° C, except for the Lactase F sample which is incubated at 35 ° C, and the samples of Biopectinase 300L and Biopectinase 0K70L, which are incubated at 40 ° C. Samples are taken after certain time intervals, and their isoflavone content is measured. Table 2 below shows the distribution of the isoflavones during the cure of the experiment.

TABLE 2 6"-0MaL 6" -0Ac 6"-0MaL 6" -0Ac 6"-0Mal Sample Genistin Genistin Genistein Genistein Daidzin Daidzin Daidzin Daidzein Glycline Glicitin Glicitein PERCENTAGES Residual enzyme, pH 9.0, 45'C t: 0 86 5 0 9 79 4 0 18 100 0 0 t = 2 hrs 89 2 0 9 81 1 0 18 100 0 0 t: 4 hrs 92 1 0 8 82 0 0 18 100 0 0 t: 6 hrs 93 0 0 7 82 0 0 18 100 0 0 t = 24 hrs 0 0 0 100 0 0 0 100 0 0 100 Biopectinase 300L, PH 4.5, 40'C O.lg / 100 g of serum enriched with glucoside t: 0 74 0 11 15 100 0 0 0 77 0 23 t: 0.5 hrs 46 0 0 54 46 3 0 51 75 0 25 t: l hr 22 0 0 78 24 3 0 73 66 10 24 t: 1.5 hrs 11 0 0 89 14 3 0 82 73 0 27 t: 2 hrs 6 0 0 94 7 3 0 90 70 0 30 6"-0MaL 6" -0Ac 6"-0MaL 6n-0Ac 6" -0Male Sample Genistin Genistin Genistein Genistein Daidzin Daidzin Daidzin Daidzein Glycline Glicitin Glicitein PERCENTAGES Biopectinase QK70L. pH 4.5, 4Q'C O.lg / 100 g of serum enriched with glucoside t: 0 74 0 11 15 100 0 0 0 77 0 23 t = 0.5 hrs 69 0 0 31 70 0 0 30 76 0 24 t: l hr 54 0 0 46 53 3 0 44 76 10 24 t - 1.5 hrs 44 0 0 56 43 0 4 52 75 0 25 t: 2 hrs 37 0 0 63 35 3 0 62 74 0 26 Biopectinase 100L, pH 4.5, 50'C Q.04g / 10Q g of serum enriched with glucoside t: 0 50 2 0 47 61 2 37 60 0 40 t - l hrs 25 2 0 73 31 2 67 54 0 55 t: 2 hrs 12 2 0 86 15 1 83 51 0 50 t: 3 hrs 7 2 0 92 9 1 90 0 0 100 Lactase F, pH 4.5, 35'C O.lg / 100 g of serum enriched with glucoside t: 0 47 8 0 45 45 7 0 48 74 0 26 t: 0.5 hrs 9 9 0 82 8 9 0 83 55 0 39 t : l hr 3 8 0 89 2 8 0 90 46 0 54 t: 2 hrs 0 9 O 91 0 8 0 92 32 0 68 Alpha-Gal 6001, pH 4.5, 50'C Q.lg / 100 g of serum enriched with glucoside t: 0 83 0 0 17 83 0 0 17 80 0 20 t - l hr 4 0 0 96 2 0 0 98 23 0 77 t = 2 hrs 1 0 0 99 0 0 0 100 10 14 76 t: 3 hrs 0 0 0 100 0 0 0 100 8 14 78 Enzeco Fungal Lactase, pH 4.5, 35'C Q.lg / 100 g of serum enriched with glucoside t: 0 83 1 0 16 79 3 1 17 85 0 15 t: 0.5 hrs 17 1 0 82 16 4 3 77 39 0 55 t: l hr 6 1 0 93 5 4 3 87 26 0 74 t: 2 hrs 0 1 0 99 0 4 3 92 4 0 96 G-Zyme G990, pH 4.5, 50'C 0.1g / 100 g of serum enriched with glucoside t: 0 83 0 0 17 83 0 0 17 80 0 20 t - l hr 49 1 0 51 41 0 0 59 82 0 18 t: 2 hrs 30 1 0 69 21 0 0 79 79 0 21 t: 3 hrs 18 0 0 82 11 0 0 89 69 11 19 6"-0MaL 6" -0Ac 6"-0MaL 6" -0Ac 6"-0Male Sample Genistin Genistin Genistein Genistein Daidzin Daidzin Daidzin Daidzein Glycline Glicitin Glicitein PERCENTAGES Novo Lactozyine 3000L, 50'C 0.2 g / 100 g of serum enriched with glucoside pH 4.5 t: 0 78 7 0 15 77 7 0 16 75 6 19 t: l hr 78 8 0 14 80 7 0 13 86 0 14 t : 4 hrs 77 8 0 15 80 7 0 13 84 0 16 pH 7.0 t: 0 78 7 0 15 77 7 0 16 75 6 19 t = l hr 72 8 0 20 77 7 0 16 72 0 28: hrs 68 8 0 24 74 7 0 19 61 0 39 Maxilact L2000, 5Q'C 0.2g / 100 g of serum enriched with glucoside pH 4.5 t: 0 78 7 0 15 77 7 16 75 19 t: l hr 78 7 0 15 77 7 16 75 19 t: 4 hrs 76 7 0 17 76 7 17 73 21 pH 7.0 t: 0 78 7 0 15 77 7 16 75 6 19 t: l hr 71 7 0 22 73 6 21 56 7 31 t: 4 hrs 65 7 0 28 69 5 26 52 0 48 Pfizer Neutral Lactase, 50'C 0.2 g / 100 g of serum enriched with glucoside pH 4.5 t: 0 78 7 0 15 77 7 0 16 75 6 19 t: l hr 77 7 0 16 77 6 0 17 73 7 20 t: 4 hrs 77 0 7 16 77 6 0 17 76 0 24 pH 7.0 t: 0 78 7 0 15 77 7 0 16 75 6 19 t: l hr 70 7 0 23 72 5 0 23 70 6 24 t: 4 hrs 55 7 0 38 60 6 0 34 66 0 34 6"-0MaL 6" -0Ac 6"-0HaL 6" -0Ac 6"-0Male Sample Genistin Genistin Genistein Genistein Daidzin Daidzin Daidzin Daidzein Glycline Glycline Glycitin PERCENTAGES Quest Biolactase 30,000, 5Q'C 0.2g / 100 g of serum enriched with glucoside pH 4.5 t: 0 78 O 15 77 7 O 16 75 6 19 t: l hr 0 0 94 0 6 0 94 0 0 100 t: 4 hrs 0 0 96 0 5 0 95 0 0 100 pH 7.0 t: 0 78 15 77 7 0 16 75 6 19 t: l hr 2 91 3 7 0 90 29 0 71 t: 4 hrs 0 93 0 6 0 94 0 0 100 Quest Neutral Lactase, 50'C 0.2g / 100 g of serum enriched with glucoside pH 4.5 t: 0 78 7 0 15 77 7 0 16 75 6 19 t: l hr 73 6 0 21 76 5 0 19 79 0 21 t: 4 hrs 73 6 0 21 76 5 0 19 76 0 24 pH 7.0 t: 0 78 7 0 15 77 7 0 16 75 6 19 t: l hr 2 7 O 91 7 4 0 89 15 0 15 t: 4 hrs 0 7 O 93 0 4 0 96 0 0 100 As shown by the conversion of genistin, daidzin and glycitin to genistein, daidzein and glycetein, respectively, substantially complete conversion of the isoflavone glucoside into isoflavone aglucone is achieved. The complementary enzymes increase notably the speed of the conversion, effecting the subetancialmente complete conversion within 1 hour with certain complementary enzymes. The complementary enzymes that have been found 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. It was found that the most effective complementary enzymes at a pH of 7.0 are Quest Biolactase 30,000 and Quest Neutral Lactaee.

EXAMPLE 3 In another experiment, an aglucone isoflavone serum protein material is recovered from an eoya serum enriched with aglucone ieoflavone. A first soybean whey protein enriched with aglucone isoflavone weighing 1000 g and containing 30 mg of genistein, 37 mg of daidzein and 7 mg of glycitein is concentrated by evaporation with low heating to 163 g (ratio of concentration-l: 6.1). The concentrated serum is heated to coagulate protein material in the serum, and centrifuged to further concentrate the whey protein material. 21 g of whey protein material containing 25 mg of genistein, 32 g of daidzein and 6 mg of glycitein are separated from the serum. The protein material of the coupled serum contains 82% of genietein, 88% of daidzein and 77% of glycitein in the combined material of whey and whey protein. A second sample of soy serum enriched with aglucone isoflavone weighing 400 g and containing 12 mg of genistein, 15 mg of daidzein and 3 mg of glycitein is heated to coagulate protein material in the serum without concentrating the serum. The protein material of the coagulated serum and the serum are centrifuged to concentrate the serum protein material. 8.7 g of the whey protein material is recovered containing 5 mg of genietein, 7 mg of daidzein and one mg of glycitein. The protein material of the recovered serum contains 44% genistein, or 47% daidzein and 34% glycitein in the combined whey protein material and serum. By comparing the whey protein materials of the first and second samples, it is clear that concentrating the serum enriched with aglucone isoflavone before removing the protein material from the whey results in an increased capture of the aglucone proteins in the protein material of the serum.

EXAMPLE 4 In another experiment, an isoflavone material of aglucone is recovered by extracting a protein material from the isoflavone serum of aglucone with an aqueous extract of alcohol and precipitating the isoflavone material from aglucone from the extract. 821 g of aglucone isoflavone whey protein material containing 86% dry protein base, 4.7 g of genistein, 2.2 g of daidzein and 0.36 g of glycitein are provided by converting the isoflavone and isoflavone glycoside conjugates into the serum to the iooflavone of aglucone and recovering the protein material of the uero with ieloflavone of aglucone from the serum. The whey protein material with aglucone ieoflavone was extracted with 6360 g of an 80: 20 wt% ethanol / water solution (7.7: 1 solution / whey protein material with aglucone isoflavone) at 60 ° C for 45 minutes. After extraction, the resulting suspension is cooled to 25 ° C and stirred on Whatman No. 4 filter paper under vacuum. A moist cake weighing 1584 g containing 798 g of aeolides, 0.8 g of genietein, 0.4 g of daidzein and 0.02 g of glycitein ee recovered together with 3397 g of a clear extract containing 23 g of eolides, 3.9 g of genistein, 1.8 g of daidzein and 0.34 g of glycitein. The cake is extracted a second time with 2000 g of an ethanol / water solution of 80: 20% by weight (2.3: 1 of eolium / protein material of the eeryelium with aglucone isoflavone) at 25 ° C for 5 minutes. After the second extraction, the resulting euepension is filtered again on whatman filter paper No.4. A moist cake weighing 1542 g and containing 794 g of solids, 0.3 g of genistein, 0.1 g of daidzein and 0.01 g of glycitein is recovered together with a second extract that peens 2042 g and contains 4.0 g of solids, 0.5 g of genistein , 0.10 g of daidzein and 0.01 g of glycitein. The extracts are combined and contain 94% genistein and 95% daidzein initially in the whey protein material with aglucone isoflavone. The extracts are then concentrated by evaporation in a Buchi evaporator under vacuum at 70 ° C to 1528 g (20% volume of original combined extract). 600 g of deionized water are added to the concentrated extract (4: 1 water / extract). White isoflavone precipitate forms when water is added. The precipitated suspension is heated at 70 ° C for 45 minutes. The suspension is then refrigerated at 4 ° C for 24 hours to allow precipitate of iaflavone to precipitate and precipitate, 7300 g of eobrenatant are decanted from the precipitate., and the remaining suspension is centrifuged to recover the precipitate. The recovered precipitate is again washed with 600 g of deionized water at 70 ° C for 15 minutes. The precipitate is recovered by centrifugation and dried under vacuum at 50 ° C. A dry aglucone isoflavone material weighing 7.3 g and containing 49% genietein, 19% daidzein and 4% glycitein is obtained.EXAMPLE 5 In another experiment, a material with a high content of genietein and a material with a high content of daidzein ee eparate from an isoflavone material of aglucone by reverse phase HPLC. Two grams of aglucone isoflavone material containing 55% genistein, 21% daidzein and 4% glycitein, dry base, is added to 1 liter of 50: 50% by weight of methanol / water solution. The solution is filtered through a Whatman No. 5 filter paper and then through a 0.45 μ filter. The solution was then loaded into a 0.08 cm diameter by 25 cm long HPLC column packed with Kromei packing material (16 μm Kromaeil C18, 100 fi spheres). A mobile phase consisting of a 50:50% methanol / water by weight solution is passed through a column at a speed of 64 ml per minute. The appearance of daidzein, glycitein and genistein from the effluent of the column is detected by UV absorption. Daidzein is collected in a first fraction and genistein is collected in a second fraction. The fractions of daidzein and genistein are evaporated to remove the alcohol, causing materials with high genistein content and high daidzein content to precipitate in each respective fraction. The materials of high content of genistein and high content of precipitated daidzein are recovered by centrifugation and dried in a vacuum oven. The material with high content of genistein recovered contains approximately 95% genistein and the material with high content of daidzein recovered contains approximately 45% daidzein. In the previous experiments, all percentages indicated for 6"-0Mal genistin, 6" -0Ac genistin, 6"-0Mal daidzin, 6" -0Ac-daidzin, 6"-0Mal glycitein and glycitein are calculated values. The enzyme concentration ee are calculated from gram of commercial enzyme preparation per 100 g of serum in each sample.The following describes a method to quantify the ieoflavone in soy products.The isoflavones are extracted from the soy products. Mixing 0.75 g of sample (dry powder by spraying or finely ground powder) with 50 ml of methanol / water solvent at 80/20 The mixture is stirred for 2 hours at room temperature with an orbital stirrer. The remaining undissolved materials are removed by filtration through Whatman No. 42 filter paper. Five ml of the filtrate is diluted with 4 ml of water and 1 ml of methanol.The extracted ieoflavones are separated by HPLC (high performance liquid chromatography) using a Hypersil C18 reverse phase column from Hewlett Packard. The ieoflavones are injected into the column and eluted with a solvent gradient starting with 88% methanol, 10% water and 2% glacial acetic acid. At a flow rate of 0.4 ml / min, all laeflavonee -genietine, 6"-0-acetylgenietine, 6" -0-malonylgenistine, genietein, daidzin, 6"-O-acetyldazidine, 6" -0-malonyldaidzine, daidzin , glycitin, 6"-0-malonylglicitin and glycitein and glycitein - are clearly resolved.The maximum detection is by absorbance of UV light at 260 mm.The identification of the maximum values was performed by means of the CLAR mass spectrometer. achieved using pure standards (genistin, genietein, daidzin, and daidzein) purchased from Indofine Chemical Company, Sommerville, 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 puroe patterns, it is assumed that the response factors are those of the original molecule, but corrected for molecular pee difference. Response factor for glycitin is that for genistin corrected for molecular weight difference. This method provides the quantities of each individual ieoflavone. For convenience, they can calculate total genistein, total daidzein and total glycitein, and re-estimate the aggregate weight of these compounds if all conjugated forms are converted into their respective unconjugated forms. These totals can also be measured directly by a method using acid hydrolysis to convert the conjugated forms. The above are simply preferred embodiments of the invention. Various changes and alterations can be made in the spirit and aspects of the same as set out in the appended claims, which must be interpreted in accordance with the principles of patent law, including the Equivalent Doctrine.

Claims (66)

1. NOVELTY OF THE INVENTION CLAIMS 1. - A process for producing a plant protein whey enriched with isoflavone aglucone from a whey of vegetable protein containing isoflavone conjugates, comprising: a) treating said whey protein at a temperature and pH for a sufficient period " for converting said isoflavone conjugates to isoflavone glycosides, and b) contacting an enzyme with said isoflavone glucoside in said vegetable protein whey at a temperature and pH for a period sufficient to convert at least a majority of the glycosides of isoflavone. aglucone isoflavone 2. The method according to claim 1, further characterized in that the vegetable protein whey is treated at a pH value between about 6 and about 13.7 and at a temperature between about 2 ° C and about 121 ° C to convert isoflavone conjugates to isoflavone glycoside 3.- The confolation procedure The composition according to claim 2, further characterized in that the vegetable protein whey is treated at a pH value between about 9 and about 10 and at a temperature between about 45 ° C and about 73 ° C. 4. - The method according to claim 3, further characterized in that the period for converting isoflavone conjugates to isoflavone glycosides is between about 4 and about 6 hours. 5. The method according to claim 2, further characterized in that the vegetable protein whey is treated at a pH value between about 10 and about 11 and at a temperature between about 5 ° C and about 50 ° C. 6. The method according to claim 5, further characterized in that the period for converting isoflavone conjugates to isoflavone glycosides is between about 0.5 and about 1 hour. 7. The process according to claim 1, further characterized in that at least a majority of the isoflavone conjugates are converted to isoflavone glucoside. 8. The process according to claim 7, further characterized in that at least 80% of said isoflavone conjugates are converted to glycoeido of ieoflavone. 9. The process according to claim 8, further characterized in that at least 90% of said isoflavone conjugates are converted to isoflavone glycosides. 10. The process according to claim 1, further characterized in that said enzyme is contacted with the isoflavone glycosides in the whey of vegetable protein at a temperature between about 5 ° C and about 75 ° C and a pH between about 3 to about 9. 11. The process according to claim 10, further characterized in that said enzyme is contacted with the isoflavone glycosides in the whey of vegetable protein at a temperature between about 35 ° C and about 45 ° C. 12. The process according to claim 10, further characterized in that the contacting of an enzyme with said glycoeidoe of ieoflavone in said plant protein eater comprises adding an effective amount of an enzyme complementary to said vegetable protein whey. 13. The method according to claim 12, further characterized in that said complementary enzyme comprises an saccharidase enzyme capable of cutting 1,4-glucoside bonds. 14. The method according to claim 13, further characterized in that said complementary enzyme is selected from the group that connects alpha-glucosidase enzymes, beta-glucosidase enzymes, beta-galactosidase enzymes, gluco-amylase enzymes, pectinase enzymes and combinations thereof. 15. - The method according to claim 1, further characterized in that at least 80% of said glycoside of ieoflavone is converted to the iooflavone of aglucone. 16. The method according to claim 15, further characterized in that at least 90% of said isoflavone glycosides are converted to aglucone isoflavones. 17. The process according to claim 1, further characterized in that said plant protein whey comprises soy whey. 18. The process according to claim 1, further characterized in that it consists in recovering a protein material of euero with ieoflavone of aglucone and isoflavones of aglucone from said whey vegetable protein enriched with isoflavone of aglucone. 19. The process according to claim 18, further characterized in that said whey protein material with aglucone isoflavone is recovered at least by one of ultracentrifugation, heat coagulation and elimination of water. 20. The method according to claim 18, further characterized in that said whey protein material with aglucone ieoflavone ee recovered by cooling said vegetable protein eyer and separating said whey protein material with aglucone isoflavone. 21. - The method according to claim 18, further characterized in that said whey protein material with aglucone isoflavone is recovered from whey vegetable protein. 22. The method according to claim 18, further characterized in that it comprises: a) extracting said whey protein material with aglucone isoflavone with an aqueous alcohol extraction agent to produce an extract enriched with aglucone isoflavone; and b) contacting said extract with an adsorbent material for a sufficient time to separate a material with high genistein content from said extract. 23. The process according to claim 22, further characterized in that said aqueous alcohol extraction agent includes between about 30% and about 90%. 24. The process according to claim 22, further characterized in that said aqueous alcohol extraction agent has a pH value of about the isoelectric point of said protein in said serum protein material with aglucone isoflavone. 25. The process according to claim 24, further characterized in that said aqueous alcohol extraction agent has a pH value between about 3 and about 6. The method according to claim 22, further characterized in that said Whey protein material with aglucone isoflavone is extracted with said extraction agent wherein a ratio of extraction agent to whey protein material does not exceed approximately 11: 1. 27. The method according to claim 22, further characterized in that said whey protein material with aglucone isoflavone is extracted with two portions of said aqueous alcohol extraction agent wherein a combined peeo ratio of the portions of said Extraction agent to said whey protein material does not exceed a total weight ratio of about 11: 1. 28. The method according to claim 22, further characterized in that said adsorbent material is particulate material. 29. The method according to claim 22, further characterized in that said extract with an adsorbent material comprises releasably ligating genistein in said extract with the adsorbent material. 30. The process according to claim 22, further characterized in that the extract is eluted through the adsorbent material with an eluent to separate a material with high genistein content from said extract. 31. The method according to claim 22, further characterized in that the material with a high content of genistein contains at least 40% genistein. 32. The method according to claim 31, further characterized in that the material with a high content of genistein contains at least 90% genistein. 33.- The method according to claim 18, further characterized in that it comprises: a) extracting said whey protein material with aglucone isoflavone with an aqueous alcohol extraction agent to produce an extract enriched with aglucone isoflavone; and b) contacting said extract with an adsorbent material for a sufficient time to remove a material with high daidzein content from said extract. 34.- The method according to claim 33, characterized in that said aqueous alcohol extraction agent includes between about 30% and about 90%. 35. The process according to claim 33, characterized in that said aqueous alcohol extraction agent has a pH value of about the isoelectric point of said protein in said whey protein material with aglucone isoflavone. 36.- The procedure according to claim 35, further characterized in that said aqueous alcohol extraction agent has a pH value between about 3 and about 6. The method according to claim 33, further characterized in that said whey protein material with aglucone isoflavone is extracted. with said extraction agent wherein a ratio of extraction agent to whey protein material does not exceed approximately 11: 1. 38.- The method according to claim 33, further characterized in that said whey protein material with aglucone isoflavone is extracted with two portions of said aqueous alcohol extraction agent wherein a combined weight ratio of the portions of said Extraction agent to said whey protein material does not exceed a total weight ratio of about 11: 1. The process according to claim 33, further characterized in that said adsorbent material is particulate material. 40.- The method according to claim 33, further characterized in that said extract with an adsorbent material comprises releasably linking daidzein in said extract with the adsorbent material. 41. The process according to claim 33, further characterized in that the extract is eluted through the adsorbent material with an eluent to separate a material with high genistein content from said extract. 42.- The method according to claim 33, further characterized in that the material with a high genistein content contains at least 40% daidzein. 43.- The method according to claim 18, further characterized in that it comprises: a) extracting said protein material from yiel with agofilone ieoflavone with an aqueous extraction agent of alcohol to produce an extract enriched with aglucone isoflavone; and b) contacting said extract with an adsorbent material for a sufficient time to remove a material with a high glycitein content from said extract. 44. The method according to claim 18, further characterized in that it comprises: a) extracting said serum protein material with aglucone isoflavone with an aqueous alcohol extraction agent to produce an extract enriched with aglucone isoflavone; and b) concentrating said extract enriched with aglucone isoflavone to between about 15% and about 30% of its original volume; and c) precipitating an isoflavone material from aglucone by adding water to said extract. 45. The method according to claim 44, further characterized in that said aqueous alcohol extraction agent includes between about 30% and about 90%. 46. The method according to claim 44, further characterized in that said serum protein material with aglucone isoflavone is extracted with said extraction agent wherein a ratio of extraction agent to serum protein material does not exceed approximately 11: 1. 47. The method according to claim 44, further characterized in that said whey protein material with aglucone isoflavone is extracted with two portions of said aqueous alcohol extraction agent. * where a combined peeo relationship of the portion of < * said extracting agent to said whey protein material does not exceed a total weight ratio of about 11: 1. 48. The process according to claim 44, further characterized in that said aqueous alcohol extraction agent has a pH value of approximately the isoelectric point of said protein in 20 said protein material of euero with isoflavone of aglucona. 49.- The method according to claim 48, further characterized in that said aqueous alcohol extraction agent has a pH value between about 3 and about 6. The process according to claim 44, further characterized in that Water is added to the extract, wherein a weight ratio of water to extract is between about 6: 1 and about 8: 1. 51.- The method according to claim 44, further characterized in that it comprises washing 5 with water the precipitated aglucone isoflavone material, wherein a weight ratio of water to aglucone isoflavone material is between about 0.8: 1 and about 2: 1. 52. The method according to claim 44, further characterized in that it comprises cooling the extract and water to maximize the precipitation of said aglucone isoflavone material. »53.- The procedure in accordance with the > 4 claim 44, further characterized in that it comprises: a) solvating said aglucone isoflavone material; and b) contacting said aqueous solution of alcohol containing the solvated aglucone ieoflavone material for a time sufficient to remove a material with a high genistein content from said aqueous alcohol solution. 54.- The method according to claim 44, further characterized in that it comprises: a) solvating said aglucone isoflavone material; and b) contacting said aqueous solution of alcohol containing the solvated aglucone isoflavone material for a time sufficient to remove a high-daidzein material from said aqueous alcohol solution. 55.- A serum of aglucone isoflavone vegetable protein produced by the process according to claim 1. 56.- A whey protein material with aglucone isoflavone produced by the procedure according to * 5 claim 18., 57.- A material with high content of genistein produced by the process according to claim 22. 58.- A material with high content of daidzein 10 produced by the process according to claim 33. »59.- A material with a high content of genistein produced by the process according to claim 53. 60 60.- A material with a high content of daidzein produced by the process according to claim 54. 61. An aglucone isoflavone material produced by the process according to claim 44. 20 62. A whey protein material with aglucone isoflavone comprising a protein of yielder and ieoflavones of aglucone. 63.- An isoflavone material of aglucone comprising a soy material, at least 10% of genistein and 25 at least 5% daidzein. 64.- A material with a high content of genistein comprising a soy material and at least 40% of genistein. 65.- The material with high genietein content according to claim 64, which contains at least 90% of genistein. * > 5 66. - A material with a high content of daidzein that _ comprises a soy material and at least 40% daidzein.