MXPA98003598A - An extract and isolated vegetable protein enriched with aglucone isoflavone, and procedure to produce - Google Patents

Abstract

There is described extract and isolate of vegetable protein enriched with aglucone isoflavone and procedure for its production and recovery: the aglucone isoflavone extract is made by extracting a vegetable protein material comprising glucone isoflavones with an aqueous extractant having a pH above from about the isoelectric point of the protein material to produce an aqueous extract, and reacting the glucone isoflavones with a sufficient amount of the enzyme beta-glucosidase or enzyme esterase for a sufficient period, temperature and pH to convert at least a greater part of the glucone isoflavones in the extract to aglucone isoflavones, and thereby produce the extract enriched with aglucone isoflavone, the isolates enriched with aglucone isoflavone are produced by adjusting the pH of the extract that reacts at approximately the isoelectric point of the protein material vegetable to precipitate the protein material na, and separating the protein material to produce a protein isolate enriched with agluco

Description

AN EXTRACT AND ISOLATED VEGETABLE PROTEIN ENRICHED WITH AGLUCON ISOFLAVONE. AND PROCEDURE TO PRODUCE THEM FIELD OF THE INVENTION The present invention relates to the production of an extract and an isolate enriched with aglucone isoflavones, by extraction of soluble material from ßr "plant protein material and treatment with one or more 10 beta-glucosidase enzymes under conditions such that a major part of the glucone isoflavones is converted to aglucone isoflavones, which are retained in the protein isolate.

BACKGROUND OF THE INVENTION Isoflavones are found in a variety of legume plants, including plant protein materials such as soy. These compounds include daidzin, 6 '"- 0A-20 diazidine, 6" -OMal-daidzin, daidzein, genistin, 6"-0Ac-genistin, 6" -OMal-genistin, genistein, glycitin, 6"- 0Malglycitin, glycitein, biochanin A, formononeti and cu sterol These compounds are typically associated with the inherent bitter taste of the soybean, and the approach has been remove these materials in the manufacture of commercial products, such as isolates and concentrates. For example, in a conventional process for the production of a soy protein isolate in which soy flakes are extracted with an alkaline aqueous medium, a large part of the isoflavones are solubilized in the extract, and remain solubilized in the serum, the which is usually discarded after precipitation of the protein with acid to form an isolate. The residual isoflavones left in the acid-precipitated protein isolate are usually removed by exhaustive washing of the isolate. It has been recently recognized that isoflavones contained in plant proteins such as soy, can inhibit the growth of human cancer cells, such as breast cancer cells and prostate cancer cells, as described in the following articles: "Genistein Inhibition of the Growth of Human Breast Cancer Cells, Independence from Estrogen Receptors and the Multi-Drug Resistance Gene "by Peterson and Barnes, Biochemical and Biophysical Research, Communications, Vol. 179, No. 1, p. 661-667, August 30, 1991; "Genistein and Biochanin A Inhibit the Growth of Human Prostate Cancer Cells but Not Epidemial Growth Factor Receptor Tyrosine Autophosphorylation" by Peterson and Barnes, The Prostate, Vol. 22, pp. 335-345 (1993); and "Soybeans Inhibit ammary Tumors in Models of Breast Cancer" by Barnes, et al., Lutangens and Carcinogens in the Diet, pp. 239-253 (1990). Of the above isoflavones, several exist as glucosides, or as glucones, with a bound glucose molecule.

Several of the glucones such as the 6"-OAc-genistin, contain an acetate group attached in the six position of the same molecule of glucose. Although all isoflavones, including glycosides, are of interest in medical evaluation, the specific isoflavones of greatest interest are aglucones, where the glucose molecule is not bound. These isoflavones are not as soluble in water as glucones or isoflavone glycosides. The specific isoflavones in this category are daidzein, genistein and glycetein. These agluconas have the following general formula: wherein Ri, R2, R3 and R-A can be selected from the group consisting of H, OH and OCH3. Therefore, the present is directed to the aglucona and to the enrichment of a vegetable protein isolate with these materials. Methods are known in the art for converting glucone isoflavones into aglucone isoflavones, as described in Japanese Patent Application 258,669 to Obata et al. Said processes reach only a moderate degree of conversion and thus are not convenient, particularly for large-scale commercial operations.

In addition, known methods such as that described in the '669 application teach the removal of isoflavones from the protein material and do not disclose how to prepare an extract or protein isolate enriched with aglucone isoflavone. Thus, there is a need for a process for converting at least a major part, and preferably substantially all, of the glucone isoflavones into aglucone isoflavones, and to produce an extract and isolated protein dj ^ enriched with aglucone isoflavone. Therefore, it is an object of the present invention to provide an extract and protein isolate enriched with aglucone isoflavone, and a process for producing the same. These and other objects are specifically achieved in the detailed description of the present invention indicated more ahead.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides an extract and isolate of vegetable prophein enriched with aglucone isoflavone and a process for producing these. Methods for producing said extracts comprise extracting a plant protein material comprising glucone isoflavones, with an aqueous extractant having a pH above about the isoelectric point of the vegetable protein material, and reacting the glucone isoflavones with a Sufficient amount of one or more beta-glucosidase enzymes for a period, temperature and pH sufficient to convert at least a greater part of the glucone isoflavones of the extract into aglucone isoflavones, and thereby produce the extract enriched with aglucone isoflavone. The present invention also provides methods for producing said extracts wherein supplement beta-glucosidase is added to the extract to produce extract enriched with aglucone isoflavone. The present invention also provides methods to obtain a protein isolate enriched with aglucone by adjusting the pH of the extract described above to approximately the isoelectric point of the protein material to precipitate the protein material and produce a protein isolate that is enriched with aglucone isoflavones. . The resulting aglucone isoflavone enriched isolate can then be separated and dehydrated to form a dry enriched isolate. The present invention also provides recovery methods, in relatively high proportions, of isoflavones from vegetable protein materials.

DESCRIPTION OF THE PREFERRED MODALITIES Although the present invention will be described with respect to soy products and although the process is particularly suitable for the production of extracts and isolates enriched with isoflavone from aglucone from soy material, however, the present process is generally applicable to the production of extracts and protein isolates from a variety of vegetable protein sources containing isoflavones. An example of such a source is a plant protein material comprising soy materials. The term "soy material" as used herein, refers to soy or any soy derivative. The starting material in accordance with the extract or isolate of the preferred embodiment are soy flakes, from which the oil has been removed by extraction with solvent. The flakes are extracted with an aqueous extractant having a pH above about the isoelectric point of the protein material, preferably a pH of about 6.0 to about 10.0, and most preferably a pH of about 6.7 to about 9.7. Typical alkaline reagents may be employed, if desired, to raise the pH of the aqueous extractant, such as sodium hydroxide, potassium hydroxide and calcium hydroxide. The desired isoflavone compounds are typically solubilized in the aqueous extractant. It is also convenient, for maximum recovery of these compounds in the aqueous extract, that the weight ratio of soy flakes to extract be controlled at specific levels to solubilize as much as possible of the isoflavones inherent in the protein material. The extraction of the proteins and isoflavones can be carried out in a variety of ways, including countercurrent extraction of the leaflets at a weight ratio of aqueous extractive to flakes from about 8: 1 to about 16: 1, where the initial extract to extract the leaflets and provide an aqueous extract of protein and isflavones. Alternatively, a two-step extraction process may be used in which the weight ratio of extractive to flakes in the initial step comprises approximately 10: 1, and then a second extraction of flakes with new extractive takes place at a weight ratio of extractive to flakes of approximately 6: 1, or less, so that the combined weight ratio of the extractant to the flakes in both steps does not exceed a total weight ratio of extractive to flakes of approximately 16: 1. After removal of insoluble materials, the resulting aqueous extract of protein comprising the solubilized isoflavones is reacted with one or more beta-glucosidase enzymes to convert aglucone isoflavones, most, and preferably substantially all, isoflavones in the form of glucone with a molecule of bound glucose. The optimal pH scale for the beta-glucosidase enzymes varies depending on the specific beta-glucosidase enzyme used, but typically ranges from about 4 to about 8. The pH of the extract is typically adjusted to approximately the pH scale at which the enzyme specific is more active before reaction with the enzyme. The pH is typically adjusted by the addition of an edible acid, such as acetic, sulfuric, phosphoric, hydrochloric, or any other suitable reagent. The beta-glucosidase enzyme may be naturally present in the soy material or present by microbial growth, referred to as a "residual" enzyme, or may be added to the protein extract. The enzyme added is referred to herein as "supplement enzyme". In general, if the concentration of residual enzyme in the soy material or extract is insufficient to convert a major part, and preferably substantially all of the isoflavones in the form of glucone to the aglucone form, then supplement enzyme should be added. The amount of enzyme sufficient to effect the conversion of isoflavones varies with a multitude of factors including the types of enzymes present, the distribution of enzyme concentrations, the pH of the system, and the activities of the enzymes present. Once sufficient concentrations of enzymes are present, either by residual enzymes, supplement enzymes, or both, the protein extract with solubilized isoflavones is reacted with beta-glucosidase enzymes for a sufficient period, time, and pH to convert to at least a major part, and preferably substantially all, the glucone isoflavones contained in the extract to the aglucone form. Preferred beta-glucosidase supplement enzymes include Biopectinase 100L and 300L, Biopectinase OK 70L, Lactase F, and Lactozy e. Lactase F is available from Amano International Enzyme Co., Inc., P.O. Box 1000, Troy, Virginia 22974, which has an optimum pH scale of about 4 to about 6, and Lactozyme is available from Novo Industries, Enzyme Division, Novo Alie, DK-2880 Bagsvaerd, Denmark, which has an optimal pH scale of about 7. Biopectinase 100L, Biopectinase 300L, and Biopectinase OK 70L are available from Quest International, Sarasota, Florida. Supplement enzymes are added in sufficient amounts to convert at least a major part, and preferably substantially all, the glucone isoflavones into aglucones. In cases where it is necessary to add supplement enzymes, the amount of enzyme added is from about 0.5% to about 5% by weight of the protein precipitate on dry basis. Another class of suitable enzymes is that of enzymes in this rasa. It is believed that these enzymes are well suited for the methods of the preferred embodiment described herein, as they convert the acetate and malonate conjugates to glucone isoflavones by removing the acetate and malonate groups from the isoflavone conjugates. In the preferred embodiment, both types of enzyme, beta-glucosidase and this rasa are used. The preferred embodiment methods are preferably one-step procedures and achieve very high conversion rates of isoflavones (from glucone to agglucose form), in relatively short periods, and with relative ease and economy. The term "one step" reaction procedure, as used herein, refers to a reaction procedure in which certain parameter values of the process are generally maintained during the course of the reaction process. These process parameters include pH and temperature. Very high conversion rates are such that at least a majority, and preferably substantially all, of the isoflavones in the form of glucone present in the extract of soy material, are converted into the form of aglucone. The term "a major part" refers to the degree of conversion of glucone isoflavones to aglucone isoflavones of at least about 50%. The term "substantially all" refers to the degree of conversion of glucone isoflavones to aglucone isoflavones of at least about 80%, and most preferably at least about 90%. While not wishing to be bound by any particular theory, it is believed that the surprisingly and unexpectedly high conversion rates of the processes described herein originate from a combination of process parameters used during the one-step reaction procedure. It is preferred that the pH of the reaction system be maintained, or nearly maintained, at a value of about 4 to about 8, and most preferably at a value at which the enzyme or enzymes are most active prior to the reaction with the conjugate. # or isoflavone conjugates during the reaction process in one step. It is preferred that the temperature of the reaction system be maintained, or nearly maintained, at a temperature of about 40 ° C to about 60 ° C, and most preferably at a temperature of about 60 ° C during the one-step reaction process. . In general, the periods necessary to achieve the conversion of substantially all of the glucone isoflavones to aglucones by the one-step procedures described herein, are from about 2 hours to about 24 hours. After reaction with one or more beta-glucosidase enzymes, the pH is adjusted to the isoelectric point for soy protein, generally between about 4.0 to about 5.0, and preferably between about 4.4 to about 4.6, by the addition of an acid. The adjustment of the pH to the isoelectric point causes the protein to precipitate in the form of a clot, which is enriched with less soluble aglucones. After precipitation, the precipitated clot or protein is separated from the serum, for example by centrifugation, to form a protein isolate enriched with aglucone isoflavones. In the preferred embodiment, the washing of the precipitated protein material is completely avoided, or reduced to a minimum, to substantially reduce the removal of the aglucone isoflavones from the protein precipitate to thereby provide an isolated isolate of aglucone isoflavone, although Agglucones are less soluble in water than other isoflavones. Therefore, the washing with water of the protein precipitated with acid can be completely avoided, or limited to a single wash with water during which the weight ratio of water to precipitated protein material is between about 2: 1 to about 6: 1. This lack of washing of the clot precipitated with acid provides an isolate enriched with the desired levels of isoflavones, although a more extensive wash could be effected with a lower recovery of isoflavones. The moderate amount of washing provides a protein isolate having a dry base content of about 1.5 to about 3.5 mg / gram of genistein, and a daidzein content of about 1.0 to about 3.0 mg / gram. The protein precipitated with acid is then dehydrated by a combination of centrifugation or concentration, and dried in the conventional manner. The preferred embodiment is not designed to be limited to a particular dehydration medium, although it is preferred to use conventional drying techniques such as spray drying to form a dry isolate. The methods described herein provide isolates that have increased amounts of aglucone isoflavones. The present invention also provides methods for recovering isoflavones, in very high proportions, from a vegetable protein material such as a soy material. The levels of recovery obtainable by the methods described herein are typically at least 50%, preferably 65%, and most preferably 80%, based on the total of all forms of the particular isoflavone in the plant protein material of departure. While not wishing to be bound by any particular theory, it is believed that high recoveries originate from the conversion reactions described herein, along with the various treatment operations also described. By converting glucone isoflavone conjugates, which are relatively soluble, into less soluble forms of aglucone, at a particular step of the treatment, it is possible to recover a high percentage of isoflavones from the filler in the resulting product. The following examples describe specific but not limiting embodiments of the present invention.

EXPERIMENTAL PART Samples were prepared by adding 5 grams of defatted soy flakes (flour), extracted, to 5 grams of water, and the pH was adjusted to 7 and 8. 0.25 grams of Lactase F or Lactozime were added to each of the suspensions, so that the enzyme concentration was about 5% by weight of the solids in each sample. The samples were incubated at 40 ° C and at 60 ° C. A subsample was extracted before adding the enzyme (t = 0) and after 24 hours of incubation at the target temperature. Table 1 shows the change and percentage distribution of isoflavones in soy flakes (flour), after the 24-hour incubation period with Lactase F or Lactozime. The samples were not sterilized before supplement enzyme was added, and microbial th and contaminants were not inhibited.

TABLE 1 6"-0Mal- 6" -0Ac- 6"-0Mal- 6" -0Ac- 6"-0Hal- SAMPLE GENIS-GENIS-GENIS-GENIS DFT-DAID-DAID-DAID-GLICIICIAL-GLYCICITINA TINA-TINA TE ZINA ZINA ZINA ZINA ZINA TINA TINA TEINA% t%%%%% *%% t? 16 80 0 4 16 79 1 3 22 62 16 DH 7.0, 40C, t = 24 without adding enzyme 4 48 0 48 3 51 0 46 0 42 58 Lactase F 2 39 0 59 2 41 0 57 0 30 70 Lactozyse 3 45 0 51 2 49 1 48 0 40 60 DH 7.0, 60C. t = 24 without adding enzyme 5 22 0 73 7 33 0 60 4 21 75 Lactase F 10 32 0 59 10 35 3 52 6 23 71 Lactozyie 4 22 0 74 3 33 0 62 0 23 77 DH 8.0.40C, t = 24 without adding enziRa 4 49 0 47 3 50 2 45 0 43 57 Lactase F 3 39 0 58 3 40 3 55 0 29 71 Lactozyie 3 46 0 49 4 48 0 47 0 40 60 DH 8.0.60C. t = 24 without adding enzißa 2 14 0 84 3 26 3 70 0 15 85 Lactase F 6 24 0 80 8 30 3 59 3 22 74 Lactozyse 2 14 0 84 3 24 6 67 9 16 84 These data indicate the degree of conversion obtainable by a combination of enzyme or residual enzymes and enzyme or supplement enzymes. The source of residual enzyme may be microbial th or endogenous soy enzymes. Significant conversion of isoflavone conjugates to aglucones in flakes (flour) of incubated soybeans at a pH of 8, 60 ° C and for 24 hours. The concentration of each type of isoflavone described herein is based on the total of all forms of that type of isoflavone. Another p of samples was prepared forming aqueous suspensions at 16% defatted soy flakes. The samples were adjusted in their pH to 4.5 and 7, and incubated at 45 ° C for 24 hours. Subsamples were taken at 0 and 24 hours. All samples were analyzed to determine the isoflavone content. The table shows instead the percentage distribution of isoflavones calculated in defatted flakes after incubation for 24 hours at pH 4.5 and 7, and at 45 C.

TABLE 2 6"-0Hal- 6" -0Ac- 6"-0Hal- 6" -0Ac 6"-0Hal- GENIS- GENIS-GENIS- GENIS DAID-DAID- ÜAID- DA? D-GLYCYCLICICTION TINA TINA ZINA ZINA TEAM ZINA ZINA TINA TINA TINA TEINA%% *%%%% *% \? -Q 49 46 4 49 44 3 44 33 22 flakes 45'C, H 7.0, t = 24 flakes 3 28 69 35 59 28 73 45'C. DH 4.5, t = 24 leaflets 19 42 39 17 48 31 14 37 49 These data indicate the degree of conversion obtainable by enzyme or residual enzymes in the protein material. Significant conversion of isoflavone conjugates to aglucones at a pH of 7 and a temperature of 45 ° C after incubation at 24 hours. In another series of experiments, the percent recovery of genistein and daidzein in a protein isolate derived from soybean was investigated. The percent recovery was found by determining the amount of genistein (or daidzein) in the isolate, and expressing that amount as a percentage based on the total amount of all forms of genistein (or daidzein) in the soy starting material. 100 g of defatted soybean meal were extracted with 1000 g of water, which was adjusted to a pH of 9.7 by the addition of sodium hydroxide at a temperature of * 32 ° C. This provided a weight ratio of water to flour • r 10: 1. The flour was separated from the extract and re-extracted with 600 g of aqueous extractive having a pH of 9.7 and a temperature of 32 ° C. The second extraction step provided a weight ratio of water to flour of 6: 1. The flour was separated by centrifugation, the first and second extracts were combined, and the pH was adjusted to 4.5 to form a suspension of soy serum and coagulum precipitated with acid. The suspension 10 was heated to 50 ° C, and 2% by dry weight of the clot of the Lactase F enzyme was added. The suspension was allowed to react for 16 hours at 50 ° C to ensure complete conversion of the glucone isoflavones in the form of aglucona. The clot precipitated with acid was separated from the serum by centrifugation to form an aglycone-enriched isolate. Further washing with water from the precipitated clot was avoided. The amount of genistein recovered in the isolate was 86% of the total of all forms of genistin and genistein in the soy starting material (defatted soybean meal). Similarly, the amount of daidzein recovered in the isolate was 75%. The following is a description of a method to quantify isoflavone in soy products. The isoflavones are extracted from soy products by mixing 0.75 grams of 25 sample (spray-dried or finely ground powder) with 50 ml of 80/20 methanol / water solvent. The mixture is stirred for 2 * hours at room temperature with an orbital shaker. After 2 hours, the remaining undissolved materials are removed * by filtration through Whatman filter paper No. 42. Five ml of the filtrate are diluted with 4 ml of water and 1 ml of methanol. The extracted isoflavones are separated by HPLC (high performance liquid chromatography) using a Beckman C18 reversed phase column. The isoflavones are injected onto a column and eluted with a solvent gradient starting with 88% methanol, 10% water, and 2% * 10 glacial acetic acid and ending with 98% methanol and 2% glacial acetic acid. At a flow rate of 0.4 ml / min, all isoflavones -genistine, 6"-O-Acetylfistin, 6" -O-Malonylgenistine, genistein, daidzin, 6"-O-Acetyldazidine, 6 'were clearly resolved. '-O-Malonyldaidzin, daidzin, glycitin, and its derivatives and glycitein »Peak detection is by UV absorption at 262 nm. The peaks are identified by mass spectrometer. Quantification is achieved using pure patterns (genistin, genistein, daidzin and daidzein) purchased from Indofine Chemical Company, Sommervilie, New Jersey. Response factors (integrated area / concentration) are calculated for each of the above compounds, and used to quantify unknown samples. For the conjugated forms for Since no pure patterns are available, it is assumed that the response factors are those of the original molecule, but corrected for the molecular weight difference. It is assumed that the response factor for glycitin is that of corrected genistin for the molecular weight difference. This method provides the quantities of each individual isoflavone. For convenience, total genistein, total daidzein, and total glycitein can be calculated, and represented as the added weight of these compounds if all conjugated forms are converted to their respective unconjugated forms. These totals can also be measured directly with a method, using acid hydrolysis to convert the conjugated forms. Of course, it is understood that the foregoing is only preferred embodiments of the invention and that various changes and alterations may be made without departing from the spirit and broader aspects thereof, as set forth in the appended claims, which are interpreted in accordance with the principles of patent laws, including the doctrine of equivalents.

Claims (5)

NOVELTY OF THE INVENTION CLAIMS *

1. - A vegetable protein isolate comprising a protein isolate having a genistein content on a dry basis of at least 1.5 mg / gram.

2. The plant protein isolate according to claim 1, characterized in that said protein isolate has a genistein content in dry base of about 1.5 to about 3.5 mg / gram.

3. The vegetable protein isolate according to claim 1, characterized in that said protein isolate has a daidzein content in dry base of about at least 1.0 mg / gram.

4. A plant protein isolate comprising a protein isolate having a daidzein content on a dry basis of at least 1.0 mg / gram.

5. The plant protein isolate according to claim 4, characterized in that said protein isolate has a daidzein content in dry base of about 1.0 to about 3.0 mg / gram.