ISOFLAVONE DISTRIBUTION SYSTEM
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. 60/557,199 titled ISOFLAVONE DISTRIBUTION SYSTEM filed March 29, 2004;
PCT Patent Application No. titled ISOFLAVONE DISTRIBUTION
SYTEM filed March 29, 2005 as attorney docket no. CGL04/0049WO2; 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 1 1 , 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 hydrolysis. 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 in a malonyl glycoside form. The method also includes hydrolyzing at least part of the at least one isoflavone in malonyl glycoside form to generate isoflavone in glycoside or aglycones form. The method also includes solubilizing at least part of the at least one sugar to form at least one solubilized sugar. The method also includes separating the at least one solubilized sugar
while at about pH less than 10 to form an isoflavone-containing purified protein stream and a sugar-containing stream.
The present invention also provides a method for the production of purified proteins with controlled isoflavones content. The method includes providing a solid plant material containing at least one protein, at least one sugar and at least one isoflavone in a malonyl glycoside form. The method also includes hydrolyzing at least part of the at least one isoflavone in malonyl glycoside form to generate isoflavone in glycoside or aglycones form. The method also includes solubilizing at least part of the at least one sugar with water or with an aqueous solution at about pH lower than pl+3, wherein pi is the isoelectric point of the at least one protein, to form at least one solubilized sugar. The method also includes separating the at least one solubilized sugar while at about pH less than 10 by at least one of decantation, centrifugation, gravimetric separation, filtration, and a combination thereof to form an isoflavone-containing purified protein stream and a sugar-containing stream.
The present invention also provides a method for the production of purified proteins with controlled isoflavones content. The method includes providing a solid plant material containing at least one protein, at least one sugar and at least one isoflavone in a malonyl glycoside form. The method also includes solubilizing at least a fraction of the at least one sugar, the at least one protein, and the at least one isoflavone in a malonyl glycoside form by contacting with water or with an aqueous solution at about pH higher than pl+1 wherein pi is the isoelectric point of the at least one protein. The method also includes hydrolyzing at least part of the at least one isoflavone in malonyl glycoside form after the solubilizing to generate isoflavone in glycoside or aglycones form. The method also includes separating the at least one solubilized sugar by at least one of membrane filtration while at about pH less than 10, precipitation at pH of about the isoelectric point of the protein and a combination thereof, to form an isoflavone-containing purified protein stream and a sugar-containing stream.
The present invention also provides a method for the production of purified proteins with controlled isoflavones content. The method includes providing a solid plant material containing at least one protein, at least one sugar and at least one isoflavone in a malonyl glycoside form. The method also includes solubilizing at least part of the at least one sugar, part of the protein and at least part of the isoflavones in malonyl glycoside form in water or in an aqueous solution to form an aqueous solution containing solubilized protein, solubilized isoflavones and solubilized sugar and a solid containing the non-solubilized protein. The method also includes separating the solid from the prior solution. The method also includes hydrolyzing at least part of the at least one isoflavone in malonyl glycoside form in the separated solution to generate isoflavone in glycoside or aglycones form. The method also includes separating the at least one solubilized sugar while at about pH less than 10 to form an isoflavone-containing purified protein stream and a sugar-containing stream. The present invention also provides a method for the production of purified proteins with controlled isoflavones content. The method includes providing a solid plant material containing at least one protein, at least one sugar and at least one isoflavone in a malonyl glycoside form. The method also includes solubilizing the at least one sugar, the at least one protein, and at the least one isoflavone in a malonyl glycoside form by contacting with water or with an aqueous solution at about pH higher than pl+1 wherein pi is the isoelectric point of the at least one protein. The method also includes separating a fraction of the solubilized protein to form a separated protein stream and an aqueous solution comprising sugars, residual protein and isoflavone in malonyl glycoside form. The method also includes hydrolyzing at least part of the at least one isoflavone in malonyl glycoside form in the solution to generate isoflavone in glycoside or aglycones form. The method also includes separating the at least one solubilized sugar while at about pH less than 10, to form an isoflavone- containing purified protein stream and a sugar-containing stream. The present invention also provides a purified protein from a soy- based starting material. The purified protein has a protein concentration of at least about 60 percent. The purified protein is characterized by at least one of: (1)
being substantially free of isoflavones in malonyl glycoside form; (2) having a higher concentration of isoflavones content in glycoside or aglycones form relative to the soy-based starting material; (3) being substantially free of an anti-nutritional factor. 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.
FIGURE 2 is a flow diagram of an isoflavone distribution system according to an alternative embodiment of the present invention. FIGURE 3 is a flow diagram of the isoflavone distribution system of
FIGURE 2 according to an alternative embodiment of the present invention.
FIGURE 4 is a flow diagram of the isoflavone distribution system of FIGURE 3 according to an alternative 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 in a malonyl glycoside form according to an exemplary embodiment. A purpose of the treatment may be purifying the protein in a way that enables controlling its isoflavones content. According to alternative embodiments, the plant may be an oilseed, such as soy and the plant material may 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 may also be a mixture of materials from several sources.
In addition to isoflavones in the form of malonyl glycoside, the plant material may contain isoflavones of other forms according to alternative embodiments. According to a preferred embodiment, the malonyl glycoside form presents at least about 25 percent of the total isoflavones in the plant material.
According to a preferred embodiment, the method involves the steps of hydrolyzing at least a fraction of the malonyl glycosides to generate isoflavone in glycoside or aglycones form, solubilizing the sugars in the plant material and separating the solubilized sugars at about pH less than 10 to form an isoflavone- containing purified protein stream and a sugar-containing stream.
According to alternative embodiments, the hydrolysis step can be conducted prior to sugars solubilization or after it. According to other alternative embodiments, hydrolysis may be conducted after partial separation.
According to alternative embodiments, hydrolysis may be catalyzed chemically, enzymatically or a combination of those. Chemically catalyzed hydrolysis may be conducted at alkaline pH (e.g. greater than about 9) and optionally also at elevated temperature. According to a preferred embodiment, relatively higher temperatures and relatively lower pHs are used to reach a given degree of hydrolysis (and vice versa). According to an alternative embodiment, further hydrolysis to the aglycones form may be conducted.
According to a preferred embodiment, the degree of malonyl glycosides hydrolysis determines the isoflavones concentration in the resulting purified protein product. According to a preferred embodiment, isoflavones concentration increases with increasing degree of hydrolysis - and the degree of hydrolysis is controlled by the nature and the amount of the catalyst and by the reaction conditions. Thus, in case of alkaline chemical catalysis, the degree of hydrolysis increases with pH, reaction temperature and reaction duration, which in turn increase the isoflavones content in the product. In case of enzymatically catalyzed hydrolysis, the degree of hydrolysis and the isoflavones content in the product increases with the amount and activity of the enzyme used and with the reaction duration. In order to stop the alkaline catalyzed hydrolysis, the pH and temperature may be lowered. In case of enzymatic catalysis, that may be done by enzyme deactivation or removal.
According to any preferred or alternative embodiments, various solvents may 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, according to an alternative embodiment 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 by weight.
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 according to alternative embodiments. 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, etc.
Referring to FIGURE 1 , a preferred embodiment of a method of purifying a protein is shown as (PE1). 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. Most of the sugars of the flakes co-dissolve with the protein. The insolubles (24) are separated from the protein and sugars containing solution/extract (22). In step 30, a fraction of the malonyl glycosides in the extract is hydrolyzed chemically or enzymatically. After hydrolysis, sugars of the extract are separated from the protein (step 40).
According to an alternative embodiment of PE1 (referred to in this disclosure as PE1.1), 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 the 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) may be concentrated by methods such as reverse osmosis and water evaporation to form soy molasses,
may be treated to recover valuable components out of it or both, according to a alternative embodiments. Isoflavones co-extracted from the flakes distribute between the retentate and the permeate. The greater the degree of hydrolysis, the greater is the fraction of isoflavones staying with the permeate and therefore the greater is the isoflavones concentration in the purified protein product.
Another parameter is the pH of the solution in the ultrafiltration step. Elevation of pH, particularly in the range of pH greater than about 9, increases isoflavones permeation through the membrane and thereby transfer into the permeate. For maximal isoflavones concentration in the purified protein, the pH in the ultrafiltration step is preferably less than about 10, more preferably less than about 9. Also, for maximal isoflavones concentration in the purified protein, both isoflavones co-extraction from the flakes and malonyl glycosides hydrolysis are maximized according to a preferred embodiment. Isoflavones co-extraction increases with the pH in the extraction process (step 20 in FIGURE 1). Isoflavones that permeate through the membrane may be recovered from the permeate by known methods.
According to another alternative embodiment of PE1 (referred to in this disclosure as PE1.2), the solubilized sugars are separated by protein 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, and lowering of pH according to alternative embodiments. Proteins solubility in aqueous solution is minimal at pH of the protein isoelectric point (pi). Thus, lowering the pH in the extract to about pi (which may be between about 4 and about 5 for soy proteins) precipitates most of the extract protein. The precipitated protein may be separated from the solution and forms, preferably after other treatments (such as washing and drying) the purified protein product (44 in FIGURE 1 ). The sugars and other soluble components of the extract stay in the solution (42), which may be concentrated by methods such as reverse osmosis and water evaporation to form soy molasses, may be treated to recover components out of it or both. Isoflavones co-extracted from the flakes distribute between the precipitate and the solution (supernatant). The greater the degree of hydrolysis, the greater the fraction of isoflavones
staying with precipitate and therefore the greater the isoflavones concentration in the purified protein product. For maximal isoflavones concentration in the purified protein, both isoflavones co-extraction from the flakes and malonyl glycosides hydrolysis are maximized according to a preferred embodiment. Isoflavones co- extraction increases with the pH in the extraction process (step 20 in FIGURE 1). Isoflavones that stay in the supernatant may be recovered from it by known methods. In case protein precipitation from the extract is induced by a method other than lowering the pH (e.g. extract concentration), for maximal isoflavones concentration in the purified protein, the pH in the ultrafiltration step is preferably less than about 10, more preferably less than about 9.
Another alternative embodiment of the method (PE2) is shown in FIGURE 2. Referring to FIGURE 2, defatted, desolventized and preferably also toasted soybean flakes (52) are treated for hydrolyzing a fraction of their malonyl glycosides (step 60) and for solubilizing the contained sugars (step 70), preferably solubilizing a maximal fraction of the sugars. Hydrolysis and solubilization may be done in that sequence (first hydrolyze and then solubilize), in the opposite sequence or simultaneously according to a alternative embodiments. According to a preferred embodiment, hydrolysis is enzymatically catalyzed. According to an exemplary embodiment, the conditions of sugars solubilization and the way it may be conducted are directed to minimize protein co-dissolution with the sugars. Minimal protein solubilization is achieved for example by conducting the solubilization with an aqueous solution (64) the pH at about that of the proteins isoelectric point. Other means of minimal solubilization are reduced temperature and reduced weight ratio between the aqueous solution and the flakes according to alternative embodiments. Addition of an organic solvent into the aqueous solution also minimizes protein dissolution, but also increases isoflavones dissolution, which may not be desired if isoflavones-rich protein product may be targeted. Maximal sugars dissolution and minimal protein dissolution may be achieved by conducting the sugars dissolution by means of multiple-step contacting with water or with an aqueous solution, preferably in a counter-current mode of operation. In the next step (80), the sugars-containing solution is separated from the non-solubilized proteins by means such as decantation,
centrifugation, other gravimetric separation, filtration, and membrane filtration according to alternative embodiments. According to a preferred embodiment, separation is conducted at conditions similar to those of sugars solubilization in order to minimize protein co-dissolution. The non-solubilized protein forms, as such or after additional treatment (e.g. washing and drying) the purified protein product (84). The separated, sugars-containing solution (82), may be concentrated by methods such as reverse osmosis and water evaporation to form soy molasses, treated to recover components out of it or both. Isoflavones co- extracted from the flakes distribute between the protein product and the sugars- containing solution. The greater the degree of hydrolysis, the greater the fraction of isoflavones in the precipitate and therefore the greater the isoflavones concentration in the purified protein product. For maximal isoflavones concentration in the purified protein, malonyl glycosides hydrolysis may be maximized. Isoflavones in the sugar-containing solution may be recovered from the permeate by known methods.
According to still another alternative embodiment (PE3) as shown in FIGURE 3, defatted, desolventized and preferably also toasted soybean flakes (92) are treated with water or with an aqueous solution (94) for solubilizing the contained sugars (step 100), 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 may be separated from the solution and forms, preferably after additional treatment such as washing and drying a first purified protein product (PE3P1 , stream 104 in FIGURE 3). The separated solution (102), which contains dissolved sugars, dissolved protein and dissolved isoflavones may be treated in step 110 for at least partial hydrolysis of malonyl glycosides to form glycosides or aglycones. In the next step (120) dissolved sugars are separated from protein. That may be done by means, such as membrane filtration (ultrafiltration), e.g. as in embodiment PE1.1. Another means may be proteins precipitation as in embodiment PE1.2. The separated protein forms as second purified protein
product of PE3 (PE3P2, stream 124 in FIGURE 3). The separated sugars- containing solution (122) may be treated similarly to similar solutions in embodiments PE1 and PE2.
Referring further to FIGURE 3, isoflavones distribute between three streams: the first purified protein product (PE3P1), the second purified protein product (PE3P2) and the sugars-containing solution (122). The isoflavones content of PE3P1 may be determined by the conditions affecting isoflavones co- dissolution with the sugars and proteins in step (100). Higher ratio between the aqueous solution and the flakes in that step and elevated temperature there decrease isoflavones content in PE3P1 . The co-dissolved isoflavones distribute between PE3P2 and the sugars solution. The greater the fraction of malonyl glycosides hydrolyzed, the greater the isoflavones content of PE3P2 and vice versa. In general (with all other parameters being equal) smaller fractions of proteins in PE3P2 (compared with the initial content in the treated flakes), leads to higher isoflavones concentration in PE3P2. Embodiment PE3 enables production of particularly isoflavones-rich protein products.
Embodiment PE4 shows an alternative embodiment of PE3. According to this embodiment, there is added a step of partial hydrolyzing malonyl glycosides prior to the separation of non-solubilized protein from the sugars and protein solution (140). That added step may be conducted prior to sugars solubilization, after it or simultaneously with it according to alternative embodiments. That added step affects isoflavones split or distribution between the first purified product PE4P1 and the sugar solution. The greater the degree of hydrolysis, the greater the isoflavones content in PE4P1. Another alternative embodiment, PE5 (similar to PE1 ) involves extraction of the protein (and sugars), hydrolysis of the malonyl glycosides to glycosides or aglycones and separation of dissolved sugars from protein. One difference is in adding a separation step prior to the hydrolysis step. 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. The amount of isoflavones in that second protein product is selectively determined by the degree of hydrolysis. In case of hydrolysis approaching maximum, the second protein product is relatively rich in isoflavones. In this embodiment, the isoflavones/protein weight ratio in the second protein product is greater than in the starting plant material, e.g. soybean meal.
Embodiment PE6 is an alternative embodiment of PE5. According to this embodiment, there is added a step of partial hydrolyzing malonyl glycosides prior to the first separation of protein. That added step may be conducted prior to extraction, after it or simultaneously with it according to alternative embodiments. That added step affects isoflavones split between the first purified product and the sugar solution. The greater the degree of hydrolysis, the greater the isoflavones content in the first product.
Isoflavones concentration in the protein products of the various embodiments may be controlled by at least the following parameters according to any preferred or alternative embodiments: (i) isoflavones extraction from the starting materials (isoflavones solubilization), which increases with pH at the range of pH greater than about 8; (ii) hydrolysis of malonyl glycosides, which increases isoflavones content of the purified protein content; and (iii) the pH of the dissolved-sugars separation, where increasing the pH, particularly in the range greater than about 8, increases isoflavones concentration in the separated sugar streams and decreases isoflavones content in protein products. *. . *. . *
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.
Comparative Example 20g of defatted and flash-desolventized soybean flakes (white flakes) were ground to small particles and added with 200g 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 30C. 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.
A fraction of the extract was ultrafiltered on a membrane with a molecular weight cut off of 30,000 (30KD). The permeate was analyzed. The results showed that the protein was substantially completely blocked on the membrane while at least 60 percent of the isoflavones permeated.
Example 1 NaOH was added to another fraction of the extract formed in the Comparative Example to bring the pH to 1 1.2. The vial was shaken at 40C for 2 hours, whereby malonyl glycosides in the solution were hydrolyzed to glycosides. Then the solution was cooled to ambient temperature and analyzed.
A fraction of the solution after hydrolysis was ultrafiltered as such on a membrane with a molecular weight cut off of 30KD. A second fraction was ultrafiltered after HCI addition to lower the pH to 8.2. The permeates were analyzed.
The results showed that the protein was practically completely blocked on the membrane at both pH levels. Isoflavones permeation was dependent on the pH, greater than 80 percent and about 30 percent for the tests at pH of 1 1.2 and 8.2, respectively. These results show that hydrolyzed isoflavones are left at pH 8.2 in the retentate of the ultrafiltration with the protein.
Example 2 An extract was formed, hydrolyzed, and analyzed as in Example 1. HCI was added to a fraction of the hydrolyzed extract and the pH was lowered to 4.3. Proteins precipitated out and were separated by centrifugation. Analysis showed that practically all the protein in the hydrolyzed extract was precipitated along with about 55 percent of the isoflavones.
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.