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(12) NEW EUROPEAN PATENT SPECIFICATIONAfter opposition procedure

(45) Date of publication and mention of the opposition decision: 26.01.2011 Bulletin 2011/04

(45) Mention of the grant of the patent: 07.06.2006 Bulletin 2006/23

(21) Application number: 02767015.7

(22) Date of filing: 10.10.2002

(51) Int Cl.:A23J 1/14 (2006.01) A23J 3/14 (2006.01)

(86) International application number: PCT/CA2002/001526

(87) International publication number: WO 2003/030652 (17.04.2003 Gazette 2003/16)

(54) FLAX PROTEIN ISOLATE AND PRODUCTION

LEINSAAT-PROTEINISOLAT UND VERFAHREN ZUR HERSTELLUNG

ISOLAT DE PROTEINE DE LIN ET PRODUCTION DE CELUI-CI

(84) Designated Contracting States: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LI LU MC NL PT SE SK TRDesignated Extension States: AL LT LV MK RO SI

(30) Priority: 10.10.2001 US 327775 P28.11.2001 US 333492 P

(43) Date of publication of application: 07.07.2004 Bulletin 2004/28

(73) Proprietor: Burcon Nutrascience (MB) Corp.Winnipeg,Manitoba R3T 1P9 (CA)

(72) Inventors: • GREEN, Brent, Everett

Winnipeg, Manitoba R3L 1G4 (CA)• MARTENS, Ronald, W.

Altona, Manitoba R0G 0B0 (CA)• TERGESEN, Johann, Franz

Vancouver, British Columbia V5Y 2H2 (CA)• MILANOVA, Radka,

Burcon Nutrascience (MB) Corp.Winnipeg, Manitoba R3T 1P9 (CA)

(74) Representative: Smart, Peter JohnBeck Greener Fulwood House 12 Fulwood PlaceLondonWC1V 6HR (GB)

(56) References cited: WO-A-00/54608 US-A- 4 208 323US-A- 4 285 862 US-A- 5 844 086US-A- 5 925 401 US-A- 6 005 076

US-A1- 2 573 072 US-A1- 4 091 121US-A1- 4 208 323 US-A1- 4 285 862

• DEV D K ET AL: "Nitrogen extractability and buffer capacity of defatted linseed (Linum usitatissimum L.) flour." JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE 1986 SEKTION NAHRUNGSGÜTERWIRTSCHAFT UND LEBENSMITTELTECH., HUMBOLDT-UNIV., 1020 BERLIN, vol. 37, no. 2, pages 199-205, XP002222166

• DEV D K ET AL: "Preparation and functional properties of linseed protein products containing differing levels of mucilage." JOURNAL OF FOOD SCIENCE 1988 SEKT. NAHRUNGSGÜTERWIRTSCHAFT UND LEBENSMITTELTECH., HUMBOLDT UNIV. ZU BERLIN, 1020 BERLIN, vol. 53, no. 6, pages 1834-1837, 1857, XP002222167

• DEV D K ET AL: "Functional and microstructural characteristics of linseed (Linum usitatissimum L.) flour and a protein isolate." LEBENSMITTEL-WISSENSCHAFT UND -TECHNOLOGIE 1986 SEKTION NAHRUNGSGÜTERWIRTSCHAFT & LEBENSMITTELTECH., HUMBOLDT-UNIV., 1020 BERLIN, vol. 19, no. 4, pages 331-337, XP001128724

• MADHUSUDHAN ET AL. J. AGRIC. FOOD CHEM. no. 33, 1985, pages 673 - 677

• VASSEL ET AL. J. BIOLOGICAL CHEMISTRY vol. 159, no. 3, 1945, pages 571 - 584

• DEV ET AL. LEBENSMITTEL - WISSENSCHAFT UND TECHNOLOGIE no. 19, 1986, pages 331 - 337

• AGRICULTURE AND AGRI-FOOD CANADA vol. 13, no. 15, 2000,

• PAINTER ET AL. INDUSTRIAL AND ENGINEERING CHEMISTRY vol. 38, no. 1, 1946, pages 95 - 98

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EP 1 434 493 B2

• ’Declaration of Brent E. Green’, 25 August 2005, WARREN, MANITOBA

• JIM LOGIE: ’Flax Protein Isolate & Production and Vassel and Nesbitt’s Paper’, 12 September 2009

• B. GREEN: ’Vassel and Nesbitt Flax Isolate’, 12 September 2009

• ’Vassel and Nesbitt Flax Isolate #2’, 12 September 2009

Remarks: The file contains technical information submitted after the application was filed and not included in this specification

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Description

FIELD OF INVENTION

[0001] The present invention relates to a novel proteinisolate derived from any flax oil seed, including the lowlinolenic acid variety linola oil seed, and the productionthereof.

BACKGROUND OF INVENTION

[0002] In US Patent No. 4,285,862 (Murray IA), thereis described the provision of a protein isolate in the formof an amorphous, viscous, sticky, gluten-like proteinmass (PMM), or a dried form of the mass. The amorphousprotein mass is formed settling an aqueous dispersionof protein micelles consisting of homogeneous am-phiphilic protein moieties. The aqueous dispersion isformed by a procedure described in detail in USP4,208,323 (Murray IB) wherein protein is extracted froma protein source material using a food grade salt solutionunder controlled conditions, the protein concentration ofthe resultant extract is increased while maintaining thesame salt concentration, and the concentrated proteinsolution is diluted, thereby forming the aqueous disper-sion of protein micelles. There is no suggestion in thisprior art that the procedures described therein may beapplied or may be modified to apply to the recovery of aflax protein isolate from flax oil seed meal.WO 00/54608 describes a method of recovering proteinfrom lupine seeds using an isoelectric precipitation pro-cedure, which is stated to be applicable to flax oil seeds,amongst others. The procedure is said to give a proteinproduct containing at least 90 wt% protein on a dry weightbasis, the protein being substantially undenatured.

SUMMARY OF INVENTION

[0003] The present invention provides certain proteinisolates which may be produced from any flax oil seedincluding a low linolenic acid mutant known as linola oilseed and a procedure for preparation of the same. Aprotein isolate is defined as a protein containing at leastabout 90 wt% protein at a Kjeldahl nitrogen conversionrate of N x6.25. The term "protein content" as used hereinrefers to the quantity of protein in the protein isolate ex-pressed on a dry weight basis. Such protein isolates andtheir preparation are not described in the Murray IA andIB patents.[0004] Linola oil seed is a mutant of flax oil seed inwhich the fatty acid composition has been changed andlinolenic acid (C18:3) has been substantially reducedfrom about 50% in conventional flax oil seed to about2%, through traditional breeding procedures. Thesemodifications were made to provide from the resultinglinola oil seed an edible polyunsaturated oil substantiallysimilar to sunflower oil in fatty acid composition.[0005] As far as the applicants are aware, there has

not previously been specifically described the prepara-tion of protein isolates from flax oil seed or linola oil seed.The applicants are aware of attempts to provide flax pro-tein products, such as described in USP 5,925,401,wherein a flax product containing 35 to 60 wt% flax pro-tein is provided, well below the protein content requiredto qualify as an isolate.[0006] Accordingly, in one aspect of the present inven-tion, there is provided a substantially undenatured flaxoil seed protein isolate having a protein content of at least100 wt%, as determined by Kjeldahl nitrogen x6.25 (Nx6.25) on a dry weight basis. The flax protein isolate maybe provided in a dry powdered form. The flax protein iso-late also may be provided in the form of dried supernatantfrom the precipitation of flax protein micelles. In addition,the flax protein micelles may be in the form of a driedcombination of concentrated supernatant from the pre-cipitation of flax protein micelles and precipitated flax pro-tein micelles. The flax oil seed protein isolate may bederived from linola, a low linolenic acid variety of flaxseed oil. The flax protein isolate is provided in a substan-tially undenatured form.In another aspect of the present invention, there is pro-vided a substantially undenatured flax oil seed proteinisolate having a protein content of at least 100% as de-termined by Kjeldahl nitrogen x6.25 (N x6.25) on a dryweight basis in the form of a wet protein micellar mass.The flax protein isolate may be derived from linola, a lowlinolenic acid variety of flax oil seed. The flax protein iso-late is provided in a substantially undenatured form.[0007] In another aspect of the present invention, thereis provided a process of preparing a flax protein isolate,which comprises (a) extracting a flax oil seed meal tocause solubilization of protein in said oil seed meal andto from an aqueous protein solution, (b) separating theaqueous protein solution from the residual oil seed meal,(c) increasing the protein concentration of said aqueousprotein solution while maintaining the ionic strength sub-stantially constant by using a selective membrane tech-nique to provide a concentrated protein solution, (d) di-luting said concentrated protein solution into chilled waterto cause the formation of protein micelles, (e) settling theprotein micelles to form an amorphous, sticky, gelati-nous, gluten-like protein micellar mass, and (f) recoveringthe micellar mass from supernatant having a protein con-tent of at least 100 wt%, as determined by Kjeldahl nitro-gen x 6.25 on a dry weight basis. The process may beeffected by: (a) using an aqueous salt solution having anionic strength of at least 0.10, preferably 0.15 to 0.16,and a pH of 4 to 7, preferably 5.3 to 6.2; (b) extractingthe flax oil seed meal with water and subsequent theretoadding salt to the resulting aqueous protein solution hav-ing an ionic strength of at least 0.10, preferably 0.15 to0.6. Preferably, the aqueous protein solution has a pro-tein content of 5 to 30 g/L, more preferably 10 to 25 g/L.The protein concentrating step may be effected to pro-vide a concentrated protein solution having a concentra-tion of at least 50g/L, preferably at least 100 g/L. The

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concentrated protein solution may be warmed to a tem-perature of at least 20 °C, preferably 25 to 40 °C, to de-crease the viscosity of the concentrated protein solutionbut not beyond a temperature above which the temper-ature of the concentrated protein solution does not permitmicelle formation upon dilution. The concentrated proteinsolution may be diluted by 15 fold or less, preferably 10fold or less, by adding the concentrated protein solutioninto a body of water having the volume required toachieve the desired degree of dilution and preferably hav-ing a temperature of less than 10 °C. The recovered pro-tein micellar mass may be dried to a proteinaceous pow-der. The process may be effected in a batch operation,a continuous operation or a semi-continuous manner.The process may be applied to linola oil seed meal.[0008] Supernatant from the settling of the protein mi-cellar mass may be processed to recover further flax pro-tein isolate. The supernatant may be concentrated usinga membrane technique and the concentrated superna-tant dried. Alternatively, the concentrated supernatantmay be mixed with the protein micellar mass and themixture is dried. The supernatant may be concentratedto a protein content of 100 to 400 g/L, preferably 200 to300 g/L, using a membrane technique, preferably usinga membrane having a molecular weight cut-off of 3,000to 10,000 daltons.[0009] The flax protein isolate product in the form ofprotein micellar mass as described herein as "gluten-like". The description is intended to indicate the appear-ance and feel of the isolate are similar to those of vitalwheat gluten and is not intended to indicate chemicalidentity to gluten.[0010] The flax protein isolate produced according tothe process herein may be used in conventional applica-tions of protein isolates, such as, protein fortification ofprocessed foods, emulsification of oils, body formers inbaked goods and foaming agents in products which en-trap gases. In addition, the protein isolate may be formedinto protein fibers, useful in meat analogs, may be usedas an egg white substitute or extender in food productswhere egg white is used as a binder. The flax proteinisolate may be used as nutritional supplements. Otheruses of the flax protein isolate are in pet foods, animalfeed and in industrial and cosmetic applications and inpersonal care products.[0011] Flax oil seed also is referred to as linseed oilseed.

BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 is a schematic flow sheet of a proce-dure for producing a flax oil seed protein isolate in ac-cordance with one embodiment of the invention.

GENERAL DESCRIPTION OF INVENTION

[0013] The novel protein isolates provided herein areprepared by following generally the procedure described

in US Patent 4,208,323, preferably under the specificconditions described herein. The process may be effect-ed as a series of batch steps or as a continuous or semi-continuous process.[0014] The initial step of the process of providing theflax or linola protein isolates involves solubilizing protein-aceous material from flax or linola oil seed meal. Theproteinaceous material recovered from flax or linola seedmeal may be the protein naturally occurring in flax orlinola seed or the proteinaceous material may be a pro-tein modified by genetic manipulation but possessingcharacteristic hydrophobic and polar properties of thenatural protein. The flax or linola meal may be any flaxor linola meal resulting from the removal of flax or linolaoil from flax or linola oil seed with varying levels of non-denatured protein, resulting, for example, from hot hex-ane extraction or cold oil extrusion methods. The removalof flax or linola oil from flax or linola oil seed usually iseffected as a separate operation from the protein isolaterecovery procedure described herein.[0015] Protein solubilization is effected most efficientlyby using a salt solution since the presence of the saltenhances the removal of soluble protein from the oil seedmeal. The salt usually is sodium chloride, although othersalts, such as, potassium chloride, may be used. The saltsolution has an ionic strength of at least about 0.10, pref-erably at least about 0.15, generally up to about 2.0 toenable solubilization of significant quantities of protein tobe effected. As the ionic strength of the salt solution in-creases, the degree of solubilization of protein in the oilseed meal initially increases until a maximum value isachieved. Any subsequent increase in ionic strengthdoes not increase the total protein solubilized. The ionicstrength of the food grade salt solution which causesmaximum protein solubilization varies depending on thesalt concerned and the oil seed meal chosen.[0016] In view of the greater degree of dilution requiredfor protein precipitation with increasing ionic strengths,it is usually preferred to utilize an ionic strength valueless than about 1.0 and more preferably a value of about0.15 to about 0.6.[0017] In a batch process, the salt solubilization of theprotein is effected at a temperature of above about 0°Cand preferably up to about 35°C, preferably accompaniedby agitation to decrease the solubilization time, which isusually about 10 to about 90 minutes. It is preferred toeffect the solubilization to extract substantially the max-imum amount of protein from the oil seed meal, so as toimprove product yield. The upper preferred temperaturelimit of about 35°C is chosen since the process becomesuneconomic at higher temperature levels in a batchmode.[0018] In a continuous process, the extraction of theprotein from the flax or linola oil seed meal is carried outin any manner consistent with effecting a continuous ex-traction of protein from the flax or linola oil seed meal. Inone embodiment, the flax or linola oil seed meal is con-tinuously mixed with a salt solution and the mixture is

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conveyed through a pipe or conduit having a length andat a flow rate for a residence time sufficient to effect thedesired extraction in accordance with the parameters de-scribed herein. In such continuous procedure, the saltsolubilization step is effected rapidly, in a time of up toabout 10 minutes, preferably to effect solubilization toextract substantially the maximum amount of protein fromthe flax or linola oil seed meal. The solubilization in thecontinuous procedure preferably is effected at elevatedtemperatures, generally up to about 60°C or more.[0019] The aqueous food grade salt solution and theflax or linola oil seed meal have a natural pH of about 5to about 7 to enable a protein isolate to be formed by themicellar route, as described in more detail below. Theoptimal pH value for maximum yield of flax or linola pro-tein isolate varies depending on the flax or linola oil seedmeal chosen.[0020] At and close to the limits of the pH range, proteinisolate formation occurs only partly through the micelleroute and in lower yields than attainable elsewhere in thepH range. For these reasons, pH values of about 5.3 toabout 6.2 are preferred.[0021] The pH of the salt solution may be adjusted toany desired value within the range of about 4 to about 7for use in the extraction step by the use of any convenientacid, usually hydrochloric acid, or alkali, usually sodiumhydroxide, as required.[0022] The concentration of oil seed meal in the foodgrade salt solution during the solubilization step may varywidely. Typical concentration values are about 5 to about15% w/v.[0023] The protein extraction step with the aqueoussalt solution has the additional effect of solubilizing fatswhich may be present in the flax meal, which then resultsin the fats being present in the aqueous phase. It is knownthat flax or linola oil seed meal contains significant quan-tities of a mucilage material which enters the aqueousflax or linola protein solution, tending to make the solutionsomewhat viscous. Such initial relatively high viscositytends to inhibit the degree to which the flax or linola pro-tein solution can subsequently be concentrated, accord-ing to the procedure described below.[0024] The protein solution resulting from the extrac-tion step generally has a protein concentration of about5 to about 30 g/L, preferably about 10 to about 25 g/L.[0025] The aqueous phase resulting from the extrac-tion step then may be separated from the residual flax orlinola oil seed meal, in any convenient manner, such asby employing vacuum filtration, followed by centrifuga-tion and/or filtration to remove residual meal. The sepa-rated residual meal may be dried for disposal.[0026] Where the flax or linola seed meal contains sig-nificant quantities of fat, then the defatting steps de-scribed in US Patents Nos. 5,844,086 and 6,005,076,assigned to the assignee hereof and the disclosures ofwhich are incorporated herein by reference may be ef-fected on the separated aqueous protein solution and onthe concentrated aqueous protein solution discussed be-

low.[0027] As an alternative to extracting the flax or linolaoil seed meal with an aqueous salt solution, such extrac-tion may be made using water alone, although the utili-zation of water alone tends to extract less protein fromthe flax or linola oil seed meal than the aqueous salt so-lution. Where such alternative is employed, then the salt,in the concentrations discussed above, may be added tothe protein solution after separation from the residual flaxor linola oil seed meal in order to maintain the protein insolution during the concentration step described below.[0028] The aqueous protein solution then is concen-trated to increase the protein concentration thereof whilemaintaining the ionic strength thereof substantially con-stant. Such concentration generally is effected to providea concentrated protein solution having a protein concen-tration of at least about 50 g/L, preferably at least about100 g/L.[0029] The concentration step may be effected in anyconvenient manner consistent with batch or continuousoperation, such as by employing any convenient selec-tive membrane technique, such as ultrafiltration or diafil-tration, using membranes, such as hollow-fibre mem-branes or spiral-wound membranes, with a suitable mo-lecular weight cut-off, such as about 2000 to about 50,000daltons, having regard to differing membrane materialsand configurations, and, for continuous operation, di-mensioned to permit the desired degree of concentrationas the aqueous protein solution passes through the mem-branes.[0030] The concentration step may be effected at anyconvenient temperature, generally about 15° to about60°C, and for the period of time to effect the desired de-gree of concentration. The temperature and other con-ditions used to some degree depend upon the membraneequipment used to effect the concentration and the de-sired protein concentration of the solution.[0031] As is well known, ultrafiltration and similar se-lective membrane techniques permit low molecularweight species to pass therethrough while preventinghigher molecular weight species from so doing. The lowmolecular weight species include not only the ionic spe-cies of the food grade salt but also low molecular weightmaterials extracted from the source material, such as,carbohydrates, pigments and anti-nutritional factors, aswell as any low molecular weight forms of the protein.The molecular weight cut-off of the membrane is usuallychosen to ensure retention of a significant proportion ofthe protein in the solution, while permitting contaminantsto pass through having regard to the different membranematerials and configurations.[0032] Depending on the temperature employed in theconcentration step, the concentrated protein solutionmay be warmed to a temperature of at least about 20°,and up to about 60°C, preferably about 25° to about 40°C,to decrease the viscosity of the concentrated protein so-lution to facilitate performance of the subsequent dilutionstep and micelle formation. The concentrated protein so-

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lution should not be heated beyond a temperature abovewhich the temperature of the concentrated protein solu-tion does not permit micelle formation on dilution bychilled water. The concentrated protein solution may besubject to a further defatting operation, if required, asdescribed in US Patents Nos. 5,844,086 and 6,005,076.[0033] The concentrated protein solution resultingfrom the concentration step and optional defatting stepthen is diluted to effect micelle formation by mixing theconcentrated protein solution with chilled water havingthe volume required to achieve the degree of dilution de-sired. The concentrated protein solution is diluted byabout 15 fold or less, preferably about 10 fold or less.[0034] The chilled water with which the concentratedprotein solution is mixed has a temperature of less thanabout 15°C, generally about 3° to about 15°C, preferablyless than about 10°C, since improved yields of proteinisolate in the form of protein micellar mass are attainedwith these colder temperatures at the dilution factorsused.[0035] In a batch operation, the batch of concentratedprotein solution is added to a static body of chilled waterhaving the desired volume, as discussed above. The di-lution of the concentrated protein solution and conse-quential decrease in ionic strength causes the formationof a cloud-like mass of highly associated protein mole-cules in the form of discrete protein droplets in micellarform. In the batch procedure, the protein micelles areallowed to settle in the body of chilled water to form anaggregated, coalesced, dense, amorphous, sticky glu-ten-like protein micellar mass (PMM). The settling maybe assisted, such as by centrifugation. Such induced set-tling decreases the liquid content of the protein micellarmass, thereby decreasing the moisture content generallyfrom about 70% by weight to about 95% by weight to avalue of generally about 50% by weight to about 80% byweight of the total micellar mass. Decreasing the mois-ture content of the micellar mass in this way also de-creases the occluded salt content of the micellar mass,and hence the salt content of dried isolate.[0036] Alternatively, the dilution operation may be car-ried out continuously by continuously passing the con-centrated protein solution to one inlet of a T-shaped pipe,while the diluting water is fed to the other inlet of the T-shaped pipe, permitting mixing in the pipe. The dilutingwater is fed into the T-shaped pipe at a rate sufficient toachieve the desired degree of dilution.[0037] The mixing of the concentrated protein solutionand the diluting water in the pipe initiates the formationof protein micelles and the mixture is continuously fedfrom the outlet from the T-shaped pipe into a settling ves-sel, from which, when full, supernatant is permitted tooverflow. The mixture preferably is fed into the body ofliquid in the settling vessel in a manner which minimizesturbulence within the body of liquid.[0038] In the continuous procedure, the protein mi-celles are allowed to settle in the settling vessel to forman aggregated, coalesced, dense, amorphous, sticky,

gluten-like protein micellar mass (PMM) and the proce-dure is continued until a desired quantity of the PMM hasaccumulated in the bottom of the settling vessel, where-upon the accumulated PMM is removed from the settlingvessel.[0039] By the utilization of a continuous process forthe recovery of flax or linola protein isolate as comparedto the batch process, the initial protein extraction stepcan be significantly reduced in time for the same level ofprotein extraction and significantly higher temperaturescan be employed in the extraction step. In addition, in acontinuous operation, there is less chance of contami-nation than in a batch procedure, leading to higher prod-uct quality and the process can be carried out in morecompact equipment.[0040] The settled isolate is separated from the resid-ual aqueous phase or supernatant, such as by decanta-tion of the residual aqueous phase from the settled massor by centrifugation. The PMM may be used in the wetform or may be dried, by any convenient technique, suchas spray drying, freeze drying or vacuum drum drying,to a dry form. The dry flax or linola protein isolate has ahigh protein content, in excess of 100 wt% protein (cal-culated as Kjeldahl N x6.25), and is substantially unde-natured (as determined by differential scanning calorim-etry). The dry flax protein isolate isolated from fatty oilseed meal also has a low residual fat content, when theprocedures of USPs 5,844,086 and 6,005,076 are em-ployed, which may be below about 1 wt%.[0041] In accordance with one aspect of the invention,it has now been found that the supernatant from the PMMformation and settling step contains significant amountsof flax or linola protein, not precipitated in the dilution step.[0042] In such procedure, the supernatant from the di-lution step, following removal of the PMM, may be con-centrated to increase the protein concentration thereof.Such concentration is effected using any convenient se-lective membrane technique, such as ultrafiltration, usingmembranes with a suitable molecular weight cut-off per-mitting low molecular weight species, including the foodgrade salt and other non-proteinaceous low molecularweight materials extracted from the source material, topass through the membrane, while retaining flax proteinin the solution. Ultrafiltration membranes having a mo-lecular weight cut-off of about 3000 to 10,000 daltonshaving regard to differing membranes and configura-tions, may be used. Concentration of the supernatant inthis way also reduces the volume of liquid required to bedried to recover the protein, and hence the energy re-quired for drying. The supernatant generally is concen-trated to a protein content of about 100 to 400 g/L, pref-erably about 200 to about 300 g/L, prior to drying.[0043] The concentrated supernatant may be dried byany convenient technique, such as spray drying, freezedrying or vacuum drum drying, to a dry form to providea further flax protein isolate. Such further flax protein iso-late has a high protein content, usually in excess of about90 wt% protein (calculated as Kjeldahl N x6.25) and is

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substantially undenatured (as determined by differentialscanning calorimetry). If desired, the wet PMM may becombined with the concentrated supernatant prior to dry-ing the combined protein streams by any convenienttechnique to provide a combined flax protein isolate. Thecombined flax protein isolate has a high protein content,in excess of about 90 wt% (calculated as Kjeldahl Nx6.25) and is substantially undenatured (as determinedby differential scanning calorimetry).[0044] In another alternative procedure, a portion onlyof the concentrated supernatant may be mixed with atleast part of the PMM and the resulting mixture dried.The remainder of the concentrated supernatant may bedried as any of the remainder of the PMM. Further, driedPMM and dried supernatant also may be dry mixed inany desired relative proportions.[0045] By operating in this manner, a number of flaxprotein isolates may be recovered, in the form of driedPMM, dried supernatant and dried mixtures of variousproportions by weight of PMM and supernatant, generallyfrom about 5:95 to about 95:5 by weight, which may bedesirable for attaining differing functional and nutritionalproperties.[0046] As an alternative to dilution of the concentratedprotein solution into chilled water and processing of theresulting precipitate and supernatant as describedabove, protein may be recovered from the concentratedprotein solution by dialyzing the concentrated protein so-lution to reduce the salt content thereof. The reductionof the salt content of the concentrated protein solutionresults in the formation of protein micelles in the dialysistubing. Following dialysis, the protein micelles may bepermitted to settle, collected and dried, as discussedabove. The supernatant from the protein micelle settlingstep may be processed, as discussed above, to recoverfurther protein therefrom. Alternatively, the contents ofthe dialysis tubing may be directly dried. The latter alter-native procedure is useful where small laboratory scalequantities of protein are desired.[0047] An alternative procedure for production of theflax protein isolate is to utilize an iso-electric precipitationprocedure. In such a procedure, extraction of the oil seedmeal is effected under alkaline conditions, followingwhich the pH of the protein solution is adjusted to a lowervalue, particularly the pH of the iso-electric point of thetargeted protein, at which pH value the protein has a neu-tral change and precipitates out of solution. The precip-itates may be washed to remove contaminants by resus-pending the precipitate in water and reprecipitating theprotein.

DESCRIPTION OF PREFERRED EMBODIMENT

[0048] Referring to Figure 1, there is illustrated sche-matically a flow sheet of a batch process carried out inaccordance with one embodiment to the invention. Flaxoil seed meal, which may be linola oil seed meal, andaqueous extraction medium are fed by line 10 to an ex-

traction vessel 12 wherein the oil seed meal is extractedand an aqueous protein solution is formed. The slurry ofaqueous protein solution and residual oil seed meal ispassed by line 14 to a vacuum filter belt 16 for separationof the residual oil seed meal which is removed by line18. The aqueous protein solution then is passed by line20 to a clarification operation 22 wherein the aqueousprotein solution is centrifuged and filtered to removefines, which are recovered by line 24.[0049] The clarified aqueous protein solution ispumped by line 26 through ultrafiltration membrane 28to produce a concentrated protein solution as the reten-tate in line 30 with the permeate being recovered by line32. The concentrated protein solution is passed into aprecipitation vessel 34 containing cold water fed by line36. Protein micellar mass formed in the precipitation ves-sel 34 is removed by line 38 and passed through a spraydryer 40 to provide dry flax protein isolate 42.[0050] Supernatant from the precipitation vessel 34 isremoved by line 44 and pumped through ultrafiltrationmembranes 46 to produce a concentrated protein solu-tion as the retentate in line 48 with the permeate beingremoved by line 50. The concentrated protein solution ispassed through a spray dryer 52 to provide further dryflax protein isolate 54.[0051] As an alternative, the concentrated protein so-lution in line 48 may be passed by line 56 to mix with theprotein micellar mass before the mixture then is dried inspray dryer 40.

EXAMPLES

Comparative Example 1:

[0052] This Comparative Example illustrates the re-covery of linola protein from linola oil seed.[0053] Linola oil seed was cold pressed and the oilrecovered. 16.8 kg of crushed meal was added to 335 Lof 0.15 M NaCl solution (5% w/v extraction concentrationat 13°C) and the mixture agitated for 60 mins, followedby a 60 min. settling period. 190 L of extract was decantedand filtered through 20 Pm filter pads to provide 180 L ofan aqueous protein solution having a protein content of6 g/L.[0054] The aqueous solution was reduced in volumeto 11 L by concentration on an ultrafiltration system using30,000 daltons molecular weight cut-off. The resultingconcentrated solution had a protein content of 6 g/L,which represented a yield of 51 wt% of the protein orig-inally extracted from the linola meal.[0055] The concentrated protein solution at a temper-ature of 30°C was added to water at 4°C at a dilution ratioof 1:10. A white cloud formed immediately and was al-lowed to settle for 16 hours. 93 L of supernatant wasdecanted leaving 12 L of precipitated, viscous, sticky pro-tein mass (PMM). An aliquot of PMM was freeze driedto determine protein content. The freeze dried PMM wasfound to have a protein content of 92 wt% (N x6.25) d.b..

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The overall yield of protein from the protein extractedfrom the linola meal was 27 wt%.

Example 2:

[0056] This Example illustrates the recovery of flax pro-tein from flax oil seed meal.[0057] 17.5 kg of commercial flax oil seed meal wasadded to 350 L of 0.5M NaCl solution (5% w/v) at 20°Cand the mixture was agitated for 60 minutes followed by60 minutes settling time. The resulting protein extract so-lution had a protein concentration of 8.5 g/L. A further17.5 kg batch of commercial flax oil seed meal was proc-essed in the same way and the resulting protein extractsolution had a protein concentration of 7.9 g/L. The twoextract solutions were decanted and filtered using 20 Pmfilter pads in a filter press and the filtrates combined.[0058] The filtered aqueous protein solution then wasconcentrated on an ultrafiltration system using 5,000 dal-tons molecular weight cut-off to provide 11 L of a con-centrated aqueous protein solution having a protein con-tent of 120 g/L.[0059] The concentrated protein solution at a temper-ature 31°C was added to tap water at 4°C at a dilutionratio of 1:10. A white cloud formed immediately and wasallowed to settle for 16 hours at 4°C. 105 L of supernatantwas decanted leaving 10 L of precipitated, viscous, stickyprotein mass (PMM). The PMM was centrifuged at10,000 g for five minutes to provide a dense white mass,which then was freeze dried.[0060] 178 g of dried protein isolate was recovered,corresponding to an overall yield of protein extracted fromthe flax oil seed meal of 6 wt%. The freeze dried PMMwas found to have a protein content of 109 wt% (N x6.25) d.b..

Example 3:

[0061] This Example illustrates the effect of pH on lino-la extraction.[0062] Linola oil seed meal was extracted in a 5% w/vsolution with the extraction pH adjusted with either NaOHor HCl to the derived pH levels of 4, 5, 6, 7, 8, 9, 10, 11and 12. All extractions were performed at room temper-ature and effected in an orbital shaker for 30 minutes at230 RPM. Following the mixing period, the spent mealwas separated from the extract and samples taken forprotein content analysis.The results obtained are set forth in the following Table I:

TABLE I

Extraction pH Extraction Protein

12 0.942%

11 0.708%

10 0.522%

[0063] As may be seen, the extractions are higher pHyielded more protein than the lower pH extractions. Ex-tractions at pH 5.0 and 4.0 were quite cloudy in appear-ance, indicating some precipitation.

SUMMARY OF DISCLOSURE

[0064] In summary of this disclosure, the present in-vention provides novel flax and linola protein isolates andprocedures for their preparation. Modifications are pos-sible within the scope of the invention.

Claims

1. A substantially undenatured flax oil seed protein iso-late having a protein content of at least 100 wt% asdetermined by Kjeldahl nitrogen � 6.25 (N � 6.25)on a dry weight basis.

2. The flax seed protein isolate claimed in claim 1 in adry powdered form.

3. The flax protein isolate claimed in claim 1 or 2 in theform of dried supernatant from the precipitation offlax protein micelles.

4. The flax protein isolate claimed in claim 1 or 2 in theform of a dried combination of concentrated super-natant from the precipitation of flax protein micellesand precipitation flax protein micelles.

5. The flax oil seed protein isolate as claimed in claim1 or claim 2, in the form of a wet protein micellarmass.

6. The flax seed protein isolate claimed in any of claims1 to 5 derived from the low linolenic acid variety offlax called linola.

7. A process of preparing a flax protein isolate, whichcomprises:

(a) extracting a flax oil seed meal to cause sol-ubilisation of protein in said oil seed meal and

(continued)

Extraction pH Extraction Protein

9 0.616%

8 0.514%

7 0.330%

6 0.264%

5 0.165%

4 0.188%

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to form an aqueous protein solution,(b) separating the aqueous protein solution fromthe residual oil seed meal,(c) increasing the protein concentration of saidaqueous protein solution while maintaining theionic strength substantially constant by using aselective membrane technique to provide a con-centrated protein solution,(d) diluting said concentrated protein solution in-to chilled water to cause the formation of proteinmicelles,(e) settling the protein micelles to form an amor-phous, sticky, gelatinous, gluten-like protein mi-cellar mass, and(f) recovering the micellar mass from superna-tant having a protein content of at least 100 wt%,as determined by Kjeldahl nitrogen � 6.25 on adry weight basis.

8. The process claimed in claim 7, wherein said extract-ing of flax oil seed meal is effected by:

(a) using an aqueous salt solution having an ion-ic strength of at least 0.10, preferably 0.15 to0.6, and a pH of 4 to 7, preferably 5.3 to 6.2;(b) extracting the flax oil seed meal with waterand subsequent thereto adding salt to the re-sulting aqueous protein solution to provide anaqueous protein solution having an ionicstrength of at least 0.10, preferably 0.15 to 0.6.

9. The process claimed in claim 7 or 8, wherein saidaqueous protein solution has a protein content of 5to 30 g/L, preferably 10 to 25 g/L.

10. The process claimed in any one of claims 7 to 9,wherein said protein concentrating step is effectedto provide a concentrated protein solution having aconcentration of at least 50 g/L, preferably at least100 g/L.

11. The process claimed in any one of claims 7 to 10,wherein said concentrated protein solution iswarmed to a temperature of at least 20°C, preferably25 to 40 °C, to decrease the viscosity of the concen-trated protein solution but not beyond a temperatureabove which the temperature of the concentratedprotein solution does not permit micelle formationupon dilution.

12. The process as claimed in any one of claims 7 to 11,wherein said concentrated protein solution is dilutedby 15 fold or less, preferably 10 fold or less, by addingthe concentrated protein solution into a body of waterhaving the volume required to achieve the desireddegree of dilution and preferably having a tempera-ture of less than 10 °C.

13. The process claimed in any one of claims 7 to 12,wherein the recovered protein micellar mass is driedto a proteinaceous powder.

14. The process claimed in any one of claims 7 to 13,wherein supernatant from the settling step is proc-essed to recover further flax protein isolate.

15. The process claimed in claim 14, wherein the super-natant is concentrated, preferably to a protein con-tent of 100 to 400 g/L, more preferably 200 to 300g/L, using a membrane technique, preferably usinga membrane having a molecular weight cut-off of3,000 to 10,000 daltons and the concentrated super-natant is dried to provide a further flax protein isolatehaving a protein content of at least 90 wt%, as de-termined by Kjeldahl nitrogen � 6.25 on a dry weightbasis.

16. The process claimed in claim 14, wherein the super-natant is concentrated, preferably using a mem-brane having a molecular weight cut-off of 3,000 to10,000 daltons, preferably to a protein content of 100to 400 g/L, more preferably 200 to 300 g/L, the con-centrated supernatant mixed with the protein micel-lar mass and the mixture is dried to provide a furtherflax protein isolate having a protein content of at least90 wt%, as determined by Kjeldahl nitrogen � 6.25on a dry weight basis.

17. The process claimed in any one of claims 7 to 16,wherein steps (a) to (f) are effected in a batch oper-ation, a continuous operation, or a semi-continuousmanner.

18. The process claimed in any one of claims 7 to 17,wherein said flax oil seed meal is linola oil seed meal.

Patentansprüche

1. Leinölsamen-Proteinisolat, das im Wesentlichennicht denaturiert ist, mit einem Proteingehalt vonmindestens 100 Gew.-%, wie mittels Kjeldahl-Stick-stoff x 6,25 (N x 6,25) bestimmt, auf Trockenge-wichtsbasis.

2. Leinsamen-Proteinisolat nach Anspruch 1 in Trok-kenpulverform.

3. Lein-Proteinisolat nach Anspruch 1 oder 2 in Formeines getrockneten Überstands aus der Ausfällungvon Leinproteinmizellen.

4. Lein-Proteinisolat nach Anspruch 1 oder 2 in Formeiner getrockneten Kombination aus konzentriertemÜberstand aus der Ausfällung von LeinProteinmizel-len und Ausfällungs-Leinproteinmizellen.

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5. Leinölsamen-Proteinisolat nach Anspruch 1 oderAnspruch 2 in Form einer nassen Proteinmizellen-masse.

6. Leinsamen-Proteinisolat nach einem der Ansprüche1 bis 5, das von der Leinart mit niedriger Linolensäu-re mit Namen Linola stammt.

7. Verfahren zur Herstellung eines Lein-Proteinisolats,umfassend:

(a) Extraktion eines Leinölsamenmehls, um dieSolubilisierung von Protein im Ölsamenmehl zubewirken und eine wässrige Proteinlösung zubilden,(b) Trennen der wässrigen Proteinlösung vomrestlichen Ölsamenmehl,(c) Erhöhen der Proteinkonzentration der wäss-rigen Proteinlösung, während die Ionenstärkemittels eines selektiven Membranverfahrens imWesentlichen konstant gehalten wird, um einekonzentrierte Proteinlösung zur Verfügung zustellen,(d) Verdünnen der konzentrierten Proteinlösungin gekühltem Wasser, um die Bildung von Pro-teinmizellen zu bewirken,(e) Ablagerung der Proteinmizellen zur Bildungeiner amorphen, klebrigen, gelatinösen, gluten-ähnlichen Proteinmizellenmasse und(f) Wiederherstellen der Mizellenmasse ausdem Überstand mit einem Proteingehalt vonmindestens 100 Gew.-%, wie mittels Kjeldahl-Stickstoff x 6,25 bestimmt, auf Trockenge-wichtsbasis.

8. Verfahren nach Anspruch 7, wobei die Extraktionvon Leinölsamenmehl bewirkt wird durch:

(a) Verwendung einer wässrigen Salzlösung miteiner Ionenstärke von mindestens 0,10, vor-zugsweise 0,15 bis 0,6, und einem pH-Wert von4 bis 7, vorzugsweise 5,3 bis 6,2;(b) Extraktion des Leinölsamenmehls mit Was-ser und anschließend daran Zugabe von Salzzu der sich ergebenden wässrigen Proteinlö-sung, um eine wässrige Proteinlösung mit einerIonenstärke von mindestens 0,10, vorzugswei-se 0,15 bis 0,6, zur Verfügung zu stellen.

9. Verfahren nach Anspruch 7 oder 8, wobei die wäss-rige Proteinlösung einen Proteingehalt von 5 bis 30g/l, vorzugsweise 10 bis 25 g/l, aufweist.

10. Verfahren nach einem der Ansprüche 7 bis 9, wobeider Proteinkonzentrationsschritt durchgeführt wird,um eine konzentrierte Proteinlösung mit einer Kon-zentration von mindestens 50 g/l, vorzugsweise min-destens 100 g/l, zur Verfügung zu stellen.

11. Verfahren nach einem der Ansprüche 7 bis 10, wobeidie konzentrierte Proteinlösung auf eine Temperaturvon mindestens 20°C, vorzugsweise 25 bis 44°C,erwärmt wird, um die Viskosität der konzentriertenProteinlösung zu senken, aber nicht über eine Tem-peratur, über der die Temperatur der konzentriertenProteinlösung bei Verdünnung keine Mizellenbil-dung erlaubt.

12. Verfahren nach einem der Ansprüche 7 bis 11, wobeidie konzentrierte Proteinlösung durch Zugabe derkonzentrierten Proteinlösung in eine Wassermasse,die ein Volumen aufweist, das erforderlich ist, umden gewünschten Verdünnungsgrad zu erzielen,und vorzugsweise eine Temperatur von unter 10°Chat, um das 15-fache oder weniger, vorzugsweiseum das 10-fache oder weniger, verdünnt ist.

13. Verfahren nach einem der Ansprüche 7 bis 12, wobeidie aufbereitete Proteinmizellenmasse zu einemproteinhaltigen Pulver getrocknet wird.

14. Verfahren nach einem der Ansprüche 7 bis 13, wobeider Überstand vom Ablagerungsschritt aufbereitetwird, um weiteres Lein-Proteinisolat zu erhalten.

15. Verfahren nach Anspruch 14, wobei der Überstandvorzugsweise auf einen Proteingehalt von 100 bis400 g/l, besonders bevorzugt 200 bis 300 g/l, mittelseines Membranverfahrens, vorzugsweise mittels ei-ner Membran mit einer Molekulargewichtstrenn-grenze von 3.000 bis 10.000 Dalton konzentriert undder konzentrierte Überstand getrocknet wird, umweiteres Lein-Proteinisolat mit einem Proteingehaltvon mindestens 90 Gew.-%, wie mittels Kjeldahl-Stickstoff x 6,25 bestimmt, auf Trokkengewichtsba-sis zur Verfügung zu stellen.

16. Verfahren nach Anspruch 14, wobei der Überstandkonzentriert wird, vorzugsweise unter Verwendungeiner Membran mit einer Molekulargewichtsgrenzevon 3.000 bis 10.000 Dalton, vorzugsweise auf einenProteingehalt von 100 bis 400 g/l, besonders bevor-zugt 200 bis 300 g/l, wobei der konzentrierte Über-stand mit der Proteinmizellenmasse gemischt unddie Mischung getrocknet wird, um ein weiteres Lein-Proteinisolat bereitzustellen mit einem Proteingehaltvon mindestens 90 Gew.-%, wie mittels Kjeldahl-Stickstoff x 6,25 auf Trockengewichtsbasis be-stimmt.

17. Verfahren nach einem der Ansprüche 7 bis 16, wobeidie Schritte (a) bis (f) in einer Batch-Vorgehenweise,einer kontinuierlichen Vorgehensweise oder einersemikontinuierlichen Vorgehensweise durchgeführtwerden.

18. Verfahren nach einem der Ansprüche 7 bis 17, wobei

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das Leinöl-Samenmehl Linolaöl-Samenmehl ist.

Revendications

1. Isolat de protéine de graine de lin substantiellementnon dénaturé ayant un taux de protéine d’au moins100 % en masse, déterminé par l’azote de Kjeldahl� 6,25 (N � 6,25) par rapport à la masse sèche.

2. Isolat de protéine de graine de lin selon la revendi-cation 1 sous la forme d’une poudre sèche.

3. Isolat de protéine de graine de lin selon la revendi-cation 1 ou 2 sous la forme d’un surnageant séchéprovenant de la précipitation des micelles de protéi-ne de lin.

4. Isolat de protéine de graine de lin selon la revendi-cation 1 ou 2 sous la forme d’une combinaison sé-chée constituée par un surnageant concentré pro-venant de la précipitation des micelles de protéinede lin et des micelles de protéine de lin précipitées.

5. Isolat de protéine de graine de lin selon la revendi-cation 1 ou la revendication 2 sous la forme d’unemasse micellaire protéinique humide.

6. Isolat de protéine de graine de lin selon l’une quel-conque des revendications 1 à 5, dérivé de la variétéde lin à faible taux d’acide linolénique dite linola.

7. Procédé de préparation d’un isolat de protéine delin, qui comprend :

(a) l’extraction d’une farine de graine de lin pourprovoquer la solubilisation de la protéine dansladite farine de graine de lin et pour former unesolution aqueuse de protéine,(b) la séparation de la solution aqueuse de pro-téine de la farine de graine de lin résiduelle,(c) l’augmentation de la concentration de la pro-téine de ladite solution aqueuse de protéine touten maintenant la force ionique substantielle-ment constante par utilisation d’une techniquede membrane sélective aux fins d’obtenir unesolution de protéine concentrée,(d) la dilution de ladite solution de protéine con-centrée dans de l’eau réfrigérée pour provoquerla formation de micelles de protéine,(e) la sédimentation des micelles de protéinepour former une masse micellaire de protéinedu type gluten, amorphe, collante, gélatineuse,et(f) la récupération de la masse micellaire, sépa-rée du surnageant, ayant un taux de protéine,déterminé par l’azote de Kjeldahl � 6,25 (N �6,25) par rapport à la masse sèche, d’au moins

100 % en masse.

8. Procédé selon la revendication 7, dans lequel laditeextraction de la farine de graine de lin est effectuéepar :

(a) l’utilisation d’une solution aqueuse salineayant une force ionique d’au moins 0,10, de pré-férence de 0,15 à 0,6, et un pH de 4 à 7, depréférence de 5,3 à 6,2 ;(b) l’extraction de la farine de graine de lin avecde l’eau, et ensuite l’addition d’un sel à la solu-tion aqueuse de protéine obtenue pour obtenirune solution aqueuse de protéine ayant une for-ce ionique d’au moins 0,10, de préférence de0,15 à 0,6.

9. Procédé selon la revendication 7 ou 8, dans lequelladite solution aqueuse de protéine a un taux de pro-téine de 5 à 30 g/l, de préférence de 10 à 25 g/l.

10. Procédé selon l’une quelconque des revendications7 à 9, dans lequel ladite étape de concentration dela protéine est effectuée de manière à obtenir unesolution concentrée de protéine ayant une concen-tration d’au moins 50 g/l, de préférence d’au moins100 g/l.

11. Procédé selon l’une quelconque des revendications7 à 10, dans lequel ladite solution de protéine con-centrée est réchauffée à une température d’au moins20°C, de préférence de 25 à 40°C, pour diminuer laviscosité de la solution de protéine concentrée, maissans aller au-delà d’une température au-dessus delaquelle la température de la solution concentrée deprotéine ne permet pas de formation de micelle pardilution.

12. Procédé selon l’une quelconque des revendications7 à 11, dans lequel ladite solution de protéine con-centrée est diluée de 15 fois ou moins, de préférencede 10 fois ou moins, par addition de la solution deprotéine concentrée dans une masse d’eau ayant levolume requis pour obtenir le degré de dilution désiréet ayant de préférence une température inférieure à10°C.

13. Procédé selon l’une quelconque des revendications7 à 12, dans lequel la masse micellaire de protéinerécupérée est séchée pour donner une poudre pro-téinique.

14. Procédé selon l’une quelconque des revendications7 à 13, dans lequel le surnageant de l’étape de sé-dimentation est traité de façon à récupérer un nouvelisolat de protéine de lin.

15. Procédé selon la revendication 14, dans lequel le

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surnageant est concentré, de préférence jusqu’à untaux de protéine de 100 à 400 g/l, plus préférable-ment de 200 à 300 g/l, en utilisant une technique demembrane, de préférence en utilisant une membra-ne ayant un seuil de coupure des masses molécu-laires de 3 000 à 10 000 daltons, et le surnageantconcentré est séché pour obtenir un nouvel isolat deprotéine de lin ayant un taux de protéine, déterminépar l’azote de Kjeldahl x 6,25 par rapport à la massesèche, d’au moins 90 % en masse.

16. Procédé selon la revendication 14, dans lequel lesurnageant est concentré, de préférence en utilisantune membrane ayant un seuil de coupure des mas-ses moléculaires de 3 000 à 10 000 daltons, de pré-férence jusqu’à un taux de protéine de 100 à 400 g/l,plus préférablement de 200 à 300 g/l le surnageantconcentré est mélangé avec la masse micellaire pro-téinique et ce mélange est séché pour obtenir unnouvel isolat de protéine de lin ayant un taux de pro-téine, déterminé par l’azote de Kjeldahl x 6,25 parrapport à la masse sèche, d’au moins 90 % en mas-se.

17. Procédé selon l’une quelconque des revendications7 à 16, dans lequel les étapes (a) à (f) sont effectuéesselon un mode discontinu, un mode continu, ou unmode semi-continu.

18. Procédé selon l’une quelconque des revendications7 à 17, dans lequel ladite farine de graine de lin estune farine de graine de linola.

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the Europeanpatent document. Even though great care has been taken in compiling the references, errors or omissions cannot beexcluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• US 4285862 A, Murray IA [0002]• US 4208323 A, Murray IB [0002] [0013]• WO 0054608 A [0002]

• US 5925401 A [0005]• US 5844086 A [0026] [0032] [0040]• US 6005076 A [0026] [0032] [0040]