HK1143380A - Production of 2s canola protein involving ion exchange - Google Patents

Description

Production of 2S canola protein involving ion exchange

Technical Field

The present invention provides a procedure for producing canola (canola)2S protein in substantially pure form by a process involving the use of ion exchange chromatography.

Background

Canola protein isolates having a protein content of at least 100 wt% (N x 6.25.25) d.b. may be formed from canola oil meal (canola oil seed meal) as described in co-pending U.S. patent application serial No. 10/137,391 (U.S. patent application publication No. 20030125526a1) filed on day 3, 5, 2002, 10/476,230 (U.S. patent application publication No. 20040254353a1) filed on day 9, 6, 2004, and the corresponding PCT publication No. WO 02/089597, both of which are assigned to the assignee of the present application and the disclosures of which are incorporated herein by reference. The procedure involves a multi-step process comprising extracting canola oil meal using a salt solution, separating the resulting aqueous protein solution from residual oil meal, increasing the protein concentration of the aqueous solution to at least about 200g/L while maintaining the ionic strength substantially constant by using a selective membrane technique, diluting the resulting concentrated protein solution into cold water to cause the formation of protein micelles (PMM), settling the protein micelles to form an amorphous, sticky, gelatinous, gluten-like protein micellar mass, and recovering the protein micellar mass from a supernatant having a protein content of at least about 100 wt%, as measured by Kjeldahl nitrogen (N x 6.25.25). Protein content as used herein is determined on a dry weight basis. The recovered PMM may be dried.

In one embodiment of the above process, the supernatant from the PMM settling step is treated to recover a protein isolate comprising dried protein from the wet PMM and supernatant. This procedure can be achieved by initially concentrating the supernatant using ultrafiltration membranes, mixing the concentrated supernatant with wet PMM and drying the mixture. The resulting canola protein isolate has a high purity of at least about 90 wt% protein (N x 6.25.25), preferably at least about 100 wt% protein (N x 6.25.25).

In another embodiment of the above method, the supernatant from the PMM settling step is treated to recover the protein from the supernatant. This procedure can be achieved by initially concentrating the supernatant using ultrafiltration membranes and drying the concentrate. The resulting canola protein isolate has a high purity of at least about 90 wt% protein (N x 6.25.25), preferably at least about 100 wt% protein (N x 6.25.25).

The procedure described in the aforementioned U.S. patent application is essentially a batch procedure. In co-pending U.S. patent application No. 10/298,678 (U.S. patent application publication No. 20040039174a1), filed on 19/11/2002, and corresponding published international application No. WO 03/043439 (assigned to the assignee of the present application and the disclosure of which is incorporated herein by reference), a continuous process for preparing canola protein isolates is described. According to them, canola oil seed meal is continuously mixed with a salt solution, the mixture is transported through a pipeline while extracting protein 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 transported through a selective membrane operation 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 cause the formation of protein micelles, and the protein micelles are continuously allowed to settle while the supernatant continuously overflows until a desired amount of PMM has accumulated in a deposition vessel. The PMM is removed from the deposition vessel and may be dried. The PMM has a protein content of at least about 90 wt% (determined by Kjeldahl nitrogen method) (N x 6.25.25), preferably at least about 100 wt% (N x 6.25.25).

The overflow supernatant may be treated to recover canola protein isolate therefrom, as described in the aforementioned U.S. patent application serial nos. 10/137,391 and 10/471,230.

Canola seeds are known to contain about 10 to about 30 wt% protein, and several different protein components have been identified. These proteins are distinguished by different sedimentation coefficients (S). These known and identified proteins include the 12S globulin, known as cruciferin (cruciferin), the 7S globulin, and the 2S albumin, known as napin.

Canola (Canola) is also known as rapeseed or oilseed rape (oil seed rape).

Compositions of PMM canola protein isolate and supernatant-derived canola protein isolate are described in co-pending U.S. patent application Ser. No. 10/413,371 (U.S. patent application publication No. 20040034204), filed on 25/2003, and 10/510,766, filed on 15/2003, and the corresponding published PCT publication No. WO 03/08876, assigned to the assignee of the present application and the disclosures of which are incorporated herein by reference. The supernatant-derived canola protein isolate includes predominantly 2S protein and lesser amounts of 7S protein and trace amounts of 12S protein. The 2S protein is a low molecular weight albumin. The PMM product comprises mainly 7S protein, of which 2S protein and 12S protein are relatively minor components. The 7S and 12S proteins are higher molecular weight globulins, with the 7S molecule being the half molecule of the 12S protein.

As described therein, the supernatant-derived canola protein isolate exhibits the following protein profile:

about 60 to about 95% wt% 2S protein,

about 5 to about 40 wt% of 7S protein, and

0 to about 5 wt% of 12S protein, preferably

About 70 to about 95 wt% 2S protein,

about 5 to about 30 wt% of 7S protein, and

0 to about 2 wt% of 12S protein.

The PMM canola protein isolate exhibits a protein profile as follows:

about 60 to about 98 wt% of 7S protein,

about 1 to about 15 wt% of 12S protein, and

0 to about 25 wt% of 2S protein, preferably

About 88 to about 98 wt% of 7S protein,

about 1 to about 10 wt% of a 12S protein, and

0 to about 6 wt% 2S protein.

It has been found that supernatant-derived canola protein isolates consisting predominantly of 2S protein exhibit superior functional properties for certain applications as compared to PMM-derived canola protein isolates consisting predominantly of 7S protein. In the procedure described in the prior application, in order to produce supernatant-derived canola protein isolate, it is necessary to effectively undergo PMM formation and supernatant preparation steps in order to fractionate the canola proteins.

In U.S. patent application No. 11/038,086 (U.S. patent application publication No. US 2005-0181112a1), filed on 21/7/2005, assigned to the assignee of the present application and the disclosure of which is incorporated herein by reference (WO 2005/067729), a procedure is described in which the supernatant from the PMM precipitate is heat-treated before or after membrane treatment to precipitate 7S protein and leave a protein solution enriched in 2S protein. The remaining solution may be spray dried to recover the 2S protein as a dry substance.

The 2S protein, which contains a minimum ratio of 7S and 12S proteins, demonstrates increased solubility (including at acidic pH) over untreated 2S protein and is capable of providing improved clarity to aqueous solutions and clear protein fortified beverages with soft drinks and sports drinks.

Summary of The Invention

The present invention utilizes an alternative procedure involving ion exchange to produce substantially pure 2S canola protein substantially free of 7S and 12S canola proteins.

In ion exchange chromatography, charged ion exchange media are used to bind oppositely charged molecules, while similarly charged and uncharged materials are not retained. This makes ion exchange chromatography a useful tool for purifying charged molecules such as proteins. The two main species of canola proteins have significantly different isoelectric points. The isoelectric point of 7S/12S globulin ranges from about 6 to 7, whereas for 2S albumin this value is approximately 11. This difference is exploited herein to separate proteins from each other by ion exchange chromatography.

The present invention provides an ion exchange process in which 2S protein is captured by binding the 2S protein to a cation exchange medium while allowing other proteins and impurities to be eluted. The 2S protein is then released from the cation exchange media by exposing the cation exchange media to saline of suitably high salt concentration.

In accordance with one aspect of the present invention, there is provided a method of producing substantially pure 2S canola protein, comprising solubilizing canola protein from canola oil seed meal to form a canola protein solution, separating the canola protein solution from residual canola oil seed meal, contacting the canola protein solution with a cation exchange medium under conditions in which the 2S canola protein binds to the cation exchange medium in preference to other canola proteins, separating bound 2S canola protein from unbound canola protein and impurities, and separating bound 2S canola protein from the cation exchange medium.

Detailed Description

As described above, ion exchange chromatography is performed on the canola protein solution to preferentially bind 2S canola protein to the ion exchange medium and then recovering 2S canola protein in substantially pure form from the ion exchange medium.

The procedure may be carried out in any convenient manner. In a preferred aspect of the invention, an aqueous solution of canola protein is contacted with a cation exchange medium at a pH of about 5 to 6, wherein both species of protein are positively charged. Salt concentrations are used to limit binding of the less positively charged 7S/12S canola protein as well as impurities such as sinapine.

The aqueous canola protein solution may most conveniently be formed by extraction from canola oil seed meal. The extraction is achieved using an aqueous salt solution having a desired salt concentration and pH effective to ensure preferential binding of the 2S protein to the cation exchange medium. The salt solution typically has a salt concentration in the range of about 0.25 to 0.35M NaCl, and the aqueous canola protein solution has a pH in the range of about 5 to about 6.

Extraction of the canola oil meal may be accomplished outside the desired pH range, and the pH of the canola protein solution may then be adjusted to a pH range of about 5 to about 6, if necessary, using any convenient acid or base.

In an alternative, the protein may be extracted from the canola oil meal by using a salt solution of lower salt concentration and then adding additional salt to the desired concentration. However, it is preferred to effect extraction with brine of the concentration required for ion exchange, since the extract solution is in a form that can be applied directly to the cation exchange medium and used for isolation of 2S canola protein immediately after formation. Thus, there is little time for oxidation reactions to occur or for phenolic compounds to bind to proteins.

The canola protein extract solution is applied to a cation exchange medium which may be provided in any convenient form, such as in the form of resin beads or a membrane adsorber. In a membrane adsorber, ion exchange groups are bound to a microporous membrane. The use of membranes instead of resin packed columns allows higher flow rates to be used and faster processing to be achieved.

Contact of the canola protein extract solution with a cation exchange medium in the presence of appropriate pH and salt levels causes the 2S protein to be preferentially adsorbed as compared to the less positively charged 7S/12S protein. After the cation exchange medium is separated from the canola protein extract solution, the 2S protein may be removed from the cation exchange medium by contact with an aqueous salt solution having a higher salt concentration than the aqueous canola protein salt solution, such as about 0.55 to about 0.70M NaCl.

The eluted 2S protein solution has a high salt concentration and is desalted by any convenient means, such as diafiltration, prior to drying the protein. This procedure produced a high purity 2S canola protein isolate substantially free of 7S/12S protein and having a protein content of at least about 100 wt% (N x 6.25.25) on a dry weight basis (d.b.).

Canola 7S/12S protein may be recovered from canola protein extracts after contact with an ion exchange medium in an undenatured form other than when isoelectric point or thermal precipitation is used to separate these proteins from 2S protein.

Examples

Example 1

This example illustrates the preparation of substantially pure 2S canola protein using a cation exchange column.

(a) Protein extraction:

typically, a series of 15% w/v extractions of canola oil meal are carried out using 150ml of brine per 22.5g of meal. These samples were stirred at room temperature for 30 minutes using a magnetic stir bar. In each case, the extract was separated from the used oil meal by centrifugation at 10200g for 10 minutes and then further clarified by successive filtration using 25 μm pore size filter paper and 0.45 μm pore size syringe filters. The protein content of the clarified extract was determined by LECO analysis (LECO FP 528 nitrogen determinator) and its protein profile determined by Size Exclusion (SEC) HPLC. In each run, the salt concentration of the salt solution can vary between 0.26 and 0.35M NaCl.

Since the extraction salt concentration is controlled, this has some effect on the protein content and profile of the initially clarified extract (table 1 below). Higher salt concentrations are desirable in terms of protein yield and extraction of 2S protein, but have a negative impact on the separation, as shown below.

TABLE 1 analysis of extracts prepared at different salt levels

Concentration of NaCl (M)	pH	Protein concentration (%)	HPLC protein Peak area% attributable to 2S
				0.26	5.60	2.62	n.d.*
0.29	5.67	2.71	n.d.*
				0.30	5.61	n.d.*	36.9
0.35	5.61	2.82	37.4

n.d. ═ not determined

(b) Chromatography:

the samples were subjected to cation exchange (CIEX) chromatography using an SP Sepharose XL (20ml) column and operated using a gradifac LPLC system (Pharmacia Biotech) capable of peak collection. In each run, 10ml of clarified extract was applied to the column via the sample loop in the system. At the saline concentrations used, 2S protein was bound to the column and 7S and 12S canola proteins and other impurities were passed through the (pass through) column. The void material (void material) is captured and then saline at a higher salt concentration than the extract is applied to the column to release the bound 2S protein. The salt concentration used was adjusted as the run proceeded in order to achieve the best separation of the proteins and to ensure release of the binding material. The procedure for the samples Gradifrac used is given in Table-2 below:

TABLE 2 sample Gradifrac procedure for CIEX isolation of 2S from extracts

Note that: as with the developed method, there may be some small variation in the segment volume (segment volume) employed.

Note that: once it was determined that all 2S protein was released in the elution step, the column wash with 1M NaCl between runs was eliminated.

Daily, fractions eluted from all runs were combined and refrigerated at-60 ℃, with the exception of the product from runs 20 to 26, and the combined fractions were refrigerated for desalting the next day. Table 3 below lists the salt concentrations used for extraction and elution in each production run.

TABLE 3 salt concentrations for extraction and 2S protein elution in each production run

Production operation	Concentration of NaCl in the extract (M)	NaCl concentration (M) for 2S elution
			123 to 89 to 1314 to 26	0.300.300.350.290.26	0.550.600.650.650.65

The salt concentrations used for extraction/protein separation and 2S protein elution were fine-tuned as the production run proceeded. In the first run, the salt concentration used was 0.30M/0.55M. The void material was collected as overlapping doublets (doubllet peak) and the first peak was found to contain almost all 7S/12S, a small amount of unbound 2S protein and most of the impurities visible in the extract, except a portion of sinapine. The second peak in the doublet, which takes a slightly longer time to emerge from the column, was found to contain a significant amount of sinapine and very small amounts of protein and other impurities. Elution with 0.55M NaCl failed to elute all of the 2S protein from the column because a significant 2S protein peak was obtained when the column was washed with 1M NaCl.

For the second run, the elution salt level was increased to 0.60M to better release the 2S protein. This time, a smaller 2S protein peak was found when the column was washed. In the third run, the elution step was increased to 0.65M NaCl and this level was found to effectively eliminate the peaks seen when washing the column. Increasing the initial salt level in the third run to 0.35M NaCl, it was desirable to bring the two gap peaks closer together. The separation between the two peaks is reduced but the two peaks are still obtained. Likewise, operation at this higher salt level increases the amount of 2S protein that is unable to bind to the column and can be found in the void.

Subsequently, the initial salt level was reduced to 0.29M and then to 0.26M, successively reducing the level of 2S protein lost to the void (table 4 below). We fear that lowering this initial salt level will result in some binding of the 7S/12S protein or sinapine to the column, which would be highly undesirable as it would complicate the 2S elution step. However, at 0.26M NaCl, 2S protein was the only species observed to bind to the column.

TABLE 4 2S content in Total void Material based on extract salt concentration

Concentration of NaCl (M)	2S Peak area (count)
		0.260.290.300.35	66769115540n.d.*224545

^*n.d. ═ not determined

(c) Desalting of eluted 2S protein:

the frozen sample of eluted 2S protein was placed in a refrigerator overnight to be thawed. The next day the still frozen container was placed in a warm water bath where the sample was warmed just until the ice crystals melted. All thawed samples were then filtered through 25 μm pore size filter paper and combined into a single large sample. The resulting 2S protein was desalted by concentration and diafiltration on a Vivaflow 5000 MWCO Hydrosart ultrafiltration membrane unit. The total volume of the collected 2S protein fractions was about 1500 ml. The combined 2S protein solution was concentrated down to 25 to 30ml and then 300ml water was added for diafiltration. The sample was re-concentrated to 25 to 30ml and 300ml water was added again, and then the sample was re-concentrated again. As can be seen from the results contained in table 5 below, desalting was effectively performed using two steps of about 10 diafiltration volumes.

TABLE 5 reduction of salt content by diafiltration

2S sample	Concentration (mS)	pH	Protein concentration (%)
				Combined GradiFrac fractions Final retentate after addition of DF1 Water followed by DF2 Water	49.65.680.943n.d.*	5.835.284.92n.d.*	0.31n.d.*n.d.*5.84

^*n.d. ═ not determined.

The retentate was then freeze dried.

(d) And (3) final product:

the final product was evaluated for dry color (dry color) using a Minolta CR-310 colorimeter, and the solution was also formulated for wet color (wet color) analysis. Protein powder (0.5g) was mixed with water (10ml) using a vortex mixer. The sample was then centrifuged at 7800g for 10 minutes (mainly to remove air) and the protein content of the supernatant determined by LECO. An aliquot (aliquot) (8ml) of the supernatant was transferred to a small beaker and sufficient water was added to adjust the protein content to 5%. The sample was then photographed and an aliquot of the sample was used for protein profiling (SEC HPLC). Some samples were also diluted to 3.5% protein and another photograph taken. The protein content of the dry powder was determined by LECO, but not enough sample was obtained for moisture content analysis. Therefore, the protein content is expressed only on the basis of the wet substance.

A total of 1.63g of final product was collected in this study. The protein content of the wet mass based powder was 105.82% w/w (N x 6.25.25). If expressed on a dry matter basis (which is standard), the protein content will be still higher. Chromatographic analysis of the rehydrated product revealed that 96.1% of the peak area detected at 280nm could be attributed to 2S and 3.8% of the peak area could be attributed to napin (pro-napin). Therefore, 99.9% of the peak area was attributable to the target protein. No 7S or 12S protein was detected.

The color score of the dry product is given in table 6.

TABLE 6 Dry color of 2S isolated from the extract by cation exchange

Sample (I)	L*	a*	b*
				2S	83.05	-2.61	15.49

The wet color of the rehydrated product in water appeared green and the clarity of the solution was excellent. We believe that the clarity of the solution should remain fairly stable under most conditions due to the complete absence of 7S/12S protein.

The yield of the product appeared to be quite good. Since the separation conditions were modified several times and it was known that 2S protein loss occurred, especially in the first run, due to incomplete binding to the resin and incomplete elution, it was difficult to calculate a representative yield. By the final run, all 2S protein was eluted, but a small amount remained unbound to the column. As mentioned above, reducing the initial salt content or permitting the problem to be solved, provided it does not allow other species to bind to the column. If 1.63g of 2S protein is considered to have been produced from 260ml (26X 10ml injection) of clarified extract, it can then be extrapolated to 6.3kg of 2S from 1000L of clarified extract.

Example 2

This example illustrates the production of substantially pure 2S canola protein using a cation exchange membrane.

(a) Protein extraction:

10% w/v extraction of 30g of canola oil meal was accomplished using 300ml of 0.3M NaCl by mixing the meal with brine and stirring the sample for 30 minutes at room temperature using a magnetic stir bar. The extract was then separated from the spent meal by centrifugation at 10,200g for 10 minutes and further clarified by successive filtration using 25 μm pore size filter paper and 0.45 μm pore size filter disc (filter disk). The protein profile of the extract was determined by SEC HPLC.

0.26M NaCl was identified as the best choice for the salt level of the extraction solution in example 1. This salt level was initially taken in preliminary experiments with membrane adsorbers (data not shown), but a small amount of 7S/12S protein was found to be bound by the membrane with some sinapine. Increasing the salt content of the extraction solution to 0.3M NaCl limits 7S/12S protein binding. The protein profile of the 0.3M NaCl extract was such that 64.6% of the peak area of the protein was attributable to 7S/12S and 35.4% to 2S.

(b) Ion exchange:

ion exchange separation was performed using two Sartobind S75(Sartorius AG, Goettingen, germany) strong acid cation adsorber membrane units connected in series. Peristaltic pumps were used to push the various solutions through the membrane unit. The sample isolation protocol is given in table 7.

TABLE 7 sample protocol for separation of 2S from extracts using cation exchange membrane adsorber

Step (ii) of	Solutions of	Volume (ml)	Flow rate (ml/min)
				Equilibration sample loading membrane rinse 2S elution	0.3M NaCl clear extract 0.3M NaCl0.67M NaCl	40105030	20202020

Approximately 32 runs were completed over two consecutive days. The 2S protein fractions eluted from all runs were pooled daily and refrigerated until desalted. The protein profile of the void/wash and eluted fractions was determined by SEC HPLC.

A small portion of 2S (7.7% of the protein peak area) was found to be lost in the void fraction, perhaps due to overloading of the system. The eluted fraction was almost completely 2S (99.6% of the protein peak area). On the first day of the production run, 0.65M NaCl was used as elution buffer, and at the end of the day, the membrane adsorber was washed with 1M NaCl (40 ml). Analysis of the 1M NaCl eluate (SEC HPLC) showed that a small amount of 2S was not eluted by 0.65M NaCl. The next day of the separation run, 0.67M NaCl was used as elution buffer. The membrane was washed with 1M NaCl, which did not release any 2S protein, indicating that 0.67M NaCl was sufficient to recover all bound 2S protein.

(c) Desalting of the isolated 2S protein:

the eluted 2S protein was desalted by concentration and diafiltration on a Vivaflow 5000 MWCO Hydrosart ultrafiltration membrane unit. The volume of all collected 2S protein fractions was about 1000 ml. It is concentrated down to 25 to 30ml and then 300ml water is added for diafiltration. The sample was re-concentrated to 25 to 30ml and 400ml water was added again, and then the sample was re-concentrated again. After the second diafiltration step, the retentate was freeze-dried.

Conductivity was measured for various samples using a conductivity meter. The goal of diafiltration is to reduce the conductivity of the sample to below 1 mS. The permeate was checked for protein profile by SEC HPLC.

Both diafiltration steps effectively reduced the conductivity of the 2S protein sample (table 8). No protein was detected in the ultrafiltration or diafiltration permeate.

TABLE 8 reduction of salt content by diafiltration

2S sample	Conductivity (mS)
		Final retentate after addition of DF1 Water to eluate followed by DF2 Water	49.87.560.79n.d.*

^*n.d. ═ not determined.

(d) And (3) final product:

the final product was evaluated for dry color using a Minolta CR-310 colorimeter, and the solution was also prepared for wet color analysis. Protein powder (0.6g) was mixed with water (10ml) using a vortex mixer. The sample was then centrifuged at 7800g for 10 minutes and the protein content of the supernatant determined by LECO. An aliquot (8ml) of the supernatant was transferred to a small beaker and sufficient water was added to adjust the protein content to 5%. The sample was then photographed and an aliquot of the sample was used for protein profiling (SEC HPLC). Some samples were also diluted to 3.5% and another photograph taken. The protein content of the dry powder was determined by LECO, but not enough sample was obtained for moisture content analysis. Therefore, the protein content is expressed only on the basis of the wet substance.

A total of 1.54g of final product was collected in this study. The purity of the product was very good with a protein content of 101.99% w/w (N x 6.25.25) in the wet mass based powder. As previously mentioned, not enough sample was produced for moisture content analysis, and therefore the protein content could not be expressed on a dry matter basis. Chromatographic analysis of the rehydrated product revealed that 99.85% of the peak area detected at 280nm could be attributed to the 2S protein. No 7S or 12S protein was detected.

The dry color values determined for the membrane absorber 2S are given in table 9.

TABLE 9 Dry color of 2S separated from the extract by cation exchange in a Membrane adsorber

Sample (I)	L*	a*	b*
				2S	84.21	-2.40	15.09

The wet color and clarity of the rehydrated product in water indicated that the 2S protein was produced by column chromatography. We believe that the clarity of the solution should remain fairly stable under most conditions due to the complete absence of 7S/12S protein.

Summary of the disclosure

In summary of this disclosure, the present invention provides a method for recovering high purity 2S canola protein using ion exchange chromatography. Modifications are possible within the scope of the invention.

Claims (9)

1. A method of producing a substantially pure 2S canola protein, comprising:

solubilizing canola protein from canola oil meal to form a canola protein solution,

separating the canola protein solution from residual canola oil meal,

contacting the canola protein solution with a cation exchange medium under conditions in which the 2S canola protein binds to the cation exchange medium in preference to other canola proteins,

separating the bound 2S canola protein from unbound canola protein and impurities, and

separating the bound 2S canola protein from the cation exchange medium.

2. The process according to claim 1, wherein said solubilizing canola proteins from canola oil seed meal is effected using aqueous saline at salt concentration and pH conditions that favor selective binding of said 2S protein to said cation exchange medium.

3. The method according to claim 2, wherein the aqueous salt solution has a salt concentration of about 0.25 to about 0.35M and a pH of about 5 to about 6.

4. The method according to claim 3, wherein the pH of the canola protein solution is about 5 to about 6.

5. The method according to claim 1, wherein the cation exchange media is in the form of resin beads or a membrane adsorber.

6. The method according to claim 1, wherein said bound 2S protein is separated from the cation exchange media by contacting said cation exchange media with an aqueous salt solution at a concentration sufficient to break bonds between said 2S protein and said ion exchange media.

7. The method according to claim 6, wherein the aqueous salt solution used to separate the bound 2S protein from the cation exchange medium has a salt concentration of about 0.55 to about 0.70M.

8. A method of producing a substantially pure 2S canola protein, comprising:

contacting canola oil meal with an aqueous salt solution having a sodium chloride concentration of about 0.25 to about 0.35M to form an aqueous canola protein solution having a pH of about 5 to about 6,

contacting the aqueous canola protein solution with a cation exchange medium to bind 2S canola protein contained in the aqueous canola protein solution to the cation exchange medium, wherein the binding of the 2S canola protein to the cation exchange medium is in preference to other canola proteins contained in the aqueous canola protein solution including 7S and 12S canola proteins and in preference to non-protein species contained in the aqueous canola protein solution,

washing the cation exchange medium to remove unbound canola protein and impurities from the cation exchange resin,

contacting the washed cation exchange medium with an aqueous salt solution having a sodium chloride concentration of about 0.55 to about 0.70M to separate the bound 2S canola protein from the cation exchange medium,

collecting the isolated 2S canola protein as an aqueous salt solution thereof,

desalting the aqueous salt solution of the 2S protein, and

drying the 2S protein.

9. The method of claim 8 wherein the cation exchange media is a membrane adsorber in which cation exchange groups are bound to a microporous membrane.