RU2206230C2 - Soybean protein isolate enriched with aglyconisoflavons (variants) - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: the suggested protein material containing gluconisoflavons is extracted with watery extragent at pH corresponding to isoelectric point of protein material. Watery extract is formed. Gluconisoflavons are subjected to reaction with either beta-glucosidase or esterase enzyme. To obtain isolates enriched with aglyconisoflavons, pH value must reach isoelectric point of plant protein material as a result of which deposition of protein material occurs. Then the latter is separated by obtaining protein isolate enriched with aglyconisoflavons. Soybean protein isolate contains (against a dry matter), at least, 1.5 mg/g genistein or, at least, 1.0 mg/g daidzein. The present innovation enables to obtain soybean protein isolate enriched with substances capable to deteriorate the growth of cancer cells. EFFECT: higher efficiency. 4 cl, 2 ex, 2 tbl

Description

 This application is a continuation of application US 08/307752, filed April 12, 1996.

 The field of technology.

 The present invention relates to a method for producing an extract and isolate enriched in agluconisoflavones by extracting a soluble substance from a plant protein material and treating with one or more beta-glucosidase enzymes under such conditions that most gluconisoflavones are converted to agluconisoflavones that remain in the protein isolate.

State of the art
Isoflavones are found in various leguminous plants, including plant-based protein materials such as soy. These compounds include daidzin, 6 "-OAC-daidzin, 6" -OMal dazin, daidzein, genistin, 6 "-OAC-genistin, 6" -OMal-genistin, genistein, glycithin, 6 "-OMAL-glycithin, glycitein , biochanin A, formononetin and caumesterol. The presence of these compounds is usually attributed to the characteristic bitter taste of soy, and the production of commercial products, such as isolates and concentrates, has the task of removing them. aqueous alkaline medium, a large amount of oflavonone dissolves in the extract and remains in the dissolved state in serum, which is usually discarded after acid precipitation of the protein.Isoflavones remaining in the acid-precipitated protein isolate are usually removed by washing the isolate.

 It has recently been discovered that isoflavones contained in plant-based protein materials, such as soy, can slow the growth of human cancer cells, such as breast or prostate cancer cells, as described in: Genistein Inhibition of the Growth of Human Breast Cancer Cells, Independence from Estrogen Receptors and the Multi-Drag Resistance Gene ", authored by Peterson and Bames, Biochemical and Biophisical Research. Communications. Vol 179, 1, pp. 661-667, August 30, 1991; "Genistein and Biochanin A Inhibit the Growth of Human Prostate Cancer Cells but Not Epidermal Growth Factor Receptor Tyrosine Autophosphorylation, Peterson and Barnes. The Prostate. Vol 22, pp.335-345 (1993); and Soybeans Inhibit Mammary Tumors in Models of Breast Cancer, Bames et al., Mutagens and Carcinogens in the Diet. Pp. 239-253 (1990).

где R₁, R₂, R₃ и R₄ могут представлять собой Н, ОН, ОСН₃. Настоящее изобретение относится к аглюконам и обогащению ими растительного белкового изолята.Some of these isoflavones exist in the form of glucosides, or glucones, with an attached glucose molecule. Some of the glucones, such as 6'-OAc-genistin, contain an acetate group attached in the sixth position to the glucose molecule itself. Although all isoflavones, including glucosides, are of interest to medicine, aglucones, compounds to which the molecule is not attached, are of most interest. glucose. Such isoflavones are less soluble in water than glucons or isoflavon glucosides. Special isoflavones of this kind are daidzin, genistein and glycitein. These aglucones have the general formula

where R ₁ , R ₂ , R ₃ and R ₄ may represent H, OH, OCH ₃ . The present invention relates to aglucones and their enrichment of a vegetable protein isolate.

 The prior art methods for the conversion of gluconisoflavones to agluconisoflavones, as described in Japanese application 258669, Obata and others. In such processes only a moderate degree of conversion is achieved, therefore, they are not beneficial, especially for large-scale commercial production. In addition, well-known processes, such as in the '669 application, describe the removal of isoflavones from protein material and do not describe the preparation of a protein extract or isolate enriched in agluconisoflavone. Thus, there is a need for a process for converting at least a large part and preferably all gluconisoflavones to agluconisoflavones and to obtain a protein extract and isolate enriched in agluconisoflavones.

 The aim of the present invention was to obtain a protein extract and isolate enriched in agluconisoflavone, and the creation of a method for producing such an extract and isolate. This and other aspects are discussed in detail in the detailed description of the invention.

 A brief description of the invention.

 According to the present invention, a plant protein extract and an isolate enriched in agluconisoflavone are prepared, and a process for their preparation is developed. Methods for producing such extracts include extracting the plant protein material containing gluconisoflavones with an aqueous extractant with a pH above the isoelectric point of the plant protein material, then reacting the gluconisoflavones with a sufficient amount of one or more beta-glucosidase enzymes under conditions (time period, temperature and pH) suitable for converting at least most of the gluconisoflavones contained in the extract into agluconisoflavones, resulting in ext act aglucone enriched. According to the present invention also developed methods for producing such extracts. In addition, the present invention also provides methods for producing a protein isolate enriched in agluconisoflavone by approximating the pH of the obtained extract to the isoelectric point of the protein material, whereby the protein material precipitates and a protein isolate enriched in agluconisoflavones is obtained. The resulting isolate, enriched in aglugonisoflavone, can be isolated and dried to form a dry enriched isolate. In addition, the present invention provides methods for the isolation of isoflavones in relatively high proportions from plant protein materials.

 Description of preferred embodiments of the invention.

 Although the present invention is described as applied to soy products, and although the method is particularly suitable for producing extracts and isolates enriched in agluconisoflavones from soy material, the present method is generally applicable for producing protein extracts and isolates from various plant protein sources, containing isoflavones. An example of such a source is plant protein material containing soy, or soy material. The term “soy material” as used herein refers to soy or soy derivatives.

 The starting material in accordance with a preferred embodiment is soybean flakes, the oil from which was removed by liquid extraction. The flakes are extracted with an aqueous extractant having a pH above the isoelectric point of the protein material, preferably the pH is between 6.0 and 10.0, more preferably between 6.7 and 9.7. If it is desired to increase the pH of the aqueous extractant, conventional alkaline reagents such as sodium, potassium and calcium hydroxides can be used. The desired isoflavone compounds are usually dissolved in the aqueous extract. To increase the yield of these compounds in the aqueous extract, certain weight ratios of the flakes are maintained in relation to the extract in order to dissolve as many isoflavones as possible.

 The extraction of protein and isoflavones can be carried out in various ways, including countercurrent extraction of flakes at a weight ratio of water extractant to flakes from 8: 1 to 16: 1, and the initial extract is used to extract flakes and obtain an aqueous extract of protein and isoflavones.

 Alternatively, a two-stage extraction process can be used, wherein the weight ratio of extractant to flakes in the first stage is about 10: 1, and the second extraction of flakes is carried out with a fresh portion of extractant at a weight ratio of extractant to flakes of about 6: 1 or less, so that the total weight the ratio of extractant to flakes in two stages does not exceed 16: 1.

 After removal of insoluble materials, the resulting aqueous protein extract containing dissolved isoflavones is reacted with one or more beta-glucosidase enzymes to convert most, and preferably almost all of the isoflavones in gluconic form, with the glucose molecule attached, to agluconisoflavones. The optimal pH value for beta-glucosidase enzymes will vary depending on the enzyme, but usually ranges from 4 to 8. The pH value of the extract before the reaction approaches the pH value at which the specific enzyme is most active, usually using an available acid, such as acetic acid, sulfuric, phosphoric, hydrochloric or any other.

 Beta glucosidase enzyme can be present in soy material from the very beginning or can be grown microbiologically, here it will be called the "residual" enzyme. The enzyme can also be added to the protein extract, in which case it will be called hereinafter "supplemental". In general, if the amount of residual enzyme in the soy material or extract is not enough to convert most, preferably all, isoflavones present in the glucon form to an aglucon form, an additional enzyme is required. The amount of enzyme sufficient to carry out the conversion of isoflavones depends on many factors, including the type of enzyme, the distribution of the concentration of the enzyme, the pH of the system, the activity of the enzymes present. If there is a sufficient concentration of enzymes, residual, additive, or both types, the protein extract with the isoflavones dissolved in it reacts with beta-lucosidase enzymes for a sufficient period of time, at a temperature and pH suitable for conversion of at least the majority, and preferably all gluconisoflavones contained in the extract in aglucon form.

 Preferred additional beta-glucosidase enzymes are Biopectinase 100L and 300L, Biopectinase OK 70L, Lactase F and Lactosim. Lactase F was obtained from Amano International Enzyme Co., Inc., P.O. Box 1000, Troy, VA 22974, the optimum pH is from 4 to 6, Lactose was obtained from Novo Industries, Ensyme Division, Novo Alle, DK-2880 Bagsvaerd, Denmark, and has an optimum pH of about 7. Biopectinase 100L, Biopectinase 300L and OK 70L Biopectinase were obtained from Quest International, Sarasota, Florida. Additional enzymes are introduced in amounts sufficient to convert at least the majority, and preferably all gluconisoflavones, to aglucones. In cases where the introduction of additional enzymes is necessary, the amount of added enzyme is from 0.5% to 5% by weight of the protein precipitate on a dry substrate.

 Esterase is another class of enzymes suitable for use. These enzymes are well suited to the described method, as they convert acetate and malonate conjugates into gluconisoflavones by cleavage of the acetate and malonate groups. In the most preferred embodiment, both glucosidases and esterases are used. The method according to the preferred embodiment is preferably one-step and allows to achieve very high degrees of conversion of isoflavones (the gluconic form is converted to agluconic) in a relatively short period of time, quite easily and economically. The term "one-step reaction" here means that the values of certain process parameters remain practically unchanged throughout the entire course of the reaction. These parameters include pH and temperature.

 Very high conversion values are achieved, such that at least a large part, and preferably all of the isoflavones present in the extract of soy material in the glucon form, are converted to the aglucon form. The term "bulk" means that the degree of conversion is at least 50%. The term “substantially all” means that gluconisoflavones are converted to agluconisoflavones with a conversion of at least 80%, and more preferably 90%.

Obviously, unexpectedly and unpredictably from the point of view of any theory, high degrees of conversion became possible as a result of a combination of parameters used in a one-stage reaction. The pH of the reaction system is preferably kept constant, or near a constant value, in the range from 4 to 8, and most preferably, at a value at which the enzyme (s) is most active in the reaction with the isoflavone conjugate (s). The temperature of the reaction system is preferably kept constant or close to a constant value, and ranges from 40 ^{° C.} to 60 ^{° C.} , most preferably 60 ^{° C.} during a one-step process. The time period required for the almost complete conversion of gluconisoflavones to aglucones in a one-step reaction is from 2 to 24 hours.

 After reaction with one or more beta-glucosidase enzymes, the pH is brought closer to the isoelectric point of the soy protein, which is generally from 4.0 to 5.0, preferably 4.4 to 4.6, by the addition of acid. As pH approaches the isoelectric point, protein precipitates enriched in less soluble aglucones. After precipitation, the protein precipitate is separated from the serum by centrifugation, resulting in a protein isolate enriched in agluconisoflavones.

 In a preferred embodiment, washing the precipitated protein material is either not carried out at all or is carried out minimally in order to significantly reduce the leaching of agluconisoflavones from the protein precipitate, despite the fact that the aglucones are less soluble in water than other isoflavones. The acid-precipitated protein is either not washed at all or limited to one washing with water, and the weight ratio of water to precipitated protein material is from 2: 1 to 6: 1. Ignoring the stage of washing the acid-precipitated protein material makes it possible to obtain an isolate enriched with the desired amount of isoflavones; when washing, a lower yield of isoflavones can be obtained. Moderate washing allows you to get a protein isolate with a solids content of from 1.5 to 3.5 mg / g of genistein, and 1.0-3.0 mg / g of daidzein.

 Water is then removed from the acid-precipitated protein by a combination of centrifugation or concentration, and it is dried using conventional techniques. The preferred embodiment is not limited to special methods of removing water, however, it is preferable to use traditional drying technology, for example spray drying, to obtain a dry isolate. As a result of the above method, isolates with a high content of agluconisoflavones are obtained.

 The present invention also discloses a process for the isolation of significant amounts of isoflavones from plant protein material such as soy. The specified method allows you to select usually at least 50%, preferably 65%, more preferably 80%, of the total number of all forms of the selected isoflavone in the original plant protein material. Obviously, high product yields are obtained as a result of the above conversion reactions in combination with various process operations, although they are not explained by any theory. By converting at a certain stage of the process conjugates of gluconisoflavones, which are relatively well soluble, into less soluble agluconic forms, it is possible at the final stage to isolate a large amount of isoflavones.

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

 Examples.

Samples were prepared by adding 5 g of extracted, defatted flakes (flour) to 5 g of water, the pH of the mixture was brought closer to 7-8. Then, 0.25 g of Lactase F or Lactosim was added to each suspension so that the concentration of the enzyme was about 5% by weight of the solids in each sample. The samples were kept at 40 ^o C and 60 ^o C. Samples were taken before the enzyme was added (t = 0) and after incubation for 24 hours at the selected temperature. The change and percentage distribution of isoflavones in soybean flakes (flour) after 24 hours with Lactase F or Lactozyme is shown in Table 1. Before the addition of the additional enzyme, the samples were not sterilized, so the microbial growth and growth on the contaminants did not stop.

These data demonstrate the degree of conversion achieved using a combination of residual enzyme (s) and additional enzyme (s). The source of residual enzymes may be microbial growth, or they may be endogenous soy enzymes. A significant degree of conversion of isoflavone conjugates to aglucones is observed for samples that were aged at pH 8, at 60 ^o C for 24 hours. The concentration of each of the isoflavone types defined here is based on the total concentration of all forms of this isoflavone type.

Another set of samples was prepared by preparing a 16% aqueous suspension of defatted soybean flakes. The pH of the samples was adjusted to 4.5 and 7 and kept at 45 ^o C for 24 hours. Samples were taken before the experiment and after 24 hours. All samples were tested for isoflavone content. Table 2 shows the change in the percentage distribution of isoflavones in fat-free flakes after aging for 24 hours at pH 4.5 and 7 at 45 ^o C.

These data show the degree of conversion achieved when residual enzyme (s) are present in the protein material. A significant degree of conversion of isoflavone conjugates to aglucones is observed at pH 7 and a temperature of 45 ^o C after incubation for 24 hours.

In another series of experiments, the percentage yield of genistein and daidzein in the protein isolate obtained from soy was determined. The percentage yield was determined by the amount of genistein (daidzein) in the isolate and this amount was presented as a percentage of the total amount of all forms of genistein (daidzein) in the original soybean material. 100 g of skimmed soy flour was extracted with 1,000 g of water, the pH of which was adjusted to 9.7 by adding sodium hydroxide at a temperature of 32 ^° C, so that the weight ratio of water to flour was 10: 1. The flour was separated from the extract and re-extracted with 600 g of an aqueous extract with a pH of 9.7 at a temperature of 32 ^{° C.} The second extraction stage was carried out at a weight ratio of water to flour of 6: 1. The flour was separated by centrifugation, the first and second extracts were combined, the pH of the solution was adjusted to 4.5, resulting in the formation of serum and precipitate. The serum was heated to 50 ^o C, Lactase F was added in an amount of 2% of the dry weight of the precipitate. The mixture was reacted for 16 hours at a temperature of 50 ^{° C.} to achieve complete conversion of gluconisoflavones to aglucones. The precipitate formed by acidification was separated from the serum by centrifugation, resulting in the formation of an isolate enriched in aglucones. The washing step of the precipitate was not carried out. The amount of genistein in the isolate was 86% of the total amount of all forms of genistin and genistein in the original soy material. Similarly, the amount of daidzein in the isolate was 75%.

 The following is a description of the method for quantitative determination of isoflavones in soy products. Isoflavones are extracted from soy products by mixing 0.75 g of a sample (spray dried or finely ground powder) with 50 ml of a 80/20 methanol / water mixture. The mixture was stirred for 2 hours at room temperature using a circular stirrer. Then, after 2 hours, insoluble matter was filtered through Whatman 42 paper filter. Five ml of the filtrate was diluted with 4 ml of water and 1 ml of methanol.

 The extracted isoflavones were separated by HPLC on a Beckman C18 reverse phase column. Isoflavones were injected into the column and removed with a solvent gradient, starting with 88% methanol, 10% water and 2% glacial acetic acid and up to 98% methanol and 2% glacial acetic acid. At a flow rate of 0.4 ml / min, all isoflavones are genistin, 6 "-O-acetylgenistin, 6" -O-malonylgenistin, genistein, daizin, 6 "-O-acetyldaidzin, 6" -O-malonildaidzin, daidzein, glycithin, its derivatives and glycitein are clearly separated. Peaks are determined by measuring UV absorption at 262 mm. Peaks were identified using mass spectrometry.

 Quantification was performed using pure standards (genistin, genistein, daidzin and daidzein) obtained from Indoline Chemical Company, Sommerville, NJ. Resolution parameters (integrable area / concentration) were calculated for each compound and used in the quantitative analysis of samples with unknown contents. For conjugated forms for which there are no pure standards, the resolution parameters are taken to be the same as for the "parent" molecule, adjusted for the difference in molecular weights. The resolution parameter for glycithin is taken equal to the parameter for genistin, adjusted for the difference in molecular weights.

 This method allows you to quantify each individual isoflavone. For convenience, you can calculate the total amount of genistein, daidzein and glycitein and present the combined weight of these compounds as if all conjugated forms were converted to the corresponding unconjugated ones. These amounts can also be determined directly using acid hydrolysis to convert the conjugated forms.

 It is understood that the foregoing is preferred embodiments of the invention and does not limit it, and various changes and additions are possible within the scope of the invention without going beyond the scope of the invention limited by the patent claims, which should be interpreted in accordance with the principles of patent law, including the doctrine of equivalents.

Claims (5)

 1. Soy protein isolate containing, calculated on the dry matter of genistein, at least 1.5 mg / g  2. Soy protein isolate according to claim 1, containing in terms of dry matter of genistein from 1.5 to 3.5 mg / g  3. Soy protein isolate according to claim 1, additionally containing, in terms of the dry matter of daidzein, at least 1.0 mg / g.  4. Soy protein isolate containing, on a dry matter basis, daidzein, at least 1.0 mg / g.  5. Soy protein isolate according to claim 4, containing, in terms of dry matter, daidzein from 1.0 to 3.0 mg / g.

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