# High pressure and heat-induced potato protein nanocomplexes as carriers for curcumin: influence of pH and curcumin addition pathway

Paula Khati, Sylvie Marchesseau, Charles Cunault, Laetitia Picart-Palmade, Dominique Chevalier-Lucia

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## To cite this version:

Paula Khati, Sylvie Marchesseau, Charles Cunault, Laetitia Picart-Palmade, Dominique Chevalier-Lucia. High pressure and heat-induced potato protein nanocomplexes as carriers for curcumin: influence of pH and curcumin addition pathway. International Journal of Food Science and Technology, 2025, 60 (1), pp.vvae056. 10.1093/ijfood/vvae056. hal-04993811

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# High pressure and heat-induced potato protein nanocomplexes as carriers for curcumin: influence of pH and curcumin addition pathway

Paula Khati<fnref n="1" />, Sylvie Marchesseau<fnref n="1" />, Charles Cunault<fnref n="1" />,<fnref n="2" />, Laetitia Picart-Palmade<fnref n="1" />, and Dominique Chevalier-Lucia<fnref n="1" />,∗

## Abstract

This study aimed to assess the potential of high hydrostatic pressure (HHP) and heat treatment (HT) in facilitating the formation of nanoassemblies of potato proteins (PPs) and curcumin to protect this sensitive bioactive compound. Curcumin was introduced into patatin-rich protein dispersions (4% wt/wt) according to two pathways: before (pathway 1) or after (pathway 2) HHP treatment (400 or 600 MPa for 8 hr at pH 6 and pH 7) or HT (55 °C for 5 min at pH 6) to optimize curcumin-protein complexation efficiency (CE). Treatments increased PP surface hydrophobicity which facilitated curcumin binding and protein aggregation, as confirmed by protein particle size increase. The highest CE was obtained with pathway 1 at 600 MPa pH 7, while HT limited curcumin complexation. Whatever the pathway, the antioxidant activity of the processed dispersions treated at 600 MPa for 8 hr or 55 °C for 5 min was increased compared to untreated ones and preserved after 10 days of storage at 4 °C. These nanocomplexes could potentially be applied in functional foods.

**Keywords:** potato protein, high hydrostatic pressure, heat treatment, curcumin complexation, aggregation, nanocomplexes, functional food

## Graphical abstract

<figure><img src="image_4.png" /></figure>

<footnote n="1">¹IATE, Université Montpellier, INRAE, L'Institut Agro Montpellier, Montpellier, France</footnote>

<footnote n="2">²ATA, Université Montpellier, Montpellier, France</footnote>

<footnote>*Corresponding author: Université de Montpellier, Campus Triolet, CC023, Place Eugène Bataillon, 34095 Montpellier, cedex 5, France.</footnote>

<footnote>Email: dominique.chevalier-lucia@umontpellier.fr</footnote>
High hydrostatic pressure (HHP) has been put forward as a nonthermal sustainable alternative process to conventional food processes, such as thermal treatments, in the preservation of food products. However, newer applications of HHP treatment in the food industry include the physicochemical and structural modification of biological macromolecules like proteins and lipids (Picart-Palmade et al., 2019; Wang et al., 2016a). Numerous studies have reported the effects of high-pressure treatment on the structure and functional properties of various proteins like meat, bovine milk, and soy proteins (Huppertz et al., 2004; Puppo et al., 2005; Song et al., 2013). The encapsulation of labile compounds in high-pressure-treated protein nanoparticles has been investigated for some food proteins, such as whey proteins (Blayo et al., 2014; Relkin et al., 2014). Despite the potential benefits of hydrostatic high pressure, there is a notable lack of research assessing plant-based proteins as vehicles for hydrophobic active compounds (Fernandes et al., 2024). Long-duration HHP treatments have been shown to enable the formation of soluble potato protein (SPP) nanoaggregates presenting varying degrees of denaturation and structure loss (Bahri et al., 2024; Khati et al., 2024), and these changes in protein properties may be advantageous in facilitating interactions with hydrophobic bioactive molecules.

The objective of this work was to evaluate the efficiency of HP-induced PP nanoaggregates to bind and protect curcumin. Two curcumin addition pathways were tested: curcumin was added to patatin-rich protein dispersions (4% wt/wt) before (pathway 1) or after (pathway 2) high-pressure treatment (HPT) (400 and 600 MPa for 8 hr at pH 6 and pH 7) or heat treatment (HT) (55 °C for 5 min at pH 6) in order to optimize the curcumin complexation efficiency. These specific processing parameters were selected since it was previously demonstrated that they enabled the production of nano-sized PP aggregates (Khati et al., 2024). Physico-chemical properties (surface hydrophobicity, particle size, and zeta potential) of the particles were investigated as well as the curcumin complexation efficiency and loading capacity according to the complexation processing pathway. The antioxidant activity of the curcumin-protein nanocomplexes was also evaluated just after HPT or HT and after storage (4 °C for 10 days).

# Materials and methods

## Materials

The commercial patatin-rich PP isolate (PPI, Solanic 200, batch 866299) was provided by AVEBE (Veendam, The Netherlands) according to their patented process (Giuseppin et al., 2014). The powder was kept at 4 °C in an air-tight container. The powder had a protein content of 84.6±0.1 (g of protein per 100 g of powder), measured using the Kjeldhal method (KjelDigester K-436 mineralization unit, and KjelFlex K-360 distillation unit, Büchi, Switzerland) with a nitrogen conversion factor of 6.25 (Schmidt et al., 2019). All other reagents and chemicals were of analytical grade. The ammonium persulfate, curcumin powder (purity ≥65%), 1-anilinonaphthalene-8-sulfonic acid (ANS), sodium phosphate dibasic anhydrous, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and BCA protein assay kit were obtained from Sigma Aldrich (Saint-Quentin-Fallavier, France). Bis-Tris, absolute ethanol, potassium chloride, sodium chloride, and potassium phosphate monobasic were purchased from VWR International (Rosny-sous-Bois, France). Dimethyl sulfoxide (DMSO) and ammonium sulfate were from Merck (Darmstadt, Germany). Milli-Q ultrapure water (Millipore) was used to prepare all aqueous solutions and dispersions.

## Preparation of curcumin stock solution

Curcumin powder was solubilized in absolute ethanol in a tinted glass container at 27.15 mM (10 g/L) and centrifuged at 8,000 g for 15 min at 20 °C (Sorvall RC5B Plus, SS34 rotor, Marshall Scientific, New Hampshire, USA) in order to remove insoluble particles. This stock solution was stored at 4 °C for a maximum of 2 days and the concentration of curcumin was verified after centrifugation and before every use using an excitation wavelength of 427 nm and a molar extinction coefficient of 61,864 L mol⁻¹ cm⁻¹ in ethanol (Majhi et al., 2010) and found to be 11.37 ± 0.5 mM.

## Preparation of protein dispersions

Potato protein dispersions were prepared following the method outlined in Khati et al. (2024). Initially, PP isolate powder was dispersed in 20 mM Bis-Tris buffer at pH 6 or 7, using gentle magnetic stirring (350 rpm) for 1 hr at room temperature to achieve 4% (wt/wt) protein dispersions. After overnight storage at 4 °C to ensure complete protein hydration, the dispersions were returned to room temperature and gently stirred for an additional 30 min. Subsequently, centrifugation at 30,000 g for 15 min at 20 °C (Sorvall RC5B Plus, SS34 rotor, Marshall Scientific, New Hampshire, USA) was performed to eliminate insoluble particles. The protein concentration in the supernatants was determined using a BCA assay kit with bovine serum albumin (BSA) as the standard, yielding 40.74 ± 1.40 g/kg (41.20 g/L) of SPP. These SPP dispersions were refrigerated at 4 °C and used within a maximum of 2 days, with a brief 5-min stirring at 300 rpm at room temperature before each use. The resulting supernatant will be referred to as the SPP dispersion.

### High-pressure treatment

The S-IL-070-550-09W system from Stansted Fluid Power Ltd., Harlow, Essex, UK, was employed to conduct HPTs. Each treatment involved sealing 50 ml volumes of SPP dispersions (4% wt/wt pH 7 or pH 6), with or without curcumin, in double-layered polyethylene bags. These bags were then placed into the HP chamber and subjected to pressures of either 400 or 600 MPa at a temperature of 20 °C. The pressurization and depressurization rates were maintained at 250 MPa/min, and the pressure was maintained for 8 hr, a duration established from prior studies (Bahri et al., 2024; Khati et al., 2024). Postpressure release, no changes in pH were detected under any of the applied conditions. Samples were analyzed immediately after treatment.

### Heat treatment

Volumes of 4 ml of SPP dispersions (4% wt/wt pH 6), whether containing curcumin or not, were filled into 15 ml polypropylene tubes fitted with polyethylene screw caps. These tubes underwent HT in a water bath at 55 °C for 5 min, a temperature just below the denaturation point of patatin (60 °C). Following treatment, the tubes were promptly cooled to approximately 8 °C in an ice bath for 1 min. Samples were analyzed immediately after treatment.

## Complexation of treated potato proteins with curcumin

To prepare the SPP-curcumin complexes, a volume of curcumin stock solution was added dropwise to 4% (wt/wt) untreated or treated SPP dispersions at a 4:1 protein-to-curcumin molar ratio. The molar concentration of PP was estimated based on the reported molecular weight of patatin, which is 44 kDa (Racusen & Weller, 1984). This molar ratio was selected based on previous works (David & Livney, 2016) and to limit ethanol addition in the
<figure><img src="image_5.png" /><figcaption>**Figure 1.** Schematic representation of the two pathways carried out to study the complexation of curcumin to SPP nanoaggregates. Each pathway was tested for SPP dispersions (4%, wt/wt) at pH 7 and pH 6. The protein-to-curcumin molar ratio was 4:1. SPP = soluble potato protein.</figcaption></figure>

protein dispersions. The final concentration of ethanol in the SPP dispersions never exceeded 2% (vol/vol) in order to prevent any additional effect of organic solvent on protein solubility, structure, and interactions (Esmaili et al., 2011). Two sets of SPP-curcumin complexes were prepared by following two different pathways outlined in Figure 1 and were immediately analyzed or freeze-dried.

### Pathway 1: HPT or HT of SPP dispersions in the presence of curcumin

Curcumin was gradually added to pH 6 or 7 SPP dispersions to achieve a 4:1 molar ratio, followed by gentle magnetic stirring (150 rpm) in the dark at room temperature for 30 min. The dispersion was then subjected to HPT (400 or 600 MPa for 8 hr) or HT (55 °C for 5 min).

### Pathway 2: HPT or HT of SPP dispersions followed by contact with curcumin

SPP dispersions at pH 6 or 7 were subjected to HPT (400 or 600 MPa for 8 hr) or HT (55 °C for 5 min) without curcumin. After treatment, curcumin was added dropwise to the treated dispersions (4:1 ratio) and stirred magnetically (150 rpm) in the dark at room temperature for 30 min to initiate complexation.

### Particle size distribution and zeta potential

The particle size distribution of SPP dispersions, whether containing curcumin or not, was assessed at 25 °C using four-sided 1 × 1 cm polystyrene cuvettes (Sarstedt, Numbrecht, Germany) with a minimum of 12 runs per measurement employing the Zetasizer nano-ZS instrument (Malvern Instruments, Orsay, France). To minimize multiple scattering effects during measurement, samples were diluted to 0.5% or 1% (wt/wt) using 20 mM Bis-Tris buffer at appropriate pH. The dispersant viscosity and refractive index were 0.89 mPa.s and 1.33, respectively, while the refractive index

for PPs, commonly used for plant-based proteins, was 1.45 (Liu & Tang, 2014). Data analysis utilized the non-negative least squares algorithm. Results were averaged and a weighted arithmetic mean diameter was calculated from the intensity or number frequency distributions. Measurements were conducted in triplicate.

The zeta potential was measured in the same equipment in disposable measuring cells (DTS 1070 Zetasizer nano series capillary cells) with a dispersant viscosity of 0.8872 mPa.s and an equilibration time of 2 min. Samples were not diluted prior to measurement, and analysis was conducted in triplicate for every sample.

### Surface hydrophobicity of potato protein aggregates

8-Anilino-1-naphthalenesulfonic acid (ANS) is the most widely used technique for the determination of protein surface hydrophobicity. Average surface hydrophobicity was determined for untreated and HPT and HT SPP dispersions without curcumin using the ANS probe (Kato & Nakai, 1980), following the method described by Elahi and Mu (2017). SPP dispersions were diluted to concentrations ranging from 0.1 to 0.02 g/L with a 20 mM Bis-Tris buffer of the same pH. Volumes of 20 µl of an 8 mM ANS solution prepared in 10 mM sodium phosphate buffer (pH 7) were added to 4 ml of the diluted SPP dispersion and vortexed. The tubes were kept in the dark at room temperature for 10 min before analysis. Fluorescence intensity (FI) measurements were made in triplicate using a spectrofluorimeter (Cary Eclipse, Varian) with excitation and emission wavelengths of 390 and 473 nm, respectively. The fluorescence emission spectra of protein samples without ANS (referred to as protein blanks) and those of buffer in the presence of ANS (referred to as the buffer blanks) were also recorded. The corrected FI (net FI) for each sample was determined by subtracting the FI of the protein blank and the FI of the buffer blank from the sample fluorescence. Linear regression analysis
was employed to calculate the initial slope of the plot of net FI against protein concentration, serving as an indicator of protein surface hydrophobicity.

## Ammonium sulfate precipitation

To quantify fully or partially denatured proteins in SPP dispersions (with or without curcumin) and assess aggregation efficiency, the ammonium sulfate precipitation method described by Khati et al. (2024) was used. Briefly, 1 ml of untreated or treated protein dispersion (with or without curcumin) was added to 9 ml of 20 mM Bis-Tris buffer with 0.5 M ammonium sulfate at pH 6 or 7 in 12 ml polypropylene copolymer (PPCO) tubes (3420-1613, Nalgene, Rochester, USA). After 2 hr in the dark at 20 °C, the tubes were manually agitated and centrifuged at 12,061 g for 20 min at 20 °C (Sorvall RC-5B-plus centrifuge, SS34 rotor). Supernatants were collected and their absorbance spectra were recorded from 200 to 600 nm using a UV spectrophotometer (UV 1900, Shimadzu UV-Vis, Japan). Protein quantification was based on absorbance reading at 280 nm. Results were expressed as the ratio of precipitated proteins in treated SPP dispersions to total protein in untreated SPP dispersions at the corresponding pH. Analyses were conducted in triplicate. For samples containing curcumin, it has been checked that the presence of curcumin did not alter the absorbance value at 280 nm, and the pellets retrieved after centrifugation were freeze-dried and stored for further analysis.

## Complexation efficiency and load capacity

Total soluble curcumin in protein dispersions was quantified by freeze-drying 2 ml aliquots of the treated or untreated SPP dispersions, whether curcumin was added before or after treatment. An equivalent volume of DMSO was then added to extract the bioactive compound, as curcumin is highly soluble in DMSO and stable against auto-oxidation in this solvent (Tsaplev et al., 2020). The mixture was agitated for 40 min at room temperature, a contact time optimized through preliminary tests, and then centrifuged at 4,500 g for 15 min. The supernatant was collected, and the curcumin concentration was determined by measuring absorbance at 427 nm using a molar extinction coefficient of 49,000 L mol⁻¹ cm⁻¹ (Khumsupan et al., 2011). Each sample was analyzed in triplicate.

The amount of curcumin bound to fully or partially denatured/aggregated proteins (BC) was quantified on the freeze-dried pellets after sulfate ammonium precipitation, using the same method. Triplicate samples were used for each analysis. Control samples without curcumin were subjected to the same analyses to correct the spectra of the samples containing curcumin accordingly.

The curcumin complexation efficiency (CE %) was calculated as follows (Equation 1):

$$CE (\%) = \frac{\text{quantity of complexed curcumin (BC)}}{\text{quantity of total soluble curcumin (TC)}} \quad (1)$$

The curcumin load amount (LA) was calculated as follows (Equation 2):

$$LA \left( \frac{\mu g \text{ curcumin}}{\text{mg protein}} \right) = \frac{\text{quantity of complexed curcumin (BC)}}{\text{quantity of precipitated proteins}} \quad (2)$$

## Antioxidant activity

The antioxidant activity of blank or curcumin-loaded SPP dispersions was measured using the Trolox equivalent antioxidant

capacity (TEAC) assay (Re et al., 1999). This assay is based on the reduction of the blue/green ABTS•+ radical cation by antioxidants in the sample, resulting in a decrease in colour intensity. This change was measured spectrophotometrically at 734 nm (UV 1900, Shimadzu UV-Vis, Japan). Trolox, a vitamin E analogue, was used as the antioxidant standard. A 2.5 mM Trolox stock solution was prepared in 5 mM phosphate-buffered saline (PBS) at pH 7 and stored at -18 °C, with aliquots thawed for daily use. The ABTS•+ radical cation solution was prepared by reacting 7 mM ABTS in ultrapure water with 2.45 mM potassium persulfate for 12-16 hr in the dark at room temperature. This ABTS•+ solution was then diluted with 10 mM PBS at pH 7 to an absorbance of 0.700 (±0.02) at 734 nm and maintained at 30 °C. For the assay, 1 ml of the diluted ABTS•+ solution was mixed with 10 µl of the SPP dispersions (with or without curcumin) diluted 1:10 in 10 mM PBS or Trolox standard solution (0-500 µM) and incubated at 30 °C. Absorbance at 734 nm was recorded after 6 min. Each experiment included appropriate solvent and sample blanks, and all measurements were performed in triplicate. The inhibition of ABTS•+ expressed in percentage was calculated using (Equation 3):

$$\left( \frac{A_b - A_s}{A_b} \right) \quad (3)$$

where A_b is the absorbance of ABTS•+ without any scavenger (blank), and A_s is the absorbance with the SPP sample (with or without curcumin). These inhibition percentages were plotted against concentration to generate a Trolox standard curve. The slope of this curve (r² > 0.9855) was used to convert the inhibition percentages to TEAC.

## Statistical analysis

All values are expressed as mean ± SD of at least triplicate experiments. XLSTAT software (2021.2.1, New York, USA) was used to conduct one-way analysis of variance and means were compared for significant difference by Tukey's post hoc test with a confidence interval of 95%.

## Results and discussion

### Physico-chemical characterization of SPP aggregates without curcumin

Mean diameters of SPP particles, calculated from size distributions in light intensity, are summarized in Table 1 for pH 6 and pH 7 SPP dispersions (4%, wt/wt) HPT at 400 and 600 MPa for 8 hr or HT (55 °C for 5 min). A significant increase in particle size was observed in HP-treated SPP dispersions (pH 6 and pH 7, without curcumin) compared to untreated dispersions, linked to pressure-induced denaturation and consequent aggregation of SPP as previously described by Khati et al. (2024). The selected HPT has resulted in the generation of SPP nanoaggregates (<300 MPa). At the same time, heat-treated SPP dispersion was characterized by aggregates with similar mean diameters around 150 nm (Table 1).

The surface hydrophobicity and overall surface charge (zeta potential) of various nanoaggregates were simultaneously investigated to determine their potential to bind curcumin, a hydrophobic bioactive molecule. As illustrated in Figure 2A, the untreated SPP dispersion at pH 6 demonstrated significantly higher (p < .05) surface hydrophobicity compared to the dispersion at pH 7. This finding suggests that, as the protein's charge and electrostatic repulsion decrease near the isoelectric point of SPP (pH 5), there is a greater exposure of hydrophobic residues on the SPP surface.
<table><caption>**Table 1.** Effect of treatment conditions and curcumin addition pathway (pathway 1: curcumin addition before treatment; pathway 2: curcumin addition after treatment) on the weighted arithmetic mean particle size calculated from size distributions in intensity of pH 6 and pH 7 soluble potato protein dispersions.</caption><thead><tr><th rowspan="2">pH</th><th rowspan="2">Treatment</th><th rowspan="2">Without curcumin</th><th colspan="2">With curcumin</th></tr><tr><th>Pathway 1</th><th>Pathway 2</th></tr></thead><tbody><tr><td rowspan="3">7</td><td>Untreated</td><td>48.9 ± 0.8<sup>a</sup></td><td>49.0 ± 0.5<sup>a</sup></td><td></td></tr><tr><td>400 MPa for 8 hr</td><td>150.0 ± 0.9<sup>b</sup></td><td>151.1 ± 9.1<sup>b</sup></td><td>151.3 ± 17.8<sup>b</sup></td></tr><tr><td>600 MPa for 8 hr</td><td>173.2 ± 16.1<sup>b</sup></td><td>52.5 ± 1.6<sup>a</sup></td><td>208.2 ± 9.0<sup>c</sup></td></tr><tr><td rowspan="4">6</td><td>Untreated</td><td>67.3 ± 1.1<sup>a</sup></td><td>55.7 ± 3.5<sup>a</sup></td><td></td></tr><tr><td>400 MPa for 8 hr</td><td>290.2 ± 24.3<sup>d</sup></td><td>387.2 ± 16.7<sup>f</sup></td><td>126.3 ± 2.4<sup>b</sup></td></tr><tr><td>600 MPa for 8 hr</td><td>148.3 ± 11.2<sup>b,c</sup></td><td>347.3 ± 2.8<sup>e</sup></td><td>167.0 ± 1.9<sup>c</sup></td></tr><tr><td>55 °C for 5 min</td><td>152.2 ± 1.7<sup>b,c</sup></td><td>299.5 ± 12.5<sup>d</sup></td><td>168.7 ± 0.7<sup>c</sup></td></tr></tbody></table>

<figure><img src="image_6.png" /><figcaption>**Figure 2.** Surface hydrophobicity (A) and overall protein charge (ζ-potential) (B) of SPP dispersions (4%, wt/wt) at pH 7 (white) and pH 6 (grey), untreated, HP-treated, and heat-treated. Each data point represents the mean ± SD of triplicates. Different letters indicate significant differences between samples (p < .05).</figcaption></figure>

At a given pH, all processed SPP dispersions exhibited significantly higher (p < .05) surface hydrophobicity compared to their respective untreated dispersion. Additionally, a gradual but significant (p < .05) increase in surface hydrophobicity was observed with increasing pressure levels. For the same patatin-rich PP isolate (4%, pH 7) pressurized at 400 MPa for up to 48 hr, a previous study found that the highest surface hydrophobicity occurred after 8 hr of pressurization (Bahri et al., 2024). The increase in SPP surface hydrophobicity after HPT was attributed to the impact of pressurization on the conformation and flexibility of SPP proteins. Pressurization induced partial unfolding of the compact folded structure of untreated SPP by disrupting weak hydrogen bonds and van der Waals forces, leading to a more open and flexible conformation (Bahri et al., 2024; Balny, 2004; Elahi & Mu, 2017). The extent of exposure of hydrophobic amino acid residues or surface hydrophobic cavities of protein particles after pressurization depended on pressure level rather than pH dispersion. For instance, surface hydrophobicity increased by 57% for pH 7 and by 53% for pH 6 SPP dispersions at 600 MPa. Similar increases in surface hydrophobicity after HPT up to 400 MPa were observed for lab-extracted patatin and other plant-based proteins, such as soybean and walnut (Elahi & Mu, 2017; Qin et al., 2013; Wang et al., 2008). However, in these studies, higher pressure levels (>400 MPa) resulted in decreased surface hydrophobicity, as the exposed aromatic amino acid groups tended to associate and aggregate into more stable structures after depressurization. In the present study, even at 600 MPa, SPP protein unfolding during pressurization and refolding after depressurization likely induced conformational changes, causing a more extensive relocation of

originally buried hydrophobic groups to the surface of protein particles. This behaviour was similarly observed in rapeseed protein by He et al. (2014). The surface hydrophobicity of high-pressure-induced nanoaggregates (600 MPa for 8 hr pH 7 and 400 MPa for 8 hr pH 6) is similar to that of the heat-induced aggregates obtained at 55 °C for 5 min suggesting similar surface interaction properties.

In addition to surface hydrophobicity, the overall protein charge (ζ-potential) reflects its surface properties and its ability to interact with other molecules (other proteins or hydrophobic compounds). Figure 2B shows the ζ-potential of SPP dispersions at pH 6 and pH 7 before and after HPT or HT. Both untreated SPP dispersions were negatively charged, as the pH levels were above the isoelectric point of SPP (pH 5). At pH 7, the untreated SPP dispersion exhibited a significantly higher negative ζ-potential (−15.00 ± 0.92 mV) compared to pH 6 (−11.8 ± 0.06 mV) (p < .05). Closer to the isoelectric point, the reduced electrostatic repulsion favours protein-protein interactions, leading to the higher initial particle size observed for untreated SPP at pH 6 compared to pH 7 (Table 1). HPT (400 or 600 MPa for 8 h) and HT (55 °C for 5 min) induced a significant (p < .05) increase in the negative net charge of SPP dispersions at both pH levels. This trend was already reported for different plant proteins from soy, quinoa, and sweet potato, processed by HPT or thermal treatments (Song et al., 2013; Zhao et al., 2018, 2023). The present results (Figure 2B) confirmed the effect of HPT and HT on the partial unfolding of SPP, exposing more internally buried charged amino acids on the protein surface, leading to enhanced ζ-potential and protein-protein electrostatic interactions (Tang & Ma, 2009). Within each

<footnote>a–f Different letters in the same column indicate significant differences (P < .05).</footnote>
pH group, the processing-induced changes in ζ-potential were similar across different treatment conditions, resulting in nearly equal increases in negative charge. Notably, HP-treated samples at pH 7 exhibited the highest ζ-potential in absolute value, indicating the greatest stability.

## Characterization of curcumin-SPP complexes: influence of the curcumin addition pathway

As it was confirmed that the selected HPT and HT generated SPP nanoaggregates, the curcumin binding to these nanoparticles was investigated according to two curcumin addition pathways: the addition of curcumin to SPP dispersions before HPT and HT (pathway 1) and the addition of curcumin to SPP dispersions post-HPT and -HT (pathway 2) (Figure 1).

### Particle size distribution and quantification of denatured proteins

The weighted arithmetic mean diameter of curcumin-loaded untreated SPP at both pH 6 and pH 7 did not significantly differ ($p < .05$) from that of the untreated dispersions without curcumin (Table 1). The presence of free curcumin, as well as curcumin potentially bound to SPP proteins via hydrophobic interactions in untreated SPP dispersions (pH 6 and pH 7), did not significantly alter the mean particle diameters or the size distribution curves (data not shown). The addition of low-molecular-weight hydrophobic compounds like curcumin to native protein dispersions typically does not result in significant changes in protein size (Blayo et al., 2014; David et al., 2015). Moreover, since curcumin alone was not detected by DLS and no additional peaks were observed in the particle size distributions, the presence of free curcumin micelles can be ruled out. The fraction of partially or fully denatured proteins in untreated SPP dispersions, with or without curcumin, at pH 6 and pH 7, was lower than 10%, indicating a low impact of PP isolate processing on protein denaturation.

When curcumin was added to SPP dispersion at pH 6 before applying HPT or HT (pathway 1), the mean diameter of protein particles formed in the presence of curcumin was significantly larger ($p < .05$) than those formed without curcumin under the same processing conditions (Table 1). This increase in particle size was associated with a significantly greater quantity ($p < .05$) of fully or partially denatured proteins precipitated by 0.5 M ammonium sulfate (Table 2). At pH 6, close to the isoelectric point of SPP, protein-protein interactions were strengthened, likely with a synergistic effect from curcumin due to its hydrophobicity, promoting the formation of larger aggregates and a higher aggregation yield. At pH 7, using the same curcumin addition pathway, significantly smaller ($p < .05$) protein particles combined with a higher fraction of fully or partially denatured proteins were obtained at 600 MPa compared to those without curcumin. At this pH and pressure level, SPP proteins underwent pressure-induced unfolding, but, during depressurization, curcumin appeared to induce the formation of a large quantity of more compact aggregates.

When curcumin was added to SPP dispersions post-HPT and -HT (pathway 2) at pH 7, the curcumin-loaded SPP dispersions treated at 600 MPa showed a 20% increase in particle mean size compared to HP-treated SPP dispersions without curcumin (Table 1). This suggests successful complexation of curcumin onto the hydrophobic surfaces of these HP-induced SPP aggregates, potentially along with self-aggregation phenomena. Similar results were observed for curcumin-loaded yeast protein complexes (Sun et al., 2023). At pH 6, the particle diameter of SPP

aggregates obtained at 600 MPa or by HT was not significantly altered ($p < .05$) by curcumin addition. For pathway 2, the largest structures without curcumin were observed at 400 MPa and pH 6 in accordance with Khati et al. (2024). The presence of curcumin destabilized the protein assembly structure, likely promoting interactions between curcumin and smaller protein assemblies (126 nm). Curcumin can intercalate into complex aggregates, as seen with amyloids (Radbakhsh et al., 2021).

### Curcumin-SPP aggregate complexation efficiency and LA

To investigate the influence of processing as well as the curcumin addition pathway on the performance of the complexation process, the CE and the LA of curcumin in SPP complexes were quantified (Figure 3).

Regardless of the treatment conditions applied, the SPP aggregates induced by HT were significantly ($p < .05$) less effective at complexing curcumin (CE and LA) compared to the various protein aggregates obtained by HPT (Figure 3). Additionally, for these HT aggregates, the pathway of curcumin addition had no significant impact on the CE or LA (Figure 3A and B). Despite HT-SPP aggregates having sizes and surface hydrophobicity comparable to HPT-SPP aggregates (Figure 2), their poor complexation performance could be attributed to the rapid aggregation kinetics. This rapid aggregation likely did not allow for strong modification or unfolding of the protein before aggregation (Khati et al., 2024).

For complexes induced by HPT, significant variations in CE were observed among different treatment conditions and curcumin addition pathways (Figure 3A). Pressure level, pH, and curcumin addition pathway all had a significant impact on CE. At both studied pH levels and for both pathways, CE was significantly higher for aggregates obtained at 600 MPa compared to 400 MPa, reaching approximately 68% at pH 7 and 52% at pH 6 for the 600 MPa-treated SPP. Additionally, for a given pressure and pH condition, CE was significantly higher when curcumin was added before the HP-SPP processing (pathway 1) compared to when curcumin was added after SPP processing (pathway 2). Furthermore, the effect of pH on CE depended on the pressure level applied. There was no significant difference in CE at 400 MPa (for a given pathway), unlike the significant differences observed at 600 MPa. At this higher pressure level, CE was significantly higher at pH 7 compared to pH 6 for both pathways (Figure 3A). It has been reported that the encapsulation efficiency of curcumin in protein nanocomplexes is significantly influenced by pH. At pH 7, Taghavi Kevij et al. (2019) found that only 21.7% of curcumin was complexed with whey protein isolate, while Mohammadian et al. (2019) reported an even lower (10%) encapsulation efficiency at pH 3.2. These differences likely stem from varying protein aggregation states across different pH levels (Tang, 2020). Taghavi Kevij et al. (2019) also noted that whey protein isolate exhibited improved encapsulation efficiency at alkaline pH, attributed to structural protein unfolding, which enhances curcumin complexation.

In addition to CE, considering the LA of curcumin is crucial as it accounts for the quantity of SSP aggregates produced (Table 2). LA reflects the carrier system's capacity to incorporate and retain a bioactive compound. Figure 3B presents the LA of different samples for both pathways. The respective effects of the studied process parameters (pH, pressure level, pathways) on curcumin LA were less pronounced than those on CE and not always statistically significant. The highest curcumin loading capacity was observed in the pH 7 sample HP treated at 600 MPa with post-treatment curcumin addition (pathway 2), reaching $1.45 \times 10^{-3}$ µg
<table><caption>**Table 2.** Effect of treatment conditions, soluble potato protein (SPP) dispersions pH, and curcumin addition pathway (pathway 1: curcumin addition before high-pressure treatment (HPT) or heat treatment (HT); pathway 2: curcumin addition after HPT or HT) on the per cent of SPP precipitated post-treatment by 0.5 M ammonium sulphate.</caption><thead><tr><th rowspan="2">pH</th><th rowspan="2">Sample</th><th rowspan="2">Without curcumin</th><th colspan="2">With curcumin</th></tr><tr><th>Pathway 1</th><th>Pathway 2</th></tr></thead><tbody><tr><td rowspan="3">7</td><td>Untreated</td><td>5.3 ± 0.5<sup>a</sup></td><td>9.0 ± 0.3<sup>b</sup></td><td></td></tr><tr><td>400 MPa for 8 hr</td><td>53.7 ± 0.7<sup>d</sup></td><td>39.6 ± 0.8<sup>c</sup></td><td>40.5 ± 0.9<sup>c</sup></td></tr><tr><td>600 MPa for 8 hr</td><td>68.2 ± 0.2<sup>e</sup></td><td>75.0 ± 0.2<sup>g</sup></td><td>72.9 ± 0.3<sup>f</sup></td></tr><tr><td rowspan="4">6</td><td>Untreated</td><td>3.5 ± 0.6<sup>a</sup></td><td>6.7 ± 2.7<sup>a</sup></td><td></td></tr><tr><td>400 MPa for 8 hr</td><td>51.3 ± 0.4<sup>c</sup></td><td>60.9 ± 0.5<sup>d</sup></td><td>43.9 ± 0.5<sup>b</sup></td></tr><tr><td>600 MPa for 8 hr</td><td>70.7 ± 0.3<sup>e</sup></td><td>76.8 ± 0.1<sup>f</sup></td><td>74.3 ± 0.3<sup>f</sup></td></tr><tr><td>55 °C for 5 min</td><td>42.8 ± 0.3<sup>b</sup></td><td>48.5 ± 3.4<sup>c</sup></td><td>41.1 ± 0.7<sup>b</sup></td></tr></tbody></table>

a-g Different letters in the same column indicate significant differences.

<figure><img src="image_7.png" /><figcaption>**Figure 3.** Influence of high-pressure treatment and heat treatment on complexation efficiency (%) (A) and load amount (µg curcumin/mg SPP) (B) of curcumin in SPP aggregates for pathway 1 (white) and pathway 2 (grey). Samples that do not share a letter within each measured parameter are significantly different (p < .05).</figcaption></figure>

curcumin/mg SPP, which was approximately three times higher than the LA obtained with pathway 1 under the same conditions. However, for other process parameters tested, the positive impact of pathway 2 on LA compared to pathway 1 was less pronounced (at pH 6, 400 or 600 MPa) or not significantly different (pH 7, 400 MPa). Moreover, it can be seen in Figure 3B that when the curcumin is added before HPT (pathway 1), the values of LA of curcumin were quite similar regardless of the other processing conditions. Similarly, the effect of pH on LA of curcumin did not follow a marked trend and depended on the other treatment conditions (Figure 3B). Therefore, considering both curcumin CE and LA, SPP dispersions at pH 6 or pH 7 treated at the highest pressure (600 MPa) were the most effective curcumin nanocarriers, followed by those treated at 400 MPa. The enhanced loading capacity could be linked to the increased accessible hydrophobic sites on the protein surface, facilitating curcumin binding via hydrophobic interactions, as shown by the rise in surface hydrophobicity (Figure 2A) (Sponton et al., 2018). Additionally, different association phenomena after pressure release may lead to denser and more compact aggregate structures or more loosely packed structures of re-assembled SPP that allow better retention of the bioactive compound (Chevalier-Lucia et al., 2011; Liu et al., 2019). The different results obtained for curcumin LA could be attributed to variations in the denaturation/aggregation state of proteins after HPT at different pH levels (Tang, 2020). This suggestion was also supported by the results obtained for HT-SPP aggregates,

which have demonstrated a low capacity to complex curcumin despite a high surface hydrophobicity (Figure 2A). Insufficient structural unfolding (partial unfolding) may have hindered optimal curcumin binding through hydrophobic interactions, and the fast-rate denaturation and aggregation (5 min treatment) may have limited the interactions between the hydrophobic groups of unfolded SPP and curcumin molecules. By performing complexation under conditions that cause proteins to unfold or denature, curcumin can also be incorporated into the interior of protein-assembled nanoparticles (Liu et al., 2019). Further investigation into the protein structures is therefore needed to confirm their structural morphology at different pH levels and under varying treatment parameters.

## Antioxidant activity

The antioxidant activity (AA) of the curcumin-loaded SPP dispersions (untreated or treated by HPT or HT) was determined. Measurements were performed right after processing and following a 10-day storage period at 4 °C in the absence of light (Figure 4). The antioxidant activity in the curcumin-loaded SPP dispersions was significantly higher compared to SPP dispersion without curcumin (data not shown). It is important to note that the antioxidant activity presented in Figure 4 represents the combined AA of the curcumin and of the SPP. Indeed, the phenolic hydroxyl group in curcumin effectively scavenges radicals by donating its hydrogen atom (Feng & Liu, 2009). The antioxidant activity of
<figure><img src="image_8.png" /><figcaption>**Figure 4.** Trolox equivalent antioxidant activity of untreated and treated curcumin-loaded SPP dispersions at pH 7 (upper graph) or pH 6 (lower graph), according to pathway 1 and pathway 2 at day 0 (white) and day 10 of storage at 4 °C away from light (grey). Different letters above the bars in each graph denote a significant (p < .05) mean difference.</figcaption></figure>

proteins primarily stem from amino acids, such as tryptophan, tyrosine, phenylalanine, and histidine, which contain hydrogen donor groups. Additionally, amino acids, such as methionine, cysteine, and proline, are recognized as endogenous protein antioxidants (Levine et al., 1996; Zhang et al., 2008). Moreover, the presence of possible traces of phenolic compounds in the SPP isolate could contribute to the overall AA measured.

The AA of the untreated curcumin-loaded SPP dispersion at pH 7 was slightly but significantly (p < .05) higher than at pH 6 at day 0 and day 10 of storage, indicating greater stability of curcumin at neutral pH. Martínez-Guerra et al. (2019) found that curcumin is more stable at basic pH values in aqueous media than under acidic conditions. Curcumin-loaded SPP dispersions treated with HT or HPT exhibited significantly higher AA than untreated samples across most pH and pathways tested (except for pH 7, 400 MPa, 8 hr). The low curcumin complexation and load amount observed after HT suggest that the increased AA seen could be attributed to intrinsic changes in SPP rather than curcumin preservation.

In contrast, HPT consistently showed the highest antioxidant activities at day 0 at 600 MPa compared to 400 MPa, consistent

with curcumin CE and LA displayed by SPP aggregates at this pressure level (Figure 3). Furthermore, across different curcumin addition pathways, AA levels were consistently higher with pathway 2, where curcumin was added after SPP processing, suggesting better preservation compared to pathway 1. During a 10-day storage period, the evolution of AA did not show a clear trend, with stability, increase, or decrease depending on process parameters compared to D0 samples. However, AA after storage never significantly dropped below the initial value of untreated curcumin-loaded SPP, indicating effective protection of curcumin's antioxidant activity by SPP aggregate complexes. Similar protective effects of curcumin through protein complexation have been observed with casein, soy, and corn protein hydrolysate (Esmaili et al., 2011; Tapal & Tiku, 2012; Wang et al., 2016b).

## Conclusion

In this study, the complexation of processed 4% (wt/wt) SPP dispersion at pH 6 or 7 with curcumin at a 4:1 molar ratio (protein: curcumin) was investigated, with curcumin added either before or after HPT or HT. The processing-induced structural
modifications in SPP facilitated curcumin binding to SPP. Curcumin was found to interact with HP-treated SPP through a simple complexation process, binding to the exposed hydrophobic regions of unfolded or denatured SPP to form nanocomplexes of different diameters. Complexation efficiency depended on the treatment parameters but was maximal for aggregates formed by HP treatment at 600 MPa from a 4% SPP dispersion at pH 7. On the other hand, SPP aggregates induced by thermal treatment are significantly less efficient at complexing curcumin (CE and LA) than those obtained by HPT. The antioxidant activity of SPP dispersions treated with curcumin increases after complexation and remains preserved during storage (10 days at 4 °C). This shows that complexation during processing could be an effective way of forming nanocomplexes to protect hydrophobic bioactive compounds like curcumin and enhance the value of foods. Surface hydrophobicity alone does not fully account for complexation efficiency; rather, the interaction of various process parameters, particularly the dynamics of pressure application and release (involving protein unfolding and refolding), plays a significant role. By adjusting the physicochemical properties of food proteins, such as size, charge, and structure, via processing, novel PP nanocarriers can be generated for the transport and protection of bioactive compounds, such as curcumin. These promising results open up new applications for the valorization of PPI in the development of natural nano-assemblies for the protection and delivery of hydrophobic bioactive molecules in functional foods or nutraceuticals. This work aligns with a sustainable approach, promoting the use of HHP processing, a recognized sustainable technology. It also enhances the value of PPs, which are conventionally extracted from PFJ and are often considered low-value waste. Further research on the fate of SPP-curcumin complexes when incorporated into food matrices and/or during stabilization treatments remains crucial. These studies should focus on assessing the impact of the matrix on complexation efficiency, curcumin protection, and stability under processing and storage conditions.

## Practical applications

Potato fruit juice, a waste product of the starch industry, contains up to 2% of crude high biological value complete proteins that are essentially wasted nutrients, and though their extraction and isolation have been exhaustively studied, their properties and applications in the food industry remain insufficiently documented. The potential to use PPs as nanocarriers for the transport and delivery of hydrophobic bioactive molecules and formulate functional foods with added health benefits is till now scarcely explored. High-pressure processing is employed as an alternative to thermal aggregation, to develop submicron protein structures to act as curcumin carriers. This study provides fundamental information on the influence of curcumin addition pathways and initial SPP dispersion pH on both the properties of the SPP-curcumin nanoassemblies formed as well as their ability to retain curcumin.

## Data availability

Data are available on request from the authors.

## Author contributions

Paula Khati (Data curation, Formal analysis, Investigation, Methodology, Visualization [equal], Writing—original draft [lead]), Charles Cunault (Methodology, Resources [equal], Writing—original draft [supporting]), Sylvie Marchesseau (Conceptualization, Writing—review & editing [equal]), Laetitia Palmade

(Conceptualization, Formal analysis, Supervision, Validation, Writing—review & editing [equal], Project administration [lead]), and Dominique Chevalier-Lucia (Conceptualization, Formal analysis, Supervision [equal], Validation [equal], Writing—review & editing [equal], Project administration [lead]).

## Funding

This research was funded by a doctoral grant from the French Ministry of Higher Education and Research.

## Conflicts of interest

None declared.

## Acknowledgements

The authors extend their gratitude to AVEBE (Vendaam, The Netherlands) for generously supplying the potato protein isolate powder. They also thank Pascale Savoyant and Nicolas Guier from ATA Montpellier University for their invaluable assistance with the high-pressure treatment of the samples.

## References

* Alting, A. C., Pouvreau, L., Giuseppin, M. L. F., & van Nieuwenhuijzen, N. H. (2011). 12-potato proteins. In G. O. Phillips & P. A. Williams (Eds.), *Handbook of food proteins* (pp. 316–334). Woodhead Publishing. https://doi.org/10.1533/9780857093639.316

* Anand, P., Kunnumakkara, A. B., Newman, R. A., & Aggarwal, B. B. (2007). Bioavailability of curcumin: Problems and promises. *Molecular Pharmaceutics*, *4*, 807–818. https://doi.org/10.1021/mp700113r

* Bahri, A., Charpentier, C., Khati, P., Parc, R. L., Chevalier-Lucia, D., & Picart-Palmade, L. (2024). Long-time high-pressure processing of a patatin-rich potato proteins isolate: Impact on aggregation and surface properties. *International Journal of Food Science & Technology*, *59*, 4680–4692. https://doi.org/10.1111/ijfs.17192

* Balny, C. (2004). Pressure effects on weak interactions in biological systems. *Journal of Physics: Condensed Matter*, *16*, S1245. https://doi.org/10.1088/0953-8984/16/14/036

* Banwo, K., Olojede, A. O., Adesulu-Dahunsi, A. T., Verma, D. K., Thakur, M., Tripathy, S., Singh, S., Patel, A. R., Gupta, A. K., Aguilar, C. N., & Utama, G. L. (2021). Functional importance of bioactive compounds of foods with potential health benefits: A review on recent trends. *Food Bioscience*, *43*, 101320. https://doi.org/10.1016/j.fbio.2021.101320

* Benzaria, A., Maresca, M., Taieb, N., & Dumay, E. (2013). Interaction of curcumin with phosphocasein micelles processed or not by dynamic high-pressure. *Food Chemistry*, *138*, 2327–2337. https://doi.org/10.1016/j.foodchem.2012.12.017

* Blayo, C., Puentes-Rivas, D., Palmade, L., Chevalier-Lucia, D., Lange, R., & Dumay, E. (2014). Binding of retinyl acetate to whey proteins or phosphocasein micelles: Impact of pressure-processing on protein structural changes and ligand embedding. *Food Research International*, *66*, 167–179. https://doi.org/10.1016/j.foodres.2014.09.005

* Chang, C., Meikle, T. G., Su, Y., Wang, X., Dekiwadia, C., Drummond, C. J., Conn, C. E., & Yang, Y. (2019). Encapsulation in egg white protein nanoparticles protects anti-oxidant activity of curcumin. *Food Chemistry*, *280*, 65–72. https://doi.org/10.1016/j.foodchem.2018.11.124

* Chen, F.-P., Li, B.-S., & Tang, C.-H. (2015a). Nanocomplexation between curcumin and soy protein isolate: Influence on curcumin stability/bioaccessibility and in vitro protein digestibility.
Journal of Agricultural and Food Chemistry, 63, 3559-3569. https://doi.org/10.1021/acs.jafc.5b00448

Chen, F.-P., Li, B.-S., & Tang, C.-H. (2015b). Nanocomplexation of soy protein isolate with curcumin: Influence of ultrasonic treatment. *Food Research International*, 75, 157-165. https://doi.org/10.1016/j.foodres.2015.06.009

Chen, F.-P., Liu, L.-L., & Tang, C.-H. (2020). Spray-drying microencapsulation of curcumin nanocomplexes with soy protein isolate: Encapsulation, water dispersion, bioaccessibility and bioactivities of curcumin. *Food Hydrocolloids*, 105, 105821. https://doi.org/10.1016/j.foodhyd.2020.105821

Chen, L., Remondetto, G., & Subirade, M. (2006). Food protein-based materials as nutraceutical delivery systems. *Trends in Food Science & Technology*, 17, 272-283. https://doi.org/10.1016/j.tifs.2005.12.011

Chen, Z., Xia, Y., Liao, S., Huang, Y., Li, Y., He, Y., Tong, Z., & Li, B. (2014). Thermal degradation kinetics study of curcumin with nonlinear methods. *Food Chemistry*, 155, 81-86. https://doi.org/10.1016/j.foodchem.2014.01.034

Chevalier-Lucia, D., Blayo, C., Gràcia-Julià, A., Picart-Palmade, L., & Dumay, E. (2011). Processing of phosphocasein dispersions by dynamic high pressure: Effects on the dispersion physicochemical characteristics and the binding of α-tocopherol acetate to casein micelles. *Innovative Food Science & Emerging Technologies*, 12, 416-425. https://doi.org/10.1016/j.ifset.2011.07.007

Coelho, M. S., Fernandes, S. S., & Salas-Mellado, M. D. L. M. (2019). Chapter 9—association between diet, health, and the presence of bioactive compounds in foods. In M. R. S. Campos (Ed.), *Bioactive compounds* (pp. 159–183). Woodhead Publishing. https://doi.org/10.1016/B978-0-12-814774-0.00009-8

David, S., & Livney, Y. D. (2016). Potato protein based nanovehicles for health promoting hydrophobic bioactives in clear beverages. *Food Hydrocolloids*, 57, 229-235. https://doi.org/10.1016/j.foodhyd.2016.01.027

David, S., Zagury, Y., & Livney, Y. D. (2015). Soy β-Conglycinin—curcumin Nanocomplexes for enrichment of clear beverages. *Food Biophysics*, 10, 195-206. https://doi.org/10.1007/s11483-014-9386-8

Dominguez Díaz, L., Fernández-Ruiz, V., & Cámara, M. (2020). The frontier between nutrition and pharma: The international regulatory framework of functional foods, food supplements and nutraceuticals. *Critical Reviews in Food Science and Nutrition*, 60, 1738-1746. https://doi.org/10.1080/10408398.2019.1592107

Edelman, R., Engelberg, S., Fahoum, L., Meyron-Holtz, E. G., & Livney, Y. D. (2019). Potato protein- based carriers for enhancing bioavailability of astaxanthin. *Food Hydrocolloids*, 96, 72-80. https://doi.org/10.1016/j.foodhyd.2019.04.058

Elahi, R., & Mu, T.-H. (2017). High hydrostatic pressure (HHP)-induced structural modification of Patatin and its antioxidant activities. *Molecules*, 22, 438. https://doi.org/10.3390/molecules22030438

Esmaili, M., Ghaffari, S. M., Moosavi-Movahedi, Z., Atri, M. S., Sharifizadeh, A., Farhadi, M., Yousefi, R., Chobert, J.-M., Haertlé, T., & Moosavi-Movahedi, A. A. (2011). Beta casein-micelle as a nano vehicle for solubility enhancement of curcumin; food industry application. *LWT - Food Science and Technology*, 44, 2166-2172. https://doi.org/10.1016/j.lwt.2011.05.023

Ezhilarasi, P. N., Karthik, P., Chhanwal, N., & Anandharamakrishnan, C. (2013). Nanoencapsulation techniques for food bioactive components: A review. *Food and Bioprocess Technology*, 6, 628-647. https://doi.org/10.1007/s11947-012-0944-0

Fathi, M., Donsi, F., & McClements, D. J. (2018). Protein-based delivery systems for the nanoencapsulation of food ingredients. *Comprehensive Reviews in Food Science and Food Safety*, 17, 920-936. https://doi.org/10.1111/1541-4337.12360

Feng, J.-Y., & Liu, Z.-Q. (2009). Phenolic and enolic hydroxyl groups in curcumin: Which plays the major role in scavenging radicals? *Journal of Agricultural and Food Chemistry*, 57, 11041-11046. https://doi.org/10.1021/jf902244g

Fernandes, J. G., Rodrigues, R. C., Pereira, L., Stringheta, P. C., Campelo, P. H., & Martins, E. (2024). Encapsulation of hydrophobic active ingredients in plant proteins: Modulation of interfacial properties and encapsulation efficiency. *Current Opinion in Food Science*, 57, 101170. https://doi.org/10.1016/j.cofs.2024.101170

Fu, Y., Liu, W.-N., & Soladoye, O. P. (2020). Towards potato protein utilisation: Insights into separation, functionality and bioactivity of patatin. *International Journal of Food Science & Technology*, 55, 2314-2322. https://doi.org/10.1111/ijfs.14343

Galves, C., Galli, G., Miranda, C. G., & Kurozawa, L. E. (2021). Improving the emulsifying property of potato protein by hydrolysis: An application as encapsulating agent with maltodextrin. *Innovative Food Science & Emerging Technologies*, 70, 102696. https://doi.org/10.1016/j.ifset.2021.102696

Ghosh, S., Banerjee, S., & Sil, P. C. (2015). The beneficial role of curcumin on inflammation, diabetes and neurodegenerative disease: A recent update. *Food and Chemical Toxicology*, 83, 111-124. https://doi.org/10.1016/j.fct.2015.05.022

Giuseppin, M. L. F., Laus, M. C., & Schipper, J. (2014). Potato protein isolates. World Intellectual Property Organization Patent WO2014011042A1 https://patents.google.com/patent/WO2014011042A1/en

Gonçalves, R. F. S., Zhou, H., Vicente, A. A., Pinheiro, A. C., & McClements, D. J. (2024). Plant-based bigels for delivery of bioactive compounds: Influence of hydrogel:Oleogel ratio and protein concentration on their physicochemical properties. *Food Hydrocolloids*, 150, 109721. https://doi.org/10.1016/j.foodhyd.2023.109721

Granato, D., Barba, F. J., Kovačević, D. B., Lorenzo, J. M., Cruz, A. G., & Putnik, P. (2020). Functional foods: Product development, technological trends, efficacy testing, and safety. *Annual Review of Food Science and Technology*, 11, 93-118. https://doi.org/10.1146/annurev-food-032519-051708

Hardy, G. (2000). Nutraceuticals and functional foods: Introduction and meaning. *Nutrition*, 16, 688-689. https://doi.org/10.1016/S0899-9007(00)00332-4

He, R., He, H.-Y., Chao, D., Ju, X., & Aluko, R. (2014). Effects of high pressure and heat treatments on physicochemical and gelation properties of rapeseed protein isolate. *Food and Bioprocess Technology*, 7, 1344-1353. https://doi.org/10.1007/s11947-013-1139-z

Hewlings, S. J., & Kalman, D. S. (2017). Curcumin: A review of its effects on human health. *Food*, 6, 92. https://doi.org/10.3390/foods6100092

Huppertz, T., Fox, P. F., & Kelly, A. L. (2004). High pressure treatment of bovine milk: Effects on casein micelles and whey proteins. *Journal of Dairy Research*, 71, 97-106. https://doi.org/10.1017/S002202990300640X

Jiang, T., Liao, W., & Charcosset, C. (2020). Recent advances in encapsulation of curcumin in nanoemulsions: A review of encapsulation technologies, bioaccessibility and applications. *Food Research International*, 132, 109035. https://doi.org/10.1016/j.foodres.2020.109035

Kapoor, A. C., Desborough, S. L., & Li, P. H. (1975). Potato tuber proteins and their nutritional quality. *Potato Research*, 18, 469-478. https://doi.org/10.1007/BF02361913

Kato, A., & Nakai, S. (1980). Hydrophobicity determined by a fluorescence probe method and its correlation with surface properties of proteins. *Biochimica et Biophysica Acta (BBA) - Protein Structure*, 624, 13-20. https://doi.org/10.1016/0005-2795(80)90220-2

Kharat, M., & McClements, D. J. (2019). Recent advances in colloidal delivery systems for nutraceuticals: A case study – Delivery by
design of curcumin. *Journal of Colloid and Interface Science*, 557, 506-518. https://doi.org/10.1016/j.jcis.2019.09.045

Khati, P., Le Parc, R., Chevalier-Lucia, D., & Picart-Palmade, L. (2024). High pressure and heat-induced potato protein aggregates: A comparison of aggregation kinetics, structure modifications, and protein interactions. *Food Hydrocolloids*, 151, 109838. https://doi.org/10.1016/j.foodhyd.2024.109838

Khumsupan, P., Ramirez, R., Khumsupan, D., & Narayanaswami, V. (2011). Apolipoprotein E LDL receptor-binding domain-containing high-density lipoprotein: A nanovehicle to transport curcumin, an antioxidant and anti-amyloid bioflavonoid. *Biochimica et Bio-physica Acta (BBA) - Biomembranes*, 1808, 352-359. https://doi.org/10.1016/j.bbamem.2010.09.007

Khurana, A., & Ho, C.-T. (1988). High performance liquid chromatographic analysis of Curcuminoids and their photo-oxidative decomposition compounds in *curcuma longa* L. *Journal of Liquid Chromatography*, 11, 2295-2304. https://doi.org/10.1080/01483918808067200

Knorr, D., Kohler, G. O., & Betschart, A. A. (1977). Potato protein concentrates: The influence of various methods of recovery upon yield, compositional and functional characteristics. *Journal of Food Processing and Preservation*, 1, 235-247. https://doi.org/10.1111/j.1745-4549.1977.tb00326.x

Langyan, S., Yadava, P., Khan, F. N., Dar, Z. A., Singh, R., & Kumar, A. (2022). Sustaining protein nutrition through plant-based foods. *Frontiers in Nutrition*, 8, 772573. https://doi.org/10.3389/fnut.2021.772573

Levine, R. L., Mosoni, L., Berlett, B. S., & Stadtman, E. R. (1996). Methio-
nine residues as endogenous antioxidants in proteins. *Proceedings
of the National Academy of Sciences of the United States of America*, 93,
15036-15040. https://doi.org/10.1073/pnas.93.26.15036

Liu, F., & Tang, C.-H. (2014). Emulsifying properties of soy protein nanoparticles: Influence of the protein concentration and/or emulsification process. *Journal of Agricultural and Food Chemistry*, 62, 2644-2654. https://doi.org/10.1021/jf405348k

Liu, L.-L., Li, X.-T., Zhang, N., & Tang, C.-H. (2019). Novel soy β-conglycinin nanoparticles by ethanol-assisted disassembly and reassembly: Outstanding nanocarriers for hydrophobic nutraceuticals. *Food Hydrocolloids*, 91, 246-255. https://doi.org/10.1016/j.foodhyd.2019.01.042

Maheshwari, R. K., Singh, A. K., Gaddipati, J., & Srimal, R. C. (2006). Multiple biological activities of curcumin: A short review. *Life Sciences*, 78, 2081-2087. https://doi.org/10.1016/j.lfs.2005.12.007

Majhi, A., Rahman, G. M., Panchal, S., & Das, J. (2010). Binding of curcumin and its long chain derivatives to the activator binding domain of novel protein kinase C. *Bioorganic & Medicinal Chemistry*, 18, 1591-1598. https://doi.org/10.1016/j.bmc.2009.12.075

Marchiani, A., Rozzo, C., Fadda, A., Delogu, G., & Ruzza, P. (2014). Curcumin and curcumin-like molecules: From spice to drugs. *Current Medicinal Chemistry*, 21, 204-222. https://doi.org/10.2174/09298673210213206115810

Martínez-Guerra, J., Palomar-Pardavé, M., Romero-Romo, M., Coro-
na-Avendaño, S., Rojas-Hernández, A., & Ramírez-Silva, M. T.
(2019). New insights on the chemical stability of curcumin in
aqueous Media at Different pH: Influence of the experimen-
tal conditions. *International Journal of Electrochemical Science*, 14,
5373-5385. https://doi.org/10.20964/2019.06.24

McClements, D. J., Decker, E. A., Park, Y., & Weiss, J. (2009). Structural design principles for delivery of bioactive components in nutraceuticals and functional foods. *Critical Reviews in Food Science and Nutrition*, 49, 577-606. https://doi.org/10.1080/10408390902841529

Mehryar, L., Esmaiili, M., Zeynali, F., Imani, M., & Sadeghi, R. (2021). Fabrication and characterization of sunflower protein isolate nanoparticles, and their potential for encapsulation and sustainable release of curcumin. *Food Chemistry*, 355, 129572. https://doi.org/10.1016/j.foodchem.2021.129572

Mohammadian, M., Salami, M., Momen, S., Alavi, F., Emam-Djomeh, Z., & Moosavi-Movahedi, A. A. (2019). Enhancing the aqueous solubility of curcumin at acidic condition through the complexation with whey protein nanofibrils. *Food Hydrocolloids*, 87, 902-914. https://doi.org/10.1016/j.foodhyd.2018.09.001

Ohara, K., Mizukami, W., Tokunaga, A., Nagaoka, S., Uno, H., & Mukai, K. (2005). Kinetic study of the mechanism of free-radical scavenging action in curcumin: Effects of solvent and pH. *Bulletin of the Chemical Society of Japan*, 78, 615-621. https://doi.org/10.1246/bcsj.78.615

Otchere, E., McKay, B. M., English, M. M., & Aryee, A. N. A. (2023). Current trends in nano-delivery systems for functional foods: A systematic review. *PeerJ*, 11, e14980. https://doi.org/10.7717/peerj.14980

Picart-Palmade, L., Cunault, C., Chevalier-Lucia, D., Belleville, M.-P., & Marchesseau, S. (2019). Potentialities and limits of some non-thermal technologies to improve sustainability of food processing. *Frontiers in Nutrition*, 5, 130. https://doi.org/10.3389/fnut.2018.00130

Puppo, M. C., Speroni, F., Chapleau, N., de Lamballerie, M., Añón, M. C., & Anton, M. (2005). Effect of high-pressure treatment on emulsifying properties of soybean proteins. *Food Hydrocolloids*, 19, 289-296. https://doi.org/10.1016/j.foodhyd.2004.07.001

Qin, Z., Guo, X., Lin, Y., Chen, J., Liao, X., Hu, X., & Wu, J. (2013). Effects of high hydrostatic pressure on physicochemical and functional properties of walnut (Juglans regia L.) protein isolate. *Journal of the Science of Food and Agriculture*, 93, 1105-1111. https://doi.org/10.1002/jsfa.5857

Racusen, D., & Weller, D. L. (1984). Molecular weight of patatin, a major potato tuber Protein1. *Journal of Food Biochemistry*, 8, 103-107. https://doi.org/10.1111/j.1745-4514.1984.tb00318.x

Radbakhsh, S., Barreto, G. E., Bland, A. R., & Sahebkar, A. (2021). Curcumin: A small molecule with big functionality against amyloid aggregation in neurodegenerative diseases and type 2 diabetes. *BioFactors*, 47, 570-586. https://doi.org/10.1002/biof.1735

Ralet, M.-C., & Guéguen, J. (1999). Potato proteins: Composition, recovery and functional properties. *Sciences des Aliments*, 19, 147-165

Ralet, M.-C., & Guéguen, J. (2000). Fractionation of potato proteins: Solubility, thermal coagulation and emulsifying properties. *LWT - Food Science and Technology*, 33, 380-387. https://doi.org/10.1006/fstl.2000.0672

Ralet, M.-C., & Guéguen, J. (2001). Foaming properties of potato raw proteins and isolated fractions. *LWT - Food Science and Technology*, 34, 266-269. https://doi.org/10.1006/fstl.2000.0684

Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. *Free Radical Biology and Medicine*, 26, 1231-1237. https://doi.org/10.1016/S0891-5849 (98)00315-3

Relkin, P., Shukat, R., & Moulin, G. (2014). Encapsulation of labile compounds in heat- and high-pressure treated protein and lipid nanoparticles. *Food Research International*, 63, 9-15. https://doi.org/10.1016/j.foodres.2014.03.038

Rezaei, A., Fathi, M., & Jafari, S. M. (2019). Nanoencapsulation of hydrophobic and low-soluble food bioactive compounds within different nanocarriers. *Food Hydrocolloids*, 88, 146-162. https://doi.org/10.1016/j.foodhyd.2018.10.003
Sabaté, J., & Soret, S. (2014). Sustainability of plant-based diets: Back to the future123. *The American Journal of Clinical Nutrition*, 100, 476S-482S. https://doi.org/10.3945/ajcn.113.071522

Schmidt, J. M., Damgaard, H., Greve-Poulsen, M., Sunds, A. V., Larsen, L. B., & Hammershøj, M. (2019). Gel properties of potato protein and the isolated fractions of patatins and protease inhibitors – Impact of drying method, protein concentration, pH and ionic strength. *Food Hydrocolloids*, 96, 246–258. https://doi.org/10.1016/j.foodhyd.2019.05.022

Sharma, R. A., Gescher, A. J., & Steward, W. P. (2005). Curcumin: The story so far. *European Journal of Cancer*, 41, 1955–1968. https://doi.org/10.1016/j.ejca.2005.05.009

Shavronskaya, D. O., Noskova, A. O., Skvortsova, N. N., Adadi, P., & Nazarova, E. A. (2023). Encapsulation of hydrophobic bioactive substances for food applications: Carriers, techniques, and biosafety. *Journal of Food Processing and Preservation*, 2023, 1–21. https://doi.org/10.1155/2023/5578382

Song, X., Zhou, C., Fu, F., Chen, Z., & Wu, Q. (2013). Effect of high-pressure homogenization on particle size and film properties of soy protein isolate. *Industrial Crops and Products*, 43, 538–544. https://doi.org/10.1016/j.indcrop.2012.08.005

Sponton, O. E., Perez, A. A., Ramel, J. V., & Santiago, L. G. (2018). Protein nanovehicles produced from egg white. Part 2: Effect of protein concentration and spray drying on particle size and linoleic acid binding capacity. *Food Hydrocolloids*, 77, 863–869. https://doi.org/10.1016/j.foodhyd.2017.11.030

Sun, H., Sun, Y., Tang, X., Cui, Y., Meng, D., Zhang, Y., Li, K., Guo, H., Chen, H., & Yang, R. (2023). The interaction mechanism and the functionality of yeast protein with hydrophilic and hydrophobic bioactive molecules. *Food Bioscience*, 52, 102448. https://doi.org/10.1016/j.fbio.2023.102448

Taghavi Kevij, H., Mohammadian, M., & Salami, M. (2019). Complexation of curcumin with whey protein isolate for enhancing its aqueous solubility through a solvent-free pH-driven approach. *Journal of Food Processing and Preservation*, 43, e14227. https://doi.org/10.1111/jfpp.14227

Tang, C.-H. (2020). Nanocomplexation of proteins with curcumin: From interaction to nanoencapsulation (a review). *Food Hydrocolloids*, 109, 106106. https://doi.org/10.1016/j.foodhyd.2020.106106

Tang, C.-H., & Ma, C.-Y. (2009). Effect of high pressure treatment on aggregation and structural properties of soy protein isolate. *LWT - Food Science and Technology*, 42, 606–611. https://doi.org/10.1016/j.lwt.2008.07.012

Tanger, C., Quintana Ramos, P., & Kulozik, U. (2021). Comparative assessment of thermal aggregation of whey, potato, and pea protein under shear stress for icroparticulation. ACS Food Science & Technology, 1, 975–985. https://doi.org/10.1021/acsfoodscitech.1c00104

Tapal, A., & Tiku, P. K. (2012). Complexation of curcumin with soy protein isolate and its implications on solubility and stability of curcumin. *Food Chemistry*, 130, 960–965. https://doi.org/10.1016/j.foodchem.2011.08.025

Temple, N. J. (2022). A rational definition for functional foods: A perspective. *Frontiers in Nutrition*, 9, 957516. https://doi.org/10.3389/fnut.2022.957516

Tringali, C. (2011). *Bioactive compounds from natural sources, second edition: Natural products as lead compounds in drug discovery*. CRC Press. https://doi.org/10.1201/b11196

Tsaplev, Y. B., Lapina, V. A., & Trofimov, A. V. (2020). Curcumin in dimethyl sulfoxide: Stability, spectral, luminescent and acid-base properties. *Dyes and Pigments*, 177, 108327. https://doi.org/10.1016/j.dyepeg.2020.108327

van Koningsveld, G. A., Walstra, P., Gruppen, H., Wijngaards, G., van Boekel, M. A. J. S., & Voragen, A. G. J. (2002). Formation and stability of foam made with various potato protein preparations. *Journal of Agricultural and Food Chemistry*, 50, 7651–7659. https://doi.org/10.1021/jf025587a

van Koningsveld, G. A., Walstra, P., Voragen, A. G. J., Kuijpers, I. J., van Boekel, M. A. J. S., & Gruppen, H. (2006). Effects of protein composition and enzymatic activity on formation and properties of potato protein stabilized emulsions. *Journal of Agricultural and Food Chemistry*, 54, 6419–6427. https://doi.org/10.1021/jf061278z

Waglay, A., & Karboune, S. (2016). Chapter 4 - potato proteins: functional food ingredients. In J. Singh & L. Kaur (Eds.), *Advances in potato chemistry and technology* (Second ed., pp. 75–104). Academic Press. https://doi.org/10.1016/B978-0-12-800002-1.00004-2

Wang, C.-Y., Huang, H.-W., Hsu, C.-P., & Yang, B. B. (2016a). Recent advances in food processing using high hydrostatic pressure technology. *Critical Reviews in Food Science and Nutrition*, 56, 527–540. https://doi.org/10.1080/10408398.2012.745479

Wang, J.-M., Yang, X.-Q., Guo, J., & Lin, Y. (2015). Amphiphilic zein hydrolysate as a novel nano-delivery vehicle for curcumin. *Food & Function*, 6, 2636–2645. https://doi.org/10.1039/C5FO00422E

Wang, L., Ke, L., Rao, P., & Zhang, Y. (2021). Fabrication and characterization of curcumin-loaded nanoparticles using protein from brewers' spent grain. *LWT*, 150, 111992. https://doi.org/10.1016/j.lwt.2021.111992

Wang, X.-S., Tang, C.-H., Li, B.-S., Yang, X.-Q., Li, L., & Ma, C.-Y. (2008). Effects of high-pressure treatment on some physicochemical and functional properties of soy protein isolates. *Food Hydrocolloids*, 22, 560–567. https://doi.org/10.1016/j.foodhyd.2007.01.027

Wang, Y.-H., Yuan, Y., Yang, X.-Q., Wang, J.-M., Guo, J., & Lin, Y. (2016b). Comparison of the colloidal stability, bioaccessibility and antioxidant activity of corn protein hydrolysate and sodium caseinate stabilized curcumin nanoparticles. *Journal of Food Science and Technology*, 53, 2923–2932. https://doi.org/10.1007/s13197-016-2257-1

Wu, C., Li, L., Zhong, Q., Cai, R., Wang, P., Xu, X., Zhou, G., Han, M., Liu, Q., Hu, T., & Yin, T. (2019). Myofibrillar protein-curcumin nanocomplexes prepared at different ionic strengths to improve oxidative stability of marinated chicken meat products. *LWT*, 99, 69–76. https://doi.org/10.1016/j.lwt.2018.09.024

Zhang, S. B., Wang, Z., & Xu, S. Y. (2008). Antioxidant and antithrombotic activities of rapeseed peptides. *Journal of the American Oil Chemists' Society*, 85, 521–527. https://doi.org/10.1007/s11746-008-1217-y

Zhao, Y., Yuan, Y., Yuan, X., Zhao, S., Kang, Z., Zhu, M., He, H., & Ma, H. (2023). Physicochemical, conformational and functional changes of quinoa protein affected by high-pressure homogenization. *LWT*, 173, 114343. https://doi.org/10.1016/j.lwt.2022.114343

Zhao, Z.-K., Mu, T.-H., Zhang, M., & Richel, A. (2018). Chemical forces, structure, and gelation properties of sweet potato protein as affected by pH and high hydrostatic pressure. *Food and Bioprocess Technology*, 11, 1719–1732. https://doi.org/10.1007/s11947-018-2137-y