RU2309603C2 - Method for production of plant whey material enriched with aglucon isoflavone (variants), method for production of material with high content of genisteine and daidzeine - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to plant whey material enriched with aglucon isoflavone, whey protein material, material with high content of genisteine, material with high content of daidzeine, aglucon isoflavone-based material and methods for production thereof from plant protein whey. Isoflavone conjugates containing in plant protein whey are converted into isoflavone glucosides by whey treatment at temperature, pH and for time sufficient for conversion. Isoflavone glucosides are converted to aglucon isoflavones by enzymatic reaction to produce aglucon isoflavone-enriched plant protein whey. Aglucon isoflavone-containing whey protein material is obtained from aglucon isoflavone-enriched plant protein whey. Material with high content of genisteine, material with high content of daidzeine, aglucon isoflavone-based material are isolated from alcohol extract, containing aglucon isoflavones of whey protein material.

EFFECT: new plant whey material.

51 cl, 2 tbl, 5 ex

Description

The present invention relates to plant protein whey enriched in agluconisoflavone, a whey protein material based on agluconisoflavone, a material based on agluconisoflavone, a material with a high content of genistein, a material with a high content of daidzein and methods for their preparation from vegetable protein whey.

Isoflavones are found in various plants from the legume family, including plant-based protein materials, such as soy. These compounds include daidzin, 6 '' - OAc daidzin, 6 '' - OMal daidzin, daidzein, genistin, 6 '' - OAc genistin, 6 '' - OMal genistin, genistein, glycithin, 6 '' - OAc glycithin, 6 '' - OMal glycithin, glycitein, biochanin A, formononetin and caumestrol. Usually the bitter taste of soy is associated with the content of these compounds.

In the manufacturing process of commercial products such as plant protein isolates and concentrates, particular attention is paid to the removal of these materials. For example, in a traditional soy protein isolate production process in which soy flakes are extracted in an aqueous alkaline medium, most of the isoflavones are dissolved in the extract along with soy protein. Protein is precipitated from the extract by acidification and separated to form an isolate, while the remaining serum contains most of the dissolved isoflavones. Isoflavones remaining in the acid-precipitated protein isolate are usually removed by thorough washing of the isolate. Serum and washings are usually discarded. Isoflavones contained in plant protein whey contain isoflavone glucosides (glucons), isoflavone conjugates, and agluconisoflavones. Isoflavone glucosides contain a glucose molecule attached to the isoflavone backbone of the compound. Isoflavone conjugates contain additional backbones attached to the glucose molecule, for example, 6 '' - OAc genistin contains an acetate group attached to the glucose molecule in the sixth position. Agluconisoflavones consist of an isoflavone skeleton, without the addition of a glucose molecule.

Soya serum contains three “groups” of isoflavone compounds, including, respectively, glucoside, conjugate and agluconic moieties: the group of genistein, diadzein and glycitein. The genistein group includes glucosidegenistin, conjugates of 6 ″ OMal genistin (6 ″ genistin malone ester) and 6 ″ OAc genistin (6 ″ acetic genistine ester) and aglucongenistein. The daidzein group includes glucosidine diazine 6 "-OMal daidzin and 6" - OAc daidzin and aglucondaidzein. The glycitein group includes glycitin glucoside, conjugate 6 "- OMal glycithin and glycitein aglucon.

All isoflavones are of interest for medical evaluations, however, aglucones are specific isoflavones of particular interest. Genistein and daidzein can significantly reduce the risk of cardiovascular disease (Plant and Mammalian Estrogen Effects on Plasma Lipids of Female Monkeys, Circulation, vol. 90, p. 1259 (October 1994)). Genistein and daidzein may also be able to relieve symptoms of conditions caused by low or altered levels of endogenous estrogen in women in conditions such as menopause or premenstrual syndrome. In addition, it has recently been discovered that agglutinisoflavones contained in plant materials such as soy are capable of stopping the growth of cancer cells in humans, such as breast or prostate cancer cells (Peterson, Bames, "Genistein Inhibition of the Growth of Human Breast Cancer Cells, Independence from Estrogen Receptors and the Multi-Drug Resistance Gene ", Biochemical and Biophysical Research Communications, vol. 179. No 1, pp.661-667, August 30, 1991; Peterson, Bames," Genistein and Biochanin A Inhibit the Growth of Human Prostate Cancer Cells but not Epidermal Growth Factor Receptor Tyrosine Autophosphorylation ", The Prostate, vol 22, pp. 335-345 (1993); and Barnes et al.," Soybeans Inhibit Mammary Tumors in Models of Breast Cancer ", Mutagens and Carcinogens in the Diet. pp. 239-253 (1990)).

As stated above, agluconisoflavones include daidzein, genistein and glycitein. These aglucones have the following general formula:

where R ₁ , R ₂ , R ₃ and R ₄ are groups H, OH or OCH ₃ . Genistein has this formula in which R ₁ = OH, R ₂ = H, R ₃ = OH and R ₄ = OH, daidzein has a formula in which R ₁ = OH, R ₂ = H, R ₃ = H and R ₄ = OH, glycitein has the formula in which R ₁ = OH, R ₃ = OCH ₃ , R ₃ = H and R ₄ = OH.

The present invention relates to aglucones and enrichment of plant protein whey and whey protein material with these compounds, and in particular, a material with a high content of genistein, a material with a high content of daidzein and a material containing agluconiisoflavone. This invention also relates to methods for producing aglucone enriched plant protein whey, aglucone enriched plant whey protein material, a high content of genistein, a high daidzein content and a material containing agluconisoflavone.

The general process for converting isoflavone conjugates contained in a plant protein to agluconisoflavones is known, it is described in U.S. Patent Application No. 08/477102, filed June 7, 1995, and is currently under consideration and belongs to the assignee of the present invention. Methods for converting isoflavone glucosides to agluconisoflavones are also known. The method for converting isoflavone glucosides to agluconisoflavones in plant protein whey is described in PCT / US94 / 10699, the application is currently under consideration and belongs to the assignee of the present invention.

In the field of conversion of isoflavone glucosides to agluconisoflavones, other methods are known, for example, Obata and others in the application for invention in Japan 258669. Such processes do not involve the conversion of isoflavone conjugates to agluconisoflavones or to obtain material with a high content of genistein, a material with a high content of daidzein, or material containing agluconisoflavone. In addition, such methods can only achieve a moderate degree of conversion of glycosides to aglucones and require a significant period of time to achieve this moderate degree of conversion. Thus, these methods are undesirable for large-scale commercial production.

The aim of the present invention was the production of plant protein whey enriched in agluconisoflavone and the development of a method for its preparation from plant protein whey.

Another objective of the present invention was to obtain whey protein material containing agluconisoflavone, and a method for its production from plant protein whey.

The next objective of the present invention was to obtain a material with a high content of genistein and a method for its preparation from plant protein whey.

A further objective of the present invention was to obtain a material with a high content of daidzein and a method for its preparation from plant protein whey.

Another objective of the present invention was to obtain a material containing agluconisoflavone, and a method for its preparation from plant protein whey.

These and other objectives of the invention have been achieved, as described below, in the detailed description of the invention.

SUMMARY OF THE INVENTION

These goals were achieved by the fact that in the method for producing plant whey material enriched with agluconisoflavone from plant whey material containing isoflavone conjugates, according to the invention, the plant whey material is treated at a temperature of 2 ° C-121 ° C and a pH of 6-13.7 to until more than 50% of the isoflavone conjugates turn into isoflavone glucosides; and said isoflavone glucosides are fermented in said whey material at a temperature of 5 ° C-75 ° C and pH 3-9 until more than 50% of the isoflavone glucosides are converted to agluconisoflavones.

Preferably, the plant whey material is treated at a pH of 9 to 10 and a temperature of 45 ° C to 73 ° C for 4 to 6 hours.

More preferably, the plant whey material is treated at a pH of 10 to 11 and a temperature of 5 ° C to 50 ° C for 0.5 to 1 hour.

In a preferred embodiment of the method, at least 80% or even 90% of the isoflavone conjugates are converted to isoflavone glucosides.

Mentioned isoflavone glucosides are subjected to fermentation in plant whey material, preferably at a temperature of from 35 ° C to 45 ° C.

In this case, fermentation involves the addition of an effective amount of an additional enzyme to the plant whey material.

A saccharidase capable of cleaving 1,4-glucosidic bonds is preferably used as an additional enzyme.

An additive enzyme selected from the group consisting of α-glucosidases, β-glucosidases, β-galactosidases, gluco-amylases, pectinases and mixtures thereof is used.

At the same time, at least 80% or 90% of the isoflavone glucosides are converted to agluconisoflavones.

Soy whey can be used as a plant whey material.

The method preferably includes the selection containing agluconisoflavones whey protein material containing protein and agluconisoflavones from the said plant whey material enriched in agloconisoflavone.

The whey protein material containing agluconisoflavones is preferably isolated by at least one method from the group consisting of ultrafiltration, heat coagulation and dehydration.

The whey protein material containing agluconisoflavones is preferably obtained by freezing the plant whey material and separating the whey protein material containing agluconisoflavones from the cooled whey.

Moreover, the whey protein material containing agluconisoflavones is preferably isolated from concentrated plant whey material.

This goal is also achieved by the fact that in the method of obtaining a material with a high content of genistein from plant whey material containing isoflavone conjugates, according to the invention, plant whey material is processed at a temperature of 2 ° C-121 ° C and a pH of 6-13.7 until until more than 50% of the isoflavone conjugates are converted to isoflavone glucosides, and said isoflavone glucosides are fermented in said whey material at a temperature of 5 ° C-75 ° C and pH 3-9 until more than 50% of isofl onglucosides will not turn into agluconisoflavones, then isolate whey protein material containing agluconisoflavones containing protein and agluconisoflavones from said plant whey enriched with agluconisoflavone, and then extracting whey protein material containing agluconisoflavones with an aqueous-alcoholic extractant is carried out to obtain an extract, the extract is carried out; the resulting extract with adsorbent material until the final the material will not separate from the extract.

In this case, a water-alcohol extractant containing from 30% to 90% alcohol is used.

Preferably, a water-alcohol extractant having a pH value near the isoelectric point of said protein is used in this whey protein material containing agluconisoflavone, in particular a water-alcohol extractant having a pH of 3 to 6 is used.

The whey protein material containing agluconisoflavones is preferably extracted with an extractant, and the weight ratio of extractant to whey protein material does not exceed 11: 1.

Moreover, the whey protein material containing agluconisoflavones is preferably extracted twice with a water-alcohol extractant, the total weight ratio of the amounts of the combined servings of the extractant and whey protein material not exceeding 11: 1.

Particulate adsorbent material may be used.

The contact of the extract with the adsorbent material preferably includes reversible binding of genistein to the adsorbent material.

The extract is preferably passed through an adsorbent material with an eluent to separate the high genistein material from the extract.

A high genistein material contains at least 40% genistein, preferably at least 90% genistein.

The goal is also achieved by the following invention - a method of obtaining a material with a high content of daidzein.

In the method for producing material with a high daidzein content from plant whey material containing isoflavone conjugates, according to the invention, plant whey material is treated at a temperature of 2 ° C-121 ° C and pH 77 6-13.7 until more than 50% of the isoflavone conjugates will not turn into isoflavone glucosides, and said isoflavone glucosides are fermented in said whey material at a temperature of 5 ° C-75 ° C and pH 3-9 until more than 50% of the isoflavone glucosides is converted to agluconisofl won, then isolating whey protein material containing agluconisoflavones containing protein and agluconisoflavones from said plant whey enriched with agluconisoflavone is extracted, and extracting whey protein material containing agluconisoflavones with an aqueous-alcoholic extractant to obtain an extract enriched with agluconisoflavone contact material, and adsorbent until material with a high content of daidzein is not will separate from the extract.

In this case, it is preferable to use a water-alcohol extractant containing from 30% to 90% alcohol, having a pH value near the isoelectric point of said protein in this whey protein material containing agluconisoflavone. In particular, a water-alcohol extractant having a pH value of from 3 to 6 is used.

The whey protein material containing agluconisoflavones is preferably extracted with an extractant at a weight ratio of the amounts of extractant and whey protein material not exceeding 11: 1.

In particular, the whey protein material containing agluconisoflavones is preferably extracted twice with a water-alcohol extractant, and the total weight ratio of the quantities of the extractant and whey protein material does not exceed 11: 1.

Particulate adsorbent material may be used.

Moreover, the contact of the extract with the adsorbent material also includes reversible binding of the daidzein extract with the adsorbent material.

The extract is preferably passed through an adsorbent material using an eluent to isolate a high daidzein material from the extract.

A high daidzein content material preferably contains at least 40% daidzein.

The goal was also achieved by the following invention - a method for producing plant whey material enriched in agluconisoflavone.

In the method for producing plant whey material enriched in agluconisoflavone from plant whey material containing isoflavone conjugates according to the invention, the plant whey material is treated at a temperature of 2 ° C-121 ° C and a pH of 6-13.7 until more than 50% of the conjugates isoflavone will not turn into isoflavone glucosides; and said isoflavone glucosides are fermented in said whey material at a temperature of 5 ° C-75 ° C and pH 3-9 until more than 50% of the isoflavone glucosides are converted to agluconisoflavones, whey protein containing agluconisoflavones containing protein and agluconisoflavones are isolated, from said plant whey material enriched with agluconisoflavone, the whey protein material containing agluconisoflavones is extracted with an aqueous-alcoholic extractant to to obtain the extract enriched in agluconisoflavone, and the resulting extract is contacted with the adsorbent material until the material containing glycitein is separated from the extract.

In this case, the method preferably includes extraction of the agluconisoflavone-containing whey protein material with an aqueous-alcoholic extractant to obtain an extract enriched in agluconisoflavone, and concentrating the resulting extract to 15% -30% of its original volume, and then precipitating the material containing agluconisoflavone by adding water to the extract .

In this case, the water-alcohol extractant contains from 30% to 90% alcohol.

The whey protein material containing agluconisoflavones is preferably extracted with an extractant in a weight ratio of extractant to whey protein material not exceeding 11: 1.

The whey protein material containing agluconisoflavones is preferably extracted twice with a water-alcohol extractant, and the total weight ratio of the quantities of extractant to whey protein material does not exceed 11: 1.

In this case, it is preferable to use a water-alcohol extractant having a pH near the isoelectric point of the protein in this agluconisoflavone whey protein material, in particular, a water-alcohol extractant having a pH from 3 to 6 is used.

Preferably, water is added to the extract in a weight ratio of water: extract of 6: 1 to 8: 1.

The precipitated material containing agluconisoflavones is preferably washed with water, wherein the weight ratio of water: agluconisoflavones containing material is from 0.8: 1 to 2: 1.

The extract and water are cooled to achieve the most complete precipitation of the agluconisoflavone-containing material.

The method preferably includes dissolving the agluconisoflavone-containing material in an aqueous-alcoholic solution and contacting the aqueous-alcoholic solution containing the dissolved agluconisoflavone-containing material with the adsorbent material until the material with a high content of genistein or daidzein is separated from the solution.

Description of preferred embodiments of the invention

The starting material for the implementation of the method is plant protein whey, which is an aqueous solution of soluble proteins, isoflavones and other water-soluble compounds remaining after removal of protein clots from the plant protein extract. In a preferred embodiment, the starting material is soya whey, since this method is particularly suitable for producing agluconisoflavone enriched whey, agluconisoflavone whey protein material, high genistein material, high daidzein material and agluconisoflavone containing material from soy products.

The starting plant protein material contains isoflavone conjugates, isoflavone glucosides, and agluconisoflavones. For example, soya serum contains: isoflavonglucosides - genistin, daidzin and glycitin; conjugates of isoflavone - 6 "- malonic esters of genistin, daidzin and glycithin and 6" - acetic esters of genistin and daidzin; and agluconisoflavones - genistein, daidzein and glycytein. In the initial whey material of soybeans, isoflavones are predominantly in the form of isoflavone conjugates.

Plant protein whey starting material can be obtained as a by-product of a conventional plant protein isolate manufacturing process. A vegetable protein source, for example, such as soy, defatted by solvent extraction, can be extracted with an aqueous extractant having a pH above the isoelectric point of the source protein to form an extract containing protein, isoflavones and other compounds recovered from the vegetable protein source during extraction. The extract is separated from the plant material insoluble in the extract. Then, the pH of the resulting extract containing soluble proteins and isoflavones is adjusted to approximately the isoelectric point of the protein in order to precipitate the protein from the extract, and for soy protein this pH is 4.4-4.6. The precipitated protein is separated to obtain a plant protein isolate, leaving the original plant protein whey material. Most of the isoflavones remain in a soluble state in serum. To increase the isoflavone content in serum, additional washing of the precipitated protein may be required, with each serving of washing liquid being added to the serum.

The starting vegetable protein whey material may be a spray dried vegetable protein whey dissolved in water. To facilitate the work, plant protein whey is dried by spray drying to obtain whey protein (the protein remaining dissolved in whey after precipitation of the protein isolate), isoflavones and other compounds in the form of solid materials. Spray dried material is added to water to reconstitute the original plant protein whey material. In a preferred embodiment, the solution contains about 2-10 g of whey dried material per 100 g of water, so that the serum is not too viscous and nevertheless contains enough isoflavones to create the desired serum enriched in agluconisoflavone, whey protein material, containing agluconisoflavone, a material with a high content of genistein, a material with a high content of daidzein and a material containing agluconisoflavone.

At the first stage of the process, the isoflavone conjugates contained in the plant protein whey source material are converted to isoflavone glucosides to produce the plant protein whey enriched therein. The degree of conversion was found to depend on the pH and temperature of the serum.

To convert isoflavone conjugates to isoflavone glucosides, the pH should be between 6 and 13.5. If necessary, the pH of the plant protein whey should be adjusted to the desired value. Soy protein whey usually has a pH value of 4.4-4.6, and to adjust the pH to the desired value, a base or basic reagent should be added to it. The pH value can be increased with any suitable base, alkaline reagent or basic reagent that increases the pH of the system, including potassium, calcium, sodium hydroxides. It turned out that the conversion occurs faster in a relatively stronger alkaline environment, mainly at pH 9-11. It is necessary to maintain the pH below 12, since isoflavon glucosides - genistin, glycithin and, in particular, daidzin - disintegrate at pH 12 and above. The reaction proceeds more slowly at lower pH values, for example at pH 6, however, the reaction will proceed at elevated temperature and / or elevated pressure.

The temperature at which the conversion of isoflavone conjugates to isoflavone glucosides occurs is in the range from 2 ° C to 121 ° C. The temperature at which conversion occurs faster depends on the pH of the serum. As shown, conversion is easier at lower temperatures if the pH is high enough. For example, with a serum pH of about 11, the conversion is fast and efficient at temperatures from 5 ° C to 50 ° C. When the pH of the whey is about 9, the conversion effectively occurs at a temperature of from 45 ° C to 73 ° C. If the pH of the whey is sufficiently low, conversion occurs at elevated temperatures. For example, at a serum pH of 6, conversion occurs at a temperature of 80 ° C to 121 ° C. In a preferred embodiment, the conversion occurs at 35 ° C. at a serum pH of about 11. In another preferred embodiment, the conversion occurs at 73 ° C. and a serum pH of about 9.

The time required for the almost complete conversion of isoflavone conjugates to isoflavone glucosides depends on the pH and temperature of the plant protein whey. The required time ranges from 15 minutes to 24 hours. Conversion occurs faster at elevated pH and elevated temperatures. At a pH of about 9-10, almost complete conversion is achieved in about 4-6 hours at 73 ° C. At a pH of about 10-11, almost complete conversion is achieved in about 30 minutes - 1 hour at 35 ° C. In a most preferred embodiment, the isoflavone conjugates are converted to isoflavone glucosides in about 45 minutes at a pH of about 11 and a temperature of about 35 ° C.

The first stage of conversion occurs with amazing efficiency, 80-100% of isoflavone conjugates are converted to isoflavone glucosides. Typically, the degree of conversion is at least 95%. Such high conversion rates are particularly attractive for use on an industrial scale.

In the second stage of the process, the isoflavone glucosides obtained in the first stage and the isoflavone glucosides previously found in serum are converted by enzymatic reaction to agluconisoflavones. The process allows to obtain plant protein whey enriched in agluconisoflavone from whey enriched in isoflavone glucoside.

It turned out that the second stage of the process depends on the concentration of enzymes in the serum and their characteristics. The conversion requires enzymes capable of cleaving the glucosidic bond in isoflavone glucosides between the isoflavone backbone and the glucose molecule. In a preferred embodiment, these enzymes are saccharidases capable of cleaving 1,4-glucosidic bonds. The concentration of enzymes necessary for the conversion of isoflavone glucosides to agluconisoflavones depends on a number of factors, including: the type of enzymes present in the serum, distribution of enzyme concentrations, enzyme activity, isoflavone glucoside concentration, pH and serum temperature during the process.

Enzymes can be present in plant protein whey from the very beginning, appear as a result of microbial processes in serum, or be added as an additive. Enzymes that are in serum from the very beginning, or that have arisen as a result of microbial processes, will hereinafter be called “residual” enzymes, added enzymes will be called “incremental”.

A suitable enzyme must be present in serum in order to convert at least a large portion, and more preferably, substantially all of the isoflavone glucosides to agluconisoflavones. Generally, if residual enzymes are unable to carry out the conversion, additional enzymes should be added to the serum. As noted above, a number of factors determine whether enzymes are present in the right concentration to effect conversion.

If an additional enzyme is introduced into the system, it must be added in such quantities that the total concentration of the present enzymes is 0.1-10% by weight relative to the weight of the whey, based on dry weight. In a preferred embodiment of the invention, the additional enzyme is introduced into the whey regardless of whether a suitable residual enzyme is present in it, since the addition of the additional enzyme significantly reduces the time required to achieve almost complete conversion of glucosides to aglucones.

Additional enzymes are selected based on optimal activity under conditions selected by pH and temperature, as well as on the basis of price. Enzymes capable of cleaving an isoflavone glucoside bond between an isoflavone backbone and a glucose backbone, such as, for example, saccharidases capable of cleaving 1,4-glucosidic bonds, are used as additives. Preferred additional enzymes are commercially available enzymes of α- and β-glucosidase, β-galactosidase, glucoamylase and pectinase. Especially preferred are enzymes such as Biopectinase 100L (which is mainly used at pH 3-6), Biopectinase 300L (optimal pH is about 3 to 6), Biopectinase OK 70L (optimal pH is about 3 to 6), Biolactase 30,000 (optimal pH from about 3 to 6), Neutral Lactase (optimal pH from about 6 to 8), all of these enzymes are from Quest International, 1833 57th Street, Post Office Box 3917, Sarasota, Florida 34243. Also especially preferred are Lactase F (optimal value pH of about 4 to 6), Lactase 50,000 (optimal pH of about 4 to 6), which were obtained from Amano International Enzyme Co., Inc., Post Office Box 1000, Troy, Virginia 22974. Other particularly preferred enzymes are: G-Zim G990 (optimum pH from about 4 to 6) and Enseco Fangal Lactase Concentrate (optimal pH from about 4 to 6), obtained from Enzyme Development Corporation, 2 Pen Plaza, Suite 2439. New York, NY 10121; Lactosim 3000L (optimum pH from about 6 to 8), Alpha-Gal 600L (optimal pH from about 4 to 6.5), obtained from Novo Nordisk Bioindustriels, Inc., 33 Tener Road, Danbury, Connecticut 06813; Neutral Lactase (optimum pH from about 6 to 8), obtained from Fizer Food Science Group, 205 East 42nd Street, New York, NY 10017; and Maxilast L2000 (optimum pH from about 4 to 6), obtained from Jist Brokadz Ingredient, Inc., King of Prussia, PA 19406.

If enzymes are present in the system in sufficient concentrations (residual, additive, or both), they are in contact with serum isoflavone glucosides at a temperature, pH, and for a period of time sufficient to convert at least most (preferably, completely all) isoflavone glucosides to agluconisoflavones. If necessary, the pH of the serum enriched with isoflavone glucosides should be adjusted to a pH value at which the enzymes react more actively with isoflavone glucosides. The pH at which the combined mixture of residual and additional enzymes reacts with isoflavone glucosides is approximately 3 to 9.

Residual enzymes have been shown to be active in the pH range of about 7 to 9, although it is likely that the pH of the serum decreases during the course of the reaction. Supplemental enzymes are active at the optimum pH value set by the enzyme manufacturers, as shown above for several specific enzymes. Typically, additional enzymes are active either in a neutral environment at a pH of from about 6 to 8, or in an acidic environment at a pH of from about 4 to 6. It has also been shown that acidic enzymes are active at a pH of about 3.

The pH of the serum can be changed, in most cases lowered from a relatively high or alkaline pH of the first stage, by adding one or more suitable acids, such as acetic, sulfuric, phosphoric, hydrochloric acid, or any other suitable reagent. Preferably, the reagent used should be a food grade acid reagent or acid.

The temperature range in which isoflavone glucosides are converted to agluconisoflavones ranges from about 5 ° C to 75 ° C. Temperature significantly affects the activity of enzymes, and, thus, the conversion rate. Additional enzymes are able to be active at temperatures above 72.5 ° C, for example, Alpha-Gal 600L is active at 75 ° C, however, it is better to carry out the reaction at lower temperatures to avoid deactivation of the enzymes. In a preferred embodiment, the conversion is carried out between about 35 ° C and about 45 ° C.

Preferably, the enzymatic reaction is carried out at the same temperature as the first stage, so there is no need to change the temperature of the serum after the first stage. Most preferably, both the first and second stages are carried out at 35 ° C.

It is also desirable that a constant temperature be maintained during the conversion of isoflavone glucosides to agluconisoflavones. However, in some cases it may be desirable to raise, lower, or otherwise vary the temperature during the reaction.

The time required for the second stage depends on factors associated with the enzymes, in particular, the concentration, temperature and pH of the serum. In most cases, the full degree of conversion is possible in 24 hours, however, the introduction of additional enzymes is preferable, which significantly increases the reaction rate. The selected additional enzyme, the concentration of enzymes, pH and temperature preferably lead to the fact that almost complete conversion is achieved in 2 hours, and most preferably in 1 hour.

The degree of conversion of isoflavone glucosides to agluconisoflavones in the second stage is astounding and typically ranges from at least 80% to 100%. Typically, a degree of conversion of isoflavone glucosides to agluconisoflavones of at least 95% is achieved.

After the conversion of isoflavone glucosides to agluconisoflavones, serum enriched with agluconisoflavones can be used as intended without prior drying or protein removal, or alternatively, agluconisoflavone whey protein material can be isolated to concentrate agluconisoflavones in the protein material. Agluconisoflavone whey protein material is a material containing protein, agluconisoflavones and residual plant compounds that can be precipitated and separated from plant protein whey. Protein material enriched in agluconisoflavones can be isolated using conventional methods such as ultrafiltration, heat coagulation, and dehydration. The resulting agluconisoflavone whey protein material can be dehydrated and dried by conventional methods.

Agluconisoflavone whey protein material can also be obtained from whey by cooling it. Agluconisoflavone whey protein material is insoluble in chilled whey and can be separated from it in the form of a precipitate by centrifugation. Preferably, to precipitate the protein material, the whey is cooled to 4 ° C.

In a preferred embodiment, the whey is concentrated to increase the yield of agluconisoflavone whey protein material. It was found that the increase in the ratio of the amount of solid phase to liquid, achieved by concentration of serum, increases the recovery of agluconisoflavone whey protein material. Serum can be concentrated by heating, holding under reduced pressure, or both. Preferably, the serum is concentrated so that the ratio of the amount of solid phase to liquid is from about 1: 3 to 1: 6, most preferably 1: 3.

Materials with a high content of genistein and daidzein can be obtained from isolated agluconisoflavone whey protein material. A high genistein material is a material containing at least 40% genistein, most preferably at least 90% genistein, the remainder is plant material (residual soy material if the high genistein material is obtained from soy serum) . Material with a high daidzein content contains at least 40% daidzein and residual plant material.

To obtain materials with a high content of genistein and daidzein, the agluconisoflavone whey protein material is first washed to remove unwanted salts and sugars, then dried. For washing, the agluconisoflavone whey protein material is diluted with water to a solids content of from about 1% to 6%, most preferably up to 2%. The wash water may have any temperature, however, it is preferable that the temperature be from 25 ° C to 75 ° C, preferably about 60 ° C. After washing, the agluconisoflavone whey protein material is separated from the washings and dried. In a preferred embodiment, the material is isolated by centrifugation and decantation of the supernatant.

To extract agluconisoflavones from whey protein and produce an extract enriched in agluconisoflavones, the agluconisoflavone whey protein material can then be extracted with a water-alcohol extractant. As the alcohol component of the extractant, it is preferable to use low molecular weight alcohols, such as methanol, and, in particular, ethanol. It turned out that agluconisoflavones are soluble at almost any alcohol concentration in the extractant. Agluconisoflavones are particularly soluble if the extractant contains from about 30 to 90% alcohol, most preferably from about 60 to about 80% alcohol. Although an aqueous alcohol solution is the preferred solvent, other solvents may be used to extract agluconisoflavones from whey protein material, including water, acetonitrile, methylene chloride, acetone, ethyl acetate, and mixtures thereof.

Extraction is carried out using a minimum amount of extractant. It is preferable that the ratio by weight of extractant to agluconisoflavone whey protein material does not exceed 11: 1. In one embodiment of the invention, the material can be isolated by countercurrent extraction, in which the ratio by weight of extractant to material is from about 6: 1 to 8: 1. In another embodiment, the material can be extracted in two portions of the extractant, and the total ratio of extractant to material does not exceed 11: 1 by weight.

Although extraction can be carried out at any pH, it is preferable that the extractant has a pH close to the isoelectric point of the protein in the agluconisoflavone whey protein material in order to minimize the solubility of the protein in the extractant. Preferably, the extractant has a pH of about 3 to 6, for soya whey protein, about 4.5 is best.

The extraction can be carried out at any temperature up to the boiling point of the extractant, preferably between about 25 ° C and 70 ° C. To reduce the time required for the extraction of agluconisoflavones from agluconisoflavone whey protein material, extraction is best carried out at a temperature above room temperature, most preferably at about 60 ° C.

After extraction, materials with high contents of genistein and daidzein can be separated from the extract enriched with agluconisoflavone by contacting the extract with adsorbent for a time sufficient to separate materials with high contents of genistein and daidzein from the extract. In a preferred embodiment, materials with high levels of genistein and daidzein are separated from the extract by reverse phase high performance liquid chromatography (HPLC, HPLC). Genistein and daidzein are separated from other isoflavones and impurities in the extract by eluting the extract through particles of adsorbent material that reversibly binds genistein, daidzein, other isoflavones and impurities into specific compounds, thus allowing each component to be separated.

First, the agluconisoflavone enriched extract is filtered to remove insoluble particles that may clog the chromatographic column. The HPLC column is prepared as follows: the standard HPLC column is filled with particles of adsorbent material that can reversibly bind genistein, daidzein, other isoflavones and contaminants to specific compounds. Any material for HPLC can serve as an adsorbent, however, the preferred material is selected taking into account the criteria of capacity, separation efficiency and price. One preferred material is Cromasil C 18 16 μm, 100 Å, manufactured by Eka Nobel, Nobel Industries, Sweden.

The filtered extract is passed through a filled HPLC column until the binding material of the column is completely saturated with isoflavones, which is manifested in the appearance of isoflavones in the eluate leaving the column. Then the column is washed with a polar eluent to effect separation. In a preferred embodiment, the eluent is an aqueous solution of alcohol. A water-alcohol eluent may contain from about 30 to 90% alcohol, it is best to use an eluent with an alcohol content of about 50% to ensure both good separation and good solubility of isoflavones. Methanol and ethanol are mainly used as alcohol; ethanol is best used if materials with high contents of genistein and daidzein will be used for the preparation of food products or medicines.

Materials with a high content of genistein and daidzein are collected in the eluate leaving the column. Daidzein is the first to leave the column, then the glycitein fraction, followed by the more polar genistein fraction. As the column leaves, the daidzein and genistein fractions are collected, the glycitein fraction can also be collected, if necessary.

Alcohol from the fractions can be removed by evaporation, after which materials with a high content of genistein and daidzein, as well as glycitein, can be isolated by conventional methods, such as centrifugation or filtration. The resulting material with a high content of genistein contains at least 40% of genistein, more preferably at least 90% of genistein, and the remaining plant materials (residual soy materials if genistein is obtained from soya serum). The resulting material with a high daidzein content contains at least 40% genistein and the remaining plant materials, which usually include a significant amount of glycitein.

In another embodiment, the agluconisoflavone material is obtained from an extract enriched in agluconisoflavone. An agluconisoflavone material is a material containing at least 10% genistein and at least 5% daidzein, as well as other isoflavones and remaining plant compounds.

After extraction of the agluconisoflavone whey protein material, the extract enriched in agluconisoflavone can be concentrated to facilitate the precipitation of the agluconisoflavone from the extract. The extract can be concentrated by heating, keeping under reduced pressure, or both. In a preferred embodiment, the extract is concentrated to about 15-30% of the original volume.

Agluconisoflavone material precipitates from the extract by adding water to the extract. In a preferred embodiment, about 6 to 8 parts of water is added to one part of the concentrated extract. When water is added to the extract, a certain amount of agluconisoflavone material precipitates.

To obtain the maximum yield of agluconisoflavone material from the extract, the extract and water are thoroughly mixed and then cooled. The extract and water are mixed for some time, preferably from about 0.5 to 1 hour. In a preferred embodiment, the extract and water are mixed at a temperature of from about 50 ° C to 75 ° C, most preferably at about 70 ° C. After the water and extract are thoroughly mixed, the mixture is cooled to precipitate agluconisoflavone material. Preferably, the mixture is cooled to about 5 ° C.-20 ° C., most preferably about 10 ° C., for a time sufficient to substantially completely precipitate the agluconisoflavone material. The precipitated agluconisoflavone material can then be separated from the extract-water mixture by conventional methods such as centrifugation and filtration.

The isolated agluconisoflavone material can then be washed with water. In a preferred embodiment, the agluconisoflavone material is washed with water at a temperature of about 70 ° C. for about 5 minutes, wherein the ratio by weight of the wash water and the material is from about 0.8: 1 to 2: 1. The agluconisoflavone material is separated from the wash water by conventional methods, such as filtration or centrifugation, and dried. The resulting agluconisoflavone material usually contains at least 20% genistein and at least 10% daidzein, the remainder is material formed from residual plant materials, including other agluconisoflavones. If the agluconisoflavone material is isolated from soybean whey, the remaining plant materials are soybean materials.

The resulting agluconisoflavone material can be further purified to obtain a high content of genistein containing at least 40%, and more preferably at least 90%, of genistein and a high content of daidzein containing at least 40% daidzein. The agluconisoflavone material may be dissolved in a water-alcohol solvent. As the alcohol component of the solvent, it is preferable to use low molecular weight alcohols, especially ethanol, its low toxicity makes it possible to use the material in the food and medical industries. The alcohol content in the solvent is preferably from about 30 to 90%, with 80% being most preferred, since such a content provides good dissolution of the agluconisoflavone material.

An aqueous alcoholic solution containing dissolved agluconisoflavone material may be contacted with the adsorbent material for a period of time sufficient to isolate materials with high contents of genistein and daidzein from the aqueous-alcoholic solution. In a preferred embodiment, materials with high contents of genistein and daidzein are isolated from the water-alcohol solution by reverse phase HPLC. An HPLC column was prepared as described above, an aqueous-alcoholic solution containing agluconisoflavone material was applied to the column, and materials with high genistein and daidzein contents were eluted from the column as described above. The resulting material with a high content of genistein contains at least 40% genistein, more preferably at least 90% genistein, and the remaining plant materials (residual soy materials if genistein is obtained from soya serum). The resulting material with a high daidzein content contains at least 40% genistein and the remaining plant materials.

experimental part

The present invention is illustrated in more detail using the following examples, soy whey is used as a plant protein whey. The examples are illustrative and do not in any way limit the scope of the invention.

As shown above, soya serum contains "groups" of isoflavones: genistein, diadzein and glycitein having the corresponding glucosides, conjugates and aglycones, while the genistein group includes conjugates 6 '' - OMal-genistin and 6 '' - OAc-genistin, glucosidegenistin and aglucongenistein. The daidzein group includes conjugates 6 '' - OMal-daidzin and 6 '' - OAc-daidzin, glucosiddaidzin and aglucondaidzein. The glycitein group includes a 6 ″ conjugate — OMal-glycithin, glucosid glycitin, and aglucon glycitein. In the tables below, the relative concentrations of isoflavones are measured as a percentage in the isoflavones group. For example, for the genistein group:% genistin +% 6`` - OMal-genistin +% 6 '' - OAc-genistin +% genistein = 100%. An increase in the degree of conversion of conjugates to glucosides and glucosides to aglucones can be determined by comparing the percentages of each type of compound in the isoflavone group.

Example 1

In the first experiment, the degree of conversion of isoflavone conjugates to isoflavone glucosides was determined. An increase in the degree of conversion is determined by a quantitative decrease in the percentage of malonic and acetic esters in the isoflavone group with a corresponding quantitative increase in the percentage of glucosides of the same group.

The effect of pH in the first step in the conversion of isoflavone conjugates to isoflavone glucosides was measured at two different temperatures. Soya whey was spray dried, then dissolved in water to obtain a soya whey suspension containing 2% solids by weight. Serum was divided into two groups of four samples. The pH of each sample was adjusted to 6.0, 7.0, 9.0, and 11.0, respectively. The groups of samples were kept in a thermostat for 24 hours, one at 45 ° C, the other at 72.5 ° C. The analysis of each sample to determine the content of isoflavone was carried out periodically at 0, 2, 4, 6, 8, and 24 hours. Table 1 shows the changes in the composition and distribution of isoflavones during the reaction.

The first stage of conversion occurs most rapidly and fully at a higher, alkaline pH and elevated temperatures, as evidenced by the relative decrease in the concentrations of 6 '' OMal and 6 '' OAc isoflavone conjugates and a corresponding increase in the concentrations of genistin, daidzin and glycitin glucosides. Almost complete conversion of isoflavone conjugates to isoflavone glucosides occurs at pH 9 and 11 at a temperature of both 45 ° C and 72.5 ° C. The transformation in the samples also occurs almost completely at pH 6 and 7 at 72.5 ° C.

Example 2

In the second experiment, the degree of conversion of isoflavone glucosides to agluconisoflavones was determined. To study the second stage, isoflavon glucoside-enriched serum obtained in the first stage was used. The degree of conversion was determined by a quantitative decrease in the percentage concentration of glucosides of the isoflavone group, accompanied by a corresponding increase in the percentage concentration of aglucones of the same group of isoflavones.

Soya serum was converted to isoflavone glucoside enriched serum by adjusting the pH of the serum to 11.0 and keeping it at 35 ° C for 30 minutes. One of the samples of serum enriched in glucosides was held for 24 hours at 45 ° C to determine the degree of conversion of isoflavone glucosides to agluconisoflavones using residual serum enzymes. The following commercial additive enzymes were introduced into other samples of glucoside-enriched serum: Biopectinase 100L, Biopectinase 300L, Biopectinase OK70L, Lactase F, Alpha-Gal 600L, G-Zim G990, Quest Biolactase 30,000, Novo Lactosim 3000L, Maxilast L2000, Maxileko L2000, Lactase, Quest Neutral Lactase and Pfizer Neutral Lactase. The pH of the samples into which Alfa-Gal 600L, G-Zim G990, Biopectinase 100L, Biopectinase 300L, Biopectinase OK70L, Lactase F and Enseko Fangal Lactase were introduced was adjusted to 4.5 before the enzyme was added. The pH of the samples into which Novo Novo Lactosim 3000L, Maxilast L2000, Pfizer Neutral Lactase, Quest Biolactase 30,000 and Quest Neutral Lactase were introduced was adjusted to 4.5 and 7.0 before the enzyme was added. Then the samples with additional enzymes were incubated at 50 ° C (except for the sample with Lactase F, which was held at 35 ° C, and samples with Biopectinase 300L and Biopectinase OK70L, which were kept at 40 ° C). Intermediate samples were taken after a certain time and the content of isoflavone was determined. Table 2 shows the distribution of isoflavones during the reaction.

Concentrations per 100 g of serum enriched with glucoside: Biopectinase 300L, Biopectinase OK70L, Lactase F, Alfa-Gal 600L, Enseko Fangal Lactase, G-Zim G990 - 0.1 g, Biopectinase 100L - 0.04 g, Biopectinase 100L - 0.04 g, Novo Lactoz , Maxilast L2000, Pfizer Neutral Lactase, Quest Biolactase 30.000, Quest Neutral Lactase - 0.2 g.

As can be seen from the data on the conversion of genistin, daidzin and glycithin to genistein, daidzein and glycitein, respectively, almost complete conversion of isoflavone glucosides to agluconisoflavones is achieved. Additional enzymes significantly increase the reaction rate, providing, in the case of certain additional enzymes, almost complete conversion in 1 hour. At pH 4.5, the most effective were: Biopectinase 100L, Biopectinase 300L, Lactase F, Alpha-Gal 600L, G-Zim G990, Quest Biolactase 30.000 and Enseko Fangal Lactase. At pH 7.0, Quest Biolactase 30.000 and Quest Neutral Lactase were the most effective.

Example 3

In another experiment, agluconisoflavone whey protein material was obtained from soy whey enriched in agluconisoflavone. The first sample of soy whey enriched in agluconisoflavone, weighing 1000 g, containing 30 mg of genistein, 37 mg of daidzein and 7 mg of glycitein, was concentrated by evaporation with gentle heating to 163 g (concentration ratio 1: 6.1). The concentrated serum was heated to coagulate the protein material and centrifuged to further concentrate the whey protein material. 21 g of whey protein material containing 25 mg of genistein, 32 mg of daidzein and 6 mg of glycitein were separated from serum. The resulting whey protein material (mixed whey and whey protein material) contained 82% genistein, 88% daidzein and 77% glycitein.

A second sample of soy whey enriched in agluconisoflavone weighing 400 g, containing 12 mg of genistein, 15 mg of daidzein, 3 mg of glycitein, was heated to coagulate the protein material without concentrating the serum. Coagulated whey protein material and serum were centrifuged to further concentrate whey protein material. Received 8.7 g of whey protein material containing 5 mg of genistein, 7 mg of daidzein and 1 mg of glycitein. The resulting whey protein material (mixed whey and whey protein material) contained 44% of genistein, 47% of daidzein and 34% of glycitein.

A comparison of the whey protein material of the first and second samples shows that the concentration of serum enriched in agluconisoflavone before separation of the whey protein material leads to an increase in the content of agluconisoflavones in the whey protein material.

Example 4

In another experiment, an agluconisoflavone material is obtained by extraction of an agluconisoflavone whey protein material with a water-alcohol extractant and precipitation of an agluconisoflavone material from the extract.

By converting the isoflavone and isoflavone glucoside conjugates contained in serum to agluconisoflavones and to obtain agluconisoflavone whey protein material from serum, 821 g of agluconisoflavone whey protein material containing 86% protein based on dry weight, 4.7 g of genistein and 2.2 g of dizine . Agluconisoflavone whey protein material was extracted with a solution of ethyl alcohol / water with a ratio of 80/20 percent by weight (ratio of solution / agluconisoflavone whey protein material was 7.7 / 1) for 45 minutes at 60 ° C. After extraction, the resulting solution was cooled to 25 ° C and filtered through a No. 4 filter from Whatman paper under vacuum. 1584 g of wet sediment containing 798 g of solids, 0.8 g of genistein, 0.4 g of daidzein and 0.02 g of glycitein, and 3397 g of a pure extract containing 23 g of dry substances, 3.9 g of genistein, 1.8 g of daidzein and 0.34 g of glycitein were isolated.

The precipitate was re-extracted with ethyl alcohol / water with a ratio of 80/20 percent by weight (ratio of solution / starting agluconisoflavone whey protein material 2.3 / 1) for 5 minutes at 25 ° C. After repeated extraction, the resulting suspension was again filtered through a No. 4 filter from Whatman paper. 1542 g of wet sediment containing 794 g of solids, 0.3 g of genistein, 0.1 g of daidzein and 0.01 g of glycitein, and 2042 g of the second extract containing 4.0 g of solids, 0.5 g of genistein, 0.3 g of daidzein and 0.01 g of glycitein were isolated. The extracts were combined, together they contained 94% of genistein and 95% of daidzein, originally contained in agluconisoflavone whey protein material.

Then, the extracts were concentrated by evaporation on a Bushi evaporator under vacuum at 70 ° C to 1528 g (20% of the combined extract volume). To the concentrated extract was added 6000 g of deionized water (water / extract ratio 4/1). When water is added, a white precipitate of isoflavones forms. The suspension of the precipitate was heated for 45 minutes at 70 ° C. Then the solution was kept in a refrigerator at 4 ° C for 24 hours to form and settle a precipitate of isoflavones. 7300 g of the supernatant was decanted from the precipitate, the remaining solution was centrifuged to separate the precipitate. The resulting precipitate was again washed with 600 g of deionized water at 70 ° C. for 15 minutes. The precipitate was separated by centrifugation and dried under vacuum at 50 ° C.

The result was a dry agluconisoflavone material weighing 7.3 g, containing 49% genistein, 19% daidzein and 4% glycitein.

Example 5

In another experiment, material with a high content of genistein and a material with a high content of daidzein are isolated from the agluconisoflavone material by reverse phase HPLC. 2 g of agluconisoflavone material containing 55% genistein, 21% daidzein and 4% glycitein based on dry weight is added to 1 liter of methanol / water solution with a ratio of 50/50 percent by weight. The solution is filtered through Whatman paper filter No. 5, then through a 0.45 μm filter.

Then, the solution was placed on an HPLC column 25 cm long and 2 '' in diameter, filled with Kromasil material (Kromasil C18, 16 μm, granule size 100 Å). The mobile phase, consisting of a methanol / water solution with a ratio of 50/50 percent by weight, is passed through the column at a rate of 64 ml / min. The appearance of daidzein, glycitein and genistein in the eluate is determined by UV absorption. Daidzein is collected in the first fraction, genistein in the second. Fractions containing daidzein and genistein are evaporated to remove alcohol, which causes precipitation of materials with a high content of genistein and daidzein in each of the fractions. Precipitated materials with a high content of genistein and daidzein are separated by centrifugation and dried in a vacuum thermostat. The resulting material with a high content of genistein contains about 95% of genistein, and the resulting material with a high content of daidzein contains about 45% of daidzein.

In the experiments described, all percentages given for 6 '' - OMal-genistin, 6 '' - OAc-genistin, 6 '' - OMal-daidzin, 6 '' - Oasdaidzin, 6 '' - OMal-glycithin and glycitein, represent calculated values. Percentages showing the concentration of enzymes are calculated based on the amount of commercial enzyme per 100 g of whey solids or 100 g of serum in each sample.

The following is a description of a method for the quantitative determination of isoflavones in soy products. Isoflavones are extracted from soy products by mixing 0.75 g of a sample (spray dried or as a finely ground powder) with 50 ml of methanol / water solvent, 80/20 ratio. The mixture is shaken for 2 hours at room temperature on an orbital shaker. After two hours, insoluble matter is removed by filtration through filter No. 42 from Whatman paper. 5 ml of the filtrate is diluted with 4 ml of water and 1 ml of methanol.

The extracted isoflavones were separated by HPLC using a Huppersil C18 reverse phase column from Hewlett Packard. Isoflavones are applied to the column and washed with a gradient of solvent, starting with 88% methanol, 10% water and 2% glacial acetic acid. At a flow rate of 0.4 ml / min, all isoflavones are genistin, 6 '' is OMal-genistin, 6 '' is OAc-genistin, genistein, daidzin, 6 '' is OMal-daidzin, 6 '' is OAc-daidzin, daidzein, glycithin, 6 '' - OMal-glycithin, glycitein - are clearly separated. Peaks are detected by UV absorption at 260 nm. Peaks are identified using an HPLC mass spectrometer.

Quantification is performed according to pure standards (genistin, genistein, daidzin and daidzein) manufactured by Indofine Chemical Campani, Sommerville, New Jersey. Characteristic resolution parameters (peak area / concentration) are calculated for each of the above substances and used for quantification in unknown samples. For conjugate forms for which pure standards are not available, the characteristic parameters are taken to be the same as for the original molecule, but a correction for the difference in molecular weights is introduced.

This method allows you to quantify each individual isoflavone. For convenience, the total content of genistein, the total content of daidzein and the total content of glycitein can be calculated and the total weight of these compounds is presented, as if all conjugated forms were converted to unconjugated. The total content can also be determined directly by acid hydrolysis, converting the compounds into an unconjugated form.

All of the above are only preferred embodiments of the invention. Various changes and additions are possible within the scope of the invention, as described in the claims, which should be considered in light of the Patent Law and the doctrine of equivalents.

Claims (51)

1. The method of obtaining plant whey material enriched in agluconisoflavone from plant whey material containing isoflavone conjugates, characterized in that the plant whey material is processed at a temperature of 2-121 ° C and a pH of 6-13.7 until more than 50% isoflavone conjugates will not turn into isoflavone glucosides; and said isoflavone glucosides are fermented in said whey material at a temperature of 5-75 ° C. and a pH of 3-9 until more than 50% of the isoflavone glucosides is converted to agluconisoflavones. 2. The method according to claim 1, characterized in that the plant whey material is processed at a pH value of from 9 to 10 and a temperature of 45-73 ° C. 3. The method according to claim 2, characterized in that the plant whey material is processed for 4 to 6 hours 4. The method according to claim 1, characterized in that the plant whey material is processed at a pH from 10 to 11 and a temperature of 5-50 ° C. 5. The method according to claim 4, characterized in that the plant whey material is processed for from 0.5 to 1 hour 6. The method according to claim 1, characterized in that at least 80% of the isoflavone conjugates are converted to isoflavone glucosides. 7. The method according to claim 6, characterized in that at least 90% of the isoflavone conjugates are converted to isoflavone glucosides. 8. The method according to claim 1, characterized in that the said isoflavone glucosides are subjected to fermentation in plant whey material at a temperature of from 35 to 45 ° C. 9. The method according to claim 1, characterized in that the fermentation comprises adding an effective amount of an additional enzyme to the plant whey material. 10. The method according to claim 9, characterized in that as an additional enzyme use a saccharidase capable of cleaving 1, 4-glucosidic bonds. 11. The method according to claim 10, characterized in that they use an additional enzyme selected from the group comprising α-glucosidase, β-glucosidase, β-galactosidase, glucoamylase, pectinase and mixtures thereof. 12. The method according to claim 1, characterized in that at least 80% of the isoflavone glucosides are converted to agluconisoflavones. 13. The method according to p. 12, characterized in that at least 90% of the isoflavone glucosides are converted to agluconisoflavones. 14. The method according to claim 1, characterized in that soy whey is used as the plant whey material. 15. The method according to claim 1, characterized in that it includes the selection containing agluconisoflavones of whey protein material containing protein and agluconisoflavones from said plant whey enriched in agluconisoflavone. 16. The method according to clause 15, wherein the whey protein material containing agluconisoflavones is isolated using at least one method from the group comprising ultrafiltration, coagulation under the influence of heat and dehydration. 17. The method according to clause 15, wherein the whey protein material containing agluconisoflavones is obtained by freezing the plant whey material and separating the whey protein material containing agluconisoflavones from the cooled whey. 18. The method according to clause 15, wherein the whey protein material containing agluconisoflavones is isolated from concentrated plant whey material. 19. The method according to p. 15, characterized in that it includes the extraction containing agluconisoflavones of whey protein material with a water-alcohol extractant to obtain an extract enriched in agluconisoflavone, and concentrating the resulting extract to 15-30% of its original volume, and then precipitating the material, containing agluconisoflavone by adding water to the extract. 20. The method according to claim 19, characterized in that the water-alcohol extractant contains from 30 to 90% alcohol. 21. The method according to claim 19, characterized in that the whey protein material containing agluconisoflavones is extracted with the extractant at a weight ratio of the amounts of extractant and whey protein material not exceeding 11: 1. 22. The method according to claim 19, characterized in that the whey protein material containing agluconisoflavones is extracted twice with a water-alcohol extractant, and the total weight ratio of the amount of extractant to whey protein material does not exceed 11: 1. 23. The method according to claim 19, characterized in that they use a water-alcohol extractant having a pH value near the isoelectric point of the protein in a given agluconisoflavone whey protein material. 24. The method according to item 23, wherein the use of a water-alcohol extractant having a pH value of from 3 to 6. 25. The method according to claim 19, characterized in that water is added to the extract at a weight ratio of water: extract of 6: 1 to 8: 1. 26. The method according to claim 19, characterized in that the precipitated material containing agluconisoflavones is washed with water, and the weight ratio of water: material containing agluconisoflavones is from 0.8: 1 to 2: 1. 27. The method according to claim 19, characterized in that the extract and water are cooled to achieve the most complete precipitation of the material containing agluconisoflavones. 28. The method according to claim 19, characterized in that it comprises dissolving the agluconisoflavone-containing material in an aqueous-alcoholic solution and contacting the aqueous-alcoholic solution containing the dissolved agluconisoflavone-containing material with the adsorbent material until the material with a high content of genistein will separate from the solution. 29. The method according to claim 19, characterized in that it comprises dissolving the agluconisoflavone-containing material in an aqueous-alcoholic solution and contacting the aqueous-alcoholic solution, including the dissolved agluconisoflavone-containing material, with the adsorbent material until the material with a high daidzein content will separate from the solution. 30. A method of obtaining a material with a high content of genistein from plant whey material containing isoflavone conjugates, characterized in that the plant whey material is processed at a temperature of 2-121 ° C and a pH of 6-13.7 until more than 50% of isoflavone conjugates will not turn into isoflavone glucosides, and said isoflavone glucosides are fermented in said whey material at a temperature of 5-75 ° C. and pH 3-9 until more than 50% of the isoflavone glucosides is converted to agluconisoflavo s, then isolate the serum protein material containing agluconisoflavones containing protein and agluconisoflavones from said plant whey material enriched with agluconisoflavone, and then extract the whey protein material containing agluconisoflavones with an aqueous-alcoholic extractant to obtain an extract enriched in agluconisoflavone and contact the resulting extract adsorbent material until the final material separates from the extract. 31. The method according to p. 30, characterized in that use a water-alcohol extractant containing from 30 to 90% alcohol. 32. The method according to p. 30, characterized in that they use a water-alcohol extractant having a pH value near the isoelectric point of said protein in this whey protein material containing aglucoisoflavone. 33. The method according to p, characterized in that use a water-alcohol extractant having a pH of from 3 to 6. 34. The method according to p. 30, characterized in that the whey protein material containing agluconisoflavones is extracted with the extractant, while the weight ratio of the extractant and whey protein material does not exceed 11: 1. 35. The method according to p. 30, characterized in that the whey protein material containing agluconisoflavones is extracted twice with a water-alcohol extractant, the total weight ratio of the quantities of the combined servings of the extractant and whey protein material not exceeding 11: 1. 36. The method according to p. 30, characterized in that the use of adsorbent material in the form of particles. 37. The method according to p. 30, characterized in that the contact of the extract with the adsorbent material includes reversible binding of genistein to the adsorbent material. 38. The method according to p. 30, characterized in that the extract is passed through an adsorbent material with an eluent to separate material with a high content of genistein from the extract. 39. The method according to p. 30, characterized in that the material with a high content of genistein contains at least 40% of genistein. 40. The method according to § 39, characterized in that the material with a high content of genistein contains at least 90% of genistein. 41. A method of obtaining material with a high content of daidzein from plant whey material containing isoflavone conjugates, characterized in that the plant whey material is processed at a temperature of 2-121 ° C and a pH of 6-13.7 until more than 50% of isoflavone conjugates will not turn into isoflavone glucosides, and said isoflavone glucosides are fermented in said whey material at a temperature of 5-75 ° C. and a pH of 3-9 until more than 50% of the isoflavone glucosides is converted to agluconisoflavone then, whey protein material containing agluconisoflavones containing protein and agluconisoflavones is isolated from said plant whey material enriched with agluconisoflavones and the whey protein material containing agluconisoflavones is extracted with an aqueous-alcoholic extractant to obtain an extract rich in agluconisoflavone with contact material and contact is made adsorbent until material with a high content of daidzein divisible from the extract. 42. The method according to paragraph 41, wherein using a water-alcohol extractant containing from 30 to 90% alcohol. 43. The method according to paragraph 41, wherein using a water-alcohol extractant having a pH value near the isoelectric point of said protein in a given whey protein material containing aglucoisoflavone. 44. The method according to item 43, wherein using a water-alcohol extractant having a pH value of from 3 to 6. 45. The method according to paragraph 41, wherein the whey protein material containing agluconisoflavones is extracted with an extractant at a weight ratio of the amounts of extractant and whey protein material not exceeding 11: 1. 46. The method according to paragraph 41, wherein the whey protein material containing agluconisoflavones is extracted twice with a water-alcohol extractant, and the total weight ratio of the quantities of the extractant and whey protein material does not exceed 11: 1. 47. The method according to paragraph 41, wherein using an adsorbent material in the form of particles. 48. The method according to paragraph 41, wherein the contact of the extract with the adsorbent material also includes reversible binding of daidzein extract with the adsorbent material. 49. The method according to paragraph 41, wherein the extract is passed through an adsorbent material using an eluent to isolate a material with a high daidzein content from the extract. 50. The method according to paragraph 41, wherein the material with a high content of daidzein contains at least 40% daidzein. 51. A method of obtaining plant whey material enriched in agluconisoflavone from plant whey material containing isoflavone conjugates, characterized in that the plant whey material is processed at a temperature of 2-121 ° C and a pH of 6-13.7 until more than 50% isoflavone conjugates will not turn into isoflavone glucosides; and said isoflavone glucosides are fermented in said whey material at a temperature of 5-75 ° C. and a pH of 3-9 until more than 50% of the isoflavone glucosides is converted to agluconisoflavones, whey protein material containing agluconisoflavones containing protein and agluconisoflavones are isolated from said agluconisoflavone enriched plant whey material is subjected to extraction whey protein material containing agluconisoflavones with an aqueous-alcoholic extractant for Acquiring extract enriched aglucone isoflavones, and contacting the resulting extract with an adsorbent material so long as the material containing glycitein, is separated from the extract.