MXPA98010748A - Process for treating plant proteins and nutritional products made therefrom - Google Patents

Abstract

The present invention relates generally to a process of isolating isoflavones/phytoestrogens from a plant protein and producing plant proteins for use in nutritional products that have reduced levels of phytoestrogens, manganese and/or nucleotides. More specifically, this invention is directed to a process of using ion exchange technology to remove phytoestrogens, manganese or nucleotides from plant proteins. Either the removed phytoestrogens or the treated plant protein may be retained for further processing. The phytoestrogens have utility as pharmaceuticals. The treated plant protein has utility in nutritional products. This invention is also directed to isoflavones prepared by the process, to plant protein products resulting from the inventive process, and to nutritional products that use the plant protein product as a source of amino nitrogen.

Description

PROCEDURE FOR TREATING PLANT PROTEINS AND NUTRITIONAL PRODUCTS FACTS OF THE SAME FIELD OF THE INVENTION The present invention relates generally to a process for treating plant proteins for use in nutritional products. The procedure uses ion exchange technology to remove phytoestrogens (also known as "isoflavones"), manganese, nucleotides, nucleosides and RNA from plant proteins, to produce a plant protein product that has reduced levels of phytoestrogens, manganese and acids nucleic The invention is also directed to nutritional products that use the plant protein product as a source of amino-nitrogen. The same procedure can also be used to extract phytoestrogens from plant materials, and to compositions < They contain the plant phytoestrogens isolated.

BACKGROUND Phytoestrogens or plant estrogens occur in a variety of plants, including plant protein materials such as those derived from soy. Phytoestrogens are defined as plant substances that are structurally and functionally similar to the 17-oestradiol gonadal steroid or that produce estrogenic effects. There are three main points of non-steroidal diet estrogens, which are: 1) isoflavones, 2) coumestans and 3) mycostrogens (fungi). The structural similarity between these substances and endogenous mammalian estrogens has been studied. A review of phytoestrogens and their effects on mammals is reported by Kaldas and Hughes in a paper entitled, "Reproductive and General Metabolic Effects of Phytoestrogens in Mammals," Reproductive Toxicology, Vol. 3, p. 81-89, 1989. The teachings in this article are in the public domain and do not need to be repeated here. As used in this specification and the appended claims, the term "isoflavones" is equivalent to the term "phytoestrogens" since that term is defined and used in the art (eg, the article by Kaldas et al.). Flavonoids and isoflavones are produced through numerous legumes and fats, including many plants commonly consumed by humans and livestock. Soy isoflavones include compounds such as daidzin, genistin, daidzein and genistein. A general structural formula for these compounds that is: Compound R R1 daidzein H H genistein H OH daidzin G H qenistin G OH where G = glucosyl It has recently been recognized that isoflavones contained in plant proteins can have a damaging impact on mammals consuming plant protein, see, Kaldas et al., Supra. The concentration of isoflavones in isolates or concentrates of plant proteins, such as isolates of soy protein, can be as high as 3,000 μg / g of protein. Isoflavones also provide bitter or "bean-like" flavor to vegetable proteins (see, Ewan et al., Infra) can reduce the bioavailability of essential minerals and may influence the nutritional value of proteins (see, Kaldas et al., supra). The consumption of isoflavones by man and cattle has also been related to reproductive systems compromised in mammals. There is some concern that the consumption of soy-based infant formulas containing soy isoflavones can have an undesirable physiological impact on the development of the baby's neuro-endocrine system. This concern is based in part on the evidence that soy-based animal feed can cause problems of effectiveness in a leopard species ("chita"). Setchell et al., 1987: "Gastroenterology" 93: 225-33. In contrast, it has also been suggested that isoflavones may have positive effects of a pharmaceutical nature, in some cases. Estrogens have two opposite effects on cancer, depending on the dose. Large doses inhibit breast cancer tumor development, while small doses seem to promote tumor growth. This duality extends to phytoestrogens or isoflavones. Isoflavones can stimulate or inhibit tumor growth. Setchell KDR, and Welch, MB J. Chrom. 386 (1987) 315-323: "Naturally Ocurring Non-Steroidal Estrogens of Dietary Origin". In McLachlan J.A., ed., "Estrogens in the Environment", New York: Elsevier Press; 1985: 69-85 and Setchell, al., "Nonsteroidal Estrogens of Dietary Origin: Possible Roles in Hormone-Dependent Diasease", Am. J. Clin. Nutr. 1984; 40: 569-578. One mechanism through which isoflavones can manifest their anti-tumor effect is the blocking of estrogen receptors and the separation of the response mediated by the receptor. In this way, the ability of endogenous estrogens to support tumor growth can be reduced. There is also indirect, demographic support for a isoflavone-mediated reduction in cancer of hormone-responsive tissues from the observation that women in countries that consume vegetarian diets have a lower incidence of breast cancer compared to countries that consume meat. . Adlercreutrz et al., "Determination of Urinary Lignans and Phytoestrogen Metabolites, Potential Antiestrogens and Anticarcinogens, in Uriñe of Women on Various Habitual Diets", Steroid Biochem. 1986; 25: 791-797. Isoflavones may also have anti-viral and fungal properties. Isofiavones have also been applied in the reduction of serum cholesterol levels in humans, positive immunological effects and activity as an antioxidant. A beneficial final isoflavone effect is the relief of vasomotor symptoms in menopausal women. Historically, the Chinese have used herbal medicine to treat "hot flashes." In this way, a procedure that easily isolates and concentrates isoflavones from the plant material can be of great value to the scientific community and the pharmaceutical industry. The presence of high levels of manganese in body tissues has been suspected in the development of criminal behavior. See, Gottschalk et al., "Abnormalities in Hair Trace Elements as Indicators of Aberrant Behaivor", Compr. Psychiatry 1991; 32: 229-237, and Scientific America, March 1995, p. 104-105. In addition, it has also been reported that the learning disability in children may be associated with increased levels of manganese in the hair as reported by Collipp et al., In an article entitled, "Manganese in Infant Formula and Learning Disabilities", Ann. Nutritional Metals, 27: 488-494, 1983. Isolates of typical plant proteins contain up to 1000 μg of manganese per gram of protein. In this way, there is a need for procedures that economically and on a commercial scale provide for the reduction of isoflavones and manganese content in the plant protein. The use of nucleotides and nucleosides (or nucleotide equivalents as defined below) in nutritional formulas has received much attention in recent years. It has been suggested that certain levels and ratios of the various nucleic acids can have a positive impact on the immune system of mammals and still prevent certain ailments, such as diarrhea. The problem with using the plant protein in such nutritional formulas is that the plant protein typically contains a very high inherent level of nucleic acids that may not be the most useful form (eg, as RNA) and can not be in the relationships more desirable. In addition, the high level of variation in nucleic acid content causes problems in commercial manufacturing. Typical plant protein isolates contain up to about 15 mg of nucleotide equivalents per gram of protein. In this way, the nutritional industry desires a source of plant protein that has substantially reduced levels of inherent nucleic acids. Ion exchange technology has been known for many years. Ion exchange resins are typically synthetic, insoluble, entangled polymers, which carry acidic or basic side groups. They have high exchange capacities and can be used for an almost unlimited number of reactions.

The ion exchange resins are used in water treatment, extraction, separation, analysis and catalysis. The ion exchange resins have an open, extended molecular structure that includes electrically charged ionic groups. A cation exchanger exchanges positive ions and, therefore, has negative ions developed in its structure. An anion exchanger has positive ions in its structure. The ions of the crystal structure network are referred to as the fixed ions; the smaller ions of opposite charge that can change sites with ions in the solution are called counterions. Common problems encountered with protein-ion exchange procedures include poor protein recovery (ie, protein adhered to the resin) and the inability of the protein slurry to pass through the resin bed resulting in high protein pressure drop through the resin bed. The process, which is described herein, satisfies the need in the nutritional industry for an economically and commercially viable process, which clearly separates isoflavones from the plant protein, leaving an economical source of isoflavones as well as a high production of a plant protein product that has highly reduced levels of soflavones, manganese and nucleotides. The patent of E.U.A. No. 5,352,384 to Shen discloses a process for producing a vegetable protein fiber enriched with isoflavones. This patent describes the use of a glucosidase to convert the glucone isoflavones (ie, daidzena) into a protein slurry to the aglucone isoflavones. The fiber fraction is then recovered from the slurry through centrifugal action to provide a fiber rich in aglucone. An article written by Ewan et al., In Journal of Food Science, Vol. 57, No. 2, 1992, entitled "Isoflavone Aglucones and Volatile Organic Compounds in Soybeans; Effects of Soaking Treatments", describes the beneficial effects of soy soaking. in moderately alkaline NaHCO3 solutions at elevated temperatures, to make soy milk with improved flavor. This publication does not suggest my describes the use of an ion exchange resin to remove isoflavones, manganese and nucleic acids from the plant protein. In an article published in volume 47 (1982) of the Journal Of Food Science, p. 933-940, by J. How and C. Morr, entitled "Removal of Phenolic Compounds from Soy Protein Extracts Using Activated Charcoal", reports submitting strains of soy protein to activated carbon and ion exchange procedure treatments to remove compounds phenolics that have been reported as possible responsible for the adverse color and flavor characteristics of soy protein products. The protein strata were subjected to a two-step sequential ion exchange treatment prior to the precipitation of the protein. The protein extract was pumped as "downflow" through a cation exchange column in the Na + form and then an anion exchanger in the form of hydroxyl and chloride to remove the polyvalent anions including phenolic acids, phytate and other . 5 The patent of E.U.A. No. 5,248,804 to Nardelli et al., Describes a process for the removal of phytate from plant protein using ion exchange resins. The procedure uses a macroporous anion exchange resin (base of weak or strong base), which has been conditioned through, 1) the conversion to the hydroxide form; 2) the conversion to the chloride or sulfate form; and 3) subsequently the conversion of the strong base sites to the carbonate form and the weak base sites to the free base form. The plant protein containing phytate is then contacted with the treated resin to remove the phytate. 15 Phytate comprises salts of phytic acid. Phytic acid is also known as inositol hexaphosphate. In this way, by using an aircraft exchange resin, the highly anionic phosphate groups provide the handling through which the resin can extract the phytate from the protein slurry. In In contrast, the isoflavones and nucleotides are neutral molecules and can not be expected to bind to the resin or exchange with the anions in the resin. The patent of E.U.A. No. 5,492,899 to Masor et al., Describes a formula for infants with ribonucleotides. This patent teaches the use of certain levels and ratios of nucleotide equivalents in formulas for infants and describes an ethnic analytic to identify and quantify the nucleotide equivalents in a nutritional matrix. As used herein and in the claims of this invention, the term "nucleotide" is the same as the term "nucleotide equivalent", as defined in the US patent. 5,492,899. The patent of E.U.A. 5,492,899 defines nucleotide equivalents such as polymeric RNA, ribo-nucleosides, ribo-nucleosides containing adducts and mono-, di- and triphosphate ribonucleotides.

COMPENDIUM OF THE INVENTION The present invention comprises a process by which plant proteins with a low content of isoflavones, with a low manganese content and / or with a low nucleotide content can be manufactured. The invention further comprises the same protein isolates with a low content of isoflavones, with a low content of manganese and with a low content of nucleotides, and said protein isolates are produced according to the process of the present invention. The present invention further comprises nutritional products made with the protein isolates produced according to the invention. An additional benefit of the process of this invention is that, not only isoflavones and manganese can be removed through the ion exchange column, but also a substantial portion of the inherent nucleic acids. A further aspect of the invention is that the same isoflavones can be isolated through further treatment of the exchange resin with an isoflavone releasing agent. These, and other aspects of the invention are specifically described in detail in the description set forth below. Thus, in a first broad aspect, the invention provides a method for treating a plant protein comprising: a) providing a slurry of plant protein containing isoflavones, manganese or nucleotides; b) providing at least one anion exchange resin capable of binding isoflavones, manganese or nucleotides present in said protein slurry; c) contacting said slurry with the anion exchange resin; and d) separating said slurry from the anion exchange resin, whereby the plant protein slurry has a reduced content of isoflavones, manganese or nucleotides. In a preferred embodiment, the exchange resin is preconditioned by exposing the resin prior to contacting step c) to an agent that places on the resin an exchangeable anion that: i) does not change the pH of the protein slurry in contact outside from the scale of 6.0 to 9.5; and ii) does not add an objectionable anion to the contact protein slurry in step d). More specifically, a resin having strong base sites and weak base sites can be preconditioned by subjecting it to the steps of: i) conversion to a hydroxide form; ii) conversion to a chloride or sulfate form; and iii) conversion of at least some of the strong base sites to the carbonate form and at least some of the weak base sites to the free base form. In this way, the agents useful for this pre-conditioning include sodium hydroxide and potassium hydroxide for step i); hydrochloric acid, sulfuric acid and sodium chloride for step ii); and sodium carbonate, and sodium carbonate and ammonium hydroxide for step ii). The step of contacting may comprise placing the anion exchange resin in the slurry; or, alternatively, passing the slurry through a structure (such as a vertical column), which contains the anion exchange resin and has at least one inlet and one outlet. Preferably, the entrance is located lower than the exit in said vertical column. The process may optionally comprise the additional steps of reconditioning the exchange resin using steps similar to those described above, and reusing the resin for further separation. Other optional steps include heat treatments of the protein and / or hydrolysis of the protein before contact with the exchange resin. Another optional additional step involves treating the exchange resin with at least one isoflavone releasing agent; and separating the isofiavone releasing agent containing isoflavones from said anion exchange resin and collecting the isoflavones. Representative isoflavone release agents include, for example, aqueous solutions of acids (e.g., HCl), aqueous solutions of bases (e.g., NaOH or KOH), alcohols (e.g., methanol or ethanol), mixtures of alcohol / water, solvent is that organic and mixtures thereof. The present invention also relates to a plant protein composition having a reduced content of at least one component of isoflavones, manganese or nucleotide selected from the group consisting of: (i) less than 30 μg of isoflavones per gram of protein; (ii) less than 450 μg of manganese per gram of protein; Y (iii) less than 10 mg of nucleotides per gram of protein. Said plant protein can be made, but not necessarily, through the method of the present, since until now there has been no extract of plant protein having phytoestrogen levels as low as those of the present invention. Most preferably, the plant protein composition comprises less than 20 μg of isoflavones per gram of protein; and less than 400 μg of manganese per gram of protein. Also described are cattle feeds, infant formulas, and nutritional products utilizing the plant protein according to the invention. Infant formulas, for example, contain less than 600 μg of isoflavones per liter of ready-to-eat formula, most preferably less than 200 μg, and preferably less than 100 μg. Finally, the invention relates to compositions containing isolated isoflavones according to the method of the present invention.

DETAILED DESCRIPTION Typically, the process of this invention is conducted by placing the anion exchange resin in a bed, column or reactor through which the protein slurry is passed. The bed, column or reactor has at least one inlet and at least one outlet, and preferably operates as a vertical column in the "upflow" mode. In another embodiment, the preconditioned resin can be added to a tank containing the protein slurry and after appropriate period for the reaction to occur, the resin containing the entrapped isoflavones is filtered from the slurry. The anion exchange resin is typically a macroporous resin and is preferably a macroporous type I or II resin. In a preferred embodiment, the anion exchange resin is selected from weak base anion exchange resins, strong base anion exchange resins and mixtures thereof. Representative examples of the anion exchange resins in the present invention include Amberlite® RA95, IRA-910, and IRA-900 sold by Rohm and Haas Company, Dowex ™ -22 and MSA ™ -1 sold by Dow Chemical and Purolite ™. A510 and A500 sold by Purolite Company. As used herein and in the claims, the term "resin" means that it includes gels, which those skilled in the art could understand to be useful in the process described herein. Representative examples of such gels are Amberlite® IRA 410 (Type II gel, strong base anion) sold by Rohm and Haas Comany, IRA 402 is a Type I strong base anion exchange gel that is not macroporous that could be useful. Representative counterions useful in the anion exchange resin according to this invention include acetate, citrate, chloride, bisulfate, carbonate and bicarbonate. Since most of the anion exchange resins are supplied in the chloride form, it is possible to use said chloride resins directly without pre-treatment. As discussed below, a preferred process for resin pre-treatment washes the chloride resin with a caustic substance to clean the resin, then a wash is conducted with HCl to clean and control the growth of microbes and then the resin is converted to the carbonate and / or bicarbonate form. In the production of plant protein using the process according to this invention, the anion that is released from the resin as a result of the entrapment of the isoflavones, manganese or nucleotides is important for the quality of the finished product. That is, the resulting protein must not be denatured, must not contain unacceptable levels of free hydroxyl groups or other offensive anions (ie, chloride) that can produce a protein product that is unacceptable for use in a nutritional product. For example, the typical soy protein isolate contains sufficient levels of soflavones, manganese and nucleotides, that treatment with an anion exchange resin having chloride as the counter ion, can produce a resulting protein that has excessive levels of chloride. In a similar way, true against ion is hydroxyl, the resulting product may need to be treated with acid to neutralize the basic product, thereby unacceptably increasing the mineral charge associated with the protein. In a preferred embodiment of this invention, the anion exchange resin utilizes a counterion, such as carbonate with bicarbonate, which avoids the aforementioned problems. As used in the specification and the appended claims, the term "carbonate" means carbonate and bicarbonate. The proteins that can be used in the method of this invention include any plant protein that contains detectable levels of isoflavones, manganese and nucleotides. More specifically, the protein can be obtained from soybeans, corn, wheat, peas, beans, cottonseed, peanuts, carrots, alfalfa, apples, barley, silky herbs, cloves, coffee, garlic, hops, marijuana, oats, algae, orchard grass, parsley, rice, rye, sage, ajinjolí, yeast, mushrooms, potatoes, hydrolyzates thereof and mixtures thereof. It is preferred that the protein be a concentrate or protein isolate, where the levels of fats, oils and carbohydrates have been reduced. It has been determined that the presence of fats and oils reduces the efficiency of the process of the invention. An intact protein is not required in the final product, the method of the invention can be used in protein hydrolysates and / or isolates, too. Chemical agents useful for converting the resin to the form that hydroxide include sodium hydroxide, calcium hydroxide, potassium hydroxide, and magnesium hydroxide. The most preferred agent is sodium hydroxyl. Chemical agents useful for converting the resin to the chloride or sulfate form include hydrochloric acid, sulfuric acid, and sodium chloride. The preferred agent is hydrochloric acid. Useful chemical agents for converting the resin to the carbonate or free base form include any of the weak base salts such as sodium carbonate, sodium bicarbonate and ammonium hydroxide. Sodium bicarbonate is the most preferred agent for protein recovery because it produces a protein effluent at a pH scale of 6.6-9.5. For the recovery of isoflavones, this is not crucial. Those skilled in the art of ion exchange technology will appreciate that the protein slurry containing the isoflavones, manganese or nucleotides, although in contact with the anion exchange resin, must be at a pH that does not denature the protein, which can cause the filling of the column. In addition, adjusting the pH beyond neutral will add significant levels of anions to the slurry, which will compete for counterion sites. Typically, a pH of about 5.5 to 10 is satisfactory. Preferably, the pH of the protein slurry feed may vary from 6.0 to 8.0. The pH of the protein slurry effluent (leaving the column or bed) should be close to the pH at which the protein will be used in a final product. Thus, if a plant protein treated in accordance with this invention is to be used in an infant formula, the pH of the effluent should be from about 6.0 to 7.5. In a preferred embodiment, the plant protein feed to the resin should be as free as possible from added anions (ie, -OH, -Cl, and the like). The addition of acids, bases, salts and the like to the feed of protein slurry reduces the efficiency of the column to remove isoflavones, manganese or nucleotides from the protein slurry. As those skilled in the art will appreciate, the exchange resins have a limited capacity and can be regenerated to an active state after exhaustion or near depletion. In this way, as contemplated in this invention, the exchange resins after making contact with the plant protein are regenerated or reconditioned through known steps to the anionic form or very preferably through the steps comprising: 1) separating the resin from any residue (ie, protein) and convert to the hydroxide form, for example, through the use of sodium hydroxide; 2) convert the resin to the chloride or sulphate form; and 3) convert the strong base sites on the resin to the carbonate form and convert the weak base sites to the free base form. Those skilled in the ion exchange resin art will appreciate that non-aqueous and alcohol-water regenerations can be used. A preferred embodiment of the process according to the present invention includes the step of homogenizing the plant protein slurry before contacting the resin. It has been found that homogenization or similar treatments thereto in the process of this invention increases the effective removal of isoflavones, manganese and nucleotides from the slurry. In addition, the homogenization of the protein slurry before contact with the resin reduces the pressure drop across the bed or column of the resin, which facilitates the easy and economical production of a plant protein to be used in nutritional products. . In some embodiments, the objective is to recover the isoflavones or phytoestrogen compounds separated from the plant protein material. In this case, the resin is treated with an isoflavone release agent, which causes the isoflavones to elute from the resin. Representative isoflavone release agents that are useful in the present invention include alcohols such as ethanol, methanol, propanol, pentanol and the like; organic solvents such as heptane, decane, cyclohexane, benzene, toluene and the like; alkaline solutions based on water such as NaOH, KOH, and ammonium hydroxide; water-based acid solutions such as HCl, and the like. In general, the isoflavone release agent must separate the isoflavones from the resin and solubilize the isoflavones. Those skilled in the art can readily determine the appropriate isoflavone release agents without undue experimentation. The present invention is also directed to a plant protein isolate having specific levels of isoflavones and to a plant protein that has been subjected to the process described herein and to nutritional products that are made from said proteins. Feeds for animals that are substantially free of isoflavones are also contemplated here. More specifically, the present invention relates to a plant protein containing less than about 30 μg of isoflavones per gram of protein, less than about 450 μg of manganese and less than about 10 μg of nucleotide equivalents per gram of protein. In a highly preferred embodiment, the protein is derived from soy and contains less than 20 μg of isoflavones per gram of protein. In a highly preferred embodiment, the plant protein contains less than 10 μg of isofiavones per gram of protein, less than 5 mg of nucleotides per gram of protein and less than 200 μg of manganese per gram of protein. The following examples describe specific, but not limiting, modalities of the present invention. The aspects of the present invention, which are believed to be novel, are set forth with particularity in the appended claims and should be understood as a structure and form of operation through the following detailed examples.

EXAMPLE 1 Procedure for Treating Plant Protein The process according to the present invention was used to isolate isoflavones and to produce a total of 221 kg of a soybean isolate powder with a low content of isoflavones, a low content of manganese and a low nucleotide content, which was used in the manufacture of a formula for infants. A total of six (6) manufacturing operations were required to produce the required soy protein isolate. A substantial amount of experimentation was conducted on a 50 liter scale to result in the best mode, described herein. The soy protein starting material used in this example was obtained from Archer Daniels Midland, Inc. (ADM) of Decatur, IL, in curdled form. The curd or milk protein was from the commercially available soy protein isolate product known as Ardex F®. In a typical commercial process, soy proteins were extracted at a slightly alkaline pH of defatted soy flakes or defatted soybean meal. The protein fraction was then precipitated from the extract by adjusting the pH to the isoelectric point of the proteins (pH 3.8 to 6.0). Since most proteins are insoluble at this pH, a curd is formed and the protein curd can be separated from soluble sugars, salts, etc., through centrifugation. To complete the purification, the protein curd is washed with water at least once at this isoelectric pH, then the protein is spray dried either as such or after resuspending at a neutral pH. In the following experiments, ADM supplied the isoelectric curd at a total solids content of 10 to 14% and at a pH of about 4.5. The supplied soybean curd was diluted to a total solids content of approximately 6.5% with water and placed in steam jacketed containers. Each batch of the protein slurry in water weighed approximately 908 kg. The slurry was then heated to about 49 ° C and neutralized to a pH of 6.8 with NaOH. The slurry was then filtered through a 60 mesh sieve, processed and homogenized to UHTST (short time at ultra high temperature). Steam injection to UHTST was 152 ° C and was maintained for 10 seconds. It was determined that the anion exchange exposure after treatment to UHTST produces a protein with undesired organoleptic properties. The slurry was then cooled to 55 ° C and homogenized at 6895 kPa. The slurry was then transferred to the ion exchange system. One aspect of the present invention resides in the discovery that ultra-high temperature short time (UHTST) treatments need to be conducted prior to contacting the slurry with the resin to avoid deterioration of the slurry during extended processing times. The process is conducted at temperatures where rapid microbiological growth can occur. Representative of the UHTST conditions useful in the present invention are temperatures of 120 ° C to 155 ° C and times of 1 to 60 seconds. Lower temperatures are associated with longer maintenance times. This treatment at UHTST, before contacting the slurry with the resin, provides microbiological stability, while minimizing the degradation of nutrients. The ion exchange system comprised a column lined with rubber, stainless steel having inlet and outlet ports and a height of 401 cm and a diameter of 30.5 cm. 70 liters of the anion exchange resin Amberlite® IRA-910 from Rohm and Haas Co. of Philadelphia, Pennsylvania were placed in the column. IRA-910 is a strongly basic macroreticular anion exchange resin. The basicity of this resin is derived from the functionality of quaternary ammonium with a slightly lower basic strength than an anion exchange resin of type 1. This resin is supplied in the chloride form and is approved by the United States Food and Drug Administration (FDA) (after the condition cyclization) to be used in the processing of edible products. Before making contact with the protein curd, the resin was preconditioned. The resin was preconditioned through contact in an upflow mode with 6 weight% NaOH at a flow rate of 4.6 to 5.7 liters per minute for 30 minutes. The resin bed was then washed with deionized water for 10 to 15 minutes in the upflow mode. The resin was then contacted with 1.0% by weight of HCl in a downflow mode at 16 liters per minute. The resin was then washed with deionized water in the downflow mode for about 30 minutes. 2.8 kg of sodium bicarbonate was added to approximately 196 liters of water and stirred to dissolve. This solution was then pumped into the column in a downflow mode at approximately 4 liters per minute. The bed was again rinsed with deionized water until the conductivity of the effluent was 300 μmhos or less. The resin bed was then washed countercurrent to remove the air and reclassify the resin. The resin bed was allowed to settle naturally and water was drained from the column. The column is now ready for the service cycle after draining the water towards the top of the resin bed. The protein slurry was pumped as an upflow through the ion exchange column at a flow rate of 3.6 to 3.8 kg per minute. The inlet temperature of the slurry was 55-60 ° C and the contact time was at least 20 minutes. The protein slurry leaving the column was cooled, samples were taken and then spray-dried using conventional techniques and equipment. The column before the next batch was regenerated with 6% NaOH, 1% HCl and 1.5% NaHCO3 (sodium bicarbonate), as described above for the initial preparation of the resin bed. All solutions were prepared with deionized water.

RESULTS A total of six batches were manufactured to produce a total of approximately 221 kg of soybean meal powder with ion exchange. Three (3) samples were taken at various times during the processing of each batch: 1) the protein slurry fed to the ion exchange column; 2) effluent from the column; and 3) dry powder. The samples were analyzed for mineral profiles of sodium, potassium, phosphorus, chlorine, calcium, magnesium, manganese, aluminum and fluorine. The samples were also analyzed for isoflavones and nucleotides. In order to make comparisons between liquids and possible powders, the dust concentration was normalized to 6.5% as a total solids content. The mean levels and normal deviations for each analyte before and after the ion exchange were calculated for the six operations. The results presented in Table I. Note that the reduction is expressed as a positive value, while a negative value represents an increase in the analyte concentration.

TABLE I Profile Mineral Mineral% Phosphorus Reduction 73.3 ± 3.4 Calcium 16.5 + 4.2 Magnesium 11.4 ± 5.1 Sodium -6.3 ± 3.9 Potassium -7 ± 21 Manganese 31 ± 10 Aluminum 6 ± 15 Chloride -270 ± 110 Fluorine 48 ± 29 The most significant reduction in the concentration of the ion exchange treatment was observed in the total phosphorus, fluorine and manganese. The reduction in phosphorus is consistent with the teachings of the U.S. patent. 5,248,804, since a large portion of the phosphorus inherent in soy exists as a phytate salt. In contrast, the effluent showed a significant increase in chloride. This is consistent with the fact that HCl is one of the regenerators used after rinsing with a caustic solution and the strong base resin has some weak base sites.

The profiles of calcium, magnesium, manganese, fluorine and aluminum before and after the treatment showed a reduction. Of this group, manganese showed a significant reduction (31 ± 10%). Surprisingly, when compared to other multivalent metals, aluminum (+3 charge) remained essentially unchanged. In addition, the removal of calcium and magnesium can be explained as adsorption or chelation with phytate. Those monovalent actions, sodium and potassium, were relatively unaffected by the ion exchange treatment (-6.3 ± 3.9% and -7 ± 21%, respectively). The negative values actually indicate a slight consumption of both sodium and potassium. These data support the typical behavior of the anion exchange resin since monovalent cations can not be exchanged or adsorbed by the anionic resin. An important benefit of the process of the present invention is that high levels of protein are recovered from the treated plant protein isolates. This means that very little protein is lost in the column per bed of resin. In these experiments, about 90 percent of the protein entering the resin column was recovered in the effluent. It is important to note that the overall efficiency of the process of this invention is improved when the solubility and homogeneity of the protein slurry is improved. In this way, pre-filtration (through a 60-mesh filter) and homogenization greatly reduce the pressure drop across the column, which increased the efficiency of the process of the invention. In comparison, the process without pre-filtration and homogenization resulted in an initial pressure drop of approximately 138 kPa, while pre-filtration and homogenization resulted in an initial pressure drop of approximately 14 to 35 kPa. After approximately 4 to 6 hours of operation without pre-filtration and homogenization, pressure drops of 276 to 414 kPa were experienced, while with pre-filtration and homogenization, the pressure drops were approximately 55 to 83 kPa. The method of this invention was also very effective in removing nucleotides. The analytical procedures used are as described in the patent of E.U.A. 5,492,899 to Masor et al. The total removal of potentially available nucleotides (TPAN) was found to be approximately 57.4 ± 7.2%. The isoflavones were almost completely removed through the process of the present invention. Table II establishes the specific isoflavones, the level of the feed slurry, the level in the effluent and the level in the powder.

TABLE II % Reduction ISOFLAVONES INCREASING EFFLUENT POWDER AUMENT. FOOD μgte * μg * μg / g VS. POWDER VS. EFFLUENT Daidzin 4.12 + 0.8 0.51 ± 0.21 0.68 ± 0.37 83.5 ± 6.8 87.6 Genistina 10.0 ± 2.8 0.82 ± 0.55 0.87 ± 0.72 91.4 ± 5.9 91.8 Daidsein 3.0 ± 6.5 0.10 ± 0.0 0.10 ± 0.0 97.4 ± 4.3 97.4 Genistein 3.7 ± 1.4 0.10 ± 0.0 0.10 ± 0.0 97.3 ± 1.4 97.3 EXAMPLE II Isolation of Isoflavones - Washing with Alcohol In this experiment, a soy curd isoflavone was isolated using the ion exchange resin previously described and then rinsing or releasing the isoflavones from the resin with an alcohol / water solution. In a laboratory column, Amberlite® IRA-910 (80 liters) was pre-conditioned as described in Example I and the soy curd, as described in Example I, was passed over the column until the levels of phosphorus in the effluent approached the rupture (ie, feed levels). The column was rinsed with warm water (49 ° C) to remove the trapped protein and then with cold water (19 ° C). Then, a solution is pumped 50% by weight of ethanol and water through the column to approximately four liters per minute in the downflow mode. The alcohol / water solution was recirculated through the column for about one hour (3 volumes the bed). During recirculation, the alcohol content was diluted to approximately 5% through mixing with water in the column. Approximately 100 liters of the solution rich in isoflavones were recovered. The analytical procedure for the analysis of isoflavones is established in Example IV. The soy protein feed contained 2.7% by weight of daidzin and 0.6% of genistin. The column solution contained 850 μg / L of daidzin and 380 μg / L of genistin. The extraction can be improved by soaking the resin in the isoflavone release agent (i.e., an alcohol solution) and increasing the alcohol percentage to about 80% by weight. Increases in the production of isoflavones can also be observed when the isoflavone release agent is heated to approximately 49 ° C.

EXAMPLE III Insulation of Isoflavones - Regeneration In this experiment, the content of the regeneration effluent was analyzed. The procedure of Example I was followed, except that during the regeneration of the resin in the NaOH treatment, 240 grams of sample was removed. The analysis of this sample for isoflavones is established in Table III.

TABLE III Isoflavones of Regeneration Daidzin Genistin Daidzein Genistein μg / g 2.6 1.2 < 0.1 < 0.1 nanomoles / g 6.2 2.8 < 0.2 < 0.2 As the data of Examples II and III indicate, the method according to this invention provides effective economic means for the isolation and concentration of isoflavone compounds.

EXAMPLE IV Nutritional Product Using Soy Protein with a Low Isoflavone Content The soy protein produced in Example I was used to produce an infant formula. Then a control product and infant formula according to this invention were analyzed for the isoflavone content. The procedure used to produce the experimental and control products was that described in the patent of E.U.A. 5,021,245 of Borsche et al., Except that the fiber was omitted. Typically, infants formulas based on plant protein contain 1.5 to 2.0% by weight of protein as a food (ready-to-eat or "RTF"). A preferred embodiment is 1.6 to 1.8% by weight of protein as a feed. Thus, as described below, an infant formula made with a plant protein treated in accordance with this invention will generally have an isoflavone content of less than 600 μg / liter of formula. (30 μg of isoflavones per gram of protein x 20 grams of protein per liter of formula (RTF) = 600 μg of soflavones per liter of the RTF formula). An HPLC (high pressure liquid chromatography) method, as described below, was used to quantify the main soy soflavones (genistin, daidzin, genistein, and daidzein) using a method adapted from the following three articles (3) , whose teachings are known in the art. 1) Setchell, KDR and Welch, MB J. Chrom. 386 (1987) 315-323 2) Wang, G., Kuian, SS, Francis, OJ, Ware, GM and Corman, AS J. Agrie. Food Chem. 38 (1990) 185-190 3) Barnes, S., Kirk M., and Coward, L. J. Agrie. Food Chem. 42 (1994) 2466-2474. Samples of the formula for experimental infants and ready-to-eat control were obtained and 20 ml of each were loaded with a tared 250 ml round bottom flask. Then 80 ml of ethyl alcohol was added and the mixture was stirred. A condenser was attached to the flask and the samples were refluxed at 80 ° C for two hours. The mixtures were then cooled to room temperature and transferred quantitatively to a 100 ml volumetric flask. The precipitate and the flask were rinsed with 15 ml of 80% alcohol (v / v). The volumetric flasks were brought to a volume with 80% alcohol and the samples were then mixed thoroughly. The samples were filtered through Whatman ™ No. 41 paper and then 15 ml of each filtrate was placed in a 15 ml conical graduated capped glass test tube. Each tube was placed in a hot water bath and a stream of nitrogen for the vapor was used to each sample to 3 ml. Then, the tubes were cooled to room temperature and 1 ml of methanol was added to each tube and then diluted to 10 ml with water and mixed well. Then, 1.5 ml of each sample was filtered through a 0.45 μm polypropylene membrane into an auto HPLC vial. Test analysis for soflavones using reverse phase HPLC was conducted with the HPLC system as follows: Vydac ™ C18 Pharmaceutical column, 250x4.6 mm, 5 μm Detection UV absorbance at 254/280 nm Injection 50 mcL Ambient temperature Flow rate 0.8 ml / min Operating time 120 minutes Eluent A 950 volumes of water; 50 volumes of CH3CN; 1 volume of trifluoroacetic acid Eluent B 400 volumes of water; 600 volumes of CH3CN; 1 volume of TFA Gradient Program: Time (minutes) 0 5 95 100 102 120% Eluent B 0 0 60 100 100 0 The results, set forth in Table IV, indicate that the method of the invention can be used to produce a nutritional product having greatly reduced levels of isoflavones.

TABLE IV Soy isoflavones Control μg / g Experimental μg / g * Daidzin 11.6 < 1.0 Daidzein 1.0 < 1.0 Genistina 19.4 < 1.0 Genistein 2.2 < 1.0 TOTAL 34 \ 2 < 1.0 N / A * a detection limit.

EXAMPLE V Tolerance Study At the time of submitting this application, a clinical study of the physiological effects of estrogens or isoflavones on plants in infant formula made in accordance with this invention was performed. Before this extensive study, a smaller tolerance study was conducted to determine the total tolerance of soy formulas with a reduced content of soflavones in healthy babies. The tolerance study was a three-week, double-masked, randomized study using 145 healthy babies with an age of two to five weeks. The babies were fed a formula based on normal milk for a week-long baseline period, and then fed a normal soy formula for two weeks, a formula based on soy isolate that fitated with a low isoflavone content, a formula based on hydrolyzed soy isolate with a reduced content of phytate and isoflavones or a formula using the protein produced in Example I. The main output variables were characteristics of evacuation, formula consumption and incidence of shedding and vomit. The secondary variables were weight gains and parenteral responses to the feeding tolerance questionnaire. The formula intake and the incidence of throwing and vomiting did not differ between the groups at the baseline or during the study period. The consistency of the average evacuation range was milder for babies fed the hydrolyzed formula compared to the other experiments. The parents associated watery and more frequent stools with the hydrolyzed formula. Babies fed the formula using the protein produced in Example I had less constipation than the baseline. The mean weight gains were similar for all the study groups. The conclusion of this study was that the removal of phytate and / or isoflavones from the soy-based formula had a minimal impact on tolerance.

INDUSTRIAL APPLICABILITY The process described in this invention is a very effective, inexpensive and reliable method for the commercial removal of isoflavones, manganese and nucleotides from plant proteins. The process produces a plant protein product having highly desirable characteristics such as an isoflavone content of less than 30 μg / g of protein, less than 450 μg of manganese per gram of protein and less than 10 mg of nucleotides per gram of protein . The protein resulting from the treatment with the process described herein also has a better taste (less "bean type" favor), an improved color (lighter) and improved functionality (i.e., ability to form a stable emulsion). In addition, the isoflavones recovered from the regeneration / alcohol release process after contacting the resin are valuable as potential cancer compounds. Commercial scale use of the process of this invention is improved when the protein slurry is pre-filtered and homogenized before contacting the resin bed. Macroporous resins in the bicarbonate form are most preferably used. As a result of the present inventor's advance to the state of the art, the nutritional industry will now be able to economically produce products containing reduced levels of isoflavones, manganese and nucleotides. Finally, humans and animals that consume the products produced according to this invention will benefit from abstinence from deleterious elements contained in the plant proteins. Since certain representative embodiments and details have been presented for the purpose of illustrating the invention, it will be apparent to those skilled in the art that various changes and modifications may be made therein without departing from the spirit or scope of the invention, as established in the appended claims.

Claims (24)

1. - A method for treating a plant protein, comprising: a) providing a slurry of the plant protein containing isoflavones, manganese or nucleotides; b) providing at least one anion exchange resin capable of binding isoflavones, manganese or nucleotides present in said protein slurry; c) contacting the slurry with said anion exchange resin; and d) separates the slurry from the anion exchange resin, whereby the plant protein slurry has a reduced content of isoflavones, manganese with nucleotides.

2. A method according to claim 1, characterized in that it further comprises the steps of exposing the resin before the contact of step c) to an agent that places on the resin an exchangeable anion that: i) does not change the pH of the Protein grouting in contact outside the range of 6.0 to 9.5; and ii) do not add an objectionable anion to the contact protein slurry in step d).

3. The process according to claim 1, wherein the step of providing at least one anion exchange resin comprises providing an anion exchange resin containing strong base sites and weak base sites, and having been subjected to the steps of: i) converting to a hydroxide form; ii) convert to a chloride or sulfate form; and iii) converting at least some of the strong base sites to the carbonate form and at least some of the weak base sites to the free base form.

4. A process according to one of claims 1 to 3, characterized in that it further comprises the steps of: e) after step d), contacting said anion exchange resin, at least one release agent isoflavones; and f) separating the softening agent containing isoflavones from said anion exchange resin and collecting the soflavones.

5. A method according to one of claims 1 to 3, characterized in that it comprises the steps of: e) after determining step d), re-conditioning the resin: i) exposing the resin to an agent, which separates the resin surface of the residue and convert the resin to a hydroxide form; ii) then exposing the resin to an agent, which converts the resin to either a chloride form or a sulfate form; and iii) then exposing the resin to an agent, which converts at least some of the resin to a carbonate form; and f) bringing the additional plant protein slurry into contact with the re-aconclicioned resin; g) Separate the additional slurry from the resin.

6. A process according to any of claims 1 to 5, characterized in that it further comprises the homogenization of the slurry of the protein that it plants before contacting the slurry with the anion exchange resin.

7. The process according to any of claims 1 to 5, characterized in that it further comprises a short time treatment of ultra high temperature before contacting the slurry with the anion exchange resin.

8. A process according to any of claims 1 to 7, wherein step c) comprises placing the anion exchange resin in the slurry.

9. A process according to any of claims 1 to 7, wherein steps c) and d) comprise passing the slurry through a structure, which contains the anion exchange resin and has at least one entrance and one exit.

10. A method according to claim 1, wherein the structure is a vertical column and wherein the slurry enters said vertical column through the inlet and expels said volume through the outlet, the entrance being located more down that exit.

11. A process according to one of claims 3 to 10, wherein said anion exchange resin is a macroporous resin and the carbonate is bicarbonate.

12. A composition comprising a protein plant having a reduced content of isoflavones, manganese or nucleotides, said plant protein made according to the method of any of claims 1 to 11.

13. A plant protein composition according to claim 12, wherein said protein has a reduced content of at least one component of isoflavones, manganese or nucleotide selected from the group consisting of: (i) less than 30 μg of isoflavones per gram of protein; (ii) less than 450 μg of manganese per gram of protein; Y (iii) less than 10 mg of nucleotides per gram of protein.

14. A plant protein composition according to claim 13, wherein said protein comprises less than 20 μg of isoflavones per gram of protein; and less than 400 μg of manganese per gram of protein.

15. A plant protein composition comprising protein and less than 30 μg of isoflavones per gram of protein.

16. A plant protein composition according to claim 15, wherein said isoflavones are present in less than 10 μg per gram of protein.

17. - A plant protein composition according to claim 15 or 16, characterized in that said protein composition contains less than 250 μg of manganese per gram of protein.

18. A plant protein composition according to one of claims 15 to 16, comprising in less than 20 μg of isoflavones per gram of protein; and less than 200 μg of manganese per gram of protein.

19. A plant protein composition according to one of claims 12 to 18, wherein said composition is a livestock feed, a nutritional product or an infant formula.

20. A plant protein composition according to claim 19, wherein said composition is a formula for infants that contains less than 200 μg of isoflavones per liter of ready-to-eat formulas.

21. An isoflavone isolate obtained according to the method of claim 4.

22. An isoflavone isolate obtained according to claim 21, wherein the protein slurry is selected from soybean or peas.

23. The process according to claim 4, wherein the isoflavone release agent is selected from aqueous solutions of acids, aqueous solutions of bases, alcohols, alcohol / water mixture, organic solvents, and mixtures thereof.

24. A process according to claim 3 or 5, wherein the agent used in step i) is selected from sodium hydroxide and potassium hydroxide, and wherein the agent employed in step ii) is selected from acid hydrochloric acid, sulfuric acid and sodium chloride; and wherein the agent employed in step iii) is selected from sodium carbonate, sodium bicarbonate and ammonium hydroxide.