PROTEIN ISOLATE AND PROCESS FOR THE PRODUCTION THEREOF

Abstract

A process for producing a protein isolate from an oilseed meal, and the isolate thus obtained, said isolate comprising proteins and an amount of 4 wt. % or less of phytic acid, said amount of phytic acid being by weight of proteins in said isolate. The process may comprise the following steps: a) providing an oilseed meal; b) mixing the oilseed meal with a first aqueous solvent to form a slurry at a pH ranging from 6 to 7.8, said slurry having a solid phase; c) separating said solid phase from said slurry, d) mixing said separated solid phase with a second aqueous solvent at a pH ranging from 1 to 3.5, preferably from 2 to 3, to form a mixture said mixture having a liquid phase; e) separating said liquid phase from said mixture formed in step d); f) f1) mixing the separated liquid phase to a phytase at a temperature and a pH suitable for phytase activity to obtain a mixture having a liquid phase and a solid phase; and/or f2) mixing the separated liquid to a salt, to obtain a resulting liquid composition having a molar concentration of said salt ranging from 0.05M to 0.5M, at a temperature ranging from 40 C. to 70 C., to obtain a mixture having a liquid phase and a solid phase; g) precipitating a solid phase from the liquid of step f) for example by a cooling down step of the mixture to a temperature of 30 C. or less; h) separating said solid precipitate from the liquid of step g) said liquid comprising a water-rich liquid phase and an oil-rich liquid phase; i) separating said water-rich liquid phase from said oil-rich liquid phase, j) subjecting said water-rich liquid phase obtained in step i) to one or several membrane filtration(s) to obtain a protein isolate; and k) optionally, drying said protein isolate to obtain a dry protein isolate.

Claims

1. A process for producing a protein isolate from an oilseed meal, said isolate comprising proteins and an amount of 4 wt. % or less of phytic acid, said amount of phytic acid being by weight of proteins in said isolate, said process comprising the following steps: a. providing an oilseed meal; b. mixing the oilseed meal with a first aqueous solvent to form a slurry at a pH ranging from 6 to 7.8, said slurry having a solid phase; c. separating said solid phase from said slurry; d. mixing said separated solid phase with a second aqueous solvent at a pH ranging from 1 to 3.5, to form a mixture said mixture having a liquid phase; e. separating said liquid phase from said mixture formed in step d); f. f1) mixing the separated liquid phase with a phytase at a temperature and a pH suitable for phytase activity to obtain a mixture having a liquid phase and a solid phase; and/or f2) mixing the separated liquid with a salt, to obtain a resulting liquid composition having a molar concentration of said salt ranging from 0.05M to 0.5M, at a temperature ranging from 40 C. to 70 C., to obtain a mixture having a liquid phase and a solid phase; g. precipitating a solid phase from the liquid of step f); h. separating said solid precipitate from the liquid of step g) said liquid comprising a water-rich liquid phase and an oil-rich liquid phase; i. separating said water-rich liquid phase from said oil-rich liquid phase, j. subjecting said water-rich liquid phase obtained in step i) to one or several membrane filtration(s) to obtain a protein isolate; and k. optionally, drying said protein isolate to obtain a dry protein isolate.

2. The process according to claim 1, wherein the oilseed meal is selected from the group consisting of rapeseed, canola, soybean, flax, lupine, sunflower, safflower, cotton, mustard and hemp seed meals, and mixtures thereof.

3. The process according to claim 1, wherein the meal is a partially defatted meal, a defatted meal or a protein enriched meal.

4. The process according to claim 1, wherein the oilseed meal is a cold-press oilseed meal which has been cold-pressed at a temperature of 85 C. or less.

5. The process according to claim 1, wherein said salt is sodium chloride.

6. The process according to claim 1, wherein the ratio of the oilseed meal to the aqueous solvent ranges from 1:5 to 1:20 (w/w).

7. The process according to claim 1, wherein any one of steps c), e), h), i) and/or j) is repeated.

8. The process according to claim 1, wherein step j) comprises the following steps: j1) optionally subjecting the liquid phase obtained in step i) to at least one microfiltration step and harvesting a permeate, j2) subjecting the liquid phase obtained in step i) or the permeate of step j1) to at least one ultrafiltration step, optionally followed by at least one diafiltration step, and harvesting the protein isolate.

9. The process according to claim 1, wherein steps a) to e), i) and j) are conducted at room temperature.

10. A process for producing a protein isolate from an oilseed meal, said isolate comprising proteins and an amount of 3 wt. % or less of phytic acid, said amount of phytic acid being by weight of proteins in said isolate, said process comprising the following steps: i. providing an oilseed meal; ii. mixing said oilseed meal with an aqueous solvent at a pH ranging from 1 to 3.5 to form a mixture said mixture having a liquid phase; iii. separating said liquid phase from said mixture formed in step ii); iv. mixing the separated liquid phase with a phytase at a temperature and a pH suitable for phytase activity to obtain a mixture having a liquid phase and a solid phase; and/or v. precipitating a solid phase from the liquid of step iv) by a cooling down step of the mixture to a temperature of 30 C. or less; vi. separating said solid precipitate from the liquid of step v), said liquid comprising a water-rich liquid phase and an oil-rich liquid phase; vii. separating said water-rich liquid phase from said oil-rich liquid phase, viii. subjecting said water-rich liquid phase obtained in step vii) to one or several membrane filtration(s) to obtain a protein isolate; and ix. optionally, drying said protein isolate to obtain a dry protein isolate.

11. A process for producing a protein isolate from an oilseed meal, said isolate comprising proteins and an amount of 3 wt. % or less of phytic acid, said amount of phytic acid being by weight of proteins in said isolate, said process comprising the following steps: A. providing an oilseed meal; B. mixing the oilseed meal with a first aqueous solvent to form a slurry at a pH ranging from 6 to 7.8, said slurry having a liquid phase; C. separating said liquid phase from said slurry, D. adjusting the pH of said separated liquid phase at a pH ranging from 2 to 4, to form a mixture said mixture having a liquid phase and a solid precipitate; E. separating said liquid phase from said solid precipitate; F. subjecting the liquid phase obtained in step E. to a microfiltration step, and recovering a permeate; G. subjecting said permeate recovered in step F. to an ultrafiltration step which is followed by at least one diafiltration step carried out with an aqueous solution of a salt to obtain said protein isolate; and H. optionally, drying said protein isolate to obtain a dry protein isolate.

12. A protein isolate obtainable by the process according to claim 1, having has least less than 4% of phytic acid by weight of proteins in said isolate, a purity of at least 85% (N6.25) on a dry protein isolate and an albumin content of at least 90% of the total protein content.

13. The process according to claim 1, comprising mixing said separated solid phase with a second aqueous solvent at a pH ranging from 2 to 3, to form a mixture said mixture having a liquid phase.

14. The process according to claim 1, wherein said precipitating a solid phase from the liquid of step f) comprises a cooling down step of the mixture to a temperature of 30 C. or less.

15. The process according to claim 1, wherein the oilseed meal is selected from the group consisting of rapeseed, canola and sunflower seed meals and mixtures thereof.

16. The process according to claim 1, wherein the oilseed meal is a cold-press oilseed meal which has been cold-pressed at a temperature of 60 C. or less.

17. The process according to claim 1, wherein the ratio of the oilseed meal to the aqueous solvent ranges from 1:6 to 1:10 (w/w).

18. The process according to claim 10, comprising mixing said oilseed meal with an aqueous solvent at a pH ranging from 2 to 3, to form a mixture said mixture having a liquid phase;

19. The process according to claim 11, comprising adjusting the pH of said separated liquid phase at a pH ranging from 3 to 3.8, to form a mixture said mixture having a liquid phase and a solid precipitate.

20. A protein isolate obtainable by the process according to claim 1, having has least less than 4% of phytic acid by weight of proteins in said isolate, a purity of at 90% (N6.25) on a dry protein isolate and an albumin content of at least 90% of the total protein content.

Description

EXAMPLES

[0134] The following analysis results (determination of protein content, of phytic acid, solubility, SE-HPLC) and described in the examples were repeated at least twice. Thus the results provided are their average.

Examples 1 to 3: Napin Production from a Globulin-Poor Rapeseed Meal

[0135] These two examples are carried out on a partially defatted dehulled rapeseed meal which was obtained as follows:

[0136] 1) Dehulled Rapeseed Cold Press Meal Production

[0137] For dehulled rapeseed cold press meal production, whole seeds were firstly dehulled with a ripple flow machine to open the seed. The mixture containing free hulls was separated with fluid bed separator to remove the hulls. The residual whole seeds in the kernelrich phase were separated with Denis D50 separator and whole seeds were recycled to the ripple flow machine for dehulling. After Denis D50 separation, the dehulling level was characterized by image analysis. A target of 80% of dehulling was fixed for rapeseed cold press meal production. The kernel-rich meal was pressed with a MBU20 press (OLEXA) to get dehulled rapeseed cold press meal with residual oil content of around 15% without exceeding 60 C. on meal at the end of the pressing step.

[0138] 2) Starting Material Production for Example 1 to 3

[0139] For the production of the oilseed meal 150 kg of rapeseed cold press meal (Table 1) was mixed with 55 C. tap water, in a 1:8 meal:water ratio (w/w) to form a slurry. The slurry was agitated at 160 rpm during 10 minutes and phytase Maxamyl P (DSM) was added (in ratio of 1% of meal) without pH adjustment. After one hour of phytase digestion, the pH of the slurry was adjusted to 6.8 with NaOH (1 M) and agitated again at 160 rpm. After 20 minutes at pH 6.8, the slurry was decanted with a centrifuge decanter at 4600 g (Z23, Flottweg). In the following examples, the insoluble fraction from decantation was used for napin extraction and was called wetcake.

TABLE-US-00001 TABLE 1 Starting Cold Press Rapeseed Meal Composition DM content 91.7% Proteins/DM 34.2% Lipids/DM 21.7%

[0140] The wet cake (32% of dry matter content) was mixed again with water at room temperature (1:3.5 wet-cake:water ratio) under 160 rpm agitation during 15 minutes. The suspension was decanted with a centrifuge decanter at 4 600 g (Z23, Flottweg). The liquid phase was clarified by a disc stack separator at 17 000 g (Easy Scale, GEA). The solid phases of the second decantation called D2 (25% of the suspension) and sludge from disk stack clarification called C2 (7% of decanted extract) constitute the starting material for Napin Isolate Process described in example 1 to 3. Composition of the starting materials D2 and C2 is given in Table 2.

TABLE-US-00002 TABLE 2 D2 C2 DM content .sup.27% 7% Protein content 7% 3% Phytic acid/proteins 24.3% 20.6%

Example 1: Lab Scale Examples and Comparative Examples for Phytic Acid Removal

[0141] A mass of 120 g of lyophilized wet cake D2 was mixed in 1.6 L of water. The pH was adjusted to a value of 2 with chlorhydric acid HCl at a concentration of 1 M. The final solid:liquid ratio was 1:7. pH was maintained at 2 for 30 minutes. Then, a centrifugation step (Thermoscientific Lynx 6000 centrifuge) followed by a filtration step (Whatman filter no 1) took place.

[0142] The next step consisted in adjusting the pH, the ionic strength, and/or adding phytase and/or heating at 55 C. pH adjustment was made using sodium hydroxide NaOH 1M. The ionic strength was obtained by adding NaCl to the required concentration. The phytase used was the phytase described in the following examples. Selected conditions are shown in Table 3. The aqueous extract was maintained in the chosen conditions during 30 minutes or 60 minutes if phytase or heating was applied.

[0143] A second centrifugation step allowed the removal of insoluble particles formed during the adjustment step. Then, the pre-purified extract was purified using an ultrafiltration system (GE Healthcare kta Flux 6, 3 kDa cutoff PS membrane4500 cm.sup.2hollow fiber). Three diavolumes were carried out maintaining the pH and the ionic strength of the extract followed by 3 diavolumes in ultrapure water. The final retentate was lyophilized and characterized, Table 3.

TABLE-US-00003 TABLE 3 Selected conditions Albumin characterization Ionic Purity Estimated purity Napin content Phytic acid/ Solubility No pH strength* Heating Phytase as is (no salt) (% protein) Protein at pH 5 1 2 0M No No 65% / 91.2 5.1% 25% 2 2 0.5M.sup. No No 47% 71% 95.5 1.4% 94% 3 4.5 0M No No 69% / 91.2 5.6% 14% 4 4.5 0.5M.sup. No No 49% 81% 96.1 1.8% 92% 5 4.5 0M Yes No 92% / 96.5 3.2% 86% 6 4.5 0M Yes Yes 99.8 / 96.9 1.0% 100% 7 2 0.5M.sup. Yes No 72% >100% 96.7 1.0% 96% *= to added [NaOH]

[0144] As can be seen from these examples: [0145] The comparison of selected conditions 1 and 2 shows that an ionic strength of 0.5 M allows the decrease of phytic acid in albumins and the increase of albumin solubility at pH 5 [0146] The comparison of selected conditions 1 and 3, and 2 and 4, shows that adjustment of pH has no influence on phytic acid content or purity [0147] The comparison of selected conditions 3 and 5 show that heating at 55 C. allows a strong increase of purity of albumins with a slight decrease of phytic acid content and an increase in solubility at pH 5 [0148] The comparison of selected conditions 5 and 6 show that the addition of phytase with heating at 55 C. allows a strong increase of purity of albumins with a strong decrease of phytic acid content and a good solubility at pH 5 [0149] The comparison of selected conditions 2 and 7 show that an ionic strength of 0.5 M combined with heating allows both a strong increase a strong increase of purity of albumins with a strong decrease of phytic acid content

[0150] Moreover, for all selected conditions, the observed albumin proportion is higher than 90%. Hence the following process steps are particularly advantageous in order to considerably increase purity and strongly decrease the phytic acid content: [0151] (1) The use of a phytase at 55 C., at pH 4.5 after extraction; or [0152] (2) The use of salt at 0.5 M at 55 C. after extraction.

Example 2: Pilot Scale's Napin Isolate Production Using a Phytase for Phytic Acid Reduction

[0153] A quantity of 244 kg of wet cake from second decantation step (D2) was mixed with 50 kg of sludge from clarification step (C2). Tap water at ambient temperature was added in a ratio of 1:3 according to wet cake D2. The slurry was agitated at 160 rpm and pH was adjusted at 2 with phosphoric acid (1M). After 20 minutes at pH 2, the slurry was decanted with a centrifuge decanter (Z23,Flottweg) at 4 600 g at room temperature. The decanted liquid phase was reheated to 55 C. with hot water (60 C.). Phytase Maxamyl P (DSM) was added (1% of dry matter of decanted liquid) and the pH was adjusted to 4.3 with NaOH (1 M). After one hour of phytase digestion, the solution was cooled down to 30 C. to form a precipitate. The precipitated solution was clarified at 30 C. (17000 g) (Easyscale, GEA) and the clarified extract was skimmed with 3 phases disk stack skimmer (ASE40, GEA) at 55 C. for partial oil removal.

[0154] The skimmed liquid phase (heavy phase) was microfiltered using a MF system (Pall, 0.8 m cutoff ceramic GP membrane-4.56 m.sup.2) at TMP of 10.2 bar. The heavy phase was concentrated 11.2 times and the retentate was diafiltered with 4.3 diafiltrations volume with acidic water (adjusted by phosphoric acid at pH 4.3) to keep constant the permeate pH. The total microfiltration and diafiltration permeates were pooled and ultrafiltered with a UF system (Koch, 5 kDa cutoff PES membrane-32 m.sup.2) at TMP of 10.2 bar. The MF permeate was concentrated 6.6 times and the UF retentate was diafiltered with 7 diavolumes with water at 55 C. During diafiltration, the pH of the retentate was not controlled and increased from 4.3 to 7.3. The final retentate after ultrafiltration/diafiltration steps was concentrated using a second smaller UF skid (Koch, 5 kDa cutoff PES membrane-4.3 m.sup.2) at 55 C. and TMP of 10.2 bar. The retentate was concentrated 5.2 times and diafiltered with 2.4 diavolumes with water at 55 C.

[0155] The retentate from the second smaller skid UF was freeze dried (Delta2, 24 LSC, Christ). A total of 2170 g of powder were obtained after freeze-drying. The color of the obtained powder was light brown (FIG. 1). This dark color is due to the increase of pH at the diafiltration step. The control of pH during diafiltration will be improved in examples 2, 3, 4.

[0156] The proximate composition of the powder is shown in Table 4.

TABLE-US-00004 TABLE 4 NAPIN Isolate Protein (Nx6.25)/DM (%) 103 Napin content/Protein 97% Lipids/DM (%) 0.5 Ash/DM (%) 0.9 Polyphenols/DM (%) 1.4 Phytic Acid (g/100 g proteins) - 2 measured as described below Histidine (g/100 g proteins) 4.1 (FAO: 1.5) Isoleucine (g/100 g proteins) 2.9 (FAO: 3) Leucine (g/100 g proteins) 6.8 (FAO: 5.9) Lysine (g/100 g proteins) 8.4 (FAO: 4.5) Methionine + Cysteine (g/100 g proteins) 7.9 (FAO: 2.2) Phenylalanine + Tyrosine (g/100 g proteins) 4.5 (FAO: 3.8) Threonine (g/100 g proteins) 3.3 (FAO: 2.3) Tryptophan (g/100 g proteins) 1.3 (FAO: 0.6) Valine (g/100 g proteins) 4.5 (FAO: 3.9)

[0157] The process at pilot scale allowed reaching a purity >100% with a phytic acid content at 2% on proteins and a very high solubility (92-95%), whatever the pH, with a well-balanced amino-acid profile. The usual nitrogen to protein conversion factor is 6.25 (Nx6.25). But this factor depends on the amino acid composition and therefore depends on the source of protein. For rapeseed, the more accurate conversion factor is between 5.2 and 5.7. But the commercial way to express the protein content remains to use the 6.25 factor (as it is used in the Novel Food for rapeseed isolate2014) and this is the factor used to determine protein content.

Example 3: Lab Scale's Napin Isolate Production Using a Phytase for Phytic Acid Reduction

[0158] For this example, the same starting material as in example 1 was used. A quantity of 1.5 kg of wet cake from D2 was mixed with 418 g of sludge from C2. Water at ambient temperature was added in ratio of 1:3 according to wet cake D2. The slurry was agitated at 160 rpm in a 1 L Erlenmeyer and pH was adjusted to 2 with phosphoric acid (2M). After 30 minutes at pH 2, the slurry was centrifuged under 15 000 g during 30 minutes (Thermoscientific Lynx 6000 centrifuge). The supernatant was filtered using a Whatman filter and the filtrate was centrifuged again at 15 000 g during 15 minutes. The supernatant from the second centrifugation was heated to 55 C. The pH of the heated liquid was readjusted to 4.5 with NaOH (2M). Phytase Maxamyl P was added (1% of the total amount of wet cake D2 and sludges C2). The slurry was agitated at 160 rpm in a 1 L Erlenmeyer. After one hour of phytase digestion, the solution was cooled down to 30 C. and a precipitate was observed. The precipitated solution was centrifuged under 15000 g during 15 minutes and the supernatant was filtered again using a Whatman filter.

[0159] The filtrate after phytase digestion was ultrafiltered with UF system (GE Healthcare kta Flux 6, 3 kDa cutoff PS membrane4500 cm.sup.2hollow fiber). The feed flux is set to 1.5 L/min and the TMP to 1.5 bar. The liquid was concentrated 13.5 times and the UF retentate was diafiltered with 7 diavolumes with water.

[0160] The final retentate after diafiltration was freeze dried and 19.7 g of powder were obtained.

TABLE-US-00005 TABLE 5 Protein/DM Napin content/Protein Phytic Acid/Protein 99% 99% 2.0%

[0161] As shown in Table 5, the purity (% of proteins on dry matter) of the powder was high (99%) and the phytic acid content on proteins was low (2.0%). The solubility of the powder was higher than 99%.

[0162] The color of the freeze dried napin was very light (FIG. 1).

Example 4: Pilot Scale of Napin Isolate Production with Hexane-Extracted Rapeseed Meal without Phytase and with Salt for Phytic Acid Reduction

[0163] In this example a defatted (dehulled) rapeseed meal by hexane extraction was made out from dehulled cold press meals used for napin isolate production. The starting meal composition is given on Table 6.

TABLE-US-00006 TABLE 6 Cold Pressed Rapeseed Meal Composition DM content 94.5% (94.45%) Proteins/DM 35.3% (35.32%) Ash/DM 7.7% (7.69%)

[0164] The process described below leads to two protein fractions: on the one hand, a cruciferin fraction, obtained by isoelectric precipitation and on the other hand, a napin fraction, obtained by membrane purification. The focus will be on napin fraction production.

[0165] A quantity of 111 kg of defatted rapeseed meal was mixed with water in a ratio meal:water of 1:8 at ambient temperature. The slurry was agitated at 160 rpm for 10 minutes and the pH was adjusted to 7 with NaOH (1M). After 45 minutes of extraction at pH 7, the slurry was decanted with a centrifuge decanter at 4 600 g (Z23, Flottweg) at room temperature. The decanted liquid phase was heated to 55 C. and clarified with a disk stack clarifier (EasyScale, GEA) at 17 000 g. The pH of the clarified liquid phase was adjusted under agitation at 160 rmp- to 3.5 by addition of phosphoric acid (1M) to precipitate the cruciferin fraction. The bulk liquid was clarified again with a disk stack clarifier at 17 000 g to separate the precipitate (sludge) from the liquid phase. The sludge phase was rich in cruciferin and the liquid phase was rich in napin (C2). The sludge phase was washed by dilution in hot water (55 C.) at pH 3.5 before being clarified again to produce washed sludges containing the cruciferin fraction and a diluted liquid phase (C3).

[0166] The C2 and C3 liquid phases constitutes the starting material for the napin process. Clarified liquid phases (C2 and C3) were pooled and microfiltered using a MF system (Pall, 0.1 m cutoff ceramic GP membrane-6.65 m.sup.2) at TMP of 10.2 bar. The liquid was concentrated 9.6 times under controlled pH at 3.5. The microfiltration permeate was ultrafiltered with a first UF system (Koch, 5 kDa cutoff PES membrane-32 m.sup.2) at TMP of 10.2 bar. The MF permeate was concentrated 6.2 times and the UF retentate was diafiltered with 4 diafiltration volumes with water at 55 C.

[0167] The ultrafiltered retentate was stored over night at 4 C. and pasteurized (Actini) the day after with a continuous tubular pasteurizer at 75 C. during 15 seconds. The hot water in the heating tubular section was under 76 C. The pasteurized retentate after ultrafiltration/diafiltration steps was concentrated using a second smaller UF skid (Koch, 5 kDa cutoff PES membrane-4.3 m.sup.2) at 20 C. and TMP at 10.2 bar. The retentate was concentrated 3.6 times and diafiltered with 2 diafiltration volumes with a 0.1M NaCl solution following by 2 diavolumes with water at 20 C.

[0168] The retentate from the second smaller skid UF was freeze dried (Delta2, 24 LSC, Christ). A total of 970 g of powder was obtained at the end of this step. The color of the powder was light beige as shown in FIG. 2.

TABLE-US-00007 TABLE 7 Protein/DM Napin content/Protein Phytic Acid/Protein 92% 92% 3.6%

[0169] As shown in Table 7, the purity or the powder was high (92%) and composed at 92% of albumins, and the phytic acid content was slightly high. The solubility at pH 5 was 78%.

Example 5: Lab Scale's Napin Isolate Production Using Salt and Heating for Phytic Acid Reduction

[0170] The starting material would be a dehulled cold press meal.

[0171] The objective of this example is to describe an alternative to the phytase step. Salt can be used instead of phytase to significantly reduce the phytic acid content. The acidic extraction is done directly on the cold press meal, without previous extraction carried out at a pH between 6 and 7.8.

[0172] The cake is mixed with water to form a slurry and the pH is adjusted to 2 with phosphoric acid 1M (or HCl 1M). The slurry is mixed for 30 minutes with a magnetic stirrer in a 1 L Erlenmeyer. The suspension is centrifuged (Thermoscientific Lynx 6000 centrifuge) for 30 minutes at 15 000 g and the supernatant is filtered using a Whatman filter. A second centrifugation step to clarify the extract is carried out for 15 minutes at 15000 g and filtered again.

[0173] NaCl is added to the clarified extract to reach a concentration of NaCl at 0.5M. The mixture is heated to 55 C. with a magnetic stirrer in a 1 L Erlenmeyer. These conditions are maintained for 60 minutes. Then, the mixture is cooled down to room temperature. A centrifugation step is carried out for 15 minutes at 15000 g. A solid residue is formed during this step.

[0174] The supernatant is filtered and then diafiltered on a UF system (GE Healthcare kta Flux 6) with a 3 kDa cut-off PS hollow fiber membrane 4800 cm.sup.2 from GE Healthcare at room temperature. The feed flux is set to 1.5 L/min and the TMP to 1.5 bar. The first three diavolumes are achieved with salted ultrapure water at a sodium chloride concentration of 0.5 M and pH is maintained at 2 with HCl 1 M. A number of 9 diavolumes are done with ultrapure water only without pH control.

[0175] The retentate obtained is freeze-dried.

[0176] The product obtained after freeze-drying is put in a bowl and a mortar is used to produce a fine powder which is then analysed.

[0177] As shown in Table 8, the final powder is characterized by a purity measured as higher than 100% (% proteins/dry matter) and a proportion of at least 94% (possibly 96.7%) of napins with a low phytic acid content: 2.7% on proteins. The solubility at pH 5 is also high with 97%. The color of the powder was very light as shown in FIG. 3.

TABLE-US-00008 TABLE 8 Napin content/ Phytic Acid/ Solubility Protein/DM Protein Protein at pH 5 >100% 94% 2.7% 97%

Comparative Example 1: Lab Scale's Napin Isolate Production According to Cheung et al. (2015), Cf. Supra

[0178] The starting material was a dehulled cold press meal, no hexane was used.

[0179] The process was applied as described by Cheung et al.

[0180] 180 g of meal was mixed with 1.8 L of water and 0.75% (w/V) of sodium chloride. The pH was adjusted to a value of 3 with hydrochloric acid at a concentration of 1 M. These conditions were maintained 90 minutes. The suspension is centrifuged (Thermoscientific Lynx 6000 centrifuge) for 20 minutes at 4 C. and at 17 500 g and the supernatant is filtered using a Whatman filter.

[0181] An isoelectric precipitation is carried out by adjusting the pH between 6.8 and 7 with 1 M sodium hydroxide. These conditions are maintained 20 minutes. The suspension is centrifuged (Thermoscientific Lynx 6000 centrifuge) for 20 minutes at 4 C. and at 17 500 g and the supernatant is filtered using a Whatman filter.

[0182] The obtained extract is concentrated and diafiltered on a UF system (GE Healthcare kta Flux 6) with a 5 kDa cut-off PS hollow fiber membrane 2000 cm.sup.2 from GE Healthcare at room temperature. The feed flux is set to 1 L/min and the TMP between 1.5 and 2 bar. A number of 3 diavolumes are done with ultrapure water only without pH control in order to remove salt and polyphenols.

[0183] The retentate obtained is freeze-dried.

[0184] The product obtained after freeze-drying is put in a bowl and a mortar is used to produce a fine powder which is then analysed.

[0185] As shown in Table 9, the final powder is characterized by a purity measured as 68% (% proteins/dry matter) and a proportion of 94% of napins with a phytic acid content higher than claimed with 9.3% on proteins. The solubility at pH 5 was high with more than 100%.

TABLE-US-00009 TABLE 9 Napin content/ Phytic Acid/ Solubility Protein/DM Protein Protein at pH 5 68% 94% 9.3% >100%

[0186] This comparative example shows that the prior art is not adapted to the treatment of, inter alia, cold pressed oilseed meal. This is due, as shown in Example 1, to the use of an insufficient amount of salt or phytase at the proper stage (e.g. diafiltration stage), in particular in order to reduce the phytic acid content in the final product.

Comparative Example 2: Lab Scale's Napin Isolate Production According to Akbari and Wu (2015), Conf. Supra

[0187] The starting material was a dehulled cold press meal, no hexane was used.

[0188] The process was applied as described by Akbari and Wu (Food Science and Technology, 2015).

[0189] The first extraction step consists in mixing 150 g of meal with 1.3 L of water. The pH was adjusted to a value of 4 with hydrochloric acid at a concentration of 1 M. These conditions were maintained 120 minutes. The suspension is centrifuged (Thermoscientific Lynx 6000 centrifuge) for 20 minutes at 5 C. and at 15 000 g and the supernatant is filtered using a Whatman filter.

[0190] A second extraction step was carried out. 1.2 L of water was added to the wet meal and mixed. The pH was adjusted to 12.5 with sodium hydroxide at a concentration of 1 M. The conditions are maintained during 60 minutes. The suspension is centrifuged (Thermoscientific Lynx 6000 centrifuge) for 20 minutes at 5 C. and at 15 000 g and the supernatant is filtered using a Whatman filter. Then the pH of the extract is adjusted to a value between 4 and 4.5 with hydrochloric acid at a concentration between 0.1 and 1 M. The suspension is centrifuged (Thermoscientific Lynx 6000 centrifuge) for 20 minutes at 5 C. and at 15 000 g and the supernatant is filtered using a Whatman filter.

[0191] The two extracts are pooled and the pH is adjusted to a value of 4 with hydrochloric acid at a concentration of 1 M.

[0192] The obtained extract is concentrated to a FCV value of 10.6 and diafiltered on a UF system (GE Healthcare kta Flux S) with a 10 kDa cut-off PS hollow fiber membrane 140 cm.sup.2 from GE Healthcare at room temperature. The feed flux is set to 50 mL/min and the TMP between 1.5 and 2 bar. One diavolume was carried out with ultrapure water. However, the purification was stopped because a precipitate formed.

[0193] The retentate obtained is freeze-dried.

[0194] The product obtained after freeze-drying is put in a bowl and a mortar is used to produce a fine powder which is then analysed.

[0195] As shown in Table 10, the final powder is characterized by a purity measured as 68% (% proteins/dry matter) and a proportion of 80% of napins with a very high phytic acid content with 19.6% on proteins. The solubility at pH 5 was low with 66%.

TABLE-US-00010 TABLE 10 Napin content/ Phytic Acid/ Solubility Protein/DM Protein Protein at pH 5 56% 80% 19.6% 66%

[0196] In this comparative example, the process applied was not successful because of a precipitation of the proteins during the diafiltration step. This result can be explained from the difference of raw material used: in the article of Akbari and Wu (2015) a hexane-deoiled meal is used whereas, in this example, the main raw material is a cold-pressed meal. This shows the particular suitability of the process of the invention to extract from cold pressed meals.

[0197] Measurement of Phytic Acid

[0198] The method to determine the percentage of phytic acid in a protein extract or isolate was adapted from Garcia-Estepa et al. (1999, Food International Research) and was applied directly to solid samples such as napin isolates and meals. For each batch of analysis, a blank measurement is carried out with all the reactants excepting the sample to be measured.

[0199] 1. Extraction

[0200] Weight between 250 and 500 mg of sample in a 25-mL beaker.

[0201] Record the exact mass.

[0202] Add 20 mL of a solution of HCl 0.4 M+10% Na.sub.2SO.sub.4.

[0203] Stir for at least 2 h at room temperature.

[0204] Centrifuge the suspension for 30 minutes at 10 000 g.

[0205] Filter the supernatant.

[0206] 2. Reaction

[0207] In a 15 mL centrifuge tube, mix the following solutions: [0208] 2.5 mL of the filtered supernatant [0209] 2.5 mL of a FeCl.sub.3 solution at a concentration of 20 mM [0210] 2.5 mL of a HCl 0.4 M+10% Na.sub.2SO.sub.4 solution [0211] 2.5 mL of sulfosalicylic acid at a concentration of 20% (masse/volume)

[0212] Stir, (the mixture should be of a purplish/burgundy color).

[0213] The centrifuge tubes are plunged in a 100 C. water bath for 15 to 20 minutes. During this step a precipitate is formed between sulfosalicylic acid, Fe.sup.3+ ions and phytic acid.

[0214] Allow the samples to cool down at room temperature.

[0215] Centrifuge the samples for 30 minutes at 10000 g.

[0216] 3. Recovery of Fe.sup.3+ Ions

[0217] The following steps are useful to recover the maximum free ions Fe.sup.3+. [0218] The supernatant is filtered on a 0.22 m filter in a 25 mL volumetric flask. [0219] A volume of 4 mL of distilled water is added to the tubes containing the precipitates. [0220] The tubes are stirred vigorously to put the precipitate in suspension. A vortex can be used. [0221] The samples are centrifuged for 10 minutes at 10 000 g.

[0222] The above steps with an asterisk * are repeated in the same order three times for each samples. Water is added to obtain a 25 mL solution for each sample.

[0223] 4. Dosage of Free Ions Fe.sup.3+

[0224] For each sample, 10 mL of the previous solution is taken into a 25 mL beaker. 10 mL of water is added.

[0225] The pH of each solution is adjusted to 2.50.5 by addition of glycine (provided by Sigma Aldrich with a purity of at least 99%).

[0226] The solution is then heated in a water bath to a temperature ranging from 70 to 80 C.

[0227] The dosage is done directly after the water bath, by addition of a 2 mM of Ethylene diamine tetra acetic acid (EDTA) solution placed in a burette beforehand.

[0228] The equivalent volume is reached when the solution changes color from a burgundy color to yellow-green.

[0229] The equivalent volume is recorded as precisely as possible.

[0230] Calculations The EDTA dosage allows the quantification of free Fe.sup.3+ ions in the medium, that is to say, the ions that are not involved in the precipitate with phytic acid.

n(Fe.sup.3+).sub.free=V.sub.eq*[EDTA]*2.5

[0231] With [EDTA] the concentration in mmol/L, V.sub.eq the equivalent volume equivalent in L and 2.5 corresponds to the 10 mL taken from the 25 mL volumetric flask (step 4).

[0232] The amount of total Fe.sup.3+ introduced in the medium is obtained with the dosage of a blank, that is to say, following all the steps but with no sample. The following formula gives the amount of Fe.sup.3+ in the precipitate.

n(Fe.sup.3+).sub.prcipitate=n(Fe.sup.3+).sub.totaln(Fe.sup.3+).sub.free

[0233] This formula can also be written as:

n(Fe.sup.3+).sub.prcipitate=n(Fe.sup.3+).sub.blankn(Fe.sup.3+).sub.free in the sample

[0234] In the literature, it is usually admitted that 6 phosphorus bind to 4 ions Fe.sup.3+.

[00001]$\frac{n\left(\mathrm{Phosphorus}\right)}{n\left({\mathrm{Fe}}^{3+}\right)}=\frac{6}{4}$

[0235] However, it is supposed that one molecule of phytic acid contains 6 phosphorus.

[00002]$n\left(\mathrm{Phytic}.Math..Math.\mathrm{Acid}\right)=\frac{n\left(\mathrm{Phosphorus}\right)}{6}$

[0236] The combination of the last two formulas is:

[00003]${n\left(\mathrm{Phytic}.Math..Math.\mathrm{Acid}\right)}_{\mathrm{precipitate}}=\frac{{n\left({\mathrm{Fe}}^{3+}\right)}_{\mathrm{precipitate}}}{4}$

[0237] As 2.5 mL were taken from the initial volume of 20 mL (extraction step 1), the molar concentration of phytic acid in the extract is 8 times the concentration of Fe.sup.3+ ions in the precipitate.

n(Phytic Acid).sub.extract=8*n(Phytic Acid).sub.precipitate

[0238] The mass of phytic acid in the extract is:

m(Phytic Acid).sub.extract=n(Phytic Acid)*M(Phytic Acid)

[0239] M (Phytic Acid) corresponds to the molecular weight of phytic acid, which is equal to 660 g/mol under the IP6 form.

[0240] The phytic acid content is usually expressed in mg/g of protein or in mg/g of dry matter corresponding to:

[00004]$\mathrm{Phytic}.Math..Math.\mathrm{acid}.Math..Math.\mathrm{content}.Math..Math.\mathrm{in}.Math..Math.\mathrm{mg}.Math.\text{/}.Math.g.Math..Math.\mathrm{of}.Math..Math.\mathrm{protein}=\frac{{m\left(\mathrm{Phytic}.Math..Math.\mathrm{Acid}\right)}_{\mathrm{extract}}}{{m\left(\mathrm{Protein}\right)}_{\mathrm{extract}}}$$\mathrm{Phytic}.Math..Math.\mathrm{acid}.Math..Math.\mathrm{content}.Math..Math.\mathrm{in}.Math..Math.\mathrm{mg}.Math.\text{/}.Math.g.Math..Math.\mathrm{of}.Math..Math.\mathrm{dry}.Math..Math.\mathrm{matter}=\frac{{m\left(\mathrm{Phytic}.Math..Math.\mathrm{Acid}\right)}_{\mathrm{extract}}}{{m\left(\mathrm{Dry}.Math..Math.\mathrm{matter}\right)}_{\mathrm{extract}}}$

[0241] Kjeldahl Method Used for Protein Content Determination

[0242] The Kjeldahl method is used for the determination of the protein content in samples under liquid form to assess the solubility of isolates or under solid form to determine the protein content in a sample and is described NF EN ISO 5983-2 Oct. 2009.

[0243] 1. Preparation of an Isolate Sample Powder

[0244] 250 mg of the powder is weighted in a 25 mL beaker; the exact mass is recorded.

[0245] 20 mL of water is added to the powder and the slurry is maintained under agitation at room temperature for 30 minutes.

[0246] The solution is then transferred into a 25 mL volumetric flask and water is added to obtain a 25 mL solution.

[0247] A 1 mL sample is taken into a Kjeldahl flask.

[0248] 2. Preparation of a Solid Sample (Meal)

[0249] Weigh between 20 and 40 mg of meal in a Kjeldahl Weighing Boat N-free provided by Bchi. Record the exact weight.

[0250] Put the boat and the sample in a Kjeldahl flask.

[0251] 3. General Procedure

[0252] In the Kjeldahl flask, introduce 4 mL sulfuric acid at 96% and approximately 0.2 g of catalyst CuSe from AppliChem (Gatersleben, Germany).

[0253] As control, at least one flask is prepared with no sample but with sulfuric acid and catalyst.

[0254] Then, the mineralization step is carried out in several steps in a Buchi SpeedDigester K-439 (Rungis, France): [0255] Preheating to 150 C. [0256] Heating for 15 minutes at 150 C. [0257] Heating for 90 minutes at 450 C.

[0258] These steps are done to decompose organic substances: in particular, nitrogen is reduced as NH.sub.4.sup.+.

[0259] The samples are allowed to cool down for 30 minutes.

[0260] The next step is the distillation: sodium hydroxide 32% is added to the sample to convert nitrogen to its NH.sub.3 form which is distilled, then converted back to NH.sub.4.sup.+ with 3% boric acid and then back titrated with 0.01 M HCl and 3% boric acid in a Kjelflex K-360 from Bchi associated with a Titrino Plus 877 from Metrohm (Herisau, Suisse). The equivalent volume is used in the following calculation.

[0261] 4. Calculation of the Protein Content

[0262] Hence, the total nitrogen content (NTK in g/L) is determined according to the following formula:

[00005]$\begin{array}{cc}\mathrm{NTK}=\frac{\left(V_{\mathrm{assay}}-V_{\mathrm{blank}}\right)*M\left(N\right)*C_n\left(\mathrm{HCl}\right)}{V_{\mathrm{sample}}}& \left(1\right)\end{array}$

[0263] V.sub.assay and V.sub.blank are the volumes of HCl at a concentration of C.sub.n(HCl) equal to 0.01 M (in mL) used for the back titration. M(N) represents the molecular mass of nitrogen, which is 14 g/mol, and V.sub.sample is the volume of extract used as sample for the analysis. For the meal analysis, V.sub.sample is replaced by the mass of meal introduced in the flask (in mg) and the result becomes a rate of nitrogen in %.

[0264] The total nitrogen content is then converted into proteins thanks to a coefficient equal to 6.25.

Protein content=NTK*6.25

[0265] It is understood by the skilled person that this measure of protein content is proportional to the amount of nitrogen in the sample.

[0266] Measure of the Protein Solubility

[0267] In order to measure the solubility, the sample is suspended in deionised water at a protein concentration of 1%. The protein content of this solution is measured by the Kjeldahl method above described. 4 aliquots of 25 mL each are prepared and their pH adjusted with NaOH [1M] or with HCl [1M] in water to the desired pH. These aliquots are stirred for at least 20 minutes and then centrifugated (15 000 g for 10 minutes). The supernatant are analysed using the Kjeldahl method. Solubility is the ratio between the protein content measured in the deionised water suspension and the one measured after pH adjustment and centrifugation.

[0268] Measure of Napin Content

INTRODUCTION

[0269] Napin content on isolates was evaluated by size exclusion high performance liquid chromatography (SE-HPLC). This analytical technique is used to separate and quantify specific proteins. The separation in SE-HPLC is done according to their molecular size by using a gel composed of porous beads as sieving medium. Smaller molecules will be transported by diffusion inside the beads in an accessible volume related to both their molecular size and bead pore diameters while larger ones will only be transported by a convective flow of eluent between the beads. As a consequence, larger molecules will be eluted first and smaller ones later.

[0270] SE-HPLC Device & Eluent Preparation

[0271] A Biosep-SEC-s2000 3007.8 mm provided by Phenomenex was used. This column was chosen as, according to the supplier, its separation range is between 1 and 300 kDa, which cover the sizes of the proteins under study. Inside the column, the gel is composed of silica with a particle size of 5 m and a pore size of 145 .

[0272] The chromatographic system is a UFLC machine from Shimadzu which is equipped with Photodiode Array (PDA) detector and a column oven set at 35 C. Detection was set to 214 nm and data were processed with the LabSolution software.

[0273] Eluent was prepared with 45% acetonitrile provided by Biosolve BV (Valkenswaard, the Netherlands) and 55% Ultra-Pure Water. 0.1% of trifluoroacetic acid (TFA) was added to the mixture. Elution was set with a 0.6 mL/min flow and lasted 30 minutes. The mixture of water and acetonitrile is commonly used to study proteins and their possible aggregates.

[0274] Samples Preparation

[0275] A solution of 5 g/L of isolate in pure water was prepared by dissolving 125 grs of powder in 25 mL of pure water and maintained under agitation at room temperature for 30 minutes. 10 mL of the solution was filtered on 0.22 m syringe filter and introduced in vials. A volume of 5 L sample was injected.

[0276] Results

[0277] Typical results obtained with SE-HPLC analysis from rapeseed isolate is given in FIG. 4. The first peak corresponds to cruciferin and the second peak to the napin.

[0278] The area under the curve (A) of each peak is calculated and thus allows to return to the napin content on the isolate. This calculation is simple as the mass extinction coefficient of both proteins, measured thanks to calibration curves on napin and cruciferin isolates, is the same. Napin content in isolate is given by the following equation (eq.1):

[00006]$\begin{array}{cc}\mathrm{Napin}.Math..Math.\mathrm{content}.Math..Math.\left(\%\right)=100\frac{A_{\mathrm{napin}}}{A_{\mathrm{napin}}{A}_{\mathrm{cruciferin}}}& \left(\mathrm{eq}.Math..Math.1\right)\end{array}$

[0279] FIG. 4 shows the correlation between the peaks observed by SE-HPLC and SDS-PAGE analysis in reducing conditions. The first peak corresponds to a high molecular weight protein, cruciferins, which is composed six subunits which are in turn composed of two main polypeptides, an -chain (around 30 kDa) and a -chain (around 20 kDa), visible on the SDS-PAGE gel.

[0280] The second peak is consistent with a low molecular weight protein, napins, which is composed of two main subunits, one small with 4 kDa, the other long with 9 kDa, associated together by a disulfide bond. FIG. 5 is a chromatogram obtained after preparing a solution of 10 g/L of napin isolate. Hence, one main peak is clearly visible at the same retention time as in FIG. 4.

[0281] The separated proteins are subunits still associated by their disulfide bridge as the elution conditions allow the dissociation of electrostatic and ionic interaction but not of the disulfide bridges.

[0282] It will be understood that these are examples of suggested operating conditions and the composition of the liquid to be separated can necessitate operating at different condition or using separation techniques other than a centrifuge to separate solids from liquids or two immiscible liquid phases.