A way of turning isoflavonoid conjugates in isoflavonones the aglycones

 

(57) Abstract:

The invention relates to a method of transforming isoflavonoid conjugates in isoflavonones the aglycones by processing a mixture containing isoflavone conjugates and water at a pH of about 6-13,5, a temperature of about 2-121oWith over a period of time sufficient to convert most of the conjugates in isoflavone conjugates, then contact with the enzyme capable of decomposing due isoflavonoid glycoside with isoflavonoid the glikona at pH of about 3 to 9 and a temperature of about 5-75oWith over a period of time sufficient to convert these isoflavonoid of glikona in isoflavonones the aglycones, as well as way of turning isoflavonoid conjugates and isoflavonoid of glikona in isoflavonones the aglycones after removal isoflavonoid conjugates and isoflavonoid of glikona from material of vegetable protein. 2 C. and 13 C.p. f-crystals, 11 PL.

This is a partial continuation of parallel applications 08/477102, filed June 7, 1995.

THE SCOPE OF THE INVENTION

The invention relates to a two-stage process of transformation isoflavonoid conjugates in the materials of vegetable protein in aglycone isoflavon. In addition, this invention is BR>
Isoflavones are found in a variety of leguminous plants, including vegetable protein materials such as soy beans. These compounds include daidzin, 6"-SLA - daidzin, 6"-OML-daidzin, daidzein, genistin, 6"-SLA-genistin, 6"-OML-genistin, genistein, glycitin, 6"-SLA-glycitin, glycitein, biochanin And, formononetin and coumestrol. Usually these compounds are associated with the inherent soybean bitter aftertaste. In the production of commercial products such as isolates (selected material) and concentrates, the goal was to remove these materials. For example, in the traditional manufacturing process of soy protein isolate in which soy flakes are extracted with water in an alkaline environment, many isoflavones are soluble in the extract and remain soluble in the extract, which is usually released after acid precipitation of the protein with the formation of the isolate. Residual isoflavones, remaining in the acid precipitated protein isolate, usually removed by exhaustive washing of the isolate.

Recently it was recognized that the isoflavones contained in vegetable proteins such as soy beans, can inhibit the growth of cancer cells, such as cancer cells and breast cancer cleto is a ne, independence from estrogen receptors and gene resistance to the set of drugs" Peterson and Barnes, "Biochemical and Biophysical Research Communications, volume 179, issue 1, pp. 661-667, August 30, 1991; "Genistein and biochanin And slow the growth of cancer cells of the prostate man, but not autophosphorylation tyrosine factor receptor epidermal growth" Peterson and Barnes, "The Prostate, vol 22, PP 335-345 (1193); and "soybeans inhibit the growth of mammary tumors in models of breast cancer" Barnes and others, "Mutagens and Carcinogens in the Diet", pages 239-253 (1990).

From the above several isoflavones exist as glycosides or as glikona attached to a molecule of glucose. Several glikona, such as 6"-SLA-genistin or 6"-OMaL-genistin contain an acetate group or molinillo group, respectively attached to the six position of the glucose molecules. Connections of this kind, i.e. with additional components attached to the glucose component, usually referred to as "conjugates". Although all of isoflavones, including glycosides, are of interest for medical evaluation, specifically the most interesting isoflavones is the aglycones, which do not have glucose attached component. Specific isoflavones in this category is 2, R3and R4you can choose from the group consisting of H, HE and co3. Therefore, the present invention is directed to aglycones and enriched vegetable protein material such substances.

In the technique known methods of transformation of isoflavones in isoflavonones the aglycones, such as described in published Japanese patent application 258669 name Abati and other Such processes can achieve only a moderate degree of conversion and therefore they are undesirable, especially for large-scale commercial operations. In addition, in the known processes, such as described in the application 258669 speaks about the destruction of the isoflavones from the protein material, and does not describe how to prepare the enriched isoflavonoid the aglycones material of vegetable protein.

Known processes for the production of specific enriched isoflavonoid the aglycones derivatives of vegetable protein, such as protein extracts, whey protein concentrates protein by transformation isoflavonoid of glikona in isoflavonones the aglycones, such as described in this now published patent applications PCT/US94/10697, PCT/US94/10699 and PCT/US94/10696, all of which are owned by the assignee of the present request is but in principle all isoflavonoid conjugates in isoflavonones the aglycones to obtain enriched isoflavonoid the aglycones of the vegetable protein material.

SUMMARY OF INVENTION

Therefore, the aim of the present invention is to provide enriched isoflavonoid the aglycones material and a new method of obtaining it from a vegetable protein material containing isoflavone conjugates.

The present invention provides methods of turning isoflavonoid conjugates in isoflavonones the aglycones. A mixture is formed containing isoflavone conjugates and water, and it is handled when the pH value is approximately 6-13,5 and at a temperature of about 2-121oWith to make the most isoflavonoid conjugates in isoflavone glikona. Isoflavone glikona or obtained from isoflavonoid conjugates, either initially present in the mixture, brought into contact with an enzyme capable of decomposing due isoflavone glycosides at pH of about 3 to 9 and at a temperature of about 5-75oWith to turn isoflavonoid of glikona in the mixture in isoflavonones the aglycones. The present invention includes process changes where appropriate isoflavone conjugates, isoflavone glikona or isoflavone the aglycones present in material of vegetable protein, are removed from the plant material before, during labretonie

The new process is a two - step method of turning at least most isoflavonoid conjugates in isoflavonones the aglycones. Preferably isoflavone conjugates are present in vegetable protein or plant material. It was found that this process provides almost complete transformation isoflavonoid conjugates and isoflavonoid of glikona in isoflavonones the aglycones. The first stage involves the transformation of isoflavonoid conjugates in isoflavone glycosides, which are also called isoflavonoid the glikona. The second stage involves the transformation of isoflavonoid of glikona, or resulting from the first stage, either originally present in the plant material, in isoflavonones the aglycones. Some materials of vegetable protein, especially soy protein materials, a significant portion of the total content of isoflavon in the vegetable protein material is present in the form isoflavonoid conjugates, so the transformation isoflavonoid conjugates in isoflavone glikona to becoming isoflavonoid of glikona in isoflavonones the aglycones can significantly increase the number isoflavonoid aglycones obtained from material of vegetable protein.

There are several variants of embodiments of the present invention.

In the first embodiment isoflavone conjugates into the form isoflavonoid aglycones, whereas compounds isoflavon remain in the vegetable protein material. Thus obtained isoflavone the aglycones can be left in the vegetable protein material or if desired, can be removed. Form isoflavonoid aglycones can be removed by solvent or non-aqueous leaching or extraction methods. Among the suitable solvents for such transactions include, but not be limited to, acetone, ethanol, and other similar organic solvents. Further, it is believed that the methods of water extraction can be applied in some areas the use of the second embodiment isoflavone conjugates into isoflavone glikona being in the vegetable protein material. Then isoflavone glikona are removed from the vegetable protein material by water leaching or extraction methods. Water leaching can be performed by wetting of materials, plant or other effect of water on plant materials, or by immersing them in water or in a mixture with other compatible water solvents such as ethanol or other alcohols to leaching relatively soluble isoflavone glikona. The pH value of the resulting aqueous system is from about 4 to 11 and most preferably about 7. After removal isoflavone glikona into the form isoflavonoid aglycones.

In the third example embodiment of the invention isoflavone conjugates are removed from the vegetable protein material prior to performing any operations transformation. Since the form isoflavonoid conjugate relatively soluble in water, isoflavone conjugates can be removed from the vegetable protein material by water leaching or extraction methods as described above. After such removal, isoflavone conjugates then turn in the form of glycone and then in the form of aglycone.

Depending on the type of material of vegetable protein, with the plants in finely divided form. It may be desirable to make the connection isoflavon contained in plant material available for the various reagents used at any stage or at both stages and are described in more detail below. The material can be permalot, fragmented or otherwise processed traditional known in the art methods. If the plant material is in such condition that the connection isoflavon in plant material easily accessible to external reagents or reactive substances, such as small patches of leaves of some plants may no longer be necessary to subject the material to plants such processing.

At the first stage or the first operation of turning isoflavone conjugates in the vegetable protein material into isoflavone glycosides through a chemical reaction. It was found that the reaction proceeds most rapidly and effectively when specific combinations of ranges of pH and temperature. Preferably the vegetable protein material is introduced into the reaction vessel or in another suitable container with enough water. The amount of water is not critical, if only formed a relatively homogeneous mixture or dispersion is Erno 6-13,5. It was found that the transformation isoflavonoid conjugates in isoflavone glikona catalyzed by alkali, and therefore it is most preferable to use a high pH value to achieve rapid transformation. The most preferred pH value for the first stage is about pH 11. The pH value can be adjusted by adding any suitable alkali, caustic substance or an alkaline reagent, which will increase the pH of the system, such as sodium hydroxide.

The preferred temperature for the first stage is approximately 2-121oC. the Most preferred temperature depends on the pH. The inventors have found that the transformation can occur quickly at lower temperatures, if the pH is relatively high. At pH of about 9 transformation occurs efficiently in the temperature range of about 45-75oWith the most preferred temperature is a temperature of about 73oC. At a pH of about 11, the preferred temperature is about 5-50oS, and the 35oParticularly preferred. If the pH is relatively low, the conversion may occur at higher temperatures. For example, at pH about 6 transformation can take place in the range rate is annoy temperature during the whole period of transformation from isoflavonoid conjugates in isoflavone glikona. However, in some cases it may be desirable to raise or lower the temperature in the first stage of transformation.

The period of time required for turning isoflavonoid conjugates in isoflavone glycosides in the first stage depends primarily on the used ranges of pH and temperature. Such periods usually range from about 15 minutes to several hours or more. The transformation can occur faster at higher pH and higher temperatures. At pH of about 9 turning almost ends after about 4-6 hours at about 73oC. In a particularly preferred embodiment for the first stage of the transformation uses a specific combination of process parameters. These parameters are: pH of about 11, a temperature of about 5-50oWith time and turning about 15-45 minutes

The first stage of transformation isoflavon extremely effective, transforming at least a majority and preferably almost all isoflavone conjugates in isoflavone glikona. The degree of transformation isoflavonoid conjugates in isoflavone glikona during the first stage is usually about 80-100%. When using the previously described predpochtitelnei for large-scale commercial operations.

The inventors have found that it is most preferable to carry out the first stage of transformation in the aqueous system. The system can be compatible with other water components, such as certain alcohols, for example methanol. In General, the first phase transformations do not require frequent stirring or special restrictions on the environment. If a system contains components other than water may be necessary to remove these components, or enough to dilute them by adding the additional amount of water. The reason for this is that some components may adversely affect the second stage of transformation, described in detail below.

The second stage of transformation involves the transformation of all isoflavonoid of glikona in the mix, including isoflavone glikona initially present in the vegetable protein material to the first stage of transformation, as well as isoflavone glikona obtained at the first stage of transformation, isoflavone glikona. The transformation is carried out by bringing isoflavonoid of glikona in the mixture in contact with an enzyme capable of decomposing due isoflavonoid glycoside temperature and pH value, and for p the concentration of enzymes, present in the mixture, and their characteristics. These enzymes are naturally present in material of vegetable protein may be present due to the growth of microbes in the material or may be added to the vegetable protein material. The enzyme that is naturally present in the plant material or the material of soy beans, or which is present due to the growth of microbes, called here a "residual enzyme". Add an enzyme called here "additional enzyme". In General, if the concentration of residual enzyme in the vegetable protein material is not enough to make a majority, and preferably almost all of the isoflavones in the form of glikona in the form of aglycones, it is necessary to add more enzyme.

The amount of enzyme required to implement the conversion in the second stage depends on a variety of factors, including the types of enzymes present, the distribution of the concentrations of the enzymes, the pH of the system, activity of the present enzyme and the temperature of the system.

If the enzyme is added, its preferred amount is usually such an amount that the total concentration present CII enzyme present in the system, by residual enzymes, additional enzymes, or both, isoflavone glikona brought into contact with the enzymes in temperature, pH value and for a period of time sufficient to convert most, and preferably almost all isoflavonoid of glikona in the mixture in the form isoflavonoid aglycones.

Preferred additional enzymes are selected based on the value of pH of the mixture and include almost all enzymes sacharides, i.e. enzymes that can break down communication 1,4-glycoside. Such enzymes can be obtained for example from aspergillus niger, aspergillus oryzae, kluyveromyces lactis and kluyveromyces fragilis. Preferred additional enzymes are commercially supplied enzymes alpha - and beta-galactosidase and the enzymes pectinase. Particularly preferred commercially supplied the following enzymes: biomachines 100L (which is preferably used in the range of pH 3-6), biomachines 300L (optimum range of pH 3-6), biomachines OK 70L (optimum range of pH 3-6), biolatina 30,000 (optimum range of pH 3-6), neutral lactase (the optimal pH range of about 6-8), all of which you can buy from a company Quest International address 1833 57 page is used in the range of about pH 4-6) and lactase 50,000 (optimum range of pH 4-6), both can be purchased from the company Amano International Enzyme Co. , Inc., P.O.Box 1000, Troy, Virginia 22974. Other particularly preferred enzymes include lactose 3000L (which is preferably used in the range of pH 6-8) and alpha-Gal 600 (which is preferably used in the range of pH 4-6,5), which you can buy from a company Novo Nordisk Bioindustrials, Inc., 33 Turner road, Danbury, Connecticut 06813; maxilact L2000 (which is preferably used in the range of pH 4-6), purchased from the company Gist Brocades Food Ingredients, Inc., King of Prussia, Pennsylvania, 19406; neutral lactase (which is preferably used in the range of about pH 6-8), bought from from Pfizer Food Science Group, 205 East 42 street, new York, new York 10017; and concentrate enseco fungal lactase (which is preferably used in the range of pH 4-6), purchased the firm Enzyme Development Corporation, 2 Penn Plaza, Suite 2439, new York, new York 10121.

It was also found that some enzymes Glyco-amylase can be used instead of the previously mentioned enzymes or in addition to them. An example of a suitable Glyco-amylase is G-Zyme G990 (which is preferably used in the range of pH 4-6), purchased the firm Enzyme Development Corporation.

Before the WTO the first container or reaction vessel. Moreover, there is no need to use labor-intensive, time-consuming or costly operations on the separation or processing of the reaction mixture or any part thereof. Instead, the product from the first stage of transformation can be used directly as starting material for the second stage of the transformation. The only parameters that need to manage or track, are pH and temperature.

The second stage of the transformation can be carried out at a pH of about 3-9. The preferred pH range for the second stage of transformation is 3-8. Used the pH value depends primarily on the type of enzyme used and therefore must be chosen accordingly. The pH value can be adjusted, in most cases, be reduced from a relatively high or alkaline pH of the first stage, in different ways, such as adding one or more suitable acids, for example acetic acid, sulfuric acid, phosphoric acid, hydrochloric acid or any other suitable reagent. It is believed that in most cases it is preferable to use an acid reagent food type or acid.

The second stage of the transformation can be carried out Tr>C. the Most preferred temperature is about 35-55oC. Preferably, the reaction system was maintained at a relatively constant temperature during the whole period of transformation from glucono isoflavon in aglycone isoflavon. However, it is envisaged that in some cases it may be desirable to increase, decrease or otherwise change the temperature during the period of time for the second phase of the transformation.

The time required for carrying out the second stage of the transformation depends primarily on various related factors and enzymes from the temperature and pH of the system. In most cases, you can achieve significant transformation within 24 hours the Time required to implement the second stage of transformation, it is possible to significantly reduce the additional enzyme, and it can be only approximately 1-3 hours

In the second stage of conversion to convert at least a majority isoflavonoid of glikona in the mixture in isoflavonones the aglycones. The degree of transformation isoflavonoid of glikona in isoflavonones the aglycones during the second phase is generally at least 80 to 100% and preferably about 90-100%. When using previously described the transformation on a reliable basis is a wonderful and desirable for commercial applications.

After completing the second phase of transformation is relatively insoluble isoflavone the aglycones are removed from the system, preferably by centrifugation or filtration. When the aglycones are removed, they can then be separated from any particles or other solid material by extraction with a suitable solvent. Examples of such solvents are not limited to, are acetone and/or alcohol, such as ethanol. Also provides that to the system or mixture you can add one or more hlopeobraznoj substances before, during or after the second stage of transformation to facilitate deposition isoflavonoid aglycones.

The following examples describe specific, but not limiting embodiments of the present invention.

EXPERIMENTAL PART

The present invention is illustrated in more detail by the following examples, using the material of soybean as plant material. Examples of intentionally illustrative, and should not be interpreted as limiting or otherwise restricting in any way the scope of the invention.

Materials of vegetable protein such as soy protein isolates, concentrates or flour contain "collection" genistein, daize shall contain the conjugates of 6"-L-genistin and 6"-SLA-genistin, genistin of glycone, genistein aglycone; family daidzein contains the conjugates of 6"-L-daidzin and 6"-SLA-daidzin, daidzin of glycone and daidzein aglycone; and family glycitein contains conjugate 6"-L-glycitin, glycitein of glycone and glycitein aglycone. In the following examples, the relative concentrations of isoflavones are measured as a percentage of the family of isoflavones. For example, the collection genestein: %6"-L - genistin + %6"-SLA-genistin + %of genistin +%genistein = 100%. The degree of transformation of the conjugates in glikona and glikona in the aglycones can be determined by comparing the percentage of each form of matter in the family of isoflavones.

EXAMPLE 1

Samples containing vegetable protein material was prepared by formation of a water mixtures of primary serum soybeans. In the first series of experiments the first stage of the transformation was carried out by setting the pH values of all samples of 9.0 and shutter speed for 3.5 h at 72,5oC. After cooling, samples and determination of their pH 7.0 were formed three groups of samples before carrying out the second stage of transformation. In the first group, the pH was left at the value of 7.0. For the second group of pH was established by 8.0. And in the third group, the pH was set at 9.0. or when 55oC. All the samples were kept for 24 h, and periodic analysis was performed after 0, 2, 4, 6 and 24 h

In the above table. 1 percentages transformation isoflavonoid conjugates in isoflavonones the aglycones in the material of soybeans with the application described here is the process.

Complete or almost complete conversion isoflavonoid conjugate to the aglycones occurred between 6 and 24 hours exposure at all pH as at 45oWith, and at 55oC. without wishing to be bound by any particular theory, we believe that a delay period that occurred prior to transformation into the form of aglycone and observed from the above data, is a consequence of the period of time in the course of biosynthesis, i.e. the formation of residual enzyme soy or microbes. The delay period may also be the consequence of the receipt of the second participant reactions, depletion of another substrate of the enzyme and/or binding of the enzyme and/or substrate.

EXAMPLE 2

In the second series of experiments the first stage of the transformation was carried out by setting the pH of all samples 11.0 and warming to 50oC for 1 h Then the samples were cooled and the pH was set to either 4.0 or 4.5. In the first group were added eff is brazzi was aged for 1 hour at 50oWith or at 60oC.

The above table. II indicates the percentage of transformation isoflavonoid conjugates in isoflavonones the aglycones in the material of soy beans in the application described here is the process.

EXAMPLE 3

In the third series of experiments the first stage of the transformation was carried out by setting the pH values of all samples 11.0 and heated to 20oC for 1 hour. Then the samples were cooled, and formed three groups according to set pH values of 4.0, 4.5 and 5.0mm. In each sample was added biomachines 100L to a concentration of 0.04 g bioactivity per 100 g of initial serum. Then all the samples were kept for 1 h at 50oC.

In the presented table. III Dan percentage conversion isoflavonoid conjugates in isoflavonones the aglycones in the material of soy beans in the application described here is the process.

EXAMPLE 4

In the fourth series of experiments, the first stage of the transformation was carried out by setting the pH values of all samples by 8.0 and a temperature of 72.5oC for 24 h After a period of 24 h, the samples were removed.

In the presented table. IV shows the percentage conversion of isoflavonoid conjugates in isoflavonones the aglycones in the material from which sperimental the second phase of the transformation was carried out using many different commercially supplied enzyme beta-galactosidase. The primary serum obtained from material of soybeans, was suspended with water to a solids content of 5%. The pH value was set at 11, and the temperature was maintained at 25oC for 45 min to turn isoflavonoid conjugates in isoflavone glikona. Then samples were formed for defining transformations of each enzyme at concentrations of 2 and 10%, temperature 50oC and at pH values of 4.5 and 7.0, the degree of conversion was measured at t=0.1 and 4 hours.

The above table. V gives the percentage conversion and the resulting distribution of isoflavones.

EXAMPLE 6

In the sixth series of experiments, the second phase transformations were carried out on material obtained after the first stage of transformation in the previously described fifth series of experiments. The second phase of the transformation was carried out using bioactivity 100L and lactase F at 50oC and at pH of 3.0 at concentrations of 5 and 10%.

In the above table. VI shows the percent conversion obtained with the use of these enzymes.

EXAMPLE 7

In another series of experiments the first stage of the transformation was carried out by setting the pH values of water samples containing 10% suspension sprayed dry lane is enriched by a glycoside of the samples was set to 4.5, and they were divided into samples of 150, Then the samples were added three commercially available enzyme alpha-Gal 600, G-Zyme 990 and biolatina 30.000. To each sample was added 0.15 grams of one type of enzyme. Then the samples were kept at 50oWith over 4 hours of Measuring the degree of conversion isoflavonoid glycosides in isoflavonones the aglycones were conducted on each withstand the sample after 0, 1, 2, 3 and 4 hours after addition of the enzyme.

In the above table. VII given the percent conversion obtained with the use of specific enzymes.

EXAMPLE 8

In the eighth series of experiments isoflavone conjugates in the source material of the primary serum turned into isoflavone glycosides using different combinations of process parameters. The first stage of the transformation was achieved at a temperature of 121oC and at pH values equal to 6, and 7.1 and 7.5; at a temperature of 80oC and at pH values equal to 6, and 7.1 and 7.5; and at a temperature of 20oC and at pH values equal to 11.5, 12, 13 and 13.7. The first stage of transformation was also achieved at relatively low temperatures, such as the 6oC. In the above table. VIII given the percent conversion obtained at the previously mentioned conditions of pH and temperature. Pravr Tignall, using a longer time conversion.

EXAMPLE 9

In another series of experiments the first stage of transformation isoflavonoid conjugates in isoflavone glycosides in a mixture of primary serum were performed at temperatures of 2oAnd 6oAt pH 11 and 12.3. The results of this test are given in table. IX.

EXAMPLE 10

In the tenth series of experiments, the second stage of transformation isoflavonoid glycosides in isoflavonones the aglycones were carried out at a temperature of 8oC and at pH values of 4.5 and 7 using the enzyme lactase 50.000 Amano and neutral lactase Quest respectively. Rich glycoside mixture of serum were formed by the passage of the primary serum through the first stage of transformation at pH 11, and the temperature of the 35oC for 45 minutes After the formation of the rich glycoside mixture was held for the second stage of the transformation.

Table X gives the percent conversion at this temperature and pH.

EXAMPLE 11

In the eleventh series of experiments, the second stage of transformation, in which isoflavone glycosides into aglycones, were carried out at a temperature of 35oC and pH values of 4.0 and 4.5. The first batch of primary samples serum, Soder>oC. Then the resulting rich in glycosides mixture passed through the second phase transformations using 5% bioactivity 100L, pH 4 and temperature of the 35oC. the Second party primary samples serum was turned into rich in glycosides samples, as previously noted, after which he used 2% lactase F at pH 4, and the temperature of the 35oC.

In the above table. XI shows the resulting percent conversion.

All percentages listed for 6"-L-genistin, 6"-SLA-genistin, 6"-L-daidzin, 6"-SLA-daidzin, glycitin, 6"-L-glycitin and glycitein are calculated values. The percentage specified for the concentration of the enzyme was calculated as grams of the drug commercial enzyme per 100 g of solids in each sample.

The description of the method for quantitative determination of isoflavones in soy products. Isoflavones extracted from soy products by mixing 0.75 g of sample (dry or sprayed finely milled powder with 50 ml of solvent 80/20 methane/water. The mixture was shaken for 2 h at room temperature using an orbital vibrator. After 2 hours, the remaining any insoluble materials are removed filtrat the x2">

Extracted isoflavones are separated using HPLC (high performance liquid chromatography (HPLC) using a reverse-phase column With 18 Hypersil Hewlett-Packard. Isoflavones injected into the column and suiryudan with gradient of solvent ranging from 88% methanol, 10% water and 2% glacial acetic acid to 98% methanol and 2% glacial acetic acid. At a flow of 0.4 ml/min all isoflavones - genistein, 6"-0-acetylcystein, 6"-0-milorganite, genistein, daidzin, 6"-0-acetyldigoxin, 6"-0-malonaldehyde, daidzin, glycitin and its derivatives and glycitein obviously dissolve (dissolve). Peak detection is when the absorption in the UV at 260 mm Identification of the peaks was carried out by HPLC-mass spectrometer.

Quantification is achieved using pure standards (genistin, genistein, daidzin and daidzein), purchased the company Indofine Chemical Company, Sommerville, new Jersey. The factors response (answer) (integrated area/concentration) are calculated for each of these substances and are used to determine the number of unknown samples. For conjugated forms, for which there is no pure standards, the factors of the reaction was taken as the factors of the parent molecule, but corrected by ofanim on the difference of molecular weights.

This method gives the amount of each separate isoflavon. For convenience, you can calculate the total number of genistein, the total number of daidzein and the total number glycitein, and they represent the cumulative weight of these compounds, if all conjugated forms turned into their corresponding non conjugated forms. This total number can also be measured directly by the method using acid hydrolysis for the conversion of conjugated forms.

The above is merely the preferred embodiments of the invention. It is possible to make various changes without departing from the meaning and broader aspects of the invention, as set forth in the attached claims, which should be interpreted in accordance with the principles of patent law including the Doctrine of equivalents.

1. A way of turning isoflavonoid conjugates in isoflavonones the aglycones, which includes the formation of a mixture containing isoflavone conjugates and water, characterized in that the processing of the mixture is carried out at a pH value of about 6-13,5 and at a temperature of about 2-121oWith over a period of time sufficient to convert most isoflavonoid of conjugatesand, with isoflavonoid the glikona in the above-mentioned mixture at pH value of about 3 to 9 and at a temperature of about 5-75oWith over a period of time sufficient to convert these isoflavonoid of glikona in isoflavonones the aglycones.

2. The method according to p. 1, characterized in that the said mixture containing isoflavone conjugates and water, is a water slurry of a vegetable protein material containing isoflavone conjugates.

3. The method according to p. 1, characterized in that isoflavone glikona exposed to the enzyme, are isoflavone glikona obtained from isoflavonoid conjugates and isoflavonoid of glikona present in this mixture before turning isoflavonoid conjugates in isoflavone glikona.

4. The method according to p. 2, characterized in that it comprises the formation of a water suspension of the vegetable protein material containing isoflavone conjugates, processing said suspension at a pH value of about 6-13,5 and at a temperature of about 2-121oWith over a period of time sufficient to convert most isoflavonoid conjugates in isoflavone glikona, Department isoflavonoid of glikona from material retitrement communication isoflavonoid glycoside, with isoflavonoid the glikona in the above-mentioned mixture at pH values of about 3 to 9 and at a temperature of about 5-75oWith over a period of time sufficient to convert isoflavonoid of glikona in isoflavonones the aglycones.

5. The method according to p. 4, characterized in that isoflavone glikona separated from material of vegetable protein, contain isoflavone glikona formed by transformation from isoflavonoid conjugates and isoflavonoid of glikona present in material of vegetable protein to becoming isoflavonoid conjugates in isoflavone glikona.

6. A way of turning isoflavonoid conjugates and isoflavonoid of glikona in isoflavonones the aglycones after removal isoflavonoid conjugates and isoflavonoid of glikona from material of vegetable protein, characterized in that it involves the removal of isoflavonoid conjugates and isoflavonoid of glikona from a vegetable protein material by leaching, the formation of a mixture isoflavonoid conjugates, isoflavonoid of glikona and water, processing said mixture at a pH value of about 6-13,5 and at a temperature of about 2-121oWith over a period of time sufficient to convert most isoflavonoid conju is lycosid, with isoflavonoid the glikona in the above-mentioned mixture at pH value of about 3 to 9 and at a temperature of about 5-75oWith over a period of time sufficient to convert isoflavonoid of glikona in isoflavonones the aglycones.

7. The method according to p. 6, characterized in that the leaching is carried out at a pH value of about 4-11.

8. The method according to p. 7, characterized in that the leaching is carried out at a pH value of about 7.

9. The method according to p. 1, 2, 4 or 6, characterized in that this mixture isoflavonoid conjugates and water or the aqueous suspension is subjected to processing when the pH value of about 9 and at a temperature of approximately between 45 and 75oC.

10. The method according to p. 1, 2, 4 or 6, characterized in that this mixture isoflavonoid conjugates and water or the aqueous suspension is subjected to processing when the value of pH of about 11 and at a temperature of about 5-50oC.

11. The method according to p. 2, 4 or 6, characterized in that the bringing into contact of the enzyme with isoflavonoid the glikona in the above-mentioned mixture or suspension includes adding some additional enzyme in this mixture or suspension, effective for splitting ties isoflavonones of glycoside in isoflavones is ment sacharides, capable of decomposing due 1,4-glycoside.

13. The method according to p. 12, wherein the additional enzyme is selected from the group consisting of enzymes alpha-galactosidase, the enzyme beta-galactosidase, enzyme glucoamylase, enzymes pectinase and combinations thereof.

14. The method according to p. 11, wherein the additional enzyme is added to this mixture or suspension at a concentration of about 0.1-10% by weight of the vegetable protein material on a dry basis.

15. The method according to p. 2 or 6, characterized in that it further includes the Department isoflavonoid aglycones from the above-mentioned mixture or suspension.

 

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The invention relates to a new microbiological method for the terminal oxidation of the alkyl groups in the carboxyl group

The invention relates to agriculture

FIELD: organic chemistry, medicine, gynecology.

SUBSTANCE: invention relates to novel tetracyclic heterocompounds of the formula (I): wherein X, Y, Z, R1 - R4, n and m has values given in the invention description and used as selective modulating agents for estrogen receptors. Also, invention relates to a method for synthesis of these compounds and pharmaceutical compositions comprising thereof, and their using in treatment and/or prophylaxis of disorders mediated by one or more estrogen receptors. Proposed compounds are useful in treatment and/or prophylaxis of disorders associated with depleting estrogen and comprising such disorders as rush of blood, vaginal dryness, osteopenia and osteoporosis, hormone-dependent cancer and hyperplasia of breast, endometrium, uterus cervix and prostate, endometriosis, uterus fibroma, osteoarthritis that can be used as contraceptive agents both separately and in combination with progestogen or progestogenous antagonist.

EFFECT: valuable medicinal properties of compounds and pharmaceutical compositions.

25 cl, 7 tbl, 171 ex

FIELD: chemistry.

SUBSTANCE: invention relates to the new isoflavon derivative of tectorigenine compound of formula (II) , where R1 represents H, NH2 or SO3M; R2 represents OR'; R3 represents H or -CH2NR"; wherein R' represents H, -CH2COONa or -CH2CH2NMe2; NR" represents or -NMe2; M represents H, Na, K or N+H(CH2CH2OH)3, it also relates to virus inactivating agent having this derivative as an active ingredient and to the method for preparation of the injections on its basis.

EFFECT: developing of production method for the injections on basis of tectorigenine.

20 cl, 10 tbl, 2 dwg,10 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to a method of separating a mixture of optical isomers of pinocembrin, particularly a pinocembrin racemate, involving separation using a chiral primary amine or a chiral sulphinamide as a separating agent.

EFFECT: increased efficiency of separation.

13 cl, 6 ex

FIELD: medicine; pharmaceuticals.

SUBSTANCE: invention relates to daidzein derivative of formula (I) or a pharmaceutically acceptable salt thereof, where R1 and R2 each independently denote H or C1-10alkyl; n equals 1, 2, 3, 4 or 5 and daidzein derivative does not include 7-O-N,N-dimethyl-aminobutyryl-daidzein, as well as to a method for production thereof, a pharmaceutical composition based on disclosed compounds and use thereof for treating vascular-cardiac diseases.

EFFECT: as compared to traditional hydrochloride daidzein derivative, produced according to this invention 7-O-N,N-diethyl-aminoacetyl daidzein hydrochloride has better solubility and best effect in treating cardiovascular diseases.

11 cl, 7 tbl, 5 ex

FIELD: biotechnology, organic chemistry, microbiology, pharmacy.

SUBSTANCE: invention describes a new highly productive strain of fungus Aspergillus terreus № 44-62 that is deposited in the Collection of the company Metkinen Oy, Littoinen, Finland, producing lovastatin. Also, invention relates to a method for isolation of lovastatin and a method for lactonization of statins, such as lovastatin and simvastatin. Method for isolation of lovastatin involves its extraction from raw obtained in culturing the above said fungus-producer, concentrating extract, lactonization of lovastatin in the absence of solvent, clearing and crystallization of the end product. The lactonization process of statins carrying out in the absence of solvent provides preparing their lactones in crystalline form directly and practically without impurities of dimmers and acid form. Invention provides highly profitable manufacturing lovastatin and allows preparing the end product corresponding to Pharmacopoeia purity, higher yield (above 70%) and low cost.

EFFECT: improved preparing method and enhanced quality of product.

48 cl, 2 dwg, 10 ex

FIELD: biotechnology, pharmacy.

SUBSTANCE: statin compounds from fermentation solution are purified by extraction and crystallization. Fermentation broth is subjected for preliminary treatment that involves preliminary alkaline treatment followed by extraction of nonpolar impurities. After preliminary treatment the statin compound is extracted into hydrophobic solvent that is separated after extraction. Then solution of hydrophobic organic solvent is concentrated and washed out, if necessary, with a base-containing aqueous solution. Then extracted statin compound is purified by crystallization. Invention provides isolating statins from fermentation broth with high degree of effectiveness and under pharmaceutically acceptable level of purity.

EFFECT: improved purifying method.

28 cl, 8 ex

FIELD: distillation industry, in particular, process for preparing of raw grain material for alcoholic fermentation.

SUBSTANCE: method involves grinding grain; mixing ground grain with water and performing thermal processing of batch; introducing phytase into batch at mixing stage, said mixing process being carried out in two stages, first stage including heating batch to temperature of 45-55 C during 15-20 min, and second stage including heating batch in flow and holding at temperature of 60-70 C during 1.5-2 hours.

EFFECT: intensified process for preparing of grain for alcoholic fermentation, increased yield of ready product and provision for adjustment of dough batch acidity within wide range of values.

1 tbl, 1 ex

FIELD: biotechnologies.

SUBSTANCE: ferment lovastatin esterase is proposed, which is immobilised on a water-insoluble hard carrier activated with a bifunctional agent. At the same time the hard carrier represents a modified di-(C1-6alkyl)amino-C1-6alkylcellulose, in another version the hard carrier represents a silica gel modified with amino-C1-6alkyl-tri(C1-6alkoxy)silane, and the bifunctional agent that activates the hard carrier represents O-sulfonate of cyanuric acid or acid halide of cyanuric acid. In the third version the hard carrier represents agarose, and the bifunctional agent is a compound that corresponds to the formula , as defined in the formula. Methods (versions) are proposed to immobilise the ferment lovastatin esterase on specified water-insoluble hard carriers. According to the methods, in process of mechanical mixing the bifunctional activating agent is brought in contact with the hard carrier in the dissolvent. The activated hard carrier is separated by filtration, then dried and suspended in a water mixture, containing the ferment lovastatin esterase, with performance of ferment immobilisation. The suspended substance is separated by filtration, washed with the buffer solution and dried. Also the method is proposed to treat simvastatin, including treatment of the simvastatin salt solution, containing the remaining amount of the lovastatin salt, immobilised witht the ferment lovastatin esterase, and a biocatalysed flow reactor is proposed with a layer for realisation of this method. The reactor comprises a reactor body (1) with the inner space (2), connected with a liquid inlet (3) and connected with a liquid outlet (4), in the inner space there is a perforated plate supporting the layer (5), containing the ferment lovastatin esterase, immobilised on the water-insoluble hard carrier.

EFFECT: immobilised lovastatin esterase according to the invention demonstrates at least 5 times higher hydrolytic activity in respect to lovastatin and lovastatin salts in presence of simvastatin and simvastatin salts than in respect to simvastatin and simvastatin salts.

18 cl, 2 dwg, 8 tbl, 20 ex

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to biotechnology and agriculture. Disclosed are a pesticidal composition comprising an isolated strain of Burkholderia sp. NRRL No. B-50319, having pesticidal activity, as well as isolated pesticidal compounds such as templazole B, templamide A and B, derived from Burkholderia sp. NRRL No. B-50319. Said composition is used in a method of controlling plant pests, a method to combat emergence and/or growth of monocotyledonous or dicotyledonous sedge weeds as well as for seed coating. Also provided are methods for producing and isolating a compound selected from templazole A and B, templamide A and B, FR901465 and FR90128, by culturing Burkholderia sp. NRRL No. B-50319 and isolating said compound from culture supernatant.

EFFECT: said compounds are used to prepare compositions for controlling plant pests and/or control appearance and/or growth of monocotyledonous or dicotyledonous sedge weeds.

12 cl, 10 dwg, 12 tbl, 13 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology. Disclosed is an industrial method of producing compactin. Method comprises culturing Penicillium citrinum VKPM F-1099 producer strain under conditions of aeration in a nutrient medium containing carbohydrate and nitrogen sources and mineral salts. Obtained culture fluid is acidified with mineral acid to pH 2.5-3.5 and held for 1-2 hours. Compactin is extracted from mycelium with butyl acetate in three steps. Extracts are purified with activated carbon. Purified extract is concentrated by distillation of butyl acetate at low pressure. Method then includes adding mineral acid and heating to 85-90 °C at low pressure to complete lactonisation of compactin. Compactin-Lactone crystallisation is carried out. Method then includes recrystallisation of compactin-lactone from aqueous solution of isopropanol at room temperature and periodic stirring for 1-2 hours, then at 6-10 °C for at least 4 hours with extraction of compactin. Product is obtained with compactin content of not less than 97 %, sum of impurities of not more than 1.0 %, single maximum dimer impurity of not more than 0.5 %.

EFFECT: output of product is not less than 65 %.

1 cl, 4 ex

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