HK1167575B - Production of soluble soy protein product from soy protein micellar mass - Google Patents

Description

Preparation of soluble soy protein products from soy protein micellar mass

Reference to related applications

This application claims priority from 61/202,055 filed on 26/1/2009 and U.S. provisional patent application No. 61/272,289 filed on 8/9/2009, according to 35USC 119 (e).

Technical Field

The present invention relates to the production of soy protein products. The present invention particularly relates to the preparation of a soluble soy protein product ("S200 Ca") from a soy protein micellar mass.

Background

In 61/107,112 (7865-. The soy protein product is particularly useful for protein fortification of soft drinks and sports drinks and other acidic aqueous systems without precipitation of the protein. The protein product is produced by: the pH of the aqueous soy protein solution is adjusted to a pH of about 1.5 to about 4.4, preferably about 2.0 to about 4.0, by extracting the soy protein source with an aqueous calcium chloride solution at natural pH, optionally diluting the resulting aqueous soy protein solution, 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 found that the process stream resulting from precipitation of a soy protein micellar mass can be further treated to provide a soy protein product having a protein content of at least about 60 wt% (N x 6.25) d.b., which is soluble in acidic media and produces a clear, heat stable solution at low pH values and thus can be used in particular for protein fortification of soft drinks and sports drinks and other aqueous systems without precipitation of protein. 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 x 6.25) d.b..

According to one aspect of the present invention, there is provided a process for preparing a soy protein product having a protein content of at least about 60 wt% (N x 6.25) on a dry weight basis, comprising:

adding a calcium salt or other divalent salt, preferably calcium chloride, to the supernatant from the soy protein micellar pellet to provide a conductivity of about 2mS to about 30mS, preferably about 8 to about 15mS,

removing the precipitated phytate material from the resulting solution to leave a clear solution,

the pH of the clear solution is optionally adjusted to about 1.5 to about 4.4, preferably about 2.0 to about 4.0, for example by the addition of hydrochloric acid,

concentrating the optionally pH adjusted clear solution to a protein content of about 50 to about 400g/L, preferably about 100 to about 250g/L, to produce a clear concentrated soy protein solution,

optionally diafiltering the clarified soy protein solution, e.g., with about 2 to about 40 volumes of water, preferably about 5 to about 25 volumes of water,

optionally a decolourisation step, e.g. treatment with granulated activated carbon, and

the concentrated protein solution is dried.

The supernatant may be partially concentrated to an intermediate concentration before the calcium salt is added. The precipitate formed is removed, the resulting solution is optionally acidified as described above, further concentrated to a final concentration, then optionally diafiltered and dried.

Alternatively, the supernatant may be first concentrated to a final concentration, calcium salt added to the concentrated supernatant, the resulting precipitate removed, the solution optionally acidified, then optionally diafiltered and dried.

One option in the above process is to omit acidification and perform the solution treatment at natural pH. In this option, calcium salt is added to the supernatant, partially concentrated supernatant or concentrated supernatant to form a removed precipitate. The resulting solution was then treated as described above without an acidification step.

When the supernatant is partially concentrated before the addition of the calcium salt and completely concentrated after removal of the precipitate, the supernatant is first concentrated to a protein concentration of about 50g/L or less and then, after removal of the precipitate, concentrated to a concentration of about 50 to about 400g/L (preferably about 100 to about 250 g/L).

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 x 6.25) d.b..

In another aspect of the present invention, we have found that an equivalent product can be produced from soy by treating a soy protein solution derived from the extraction of the sodium salt of a soy protein source material, by concentrating the soy protein solution, optionally diafiltering the concentrated soy protein solution, optionally adjusting the pH of the solution to about 2 to about 4, and drying the acidified solution. According to this aspect of the invention, there is provided a process for preparing a soy protein product having a protein content of at least about 60 wt% (N x 6.25) dry weight, comprising:

extracting the soy protein source to solubilize soy protein in the source material and form an aqueous soy protein solution having a pH of about 5 to about 7,

concentrating the aqueous soy protein solution to a concentration of about 50 to about 400g/L to form a concentrated soy protein isolate,

optionally diafiltering the soy protein solution before or after complete concentration,

optionally adjusting the pH of the concentrated and diafiltered soy protein solution to about 2 to about 4 to provide a clear acidified soy protein solution, and

the soy protein solution is 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 x 6.25) d.b..

It has also been found that soy protein isolate formed as a protein micellar mass and soy protein isolate obtained from supernatant from protein micellar mass precipitation are soluble in acidic media and can be used to provide aqueous solutions of acceptable clarity.

While the present invention is primarily directed to the production of soy protein isolate, it is also contemplated that a lower purity soy protein product having similar properties to soy protein isolate may be provided. Such lower purity products may have a protein concentration of at least about 60 wt% (N × 6.25) d.b..

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

The soy protein products provided herein can be provided as aqueous solutions thereof that have high clarity at acid pH values and are heat stable at these pH values.

In another aspect of the invention, there is provided an aqueous solution of the soy product provided herein, which is heat stable at low pH. 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 products produced according to the methods herein lack the characteristic soy flavor of soy protein isolates and are not only suitable for protein fortification of acidic media, but are also useful in a variety of conventional applications of protein isolates, including but not limited to protein fortification of processed foods and beverages, emulsification of oils, as a bulk forming agent in baked goods, and as a foaming agent in gas-entrapped products. In addition, soy protein products can be formed into protein fibers, can be used in meat analogs, and as protein substitutes or supplements in food products where protein is used as a binder. The soy protein product can be used for nutritional supplementation. Other uses of soy protein products are in pet food, animal feed, industrial and cosmetic applications, and personal care products.

Detailed Description

The initial step of the process of providing a soy protein product comprises solubilizing soy protein from a soy protein source. The soy protein source may be soy, or any soy product or by-product derived from the processing of soy, including but not limited to soy flour (meal), soy flakes, soy flour (grits), and soy flour (flour). The soy protein source may be used in a full fat, partially defatted or fully defatted form. Where the soy protein source contains a substantial amount of fat, a degreasing step is typically required in 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 having the characteristic hydrophobicity and polarity of the native protein.

Protein solubilization can be carried out by using food grade sodium salt solutions (e.g., food grade sodium chloride solutions). Non-food grade chemicals may be used when the soy protein isolate is used for non-food applications. Other monovalent salts, such as potassium chloride, may also be used. As the salt solution concentration increases, the solubility of the protein from the soy protein source initially increases until a maximum value is reached. Any subsequent increase in salt concentration did not increase the total dissolved protein. The concentration of the salt solution that causes maximum protein solubilization varies with the salt involved. The choice of the concentration of the sodium salt solution is also influenced by the proportion of protein that is desired to be obtained by the micellar route. Higher salt concentrations (preferably from about 0.5M to about 1.0M) generally result in more protein micellar mass when the concentrated soy protein solution is diluted into cold water. The extraction may be performed with a higher concentration of sodium chloride solution, or alternatively, with a solution of less than 0.5M sodium chloride (e.g., 0.10M or 0.15M sodium chloride), and then, after removal of the soy protein source, additional salt is added to the soy protein solution.

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

In a continuous process, the extraction of protein from the soy protein source is performed in any manner consistent with the continuous extraction of protein from the soy protein source. In one embodiment, the soy protein source is continuously mixed with a food grade salt solution, and the mixture is conveyed through a tube 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 this continuous process, the salt solubilization step is rapidly conducted in up to about 10 minutes, preferably to effect solubilization to substantially extract as much protein from the soy protein source as is practicable. Dissolution in a continuous process is carried out at a temperature between about 1 ℃ and about 100 ℃, preferably between about 15 ℃ and about 35 ℃.

The extraction may be carried out at the natural pH of the soy protein source/salt solution system (typically about 5 to about 7). Alternatively, for use in the extraction step, the pH of the extraction may be adjusted to any desired value in the range of from about 5 to about 7 using any suitable acid (typically hydrochloric acid) or base (typically sodium hydroxide) as desired.

The concentration of the soy protein source in the food grade 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 the aqueous salt solution has the additional effect of solubilizing fats that may be present in the soy protein source, which in turn causes the fats to be present in the aqueous phase.

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

The aqueous salt solution may contain an antioxidant. The antioxidant may be any suitable antioxidant, such as sodium sulfite or ascorbic acid. The amount of antioxidant used may vary from about 0.01 to about 1% by weight of the solution, preferably about 0.05% by weight. 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 suitable manner, for example, using a decanter centrifuge followed by disc centrifugation and/or filtration to remove residual soy protein source material. The separated residual soy protein source may be dried. Alternatively, the separated residual soy protein source may be treated to recover some residual proteins, such as by a conventional isoelectric precipitation process or any other suitable process to recover such residual proteins.

Where the soy protein source contains significant amounts of fat, as described in U.S. patent nos. 5,844,086 and 6,005,076, assigned to the assignee herein, the disclosures of which are incorporated herein by reference, the defatting step described therein can be performed on the isolated aqueous protein solution. Alternatively, degreasing of the separated aqueous protein solution may be accomplished by any other suitable method.

The aqueous soy protein solution may be treated with an adsorbent, such as powdered activated carbon or granular activated carbon, to remove color and/or odor compounds. This adsorbent treatment may be carried out under any suitable 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 utilized. The adsorbent may be removed from the soy protein solution by any suitable method, such as by filtration.

Instead of extracting the soy protein source with aqueous salt solution, this extraction can be performed with water alone. When this alternative is utilized, then the salt can be added to the protein solution after separation from the residual soy protein source at the concentrations discussed above. When the first fat removal step is carried out, the salt is generally added after this operation is completed.

Another alternative process is to extract the soy protein source with a food grade salt solution at a relatively high pH above about 7, generally up to about 11. The pH of the extraction system can be adjusted to the desired base value with any suitable food grade base, such as aqueous sodium hydroxide. Alternatively, the soy protein source may be extracted with a salt solution at a relatively low pH, below about pH 5, typically at a minimum of about pH 3. The pH of the extraction system can be adjusted to the desired acid value by using any suitable food grade acid, such as hydrochloric acid or phosphoric acid. When such an alternative is utilized, then the aqueous phase resulting from the soy protein source extraction step is separated from the residual soy protein source in any suitable manner, for example, using a decanter centrifuge followed by disc centrifugation and/or filtration to remove the residual soy protein source. The separated residual soy protein source may be dried for disposal or further processed to recover residual protein, as discussed above.

The aqueous soy protein solution resulting from the high or low pH extraction step is then adjusted to a pH in the range of about 5 to about 7, as discussed above, prior to further processing as discussed below. This pH adjustment can suitably be carried out with any suitable acid (e.g. hydrochloric acid) or base (e.g. sodium hydroxide). If desired, the protein solution may be clarified by any suitable process, such as centrifugation or filtration, after pH adjustment and prior to further processing.

If the purity is sufficient, the resulting aqueous soy protein solution may be directly dried to produce a soy protein product. To reduce the impurity 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. This concentration is generally carried out to provide a concentrated protein solution having a protein concentration of from about 50g/L to about 400g/L, preferably from about 100 to about 250 g/L.

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

Ultrafiltration and similar selective membrane techniques are known to allow low molecular weight species to pass through the membrane while preventing higher molecular weight species from passing through. 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, such as trypsin inhibitors, which are themselves low molecular weight proteins. The cut-off molecular weight of the membrane is typically selected according to the different membrane materials and structures to ensure that a significant proportion of the protein is retained in solution while allowing contaminants to pass through.

The protein solution may be subjected to a diafiltration step, before or after complete concentration, preferably using a saline solution of the same molar concentration and pH as the extraction solution. If it is desired to reduce the salt content of the retentate, the diafiltration solution used may be a saline solution of the same pH but of lower salt concentration than the extraction solution. However, the salt concentration of the diafiltration solution must be selected so that the salt level in the retentate remains high enough to maintain the desired protein solubility. 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 a diafiltration operation, additional quantities of contaminants may be removed from the aqueous 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. According to one aspect of the invention, if the retentate is to be dried without further processing, diafiltration may be effected until the retentate is sufficiently pure to provide the desired protein concentration when dried, preferably to provide an isolate having a protein content of at least about 90 wt% (N × 6.25) on a dry weight basis. This diafiltration may be performed 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 a molecular weight cut-off in the range of about 3,000 to about 1,000,000 daltons, preferably about 5,000 to about 100,000 daltons, in terms of different membrane materials and configurations.

The concentration step and diafiltration step may be performed 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 x 6.25) d.b., for example at least about 60 wt% protein (N x 6.25) d.b. The contaminants may be only partially removed by partially concentrating and/or partially diafiltering the aqueous soy protein solution. This protein solution can 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 suitable antioxidant, such as sodium sulfite or ascorbic acid. The amount of antioxidant used in the diafiltration medium depends on the material used and may vary from about 0.01 to about 1% by weight, preferably about 0.05% by weight. 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 suitable temperature (generally about 2 ℃ to about 60 ℃, preferably about 20 ℃ to about 35 ℃) for a period of time to achieve the desired concentration and degree of diafiltration. The temperature and other conditions used will depend, in part, on the membrane equipment used to perform 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, the Kunitz inhibitor, which is a heat labile molecule with a molecular weight of about 21,000 daltons, and the Bowman-Birk inhibitor, which is a hotter stable molecule with a molecular weight of about 8,000 daltons. The level of trypsin inhibitor activity in the final soy protein isolate can be controlled by manipulating various process variables.

For example, the concentration and/or diafiltration steps may be operated in a manner that facilitates removal of trypsin inhibitors in the permeate as other contaminants. Removal of trypsin inhibitors may be facilitated by using a larger pore size (e.g., about 30,000 to about 1,000,000Da) membrane, operating the membrane at elevated temperatures (e.g., about 30 to about 60 ℃), and utilizing a larger volume (e.g., about 20 to about 40 volumes) of diafiltration medium.

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

This addition of the reducing agent can be carried out at different 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 membrane treatment steps described above.

If it is desired to retain active trypsin inhibitors in the concentrated protein solution, this can be achieved by using concentration and diafiltration membranes with smaller pore sizes, operating the membranes at lower temperatures, using a smaller volume of diafiltration medium and not using a reducing agent.

If desired, the concentrated and optionally diafiltered protein solution may be subjected to further defatting procedures, 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 suitable method.

The concentrated and 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. This adsorbent treatment may be carried out under any suitable conditions, typically at the 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 utilized. The adsorbent may be removed from the soy protein solution by any suitable method, 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. This pasteurization may be carried out under any desired pasteurization conditions. The concentrated and optionally diafiltered protein solution is typically heated to a temperature of about 55 ℃ to about 70 ℃, preferably about 60 ℃ to about 65 ℃, for about 30 seconds to about 60 minutes, preferably about 10 minutes to about 15 minutes. The pasteurized, concentrated protein solution may then be cooled, preferably to a temperature of about 25 ℃ to about 40 ℃, for further processing as described below.

According to one aspect of the invention, the concentrated and diafiltered soy protein solution is dried to obtain a soy protein product. Alternatively, the pH of the concentrated and diafiltered soy protein solution may be adjusted to a pH of about 2.0 to about 4.0, preferably about 2.9 to about 3.2. The pH adjustment may be carried out in any suitable manner, for example by addition of hydrochloric acid or phosphoric acid. The resulting acidified soy protein solution is then dried. As another alternative, the pH-adjusted soy protein solution may be subjected to a heat treatment to inactivate heat-labile anti-nutritional factors (such as the trypsin inhibitors mentioned above). This heating step also provides the additional benefit of reducing the microbial load. The protein solution is typically heated to a temperature of from about 70 c to about 100 c, preferably from about 85 c to about 95 c, for a period of from about 10 seconds to about 60 minutes, preferably from about 30 seconds to about 5 minutes. The heat treated acidified soy protein solution may then be cooled to a temperature of about 2 ℃ to about 60 ℃, preferably about 20 ℃ to about 35 ℃. The resulting acidified heat treated soy protein solution is then dried.

If desired, the ionic strength of the concentrated and optionally diafiltered protein solution may be increased by the addition of salt to promote the formation of protein micellar mass upon dilution, as an alternative to the ionic strength adjustment operation described above.

Depending on the temperature used in the concentration step and optional diafiltration step and whether a pasteurization step is performed, the concentrated protein solution may be heated to a temperature of at least about 20 ℃, up to about 60 ℃, preferably about 25 ℃ to about 40 ℃, to reduce the viscosity of the concentrated protein solution to facilitate performance of the subsequent dilution step and micelle formation. The concentrated protein solution should not be heated above a temperature at which micelle formation does not occur upon dilution with cooling water.

Then, the concentrated protein solution obtained from the concentration step, the optional diafiltration step, the optional ionic strength adjustment step, the optional defatting step, the optional adsorbent treatment step, and the optional pasteurization step is diluted to form micelles by mixing the concentrated protein solution with cooling water having a volume to achieve the desired dilution. The dilution of the concentrated protein solution may vary depending on the proportion of soy protein desired to be obtained by the micellar route and the proportion from the supernatant. Generally, with lower dilutions, a greater proportion of the soy protein remains in the aqueous phase.

Where it is desired to provide the maximum proportion of protein by the micellar route, the concentrated protein solution is diluted from about 5-fold to about 25-fold, preferably from about 10-fold to about 20-fold.

The cooling water mixed with the concentrated protein solution has a temperature of less than about 15 ℃, typically from about 1 ℃ to about 15 ℃, preferably less than about 10 ℃, because at the dilution factor used, increased yields of protein isolate in the form of protein micellar mass are achieved with these cooler temperatures.

In a batch operation, a batch of concentrated protein solution is added to a static body having a desired volume of cooling water as discussed above. Dilution of the concentrated protein solution and subsequent reduction in ionic strength causes the formation of a cloud of highly cross-linked protein molecules in the form of micellar-type discrete protein droplets. In the batch process, protein micelles are allowed to settle in a body of cooling water to form aggregated, coalesced, thick, amorphous, cohesive gluten-like protein micelles (PMM). Sedimentation may be assisted by, for example, centrifugation. This induced settling reduces the liquid content of the protein micellar mass, thereby reducing the moisture content of the total micellar mass, typically from about 70% to about 95% by weight, to a value typically from about 50% to about 80% by weight. Reducing the moisture content of the micellar mass in this way also reduces the sorbate salt content of the micellar mass and thus the salt content of the dried protein product.

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. 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 protein micelles, and the mixture is continuously fed from the outlet of the T-tube into a settling vessel, from which the supernatant liquid is allowed to overflow when full. The mixture is preferably fed into the liquid body in the settling vessel in such a way as to minimize turbulence in the liquid body.

In a continuous process, protein micelles are allowed to settle in a settling vessel to form an aggregated, coalesced, thickened, amorphous, cohesive gluten-like Protein Micellar Mass (PMM), the process is continued until the desired amount of PMM has accumulated at the bottom of the settling vessel, and the accumulated PMM is then removed from the settling vessel. Instead of sedimentation by precipitation, the PMM can be continuously separated by centrifugation.

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

The settled micellar mass is separated from the residual aqueous phase or the supernatant by, for example, decanting the residual aqueous phase from the settled mass, or by centrifugation. The PMM may be used in wet form or may be dried to a dry form by any suitable technique, such as spray drying or freeze drying. The dried PMM has a high protein content of more than about 90% by weight protein, preferably at least about 100% by weight (calculated as N x 6.25) d.b., and is substantially undenatured. Alternatively, the wet PMM may be adjusted to a pH of from about 2.0 to about 4.0, preferably from about 2.9 to about 3.2. The pH adjustment may be carried out in any suitable manner, for example by addition of hydrochloric acid or phosphoric acid. The resulting acidified soy protein solution is then dried. As another alternative, the pH-adjusted soy protein solution may be subjected to a heat treatment to inactivate heat-labile anti-nutritional factors (such as the trypsin inhibitors mentioned above). This heating step also provides the additional benefit of reducing the microbial load. The protein solution is typically heated to a temperature of from about 70 c to about 100 c, preferably from about 85 c to about 95 c, for a period of from about 10 seconds to about 60 minutes, preferably from about 30 seconds to about 5 minutes. The heat treated acidified soy protein solution may then be cooled to a temperature of about 2 ℃ to about 60 ℃, preferably about 20 ℃ to about 35 ℃. The resulting acidified, heat treated soy protein solution is then dried.

In one aspect of the invention, a calcium salt or other divalent salt (preferably calcium chloride) is added to the supernatant (which may be first concentrated or partially concentrated in the manner described below) to provide a conductivity of from about 2mS to about 30mS, preferably from 8mS to about 15 mS. The calcium chloride added to the supernatant may be in any desired form, for example, a concentrated aqueous solution thereof.

The addition of calcium chloride has the effect of depositing phytic acid from the supernatant in the form of calcium phytate. The precipitated phytate is recovered from the supernatant, for example by centrifugation and/or filtration, to leave a clear solution.

The pH of the clear solution may then be adjusted to a value of from about 1.5 to about 4.4, preferably from about 2.0 to about 4.0. The pH adjustment may be carried out in any suitable manner, for example by addition of hydrochloric acid or phosphoric acid. If desired, once the precipitated phytate material has been removed, the acidification step may be omitted from the different options described herein (except for the heat treatment mentioned below).

The pH-adjusted clear acidified aqueous soy protein solution may be heat treated to inactivate heat-labile anti-nutritional factors such as the trypsin inhibitors mentioned above. This heating step also provides the additional benefit of reducing the microbial load. The protein solution is generally heated to a temperature of from about 70 ℃ to about 100 ℃, preferably from about 85 ℃ to about 95 ℃ for from about 10 seconds to about 60 minutes, preferably from about 30 seconds to about 5 minutes. The heat treated acidified soy protein solution may then be cooled to a temperature of about 2 ℃ to about 60 ℃, preferably about 20 ℃ to about 35 ℃, for further processing as described below.

If not already concentrated, the clear solution, optionally pH-adjusted and optionally heat-treated, is concentrated to increase its protein concentration. This concentration is carried out using any suitable 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 to 1,000,000 daltons, preferably about 5,000 to about 100,000 daltons, can be used in accordance with different membrane materials and configurations. 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 400g/L, preferably about 100 to about 250 g/L. This concentration operation can be carried out either in batch mode or in continuous operation as described above.

When the supernatant is fully concentrated after partial concentration and removal of the precipitate prior to addition of the calcium salt, the supernatant is first concentrated to a protein concentration of about 50g/L or less and then, after removal of the precipitate, concentrated to a protein concentration of about 50 to about 400g/L, preferably about 100 to about 250 g/L.

The protein solution may be subjected to a diafiltration step, preferably using water or a diluted salt solution, before or after partial or complete concentration. The diafiltration solution may be at its natural pH, which is equal to the pH of the protein solution being diafiltered or any pH therebetween. 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 a diafiltration operation, further quantities of contaminants are removed from the aqueous 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 protein solution has been sufficiently purified. This diafiltration may be performed using the same membrane as the concentration step. However, if desired, diafiltration may be effected using a separate membrane, such as a membrane having a molecular weight cut-off in the range of about 3,000 to about 1,000,000 daltons, preferably about 5,000 to about 100,000 daltons, depending on the different membrane materials and configuration.

The concentration step and diafiltration step may be performed 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 x 6.25) d.b., for example at least about 60 wt% protein (N x 6.25) d.b. The contaminants may be only partially removed by partially concentrating and/or partially diafiltering the aqueous soy protein solution. This protein solution can 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 suitable antioxidant, such as sodium sulfite or ascorbic acid. The amount of antioxidant used in the diafiltration medium depends on the material used and may vary from about 0.01 to about 1% by weight, preferably about 0.05% by weight. The antioxidant serves to inhibit oxidation of any phenolics present in the concentrated soy protein isolate solution.

The concentration step and diafiltration step may be carried out at any suitable temperature (typically about 2 ℃ to about 60 ℃, preferably about 20 ℃ to about 35 ℃) for a period of time to achieve the desired concentration and degree of diafiltration. The temperature and other conditions used will depend, in part, on the membrane equipment used to perform the membrane treatment, the desired protein concentration of the solution, and the efficiency of contaminant removal for the permeate.

As described above, the level of trypsin inhibitor activity in the final soy protein product can be controlled by manipulating various process variables.

As previously mentioned, heat treatment of acidified aqueous soy protein solutions can be used to inactivate heat-labile trypsin inhibitors. The partially or fully concentrated acidified soy protein solution may also be heat treated to inactivate heat labile trypsin inhibitors.

In addition, the concentration and/or diafiltration steps may be operated in a manner that facilitates removal of trypsin inhibitors in the permeate as other contaminants. Removal of trypsin inhibitors may be facilitated by using a larger pore size (e.g., about 30,000 to 1,000,000Da) membrane, operating the membrane at elevated temperatures (e.g., about 30 to about 60 ℃), and utilizing a larger volume (e.g., about 20 to about 40 volumes) of diafiltration medium.

Acidification and membrane treatment of the diluted protein solution at lower pH (e.g., about 1.5 to about 3) can reduce trypsin inhibitor activity relative to treating the solution at higher pH (e.g., about 3 to about 4.4). When concentrating and diafiltering the protein solution at the lower end of the pH range, it may be desirable to increase 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 suitable 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 disrupts or rearranges the disulfide bonds of the inhibitor. Suitable reducing agents include sodium sulfite, cysteine and N-acetyl cysteine.

This addition of the reducing agent can be carried out at different 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 clarified aqueous soy protein solution after removal of residual soy protein source material, may be added to the diafiltered retentate prior to dilution, may be added to the supernatant, may be added to the concentrated and diafiltered calcium-modified supernatant prior to drying, or may be dry blended with the dried soy protein product. The addition of the reducing agent may be combined with the above-described heat treatment step and film treatment step.

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 using a reducing agent, operating the concentration and diafiltration steps at the upper end of the pH range (e.g., about 3 to about 4.4), using 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 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. This adsorbent treatment may be carried out under any suitable conditions, typically at the 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 utilized. The adsorbent may be removed from the soy protein solution by any suitable method, such as by filtration.

The pH of the concentrated and optionally diafiltered and optionally adsorbent treated protein solution may be adjusted to about 2.0 to about 4.0 if a pH adjustment step has not been utilized. The protein solution treated with pH adjustment, concentration and optionally diafiltration and optionally adsorbent may also be heat treated as described above to reduce the level of trypsin inhibitor activity.

The concentrated and optionally diafiltered and optionally adsorbent treated protein solution is dried to a dry form by any suitable technique, such as spray drying or freeze drying. The dried soy protein product has a protein content of at least about 60 wt% (N x 6.25) d.b., preferably more than about 90 wt% (N x 6.25) d.b., more preferably at least about 100 wt%. The soy protein product has a low phytic acid content, typically less than about 1.5% by weight.

In one embodiment of the invention, the supernatant from the formation of PMM can be directly processed to form a soy protein product using the above steps, while omitting the addition of calcium chloride. The soy protein product so formed has a protein content of 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%.

The soy protein products produced herein are soluble in an acidic aqueous environment, making the products ideal for incorporation into beverages (including both carbonated and uncarbonated beverages) to provide protein fortification thereto. The beverage has a wide acidic pH range of about 2.5 to about 5. The soy protein product provided herein can be added to such beverages in any suitable amount to provide protein fortification to such beverages, for example, at least about 5g of 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 mixed with the dried beverage prior to reconstituting the beverage by dissolving in water. In some instances, where components present in a beverage may adversely affect the ability of the composition to remain dissolved in the beverage, it may be necessary to alter the normal beverage formulation to allow for the composition of the present invention.

Examples

Example 1：

This example illustrates the production of a protein micellar mass (S300), a protein isolate obtained from supernatant (S200), and a protein isolate obtained from calcium modified supernatant (S200Ca) from soy.

'a' kg defatted minimally heat treated soy flour was added to 'b' L of 'c' M NaCl solution at ambient temperature and stirred for 60 minutes to provide an aqueous protein solution. Residual soy flour was removed and the resulting protein solution was clarified by centrifugation and filtration to produce a'd' L filtered protein solution having a protein content of 'e'% by weight.

The protein extract solution was reduced to 'f' kg by concentration on a 'g' membrane with a molecular weight cut off of 'h' daltons, yielding a concentrated protein solution with a protein content of 'i'% by weight.

The conductivity of the concentrated protein solution was 'j' mS. Concentrated sodium chloride solution was added to the retentate to increase the conductivity to 'k' mS. Then, the concentrated protein solution of 'l' c was diluted'm' into cold RO water having a temperature of 'n' c. A white turbidity immediately formed. The supernatant was removed and the precipitated, sticky mass (PMM) was recovered by centrifugation in a yield of 'o' wt% of the filtered protein solution. The dried PMM was found to give a protein with a protein content of 'p'% (N × 6.25) d.b.. The product was named 'q' S300.

The parameters 'a' to 'q' are listed in the following table 1:

TABLE 1Parameters of production S300

q	S005-J27-08A	S005-K19-08A
			a	10	10
b	200	200
			c	0.15	0.50
d	185	165
			e	0.70	1.34
f	5.28	12.06
			g	PES	PES
h	100,000	100,000
			i	21.28	17.51
j	9.45	24.9
			k	21.4	24.9
l	27.8	30
			m	1∶10	1∶5
n	1.6	4
			o	18.5	20.8

p

91.31

99.66

Supernatants from both experiments were treated differently. The supernatant from the S005-J27-08A assay was treated without calcium modification. In this experiment, 65L of supernatant was concentrated to a volume of 5L on a PES membrane with a molecular weight cutoff of 10,000 daltons, and then diafiltered on the same membrane with 25L of reverse osmosis purified water. The diafiltered retentate had a protein concentration of 12.60% by weight. With additional protein recovered from the supernatant, the overall recovery of the filtered protein solution was 69.2%. The diafiltered retentate was dried to form a product having a protein content of 98.76% (N × 6.25) d.b.. The product was named S005-J27-08AS 200.

The supernatant from run S005-K19-08A was treated with calcium modification. To 65L of the supernatant was added 0.336kg of CaCl₂This increases the conductivity of the solution from 6.31mS to 12.65 mS. Tong (Chinese character of 'tong')The precipitate formed was removed by centrifugation and the pH of the centrate was then adjusted to 3 with dilute HCl. The acidified centrate was then concentrated from a 66L volume to a 5L volume on a PES membrane with a 10,000 dalton cut-off. The concentrate was then diafiltered on the same membrane using 25L of reverse osmosis purified water adjusted to pH 3 with dilute HCl. With additional protein recovered from the supernatant, the overall recovery of the filtered protein solution was 37.1%. The diafiltered retentate was dried to produce a product having a protein content of 98.01% (N × 6.25) d.b.. The product is named as S005-K19-08A S200 Ca.

The color of the dry powdered product was evaluated in reflectance mode using a HunterLab ColorQuest XE instrument. The color values are listed in table 2 below:

TABLE 2HunterLab score of dried product

As can be seen from table 2, the dry color of all products was very light.

Example 2：

This example contains an evaluation of the thermal stability in water of the soy protein isolate (S300, S200Ca) produced by the method of example 1.

A2% w/v protein solution of each product in water was produced and the pH was adjusted to 3. The clarity of these solutions was evaluated by measuring turbidity in transmission mode with a HunterLab ColorQuest XE instrument. The solution was then heated to 95 ℃ and held at this temperature for 30 seconds, and then immediately cooled to room temperature in an ice bath. Then, the clarity of the heat-treated solution was measured again.

The clarity of the protein solution before and after heating is listed in table 3 below:

TABLE 3Effect of Heat treatment on clarity of different samples

As can be seen in table 3, the S200 and S200Ca samples gave very clear solutions in water at pH 3. The solution of the S300 sample was not as clear. All samples were thermally stable and the turbidity level remained essentially constant or actually improved upon heating.

Example 3：

This example includes an evaluation of the solubility in water of the soy protein isolate (S300, S200Ca) produced by the method of example 1. Solubility was tested according to protein solubility (referred to as the protein method, Morr et al, modification of the J.food Sci.50: 1715-1718 procedure) and total product solubility (referred to as the pellet method).

Enough protein powder to provide 0.5g 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 fine paste was formed. Additional water was then added to bring the volume to about 45 ml. The contents of the beaker were then stirred slowly for 60 minutes using a magnetic stirrer. The pH was measured immediately after the protein was dispersed and adjusted to the appropriate level (2, 3,4, 5, 6 or 7) with dilute NaOH or HCl. Samples were also prepared at neutral pH. For the pH adjusted samples, the pH was measured and corrected twice during 60 minutes of stirring. After stirring for 60 minutes, the samples were made up to a total volume of 50ml with RO water, giving a 1% w/v protein dispersion. The protein content of the dispersion was measured with a LECO FP528 nitrogen detector. An aliquot (20ml) of the dispersion was then transferred to a pre-weighed centrifuge tube that had been dried in a 100 ℃ oven and then cooled in a desiccator, and the tube was capped. The sample was centrifuged at 7800g for 10 minutes to settle the insoluble material and give 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 pellet 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 (pellet method) (%) × (1- (weight of dried insoluble pellet material/((weight of 20ml dispersion/weight of 50ml dispersion) × initial weight of dried protein powder)) × 100 @ pellet method) (%) (weight of dried insoluble pellet material/((weight of 20ml dispersion/weight of 50ml dispersion) ×)) × 100 ×)

The natural pH of the protein isolate produced in example 1 in water (1% protein) is shown in table 4:

TABLE 4Natural pH of a protein solution prepared at 1% protein in water

Batches of	Product(s)	Natural pH
			S005-J27-08A	S300	6.67
S005-K19-08A	S300	6.76
			S005-J27-08A	S200	6.70
S005-K19-08A	S200Ca	3.29

The solubility results obtained are listed in tables 5 and 6 below:

TABLE 5Solubility of the product at different pH values based on the protein method

TABLE 6Solubility of the product at different pH values based on the pellet method

As can be seen from the results in tables 5 and 6, the S300 product was very soluble at pH 2, 3 and 7. S200 is very soluble at pH 2 to 4 and 7. S200Ca was very soluble in the pH range 2 to 4.

Example 4：

This example includes the clarity evaluation in water of the soy protein isolate (S300, S200Ca) produced by the method of example 1.

The clarity of a 1% w/v protein solution prepared as described in example 3 was evaluated by measuring absorbance at 600nm, with lower absorbance fractions indicating greater clarity. Analysis of the sample in transmission mode on a HunterLab ColorQuest XE instrument also provides a percent haze reading, which is another measure of clarity.

The clarity results are set forth in tables 7 and 8 below:

TABLE 7Clarity of protein solutions at different pH values evaluated by A600

TABLE 8Clarity of protein solutions at different pH values as assessed by the HunterLab assay

As can be seen from the results of tables 7 and 8, the solution of S300 was clear at pH 2 and slightly turbid at pH 3. Solutions of this product are very cloudy at higher pH values. Solutions of S200 and S200Ca were clear in the pH range 2 to 4, as were solutions of S200 at native pH and pH 7.

Example 5：

This example contains an evaluation of the solubility of the soy protein isolate (S300, S200Ca) produced by the method of example 1 in soft drinks (Sprite) and sports drinks (Orange Gatorade). Solubility was determined by adding protein to the beverage without pH correction and adjusting the pH of the protein fortified beverage to the initial beverage level.

In evaluating solubility without pH correction, a sufficient amount of protein powder to provide 1g of protein was weighed into a beaker, 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 the solution was then stirred slowly on a magnetic stirrer for 60 minutes to give a 2% protein w/v dispersion. The protein content of the samples was analyzed using a LECO FP528 nitrogen detector, then aliquots of the protein-containing beverages were centrifuged at 7800g for 10 minutes and the protein content of the supernatants measured.

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

In evaluating solubility with 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 provide 1g of protein was weighed into a beaker and a small amount of beverage was added and stirred until a fine paste was formed. Additional beverage was added to bring the volume to about 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 initial 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. The protein content of the samples was analyzed using a LECO FP528 nitrogen detector, then aliquots of the protein-containing beverages were centrifuged at 7800g for 10 minutes and the protein content of the supernatants measured.

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

The results obtained are listed in table 9 below:

TABLE 9Solubility of the product in Sprite and Orange Gatorade

As can be seen from the results of table 9, S200Ca is a product having the best solubility in Sprite and Orange Gatorade. This is an acidified product and therefore has little effect on the pH of the beverage. The remaining products are not acidified and therefore their solubility is increased by pH correction of the beverage. The solubility of the S300 product was good after pH correction, but the solubility of S200 was unexpectedly low in view of the solubility results obtained in water in example 3.

Example 6：

This example contains an assessment of the clarity of the soy protein isolate (S300, S200Ca) produced by the method of example 1 in soft drinks and sports drinks.

The clarity of the 2% w/v protein dispersions prepared in the soft drink (Sprite) and sports drink (Orange Gatorade) of example 5 was evaluated using the method described in example 4. For absorbance measurements at 600nm, the appropriate beverage was used as a blank in the spectrophotometer before the measurements were taken.

The results obtained are shown in tables 10 and 11 below:

watch 10Clarity of the product in Sprite and Orange Gatorade (A600)

TABLE 11HunterLab turbidity readings of the product in Sprite and Orange Gatorade

As can be seen from the results of tables 10 and 11, the S200Ca product had the lowest effect on clarity in Sprite and Orange Gatorade. However, S200Ca in Sprite was slightly hazy, especially when tested with pH correction. Sprite and Orange Gatorade with S300 and S200 were very turbid whether or not pH correction was used.

Example 7：

This example illustrates the production of a soy protein isolate obtained from the concentrated retentate (S500) from the sodium chloride extraction.

12.5kg of defatted minimally heat treated soy flour was added to 125L of 0.15M NaCl solution at ambient temperature and stirred for 30 minutes to provide an aqueous protein solution. Residual soy flour was removed and the resulting protein solution was clarified by centrifugation and filtration to yield 97L of filtered protein solution having a protein content of 1.14% by weight.

The volume of the protein extract solution was reduced to 7L by concentration on a PVDF membrane having a molecular weight cutoff of 5,000 daltons, resulting in a concentrated protein solution having a protein content of 14.83% by weight.

The concentrated protein solution was then diafiltered with 14L of 0.075M NaCl solution. The diafiltered retentate had a final weight of 6.14kg and a protein content of 14.16% by weight, with a yield of 78.4% by weight of the filtered protein solution. The diafiltered retentate was dried to form a product having a protein content of 95.45% (N × 6.25) d.b.. The product was named S005-L17-08AS 500.

A3.2% w/v protein solution of S500 was prepared in water and the pH was lowered to 3 with dilute HCl. The color and clarity were then evaluated with a HunterLab ColorQuest XE instrument operating in transmission mode.

Color and clarity values are listed in table 12 below:

TABLE 12HunterLab fraction at pH 3 of a 3.2% protein solution of S005-L17-08A S500

Sample (I)	L*	a*	b*	Turbidity (%)
					S500	94.86	-1.15	15.45	22.0

As can be seen from Table 12, the S500 solution was very light in color at pH 3, but the solution was also cloudy.

The color of the dried powder was also evaluated in reflectance mode using a HunterLab ColorQuest XE instrument. Color values are listed in table 13 below:

watch 13HunterLab score of dried S005-L17-08A S500

As can be seen from Table 13, the dry color of the product was very light.

Example 8：

This example includes the evaluation of the thermal stability in water of the soy protein isolate (S500) produced by the method of example 7.

A2% w/v protein solution of the product in water was produced and the pH was adjusted to 3. The clarity of this solution was evaluated by measuring turbidity in transmission mode with a HunterLab ColorQuest XE instrument. The solution was then heated to 95 ℃ and held at this temperature for 30 seconds, and then immediately cooled to room temperature in an ice bath. Then, the clarity of the heat-treated solution was measured again.

The clarity of the protein solution before and after heating is set forth in table 14 below:

TABLE 14Effect of Heat treatment on the clarity of the S005-L17-08A S500 solution

As can be seen in table 14, the S500 sample gave a very clear solution in water at pH 3. The samples were thermally stable and the turbidity changed only slightly upon heating.

Example 9：

This example comprises an evaluation of the solubility in water of the soy protein isolate (S500) produced by the method of example 7. Solubility was tested according to protein solubility (referred to as the protein method, Morr et al, modification of the J.food Sci.50: 1715-1718 procedure) and total product solubility (referred to as the pellet method).

Enough protein powder to provide 0.5g 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 fine paste was formed. Additional water was then added to bring the volume to about 45 ml. The contents of the beaker were then stirred slowly for 60 minutes using a magnetic stirrer. The pH was measured immediately after the protein was dispersed and adjusted to the appropriate level (2, 3,4, 5, 6 or 7) with dilute NaOH or HCl. Samples were also prepared at neutral pH. For the pH adjusted samples, the pH was measured and corrected twice during 60 minutes of stirring. After stirring for 60 minutes, the samples were made up to a total volume of 50ml with RO water, giving a 1% w/v protein dispersion. The protein content of the dispersion was measured with a LECO FP528 nitrogen detector. An aliquot (20ml) of the dispersion was then transferred to a pre-weighed centrifuge tube that had been dried in a 100 ℃ oven and then cooled in a desiccator, and the tube was capped. The sample was centrifuged at 7800g for 10 minutes to settle the insoluble material and give 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 pellet 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 (pellet method) (%) × (1- (weight of dried insoluble pellet material/((weight of 20ml dispersion/weight of 50ml dispersion) × initial weight of dried protein powder)) × 100 @ pellet method) (%) (weight of dried insoluble pellet material/((weight of 20ml dispersion/weight of 50ml dispersion) ×)) × 100 ×)

The natural pH of the protein isolate produced in example 7 in water (1% protein) is shown in table 15:

watch 15Natural pH of S500 solution prepared at 1% protein in water

Batches of	Product(s)	Natural pH
			S005-L17-08A	S500	6.61

The solubility results obtained are listed in tables 16 and 17 below:

TABLE 16S500 solubility at different pH values based on the protein method

TABLE 17S500 solubility at different pH values based on the pellet method

As can be seen from the results in tables 16 and 17, the S500 product is very soluble at pH 2, 3 and 7 and at natural pH.

Example 10：

This example comprises an evaluation of the clarity in water of the soy protein isolate (S500) produced by the method of example 7.

The clarity of a 1% w/v protein solution prepared as described in example 9 was evaluated by measuring absorbance at 600nm, with lower absorbance fractions indicating greater clarity. Analysis of the sample in transmission mode on a HunterLab ColorQuest XE instrument also provides a percent haze reading, which is another measure of clarity.

The clarity results are set forth in tables 18 and 19 below:

watch 18Clarity of S500 solutions at different pH values evaluated by A600

Watch 19Clarity of S500 solutions at different pH values evaluated by HunterLab analysis

As can be seen from the results of tables 18 and 19, the solution of S500 has excellent clarity at pH 2, 3 and 7 and at natural pH.

Example 11：

This example contains an evaluation of the solubility of the soy protein isolate (S500) produced by the method of example 7 in soft drinks (Sprite) and sports drinks (Orange Gatorade). Solubility was determined by adding protein to the beverage without pH correction and adjusting the pH of the protein fortified beverage to the initial beverage level.

In evaluating solubility without pH correction, a sufficient amount of protein powder to provide 1g of protein was weighed into a beaker, 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 the solution was then stirred slowly on a magnetic stirrer for 60 minutes to give a 2% protein w/v dispersion. The protein content of the samples was analyzed using a LECO FP528 nitrogen detector, then aliquots of the protein-containing beverages were centrifuged at 7800g for 10 minutes and the protein content of the supernatants measured.

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

In evaluating solubility with 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 provide 1g of protein was weighed into a beaker and a small amount of beverage was added and stirred until a fine paste was formed. Additional beverage was added to bring the volume to about 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 initial 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. The protein content of the samples was analyzed using a LECO FP528 nitrogen detector, then aliquots of the protein-containing beverages were centrifuged at 7800g for 10 minutes and the protein content of the supernatants measured. The results obtained for solubility (% protein in supernatant/% protein in initial dispersion) × 100 are listed in table 20 below:

watch 20Solubility of S500 in Sprite and Orange Gatorade

As can be seen from the results in table 20, S500 is not very soluble in the beverage without adjusting the pH. This may be due in part to the fact that S500 is not an acidified product. Correcting the pH did increase the solubility of S500 in both beverages, although the protein was still not completely soluble.

Example 12：

This example contains an assessment of the clarity of the soy protein isolate (S500) produced by the method of example 7 in soft drinks and sports drinks.

The clarity of the 2% w/v protein dispersions prepared in the soft drink (Sprite) and sports drink (Orange Gatorade) of example 11 was evaluated by the method described in example 10. For absorbance measurements at 600nm, the appropriate beverage was used as a blank in the spectrophotometer before the measurements were taken.

The results obtained are shown in tables 21 and 22 below:

TABLE 21Clarity of S500 in Sprite and Orange Gatorade (A600)

TABLE 22S500 HunterLab turbidity readings in Sprite and Orange Gatorade

As can be seen from the results of tables 21 and 22, Sprite and Orange Gatorade with addition of S500 are very turbid, and only a slight improvement is possible by correcting the pH.

Summary of the disclosure

In summary of the present disclosure, soy protein isolates are produced that provide a thermally stable and clear aqueous solution at acid pH. Modifications are possible within the scope of the invention.

Claims (1)

1. A process for preparing a soy protein product having a protein content of at least 60 wt% N x 6.25 on a dry weight basis, characterized in that:

(a) (ii) adding a calcium salt or other divalent salt to the supernatant from the precipitation of the soy protein micellar mass to provide a conductivity of 2mS to 30mS, or

(ii) Partially concentrating the supernatant from the soy protein micellar pellet to a protein concentration of less than 50g/L, and adding a calcium salt or other divalent salt to the partially concentrated supernatant to provide a conductivity of 2mS to 30mS, or

(iii) Concentrating the supernatant from the soy protein micellar pellet to a protein concentration of 50 to 400g/L, and adding a calcium salt or other divalent salt to the concentrated supernatant to provide a conductivity of 2 to 30mS,

(b) removing the precipitate from the solution obtained in step (a) to leave a clear solution,

(c) the pH of the clear solution is adjusted to 1.5 to 4.4 to form a clear acidified protein solution without precipitation of protein material,

(d) (i) in the case of step (a) (i), concentrating the pH adjusted clarified solution to a protein content of 50 to 400g/L to provide a clarified concentrated soy protein solution or (ii) in the case of step (a) (ii), further concentrating the pH adjusted clarified solution to a protein content of 50 to 400g/L to provide a clarified concentrated soy protein solution,

(e) optionally diafiltering the clarified concentrated protein solution, and

(f) the concentrated solution was dried.

2. The process of claim 1 characterized in that said soy protein product has a protein content of at least 90 wt% N x 6.25 on a dry weight basis.

3. The process of claim 2 characterized in that the soy protein product has a protein content of at least 100 wt% N x 6.25 on a dry weight basis.

4. The method according to claim 1, characterized in that the concentration steps (a) (iii) and/or (d) (i) are carried out to a protein content of 100 and 250 g/L.

5. The method according to claim 1, characterized in that the pH of the clear solution is adjusted to 2.0 to 4.0.

6. The process of claim 1, characterized in that the concentration step (d) (i), (a) (iii) or (a) (ii) and/or the further concentration step (d) (ii) is performed by ultrafiltration and/or the optional diafiltration step is performed using a membrane having a molecular weight cut-off of 3,000 to 1,000,000 daltons.

7. The method of claim 6, characterized in that said molecular weight cut-off is between 5,000 and 100,000 daltons.

8. The process according to claim 1, characterized in that the soy protein solution is subjected to a diafiltration step using water, acidified water, dilute salt solution or acidified dilute salt solution, using 2 to 40 volumes of diafiltration solution.

9. The process according to claim 8, characterized in that said diafiltration step is at least partly carried out in the presence of an antioxidant.

10. The process according to claim 8, characterized in that said diafiltration step is carried out using 5 to 25 volumes of diafiltration solution.

11. The process of claim 1, characterized in that the clear acidified soy protein solution is subjected to a heat treatment step to inactivate heat labile anti-nutritional factors at a temperature of 70 ℃ to 100 ℃ for 10 seconds to 60 minutes; the heat treatment step also optionally pasteurizes the clarified acidified protein solution; and optionally cooling the heat treated clear acidified soy protein solution to a temperature of 2 ℃ to 60 ℃ for further processing.

12. The method of claim 11, characterized in that the heat-labile antinutritional factor is a heat-labile trypsin inhibitor; the heat treatment step is performed at a temperature of 85 ℃ to 95 ℃ for 30 seconds to 5 minutes; and optionally cooling the heat treated clear acidified soy protein solution to a temperature of 20 ℃ to 35 ℃ for further processing.

13. The process of claim 1, characterized in that a reducing agent is added to the supernatant, and/or the concentration and/or optional diafiltration step, and/or the concentrated and optional diafiltered soy protein solution before drying, and/or the dried soy protein product, to disrupt or rearrange the disulfide bonds of trypsin inhibitors to achieve a reduction in trypsin inhibitor activity.