Patent ID: 12193455

EXAMPLES

Description of Analytical Methods

Dry Matter Content

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).

DM⁡(%)=mdmw×1⁢0⁢0(Equation⁢1)wherein:DM—dry matter content (%),md—mass of powder after drying (mg),mw—mass of powder before drying (mg).
Kjeldahl Method Used for Protein Content Determination

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.

1. Preparation of an Isolate Sample Powder

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

2. Preparation of a Solid Sample (Meal)

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

Put the boat and the sample in a Kjeldahl flask.

3. General Procedure

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).

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

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

These steps are done to decompose organic substances: in particular, nitrogen is reduced as NH4+.

The samples are allowed to cool down for 30 min.

The next step is the distillation: sodium hydroxide 32% is added to the sample to convert nitrogen to its NH3form which is distilled, then converted back to NH4+with 3% boric acid (w/v) and then back titrated with 0.01 mol·L−1HCl and 3% boric acid in a Kjelflex K-360 from Buchi associated with a Titrino Plus 877 from Metrohm (Herisau, Suisse). The equivalent volume is used in the following calculation.

4. Calculation of the Protein Content

Hence, the total nitrogen content (NTK in g·L−1) is determined according to the following formula:

NTK=(Vassay-Vblank)×M⁡(N)×Cn(HC⁢l)Vsample(1)

Vassayand Vblankare the volumes of HCl at a concentration of Cn (HCl) equal to 0.01 mol·L−1(in mL) used for the back titration. The molecular mass of nitrogen is 14 g·mol−1, and Vsampleis the volume of extract used as sample for the analysis. For solid sample analysis, Vsampleis 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

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

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

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·L−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·L−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).

Protein/DMDM(%)=(CprotCpow×DM100)×100(Equation⁢2)wherein:Protein/DM—purity of protein powder on dry matter base (%),Cprot—concentration of proteins in prepared solution according the Kjeldahl method (g·L−1),Cpow—concentration of powder in prepared solution (g·L−1),DM—dry matter content of powder (%).
Protein Solubility at Room Temperature

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

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·L−1NaOH or 0.1 mol·L−1HCl 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).

S⁢o⁢lp⁢H=(Cs×VsCi×Vi)×100(Equation⁢3)wherein:SolpH—protein solubility at given pH (%),Cs—protein concentration in supernatant (g·L−1),Vs—final volume of solution after adjustment of pH (mL),Ci—initial protein concentration (g·L−1),Vi—initial volume of solution (mL).

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

Phytic Acid Content

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 Fe3+ions and titration.

1. Extraction:

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·L−1HCl+10% Na2SO4(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.

2. Reaction:

In a 15 mL centrifuge tube a reaction mixture composed of 2.5 mL of 20 mmol·L−1FeCl3, 2.5 mL of 0.4 mol·L−1HCl+10% Na2SO4(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 15 to 20 min. During this step a precipitate is formed between sulfosalicylic acid, Fe3+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.

3. Recovery of Fe3+Ions:

The following steps are useful to recover the maximum free ions Fe3+:The supernatant is filtered on a 0.22 μm filter in a 25 mL volumetric flask.A volume of 4 mL of distilled water is added to the tubes containing the precipitates.The tubes are stirred vigorously to put the precipitate in suspension. A vortex can be used.The samples are centrifuged for 10 min at 10000 g.

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.

4. Dosage of Free Ions Fe3+:

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·L−1) placed in a burette beforehand.

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

The equivalent volume is recorded as precisely as possible.

Calculations:

The EDTA dosage allows the quantification of free Fe3+ions in the medium. These free Fe3+ions are the ones which have not precipitated with the phytic acid present in the solution.

n⁡(Fe3+)f⁢r⁢e⁢e=Ve⁢q×CE⁢D⁢T⁢A×Vs⁢o⁢lVswherein:CEDTA—concentration of EDTA (mmol·L−1),Veq—equivalent volume (L),Vsol—volume of initial solution (L),Vs—volume of taken sample from initial solution (L).

The total amount of Fe3+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 Fe3+in the precipitate.
n(Fe3+)precipitate=n(Fe3+)total−n(Fe3+)free
This formula can also be written as:
n(Fe5+)precipitate=n(Fe3+)blank−n(Fe3+)free in the sample

In the literature, it is usually admitted that 6 phosphorus bind to 4 ions Fe3+

n(Phosphorus)n(Fe3+)=64

However, if one molecule of phytic acid contains 6 phosphorus,

n⁡(Phytic⁢Acid)=n⁡(Phosphorus)6
then the combination of the last two formulae is:

n⁡(Phytic⁢Acid)precipitate=n⁡(Fe3+)precipitate4

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 Fe3+ions in the precipitate.
n(Phytic Acid)extract=8×n(Phytic Acid)precipitate

The mass of phytic acid in the extract is:
m(Phytic Acid)extract=n(Phytic Acid)×M(Phytic Acid)

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

The phytic acid content is usually expressed in mg·g−1of protein or in mg·g−1of dry matter corresponding to:

Phytic⁢acid⁢content⁢⁢in⁢mg·g-1⁢of⁢protein=m⁡(Phytic⁢Acid)extractm⁡(Protein)extractPhytic⁢acid⁢content⁢⁢in⁢mg·g-1⁢of⁢dry⁢matter=m⁡(Phytic⁢Acid)extractm⁡(Dry⁢matter)extract
Chlorogenic Acid Content

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).

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·L−1/NaCl 0.5 mol·L−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·L−1/NaCl 0.5 mol·L−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·L−1/NaCl 0.5 mol·L−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·min−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 (seeFIG.5) are conducted using a quadrupole mass spectroscopy detector (m/z 355.3+) and a diode array detector at 325 nm of analytical wavelength.

A set of calibration curves of chlorogenic acid isomers (seeFIG.6) were linear for a range of concentration from 0.05 to 5.0 g·L−1for 3-CQA (R2=1.000) and 0.01 to 1.0 g·L−1for 3-, 4- and 5-CQA (R2=1.000) (seeFIG.6). The limit of quantification defined as signal-to-noise ratio>10 was 2.2 mg·μL−1, 5.0 mg·L−1, 3.0 mg·μL−1for 3-, 4- and 5-CQA, respectively.

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

CC⁢Q⁢A/Protein/DM⁢(%)=(CC⁢Q⁢ACp⁢o⁢w×Protein/DM1⁢0⁢0)×1⁢0⁢0wherein:CCQA—concentration of chlorogenic acid isomer in a sample (g·L−1),Cpow—concentration of powder in prepared solution (g·L−1),Protein/DM—purity of protein powder on dry matter base (%).
Colour Measurement

50 mg of protein powder are weighted, dissolved in 0.01 mol·L−1NaOH 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:

Δ⁢E=(Ls⁢a⁢m⁢p⁢l⁢e*-Lstandard*)+(as⁢a⁢m⁢p⁢l⁢e*-astandard*)+(bs⁢a⁢m⁢p⁢l⁢e*-bstandard*)
(as described in Salgado et al., LWT—Food Science and Technology 45 (2012) 65-72)

As a standard the sample of Example 1 was used.

Example 1: A Sunflower Protein Isolate from an Industrial Meal

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

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 1Starting composition of thesunflower seed industrial meal.DM (%)91.2Proteins/DM (%)36.8Phytic acid/Protein (%)13.7Lipids/DM (%)n.d.DM = Dry Matter content.

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

Solid/Liquid Extraction: 200 g of the powder thus obtained was mixed with a solution of NaCl (1.0 mol·L−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·L−1). The mixture was stirred at 800 rpm at 20° C. during 30 min. After a centrifugation conducted at 15,000 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.

Membrane purification: The ultrafiltration step was carried out using a UF system (GE Healthcare, 3 kDa cut-off hollow fiber cartridge—4 800 cm2) 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·L−1). Subsequently, the pH of the retentate was adjusted to 9 using an aqueous solution of NaOH (1 mol·L−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 2Composition of sunflower protein isolate.DM (%)93.5Proteins/DM (%)99.9C3-CQA/Protein (%)<0.20 (peak not detected)C4-CQA/Protein (%)<0.20 (peak not detected)C5-CQA/Protein (%)<0.20 (0.056)*Phytic acid/Protein (%)1.35*outside calibration range.DM = dry matter content.

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.

Protein solubility of the isolatepH(%)776.8440.83.579.3389.6

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

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 3Composition of the sunflower cold pressmeal used in both examples.DM (%)89.3Proteins/DM (%)42.8Phytic acid/Protein (%)24.8Lipids/DM (%)14.6
1. Low-Temperature Production of Sunflower Isolate
Washing Steps

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·L−1). Then, the mixture was centrifuged at 4,000 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 4,000 g during 10 min at 20° C., the pellet was collected and stored at 4° C. overnight.

Solid/Liquid Extraction

The collected pellet from the previous steps was mixed with an aqueous solution of NaCl (0.5 mol·L−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·L−1). Then, the mixture was centrifuged at 4,000 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

A microfiltration (MF) step was carried out using a MF system (Millipore, 0.22 μm cut-off Pellicon XL Durapore PVDF membrane—0.1 m2). 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·L−1. The total microfiltration permeates were pooled and stored at ambient temperature until ultrafiltration step.

The ultrafiltration (UF) step was carried out using a UF system (GE Healthcare, 3 kDa cut-off hollow fiber cartridge—4,800 cm2) 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·L−1NaCl. Then, the pH of retentate was adjusted to 9.5 using the solution of 1 mol·L−1NaOH and the retentate was concentrated by a VRF of 2. Then, the retentate was washed with 3 diafiltrations volume of ultrapure water.

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

TABLE 4Composition of sunflower protein isolate.DM (%)92.8Proteins/DM (%)103.4C3-CQA/Protein (%)<0.20 (peak not detected)C4-CQA/Protein (%)<0.20 (peak not detected)C5-CQA/Protein (%)<0.20 (0.013)*Phytic acid/Protein (%)0.80*outside the calibration range

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.

pHProtein solubility of the isolate (%)729.5495.73.599.03101.1
2. High-Temperature Production of Sunflower Isolate
Washing Steps

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 4,000 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 4,000 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. 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.

Solid/Liquid Extraction

The washed pellet from the previous step was mixed with an aqueous solution of NaCl (0.3 mol·L−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·L−1). Then, the mixture was centrifuged at 4,000 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 4,000 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.

Membrane Purification

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

The microfiltration step was carried out using a MF system (Millipore, 0.22 μm cut-off Pellicon XL Durapore PVDF membrane 0.1 m2). 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. The ultrafiltration step was carried out using a UF system (GE Healthcare, 3 kDa cut-off hollow fiber cartridge 4,800 cm2). 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·L−1) at 55° C. The pH of retentate was subsequently adjusted to 9 using an aqueous solution of NaOH (1 mol·L−1). Then, the retentate was washed with 3 diafiltrations volume of hot water (55° C.) and concentrated by a VRF of 2.

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 5Composition of sunflower protein isolateDM (%)92.9Proteins/DM (%)100.9C3-CQA/Protein (%)<0.20 (peak not detected)C4-CQA/Protein (%)<0.20 (peak not detected)C5-CQA/Protein (%)<0.20 (0.010)*Phytic acid/Protein (%)0.78*outside the calibration range

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.

pHProtein solubility of the isolate (%)799.8436.93.5102.83101.2

Example 3: Detoxified Sunflower Isolates from Sunflower Cold Press Meal

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 6Starting composition of the cold pressmeal of dehulled sunflower seed.DM (%)89.3Proteins/DM (%)42.8Phytic acid/Protein (%)24.8Lipids/DM (%)14.6
Acidic Washing Steps

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 4,600 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 4,600 g (Z23, FlottWeg). The sludge from the rewash constitutes the starting meal for DSFI production.

Extraction

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·L−1).

The slurry was decanted with a decanter centrifuge at 4,600 g (Z23, FlottWeg) at room temperature. The decanted liquid phase was reheated at 55° C. and clarified with a disk stack clarifier at 17,000 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

Microfiltration

The (partially) skimmed liquid phase (heavy phase) was microfiltered using a MF system (Pall, 0.1 μm cutoff ceramic GP membrane—0.7 m2).

Ultrafiltration

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 m2)

The total microfiltration and diafiltration (MF+DF) permeates were pooled and ultrafiltered with a UF system (Koch, 5 kDa cutoff PES membrane—4.3 m2). The MF+DF permeates was concentrated 4.5 times at room temperature.

The UF retentate was diafiltered with 2 diavolumes of salted water NaCl (0.4 mol·L−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 7Composition of sunflower protein isolate.DM (%)98.1Proteins/DM (%)97.4C3-CQA/Protein(%)<0.20 (peak not detected)C4-CQA/Protein (%)<0.20 (peak not detected)C5-CQA/Protein(%)<0.20 (0.029)*Phytic acid/Protein(%)3.38*outside the calibration range

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.

pHProtein solubility of the isolate (%)7100.1432.03.536.9367.3
Colour Measurement

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:

ExampleL*a*b*AE184.32 ± 3.711.08 ± 1.4534.78 ± 2.66—2.184.39 ± 4.870.23 ± 0.8841.30 ± 2.746.582.281.05 ± 4.870.22 ± 0.9424.11 ± 1.3911.20339.62 ± 3.7410.08 ± 1.8049.78 ± 3.3248.01

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.)

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.

The ionic strength was adjusted to 0.03 mol·L−1by adding NaCl. The pH of the protein solutions was lowered by adding various amounts of HCl or NaOH solutions (0.01 mol·L−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.

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.

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 12,100 g provides equivalent results to remove solid particulates when compared to a speed of 15,800 g.

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 inFIG.2, table a) of Gonzalez-Perez et al. 2005 (Physicochemical properties of2S albumins and the corresponding protein isolate from sunflower(Helianthus annuus)) in JFS C: Food Chemistry and toxicology Vol. 70, Nr. 1, 2005.

TABLE 9Protein solubility (%)Isolate testedpH 3pH 3.5pH 4.0pH 7Example 183.98//67.22Example 2.196.90100.7291.0043.90Example 2.297.0093.7435.7495.97Example 360.6342.2930.0494.75Gonzalez-Perez et al.aboutabout 80aboutabout200590%%40%60%

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

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·L−1in 0.25 mol·L−1Tris-HCl buffer, pH 7/0.5 mol·L−1NaCl (v/v).

5-20 μL of sample was injected into a Biosep SEC s-2000 column (300×7.8 mm; 5 μm) from Phenomenex (Torrance, CA, 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·min−1. All solvents were HPLC grade and were supplied from Fisher Scientific (Hampton, USA). The ultrapure water (H2O) with resistivity≥18.2 MQ·cm−1was 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 (CGLOB) and albumins (CALB) 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:

CG⁢L⁢O⁢B=AG⁢L⁢O⁢BAG⁢L⁢O⁢B+AA⁢L⁢B×100(Equation.1)CA⁢L⁢B=AA⁢L⁢BAG⁢L⁢O⁢B+AA⁢L⁢B×1⁢0⁢0(Equation.2)

where AGLOBand AALBis 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 10ExampleGlobulins (wt %)Albumins (wt %)176.50 ± 2.3023.50 ± 2.302.171.80 ± 0.9328.20 ± 0.93