MXPA94008636A - Protein isolate having an increased degree of isoflavone compounds and procedure for producing the same - Google Patents

Abstract

The present invention refers to the production of an isoflavone enriched vegetable protein isolate in which the relation in material weight to the extraction agent is controlled and avoided or it is reduced to the minimum the coagulation washing of the precipitated acid protein to provide an increased degree of isoflavone in the protein isolate.

Description

ISOLATED PROTEIN THAT HAS AN INCREASED LEVEL OF ISOFLAVONA COMPOUNDS AND PROCEDURE TO PRODUCE THE SAME lnventor (s): JEROME L. SHEN, BALAGTAS F. GUEVARA AND FRANK E. SPADAFQRA; Americans with address at 5937 Keith Place, St. Louis, Missouri 63109; 9168 Fax Bridge Drive, Sunset Hills, Missouri 63127 and 2919 Accamac Street, St. Louis, Missouri 63104, E.U.A.

Causabiente: PROTEIN TECHNOLOGIES INTERNATIONAL, INC. a North American society organized and existing in accordance with the laws of the State of Missouri residing in Checkerbaard Square, St. Louis, Missouri 63164, E.U.A.

SUMMARY OF THE DESCRIPTION The present invention relates to the production of a vegetable protein isolate enriched with isoflavone in which the weight ratio of material to extractant is controlled and the washing of the precipitated protein acidic acid is prevented or minimized. provide an increased level of isoflavones in the protein isolate.

BACKGROUND OF THE INVENTION The present invention relates to a vegetable protein isolate enriched with isoflavone and to a process for producing the same. Isoflavones are present in a variety of legume plants, including plant protein materials such as soybean. These compounds for the purposes of the present invention generally include daidzin, 60AC-daidzin, daidzein, genistin, 60AC-genistin, genistein, glycitin, biovan in-A, formononetrin and coumestrol. Typically these compounds are associated with the inherent bitter taste of the soybean and the objective has been to remove these materials in the production of commercial products, such as isolates and concentrates. For example, in a conventional process for the production of a soy protein isolate, in which the soy flakes are extracted with an aqueous alkaline medium, a large part of the isoflavones are solubilized in the extract and remain solubilized in the serum. which is usually discarded after the precipitation of the acid from the protein to form an isolate. The residual isoflavones that remain in the precipitated protein isolate of the acid are usually removed by exhaustive washing of the isolate. It has been recently recognized that isoflavones contained in plant proteins such as soybean can inhibit the growth of human cancer cells, such as breast cancer cells and prostate cancer cells as described in the following articles: "Genistein Inhibition of the Growth of Human Breast Cancer Cells: Independence from Estrogen Receptors and the Multi-Drug Resistance Gene "pro Peterson and Barnes, Biochemical and Biophysical Research Communications, Vol. 179, No. 1, you pay. 661-666, August 30, 1991; "Genistein and Biochanin A Inhibit The Growth of Human Prostrate Cancer Cells But not Epidermal Growth Factor Receptor Tyrosine Atuophasphorylat ion" pro Peterson and Barnes, The Prostate 22: 335-34-5 (1993) and "Soybeans Inhibit ammory Tu ors in Models of Breast Cancer "by Barnes and others. Mutagens and Carcinoqens in the Dietoaqs. 239-253 (1990). The aforementioned interest in isoflavones has pointed out a need for protein materials, suitable for administering ion in a diet, which are abundant in these compounds, which have been tried to remove with great efforts in the prior art processes. for the production of co-branded protein materials. It is therefore an object of the present invention to provide a protein isolate enriched with isoflavone and a process for producing the same. These and other objects are specifically achieved in the detailed description of the present invention set forth below.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to a vegetable protein isolate enriched with isoflavone and to a process for producing the same which comprises extracting a plant protein material with an aqueous extraction agent having a pH above about the isoelectric point of the protein material to produce an aqueous extract of protein and isoflavones. The pH of the aqueous extract is then adjusted around the isoelectric point of the protein material in order to precipitate the protein material. The precipitated protein material is separated afterwards and the additional wash of precipitate is avoided or minimized to prevent the removal of dodehyde isoflavones and provide an isolate enriched with isoflavone. AdicianaJ mind, because the isoflavones are easily soluble in the aqueous extraction agent used to solubilize the protein, the specific weight ratios of the protein material to water are used to maximize the solubility of the protein. =? vapas during extraction.

For the purposes of the present invention, the isoflavones of interest have the following general formula. wherein R1, R2, R3, and R4- can be selected from the group consisting of H, OH, and OCH: a so or glucosides of these compounds. Specifically, these compounds and glycosides thereof which have been isolated from plant protein materials include daidzin, 60AC-daidzin, daidzein, genistin, 60AC-genistin, genistein, glycitin, biochanin-A, formononetrin and coumestral. The preferred isoflavones in the present invention for the purposes of enrichment of the isolate include daidzin, 60AC-daidzin, daidzein, genistin, 60AC-ist ina gene, genistein.

DESCRIPTION OF THE PREFERRED MODALITIES Although the present invention will be described with respect to soy products and the process is particularly suitable for the production of an isolate = X enriched with isoflavone from soybean materials, the process of the present however is applicable in general to the production of protein isolates from a variety of vegetable protein supplies that * contains isoflavone. The starting material for the present invention are soy flakes, from which the oil has been removed by solvent extraction. The flakes are extracted with an aqueous extraction agent having a pH above the isoelectric point of the protein material, preferably a pH of about 6.0-10.0 and a preferred DHI-GIUV of about .6 to 9.7. Typical alkaline reagents can emole < It is desired to raise the pH of the aqueous extraction agent which includes sodium hydroxide, potassium hydroxide, calcium hydroxide, etc. The desired isoflavone compounds are typically solubilized in the With the aim of maximizing the recovery of these tot lumps in the aqueous extract, the ratio by weight of hoiuela = to the aqueous extraction agent is controlled to specific levels in order to solubilize so many inherent isoflavones in Protein materials such as Dosible The extraction of jsofl proteins can be carried out in a variety of ways including concurrent extraction of the flakes to a ratio in DSSO V? ^ of flakes to aqueous extract of the imadap The initial extract is used to re-extract the leaflets and provide an aqueous extract to the isoflavones protein, alternatively, a two-step extraction procedure can be used. wherein the weight ratio of flakes to extracting agent in the initial step comprises approximately 6: 1 and then a second extraction of the flakes with the fresh extracting agent is carried out at a weight ratio of flakes to extraction agent of * Approximately 3: 1? about or: 1 such that the combined weight ratio of flakes to extractant in both steps does not exceed a total weight ratio of flakes extraction agent of approximately 11: 1 =? 14.1. Although, without criticism, the extraction can be carried out at temperatures of up to approximately 4 ° 6 ° C, for a period between about 5 v 60 $ minute.:. oreter, 15 minutes. The μH of the isoflavone-containing aqueous cephaloprotein extract described above, is then adjusted to approximately the isoelectric point of the protein by the addition of an edible acid, such as acetic, sulfuric, phosphoric, hydrochloric, or any other reagent. adequate acid. The isoelectric point for the SGva gene protein; to the one from around H.O to 5.0 and preferablely to | B SDGOX imadamente 4-.4 4,6. Adjusting the pH to the isoelectric point precipitates the protein in the form of a curd. Typically, in the production of a conventional protein isolate the precipitated acidic protein is separated from the remaining aqueous extract, described as the serum, and then washed or treated to remove the residual flavors. The washed isolate is then dried to form a dry isolate having a protein content, on a dry basis, exceeding 90%. Extensive washing has often been used to remove undesirable flavors, which have been attributed to various "phenolic" compounds in soybeans such as isoflavones. In the present invention, the washing of the precipitated protein material is completely avoided or reduced to a minimum in order to reduce substantially the removal of the two zones of the protein precipitate. with this an isolate enriched with isoflavone. For example, by avoiding or minimizing the washing of the precipitated ds-proteins, the recovery of isoflavones in the protein isolate = echo can be more than doubled. The washing of the acid-precipitated protein with water is therefore avoided by the foot or is limited to a water-washed mixture during which the water-to-water ratio to the orotema material is e = approximately Z °. í? 4: 1 This lack of washing of the precipitated acid curd provides an isolate enriched with the desired isoflavones. The precipitated acidic protein is then dried by a combination of centrifugation or concentration and dried in a conventional manner. The present invention is not intended to be limited by particular drying means but it is preferred to use conventional drying techniques such as spray drying to form a dry isolate. The protein isolate produced in the manner described above provides isolates having amounts Increased amounts of isoflavones, compared to a conventional isolate as illustrated in the following examples.

EXAMPLE 1 In order to illustrate the increased isoflavone levels in the proteins = produced in accordance with the present invention, a conventional protein isolate and a process for producing the same complete first to show the recovery of the desired isoflavones in a conventional procedure. 45.4 g of defatted soy flakes were placed in an extraction tank and extracted with 4-54 liters of water heated to 32 ° C to which sufficient calcium hydroxide was added to adjust the pH to -. 7 This provided an JO water weight ratio to 10: 1 leaflets. The leaflets are * separated from the extract and solved to extract with 672.4t'q of aqueous extract having a pH of 1. 7 and a temperature of 32.2 ° C. This second extraction step provided a weight ratio of water to chips of t > : l. The leaflets were removed by centrifugation, the first and second extracts were combined and adjusted to a pH of 4-5 with hydrochloric acid. The acidic precipitate was separated from the serum by centrifugation and the? Then with water in ^ P ^ a weight amount of seven times that of the starting material to provide a protein isolate. The analysis of the curd, whey, the flakes used and the starting material was completed for genistin "which includes gemstin, genistein, and 60AC-q is ispa) and daidzin (which includes daidzin, aaidzein, and 60AC-da? dz na> The analysis of these isoflavones was achieved by the procedure described by TS below # PROCEDURE FOR THE MEASUREMENT OF GENISTINE AND TOTAL DAIDZIN L. 0.25 g of soya product were weighed and added to 20 ml of extraction solution consisting of dt? ßO parts of methyl alcohol, 10 parts of water and 10 parts of HCl -H 3N. 2. An additional 20 ml of HCl was added to 4! -! and the mixture was stirred for 10 minutes. 3 »The solution was refluxed with a condenser during a ñora. 4. The solution was cooled and filtered through Whatman No. 4 filter paper. 5. An aliquot of iOml was removed to which lO l of millipore water and 0.4 ml of acetic acid were added, and 1 o. = * -. 6. Each solution was entered into a column of CLAR and measurement for the above isoflavone levels by UV absorption. The analysis of the precipitated curd, the soy whey, the flakes used and the starting material for the above isoflavones is shown in Table 1. The results are also shown as a percentage of recovery of the isof lavones scored from the level JBk contained in the starting material.

TABLE 1 Level% recovery (mg / g / on dry basis) The gene is inactivated by dzina gene i ín a daidzina Cu jada. .54 23% 15% Serum 3.24 3.30 75% 63% Ho employed women 0.21 0.19 2% ?? Paid Material t.72 1.56 The above example clearly illustrates that the desired isoflavones, in a conventional procedure, are concentrated mostly in the serum. which results in low levels of isoflavones in most commercial protein isolates.

EXAMPLE 2 45.5 V g of defatted soy flakes were placed in an extraction tank and extracted in a continuous two-stage countercurrent process with 363.2 liters of water heated to 32.2 ° C, to which sufficient calcium hydroxide was added to adjust the pH to 9.7. This # provided a weight ratio of water to 6: 1 leaflets. La1; Flakes were removed by centrifugation and the aqueous extract was adjusted to a pH of 4-5 in order to precipitate the protein, which was separated after the serum by centrifugation. The washing of the separated bath with water was avoided. The analysis of the curd, to the whey, the leaves used for the oaitide material was completed in a manner similar to that described in section 1. These results are listed in table 2, PICTURE Level% gives recovery g / gm / on dry basis) Mater? A-1 genistina da dzina genis tina daidzina What is 2.31 1.59 59% 44-% Serum 1.66 2.11 39% 53% H used rides 0.21 .26 2% 3% tlate ial of? Par ida 1.56 The recovery results described in the standard show that the desired level of isoflavone in the curd has been substantially increased as compared to Example 1 to thereby provide an isolate of soybean orotein enriched with isofiavone.

EXAMPLE 3 The precipitated acidic curd was prepared as described in Example 2 except that after precipitation of the acid gives the mixture, the curd was washed: c, n water at an effective temperature equal to a ratio of two times the weight of the leaflets. The analysis was completed as described in Example 1 and the level of recovery of the ofiavones is shown in Table 3.

TABLE 3 Level% of recovery '' (Tig gm / on dry basis i Material genistina da dzina qenistina daidzina Curd 2.03 1.37 52% 36% Serum 1.94 2.35 45% 5 Employed flakes 0.31 0.26 3% 3% Starting material 1.72 1.56 The results of recovery show a substantial increase in the recovery of isoflavone in the curd compared to the example ± to provide therewith a isolate 3c-soy protein enriched with isoflavone.

EXAMPLE 4- The precipitated acidic curd was prepared as described in Example 2 except that after the acid precipitation. the curd was the. acia with an amount of aqua at room temperature iqual to a # ratio in four times the weight of the leaflets. The recovery of isoflavones from this procedure is listed in Table 4.

TABLE h Level% recovery '' mg / cj / e-n dry case) Material genietina da dzina qenistma daidzma • $ Curd 1.60 1.12 4-6% 3i% Serum 2.20 2.63 51% 66% Ho juei - > s employees 0.31 0.26 3% 3% Material d-? Item 1.72 and ..56 After recovering from a curd isoflavone recovery compared to Example 1, the recovery is less than that described in Example 3.

EXAMPLE F. 100 grams of soy flour ds-sc rasada cen .00 grams of water were extracted to ^ n s tsmperjtbi.i de 32.2 ° C. The paste had a pH of 6. /. The paste was stirred for 5 minutes and centrifuged to remove the flour used. The extract was adjusted to a pH of 4.5 with hydrochloric acid and the curd was separated from the soybean by centrifugation for 10 minutes. The washing of the dressing is not carried out. The recovery of the isoflavones in the empty fractions was measured as described in Example 1 and is listed in Table 5.

TABLE 5 Level% recovery Mater lal genistin daidzin genis ina daidzin What is 2.? +9 1.76 t 4t?% Serum 29%. 37% Lead time 0.91 1.40 11% 17% Match material 1. 1.56 Can you see that the levels of sofiavona in the cuajaos prayed to me to eat dinner in comparison to the dal? > emo what i EXAMPLE 6 One lOOg of defatted soy flour was extracted with 600g of water at a temperature of 32.2 ° C. The pH was adjusted to 9.7 by the addition of calcium hydroxide. The paste was stirred for 15 minutes and then centrifuged for 5 minutes to separate the extract. The flour used was extracted after a second time mixing the flour with 1'OOc? Do water for 5 minutes. The second extract was separated from the flakes employed by centrifugation for 5 minutes. The first v. Aqueous extracts were combined and the pH adjusted to 4.5, the isoelectric point of the protein. The precipitated curd was recovered by centrifugation and the recovery of iodine in the curd, whey and the fractions of flakes used was measured as described in example 1. The recovery of isoflavone on the table t > .

TABLE 6 Level% recovery Material genietina daidzina qenistina daidzina Curd 2.23 1.4-6 57% 41% Serum 1.77 2.27 pl% 57%

Claims (5)

Ho ueias-empleadas 0.21 0.19 2% M a y to Part 1.72 It will be apparent to one skilled in the art that numerous changes and modifications to the present invention can be made as described herein. NOVELTY OF THE INVENTION CLAIMS

1. A process for producing an isolate of vegetable protein enriched with isoflavone characterized in that it comprises: a) extracting a plant protein material containing sofiavones with an acid extraction agent having a pH above about the isoelectric point of the material p? R produce an extract ai. of proteins to isoflavones, b) adjust the pH of the aqueous extract to enhance the isoelectric point of the material (in order to precipitate the protein material a, and c) separate said precipitated protein material and avoid further washing of said material precipitated with water to provide a protein isolate enriched with soflavone.

2. A method according to claim 1, characterized in accordance with > This is because the extiation is carried out at a pH of about 6.0-lU.O. 3"A method according to claim 2, further characterized because the extraction is carried out at a pH of approximately 6.7-9.7. 4-. A process according to claim 1, further characterized in that the pH of the extract is adjusted to about 4.4 to 4-.e > . 5. A process according to claim 1, further characterized in that the plant orotein material is extracted with said extraction agent and in a ratio of extract to material of approximately 5: 1 to 12: 1. 6. A process according to claim 1, further characterized in that the extraction of the plant protein material comprises a double extraction, in such a way that the combined weight ratio of extraction agent to material of both extractions does not exceed a ratio in Total Weight Approximately 11: 1 to 14: 1. 7. A process for producing a plant protein isolate enriched with isoflavin also characterized in that it comprises: a) extracting a plant protein material containing isoflavones with a Acuse agent that has a high pH around the isoelectric point of the material to produce an e < aqueous protein and isofiavone treatment, b) adjust the pH of the aqueous extract to around the isoelectric point of the protein material in order to precipitate the orotema material, and c) separate said precipitated protein material and wash said material with water in a weight amount which is about four times the weight of protein material to provide a protein isolate enriched with isofl von. 6. A process according to claim 7, further characterized in that the extraction is carried out at a pH of about 6.0-10.0. 9. The process according to claim 6, further characterized in that the extraction is carried out at a pH of about 6.7-9.7. 10. A procedure in accordance with the complaint, characterized in that the oH of the F extract is adjusted to approximately 4.4 to 4.6. 11. A process according to claim 7, further characterized in that the protein material is extracted with said extract in a ratio of extract to material of approximately 12: 1 Sil. 12. A process according to claim 7, further characterized by extraction of the plant crotim material comprises a double extraction, such that the ratio in combined passage of extraction agent to material from both extractions does not exceed a ratio in total weight of approximately 11: 1 to 14-: i. 1

3. A method according to claim 7, further characterized in that the precipitated protein material is washed with water in an amount by weight that is less than approximately twice the weight of the protein material. 1

4. A method according to claim 7, further characterized in that it includes the step of drying the isolate enriched with isoflavone. 1

5. The isoflavone-enriched isolate produced by the method according to claim 14-, In testimony of which I sign the foregoing in this Mexico City, D.F. to the 7 days of the month of November of 1994-. # By: PROTEIN TECHNOLOGIES INTERNATIONAL, INC MK / ep