Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/18—Ion-exchange chromatography
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
  • A23J1/14—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

• the present invention provides a procedure for the production of canola 2S protein in substantially pure form by a process involving the use of ion-exchange chromatography.
• Canola protein isolates having protein contents of at least 100 wt % (N ⁇ 6.25) d.b. can be formed from canola oil seed meal, as described in co-pending U.S. patent application Ser. No. 10/137,391 filed May 3, 2002 (US Patent Application Publication No. 20030125526 A1), U.S. patent application Ser. No. 10/476,230 filed Jun. 9, 2004 (US Patent Application Publication No. 20040254353 A1) and corresponding PCT Publication No. WO 02/089597, both assigned to the assignee hereof and the disclosures of which are incorporated herein by reference.
• the procedure involves a multiple step process comprising extracting canola oil seed meal using a salt solution, separating the resulting aqueous protein solution from residual oil seed meal, increasing the protein concentration of the aqueous solution to at least about 200 g/L while maintaining the ionic strength substantially constant by using a selective membrane technique, diluting the resulting concentrated protein solution into chilled water to cause the formation of protein micelles, settling the protein micelles to form an amorphous, sticky, gelatinous, gluten-like protein micellar mass (PMM), and recovering the protein micellar mass from supernatant having a protein content of at least about 100 wt % as determined by Kjeldahl nitrogen (N ⁇ 6.25).
• protein content is determined on a dry weight basis.
• the recovered PMM may be dried.
• the supernatant from the PMM settling step is processed to recover a protein isolate comprising dried protein from the wet PMM and supernatant.
• This procedure may be effected by initially concentrating the supernatant using ultrafiltration membranes, mixing the concentrated supernatant with the wet PMM and drying the mixture.
• the resulting canola protein isolate has a high purity of at least about 90 wt % of protein (N ⁇ 6.25), preferably at least about 100 wt % protein (N ⁇ 6.25).
• the supernatant from the PMM settling step is processed to recover a protein from the supernatant.
• This procedure may be effected 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 ⁇ 6.25), preferably at least about 100 wt % protein (N ⁇ 6.25).
• canola oil seed meal is continuously mixed with a salt solution, the mixture is conveyed through a pipe 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 conveyed through a selective membrane operation to increase the protein content of the aqueous protein solution to at least about 200 g/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 permitted to settle while the supernatant is continuously overflowed until the desired amount of PMM has accumulated in the settling vessel.
• the PMM is removed from the settling vessel and may be dried.
• the PMM has a protein content of at least about 90 wt % as determined by Kjeldahl nitrogen (N ⁇ 6.25), preferably at least about 100 wt % (N ⁇ 6.25).
• the overflowed supernatant may be processed to recover canola protein isolate therefrom.
• Canola seed is known to contain about 10 to about 30 wt % proteins and several different protein components have been identified. These proteins are distinguished by different sedimentation coefficients (S). These known and identified proteins include a 12S globulin, known as cruciferin, a 7S globulin and a 2S albumin, known as napin.
• S sedimentation coefficients
• Canola is also known as rapeseed or oil seed rape.
• the supernatant-derived canola protein isolate exhibits a protein profile which is:
• the PMM canola protein isolate exhibits a protein profile which is:
• the supernatant-derived canola protein isolate consisting predominantly of 2S protein exhibits superior functional properties for certain applications than the PMM-derived canola protein isolate consisting predominantly of 7S protein.
• the procedures described in the prior applications in order to produce the supernatant-derived canola protein isolate, it was necessary to go through the steps of PMM formation and provision of a supernatant in order, in effect, to fractionate the canola proteins.
• the 2S protein having a minimal proportion of the 7S and 12S proteins demonstrates increased solubility over the untreated 2S protein (including at acid pH values) and is able to provide improved clarity in aqueous solution and with soft drinks and sport drinks, providing clear protein fortified beverages.
• the present invention utilizes an alternative procedure involving ion exchange to prepare substantially pure 2S canola protein substantially free from 7S and 12S canola proteins.
• ion exchange chromatography In ion exchange chromatography, charged ion-exchange medium is 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 major classes of canola proteins have significantly different isoelectric points.
• the 7S/12S globulins have an isoelectric point in the range of about 6 to 7, while for the 2S albumin the value is approximately 11. This difference is utilized herein to separate the proteins from each other by ion exchange chromatography.
• An ion exchange process is provided herein in which 2S protein is captured by binding it to the cation-exchange medium, while permitting other proteins and impurities to be washed away.
• the 2S protein then is released from the cation-exchange medium by exposure of the cation-exchange medium to saline at a suitably high salt concentration.
• a method of producing substantially pure 2S canola protein which comprises solubilizing canola proteins 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 wherein the 2S canola protein is bound to the cation-exchange medium in preference to other canola proteins, separating the bound 2S canola protein from unbound canola proteins and impurities, and separating the bound 2S canola protein from the cation-exchange medium.
• ion-exchange chromatography is effected on canola protein solution to preferentially bind the 2S canola protein to the ion exchange medium and the 2S canola protein is subsequently recovered in substantially pure form from the ion exchange medium.
• an aqueous solution of canola protein is contacted with a cation exchange medium at a pH of about 5 to 6, where both classes of protein are positively charged.
• a salt concentration is utilized to limit binding of the less positively charged 7S/12S canola proteins as well as impurities, such as sinapine.
• the aqueous canola protein solution may be most conveniently formed by extraction from canola oil seed meal.
• the extraction is effected using an aqueous salt solution having a desired saline concentration and pH value to be effective in ensuring preferential binding of 2S protein to the cation exchange medium.
• the salt solution generally has a salt concentration in the range of about 0.25 to 0.35 M NaCl and the pH of the aqueous canola protein solution is in the range of about 5 to about 6.
• Extraction of the canola oil seed meal may be effected outside the desired pH range and the pH of the canola protein solution then may be adjusted to the pH range of about 5 to about 6 with any convenient acid or base, as necessary.
• the protein may be extracted from the canola oil seed meal by using a saline solution of lower salt concentration and then additional salt is added to the desired concentration.
• a saline solution of lower salt concentration it is preferred to effect the extraction with saline at the concentration required for the ion exchange, since the extract solution is in a form for direct application to the cation exchange medium for the isolation of the 2S canola protein immediately after formation. Therefore, there is little time for oxidation reactions to occur or the binding of phenolics to protein.
• the canola protein extract solution is applied to the cation exchange medium, which may be provided in any convenient form, such as in the form of resin beads or a membrane adsorber.
• the ion exchange groups are bound to microporous membranes.
• membranes instead of resin-packed columns allows the use of higher flow rates and results in faster processing.
• the contact of the canola protein extract solution with the cation exchange medium causes the 2S protein to be adsorbed in preference to the less positively charged 7S/12S proteins.
• the 2S protein may be removed from the cation exchange medium by contact with an aqueous saline solution having a higher salt concentration than that of the aqueous canola protein saline solution, such as about 0.55 to about 0.70 M NaCl.
• the eluted solution of 2S protein has a high salt concentration and is desalted by any convenient manner, such as diafiltration, before drying the protein.
• the procedure produces a high purity 2S canola protein isolate substantially free from the 7S/12S proteins and having a protein content of at least about 100 wt % (N ⁇ 6.25) on a dry weight basis (d.b.).
• the canola 7S/12S proteins may be recovered from the canola protein extract after contact with the ion exchange medium in an undenaturated form, in contrast to the form when isoelectric or heat precipitation are utilized to separate the proteins from 2S protein.
• This Example illustrates the preparation of substantially pure 2S canola protein using a cation exchange column.
• a series of 15% w/v extractions of canola oil seed meal was carried out using typically 150 ml saline per 22.5 g meal. The samples were stirred for 30 minutes at room temperature using a magnetic stir bar. In each instance, the extract was separated from the spent meal by centrifugation at 10,200 g for 10 minutes and then further clarified by successive filtration with 25 ⁇ m pore size filter paper and 0.45 ⁇ m pore size syringe filters. The protein content of the clarified extract was determined by LECO analysis (LECO FP 528 Nitrogen Determinator) and the protein profile determined by size exclusion (SEC) HPLC. In the various runs, the salt concentration in the saline solution varied from 0.26 to 0.35 M NaCl.
• the concentration of salt used for the extraction/protein separation and the elution of the 2S protein were fine tuned as the production runs proceeded.
• the salt concentrations used were 0.30M/0.55M.
• the void material was collected as overlapping doublet peaks, with the first peak found to contain almost all of the 7S/12S, a small amount of unbound 2S protein and most of the impurities seen in the extract except a portion of the sinapine.
• the second peak in the doublet which took slightly longer to emerge from the column, was found to contain a notable 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 as a notable 2S protein peak was obtained when cleaning the column with 1 M NaCl.
• the elution salt level was raised to 0.60 M to better release the 2S protein. This time a smaller 2S protein peak was found when the column was cleaned.
• the elution step was increased to 0.65 M NaCl and this level was found to effectively eliminate the peak seen when cleaning the column.
• the initial salt level in the third trial was increased to 0.35 M NaCl in the hopes of bringing the two void peaks closer together. The separation between the doublet peaks was reduced, but a doublet was still obtained. Also, operating at this higher salt level increased the amount of 2S protein that did not bind the column and was found in the void.
• the sample was reconcentrated to 25 to 30 ml, and a further 300 ml of water was added and then the sample reconcentrated again.
• the desalting was conducted effectively with two steps of approximately 10 diafiltration volumes.
• the dry colour of the final product was assessed using a Minolta CR-310 Chroma meter and a solution was also prepared for wet colour analysis.
• Protein powder 0.5 g was combined with water (10 ml) using a vortex mixer. The sample was then centrifugated at 7800 g for 10 min (mainly to remove air) and the protein content of the supernatant determined by LECO. An aliquot (8 ml) 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 used for protein profile analysis (SEC HPLC). Some sample was also diluted to 3.5% protein and another photograph taken. The protein content of the dry powder was tested by LECO but there was not enough sample obtained to do a moisture content analysis. Therefore, the protein content was only expressed on a wet basis.
• This Example illustrates the use of a cation exchange membrane to produce substantially pure 2S canola protein.
• 10% w/v extractions of 30 g of canola oil seed meal were effected using 300 ml of 0.3 M NaCl by combining the meal and saline and stirring the samples for 30 minutes at room temperature using a magnetic stir bar.
• the extract was then separated from the spent meal by centrifugation at 10,200 g for 10 minutes and further clarified by successive filtration with 25 ⁇ m pore size filter paper and 1 ⁇ m and 0.45 ⁇ m pore size filter disks.
• the protein profile of the extract was determined by SEC HPLC.
• Example 1 0.26 M NaCl was identified in Example 1 as the best choice of salt level for the extraction solution. This salt level was initially adopted in preliminary experiments with the membrane adsorber (data not shown), but small amounts of 7S/12S proteins and some sinapine were found to be bound by the membrane. Increasing the salt content of the extraction solution to 0.3 M NaCl limited the 7S/12S proteins binding. The protein profile of the 0.3 M NaCl extract was 64.6% of protein peak area due to 7S/12S and 35.4% due to 2S.
• Eluted 2S protein was desalted by concentration and diafiltration on a Vivaflow 5000 MWCO Hydrosart ultrafiltration membrane unit. The volume of all the collected 2S protein fractions was approximately 1000 ml. This was concentrated down to 25 to 30 ml and then 300 ml of water was added for diafiltration. The sample was reconcentrated to 25 to 30 ml and a further 400 ml of water was added and the sample reconcentrated again. After the second diafiltration step, the retentate was freeze dried.
• the conductivity of various samples was measured using a conductivity meter.
• the goal of diafiltration was to reduce the conductivity of the sample below 1 mS.
• the permeates were checked for protein profile by SEC HPLC.
• the dry colour of the final product was assessed using a Minolta CR-310 Chroma meter and a solution was also prepared for wet colour analysis.
• Protein powder (0.6 g) was combined with water (10 ml) using a vortex mixer. The sample was then centrifuged at 7800 g for 10 minutes and the protein content of the supernatant determined by LECO. An aliquot (8 ml) 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 used for protein profile analysis (SEC HPLC). Some sample was also diluted to 3.5% and another photograph taken. The protein content of the dry powder was tested by LECO but not enough sample was obtained to do a moisture content analysis. Therefore, the protein content was only expressed on a wet basis.
• the present invention provides a method of recovering high purity 2S canola protein using ion exchange chromatography. Modifications are possible within the scope of this invention.

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• 2008
  • 2008-08-01 US US12/452,639 patent/US20100184956A1/en not_active Abandoned
  • 2008-08-01 RU RU2010107609/10A patent/RU2490274C2/ru not_active IP Right Cessation
  • 2008-08-01 WO PCT/CA2008/001425 patent/WO2009018660A1/en not_active Ceased
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