HK1093661B - Oil seed meal preparation - Google Patents

Description

Preparation of oil seed meal

Technical Field

The present invention relates to the preparation of oil seed meal from which protein is recovered.

Background

In co-pending U.S. patent application nos. 10/137,391 (WO 02/089597), filed on 3.5.2002, and 10/476,830, filed on 3.3, all assigned to the assignee hereof, the disclosure of which is incorporated herein by reference, processes are described for producing high purity protein isolates containing at least about 100 wt% protein as measured by the kjeldahl method or equivalent methods for nitrogen (N) content and multiplied by a scaling factor of 6.25. The term "protein content" as used herein refers to the amount of protein in the isolated protein, on a dry weight basis. In the above-mentioned U.S. patent application, the isolated protein is prepared by the following process: the oilseed meal is extracted with a food grade salt solution, the resulting protein solution is subjected to a preliminary treatment with a colorant adsorbent, and if desired, concentrated to a protein content of at least about 200g/L, and the concentrated protein solution is then diluted with cold water to form protein micelles, which are allowed to settle to form aggregates, thick amorphous, sticky gluten-like protein isolate clumps, known as "protein micellar masses" or PMMs, which are separated from the residual aqueous phase and used as such or dried.

In one embodiment of the above process, and as described in detail in U.S. patent application nos. 10/137,391 and 10/476,830, the supernatant from the PPM precipitation step is treated to recover the isolated protein comprising dried protein from the wet PMM and supernatant. This recovery method can be achieved as follows: the supernatant was first concentrated with an ultrafiltration membrane, and then the concentrated supernatant was mixed with wet PMM and the resulting mixture was dried. The resulting canola (canola) protein isolate has a high purity of at least about 90 wt%, preferably at least about 100 wt% protein (N X6.25).

In another embodiment of the above process, and as specifically described in applications Nos. 10/137,391 and 10/476,830, the supernatant from the PPM precipitation step is treated to recover the protein isolate from the supernatant. This recovery method can be achieved as follows: the supernatant was first concentrated with an ultrafiltration membrane and the resulting concentrate was then dried. The resulting canola protein isolate has a high purity of at least about 90 wt%, preferably at least about 100 wt% protein (N x 6.25).

The process described in the above-mentioned us patent application is essentially a batch process. In co-pending U.S. patent application No. 10/298,678 (WO 03/043439), filed on 19/11/2002 (assigned to the assignee hereof, the disclosure of which is incorporated herein by reference), a continuous process for the preparation of canola protein isolate is described. According to this patent application, canola oil seed meal is continuously mixed with a salt solution, the resulting mixture is piped while protein is being extracted from the canola oil seed meal to form an aqueous protein solution, the aqueous protein solution is continuously separated from residual canola oil seed meal, the aqueous protein solution is continuously conveyed through a selective membrane for treatment to increase the protein content of the aqueous protein solution to at least about 200g/L while maintaining the ionic strength substantially constant, the resulting concentrated protein solution is continuously mixed with chilled water to promote formation of protein micelles, the protein micelles are continuously precipitated, and the supernatant is continuously overflowed until a desired amount of PMM has accumulated in the precipitation tank. The PMM is removed from the precipitation tank and may be dried. The protein content of the PMM is at least about 90 wt% (N.times.6.25), preferably at least about 100 wt%.

The meal that is leached in the initial step of the protein isolate preparation process contains many components that can cause the isolated protein to develop taste and color. For example, the husk particles in the meal contain some phenolic compounds that leach into the extract. Such phenolic compounds are susceptible to oxidation to form colored compounds.

Other compounds that may contribute to the quality of meal and its products are sinapiside and its degradation products. The degradation of sinapioside is catalyzed by degrading enzymes known as myrosinase, which break down sinapioside into isothiocyanates, thiocyanates, nitriles and elemental sulfur. The degradation products of sinaposide reduce the value of sinaposide-containing plants when used as human food or animal feed.

Canola is also known as oilseed rape.

Summary of The Invention

In the present invention, canola oil seeds are heat treated to inactivate myrosinase and subjected to a dehulling (dehulll) process, followed by crushing the dehulled oil seeds to remove oil therefrom. The process minimizes the presence of components in the meal that adversely affect the color and taste profile of protein derived from oil seed meal separation using the above-described process. The heat treatment methods provided herein can also be used to inactivate other enzymes that may be present in the oilseeds.

Inactivation of myrosinase and other enzymes present in canola oil seeds may be achieved by any convenient means suitable for inactivation of the enzymes. Most conveniently, the inactivation is carried out using steam at about 90 ℃ for a minimum of 10 minutes, but other temperatures, times and methods may be used, for example treatment using infrared, microwave or radio frequency. An important feature is that various enzymes, including myrosinase, are inactivated.

Accordingly, one aspect of the present invention provides a method of forming canola oil seed meal, the method comprising heat treating canola oil seeds to inactivate various enzymes therein, dehulling the canola oil seeds, and removing canola oil from the heat-treated and dehulled oil seeds to provide canola oil seed meal.

The canola oil seed meal produced by the process of the present invention may then be treated to recover canola protein isolates therefrom having a protein content of at least about 90 wt% (N x6.25), preferably at least 100 wt%. The canola protein isolation process used is preferably one of the processes described in the above-mentioned U.S. patent applications.

Brief description of the drawings

FIG. 1 is a process flow diagram of a method for producing dehulled and de-oiled canola oil seeds according to a preferred embodiment of the present invention;

FIG. 2 is a process flow diagram of a method for obtaining dehulled and de-oiled canola oil seeds according to a less preferred embodiment of the present invention;

FIG. 3 is a flow diagram for the preparation of canola protein isolate from dehulled and deoiled canola oil seeds prepared according to the method of FIG. 1 or FIG. 2;

fig. 4 is a pictorial sample of a heat treatment temperature profile for canola oil seed fraction and dehulled seed kernel fraction.

Detailed Description

The present invention relates to the processing of canola oil seeds to produce canola oil seed meal from which canola protein isolates can be prepared.

The process comprises heat treatment of canola oil seeds to inactivate myrosinase and other enzymes present in the seeds and dehulling the seeds. Dehulling may be done after heat treatment or before heat treatment. The treated seeds are then subjected to a de-oiling step, leaving canola oil seed meal.

The heat treatment may conveniently be effected by heating with steam for a minimum of about 5 minutes, preferably about 10 minutes, at about 90 ℃. As noted above, other temperatures, times, and methods may be used, such as infrared, microwave, or radio frequency processing. After heat treatment, the oilseeds are typically cooled to ambient temperature for further processing.

In one embodiment of the invention shown in fig. 1, canola oil seeds are first deactivated in a cooker by steam injection at about 90 ℃ for about 10 minutes. The deactivated oilseeds are then cooled to ambient temperature, for example by using a fluid bed dryer. The cooled deactivated canola oil seeds are then conveyed to a crusher where the canola hulls are crushed and the crushed canola hulls are separated from the canola seed kernels, for example by air classification. Canola kernels are separated into a larger fraction (oversize) and a smaller fraction (undersize), for example by using a vibrating screen. In the illustrated example of fig. 1, the separation step uses a 14 mesh screen.

The oversize fraction is often also accompanied by a large amount of residual uncrushed shell, which is usually recycled to the crusher several times to remove the residual shell. Once the oversize fraction has been dehulled, it may be processed by flaking the kernels and solvent leaching the green embryos to recover canola oil and produce canola oil seed meal. The recovered meal is typically subjected to desolventization.

The undersize fraction is treated, for example by air classification, to remove residual shells. The undersize fraction, once dehulled, may be processed by flaking the kernels and solvent leaching the green embryos to recover canola oil and produce canola oil seed meal. The remaining meal is typically desolventized. The oversize and undersize of the canola seed kernels may be combined prior to the flaking step.

In another embodiment of the invention shown in FIG. 2, the inactivation of the enzyme is performed after dehulling. Canola oil seed meal is fed to a crusher where canola hulls are crushed and the crushed canola hulls are separated from canola seed kernels. Canola kernels are separated into a larger fraction (oversize) and a smaller fraction (undersize), for example by using a vibrating screen. The oversize fraction is often also accompanied by a large amount of residual uncrushed shell, which is usually recycled to the crusher several times to remove the residual shell.

The oversize fraction and undersize fraction were each deactivated in the cooker by steam injection at about 90 ℃ for 10 minutes. The deactivated two parts are then cooled separately, for example by using a fluid bed dryer.

The cooled two portions are then processed to recover the canola oil and produce a canola oil seed meal. The oversize fraction is blanked and the residual shell removed, for example by air separation, and the green stock is then solvent leached. The remaining meal may be desolventized.

And (4) rolling the bottom material sieving part, and leaching the green blank with a solvent. The remaining meal may be desolventized.

The residue meal produced by these procedures is further processed using the methods described in the above-mentioned U.S. patent application to recover canola protein isolate therefrom, as described in more detail below.

As outlined in the above-mentioned us patent applications, PMM-derived canola protein isolate and supernatant-derived canola protein isolate may be isolated from canola oil seed meal by either batch processes or continuous processes or semi-continuous processes, respectively.

The initial step of the process for producing canola protein isolates involves solubilizing proteinaceous material from canola oil seed meal. The proteinaceous material recovered from canola oil seed meal may be the protein naturally occurring in canola seeds, or the proteinaceous material may be a protein modified by genetic manipulation but still possessing the characteristic hydrophobic and polar properties of the natural protein. The canola meal may be any canola meal resulting from the removal of canola oil from canola oil seeds and may contain varying levels of undenatured protein due to, for example, the use of a hot hexane leach process or a cold oil extrusion process. Removal of canola oil from canola oil seeds is typically accomplished by a separate operation from the protein isolate recovery process described herein.

The solubilization of proteins is achieved according to the invention by using a salt solution. The salt is typically sodium chloride, but other suitable salts, such as potassium chloride and calcium chloride, may also be used. The ionic strength of the salt solution is at least about 0.10, preferably at least about 0.15, so that solubilization of substantial amounts of protein is achieved. With the increasing ionic strength of the salt solution, the degree of solubilization of the protein in the oil seed meal begins to increase and finally reaches a maximum. Any subsequent increase in ionic strength did not increase the total protein solubilized. The ionic strength of the salt solution that causes maximum protein solubilization varies depending on the oil seed meal selected.

It is generally preferred to use an ionic strength value of less than about 0.8, more preferably from about 0.15 to about 0.6, in view of the higher degree of dilution required to precipitate the protein as the ionic strength increases.

In a batch process, the dissolution of the protein in the salt solution is achieved at a temperature of at least about 5 ℃, preferably at most about 35 ℃, preferably while stirring, to reduce the dissolution time, which is typically from about 10 minutes to about 60 minutes. Preferably, the solubilization is performed to sufficiently extract as much protein from the oil seed meal as possible to achieve an overall higher yield.

The lower temperature limit is chosen to be about 5 c because dissolution is too slow to proceed below this temperature, while the preferred upper temperature limit is chosen to be about 35 c because the process becomes uneconomical at higher temperatures in batch mode.

In a continuous process, leaching protein from canola oil seed meal is carried out by any means suitable to effect continuous leaching of protein from canola oil seed meal. In one embodiment, the canola oil seed meal is continuously mixed with a salt solution and the resulting 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 leaching action in accordance with the parameters described herein. In such a continuous process, the salt solubilization step is accomplished rapidly in a time of up to about 10 minutes, preferably such solubilization is carried out to substantially extract as much protein from the oil seed meal as possible. The dissolution process in a continuous process is preferably carried out at elevated temperatures, preferably above about 35 c, usually up to about 65 c.

The aqueous salt solution and the canola oil seed meal have a natural pH of from about 5 to about 6.8 to form the protein isolate by the micellar route, as described in more detail below.

At and near the upper and lower limits of the pH range, the formation of the protein isolate occurs only partially through the micellar pathway, with lower yields of protein isolate than can be achieved elsewhere in the pH range. For these reasons, the pH is preferably from about 5.3 to about 6.2.

The pH of the salt solution can be adjusted as needed to any desired value in the range of about 5 to about 6.8 for the leaching step by using any suitable acid (typically hydrochloric acid) or base (typically sodium hydroxide).

The oil seed meal concentration in the salt solution may vary greatly during the dissolution step. Typical concentration values are about 5% w/v to about 15% w/v.

The protein leaching step with aqueous salt solution has the additional effect of dissolving lipids that may be present in the canola meal, which may result in lipids being present in the aqueous phase.

The concentration of the protein solution resulting from the leaching step is generally from about 5 to about 40g/L, preferably from about 10 to about 30 g/L.

The aqueous phase from the leaching step may then be separated from the residual canola meal by any convenient means, for example by centrifugation using decantation followed by disc centrifugation and/or filtration to remove the residual meal. The separated residue may be dried for disposal.

The color of the final canola protein isolate can be improved to lighter and less intense yellow by mixing powdered activated carbon or other pigmented adsorbent with the aqueous protein solution separated and then conveniently removing the adsorbent by filtration to provide a protein solution. The pigment can also be removed by dialysis.

This pigment removal step may be carried out under any suitable conditions, typically at the ambient temperature of the separated aqueous protein solution, using any suitable pigment adsorbent. For powdered activated carbon, the amount is about 0.025% to about 5% w/v, preferably about 0.05% to about 2% w/v.

In the case of canola seed meal containing large amounts of oil and fat, as described in U.S. Pat. nos. 5,844,086 and 6,005,076 (assigned to the assignee hereof, the disclosures of which are incorporated herein by reference), the oil removal steps described in these two patents can then be carried out on the separated aqueous protein solution and the concentrated aqueous protein solution discussed below. When the color-improving step is performed, this step may be performed after the first oil-removing step.

Another approach is to leach the oil seed meal with a salt solution at a relatively high pH, i.e., above about 6.8, and typically up to about 9.9. The pH of the sodium chloride solution can be adjusted to the desired alkaline value by using any suitable food grade base, such as aqueous sodium hydroxide. Alternatively, the oil seed meal may be leached with a sodium chloride solution at a relatively low pH, i.e., below about pH5, typically at a minimum of about pH 3. Where such an alternative is employed, the aqueous phase resulting from the oil seed meal leaching step may then be separated from the residual canola meal by any convenient means, such as by centrifugation using decantation followed by disc centrifugation to remove the residual meal. The separated residue may be dried for disposal.

The aqueous protein solution obtained in the high or low pH leaching step is then adjusted to a pH in the range of about 5 to about 6.8, preferably in the range of about 5.3 to about 6.2, as described above, followed by further processing as discussed below. The pH adjustment can be suitably carried out using any suitable acid (e.g., hydrochloric acid) or base (e.g., sodium hydroxide).

The aqueous protein solution is then concentrated to increase the protein concentration in the solution while maintaining the ionic strength in the solution substantially constant. This concentration is typically performed to provide a concentrated protein solution having a protein concentration of at least about 50g/L, preferably at least about 200g/L, and more preferably at least about 250 g/L.

The concentration step may be carried out by any convenient means suitable for batch or continuous operation, such as by employing any suitable selective membrane technique (e.g., ultrafiltration or dialysis), using membranes such as hollow fiber membranes or spiral cross-flow membranes having a suitable molecular weight cutoff, depending on the membrane material and configuration, of from about 3,000 to about 100,000 daltons, preferably from about 5,000 to about 10,000 daltons, and for continuous operation, the membranes are also sized so as to achieve the desired degree of concentration of the aqueous protein solution as it permeates through the membrane.

The concentrated protein solution may then be subjected to a dialysis step using an aqueous sodium chloride solution of the same molar concentration and pH as the leach solution. This dialysis operation may be performed by using from about 2 to about 20 volumes of dialysis solution, preferably from about 5 to about 10 volumes of dialysis solution. In a dialysis operation, other contaminants are removed from the aqueous protein solution by passing the permeate through a membrane. The dialysis operation can be carried out until no other significant amounts of phenolics and visible colour are present in the permeate. Depending on the membrane material and configuration, the dialysis operation may be performed using a membrane having a molecular weight cut-off in the range of about 3,000 to about 100,000 daltons, preferably about 5,000 to about 10,000 daltons.

An antioxidant may be present in the dialysis medium during at least a portion of the dialysis step. The antioxidant may be any suitable antioxidant, such as sodium sulfite or ascorbic acid. The amount of antioxidant employed in the dialysis medium will depend on the materials employed and can range from about 0.01 wt% to about 1 wt%, preferably about 0.05 wt%. The antioxidant serves to inhibit oxidation of phenolics present in the concentrated canola protein isolate solution.

The concentration step and dialysis step can be carried out at any suitable temperature, typically from about 20 ℃ to about 60 ℃, preferably from about 20 ℃ to about 30 ℃, for a time sufficient to achieve the desired degree of concentration. The temperature and other conditions used will depend to some extent on the membrane equipment used to perform the concentration operation and the desired protein concentration of the solution.

Concentrating the protein solution to a preferred concentration of greater than about 200g/L in the concentration step not only increases the process yield to a level of greater than about 40%, preferably greater than about 80% (based on the proportion of leached protein recovered as dry protein isolate), but also reduces the salt concentration of the final protein isolate after drying. The ability to control the salt concentration of the isolated protein is important for the use of the isolated protein, and variations in salt concentration can affect functional and organoleptic properties in specific food applications.

It is well known that ultrafiltration and similar selective membrane techniques allow low molecular weight species to pass through, while preventing higher molecular weight species from passing through. The low molecular weight materials include not only the ions of the food grade salt but also low molecular weight materials leached from the starting material, such as carbohydrates, pigments and anti-nutritional factors, as well as any low molecular weight forms of protein. Depending on the membrane material and configuration, the molecular weight cut-off of the membrane is typically selected to ensure that a substantial proportion of the protein is retained in solution while allowing contaminants to pass through.

The concentrated and optionally dialyzed protein solution may be subjected to further oil removal if desired, as described in U.S. Pat. Nos. 5,844,086 and 6,005,076.

As an alternative to the above described bleaching operation, the concentrated and optionally dialyzed protein solution may be subjected to a bleaching operation. Here, powdered activated carbon and Granular Activated Carbon (GAC) can be used. Another material that may be used as a color adsorbent is polypropylenepoyrrolidone.

The color adsorbent treatment step may be carried out under any convenient conditions, typically at ambient temperature of the canola protein solution. For powdered activated carbon, the amount may be from about 0.025% to about 5% w/v, preferably from about 0.05% to about 2% w/v. When polypropylenepoyrrolidone is used as the color adsorbent, it can be used in an amount of about 0.5% to about 5% w/v, preferably about 2% to about 3% w/v. The color adsorbent may be removed from the canola protein solution by any convenient means, such as by filtration.

The concentrated and optionally dialyzed protein solution obtained from the optional decolorization step may be pasteurized to kill any bacteria that may have been present in the original meal for preservation or other reasons and leached from the meal into the canola protein isolate solution in the leaching step. The pasteurization may be carried out under any desired pasteurization conditions. Generally, the concentrated and optionally dialyzed protein solution is heated to a temperature of about 55 ° to about 70 ℃, preferably about 60 ° to about 65 ℃, for a sterilization time of about 10 to about 15 minutes, preferably about 10 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.

Depending on the temperature employed in the concentration step, the concentrated protein solution may be heated to a temperature of at least about 20 ℃ and up to about 60 ℃, preferably to about 25 ℃ to about 40 ℃, to reduce the viscosity of the concentrated protein solution, facilitating the subsequent dilution step and micelle formation. The concentrated protein solution should not be heated above a temperature above which the concentrated protein solution will not form micelles when diluted with cold water. As described in the above-mentioned U.S. patent nos. 5,844,086 and 6,005,076, the concentrated protein solution may be subjected to further oil removal if desired.

The concentrated protein solution is then diluted to form micelles by mixing the concentrated protein solution obtained in the concentration step and the optional dialysis step, the optional decolorization step, the optional pasteurization step, and the optional oil removal step with cold water in the volume required to achieve the desired degree of dilution. The degree of dilution of the concentrated protein solution may vary depending on the ratio of canola protein desired to be obtained by the micellar route to the canola protein obtained from the supernatant. Generally, the higher the dilution level, the higher the proportion of canola protein retained in the aqueous phase.

When it is desired to provide the maximum proportion of protein by the micellar route, the concentrated protein solution is diluted about 15-fold or less, preferably about 10-fold or less.

The temperature of the chilled water mixed with the concentrated protein solution is less than about 15 ℃, typically from 3 ℃ to about 15 ℃, preferably less than about 10 ℃ because at the dilution factor used, the yield of isolated protein in the form of protein micellar mass obtained with said lower temperature is increased.

In a batch operation, a batch of concentrated protein solution may be added to a static body of cold water having the desired volume described above. Dilution of the concentrated protein solution and subsequent reduction in ionic strength results in the formation of a cloud of highly bound protein molecules in the form of micellar discrete protein droplets. In a batch process, protein micelles are allowed to settle in a cold body of water to form an aggregated, coalesced, thick, amorphous, cohesive, gluten-like Protein Micellar Mass (PMM). Settling may be assisted, for example, by centrifugation. This induced settling process reduces the liquid content, and thus the moisture content, of the protein micellar mass from typically about 70% to about 95% by weight to a value typically about 50% to about 80% by weight, based on the weight of the total micellar mass. Reducing the moisture content of the micellar mass in this way also reduces the salt content entrained in the micellar mass and thus the salt content of the dried isolated protein.

Alternatively, the dilution operation may be carried out continuously as follows: the concentrated protein solution was continuously fed into one inlet of the T-piece while dilution water was fed into the other inlet of the T-piece to mix them in the tubing. The flow rate of dilution water into the tee is sufficient to achieve the desired degree of dilution of the concentrated protein solution.

Mixing the concentrated protein solution and dilution water in a pipe initiates formation of protein micelles, and the resulting mixture is fed from the outlet of the T-pipe into a settling tank which is filled with supernatant liquid. The mixture is preferably injected into the liquid in the settling tank in a manner that minimizes turbulence within the liquid.

In a continuous process, protein micelles are allowed to settle in a settling tank to form an aggregated, coalesced, concentrated, amorphous, sticky gluten-like Protein Micellar Mass (PMM), and the process continues until a desired amount of PMM has accumulated at the bottom of the settling tank, at which point the accumulated PMM is removed from the settling tank. In a batch process, settling may be aided, for example, by centrifugation.

The combination of the two process parameters of concentrating the protein solution to a preferred protein content of at least about 200g/L and using a dilution factor of less than about 15 results in higher yields, and often very high yields, in recovering the protein from the original meal extract in the form of protein micellar mass, and in higher purity of the isolated protein in terms of protein content than is achieved using any of the known prior art methods of forming isolated proteins discussed in the above-mentioned U.S. patents.

When the canola protein isolate is recovered by employing a continuous process, the time for the initial protein leaching step is significantly reduced for the same protein leaching level, and considerably higher temperatures can be used in the leaching step, as compared to a batch process. Furthermore, in a continuous process, the chance of contamination is lower than in a batch process, which results in a higher product quality and a continuous process can be carried out in a more compact plant.

The precipitated protein isolate can be separated from the residual aqueous phase or supernatant, for example, by decanting the residual aqueous phase from the precipitated pellet, or by centrifugation. The PMM may be used in wet form or it may be dried to a dry form using any convenient technique, such as spray drying, freeze drying or vacuum drum drying. The dried PMM has a high protein content, in excess of about 90 wt% protein, preferably at least about 100 wt% protein (calculated as Kjeldahl N × 6.25), and is substantially undenatured (as determined by differential scanning calorimetry). The dried PMM isolated from the fat-rich oilseed meal also has a low residual fat content, which can be less than about 1 wt%, when the processes of U.S. patent nos. 5,844,086 and 6,005,076 are employed as needed. The canola protein isolate may contain a reduced amount of phytic acid, preferably less than about 1 wt%, compared to leaching the meal with aqueous sodium chloride solution under the same reaction conditions.

The supernatant from the PMM formation and precipitation step contains a significant amount of canola protein which has not been precipitated in the dilution step and is therefore treated to recover canola protein isolate therefrom. The supernatant from the dilution step is concentrated after removal of the PMM to increase the protein concentration therein. The concentration process may be carried out by employing any convenient selective membrane technique (e.g., ultrafiltration) using a membrane having a suitable molecular weight cut-off that allows low molecular weight materials (including salts and other non-proteinaceous low molecular weight materials leached from the proteinaceous starting material) to pass through the membrane while retaining the canola protein in solution. Depending on the membrane material and configuration, ultrafiltration membranes having a molecular weight cut-off of about 3,000 to 100,000 daltons may be used. Concentrating the supernatant in this manner also reduces the volume of liquid to be dried for protein recovery. Typically, the supernatant is concentrated to a protein concentration of about 100 to about 400g/L, preferably about 200 to about 300g/L, prior to drying. As described above for the protein solution concentration step, this concentration operation may be carried out in a batch mode, or in a continuous operation.

The concentrated supernatant may be dried to a dry form by any convenient technique, such as spray drying, freeze drying or vacuum drum drying, to produce additional canola protein isolate. Such additional canola protein isolates have a high protein content in excess of about 90 wt% protein, preferably at least about 100 wt% protein (calculated as kjeldahl N x6.25), and are substantially undenatured (as determined by differential scanning calorimetry).

If desired, at least a portion of the wet PMM and at least a portion of the concentrated supernatant may be combined prior to drying the combined protein stream using any convenient technique to provide a combined canola protein isolate composition according to one embodiment of the invention. The relative proportions of proteinaceous material mixed together can be selected so that the resulting canola protein isolate composition has a desired 2S/7S/12S protein profile. Alternatively, the dried protein isolates may be combined in any desired ratio to obtain any desired specific 2S/7S/12S protein profile in the mixture to provide a composition according to the invention. The combined canola protein isolate compositions have a very high protein content, in excess of about 90 wt%, preferably at least about 100 wt% (calculated as kjeldahl N x6.25), and are substantially undenatured (as determined by differential scanning calorimetry).

In another alternative process, in which only a portion of the concentrated supernatant is mixed with only a portion of the PMM, the remaining concentrated supernatant may be dried as any remaining PMM. In addition, as discussed above, the dried PMM and the dried supernatant can also be dry mixed in any desired relative proportions.

By operating in this manner, a plurality of canola protein isolates can be recovered in the form of a dried PMM, a dried supernatant and a dried mixture of PMM-derived canola protein isolate and supernatant-derived canola protein isolate in various weight ratios, typically from about 5: 95 to about 95: 5 (by weight), as may be desired to achieve different functional and nutritional forms depending on the different ratios of 2S/7S/12S protein in the composition.

As an alternative to diluting the concentrated protein solution into chilled water and treating the resulting precipitate and supernatant as described above, the protein may be recovered from the concentrated protein solution by concentrating the protein solution to reduce its salt content by dialysis. The reduction in the salt content of the concentrated protein solution results in the formation of protein micelles in the dialysis tubing. After dialysis, the protein micelles may be allowed to settle, collected and dried as discussed above. The supernatant from the protein micelle settling step may be treated as discussed above to recover further protein therefrom. Alternatively, the contents of the dialysis tubing can be dried directly. The latter alternative is useful when it is desired to obtain laboratory-scale quantities of protein.

Examples

Example 1:

this example describes the preparation of canola oil seed meal and subsequent processing to obtain canola protein isolates.

125kg of Carnola seed of Argentina variety was treated as shown in FIG. 1. The seeds were first heat treated at 90 ℃ in a steam heated cooker for a 10 minute residence time to inactivate myrosinase and other enzymes. After cooling the resulting 115.8kg of deactivated canola oil seeds in a fluid bed dryer, the seeds were broken and the hulls were partially removed by air separation.

The larger canola seed kernels (oversize) were separated by 14 mesh vibratory screening and the oversize was recycled to the crusher 4 times to obtain 42.4kg of material, predominantly canola seed kernels, with a small portion of husk. The undersize (36kg) was finally air-separated to remove the residual husk. The final kernel (35.3kg) or undersize fraction was pressed into a green embryo by a padding machine, then 34.1kg of the canola embryo was transferred to a soxhlet extractor and the oil was extracted with solvent, while the oversize was discarded.

Dehulled and deoiled meal (16.17kg) after oil leaching was used as a raw material for protein leaching as described in example 2 below. The de-shelled canola meal is designated SD 024.

Two additional dehulled and deoiled canola meal fractions were obtained from a second 130.4kg batch of argentina canola seeds according to the process of figure 2. For this batch, the seeds were first broken and the hulls were removed by an air separation section.

The larger canola seed kernels were separated by 14 mesh vibrating screens and the resulting oversize material was cycled 4 times to obtain 52.2kg of canola seed kernels and canola hulls. After the final sieving through the vibrating sieve, the undersize (49.2kg) and oversize were heat treated with steam at 90 ℃ for 10 minutes. Both parts are cooled in a fluid bed dryer. The final kernels are pressed in a flaking mill into green embryos. The green stock obtained from the undersize (38.1kg) was directly solvent leached to remove oil using a soxhlet extractor to produce (11.35kg) an oil-removed meal, designated SD 029. The green bodies obtained from the oversize were again air separated and then solvent leached to remove oil using a soxhlet extractor to produce an oil-removed meal (11.37kg) designated SD 027.

The temperature profile at deactivation of canola oil seeds used to prepare samples SD024 ("# 1 batch"), SD029 ("# 2 batch undersize"), and SD027 ("# 2 batch oversize") is shown in fig. 4.

In the process, a total of 35.3kg dehulled seed kernels (undersize) were recovered from 112.3kg of #1 batches of deactivated canola seeds, in a total yield of 31.43 wt%. From 130.4kg of batch #2 canola, 38.1kg of dehulled and green embryo-pressed powder (undersize) were produced in total, giving a yield of 29.2 wt%. The relatively low yield of the shelled canola may be due in part to the use of coarse rolls in the crusher resulting in less canola seed breakage. Using finer pitch rolls (18 grooves per inch) will make the nip between the rolls narrower and smaller seeds can be broken. Larger and more uniform seeds also improve dehulling yield and consistency.

The air separation condition is adjusted to effectively separate the husk and the kernel. Differential air pressure was set at 0.4-0.8 inches of water to achieve effective separation. Higher pressure differentials can result in excess endosperm being removed with the hull portion.

The kernel fraction recovered from the winnowing process is comprised of a plurality of particle sizes, with a lower proportion of husk fragments being present in the finer canola fraction. Thus, the smaller dehulled seed kernel fraction can be recovered from the larger seed kernels and hulls by screening them through a 14 mesh vibrating screen. The optimum screen size may be preselected by manual screening tests prior to setting up the shaker apparatus.

The oil cells are ruptured by passing the endosperm portion of the dehulled endosperm through a set of smooth rolls of a Lauhauf embryo press for the flaking operation.

Using a nip setting of 0.08mm, it was possible to effectively mill both batches #1 and #2 of dehulled kernels, resulting in green embryos of 0.101-0.125mm thickness. However, the embryos produced by batch #2 were brittle and easily crumbled compared to batch # 1. This result indicates that inactivating the canola seeds prior to dehulling results in a more stable green embryo.

After the deoiling treatment, the residual oil content of the deoiled canola meal of batch #1 was 1.50 wt%. The undersize and oversize of batch #2 contained 1.87 wt% and 1.23 wt% oil, respectively.

Example 2:

this example illustrates the preparation of canola protein isolate from deoiled meal prepared in accordance with the method of example 1.

Canola meal after dehulling, degreasing and inactivation of myrosinase enzyme as described in example 1 was treated in accordance with the method of fig. 3 to produce canola protein isolates.

'a' kg of canola meal after dehulling, degreasing and inactivating myrosinase was added to 'b' L of 0.15M NaCl solution at ambient temperature and stirred for 30 minutes to make an aqueous protein solution. Residual canola meal is removed by filtration through cheesecloth (cheese cloth) or by other suitable filtration methods. The resulting protein solution was clarified by centrifugation to produce 'c' L of clarified protein solution having a protein content of'd' g/L.

The volume of an aliquot of the 'e' L protein leach solution was reduced to 'f' L by concentration in an ultrafiltration system using a membrane with a molecular weight cut-off of 'g' daltons. The protein content of the resulting concentrated protein solution was ` h ` g/L. The concentrated protein solution was then dialyzed against a 'j' L0.15M sodium chloride solution containing 0.05% by weight ascorbic acid using a membrane having a molecular weight cut-off of 'i' daltons, the final volume of the dialyzed protein solution being 'k' L, the protein content being 'L' g/L.

The dialyzed protein solution was diluted at'm' deg.C to 'o' deg.C in water at a ratio of 'n'. A white cloud formed immediately and was allowed to settle. The upper dilution water was removed and the precipitated, viscous sticky mass (PMM) was recovered from the bottom of the precipitation tank, in a yield of 'p' wt% of the leached protein. The protein content of the protein from dried PMM was determined to be 'q'% (N × 6.25) d.b. The resulting article is designated as 'r'.

The parameters 'a' to 'r' are given in table I below:

TABLE I

	BW-SD024-B03-03AC300	BW-SD029-B10-03AC300	BW-SD027-B17-02AC300
				a	5	5	5
b	50	50	50
				c	38.3	39	36
d	25.7	21.6	23.1
				e	38.3	39	36
f	2.5	3.5	2.5
				g	10000	10000	10000
h	218.3	218.9	232.0
				i	10000	10000	10000
j	50	35	17.5
				k	1.8	3.5	2.5
l	266.7	218.9	232.0
				m	30.5	31	31.4
n	1∶10	1∶10	1∶10
				o	1.7	2	2.2
p	40.2	55.6	57.3
				q	106.7	110.1	107.6

The volume of dilution water removed was reduced by ultrafiltration to a protein concentration of't' g/L using a membrane with a molecular weight cut-off of's' daltons. The concentrate is dried. Plus additional protein recovered from the supernatant, the total protein recovery accounted for 'u' wt% of the leached protein. The protein content of the resulting dried protein was 'v'% (N × 6.25) d.b.

The resulting article is designated 'w'. The parameters s to w are given in table II below:

TABLE II

w	BW-SD024-B03-03AC200	BW-SD029-B10-03AC200	BW-SD027-B17-02AC200
				s	10000	10000	10000
t	20.7	52.1	118.0
				u	46.7	70.6	78.6
v	103.8	103.6	106.2

Example 3:

this example describes the results obtained according to the method of example 2.

(a) Leaching and separating steps:

table III below shows the apparent leaching rates of three different meals. The apparent leach rate represents the percentage of protein that can be recovered if the total brine volume can be recovered. However, recovery rates may vary due to differences in the meal and/or different liquid holdup in the meal. When the actual volume after the clarification process operation is taken into account in the calculation, the result is then the protein yield. The apparent leaching rate was higher than 40% for all three cases. For the SD024 and SD027 pulps, their apparent leaching rates were of the same quantitative grade, 47.5 wt% and 46.1 wt%, respectively. The apparent leaching rate of SD029 dregs is slightly lower. The process of dehulling and heat treating the meal had no significant effect on the apparent extraction rate, since the apparent extraction rate values were in the same range for the low temperature desolventized meal or meal (data not shown).

TABLE III apparent Leaching Rate and protein yield in the filtered liquid

	Apparent leaching rate (wt%)	Protein yield after filtration step (wt%)
			BW-SD024-B03-03A	47.5％	36.4％
BW-SD029-B10-03A	41.3％	38.0％
			BW-SD027-B17-03A	46.1％	33.1％

(b) Ultrafiltration #1 and # 2:

protein recovery (table IV) for SD029 and SD027 meal was similar to that typically observed when ultrafiltration #1 was performed on other meals using PVDF 5 spiral cross-flow membranes. The lower value of 55 wt% for SD024 meal was due to some protein loss in the permeate. The chromatogram of the permeate showed a significant amount of 2S protein in batch BW-SD 024-B03-03A. This protein loss is thought to be due to the new membrane used.

TABLE IV protein recovery and protein yield in retentate from Ultrafiltration #1

	Recovery of protein in retentate (wt%)	Post-ultrafiltrate protein yield (wt%)
			BW-SD024-B03-03A	55％	17.78％
BW-SD029-B10-03A	72％	27.38％
			BW-SD027-B17-03A	70％	23.15％

For ultrafiltration #2, the protein recovery was 75 wt% (SD024), 90 wt% (SD029), and 100 wt% (SD 027).

(c) Protein distribution in finished product:

tables V and VI below show the protein profiles of the final PMM-derived isolate and supernatant-derived isolate. The protein peak of the SEC chromatogram as a whole was considered to be 100 wt%. This means that, for example, if there is 80 wt% of 7S, 80 wt% of the total peak area of all protein peaks belongs to the 7S protein.

TABLE V protein profiles of PMM-derived isolates obtained from different meals

	12S(wt)	7S(wt)	2S(wt)
				BW-SD024-B03-03A	17.5％	81.3％	1.5％
BW-SD029-B10-03A	9.6％	81.3％	9.1％
				BW-SD027-B17-03A	7.9％	82.4％	9.7％

It can be seen that the protein distribution in PMM follows the same pattern as previously observed (see co-pending U.S. patent application No. 10/413,371 (WO 03/088760), assigned to the assignee hereof, and the disclosure of which is incorporated herein by reference, filed on 15/4/2003), with 7S being the major protein in PMM. The amount of 2S in PMM obtained from SD024 meal was found to be low and thus the concentration of 12S was higher due to losses in the protein filtration membrane.

TABLE VI protein profiles of supernatant-derived protein isolates obtained from different meals

	12S(wt)	7S(wt)	2S(wt)
				BW-SD024-B03-03A	6.8％	81.7％	11.5％
BW-SD029-B10-03A	1.5％	16.7％	82.9％
				BW-SD027-B17-03A	0.7％	9.6％	89.7％

Due to the loss of 2S from SD024 meal, the yield as finished product wt% of leached protein was significantly lower than that of D027 or SD029 meal. The composition of the supernatant-derived isolate was similar to that of the PMM-derived isolate. Because of the dilution, the amount of 2S protein remaining in the supernatant solution is insufficient and thus 2S is not the major protein component. Since 7S is also present in the supernatant, only in low concentrations, the absence of 2S results in 7S being the major protein in the supernatant-derived protein isolate. However, for the operations carried out after SD029 and SD027 meal, the composition of the supernatant-derived isolate was found to be within the normal range observed previously for supernatant-derived isolates.

The above results show that generally speaking, dehulling and heat treatment processes of the meal do not affect the composition of the canola protein isolate obtained.

(d) Color of canola protein isolate:

tables VII and VIII below show the "L", "a" and "b" color values for the dry or reconstituted preparations (dry powder resuspended in 0.1M saline and adjusted with agitation for about 1 hour) measured using a Minolta CR-310 colorimeter for the dry preparation and a Hunter Lab DP-9000 colorimeter for the reconstituted preparation. The value of "L" ranges from 0 to 100, indicating the lightness of the product (white when L ═ 100). The "a" value (-60 to +60) represents the green-red space. The more negative the "a" value, the greener the article, and the more toward +60 the "a" value, the redder the article. The "b" value (-60 to +60) represents the blue-yellow space. The more negative the "b" value, the more blue the article, and the more toward +60 the more yellow the article.

By comparing the brightness of the dried and reconstituted products, it was found that the product obtained from the meal batch with heat treated seeds had the highest L value. These products were significantly brighter than the products obtained from the #2 batch meal which was heat treated only after seed breakage. This result indicates that myrosinase enzyme is active and has sufficient time to catalyze the degradation of sinapine before it is finally inactivated. It is believed that the degradation products of sinapin resulted in a darker color of the PMM-derived isolate and the supernatant-derived isolate obtained from such meal.

The protein isolate obtained from SD024 meal more tended to be green, whereas the protein isolates obtained from SD027 and SD029 had higher "a" values and therefore had a redder color. The blue-yellow spaces of the dry powder and liquid samples did not show the same trend. For example, the "b" value for a dry preparation of SD024 PMM-derived protein isolate was minimal in all three different runs, while the "b" value for a liquid sample of SD024 PMM-derived protein isolate was the highest. Both the PMM-derived isolate and supernatant-derived isolate of SD027 meal were observed as the darkest yellow powders. The preparation obtained from the PMM-derived isolate of SD024 meal and the supernatant-derived isolate of SD029 meal was the least yellow.

From the liquid color analysis, the PMM-derived protein isolate with the darkest yellow color was the protein isolate obtained from SD024 meal. For supernatant-derived protein isolates, the darkest yellow was obtained from SD 027.

TABLE VII- -L, a, b color values of powders from Dry products

TABLE VIII- -L, a, b color values of reconstituted liquids

Example 4:

this example describes the inactivation of enzymes by radio frequency treatment.

A batch of canola seeds having a moisture content of about 9% was divided into three 2kg samples. One of the samples was used as a control sample and was not further processed.

Two 2kg samples of canola seeds were exposed to radio frequency treatment. Exposure to radio frequency causes a rapid increase in the overall temperature of the canola seed sample. One sample was heated from ambient temperature to 90 ℃ in about 160 seconds and held at 90 ℃ for 5 minutes. Another sample was heated from ambient temperature to 90 ℃ in about 160 seconds and held at 90 ℃ for 10 minutes.

After both samples were maintained at 90 ℃, they were cooled to 30 ℃ by storage in a griddle and in a 4 ℃ cooling chamber for about 10 minutes.

The activity of myrosinase was determined by assaying the sinaposide degradation product glucose. The detection method comprises the following steps: an aliquot of 100g canola seed was homogenized with 250ml tap water in a Silverson homogenizer at 6000rpm until a slurry mixture was formed. The mixture was allowed to stand for 20 minutes and then centrifuged at 10000Xg for 5 minutes. The supernatant obtained in this step was decanted and glucose was detected using a Diastix glucose monitoring strip (Bayer).

All three seed samples of the heat treated and control samples were tested for glucose. The results are shown in table IX below.

TABLE IX

	Glucose level in supernatant
		Control sample	6mmol/l
Canola seeds heated at 90 ℃ for 5 minutes	＜5mmol/l
		Canola seeds heated at 90 ℃ for 10 minutes	Not detected out

No glucose was detected from canola seed samples heat treated at 90 ℃ for 10 minutes. This suggests that the use of radiofrequency is an effective means of inactivating myrosinase.

Example 5:

this example illustrates the preparation of enzyme-inactivated canola meal for use in producing a sample of isolated protein in an amount sufficient for organoleptic analysis.

Three tons of canola seeds are processed in succession to produce enzyme-inactivated canola meal. Enzyme inactivation was performed using a two-pan Simon-Rosedown cooker. Prior to beginning operation, the cooker is preheated. Steam pressure was adjusted during the run to maintain the desired seed temperature. The temperature in the pan was 60 deg.C (. + -. 5 deg.C) for the upper pan and 82-86 deg.C for the lower pan. The feed rate of canola seeds to the cooker was-300 kg/hr and the residence time in the lower tray was-12 minutes. The inactivated seeds were then transferred to a grain dryer and rapidly cooled to < 60 ℃.

Canola seeds are very dry after inactivation and need to be tempered. The moisture content of the seeds is 5.74%, and the moisture content is increased to 8.0% by spraying 3% water (w/w) for blending. The water and oilseeds are mixed for approximately 15 minutes and then transferred to portabin, capped and allowed to equilibrate for a minimum of 12 hours.

Thin, thin green embryos with large surface areas are prepared for cooking/pre-pressing by subjecting the seeds to a flaking operation through a flaking mill, which breaks the oil cells. The thickness of the green blank is between 0.18 and 0.23 mm. The feed rate was controlled to balance the press rate, which was about 130 kg/hr.

Cooking is performed to further disrupt the oil cells, make the green body pliable, and increase the efficiency of the screw press by reducing the viscosity of the contained oil. Prior to beginning operation, the cooker is preheated. Steam pressure was adjusted during the run to maintain the desired green temperature. The temperature in the pan was 42 deg.C (+ -2 deg.C) for the upper pan and 65 deg.C (+ -3 deg.C) for the lower pan.

The pressing process removes approximately 2/3-3/4 of the oil and produces a material suitable for solvent leaching. The material needs to be resistant to extrusion to prevent clogging in the leaching equipment and also needs to be porous for good mass transfer and drainage. The pressed green and cooked seeds were pressed with a Simon-Rosedown Prepress. Discarding the coarse pressed oil.

The oil in the cake was removed by solvent leaching by contacting the cake with isohexane. There are two mechanisms in play: the oil is leached into the solvent and the residue (isohexane-solid) is washed with a gradually thinning solvent-mixed oil (hexane-oil). Leaching is typically a continuous countercurrent process.

The canola seed press cake was extracted with isohexane in a Crown Iron Works loop extractor (type II) for a total residence time of about 100 minutes (ring in to ring out) and a solvent to solids ratio of about 3.2: 1 (w: w). The crude oil is desolventized in a climbing film evaporator and a stripper. The oil was discarded.

The desolventization of the slag (hexane-solids) was carried out in a steam jacketed Schnecken screw and a two pan desolventizer-oven. The temperature in the pan was < 50 ℃ at the Schnecken exit, 50 ℃ (± 5 ℃) in the desolventizer pan, 45 ℃ (± 5 ℃) in the oven pan.

Vacuum drying is performed to stop desolventizing of the leached canola meal. Approximately 150kg of each batch of deoiled canola meal was fed into a Littleford reactor. The meal was then heated to 47 ℃ (± 2 ℃) under vacuum of 23-25 mmHG. The meal was held at this temperature for 2 hours and then discharged into a plastic lined fiberboard bucket. In total 1317.3kg of enzymatically inactivated de-oiled and vacuum desolventized canola meal were produced.

Example 6:

this example illustrates the preparation of canola protein isolate from the deoiled enzyme-inactivated meal of example 5 and from a commercially available low temperature desolventized meal. Canola protein isolates will be used to compare color and flavor.

The deoiled enzyme deactivated meal of example 5 is designated as SA034 and the commercial meal is designated as AL 022.

'a' kg of canola meal was added to 'b' L of 0.15M NaCl solution at ambient temperature and stirred for 30 minutes to make an aqueous protein solution. Residual canola meal is removed by vacuum filtration (for BW-AL022-B24-03A) or decantation centrifugation (for BW-SA034-E06-04A C300) and disk centrifugation. The resulting protein solution was clarified by filtration through a filter press to yield 'c' L of a clarified protein solution having a protein content of'd' g/L.

The volume of an aliquot of the 'e' L protein leach solution was reduced to 'f' L by concentration in an ultrafiltration system using a membrane with a molecular weight cut-off of 'g' daltons. The protein content of the resulting concentrated protein solution was ` h ` g/L. The concentrated protein solution was then dialyzed against 'j' L of a 0.05 wt% ascorbic acid in 'k' MNaCl solution in a final volume of 'L' L, having a protein content of'm' g/L, using a membrane having a molecular weight cut-off of 'i' daltons in a dialysis system.

The concentrated solution was diluted to 'p' deg.C in water at 'n' deg.C in the proportion of 'o'. A white cloud formed immediately and was allowed to settle. The supernatant dilution water was removed and the precipitated, viscous sticky mass (PMM) was recovered from the bottom of the precipitation tank, in a yield of 'q' wt% of the leached protein. The protein content of the dried PMM-derived protein was determined to be 'r'% (N.times.6.25) d.b.. The resulting product is designated as's'.

	BW-AL022-B24-03A C300	BW-SA034-E06-04A C300
			a	150	150
b	1000	1500
			c	1180	1265
d	12.2	15.7
			e	1180	1265
f	45	65
			g	10000	5000
h	283	213
			i	10000	5000
j	235	325
			k	0.15	0.1
l	35.35	57.5
			m	316	248
n	31.9	29.6
			o	1∶15	1∶10
p	3.7	3.1
			q	48.7	33.6
r	102.8	100.9

The volume of dilution water removed was reduced by ultrafiltration to a protein concentration of 'u' g/L using a membrane with a molecular weight cut-off of't' daltons. The concentrate is dried. Plus additional protein recovered from the supernatant, the total protein recovery accounted for 'v' wt% of the leached protein. The protein content of the resulting dried protein was 'w'% (N × 6.25) d.b.

The resulting article is designated 'x'.

x	BW-AL022-B24-03A C200	BW-SA034-E06-04A C200
			t	10000	100000
u	158.7	192.1
			v	78.2	56.4
w	104.4	94.7

Example 7:

this example describes the results obtained according to the method of example 6.

(a) Sensory analysis

The canola protein isolate was submitted for organoleptic analysis. The sensory panel consisted of 11 trained panelists. Each panelist was asked which sample had the least flavor and which sample it would prefer.

The canola protein isolate obtained according to the method of example 6 was resuspended in 0.05M saline solution at a concentration of 5% w/v. The protein powder was completely dissolved before starting the organoleptic evaluation.

Table X below shows the sensory analysis results for PMM articles. It appears that the protein isolate derived from the enzyme-inactivated meal is the least flavored product and is also the preferred product. 64% of the panellists found the least PMM flavour from the enzyme-inactivated meal, while 27% found the least PMM flavour from the cold meal. 9% of the panelists were unable to find what the difference was between the two preparations.

When panelists were asked which product they would prefer, 64% of them suggested PMM preferably derived from enzyme-inactivated meal, 18% of them preferably derived from low temperature meal, and 18% of them neither product.

TABLE X-C300 organoleptic analysis of articles

	Has minimal flavor	Preferred articles
			BW-AL022-B24-03A C300	3	2
BW-SA034-E06-04A C300	7	7
			No difference can be found	1	2

Table XI below shows the results of sensory analysis of the supernatant-derived protein isolate. It appears that the protein isolate derived from the enzyme-inactivated meal is the least flavored product and is also the preferred product. 55% of the panellists found the least flavour of the protein derived from the supernatant of the enzyme-inactivated meal, while 27% of the panellists found the least flavour of the product obtained from the cold meal. 9% of the panelists were unable to find what the difference was between the two preparations.

When the panelists were asked which product they would prefer, 82% of them suggested that the supernatant-derived protein obtained from the enzyme-inactivated meal was preferred, 9% of them were preferably from the meal at low temperature, and 9% of them were both less preferred.

TABLE XI- -sensory analysis of C200 preparations

	Has minimal flavor	Preferred articles
			BW-AL022-B24-03A C200	3	1
BW-SA034-E06-04A C200	6	9
			No difference can be found	2	1

(a) Colour analysis

Table XII below shows the "L", "a" and "b" color values for the reconstituted preparation (5% w/v preparation in 0.05M saline) as measured using a Hunter Lab DP-9000 colorimeter. The value of "L" ranges from 0 to 100, indicating the lightness of the product (white when L ═ 100). The "a" value (-60 to +60) represents the green-red space. The more negative the "a" value, the greener the article, and the more toward +60 the "a" value, the redder the article. The "b" value (-60 to +60) represents the blue-yellow space. The more negative the "b" value, the more blue the article, and the more toward +60 the more yellow the article.

By comparing the brightness of the liquid samples, it appears that the L-value of the product from enzyme-inactivated meal is significantly higher for both PMM and supernatant-derived protein isolates than for the product from low-temperature meal. This means that in both cases the enzyme-inactivated meal produced a lighter colored isolated protein.

The PMM isolate and supernatant isolate both followed the same trend for the green-red space as well as the blue-yellow space. Using enzyme-inactivated meal as the starting material, the "a" value was slightly lower than that of the low-temperature meal, i.e. the samples tended to be more green. The "b" value increased when enzyme-inactivated meal was used, i.e. the sample was more yellow than the sample obtained from low temperature meal.

TABLE XII- -L, a, b color values of reconstituted liquid

A summary of the present disclosure is now provided. The present invention provides a process for producing canola protein isolates having improved color and taste by first heat inactivating myrosinase and other enzymes in canola seeds and then further treating the seeds. Various modifications are possible within the scope of the invention.

Claims (38)

1. A process for the preparation of a canola protein isolate having a protein content of at least 90 wt% (Nx6.25) from intact canola oil seeds, which comprises:

heat-treating intact canola oil seeds to inactivate enzymes therein, said heat-treatment being carried out at 90 ℃ for 5 to 10 minutes,

dehulling the heat-treated canola oil seeds,

removing canola oil from the heat-treated and dehulled oilseeds to provide a canola oil seed meal; and

treating said canola oil seed meal to recover canola protein isolates therefrom.

2. The method of claim 1, wherein said heat treated and dehulled oilseeds are subjected to a ginning step prior to said deoiling step.

3. The method according to claim 1 or 2, wherein the heat treatment, dehulling and degreasing steps are carried out as follows:

heat treating intact canola oil seeds to inactivate enzymes therein,

cooling the heat-treated canola oil seeds,

crushing the hulls of the heat-treated canola oil seeds,

removing broken hulls from canola oil seeds, and

canola oil is removed from canola oil kernels by solvent leaching to leave a meal.

4. A method according to claim 3, characterised in that the oversize fraction and the undersize fraction are separated from the crushed husk, said oversize fraction being recycled to the crushing and separating step, said undersize fraction being subjected to an air separation treatment for further removal of the husk, and the recycled oversize fraction and/or the air separated undersize fraction being subjected to a flaking prior to said solvent leaching step.

5. The process of claim 1, characterized in that said canola oil seed meal is treated to recover therefrom a canola protein isolate having a protein content of at least 100 wt% (Nx 6.25).

6. The method according to any one of claims 1 to 5, characterized in that the deactivation treatment is carried out by heating with steam or radio frequency radiation.

7. The process of claim 1, characterized in that said canola oil seed meal is treated by the steps of:

(i) extracting canola oil seed meal with an aqueous salt solution to solubilize canola protein in the canola oil seed meal to form an aqueous canola protein solution having a pH of 5 to 6.8,

(ii) separating the aqueous protein solution from the residual canola oil seed meal,

(iii) increasing the protein concentration in the aqueous protein solution while maintaining the ionic strength substantially constant by using a selective membrane technique to provide a concentrated protein solution,

(iv) diluting the concentrated canola protein isolate in cold water at a temperature below 15 ℃ results in the formation of dispersed protein particles in the aqueous phase in the form of micelles,

(v) allowing the protein micelles to settle to form an amorphous, viscous, gelatinous gluten-like protein micelle mass, and

(vi) recovering a protein micellar mass from the supernatant, the protein content of the protein micellar mass being at least 90 wt% on a dry weight basis as determined by Kjeldahl nitrogen x 6.25.

8. The method of claim 7, characterized in that in step (iii) the concentrated protein solution is produced by ultrafiltration at a concentration of at least 200 g/L.

9. A process according to claim 7 characterised in that steps (i) to (vi) are carried out in a batch mode and the extraction of the oil seed meal is carried out using an aqueous salt solution having an ionic strength of at least 0.10 and a pH of from 5 to 6.8, the aqueous protein solution having a protein content of from 5 to 40 g/L.

10. The process according to claim 9, characterized in that said leaching of said oil seed meal is effected by agitating said aqueous salt solution for 10 to 60 minutes, while the concentration of oil seed meal in said aqueous salt solution is 5 to 15% w/w during the performance of said leaching step.

11. A process according to claim 9, characterized in that said steps (i) to (vi) are carried out in a continuous manner, said leaching step being carried out by:

(i) continuously mixing the oil seed meal with a brine solution having an ionic strength of at least 0.10 and a pH of from 5 to 6.8 at a temperature of from 5 ℃ to 65 ℃, and

(ii) continuously transporting the mixture through a pipeline over a period of up to 10 minutes while leaching protein from the oil seed meal to form an aqueous protein solution having a protein content of from 5 to 40 g/L.

12. The method of claim 9 or 11, characterized in that the ionic strength is 0.15-0.6, the pH is 5-6.2 and the protein content of the aqueous protein solution is 10-30 g/L.

13. The method of claim 11 characterized in that in said mixing step the oil seed meal concentration in said aqueous salt solution is between 5 and 15% w/v.

14. The process according to claim 7, characterized in that said leaching of said oil seed meal is effected by using an aqueous salt solution having an ionic strength of at least 0.10 and a pH of 3-5 or 6.8-9.9, the pH of the aqueous protein solution being adjusted to 5-6.8 after separation of the aqueous protein solution from the residual oil seed meal.

15. The method of claim 7, characterized in that after separating the aqueous protein solution from the residual oil seed meal, the aqueous protein solution is subjected to a depigmentation step.

16. The method of claim 15, characterized in that said pigment removal step is carried out in the following manner:

(a) dialyzing the aqueous protein solution; or

(b) The pigment adsorbent is mixed with the aqueous protein solution, and then the pigment adsorbent is removed from the aqueous protein solution.

17. The method of claim 16, characterized in that the pigment adsorbent is powdered activated carbon.

18. A process according to claim 7 characterised in that the oil seed meal is leached with water and then salt is added to the aqueous protein solution obtained to obtain an aqueous protein solution having an ionic strength of at least 0.10.

19. The method of claim 7, characterized in that the concentrated protein solution is warmed to a temperature of at least 20 ℃ to reduce the viscosity of the concentrated protein solution but not to a temperature at which the concentrated protein solution is incapable of forming micelles.

20. The method of claim 17, characterized in that the temperature is between 25 ℃ and 40 ℃.

21. A method according to any one of claims 7-19, characterized in that the concentrated protein solution is dialyzed against a brine solution having the same molar concentration and pH as the leaching solution.

22. The method according to claim 21, characterized in that 2-20 volumes of dialysis solution are used.

23. The method of claim 21, characterized in that dialysis is performed using 5-10 volumes of dialysis solution until no more phenolics and visible color are present in the dialysis solution.

24. The process according to claim 21, characterized in that an antioxidant is present in the dialysis medium during at least a part of the dialysis step.

25. The method of claim 24, characterized in that the antioxidant is sodium sulfite or ascorbic acid.

26. The method according to any one of claims 21-25, characterized in that the concentrated and dialyzed protein solution is subjected to a pigment removal step.

27. The method of claim 26, characterized in that the pigment removal step is performed using 0.025-5% w/v powdered activated carbon or using 0.5-5% w/v polyvinylpyrrolidone.

28. The method according to any one of claims 7 to 20, characterized in that the concentrated protein solution is subjected to a pigment removal step.

29. The method according to any one of claims 21 to 25, characterized in that the concentrated and dialyzed protein solution is subjected to a pasteurization step.

30. The method according to claim 29, characterized in that the pasteurization step is carried out at a temperature of 55 ℃ to 70 ℃ for 10 to 15 minutes, after which the pasteurized solution is cooled to a temperature of 25 ℃ to 40 ℃.

31. The method according to any one of claims 7 to 20, characterized in that the protein solution is subjected to a pasteurization step.

32. The method of claim 7, characterized in that steps (i) and (vi) are accomplished in a batch mode of operation, wherein the concentrated protein solution is diluted 15-fold or less by adding the concentrated protein solution to a body of water having the volume necessary to achieve the desired degree of dilution.

33. The method of claim 7, characterized in that said steps (i) to (vi) are operated in a continuous manner, and said concentrated protein solution is continuously mixed with said chilled water to dilute the concentrated protein solution 15-fold or less.

34. The method of claim 32 or 33, characterized in that the concentrated protein solution is diluted 10-fold or less in water at a temperature below 10 ℃.

35. The process according to any one of claims 7 to 21, characterized in that the recovered protein micellar mass is dried to a salted protein powder.

36. The process of any one of claims 7 to 21, characterized in that after recovery of the protein micellar mass from the supernatant, the supernatant is treated in a batch, semi-continuous or continuous manner to recover additional amounts of the canola protein isolate therefrom.

37. The method of claim 36, characterized in that said additional amount of isolated protein is obtained as follows:

(a) concentrating the supernatant to a protein concentration of 100-400g/L and drying the concentrated supernatant;

(b) concentrating the supernatant to a protein concentration of 100-400g/L, mixing the concentrated supernatant with the recovered protein micellar mass, and drying the resulting mixture; or

(c) Concentrating the supernatant to a protein concentration of 100-400g/L, mixing a portion of the concentrated supernatant with at least a portion of the recovered protein micellar mass, and drying the resulting mixture.

38. The process of claim 37, characterized in that in (c), the concentrated supernatant remaining therein is dried, and any remaining recovered protein micellar mass is dried.