CROSS-REFERENCE TO RELATED APPLICATIONS

The following patent applications are cross-referenced and are hereby incorporated by reference in their entirety: U.S. Patent Application No. 60/557,181 titled ISOFLAVONE DISTRIBUTION SYSTEM filed March 29, 2004; U.S. Patent Application No. 60/557,199 titled ISOFLAVONE DISTRIBUTION SYSTEM filed March 29, 2004; PCT Patent Application No. titled ISOFLAVONE DISTRIBUTION SYTEM filed March 29, 2005 as attorney docket no. CGL04/0049WO1 ; PCT Patent Application No. titled PROTEIN PURIFICATION SYTEM filed March 29, 2005 as attorney docket no. CGL04/0093WO1 ; PCT Patent Application No. PCT/US05/004160 titled PHENOLIC COMPOUND PURIFICATION filed February 9, 2005; PCT Patent Application No. PCT/US05/04153 titled PHENOLIC COMPOUND PURIFICATION filed February 9, 2005; PCT Patent Application No. PCT/US05/04166 titled CYCLITOL SEPARATION METHOD filed February 9, 2005; U.S. Patent Application No. 60/660,807 titled FOOD OR FEED INGREDIENT AND METHOD filed March 11 , 2005 as attorney docket no. CGL04/0293USP2.

FIELD OF THE INVENTION

The present invention generally relates to a phenolic distribution system. The present invention more particularly relates to an isoflavone production and enrichment system. The present invention more particularly relates to a system and method for controlling isoflavones content of purified proteins. The present invention more particularly relates to a system and method for controlling isoflavones content of purified protein products by means of controlling the pH of sugars separation from protein.

BACKGROUND OF THE INVENTION

Various plant protein sources contain phenolic compounds such as isoflavones, e.g. soybeans. Typically, soybeans are dehulled, flaked, extracted to separate oil, and desolventized to form defatted flakes containing about 45 percent protein. After roasting for deactivation of anti-nutritional factors (ANF), those flakes are incorporated as a protein source in animal feed (soybean meal, SBM). A fraction of defatted soybeans is further purified to higher protein concentrations for applications such as food and fish feed in aquaculture.

One criterion for protein purity is protein concentration in the product, which is important in some applications, e.g. food/feed for infants, young animals, fish, etc. Another criterion is the content of components that interfere with optimal food/feed utilization of the protein. Those are generally referred to as anti-nutritional factors (ANF). Oligosaccharides present in defatted soybean are ANF.

There are two conventional industrial approaches to purification of proteins. According to one such conventional approach, sugars (mainly mono-, di-, tri- and tetra- saccharides) and other water-soluble components are washed out of SBM at conditions that minimize protein dissolution. Typically, those conditions are either conducting the washing at a pH of about the isoelectric point or washing with an aqueous ethanol solution of 60-80 percent ethanol. The product of this approach is referred to in this disclosure as "protein concentrate."

The other conventional industrial method of protein purification involves the following steps: (i) extraction (dissolution) of the protein and of the other soluble components (e.g. from a non-toasted source) into a slightly alkaline aqueous solution (typically, no organic solvent); (ii) separation of the extract from the insolubles (e.g. fibers and other non-protein insoluble components); and (iii) separation of the protein in the extract from other soluble components. Such separation typically uses precipitation at about the isoelectric point or ultrafiltration. The product of this approach is referred to in this disclosure as "protein isolate."

The sugars and other soluble components separated from the protein during purification typically end up in an aqueous solution, which is a co- product of purification. The aqueous solution (referred to, in many cases, as soy solubles, soy molasses, soy whey, etc.) contains isoflavones. There are three families of isoflavones: genistin, daidzin and glycitin. Each family may appear in four forms: aglycones, glycosides, malonyl glycosides, and acetyl glycosides (the malonyl and acetyl glycosides are also referred to in this disclosure as conjugates). The majority of the isoflavones in those solutions is of the genistin family. Most of the isoflavones in those solutions are in the forms of malonyl glycosides and glycosides.

According to conventional methods, the isoflavones in the co- product solution are removed from the purified proteins (so that the ratio between isoflavones and proteins in the purified protein products are lower than that ratio in the protein source, e.g. soybean or defatted and desolventized soybean). However, such conventional methods of protein purification have several disadvantages including that they do not enable controlling the distribution of isoflavones between the purified protein and the co-product solution.

Accordingly, there is a need for a method that controls the distribution of isoflavones between the purified protein and the co-product solution. There is also a need for a method for the production of purified proteins with selectively controlled isoflavones content. There is also a need for an isoflavone distribution system that does not substantially interfere with protein purification or substantially increase protein losses during such purification. It would be advantageous to provide an isoflavone distribution system filling any one or more of these needs or having other advantageous features.

SUMMARY OF THE INVENTION

The present invention provides a method for the production of purified proteins with controlled isoflavones content. The method includes providing a plant material containing at least one protein, at least one sugar and at least one isoflavone. The method also includes solubilizing the at least one sugar; and (c) separating the at least one solubilized sugar while at pH of between about pi + 0.2 and about pi + 7.5 , where pi is the isoelectric point of the protein, to form an isoflavone-containing purified protein stream and a sugar-containing stream. The present invention also provides an isoflavones-containing purified product. The product is produced by the process including providing a plant material containing at least one protein, at least one sugar and at least one isoflavone. The process also includes solubilizing the at least one sugar; and (c) separating the at least one solubilized sugar while at pH of between about pi + 0.2 and about pi + 7.5 , where pi is the isoelectric point of the protein, to form an isoflavone-containing purified protein stream and a sugar-containing stream.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 is a flow diagram of an isoflavone distribution system according to an exemplary embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED AND OTHER EXEMPLARY EMBODIMENTS

FIGURE 1 shows a method of treating a plant material having a protein, a sugar, and a phenolic compound such as isoflavone. A purpose of the treatment is purifying the protein in a way that enables controlling its isoflavones content. According to alternative embodiments, the plant can be an oilseed, such as soy and the plant material can be a defatted oilseed, such as soybean meal, defatted soybean flour, defatted soybean flakes, flash-desolventized defatted soy flakes, soy molasses, soy whey, soy solubles and various combinations of those. It can also be a mixture of materials from several sources.

According to a preferred embodiment, the method or treatment involves the steps of solubilizing the sugar and separating it while at pH of between about pi + 0.2 and about pi + 7.7 (preferably between about pi + 0.5 and about pi + 7), where pi is the protein's isoelectric point, to form an isoflavone- containing purified protein stream and a sugar-containing stream.

For soy proteins, pi is between about 4 and about 5.

Various solvents can be used for the solubilization of the sugars, such as water and aqueous solutions. Those aqueous solutions may contain water-soluble organic solvents, e.g. ethanol. Yet, in cases where isoflavones-rich proteins are desired, the content of a polar organic solvent in the aqueous solution may be limited, e.g. less than about 50 percent by weight, more preferably less than about 30 percent.

According to a preferred embodiment, the desire is to maximize the solubilization of the sugars contained. There are, however, various options with regards to co-dissolution of proteins. Selecting the preferred option among those depends on protein purity requirements, on desired isoflavones/protein ratio and on the method selected for the separation of the dissolved sugar.

An exemplary embodiment of the method (PE1) is shown in FIGURE 1. Referring to FIGURE 1 , defatted and flash-desolventized soybean flakes (12) are extracted (in step 20) with a slightly alkaline aqueous solution (14) at pH of about 8. The weight ratio between the aqueous solution and the flakes and all other parameters are adjusted for maximal protein extraction according to a preferred embodiment. Most of the flake sugars co-dissolve with the protein. The insolubles (24) are separated from the protein and sugars containing solution/extract (22). In step 30, the pH of the solution is adjusted. Then, sugars of the extract are separated from the protein (step 40).

According to an alternative embodiment of PE1 , the separation uses membrane separation, such as ultrafiltration, where the membrane's molecular weight cut off is selected to block the permeation of most of the protein. Most of the sugars and other soluble components of the extract permeate through membrane. The proteins that are retained on the membrane and optionally further treated, e.g. by wash/diafiltration and drying to form the protein product (44). The permeate that contains the sugars and other soluble components (42) can be concentrated by methods such as reverse osmosis and water evaporation to form soy molasses, can be treated to recover components out of it or both. Isoflavones co-extracted from the flakes distribute between the retentate and the permeate. Lowering the pH increases the fraction of isoflavones staying with permeate and therefore the isoflavones concentration in the purified protein product. Isoflavones that permeate through the membrane can be recovered from the permeate by known methods.

According to another alternative embodiment of PE1 , proteins are separated from dissolved sugars by precipitation. Such precipitation can be induced by methods such as concentrating the extract (by means of reverse osmosis, water evaporation or both), lowering the temperature, solvent addition, acidulation, etc. according to alternative embodiments. Acidulation affects both protein solubility and isoflavones retention with the proteins.

According to still another alternative embodiment, a combination of precipitation and ultrafiltration is used, e.g. precipitation of part of the protein, separation of the precipitate from the supernatant and ultrafiltration of the latter.

According to another alternative embodiment of the method (PE2) as shown in FIGURE 1 , defatted, desolventized and preferably also toasted soybean flakes are treated for solubilizing the contained sugars, preferably solubilizing a maximal fraction of the sugars. In the next step, the sugars- containing solution is separated from the non-solubilized proteins by means such as decantation, centrifugation, other gravimetric separation, filtration, and membrane filtration. More of the isoflavones are retained with the protein on decreasing the pH towards pi.

According to still another alternative embodiment (PE3) of the method as shown in FIGURE 1 , defatted, desolventized and preferably also toasted soybean flakes are treated with water or with an aqueous solution for solubilizing the contained sugars, preferably solubilizing a maximal fraction of the sugars. Unlike in PE2, the conditions of sugars solubilization are such that a fraction of the protein contained co-dissolve with the sugars. Unlike in PE1 , the conditions of sugars solubilization are such that only a limited fraction of the protein contained co-dissolve rather than a maximal fraction. The non-solubilized protein is separated from the solution and forms, preferably after additional treatment such as washing and drying a first purified protein product. The pH of the separated solution (which contains dissolved sugars, dissolved protein and dissolved isoflavones) is adjusted and dissolved sugars are separated from the protein. That can be done by means, such as membrane filtration (ultrafiltration), or by precipitation.

Another alternative embodiment of the method (PE4) is shown in FIGURE 1. PE4 is similar to PEI in that PE4 involves extraction of the protein (and sugars), pH adjustment and separation of dissolved sugars from protein. The difference is in adding a separation step prior to the pH adjustment. In that separation step, part of the protein is separated from the extract to form, as such, or after further treatment, a first protein product. This separation reduces the amount of protein left in the solution and separated from sugars in the second separation (e.g. by ultrafiltration or precipitation) to form a second protein product, as such, or after further treatment.

In cases of producing two protein products, the process enables affecting the ratio between isoflavones glycoside and isoflavones malonyl glycosides there. One of the products has a higher proportion of glycosides, compared with the ratio in the soybean, and the other higher proportion of malonyl glycosides. * * *

EXAMPLES While the invention will now be described in connection with certain embodiments in the following examples so that aspects thereof may be more fully understood and appreciated, the examples are not intended to limit the invention to these particular examples.

Example 1

2Og of defatted and flash-desolventized soybean flakes (white flakes) were ground to small particles and added with 20Og water to a vial. The pH of the solution was adjusted to 8.2 by the addition of NaOH and the vial was shaken for 2 hours at 3OC. The insolubles were separated from the solution by centrifugation followed by filtration through a 0.45 micron filter. The solution (extract) was analyzed for isoflavones.

Samples of the extract were introduced to several vials. The pH in those was adjusted to various levels by HCI addition. Where precipitate was formed, it was removed by centrifugation and filtration on a 0.45 micron filter. The clear solutions were analyzed for their isoflavones concentrations, which were compared with those of the extract. The total isoflavones concentrations in the clear solutions at pH 6.0, 5.4, 5.0 and 4.2 were about 75 percent, 60 percent, 50 percent and 45 percent of the concentration in the extract at pH 8.2. The concentration of the isoflavones in glycoside form at pH 6.0, 5.4 and 5.0 were about 40 percent, 30 percent and 25 percent of their concentration in the extract at pH 8.2. For the malonyl glycosides the figures at pH 6.0, 5.4, 5.0 and 4.2 were 90 percent, 75 percent, 60 percent and 40 percent of the concentration in the extract at pH 8.2. these results show that, on extraction of proteins, sugars and isoflavones at slightly alkaline solution, a large fraction of the extracted protein can be separated out of the extract with most of the glycosides but a relatively small fraction of the malonyl glycosides. The rest of the protein can be recovered from the extract in a form that is rich in isoflavones, particularly those in malonyl glycoside form.

The extract and the clear solutions were ultrafiltered on membranes with a molecular weight cut off of 30,000 Dalton. Also ultrafiltered was an extract to which NaOH was added to adjust the pH to 10.8. The permeate was analyzed. The results showed that the protein was practically completely blocked on the membrane. The concentration of isoflavones in the permeate were compared to their concentration in the extract. The isoflavones concentrations in the permeates at pH 10.8, 8.2, 6.0, 5.0 and 4.2 were about90 percent, 60 percent, 50 percent, 40 percent and 25 percent of the concentration in the extract at pH 8.2. * * *

While the preferred and other exemplary embodiments described in this disclosure are presently preferred, it should be understood that these embodiments are offered by way of example only. The invention is not limited to a particular embodiment, but extends to various modifications, combinations, and permutations.