# Metal-Chelating Peptides Derived from Sunflower Meal Protein: Preparation, Isolation, Identification, and Antioxidant Properties

Jairo Andrés Camaño Echavarría, Christelle Mathé, Philippe Arnoux, Loïc

Stefan, Cédric Paris, Katalin Selmeczi, Chibuike Udenigwe, Laetitia

Canabady-Rochelle

► To cite this version:

Jairo Andrés Camaño Echavarría, Christelle Mathé, Philippe Arnoux, Loïc Stefan, Cédric Paris, et al. Metal-Chelating Peptides Derived from Sunflower Meal Protein: Preparation, Isolation, Identification, and Antioxidant Properties. ACS Food Science & Technology, 2025, 5 (6), pp.2336-2350. 10.1021/acsfoodscitech.5c00191. [hal-05151504](https://hal.univ-lorraine.fr/hal-05151504v1)

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Submitted on 4 Dec 2025

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1 Metal-Chelating Peptides Derived from Sunflower Meal

2 Protein: Preparation, Isolation, Identification and Antioxidant Properties

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5 Jairo Andrés Camaño Echavarría¹*, Christelle Mathe¹, Philippe Arnoux¹, Loïc Stefan², Cédric

6 Paris³, Katalin Selmeczi⁴, Chibuike Udenigwe⁵, Laetitia Canabady-Rochelle¹*

7 *Corresponding authors:

8 E-mail:

9 jairo-andres.camano-echavarria@univ-lorraine.fr

10 laetitia.canabady-rochelle@univ-lorraine.fr

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12 ¹Université de Lorraine, CNRS, LRGP, F-54000 Nancy, France

13 ²Université de Lorraine, CNRS, LCPM, F-54000 Nancy, France

14 ³Université de Lorraine, LIBIO, F-54000 Nancy, France

15 ⁴Université de Lorraine, CNRS, L2CM, F-54000 Nancy, France

16 ⁵School of Nutrition Sciences, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada

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Sunflower meal, a by-product of sunflower seed oil extraction, contains approximately 30-50 % proteins.

Recognized for its high protein content and bioavailability, it is suitable for bioactive peptides production. Yet, research exploring the potential of sunflower proteins for generating metal-chelating peptides (MCPs) is sparse. Therefore, this study aimed to evaluate sunflower meal as a protein source to produce metal-chelating peptides. After protein extraction to obtain a sunflower protein isolate, single and sequential enzymatic treatments were applied to produce hydrolysates using Protamex® (Prot) and Protamex followed by Flavourzyme® (Prot+Flav), respectively. Upon sequential treatment, effectively large number of peptide bonds were cleaved, releasing mainly small-sized peptides. Prot hydrolysates exhibited the highest Fe²⁺-chelating properties, inhibition of Cu²⁺-induced reactive oxygen species (ROS) production, and ABTS scavenging activities. Besides, sequential hydrolysis with both enzymes enhanced the inhibition of Fe³⁺-induced ROS production and the reducing power. The Cu²⁺-chelating peptides present in hydrolysates were separated using immobilized metal ion affinity chromatography (IMAC-Cu²⁺) and identified by LC-MS/MS analysis. MS/MS analysis of enriched Cu²⁺-binding peptide fractions unveiled twenty-nine potential His-containing MCPs from SMPI hydrolysate. The molecular weight of Cu²⁺-identified peptides ranged from 0.8-1.7 kDa, with the larger-sized peptides (> 1 kDa) presenting the most effective bioactive properties. His, Glu and Asp residues were crucial for metal-chelating and antioxidant properties. Due to their high potential to bind Cu²⁺, Fe²⁺ and Fe³⁺, sunflower meal peptides could serve as potential MCPs candidates for use as food or pharmaceutical agents to prevent metal-induced oxidation and related-diseases.

**Key words:** bioactive peptides, protein hydrolysate, enzymatic hydrolysis, sunflower seed, peptide-metal complex
47 **1. Introduction**

Sunflower (Helianthus annuus L.) is the 3rd ranking oilseed after soybean and rapeseed in terms of volumes
with a worldwide production of 47 million metric tons in 2023. ¹ Obtained after sunflower seed oil extraction,
the meal residue contains approximately 30-50 % proteins. Due to its high protein content and bioavailability,
sunflower meal is a potential source of biomaterials for a broad range of food applications, particularly for
bioactive peptides production. Initially encrypted within primary protein sequence under an inactive form,
bioactive peptides can be released by proteolysis.² They have gained considerable attention owing to their
diverse health benefits. Sunflower meal protein produced hydrolysates were reported to possess immune-
modulating³, angiotensin-converting inhibiting⁴, antioxidant and anti-inflammatory activities.⁵
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In food protein hydrolysates, metal-chelating peptides (MCPs) can be generated from a variety of protein
sources, such as milk, eggs and fish.² Based on a sustainable development perspective and utilization of
biological resources, many studies have focused on by-product and plant proteins. This presents tremendous
potential for the production of MCPs due to their high protein content and their potential high-added value
after proteolysis.⁵ Several plant-based proteins such as rapeseed⁶, defatted walnut flake⁷ and chia seed⁸ were
reported for producing zinc, iron and copper-chelating peptides, respectively. Meanwhile, MCPs were rarely
investigated in sunflower protein hydrolysate.⁹⁻¹¹
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MCPs are characterized by their capacity to form coordination complexes with metals such as Ca²⁺, Zn²⁺,
Fe²⁺/³⁺ and Cu²⁺. These biofunctional peptides have widely been proposed as potent alternatives to combat
metal-related diseases.². Indeed, MCPs can be used as delivery agents for enhancing the stability, absorption,
and bioavailability of minerals.¹² Additionally, by complexing Fe²⁺ and Cu²⁺, MCPs could efficiently mitigate
oxidative damage associated with chronic diseases. Indeed, iron and copper ions have the potential to catalyze
the production of reactive oxygen species (ROS) via the Fenton and Haber Weiss reactions, triggering radical
chain reactions that can damage biomolecules and thus, induce various diseases (degenerative, neurological,
cardiovascular and cancer).¹³
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MCPs can be separated using immobilized metal ion affinity chromatography (IMAC). This separation
technique is based on the affinity of some peculiar amino acids (AA) residues present in peptides for some
transition metal ions (e.g., Cu²⁺, Ni²⁺ or Co²⁺) immobilized onto the chromatographic phase. Due to its high
selectivity, high binding capacity and high recovery, IMAC is a powerful purification methodology for MCPs
in food-derived protein hydrolysates.⁶ Zu et al.¹⁴ and Xie et al.⁶ purified and identified zinc-chelating peptides
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from wheat germ and rapeseed hydrolysates, respectively, using IMAC-Zn²⁺. More recently, Bao et al.¹⁰ used IMAC-Ca²⁺ to purify calcium-binding peptides from sunflower meal hydrolysates, whereas Latha et al.⁸ separated IMAC-Cu²⁺ and identified metal-chelating peptides from chia seed proteins.

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To the best of our knowledge, there are only a few reports on the use of sunflower meal for producing MCPs.⁹⁻¹¹. Although Cu²⁺-chelating peptides were produced from sunflower meal protein hydrolysate¹¹, the peptide sequences or their metal-related antioxidant activities were not identified. Besides, there are no reports on purification and identification of Cu²⁺-chelating peptides from sunflower meal protein hydrolysate generated from sequential enzymatic treatment. In contrast to previous studies that have primarily reported the antioxidant properties of sunflower protein hydrolysates, our work provides a comprehensive and in-depth characterization of their metal-chelating profile. By combining a sequential enzymatic hydrolysis approach with physicochemical characterization, peptide identification using IMAC-Cu²⁺ followed by MS/MS, and antioxidant activity assessment, this work provides novel insights into the metal-chelating profile of this by-product. This study not only highlights the biofunctional potential of these peptides, but also establishes relationship between their sequences and metal-related antioxidant properties.

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## 2. Materials & Methods

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### 2.1. Chemicals and Equipment

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Sunflower meal (SM) was provided by SAS IMPROVE (Dury, France). Protamex® (Prot, protease from Bacillus sp., ≥ 1.5 U/g), Flavourzyme® (Flav, protease from Aspergillus oryzae, ≥ 500 U/g), 37% hydrochloric acid (HCl), 97% sulfuric acid (H₂SO₄), nitric acid (HNO₃), ammonium bicarbonate, o-phthal dialdehyde (OPA), N,N-dimethyl-2-mercaptoethyl ammonium, sodium tetraborate, sodium dodecyl sulfate, glycine, monobasic potassium phosphate, and all the antioxidant testing chemicals, including ethylenediaminetetraacetic acid (EDTA), carnosine, 3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p'-disulfonic acid monosodium salt hydrate 97% (ferrozine), ferrous chloride solution (FeCl₂), 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox®), potassium persulfate, potassium ferricyanide, trichloroacetic acid (TCA), ferric chloride (FeCl₃), iron(III) ferrocyanide Fe₄[Fe(CN)₆]₃ (Prussian blue), ascorbic acid were purchased from Sigma-Aldrich (St Louis, MO, USA). All the other reagents were analytical grade and purchased from VWR

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International (Atlanta, USA): copper (II) sulfate pentahydrate, potassium sulfate, sodium phosphate monobasic-NaH<sub>2</sub>PO<sub>4</sub>, sodium phosphate dibasic-Na<sub>2</sub>HPO<sub>4</sub>, Na<sub>2</sub>HPO<sub>4</sub>·12H<sub>2</sub>O, sodium chloride, 99% HEPES (C<sub>8</sub>H<sub>18</sub>N<sub>2</sub>O<sub>4</sub>S), and copper (II) chloride hydrate. The PolyEthylenGlycol molecular standards (PEG: 200, 600, 1000, 3000 and 8000 Da) were purchased from Sigma-Aldrich (St Louis, MO, USA).

All the spectrophotometric measurements were performed using a MRX Multiskan Go microplate reader (ThermoFicher Scientific, Vantaa, Finland) and 96-well microplates NunclonTM delta surface (Thermo scientific, Roskilde, Denmark).

## 2.2. Production of Sunflower meal protein isolate

Sunflower meal protein isolate (SMPI) was obtained using the method reported by Kalpana et al.<sup>15</sup> based on NaCl extraction to solubilize the majority of globulins proteins, and avoid undesirable colour change of the extract due to the oxidation of phenolic compounds and their interaction with proteins. Sunflower meal (30 g) was dissolved into a 1:10 (g/ mL) meal-to-ultra-pure water ratio, added with 10 % (w/v) of NaCl. Then, this former solution was stirred (60 minutes, 40 °C, and pH 6). After solubilization, the samples were cloth filtrated in order to remove solidified residual fibrous. The resulting solution was centrifuged (Centrifuge Universal 320, Hettich, Tuttlingen, Germany) for 10 minutes, at 20 °C and 3,300 g. Afterward, the resulting supernatant composed of solubilised proteins was recovered and filtrated by vacuum filtration to remove the salt residues remaining from solubilisation. The filtrated supernatant was precipitated by adjusting the pH at 4.5 within 10 min, corresponding to the isoelectric point of sunflower proteins.<sup>9</sup> This suspension was kept for 30 minutes at room temperature (25 °C), and then centrifuged (3 300 g, 10 minutes, 20 °C). The pellet was finally dried using a convective oven (Thermo ScientificTM, USA) at 55 °C overnight. SMPI was stored at -20 °C until further use.

## 2.3. Proteolysis

Enzymatic hydrolysis. Sunflower meal protein isolate hydrolysates were obtained as previously described with some modification.<sup>16</sup> First, SMPI was dissolved in 250 mL of 50 mM ammonium bicarbonate buffer solution, and then hydrolysed with Protamex® (Prot), Flavourzyme® (Flav), and Protamex® followed by Flavourzyme® (Prot+Flav) using their optimal conditions, respectively. Hydrolysis parameters were as follows: 2 % (w/v) SMPI concentration, 1 % (w/w) enzyme/substrate ratio, pH 8, 2 h, and 55 °C for Protamex® while pH 7, 2 h, and 55 °C were used for Flavourzyme®. Upon hydrolysis, pH was maintained at the desired values by adding 5 M NaOH. After each hydrolysis step, each protease was heat inactivated (95 °C, 15 min). In sequential
treatment, Protamex® was inactivated just before Flavourzyme® treatment. Then, hydrolysates were centrifuged (10 000 g, 15 min, 25 °C) and the whole hydrolysates fractions were freeze-dried (LABCONCO, Kansas City, USA) and stored at -20 °C for further analysis.

Ultrafiltration. Considering our initial results, the two hydrolysates with the highest degree of hydrolysis (Prot and Prot+Flav) were further dissolved (250 mL; 20 mg/mL in ultrapure water) and submitted to ultrafiltration under magnetic stirring and 5 bar of nitrogen pressure.¹⁷ The ultrafiltration unit (UF stirred cell 47-mm system XFUF04701; Millipore, Molsheim, France) was initially equipped with a 10-kDa molecular weight cut-off membrane (MWCO), followed by a 1-kDa MWCO membrane (Millipore, Billerica, USA), conditioned according to the manufacturer's specification, before ultrafiltration. The 10-kDa MWCO membrane was used as a first step to remove larger peptides, prevent potential fouling and improve the efficiency of the subsequent 1-kDa MWCO ultrafiltration. This sequential ultrafiltration approach facilitated the effective separation of low-molecular-weight peptides while maintaining optimal membrane performance. The ultrafiltrated fractions of hydrolysates (≤ 1 kDa Prot and ≤ 1 kDa Prot+Flav fractions) were freeze-dried and kept at -20 °C until further analysis.

## 2.4. Physicochemical characterization

### 2.4.1. OPA quantification and degree of hydrolysis

The peptide concentration expressed as mmol equivalent of primary amine groups per gram of hydrolysate (mmol eq. NH₂/g) was evaluated by o-phthal dialdehyde (OPA) quantification as previously reported.¹³ The degree of hydrolysis (DH) was determined exclusively for the whole hydrolysates (non-ultrafiltrated ones) through OPA quantification and expressed as the ratio of free amino groups present in the hydrolysate to the total amino groups present in the sample. The latter was derived from the complete hydrolysis of sunflower protein (6 N HCl, ratio 1:1 w/v, 130 °C, 24 h), with the assumption that all peptide bonds of the protein were cleaved during acid digestion.¹⁶ The DH was determined by the Equation 1.

$$ \text{Degree of hydrolysis (\%)} = \left[ \frac{\text{NH}_2 \times \text{NH}_2 0}{\text{NH}_2 \text{T} - \text{NH}_2 0} \right] \times 100 \quad (1) $$

Where: NH₂ X is number of free-NH₂ groups at final time of enzymatic hydrolysis, NH₂ 0 is number of free-NH₂ groups before hydrolysis, and NH₂ T is NH₂ after complete hydrolysis.
159 **2.4.2. Molecular weight distribution**

160 **2.4.2.1. SDS-PAGE profile**

161 The molecular weight (MW) profile of SM, SMPI and SMPI hydrolysates were studied using sodium dodecyl
162 sulphate polyacrylamide gel electrophoresis (SDS-PAGE), following the procedure described by Zilic et al.¹⁸
163 in a Mini-Protean tetra cell electrophoresis system (Bio-Rad Laboratories, Hercules, USA). Samples were
164 prepared at 20 mg/mL in ultrapure water and the solubilized protein solutions were mixed at a 1:4 (v/v) ratio
165 with sample buffer. This sample buffer consisted of 0.5 M Tris-HCl, pH 6.8, with 10% (w/v) SDS, 50% (v/v)
166 glycerol, and 0.05% (w/v) bromophenol blue along with 0.5% (w/v) 2-mercaptoethanol as a reducing agent.
167 These mixtures were heated at 100 °C for 5 minutes. Subsequently, 10 µL of each sample (equivalent to 40 µg
168 of protein) or MW markers (Catalog No: 26619, Thermo Scientific™, Vilnius, Lithuania; range: 10–250 kDa)
169 were loaded onto the polyacrylamide gel, consisting in a 7.5 % separation gel and a 5 % stacking gel.¹⁸
170 Electrophoresis was carried out at a voltage of 120 V for 15 minutes, followed by 180 V for 1 hour. After
171 electrophoresis, the gel was stained for 1 hour using a solution containing 0.16% (w/v) Coomassie blue R-250,
172 7.5% (v/v) acetic acid, and 5% (v/v) ethanol. Then, the gel was destained for 1 hour using a solution of 30%
173 (v/v) ethanol and 7.5% (v/v) acetic acid.

174 **2.4.2.2. Size exclusion chromatography**

175 The molecular weight (MW) distribution of the whole hydrolysates (Prot and Prot+Flav) and their ≤ 1 kDa
176 ultrafiltrates (≤ 1 kDa Prot and ≤ 1 kDa Prot+Flav) was analyzed using size-exclusion chromatography (SEC)
177 with a 515 HPLC pump (Waters, Milford, MA, USA), a degasser, and a RID 10-A Shimadzu detector, as
178 previously described.16 The system featured a Phenomenex PolySep-GFC-P 2000 column (300 × 7.8 mm - 8
179 µm – 200 A - separation range 100 Da – 10 kDa) safeguarded by a guard column (35 × 7.8 mm) linked to a
180 Phenomenex PolySep-GFC-P 1000 column (300 × 7.8 mm - 8 µm – 100 A - separation range 20 Da – 3 kDa).
181 The four previous hydrolysates (5 mg/mL) were loaded (200 µL) and eluted using an aqueous buffer containing
182 0.1 M sodium nitrate (NaNO₃) and 0.2 % (w/v) sodium azide (NaN₃) at a flow rate of 0.7 mL/min at room
183 temperature (25 °C). The polyethylene glycol (PEG) standard solutions (200, 600, 1000, 3000, and 8000 Da)
184 were similarly injected for SEC calibration. The chromatograms were analyzed using ASTRA® software
185 (Wyatt Technology, Goleta, USA).
186 **2.5. Metal-chelating peptides characterization**

187 **2.5.1. Antioxidant activities**

188 **2.5.1.1. Ascorbate tests**

189 The ability of hydrolysates to inhibit the ROS generation catalyzed by transition metals (i.e., Cu²⁺, Fe³⁺) was 190 investigated by the ascorbate test. In the presence of oxygen, these metals are able to catalyze the ascorbate 191 oxidation and the metal-catalyzed Fenton-type reaction to produce ROS (O₂⁻, H₂O₂ and OH⁻).¹⁹ In this assay, 192 Cu²⁺ and Fe³⁺ are reduced into Cu⁺ and Fe²⁺ by ascorbate, respectively. Yet, the resulting metal species are 193 unstable and easily oxidized back to Cu²⁺ and Fe³⁺ forms, following the redox cycle of the metal ions.²⁰ 194 Ascorbate (AscH⁻) solution was prepared at 5 mM using 50 mM HEPES buffer at pH 7.4. Stock solution of 195 Cu²⁺ and Fe³⁺ were prepared at 2 mM using CuCl₂ (in ultrapure water) and FeCl₃ (in 0.1 M HNO₃ acidic 196 solution to maintain the Fe³⁺ in solution). EDTA (positive control in Cu²⁺ assay, and negative control in Fe³⁺ 197 assay), and deferoxamine (DFO, positive control in Fe³⁺ assay) were prepared at 2 mM in order to reach a final 198 concentration of the components at 100 µM, with 1: 1: 1 AscH⁻: ligand: Cu²⁺/Fe³⁺ ratio, whereas, SMPI 199 hydrolysates were prepared at 5 mM (eq NH₂ according to OPA assay), with 1: 5: 1 AscH⁻: ligand: Cu²⁺/Fe³⁺ 200 ratio. The reaction was initiated by the introduction of ascorbate, and the kinetic study of the ascorbate 201 concentration was carried out by following the absorbance at 265 nm.¹⁹ The results were reported as ascorbate 202 consumed after 2 and 10 min for Cu²⁺ and Fe³⁺, respectively (ε = 14 500 M⁻¹ cm⁻¹). Indeed, ascorbate 203 consumption has a direct correlation with the levels of Cu²⁺ and Fe³⁺ in solution, which can be complexed in 204 the presence of hydrolysates/positive controls. Note that all the measurements were carried out in triplicate. 205

205 **2.5.1.2. Ferrozine test**

206 The Fe²⁺-chelating ability of hydrolysates was determined by ferrozine test as previously reported using EDTA 207 (0-0.04 mg/mL) as positive control.²¹ Briefly, 7.5 µL of FeCl₂ (2 mM) was mixed with 277.5 µL of control or 208 hydrolysate solution (0 to 1 mg/mL in ultrapure water), followed by the addition of 15 µL of ferrozine solution 209 (5 mM) after a 3-minute incubation at room temperature (25 °C). The mixture was allowed to stand for 10 210 minutes after shaking. The absorbance of the solution was measured at 562 nm. The iron-chelating activity of 211 the hydrolysates was determined by the Equation 2.

$$ \text{Iron chelating activity (\%)} = \left[ \frac{A_0 - A_s}{A_0} \right] \times 100 \qquad (2) $$
A₀ is the absorbance of the blank (sample volume replaced by ultrapure water), and Aₛ is the absorbance of the sample (control/hydrolysates). The half-maximal effective concentration (EC₅₀) was determined as the hydrolysate concentration required for 50 % iron chelation activity.

### 2.5.1.3. ABTS radical scavenging activity

The ABTS radical scavenging activity was determined as previously reported.²¹ 7 mM ABTS stock solution was prepared in phosphate buffer (4 mM, pH 7.4). The solution was maintained in total darkness at room temperature for 16 h before use. Then, an aliquot of the ABTS stock solution was diluted with the buffer to obtain an initial absorbance value of 0.70 ± 0.02 at 734 nm. 150 µL hydrolysate (0-0.4 mg/mL) or Trolox® (0-0.03 mg/mL used as positive control) were mixed with 150 µL ABTS radical, shaking for 10 s. After 10 minutes of incubation at 25 °C, the absorbance of the reaction was measured at 734 nm. The ABTS radical scavenging activity was calculated with the Equation 3.

ABTS radical scavenging (%) = $\left[ \frac{A_0 - A_s}{A_0} \right] \times 100$ (3)

Where A₀ represents the initial absorbance of ABTS radical solution (sample volume replaced by buffer), and Aₛ indicates the absorbance of the remaining radical (with samples).

The EC₅₀ was determined as hydrolysate/control concentration required for scavenging 50 % of ABTS radical. In addition, the TEAC index (Trolox® Equivalent Antioxidant Capacity) was determined from the calibration curve and results were expressed as µmol Trolox® equivalent per gram of sample (µmol TE/g sample).

### 2.5.1.4. Reducing power

The reducing power was measured as previously reported.²¹ Hydrolysates (0-3 mg/mL) and positive control (ascorbic acid, 0-1 mg/mL) were prepared in phosphate buffer (0.2 M, pH 6.6). An aliquot of each former dilution (70 µL) was mixed with 35 µL of 1%(w/v) potassium ferricyanide solution and, incubated at 50 °C for 20 min. Then, 135 µL of distilled water, 33 µL of 10% (v/v) trichloroacetic acid solution and 27 µL of 0.1 (w/v) FeCl₃ were added. The reduction product, i.e., ferrocyanide, was further stabilized with ferric ions in order to produce Prussian blue. The absorbance was determined at 700 nm after 10 min of incubation at 25 °C. The ability of hydrolysates to reduce Fe³⁺ was expressed in percentage (%) and calculated following Equation 4.

$$ \text{Reducing capacity (\%)} = 100 - \left[ \frac{A_0 - A_s}{A_0} \right] \times 100 \qquad (4) $$
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Where A₀ is absorbance of a 66 µM Prussian blue solution in the same reaction medium, and Aₛ is sample absorbance.

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Results were expressed as effective concentration of hydrolysates able to reach 50 % the maximal reducing power capacity (EC₅₀).

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### 2.5.2. IMAC experiments

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The Cu²⁺-chelating peptides present in whole and ≤ 1 kDa fractions of hydrolysates were fractionated by IMAC-Cu²⁺ using a HPLC system (LC-20AD, Shimadzu, Japan) at 1 mL/min flow rate, an oven temperature set at 25 °C and a Diode Array Detector (SPD-M20A, Shimadzu, Japan). MCPs were separated on a 1 mL HisTrap™HP column (7 x 250 mm) prepacked with Ni Sepharose High Performance (Cytiva, Marlborough, MA, USA). Experiments were carried out according to Irankunda et al. with modifications.²² The method included five steps described below in details: (i) Cu²⁺ regeneration (i.e., Ni²⁺ ions were removed and replaced by Cu²⁺ ions), (ii) equilibration, (iii) injection followed by unbound compounds removal, (iv) elution, and (v) washing.

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First, 10 column volumes (CV) of ultrapure water were used to remove the ethanol remaining in the column (storage conditions). Then, the column was stripped using 50 mM EDTA solution to remove Ni²⁺ immobilized ions, followed by Cu²⁺ regeneration step using 10 CV of a 0.1 M CuSO₄·5H₂O solution. Then, 20 CV of equilibration buffer (PBS 1X pH 7.4: Na₂HPO₄·12H₂O 6.7 mM; KH₂PO₄ 1.25 mM; NaCl 300 mM) were used to remove the unbound copper ions and equilibrate the column. Finally, 50 µL of hydrolysates (20 mM eq NH₂ according to OPA assay) were loaded onto IMAC-Cu²⁺ column. The first fraction (F1) labeled non-copper-chelating peptides was eluted with the equilibration buffer after 5 min in order to remove peptides without metal-chelating properties, then the Cu²⁺-chelating peptides (F2-F4) were eluted with an imidazole gradient (0-50 mM, prepared in PBS 1X pH 7.4) for 60 min. Finally, a 300 mM imidazole solution was used to wash the columns before the next run. The absorbance of the eluate was monitored at 280 nm. The F1-F4 fractions were collected using a fraction collector (FRC-10A, Shimadzu, Japan) for further LC-MS/MS identification analysis of Cu²⁺-chelating peptides in hydrolysates. IMAC-Cu²⁺ experiments were performed in triplicate to ensure reproducibility. The retention time for each fraction (RT) was determined using the highest intensity point of the peak. This ensures consistency in identifying the elution profile of each sample. Retention times are reported as the mean of the three independent runs with their standard deviations.

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265 **2.5.3. Mass spectrometry identification and sequence analysis**

266 LC-MS/MS analysis. To identify the peptide sequences present in the produced hydrolysates, the IMAC-Cu²⁺
267 collected fractions (F1-F4) were analysed on a Thermo Scientific UHPLC-HRMS system composed of a
268 Vanquish™ liquid chromatography unit coupled to a photodiode array detector and an Orbitrap ID-X™
269 Tribrid™ high-resolution mass spectrometer operating in electrospray positive ionization mode (ESI⁺) as
270 previously reported.²³

271 Peptide sequences identification. The Thermo Xcalibur raw LC-MS/MS data files resulting from the fractions
272 (F1-F4) collected from the hydrolysates separated in IMAC- Cu²⁺ were processed in the Andromeda search
273 engine of a commonly used proteomic software tools MaxQuant™ (version 2.4.13.0).²⁴ The peptides
274 sequences were searched against a restricted sunflower seed proteins database (*Helianthus annuus*) containing
275 30 entries obtained from UniProtKB (https://www.uniprot.org/, accessed on 27 January 2024, Supplementary
276 data, Table SD1). The parameters of database searches were set up as previously described (Table SD2).²⁴
277 Before searching peptides sequences from the collected fraction of hydrolysates, this method was first
278 validated using a synthetic peptides hydrolysate, mixing 8 pure peptides at 1 mM each. These metal-chelating
279 peptides (i.e., *AIPTGTAH*, *FATCIARH*, *KFPILEH*, *KFPILEHL*, *KFPILEHLR*, *WLHNDGN*,
280 *WLHNDGNTE*, *WVSFKTN*) were obtained from 11S globulin seed storage protein G3 (P19084,
281 11S3_HELAN) present in sunflower seed. All the 8 peptides were identified (Supplementary data, Figure
282 SD1). Finally, the AA composition of identified MCPs present in SMPI hydrolysates were computed through
283 ExPASy ProtParam server (https://web.expasy.org/protparam/).

284 **2.6. Statistical analysis**

285 To evaluate the effect of enzymatic treatments and 1-kDa ultrafiltration process on physical-chemical and
286 biofunctional properties of SMPI hydrolysates, results were statistically analyzed by one-way analysis of
287 variance (ANOVA). All the measurements of OPA quantification and biofunctional properties (Cu²⁺/Fe³⁺
288 ascorbate test, Fe²⁺-chelating, radical scavenging, reduction power and Cu²⁺-binding abilities) were obtained
289 in quintuplicate and data presented as means ± standard deviation. In our study, we performed three replicates
290 for each IMAC-Cu²⁺ experiment. Significant differences between means of samples were determined by
291 Fisher's multiple range test. Differences at *p*-value < 0.05 were considered significant. Statistical calculations
292 were conducted using the software Statgraphics Centurion XVI version 16.1.03 (Statgraphics Technologies
293 Inc, Virginia, USA).
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# 3. Results and Discussion

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## 3.1. Physicochemical characterization of the protein and hydrolysates

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### 3.1.1. Protein content of sunflower meal protein isolate

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The sunflower meal (SM) proteins were extracted using a NaCl extraction at pH 6, followed by isoelectric precipitation. The protein content initially present in SM (46.10 ± 0.9 %) increased up to 90.90 ± 2.7 % in SMPI, fulfilling the requirements of protein isolates (> 90 % of protein). Helianthinin (i.e., 11S globulin) is the main storage protein in sunflower meal. The extractability of such protein increases by using NaCl, and upon the isoelectric precipitation at pH 4.5, the 11S globulin can be isolated, resulting in a protein content increase. SMPI obtained has a suitable protein content, and should be further considered as an alternative to obtain hydrolysates. Indeed, the SMPI produced in this study exhibits a similar protein content (90.90 ± 2.7 %) compared to that reported by Alexandrino et al.(92.08 %-98.6 %).²⁵

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The degree of hydrolysis (DH), defined as the percent of peptide bonds cleaved in a protein hydrolysate, is the first parameter used to characterize a hydrolysate. It provides primary information about the peptide size.²⁶ In our study, we were interested in small-sized peptides, commonly reported for their bioactivity. The DH parameter can only be measured for whole hydrolysate, i.e., before ultrafiltration.

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Hydrolysates were prepared with Protamex® and Flavourzyme®, i.e., complex mixtures of endo-peptidases and endo/exopeptidases, respectively. Due to their low cost, these enzymes are commonly used in the food industry to produce food-derived bioactive peptides. To enhance the hydrolysis of SMPI, a sequential enzymatic treatment was applied (Prot+Flav). After 2 h of hydrolysis, SMPI hydrolysates obtained using Protamex® or Flavourzyme® alone had a DH of 31 and 23 %, respectively (Table 1). This demonstrates that the endopeptidases contained in Protamex® can hydrolyze more SMPI compared with Flavourzyme®. As endopeptidase, Protamex® is an enzyme widely used in agro-industrial by-products hydrolysis, with an extensively broad specificity in peptide bond cleavage. Flavourzyme® cleaves peptide bonds between Leu and Pro or between two Pro residues.²⁷ Similar trend was reported in other studies, where the DH values of SMPI hydrolysates hydrolysed with Protamex® or Flavourzyme® alone were comprised within 10.37-20.05 %⁴ and 5.93-10.55 %¹⁰, respectively.

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Moreover, considering the specificity of each enzyme, the Flav treatment as endo/exopeptidases was carried out sequentially after the Prot treatment as endopeptidase only to improve the DH. The DH of Prot+Flav
322 hydrolysate increased up to 38 %, which was significantly higher than that of single enzymatic treatments.

323 Thus, the number of N-terminal sites available for the activity of exopeptidases present in Flavourzyme® was
324 increased by the first hydrolysis step with Protamex®.

325 Among the produced hydrolysates, Prot and Prot+Flav hydrolysates exhibit the highest DH values and were,
326 therefore, selected for further analysis with their respective ≤ 1 kDa fractions.

327 Peptide quantification, as determined by OPA assay, refers to the measurement of free amino groups released
328 during protein hydrolysis and it is expressed as mmol eq. NH₂/g of hydrolysate. This value is directly
329 proportional to the concentration of peptides in the hydrolysate, since a higher production of peptides will
330 result in a greater number of exposed amino groups. The peptide molar concentration increased from 0.39 to
331 0.89, 0.67 and 1.23 mmol eq. NH₂/g with hydrolysis of the SMPI by Prot, Flav, and Prot+Flav, respectively
332 (Table 1). The peptide molar concentration notably increased in both ≤ 1 kDa fractions up to 1.16 and 3.33 for
333 Prot and Prot+Flav, respectively. However, the most important difference was found in the sequential
334 treatment, where Prot+Flav hydrolysate exhibits the highest amount of small-sized peptides.

335 **3.1.2. Molecular weight distribution of peptides**

336 **3.1.2.1. SDS-PAGE**

337 The MW distribution of protein constituents of the SM, SMPI and hydrolysates (Prot and Prot+Flav) was
338 characterized by SDS-PAGE between 10-250 kDa (Figure 1A). First, the SM electrophoretic pattern shows
339 four major, high-intensity band regions at approximately 35, 25, 20 and 10-15 kDa, corresponding to the 11S
340 globulin subunits (α, α', β) and 2S albumins, respectively. The polypeptide composition of sunflower meal
341 proteins presents a similar pattern to those reported in the literature.³ Compared to sunflower meal (lane SM),
342 notable changes in MW distribution of the protein isolate (lane SMPI) are observed, especially for the 2S
343 albumins region. This band, representing residual 2S albumins, is of lower intensity due to the protein
344 extraction process used, as 11S globulins are isolated from 2S albumins at their isoelectric point (pH 4.5).¹⁸
345 Finally, the SDS-PAGE profile of Prot and Prot+Flav hydrolysates shows that the band intensity of 11S
346 globulin subunits decreased, related to their partial Prot hydrolysis, producing peptides with MW range of 10-
347 25 kDa (lane Prot). Finally, for the sequential treatment (lane Prot+Flav), bands with MW higher than 10 kDa
348 disappeared while the bands corresponding to a high number of oligopeptides (<10 kDa) increased, due to the
349 high proteolytic activity of exopeptidases in Flavourzyme®. These results are in line with those previously
350 discussed for DH.
351 **3.1.2.2. Size exclusion chromatography**

352 The size exclusion chromatography (SEC) was used to estimate the MW of the hydrolysates (Prot and Prot+Flav) and their ≤ 1 kDa fractions (Figure 1B-C). Indeed, the peptides MW distribution plays a crucial role in their metal-chelating and antioxidant properties. The enzymatic treatments hydrolyzed the high MW protein of sunflower meal (above 20 kDa) into lower MW peptides (<10 kDa). The whole hydrolysates Prot and Prot+Flav exhibit the highest MW distribution in the range of > 3 kDa (45 %) and 0.2-0.6 kDa (64%), respectively. This finding can be explained by a higher production of small-sized peptides in Prot+Flav hydrolysate (DH: 38.80 %) compared to Prot one (DH: 30.96 %). Besides, in regard to the ≤ 1 kDa MWCO membrane ultrafiltration applied, the highest MW distribution was found from 0.2 to 0.6 kDa, with 76 and 86 % for ≤ 1 kDa Prot and ≤ 1 kDa Prot+Flav, respectively. Thus, it is possible to obtain protein hydrolysates from sunflower meal, enriched with small-sized peptides. Moreover, the SEC analysis is consistent with the DH and SDS-PAGE results. Our findings are in accordance with Sentís-Moré et al.⁴, who reported a Protamex® sunflower hydrolysates, with MW distribution from 0.5 Da to 5 kDa. In contrast, Velliquette et al.³ reported sunflower peptides with MW distribution ranging from 10 to 25 kDa after Flavourzyme® only.

365 In the present study, SM proteins were hydrolyzed using Protamex®, an enzyme that has previously been applied to this by-product. Previous work using Protamex® to hydrolyze SM protein focused primarily on the evaluation of physicochemical parameters (i.e., hydrolysis yield and molecular weight distribution), without reporting any information on the resulting bioactive properties. Besides, while the use of sequential enzymatic hydrolysis on SM protein has been investigated previously, to our knowledge the specific combination of Protamex® and Flavourzyme® used in our study has not been reported previously. Therefore, by evaluating the biofunctional characterization of these hydrolysates, this study allows to establish a relationship between enzymatic treatment, peptide composition and antioxidant mechanisms. This highlights our approach over previous studies and reinforces the potential of sunflower meal as a valuable source of MCPs.
374 **3.2.** Biofunctional characterization of MCPs in SMPI hydrolysates

375 **3.2.1.** Antioxidant activities

376 **3.2.1.1.** Inhibition of Cu²⁺-induced ROS production

377 Ascorbate test was used to evaluate the effect of Cu²⁺/Fe³⁺ complexation by MCPs present in hydrolysates on
378 the inhibition of ROS production (i.e., O₂⁻, HO•) generated through Fenton-type reaction at physiological pH
379 7.4 (Figure 2A).¹⁹,²⁰

380 The ascorbate test results for the inhibition of Cu²⁺-induced ROS production are presented for all the obtained
381 hydrolysates and expressed as ascorbate consumed after 3 min (Figure 2B). The negative control (buffer)
382 displays the highest ascorbate consumption, associated with its oxidation into dehydroascorbic acid, with a
383 value of 46.5 µM, indicating the maximal ROS production. In the same test, the positive control, i.e., EDTA,
384 clearly showed the lowest ascorbate consumption, which is related with its strong Cu²⁺-chelating ability.
385 Compared to the negative control, all the whole and ≤ 1 kDa fraction samples significantly (*p*-value < 0.05)
386 decreased the absorbate consumption. This indicates that, after Cu²⁺ complexation by peptides present in
387 hydrolysates, Cu²⁺ was unable to catalyze the Fenton-type reaction to produce ROS in the redox cycle. The
388 differences observed between hydrolysates can be attributed to the differences in peptide composition (peptide
389 length, amino acid sequences). Interestingly, Prot hydrolysates (whole and ≤ 1 kDa fraction) present the most
390 potent Cu²⁺-chelating peptides. Copper-chelating ability could be associated with various amino acid residues
391 such as His, Cys and Arg, as reported in sunflower protein¹¹, ²⁸ and rice-bran-derived albumin hydrolysates.²⁹
392 Interestingly, the DH increase in the Prot+Flav sequential treatment does not enhance the Cu²⁺-chelating
393 ability, suggesting that Cu²⁺-chelating sites of peptides have been destroyed upon sequential hydrolysis.
394 Similar results were reported by Carrasco-Castilla et al. who produced lectin hydrolysates, whose Cu²⁺-
395 chelating ability decreased after extensive hydrolysis.³⁰ Yet, Peng et al. reported that Cu²⁺-chelating ability of
396 whey protein hydrolysates increased with increasing DH (from 19.3 to 38.5 %).³¹ Moreover, small-sized
397 peptides obtained after the 1 kDa MWCO ultrafiltration possessed a poor ROS inhibition associated with a
398 lower Cu²⁺-chelating ability (higher ascorbate consumed).

399 **3.2.1.2.** Inhibition of Fe³⁺-induced ROS production

400 The inhibition of Fe³⁺-induced ROS production of all hydrolysates expressed as ascorbate consumed after 10
401 min (Figure 2C) was different from the Cu²⁺-induced ROS production. Here, EDTA, used as negative control
agent, showed the maximum ascorbate consumption (54 µM) while the ascorbate consumption decreased drastically to 3 µM (lowest value) for DFO, a positive control highly recognized as a Fe³⁺-chelating agent. Regarding the hydrolysates, the ascorbate consumption decreased notably to the range 8-14 µM, suggesting that all the produced hydrolysates and their ≤ 1 kDa fractions contain Fe³⁺-chelating peptides. The ability to complex Fe³⁺ to prevent ROS production is notably associated with the abundance of small-sized peptides in the hydrolysates. For instance, the whole Prot+Flav and both ≤ 1 kDa fractions hydrolysates, all characterized by their high peptide concentration expressed as mmol eq. NH₂/g of powder (OPA assay, Table 1, section 3.1.1) and correlated with their higher small-sized peptide (Figure 1C) compared to Prot hydrolysate, showed greater potential to prevent Fe³⁺-induced ROS production than the whole Prot hydrolysate. Yet, this observed difference is only significant for the ≤ 1kDa Prot+ Flav hydrolysate. The Fe³⁺-chelating ability is mostly influenced by factors such as MW, AA composition and their position within the sequence. Peptide sequences with abundance of negatively charged polar residues (Asp and Glu) can complex Fe³⁺ through carboxylate groups.¹⁹ Additionally, the MW of peptides with Fe³⁺-chelating ability usually ranges from 300-1500 Da, associated with a higher amount of carboxyl group available at each terminus of a peptide compared to larger-sized peptides.³² Verma et al. reported a range of highest to lowest extent of Fe³⁺-AA interaction as follows: Glu > Asp > His > Cys > Tyr > Gln > Lys > Asn > Ser > Trp, Leu, Arg, Ala.³² These AA residues have been reported in the literature for sunflower meal and sunflower meal protein hydrolysate. ⁹,³³ Based on our results, MCPs present in hydrolysates can complex Cu²⁺/Fe³⁺ in solution, leading to the inhibition of metal-induced ROS generation.

#### 3.2.1.3. Fe²⁺-chelating activity

The ferrous state of iron (Fe²⁺), one of the most important pro-oxidant agents, catalyzes ROS production through the Fenton reaction. Therefore, food-derived peptides endowed with iron-chelating properties may contribute to delay peroxidation and subsequently prevent cells and tissues damages.³⁴ The Fe²⁺-chelating activity of the hydrolysates and their ≤ 1 kDa fractions was assessed by ferrozine test and expressed as EC₅₀ (mg/mL) (Figure 3A-B). The lower the EC₅₀ value, the higher the Fe²⁺-chelating activity. For the whole hydrolysates, Prot hydrolysate showed higher Fe²⁺-chelating activity than Prot+Flav ones. Indeed, the EC₅₀ value increased significatively from 0.09 (Prot) to 0.82 mg/mL (Prot+Flav) after the sequential enzymatic treatment, meaning lower Fe²⁺-chelating activity of the hydrolysate. Also, this suggests that sequential treatment using Flavourzyme® could hydrolyze a high number of MCPs peptides already produced by Prot. Regarding the two ≤ 1 kDa fractions, the same trend was found for both enzymatic treatments. Indeed, the
EC₅₀ value of ≤ 1 kDa Prot+Flav could not be determined, as the activity was lower than 7 % in the range of concentration investigated (0-1 mg/mL). The Fe²⁺-chelating activity was related with the presence of specific AA residues in the peptide sequence, such as negatively charged amino acids (Asp, Glu) and neutral polar amino acids (His, Cys).³² Indeed, these AA were widely reported in sunflower meal and sunflower meal hydrolysates.⁹, ³³

Our results demonstrate that the Fe²⁺-chelating activity of hydrolysates is notably influenced by the MW distribution of peptides, where small-sized peptides (≤ 1 kDa) may not have enhanced this property. The high Fe²⁺-chelating activity of Prot hydrolysate would be rather associated with the higher content of large-sized peptides than in Prot+Flav hydrolysate, where the enzymes can expose more efficient sites available for Fe²⁺-chelation. Tang et al. found that the Fe²⁺-chelating activity was affected negatively by significant decreases in peptide chain length due to extended hydrolysis in hemp (*Cannabis sativa* L.) protein isolate.³⁵ Similarly, in He et al. the whole hydrolysates and its 5-10 kDa fraction obtained from rapeseed protein exhibited the highest Fe²⁺-chelating activity (lower EC₅₀ values).³⁶

In our study, Prot hydrolysates (whole and ≤ 1 kDa fraction) exhibited a stronger Fe²⁺-chelating properties (lower EC₅₀ values, EC₅₀: 0.09-0.10 mg/mL) compared to those reported in the literature for other hydrolysates from plant-derived proteins, including rapeseed protein (EC₅₀ values > 3 mg/mL³⁶), tree peony seed protein hydrolyzed by Alcalase, Neutrase and Papain enzymes (EC₅₀: 0.99-1.18 mg/mL³⁴), mung bean protein (EC₅₀: 0.22-0.55 mg/mL³⁷), and *Lentinus edodes* protein (EC₅₀: 0.99-2.7 mg/mL²⁷). Taken together, our findings suggest that Prot hydrolysates from sunflower protein could be good metal-chelating agents to prevent the oxidative damages caused by ferrous ion in food and biological systems.

#### 3.2.1.4. ABTS radical scavenging activity

The ability of hydrolysates to donate a hydrogen atom or an electron to scavenge free radicals was determined by measurement of ABTS radical scavenging activity (Figure 3C-D). All the hydrolysates exhibited dose-dependent ABTS radical scavenging activity from 0.02-0.4 mg/mL. The highest activity was reached at 0.4 mg/mL (> 87 %) (Figure 3C). With extended hydrolysis in Prot+Flav, a slight increase (*p*-value < 0.05) in EC₅₀ value is observed, meaning a loss of ABTS radical scavenging activity (Figure 3D). Thus, during the sequential use of Flavourzyme®, some ABTS radical scavenging peptides produced in Prot may be cleaved further, leading to the decrease of this antioxidant property. Additionally, among the ≤ 1 kDa fractions, no significant
460 difference was observed (*p*-value > 0.05), suggesting that neither sequential treatment nor ≤ 1 kDa ultrafiltration
461 enhanced the ABTS radical scavenging activity.

462 The ABTS radical scavenging activity of hydrolysates could be attributed to peptides containing Phe, Tyr, Trp,
463 Cys and His, with their benzyl, phenolic, indoyl, sulfhydryl, and imidazole groups, respectively.³⁸ In our study,
464 the EC₅₀ values of all the studied hydrolysates were lower (better activity) than those of peptides from
465 vegetable-derived proteins hydrolysed by Protamex® (tree peony seed protein, EC₅₀: 1.81 mg/mL³⁴ and mung
466 bean proteins, EC₅₀: 0.24 mg/mL).³⁷

467 Furthermore, data were also reported as µM Trolox equivalent per gram of sample (µmol TE/g) to compare
468 our results with other studies (Figure 3D). In TEAC analysis, the higher the value, the higher the ABTS radical
469 scavenging activity. Overall, SMPI hydrolysates and their ≤ 1 kDa fractions scavenged the ABTS radical
470 effectively resulting in antioxidant activity. The TEAC value of Prot hydrolysate, which displays the highest
471 radical scavenging activity, is higher than those of peptides from hydrolysate of hemp seeds (124-240 µM
472 TE/g³⁹), and similar to those of colocynth seeds (290-525 µM TE/g).⁴⁰ In contrast, higher values were reported
473 for canola proteins (5.45 mM TE/g).⁴¹

### 3.2.1.5. Reducing power

475 The ability of hydrolysates to reduce ferric ions (Fe³⁺) into ferrous ions (Fe²⁺) was evaluated using the
476 Fe³⁺/ferricyanide assay, commonly used for measuring the antioxidant properties of food protein hydrolysates
477 in reference to ascorbic acid as positive control. The lower the EC₅₀, the higher the reducing power. All the
478 hydrolysates exhibited reducing power in a concentration-dependent manner (Figure 3E-F). Indeed, peptides
479 present in hydrolysates could present AA residues that act as an extra source of electrons and protons (Phe,
480 Try, Trp, Asp, and Gln).³⁴ Compared to Prot hydrolysates, the reducing power of Prot+Flav hydrolysates
481 increased markedly with increasing DH (lower EC₅₀, Figure 3F). Whatever the hydrolysate the hydrolysate,
482 the strongest reducing power was observed in ≤ 1 kDa Prot+Flav, related to its highest peptide concentration
483 (meaning smaller-sized peptides, **Table 1**). Similarly, the reducing power of several protein hydrolysates were
484 reported to increase when the number of small-sized peptides or the DH increased (tree peony seed protein³⁴;
485 mung bean protein³⁷; sunflower meal).⁴²

### 3.2.2. Cu²⁺-chelating peptides separation by IMAC

486 The Cu²⁺-chelating peptides present in the hydrolysates and their corresponding ≤ 1 kDa fractions were
487 separated/fractionated using IMAC-Cu²⁺. This method, based on peptides-metal interaction, provides several
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advantages due to its high-binding capacity and high recovery. The Cu²⁺-chelating peptides were eluted using imidazole gradient. Yet, due to imidazole absorbing at 214 nm, which is also the wavelength used for monitoring peptide bonds, the peptides were followed at 280 nm. The IMAC-Cu²⁺ chromatogram shows the injection phase (0-5 min) and elution phase (5-65 min) of the hydrolysates (Figure 4A-D).

The imidazole gradient was applied (0-50 mM, 60 min, elution phase Figure 4) and Cu²⁺-chelating peptides bound onto IMAC-Cu²⁺ phase were eluted in several peaks, i.e., the fractions F2, F3, and even the fraction F4 for ≤ 1 kDa Prot+Flav only (Table 2). Among the hydrolysates, the retention time (RT) of the Cu²⁺-chelating peptides fractions were not significantly different for F2 (RT: ~11 min) and F3 (RT: ~14 min) (*p*-value > 0.05). Thus, the mechanism of peptide-Cu²⁺ interaction in IMAC-Cu²⁺ column may be similar in the four hydrolysates and their difference in chelation capacity may rather be explained by the number of chelating peptides.

### 3.2.3. Identification of Cu²⁺-chelating peptides by LC-MS/MS

The various fractions F1, F2, F3 and F4 were collected and subjected to LC-MS/MS analysis to identify the Cu²⁺-chelating peptides sequences present in the hydrolysates. Figure 5A shows the total number of identified peptides in each fraction, with the highest number of Cu²⁺-chelating peptides identified in Prot, followed by Prot+Flav and ≤ 1 kDa fractions. This observation strongly supports the high Cu²⁺-chelating profile of Prot hydrolysate. The peptides sequences identified in the unbound peptides fraction F1 and in the enriched MCPs fractions F2-F3 are displayed in Table SD3 and Table 3, respectively. Interestingly, all the peptide sequences identified in F2 and F3 contain at least one His residue (Table 2), which plays a pivotal role in the Cu²⁺-binding ability of hydrolysates due to its imidazole ring.² In comparison, His-containing peptides were not identified in the unbounding peptides fractions F1 (Table SD3). On the contrary, there may be other His-containing MCPs present in the F4 of ≤ 1 kDa Prot+Flav fraction, but they were not identified due to their low concentration compared to the whole hydrolysates and the sensitivity of analysis. Furthermore, the sequential treatment (Prot+Flav) does not show a better Cu²⁺-chelating profile than the single treatment (Prot). Indeed, MS/MS analysis of F2 indicates that 7 new MCPs were generated in Prot+Flav after Flav treatment. However, 10 MCPs already present in Prot were hydrolysed, resulting in a lower total number (Prot+Flav: 12) of MCPs available to bind Cu²⁺ ions compared to Prot hydrolysate (Prot: 15) (Table 3).

To better understand the Cu²⁺-chelating profile of the identified sequences, the AA composition of peptide sequences in F2 (fraction with the highest number of sequences) of all the hydrolysates were determined by
the free-accessed ProtParam tool (https://web.expasy.org/protparam/). In our study, we focused on characterizing the AA composition of the metal-chelating peptide fraction itself (IMAC-Cu²⁺ fraction) rather than the total AA composition of sunflower protein and its hydrolysate, already well-documented in the literature. This AA analysis provides valuable insights into the functional properties of the peptides within this specific fraction, allowing a deeper understanding of their metal chelating potential.

All the four hydrolysates contain His residue as the major amino acid in their sequences (>10 %), being the main AA responsible for Cu²⁺-binding properties of the hydrolysates (Figure 5B). In Prot hydrolysate, the peptide sequences contain higher His content than the Prot+Flav hydrolysate, which could explain its high Cu²⁺-chelating properties (see Figure 2B inhibition of Cu²⁺-induced ROS production). Negatively charged (Asp and Glu) and positively charged amino acids (Arg and His) are the major potential metal-binding AA residues present in the identified sequences (Figure 5B). Also, the peptide sequences contain high amount of Glu, an important AA for Fe³⁺-chelating properties, explaining the results of the inhibition of Fe³⁺-induced ROS production by the hydrolysates. Besides, Figure 5C reveals high amount of EAA, HAA, NCAA, and PCAA in the identified MCPs, which have highly been associated with the antioxidant properties of food protein hydrolysates.³⁸

In comparison to the literature, several peptides identified from plant-derived proteins have been reported to contain Histidine (His) residue (Table 3) as identified in our work. His-containing peptides are widely known for their strong metal-chelating properties. Some examples include KPHK, EPSH, HADAD, and NAPLPPPLKH: these peptides identified from chia seed,⁸ rapeseed,⁶ mug bean⁴⁴ and wheat germ¹⁴ protein hydrolysates, respectively showed affinity for metal ions and potential bioactive properties. Similarly, MCPs derived from agro-industrial by-product (e.g., SGSTGH, HLRQEEKEEVTVGSLK and FDHIVY) have also been identified as bioactive peptides, which supports the potential of protein-rich waste materials for the production of value-added functional peptides (Table 3).

More, another interesting comparison comes from the position of His within the peptide sequence. In our study, His residues were mostly found in the first, second and third positions from the amino terminus (e.g., HNDGNTELVVVVF, PHYA and WVHNDGNAE) or in the first, second and third positions from carboxylate terminus (e.g., GVDFIRH, DFIRHR, GTAHWL). Indeed, the different positions of His residue in the structure of MCPs may define the stability of the formed peptide-Cu²⁺ complex.⁴⁷. For instance, MCPs with His residue in the first and third positions from the amino terminus could possess better stability in the formed peptide-Cu²⁺ complex.⁴⁸ **Figure 6A-B** displays the sequenced mass spectra of two of the most
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interesting sequences of Cu²⁺-chelating peptides identified from SMPI hydrolysates loaded into MaxQuant
Viewer.

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Product oxidation is one of the main challenges in the food industry. Metal ions such as Cu²⁺ and Fe²⁺ can lead to lipid-rich food oxidation via Fenton and Haber-Weiss reaction. Oxidation reaction leads to color changes, deterioration of nutritional properties, off-flavors up to texture changes. Due to their ability to bind these transition metals, MCPs identified in our study could offer promising applications as food or pharmaceutical bioactive agents to prevent metal-induced oxidation as previous work on inhibition of the lipid oxidation on food emulsions by MCPs.⁴⁹ Finally, by valorising sunflower meal by-products to release MCPs, our approach minimizes the waste and also promotes a circular economy model in the food and agricultural fields, encouraging the development of high-value ingredients from renewable sources.

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This study reported the physicochemical properties, metal-chelating and antioxidant activities of SMPI hydrolysate obtained by single (Prot) and sequential (Prot+Flav) enzymatic treatments and their ≤ 1 kDa fractions. For the first time, Cu²⁺-chelating peptides were identified from SMPI hydrolysate using IMAC-Cu²⁺ and their metal-chelating and metal-related antioxidant activities characterized. Upon sequential treatment, large number of peptide bonds were cleaved effectively, releasing mainly small-sized peptides. Prot hydrolysates contained higher number of larger-sized peptides compared to Prot+Flav. Some similarities were observed between the Fe²⁺-chelating, inhibition of Cu²⁺-induced ROS production and ABTS radical scavenging data. Indeed, the Prot hydrolysate and its corresponding ≤ 1 kDa fraction exhibited the highest activities in these assays while the Prot+Flav and its ≤ 1 kDa fraction enhanced the Fe³⁺-induced ROS and the reducing power. Moreover, both MW distribution and AA residues of the sequences play a key role in metal-chelating and antioxidant properties of peptides from SMPI. The molecular weight of Cu²⁺-identified peptides ranged from 0.8-1.7 kDa, with the larger-sized peptides (> 1 kDa) presenting the most effective bioactive properties. Besides, the presence of His, Glu, Asp, and Arg in the peptide sequences identified improved the metal-chelating and antioxidant properties. Taken together, sunflower meal by-product is an interesting protein source to generate protein hydrolysates endowed with metal-chelating and antioxidant agents. According to the structural characteristics of the sequences, the peptides identified in our study could serve as antioxidant and MCPs candidates to be used as food or pharmaceutical active agents to prevent metal-induced oxidation and diseases. Further works should deeply investigate the potential bioavailability of these MCPs in the context of food applications including studies of gastrointestinal digestion and absorption.

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Note that the pH, temperature and ionic strength variations may affect the stability and binding affinity of peptides to metals, since protonation or deprotonation of coordinating AA residues may modify the coordination properties and functionality. Therefore, future studies will be required to evaluate the stability of peptide-metal complexes over a range of pH and temperatures values and in the presence of physiologically relevant ions. These investigations would lead to a better understanding of the stability of the complexes under variable conditions, while also providing valuable insights for their practical application.

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As future perspective, the use of molecular dynamic simulation and molecular docking of the peptide-Cu²⁺ complexes should be performed as theoretical approaches to gain a better insight into the interaction of peptides with metal ions prior to in vitro evaluation of peptides.

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**Abbreviations used:**

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600

AA, amino acids; AAA, aromatic amino acid; ABTS, 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt; ANOVA, one-way analysis of variance; Asch, ascorbate; BCAA, branched chain amino acids; CV, column volumes; DFO, deferoxamine; DH, degree of hydrolysis; EAA, essential amino acids; EC₅₀, half maximal effective concentration; EDTA, ethylenediaminetetraacetic acid; ESI⁺, electrospray positive ionization mode; Flav, Flavourzyme®; HAA, hydrophobic amino acids; HPLC, high-performance liquid chromatography; IMAC, immobilized metal affinity chromatography; LC-MS/MS, liquid chromatography tandem mass spectrometry; MCPs, metal-chelating peptides; MW, molecular weight; MWCO, molecular weight cut-off; NCAA, negatively charged amino acids; OPA, O-phthal dialdehyde; PCAA, positively charged amino acids; PEG, polyethylene glycol; Prot, Protamex®; Prot+Flav, Protamex® followed by Flavourzyme®; ROS, reactive oxygen species; RT, retention time; SCAA, sulphur containing amino acids; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SEC, size exclusion chromatography; SM, sunflower meal by-product; SMPI, sunflower meal protein isolate; TCA, trichloroacetic acid; TEAC, Trolox® equivalent antioxidant capacity; Trolox®, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.

601

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603

604
605 **Author Contributions**

606 J.A.C.E.: Conceptualization, Methodology, Data curation, Software, Formal analysis, Resources, Writing –

607 original draft. C.M.: Methodology, Writing - review & editing. P.A.: Methodology, Data curation. C.P.:

608 Methodology, Data curation, Writing - review & editing. L.S.: Methodology, Writing - review & editing. K.S.:

609 Methodology, Writing - review & editing. C.C.U.: Methodology, Writing - review & editing. L.C-R.:

610 Conceptualization, Project administration, Data curation, Formal analysis, Funding acquisition, Supervision,

611 Visualization, Writing – review & editing.

612

613 **Supporting information description**

614 Protein data base used for peptide identification, parameters of database searches used for peptide

615 identification, peptides sequences identified from sunflower meal protein hydrolysates, MaxQuant™

616 identification successfully carried out on a synthetic mixture containing 8 peptides at 1 mM.

617

618 **Acknowledgments**

619 The authors thank the French ministry government for the MESR grant, the ANR JCJC MELISSA (2020-

620 2024), the “Impact Biomolecules” project of the “Lorraine Université d’Excellence”(Investissements

621 d'avenir-ANR project number 15-004). The authors thank the PASM platform for the use of the Orbitrap LC-

622 MS system (Plateau d'Analyse Structurale et Métabolomique - SF4242 EFABA, F-54505 Vandoeuvre-lès-

623 Nancy, France).

624
625 **4. References**

626 (1) Li, Z.; Xiang, F.; Huang, X.; Liang, M.; Ma, S.; Gafurov, K.; Gu, F.; Guo, Q.; Wang, Q. Properties and Characterization of Sunflower Seeds from Different Varieties of Edible and Oil Sunflower Seeds. *Foods* **2024**, *13* (8), 1188. DOI: 10.3390/foods13081188.

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822 Tables

823 **Table 1.** The degree of hydrolysis (DH) and peptide concentration of hydrolysates prepared by different
824 enzymatic treatments determined by OPA assay. The DH can be measured only before ultrafiltration.

825

<table><thead><tr><th>Samples</th><th>Degree of hydrolysis<br>(%)</th><th>Peptide concentration*<br>(mmol eq. NH<sub>2</sub>/g)</th></tr></thead><tbody><tr><td>SMPI</td><td>--</td><td>0.39 ± 0.11<sup>a</sup></td></tr><tr><td>Prot</td><td>30.96 ± 1.34<sup>a</sup></td><td>0.89 ± 0.04<sup>b</sup></td></tr><tr><td>Flav</td><td>23.33 ± 1.69<sup>b</sup></td><td>0.67 ± 0.04<sup>c</sup></td></tr><tr><td>Prot+Flav</td><td>38.80 ± 0.87<sup>c</sup></td><td>1.23 ± 0.02<sup>d</sup></td></tr><tr><td>≤ 1 kDa Prot</td><td>--</td><td>1.16 ± 0.03<sup>b</sup></td></tr><tr><td>≤ 1 kDa Prot + Flav</td><td>--</td><td>3.33 ± 0.01<sup>e</sup></td></tr></tbody></table>

826 *Peptide quantification refers to the measurement of free amino groups released during proteolysis as an indicator of
827 peptide production. Higher values correspond to increased peptide concentrations, reflecting a higher degree of protein
828 hydrolysis. Different letters indicate in the same column indicate the significant difference between the hydrolysates and
829 their ≤ 1 kDa fractions (*p*-value < 0.05). n=5. DH: degree of hydrolysis; Prot: Protamex® hydrolysate; Prot+Flav:
830 Protamex® followed by Flavourzyme® hydrolysate; ≤ 1 kDa: 1 kDa ultrafiltrated hydrolysate.

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<table><caption>**Table 2.** Reproducibility of IMAC-Cu²⁺ separation experiments: Retention times (mean ± standard deviation) of peptide fractions obtained from sunflower meal protein hydrolysates.</caption><thead><tr><th rowspan="2">Sample</th><th colspan="4">Retention time of a given fraction (min)</th></tr><tr><th>F1</th><th>F2</th><th>F3</th><th>F4</th></tr></thead><tbody><tr><td>Prot</td><td>1.28 ± 0.1<sup>a</sup></td><td>10.9 ± 0.3<sup>a</sup></td><td>14.65 ± 0.2<sup>a</sup></td><td>NO</td></tr><tr><td>Prot+Flav</td><td>1.41 ± 0.3<sup>a</sup></td><td>10.8 ± 0.3<sup>a</sup></td><td>14.20 ± 0.4<sup>a</sup></td><td>NO</td></tr><tr><td>≤ 1 kDa Prot</td><td>1.21 ± 0.1<sup>a</sup></td><td>10.7 ± 0.2<sup>a</sup></td><td>14.1 ± 0.3<sup>a</sup></td><td>NO</td></tr><tr><td>≤ 1 kDa Prot+Flav</td><td>1.24 ± 0.2<sup>a</sup></td><td>10.8 ± 0.2<sup>a</sup></td><td>14.5 ± 0.4<sup>a</sup></td><td>20.9 ± 0.5</td></tr></tbody></table>

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Different letters in the same column indicate the significant difference between the hydrolysates and their ≤ 1 kDa fractions (*p*-value < 0.05). n=3. F1: fraction collected before the elution gradient (0–5 min in the chromatogram), containing unbound peptides with no affinity for Cu²⁺ ions; F2: first fraction of Cu²⁺-bound peptides eluted using imidazole; F3: second fraction of Cu²⁺-bound peptides obtained upon further elution with imidazole, F4: third fraction of Cu²⁺-bound peptides, observed only in the ≤ 1 kDa Prot+Flav hydrolysate, collected after additional imidazole elution. Prot: Protamex® hydrolysate; Prot+Flav: Protamex® followed by Flavourzyme® hydrolysate; ≤ 1 kDa: 1 kDa ultrafiltrated hydrolysate; NO: not observed.

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<table><caption>**Table 3.** Cu²⁺-chelating peptides identified from the F2-F3 sunflower meal protein isolate hydrolysates fractions using IMAC-Cu²⁺ separation followed by LC-MS/MS analysis.</caption><thead><tr><th rowspan="2">Sequence</th><th colspan="4">Intensity (x 10<sup>4</sup>)</th><th rowspan="2">Length</th><th rowspan="2">Mass (g/mol)</th><th rowspan="2">Score</th><th rowspan="2">Entry</th></tr><tr><th>Prot</th><th>Prot+Flav</th><th>≤ 1 kDa Prot</th><th>≤ 1 kDa Prot+Flav</th></tr></thead><tbody><tr><td colspan="9" style="text-align: center; font-weight: bold;">Fraction 2 (RT ≈ 11 min)</td></tr><tr><td>AGGISSEHIQGQQQ</td><td>NI</td><td>1553.80</td><td>NI</td><td>NI</td><td>14</td><td>1438.68</td><td>12.87</td><td>A0A251UYX1</td></tr><tr><td>AIQSPH</td><td>61.49</td><td>NI</td><td>30.21</td><td>NI</td><td>6</td><td>651.33</td><td>4.66</td><td>P19084</td></tr><tr><td>AVLAPH</td><td>49.70</td><td>NI</td><td>26.83</td><td>NI</td><td>6</td><td>606.35</td><td>1.42</td><td>A0A251T566</td></tr><tr><td>DFIRH</td><td>432.36</td><td>165.68</td><td>231.62</td><td>NI</td><td>5</td><td>686.35</td><td>10.80</td><td>A0A251TKL9</td></tr><tr><td>FPILEH</td><td>NI</td><td>37.99</td><td>NI</td><td>72.46</td><td>6</td><td>754.40</td><td>1.42</td><td>P19084</td></tr><tr><td>FPILEHL</td><td>NI</td><td>253.26</td><td>NI</td><td>124.96</td><td>7</td><td>867.49</td><td>3.24</td><td>P19084</td></tr><tr><td>FSPH</td><td>163.85</td><td>NI</td><td>103.38</td><td>NI</td><td>4</td><td>486.22</td><td>8.08</td><td></td></tr><tr><td>FVTPEEEQQQM</td><td>76.69</td><td>NI</td><td>NI</td><td>NI</td><td>12</td><td>1501.65</td><td>1.19</td><td></td></tr><tr><td>GENDQRGHIIF</td><td>36.28</td><td>507.19</td><td>NI</td><td>NI</td><td>11</td><td>1284.62</td><td>0.51</td><td>A0A251TUS1</td></tr><tr><td>GENDQRGHIIFVQ</td><td>NI</td><td>130.80</td><td>NI</td><td>NI</td><td>13</td><td>1511.75</td><td>1.52</td><td></td></tr><tr><td>GISSEHIQGQ</td><td>NI</td><td>NI</td><td>NI</td><td>21.07</td><td>10</td><td>1054.50</td><td>0.46</td><td>A0A251UYX1</td></tr><tr><td>GIVSPH</td><td>93.97</td><td>NI</td><td>58.57</td><td>12.87</td><td>6</td><td>608.33</td><td>4.66</td><td>A0A251TUS1</td></tr><tr><td>GVDFIRH</td><td>252.44</td><td>78.13</td><td>118.11</td><td>79.36</td><td>7</td><td>842.44</td><td>12.16</td><td></td></tr><tr><td>HNDGNTELVVVVF</td><td>NI</td><td>239.82</td><td>NI</td><td>NI</td><td>13</td><td>1441.72</td><td>4.72</td><td>A0A251TKL9</td></tr><tr><td>HVDQETASKL</td><td>94.04</td><td>NI</td><td>NI</td><td>NI</td><td>10</td><td>1126.56</td><td>1.84</td><td>A0A9K3HVJ0</td></tr><tr><td>NIDNPSHADFVNPQ</td><td>216.29</td><td>NI</td><td>NI</td><td>NI</td><td>14</td><td>1566.71</td><td>1.74</td><td>A0A9K3NGE6</td></tr><tr><td>PHYA</td><td>99.76</td><td>NI</td><td>NI</td><td>NI</td><td>4</td><td>486.22</td><td>2.71</td><td>A0A9K3JEW0</td></tr><tr><td>RGENDQRGHIIF</td><td>674.32</td><td>NI</td><td>NI</td><td>NI</td><td>12</td><td>1440.72</td><td>2.73</td><td>A0A251TUS1</td></tr><tr><td>SEVFH</td><td>NI</td><td>NI</td><td>NI</td><td>45.03</td><td>5</td><td>617.28</td><td>1.94</td><td>A0A9K3HVJ0</td></tr><tr><td>SEVFHVD</td><td>161.31</td><td>155.86</td><td>NI</td><td>NI</td><td>7</td><td>831.38</td><td>3.24</td><td></td></tr><tr><td>SFLAGGISSEHIQGQQQ</td><td></td><td>395.13</td><td>NI</td><td>NI</td><td>17</td><td>1785.86</td><td>2.25</td><td>A0A251UYX1</td></tr><tr><td>SPHWTIN</td><td>25.57</td><td>121.55</td><td>28.07</td><td>49.29</td><td>7</td><td>853.41</td><td>11.11</td><td>P19084</td></tr><tr><td>WVHNDGNAE</td><td>21.03</td><td>NI</td><td>NI</td><td>NI</td><td>9</td><td>1040.43</td><td>0.52</td><td>A0A9K3NGE6</td></tr><tr><td>YANQLDPNHRRF</td><td>NI</td><td>54.97</td><td>NI</td><td>NI</td><td>12</td><td>1529.75</td><td>1.19</td><td>A0A251TUS1</td></tr><tr><td colspan="9" style="text-align: center; font-weight: bold;">Fraction 3 (RT ≈ 14 min)</td></tr><tr><td>ALFSPH</td><td>304.06</td><td>NI</td><td>72.077</td><td>NI</td><td>6</td><td>670.34</td><td>14.67</td><td>A0A251UYX1</td></tr><tr><td>DFIRHR</td><td>151.10</td><td>NI</td><td>45.85</td><td>6.16</td><td>6</td><td>842.45</td><td>4.12</td><td>A0A251TKL9</td></tr><tr><td>GTAHWL</td><td>60.17</td><td>NI</td><td>NI</td><td>NI</td><td>6</td><td>683.34</td><td>1.42</td><td>P19084</td></tr><tr><td>GVDFIRHR</td><td>484.99</td><td>22.71</td><td>161.79</td><td>24.31</td><td>8</td><td>998.54</td><td>3.60</td><td>A0A251TKL9</td></tr><tr><td>NPSHADFV</td><td>NI</td><td>NI</td><td>NI</td><td>16.08</td><td>8</td><td>885.40</td><td>0.60</td><td>A0A9K3NGE6</td></tr><tr><td colspan="9" style="text-align: center; font-weight: bold;">Metal-chelating peptides previously reported in hydrolysates from plant-derived proteins or from byproducts</td></tr><tr><td>EFEGGSIEH</td><td></td><td>Sunflower seed hydrolysate<sup>43</sup></td><td></td><td></td><td>9</td><td>1004.01</td><td>NR</td><td>Putative 11-S</td></tr><tr><td>KPHK</td><td></td><td>Chia seed protein hydrolysate<sup>8</sup></td><td></td><td></td><td>4</td><td>508.61</td><td>NR</td><td>NR</td></tr><tr><td>EPSH</td><td></td><td>Rapeseed protein hydrolysate<sup>6</sup></td><td></td><td></td><td>4</td><td>468.47</td><td>NR</td><td>Cruciferin precursor</td></tr><tr><td>HADAD</td><td></td><td>Mug bean protein hydrolysate<sup>44</sup></td><td></td><td></td><td>5</td><td>527.49</td><td>NR</td><td>NR</td></tr><tr><td>NAPLPPPLKH</td><td></td><td>Wheat germ hydrolysate<sup>14</sup></td><td></td><td></td><td>10</td><td>1083.28</td><td>NR</td><td>NR</td></tr><tr><td>SGSTGH</td><td></td><td>Fish skin gelatin hydrolysate<sup>12</sup></td><td></td><td></td><td>6</td><td>545.24</td><td>NR</td><td>Collagen a3(I)</td></tr><tr><td>HLRQEEKEEVTVGSLK</td><td></td><td>Oyster protein hydrolysate<sup>45</sup></td><td></td><td></td><td>16</td><td>1882.08</td><td>NR</td><td>NR</td></tr><tr><td>FDHIVY</td><td></td><td>Tilapia bone hydrolysate<sup>46</sup></td><td></td><td></td><td>6</td><td>793.38</td><td>NR</td><td>I3IYK1</td></tr></tbody></table>

Prot: Protamex® hydrolysate; Prot+Flav: Protamex® followed by Flavourzyme® hydrolysate; ≤ 1 kDa: 1 kDa ultrafiltrated hydrolysate; NI: not identified; NR: not reported. F2: first fraction of Cu²⁺-bound peptides eluted using imidazole; F3: second fraction of Cu²⁺-bound peptides obtained upon further elution with imidazole.
891 **Figures captions**

892 **Fig. 1.** Molecular weight distribution of protein and peptides hydrolysates from SMPI. (A) SDS-PAGE profile
893 (12.5 %) of sunflower meal, sunflower meal protein isolate, and whole protein hydrolysates (Prot and Pro).
894 (B) and (C) size exclusion chromatographic profile of sunflower meal protein isolate hydrolysates and their ≤
895 1 kDa fractions. M: Marker; SM: sunflower meal; SMPI: sunflower meal protein isolate; Prot: Protamex® hydrolysate; Prot+Flav:
896 Protamex® followed by Flavourzyme® hydrolysate; ≤ 1 kDa: 1 kDa ultrafiltrated hydrolysate; PEG: Polyethylene glycol.

897 **Fig. 2.** (A) The process for generating reactive oxygen species (ROS) via the Fenton reaction cycle involves
898 molecular oxygen and ascorbate in the presence of copper and iron ions. (B-C) The inhibition of Cu²⁺/Fe³⁺-
899 induced ROS production by chelating copper assessed by measuring ascorbate absorbance intensity at 265 nm.
900 Error bar corresponds to the standard deviation. Different letters indicate the significant difference between samples (*p*-value < 0.05).
901 n=3. Asch: ascorbate; DFO: deferoxamine; Prot: Protamex® hydrolysate; Prot+Flav: Protamex® followed by Flavourzyme®
902 hydrolysate; ≤ 1 kDa: 1 kDa ultrafiltrated hydrolysate.

903 **Fig. 3.** Antioxidant activities of Prot, Prot+Flav, ≤ 1 kDa Prot and ≤ 1 kDa Prot+Flav hydrolysates. Fe²⁺-chelating activity (A), ABTS radical scavenging activity (C), and reducing power ability (E) as a function of hydrolysate concentration. Half efficient concentration EC₅₀ (mg/mL) values determined for Fe²⁺-chelating (B), ABTS radical scavenging (D), and reducing power activities (F). Error bar corresponds to the standard deviation.
904
905
906 Different letters indicate significant difference between samples (*p*-value < 0.05). n=5. TEAC: Trolox equivalent antioxidant capacity;
907 Prot: Protamex® hydrolysate; Prot+Flav: Protamex® followed by Flavourzyme® hydrolysate; ≤ 1 kDa Prot: Protamex® hydrolysate;
908
909 Prot+Flav: Protamex® followed by Flavourzyme® hydrolysate; ≤ 1 kDa: 1 kDa ultrafiltrated hydrolysate.

910 **Fig. 4.** The fractionation of Cu²⁺-chelating peptides of sunflower meal protein isolate hydrolysates obtained
911 by different enzymatic treatments. The unbounding (injection phase) and bound peptides (elution phase)
912 present in Prot (A), Prot+Flav (B), ≤ 1 kDa Prot (C) and ≤ 1 kDa Prot+Flav (D). Prot: Protamex® hydrolysate;
913 Prot+Flav: Protamex® followed by Flavourzyme® hydrolysate; ≤ 1 kDa Prot: Protamex® hydrolysate; Prot+Flav: Protamex® followed
914 by Flavourzyme® hydrolysate; ≤ 1 kDa: 1 kDa ultrafiltrated hydrolysate.

915 **Fig. 5.** Results obtained from the MS/MS identification of peptides present in sunflower meal protein isolate
916 hydrolysates. (A) Number of peptides identified in F1, F2 and F3 fraction in whole and ≤ 1 kDa fraction
917 hydrolysates. (B) and (C) amino acid composition of Cu²⁺-chelating peptides identified in the F2 fractions of
918 all the hydrolysates. Prot: Protamex® hydrolysate; Prot+Flav: Protamex® followed by Flavourzyme® hydrolysate; ≤ 1 kDa Prot:
919 Protamex® hydrolysate; Prot+Flav: Protamex® followed by Flavourzyme® hydrolysate; ≤ 1 kDa: 1 kDa ultrafiltrated hydrolysate. AAA:
920 Aromatic amino acids= phenylalanine, tryptophan, tyrosine; NCAA: Negatively charged amino acids= aspartic acid, glutamic acid,
921 asparagine, glutamine; PCAA: Positively charged amino acids= arginine, histidine, lysine; HAA: Total Hydrophobic amino acids=
922 alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan, proline, methionine and cysteine; SCAA: Sulphur containing
923 amino acids= methionine and cysteine; EAA: Essential amino acids= tryptophan, lysine, histidine, phenylamine, leucine, isoleucine,
924 methionine, valine, threonine; BCAA: Branch chain amino acids= isoleucine; leucine, valine.

925 **Fig. 6.** Sequenced mass spectra of two different Cu²⁺-chelating peptides structures obtained from sunflower
926 meal protein isolate hydrolysates and processes by MaxQuant software. (A) HNDGNTELVVVVF (F2:
927 Prot+Flav) and (B) SPHWTIN (F2: all the hydrolysates), containing His residue in the first and third position
928 from amino terminus, respectively. Prot: Protamex® hydrolysate; Prot+Flav: Protamex® followed by Flavourzyme®
929 hydrolysate; ≤ 1 kDa: 1 kDa ultrafiltrated hydrolysate.

930

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934

935
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>
(A) Inhibitory mechanism of metal-induced ROS

AsH⁻: Ascorbate

DHAsH⁺: Dehydroascorbic acid

Mⁿ⁺¹: Cu²⁺/Fe³⁺

<figure><img src="image_5.png" /></figure>

Inhibition

MCP: Metal-Chelating Peptide

<figure><img src="image_6.png" /></figure>

(B) Cu²⁺-induced ROS

<figure><img src="image_7.png" /></figure>

(C) Fe³⁺-induced ROS

<figure><img src="image_8.png" /></figure>

942

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<figure><img src="image_9.png" /><figcaption>Fig. 3.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Fig. 4.</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>Fig. 5.</figcaption></figure>

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<figure><img src="image_12.png" /><figcaption>Fig. 6.</figcaption></figure>

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<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>