WO2005077930A1

WO2005077930A1 - Phenolic compound purification

Info

Publication number: WO2005077930A1
Application number: PCT/US2005/004160
Authority: WO
Inventors: Ian Purtle; Aharon Eyal; Asher Vitner
Original assignee: Cargill, Incorporated
Priority date: 2004-02-09
Filing date: 2005-02-09
Publication date: 2005-08-25

Abstract

A process for improving the purity of a phenolic compound from a starting material of biological source is disclosed. The process comprises providing the starting material from the biological source comprising a mixture of at least one phenolic compound having a purity P1 and at least one non-phenolic compound. The process also comprises forming at least a fraction of the starting material, an alkaline aqueous solution having at least a pH of about pKa minus 1.5, wherein the pKa is that of the at least one phenolic compound, and insolubles. The process also comprises separating the insolubles from the alkaline solution, whereby there is formed a purified solution comprising at least one purified phenolic compound having a purity P2 and insolubles containing the at least on non-phenolic compound wherein P2 is greater than P1 and the insolubles contain less than about 20 percent (%) of the at least one phenolic compound present in the starting material.

Description

PHENOLIC COMPOUND PURIFICATION

CROSS-REFERENCE TO RELATED APPLICATIONS U.S. Patent Application No. 60/543067 titled "PHENOLIC PURIFICATION SYSTEM" filed 06-Feb-2004; U.S. Patent Application No.

60/543066 titled "PHENOLIC PURIFICATION SYSTEM filed 09-Feb-2004 and U.S. Patent Application No.: [to be determined] titled "PHENOLIC COMPOUND PURIFICATION" filed 09-Feb-2005 as attorney docket no. CGL04/0009/PCT; U.S. Patent Application No. 60/630137 titled "MONOSACCHARIDE PRODUCTION SYSTEM" filed 22-Nov-2004.

FIELD OF THE INVENTION The present invention generally relates to a method of purifying a phenolic compound. The present invention more particularly relates to a process for the purification of a starting material resulting from a biological source. More particularly, the present invention relates to a process for the purification of a starting material resulting from a biological source wherein the starting material comprises a mixture of at least one phenolic compound and at least one non- phenolic impurity. More particularly, the present invention relates to a process for purifying a phenolic compound such as isoflavones from plant materials such as soybean extracts.

BACKGROUND OF THE INVENTION It is generally known to provide a method of purifying phenolic compounds (such as isoflavones) by extracting a phenolic compound from a plant source such as "soy solubles." Typically, on a solvent-free basis, the isoflavones concentration in soy solubles is smaller than 5 percent (%). Soy solubles contain 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 as conjugates). Most of the isoflavones in the solubles are in the forms of malonyl glycosides and glycosides.

However, such known methods have several disadvantages. Accordingly, there is a need for a method of purifying phenolic compounds (such as isoflavones) from a variety of plant materials with suitable purity, color, flavor, solubility, shelf stability, etc. to promote their incorporation in a variety of food, beverage, dietary supplements, pharmaceutical products, etc. There is also a need for a method of purifying a phenolic compound having other advantageous features as described in this disclosure. SUMMARY OF THE INVENTION The present invention relates to a process for improving the purity of a phenolic compound from a starting material of a biological source. The process comprises providing the starting material from the biological source comprising a mixture of at least one phenolic compound having a purity P1 and at least one non-phenolic compound. The process also comprises forming at least a fraction of the starting material, an alkaline aqueous solution having at least a pH of about pKa minus 1.5, wherein the pKa is that of the at least one phenolic compound, and insolubles. The process also comprises separating the insolubles from the alkaline solution, whereby there is formed a purified solution comprising at least one purified phenolic compound having a purity of P2 and insolubles containing the at leas one non-phenolic compound wherein P2 is greater thatn P1 and the insolubles contain less than about 20 percent (%) of the at least one phenolic compound present in the starting material.The present invention also related to a process for improving the purity of a phenolic compound from a starting material of biological source. The starting material comprises a mixture of at least one phenolic compound and at least one non-phenolic compound. The starting material is characterized in that, on adjusting the total concentration of the solutes of the starting material, in an aqueous medium at a pH of up to about 8, to a concentration of between about 10 percent (%) and about 40 percent (%), the non-phenolic compound presents as a first precipitate, and in that weight ratio, R1 , of the first parcipitate, when dried, to the phenolicompound in the starting material, is at least 1.5:1. The process comprises forming at least a fraction of the starting material, an alkaline aqueous solution having a pH of at least about pKa- 1.5, wherein the pKa is that of the at least one phenolic compound, a and total solutes concentration of between about 1 percent (%) and about 40 percent (%), 5 and insolubles containing the non-phenolic compound, wherein a weight ratio, R2, of the insolubles, when dried, to phenolic compound in the starting material is at least about 10 percent (%) of R1 , and wherein the insolubles contain less than about 20 percent (%) of the phenolic compound present in the starting material.

BRIEF DESCRIPTION OF THE DRAWINGSo FIGURE 1 is a flow diagram of a method of purifying a phenolic compound according to an exemplary embodiment.

DETAILED DESCRIPTION OF THE PREFERRED AND OTHER EXEMPLARY EMBODIMENTS A process for the purification of a starting material resulting from as biological source is shown as a method of purifying a phenolic compound in FIGURE 1 according to an exemplary embodiment. The starting material contains a phenolic compound according to a preferred embodiment, preferably a polyphenol, more preferably a flavonoid, most preferably an isoflavone. The starting material also contains at least one non-phenolic compound according to ao preferred embodiment. Preferably, the starting material is an extract of plant material, preferably an extract of soy material.

Referring to FIGURE 1 , the starting material is soy solubles (22) resulting from a process for protein purification (20). According to a preferred embodiment, such protein purification process, a soy material (12), preferably5 soybean meal (SBM) or defatted and flash-desolventized soy flakes (DFWF) is contacted with water (14) and optionally also with an organic solvent. After the contact and other treatments, purified soy proteins (24) are separated using methods such as filtration, centrifugation, precipitation, decantation and membrane filtration, e.g. ultrafiltration. According to any preferred or alternative embodiments, the polyphenol (e.g. isoflavone) may be separated from soy solubles by adjusting the solution concentration of the soy solubles to between about 10 percent (%) and 40 percent (%), more preferably between about 15 percent (%) and 30 percent (%). Such adjustment of concentration induces the precipitation of a solid with increased polyphenol (e.g. isoflavone) purity compared to the soy solubles, typically about 4-7 percent (%) on a solvent-free basis. Typically, polyphenol (e.g. isoflavone) in such precipitate are further purified to 40 percent (%) purity in a costly process such as chromatographic separation. One embodiment of the method of purifying the phenolic compound

(e.g. isoflavone) includes purifying polyphenols (e.g. isoflavone) in the precipitate so that that precipitate is a suitable starting material.

Impurities removed from the soy material during protein purification (20), end up in an aqueous solution, sometimes referred to as soy solubles, soy molasses or soy whey (22). Such solution contains polyphenols (e.g. isoflavone) and many other components, including various carbohydrates (main components), soluble proteins and/or peptides, saponins, mineral salts (ashes), amino acids and other solutes. The concentration of the solution depends on the method of the protein purification. In those cases where an organic solvent is used for protein purification, it is evaporated from the co-product solution along with water to reach a solution of about 50 percent (%) concentration, typically referred to as soy molasses. In cases of using no organic solvent, the solution is dilute, particularly when membrane filtration is used for separating the purified proteins. In the latter case, the solutes are of low molecular weight and end up in the membrane permeate. The permeate is of few percent concentration and is concentrated by reverse osmosis (RO) and/or evaporation according to a preferred embodiment. Other soy solubles suitable for use as a starting material may be obtained by extraction of solubles from defatted soybean germ according to alternative embodiments. According to an exemplary embodiment, the starting material (e.g. soy solubles or a precipitate obtained from those) is combined with a basic compound in an aqueous medium to form an aqueous solution with pH greater than at least about the isoflavones pKa minus about 1.5. In cases where there are several isoflavones, the pKa is that of the stronger acid isoflavone, according to a preferred embodiment. The conditions are adjusted such that insolubles are formed. (The term "insolubles" as used in thfe disclosure means material that does not dissolve in the solution at the selected conditions.)

Referring further to FIGURE 1 , in step (30), an alkali (26) is added to the solubles (22) and the concentration of the solution is adjusted to between about 1 percent (%) and about 40 percent (%) total solids. (The term "solids" when referred to concentration in a solution may also mean "solute".) Any alkali is suitable according to any preferred or alternative embodiments. Particularly suitable alkali are ammonia and hydroxides, carbonates and bicarbonates of alkali metals. Concentration adjustment may involve dilution with water or water removal, depending on the concentration of the solubles. Water removal could use e.g. evaporation, multiple-effect evaporation, reverse osmosis and their combination. According to a preferred embodiment, water removal is conducted at a temperature not greater than about 60 degrees Celsius. If the starting material is solid, it is preferably introduced into an alkali solution. Means for facilitating dissolution are preferably applied, e.g. mixing and ultrasound.

The total solids concentration in that step is selected so that insolubles exist according to a preferred embodiment. The preferred concentration is a matter of economic optimization based on the cost of water removal and preferred conditions for the recovery of the product. The preferred concentration is typically smaller in the case of a solid starting material than in the case of starting with a solution such as soy solubles.

The insolubles formed contain non-phenolic compounds so that the solution is purified with regards to the phenolic compound (e.g. isoflavones) according to a preferred embodiment. The insolubles may contain less than about 20 percent (%) phenolic compound (e.g. isoflavones), typically less than about 10 percent (%), suitably less than about 5 percent (%) and most preferably less than about 1 percent (%) phenolic compound (e.g. isoflavones). Those insolubles are preferably separated (step 40 in FIGURE 1) by means such as centrifugation, filtration, decantation, etc. and various combinations of those. The separated insolubles (42) may be used for various applications, e.g. in animal feed.

According to a preferred embodiment, the purified solution (44) is further treated for separation of the phenolic compound (e.g. isoflavones) according to a preferred embodiment. A preferred method for such separation is lowering the pH of the solution by at least 0.2 pH unit. Thus, as shown in FIGURE 1 , after separation of the insolubles (step 40), an acid (46) is added (step 50) to the purified solution (44), whereby insolubles are formed. Those insolubles are enriched in isoflavones, i.e. their isoflavones concentration, on a solvent-free basis, is greater than that in the purified solution according to a preferred embodiment. Those isoflavones-enriched solids are separated from the isoflavones-depleted solution (step 60 in FIGURE 1) by methods such as centrifugation, filtration, decantation, etc. and various combinations of those according to an alternative embodiment. The isoflavones-enriched solid obtained is concentrated in isoflavones. The concentration of the isoflavones in the isoflavones-enriched solid is typically greater than about 30 percent (%), preferably greater than about 40 percent (%), most preferably greater than about 50 percent (%). Those solids may contain at least about 30 percent (%) of the amount of the isoflavones in the starting material (i.e. the isoflavones recovery yield is at least about 30 percent (%)), more preferably at least 50 percent (%), most preferably at least about 60 percent (%). If desired, the isoflavones-enriched solids can be further purified, e.g. by solvent extraction, chromatographic separation, ion-exchange, adsorption on resin of low polarity, re-crystallization, etc.

The following is a preferred method of conducting re-crystallization for further purification of the isoflavones-enriched insolubles. Water and alkali are added to the insolubles to form a solution having at least pH of about pKa minus 1.5, and a total solid concentration of between about 1 percent (%) and 40 percent (%). A purified solution is formed, containing the isoflavones, and further insolubles containing at least one impurity and a minimal content of isoflavones, if any. Those insolubles are removed by methods as disclosed above. Then, the pH of the purified solution is lowered, inducing thereby precipitation of a precipitate further enriched in isoflavones. That precipitate is separated from the depleted solution, and if desired, purified again by another step of re- crystallization. I

According to another embodiment, the pH is reduced by contact with a stream (e.g. solution or a solid) that has buffering capacity. According to a preferred embodiment, the contact is with a protein, preferably a purified one. Such contact results in precipitation of isoflavones and can form an isoflavones- enriched purified protein.

According to a preferred embodiment, the concentration of the solution at reduced pH (e.g. in step 50 of FIGURE 1) is about the same as that of the solution at the elevated pH (e.g. in step 30 of FIGURE 1) or smaller. Preferably, the temperature of the solution at the reduced pH is about the same as that of the solution at the elevated pH or higher. Most preferably, both temperature and concentration of the solution at reduced pH are similar to those of the solution at elevated pH.

According to a preferred embodiment, the malonyl glycoside and acetyl glycosides in the solution are hydrolyzed to form the glycoside form prior to the separation of the isoflavones-enriched solids, more preferably prior to the lowering of the pH. The hydrolysis is preferably catalyzed chemically, enzymatically or in a combination of the two. Chemical catalysis is conducted at elevated pH according to a preferred embodiment and optionally also at elevated temperature. Typically, the higher the temperature, the lower is the pH needed and vice versa. Part of the hydrolysis may take place at the elevated pH (see step 30 of FIGURE 1) according to an alternative embodiment. The method of purifying a phenolic compound (e.g. isoflavones) is suitable for the recovery of purified isoflavones from a variety of sources. As indicated above, much of the impurities in sources such as soy solubles are ones of high solubility, e.g. carbohydrates, which do not tend to co-precipitate with isoflavones. There are, however, ones that do tend to co-precipitate and their concentration is high enough to limit the isoflavones purity in the precipitate in the common industrial practice to about 4-7 percent (%). A measure of the concentration of impurities that tend to co-precipitate with isoflavones is the amount of solid formed on adjusting the concentration of the source in an aqueous medium to between about 10 percent (%) and 40 percent (%) solids at pH of up to about 8. That amount (dried solids) is compared to the amount of isoflavones in the starting material and the ratio between the two is referred to in this disclosure as "R1." The greater R1 , the greater is the amount of impurities that tend to co- precipitate with isoflavones and lower the isoflavones purity in the precipitate. Typically R1 is greater than about 1.5, in most cases greater than about 3 and in particularly problematic cases greater than about 5.

The greater R1 is, the greater is the need for removing impurities as insolubles, i.e. the greater should the amount of insolubles be at about pH > pKa minus 1.5. The amount of those insolubles, presented as the ratio between their amount (dried form), and the amount of isoflavones in the starting material (referred to in this disclosure as "R2"), is therefore compared with R1. At the chosen conditions, R2 can reach more than about 10 percent (%), preferably more than about 30 percent (%) and most preferably more than 50 percent (%) of R1. Furthermore, the insolubles to be removed are substantially free of isoflavones so that their removal presents no significant yield loss.

The starting material may contain solutes that are of commercial value. For example, soy solubles/molasses contain carbohydrates, saponins, proteins and other components. Some of those, which tend to co-precipitate with isoflavones and, in that respect are considered as impurities, could also be recovered for some applications. Preferably, the method of purifying a phenolic compound (e.g. isoflavones) also involves separation of such solutes and their commercial utilization. Such solutes can be recovered from the various streams of the process, e.g. from the starting material before the first pH adjustment, from the insolubles formed and separated at the elevated pH, from the purified solution, from the isoflavones-depleted solution and from co-products of re-crystallization. Separation of solutes from the starting material may also be introduced as a pre-treatment before pH adjustment to above about pKa minus 1.5 according to an alternative embodiment, j For example, proteins present in the starting material may be separated by adjusting the pH of the solution to about the protein isoelectric point (pi), by membrane filtration, or by a combination of those. In many cases, such removed proteins are enriched in the Bowman-Birk tryspin inhibitor (BBI) and present a source for its recovery. Thus, a preferred embodiment involves treating the starting material to remove protein at conditions such that those removed proteins are enriched in BBI. Alternatively, BBI can be recovered from other process streams, e.g. the isoflavones-depleted solution. The process may also involve separation of other solutes from the starting material or from other process streams according to alternative embodiments. For example, ashes and amino acids may be removed by means such as ion-exchange.

Carbohydrates may also be separated from the starting material and from other process streams according to another alternative embodiment. In addition or alternatively to separation, carbohydrates may be removed by degradation to other components. Such degradation may involve enzymatic hydrolysis and/or fermentation of the carbohydrates to solutes that are easy to separate, e.g. by distillation, by extraction, by crystallization, or by known methods of solid-liquid separation. Fermentation products may be recovered according to an alternative embodiment. Optionally, carbohydrates may be fermented to form enzymes suitable for the isoflavones process, e.g. ones for the hydrolysis of the malonyl and acetyl forms. 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. EXAMPLES COMPARATIVE EXAMPLE A Defatted soy flakes were extracted with slightly basic water. The solution was separated from the insolubles. (The separated solution was treated on an ultrafiltration membrane. Most of the dissolved protein was retained in the retentate, while the rest of the protein, the lower molecular weight solutes and most of the water permeated through the membrane. The permeating solution, containing proteins, several carbohydrates, several mineral salts and isoflavones, is referred to in the following as the permeate. The total solutes concentration of the permeate was about 1.5 percent (%). HCI was added to the permeate solution to bring the pH to 4.5.

Insolubles appeared. After overnight storage, the precipitate was removed by centrifugation, dried and weighed. Its weight corresponded to 4.3 percent (%) of the total solutes in the acidified solution. Then, NaOH was added to the solution to bring the pH to 11. The temperature was brought up to 55 degrees Celsius and the solution was kept at that temperature for 1 hour in order to hydrolyze the isoflavone malonyl and acetyl glycosides in the solution to their glycoside form. The solution was cooled to ambient temperature and HCI was added to it to bring the pH to neutral. The treated permeate had a total solute concentration of about 1.7 percent (%) and isoflavones content of about 0.6 percent (%) of the total solutes.

About 320g of treated permeate was concentrated to 20.5 percent (%) total solutes by water removal in an evaporator operated under vacuum at 55 degrees Celsius. The solution was cooled to ambient temperature and kept at that temperature overnight. A precipitate was formed. The precipitate was separated from the concentrated solution by centrifugation, dried, weighed and analyzed for isoflavones. The weight of the dried precipitate was 0.194g. The amount of isoflavones in the treated permeate solution was 0.033g. Thus, the ratio between the dried precipitate and the isoflavones in the starting material (R1 ) was 5.9. (The ratio between the weight of the precipitate formed and removed at pH 4.5 and the isoflavones content was about 10:1 ~ Concentration of the permeate to about 20 percent (%) total solutes without the pre-treatment at pH 4.5 would likely have resulted in R1 greater than 5.9.) The precipitate contained about 40 percent (%) of the initial isoflavones in the solution and the isoflavones concentration in the dried precipitate was 8.9 percent (%). This comparative example shows that the amount of impurities in the permeate that tend to co-precipitate with the isoflavones is relatively high and that on concentration to about 20 percent (%) an isoflavones-containing precipitate can be formed, but the concentration of isoflavones there is relatively small.

COMPARATIVE EXAMPLE B Treated permeate solution prepared as described in Comparative

Example A was concentrated to total solutes concentration of 49 percent (%). The pH in the solution was 5.3. Insolubles were observed. Mixing the solution suspended the solids and allowed taking a homogenized sample. Such homogenized sample is referred to in the following as "concentrated-treated permeate."

103g of concentrated-treated permeate was mixed with water and NaOH to bring the pH to 7 and the total solutes concentration to 14.8 percent (%). After mixing at ambient temperature for an hour, the insolubles were separated by centrifugation, and a fraction of those were dried, weighed and analyzed for isoflavones. The total weight of the precipitate was 1.06g on dry basis. The amount of isoflavones in the treated solution was about 0.25g. Thus, the ratio between the dried precipitate and the isoflavones in the starting material (R1) was about 4. The precipitate contained about 25 percent (%) of the initial isoflavones in the solution and the isoflavones concentration in the dried precipitate was 6.1 percent (%). As in Comparative Example A, diluting a concentrated-treated permeate to about 15 percent (%) results in an isoflavones-containing precipitate, but the concentration of isoflavones there is small.

COMPARATIVE EXAMPLE C A permeate was produced according to the method of Comparative Example A. The permeate was concentrated by reverse osmosis (RO) to 3 percent (%) total solids. The obtained solution was further concentrated by water evaporation to total solids concentration of 110.5 percent (%). The concentrated solution was cooled to ambient temperature. A precipitate was formed. The precipitate was separated from the concentrated solution by centrifugation, dried, weighed and analyzed for isoflavones. The weight of the dried precipitate was about 0.21 g. The amount of isoflavones in the treated solution was 0.045g. Thus, the ratio between the dried precipitate and the isoflavones in the starting material (R1) was 4.7. This shows that the amount of impurities that tend to co-precipitate with isoflavones is high also in permeates that did not go through the alkali treatment for hydrolysis of malonyls and acetyls.

EXAMPLE 1 About 440g of concentrated-treated permeate were prepared as in Comparative Example B. The concentrated-treated permeate contained 0.212 percent (%) isoflavones. The concentrated-treated permeate was mixed with about 110Og water and about 10Og NaOH solution of 1.5N concentration. The pH of the formed solution was 11. Mixing was then continued for 2 hours at ambient temperature and for additional 0.5 hour at 60 degrees Celsius. The temperature was lowered to the ambient one. A solution with total solids concentration of 14.3 and a precipitate were formed. The precipitate was removed and the clarified solution was analyzed. The precipitate was washed, dried, weighed and analyzed. The precipitate weighed about 4g. The amount of isoflavones in the initial concentrated-treated solution was about 0.93g, so that the ratio between that dried precipitate and the isoflavones in the starting material (R2) was about 4, which is similar to R1 (see Comparative Example B). Isoflavones concentrations in the clarified solution and in the washed and dried precipitate were 0.053 percent (%) and 0.03 percent (%), respectively.

Example 1 shows that a large fraction of the impurities that tend to co-precipitate with isoflavones can be removed, but with very little loss of yield. The precipitate contained less than 1 percent (%) of the amount of isoflavones in the initial concentrated-treated solution.

EXAMPLE 2 About 1600g of the clarified solution obtained in Example 1 were mixed with 30 percent (%) HCI in an amount suitable to bring the pH down from 11 to 7. Mixing was applied for 2.5 hours at ambient temperature. A precipitate was formed. The precipitate was separated, washed, dried, weighed and analyzed. Its weight was about 0.6g. Isoflavones concentrations in the isoflavones-depleted solution and in the washed and dried precipitate were 0.036 percent (%) and 63 percent (%), respectively. The precipitate contained about 40 percent (%) of the amount of isoflavones in the initial concentrated-treated permeate.

Example 2 shows that a highly-concentrated isoflavones product is formed. Isoflavones concentration there was 63 percent (%) compared with 6.1 percent (%) in Comparative Example B.

EXAMPLE 3 About 150g of concentrated-treated solution were prepared as in Comparative Example B. The solution was mixed with about 200g water and about 50g NaOH solution of 1.5N. The pH of the formed solution was 11. Mixing was then continued for 7 hours at 20C. A solution with total solids concentration of 14.7 and a precipitate were formed. The precipitate was separated by centrifugation and the clarified solution was analyzed. The precipitate was washed, dried, weighed and analyzed. Its weight was about 2.4g. The amount of isoflavones in the initial concentrated-treated solution was about 0.45g, so that the ratio between that dried precipitate and the isoflavones in the starting material (R2) was about 5 (somewhat greater than R1 as found in Comparative Example B). Isoflavones concentrations in the clarified solution and in the washed and dried precipitate were 0.081 percent (%) and 0.049 percent (%), respectively. The precipitate contained less than 1 percent (%) of the amount of isoflavones in the initial concentrated-treated solution. I In this Example, much of the cό-precipitating impurities were removed with substantially no loss of isoflavones.

EXAMPLE 4 About 515g of the clarified solution obtained in Example 3 were mixed with 1.5N HCI in an amount suitable to bring the pH down from 11 to 7. Mixing was applied for 48 hours at ambient temperature. A precipitate was formed. The precipitate was separated, washed, dried, weighed and analyzed. Its weight was about 0.67g. Isoflavones concentrations in the isoflavones- depleted solution and in the washed and dried precipitate were 0.027 percent (%) and 46 percent (%), respectively.

About 60 percent (%) of the amount of isoflavones in the initial concentrated-treated solution was recovered in a precipitate highly concentrated in isoflavones. EXAMPLE 5 5.5g of the wet precipitate formed in Comparative Example B (about 0.8g on dry basis) was mixed with about 30g water and about 0.5g of 1.5N NaOH solution. The pH of the formed solution was 11. Mixing was then continued for 2 hours at ambient temperature. A solution with total solids concentration of 0.9 percent (%) and a precipitate were formed. The precipitate was separated and the clarified solution was analyzed. The precipitate was washed, dried, weighed and analyzed. Its weight was about 0.14g. The amount of isoflavones in the starting wet precipitate was about 0.05g, so that the ratio between that dried precipitate formed at the elevated pH and the isoflavones in the starting material (R2) was about 2.8, which is about 70 percent (%) of R1 as found in Comparative Example B. Isoflavones concentrations in the clarified solution and in the washed and dried precipitate were 0.21 percent (%) and 0.47 percent (%), respectively. The precipitate contained less than 1 percent (%) of the amount of isoflavones in the initial wet precipitate.

The starting material in Example 5, i.e. a precipitate containing about 6.1 percent (%) isoflavones, was obtained from a permeate, rather than the permeate itself. Here too, much of the co-precipitating impurities are removed at the elevated pH with substantially no loss of isoflavones. EXAMPLE 6 About 29g of the clarified solution obtained in Example 5 were mixed with 30 percent (%) HCI in an amount suitable to bring the pH down from 1 1 to 7. Mixing was applied overnight at ambient temperature. A precipitate was formed. T he precipitate was separated, washed, dried, weighed and analyzed. Its weight was about 0.098g. Isoflavones concentrations in the isoflavones-depleted solution and in the washed and dried precipitate were 0.013 percent (%) and 42 percent (%), respectively.

As demonstrated in Examples 5 and 6, isoflavones-containing solid having a purity of 6.1 percent (%) were purified to 42 percent (%) purity. EXAMPLE 7 Permeate was concentrated by RO to 3 percent (%) total-solutes as in Comparative Example C. Its isoflavones concentration was 0.02 percent (%). 283g of that solution was farther concentrated to 7.5 percent (%) total solids, cooled and 1.5N HCI solution was added to bring the pH to 4.5. A precipitate was observed. It was separated by centrifugation, washed, dried, weighed and analyzed for isoflavones. Its dry weight was about 0.22g. The amount of isoflavones in the starting material was 0.057g so that the weight ratio between the precipitate and the initial isoflavones content is 3.9. The precipitate contained no detectable amount of isoflavones. The pH of the clear solution was brought to 10.5 by the addition of NaOH and the solution was concentrated by water evaporation to 12.4 percent (%) total solids. More NaOH was added to reach a pH of 11 and the solution was mixed for 1.5 hours at ambient temperature and for 3 additional hours at 17C. A precipitate appeared. It was separated by centrifugation, washed, dried, weighed and analyzed for isoflavones. Its dry weight was about 0.40g, i.e. the weight ratio between the precipitate and the initial isoflavones content (R2) was 7.0. The precipitate contained less than 1 percent (%)'of the initial amount of isoflavones.

EXAMPLE 8 About 55g of the clarified solution obtained in Example 7 were mixed with 1.5N HCI in an amount suitable to bring the pH down from 1 1 to 7. Mixing was applied overnight at 17 degrees Celsius. A precipitate was formed. The precipitate was separated, washed, dried, weighed and analyzed. Its weight was about 0.059g. Isoflavones concentrations in the isoflavones-depleted solution and in the washed and dried precipitate were 0.044 percent (%) and 27 percent (%), respectively.

EXAMPLE 9 Permeate was concentrated by RO to 16 percent (%) total-solutes and its isoflavones concentration was 0.08 percent (%). NaOH solution of 1.5N was added to 128g of that solution to bring the pH to 1 1 and the solution was mixed at ambient temperature for 2 hours. A precipitate appeared. It was separated by centrifugation, washed, dried, weighed and analyzed for isoflavones. Its dry weight was about 0.24g. The precipitate contained less than 1 percent (%) of the initial amount of isoflavones. EXAMPLE 10 About 130g of the clarified solution obtained in Example 9 were mixed with HCI in an amount suitable to bring the pH down from 1 1 to 7. Mixing was applied overnight at 18C. A precipitate was formed. The precipitate was separated, washed, dried, weighed and analyzed. Its weight was about 0.061 g. Isoflavones concentrations in the formed clarified solution and in the washed and dried precipitate were 0.087 percent (%) and 86 percent (%), respectively. . * * *

While the preferred and other exemplary embodiments illustrated in the drawings and described above 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.

Claims

CLAIMSWhat is claimed is:

1. A process for improving the purity of a phenolic compound from a starting material of biological source comprising the steps of: I (a) providing the starting material from the biological source comprising a mixture of at least one phenolic compound having a purity P1 and at least one non-phenolic compound;

(b) forming at least a fraction of the starting material, an alkaline aqueous solution having at least a pH of about pKa minus 1.5, wherein the pKa is that of the phenolic compound, and insolubles; and

(c) separating the insolubles from the alkaline solution, whereby there is formed a purified solution comprising the purified phenolic compound having a purity P2 and insolubles containing the non-phenolic compound wherein P2 is greater than P1 and the insolubles contain less than about 20 percent (%) of the phenolic compound present in the starting material.

2. The process of Claim 1 comprising the further step of: (d) adjusting the pH of the separated purified solution to less than that of the formed purified solution by at least about 0.2 pH units, inducing thereby formation of insolubles enriched in the phenolic compound and a depleted solution.

3. The process of Claim 2 comprising the further step of: (e) separating the enriched insolubles from the depleted solution.

4. The process of Claim 1 wherein the phenolic compound comprises a polyphenol.

5. The process of Claim 1 wherein the phenolic compound comprises a flavonoid.

6. The process of Claim 5 wherein the flavonoid comprises at least one of anthocyanidins, anthocyanins, f lavanols, flavonols, flavones, flavanones, and isoflavones.

7. The process of Claim 5 wherein the flavonoid comprises isoflavones comprising at least one of malonyl conjugates, glycosides and aglycones of daidzin, genistin, and glycitin.

8. The process of Claim 1 wherein the biological source is selected from at least one of a plant, an extract thereof and a processed material derived therefrom.

9. The process of Claim 8 wherein the plant comprises the family Glycine.

10. The process of Claim 8 wherein the plant comprises Glycine max.

11. The process of Claim 1 wherein the biological source comprises at least one of soybean, non-oil products of soybean processing, defatted soy flakes, soybean meal, soy molasses, soy solubles, soy whey and soy germ.

12. The process of Claim 1 wherein the biological source is an aqueous solution and is initially subjected to ultrafiltration processing to provide a permeate, the permeate comprising the starting material.

13. The process of Claim 12 wherein the permeate is further concentrated, the permeate concentrate comprising the starting material.

14. A process for improving the purity of a phenolic compound from a starting material of biological source, the starting material comprising at least one phenolic compound and at least one non-phenolic compound, the starting material being characterized in that, on adjusting the total concentration of the solutes of the starting material, in an aqueous medium at a pH of up to about 8, to a concentration of between about 10 percent (%) and about 40 percent (%), the non-phenolic compound presents as a first precipitate, and in that the weight ratio, R1 , of the first precipitate, when dried, to the phenolic compound in the starting material, is at least 1.5:1 , the process comprising: forming at least a fraction of the starting material, an alkaline aqueous solution having a pH of at least about pKa minus 1.5, wherein the pKa is that of the phenolic compound, and a total solutes concentration of between about 1 percent (%) and about 40 percent (%), and insolubles containing the non-phenolic compound, wherein a weight ratio, R2, of the insolubles, when,dried, to the phenolic compound in the starting material is at least about 10 percent (%) of R1, and wherejn the insolubles contain less than about 20 percent (%) of the phenolic compound present in the starting material.

15. The process of Claim 14 comprising the further step of separating the phenolic compound from aqueous solution.

16. The process of Claim 14 wherein the starting material is a solid.

17. The process of Claim 16 wherein the solid is a product of co- precipitation of the phenolic compound and the non-phenolic compound.

18. The process of Claim 15 wherein the starting material is a liquid. .

19. The process of Claim 15 comprising the further step of: separating the aqueous solution from the insolubles.

20. The process of Claim 19 comprising the further step of: (d) adjusting the pH of the separated purified solution to less than that of the formed purified solution by at least about 0.2 pH units, inducing thereby formation of insolubles enriched in the phenolic compound, the insolubles comprising enriched insolubles having at least about 20 percent (%) of the phenolic compound on a dry basis.

21. The process of Claim 20 comprising the further step of: (e) separating the enriched insolubles from the aqueous solution.

22. The process of Claim 14 wherein the phenolic compound is selected from a group comprising flavonoids.

23. The process of Claim 14 Wherein the starting material is formed during the production of purified soy protein.

24. The process of Claim 13 wherein the production of purified soy protein comprises extraction with a solvent.

25. The process of Claim 23 wherein the production of purified soy protein comprises dissolution of soy protein in an aqueous solution followed by separation of dissolved protein by at least one of isoelectric point precipitation and membrane separation.

26. The process of Claim 23 wherein the starting material comprises at least one of soy solubles, soy whey, soy molasses, precipitates obtained from those and solutions obtained from those.

27. The process of Claim 14 further comprising the step of hydrolyzing isoflavone malonyls to isoflavone glycoside.

28. The process of Claim 27 wherein the hydrolyzing is conducted by at least one of treatment at alkaline pH and enzymatic hydrolysis.

29. The process of Claim 14 wherein the starting material is a solution with total solutes concentration of less than about 3 percent (%).

30. The process of Claim 29 wherein a total solutes concentration of between about 10 percent (%) and about 40 percent (%) is achieved by water removal using at least one of evaporation, multiple-effect evaporation, reverse osmosis and a combination thereof.

31. The process of Claim 26 wherein the isoflavones form less than about 5 percent (%) of the starting material on a dry basis.

32. The process of Claim 21 wherein the enriched insolubles contain at least about 30 percent (%) of the at least one phenolic compound in the starting material.

33. The process of Claim 21 further comprising re-crystallization of the enriched insolubles according to the steps of: (i) addition of water and alkali to the enriched insolubles to form a solution having at least pH of about pKa minus 1.5, wherein the pKa is that of the phenolic compound and having a total solutes concentration of between about 1 percent (%) and about 40 percent (%), whereby there is formed a purified solution containing the phenolic compound and further insolubles containing the non-phenolic compound, wherein the further insolubles contain less than 20 percent (%) of the at least one phenolic compound present in the starting enriched precipitate; (ii) separating the purified solution from the further insolubles; (iii) adjusting the pH of the purified solution resulting from step

(ii) to less than that of step (i) by at least about 0.2 pH units, inducing thereby precipitation of a phenolic precipitate comprilsing a precipitate further enriched in the phenolic compound, the phenolic precipitate comprising at least 30 percent (%) phenolic compound on a dry basis.

34. The process of Claim 33 further comprising recrystallization of the phenolic precipitate.

35. The process of Claim 34 wherein the recrystallizations are conducted in a counter-current mode.

36. The process of Claim 33 further comprising purifying the phenolic precipitate by at least one of solvent extraction, chromatographic separation, ion- exchange, adsorption on resin of low polarity and a combination thereof.

37. The process of Claim 26 wherein the solute comprises at least one of carbohydrates, proteins and saponins.

38. The process of Claim 26 further comprising separating dissolved protein by at least one of adjusting the pH to about that of the protein iso-electric point, membrane separation and a combination thereof.

39. The process of Claim 38 wherein the separated protein is enriched with the Bowman-Birk Inhibitor.

40. The process of Claim 38 further comprising the step of removing ashes and amino acids by ion-exchange.

41. The process of Claim 38 further comprising removal of carbohydrates by at least one of separation and degradation.

42. The process of Claim 42 wherein the degradation comprises fermentation.

43. The process of Claim 38 wherein adjusting of the pH involves contact with protein forming thereby an isoflavones-enriched protein.

44. The process of Claim 38 wherein an organic solvent is used to facilitate the formation of the insolubles.

45. The process of Claim 38 wherein the organic solvent comprises hexane.

46. The process of Claim 38 wherein insolubles are removed- rom the starting material before the forming the solution having at least a pH of about pKa minus 1.5 at total solutes concentration wherein the at least one phenolic compound is under-saturated.

47. The process of Claim 38 wherein a weight ratio, R3, of combined insolubles removed in the process steps of forming purified phenolic compound, to the at least one phenolic compound in the starting material is at least about 30 percent (%) of R1.

48. The process of Claim 47 wherein the water removal is conducted at a temperature of not more than about 60 degrees C.

49. The process of Claim 38 wherein the first precipitate contains between about 1 percent and about 10 percent of the phenolic compound.