SUNFLOWER ALBUMIN ISOLATE AND PROCESS FOR THE PRODUCTION THEREOF
20220192221 · 2022-06-23
Inventors
Cpc classification
A23J1/14
HUMAN NECESSITIES
B01D61/14
PERFORMING OPERATIONS; TRANSPORTING
A23L11/30
HUMAN NECESSITIES
International classification
A23J1/14
HUMAN NECESSITIES
Abstract
The invention relates to a process for producing a sunflower protein isolate, said process comprising the following steps: (a) providing a sunflower seed press cake, preferably a sunflower seed cold press cake; (b) mixing said sunflower seed press cake with an aqueous NaCI solution at a pH ranging from 3 to 4.5, in order to solubilize proteins present in said sunflower seed press cake and to thus obtain a solubilised protein solution, wherein said aqueous NaCI solution has a NaCI concentration ranging from 0.2 to 0.6 mol.Math.L.sup.−1; (c) separating said solubilised protein solution from solids therein; (d) subjecting said solubilised protein solution obtained in step c) to one or several membrane filtration(s) to obtain a protein isolate, and optionally drying said protein isolate to obtain a dry sunflower protein isolate. The invention also relates to a sunflower protein isolate and its use and product which incorporate.
Claims
1. A process for producing a sunflower protein isolate, said process comprising the following steps: (a) providing a sunflower seed press cake; (b) mixing said sunflower seed press cake with an aqueous NaCl solution at a pH ranging from 3 to 4.5, in order to solubilize proteins present in said sunflower seed press cake and to thus obtain a solubilized protein solution, wherein said aqueous NaCl solution has a NaCl concentration ranging from 0.2 to 0.6 mol.Math.L.sup.−1; (c) separating said solubilized protein solution from solids therein; (d) subjecting said solubilized protein solution obtained in step c) to one or several membrane filtration(s) to obtain a protein isolate, and (e) optionally drying said protein isolate to obtain a dry sunflower protein isolate.
2. The process according to claim 1, wherein: said aqueous NaCl solution of step (b) has a NaCl concentration ranging from 0.2 to 0.3 mol.Math.L.sup.−1 and said pH is from 4.0 to 4.2, or said aqueous NaCl solution of step (b) has a NaCl concentration ranging from 0.3 to 0.6 mol.Math.L.sup.−1 and said pH is from 3.1 to 3.5.
3. The process according to claim 1, wherein step c) is carried out by centrifugation.
4. The process according to claim 1, wherein step d) comprises the following step: subjecting the solubilized protein solution obtained in step c) to at least one ultrafiltration step, optionally followed by at least one diafiltration step, and harvesting the protein isolate.
5. The process according to claim 1, wherein steps b) to d) are conducted at room temperature.
6. The sunflower protein isolate obtainable by the process according to claim 1, wherein said protein isolate comprises a protein content of at least 88 wt % by weight of the total dry matter, an albumin protein content of at least 85 wt % by weight of the total proteins; and a phytic acid content of less 4 wt % by weight of the total dry matter.
7. The sunflower protein isolate obtainable by the process according to claim 2, wherein in step (b) said aqueous NaCl solution has a NaCl concentration ranging from 0.2 to 0.3 mol.Math.L.sup.−1 and said pH is from 4.0 to 4.2, and wherein said sunflower protein isolate comprises the following features: i. a total protein content of at least 90 wt % by weight of the total dry matter, ii. an albumin content of at least 85 wt % by weight of the total proteins; and iii. a phytic acid content of less than 1% by weight of the total dry matter.
8. The sunflower isolate according to claim 7, wherein if further has further comprising at least one of the following features: a color defined by the following coordinates L*=92.2±2.9, a*=1.3±0.7, b*=11.0±1.4, ΔE=15 or less a solubility of at least 95%, in an aqueous solution at a pH ranging from 2 to 11, a cysteine content ranging from 30.5 to 49 mg.Math.g.sup.−1 of total protein in said protein isolate, a methionine content ranging from 26.5 to 43 mg.Math.g.sup.−1 of total protein in said protein isolate, and/or an amino acid content as defined in Table 4.
9. A food or beverage product comprising a sunflower seed protein isolate of claim 6.
10. The sunflower seed protein composition obtainable or obtained by the process according to claim 1, wherein after step (c) and before step (d), said sunflower protein composition has a protein content of at least 35 wt % by weight of the total dry matter (/dm); optionally said composition has less 4 wt %/dm of phytic acid and/or less than 3.5 wt %/dm phenolic compounds.
11. A feed, dietary supplement or additive, for animal feeding, comprising the sunflower seed protein composition of claim 10.
12. The process according to claim 1, wherein the sunflower seed press cake is a sunflower seed cold press cake.
13. The process according to claim 1, wherein said aqueous NaCl solution of step (b) has a NaCl concentration of 0.25 mol.Math.L.sup.−1 and said pH is of from 4.05 to 4.15, or said aqueous NaCl solution of step (b) has a NaCl concentration ranging from 0.4 to 0.5 mol.Math.L.sup.−1 and said pH is of about 3.2 to 3.4.
14. The sunflower protein isolate obtainable by the process according to claim 1, wherein said protein isolate comprises a protein content of at least 90 wt % by weight of the total dry matter, an albumin protein content of at least 85 wt % by weight of the total proteins; and a phytic acid content of less 4 wt % by weight of the total dry matter.
15. The sunflower protein isolate obtainable by the process according to claim 7, wherein said sunflower protein isolate has no detectable free chlorogenic acid monoisomers.
16. The sunflower seed protein composition obtainable or obtained by the process according to claim 1, wherein after step (c) and before step (d), said sunflower protein composition has a protein content of 35% to 55% by weight of the total dry matter (/dm); optionally said composition has less 4 wt %/dm of phytic acid and/or less than 3.5 wt %/dm phenolic compounds.
Description
[0109] Foregoing and other objects and advantages of the invention will become more apparent from the following detailed description and accompanying drawing, which refers to non-limiting examples illustrating the process according to the invention.
[0110]
[0111]
[0112]
EXAMPLES
Example 1: Sunflower Albumin Extraction
1. Materials and Methods
[0113] 1.1. Chemicals
[0114] Sodium chloride (NaCl, CAS 7647-14-201), sodium hydroxide pellets (NaOH, CAS 1310-73-2) ethylenediaminetetraacetic acid (EDTA, CAS 6381-92-6) were obtained from VWR (Darmstadt, Germany). Hydrochloric acid (HCl, CAS 7647-01-0) was from Carlo Erba (Milan, Italy). Tris(hydroxymethyl)aminomethane (Tris, CAS 77-86-1), glycine (CAS 56-40-6), iron (III) chloride (FeCl3, CAS 7705-08-0), sodium sulfate (Na2SO4, CAS 7757-82-6), 5-sulfosalicylic acid hydrate (CAS 304851-84-1) was from Fisher Scientific (Hampton, USA).
[0115] 1.2. Solid/Liquid Extraction
[0116] Sunflower cold press meal (or cake) was provided by Olead (Pessac, France). The protein, fat and phytic acid content in the meal were 42.8, 14.6, and 6.6% on dry matter basis, respectively. The meal was extracted with 1:9 solid/liquid ratio (w/w). The pH (3-6) and various concentrations of NaCl solutions (0-0.5 mol.Math.L.sup.−1) were used according to the DoE matrix. The mixture was stirred at 600 rpm during 60 min at 20° C. If necessary, the pH was readjusted. Then, the slurry was centrifuged (15 000×g, 30 min, 20° C.) and partly clarified on a Whatman filter paper. About 1600 mL of aqueous extract was obtained. The remaining solid residue was collected, stored at −80° C. and freeze dried.
[0117] 1.3. Experimental Design and Process Optimization
[0118] The design of experiment (DoE) and statistical analysis were based on response surface methodology (RSM). The influence of two factors: pH (3-6) and NaCl concentration (0-0.5 mol.Math.L.sup.−1) on three responses: extraction yield (SFA.sub.YIELD%), content (C.sub.SFA%) and phenolic contamination (SFA.sub.PHEN mg.Math.g.sup.−1) of albumins in liquid extract, as well as acid phytic (C.sub.PHYT%) and protein (C.sub.PROT%) content in remaining solid residue were taking into consideration. For design of experiments Modde 9.1.1.0 software form Sartorius Stedim Biotech (Gottingen, Germany) was used. According to generated experimental matrix, 11 experiments were performed containing three replications at the central point. The response surface curve, polynomial equation and observed/predicted plots were achieved. The mathematical relationship between factors and response was described by second-degree polynomial equation as follow:
y=β.sub.0+β.sub.1x.sub.1+β.sub.2x.sub.2+β.sub.11x.sub.1.sup.2+β.sub.22x.sub.2.sup.2+β.sub.12x.sub.1x.sub.2
where:
y—response,
β.sub.0—constant,
β.sub.1—coefficient of linear effect of pH,
β.sub.2—coefficient of linear effect of NaCl concentration,
β.sub.11—coefficient of cubic effect of pH,
β.sub.22—coefficient of cubic effect of NaCl concentration,
β.sub.12—coefficient of interactive effect of pH and NaCl concentration,
x.sub.1—uncoded pH value,
x.sub.2—uncoded NaCl concentration value.
[0119] The obtained equations were statistically verified by evaluation of regression coefficient (R.sup.2), residual standard deviation (RSD) and analyse of variance test (p-value, lack of fit). The significance level of p=0.05 was claimed.
[0120] 1.4. Multi-Objective Optimization
[0121] The multi-objective optimization of extraction process was carried out with using the predictive equations of RSM. The genetic-evolutionary algorithms were employed to identify the optimum of experimental parameters in term of pH and NaCl concentration. The non-dominated solutions were calculated including the set of constraints: SFA.sub.YIELD>70%, C.sub.SFA>90%, SFA.sub.PHEN<1.6 mg.Math.g.sup.−1%, C.sub.PHYT<4% and C.sub.PROT>40%. The selected optimal conditions were validated on additional experimental batch extraction by comparing the observed responses with prediction intervals of models (PI) and calculation of relative error (RE). The optimization runs (n=2 000) and all data exploitation were carried out using MATLAB software from MathWorks (Natick, USA).
[0122] 1.5. Protein Purification
[0123] Protein purification was performed in three principal stages: extract clarification by microfiltration, protein precipitation from extract using ammonium sulfate and protein desalting by size exclusion chromatography. The microfiltration step was carried out on Akta system from GE Healthcare (Illinois, USA) using Hydrosart membrane system (0.2 μm 200 cm.sup.2) from Sartorius (Gottingen, Germany). The 4 L of collected liquid extract was concentrated by a volumetric factor of 8 and then the retentate was washed with 2 diafiltration volumes of 0.5 mol.Math.L.sup.−1 NaCl. The total microfiltration permeates were pooled for next step. Ammonium sulfate was added to microfiltration permeates up to 65% of saturation and stirred for 30 min at a room temperature. After protein precipitation and centrifugation step (15 000×g for 30 min at 20° C.) obtained pellet was dissolved in 750 mL of deionized water. The desalting of proteins was carried out on low pressure chromatography system of Akta Pure from GE Healthcare (Illinois, USA). The sample volume of 20% bed volume of column (10 cm of height, 5 cm of diameter) was injected into G-25 Fine silica gel (GE Healthcare, Illinois, USA). The elution was performed at 10 mL.Math.min.sup.−1 with deionized water and the peak corresponding to protein recorded at 280 nm and less than 1% of conductivity was collected and freeze-dried.
[0124] 1.6. Analytical Methods
[0125] 1.6.1. Protein Characterization by Gel Electrophoresis
[0126] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Leammli method (Laemmli et al., 1970). Sunflower aqueous extract was diluted in 0.1 mol.Math.L.sup.−1 of sodium phosphate buffer at pH 7 to obtain a final concentration of 2 g.Math.L.sup.−1. Then, the sample was solubilized in 50 μL of Laemmli buffer containing 2% β-mercaptoethanol (v/v) and heated at 95° C. for 5 min. Molecular weight markers ranging 250-10 kDa (Precision Plus Protein Standards) and 26.6-6.5 kDa (Polypeptide SDS-PAGE Standard) were used (Bio-Rad, Hercules, USA). 10 μL of sample and markers were deposed to a 5% of polyacrylamide stacking gel and separated in a 17% polyacrylamide resolving gels. The migration step was carried out at 20 mA per gel. The gels were subsequently stained with Coomassie Brilliant Blue and destained overnight in 10% acetic acid solution (v/v).
[0127] 1.6.2. Determination of Albumin and Globulin Content by SE-HPLC
[0128] SE-HPLC analysis was performed according to the method of Defaix et al. (Defaix 2019). The analyses were carried out on HPLC Shimadzu LC30 system coupled with photodiode array (PDA) detector and operated by LabSolutions software, all from Shimadzu Corporation (Kyoto, Japan). 5-20 μL of sample was injected onto a Biosep SEC s-2000 column (300×7.8 mm; 5 μm) from Phenomenex (Torrance, USA). The exclusion rang of molecular weight was comprise from 1 to 300 kDa. During analysis the autosampler and column compartment were maintained at 20 and at 35° C., respectively. The mobile phase consisted in 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.cm.sup.−1 was used. The PDA signal was recorded between 190 and 400 nm with maximal absorption at 214 or 280 nm for protein or 325 nm for phenolic compound detection. To determine globulin and albumin proportion in sunflower aqueous extract the meal globulin/albumin ratio (70:30) was considered. This ratio corresponds to the mean value denoted in several articles (Mazhar et al., 1998; Baudet et al., 1977; Raymond et al., 1995). All measurements were performed in triplicate and the average value was calculated. All measurements were performed in triplicate and the average value was calculated.
[0129] 1.6.3. Determination of Soluble Proteins, Free Chlorogenic Acid Isomers and Chlorogenic Acid Bound
[0130] Sunflower protein and free chlorogenic acid isomer (3-CQA, 5-CQA et 4-CQA) content were determined using the SE-HPLC method of Albe Slabi et al. (Albe Slabi, 2019). The same HPLC system and chromatographic as presented in section 1.6.2 was used except for mobile phase composition (acetonitrile/water/formic acid (10:89.9:0.1 v/v)). The amount of covalently fixed CQAs (milligram of CQAs bound per one gram of SFAs) were quantified considering the surface of peak at 325 nm eluted at the retention time of SFAs and the average calibration slop of 3-CQA, 5-CQA et 4-CQA. The mass of soluble SFAs in liquid extract was determined basing on the concentration of total sunflower proteins and the proportion of SFA fraction in aqueous extract (section 1.6.2.). In the calculations, the same value of molar attenuation coefficient for helianthinins and SFAs was assumed, as it has been confirmed by Defaix et al. (Defaix, 2019). All analysis was carried out in triplicate and an average value was calculated.
[0131] 1.6.4. Kjeldahl Method
[0132] The measure of total nitrogen content in sample was carried out in accordance to Kjeldahl method procedures described in AOAC method 991.20 (AOAC). 0.5-2 mL of sample was mineralized in a digestion flask with 4 mL of 96% H.sub.2SO.sub.4 (v/v) and approximately 10 mg of catalyst. The mineralization step was achieved at 450° C. during 150 min. After this time, the solution was distilled with 32% NaOH (w/v) and the mixture was titrated against 0.01 mol.Math.L.sup.−1 HCl. A blank consisted of non-protein containing sample. The non-protein nitrogen in sample was determined in supernatant after protein precipitation using 50% trichloroacetic acid (w/v). A nitrogen to protein conversion Nx5.6 was used. All analyzes were repeated in triplicate. Average values of concentration and standard deviation were calculated.
[0133] 1.6.5. Quantification of Phytic Acid
[0134] Phytic acid determination was adapted from the method described by Garcia-Estepa et al. (Garcia-Estepa et al., 1999): 250-300 mg of solid sample was stirred with 20 mL of 0.4 mol.Math.L.sup.−1 HCl/10% Na2SO4 (w/v) for about 120 min at room temperature. The blank consisted of deionized water. Then, the mixture was centrifuged (10 000×g, 30 min, 20° C.) and supernatant was additionally clarified using 0.22 μm filter. In centrifuge tube 2.5 mL of 20 mmol.Math.L.sup.−1 FeCl.sub.3, 0.4 mol.Math.L.sup.−1 HCl/10% Na2SO4 (w/v), 20% sulfosalicylic acid (w/v) and filtered supernatant were mixed and heated at 100° C. for 20 min. After cooling at room temperature, the sample was centrifuged (10 000×g, 30 min, 20° C.) filtered (0.22 μm) and transferred into 50 mL volumetric flask. The pellet was washed with 4 mL of deionized water, centrifuged (10 000×g, 10 min, 20° C.) and supernatant was added after filtration (0.22 μm) into 50 mL volumetric flask. This step was repeated in triplicate. The volumetric flask was completed with deionized water. The pH of 20 mL of obtained solution was adjusted to 2.5±0.5 using glycine. After heating to 70-80° C. the sample was tittered against 2 mmol.Math.L.sup.−1 EDTA solution. The equivalent volume was reached when the solution changes color from burgundy to yellow-green. The results were expressed as phytic acid content on dry matter basis of sunflower meal (Ac Phyt/dm %). All measurements were performed in triplicate and average value was calculated.
[0135] 1.6.6. SFA Solubility
[0136] The SFA isolate was suspended in deionized water at 5.0 g.Math.L.sup.−1 (room temperature). The pH was adjusted to a given value by adding either 0.1 mol.Math.L.sup.−1 NaOH or 0.1 mol.Math.L.sup.−1 HCl and kept constant during 30 min. Then, the slurries were centrifuged (15 000×g, 20 min, 20° C.). The protein concentration in supernatant was measured by SE-HPLC according to Albe Slabi et al. (Albe Slabi et al., 2019 cf. ref 1). All analyzes were repeated in triplicate and average value of concentration and standard deviation were calculated.
[0137] 1.6.7. Colour Measurement
[0138] The solution of protein powder in deionized water was prepared at a concentration of 1% (w/v) and clarified thought 0.22 μm membrane filter. The color was recorded in CieL*a*b* scale using Lovibond PFX195 Tintometer at room temperature. The measure was performed in ten repetitions and average value of L*, a*, b* parameters with standard deviation were calculated.
[0139] 1.6.8. Amino Acid Composition
[0140] The determination of amino acid content (except tryptophan) was performed according ISO 13903:2005 procedures. Generally, protein sample (10 mg) was first hydrolysed using hydrolysis mixture solution and the hydrolysate was injected into C18 column of HPLC system. Composition in amino acids was determined by reaction with ninhydrin using photometric detection at 570 nm or 470 nm (for proline). For tryptophan quantification (EU 152/2009) sample was hydrolysed with barium hydroxide solution at 110° C. for 20 h and then injected into C18 column of HPLC system coupled with fluorescence detector (excitation 280 nm, emission 356 nm).
[0141] 1.6.9. SFA Structure Study
[0142] SFA proteins were analysed by Dynamic Light Scattering (DLS), Circular Dichroism (CD) and Differential Scanning calorimetry (DSC). DLS measurement was recorded on Zeta Sizer Nano-S from Malvern Instruments (Worcestershire, UK). 20 μL of filtered (0.22 μm) SFA solution at a concentration of 1 g.Math.L.sup.−1 at pH 4 (10 mM sodium phosphate buffer), pH 7 (10 mM sodium phosphate buffer) and pH 9 (10 mM borate buffer) were used. Samples were maintained at 25° C. during measurement. The number of acquisitions varied between 13 and 17 scans. Volumetric distribution of particle size was determined.
[0143] Circular dichroism (CD) was carried out using a Chirascan Plus device from Applied Photophysics (Leatherhead, UK). The far-UV spectra were obtained in the UV region of 180-280 nm. CD spectrophotometer was kept under constant flow of nitrogen gas and the temperature was maintained at 20° C. Samples were prepared in the same way as for DLS analysis and blank assay corresponding to appropriate buffer solution was subtracted. All spectra were repeated at least in triplicate and mean measurement was calculated. The far-UV spectra were converted into mean residue ellipticity [θ.sub.MRE] using the number of amino acids set at 130. The content of α-helix, β-sheet structures and random coils were obtained after spectrum deconvolution using CDNN software version 2.1.
[0144] DSC determination was carried out by Microcal VP-DSC from Malvern Panalytical (Worcestershire, UK) using the SFA solution at a concentration of 2 g.Math.L.sup.−1 in 10 mM sodium phosphate buffer, pH 7. The solution was filtered (0.22 μm) prior analysis. The blank assay consisted on protein-free phosphate buffer. Thermogram was acquired over a temperature range from 20 to 130° C. at the rate of 1° C./min. Thermal properties of SFA were expressed as the temperature of denaturation (T.sub.m) and the enthalpy calorimetry ΔH.sub.cal, that represents the total amount of energy emitted during the denaturation process.
[0145] 1.6.10. Functional Properties of SFA Isolate
[0146] For foaming study, 20 mL of 1% (w/v) SFA isolate solution in 10 mM sodium phosphate buffer pH 7 was mixed at 10 000 rpm during 5 min at room temperature using Ultra-Turrax T25 digital homogenizer from IKA (Staufen im Breisgau, Germany). Foaming capacity was calculated as ratio (in %) foam volume (mL) in to initial volume of SFA solution (mL). The foaming stability was expressed as the percentage of remaining foam volume (mL) after 5, 15, 30, 60 and 120 min.
[0147] The emulsifying capacity were evaluated using 5% (w/v) SFA isolate solution in 10 mM sodium phosphate buffer pH 7. For this purpose, 5 mL of above mentioned SFA solution was mixed with 2.5 mL of sunflower oil and mixed by Ultra-turax Homogenizer at 10 000 rpm at room temperature during 30 s. After this time, 2.5 mL of sunflower oil was additionally mixed at the same speed during 90 s. The mixture was then centrifuged at 1 100×g during 5 min. The ratio (in %) of emulsion volume (mL) after centrifugation and initial volume of mixture (mL) represents the emulsifying capacity. The stability of emulsion was determined after heating at 85° C. during 15 min and additional centrifugation (the same parameters). It represents the ratio (in %) of emulsion volume (mL) after and before heating. All measurement of technofunctional properties were carried out in triplicate and average value with standard deviation were calculated. The technofunctional properties of soy proteins (Solae, St. Louis, USA) measured in the same way as those of SFAs were used as a reference.
[0148] 1.6.11. Other Method for Determining Protein Solubility at Room Temperature
[0149] The solubility of the protein isolate of the invention in aqueous solution is measured as follows:
[0150] About 250 mg of protein powder is weighted, the exact mass recorded, and then dissolved in 25 mL of a distilled water in a beaker. The solution is transferred into a 50 mL volumetric flask at room temperature. The beaker is washed three times with 5 mL of distilled and the washing solutions are transferred into a 50 mL volumetric flask. Finally, the volumetric flask is completed with distilled water. A volume of 15 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−1 NaOH or 0.1 mol.Math.L−1 HCl and the stirring maintained during 30 min. After this time, the solid precipitate was separated by centrifugation at 11 000×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 (Nx5.6). The protein solubility at the given pH was calculated as follows (Equation 1).
2. Results
[0157] 2.1. Effect of pH on Sunflower Proteins Extraction
[0158] The trial study aimed at selecting the pH range that allows selective extraction of SFAs. For this purpose, the influence of pH (3-7) on extraction of helianthinins and SFAs was investigated (
[0159] The graph in
[0160] Besides, albumins (SFAs) extraction was high (around 50%) under strong acidic conditions (pH 3-5). It started to decrease at pH 6 to reach a minimum at pH 7 (25.87±2.38%).
[0161] The graph in
[0162] The SFAs extraction yield under acidic condition was only around 50%. The effect of NaCl was also investigated. To assess a synergetic effect between NaCl and pH, a design of experiments (DoE) study was implemented. Beside SFAs extraction yield, SFAs composition and polyphenol content in the liquid phase was investigated.
[0163] 2.2. Effect of pH and NaCl on SFA Yield, Content and Phenolic Contamination During Acidic Extraction
[0164] The effect of the pH (x.sub.1) (3-6) and mild NaCl concentration (x.sub.2) (0-0.5 mol.Math.L.sup.−1) on SFA extraction was studied by DoE. Responses related to the liquid extract and the solid residue co-produced after protein extraction were considered. Regarding the liquid phase, albumin extraction yield (SFA.sub.YIELD%), albumin content (C.sub.SFA%) and phenolic contamination of albumins (SFA.sub.PHEN mg.Math.g.sup.−1) were measured. Concerning the solid reside, the phytic acid (C.sub.PHYT%) and protein (C.sub.PROT%) content were considered. A full factorial design with experimental matrix consisting on 11 runs with the central point repeated in triplicate was applied. The obtained coefficients of the models (constant terms, cubic effects, interactions) and statistical model parameters (R.sup.2, RSD, p-value, lack of fit) are summarized in Table 1.
TABLE-US-00001 TABLE 1 Regression coefficient of the predicted polynomial models for sunflower albumin extraction yield (SFA.sub.YIELD), albumin content in liquid extract (C.sub.SFA), phenolic contamination of albumins (SFA.sub.PHEN) and phytic acid (C.sub.PHYT) and protein (C.sub.PROT) content in solid residue. Statistic model parameters Regression coefficients p- Lack Response β.sub.0 β.sub.1 β.sub.2 β.sub.11 β.sub.22 β.sub.12 R.sup.2 RSD value of fit SFA.sub.YIELD 3.92 25.42 117.0 −3.37 −142.8 n.s.* 0.953 3.61 0.000 0.056 C.sub.SFA −29.89 59.23 94.43 −7.13 n.s. −25.27 0.981 3.10 0.000 0.051 SFA.sub.PHEN −2.96 1.86 −1.17 −0.17 n.s. n.s. 0.937 0.15 0.000 0.311 C.sub.PHYT 20.00 −6.96 −6.75 0.79 n.s. 0.90 0.802 0.74 0.027 0.390 C.sub.PROT −3.20 20.92 15.77 −2.25 n.s. −6.35 0.942 1.31 0.001 0.154 *no significant
[0165] The albumin extraction yield (SFA.sub.YIELD%) ranges between 32.06 and 75.25%. According to statistical analysis, pH (x.sub.1), NaCl concentration (x.sub.2), and both cubic terms of pH (x.sub.11) and NaCl concentration (x.sub.22) significantly impacted the studied response. Regression coefficient (R.sup.2) was high and demonstrates that 95.3% of data fitted model. The variation between predicted/observed plot was 3.61. The model p-value (0.000) and the lack of fit (0.056) were not significant. From these results antagonist effect of pH and ionic strength could be deduced. As observed above, DoE shows that SFA extraction yield is negatively impacted at low NaCl concentration by increasing pH (minimum around 6). This also demonstrates that SFA extraction can be considerably increased by NaCl in the whole studied pH range. The maximal extractability of albumins (75%) was found in the area of pH from 3.25 to 4.35 and salt addition above 0.33 mol.Math.L.sup.−1.
[0166] Relationship of pH and salt concentration on the albumin proportion in the liquid phase (C.sub.SFA%) was also investigated. The obtained response function varied from 37.6 to 90.8%. The results of ANOVA analysis show that the significant terms are pH (x.sub.1), NaCl concentration (x.sub.2), quadratic terms of pH (x.sub.11) and interaction of pH and NaCl concentration (x.sub.12) at the 95% confidence level. The regression parameters of the obtained model (R.sup.2=0.981, RSD=3.10) confirm well-fitting predicted and actual values. Furthermore, p-value of the model (0.000) and lack of fit (0.051) confirm good model performance. The impact of both pH and NaCl was low between pH 3 and 5. Under these conditions the SFAs content was relatively constant (80-90%) whatever the ionic strength. The constant albumin proportion in relation to above-mentioned conclusion about increased SFA extraction yield in this pH region suggests that upon salt addition extraction yield of helianthinins is also improved. This finding indicates a protective effect of ionic strength against helianthinins denaturation under acid conditions. A negative effect of pH and NaCl was observed between pH 5 and 6. Obviously, this effect is reinforced by high NaCl concentration since the lowest SFA content (<40%) was observed at pH 6 and 0.5 mol.Math.L.sup.−1. Thus, selective extraction of SFAs could be only achieved in lower pH values regardless of used salt concertation.
[0167] The third response studied was the amount of CQA covalently bound to SFAs (SFA.sub.PHEN mg.Math.g.sup.−1). The response surface shows a significant effect of pH (x.sub.1), NaCl concentration (x.sub.2), cubic term of pH (x.sub.11). The responses varied in the range of phenolic contamination between 0.6481 and 2.1201 mg.Math.g.sup.−1. The plot of predicted vs actual values showing R.sup.2=0.937, RSD=0.15, p-value=0.000 and lack of fit=0.311 confirm reliable model for predictive purposes. From these results, the antagonistic effect of pH and NaCl concentration on the irreversible phenolic association with SFAs can be observed. At low NaCl concentration, the higher pH value leads to phenolic complexation. The maximum of phenol bonding to proteins was observed at pH 5-6 without salt addition (>2 mg of CQA per gram of SFAs). This effect is most pronounced under strong acidic pH. CQA (the main phenolic compounds in sunflower) has a negative net charge (pKa=3-3.5) in this pH range, while SFAs are positively charged. Hence, there is probably a high concentration of CQAs at SFA surroundings due to electrostatic interactions that might potentiate the covalent binding between these two compounds.
[0168] 2.3. Effect of pH and NaCl on Phytic Acid and Protein Content in Solid Residue after Acidic Extraction
[0169] The nitrogen content and the level of phytic acid are known to make a large part of oilseed meal value for feed applications. Phytic acid (3-10% on dry matter base of sunflower meals) is particularly pointed out due to decrease of biodisponibility of some minerals (Ca.sup.2+, Mg.sup.2+, Zn.sup.2+, Fe.sup.2+, Mn.sup.2+, Cu.sup.2+) and proteins in digestive tract by forming unabsorbable complexes (Nissar et al., 2017; Kumar et al., 2010; Cheryan et al., 1980).
[0170] Based on RSM results the content of phytic acid content in solid residue after extraction (C.sub.PHYT%) was found in the range from 3.14 to 6.69%. Among all variables, pH (x.sub.1), NaCl concentration (x.sub.2) quadratic term of pH (x.sub.11) and interaction of both (x.sub.12) were kept in model equation. The interaction between factors (x.sub.11) has no noticeable impact on the response. The obtained model fits within acceptable limits (R.sup.2=0.802, RSD=0.74) in relation to actual data. Good p-value (0.027) of the model and no significant lack of fit (0.154) were also observed. The results indicate that the content of phytic acid in the solid increases considerably near mild acidic pH and reaches the higher values around pH 3 and 6. On the other hand, the negative effect of ionic strength was also observed in the region of moderate acidic pH. The highest phytate reduction (50%) in relation to initial phytic acid content in sunflower meal (6.6%/dm) was observed in the zone of pH 4-4.5 and 0.4 mol.Math.L.sup.−1 NaCl. Surprisingly, these extraction behaviours of phytic acid coincides well with the extraction profile of SFAs.
[0171] Regression coefficients and ANOVA test were also applied to study the influence of pH and NaCl concentration on protein content in sunflower solid residue (C.sub.PROT%) ranging from 29.74-45.68%. The analysis of variance reveals the negative effect of pH (x.sub.1), NaCl concentration (x.sub.2), quadratic term of pH (x.sub.11) and pH/NaCl concentration interaction (x.sub.12). The excellent value of R.sup.2 (0.942), RSD (1.31), p-value of model (0.001) and lack of fit (0.154) indicate good accuracy of the model for simulation of protein content in solid residue. Minimum residual protein content in solid was achieved with high value of pH (about 6) and high NaCl concentration (about 0.5 mol.Math.L.sup.−1). In contrast, protein rich solid is produced using pH ranging from 4 to 5.5 without salt addition.
[0172] 2.4. Multi-Objective Optimization
[0173] The regression equations of models were used to identify the most suitable process for solid/liquid extraction. The objective of the optimization was to maximize extraction yield and content of SFAs, while minimize phenolic contamination of albumins. Simultaneously, a value-added residual solid characterized by high protein level and reduced content of in phytic acid was desired. To reach the targets, the following constraints were selected: SFA.sub.YIELD>70%, C.sub.SFA>90%, SFA.sub.PHEN<1.6 mg.Math.g.sup.−1, C.sub.PHYT<4% and C.sub.PROT>40%. The set of non-dominated solutions from multi-objective optimization was presented in
[0174] The accuracy of models to predict the process performance was verified by comparing of predicted and actual values from the extraction in the optimum of parameters. The results of model validation including prediction interval (P1), experimental value and relative error (RE %) were summarized in Table 2.
TABLE-US-00002 TABLE 2 Results of model validation for the extraction in the optimum of parameters. Factor PI C.sub.NaCl (p-value > Experimental RE pH (mol .Math. L.sup.−1) Response 0.05) value (%) 4.1 0.25 SFA.sub.YIELD (%) 68.21-75.43 61.62 −14.20 C.sub.SFA (%) 87.70-93.90 88.78 −2.23 SFA.sub.PHEN 1.37-1.67 1.55 +2.02 (mg .Math. g.sup.−1) C.sub.PHYT (%) 3.24-4.72 3.74 −6.03 C.sub.PROT (%) 40.87-43.49 40.97 −2.88
[0175] The observed responses for C.sub.SFA (88.78%), SFA.sub.PHEN (1.55 mg.Math.g.sup.−1), C.sub.PHYT (3.74%) and C.sub.PROT (40.97%) are included in the 95% probably prediction intervals. Only SFA.sub.YIELD (61.62%) was lower (SE=−14.20%) in relation to estimated values. This is probably due to extremely good replicates in the central point of DoE (reproducibility=99.3%) resulting in narrow prediction interval (±2.83%). However, experimental data of SFA yield fit to predicted values within a satisfying limit of RE≤15% that is commonly used for process prediction. Therefore, the selected condition guarantees the most sustainable albumin extraction from sunflower meal and the increase of the value of residual solid.
[0176] 2.5. Characterisation of SFA Obtained from Acid Extraction
[0177] Based on the results of multi-objective optimization, SFAs were extracted at pH 4.1 and 0.25 mol.Math.L.sup.−1 NaCl and purified by Size Exclusion chromatography. The chemical composition of the obtained SFAs were displayed in Table 3.
TABLE-US-00003 TABLE 3 Characterization of SFA isolate produced under optimal conditions (pH 4.1 and 0.25 mol .Math. L.sup.−1 NaCl). Dry matter content (%) 94.8 Protein content/dm (%) 93.3 Albumin content (%) 90.3 ± 0.1 Chlorogenic acid 3-CQA n.d.* monoisomers 5-CQA n.d. content/dm (%) 4-CQA n.d. Acid phytic content/dm (%) 0.88 Couleur L* 92.2 ± 2.9 a* 1.3 ± 0.7 b* 11.0 ± 1.4 Sulfur-containing amino acids (mg .Math. g.sup.−1 protein) 73.31 * n.d.—not detected
[0178] The proteins (93.3% proteins/dm) revealed rich in sunflower albumins (90.3±0.1%). They contained virtually no free chlorogenic acid isomers (not detected), low in phytic acid (0.88%/dm) and its colour was close to white (L*=92.2±2.9, a*=1.3±0.7, b*=11.0±1.4) (
TABLE-US-00004 TABLE 4 Amino acid composition of SFAs in comparison to the WHO requirement pattern. Amino acid WHO/FAO/UN composition of SFAs amino acid pattern Amino acid (mg .Math. g.sup.−1 protein) (mg .Math. g.sup.−1 protein) Histidine 20.26 15.00 Isoleucine 40.19 30.00 Leucine 58.31 59.00 Lysine 58.95 45.00 Methionine 34.08 16.00 Cystine 39.23 6.00 Methionine and cysteine 73.31 22.00 Phenylalanine and tyrosine 42.55 30.00 Threonine 41.80 23.00 Tryptophan 4.18 6.00 Valine 42.55 39.00
[0179] The structural and functional properties of isolated SFAs were also studied. The graph in
[0180] In order to assess information about size distribution of SFAs under various pH values (4, 7, 9) DLS analysis were performed. A single peak with size between 3.8-4.0 d. nm (99.5-100% of total sample volume) observed whatever the pH. The peak was attributed to low molecular weight proteins of 18-19 kDa. This confirms that the sample consisted mainly on SFAs (with theoretical molecular weight of 10-18 kDa) (Gonzalez et al. 2005; Gonzalez et al., 2007; Kortt et al., 1990; Berecz et al. 2010; Salgado et al., 2012; Gueguen et al., 2016) and no protein aggregation was detected.
[0181] On the other hand, the stability of SFA structure against various pH and thermal treatment was investigated. The far-UV CD spectra indicated that there is no change in SFA conformation state according variable pH values (under acidic (pH 4), neutral (pH 7) and alkaline (pH 9) conditions). The deconvolution of CD spectra revealed similar proportion of α-helix (36-38%) and β-sheet (36-37%) elements, while random coil forms were found at slightly lower level (26-27%). Also, DSC thermogram of SFA solution at pH 7 recording the peak of denaturation at the temperature of 120.8° C. with a ΔH.sub.cal of 17.6 kcal.Math.mol.sup.−1 indicated extremely high thermoresistance of produced SFAs.
[0182] The foaming (a) and emulsifying proprieties (b) of SFAs were compared with commercially available soy proteins used as a reference. Generally, SFAs present higher foaming capacity (359±14% of initial solution volume) in relation to lower value obtained for soy proteins (297±19% of initial solution volume). Also, the foam formed by SFAs (about 50% over 120 min from mixing) turned out to be more stable comparing to the more labile foam of soybean proteins (24±14% over 120 min from mixing). On the other hand, two proteins evaluated exhibited comparable ability to form emulsion (about 40-45% of initial solution volume) and the thermal stability at 85° C. of emulsions obtained by SFAs and soy isolate were similar and very high (about 95-100% of initial emulsion volume). Altogether, the obtained results met the principal conclusions that SFAs have an important potential as an alternative plant-based ingredient.
[0183] 2.6. Solubility of Sunflower Albumin Isolate at pH 4, 7 and 9 Determined by the Kjeldahl Method.
[0184] The solubility of sunflower albumin isolate at pH 4, 7 and 9 determined by the Kjeldahl method was 104.2%, 101.8% and 100.8%, respectively.
3. Conclusion
[0185] The results show that solid/liquid extraction at pH 4.1 and 0.25 mol.Math.L.sup.−1 NaCl resulted in selective production of SFAs (88.78%) with low phenolic contamination (1.55 mg.Math.g.sup.−1) and good SFA extraction yield (61.62%). Simultaneously, the optimal extraction condition allowed obtaining the protein-rich solid residue (40.97%/dm) with reduced amount of antinutritional phytate (3.74%/dm) and other non-protein compounds. The colorless SFAs isolated in the optimum of extraction parameters were perfectly soluble in water (about 100% in all pH range 2-11) and highly resistant on both pH and temperature treatments. Also, comparable functional properties (foaming and emulsifying) of SFAs to the routinely used protein products from soybean were shown. SFAs are therefore very promising as valuable protein-based ingredient which could be successfully used in various food applications. Therefore, proposed alternative process for preparation of SFAs and residual solid is an answer for sustainable valorization of sunflower meal in food and feed industry.
Example 2: A Sunflower Albumin Isolate from a Cold Press Meal from Dehulled Kernels
[0186] A sunflower albumin isolate (SFA) was obtained according to the process of the invention. The process and the analytical methods were performed as described in Example 1, unless otherwise specified. For this purpose, a cold press meal from (fully) dehulled sunflower kernels was used (see Table 5 below).
[0187] 2.1 Determination of Total Phenolic Content
[0188] Total phenolic content was measured according to ISO 14502-1: 2005 procedures (Determination of substances characteristic of green and black tea—Part 1: Content of total polyphenols in tea-colorimetric method using Folin-Ciocalteu reagent. In ISO 14502-1 International Standardization (p. 10). International Organization for Standardization Switzerland). For this purpose, solid sample was first extracted with 70% methanol (v/v) respecting the solid/liquid of 1:25 (w/v) at 70° C. during 10 min. After this time, the mixture was cooled to room temperature and centrifugated at 3 500 rpm at 20° C. for 10 min. The obtained pellet was subjected to the second extraction using the same parameters. The supernatant from both extraction steps was pooled and then analysed within 24 h.
[0189] Total phenolic content in supernatant was measured colorimetrically using Folin-Ciocalteu reagent. The calibration curve was performed using gallic acid stock solutions prepared in the concentration range from 10 to 50 g/L. 200 μL of supernatant, calibration stock solution or water (blank essay) was mixed with 1 mL of Folin-Ciocalteu reagent (previously diluted ten times with ultrapure water) and stirred energetically during 1 min. Between 1 and 8 min from stirring, 0.8 mL of sodium carbonate (7.5% w/w) was added. The mixture was left for 1 h at 20° C. After this time, the absorbance was recorded at 765 nm at 23° C. The concentration of total phenolics in supernatant was expressed in gallic acid equivalent.
[0190] Starting Material
TABLE-US-00005 TABLE 5 Starting composition of the cold press meal from dehulled sunflower kernels. DM (%) 94.1 Proteins/DM (%)(N × 5.6) 46.9 Lipids/DM (%) 8.6 Phytic acid content/DM(%) 4.9 Total phenolic content/DM(%) 3.9 DM = Dry Matter content.
[0191] Solid/Liquid Extraction
[0192] 500 g of the cold press meal from dehulled sunflower kernels was mixed with a solution of NaCl (0.25 mol.Math.L−1) in a solid/liquid ratio of 1:9 (wt %). The pH was adjusted to 4.1 using a solution of HCl (1.0 mol.Math.L−1). The mixture was stirred at 300 rpm at room temperature during 60 min. After 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. The wet residual solid was freeze-dried and then analysed. The wet residual solid was freeze-dried and then analysed.
[0193] Purification
[0194] Protein purification was performed in three principal stages: extract clarification by microfiltration, protein precipitation from extract using ammonium sulfate and protein desalting by size exclusion chromatography. The microfiltration step was carried out on Akta system from GE Healthcare (Illinois, USA) using Hydrosart membrane system (0.2 μm 200 cm2) from Sartorius (Gottingen, Germany). The 3.8 L of collected liquid extract was concentrated by a volumetric factor of 5. The microfiltration permeate was used for next step. Ammonium sulfate was added to microfiltration permeates up to 65% of saturation and stirred for 30 min at a room temperature. After protein precipitation and centrifugation step (15 000×g for 30 min at 20° C.) obtained pellet was dissolved in 190 mL of deionized water. The desalting of proteins was carried out on low pressure chromatography system of Akta Pure from GE Healthcare (Illinois, USA). The sample volume of 20% bed volume of column (10 cm of height, 5 cm of diameter) was injected into G-25 Fine silica gel (GE Healthcare, Illinois, USA). The elution was performed at 10 mL.Math.min-1 with deionized water and the peak corresponding to protein recorded at 280 nm and less than 1% of conductivity was collected and freeze-dried.
[0195] Results
[0196] The powder had a high purity (104.7% on dry matter basis) and was rich in sunflower albumins (89.0%) and had a low phytic acid content (0.7% on dry matter basis) (Table 6).
TABLE-US-00006 TABLE 6 Composition of sunflower albumin isolate. DM (%) 91.2 Proteins/DM (%)(N × 5.6) 104.7 Albumin content (%) 89.0 Phytic acid content/DM(%) 0.7 DM = dry matter content.
[0197] The protein solubility of sunflower albumin isolate at pH 4, 7 and 9, determined by the SE-HPLC method, was 100%, 97.2% and 95.9%, respectively (Table 7).
TABLE-US-00007 TABLE 7 Solubility of sunflower albumin isolate as a function of pH determined by the SE-HPLC method. Protein solubility of pH the isolate (%) 4 100.0 7 97.2 9 95.9
[0198] The protein solubility at pH 4, 7 and 9, determined by the Kjeldahl method, was 94.8%, 94.2% and 96.7%, respectively (see Table 8 below).
TABLE-US-00008 TABLE 8 Solubility of sunflower albumin isolate as a function of pH determined by the Kjeldahl method. Protein solubility of pH the isolate (%) 4 94.8 7 94.2 9 96.7
[0199] The composition of sunflower residual solid (Table 9) was 53.9% and 3.8% of protein and phytic acid content, respectively.
TABLE-US-00009 TABLE 9 Composition of sunflower residual solid. Proteins/DM (%) (N × 5.6) 53.9 Phytic acid content/DM(%) 3.8
Example 3: A Wet Residual Solid from Dehulled Kernels
[0200] A sunflower wet residual solid was obtained according to the process of the invention using a cold press meal from (fully) dehulled sunflower kernels was used (see Table 5). The process and the analytical methods were performed as described in Examples 1. The determination of total phenolic content is described in section 2.1.
[0201] Solid/Liquid Extraction: 200 g of the cold press meal from dehulled sunflower kernels was mixed with a solution of NaCl at different concentrations—0.6 mol.Math.L.sup.−1, 0.25 mol.Math.L.sup.−1 and 0.2 mol.Math.L.sup.−1—in a solid/liquid ratio of 1:9 (wt %). The pH was adjusted to different values—respectively 3.0, 4.1 and 4.5—using a solution of HCl (1.0 mol.Math.L.sup.−1). The mixture was stirred at 300 rpm at room temperature during 60 min. After this time, the mixture was centrifuged at 15 000×g during 30 min at 20° C. and the pellet that represented the wet residual meal was collected to be analysed (Table 10). the
[0202] Results
TABLE-US-00010 TABLE 10 Composition of the wet residual solid from dehulled kernels. Solid/liquid extraction conditions pH 3 4.1 4.5 NaCl concentration (mol .Math. L−1) 0.6 0.25 0.2 DM content(%) 35.7 36.7 39.0 Proteins/DM (%)(N × 5.6) 47.5 49.3 51.1 Total phenolic content/DM(%) 2.6 2.5 2.0 DM = dry matter content.
REFERENCES
[0203] 1. Albe Slabi, S., Mathé C., Framboisier, X., Defaix, C., Mesieres, O., Galet, O., & Kapel, R. (2019). A new SE-HPLC method for simultaneous quantification of proteins and main phenolic compounds from sunflower meal aqueous extracts. Analytical and bioanalytical chemistry, 1-11. [0204] 2. AOAC (1995). Method 991.20. Protein (crude) in animal feed. In Official methods of analysis of the Association of Official Analytical Chemists (19th edition). Washington, D.C.: Association of Official Analytical Chemists. [0205] 3. Baudet, J., & Mosse, J. (1977). Fractionation of sunflower seed proteins. Journal of the American Oil Chemists' Society, 54(2), A82. [0206] 4. Bongartz, V., Brandt, L., Gehrmann, M. L., Zimmermann, B. F., Schulze-Kaysers, N., & Schieber, A. (2016). Evidence for the formation of benzacridine derivatives in alkaline-treated sunflower meal and model solutions. Molecules, 21(1), 91. [0207] 5. Burnett, G. R., Rigby, N. M., Mills, E. C., Belton, P. S., Fido, R. J., Tatham, A. S., & Shewry, P. R. (2002). Characterization of the emulsification properties of 2S albumins from sunflower seed. Journal of colloid and interface science, 247(1), 177-185. [0208] 6. Cater, C. M., Gheyasuddin, S., & Mattil, K. F. (1972). Effect of chlorogenic, quinic, and caffeic acids on the solubility and color of protein isolates, especially from sunflower seed. Cereal chemistry. [0209] 7. Cheryan, M., & Rackis, J. J. (1980). Phytic acid interactions in food systems. Critical Reviews in Food Science & Nutrition, 13(4), 297-335. [0210] 8. Defaix, C., Aymes, A., Slabi, S. A., Basselin, M., Mathé C., Galet, O., & Kapel, R. (2019). A new size-exclusion chromatography method for fast rapeseed albumin and globulin quantification. Food chemistry, 287, 151-159. [0211] 9. Durante, M., Bernardi, R., Lupi, M. C., & Sabelli, P. (1989). Characterization of Helianthus annuus L. storage proteins. Journal of Agricultural and Food Chemistry, 37(4), 852-855. [0212] 10. FAO (2012). FAOSTAT-Agriculture, last update 11 Jun. 2008. FAO Statistics Division 2009, access date: 25 May 2018. http://www.fao.org/docrep/016/ap106e/ap106e.pdf. [0213] 11. Garcia-Estepa, R. M., Guerra-Hernandez, E., & Garcia-Villanova, B. (1999). Phytic acid content in milled cereal products and breads. Food research international, 32(3), 217-221. [0214] 12. Gonzalez-Perez, S., Merck, K. B., Vereijken, J. M., van Koningsveld, G. A., Gruppen, H., & Voragen, A. G. J. (2002). Isolation and characterization of undenatured chlorogenic acid free sunflower (Helianthus annuus) proteins. Journal of Agricultural and Food Chemistry, 50, 1713-1719. [0215] 13. Gonzalez-Perez, S., van Koningsveld, G. A., Vereijken, J. M., Merck, K. B., Gruppen, H., & Voragen, A. G. J. (2005). Emulsion properties of sunflower (Helianthus annuus) proteins. Journal of Agricultural and Food Chemistry, 53, 2261-2267. [0216] 14. Gonzalez-Perez, S., & Vereijken, J. M. (2007). Sunflower proteins: overview of their physicochemical, structural and functional properties. Journal of the Science of Food and Agriculture, 87, 2173-2191. [0217] 15. Gueguen, J., Walrand, S., & Bourgeois, 0. (2016). Plant proteins: Context and potentialities for human food. Cahiers de Nutrition et de diététique, 51(4), 177-185. [0218] 16. Ivanova, P., Chalova, V., Koleva, L., Pishtiyski, I., & Perifanova-Nemska, M. (2012). Optimization of protein extraction from sunflower meal produced in Bulgaria. Bulgarian Journal of Agricultural Science, 18(2), 153-160. [0219] 17. Kortt, A. A., & Caldwell, J. B. (1990). Low molecular weight albumins from sunflower seed: identification of a methionine-rich albumin. Phytochemistry, 29(9), 2805-2810. [0220] 18. Kortt, A. A., Caldwell, J. B., Lilley, G. G., & Higgins, T. J. (1991). Amino acid and cDNA sequences of a methionine-rich 2S protein from sunflower seed (Helianthus annuus L.). European Journal of Biochemistry, 195(2), 329-334. [0221] 19. Kuijpers, T. F., Narváez-Cuenca, C. E., Vincken, J. P., Verloop, A. J., van Berkel, W. J., & Gruppen, H. (2012). Inhibition of enzymatic browning of chlorogenic acid by sulfur-containing compounds. Journal of agricultural and food chemistry, 60(13), 3507-3514. [0222] 20. Kumar, V., Sinha, A. K., Makkar, H. P., & Becker, K. (2010). Dietary roles of phytate and phytase in human nutrition: A review. Food chemistry, 120(4), 945-959. [0223] 21. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nnature, 227(5259), 680. [0224] 22. Laisney, J. (1996). Process for Obtaining Oils and Fats—Oils from Seed and Kernels—General Principles of Trituration. In: Oils and Fats. A Comprehensive Treatise, vol. 1. Paris, France: Lavoisier Publishing, 1pp, 715-751. [0225] 23. Malik, M. A., & Saini, C. S. (2017). Polyphenol removal from sunflower seed and kernel: Effect on functional and rheological properties of protein isolates. Food Hydrocolloids, 63, 705-715. [0226] 24. Mazhar, H., Quayle, R., Fido, R. J., Stobart, A. K., Napier, J. A., & Shewry, P. R. (1998). Synthesis of storage reserves in developing seeds of sunflower. Phytochemistry, 48(3), 429-432. [0227] 25. Nioi, C., Kapel, R., Rondags, E., & Marc, I. (2012). Selective extraction, structural characterisation and antifungal activity assessment of napins from an industrial rapeseed meal. Food chemistry, 134(4), 2149-2155. [0228] 26. Nissar, J., Ahad, T., Naik, H. R., & Hussain, S. Z. (2017). A review phytic acid: As antinutrient or nutraceutical. Journal of Pharmacognosy and Phytochemistry, 6(6), 1554-1560. [0229] 27. Ordóñez, C., Asenjo, M. G., Benitez, C., & González, J. L. (2001). Obtaining a protein concentrate from integral defatted sunflower flour. Bioresource technology, 78(2), 187-190. [0230] 28. Ozdal, T., Capanoglu, E., & Altay, F. (2013). A review on protein-phenolic interactions and associated changes. Food Research International, 51(2), 954-970. [0231] 29. Pickardt, C., Neidhart, S., Griesbach, C., Dube, M., Knauf, U., Kammerer, D. R., & Carle, R. (2009). Optimisation of mild-acidic protein extraction from defatted sunflower (Helianthus annuus L.) meal. Food Hydrocolloids, 23(7), 1966-1973. [0232] 30. Rahma, E. H., & Rao, M. N. (1979). Characterization of sunflower proteins. Journal of Food Science, 44(2), 579-582. [0233] 31. Raymond, J., Robin, J. M., & Azanza, J. L. (1995). 11 S seed storage proteins from Helianthus species (Compositae): Biochemical, size and charge heterogeneity. Plant Systematics and Evolution, 198(3-4), 195-208. [0234] 32. Richard-Forget, F. C., & Gauillard, F. A. (1997). Oxidation of chlorogenic acid, catechins, and 4-methylcatechol in model solutions by combinations of pear (Pyrus communis cv. Williams) polyphenol oxidase and peroxidase: a possible involvement of peroxidase in enzymatic browning. Journal of Agricultural and Food Chemistry, 45(7), 2472-2476. [0235] 33. Saeed, M., & Cheryan, M. (1988). Sunflower protein concentrates and isolates low in polyphenols and phytate. Journal of Food Science, 53, 1127-1131, 1143. [0236] 34. Salgado, P. R., Drago, S. R., Ortiz, S. E. M., Petruccelli, S., Andrich, O., Gonzalez, R. J., & Mauri, A. N. (2012). Production and characterization of sunflower (Helianthus annuus L.) protein-enriched products obtained at pilot plant scale. LWT-Food Science and Technology, 45(1), 65-72. [0237] 35. Sosulski, F. W., & Bakal, A. (1969). Isolated proteins from rapeseed, flax and sunflower meals. Canadian Institute of Food Technology Journal, 2(1), 28-32. [0238] 36. Wildermuth, S. R., Young, E. E., & Were, L. M. (2016). Chlorogenic Acid Oxidation and Its Reaction with Sunflower Proteins to Form Green-Colored Complexes. Comprehensive Reviews in Food Science and Food Safety, 15(5), 829-843. [0239] 37. Yoruk, R., & Marshall, M. R. (2003). Physicochemical properties and function of plant polyphenol oxidase: a review 1. Journal of Food Biochemistry, 27(5), 361-422.