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. 60/557,189 entitled PHYTATE REDUCED FEED SYSTEM filed March 29, 2004 as attorney docket no. CGL04/0091 USP1 ; U.S. Patent Application No. 60/557,197 entitled PHYTATE REDUCED FEED SYSTEM filed March 29, 2004 as attorney docket no. CGL04/0092USP1 ; PCT Patent Application No. entitled PHYTATE REDUCED FOOD filed March 29, 2005 as attorney docket no. CGL04/0092WO1 ; PCT Patent Application No. 60/557199 entitled ISOFLAVONE DISTRIBUTION SYTEM filed March 29, 2005 as attorney docket no. CGL04/0049WO1 ; PCT Patent Application No. entitled PROTEIN PURIFICATION SYTEM filed March 29, 2005 as attorney docket no. CGL04/0093WO1 ; PCT Patent Application No. PCT/US05/004160 entitled PHENOLIC COMPOUND PURIFICATION filed February 9, 2005 as attorney docket no. CGL04/0008WO1 ; PCT Patent Application No. PCT/US05/04153 entitled PHENOLIC COMPOUND PURIFICATION filed February 9, 2005 as attorney docket no. CGL04/0009WO1 ; PCT Patent Application No. PCT/US05/04166 entitled CYCLITOL SEPARATION METHOD filed February 9, 2005 as attorney docket no. CGL03/0489WO1 ; U.S. Patent Application No. 60/660807 entitled 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 phytate reduced food. The present invention more particularly relates to a method for the production of a purified proteinous food or feed component. The present invention more particularly relates to a method for the production of such food or feed component, which is relatively low in phytate. BACKGROUND OF THE INVENTION

Various plant protein sources contain phytate. Phytate is inositol hexa-phosphate. At low pH, phytate appears in a free acid form carrying 12 protons. As pH goes up, phytic acid dissociates and protons are replaced with cations. Phytic acid and its salts are strong complexants and form complexes with various metal ions, e.g. ions of transition metals. The term "phytate" as used in this disclosure means and includes any of those forms including the fully protonated and fully dissociated ones. The term "phytate" as used in this disclosure also means and includes various compositions of phytate, including its complexed forms. The term "phytate" as used in this disclosure also means and includes partially hydrolyzed phytate.

Phytate is undesired in various feed applications. Its phosphorous is not available to the animal on one hand, while its strong complexation capacity reduces the availability of essential minerals. Conventional industrial methods of plant protein purification are not sufficiently effective in separation of phytate. One known conventional industrial option uses an enzyme - typically phytase - in order to hydrolyze the phytate contained in the purified protein. However, such known enzyme system has several disadvantages including that the more phytate present in the purified protein, the greater are the costs of that enzymatic hydrolysis.

Typically, soybeans are dehulled, flaked, extracted to separate oil and desolventized to form defatted flakes containing about 45 percent protein. After toasting for deactivating some of the anti-nutritional factors (ANF), those flakes (e.g. soybean meal, SBM) are incorporated as a protein source in animal feed. Toasting does not eliminate phytate, which is considered an ANF. 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. Oligosaccharides and phytate present in defatted soybean are ANF.

There are two known conventional industrial approaches to protein purification. 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 selected to minimize protein dissolution. Typically, those conditions are either: (i) conducting the washing at a pH of about the isoelectric point of the protein; or (ii) washing with an aqueous ethanol solution of 60-80 percent ethanol. The product 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 of the protein or ultrafiltration. The product is referred to in this disclosure as "protein isolate."

Such two known conventional industrial approaches to purifications of protein are relatively efficient in removing sugars from the protein. However, such known approaches to purifications of protein have several disadvantages including that they can be inefficient in removing phytate. The 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.) also contains part of the phytate.

Accordingly, there is a need for a method that forms purified protein products with reduced phytate content suitable for use in food or feed. There is also a need for a relatively inexpensive system for reducing phytate in purified protein products. It would be advantageous to provide a phytate reduced feed 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 a protein-containing substance. The method includes providing a starting material comprising a protein, a sugar and a phytate. The method also includes extracting with a solvent at least a fraction of the sugar and at least a fraction of the phytate from the starting material to form at least one purified proteinous material and at least one extract. The method includes separating the at least one purified proteinous material from the at least one extract to form a separated purified proteinous material and a separated extract. The method includes incorporating the separated purified proteinous material in at least one product. The solvent comprises at least one of water and aqueous solutions. The pH of the solvent during at least part of the extraction differs from pi by at least about 0.2 pH units, wherein pi is the isoelectric point of the protein.

The present invention also provides a food comprising a protein- containing substance derived from a starting material having protein and phytate, the food comprising at least about 70 percent of the protein of the starting material and less than about 60 percent of the phytate of the starting material. The food is prepared by the process which includes providing the starting material comprising the protein, a sugar and the phytate. The process also includes extracting with a solvent at least a fraction of the sugar and at least a fraction of the phytate from the starting material to form at least one purified proteinous material and at least one extract. The process also includes separating the at least one purified proteinous material from the at least one extract to form a separated purified proteinous material and a separated extract. The process also includes incorporating the separated purified proteinous material in at least one product. The solvent comprises at least one of water and aqueous solutions. The pH of the solvent during at least part of the extraction differs from pi by at least about 0.2 pH units, wherein pi is the isoelectric point of the protein. DETAILED DESCRIPTION OF THE PREFERRED AND OTHER EXEMPLARY EMBODIMENTS

A system and method for reducing phytate in a protein source is disclosed as a phytate reduced feed or food system and method. As used in this disclosure, the term "food" means and includes food or feed that physically nourishes or sustains, and includes products or consumables that are ingested for flavor. The method involves treating a plant material that contains a protein, a sugar, and a phytate. According to an exemplary embodiment, the starting material comprises between about 30 percent and about 90 percent by weight protein on dry basis, suitably between about 35 percent and about 80 percent, more suitably between about 40 percent and about 70 percent. According to an exemplary embodiment, the starting material includes at least about 10 percent by weight sugar on dry basis, suitably at least about 15 percent, suitably at least about 20 percent. According to an exemplary embodiment, the starting material contains a phytate content of at least about 1 percent by weight of the protein content, suitably at least about 1.5 percent, suitably at least about 2.0 percent, suitably at least about 3 percent, more suitably at least 4 percent. According to alternative embodiments, the plant material may be an oilseed, such as soybean, dehulled soybean or a defatted oilseed. Other suitable starting materials include soybean meal, defatted soybean flour, defatted soybean flakes, flash- desolventized defatted soy flakes, soy molasses, soy whey and soy solubles. Other suitable starting materials include heat treated and desolventized defatted soybean flakes.

According to a preferred embodiment, sugars and phytate are extracted from the starting material. The solvent used for the extraction may be water or an aqueous solution. The term "extraction" as used in this disclosure means and includes dissolution into the solvent. Extraction can be done by contacting the starting material with the solvent. According to a preferred embodiment, a multiple-stage contacting is conducted. According to an alternative embodiment, contacting may be a single stage operation. According to a particularly preferred embodiment, the contacting includes multiple-stage extraction conducted in a counter-current mode of operation. According to other alternative embodiments, other modes could be used in cases where more than one extract is desired and when those multiple extracts are desired at different compositions.

Sugars and other soluble compounds dissolve in the solvent during the extraction to form a solution of those solubles, which is referred to in this disclosure as "extract." Less solubles components, such as phytate and protein may also dissolve, at least partially, depending on the extraction conditions. In addition to the number of contact stages and mode of operation (e.g. counter current vs. cross-current or co-current), extraction efficiency is at least partially dependent on parameters such as the relative solvent amount, the temperature, the contact duration (in each stage) and the pH.

According to a preferred embodiment, the pH of the solvent is different than the isoelectric point of the protein (pi), preferably greater, during at least part of the extraction. Thus, pH > pi preferably exists in the solvent, for example, during at least part of the contact time or in at least one stage of a multiple-stage extraction. According to a preferred embodiment, in case of multiple-stage counter-current extraction, pH greater than about pi is maintained in the last stage, i.e. in the sugars and phytate-containing solvent before it exists the extraction operation. According to a preferred embodiment, the pH in the solvent in at least part of the extraction is greater than pi by at least about 0.2 units, more preferably by at least about O.5 units, preferably by more than about one unit. According to a preferred embodiment, the pH in the solvent in at least part of the extraction is greater than pi by less than 3 units, more suitably less than 2.5 units. According to a preferred embodiment, the pH of the solvent in at least part of the extraction is between about 4.8 and about 6.5, preferably between about 5 and about 6.

According to a preferred embodiment, the solvent used in the extraction is an aqueous solution containing a reagent capable of forming strong interactions with cations of alkaline earth metals such as calcium or magnesium. Such reagent can be a complexant according to an exemplary embodiment. According to a preferred embodiment, the reagent is approved for use in feed, in food or in both. Suitable complexants of calcium cations approved for use in feed include citric acid, EDTA, etc.

According to a preferred embodiment, the solvent used in the extraction operation is an aqueous solution containing no organic solvent.

At the end of the extraction, the extract may be separated from the non-solubilized matter to form an extract stream and a solid stream according to an exemplary embodiment. According to any preferred embodiment or alternative embodiments, the separation may use methods such decantation, centrifugation, gravimetric separation, filtration, membrane filtration and various combination of those. According to a preferred embodiment, the pH of solvent during the separation step is greater than about pi.

The conditions in the extraction step are such that the separated extract contains a maximal fraction of the sugars present originally in the starting material, e.g. at least about 50 percent, more suitably at least about 60 percent, more suitably at least about 70 percent according to suitable embodiments.

The conditions in the extraction step are such that the separated extract contains a maximal fraction of the phytate present originally in the starting material, e.g. at least about 40 percent, more suitably at least about 50 percent, more suitably at least about 60 percent according to suitable embodiments.

With regards to protein co-dissolution/extraction, there are several options. According to an exemplary embodiment, protein extraction may be minimized ~ but without maintaining pH equal to pi during the whole extraction step. According to an alternative embodiment, protein extraction may be permitted -- and may be achieved by adjusting pH, relative solvent amount and other parameters, as described above. According to another alternative embodiment, protein extraction is limited, for example to less than about 70 percent of the original protein content of the starting material, suitably less than about 60 percent, suitably less than about 50 percent. The non-extracted protein is left with the insolubles obtained on separation of the extract according to an exemplary embodiment. Since most of the sugars, other solubles, and a great part of the phytate may be removed with the extract, the insolubles form purified proteinous material, at least with regards to sugars, other solubles and phytate. The concentration of the protein there depends in part on the ratio between proteins and other insolubles of the starting material and, thus, on the fraction of extracted protein. Hence, if high concentration of protein in the product is desired (rather than just low concentration of sugars and phytate), proteins co-extraction may be limited.

The purified proteinous material may be used as an ingredient in food and feed as such, or after further treatments, such as washing and drying according to any preferred or alternative embodiments.

Since a large fraction of the phytate present originally in the starting material may be extracted, but only a limited fraction of the protein, the phytate/protein ratio in the purified proteinous material may be significantly smaller than that in the starting material, suitably less than about 60 percent, preferably less than about 50 percent, more preferably less than about 40 percent. Ratios less than about 30 percent of may also be obtained.

Protein may be recovered from the separated extract, for example by means such as precipitation, membrane filtration (ultrafiltration) and a combination of those according to an exemplary embodiment. Precipitation may use means such as solvent removal (e.g. by distillation), temperature adjustment, and pH adjustment according to alternative embodiments. The latter is preferably done by lowering the pH of the extract by at least about 0.2 pH units. Protein separation by membrane filtration may use ultrafiltration membranes with suitable molecular weight cut-off, e.g. about 30,000 Dalton. According to a preferred embodiment, ultrafiltration is conducted without major modification of the extract pH, e.g. at pH similar to about that in the extract separation from the purified proteinous material. That separated protein, as such or after some more treatment, may form a second product, or may be combined with the purified protein product to form a combined composition, which is then used in the feed or food product (according to alternative embodiments).

Phytate is an ester of inositol with six phosphates (inositol hexa- phosphate). Hydrolysis cleaves phosphates off the inositol backbone. According to an exemplary embodiment, phytate is hydrolyzed in the system, e.g. by enzymatic catalysis. Phytase is a suitable enzyme for that purpose. According to an alternative embodiment, hydrolysis does not need completion in the sense that it is sufficient to hydrolyze only a fraction of the phosphate-inositol ester bonds on a given phytate molecule.

There are multiple ways (or combinations of ways) of using phytate hydrolysis in the phytate reduced feed system and method. According to an exemplary embodiment, phytate hydrolysis is conducted on the separated purified proteinous material prior to further treatments, such as washing and drying, simultaneously with such treatment or after it. According to another alternative embodiment, a mixture of phytase and the purified proteinous material (e.g. after further treatment, if applied) may be incorporated into the feed or food product.

According to another alternative embodiment, hydrolysis may be conducted during the separation or prior to it. There is, for example, the option of combining extraction with hydrolysis. According to a preferred embodiment, multiple-stage extraction is conducted, wherein hydrolysis may be conducted between extraction stages or wherein one stage or more combines extraction and hydrolysis. In a counter-current mode of extraction, hydrolysis can be conducted in the latter stages of the extraction, i.e. where the phytate content of the solid phase is already low. According to other alternative embodiments, partial hydrolysis may be conducted at an early stage of the extraction, or even before extraction starts.

When conducting hydrolysis in a medium containing solids and aqueous solution, as in an extraction stage, both dissolved and non-dissolved phytate may be hydrolyzed. According to an alternative embodiment, hydrolysis may be conducted in a system containing minimal amounts of solids so that mainly dissolved phytate is hydrolyzed. Thus, in a multiple-stage extraction, hydrolysis may be conducted on an aqueous solution separated from a stage before being introduced into the next stage. The same is true for any phytate- containing process stream, particularly in case of reuse in the process. Hydrolysis of dissolved phytate is more rapid than hydrolysis of phytate in a non-dissolved form and is therefore likely less costly. Hydrolysis in a system that is substantially free of solids, simplifies the use of enzymes in an immobilized form, e.g. hydrolyzing of dissolved phytate while the solution containing it moves through a column containing phytase immobilized on a solid support. Such use of immobilized enzymes also reduces the cost of enzymatic hydrolysis.

According to alternative embodiments, phytate hydrolysis may be combined with other hydrolysis reactions, e.g. hydrolysis of oligosaccharides and hydrolysis of peptides. Those other hydrolysis reactions can be conducted substantially simultaneously with phytate hydrolysis or sequentially in any desired order. Hydrolysis with immobilized enzymes in a solution, which is substantially free of solids, is particularly suitable.

According to a preferred embodiment, a low-phytate purified protein product is produced, which is the purified proteinous material obtained after separation of the extract, or is derived from that purified proteinous material. That product may be used as ingredient in low-phytate proteinous feed or food products. In case of co-extracting protein and recovery of that co-extracted protein from the extract, another low-phytate purified protein product is formed according to an alternative embodiment. In that case, the two products can be used separately. According to another alternative embodiment, the two products, or fractions of those, are combined to generate a combined product suitable for use in low-phytate proteinous food or feed products.

According to an alternative embodiment, the separated recovered protein may be further treated (e.g. for phytate removal). The further treatment may include recycling the separated recovered protein to a wash operation. According to a preferred embodiment, the food or feed product includes at least about 70 percent of the protein of the starting material, preferably at least about 80 percent, more preferably at least about 90 percent. According to an alternative embodiment, the food or feed product includes less than about 60 percent of the phytate of the starting material, preferably less than about 50 percent, preferably less than about 40 percent, preferably less than about 30 percent.

According to another preferred embodiment, a protein comprising food or feed product is formed and is characterized in that at least about 20 percent of its protein content (suitably at least about 40 percent, more suitably at least about 70 percent, results from the separated purified protein material.

The low-phytate purified protein produced may be particularly suitable for use in fish feed.

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.