A SUNFLOWER SEED PROTEIN ISOLATE AND A PROCESS FOR PRODUCING THE SAME

Abstract

A process for preparing a sunflower seed protein isolate and a protein isolate which is obtainable by such process. The process comprises the following steps: mixing a defatted seed meal with an aqueous NaCl solution at a basic pH; separating said solubilised protein solution from solids; diafiltering said solubilised protein solution through an ultrafiltration membrane system using an aqueous NaCl diafiltration NaCl solution and at least 2 diavolumes of said aqueous NaCl diafiltration solution, diafiltering said NaCl-diafiltered protein; concentrating said purified protein solution; and drying said purified protein concentrate to obtain a protein isolate.

Claims

1. A process for preparing a protein isolate, said protein being a sunflower seed protein, said process comprising the following steps: (a) providing an at least partially defatted seed meal, said seed meal being a sunflower seed meal and having a proportion of dry matter ranging from 80 wt % to 98 wt %; (b) mixing said at least partially defatted seed meal with an aqueous NaCl solution at a pH of about 6 to 8, in order to solubilize proteins present in said at least partially defatted seed meal and to thus obtain a solubilized protein solution, wherein said aqueous NaCl solution has a NaCl concentration ranging from 0 to 1.2 mol.Math.L-1; (c) separating said solubilized protein solution from solids therein; (d) diafiltering said solubilized protein solution through an ultrafiltration membrane system having a molecular weight cutoff of 1 to 100 kDa, said diafiltration step being effected using: an aqueous NaCl diafiltration solution having a NaCl concentration ranging from 0.1 to 0.6 mol.Math.L-1; and at least 2 diavolumes of said aqueous NaCl diafiltration solution, to obtain a NaCl-diafiltered protein solution; (e) subsequently to step (d), diafiltering said NaCl-diafiltered protein solution through an ultrafiltration membrane system with a molecular weight cutoff of 1 to 100 kDa, said diafiltration step (e) being effected using water, to obtain a purified protein solution; (f) concentrating said purified protein solution to obtain a purified protein concentrate; and (g) drying said purified protein concentrate to obtain said protein isolate; wherein said process does not contain a step of precipitation of said protein prior to step (d).

2. The process of claim 1, wherein said aqueous NaCl solution of step (b) has a NaCl concentration ranging from 0.3 to 0.5 mol.sup.−1.

3. The process of claim 1, wherein said diafiltration step of step (d) is carried out using at least 3 to up to about 30 diavolumes of said aqueous NaCl diafiltration solution.

4. The process of claim 1, wherein prior to step b) a washing step wherein said at least partially defatted seed meal is washed with water thus producing a washing mix.

5. The process of claim 4, wherein the pH of said washing mix is adjusted to range from 4 to 6.

6. The process of claim 1, wherein all steps are carried out at a temperature ranging from 50° C. to 60° C.

7. The process of claim 1, wherein the pH of the NaCl diafiltered solution of step d) is adjusted to a pH ranging from 7 to 10 before being subjected to step (e).

8. The process of claim 1, wherein prior to step d) the solubilized protein solution is microfiltrated through a filtration membrane having a nominal pore size ranging from 0.1 to 1 μm and the retentate collected is the solution submitted to step d).

9. The process of claim 1, wherein during step f) said purified protein solution is concentrated by a volumetric reduction factor of 2 or more.

10. The process of claim 1, wherein said drying step is freeze drying.

11. A sunflower seed native protein isolate, said isolate comprising sunflower globulins and albumins, said isolate having a solubility in aqueous solution which is superior, or equal, to 65% at pH 3 and superior or equal to 65% at pH 7; wherein said solubility at the given pH is calculated as follows: ${\mathrm{SoI}}_{pH}=\left(\frac{C_{s}x{V}_s}{C_{i}x{V}_i}\right)\times 100$ wherein: SOI.sub.pH protein solubility at given pH (%), C.sub.s—protein concentration in supernatant (g.Math.L.sup.−1), V.sub.s—final volume of solution after adjustment of pH (mL), C.sub.i—initial protein concentration (g.Math.L.sup.−1), V.sub.i—initial volume of solution (mL).

12. The sunflower seed native protein isolate of claim 11, wherein said sunflower seed native protein isolate has an aqueous solubility superior, or equal, to 95% at pH 3 and/or an aqueous solubility superior, or equal, to 90% at pH 7.

13. The sunflower seed protein isolate of claim 11, wherein said isolate has at most 0.2 weight % by weight of total protein in said isolate (dry matter) of at least one chlorogenic acid isomer.

14. The sunflower seed protein isolate of claim 11, wherein said isolate has at most 2 wt % of phytic acid by weight of total proteins in said isolate.

15. A food product or beverage comprising the sunflower seed protein of claim 11.

16. The process of claim 1, wherein said diafiltration step of step (d) is carried out using at least 5 to up to about 30 diavolumes of said aqueous NaCl diafiltration solution.

17. The process of claim 1, wherein during step f) said purified protein solution is concentrated by a volumetric reduction factor of 4.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0109] FIG. 1 is a view of a Petri dish containing a sunflower seed protein isolate according to example 1.

[0110] FIG. 2 is a view of a Petri dish containing a sunflower seed protein isolate according to example 2 at low temperature (20° C.).

[0111] FIG. 3 is a view of a Petri dish containing a sunflower seed protein isolate according to example 2 at high temperature (55° C.).

[0112] FIG. 4 is a view of a Petri dish containing a sunflower protein isolate from sunflower cold-pressed meal according to example 3.

[0113] FIG. 5 is a graph representing the size exclusion chromatogram of sunflower meal extract (red bold line—detection 280 nm, green thin line—detection 325 nm). The X-axis represents time in minutes. The Y-axis represents intensity in milli absorption units. The internal, boxed, FIG. represents the mass spectrum at m/z 355.3.sup.+ of sunflower extract (orange continuous line) and reference substances of chlorogenic acid isomers (black dotted line): 3-caffeyolquinic acid (3-CQA), 5-caffeyolquinic acid (5-CQA) and 4-caffeyolquinic acid (4-CQA).

[0114] FIG. 6 is a graph showing the calibration curves of reference substances of chlorogenic acid (detection 325 nm): (A) 3-caffeyolquinic acid, (B) 5-caffeyolquinic acid and (C) 4-caffeyolquinic acid. The X-axis represents concentration in g.Math.L.sup.−1. The Y-axis represents intensity in milli absorption units. Each plot represents an average of three independent repetitions for six concentration levels. Linear regression was performed to determine curve slop and correlation coefficient (R.sup.2).

EXAMPLES

Description of Analytical Methods

[0115] Dry Matter Content

[0116] Approximately 250 mg of protein powder is weighted on a dried aluminium disk and the exact mass is recorded and placed in a drying oven set at 110° C. After a minimum of 24 h, the sample is let to cool to ambient temperature in a desiccator and reweighted. The dry matter content in the sample is calculated according the formula (Equation 1).

[00001]$\begin{array}{cc}\mathrm{DM}\left(\%\right)=\frac{m_d}{m_w}\times 100& \left(\mathrm{Equation}1\right)\end{array}$

wherein:
DM—dry matter content (%),
m.sub.d—mass of powder after drying (mg),
m.sub.w—mass of powder before drying (mg).

[0117] Kjeldahl Method Used for Protein Content Determination

[0118] The Kjeldahl method is used for the determination of the protein content in liquid samples (to assess the solubility of isolates) or in solid samples to determine their protein contents and is described for example in the standard NF EN ISO 5983-2 Oct. 2009.

[0119] 1. Preparation of an Isolate Sample Powder

[0120] A 0.5 to 2 mL sample is taken into a Kjeldahl flask. The exact volume is recorded.

[0121] 2. Preparation of a Solid Sample (Meal)

[0122] Weigh between 20 and 40 mg of meal in a Kjeldahl Weighing Boat N-free provided by Büchi. Record the exact weight.

[0123] Put the boat and the sample in a Kjeldahl flask.

[0124] 3. General Procedure

[0125] In the Kjeldahl flask, introduce 4 mL of sulfuric acid at 96% (v/v) and approximately 0.2 g of catalyst Cu—Se from AppliChem Panreac (Gatersleben, Germany).

[0126] As control, at least one flask is prepared with no sample but with sulfuric acid and catalyst.

[0127] Then, the mineralization step is carried out in several steps in a Büchi SpeedDigester K-439 (Rungis, France): [0128] Preheating to 150° C. [0129] Heating for 15 min at 150° C. [0130] Heating for 90 min at 450° C.

[0131] These steps are done to decompose organic substances: in particular, nitrogen is reduced as NH.sub.4.sup.+.

[0132] The samples are allowed to cool down for 30 min.

[0133] 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 (w/v) and then back titrated with 0.01 mol.Math.L.sup.−1 HCl and 3% boric acid in a Kjelflex K-360 from Büchi associated with a Titrino Plus 877 from Metrohm (Herisau, Suisse). The equivalent volume is used in the following calculation.

[0134] 4. Calculation of the Protein Content

[0135] Hence, the total nitrogen content (NTK in g.Math.L.sup.−1) is determined according to the following formula:

[00002]$\begin{array}{cc}\mathrm{NTK}=\frac{\left(V_{assay}-V_{bl\mathrm{ank}}\right)\times M\left(N\right)\times C_n\left(\mathrm{HCI}\right)}{v_{\mathrm{sample}}}& \left(1\right)\end{array}$

[0136] V.sub.assay and V.sub.blank are the volumes of HCl at a concentration of C.sub.n(HCl) equal to 0.01 mol.Math.L.sup.−1 (in mL) used for the back titration. The molecular mass of nitrogen is 14 g.Math.mol.sup.−1, and V.sub.sample is the volume of extract used as sample for the analysis. For solid sample 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 %. The total nitrogen content is then converted into proteins thanks to a coefficient equal to 6.25.

Protein content=NTK×6.25

[0137] It is understood by the skilled person that this measure of protein content is proportional to the amount of nitrogen in the sample.

[0138] The standard factor, 6.25, to convert nitrogen to protein content was used. However, the value of this factor may differ for sunflower proteins. Consequently, the purity of powder may exceed 100% in some cases.

Purity of Protein Powder

[0139] Exactly 125 mg of protein powder is weighted, the exact mass recorded, and then dissolved in a beaker in 5 mL of a NaOH solution (0.1 mol.Math.L.sup.−1). The solution is transferred into a 25 mL volumetric flask at room temperature. The beaker is washed three times with 5 mL of a NaOH solution (0.1 mol.Math.L.sup.−1) and the washing solutions are transferred into a 25 mL volumetric flask. Finally, the volumetric flask is completed with the same NaOH solution. Final volume of the mixture was 25 mL. The concentration of proteins in the solution was measured according the Kjeldahl method (see below). The purity of protein in powder on dry matter base was calculated as follows (Equation 2).

[00003]$\begin{array}{cc}Protein/D{M}_{DM}\left(\%\right)=\left(\frac{C_{prot}}{C_{pow}\times \frac{DM}{100}}\right)\times 100& \left(\mathrm{Equation}2\right)\end{array}$

wherein:
Protein/DM—purity of protein powder on dry matter base (%),
C.sub.prot—concentration of proteins in prepared solution according the Kjeldahl method (g.Math.L.sup.−1),
C.sub.pow—concentration of powder in prepared solution (g.Math.L.sup.−1),
DM—dry matter content of powder (%).

Protein Solubility at Room Temperature

[0140] The solubility of the protein isolate of the invention in aqueous solution is measured as follows:

[0141] In a non-controlled condition of temperature (about 20° C.), about 500 mg of protein powder is weighted, the exact mass recorded, and then dissolved in 50 mL of a distilled water in a beaker. The solution is transferred into a 100 mL volumetric flask at room temperature. The beaker is washed three times with 10 mL of distilled and the washing solutions are transferred into a 100 mL volumetric flask. Finally, the volumetric flask is completed with distilled water. A volume of 20 mL of solution is placed in a 25 mL beaker and stirred on a stirring plate at approximatively 300 rpm at room temperature during 10 min. Then, the pH of the solution is adjusted to the required pH using an aqueous solution of 0.1 mol.Math.L.sup.−1 NaOH or 0.1 mol.Math.L.sup.−1 HCl and the stirring maintained during 30 min. After this time, the solid precipitate was separated by centrifugation at 15000 g during 20 min at 20° C. Subsequently, the concentration of protein in the initial solution and in the supernatant was measured according the Kjeldahl method (N×6.25). The protein solubility at the given pH was calculated as follows (Equation 3).

[00004]$\begin{array}{cc}So{l}_{pH}=\left(\frac{C_s\times V_s}{C_i\times V_i}\right)\times 100& \left(\mathrm{Equation}3\right)\end{array}$

wherein:
Sol.sub.pH—protein solubility at given pH (%),
C.sub.S—protein concentration in supernatant (g.Math.L.sup.−1),
V.sub.S—final volume of solution after adjustment of pH (mL),
C.sub.i—initial protein concentration (g.Math.L.sup.−1),
V.sub.i—initial volume of solution (mL).

[0142] For comparison purposes, solubility at a particular ionic strength (0.03 mol.Math.L.sup.−1) was also measured according to the modus operandi recited in example 4.

Phytic Acid Content

[0143] The method to determine the percentage of phytic acid in a protein extract or isolate was adapted from García-Estepa et al. (1999, Food International Research) and is applied directly to solid samples such as protein isolates and meals. For each batch of analysis, a “blank” measurement is carried out with all the reactants excepting the sample to be measured. This method consists of four stages—extraction, reaction, recovery of Fe.sup.3+ ions and titration.

[0144] 1. Extraction:

[0145] A quantity of about 250 to 500 mg of protein isolate or sunflower meal is weighted, its exact weight recorded, and then placed in a 25 mL beaker. A volume of 20 mL of a solution of 0.4 mol.Math.L.sup.−1 HCl+10% Na.sub.2SO.sub.4 (w/v) is added and the mixture is stirred at approximatively 300 rpm for minimum 120 min at room temperature. After this time, the mixture is centrifuged at 10000 g during 30 min at 20° C. and supernatant was additionally filtered (0.22 μm). The blank assay consisted of a sample containing no protein.

[0146] 2. Reaction:

[0147] In a 15 mL centrifuge tube a reaction mixture composed of 2.5 mL of 20 mmol.Math.L.sup.−1 FeCl.sub.3, 2.5 mL of 0.4 mol.Math.L.sup.−1 HCl+10% Na.sub.2SO.sub.4 (w/v) and 2.5 mL of 20% sulfosalicylic acid (w/v) is prepared. 2.5 mL of filtered sample is added to the mixture and the tube is shaken for approximatively 3 min. The color formed should be burgundy. The centrifuge tube is plunged in a 100° C. water bath for to 20 min. During this step a precipitate is formed between sulfosalicylic acid, Fe.sup.3+ ions and phytic acid. The sample is then cooled to ambient temperature and subsequently centrifuged at 10000 g during 30 min at 20° C. The supernatant is recovered.

[0148] 3. Recovery of Fe.sup.3+ Ions:

[0149] The following steps are useful to recover the maximum free ions Fe.sup.3+: [0150] The supernatant is filtered on a 0.22 μm filter in a 25 mL volumetric flask. [0151] A volume of 4 mL of distilled water is added to the tubes containing the precipitates. [0152] The tubes are stirred vigorously to put the precipitate in suspension. A vortex can be used. [0153] The samples are centrifuged for 10 min at 10000 g.

[0154] The above steps with an asterisk “*” are repeated in the same order three times for each sample. Water is added to obtain a 25 mL solution for each sample.

[0155] 4. Dosage of Free Ions Fe.sup.3+:

[0156] A volume of 10 mL of the previous solution and 10 mL of distilled water is placed into a 25 mL beaker. The pH of mixture is adjusted to 2.5±0.5 using glycine powder (purity of at least 99%). The mixture is then heated 30 min in a water bath to a temperature ranging from 70 and 80° C. The dosage is carried out directly after the water bath, by addition of drops of an Ethylene diamine tetra acetic acid (EDTA) solution (2 mmol.Math.L.sup.−1) placed in a burette beforehand.

[0157] The equivalent volume is reached when the solution changes color from a burgundy color to yellow-green.

[0158] The equivalent volume is recorded as precisely as possible.

[0159] Calculations:

[0160] The EDTA dosage allows the quantification of free Fe.sup.3+ ions in the medium. These free Fe.sup.3+ ions are the ones which have not precipitated with the phytic acid present in the solution.

[00005]${n\left(F{e}^{3+}\right)}_{free}=V_{eq}\times C_{EDTA}\times \frac{V_{sol}}{V_s}$

wherein:
C.sub.EDTA—concentration of EDTA (mmol.Math.L.sup.−1),
V.sub.eq— equivalent volume (L),
V.sub.sol—volume of initial solution (L),
V.sub.s—volume of taken sample from initial solution (L).

[0161] The total amount of Fe.sup.3+ introduced in the medium is obtained with the dosage of a blank, that is to say, a solution which contains no sample but has been processed as described above. The following formula gives the amount of Fe.sup.3+ in the precipitate.

n(Fe.sup.3+).sub.précipitate=n(Fe.sup.3+)t.sub.otal−n(Fe.sup.3+).sub.free

[0162] This formula can also be written as:

n(Fe.sup.3+).sub.précipitate=n(Fe.sup.3+).sub.blank−n(Fe.sup.3+).sub.free in the sample

[0163] In the literature, it is usually admitted that 6 phosphorus bind to 4 ions Fe.sup.3+

[00006]$\frac{n\left(\mathrm{Phosphorus}\right)}{n\left(F{e}^{3+}\right)}=\frac{6}{4}$

[0164] However, if one molecule of phytic acid contains 6 phosphorus,

[00007]$n\left(\mathrm{Phytic}\mathrm{Acid}\right)=\frac{n\left(\mathrm{Phosphorus}\right)}{6}$

[0165] then the combination of the last two formulae is:

[00008]${n\left(\mathrm{Phytic}\mathrm{Acid}\right)}_{precipitate}=\frac{{n\left(F{e}^{3+}\right)}_{precipitate}}{4}$

[0166] As 2.5 mL are taken from the initial volume of 20 mL, 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

[0167] The mass of phytic acid in the extract is:

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

[0168] M(Phytic Acid) corresponds to the molecular weight of phytic acid, which is equal to 660 g.Math.mol.sup.−1 under the IP6 form.

[0169] The phytic acid content is usually expressed in mg.Math.g.sup.−1 of protein or in mg.Math.g.sup.−1 of dry matter corresponding to:

[00009]$\mathrm{Phytic}\mathrm{acid}\mathrm{content}\mathrm{in}\mathrm{mg}.Math.g^{-1}\mathrm{of}\mathrm{protein}=\frac{{m\left(\mathrm{Phytic}\mathrm{Acid}\right)}_{extract}}{{m\left(\mathrm{Protein}\right)}_{extract}}$$\mathrm{Phytic}\mathrm{acid}\mathrm{content}\mathrm{in}\mathrm{mg}.Math.g^{-1}\mathrm{of}\mathrm{dry}\mathrm{matter}=\frac{{m\left(\mathrm{Phytic}\mathrm{Acid}\right)}_{extract}}{{m\left(\mathrm{Dry}\mathrm{matter}\right)}_{extract}}$

[0170] Chlorogenic Acid Content

[0171] Chlorogenic acid is an ester of caffeic and quinic acid and occurs in sunflower seeds mainly as three isomers: 3-caffeoylquinic acid (3-CQA), 4-caffeoylquinic acid (4-CQA) and 5-caffeoylquinic acid (5-CQA).

[0172] For quantification of their respective contents in a particular sample a method by Size Exclusion Chromatography (SEC) is chosen. This method was validated according to the ICH Guidelines, ‘Validation of analytical procedures: text and methodology Q2R1’, November 2005). For this purpose, about 25 mg of protein powder is weighted, the exact mass recorded, and then dissolved in a beaker in 1 mL of a buffer (Tris-HCl 0.25 mol.Math.L.sup.−1/NaCl 0.5 mol.Math.L.sup.−1, pH 7.0). The solution is transferred into a 5 mL volumetric flask at room temperature. The beaker is washed three times with 1 mL of a buffer (Tris-HCl 0.25 mol.Math.L.sup.−1/NaCl 0.5 mol.Math.L.sup.−1, pH 7) and the washing solutions are transferred into the 5 mL volumetric flask. Finally, the volumetric flask is completed with the same buffer (Tris-HCl 0.25 mol.Math.L.sup.−1/NaCl 0.5 mol.Math.L.sup.−1, pH 7). Subsequently, 5 μL of said solution is injected into Biosep SEC-s-2000 column (300×7.5 mm; 5 μm) which is maintained at 35° C. The mobile phase used consists of acetonitrile/0.1% formic acid (10:90 v/v) and the flow rate is set on 0.6 mL.Math.min.sup.−1. The retention times of chlorogenic acid isomers based of an aqueous extract of sunflower meal are confirmed after injection of 3-, 4- and 5-CQA standard solutions. These measures (see FIG. 5) are conducted using a quadrupole mass spectroscopy detector (m/z 355.3+) and a diode array detector at 325 nm of analytical wavelength.

[0173] A set of calibration curves of chlorogenic acid isomers (see FIG. 6) were linear for a range of concentration from 0.05 to 5.0 g.Math.L.sup.−1 for 3-CQA (R.sup.2=1.000) and 0.01 to 1.0 g.Math.L.sup.−1 for 3-, 4- and 5-CQA (R.sup.2=1.000) (see FIG. 6). The limit of quantification defined as signal-to-noise ratio >10 was 2.2 mg.Math.μL.sup.−1, 5.0 mg.Math.L.sup.−1, 3.0 mg.Math.μL.sup.−1 for 3-, 4- and 5-CQA, respectively.

[0174] The content of chlorogenic acid isomers in powder in relation to one gram of protein on dry matter base is calculated as follows:

[00010]$C_{CQA}/Protein/\mathrm{DM}\left(\%\right)=\left(\frac{C_{CQA}}{C_{pow}\times \frac{Protein/DM}{100}}\right)\times 100$

wherein:

[0175] C.sub.CQA—concentration of chlorogenic acid isomer in a sample (g.Math.L.sup.−1),

[0176] C.sub.pow— concentration of powder in prepared solution (g.Math.L.sup.−1),

[0177] Protein/DM—purity of protein powder on dry matter base (%).

[0178] Colour Measurement

[0179] 50 mg of protein powder are weighted, dissolved in 0.01 mol.Math.L.sup.−1 NaOH at a concentration of 1% (w/v) and then, the mixture is filtered on a 0.22 μm filter. The colour is recorded in CieL*a*b* scale using Lovibond PFX195 Tintometer at room temperature. To do this, a baseline calibration is performed on empty quartz cuvette. Subsequently, about 3 mL of sample is placed in cell and the colour is measured in ten replicates. From the results average values of L*, a*, b* parameters and standard deviation were determined. Additionally, the difference of colour between samples expressed by delta E (ΔE) was calculated using the following equation:

ΔE=√{square root over ((L*.sub.sample−L*.sub.standard)+(a*.sub.sample−a*.sub.standard)+(b*.sub.sample−b*.sub.standard))}

(as described in Salgado et al., LWT—Food Science and Technology 45 (2012) 65-72)

[0180] As a standard the sample of Example 1 was used.

Example 1: A Sunflower Protein Isolate from an Industrial Meal

[0181] Except if otherwise stated, percentages are mass (i.e. mass/total mass percentages).

[0182] A sunflower Isolate (SFI) according to the invention was obtained using a process comprising two main steps: 1) a neutral extraction step and 2) a purification step using ultrafiltration. For this purpose, a dehulled industrial sunflower meal, de-oiled with hexane, was used (see Table 1).

TABLE-US-00001 TABLE 1 Starting composition of the sunflower seed industrial meal. DM (%) 91.2 Proteins/DM (%) 36.8 Phytic acid/Protein (%) 13.7 Lipids/DM (%) n.d. DM = Dry Matter content.

[0183] The industrial sunflower defatted meal was first milled and sieved (pores 500 μm).

[0184] Solid/Liquid Extraction: 200 g of the powder thus obtained was mixed with a solution of NaCl (1.0 mol.Math.L.sup.−1) in a solid/liquid ratio of 1:9 (wt %). The pH was adjusted to 7.0 using a solution of NaOH (1.0 mol.Math.L.sup.−1). The mixture was stirred at 800 rpm at 20° C. during 30 min. After a centrifugation conducted at 15000 g during 30 min at 20° C., the supernatant was filtered using a Whatman filter paper (Fisherbrand, cellulose, diameter 190 mm, thickness 0.17 mm, particles retention 17-30 μm). The liquid phase was collected to be purified.

[0185] Membrane purification: The ultrafiltration step was carried out using a UF system (GE Healthcare, 3 kDa cut-off hollow fiber cartridge—4 800 cm.sup.2) at room temperature. The liquid phase collected at the extraction stage was washed with 6 diafiltrations volumes of aqueous solution of NaCl (0.5 mol.Math.L.sup.−1). Subsequently, the pH of the retentate was adjusted to 9 using an aqueous solution of NaOH (1 mol.Math.L.sup.−1) and then washed with 4 diafiltrations volumes with Ultrapure water. The final UF retentate was then collected and freeze dried. The color of the powder was light beige (FIG. 1).

TABLE-US-00002 TABLE 2 Composition of sunflower protein isolate. DM (%) 93.5 Proteins/DM (%) 99.9 C.sub.3-CQA/Protein (%) <0.20 (peak not detected) C.sub.4-CQA/Protein (%) <0.20 (peak not detected) C.sub.5-CQA/Protein (%) <0.20 (0.056)* Phytic acid/Protein (%) 1.35 *outside calibration range. DM = dry matter content.

[0186] The purity (proteins on dry matter) of the powder was high (99.9%) and the phytic acid content: 1.35% on proteins (Table 2) was relatively low. The protein solubility at pH 7 and 3 was 76.8% and 89.6%, respectively.

TABLE-US-00003 Protein solubility of the isolate pH (%) 7 76.8 4 40.8 3.5 79.3 3 89.6

Example 2: A Sunflower Protein Isolate from a Sunflower Cold Press Meal

[0187] The production of Sunflower Isolates (SFI) from a dehulled sunflower—cold press meal (Table 3) was conducted at low (1) or high (2) temperature. Both processes comprise three main steps: 1) an acidic washing to remove contaminates (phenolic and phytic acids as well as and other water-soluble molecules) followed by 2) a neutral extraction and 3) a membrane purification.

TABLE-US-00004 TABLE 3 Composition of the sunflower cold press meal used in both examples. DM (%) 89.3 Proteins/DM (%) 42.8 Phytic acid/Protein (%) 24.8 Lipids/DM (%) 14.6

[0188] 1. Low-Temperature Production of Sunflower Isolate

Washing Steps

[0189] 500 g of sunflower cold press meal were mixed with water at 20° C. according to a solid/liquid ratio 1:9 (wt %) and the mixture stirred at 600 rpm during 10 min. The pH was adjusted to 6.0 using an aqueous solution of citric acid (1 mol.Math.L.sup.−1). Then, the mixture was centrifuged at 4000 g during 10 min at 20° C. and the supernatant was disposed of. The pellet was rewashed with water (20° C.) in a solid/liquid ratio 1:1.5 (wt %) during 5 min. After re-centrifugation at 4000 g during 10 min at 20° C., the pellet was collected and stored at 4° C. overnight.

Solid/Liquid Extraction

[0190] The collected pellet from the previous steps was mixed with an aqueous solution of NaCl (0.5 mol.Math.L.sup.−1) according to a solid/liquid ratio 1:9 (wt %) and stirred at 600 rpm at 20° C. during 30 min. The pH was adjusted to 7.3 using an aqueous solution of NaOH (1 mol.Math.L.sup.−1). Then, the mixture was centrifuged at 4000 g during 10 min at 20° C. After centrifugation, the supernatant was additionally filtered with a Whatman filter paper and the liquid phase was collected. The pellet from centrifugation step was rewashed with water (20° C.) in a solid/liquid ratio 1:1.5 (wt %) during 5 min and then re-centrifuged and filtered in the same way. The collected supernatants were pooled and stored at ambient temperature until microfiltration step.

Membrane Purification

[0191] A microfiltration (MF) step was carried out using a MF system (Millipore, 0.22 μm cut-off Pellicon XL Durapore PVDF membrane—0.1 m.sup.2). The collected liquid phases from previous step were concentrated by a volumetric reduction factor (VRF) factor of 2 and the retentate was washed with 1 diafiltration volume of a NaCl solution of 0.5 mol.Math.L.sup.−1. The total microfiltration permeates were pooled and stored at ambient temperature until ultrafiltration step.

[0192] The ultrafiltration (UF) step was carried out using a UF system (GE Healthcare, 3 kDa cut-off hollow fiber cartridge—4 800 cm.sup.2) at room temperature. The collected permeates from previous step were concentrated by a VRF of 2 and the retentate was washed with 5 diafiltrations volumes of water at 0.5 mol.Math.L.sup.−1 NaCl. Then, the pH of retentate was adjusted to 9.5 using the solution of 1 mol.Math.L.sup.−1 NaOH and the retentate was concentrated by a VRF of 2. Then, the retentate was washed with 3 diafiltrations volume of ultrapure water.

[0193] The final UF retentate was collected and freeze dried. The color of the powder was light greenish (FIG. 2).

TABLE-US-00005 TABLE 4 Composition of sunflower protein isolate. DM (%) 92.8 Proteins/DM (%) 103.4 C.sub.3-CQA/Protein (%) <0.20 (peak not detected) C.sub.4-CQA/Protein (%) <0.20 (peak not detected) C.sub.5-CQA/Protein (%) <0.20 (0.013)* Phytic acid/Protein (%) 0.80 *outside the calibration range

[0194] The purity (proteins on dry matter) of the powder was high (103.4%) and the phytic acid content: 0.8% on proteins (Table 4) was low. The protein solubility at pH 7 and 3 was 29.5% and 101.1%, respectively.

TABLE-US-00006 pH Protein solubility of the isolate (%) 7 29.5 4 95.7 3.5 99.0 3 101.1

[0195] 2. High-Temperature Production of Sunflower Isolate

Washing Steps

[0196] 500 g of a sunflower cold press meal were mixed with water in a solid/liquid ratio 1:9 (wt %) and stirred at 600 rpm at 55° C. during 30 min. The pH of the resulting mix was measured. The pH value was not superior to 7.3, and no pH adjustment was made. Then, the mixture was centrifuged at 4000 g during 10 min at 40° C. and filtered by sieve filtration (screen having a mesh of 150 μm). A first liquid phase was obtained. The resulting solid was rewashed with hot water (55° C.) in a solid/liquid ratio 1:1.5 (wt %) during 5 min. After re-centrifugation at 4000 g during 10 min at 40° C. and sieve filtration (mesh of 150 μm), the collected solid constitutes the starting meal for SFI production. It was stored overnight at 4° C.

[0197] The liquid phases collected from the above washing and rewashing steps were pooled and stored at 55° C. in the oven to be analysed/quantified.

[0198] Solid/Liquid Extraction

[0199] The washed pellet from the previous step was mixed with an aqueous solution of NaCl (0.3 mol.Math.L.sup.−1) in a solid/liquid ratio 1:9 (wt %) and stirred at 600 rpm at 55° C. during 30 min. The pH was adjusted to 7.3 using an aqueous solution of NaOH (1 mol.Math.L.sup.−1). Then, the mixture was centrifuged at 4000 g during 10 min at 40° C. and filtered by sieve filtration (mesh of 150 μm). The liquid phase was collected and maintained at 55° C. in an oven. The solid collected was rewashed with hot water (55° C.) in a solid/liquid ratio 1:1.5 (wt %) during 5 min. Then it was centrifuged again at 4000 g during 10 min at 40° C. and filtered by sieve filtration. The liquid phase from this second extraction was pooled with the previous one and stored at 55° C. in the oven until subsequent microfiltration step.

[0200] Membrane Purification

[0201] The liquid phases collected from the extraction steps are then microfiltered.

[0202] The microfiltration step was carried out using a MF system (Millipore, 0.22 μm cut-off Pellicon XL Durapore PVDF membrane 0.1 m.sup.2). The pooled liquid phases from the extraction step were concentrated by a VRF of 3.5 and the retentate was washed with 2 diafiltrations volumes of hot water at 55° C. The total microfiltration permeates were pooled and stored at 55° C. in the oven until the ultrafiltration step.

[0203] The ultrafiltration step was carried out using a UF system (GE Healthcare, 3 kDa cut-off hollow fiber cartridge 4800 cm.sup.2). The pooled permeates from the microfiltration step were concentrated by a VRF of 4 and the retentate was washed with 5 diafiltrations volumes of a hot aqueous solution of NaCl (0.2 mol.Math.L.sup.−1) at 55° C. The pH of retentate was subsequently adjusted to 9 using an aqueous solution of NaOH (1 mol.Math.L.sup.−1). Then, the retentate was washed with 3 diafiltrations volume of hot water (55° C.) and concentrated by a VRF of 2.

[0204] The final UF retentate was collected and freeze dried for 72 h. The colour of the powder was beige with greenish hue (FIG. 3).

TABLE-US-00007 TABLE 5 Composition of sunflower protein isolate DM (%) 92.9 Proteins/DM (%) 100.9 C.sub.3-CQA/Protein (%) <0.20 (peak not detected) C.sub.4-CQA/Protein (%) <0.20 (peak not detected) C.sub.5-CQA/Protein (%) <0.20 (0.010)* Phytic acid/Protein (%) 0.78 *outside the calibration range

[0205] The purity (proteins on dry matter) of the powder was high (100.9%) and the phytic acid content 0.78% on proteins (Table 5) was low. The protein solubility at pH 7 and 3 was 99.8% and 101.2%, respectively.

TABLE-US-00008 pH Protein solubility of the isolate (%) 7 99.8 4 36.9 3.5 102.8 3 101.2

Example 3: Detoxified Sunflower Isolates from Sunflower Cold Press Meal

[0206] Detoxified Sunflower Isolate (DSFI) was produced from a cold press meal of dehulled sunflower seed (Table 6) at a pilot scale by using three main steps: 1) an acidic wash to remove contaminates (such as phenolic compounds and phytic acid) and other water soluble molecules, followed by 2) a neutral extraction and 3) a membrane purification in order to obtain a DSFI.

TABLE-US-00009 TABLE 6 Starting composition of the cold press meal of dehulled sunflower seed. DM (%) 89.3 Proteins/DM (%) 42.8 Phytic acid/Protein (%) 24.8 Lipids/DM (%) 14.6

[0207] Acidic Washing Steps

[0208] 60 kg of a sunflower cold press meal were washed by mixing with some acidic water (pH 2) in a ratio meal:water of 1:8 (wt %) at 55° C. in order to form a slurry having a pH of 4.8±0.2. The slurry was agitated at 160 rpm during 15 min and decanted with a decanter centrifuge at 4600 g (Z23, FlottWeg). The decanted sludge was rewashed by mixing it again with some acidic water at pH 4.8±0.2, at a temperature of 55° C., in a ratio sludge:water of 1:3.5 (wt %) to form a new slurry having a pH of 4.8±0.2. The slurry formed was agitated at 160 rpm during 15 min and decanted with a decanter centrifuge at 4600 g (Z23, FlottWeg).

[0209] The sludge from the rewash constitutes the starting meal for DSFI production.

[0210] Extraction

[0211] For DSFI production, the rewashed sludges from the second acidic wash were mixed with water in a ratio sludge:water of 1:3.5 (wt %) at 55° C. under agitation at 160 rpm during 30 min. The pH was adjusted to 7.3 by using aqueous solutions of phosphoric acid or NaOH (both at 1 mol.Math.L.sup.−1).

[0212] The slurry was decanted with a decanter centrifuge at 4600 g (Z23, FlottWeg) at room temperature. The decanted liquid phase was reheated at 55° C. and clarified with a disk stack clarifier at 17000 g (EasyScale, GEA), in order to remove fines, and skimmed with a 3-phases disk stack skimmer (ASE40, GEA) at 55° C. in order to remove the oil (at least partially).

Purification

[0213] Microfiltration

[0214] The (partially) skimmed liquid phase (heavy phase) was microfiltered using a MF system (Pall, 0.1 μm cutoff ceramic GP membrane-0.7 m.sup.2).

[0215] Ultrafiltration

[0216] The heavy phase was concentrated 7.2 times and the retentate was diafiltered with 2 diafiltration volumes with hot water at 55° C. with UF system (Koch, 5 kDa cutoff PES membrane—4.3 m.sup.2)

[0217] The total microfiltration and diafiltration (MF+DF) permeates were pooled and ultrafiltered with a UF system (Koch, 5 kDa cutoff PES membrane—4.3 m.sup.2). The MF+DF permeates was concentrated 4.5 times at room temperature.

[0218] The UF retentate was diafiltered with 2 diavolumes of salted water NaCl (0.4 mol.Math.L.sup.−1) following by 4 diafiltrations volumes of water at 20° C. The final UF retentate was freeze dried. The colour of the powder was medium greenish brown.

TABLE-US-00010 TABLE 7 Composition of sunflower protein isolate. DM (%) 98.1 Proteins/DM (%) 97.4 C.sub.3-CQA/Protein (%) <0.20 (peak not detected) C.sub.4-CQA/Protein (%) <0.20 (peak not detected) C.sub.5-CQA/Protein (%) <0.20 (0.029)* Phytic acid/Protein (%) 3.38 *outside the calibration range

[0219] The purity (proteins on dry matter) of the powder was high (97.4%) as well as the phytic acid content: 3.38% on proteins (Table 7). The protein solubility at pH 7 and 3 was 100.1% and 67.3%, respectively.

TABLE-US-00011 pH Protein solubility of the isolate (%) 7 100.1 4 32.0 3.5 36.9 3 67.3

Colour Measurement

[0220] As can be shown from the figures, the isolate present a light coloration, which is high in demand in the food industry. The L*, a*, b* parameters of these isolates are as listed in Table 8 below:

TABLE-US-00012 Example L* a* b* ΔE 1 84.32 ± 3.71  1.08 ± 1.45 34.78 ± 2.66 — 2.1 84.39 ± 4.87  0.23 ± 0.88 41.30 ± 2.74 6.58 2.2 81.05 ± 4.87  0.22 ± 0.94 24.11 ± 1.39 11.20 3 39.62 ± 3.74 10.08 ± 1.80 49.78 ± 3.32 48.01

Example 4: Solubility of the Protein Isolates of Previous Example at pH 3, 3.5, 4 and 7 at a Specific Ionic Strength (30 nM) at Room Temperature (about 20° C.)

[0221] Additional protein solubility measurements were performed with the protein isolates obtained according to examples 1, 2.1, 2.2 and 3 above. In non-controlled conditions of temperature of about 20° C., each of these proteins were dispersed to final concentration of 4.0 mg/mL in water and the pH adjusted to 8.5 by addition of small amounts of NaOH solutions.

[0222] The ionic strength was adjusted to 0.03 mol.Math.L.sup.−1 by adding NaCl. The pH of the protein solutions was lowered by adding various amounts of HCl or NaOH solutions (0.01 mol.Math.L.sup.−1) to obtain final pH at 3.0, 3.5, 4 or 7, and kept constantly (±0.05) during 2 h under agitation (100 rpm) at room temperature. Next, the samples were centrifuged for 15 min at 12100×g at 20° C.

[0223] The protein concentration in supernatant was measured by the Kjeldahl method (AOAC method 991.20, 1995). Solubility was expressed as proportion (%) of the amount of protein dissolved at pH 8.5.

[0224] The change of centrifugation speed and of measurement of protein concentration when compared to the one disclosed in Gonzalez-Perez et al. 2005 are on consequential. The solubility data are ratio of protein concentration (hence independent from the method) and a centrifugation speed of 12100 g provides equivalent results to remove solid particulates when compared to a speed of 15800 g

[0225] The results of these measurements are compiled in table 9 below which includes the solubility data of Sunflower isolate obtained using methanol extraction in order to dephenolized the isolate as described in in FIG. 2, table a) of Gonzalez-Perez et al. 2005 (Physicochemical properties of 2S albumins and the corresponding protein isolate from sunflower (Helianthus annuus)) in JFS C:Food Chemistry and toxicology Vol. 70, Nr.1, 2005.

TABLE-US-00013 TABLE 9 Protein solubility (%) Isolate tested pH 3 pH 3.5 pH 4.0 pH 7 Example 1 83.98 / / 67.22 Example 2.1 96.90 100.72 91.00 43.90 Example 2.2 97.00  93.74 35.74 95.97 Example 3 60.63  42.29 30.04 94.75 Gonzalez-Perez about about about about et al. 2005 90% 80% 40% 60%

Example 5: Analysis of the Isolate of the Invention Obtained in Examples 1, 2.1 and 2.2

[0226] SE-HPLC analysis was performed according to the method of Defaix et al. (2019). The analyses were carried out on a HPLC Shimadzu LC30 system coupled with photodiode array detector (PDA) and operated by LabSolutions software, all from Shimadzu Corporation (Kyoto, Japan). The solutions of proteins were prepared at a concentration of 5 g.Math.L.sup.−1 in 0.25 mol.Math.L.sup.−1 Tris-HCl buffer, pH 7/0.5 mol.Math.L.sup.−1 NaCl (v/v).

[0227] 5-20 μL of sample was injected into a Biosep SEC s-2000 column (300×7.8 mm, 5 μm) from Phenomenex (Torrance, Calif., USA). The exclusion range of molecular weight was between 1 and 300 kDa. During analysis the autosampler and column compartment were maintained at 20 and at 35° C., respectively. The mobile phase consisted of acetonitrile/water/trifluoracetic acid (45:54.9:0.1 v/v). The elution flow rate was set at 0.6 mL.Math.min.sup.−1. All solvents were HPLC grade and were supplied from Fisher Scientific (Hampton, USA). The ultrapure water (H.sub.2O) with resistivity ≥18.2 MΩ.Math.cm.sup.−1 was used. The PDA signal was recorded between 190 and 400 nm with maximal absorption at 214 nm for protein detection. Assuming the same value of molar extinction coefficient for sunflower globulins and albumins as it was previously demonstrated by Defaix et al. (2019), the content of globulins (C.sub.GLOB) and albumins (C.sub.ALB) in relation to total sunflower proteins, that is the total amount of globulins and albumins in the extract (as other amounts of other proteins are considered negligible) were calculated as follow:

[00011]$\begin{array}{cc}{C}_{GLOB}=\frac{A_{GLOB}}{A_{GLOB}+A_{ALB}}\times 100& \left(\mathrm{Equation}.1\right)\\ C_{ALB}=\frac{A_{ALB}}{A_{GLOB}+A_{ALB}}\times 100& \left(\mathrm{Equation}.2\right)\end{array}$

[0228] where A.sub.GLOB and A.sub.ALB is peak surface at 214 nm corresponding to globulins or albumins, respectively. All measurements were performed in triplicate and a mean value was calculated. The results are shown in Table 10.

TABLE-US-00014 TABLE 10 Example Globulins (wt %) Albumins (wt %) 1 76.50 ± 2.30 23.50 ± 2.30 2.1 71.80 ± 0.93 28.20 ± 0.93 2.2 64.44 ± 0.70 35.56 ± 0.70