AU9049098A - An aglucone isoflavone enriched vegetable protein extract and isolate and process for producing - Google Patents

Description

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t' -1- W100100 I Regulation 3.2 AUSTRALIAk Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

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Namne of Applicant: Actual Inventors: Address for service in Australia: Invention Title: PROTEIN TECHNOLOGIES INTERNATIONAL, INC.

To be advised CARTER SMITH BEADLE 2 Railway Parade Canmberwell Victoria 3124 Australia AN ACLUCONE [SOFLAVONE ENRICHED VEGETABLE PROTEIN EXTRACT AND ISOLATE AND PROCESS FOR PRODUCING The following statemient is a flil description of this invention, including the best method of performing it known to us

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AN AGLUCONE ISOFLAVONE ENRICHED

VEGETABLE

PROTEIN EXTRACT AND ISOLATE AND PROCESS FOR PRODUCING This application is a continuation application of copending U.S. application Serial No. 08/307,752 filed on April 12, 1996.

Field of the Invention The present invention relates to the production of an extract and isolate enriched with aglucone isoflavones, by extraction of soluble material from a vegetable protein material and treatment with one or more beta-glucosidase enzymes under conditions such that a majority of the glucone isoflavones are converted to aglucone isoflavones which are retained in the protein isolate.

Background of the nvention Isoflavones occur in a variety of leguminous plants, including vegetable protein materials such as soybeans. These compounds include daidzin, 6"-OAc daidzin, 6"- -Mal daidzin, daidzein, genistin, 6"-OAc genistin, 6"-OMal genistin, genistein, glycitin, 6"-OMal glycitin, glycitein, biochanin A, formononetin, and coumesterol. Typically :these compounds are associated with the inherent, bitter flavor of soybeans, and inthe S*production of commercial products, such as isolates and concentrates, the focus has been :to remove these materials. For example, in a conventional process for the production of a soy protein isolate in which soy flakes are extracted with an aqueous alkaline medium, Smuch of the isoflavones are solubilized in the extract, and remain solubilized in the whey, which is usually discarded folloving acid precipitation of the protein to form an isolate.

Residual isoflavones left in the acid precipitated protein isolate are usually removed by exhaustive washing of the isolate.

SIt has been recently recognized that the isoflavones contained in vegetable proteins such as soybeans may inhibit the growth of human cancer cells, such as breast S 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 Bames, Biochemical nd Biohsical Re rh. Communications Vol. 179, No. 1, pp. 661-667, Aug. S1991; "Genistein and Biochanin A Inhibit the Growth of Human Prostate Cancer Cells but Not Epidermal Growth Factor Receptor Tyrosine 17* -2- AutophosphorYlation" by Peterson and Barnes, DLEMA-te Vol. -22, pp. 335-345 (1993); and "Soybeans Inhibit Mammary Tumors in Models of Breast Cancer" by Barnes. et at., Mutagens and Careinoells inthDip.23-3(90) Of the above isoflavofles, several exist as glucosides, or as glucones. with a glucose molecule attached. Several of the glucones such as the 6'-OAc genistint, contain an acetate group attached to the six position of the glucose molecule itself.

While all the isoflavofles, including the glucosides are of interest. in medical evaluation, the specific isoflavones of most interet are the aglucofles, wherein the glucose molecule is not attached. These isoflavones are not as water soluble as the glucones or isoflavorle glucosides. Specific isoflavafles in this category are daidzeifl, genisteifi. and glycitein. These aglucones have the following general formula: A2 4 wherein, R-L, R 3 and R, may be selected from the group consisting of H, OH and OCH 3 It is therefore to the aglucones and enrichment of a vegetable protein isolate with these materials to which the present invention is directed.

Methods are known in the art for converting glucone isoflavones to aglucone isoflavones, such as described in Japanese Patent Application 258,669 to Obata et al. Such processes achieve only.a moderate extent of conversion and so are not desirable, particularly for large scale commercial operations. In addition, known processes such as. d escribed in the '669 application teach removing the isoflavones from the protein material and do not describe how to prepare an aglucone isoflavone enriched protein extract or isolate. Thus, there is a need for a proces of converting at leat a majority and preferably substantially all glucone isoflavolles to aglucone isoflavofles, and for producing an aglucone isoflavone enriched protein extract and isolate.

It is thecrefore an object of the present invention to provide an aglucofle isoflavonfe enriched e"tract and protein isolate, mnd a proc=s for producing the same. This, and other objects, are specifically achieved in the detailed description of the present invention set forth below.

I Summary of the Invention The present invention provides an aglucone isoflavone enriched vegetable protein extract and isolate and processes for producing such. The methods for producing such extracts comprise extracting a vegetable 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 time period, temperature, and pH sufficient to convert at least a majority of the glucone isoflavones in the extract to.aglucone isoflavones, and thereby produce the aglucone isoflavone enriched extract. The present invention also provides methods for producing such extracts wherein supplemental betaglucosidase is added to the extract to produce aglucone isoflavone enriched SI. extract. The present invention further provides methods of obtaining an aglucone enriched protein isolate by adjusting the pH of the previously described extract to about the isoelectric point of the protein material to precipitate the protein material S and produce a protein isolate which is enriched with aglucone isoflavones. The resulting aglucone isoflavone enriched isolate can then be separated and dewatered S0 to form a dried enriched isolate. The present invention, in addition, provides methods of recovering, in relatively high proportions, isoflavones from vegetable S protein materials.

Description of the Preferred Embodiments Although the present invention will be described with respect to soybean S 25 products and although the process is particularly suited for the production of aglucone isoflavone enriched extracts and isolates from soybean material, nevertheless the present process is generally applicable to the production of protein extracts and isolates from a variety of vegetable protein sources which contain isoflavones. An example of such a source is a vegetable protein material comprising soy or soybean materials. The term soybean material" as used herein refers to soybeans or any soybean derivative.

The starting material in acorda with the preferred embodiment lexact 1 d P1

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or isolate is soybean flakes, from which the oil has been removed by solvent extraction. 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 elevate 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 extract. It is also desirable, in order to maximize recovery of these compounds in the aqueous extract that the weight ratio of soybean flakes to extract is controlled to specific levels in order to solubilize as much of the inherent isoflavones in the protein material as possible.

Extraction of the proteins and isoflavones can be carried out in a variety of S ways including countercurrent extraction of the flakes at a weight ratio of aqueous extractant to flakes of about 8:1 to about 16:1, in which the initial extract is used 1 to extract the flakes and provide an aqueous extract of protein and isoflavones.

Alternatively, a two step extraction process can be used in which the weight ratio S of extractant to flakes in the initial step comprises about 10:1, and then a second extraction of the flakes with fresh extractant takes place at a weight ratio of extractant to flakes of about 6:1, or less, so that the combined weightratio of extractant to flakes in both steps does not exceed a total weight ratio of extractant to flakes of about 16:1.

After removal of insoluble materials, the resulting aqueous protein extract comprising solubilized isoflavones is then reacted with one or more betaglucosidase enzymes in order to convert a majority, and preferably substantially all, the isoflavones in glucone form, with a glucose molecule attached, to an aglucone isoflavone. The optimum pH range for the beta-glucosidase enzymes will vary depending on the specific beta-glucosidase enzyme used, but typically will vary between about 4 and about 8. The pH of the extract is typically adjusted to about the pH range at which the specific enzyme is most active prior to 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-giucosidase nzyme may be naturaly present in the soybean

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material or present from microbial growth, referred to herein as "residual" enzyme, or may be added to the protein extract. Added enzyme is referred to herein as "supplemental enzyme'. Generally, if the concentration of residual enzyme in the soybean material or extract is insufficient to convert a majority, and preferably substantially all, the isoflavones in glucone form to aglucone form, then supplemental enzyme should be added. The amount of enzyme sufficient to perform the conversion of isoflavones, varies upon a multitude of factors including the types of enzymes present, distribution of enzyme concentrations, pH of the system, and activities of enzymes present. Once sufficient concentrations of enzymes are present, either via residual enzymes, supplemental enzymes, or both, the protein extract with solubilized isoflavones is reacted with the beta-glucosidase enzymes for a time period, temperature, and pH sufficient to convert at least a majority and preferably substantially all the glucone isoflavones contained in the extract to the aglucone form.

Si'S Preferred supplemental beta-glucosidase enzymes include Biopectinase 100L and 300L, Biopectinase OK 70L, Lactase F, and Lactozyme. Lactase F is available from Amano International Enzyme Co., Inc., P.O. Box 1000, Troy, VA 22974, which has an optimum pH range of about 4 to about 6, and Lactozyme is available from Novo Industries, Enzyme Division, Novo Alle, DK-2880 Bagsvaerd, Denmark, which has an optimum pH range of about 7. Biopectinase 100L, Biopectinase 300L, and Biopectinase OK 70L are available from Quest International, Sarasota, Florida. Supplemental enzymes are added in amounts sufficient to convert at least a majority and preferably substantially all the glucone isoflavones to aglucones. In instances where it is necessary to add supplemental enzymes, the amount of enzyme added is about 0.5% to about 5% by weight of the protein precipitate on a dry basis.

Another class of suitable enzymes is that of esterase enzymes. These enzymes are believed to be well suited to the preferred embodiment processes 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 most preferred embodiment, both types of enzymes, betaglucosidase and esterase enzymes are utilized.

i; i, i: i9 -6- The processes of the preferred embodiment are preferably one-step processes and achieve very high degrees of conversion of isoflavones (from glucone form to aglucone form), in relatively short time periods, and with relative ease and economy. The term "one-step" reaction process as used herein refers to a reaction process in which certain process parameter values are generally maintained over the course of the reaction process. These process parameters include pH and temperature.

The very high degrees of conversion are such that at least a majority, and preferably substantially all. the isoflavones in glucone form present in the soybean material extract, are converted to aglucone form. The term "a majority" refers to extent of conversion of glucone isoflavones to aglucone isoflavones of at least about 50%. The term "substantially all" refers to extent of conversion of glucone isoflavones to aglucone isoflavones of at least about 80%, and most preferably at least about Although not wishing to be bound to any particular theory, it is believed that the surprisingly and unexpectedly high degrees of conversion of the processes described herein result from a combination of process parameters utilized during the one-step reaction process. It is preferred that the pH of the reaction system be maintained, or approximately so, at a value of from about 4 to about 8, and most preferably at a value at which the enzyme(s) are most active prior to reaction with the isoflavone conjugate(s) during the one-step reaction process. It is preferred i that the temperature of the reaction system be maintained, or approximately so, at a temperature of from about 40* C to about 60* C, and most preferably at a temperature of about 600 C during the one-step reaction process. Generally, the time periods necessary to achieve conversion of substantially all glucone isoflavones to aglucones via the one-step processes 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. Adjustment of the pH to the isoelectric point precipitates the protein in the form of a curd, which is enriched with theless soluble aglucones. Following i r i i :e precipitation, the curd or precipitated protein is separated from the whey such as by centrifugation to form a protein isolate enriched with aglucone isoflavones.

In the preferred embodiment, washing of the precipitated protein material is either avoided entirely, or minimized in order to substantially reduce removal of the aglucone isoflavones from the protein precipitate to thereby provide an aglucone isoflavone enriched isolate even though the aglucones are less soluble in water than other isoflavones. Washing of the acid precipitated protein with water may therefore be avoided completely, or be limited to a single washing 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 acid precipitated curd provides an isolate enriched with the desired levels of isoflavones, even though more extensive washing could be carried out with a lesser recovery of isoflavones.

The moderate amount of washing provides a protein isolate having a dry basis content of about 1.5 to about 3.5 mg/gram of gnistein, and a daidzein content of about 1.0 to about 3.0 mg/gram.

The acid precipitated protein is then dewatered by a combination of centrifugation or concentration, and is dried in a conventional manner. The preferred embodiment is not intended to be limited by a particular means of Sdewatering, although it is preferred to use conventional drying techniques such as spray drying to form a dried isolate. The processes described herein provide isolates which have increased amounts of aglucone isoflavones.

The present invention also provides methods of recovering isoflavones, in very high proportions, from a vegetable protein material such as a soybean material. The recovery levels obtainable by the processes described herein are typically at least 50%, preferably 65%, and most preferably 80%, based upon the total of all forms of the particular isoflavone in the starting vegetable protein material. Although not wishing to be bound to any particular theory, it is believed that the high recoveries stem from the conversion reactions described herein coupled with the various processing operations also described. By converting glucone isoflavone conjugates, which are relatively soluble, to less soluble aglucone forms, at a particular stage of processing, it is possible to recover in the resulting product, a high percentage of the isoflavoaes from the feed material.

_r' i: The following examples describe specit'ic but non-limiting embodiments of the present invention.

Samples were prepared by adding 5 grams of extracted, defatted soy flakes (flour) to 5 grams of water and the pH adjusted to 7 and to 8. 0.25 gramts Lactase F or Lactozyme was added to each of the suspensions such that enzyme concentration was at about 5 by weight of thie solids in each sample. Samples were incubated at 400 C and at 601 C. A subsample was withdrawni before enzyme was added and after 24 hours incubation at the target temperature.

I0 Te change and percent distribution of isoflavones in soy flakes (flour) after the 24 hour incubation period with either L.actase F or Lactozyme is shown in Table S1. The samples were not sterilized before adding supplemental enzyme and

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microbial and contaminant growth was not inhibited.

TAB3LE 1 a tact"'T 2 M 7.0. M~ t~ Lsc"..F 10 t tonY.

4S.bpu. v.oic- 4"-WUL. 2 X X X X X it 11 0 6. 14 IV9 1 3 22 62 Is 42 0 o 22 a 13 32 0 5 22 7 4 To 2 05 a 0 47 21 U3 '23 63 29 to Is

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Ta These data indicate the degree of conversion attainable by a combination of .residual enzyme(s) and supplemental enzyme(s). Source ofresiulnzmma be microbial growth or endogenous soy enzymes. Significant conversion of.

isoffavone conjugates to aglucones occurred in soy flakes (flour) incubated at a pH of 8, 6(r C, and for 24 hours. The concentration of each type of isoflavone desribed herein is based upon the total of all forms of that isoflavone type.

-9- Another set of samples were prepared by forming 16% aqueous suspenlsions Of defatted soy flakes- The sarnples werepHdjseto45ad7 and incubated at 45- C for 24 hours. Subsarnples were taken at 0 and 24 bours.

All samples were analyzed for isofl .avofle content-. Table 2 shows the cha-nge in percent distribution of calculated isoflavofles in defatted flakes after incubation fbi 24 hours at p1-I 4.5 and 7. and at 450 C.

TABLE 2 flakest 3 44 These data indicate the degree of conversion attainable by residual enzynte(s) in .the protein material. Significant conversion of isoflavofiC conjugates to aglucones i:occurred at a pHl of 7 and temperature of 45G C after 24 hours incubation.

in another series of experiments1 the percent recovery of genisteifi and daidzeirk in a protein isolate derived from Soybeans was Investigated. The percent recovery was found by determining the amount Of genistein (or daidzeifl) in the isolate, and expressing that armount as a percentage based upofiithe total amount of all forms of genistein (or daidzein) in the soybean starting material. 100 g of defatted soy flour was extracted with 1,000 g of water, which was adjusted to a pH of 9.7 by the addition of sodium hydroxide at a temperature Of 320 C. This provided a weight ratio of water to flour of 10: 1. The flour was separated from the extract and re-extracted with 600 g of aqueous extract 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 centrifulgation, the first and second extracts combined, and the pH adjusted to 4.5 to form a slurry of soy whey and acid precipitated curd. The slurry was heated to 500 C, and 2% by dry weight of the curd of the enzyme Lactas F was added. The slurry was allowed to react for 16 hours at 50" C to ensure complete conversion of the glucone isoflavones to the aglucone form. The acid precipitated curd was separated from the whey by centrifugation to form an aglucone enriched isolate. Further washing of the precipitated curd with water was avoided. The amount of genistein recovered in the isolate was 86% of the total of all forms of genistin and genistein in the starting soybean material (defatted soy flour). Similarly, the amount of daidzein recovered in the isolate was The following is a description of a method for quantifying isoflavones in S soy products. The isoflavones are extracted from soy products by mixing 0.75 I-l0 gram of sample (spray dried or finely ground powder) with 50 ml of 80/20 methanol/water solvent. The mixture is shaken for 2 hours at room temperature with an orbital shaker. After 2 hours, the remaining undissolved materials are removed by filtration through Whatman No. 42 filter paper. Five ml of the S filtrate are diluted with 4 ml of water and I ml of methanol.

The extracted isoflavones are separated by HPLC (High Performance S Liquid Chromatography) using a Beckman C18 reverse phase column. The isoflavones are injected on to the column and eluted with a solvent gradient starting with 88% methanol, 10% water, and 2% glacial acetic acid and ending with 98% methanol and 2% glacial acetic acid. At a flow rate of 0.4 ml/min, all the isoflavones genistin, 6"-0-Acetylgenistin, 6"-0-Malonylgenistin, genistein, daidzin, 6"-0-Acetyldaidzin, 6"-0-Malonyldaidzin, daidzin, glycitin and its derivatives and glycitein are clearly resolved. Peak detection is by UV absorbance at 262 mm. Identification of the peaks is performed by mass spectrometer.

Quantification is achieved by using pure standards (genistin, genistein, daidzin and daidzein) purchased from Indofine Chemical Company, Sommerville, NJ. Response factors (Integrated area/concentration) are calculated for each of the above compounds and are used to quantitate unknown samples. For the i conjugated forms for which no pure standards are available, response factors are assumed to be that of the parent molecule but corrected for molecule weight difference. The response factor for glycitin is assumed to be that for genistin corrected for molecular weight dife.ence.

X7 4- This method provides thle quantities oeah individufli is ofavofle. For coneflfle, ttalgenstefl1 total daidzeifl and total glycitein can be lc:Ulated and repreent the,-aggregate weighlt of these compoundsifalheongaefos arc converted to their Mepective unconjiigated forms.- These totals can also be measured directly by a method using acid hydrolysis to convert the conjugated forms. rfre ~4I LA t AL 4)

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.4 ii .~8 .tc. C C of course, it is understood that the foregoing ar, JMA- ibodimnfl of the invention and that various changes and altertionis can be made without departing from the spirit and broader aspects thereof as set forth in the appended claims, which are to be interpreted in accord an with the principals of patent law including the Doctrine Of Equivalents.

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Claims (2)

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

2. The -vegetable protein isolate of claim 1 wherein said Protein isolate has a dry basis genistein content of about 1.5 to m. nglgram. The vegetable protein isolate of clim 1 -wherein said protein isolate has a dry basis laidzein content of about at least 1.0 mg/gram. 1.Avegetable protein isolate comprising a protei ioaehvingadybssadzi content of about at least 1.0 mg/gram.- The vegetable protein isolate of claim 4 wherein said protein isolate has a dry basis 11 iaidzein content of about 1.0 to *3.0 mg/gram.- A vegetable protein according to any one of claims 1 to 6 sbstnilya hereinbefore described. DATED: 27 October 1998 CARTER SNUTH BEADLE Patent Attorneys for the Applicant:- PROTEIN TECHNOLOGIES INTERNATIONAL, INC.