Tuning Whey Protein Properties: Ohmic Heating Effects on Interfacial Properties and Hydrophobic and Hydrophilic Interactions

Abstract

Ohmic heating (OH) emerged as an alternative processing method for food preservation and has more recently been used to modify the functional properties of proteins. This study aimed to evaluate the effects of OH on the interfacial properties of whey proteins (WPC) and its interactions with hydrophobic and hydrophilic compounds. WPC solutions (8% w/w) were subjected to electric field intensities ranging from 0 to 50 V·cm⁻¹ until reaching 80 °C. Structural and physicochemical parameters, including free sulfhydryl content, zeta potential, surface hydrophobicity, intrinsic fluorescence, and solubility, were analyzed. Protein–ligand interactions were also evaluated using β-carotene and caffeic acid as model compounds. The results indicated that moderate electric field intensities (30 V·cm⁻¹) promoted increased surface hydrophobicity and intrinsic fluorescence, suggesting protein unfolding and exposure of hydrophobic regions. Higher electric field intensities (40–50 V·cm⁻¹) led to aggregation, reducing solubility and binding affinity to β-carotene. Conversely, OH processing increased the interaction of WPC with caffeic acid due to enhanced exposure of hydrophilic binding sites. These findings provide insights into the modulation of whey protein interfacial properties through OH and highlight its potential for tailoring protein functionality in food formulations.

1. Introduction

Whey proteins, particularly the concentrated fractions (WPC—whey protein concentrate), have broad applicability in food systems due to their solubility, emulsifying capacity, and interaction with bioactive compounds. However, the stability of these proteins may be compromised during conventional thermal processing, resulting in the loss of functionality and the formation of undesirable aggregates [1,2,3].

The use of emerging technologies in dairy protein processing has garnered increasing interest in the food industry due to their potential to modify structural and functional properties without compromising nutritional quality [4,5,6]. Among these technologies, ohmic heating (OH) has stood out as an efficient method for the thermal treatment of protein solutions, providing rapid and uniform heating [4,7]. In addition to thermal effects, the electric field applied in OH can directly influence the molecular structure of proteins, affecting their intermolecular interactions and, consequently, their functional properties [8,9]. However, the specific impacts of OH on whey proteins remain poorly understood, particularly regarding their interactions with hydrophilic and hydrophobic compounds. Therefore, investigating how OH influences these properties may offer new possibilities for the formulation of innovative protein-based products.

The primary mechanism by which electric fields and electromagnetic waves modify proteins involves the absorption of energy by polar groups, leading to the generation of free radicals, protein aggregation, or unfolding [7]. These free radicals can disrupt various intermolecular interactions, including Van der Waals forces, electrostatic and hydrophobic interactions, hydrogen bonds, disulfide bridges, and salt bridges, resulting in alterations at different structural levels of proteins and their functional properties [10,11].

Ohmic heating enables very fast and homogeneous warming of liquid systems, making it possible to apply elevated temperatures without triggering coagulation or extensive protein unfolding [12,13]. These structural changes can significantly impact protein functionality, influencing their emulsifying capacity, gel formation, and encapsulation of bioactive compounds. However, the intensity of the electric field and processing conditions play a critical role in determining the magnitude of these modifications.

Ohmic heating applied to whey proteins has been investigated for use in dairy beverages and foods [14], in the development of hydrogels [15], in cheese production combined with ultrasound technology [16], and for the evaluation of rheological and electrical properties [8,17]. However, no studies have been conducted on the effect of ohmic heating on the interfacial properties of whey proteins.

When mixing proteins with bioactive compounds, the industry must understand how this interaction influences other technological properties of the product. Understanding the interfacial properties of proteins in binding with bioactive compounds is essential for developing more stable, nutritious, and functional foods [18,19,20,21]. This approach can be applied to encapsulation, emulsification, improved bioavailability, and food fortification, contributing to innovation in the food industry and the development of healthier and more sustainable products.

Given this context, this study aims to evaluate the effects of ohmic heating on the interfacial properties of WPC, investigating its interactions with hydrophobic and hydrophilic compounds. To achieve this, different electric field intensities (0–50 V·cm⁻¹) were applied to protein solutions, analyzing structural and physicochemical parameters such as free sulfhydryl content, zeta potential, surface hydrophobicity, intrinsic fluorescence, and solubility. Additionally, protein–ligand interaction experiments were conducted to understand how OH-induced modifications affect WPC’s affinity for β-carotene and caffeic acid.

2. Materials and Methods

2.1. Ohmic Heating

Whey protein concentrate (80% protein) was sourced from Growth Supplements Produtos Alimentícios (Tijucas, Brazil). The trials were carried out using an ohmic heating device composed of a voltage generator (Tdgc2-2 2 kVA, 220 V AC/60 Hz, Variac^®, Variac Transformer Company, Pune, India), which al-lowed for accurate temperature regulation and had a working volume of 125 mL with internal dimensions of 5 × 5 × 5 cm. The system was equipped with two titanium electrodes of opposite polarity to maximize efficiency. Throughout the experiments, temperature was continuously tracked with a digital thermometer built into the apparatus [22].

The solutions were prepared at a fixed concentration of 8% (w/w), reflecting the protein levels typically found in high-protein dairy beverages. Ohmic heating was applied under different electric field strengths (0, 20, 30, 40, and 50 V·cm⁻¹). To ensure uniformity, the whey protein concentrate dispersions were continuously mixed with a magnetic stirrer. The treatments were carried out until the samples reached 80 °C, a condition selected to minimize excessive denaturation and the development of visible aggregates. Since all trials were processed at the same final temperature, the influence of the electric field be-came more evident.

2.2. Free Sulfhydryls

The quantification of free sulfhydryl (SH) groups was carried out according to the procedure described by Ellman et al. [23]. A whey protein concentrate solution (1 mg·mL⁻¹) was prepared in phosphate buffer at pH 8, to which 12 μL of Ellman’s reagent was added. The mixture was gently stirred and kept in the dark for 20 min. Following incubation, 200 μL of the sample was transferred into microplates, and absorbance was recorded at 412 nm using a microplate reader (Multiskan SkyHigh, Thermo Fisher Scientific, Waltham, MA, USA). For the blank control, the protein solution was replaced with phosphate buffer, while Ellman’s reagent was still included. The concentration of SH groups (mmol SH·g⁻¹ of protein) was calculated using Equation (1):

$$SH=1000\times {\displaystyle \frac{({Abs}_{sample}-{Abs}_{blank})}{\mathrm{13,600}}}$$

(1)

2.3. Surface Hydrophobicity

Surface hydrophobicity of the whey protein concentrate was assessed using the bromophenol blue/UV-Vis approach (Multiskan SkyHigh, Thermo Scientific, Waltham, MA, USA) [24]. Briefly, bromophenol blue was mixed with the protein solution, after which the samples were vortexed and centrifuged at 10,000× g for 10 min. Subsequently, 200 μL of the supernatant was dispensed into microplate wells, and the absorbance was recorded at 595 nm with a microplate reader (Multiskan SkyHigh, Thermo Scientific, Waltham, MA, USA). Surface hydrophobicity was calculated based on the binding of bromophenol blue (BPB) to the protein according to Equation (2):

$${BPB}_{bound}\left(\mathsf{\mu }\mathrm{g}\right)=200 \mathsf{\mu }\mathrm{g}\times {\displaystyle \frac{({Abs}_{blank}-{Abs}_{sample})}{{Abs}_{blank}}}$$

(2)

2.4. Zeta Potential

Both control and heat-treated WPC solutions (0.01 g·mL⁻¹) were diluted 15-fold, and their zeta potential was determined using a Zetasizer Nano (ZEN 3600, Malvern Instruments Ltd., Malvern, UK) [25].

2.5. Solubility

The solubility percentage of WPC was evaluated using the Bradford assay [26]. Briefly, 3 g of each protein concentrate were dissolved in 60 mL of distilled water, and the pH was adjusted to 7 with 1M HCl or NaOH. Two-milliliter aliquots were transferred into Eppendorf tubes, agitated for 20 min at room temperature (25 °C), and subsequently centrifuged at 10,000× g and 4 °C for 10 min using an Eppendorf 5804 R centrifuge (Hamburg, Germany). Finally, 200 μL of the resulting supernatant from each tube was placed into micro-plate wells, and absorbance was measured at 595 nm using a microplate reader (Multiskan SkyHigh, Thermo Fisher Scientific).

2.6. Particle Size

The particle size of the WPC samples was analyzed using dynamic light scattering (DLS) with a Malvern Zetasizer Z90 (Malvern Instruments Ltd., Malvern, UK). One-milliliter aliquots were placed in cuvettes for measurement, and the sample cell was kept at 25 °C throughout the analysis [27].

2.7. Intrinsic Fluorescence

Intrinsic fluorescence experiments for WPC and modified WPC were conducted using a spectrofluorometer (Fluoromax-PlusC, Horiba, Irvine, CA, USA), with excitation at 270 nm (Trp and Tyr excitation) and emission spectra recorded between 300 and 600 nm [28].

2.8. Formation of Modified WPC/β-Carotene and Modified WPC/Caffeic Acid Complexes

The interaction between WPC (both native and modified) and the ligands β-carotene (BC) and caffeic acid (CA) was investigated using intrinsic fluorescence spectroscopy. WPC and its modified forms were dispersed in a phosphate buffer (pH 7, 50 mM) to reach a final protein concentration of 4.3 mM. Stock solutions of BC and CA were prepared at 1 mM in ethanol and water, respectively. Protein dispersions of 3 mL were titrated with in-creasing amounts of BC or CA stock solutions, yielding final ligand concentrations from 0 to 290 μM. The absorption spectra of the resulting WPC-BC, WPC-CA, modified WPC-BC, and modified WPC-CA complexes were recorded between 300 and 600 nm using a spectrofluorometer (Fluoromax-PlusC, Horiba, USA), with the fluorescence curves provided in the Supplementary Materials (Figure S1). The intrinsic fluorescence intensity (FI) at the emission maximum was expressed as relative fluorescence intensity (RFI), calculated by RFI = FI/FI₀, where FI corresponds to the fluorescence of the protein–ligand complex and FI₀ to the fluorescence of the unbound protein (WPC or modified WPC). Generally, adding ligands to protein solutions can either increase or decrease RFI, reflecting the formation of protein–ligand complexes in solution [28,29].

The fluorescence curves were used to determine the ligand saturation concentration (β-carotene or caffeic acid) to assess whether the modified proteins exhibit greater interaction with these compounds. The data were then fitted to an exponential decay model (Equation (3)):

$$F=F_{\infty }+(F_0-F_{\infty })e^{-k\left[L\right]}$$

(3)

where F, F₀ and F_∞ represent the fluorescence intensity at a given ligand concentration, the initial fluorescence intensity, and the minimum fluorescence intensity reached when the protein is saturated by the ligand, respectively; k is the affinity constant between WPC proteins and the ligands, and [L] is the ligand concentration at each measured point (µM).

The maximum bound concentration of β-carotene/caffeic acid can be estimated by assuming that the system follows an exponential saturation model, where fluorescence reaches a plateau F∞. Thus, the ligand concentration at which fluorescence no longer changes significantly can be estimated by solving the equation for a value close to F∞, typically around 95% of the maximum effect. The maximum ligand concentration bound to the proteins (C_max) is defined as (Equation (4)):

$$C_{max}\approx {\displaystyle \frac{3}{k}}$$

(4)

Furthermore, the number of binding sites available for β-carotene/caffeic acid at saturation (n) and the apparent binding constant (Ka) can be determined using the Scatchard model. These parameters are calculated as the slope and intercept, respectively, of the linear regression between [P](1 − f_i) vs. [L](1/f_i − 1) (Equations (5) and (6)) [30]:

$$f_i={\displaystyle \frac{F-F_0}{F_{\infty }-F_0}}$$

(5)

$$\left[P\right]\left(1-f_i\right)={\displaystyle \frac{\left[L\right]}{n}}\left({\displaystyle \frac{1}{f_i}}-1\right)-{\displaystyle \frac{1}{n{K}_a}}$$

(6)

where [P] represents the protein concentration (µM).

2.9. Statistical Analysis

All experiments were carried out in triplicate. Data were analyzed using JASP soft-ware (version JASP 0.95.3), and mean values were compared through ANOVA followed by Tukey’s post hoc test, considering a significance threshold of p < 0.05.

3. Results and Discussion

3.1. Thermal and Electrical Parameters

Figure 1 presents various parameters of ohmic heating applied to the WPC solution. The temperature evolution over time reveals that higher electric field intensities (40 and 50 V·cm⁻¹) promote significantly faster heating compared to lower values, such as 20 V·cm⁻¹ (Figure 1A). This effect is expected since energy dissipation in ohmic heating is proportional to the square of the electric field and the conductivity of the solution, leading to a more efficient conversion of electrical energy into heat as the field intensity increases.

The relationship between electrical conductivity and temperature exhibits linear behavior, regardless of the applied electric field (Figure 1B). All curves overlap independently of the electric field intensity, indicating that electrical conductivity is primarily associated with the composition of the material rather than the field strength. However, at temperatures above 60 °C, the electrical conductivity for 50 V·cm⁻¹ is slightly higher than in other treatments. This may be related to structural changes in proteins (driven by a higher current flow) and the exposure of groups that facilitate electrical charge conduction.

This progressive increase in conductivity can be attributed to the reduction in medium viscosity and the greater mobility of ions as temperature rises. Since conductivity determines the efficiency of electrical-to-thermal energy conversion, this effect contributes to the faster heating of the solution at higher field intensities. Balthazar et al. [31] observed that the electrical conductivity values of sheep’s milk exhibited the same behavior regardless of the applied electric field.

The heating rate (Figure 1C) shows a significant increase as the electric field intensity rises, confirming that higher values result in greater thermal efficiency. At 50 V·cm⁻¹, the heating rate is the highest recorded, while the lowest rate occurs at 20 V·cm⁻¹. This reinforces the direct relationship between field intensity and power dissipation in the medium, optimizing the conversion of electrical energy into heat [32].

Regarding energy consumption (Figure 1D), an inverse trend is observed: higher electric field values result in lower total energy consumption. At 20 V·cm⁻¹, energy consumption is significantly higher compared to 40 and 50 V·cm⁻¹. This behavior suggests that the overall efficiency of the process improves at higher field intensities, likely due to the reduction in the electrical resistance of the solution as the temperature increases, promoting a more efficient use of the supplied energy [33].

3.2. Free Sulfhydryl

Figure 2 illustrates the relationship between the concentration of free sulfhydryl (SH) groups and the intensity of the electric field applied during the ohmic heating of WPC solutions. When proteins are heated to temperatures above 80 °C, disulfide bonds are broken [34], initiating a more intense denaturation process. A progressive increase in the number of sulfhydryl groups with increasing electric field intensity is observed, suggesting that protein denaturation occurs more intensely as the electric field strength rises. This behavior can be explained by the fact that ohmic heating induces structural changes in proteins, promoting the exposure of sulfhydryl groups previously buried within the protein chains or the breaking of disulfide bonds caused by heating, leading to protein unfolding [35]. When studying the effects of the electric field on the thermal unfolding of β-lactoglobulin [35] and whey protein isolate (WPI) [36], the authors observed that at temperatures above 80 °C, treatments involving conventional heating alone exhibited higher amounts of free sulfhydryl groups compared to ohmic heating. This may be related to less aggressive thermal effects associated with the presence of an alternating electric field [36]. Thus, our results may be more influenced by the heating rate rather than the direct action of the electric field on the various bonds that constitute the higher-order structures of proteins.

The more pronounced and significant increase (p-value < 0.05) in SH concentration between 20 and 30 V·cm⁻¹ indicates a critical denaturation stage, where internal bonds begin to break, and the protein’s tertiary structure undergoes significant modifications. Furthermore, it can be inferred that SH bonds may be more sensitive to exposure to electric fields, making them susceptible to bond cleavage when an electric current passes through the material [37].

Between electric field intensities of 30 and 40 V·cm⁻¹, no significant difference was observed in the concentration of free sulfhydryl groups. It is possible that, at this intermediate electric field range, the structural changes between the applied voltages were minimal, resulting in limited exposure of sulfhydryl groups. The energy input was likely insufficient to induce the degree of protein unfolding required to break disulfide bonds and consequently expose additional reactive sulfhydryl sites.

At higher field intensities (40 and 50 V·cm⁻¹), the SH concentration continues to increase, albeit at a slower rate. This effect may be attributed to the occurrence of secondary reactions, such as the formation of new disulfide bonds between denatured proteins. The aggregation of polypeptide chains and the consequent loss of native structure may limit the availability of free SH groups. The exposure of SH groups can be beneficial for certain applications, such as emulsification and gel formation, but excessive denaturation may lead to undesirable aggregation.

3.3. Surface Hydrophobicity

WPC proteins appear to be highly susceptible to ohmic heating treatment, with a significant increase (p-value < 0.05) in H₀ values up to 30 V·cm⁻¹ (Figure 3). Partial unfolding of proteins results in the exposure of hydrophobic residues that were originally hidden within the protein’s higher-order structures. More intense treatments (40–50 V·cm⁻¹) may induce structural rearrangements and the formation of new disulfide bonds [12], as well as secondary structure compaction and aggregation [38], thereby reducing the surface area available for hydrophobic interaction with bromophenol blue. When considering the thermal effect, protein denaturation results in greater exposure of hydrophobic regions [3], which promotes aggregation through intermolecular interactions. Additionally, it is plausible that electrical processing combines electrical, chemical, and thermal effects within a single treatment [7].

3.4. Zeta Potential

The zeta potential represents the surface electrical characteristics of particles in sus-pension. Systems with greater absolute zeta potential show enhanced intermolecular re-pulsion, decreased aggregation, and increased stability [39,40]. The zeta potential of WPC proteins showed significant variations (p-value < 0.05) with the increase in the applied electric field during ohmic heating (Figure 4). Zhang et al. [41] and Xi et al. [42] observed that higher thermal treatment intensity resulted in increased zeta potential values for whey proteins. In our study, we found no linearity in the zeta potential values. This suggests that a combination of temperature and electric current can modulate zeta potential values in a non-linear manner. Moreover, the fluctuations observed in zeta potential values may be associated with the system’s tendency to partially recover its initial state, possibly due to a competitive effect between protein structural reorganization and surface charge recombination. Wang et al. [43], observed similar results in his treatments using different intensities of pulsed electric fields.

In none of the treatments were absolute values greater than 30 mV observed. It is well known that colloidal systems exhibit electrosteric stability when zeta potential values exceed (in absolute terms) 30 mV [44]. Initially, the zeta potential of untreated WPC was 24.24 mV. However, as the electric field increased, distinct behaviors were observed: a reduction at 20 and 40 V·cm⁻¹ and an increase at 30 and 50 V·cm⁻¹. The reduction in zeta potential at 20 and 40 V·cm⁻¹, associated with weakened electrostatic repulsion, may be related to the formation of protein aggregates that neutralize surface charges and amino acid residues.

At an electric field of 50 V·cm⁻¹, the zeta potential increased significantly, becoming more negative. This effect can be explained by conformational changes in the proteins, leading to a redistribution of surface charges on the molecules. Additionally, the exposure of free amino and sulfhydryl groups due to a more abrupt structural modification may have influenced the migration of polar groups from the interior to the surface [45]. These results are consistent with the findings on electrical conductivity as a function of electric fields in our study (Figure 1C). Higher conductivity may be associated with a greater number of net charges on the protein, facilitating the transport of electric current during processing.

3.5. Intrinsic Fluorescence

Protein fluorescence spectroscopy analysis is considered a powerful non-invasive tool for assessing structural information related to the local molecular environments of polar or nonpolar fluorophores [46]. The tryptophan residues (Trp-26, Trp-60, Trp-104, and Trp-118 in α-lactalbumin; Trp-19 and Trp-61 in β-lactoglobulin; and Trp-213 and Trp-134 in bovine serum albumin) found in WPC structures are highly sensitive to alterations in their microenvironment, enabling the monitoring of conformational modifications through changes in fluorescence intensity and shifts in emission maxima [22,47,48]. The intrinsic fluorescence results (Figure 5A) indicate significant structural changes in WPC proteins subjected to ohmic heating. An increase in fluorescence intensity across different applied electric fields was observed, suggesting greater exposure of aromatic residues, such as tryptophan, tyrosine, and/or phenylalanine, to the aqueous medium. This effect may be associated with the partial unfolding of proteins, leading to the exposure of new hydrophobic groups.

Regarding the different treatments, it was observed that at 30 V·cm⁻¹, intrinsic fluorescence was highest, indicating that a greater number of aromatic amino acids were exposed at this electric field due to increased protein unfolding, leading to enhanced fluorescence emission [39]. These results align with the surface hydrophobicity findings, which show a greater number of hydrophobic sites under this treatment.

Figure 5B shows a shift in the fluorescence maximum position as a function of the applied electric field. The treatment at 30 V·cm⁻¹ resulted in a shift toward higher wavenumbers (red shift), suggesting a more polar environment around the fluorescent residues. This may be a consequence of solvent exposure due to structural changes or swelling of the globular proteins that make up whey protein concentrate [49]. Additionally, red shifts may indicate that, at this electric field intensity, the proteins were stretched (less tense structure) [50] and/or tryptophan groups moved to more hydrophilic microenvironments [51].

On the other hand, at 20 V·cm⁻¹, a shift toward lower wavenumbers (blue shift) was observed, indicating that the fluorescent residues are more shielded, possibly due to intermolecular interactions or the formation of protein aggregates. At 50 V·cm⁻¹, no change in the fluorescence maximum was detected. However, a red shift could suggest more hydrophobic microenvironments [36]. These results further support the previously discussed hypotheses regarding the conformational changes induced by ohmic heating. The increase in fluorescence suggests initial protein unfolding, while the spectral shifts indicate a complex structural reorganization, possibly linked to protein exposure followed by aggregation.

3.6. Solubility

Figure 6 presents the solubility of WPC as a function of the electric field intensity applied during ohmic heating. The observed trend suggests a nonlinear effect of the electric field on solubility. Initially, protein solubility increases as the electric field is applied, reaching a peak at approximately 20 V·cm⁻¹. This increase may be attributed to favorable structural modifications in the proteins, such as the exposure of hydrophilic groups, promoting greater interaction with water. This effect could be associated with controlled partial protein denaturation, enhancing their dispersibility in the aqueous medium.

However, for electric fields greater than 20 V·cm⁻¹, solubility begins to decrease significantly (p-value < 0.05). This reduction in solubility can be explained by the intensification of protein denaturation and possible irreversible aggregation, leading to the formation of less soluble structures [52]. The application of high-intensity electric fields may promote hydrophobic interactions and bonding between denatured proteins, forming larger aggregates and reducing their ability to dissolve in water.

The lower solubility observed at electric field intensities of 40 and 50 V·cm⁻¹ suggests that excessive thermal energy and electromagnetic interactions intensify processes such as the formation of insoluble aggregates and even partial protein gelation. These results may align with the values obtained for SH content. The increase in SH levels indicates that internal protein structures were disrupted, exposing these groups; however, their reactivity may have led to the formation of aggregates and less soluble structures [52]. This behavior is particularly relevant for industrial applications, as it can directly impact the functionality of proteins in food formulations that require high solubility, such as protein beverages and emulsions.

3.7. Particle Size

The particle size of a protein is an important factor that affects its functional properties [53]. Figure 7 presents the results for particle size and polydispersity index (PDI) (Figure 7A), as well as the particle size distribution (Figure 7B) for WPC solutions modified by ohmic heating. Regarding particle size, ohmic heating modifies proteins in different ways. A significant increase (p-value < 0.05) is observed when an electric field of 20 V·cm⁻¹ is applied. In this first stage, the increase in particle size may be associated with partial protein unfolding or expansion of the globular protein volume caused by greater interaction between the particles and the hydration water. Protein unfolding promotes the exposure of sulfhydryl groups, which in turn show an increased tendency to interact with water molecules, resulting in enhanced solubility, as observed in the solubility analysis section. In the second stage (30–40 V·cm⁻¹), particle size is smaller but still larger than the control treatment. In these treatments, it can be proposed that proteins undergo a more intense denaturation process, leading to polypeptide structure collapse and a reduction in protein size. In the third stage (50 V·cm⁻¹), the ohmic heating process was more intense, resulting in the formation of larger aggregates of denatured proteins. This is confirmed by the shift toward larger sizes in the particle size distribution results (Figure 7B).

3.8. Relative Fluorescence Intrinsic

Figure 8 shows the relative intrinsic fluorescence (RFI) for the different modified proteins as a function of ligand concentration (β-carotene or caffeic acid). When proteins or their aggregates interact with ligands, a reduction in intrinsic fluorescence can occur, a process referred to as fluorescence quenching [54,55]. In all cases, RFI decreased with increasing ligand concentration, indicating that the fluorescence of tryptophan residues was suppressed due to protein–ligand interactions.

When observing the RFI values for β-carotene, it is noticeable that at the initial concentrations, the 30 V·cm⁻¹ treatment exhibited a greater RFI reduction rate compared to the other treatments. This may be related to structural changes in the proteins at this electric field intensity, where a larger number of binding sites were exposed (see Figure 8A), facilitating interactions with β-carotene. In contrast, at 20 V·cm⁻¹, the RFI reduction rate at the initial concentrations was lower. This suggests that the milder denaturation of WPC chains was not sufficient to expose binding sites for β-carotene, combined with the low hydrophobicity of proteins after ohmic heating at this electric field intensity. After the addition of 120 µM of β-carotene, all curves exhibited similar behavior, with minor variations associated with slight structural changes caused by the attachment of additional β-carotene molecules.

For caffeic acid (Figure 8B), all curves were very similar, indicating that thermal and/or electrical modifications did not significantly affect the protein–ligand interaction.

3.9. Interaction Between Modified WPC and β-Carotene

Figure 9 presents a compilation of various results for β-carotene quenching in both modified and untreated WPC proteins. Figure 9A displays the β-carotene saturation concentration values fitted using an exponential decay model. It is observed that the native protein and the one modified at the lowest electric field studied (20 V·cm⁻¹) exhibited the highest saturation concentration. Several studies have shown that β-carotene interacts well with whey proteins due to their mutual hydrophobic characteristics [41,47]. At higher electric field intensities (30–50 V·cm⁻¹), there was a drastic reduction in β-carotene saturation concentration. These results correlate with surface hydrophobicity values (Figure 3), as higher electric fields led to a decrease in protein surface hydrophobicity due to extensive protein denaturation and aggregation (Figure 7B). This structural change caused by more intense treatments conceals hydrophobic groups within the protein structure, reducing the active binding sites for β-carotene [56,57]. Although 30 V·cm⁻¹ was one of the treatments with the highest surface hydrophobicity, its β-carotene saturation concentration was significantly lower than that of 20 V·cm⁻¹. It is possible that the net sum of hydrophobic and hydrophilic groups (free sulfhydryl groups) influenced the interaction with β-carotene due to differences in polarity and steric hindrance, weakening hydrophobic interactions [58]. This also explains the reduction in stoichiometry (Figure 9B) as the electric field increased.

The results for Ks (Figure 9C) suggest that the predominant quenching mechanism is static, indicating the formation of protein–β-carotene complexes. Ks also follows the trend observed in previous results: when the proteins were either unmodified or treated at a low electric field, their interaction properties with β-carotene improved (with an apparent increase in protein–ligand interaction at 20 V·cm⁻¹). However, as the electric field increased, the binding affinity between proteins and β-carotene decreased due to protein denaturation and the consequent reduction in hydrophobic clusters available for ligand interaction.

Figure 9D shows a correlation between the binding constant and the number of active binding sites. Based on the results, it can be proposed that a shift toward the first quadrant (upper right) is the most desirable for food processing applications requiring enhanced interaction between bioactive compounds and macromolecules. This is particularly relevant for applications such as encapsulated and emulsified systems, where improved binding can contribute to protection or controlled release modulation [41,59].

3.10. Interaction Between Modified WPC and Caffeic Acid

For caffeic acid (Figure 10A), it was observed that the different ohmic heating treatments achieved a higher saturation concentration of binding with WPC proteins.

Other studies have reported n values ranging from 0.56 to 1.72 [18,19,20,21], which are significantly lower than those observed in our results. This discrepancy may be related to protein composition. In the cited studies, the authors used purified whey proteins such as casein, beta-lactoglobulin, and alpha-lactalbumin. Since WPC consists of various types of whey proteins, its composition may provide a greater number of hydrophilic active sites per mole of protein, enhancing the interaction with caffeic acid.

The binding constants between the modified proteins and caffeic acid are shown in Figure 10C. Zhang et al. [21] observed that caffeic acid exhibits higher binding constant values with whey proteins compared to other organic acids (chlorogenic acid, p-coumaric acid, and ferulic acid), supporting our findings. When analyzing the effects of the electric field on WPC modification, it is evident that all Ka values were lower than those of the control. Cheng et al. [18] report that proteins internal binding regions can offer a high-affinity environment, enabling stable interactions with caffeic acid molecules. In contrast, some other binding sites may not fully accommodate the caffeic acid due to steric hindrance or chemical limitations, leading to lower binding affinity. However, other binding sites may be unable to fully accommodate the caffeic acid molecule due to steric or chemical constraints, resulting in relatively low affinity. Therefore, the structural modifications induced by ohmic heating may reduce this relative affinity.

When comparing Ka and n (Figure 10D), it is observed that all ohmic heating treatments shifted to the fourth quadrant (lower right). This indicates that while more active binding sites were available, the interactions were weaker. It is possible that caffeic acid interacted in regions near free sulfhydryl bonds (which are also hydrophilic) through weak Van der Waals interactions. This contrasts with the β-carotene results, where some treatments showed an increase in Ka, as hydrophobic interactions are stronger than Van der Waals forces [60].

In summary, based on the results presented earlier, it can be concluded that the variation in the electric field during ohmic heating significantly alters the techno-functional characteristics of WPC (Figure 11). Initially, at lower electric fields, such as 20–30 V·cm⁻¹, proteins undergo initial unfolding due to heat-induced denaturation, exposing hydrophobic groups, including aromatic amino acids (as evidenced by the fluorescence assay). Subsequently, at electric fields above 30 V·cm⁻¹, particularly at 50 V·cm⁻¹, structural rearrangements occur due to intermolecular interactions and the formation of new disulfide bonds, leading to the formation of irreversible aggregates.

4. Conclusions

The results demonstrate that ohmic heating significantly alters the structural and functional properties of whey protein concentrate, influencing its interaction with bioactive compounds. Moderate electric fields (30 V·cm⁻¹) promoted controlled protein unfolding, enhancing hydrophobicity and β-carotene binding. In contrast, higher intensities (40–50 V·cm⁻¹) induced aggregation, reducing solubility and ligand affinity. Conversely, interaction with caffeic acid increased in all OH-treated samples, with the strongest binding observed at 50 V·cm⁻¹ due to greater exposure of hydrophilic sites. These findings suggest that controlled OH processing at moderate intensities can be used to tailor whey protein properties for specific food applications, such as emulsification, encapsulation, and fortification. For future studies, it is recommended that more detailed scans be performed regarding the range of applied electric field intensities, reducing the increments from 10 V·cm⁻¹ to 1 V·cm⁻¹. This approach would allow for a deeper understanding of the conformational changes in the proteins and enable the precise identification of inflection points related to free sulfhydryl content, solubility, and particle size. The observations presented in this study represent an initial step toward the development of new mechanistic approaches for applying ohmic heating as a strategy to modulate the properties of whey proteins.