HK1198101B - Preparation of soy protein isolate using calcium chloride extraction - Google Patents

Description

Preparation of soy protein isolate using calcium chloride extraction

Reference to related applications

This application is a continuation-in-part application of co-pending U.S. patent application No. 12/828,212 filed on 30.6.2010, which itself claims priority from U.S. provisional patent application No. 61/213,647 filed on 30.6.2009, in accordance with 35 USC 119 (e).

Technical Field

The present invention relates to the preparation of soy protein products.

Background

In us patent application No. 12/603,087 (7865-. The soy protein product is useful for protein fortification of, inter alia, soft drinks and sports drinks, as well as other acidic aqueous systems, without protein precipitation. The soy protein product is produced by: extracting the soy protein source with an aqueous calcium chloride solution at natural pH, optionally diluting the resulting aqueous soy protein solution, adjusting the pH of the aqueous soy protein solution to a pH of about 1.5 to about 4.4, preferably about 2.0 to about 4.0, to produce an acidified clear soy protein solution, which may optionally be concentrated and/or diafiltered prior to drying.

Summary of The Invention

It has now been unexpectedly found that a soy protein product having a protein content of at least about 60 wt% (N x6.25) d.b. can be formed by a procedure which involves extracting a soy protein source with calcium chloride at low pH.

In one aspect of the invention, the soy protein source material is extracted with an aqueous calcium chloride solution at low pH and the resulting aqueous soy protein solution is optionally diluted, optionally adjusted in pH in the acidic range, and then subjected to ultrafiltration and optionally diafiltration to provide a concentrated and optionally diafiltered soy protein solution, which may be dried to provide a soy protein product.

In another aspect of the invention, the soy protein source material is extracted with an aqueous calcium chloride solution at low pH and the resulting aqueous soy protein solution is optionally diluted, optionally adjusted in pH in the acidic range, and then subjected to ultrafiltration and optionally diafiltration to provide a concentrated and optionally diafiltered soy protein solution. The concentrated and optionally diafiltered soy protein solution may then optionally be adjusted in pH in the pH range of about 1.5 to about 7, preferably about 4 to about 7, more preferably about 5 to about 7, and diluted with water to fractionate the soy protein into a globulin-rich precipitate and an albumin-rich supernatant comprising trypsin inhibitors. The precipitate formed from the dilution step may be collected and further processed or dried to provide a soy protein product, but with reduced levels of trypsin inhibitors.

In another aspect of the invention, the concentrated and optionally diafiltered and optionally pH adjusted soy protein solution prepared as described above is diluted into water. The pH of the diluted sample is adjusted to about 1.5 to about 4.4, preferably about 2.0 to about 4.0 to re-solubilize the protein precipitated by the dilution step. The diluted and pH adjusted solution may then optionally be heat treated and/or concentrated and/or diafiltered.

Soy protein products having a protein content of at least about 60 wt% (N x6.25) d.b. as provided herein are soluble at acidic pH values to provide clear and heat stable aqueous solutions thereof. The soy protein product can be used for protein fortification of, inter alia, soft drinks and sports drinks, as well as other aqueous systems, without protein precipitation. The soy protein product is preferably an isolate having a protein content of at least about 90 wt.%, preferably at least about 100 wt.% (N x6.25) d.b.

According to one aspect of the present invention, there is provided a method of producing a soy protein product having a soy protein content of at least about 60 wt% (N x6.25) on a dry weight basis, comprising:

(a) extracting the soy protein source with an aqueous calcium salt solution, typically a calcium chloride solution, at a low pH, typically from about 1.5 to about 5.0, to cause solubilization of the soy protein from the protein source and to form an aqueous soy protein solution,

(b) at least partially separating the aqueous soy protein solution from residual soy protein source,

(c) optionally diluting the aqueous soy protein solution,

(d) optionally adjusting the pH of the aqueous protein solution to a value in the range of from about 1.5 to about 5.0, preferably from about 1.5 to about 4.4, more preferably from about 2.0 to about 4.0, and different from the pH of the extraction,

(e) the aqueous soy protein solution is optionally finished to remove residual particles,

(f) optionally concentrating the aqueous soy protein solution by using a selective membrane technique while keeping the ionic strength substantially constant,

(g) optionally diafiltering the concentrated soy protein solution, and

(h) the concentrated and diafiltered soy protein solution is optionally dried.

The soy protein product is preferably an isolate having a protein content of at least about 90 wt.%, preferably at least about 100 wt.% (N x6.25) d.b.

A variation of this procedure may be used to produce a product with reduced levels of albumin and trypsin inhibitors. In this variation, the concentrated and optionally diafiltered soy protein solution is adjusted in pH in the range of about 1.5 to about 7.0, preferably about 4.0 to about 7.0, more preferably about 5.0 to about 7.0, and then diluted into water to obtain a precipitate having reduced levels of albumin and trypsin inhibitors. The precipitate may be collected and dried to give the product, or may be dissolved in water at a pH of about 1.5 to about 4.4, preferably about 2.0 to about 4.0, and then dried. Alternatively, the solution formed by dissolving the precipitate in water at a pH of about 1.5 to about 4.4, preferably about 2.0 to about 4.0, may optionally be heat treated and/or finished and/or concentrated and/or diafiltered prior to drying.

Thus, in another aspect of the present invention, a process is described for producing a soy protein product having a soy protein content of at least about 60 wt% (N x6.25) on a dry weight basis, comprising:

(a) extracting the soy protein source with an aqueous calcium salt solution, typically a calcium chloride solution, at a low pH, typically from about 1.5 to about 5.0, to cause solubilization of the soy protein from the protein source and to form an aqueous soy protein solution,

(b) at least partially separating the aqueous soy protein solution from residual soy protein source,

(c) optionally diluting the aqueous soy protein solution,

(d) optionally adjusting the pH of the aqueous protein solution to a value in the range of from about 1.5 to about 5.0, preferably from about 1.5 to about 4.4, more preferably from about 2.0 to about 4.0, and different from the pH of the extraction,

(e) the aqueous soy protein solution is optionally finished to remove residual particles,

(f) concentrating the aqueous soy protein solution by using a selective membrane technique while keeping the ionic strength substantially constant,

(g) optionally diafiltering the concentrated soy protein solution,

(h) optionally adjusting the pH of the concentrated and optionally diafiltered soy protein solution to a value in the range of about 1.5 to about 7.0, preferably about 4.0 to about 7.0, more preferably about 5.0 to about 7.0,

(i) diluting the concentrated and optionally diafiltered and pH adjusted soy protein solution to water,

(j) separating the precipitate formed from the dilution water, called supernatant, and

(k) drying the isolated soy protein precipitate.

The soy protein product is preferably an isolate having a protein content of at least about 90 wt.%, preferably at least about 100 wt.% (N x6.25) d.b.

Another variation of this procedure may be employed to produce the product. In this variant, the concentrated and optionally diafiltered and optionally pH-adjusted soy protein solution is diluted into water and the pH is adjusted after dilution, which redissolves the precipitate formed by the dilution step. The resulting pH adjusted solution is optionally heat treated and/or finished and/or concentrated and/or diafiltered to obtain the product prior to drying.

Thus, in another aspect of the present invention, a process is described for producing a soy protein product having a soy protein content of at least about 60 wt% (N x6.25) on a dry weight basis, comprising:

(a) extracting the soy protein source with an aqueous calcium salt solution, typically a calcium chloride solution, at a low pH, typically from about 1.5 to about 5.0, to cause solubilization of soy protein from the protein source and to form an aqueous soy protein solution,

(b) at least partially separating the aqueous soy protein solution from residual soy protein source,

(c) optionally diluting the aqueous soy protein solution,

(d) optionally adjusting the pH of the aqueous protein solution to a value in the range of from about 1.5 to about 5.0, preferably from about 1.5 to about 4.4, more preferably from about 2.0 to about 4.0, and different from the pH of the extraction,

(e) the aqueous soy protein solution is optionally finished to remove residual particles,

(f) concentrating the aqueous soy protein solution by using a selective membrane technique while keeping the ionic strength substantially constant,

(g) optionally diafiltering the concentrated soy protein solution,

(h) optionally adjusting the pH of the concentrated and optionally diafiltered soy protein solution to a value in the range of about 1.5 to about 7, preferably about 4.0 to about 7.0, more preferably about 5.0 to about 7.0,

(i) diluting the concentrated and optionally diafiltered and pH adjusted soy protein solution to water,

(j) adjusting the pH of the diluted sample to a value in the range of about 1.5 to about 4.4, optionally about 2.0 to about 4.0, to re-dissolve the protein precipitate formed by the dilution step,

(k) optionally concentrating the pH adjusted soy protein solution by using a selective membrane technique while maintaining the ionic strength substantially constant,

(l) Optionally diafiltering the concentrated, pH adjusted soy protein solution, and

(m) drying the concentrated and optionally diafiltered, pH adjusted soy protein solution.

The soy protein product is preferably an isolate having a protein content of at least about 90 wt.%, preferably at least about 100 wt.% (N x6.25) d.b.

Although the present description is primarily directed to the production of soy protein isolate, the concentration and/or diafiltration steps described herein may be manipulated to produce a less pure soy protein product, e.g., a soy protein concentrate having a protein content of at least about 60 wt.%, but which has substantially similar properties as the isolate.

The novel soy protein product of the present invention can be blended with powdered beverages for forming aqueous soft drinks or sports drinks by dissolving it in water. Such a blend may be a powdered beverage.

The soy protein products provided herein can be provided as aqueous solutions thereof having a high degree of clarity at acidic pH values and being heat stable at these pH values.

In another aspect of the invention, an aqueous solution of a soy product provided herein that is heat stable at low pH is provided. The aqueous solution may be a beverage, which may be a clear beverage in which the soy protein product is completely soluble and transparent, or an opaque beverage in which the soy protein product does not increase opacity. The soy protein product also has good solubility at about pH 7. An aqueous solution of the soy protein product prepared at a near neutral pH (e.g., a pH of about 6 to about 8) can be a beverage.

The soy protein products produced according to the methods herein lack the characteristic beany flavor of soy protein isolates and are not only suitable for protein fortification in acidic media, but are also useful in a wide variety of conventional applications of protein isolates, including but not limited to protein fortification of processed foods and beverages, emulsification of oils, as body formers (body formers) in baked goods and foaming agents in gas-entrapped products. In addition, soy protein products can be formed into protein fibers for meat analogs and can be used as protein (eg white) substitutes or supplements in food products where protein (eg white) is used as a binder. The soy protein product can also be used in nutritional supplements. Other uses of soy protein products are in pet food, animal feed, industrial and cosmetic applications, and personal care products.

Summary of the invention

The starting step in the process of providing a soy protein product involves solubilizing soy protein from a soy protein source. The soy protein source may be soy or any soy product or by-product obtained from the processing of soy, including but not limited to soy flour (soymeal), soy flakes, soy grits (soy grits), and soy flour (flours). The soy protein source may be used in a full fat form, a partially defatted form, or a fully defatted form. When the soy protein source contains significant amounts of fat, a degreasing step is typically required during the process. The soy protein recovered from the soy protein source may be a protein naturally occurring in soy, or the proteinaceous material may be a protein modified by genetic manipulation, but possessing the characteristic hydrophobic and polar properties of the natural protein.

The solubilization of protein from the soy protein source material is most conveniently carried out using a calcium chloride solution, although solutions of other calcium salts may be used. In addition, other alkaline earth metal compounds, such as magnesium salts, may be used. Further, extraction of soy protein from a soy protein source may be carried out using a combination of a calcium salt solution and another salt solution (such as sodium chloride). Additionally, extraction of soy protein from the soy protein source may be carried out using water or other salt solutions (such as sodium chloride), followed by the addition of calcium chloride to the aqueous soy protein solution produced in the extraction step. Then, the precipitate formed upon addition of calcium chloride was removed prior to subsequent processing.

As the concentration of the calcium salt solution increases, the degree of solubilization of protein from the soy protein source initially increases until a maximum value is reached. Any subsequent increase in salt concentration did not increase total protein solubilized. The concentration of the calcium salt solution that causes the greatest protein solubilization varies depending on the salt concerned. It is generally preferred to utilize concentration values of less than about 1.0M, more preferably, values of from about 0.10M to about 0.15M.

In a batch process, the solubilization of protein is carried out at a temperature of from about 1 ℃ to about 100 ℃, preferably from about 15 ℃ to about 65 ℃, more preferably from about 20 ℃ to about 35 ℃, preferably with agitation to reduce the solubilization time, which is typically from about 1 to about 60 minutes. Solubilization is preferably performed to extract substantially as much protein as is practical from the soy protein source to provide overall high product yield.

In a continuous process, soy protein extraction is performed from a soy protein source in any manner consistent with performing continuous extraction of soy protein from a soy protein source. In one embodiment, the soy protein source is continuously mixed with the calcium salt solution, and the mixture is conveyed through a pipe or conduit having a length and at a flow rate for a residence time sufficient to effect the desired extraction in accordance with the parameters described herein. In such a continuous procedure, the solubilization step is carried out rapidly, preferably in a time of up to about 10 minutes, to extract substantially as much protein as is practical from the soy protein source. The dissolution in a continuous procedure is carried out at a temperature between about 1 ℃ and about 100 ℃, preferably between about 15 ℃ and about 65 ℃, more preferably between 20 ℃ and about 35 ℃.

The extraction is typically carried out at a pH of about 1.5 to about 5.0. The pH of the extraction system (soy protein source and calcium salt solution) can be adjusted to any desired value for the extraction step in the range of about 1.5 to about 5.0 by using any convenient food grade acid, typically hydrochloric or phosphoric acid.

The concentration of the soy protein source in the calcium salt solution during the solubilization step can vary widely. Typical concentration values are about 5 to about 15% w/v.

The protein extraction step with an aqueous calcium salt solution has the additional effect of solubilizing fats that may be present in the soy protein source, which thus results in fats being present in the aqueous phase.

The protein solution resulting from the extraction step typically has a protein concentration of about 5 to about 50g/L, preferably about 10 to about 50 g/L.

The aqueous calcium salt solution may contain an antioxidant. The antioxidant may be any convenient antioxidant such as sodium sulfite or ascorbic acid. The amount of antioxidant employed may vary from about 0.01 to about 1 weight percent of the solution, preferably about 0.05 weight percent. The antioxidant serves to inhibit oxidation of any phenolics in the protein solution.

The aqueous phase resulting from the extraction step may then be separated from the residual soy protein source in any convenient manner, such as by a decanter centrifuge or any suitable screen, followed by disc centrifugation and/or filtration to remove residual soy protein source material. The separated residual soy protein source may be dried for disposal. Alternatively, the separated residual soy protein source may be treated to recover some residual protein. The separated residual soy protein source may be re-extracted with fresh calcium salt solution, wherein the re-extraction is performed at a pH in the range of about 1.5 to about 5.0, and the resulting protein solution, upon clarification, is combined with the initial protein solution for further processing as described below. Alternatively, the separated residual soy protein source may be treated by a conventional isoelectric precipitation procedure or any other convenient procedure to recover such residual protein.

When the soy protein source contains significant amounts of fat, the aqueous protein separated may then be subjected to the defatting step described therein, as described in U.S. patent nos. 5,844,086 and 6,005,076, the disclosures of which are incorporated herein by reference, assigned to their assignee. Alternatively, the defatting of the separated aqueous protein solution may be achieved by any other convenient procedure.

The aqueous soy protein solution may be treated with an adsorbent, such as powdered or granular activated carbon, to remove colored and/or odorous compounds. Such adsorbent treatment may be carried out under any convenient conditions, typically at the ambient temperature of the separated aqueous protein solution. For powdered activated carbon, amounts of about 0.025% to about 5% w/v, preferably about 0.05% to about 2% w/v, are employed. The adsorbent may be removed from the soy protein solution by any convenient means, such as by filtration.

The resulting aqueous soy protein solution may be diluted with from about 0.5 to about 10 volumes, preferably from about 0.5 to about 2 volumes, of aqueous diluent in order to reduce the conductivity of the aqueous soy protein solution to a value generally below about 90 mS, preferably from about 4 to about 31 mS. Although dilute salt solutions having a conductivity of up to about 3 mS, such as sodium chloride or calcium chloride, can be used, water is typically used to effect such dilution.

The temperature of the diluent mixed with the soy protein solution may be from about 2 ℃ to about 70 ℃, preferably from about 15 ℃ to about 65 ℃, more preferably from about 20 ℃ to about 35 ℃.

The pH of the optionally diluted soy protein solution may be adjusted to a value different from the extraction pH, but still within the range of about 1.5 to about 5.0, preferably about 1.5 to about 4.4, more preferably about 2.0 to about 4.0, by the addition of any suitable food grade acid (such as hydrochloric acid or phosphoric acid) or food grade base (typically sodium hydroxide) as desired.

The conductivity of the diluted and optionally pH adjusted soy protein solution is generally less than about 95 mS, preferably from about 4 to about 36 mS.

The aqueous soy protein solution may be subjected to a heat treatment to inactivate heat-labile anti-nutritional factors (such as trypsin inhibitors) present in the soy protein source material as a result of extraction from such solution during the extraction step. This heating step also provides the additional benefit of reducing microbial load. Typically, the protein solution is heated to a temperature of from about 70 ℃ to about 160 ℃, preferably from about 80 ℃ to about 120 ℃, more preferably from about 85 ℃ to about 95 ℃ for a period of from about 10 seconds to about 60 minutes, preferably from about 30 seconds to about 5 minutes. The heat treated soy protein solution may then be cooled to a temperature of about 2 ℃ to about 65 ℃, preferably about 20 ℃ to about 35 ℃, for further processing as described below.

The optionally diluted, optionally pH adjusted and optionally heat treated protein solution may optionally be finished by any convenient means (e.g. filtration) to remove any residual particles.

The resulting aqueous soy protein solution may be dried directly to produce a soy protein product. To provide a soy protein product (e.g., soy protein isolate) having a reduced impurity content and a reduced salt content, the aqueous soy protein solution may be treated prior to drying.

The aqueous soy protein solution may be concentrated to increase its protein concentration while maintaining its ionic strength substantially constant. Such concentration is typically carried out to provide a concentrated soy protein solution having a protein concentration of from about 50 to about 300g/L, preferably from about 100 to about 200 g/L.

The concentration step may be carried out in any convenient manner consistent with batch or continuous operation, for example by employing any convenient selective membrane technique (such as ultrafiltration or diafiltration) using membranes (such as hollow fibre membranes or spiral wound membranes) having a suitable molecular weight cut-off, for example from about 3,000 to about 1,000,000 daltons, preferably from about 5,000 to about 100,000 daltons, having regard to the different membrane materials and structures, and which are sized to allow the desired degree of concentration as the aqueous protein solution passes through the membrane for continuous operation.

As is well known, ultrafiltration and similar selective membrane techniques allow low molecular weight species to pass while preventing higher molecular weight species from also passing. The low molecular weight species include not only ionic species of food grade salts, but also low molecular weight materials extracted from the source material, such as carbohydrates, pigments, low molecular weight proteins, and anti-nutritional factors which are themselves low molecular weight proteins, such as trypsin inhibitors. The cut-off molecular weight of the membrane is generally selected to ensure that a significant proportion of the protein is retained in solution, while allowing contaminants to pass, taking into account the different membrane materials and structures.

The concentrated soy protein solution may then be subjected to a diafiltration step using water or dilute saline solution. The diafiltration solution may be at its natural pH or at a pH equal to or any pH value between the pH of the protein solution being diafiltered. This diafiltration may be effected using from about 2 to about 40 volumes of diafiltration solution, preferably from about 5 to about 25 volumes of diafiltration solution. In the diafiltration operation, further quantities of contaminants are removed from the aqueous soy protein solution by passage through the membrane with the permeate. This purifies the aqueous protein solution and may also reduce its viscosity. The diafiltration operation may be effected until no significant further amounts of contaminants or visible colour are present in the permeate, or until the retentate has been sufficiently purified so as to provide, when dried, a soy protein isolate having a protein content of at least about 90 wt% (N x6.25) d.b.. This diafiltration may be effected using the same membrane as the concentration step. However, if desired, the diafiltration step may be effected using separate membranes having different molecular weight cut-offs, for example membranes having molecular weight cut-offs in the range of about 3,000 to about 1,000,000 daltons, preferably about 5,000 to about 100,000 daltons, taking into account the different membrane materials and configurations.

Alternatively, a diafiltration step may be applied to the aqueous protein solution, or partially concentrated aqueous protein solution, prior to concentration. Diafiltration may also be applied at multiple points during the concentration process. When diafiltration is applied prior to concentration, or to a partially concentrated solution, the resulting diafiltration solution may be subsequently additionally concentrated. The reduction in viscosity achieved by multiple diafiltrations as the protein solution is concentrated may allow a higher final fully concentrated protein concentration to be achieved. This reduces the volume of material to be dried.

The concentration step and diafiltration step may be carried out herein in such a way that the subsequently recovered soy protein product contains less than about 90 wt% protein (N x6.25) d.b., for example at least about 60 wt% protein (N x6.25) d.b. By partially concentrating and/or partially percolating the aqueous soy protein solution, it is possible to remove the contaminants only partially. The protein solution may then be dried to provide a soy protein product having a lower level of purity. The soy protein product is still capable of producing a clear protein solution under acidic conditions.

An antioxidant may be present in the diafiltration medium during at least a portion of the diafiltration step. The antioxidant may be any convenient antioxidant, for example sodium sulfite or ascorbic acid. The amount of antioxidant employed in the diafiltration medium depends on the material employed and may vary from about 0.01 to about 1 wt%, preferably about 0.05 wt%. The antioxidant serves to inhibit oxidation of any phenolics present in the concentrated soy protein solution.

The concentration step and optional diafiltration step may be carried out at any convenient temperature (typically from about 2 ℃ to about 65 ℃, preferably from about 20 ℃ to about 35 ℃) and for a period of time to achieve the desired degree of concentration and diafiltration. The temperature and other conditions used will depend to some extent on the membrane equipment used to carry out the membrane treatment, the desired protein concentration of the solution and the efficiency of contaminant removal for the permeate.

There are two major trypsin inhibitors in soybean, namely, Kunitz inhibitor (which is a thermally unstable molecule with a molecular weight of about 21,000 daltons) and Bowman-Birk inhibitor (which is a more thermally stable molecule with a molecular weight of about 8,000 daltons). The level of trypsin inhibitor activity in the final soy protein product can be controlled by manipulating various process variables.

As described above, heat treatment of the aqueous soy protein solution may be used to inactivate heat-labile trypsin inhibitors. The partially or fully concentrated soy protein solution may also be heat treated to inactivate heat labile trypsin inhibitors. When heat treatment is applied to the partially concentrated soy protein solution, the resulting heat treated solution may be additionally concentrated thereafter.

In addition, the concentration and/or diafiltration step may be operated in a manner that facilitates removal of trypsin inhibitors and other contaminants from the permeate. Removal of trypsin inhibitors is facilitated by using membranes with larger pore sizes (e.g., about 30,000 to about 1,000,000 Da), operating the membranes at elevated temperatures (e.g., about 30 ℃ to about 65 ℃) and employing larger volumes (e.g., about 20 to about 40 volumes) of diafiltration medium.

Extraction and/or membrane treatment of protein solutions at lower pH (1.5-3.0) may reduce trypsin inhibitor activity relative to treatment of solutions at higher pH (3.0-5.0). When concentrating and diafiltering the protein solution at the lower end of the pH range, it may be desirable to raise the pH of the retentate prior to drying. The pH of the concentrated and diafiltered protein solution may be raised to a desired value, for example pH 3, by the addition of any convenient food grade base, such as sodium hydroxide. If it is desired to lower the pH of the retentate prior to drying, this may be achieved by the addition of any convenient food grade acid, such as hydrochloric or phosphoric acid.

In addition, a reduction in trypsin inhibitor activity can be achieved by exposing the soy material to a reducing agent that cleaves or rearranges the disulfide bonds of the inhibitor. Suitable reducing agents include sodium sulfite, cysteine and N-acetyl cysteine.

Such addition of the reducing agent may be carried out at various stages of the overall process. The reducing agent may be added with the soy protein source material in the extraction step, may be added to the clear aqueous soy protein solution after removal of residual soy protein source material, may be added to the concentrated protein solution before or after diafiltration, or may be dry blended with the dried soy protein product. The addition of the reducing agent may be combined with the heat treatment step and the film treatment step described above.

If it is desired to retain active trypsin inhibitors in the concentrated protein solution, this can be achieved by: eliminating or reducing the intensity of the heat treatment step, not utilizing a reducing agent, operating the concentration and diafiltration steps at the higher end of the pH range (3.0-5.0), utilizing concentration and diafiltration membranes with smaller pore sizes, operating the membranes at lower temperatures and using a smaller volume of diafiltration medium.

The concentrated and optionally diafiltered protein solution may be subjected to further defatting operations, if desired, as described in U.S. Pat. nos. 5,844,086 and 6,005,076. Alternatively, defatting of the concentrated and optionally diafiltered protein solution may be achieved by any other convenient procedure.

The concentrated and optionally diafiltered aqueous protein solution may be treated with an adsorbent, such as powdered activated carbon or granular activated carbon, to remove colored and/or odorous compounds. Such adsorbent treatment may be carried out under any convenient conditions (typically at ambient temperature of the concentrated protein solution). For powdered activated carbon, amounts of about 0.025% to about 5% w/v, preferably about 0.05% to about 2% w/v, are employed. The adsorbent may be removed from the soy protein solution by any convenient means, such as by filtration.

The concentrated and optionally diafiltered soy protein solution resulting from the optional defatting and optional adsorbent treatment steps may be subjected to a pasteurization step to reduce microbial load. Such pasteurization may be carried out under any desired pasteurization conditions. The concentrated and optionally diafiltered soy protein solution is typically heated to a temperature of about 55 c to about 70 c, preferably about 60 c to about 65 c, for a period of about 30 seconds to about 60 minutes, preferably about 10 minutes to about 15 minutes. The pasteurized concentrated and diafiltered soy protein solution may then be cooled, preferably to a temperature of about 20 ℃ to about 35 ℃, for drying or further processing.

According to one aspect of the invention, the concentrated and optionally diafiltered soy protein solution may be dried by any convenient technique (e.g., spray drying or freeze drying) to obtain a soy protein product. The protein content of the dried soy protein product is greater than about 60 wt% (N x6.25) d.b.. Preferably, the dried soy protein product is an isolate having a high protein content of greater than about 90 wt.% protein, preferably at least about 100 wt.% (N x6.25) d.b..

In another aspect of the invention, the concentrated protein solution resulting from the concentration step and optional diafiltration step, optional defatting step, optional adsorbent treatment step and optional pasteurization step is optionally pH adjusted in the range of about 1.5 to about 7, preferably about 4.0 to about 7.0, more preferably about 5.0 to about 7.0, and then diluted by mixing the concentrated protein solution with water having the volume necessary to achieve the desired dilution. When it is intended to separate the precipitated protein from the residual aqueous phase, referred to as the supernatant, as is the case in this aspect of the invention, the dilution is generally from about 5-fold to about 25-fold, preferably from about 10-fold to about 20-fold. The temperature of the water mixed with the concentrated protein solution is preferably from about 1 ℃ to about 65 ℃, preferably from about 20 ℃ to about 35 ℃.

In a batch operation, a batch of concentrated protein solution is added to a static body of water (static body of water) having the desired volume as discussed above. Dilution of the concentrated protein solution reduces the ionic strength and causes the formation of a protein precipitate. In a batch procedure, the protein precipitate is allowed to settle in the water body. Sedimentation may be assisted, for example, by centrifugation. Such induced settling reduces the moisture content and the occluded salt content of the precipitated proteins.

Alternatively, the dilution operation may be performed continuously by passing the concentrated protein solution continuously to one inlet of the T-tube while feeding dilution water to the other inlet of the T-tube, allowing mixing in the tube. The dilution water is fed into the tee at a rate sufficient to achieve the desired dilution of the concentrated protein solution.

The mixing of the concentrated protein solution and dilution water in the tube causes the formation of a protein precipitate, and the mixture is continuously fed from the outlet of the T-tube into a settling vessel, which when filled, allows the supernatant to overflow. The mixture is preferably fed into the liquid body in the settling vessel in such a way that turbulence within the liquid body is minimised.

In a continuous procedure, the protein precipitate is allowed to settle in the settling vessel and the procedure is continued until a desired amount of precipitate has accumulated at the bottom of the settling vessel, whereupon the accumulated precipitate is removed from the settling vessel. Instead of sedimentation by sedimentation, the precipitate can be continuously separated by centrifugation.

By recovering the soy protein precipitate using a continuous process, the time of the initial protein extraction step can be significantly reduced for the same level of protein extraction as compared to a batch process. In addition, there are fewer opportunities for contamination in continuous operation than in batch procedures, resulting in higher product quality, and the process can be performed in more compact equipment.

The settled precipitate is separated from the residual aqueous phase or supernatant by, for example, decanting the residual aqueous phase from the settled cake or by centrifugation. The precipitate may be washed to remove residual supernatant, e.g., with about 1 to about 10, preferably about 2 to about 3 volumes of water, and then again recovered as above. The optionally washed precipitate may be used in wet form or may be dried to dry form by any convenient technique, such as spray drying or freeze drying. The dried precipitate has a high protein content of more than about 60 wt.% protein, preferably at least about 90 wt.% protein (N × 6.25), more preferably at least about 100 wt.% (N × 6.25).

The supernatant resulting from the dilution step may be dried to provide the soy protein product. Alternatively, the supernatant may be treated to reduce its impurity content and/or its trypsin inhibitor activity by any convenient means (e.g. pH adjustment and/or heat treatment and/or membrane treatment). The treated supernatant can then be dried to provide a soy protein product.

As described above, the precipitated protein precipitate formed in the dilution step may be directly dried to obtain the protein product. Alternatively, the wet protein precipitate may be resuspended in water, for example, from about 2 to about 3 volumes, and redissolved by adjusting the pH of the sample to about 1.5 to about 4.4, preferably from about 2.0 to about 4.0, using any convenient acid (e.g., hydrochloric acid or phosphoric acid). The re-solubilized protein solution may then be dried to a dry form by any convenient technique, such as spray drying or freeze drying. The protein content of the dried protein product is greater than about 60% protein by weight, preferably at least about 90% protein by weight, more preferably at least about 100% protein by weight (N x 6.25).

As another alternative, the re-solubilized soy protein solution may be subjected to a heat treatment to inactivate any remaining heat-labile anti-nutritional factors. This heating step also provides the additional benefit of reducing microbial load. Typically, the protein solution is heated to a temperature of from about 70 ℃ to about 160 ℃, preferably from about 80 ℃ to about 120 ℃, more preferably from about 85 ℃ to about 95 ℃ for a period of from about 10 seconds to about 60 minutes, preferably from about 30 seconds to about 5 minutes. The heat treated soy protein solution may then be cooled to a temperature of about 2 ℃ to about 65 ℃, preferably about 20 ℃ to about 35 ℃, for further processing as described below.

The re-solubilized and optionally heat-treated protein solution may optionally be finished by any convenient means (e.g. filtration) to remove any residual particles.

The re-solubilized, optionally heat treated, optionally finished clear protein solution may be concentrated to increase its protein concentration. Such concentration is carried out using any convenient selective membrane technique (such as ultrafiltration or diafiltration) using a membrane having a suitable molecular weight cut-off to allow low molecular weight species (including salts, carbohydrates, pigments, trypsin inhibitors and other low molecular weight materials extracted from the protein source material) to pass through the membrane while retaining a significant proportion of the soy protein in solution. Ultrafiltration membranes having a molecular weight cut-off of about 3,000-1,000,000 daltons, preferably about 5,000 to about 100,000 daltons, can be used with due regard to different membrane materials and structures. Concentrating the protein solution in this manner also reduces the volume of liquid required to be dried to recover the protein. Prior to drying, the protein solution is typically concentrated to a protein concentration of about 50g/L to about 300g/L, preferably about 100 to about 200 g/L. Such concentration operation may be carried out in batch mode or in continuous operation as described above.

The soy protein solution may be subjected to a diafiltration step using water, either before or after complete concentration. The water may be at its natural pH or at a pH equal to the pH of the protein solution being diafiltered or any pH value in between. Such diafiltration may be effected using from about 2 to about 40 volumes of diafiltration solution, preferably from about 5 to about 25 volumes of diafiltration solution. In the diafiltration operation, further quantities of contaminants are removed from the clarified aqueous soy protein solution by passage through the membrane with the permeate. The diafiltration operation may be effected until no significant further amounts of contaminants or visible colour are present in the permeate, or until the retentate has been sufficiently purified so as to provide, on drying, a soy protein product having the desired protein content, preferably an isolate having a protein content of at least about 90 wt% (N x6.25) d.b.. Such diafiltration may be effected using the same membrane as the concentration step. However, if desired, the diafiltration step may be effected using separate membranes having different molecular weight cut-offs, for example membranes having molecular weight cut-offs in the range of about 3,000 to about 1,000,000 daltons, preferably about 5,000 to about 100,000 daltons, taking into account the different membrane materials and configurations.

The concentration step and diafiltration step may be carried out herein in such a way that the soy protein product subsequently recovered by drying the concentrated and diafiltered retentate contains less than about 90 wt% protein (N x6.25) d.b., for example at least about 60 wt% protein (N x6.25) d.b. By partially concentrating and/or partially percolating the aqueous soy protein solution, it is possible to remove the contaminants only partially. The protein solution may then be dried to provide a soy protein product having a lower level of purity. The soy protein product is still capable of producing a clear protein solution under acidic conditions.

An antioxidant may be present in the diafiltration medium during at least a portion of the diafiltration step. The antioxidant may be any convenient antioxidant, for example sodium sulfite or ascorbic acid. The amount of antioxidant employed in the diafiltration medium depends on the material employed and may vary from about 0.01 to about 1 wt%, preferably about 0.05 wt%. The antioxidant serves to inhibit oxidation of any phenolics present in the concentrated soy protein solution.

The optional concentration step and optional diafiltration step may be carried out at any convenient temperature (typically from about 2 ℃ to about 65 ℃, preferably from about 20 ℃ to about 35 ℃) and for a period of time to achieve the desired degree of concentration and diafiltration. The temperature and other conditions used will depend to some extent on the membrane equipment used to carry out the membrane treatment, the desired protein concentration of the solution and the efficiency of contaminant removal for the permeate.

As previously described, heat treatment of the redissolved aqueous soy protein solution can be used to inactivate residual heat-labile trypsin inhibitors. The partially or fully concentrated reconstituted soy protein solution may also be heat treated to inactivate heat labile trypsin inhibitors.

In addition, the concentration and/or diafiltration step may be operated in a manner that facilitates removal of trypsin inhibitors and other contaminants from the permeate. Removal of trypsin inhibitors is facilitated by using membranes with larger pore sizes (e.g., 30,000-1,000,000 daltons), operating the membranes at elevated temperatures (e.g., 30 ℃ -65 ℃) and employing larger volumes (e.g., 20-40 volumes) of diafiltration medium.

Treating the protein solution at a lower pH (1.5-3.0) reduces trypsin inhibitor activity relative to treating the solution at a higher pH (3.0-5.0). When concentrating and diafiltering the protein solution at the lower end of the pH range, it may be desirable to raise the pH of the retentate prior to drying. The pH of the concentrated and diafiltered protein solution may be raised to a desired value, for example pH 3, by the addition of any convenient food grade base, such as sodium hydroxide.

In addition, a reduction in trypsin inhibitor activity can be achieved by exposing the soy material to a reducing agent that cleaves or rearranges the disulfide bonds of the inhibitor. Suitable reducing agents include sodium sulfite, cysteine and N-acetyl cysteine.

Such addition of the reducing agent may be carried out at various stages of the overall process. The reducing agent may be added to the wet protein precipitate resulting from the dilution step, may be added to the protein solution formed by redissolving the precipitate, may be added to the concentrated solution before or after diafiltration or may be dry blended with the dried soy protein product. The addition of the reducing agent may be combined with the heat treatment step and the film treatment step described above.

If it is desired to retain residual active trypsin inhibitors in the concentrated protein solution, this can be achieved by: eliminating or reducing the intensity of the heat treatment step, not utilizing a reducing agent, operating the concentration and diafiltration steps at the higher end of the pH range (3.0-4.4), utilizing concentration and diafiltration membranes with smaller pore sizes, operating the membranes at lower temperatures and using a smaller volume of diafiltration medium.

The redissolved, optionally concentrated, and optionally diafiltered aqueous protein solution may be treated with an adsorbent, such as powdered activated carbon or granular activated carbon, to remove color and/or odor compounds. Such adsorbent treatment may be carried out under any convenient conditions (typically at the ambient temperature of the protein solution). For powdered activated carbon, amounts of about 0.025% to about 5% w/v, preferably about 0.05% to about 2% w/v, are employed. The adsorbent may be removed from the soy protein solution by any convenient means, such as by filtration.

The redissolved, optionally concentrated and optionally diafiltered aqueous soy protein solution may then be dried by any convenient technique, such as spray drying or freeze drying. The protein content of the dried soy protein product is at least about 60 wt% (N × 6.25) d.b., preferably more than about 90 wt% (N × 6.25) d.b., more preferably at least about 100 wt% (N x 6.25.25) d.b.

According to another aspect of the present invention, the mixture of concentrated protein solution and dilution water may be treated without a fractionation step. In this case, the dilution is usually about 1 to 25 times, preferably about 3 to about 12 times. The temperature of the water mixed with the concentrated protein solution is from about 1 ℃ to about 65 ℃, preferably from about 20 ℃ to about 35 ℃.

The dilution water containing the precipitated protein precipitate is adjusted to a pH of about 1.5 to about 4.4, preferably about 2.0 to about 4.0, using any convenient acid, such as hydrochloric acid or phosphoric acid. The adjustment of the pH causes the protein deposited by dilution to re-dissolve. The protein solution may be used in wet form or may be dried to dry form by any convenient technique, such as spray drying or freeze drying.

As another alternative, the protein solution formed by pH adjusting the mixture of protein precipitate and supernatant may be processed using the same steps described above for the separated precipitate that is re-solubilized by pH adjustment.

The aqueous soy protein solution, optionally concentrated, optionally diafiltered, optionally heat treated, optionally finished, optionally adsorbent treated, may then be dried by any convenient technique, such as spray drying or freeze drying. The protein content of the dried soy protein product is greater than about 60 wt.% protein, preferably at least about 90 wt.%, more preferably about 100 wt.% (N x6.25) d.b..

The soy protein products produced herein are soluble in acidic aqueous environments, making the products ideal for incorporation into beverages (both carbonated and non-carbonated) to provide protein fortification thereto. Such beverages have a wide range of acidic pH values ranging from about 2.5 to about 5. The soy protein products provided herein can be added to such beverages in any convenient amount to provide protein fortification to such beverages, for example, at least about 5g soy protein per serving. The added soy protein product dissolves in the beverage and does not impair the clarity of the beverage, even after heat treatment. The soy protein product may be blended with the dried beverage prior to reconstituting the beverage by dissolving in water. In some instances, it may be necessary to alter the normal formulation of the beverage to allow for the composition of the present invention as the components present in the beverage may adversely affect the ability of the composition to remain dissolved in the beverage.

Examples

Example 1：

This example illustrates the preparation of a clear, heat stable protein solution by extraction with calcium chloride solution at low pH.

Soy white flakes (10 g) were mixed with 0.15M calcium chloride solution (100 ml) and the pH of the sample was immediately adjusted to 4.8 and 1.5 with HCl. The sample was extracted at room temperature for 30 minutes using a magnetic stirrer. During the 30 minute extraction, the pH of the sample was monitored and adjusted 2 times. The extract was separated from the spent meal (ent meal) by centrifugation at 10,200 g for 10 minutes and the centrate (centrate) was further clarified by filtration using a filter paper with a 25 μm pore size. The clarity of the filtrate was measured using a HunterLab ColorQuest XE operated in transmission mode to provide a percent haze reading. The sample was then diluted with 1 volume of reverse osmosis purified water and the turbidity level was measured again. The pH of the diluted sample was then adjusted to 3 using either HCl or NaOH as needed. The pH adjusted samples were then analyzed for turbidity levels. The samples were then heat treated to 95 ℃ for 30 seconds, immediately cooled to room temperature in ice water, and the turbidity level was re-evaluated.

The haze values determined for the various samples are shown in tables 1 and 2.

TABLE 1 haze values for samples extracted with calcium chloride solution at pH 1.5

TABLE 2 haze values for samples extracted with calcium chloride solution at pH 4.8

As can be seen from the results presented in tables 1 and 2, the initial filtrate was somewhat turbid, but improved clarity has been obtained by using finer filters. Dilution with 1 volume of water improved the clarity of the pH 1.5 sample, but the precipitate was introduced in the pH 4.8 sample. Adjusting the pH of the diluted sample to 3 gives good clarity to the sample that was originally at pH 4.8, whereas the sample that was originally at pH 1.5 may have slight turbidity. After heat treatment, both samples were considered clear.

Example 2:

this example illustrates the preparation of a soy protein isolate according to one embodiment of the present invention.

20 kg of defatted minimally heat treated soy flour was added to 200L of 0.15M calcium chloride solution at ambient temperature and agitated for 30 minutes to provide an aqueous protein solution. Immediately after the powder was dispersed in the calcium chloride solution, the pH of the system was adjusted to 3 by adding dilute HCl. During the course of the 30 minute extraction, the pH was monitored and periodically corrected to 3. Residual soybean powder was removed by centrifugation to give 174L of a protein solution having a protein content of 3.37% by weight. The protein solution was then mixed with 174L of reverse osmosis purified water and the pH was corrected to 3. The solution was then polished by filtration to give 385L of filtered protein solution having a protein content of 1.21 wt%.

The volume of the filtered protein solution was reduced to 25L by concentration on a PVDF membrane with a molecular weight cutoff of 5,000 daltons. The concentrated protein solution was then diafiltered with 125L of reverse osmosis purified water. The protein content of the resulting diafiltered, concentrated protein solution was 14.51 wt% and indicated that 81.3 wt% of the filtered protein solution was obtained. The diafiltered, concentrated protein solution was then dried to give a product with a protein content found to be 99.18% (N × 6.25) d.b. The product is designated as S005-A13-09A S703.

S005-A13-09A S703, sufficient to supply 0.48 g of protein, was dissolved in 15 ml of reverse osmosis purified water and the Color and clarity of the solution was evaluated using a HunterLab Color Quest XE instrument operating in transmission mode. The pH of the solution was measured using a pH meter.

The pH, color and clarity values are shown in table 3 below:

TABLE 3pH and HunterLab score of the solution of-S005-A13-09A S703

As can be seen from table 3, the aqueous solution of S703 was translucent, not transparent. The relatively high level of turbidity in this sample resulted in slightly lower values of L x than expected.

The Color of the dry powder was also evaluated with a HunterLab Color Quest XE instrument in reflectance mode. Color values are shown in table 4 below:

TABLE 4HunterLab score of dry powder S005-A13-09A S703

As can be seen from Table 4, the dried product was very pale in color.

Example 3:

this example includes an evaluation of the thermal stability in water of the soy protein isolate (S703) produced by the method of example 2.

A solution of S005-A13-09AS703 was prepared by dissolving protein powder sufficient to supply 0.8 g of protein in 40 ml of RO water, and then adjusting the pH to 3. The clarity of the solution was evaluated by turbidity measurements with a HunterLab Color Quest XE instrument. The solution was then heated to 95 ℃, held at this temperature for 30 seconds, and then immediately cooled to room temperature in an ice bath. The clarity of the heat treated solution was then measured again.

The clarity of the protein solution before and after heating is shown in table 5 below:

TABLE 5Effect of Heat treatment on clarity of the S005-A13-09A S703 solution

As can be seen from the results in Table 5, the starting solution of S005-A13-09A S703 was found to be quite turbid. However, the solution is thermally stable and the turbidity level is actually slightly reduced by the heat treatment.

Example 4:

this example includes an evaluation of the solubility in water of the soy protein isolate (S703) produced by the method of example 2. Solubility was tested based on protein solubility (referred to as the protein method, a modified version of the procedure in Morr et al, J. Food Sci. 50: 1715-1718) and total product solubility (referred to as the pellet method).

Protein powder sufficient to supply 0.5 g of protein was weighed into a beaker, then a small amount of Reverse Osmosis (RO) purified water was added and the mixture was stirred until a smooth paste was formed. Additional water was then added to bring the volume to about 45 ml. The contents of the beaker were then slowly stirred for 60 minutes using a magnetic stirrer. The pH was measured immediately after protein dispersion and adjusted to the appropriate level with dilute NaOH or HCl (2, 3, 4, 5, 6 or 7). Samples were also prepared at natural pH. For the pH adjusted samples, the pH was measured during 60 minutes stirring and corrected twice. After 60 minutes of stirring, the sample was made up to a total volume of 50ml with RO water to give a 1% w/v protein dispersion. The protein content of the dispersions was measured using a Leco FP528 Nitrogen terminator (Nitrogen Determinator). An aliquot (20ml) of the dispersion was then transferred to a pre-weighed centrifuge tube that had been dried overnight in a 100 ℃ oven and then cooled in a desiccator, and the tube was capped. The sample was centrifuged at 7,800 g for 10 minutes, which sedimented insoluble material and yielded a clear supernatant. The protein content of the supernatant was measured by Leco analysis, then the supernatant and the tube cap were discarded, and the pellet material was dried overnight in an oven set at 100 ℃. The next morning the tubes were transferred to a desiccator and allowed to cool. The weight of the dry precipitate material was recorded. The dry weight of the original protein powder was calculated by multiplying the weight of the powder used by a factor ((100-moisture content of powder (%)/100). The solubility of the product was then calculated in two different ways:

1) solubility (protein method) (%) = (% protein in supernatant/% protein in initial dispersion) × 100

2) Solubility (sediment method) (%) = (1- (insoluble sediment material dry weight/((20 ml dispersion weight/50 ml dispersion weight) × protein powder initial dry weight))) × 100

The natural pH of the protein isolate produced in example 1 in water (1% protein) is shown in table 6:

table 6-natural pH of S703 solution prepared with 1% protein in water

Batches of	Product(s)	Natural pH
			S005-A13-09A	S703	3.36

The solubility results obtained are shown in tables 7 and 8 below:

TABLE 7Solubility of S703 at different pH values based on the protein method

TABLE 8Solubility of S703 at different pH values based on the precipitation method

As can be seen from the results in tables 7 and 8, the S703 product is highly soluble at pH values of 2, 3 and 7 as well as at natural pH. Solubility was slightly low at pH 4.

Example 5：

This example includes an evaluation of the clarity in water of the soy protein isolate (S703) produced by the method of example 2.

The clarity of a 1% w/v protein solution prepared as described in example 4 was evaluated by measuring absorbance at 600nm, where a lower absorbance score indicates greater clarity. Analysis of the sample in transmission mode on a HunterLab ColorQuest XE instrument also provides a percent haze reading, another measure of clarity.

Clarity results are shown in tables 9 and 10 below:

TABLE 9Clarity of the S703 solution evaluated by A600 at different pH values

Watch 10Clarity of the S703 solution as assessed by the HunterLab assay at different pH values

As can be seen from the results of tables 9 and 10, the solution of S703 was clear to slightly turbid at pH 2-3. A slightly cloudy solution was also obtained at pH 7.

Example 6：

This example includes an evaluation of the solubility of the soy protein isolate (S703) produced by the method of example 2 in soft drinks (Sprite) and sports drinks (Orange Gatorade). The protein was added to the beverage and the solubility was determined without pH correction and the solubility was again determined by adjusting the pH of the protein fortified beverage to the level of the original beverage.

In evaluating solubility without pH correction, a sufficient amount of protein powder to supply 1 g of protein was weighed into a beaker and a small amount of beverage was added and stirred until a smooth paste was formed. Additional beverage was added to bring the volume to 50ml and then the solution was stirred slowly on a magnetic stirrer for 60 minutes to give a 2% protein w/v dispersion. Samples were analyzed for protein content using a Leco FP528 nitrogen terminator (nitrogen determinator) and aliquots of the protein containing beverages were then centrifuged at 7,800 g for 10 minutes and the protein content of the supernatant measured.

Solubility (%) = (% protein in supernatant/% protein in initial dispersion) × 100

In assessing solubility by pH correction, the pH of the protein-free soft drink (Sprite) (3.39) and sports drink (Orange Gatorade) (3.19) was measured. A sufficient amount of protein powder to supply 1 g of protein was weighed into a beaker and a small amount of beverage was added and stirred until a smooth paste was formed. Additional beverage was added to bring the volume to approximately 45ml and the solution was then stirred slowly on a magnetic stirrer for 60 minutes. The pH of the protein-containing beverage was measured and then adjusted to the original protein-free pH with HCl or NaOH as needed. The total volume of each solution was then brought to 50ml with additional beverage to give a 2% protein w/v dispersion. Samples were analyzed for protein content using a Leco FP528 Nitrogen terminator (Nitrogen Determinator) and aliquots of the protein containing beverages were then centrifuged at 7,800 g for 10 minutes and the protein content of the supernatant measured.

Solubility (%) = (% protein in supernatant/% protein in initial dispersion) × 100

The results obtained are shown in table 11 below:

TABLE 11Solubility of S703 in Sprite and Orange Gatorade

As can be seen from the results of table 11, S703 is highly soluble in Sprite and Orange Gatorade. Since S703 is an acidified product, the addition of protein has little effect on the pH of the beverage.

Example 7：

This example includes an evaluation of the clarity of the soy protein isolate (S703) produced by the method of example 2 in soft drinks and sports drinks.

The clarity of the 2% w/v protein dispersions prepared in soft drinks (Sprite) and sports drinks (OrangeGatorade) in example 6 was evaluated using the method described in example 5. For absorbance measurements at 600nm, the spectrophotometer was blanked with the appropriate beverage before the measurements were taken.

The results obtained are shown in tables 12 and 13 below:

TABLE 12Clarity of S703 in Sprite and Orange Gatorade (A600)

Watch 13S703 HunterLab turbidity readings in Sprite and Orange Gatorade

As can be seen from the results in tables 12 and 13, the good solubility results obtained in Sprite and Orange Gatorade for S703 did not translate to clarity in these beverages. In fact, the resulting solution was very cloudy.

Example 8:

this example illustrates the preparation of a soy protein isolate according to a further embodiment of the present invention.

At ambient temperature will be 100g defatted Soybean white flakes to 1000 ml of 0.15M CaCl₂To the solution and stirred for 30 minutes to provide an aqueous protein solution. Immediately after wetting the flakes with calcium chloride solution, the pH of the system was adjusted to 4.5 with hydrochloric acid solution. Throughout the 30 minute extraction, the pH was monitored and periodically corrected. After the extraction step, residual soy white flakes were removed and the resulting protein solution was clarified by centrifugation and filtration to produce 578 ml of filtered protein solution having a protein content of 2.05 wt%.

530 ml of the protein extract solution was reduced to 45ml on a polyethersulfone membrane having a molecular weight cutoff of 10,000 daltons, producing a concentrated protein solution having a protein content of 19.40 weight percent. The concentrated protein solution was then split into two portions.

20ml of the concentrated protein solution was diluted at 24 ℃ into 200 ml of Reverse Osmosis (RO) purified water at 24 ℃. A white haze formed and was allowed to settle. The sample is then centrifuged to separate the protein precipitate from the supernatant fraction. 5.72g of wet protein precipitate was collected and then redissolved in 20ml of RO water, where HCl solution was added to lower the pH to 2.99. The re-solubilized protein precipitate (recovered in a 23.8 wt% yield of filtered protein solution) was freeze-dried to provide the product designated as S703-7300. The protein content of the dried product was found to be 101.75% (N × 6.25) d.b..

Another 21 ml of the concentrated protein solution was diluted into 210 ml RO water at 24 ℃. The pH of the sample was then lowered from 4.76 to 2.98 with HCl solution. The volume of 220 ml of acidified solution was reduced to 33 ml on a polyethersulfone membrane with a molecular weight cutoff of 10,000 daltons, producing a concentrated protein solution with a protein content of 9.76 wt.%. The concentrated protein solution (recovered in a 30.1 wt% yield of the filtered protein solution) was freeze-dried to provide the product designated as S703-7301. The protein content of the dried product was found to be 92.21% (N × 6.25) d.b..

Solutions of S703-7300 and S703-7301 were prepared by dissolving enough powder to provide 0.48 g of protein in 15 ml of RO water. The color and clarity of the solution were evaluated using a HunterLab ColorQuest XE operated in transmission mode. The pH of the solution was measured with a pH meter.

The pH, color and clarity values are shown in table 14 below.

TABLE 14 pH and HunterLab score of the solutions S703-7300 and S703-7301

As can be seen from the results presented in Table 14, the S703-7300 and S703-7301 solutions were translucent and light in color.

Example 9:

this example illustrates the formation of a protein precipitate upon dilution of a concentrated protein solution prepared at low pH, then pH adjusted prior to the dilution step.

100 g of defatted soy white flakes was added to 1000 ml of 0.15M CaCl at ambient temperature₂To the solution and stirred for 30 minutes to provide an aqueous protein solution. Immediately after wetting the flakes with calcium chloride solution, the pH of the system was adjusted to 3.0 with hydrochloric acid solution. Throughout the 30 minute extraction, the pH was monitored and periodically corrected. After the extraction step, residual soy white flakes were removed and the resulting protein solution was clarified by centrifugation and filtration to produce 568 ml of filtered protein solution having a protein content of 2.78 wt%.

550 ml of protein extract solution was reduced to 84ml on a polyethersulfone membrane with a molecular weight cutoff of 10,000 daltons, producing a concentrated protein solution with a protein content of 15.18 weight percent.

The ultrafiltration retentate, pH 3.11, was divided into equal portions and pH adjusted to approximately 4, 5, 6 or 7 with 6M NaOH and 0.5M HCl as needed. The protein content of the pH adjusted retentate sample was measured. An aliquot of the clarified pH-adjusted retentate sample was centrifuged at 7,800 g for 10 minutes and the protein content of the centrate was then determined. An additional aliquot of the pH adjusted retentate sample was diluted with 10 volumes of RO water, mixed with vortex, and the pH, conductivity, a600 and protein content of the diluted sample were determined. The diluted sample was clarified by centrifugation at 7,800 g for 10 minutes, and the protein content of the centrate was then determined.

Raising the pH of the retentate causes all samples to become more cloudy regardless of the final pH. Measurement of protein content before and after clarification indicated that about 20% of the protein in the sample was precipitated by pH adjustment (table 15).

TABLE 15 protein content of pH adjusted retentate samples before and after clarification

Dilution of the pH adjusted retentate samples resulted in very turbid samples, especially when the retentate was at pH 4 and higher (table 16). Analysis of the protein concentration of the samples before and after clarification showed that some of the protein precipitated at all pH values, but especially when the pH of the retentate was 4 or higher before the dilution step. The high degree of protein precipitation in the samples at pH 4-7 indicates that the dilution step introduces protein precipitation in excess of that induced by pH adjustment.

TABLE 16-Properties of pH adjusted (no clarification) retentate samples after dilution

Summary of the disclosure

In summary of the present disclosure, the present invention provides a method for producing a soy protein isolate soluble in acidic media based on the extraction of a soy protein source material at low pH using an aqueous calcium chloride solution. Modifications are possible within the scope of the invention.

Claims (37)

1. A method of producing a soy protein product having a soy protein content of at least 60 wt% (N x6.25) on a dry weight basis, characterized in that:

(a) extracting the soy protein source with an aqueous calcium salt solution, optionally containing an antioxidant, at a pH of 1.5 to less than 5.0 to cause solubilization of soy protein from the soy protein source and to form an aqueous soy protein solution,

(b) at least partially separating the aqueous soy protein solution from residual soy protein source,

(c) optionally diluting said aqueous soy protein solution, and

(A) (d) adjusting the pH of the aqueous protein solution to a value in the range of 1.5-5.0 and different from the pH of the extraction,

(e) optionally finishing the aqueous soy protein solution to remove residual particles,

(f) concentrating the aqueous soy protein solution by using a selective membrane technique while keeping the ionic strength constant,

(g) optionally diafiltering the concentrated soy protein solution,

(h) optionally adjusting the pH of the concentrated and optionally diafiltered soy protein solution to a value in the range of 1.5-7.0,

(i) diluting the concentrated and optionally diafiltered and pH adjusted soy protein solution to water,

(j) separating the precipitate formed from the dilution water, called supernatant, and

(ki) drying the isolated soy protein precipitate, or

(kii) washing the isolated soy protein with 1-10 volumes of water and recovering the washed precipitate; or

(kiii) dissolving the isolated soy precipitate in water at low pH to form a soy protein solution, which is optionally dried, or

(B) (d) optionally finishing the aqueous soy protein solution to remove residual particles,

(e) concentrating the aqueous soy protein solution by using a selective membrane technique while keeping the ionic strength constant,

(f) optionally diafiltering the concentrated soy protein solution, and

(g) the concentrated and optionally diafiltered soy protein solution is diluted to form a precipitate, which is redissolved in dilution water by pH adjustment to form a soy protein solution.

2. The method of claim 1, characterized in that said extraction step is carried out using an aqueous calcium chloride solution having a concentration of less than 1.0M.

3. The method of claim 2, characterized in that said extraction step is carried out using an aqueous calcium chloride solution having a concentration of 0.10-0.15M.

4. The process of claim 1 or 2, characterized in that said extraction step is carried out at a temperature of 15 ℃ to 65 ℃ to produce said aqueous soy protein solution having a protein content of 5 to 50 g/L.

5. The process of claim 4, characterized in that said extraction step is carried out at a temperature of 20 ℃ to 35 ℃ to produce said aqueous soy protein solution.

6. The process of claim 1 or 2, characterized in that the pH is adjusted to 1.5 to 4.4 in step (a) (d).

7. The process of claim 6, characterized in that the pH is adjusted to 2.0 to 4.0 in step (A) (d).

8. The process of claim 1 or 2, characterized in that in step (c), the aqueous soy protein solution is diluted to a conductivity of less than 90 mS.

9. The process of claim 8, characterized in that in step (c), said aqueous soy protein solution is diluted with 0.5 to 10 volumes of aqueous diluent to provide a conductivity of said soy protein solution of 4 to 31 mS.

10. The process according to claim 9, characterized in that the temperature of the aqueous diluent is between 2 ℃ and 70 ℃.

11. The method of claim 10, characterized in that the temperature is 20 ℃ to 35 ℃.

12. The method of claim 1 or 2, characterized in that the conductivity of the soy protein solution after dilution in step (c) and pH adjustment in step a (d) is less than 95 mS.

13. Method according to claim 12, characterized in that the conductivity is 4-36 mS.

14. The process according to claim 1 or 2, characterized in that the soy protein solution is concentrated in step (A) (f) or (B) (e) to produce a concentrated soy protein solution having a protein concentration of 50-300 g/L, and wherein the concentration step is carried out by ultrafiltration at a temperature of 2-65 ℃ using a membrane having a molecular weight cutoff of 3,000-1,000,000 daltons.

15. The method of claim 14 characterized in that the concentrated soy protein solution has a protein concentration of 100-.

16. The process as claimed in claim 14, characterized in that the soy protein solution is subjected to an optional diafiltration step (a) (g) or (B) (f) before or after partial or complete concentration of the soy protein solution using 2-40 volumes of water, dilute brine, acidified water or acidified dilute brine, said diafiltration being carried out until no contaminants or visible colour are present in the permeate and/or until the retentate has been sufficiently purified to provide a soy protein isolate having a protein content of at least 90 wt% (N x6.25) d.b. after subsequent processing and upon drying, wherein said diafiltration is carried out using a membrane having a molecular weight cutoff of 3,000-1,000,000 dalton, wherein an antioxidant is optionally present in the diafiltration medium during at least part of the diafiltration step.

17. The method according to claim 16, characterized in that the optional diafiltration step is performed using 5-25 volumes of water, dilute brine, acidified water or acidified dilute brine, using a membrane with a molecular weight cut-off of 5,000-100,000 daltons.

18. The process of claim 16, characterized in that the concentration and optional diafiltration steps are operated in a manner that facilitates removal of trypsin inhibitors.

19. The process of claim 16, characterized in that the concentration steps (a) (f) and (B) (e) and optional diafiltration steps (a) (g) and (B) (f) are carried out at a temperature of 2 ℃ to 65 ℃.

20. The method of claim 19, characterized in that the temperature is 20 ℃ to 35 ℃.

21. The process according to claim 19, characterized in that the concentrated and optionally diafiltered soy protein solution is pasteurized before drying, and it is carried out at a temperature of 55 ℃ to 70 ℃ for 30 seconds to 60 minutes.

22. The process of claim 1 or 2, characterized in that a reducing agent is present during the extraction step and during the concentration and/or optional diafiltration step, and/or is added to the concentrated and optionally diafiltered soy protein solution and/or to the dried soy protein product prior to drying, such that the disulfide bonds of the trypsin inhibitor are cleaved or rearranged to achieve a reduction in trypsin inhibitor activity.

23. The process of claim 1 or 2, characterized in that step (a) (h) is carried out to a pH of 4.0-7.0.

24. The method of claim 23, characterized in that step (a) (h) is carried out to a pH of 5.0-7.0.

25. The process of claim 1 or 2, characterized in that the dilution step (a) (i) is carried out 5-to 25-fold with water, and wherein the temperature of the water used for carrying out the dilution is between 1 ℃ and 65 ℃.

26. The process of claim 25, characterized in that the dilution step (a) (i) is carried out 10-to 20-fold and the temperature is 20 ℃ to 35 ℃.

27. The process according to claim 1 or 2, characterized in that in step (a) (kii) the precipitate is washed with 2-3 volumes of water.

28. The process of claim 27, characterized in that in step (a) (kii) the precipitate is washed with 2-3 volumes of water.

29. The process of claim 1 or 2, characterized in that in step (a) (kiii) the precipitate is dissolved in 1-10 volumes of water at a pH of 1.5-4.4 to form a soy protein solution which can be dried.

30. The method of claim 29, characterized in that in step (a) (kiii) the precipitate is dissolved in 2-3 volumes of water at a pH of 2.0-4.0.

31. The process according to claim 1 or 2, characterized in that the concentrated and optionally diafiltered soy protein solution is subjected to a heat treatment step to inactivate heat-labile anti-nutritional factors, said heat treatment step further subjecting each solution to pasteurization, said heat treatment being carried out at a temperature of 70-160 ℃ for 10 seconds-60 minutes, and the heat treated soy protein solution is cooled to a temperature of 2-65 ℃ for further processing.

32. The method of claim 31, characterized in that the heat treatment is carried out at a temperature of 85 ℃ to 95 ℃ for 30 seconds to 5 minutes and the heat treated soy protein solution is cooled to a temperature of 20 ℃ to 35 ℃ for further processing.

33. The process of claim 32, characterized in that the heat-treated soy protein solution, the concentrated and optionally diafiltered solution and/or the cooled soy protein solution is treated with an adsorbent to remove color and/or odor compounds.

34. The method of claim 32, characterized in that the heat-treated soy protein solution or the cooled soy protein solution is dried to form a soy protein product having a protein content of at least 90 wt% (N x6.25) d.b..

35. The process of claim 1 or 2, characterized in that step (a) (h) is carried out at a pH of 1.5 to 7.0.

36. The process of claim 35, characterized in that step (a) (h) is carried out at a pH of 5.0 to 7.0.

37. A soy protein product produced by the method of any of claims 1-36, which is blendable with a water-soluble powdered material for producing an aqueous solution of a blend, wherein the blend is optionally a powdered beverage or an aqueous solution that can be a beverage having a pH in the range of 6-8 in which the soy protein product is soluble.