Impact of high-intensity ultrasound on cowpea protein extractability, structural, and techno- functional properties

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This study compared two protein extraction methods: 1-the pH-shift method at pH 7.5, 8.0, and 9.0, and at the natural pH of the cowpea flour–water dispersion (pH 6.4); 2- pH-shift protein extraction assisted by high-intensity ultrasound (HIUS; 400 W, 60 min, pulse 2 s on/2 s off, T < 30 °C). Protein yield, structural modifications, thermal stability, and techno-functional properties were assessed. Protein content ranged from 77.85% to 83.0%. HIUS increased yield at all pH conditions, including at pH 6.4. HIUS induced partial unfolding, increased surface hydrophobicity, and decreased thermal stability as a function of pH-shift extraction. These structural changes improved solubility at pH 6.0 and enhanced emulsifying capacity, especially in I7.5-US and I8-US, forming emulsions with smaller droplet sizes. I6.4-US was particularly attractive, providing yields and solubility comparable to or higher than I9 while avoiding the use of highly alkaline solutions. Although HIUS-assisted extraction slightly reduced protein purity due to co-extraction of non-protein compounds, it represents a simpler and more sustainable approach. This strategy supports the development of plant-based protein ingredients with high functionality and reduced chemical usage. Protein extraction yield Surface hydrophobicity Denaturation degree Protein solubility Emulsifying capacity Figures Figure 1 Figure 2 Figure 3 Introduction The growing demand for plant-based proteins has prompted the exploration of alternative sources such as cowpea ( Vigna unguiculata ). Cowpea, in particular, is a legume from the Fabaceae family cultivated in tropical and subtropical regions, with a global production estimated at approximately 5.8 million tons annually (Carneiro da Silva et al., 2019). The high protein content (22–25%) and excellent nutritional profile (Avanza et al., 2013 ) make it a promising raw material not only for direct consumption in the form of whole seeds or flours, but also for the development of protein isolates as sustainable alternatives to conventional animal-derived proteins commonly employed in food formulations. The pH-shift method is one of the simplest and most widely used for obtaining protein isolates and several studies have shown that enhances protein yield and induces structural modifications (Nathia-Neves et al., 2025; Long et al., 2025 ; Fernández Sosa et al., 2021a ). These changes significantly affect techno-functional properties, such as solubility, emulsifying capacity, foaming capacity, and gelling capacity (Nathia-Neves et al., 2025; Long et al., 2025 ; Hadidi et al., 2023 ). Moreover, exposure to elevated pH conditions can compromise the nutritional integrity of proteins trough the chemical degradation of essential residues such as lysine (Nathia-Neves et al., 2025; Long et al., 2025 ), and by diminishing their digestibility (Sultan et al., 2024 ). At industrial scale, protein extraction using highly alkaline solutions entails substantial chemical input and generates large volumes of high-pH effluents, making the method environmentally unsustainable without mitigation strategies (Patel et al., 2025 ). Combining the pH-shift method with other technologies has been proposed to overcome the limitations of an alkaline protein extraction (Nathia-Neves et al., 2025; Long et al., 2025 ; Hadidi et al., 2023 ; Bernardi et al., 2021 ). One such technique is high-intensity ultrasound (HIUS), which promotes cell disruption and protein solubilization, and facilitates mass transfer. Acoustic cavitation generated by HIUS has been shown to alter the structural conformation of proteins, thereby modifying their physicochemical and techno-functional properties (Nathia-Neves et al., 2025; Long et al., 2025 ; Bernardi et al., 2021 ). Moreover, some research has reported the effect of varying energy input, treatment times, or a fixed ultrasound treatment condition combined with a pH-shift method for protein extraction from legumes like cowpea, raw pea flour, lupin, chickpea, peanut, and Faba bean, focusing on techno-functional properties (Nathia-Neves et al., 2025; Long et al., 2025 ; Bernardi et al., 2021 ; Haididi et al., 2023); Mendes Justino et al., ( 2024 ) focused on the effect of HIUS on protein structure as well as its potential applications in the food industry. Loushigam & Shanmugam ( 2023 ) informed that cowpea protein isolates extracted at pH 9.0 combined with HIUS (200 W; 10 min.) resulted in increased protein yield, solubility, water-holding capacity, foaming capacity, and stability, emulsion activity and stability, zeta potential, and in vitro protein digestibility. Quintero-Quiroz et al. ( 2022 ) informed an enhancement of protein yield and techno-functional properties of protein extraction from quinoa, lentils, and black beans at pH 9 (20 min, 320 W, seeds: solvent rate: 1:5). A comprehensive analysis of the physicochemical properties of cowpea protein isolates extracted by the pH-shift method combined with HIUS remains limited. This study aims to evaluate the impact of HIUS-assisted extraction (400 W, 60 min, pulse mode: 2 s on, 2 s off, ice bath) combined with the pH-shift method (7.5, 8.0, and 9.0) on parameters related to: a) the extraction process itself (protein extractability); b) protein structure modifications of cowpea protein isolates (CPI) (primary, secondary, and tertiary structures, thermal stability); c) the techno-functional properties of CPI (color, protein solubility, and emulsifying capacity). In addition, the impact of ultrasound extraction of cowpea protein at pH of the flour-water dispersions (6.4) was also assessed. Materials and methods Material Cowpea ( Vigna unguiculata ) seeds were provided by Estación Experimental El Sombrero-Corrientes (Instituto Nacional de Tecnología Agropecuaria-INTA) (crop 2022). Intact seeds (with seed coat) were ground using an HC-1000Y electric mill (Arcano, Tianjin, China) and sieved through an 80 ASTM mesh (177 µm). The resulting flour was defatted with hexane (10% w/v) under continuous stirring (24 h, 4 ºC). The mixture was filtered, and the flour was air-dried for 24 h at 25 ºC. The protein content was 25.60 ± 1.28% determined by the Kjeldhal method. Protein extraction Protein extraction was carried out following the method described by Fernández Sosa et al. ( 2021a ) with slight modifications. Defatted cowpea flour-water dispersions (1:10, w/v) were adjusted to pH 7.5, 8.0, and 9.0 using 2 M NaOH. Additionally, protein extraction was conducted at the natural pH (6.4) of the cowpea flour–water dispersion. The dispersions were then stirred for 1 h in the presence or absence of high-intensity ultrasound treatment (HIUS). The HIUS treatment was performed using a VCX 500 ultrasonic processor equipped with a 13 mm diameter titanium probe, model 630 − 0219 (Sonics & Materials Inc., Newtown, USA), maintaining the temperature below 30°C (400 W, 60 min, pulse mode: 2 s on, 2 s off, ice bath). Subsequently, both HIUS-treated and untreated dispersions were centrifuged (10,000 × g, 30 min, 20°C). The supernatants were precipitated at the isoelectric point (pH 4.5, 4°C, 2 h) and then centrifuged (10,000 × g, 20 min, 4°C). Then, proteins were dissolved in distilled water (pH 7.0 using 2 M NaOH), lyophilized, and stored (4°C). The CPI were named as I6.4, I6.4-US, I7.5, I7.5-US, I8, I8-US, I9, and I9-US, according to the extraction pH and the presence or absence of HIUS. Protein content and yield The protein content of CPI and cowpea flour was determined using the Kjeldahl method (N×6.25) (Association of Official Analytical Chemists [AOAC], 1990). The yield by weight and the protein recovery from total seed protein extraction were calculated (Eq. 1 and Eq. 2). \(\:Yield\:in\:weight\:\left(\%\right)=\frac{weight\:of\:sample}{weight\:of\:flour}x\:100\) (𝐸𝑞. 1) \(\:Protein\:yield\:recovery\:\left(\%\right)=\frac{Protein\:content\:of\:sample}{protein\:content\:of\:flour}x\:100\) (𝐸𝑞. 2) Water activity (A) Aw of all CPI was evaluated using an AquaLab PRE (METER Group, Pullman, USA) at 25°C. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was performed according to Laemmli's (1970) method. All gels were run in Mini-Protean Tetra Cell mini slabs (Bio-Rad, Hercules, USA) using a separating gel (12% w/v acrylamide) and a stacking gel (4% w/v acrylamide). CPI was dispersed (0.1% w/v protein) in sample buffer without (non-reduced conditions) or with 2-mercaptoethanol (reduced conditions, 2-ME) (5% v/v) and centrifuged (10,000 × g, 20 min, 25°C). Electrophoresis was performed at a constant current of 25 mA per gel. Gels were fixed and stained with Coomassie Brilliant Blue dye solution (0.2% w/v) in water/methanol/acetic acid (5:5:2) and subsequently destained using a water/methanol/acetic acid (13:5:2). Protein molecular weights were estimated using low molecular weight markers (Pharmacia Hepar Inc., Franklin, USA). Fourier transform infrared (FTIR) spectroscopy All CPI were analyzed using a Spectrum 3 FT-IR/NIR spectrometer (PerkinElmer, Waltham, USA) equipped with a universal ZnSe-ATR crystal. IR spectra (4,000–400 cm − 1 ) were registered by co-adding 64 scans (4 cm − 1 spectral resolution). The IR spectrum of the amide I band of proteins (1700–1600 cm − 1 ) was deconvolved and fitted to Gaussian profiles using the equipment software Spectrum 10. Fluorescence spectroscopy All CPI were dispersed (0.1% w/v protein) in buffer A (0.05 M Na 2 HPO 4 , NaCl 0.15 M, pH 8), stirred (30 min, 25°C), and centrifuged (10,000 × g, 30 min, 25°C). Fluorescence intensity spectra of the supernatants were measured FluoroMate FS-2 fluorescence spectrophotometer (Scinco, Seoul, South Korea) at excitation and emission wavelengths of 290 nm and 300–400 nm, respectively (slit width, 5 nm; scanning speed of 300 nm/min; 25°C). Protein concentration was determined by the Lowry method (Lowry et al., 1951 ). Surface hydrophobicity (H) H 0 of CPI was determined according to Cardamone and Puri ( 1992 ) using 1,8-aniline-naphthalene-sulfonate (ANS) as a fluorescent probe (Aldrich Chemical Co., Milwaukee, Wisconsin, USA). The emission spectra (400–600 nm) of samples in buffer A (0.01–0.03% w/v protein) equilibrated with different ANS concentrations (0.0 to 100 µM) were recorded. Fluorescence measurements were corrected by subtracting the corresponding blank to obtain the increased fluorescence (ΔFi) due to ANS binding. The ΔFi at 465 nm (λ of maximum emission of ANS-protein complex) was plotted vs. the ANS concentration (µM), and data were adjusted with the following equation: \(\:\varDelta\:Fi=\frac{A\:x\:ANS}{B+ANS}\:\) (𝐸𝑞.3) Where, the coefficients are A = ΔFi max (ΔFi max is the fluorescence intensity at saturation) and B = 1/Ka (Ka is the equilibrium-binding constant, from the fitting). H 0 is proportional to ΔFi max per mg protein and therefore estimated from Eq. 3 by dividing A by the protein concentration of each sample. Differential scanning calorimetry (DSC) DSC measurements were performed according to Peyrano et al., ( 2016 ). A Q20 differential scanning calorimeter (TA Instruments, New Castle, USA) was used to study the thermal properties of CPI. Hermetically sealed aluminum pans were prepared to hold 15–20 mg of CPI suspended in water (20% w/w protein). Samples were scanned at a rate of 5°C/min from 20 to 120°C. An empty pan was used as a reference. Denaturation temperature (T d °C) and enthalpy change of transition (ΔH J/g dry protein) were calculated with the TA Universal Analysis 2000 software. The degree of protein denaturation (DD) was calculated according to the following equation: \(\:DD\left(\%\right)=\frac{100-{\varDelta\:H}_{t}}{{\varDelta\:H}_{0}}x\:100\) (Eq. 4) Where ΔH 0 corresponds to the enthalpy change of I6.4, and ΔH t corresponds to the enthalpy changes of the other CPI obtained. Color Color was evaluated (CIELab parameters) using a Chroma Meter CR-300C colorimeter (MINOLTA, Tokyo, Japan). Lightness (L*), equilibrium between green and red (a*), and equilibrium between yellow and blue (b*), were measured and total color difference (ΔE*) and browning index (BI), were calculated following Adal et al. (2024) (Eq. 5 and Eq. 6) using the I6.4 as reference. Measurements were performed ten times for each sample, and the average value was reported. \(\:{\varDelta\:E}^{*}={({\left({\varDelta\:L}^{*}\right)}^{2}+{\left({\varDelta\:a}^{*}\right)}^{2}+{\left({\varDelta\:b}^{*}\right)}^{2})}^{1/2}\) (Eq. 5) \(\:BI=\frac{100}{0.17}x(\frac{{a}^{*}+1.75{L}^{*}}{5.645{L}^{*}+{a}^{*}-3.012{b}^{*}}-0.31)\) (Eq. 6) Protein solubility (S 0 ) S 0 was evaluated over a pH range from 2.0 to 10.0 in distilled water, following the method described by Fernández Sosa et al. ( 2021a ). S 0 was expressed as the percentage ratio between the soluble protein in the supernatants determined by the Lowry method (Lowry et al., 1951 ) and the total protein content measured by the Kjeldahl method (AOAC, 1990). Bovine serum albumin was used as a standard. Preparation of oil-in-water emulsions CPI dispersions (1.5% w/w) were stirred (1 h, 25°C) and used to prepare o:w emulsions with sunflower oil (1/5). The two phases were premixed (20,000 rpm; 1 min) with an Ultra-Turrax T10 rotor/stator homogenizer (IKA, Staufen, Germany). Then, the emulsions were further homogenized with a VCX 500 ultrasonic processor equipped with a 13 mm diameter titanium probe, model 630 − 0219 (Sonics & Materials Inc., Newtown, USA), maintaining the temperature below 30°C (375 W, 5 min, pulse mode: 30 s on, 30 s off; ice bath). Particle size distribution The particle size distribution of emulsions (freshly prepared and stored at 4°C for 24 h) was determined using an Analysette 22 NeXT laser diffraction system (Fritsch GmbH, Idar-Oberstein, Germany), without and with SDS (1% w/v). To avoid multiple scattering effects, samples were dispersed in 600 mL of water at 2,000 rpm. The optical parameters were refractive index for oil (1.47) and water (1.33), and the adsorption coefficient (0.001). The surface average diameter [D 3,2 ] and the volume average diameter [D 4,3 ] were determined. The variation of [D 4,3 ] values at different times (initial and after one day), in the absence and presence of SDS, was used to calculate the flocculation index (FI) and the coalescence index (CI), following Palazzolo et al. (2005) (Eqs. 7 and 8). \(\:FI=\frac{{D}_{\text{4,3}t}-{D}_{\text{4,3}t+SDS}}{{D}_{\text{4,3}t+SDS}}\) (Eq. 7) \(\:CI=\frac{{D}_{\text{4,3}t+SDS}-{D}_{\text{4,3}i+SDS}}{{D}_{\text{4,3}t+SDS}}\) (Eq. 8) Where D 4,3t is the D 4,3 value at a given time t, D 4,3t+SDS ​is the D 4,3 ​ value at a given time t in the presence of SDS, and D 4,3i+SDS is the initial D 4,3 ​ value in the presence of SDS. Statistical analysis CPI obtained at each extraction condition was prepared in triplicate. All analytical determinations described in Sections 2.3 to 2.12 were conducted in triplicate. Statistical differences between the samples were determined by analysis of variance (ANOVA) (Tukey’s test, p < 0.05), using InfoStat software (Di Rienzo et al., 2017 ). Results and discussion Protein extractability The protein content of the CPI extracted at pH 6.4 and by the pH-shift method ranged from 79.84% to 83.00% (Table 1 ). The yield in weight and protein yield recovery were not significantly affected by increasing the extraction pH (no significant differences among pH 7.5, 8.0, and 9.0); however, these values were higher (p < 0.05) than those obtained at pH 6.4 (Table 1 ). Similar protein contents and extraction yields have been reported for alkaline protein extraction from various legumes, including green lentil, red lentil, black lentil, mung bean, yellow pea, pigeon pea, and cowpea (Shrestha et al., 2023 ; Fernández Sosa et al., 2021a ; Gómez et al., 2021 ; Peyrano et al., 2017 ). Table 1 Protein content, yield in weight, protein yield recovery and water activity of cowpea protein isolates. Sample Protein content (%) Yield in weight (%) Protein yield recovery (%) a w I6.4 79.84 ± 0.60bcd 11.32 ± 0.00a 35.29 ± 0.27a 0.460 ± 0.001g I6.4-US 76.92 ± 0.85a 18.70 ± 0.00cd 56.19 ± 0.62c 0.546 ± 0.001h I7.5 80.39 ± 0.44bcd 17.06 ± 0.00b 53.57 ± 0.29b 0.434 ± 0.001f I7.5-US 81.65 ± 2.09cd 19.32 ± 0.46d 61.13 ± 1.57d 0.396 ± 0.000d I8 81.13 ± 2.36bcd 17.19 ± 0.05bc 54.48 ± 1.59bc 0.403 ± 0.001e I8-US 78.92 ± 1.82abc 19.37 ± 0.02d 59.68 ± 0.98d 0.350 ± 0.001b I9 83.00 ± 0.03d 17.81 ± 1.26bcd 56.47 ± 0.02c 0.337 ± 0.001a I9-US 77.85 ± 0.27ab 18.74 ± 1.12cd 56.09 ± 0.19bc 0.391 ± 0.001c Data are the mean ± standard deviation. Different letters in a column indicate significant differences (p < 0.05) between samples. The yield in weight of CPI extracted with HIUS under each pH condition was also evaluated. At pH 6.4, 7.5, and 8.0, significant increases were observed (64.19%, 13.25%, and 12.62%, respectively), along with higher protein yield recovery (59.22%, 14.11%, and 9.54%, respectively). The acoustic cavitation induced by HIUS promotes particle size reduction, thereby increasing the contact surface area between the plant matrix and the solvent, which enhances mass transfer and facilitates extraction (Quintero-Quiroz et al., 2022 ). However, the protein content of I6.4-US (76.92%) was lower than that of I6.4 (79.84%) (p < 0.05), indicating that HIUS-assisted extraction may also promote the co-extraction of non-protein constituents by further disrupting cellular structures and increasing the solubilization of intracellular compounds such as carbohydrates, phenolics, and soluble dietary fibers (Yusoff et al., 2022 ). This effect reduced the isolated protein content, despite the significantly higher protein recovery obtained. At pH 9.0, HIUS did not significantly increase weight or protein yield; in fact, the protein content decreased (Table 1 ), likely due to protein denaturation at this pH, which could limit the ability of HIUS to disrupt the protein matrix and, consequently, reduce extraction efficiency. Suchintita Das et al. ( 2023 ) reported that the sono-physical and sono-chemical effects of HIUS help break protein–polysaccharide interactions in the faba bean matrix, releasing more proteins into the solvent, and they observed results similar to ours when comparing HIUS-assisted extraction of faba bean proteins in water and in an alkaline solution (pH 10). Overall, our results demonstrate that HIUS-assisted extraction enhances the solubilization of cowpea proteins at pH 6.4, 7.5, and 8.0, achieving extraction yields comparable to or even higher than those obtained at pH 9.0. This improvement increases the overall efficiency of the process by reducing the use of highly alkaline solutions and minimizing potential nutritional drawbacks associated with undesirable chemical reactions at elevated pH values (Momen et al., 2021 ; Long et al., 2025 ). While extraction yield and protein content determine the efficiency of the isolation process, the storage stability of the resulting protein isolates is equally important for their practical application. Therefore, the water activity (a w ) of the obtained CPI was measured, since it reflects the fraction of unbound water in the food matrix and plays a key role in chemical reactions, microbial growth, and physical stability. The a w values of CPI varied significantly depending on extraction pH and ultrasound application, ranging between 0.546 and 0.337. Shih et al. ( 2016 ) reported that an Aw of 0.25 led to fewer alterations during storage compared to an Aw of 0.75, significantly affecting the physicochemical and techno-functional properties of soy protein isolates. Generally, the a w decreased with the increasing pH (Table 1 ). The application of HIUS increased a w values at pH 6.4 and pH 9.0 (p < 0.05) and decreased a w values at pH 7.5 and 8.0. The increment of aw (16–19%) could be related to the minor protein content of I6.4-US and I9-US, and as a consequence, a decrease in water-protein interaction (Table 1 ). Although the decrease of aw (9–13%) in I7.5-US and I8-US could be due to a structural reorganization that improves the water-protein interactions and decreases the unbound water in the protein matrix. Overall, these results indicate that aw is governed by both protein content and the structural characteristics of the isolates resulting from the extraction process. Modifications of the protein structure Electrophoretic profile The electrophoretic profiles of the CPI were obtained by SDS-PAGE to determine the molecular weights of the polypeptide chains, which provides information about the primary structure of the proteins (Rahman & Lamsal, 2021 ). Under non-reducing conditions (Fig. 1 A), the I6.4 presented polypeptides of 84, 66, and 56 kDa, corresponding to the 7S globulin fraction, known as vicilin, as well as polypeptides of 94, 49, 33, 30, 27, 23, and 19–17 kDa, which are associated with the albumin fraction. An additional polypeptide of 38 kDa and an increase of band intensity at 46 and 56 kDa (particularly at pH 7.5 and pH 8.0), and at 33, 30, and 27 kDa of CPI obtained at alkaline pH were observed. Also, polypeptides with molecular weights greater than 94 kDa and soluble aggregates in the stacking gel were observed. These results suggest that alkaline pH extraction not only facilitates the recovery of additional polypeptides but also induces a rearrangement of the protein structure, leading to protein denaturation. These results are consistent with those reported by Peyrano et al. ( 2016 ). Under reducing conditions (Fig. 1 B), the presence of disulfide bonds was confirmed by the absence of soluble aggregates in the stacking gel, and a decrease in band intensity above 94 kDa, as well as those at 84 and 38 kDa associated to an increase of intensity at 29 and 20 kDa bands. All electrophoretic profiles of CPI obtained by HIUS-assisted extraction at alkaline pH were similar, consistent with the inability of HIUS to break covalent or peptide bonds, leaving the polypeptide composition unaltered. Similar behavior for protein isolates obtained via HIUS-assisted extraction has been reported for quinoa, black bean, lentil (Quintero-Quiroz et al., 2022 ), and soy (Rahman & Lamsal, 2021 ). Secondary structure The secondary structure of proteins refers to the local folding patterns that depend on both the amino acid sequence and interactions between atoms in the polypeptide backbone. Experimentally, it is determined by evaluating the relative proportions of α-helix, β-sheet, β-turn, and random coil structures (Rahman & Lamsal, 2021 ). CPI exhibited the characteristic amide I band of proteins, which, due to its sensitivity to conformational changes, allows the study of folding–unfolding and aggregation processes (Fernández Sosa et al., 2021a ). Deconvolution of the amide I band enabled calculation of the relative content of secondary structural elements (α-helix, β-sheet, β-turns, and random coil) (Table 2 ). The I6.4 showed high proportions of β-sheets and β-turns, with very low α-helix and random coil contents, indicating a highly ordered and compact conformation with limited flexibility. Increasing the extraction pH significantly raised α-helix content at the expense of β-structures (β-turns and β-sheets) (Table 2 ), suggesting that alkaline conditions promoted partial unfolding and refolding of the protein backbone. Nevertheless, all CPI obtained via the pH-shift process retained a predominance of β-sheet structures, indicating that the overall organization remained largely governed by intermolecular hydrogen-bonding typical of legume storage proteins. Similar β-sheet dominance has been reported for CPI regardless of extraction pH (8.0 and 10.0) (Gómez et al., 2021 ) and for commercial soy protein isolates (Hu et al., 2013 ). Table 2 The positions and the relative content of secondary structure under the amide I region of cowpea protein isolates. Sample β-sheet (1610–1640 cm − 1 and 1680–1695 cm − 1 ) α-Helix (1650–1660 cm − 1 ) β-Turn (1660–1680 cm − 1 ) Random coil (1640–1650 cm − 1 ) I6.4 60.72 ± 0.21d 3.25 ± 1.70a 34.48 ± 1.73e 1.55 ± 0.17a I6.4-US 49.70 ± 0.38a 11.79 ± 0.99b 22.24 ± 0.16c 16.26 ± 0.39d I7.5 56.00 ± 3.81c 22.24 ± 3.46de 23.38 ± 1.45bc 1.38 ± 0.20a I7.5-US 52.07 ± 0.67ab 2.00 ± 0.68a 27.41 ± 0.22d 18.93 ± 0.50e I8 53.44 ± 0.08bc 16.93 ± 0.07c 20.99 ± 0.54bc 8.64 ± 0.54c I8-US 55.47 ± 0.13c 19.91 ± 0.77d 18.55 ± 0.03ab 6.07 ± 0.03b I9 56.00 ± 2.44c 25.06 ± 0.34e 16.63 ± 0.42a 2.31 ± 0.42a I9-US 51.68 ± 0.14bc 22.14 ± 3.40de 22.86 ± 1.02c 3.32 ± 2.25a Data are the mean ± standard deviation. Different letters in a column indicate significant differences (p < 0.05) between samples. HIUS-assisted extraction further modified CPI secondary structure in a pH-dependent manner (Table 2 ). I6.4-US exhibited increased α-helix and random coil contents, with reduced β-turns and β-sheets, indicating partial unfolding and a more flexible conformation. In I7.5-US, HIUS decreased α-helix and β-sheet contents while increasing random coil and β-turn structures, resulting in a more disordered state. Conversely, I8-US showed a more ordered structure, with higher α-helix and lower random coil contents. I9-US displayed variable effects on α-helix and β-turn contents, maintaining an inverse relationship between these elements. Hydrogen bonds and electrostatic interactions stabilize protein secondary structures, and HIUS likely disrupts these interactions, inducing conformational changes (Rahman & Lamsal, 2021 ). Comparable effects have been observed in commercial soy protein isolates treated with HIUS (400 W, 30 min) (Hu et al., 2013 ) and in whey protein concentrate, where α-helix content slightly increased while β-sheet and β-turn contents slightly decreased (450 W, 30 min) as determined by circular dichroism (Chandrapala et al., 2011 ). Adal ( 2024 ) further noted that, although proteins can partially refold after HIUS, Faba bean protein isolates do not regain the native structure of untreated samples. Tertiary structure Tryptophan residues, the primary intrinsic fluorophores in proteins, exhibit a maximum emission wavelength (λ max ) in water at 348 nm (Fernández Sosa et al., 2021a ). The I6.4 exhibited a λ max of 340.23 ± 0.58 nm (Table 3 ), which describes a polar environment (Rahman & Lamsal, 2021 ). The pH-shift extraction did not result in significant changes in λ max compared to the I6.4 (p > 0.05). However, a slight red shift tendency (higher λ max values; major exposure to a more polar environment) was observed at pH 7.5 and 8.0, whereas a slight blue shift tendency (lower λ max values; major exposure to a less polar environment) was noted at pH 9.0 (p < 0.05). Peyrano et al. ( 2016 ) reported similar behavior for CPI when the extraction pH increased from 8.0 to 10.0. Table 3 Fluorescence spectroscopy and surface hydrophobicity of cowpea protein isolates. Sample λ máx (nm) Fi (Fi/mg/mL protein) H 0 (Fi/mg/mL protein) I6.4 340.23 ± 0.58ab 1.53x10 4 ± 9.63x10 1 bc 1584 ± 11ab I6.4-US 340.90 ± 0.00b 1.34x10 4 ± 5.26x10 2 a 1706 ± 25c I7.5 340.90 ± 0.00b 1.56x10 4 ± 2.46x10 2 bc 1487 ± 11ab I7.5-US 339.90 ± 0.00a 1.66x10 4 ± 1.00x10 3 c 1957 ± 23cd I8 340.90 ± 0.00b 1.42x10 4 ± 9.46x10 1 ab 1333 ± 186a I8-US 339.90 ± 0.00a 1.42x10 4 ±1.21x10 2 ab 2190 ± 66de I9 339.90 ± 0.00a 1.55x10 4 ± 1.07x10 3 bc 2473 ± 11ef I9-US 339.90 ± 0.00a 1.51x10 4 ± 3.59x10 2 abc 2693 ± 53f Fi: Intrinsic intensity; H 0 : Surface hydrophobicity. λmax: maximum emission wavelength. Data are the mean ± standard deviation. Different letters in a column indicate significant differences (p < 0.05) between samples. The HIUS-protein extraction at pH 6.4 resulted in non-significant differences in λ max but a significant decrease in Fi, which could be attributed to an increased distance between Tryptophan and Tyrosine residues due to the unfolding process (Fernández Sosa et al., 2021a ), by reducing the energy transfer and fluorescence intensity. The HIUS-assisted protein extraction at pH 7.5 and 8.0 resulted in a significant decrease in λ max of CPI obtained, suggesting a major exposure of Tryptophan residues to a more hydrophobic environment, reaching similar values to those obtained for I9 (Table 3 ). The high alkaline concentration during protein extraction at this pH could modify the microenvironment of Tryptophan toward a more shielded one, modifying its sensitivity to treatments (Peyrano et al., 2016 ) and resulting in non-significant differences of λ max between protein isolates extracted with and without HIUS. When HIUS-assisted protein extraction was performed at alkaline pH, an additional protein unfolding and reorganization of protein structure may have occurred. This could explain the absence of significant changes in Fi spectra compared to CPI obtained without HIUS. The measurement of protein surface hydrophobicity (H 0 ) provides insight into the number of hydrophobic groups exposed on the molecular surface in a polar aqueous environment. It is commonly used to assess changes in protein conformation and stability, as well as their impact on techno-functional properties (Tang et al., 2021 ; Hu et al., 2015 ). The pH-shift extraction at pH 9.0 resulted in a significant increase in the H 0 of I9 compared to the other protein isolates (p < 0.05) (Table 3 ). Peyrano et al. ( 2016 ) also reported a significant increase in the H 0 of CPI when the extraction pH increased from 8.0 to 10.0. Besides, the HIUS-assisted protein extraction resulted in a significant increase in H 0 at pH 6.4 (7.67%), pH 7.5 (31.60%), and pH 8.0 (64.28%) (p < 0.05), meanwhile, non-significant changes were observed at pH 9.0. These results are consistent with Fi data, indicating that HIUS-assisted extraction at these pH levels induces a certain degree of protein unfolding, resulting in greater exposure of hydrophobic groups to the polar environment. In contrast, at pH 9.0, a structural refolding likely occurs, preventing further modification of this property. The H 0 values obtained also explain structural modifications that influence the a w values reported (Table 1 ). Rahman & Lamsal, ( 2021 ) reported an increase in H 0 of soy proteins induced by HIUS, which was more pronounced with increasing treatment time and power. On the other hand, the H 0 values of I9-US and I9 were not significantly different, but were the highest, and similar to that reported by Peyrano et al. ( 2016 ) for CPI obtained at pH 10.0 without HIUS. Thermal properties The thermal stability of proteins is commonly assessed by differential scanning calorimetry (DSC), where an endothermic peak reflects alterations in the higher-order structure of the protein (Garcia de Oliveira et al., 2024 ). Thermograms of all CPI exhibited a single major endothermic peak (data not shown), corresponding to the 7S globulins, the predominant fraction identified in the electrophoretic profiles. This peak was asymmetric and spanned a broad temperature range, which is attributed to the overlap of multiple denaturation events occurring in the same region, a typical feature of seed-derived proteins (Garcia de Oliveira et al., 2024 ; Lefèvre et al., 2022 ; Mession et al., 2013 ). The DSC parameters are presented in Table 4 . ΔH represents the energy required to induce denaturation, while T d indicates the temperature at which the protein denatures (Mir et al., 2019 ). The highest ΔH value was recorded for I6.4, indicating greater structural stability under this condition. Alkaline extraction resulted in a reduction in ΔH, suggesting a decreased structural integrity, possibly due to protein unfolding or conformational changes induced. The T d of I6.4 was 86.12 ± 0.06°C, which is consistent with values previously reported for cowpea protein concentrates (Garcia de Oliveira et al., 2024 ). The extraction at pH 7.5 and pH 8.0 did not significantly affect the T d ; however, extraction at pH 9.0 resulted in greater sensitivity to thermal treatment, as evidenced by a significant decrease in T d to 85.24 ± 0.04°C. This value is higher than that reported by (Peyrano et al., 2016 ) for CPI, which can be attributed to differences in protein concentration and heating rate during the calorimetric analysis (Peyrano et al., 2017 ). Table 4 Thermal properties of cowpea protein isolates. Sample ΔH (J/g dry protein) T d (°C) DD (%) I6.4 11.21 ± 0.29e 86.12 ± 0.06b - I6.4-US 8.58 ± 0.01bc 86.22 ± 0.14b 23.53 ± 0.04bc I7.5 9.36 ± 0.09cd 86.31 ± 0.21b 16.53 ± 0.76ab I7.5-US 8.13 ± 0.12ab 86.27 ± 0.18b 27.53 ± 1.08cd I8 9.87 ± 0.14d 86.71 ± 0.32bc 12.04 ± 1.24a I8-US 8.81 ± 0.01bc 87.40 ± 0.05d 21.48 ± 0.07bc I9 8.51 ± 0.45bc 85.24 ± 0.04a 24.10 ± 1.04c I9-US 7.61 ± 0.20a 86.98 ± 0.01cd 32.10 ± 1.76d ΔH: enthalpy change of transition; Td: denaturation temperature; DD: degree of protein denaturation. Data are the mean ± standard deviation. Different letters in a column indicate significant differences (p < 0.05) between samples. HIUS-assisted extraction significantly reduced ΔH across all evaluated pH values (p < 0.05), indicating enhanced protein denaturation associated with structural and conformational alterations in CPI caused by bond disruption. I9-US exhibited the lowest ΔH values and the highest DD (32.10%). As mentioned in the previous section, this isolate presented the highest H 0 , suggesting that the high concentration of OH⁻ during extraction may have already promoted prior protein denaturation. This increased exposure of hydrophobic regions upon HIUS-assisted extraction may have favored the formation of insoluble aggregates through hydrophobic and protein–protein interactions, thereby limiting the recovery of soluble proteins. As a consequence, HIUS-assisted extraction did not affect the extraction yields of I9-US and had no significant effect on T d of I6.4-US and I7.5-US; however, HIUS increased T d values of I8-US and I9-US. This suggests that HIUS may induce a distinct reorganization of denatured proteins under highly alkaline conditions, enhancing thermal stability compared to samples extracted without HIUS, with potential implications for food formulation and processing strategies. Techno-functional properties Plant proteins have gained attention as promising food ingredients owing to their functional properties, such as high solubility and emulsifying capacity, among others (Thompson et al., 2023 ; Fernández Sosa et al., 2021b ; Peyrano et al., 2017 ). The operating procedures used to extract proteins from plants influence their molecular structure and aggregation state, which is mainly related to their functional performance (Hadidi et al., 2023 ). Color Freeze-dried CPI can be used as an ingredient in a wide range of food formulations. Since color is a key sensory attribute that strongly influences consumer acceptance, the evaluation of CPI colour obtained is of considerable importance (Ma et al., 2022 ). All CPI samples obtained presented a light brown color, with positive values for both a* and b* parameters (Table 5 ). The markedly higher b* values contributed to a yellowish appearance (Fig. 2 ). The I6.4 exhibited the highest L* and the lowest BI value; meanwhile, I7.5 and I9 presented the higher differences of ΔE* and BI with non-significant differences between them (p > 0.05), suggesting that the extraction pH has a significant and variable effect on the color parameters. Table 5 Color parameters of the cowpea protein isolates. Sample a* b* L* ΔE* BI I6.4 0.65 ± 0.02a 10.89 ± 0.17a 86.85 ± 0.43f - 14.41 ± 0.47a I6.4-US 2.30 ± 0.04e 15.45 ± 0.16g 80.26 ± 0.19d 8.19 ± 0.42c 23.04 ± 0.22g I7.5 2.30 ± 0.03e 14.63 ± 0.06f 77.50 ± 0.16b 10.21 ± 0.41e 22.67 ± 0.15fg I7.5-US 2.19 ± 0.04d 13.54 ± 0.12cd 78.25 ± 0.39c 9.14 ± 0.76d 20.66 ± 0.28d I8 1.77 ± 0.10c 13.29 ± 0.29c 80.06 ± 0.38d 7.30 ± 0.61b 19.39 ± 0.53c I8-US 1.41 ± 0.01b 11.18 ± 0.17b 81.80 ± 0.25e 5.13 ± 0.55a 15.64 ± 0.30b I9 2.36 ± 0.06e 14.23 ± 0.23e 76.57 ± 0.31a 10.95 ± 0.73e 22.40 ± 0.52f I9-US 2.36 ± 0.03e 13.69 ± 0.14d 77.00 ± 0.26a 10.39 ± 0.37e 21.42 ± 0.32e ΔE*: Total color difference; BI: Browning index. Data are the mean ± standard deviation. Different letters in a column indicate significant differences (p < 0.05) between samples. HIUS-protein extraction associated with pH resulted in varied color parameter changes. I6.4-US showed a decrease in L* and an increment of BI, with ΔE* value of 8.19, which could be attributed to the extraction of non-protein components mentioned in section 3.1. I7.5-US and I8-US showed an increase in L* and a significant reduction in both BI and ΔE* (p < 0.05), and I9-US presented non-significant changes in L* and ΔE*, but a decrease in the BI was found (Fig. 2 ). Adal ( 2024 ) reported that ΔE* and BI reduction may be attributed to HIUS, since sonication induced pigment degradation and a decrease in protein particle size, which enhances multiple light scattering and consequently increases sample whiteness. Protein solubility (S 0 ) S 0 is a critical techno-functional property, as it directly influences other properties of interest to the food industry, such as gelation, emulsification, and foaming capacity (Marinacci et al., 2025 ). Consequently, numerous structural modification strategies have been employed to improve protein solubility. Understanding the pH-dependent behavior of plant protein solubility is therefore essential for developing efficient extraction and isolation methods from plant-based raw materials. All S 0 profiles of CPI exhibited a similar pattern as a function of pH, following a characteristic U-shaped curve between pH 2.0 and pH 10.0 (Fig. 3 ). Maximum S 0 (~ 100%) was observed between pH 8.0 and pH 10.0, whereas the minimum S 0 (~ 3.5%) occurred between pH 3.0 and pH 5.0. Similar results were reported by Gómez et al., ( 2021 ) and Shevkani et al., ( 2019 ). HIUS-assisted extraction significantly improved S 0 at pH 4.0 of I6.4-US (16%), I7.5-US (64%), and I9-US (64%), and at pH 6.0 of I6.4-US (64%), I7.5-US (23%), I8-US (28%) and I9-US (29%), which is particularly relevant given that most food systems have a pH close to neutrality. Characteristics of emulsions: particle size distribution The ability of plant proteins to form and stabilize emulsions is highly dependent on the nature of the proteins and on the extraction method used (McClements et al., 2022 ). The size of the dispersed particles also plays a main role in emulsion stability (Thompson et al., 2023 ). All emulsions exhibited a monomodal particle size distribution, both in the absence and presence of 1% SDS. All emulsions were formulated at pH 7.0, ensuring that proteins were almost fully dissolved in the aqueous phase during homogenization (S 0 : 86.09–95.23%). This high S 0 enhanced protein migration and adsorption at the oil–water interface, thereby promoting both emulsion formation and stabilization. No noticeable changes in the visual appearance of the formulated emulsions were observed as pH increased. Freshly prepared emulsions with pH-shift protein extraction showed a decrease in D 3.2 , D 4.3 , and FI 0 values when pH increased (Table 6 ), which is in accordance with the protein modifications mentioned in sections 3.2.2 and 3.3. Table 6 Particle size, span, flocculation index, and coalescence index of o:w emulsions stabilized with cowpea protein isolates. Sample D 3,2 D 4,3 Span FI 0 FI 1 CI 1 I6.4 0.979 ± 0.002f 1.234 ± 0.006f 1.182 ± 0.005ab 0.104 ± 0.009dA 0.126 ± 0.036A 0.000 ± 0.000a I6.4-US 0.822 ± 0.004cd 1.046 ± 0.005d 1.178 ± 0.004a 0.303 ± 0.017eB 0.075 ± 0.039A 0.246 ± 0.018b I7.5 0.902 ± 0.002e 1.159 ± 0.005e 1.180 ± 0.003ab 0.079 ± 0.002bcA 0.083 ± 0.002B 0.000 ± 0.000a I7.5-US 0.796 ± 0.009b 1.005 ± 0.007b 1.195 ± 0.005c 0.044 ± 0.007aA 0.038 ± 0.015A 0.005 ± 0.012a I8 0.830 ± 0.001d 1.049 ± 0.001d 1.189 ± 0.001bc 0.085 ± 0.004cdA 0.072 ± 0.015A 0.012 ± 0.003a I8-US 0.757 ± 0.009a 0.957 ± 0.008a 1.225 ± 0.002d 0.057 ± 0.011aA 0.051 ± 0.008A 0.000 ± 0.000a I9 0.810 ± 0.003c 1.018 ± 0.005bc 1.193 ± 0.005c 0.060 ± 0.004abA 0.067 ± 0.009A 0.000 ± 0.000a I9-US 0.823 ± 0.003d 1.027 ± 0.005c 1.175 ± 0.004a 0.058 ± 0.009aA 0.055 ± 0.015A 0.000 ± 0.000a D 3,2 : surface average diameter of freshly o:w emulsions; D 4,3 : volume average diameter of freshly o:w emulsions; FI 0 : flocculation index of freshly o:w emulsions of o:w emulsions; FI 1 : Flocculation index of o:w emulsions at one day of storage; CI 1 :Coalescence index of o:w emulsions at one day of storage of o:w emulsions. Data are the mean ± standard deviation. Different lowercase letters within a column indicate significant differences and different uppercase letters between rows indicate significant differences (p < 0.05). Emulsions formulated with I6.4-US, I7.5-US, and I8-US exhibited significantly lower D 3.2 and D 4.3 values, indicating greater efficiency in forming emulsions with smaller droplet sizes. Moreover, the FI 0 decreased in emulsions prepared with I7.5-US and I8-US. As detailed in Section 3.2.3, HIUS-assisted extraction increased H 0 , particularly at pH 7.5 and 8.0. The enhanced exposure of hydrophobic groups on the protein surface facilitated their adsorption at the oil–water interface, promoting the formation of emulsions with smaller droplet sizes and probably greater stability. The analysis of emulsion stability after 24 h revealed that, except I6.4-US, all samples exhibited a CI equal to or close to zero. Regarding the FI, a decrease was observed in I6.4-US, whereas an increase was observed in I7.5. No significant changes in FI were observed in the remaining samples, suggesting consistent stability over 24 h. Conclusions HIUS-assisted protein extraction notably improved protein yield of I6.4-US, I7.5-US, and I8-US, by enhancing mass transfer and disrupting plant cell structures, with extraction yields and protein solubility comparable to or higher than those obtained for I9, while avoiding the excessive use of highly alkaline solutions that may induce protein denaturation and reduce nutritional quality. However, it also promoted the co-extraction of non-protein components, particularly in I6.4-US. Structural analyses revealed that HIUS altered the secondary and tertiary structures of proteins by inducing partial unfolding, increasing surface hydrophobicity, and decreasing thermal stability, effects that were also dependent on pH. These conformational changes correlated with the improvement in solubility at pH 6.0 and the enhancement of emulsifying capacity, especially for I7.5-US and I8-US, where smaller emulsion droplet sizes were obtained. Overall, the combined application of HIUS and pH-shift extraction emerges as a promising strategy to obtain plant protein isolates with techno-functional properties. Additionally, HIUS-assisted extraction at pH 6.4 represents a promising alternative to pH shift method, combining efficiency, functionality, and sustainability, and supporting the development of plant-based protein ingredients with minimal chemical intervention. Abbreviations 2-ME 2-mercaptoethanol ANS 1,8-aniline–naphthalene-sulfonate ANOVA Analysis of Variance CI coalescence index CPI cowpea protein isolates D 3,2 surface average diameter D 4,3 volume average diameter ΔH enthalpy change of transition DD degree of protein denaturation FI flocculation index Fi fluorescence intensity FTIR Fourier transform infrared HIUS high-intensity ultrasound treatment H 0 surface hydrophobicity λ max maximum emission wavelength MW molecular weight S 0 protein solubility SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis T d denaturation temperature Declarations Declaration of Competing Interest The authors declare that they have no competing interests. Funding This work was supported by Universidad Nacional del Nordeste (UNNE), Argentina (Grant numbers 20F0009 and 24F010). Carlos Fernando Calgaro acknowledges CONICET (Argentina) for the award of a doctoral fellowship. Author Contribution Carlos Fernando Calgaro: Investigation, Data curation, Formal analysis, Methodology, Validation, Visualization, Writing – Original Draft Preparation.Belén Andrea Acevedo: Conceptualization, Data curation, Funding acquisition, Project administration, Resources, Supervision, Validation, Visualization, Writing – Review & Editing.Felicitas Peyrano: Conceptualization, Investigation, Data curation, Formal analysis, Methodology, Resources, Validation, Visualization, Writing – Review & Editing.Mercedes Carolina Rasia: Investigation, Data curation, Resources, Validation, Writing – Review & Editing.María Guadalupe Chaves: Conceptualization, Data curation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – Review & Editing. Acknowledgement The authors wish to thank Andrea Gisella Gomez for her kind help and suggestions during the SDS-PAGE and FTIR assays. 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Sultan, Z., Ashfaq, A., Jahan, K., Qadri, O. S., Younis, K., & Yousuf, O. (2024). pH shift extraction technique for plant proteins: A promising technique for sustainable development. Energy Nexus , 16 , 100329. https://doi.org/10.1016/j.nexus.2024.100329 Tang, S., Li, J., Huang, G., & Yan, L. (2021). Predicting protein surface property with its surface hydrophobicity. Protein and Peptide Letters, 28 (8), 938–944. https://doi.org/10.2174/0929866528666210222160603 Thompson, C. M. B., Acevedo, B. A., Añón, M. C., & Avanza, M. V. (2023). Emulsifying Capacity of Cowpea Protein Isolates. Effect of Thermal and Hydrolytic Treatment. Plant Foods for Human Nutrition , 78 (2), 366–374. https://doi.org/10.1007/s11130-023-01072-8 Yusoff, I. M., Mat Taher, Z., Rahmat, Z., & Chua, L. S. (2022). A review of ultrasound-assisted extraction for plant bioactive compounds: Phenolics, flavonoids, thymols, saponins and proteins. Food Research International , 157 , 111268. https://doi.org/10.1016/j.foodres.2022.111268 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8744459","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":585919827,"identity":"23343dd5-58ca-4649-91d1-9c175398d0e1","order_by":0,"name":"Carlos Fernando Calgaro","email":"","orcid":"","institution":"Facultad de Ciencias Exactas y Naturales y Agrimensura. Universidad Nacional del Nordeste (UNNE) and Instituto de Química Básica y Aplicada del Nordeste Argentino (IQUIBA NEA). UNNE-CONICET.","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"Fernando","lastName":"Calgaro","suffix":""},{"id":585919828,"identity":"cec49392-f306-406a-be03-d2504c4f4ef7","order_by":1,"name":"Belén Andrea Acevedo","email":"","orcid":"","institution":"Facultad de Ciencias Exactas y Naturales y Agrimensura. Universidad Nacional del Nordeste (UNNE) and Instituto de Química Básica y Aplicada del Nordeste Argentino (IQUIBA NEA). UNNE-CONICET.","correspondingAuthor":false,"prefix":"","firstName":"Belén","middleName":"Andrea","lastName":"Acevedo","suffix":""},{"id":585919829,"identity":"0c121cbb-dd2a-4581-baa8-7636afbc6e78","order_by":2,"name":"Felicitas Peyrano","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYBAC9gbmhgMMBmAWkjAPHi08BxhBWgxALBK0ACmgFokEYrWwNzYe+FHwR15+5uNjEj8YDueZszcwPnjbxiBv3oBDC8/BhoM9BgaGG26nJRv2MBwutuw5wGw4t43BcM4B7FrsJRIbDgP9wrhBOsfwAQ/D4cQNNxLYpHnbGBhn4HKY/EOwFvv5M89/OPgHpOX+A/bfQC32OLVIMIK1JDbc4GF8DLGFgY0ZqCURpxaeRJBfjJM3nEkzNpYxSE/ccCaxWXLOOYlknFrYDx/+8OOPnO389sPPJN9UWCduOH744Ic3ZTa2uLSgAVAyYADHlARxGkbBKBgFo2AUYAUAPkZcSnlAQWUAAAAASUVORK5CYII=","orcid":"","institution":"Centro de Investigación en Química e Ingeniería Teórica y Experimental (QUITEX), Universidad Tecnológica Nacional, CONICET, FRRe","correspondingAuthor":true,"prefix":"","firstName":"Felicitas","middleName":"","lastName":"Peyrano","suffix":""},{"id":585919831,"identity":"f75de744-aa31-45f2-ba06-3415e820db25","order_by":3,"name":"Mercedes Carolina Rasia","email":"","orcid":"","institution":"Facultad de Ciencias de la Alimentación, Universidad Nacional de Entre Ríos e Instituto de Ciencia y Tecnología de los Alimentos de Entre Ríos (ICTAER) CONICET-UNER.","correspondingAuthor":false,"prefix":"","firstName":"Mercedes","middleName":"Carolina","lastName":"Rasia","suffix":""},{"id":585919833,"identity":"de013d9d-909e-4ae9-87d3-ca59eb39034d","order_by":4,"name":"María Guadalupe Chaves","email":"","orcid":"","institution":"Centro de Investigación en Química e Ingeniería Teórica y Experimental (QUITEX), Universidad Tecnológica Nacional, CONICET, FRRe","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Guadalupe","lastName":"Chaves","suffix":""}],"badges":[],"createdAt":"2026-01-30 19:24:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8744459/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8744459/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101942453,"identity":"537ae525-efca-4bb9-976b-2591e7a81208","added_by":"auto","created_at":"2026-02-05 09:27:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":402195,"visible":true,"origin":"","legend":"\u003cp\u003eElectrophoresis profile of CPI A) under non-reducing ((-)2ME) and B) reducing ((+)2ME) conditions.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8744459/v1/c181f045e1a28bb38d5a3231.png"},{"id":101942440,"identity":"7df96bd3-1d63-482d-bf05-33b44d57b4ac","added_by":"auto","created_at":"2026-02-05 09:27:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":328370,"visible":true,"origin":"","legend":"\u003cp\u003eColor of the freeze-dried cowpea protein isolates.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8744459/v1/21052b56ec7d053176f7d662.png"},{"id":101942437,"identity":"5501166d-f029-4a01-afc5-ec8b52bb64cd","added_by":"auto","created_at":"2026-02-05 09:27:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":78618,"visible":true,"origin":"","legend":"\u003cp\u003eProtein solubility profile of CPI as function of pH. A) I6.4 and I6.4-US, B) I7.5 and I7.5-US, C) I8 and I8-US and D) I9 and I9-US.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8744459/v1/ea41807a4bd418aca065afc0.png"},{"id":101942476,"identity":"c4ca1fcd-7cce-4b80-8280-89d0bbbb13cb","added_by":"auto","created_at":"2026-02-05 09:27:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2067427,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8744459/v1/23092623-4a27-44a7-a8a7-0b5fb37473f7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Impact of high-intensity ultrasound on cowpea protein extractability, structural, and techno- functional properties","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe growing demand for plant-based proteins has prompted the exploration of alternative sources such as cowpea (\u003cem\u003eVigna unguiculata\u003c/em\u003e). Cowpea, in particular, is a legume from the Fabaceae family cultivated in tropical and subtropical regions, with a global production estimated at approximately 5.8\u0026nbsp;million tons annually (Carneiro da Silva et al., 2019). The high protein content (22\u0026ndash;25%) and excellent nutritional profile (Avanza et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) make it a promising raw material not only for direct consumption in the form of whole seeds or flours, but also for the development of protein isolates as sustainable alternatives to conventional animal-derived proteins commonly employed in food formulations.\u003c/p\u003e \u003cp\u003eThe pH-shift method is one of the simplest and most widely used for obtaining protein isolates and several studies have shown that enhances protein yield and induces structural modifications (Nathia-Neves et al., 2025; Long et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Fern\u0026aacute;ndez Sosa et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). These changes significantly affect techno-functional properties, such as solubility, emulsifying capacity, foaming capacity, and gelling capacity (Nathia-Neves et al., 2025; Long et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Hadidi et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, exposure to elevated pH conditions can compromise the nutritional integrity of proteins trough the chemical degradation of essential residues such as lysine (Nathia-Neves et al., 2025; Long et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), and by diminishing their digestibility (Sultan et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). At industrial scale, protein extraction using highly alkaline solutions entails substantial chemical input and generates large volumes of high-pH effluents, making the method environmentally unsustainable without mitigation strategies (Patel et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCombining the pH-shift method with other technologies has been proposed to overcome the limitations of an alkaline protein extraction (Nathia-Neves et al., 2025; Long et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Hadidi et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Bernardi et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). One such technique is high-intensity ultrasound (HIUS), which promotes cell disruption and protein solubilization, and facilitates mass transfer. Acoustic cavitation generated by HIUS has been shown to alter the structural conformation of proteins, thereby modifying their physicochemical and techno-functional properties (Nathia-Neves et al., 2025; Long et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Bernardi et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMoreover, some research has reported the effect of varying energy input, treatment times, or a fixed ultrasound treatment condition combined with a pH-shift method for protein extraction from legumes like cowpea, raw pea flour, lupin, chickpea, peanut, and Faba bean, focusing on techno-functional properties (Nathia-Neves et al., 2025; Long et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Bernardi et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Haididi et al., 2023); Mendes Justino et al., (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) focused on the effect of HIUS on protein structure as well as its potential applications in the food industry. Loushigam \u0026amp; Shanmugam (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) informed that cowpea protein isolates extracted at pH 9.0 combined with HIUS (200 W; 10 min.) resulted in increased protein yield, solubility, water-holding capacity, foaming capacity, and stability, emulsion activity and stability, zeta potential, and in vitro protein digestibility. Quintero-Quiroz et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) informed an enhancement of protein yield and techno-functional properties of protein extraction from quinoa, lentils, and black beans at pH 9 (20 min, 320 W, seeds: solvent rate: 1:5). A comprehensive analysis of the physicochemical properties of cowpea protein isolates extracted by the pH-shift method combined with HIUS remains limited.\u003c/p\u003e \u003cp\u003eThis study aims to evaluate the impact of HIUS-assisted extraction (400 W, 60 min, pulse mode: 2 s on, 2 s off, ice bath) combined with the pH-shift method (7.5, 8.0, and 9.0) on parameters related to: a) the extraction process itself (protein extractability); b) protein structure modifications of cowpea protein isolates (CPI) (primary, secondary, and tertiary structures, thermal stability); c) the techno-functional properties of CPI (color, protein solubility, and emulsifying capacity). In addition, the impact of ultrasound extraction of cowpea protein at pH of the flour-water dispersions (6.4) was also assessed.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterial\u003c/h2\u003e \u003cp\u003eCowpea (\u003cem\u003eVigna unguiculata\u003c/em\u003e) seeds were provided by Estaci\u0026oacute;n Experimental El Sombrero-Corrientes (Instituto Nacional de Tecnolog\u0026iacute;a Agropecuaria-INTA) (crop 2022). Intact seeds (with seed coat) were ground using an HC-1000Y electric mill (Arcano, Tianjin, China) and sieved through an 80 ASTM mesh (177 \u0026micro;m). The resulting flour was defatted with hexane (10% w/v) under continuous stirring (24 h, 4 \u0026ordm;C). The mixture was filtered, and the flour was air-dried for 24 h at 25 \u0026ordm;C. The protein content was 25.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28% determined by the Kjeldhal method.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eProtein extraction\u003c/h3\u003e\n\u003cp\u003eProtein extraction was carried out following the method described by Fern\u0026aacute;ndez Sosa et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e) with slight modifications. Defatted cowpea flour-water dispersions (1:10, w/v) were adjusted to pH 7.5, 8.0, and 9.0 using 2 M NaOH. Additionally, protein extraction was conducted at the natural pH (6.4) of the cowpea flour\u0026ndash;water dispersion. The dispersions were then stirred for 1 h in the presence or absence of high-intensity ultrasound treatment (HIUS). The HIUS treatment was performed using a VCX 500 ultrasonic processor equipped with a 13 mm diameter titanium probe, model 630\u0026thinsp;\u0026minus;\u0026thinsp;0219 (Sonics \u0026amp; Materials Inc., Newtown, USA), maintaining the temperature below 30\u0026deg;C (400 W, 60 min, pulse mode: 2 s on, 2 s off, ice bath). Subsequently, both HIUS-treated and untreated dispersions were centrifuged (10,000 \u0026times; g, 30 min, 20\u0026deg;C). The supernatants were precipitated at the isoelectric point (pH 4.5, 4\u0026deg;C, 2 h) and then centrifuged (10,000 \u0026times; g, 20 min, 4\u0026deg;C). Then, proteins were dissolved in distilled water (pH 7.0 using 2 M NaOH), lyophilized, and stored (4\u0026deg;C). The CPI were named as I6.4, I6.4-US, I7.5, I7.5-US, I8, I8-US, I9, and I9-US, according to the extraction pH and the presence or absence of HIUS.\u003c/p\u003e\n\u003ch3\u003eProtein content and yield\u003c/h3\u003e\n\u003cp\u003eThe protein content of CPI and cowpea flour was determined using the Kjeldahl method (N\u0026times;6.25) (Association of Official Analytical Chemists [AOAC], 1990). The yield by weight and the protein recovery from total seed protein extraction were calculated (Eq.\u0026nbsp;1 and Eq.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:Yield\\:in\\:weight\\:\\left(\\%\\right)=\\frac{weight\\:of\\:sample}{weight\\:of\\:flour}x\\:100\\)\u003c/span\u003e \u003c/span\u003e (\u0026#119864;\u0026#119902;. 1)\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:Protein\\:yield\\:recovery\\:\\left(\\%\\right)=\\frac{Protein\\:content\\:of\\:sample}{protein\\:content\\:of\\:flour}x\\:100\\)\u003c/span\u003e \u003c/span\u003e (\u0026#119864;\u0026#119902;. 2)\u003c/p\u003e\n\u003ch3\u003eWater activity (A)\u003c/h3\u003e\n\u003cp\u003eAw of all CPI was evaluated using an AquaLab PRE (METER Group, Pullman, USA) at 25\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eSodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)\u003c/h3\u003e\n\u003cp\u003eSDS-PAGE was performed according to Laemmli's (1970) method. All gels were run in Mini-Protean Tetra Cell mini slabs (Bio-Rad, Hercules, USA) using a separating gel (12% w/v acrylamide) and a stacking gel (4% w/v acrylamide). CPI was dispersed (0.1% w/v protein) in sample buffer without (non-reduced conditions) or with 2-mercaptoethanol (reduced conditions, 2-ME) (5% v/v) and centrifuged (10,000 \u0026times; g, 20 min, 25\u0026deg;C). Electrophoresis was performed at a constant current of 25 mA per gel. Gels were fixed and stained with Coomassie Brilliant Blue dye solution (0.2% w/v) in water/methanol/acetic acid (5:5:2) and subsequently destained using a water/methanol/acetic acid (13:5:2). Protein molecular weights were estimated using low molecular weight markers (Pharmacia Hepar Inc., Franklin, USA).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFourier transform infrared (FTIR) spectroscopy\u003c/h2\u003e \u003cp\u003eAll CPI were analyzed using a Spectrum 3 FT-IR/NIR spectrometer (PerkinElmer, Waltham, USA) equipped with a universal ZnSe-ATR crystal. IR spectra (4,000\u0026ndash;400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were registered by co-adding 64 scans (4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e spectral resolution). The IR spectrum of the amide I band of proteins (1700\u0026ndash;1600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was deconvolved and fitted to Gaussian profiles using the equipment software Spectrum 10.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFluorescence spectroscopy\u003c/h3\u003e\n\u003cp\u003eAll CPI were dispersed (0.1% w/v protein) in buffer A (0.05 M Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, NaCl 0.15 M, pH 8), stirred (30 min, 25\u0026deg;C), and centrifuged (10,000 \u0026times; g, 30 min, 25\u0026deg;C). Fluorescence intensity spectra of the supernatants were measured FluoroMate FS-2 fluorescence spectrophotometer (Scinco, Seoul, South Korea) at excitation and emission wavelengths of 290 nm and 300\u0026ndash;400 nm, respectively (slit width, 5 nm; scanning speed of 300 nm/min; 25\u0026deg;C). Protein concentration was determined by the Lowry method (Lowry et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1951\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eSurface hydrophobicity (H)\u003c/h3\u003e\n\u003cp\u003eH\u003csub\u003e0\u003c/sub\u003e of CPI was determined according to Cardamone and Puri (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1992\u003c/span\u003e) using 1,8-aniline-naphthalene-sulfonate (ANS) as a fluorescent probe (Aldrich Chemical Co., Milwaukee, Wisconsin, USA). The emission spectra (400\u0026ndash;600 nm) of samples in buffer A (0.01\u0026ndash;0.03% w/v protein) equilibrated with different ANS concentrations (0.0 to 100 \u0026micro;M) were recorded. Fluorescence measurements were corrected by subtracting the corresponding blank to obtain the increased fluorescence (ΔFi) due to ANS binding. The ΔFi at 465 nm (λ of maximum emission of ANS-protein complex) was plotted vs. the ANS concentration (\u0026micro;M), and data were adjusted with the following equation:\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\varDelta\\:Fi=\\frac{A\\:x\\:ANS}{B+ANS}\\:\\)\u003c/span\u003e \u003c/span\u003e(\u0026#119864;\u0026#119902;.3)\u003c/p\u003e \u003cp\u003eWhere, the coefficients are A\u0026thinsp;=\u0026thinsp;ΔFi\u003csub\u003emax\u003c/sub\u003e (ΔFi\u003csub\u003emax\u003c/sub\u003e is the fluorescence intensity at saturation) and B\u0026thinsp;=\u0026thinsp;1/Ka (Ka is the equilibrium-binding constant, from the fitting). H\u003csub\u003e0\u003c/sub\u003e is proportional to ΔFi\u003csub\u003emax\u003c/sub\u003e per mg protein and therefore estimated from Eq.\u0026nbsp;3 by dividing A by the protein concentration of each sample.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDifferential scanning calorimetry (DSC)\u003c/h2\u003e \u003cp\u003eDSC measurements were performed according to Peyrano et al., (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). A Q20 differential scanning calorimeter (TA Instruments, New Castle, USA) was used to study the thermal properties of CPI. Hermetically sealed aluminum pans were prepared to hold 15\u0026ndash;20 mg of CPI suspended in water (20% w/w protein). Samples were scanned at a rate of 5\u0026deg;C/min from 20 to 120\u0026deg;C. An empty pan was used as a reference. Denaturation temperature (T\u003csub\u003ed\u003c/sub\u003e \u0026deg;C) and enthalpy change of transition (ΔH J/g dry protein) were calculated with the TA Universal Analysis 2000 software. The degree of protein denaturation (DD) was calculated according to the following equation:\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:DD\\left(\\%\\right)=\\frac{100-{\\varDelta\\:H}_{t}}{{\\varDelta\\:H}_{0}}x\\:100\\)\u003c/span\u003e \u003c/span\u003e (Eq.\u0026nbsp;4)\u003c/p\u003e \u003cp\u003eWhere ΔH\u003csub\u003e0\u003c/sub\u003e corresponds to the enthalpy change of I6.4, and ΔH\u003csub\u003et\u003c/sub\u003e corresponds to the enthalpy changes of the other CPI obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eColor\u003c/h2\u003e \u003cp\u003eColor was evaluated (CIELab parameters) using a Chroma Meter CR-300C colorimeter (MINOLTA, Tokyo, Japan). Lightness (L*), equilibrium between green and red (a*), and equilibrium between yellow and blue (b*), were measured and total color difference (ΔE*) and browning index (BI), were calculated following Adal et al. (2024) (Eq.\u0026nbsp;5 and Eq.\u0026nbsp;6) using the I6.4 as reference. Measurements were performed ten times for each sample, and the average value was reported.\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:{\\varDelta\\:E}^{*}={({\\left({\\varDelta\\:L}^{*}\\right)}^{2}+{\\left({\\varDelta\\:a}^{*}\\right)}^{2}+{\\left({\\varDelta\\:b}^{*}\\right)}^{2})}^{1/2}\\)\u003c/span\u003e \u003c/span\u003e (Eq.\u0026nbsp;5)\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:BI=\\frac{100}{0.17}x(\\frac{{a}^{*}+1.75{L}^{*}}{5.645{L}^{*}+{a}^{*}-3.012{b}^{*}}-0.31)\\)\u003c/span\u003e \u003c/span\u003e (Eq.\u0026nbsp;6)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eProtein solubility (S\u003csub\u003e0\u003c/sub\u003e)\u003c/h2\u003e \u003cp\u003eS\u003csub\u003e0\u003c/sub\u003e was evaluated over a pH range from 2.0 to 10.0 in distilled water, following the method described by Fern\u0026aacute;ndez Sosa et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). S\u003csub\u003e0\u003c/sub\u003e was expressed as the percentage ratio between the soluble protein in the supernatants determined by the Lowry method (Lowry et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1951\u003c/span\u003e) and the total protein content measured by the Kjeldahl method (AOAC, 1990). Bovine serum albumin was used as a standard.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of oil-in-water emulsions\u003c/h2\u003e \u003cp\u003eCPI dispersions (1.5% w/w) were stirred (1 h, 25\u0026deg;C) and used to prepare o:w emulsions with sunflower oil (1/5). The two phases were premixed (20,000 rpm; 1 min) with an Ultra-Turrax T10 rotor/stator homogenizer (IKA, Staufen, Germany). Then, the emulsions were further homogenized with a VCX 500 ultrasonic processor equipped with a 13 mm diameter titanium probe, model 630\u0026thinsp;\u0026minus;\u0026thinsp;0219 (Sonics \u0026amp; Materials Inc., Newtown, USA), maintaining the temperature below 30\u0026deg;C (375 W, 5 min, pulse mode: 30 s on, 30 s off; ice bath).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eParticle size distribution\u003c/h2\u003e \u003cp\u003eThe particle size distribution of emulsions (freshly prepared and stored at 4\u0026deg;C for 24 h) was determined using an Analysette 22 NeXT laser diffraction system (Fritsch GmbH, Idar-Oberstein, Germany), without and with SDS (1% w/v). To avoid multiple scattering effects, samples were dispersed in 600 mL of water at 2,000 rpm. The optical parameters were refractive index for oil (1.47) and water (1.33), and the adsorption coefficient (0.001). The surface average diameter [D\u003csub\u003e3,2\u003c/sub\u003e] and the volume average diameter [D\u003csub\u003e4,3\u003c/sub\u003e] were determined. The variation of [D\u003csub\u003e4,3\u003c/sub\u003e] values at different times (initial and after one day), in the absence and presence of SDS, was used to calculate the flocculation index (FI) and the coalescence index (CI), following Palazzolo et al. (2005) (Eqs.\u0026nbsp;7 and 8).\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:FI=\\frac{{D}_{\\text{4,3}t}-{D}_{\\text{4,3}t+SDS}}{{D}_{\\text{4,3}t+SDS}}\\)\u003c/span\u003e \u003c/span\u003e (Eq.\u0026nbsp;7)\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:CI=\\frac{{D}_{\\text{4,3}t+SDS}-{D}_{\\text{4,3}i+SDS}}{{D}_{\\text{4,3}t+SDS}}\\)\u003c/span\u003e \u003c/span\u003e (Eq.\u0026nbsp;8)\u003c/p\u003e \u003cp\u003eWhere D\u003csub\u003e4,3t\u003c/sub\u003e is the D\u003csub\u003e4,3\u003c/sub\u003e value at a given time t, D\u003csub\u003e4,3t+SDS\u003c/sub\u003e ​is the D\u003csub\u003e4,3\u003c/sub\u003e​ value at a given time t in the presence of SDS, and D\u003csub\u003e4,3i+SDS\u003c/sub\u003e is the initial D\u003csub\u003e4,3\u003c/sub\u003e​ value in the presence of SDS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eCPI obtained at each extraction condition was prepared in triplicate. All analytical determinations described in Sections 2.3 to 2.12 were conducted in triplicate. Statistical differences between the samples were determined by analysis of variance (ANOVA) (Tukey\u0026rsquo;s test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), using InfoStat software (Di Rienzo et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eProtein extractability\u003c/h2\u003e \u003cp\u003eThe protein content of the CPI extracted at pH 6.4 and by the pH-shift method ranged from 79.84% to 83.00% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The yield in weight and protein yield recovery were not significantly affected by increasing the extraction pH (no significant differences among pH 7.5, 8.0, and 9.0); however, these values were higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than those obtained at pH 6.4 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Similar protein contents and extraction yields have been reported for alkaline protein extraction from various legumes, including green lentil, red lentil, black lentil, mung bean, yellow pea, pigeon pea, and cowpea (Shrestha et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Fern\u0026aacute;ndez Sosa et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e; G\u0026oacute;mez et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Peyrano et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProtein content, yield in weight, protein yield recovery and water activity of cowpea protein isolates.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtein content (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYield in weight (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProtein yield recovery (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ea\u003csub\u003ew\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI6.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e79.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60bcd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.460\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001g\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI6.4-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e76.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e56.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.546\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001h\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44bcd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e53.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.434\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001f\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI7.5-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e81.65\u0026thinsp;\u0026plusmn;\u0026thinsp;2.09cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e61.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.57d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.396\u0026thinsp;\u0026plusmn;\u0026thinsp;0.000d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e81.13\u0026thinsp;\u0026plusmn;\u0026thinsp;2.36bcd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e54.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.59bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.403\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI8-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e78.92\u0026thinsp;\u0026plusmn;\u0026thinsp;1.82abc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e59.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.350\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e83.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26bcd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e56.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.337\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI9-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e77.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e56.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.391\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eData are the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Different letters in a column indicate significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between samples.\u003c/p\u003e \u003cp\u003eThe yield in weight of CPI extracted with HIUS under each pH condition was also evaluated. At pH 6.4, 7.5, and 8.0, significant increases were observed (64.19%, 13.25%, and 12.62%, respectively), along with higher protein yield recovery (59.22%, 14.11%, and 9.54%, respectively). The acoustic cavitation induced by HIUS promotes particle size reduction, thereby increasing the contact surface area between the plant matrix and the solvent, which enhances mass transfer and facilitates extraction (Quintero-Quiroz et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, the protein content of I6.4-US (76.92%) was lower than that of I6.4 (79.84%) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that HIUS-assisted extraction may also promote the co-extraction of non-protein constituents by further disrupting cellular structures and increasing the solubilization of intracellular compounds such as carbohydrates, phenolics, and soluble dietary fibers (Yusoff et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This effect reduced the isolated protein content, despite the significantly higher protein recovery obtained. At pH 9.0, HIUS did not significantly increase weight or protein yield; in fact, the protein content decreased (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), likely due to protein denaturation at this pH, which could limit the ability of HIUS to disrupt the protein matrix and, consequently, reduce extraction efficiency. Suchintita Das et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) reported that the sono-physical and sono-chemical effects of HIUS help break protein\u0026ndash;polysaccharide interactions in the faba bean matrix, releasing more proteins into the solvent, and they observed results similar to ours when comparing HIUS-assisted extraction of faba bean proteins in water and in an alkaline solution (pH 10). Overall, our results demonstrate that HIUS-assisted extraction enhances the solubilization of cowpea proteins at pH 6.4, 7.5, and 8.0, achieving extraction yields comparable to or even higher than those obtained at pH 9.0. This improvement increases the overall efficiency of the process by reducing the use of highly alkaline solutions and minimizing potential nutritional drawbacks associated with undesirable chemical reactions at elevated pH values (Momen et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Long et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile extraction yield and protein content determine the efficiency of the isolation process, the storage stability of the resulting protein isolates is equally important for their practical application. Therefore, the water activity (a\u003csub\u003ew\u003c/sub\u003e) of the obtained CPI was measured, since it reflects the fraction of unbound water in the food matrix and plays a key role in chemical reactions, microbial growth, and physical stability. The a\u003csub\u003ew\u003c/sub\u003e values of CPI varied significantly depending on extraction pH and ultrasound application, ranging between 0.546 and 0.337. Shih et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) reported that an Aw of 0.25 led to fewer alterations during storage compared to an Aw of 0.75, significantly affecting the physicochemical and techno-functional properties of soy protein isolates. Generally, the a\u003csub\u003ew\u003c/sub\u003e decreased with the increasing pH (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The application of HIUS increased a\u003csub\u003ew\u003c/sub\u003e values at pH 6.4 and pH 9.0 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and decreased a\u003csub\u003ew\u003c/sub\u003e values at pH 7.5 and 8.0. The increment of aw (16\u0026ndash;19%) could be related to the minor protein content of I6.4-US and I9-US, and as a consequence, a decrease in water-protein interaction (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Although the decrease of aw (9\u0026ndash;13%) in I7.5-US and I8-US could be due to a structural reorganization that improves the water-protein interactions and decreases the unbound water in the protein matrix. Overall, these results indicate that aw is governed by both protein content and the structural characteristics of the isolates resulting from the extraction process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eModifications of the protein structure\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003eElectrophoretic profile\u003c/h2\u003e \u003cp\u003eThe electrophoretic profiles of the CPI were obtained by SDS-PAGE to determine the molecular weights of the polypeptide chains, which provides information about the primary structure of the proteins (Rahman \u0026amp; Lamsal, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Under non-reducing conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), the I6.4 presented polypeptides of 84, 66, and 56 kDa, corresponding to the 7S globulin fraction, known as vicilin, as well as polypeptides of 94, 49, 33, 30, 27, 23, and 19\u0026ndash;17 kDa, which are associated with the albumin fraction. An additional polypeptide of 38 kDa and an increase of band intensity at 46 and 56 kDa (particularly at pH 7.5 and pH 8.0), and at 33, 30, and 27 kDa of CPI obtained at alkaline pH were observed. Also, polypeptides with molecular weights greater than 94 kDa and soluble aggregates in the stacking gel were observed. These results suggest that alkaline pH extraction not only facilitates the recovery of additional polypeptides but also induces a rearrangement of the protein structure, leading to protein denaturation. These results are consistent with those reported by Peyrano et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUnder reducing conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), the presence of disulfide bonds was confirmed by the absence of soluble aggregates in the stacking gel, and a decrease in band intensity above 94 kDa, as well as those at 84 and 38 kDa associated to an increase of intensity at 29 and 20 kDa bands.\u003c/p\u003e \u003cp\u003eAll electrophoretic profiles of CPI obtained by HIUS-assisted extraction at alkaline pH were similar, consistent with the inability of HIUS to break covalent or peptide bonds, leaving the polypeptide composition unaltered. Similar behavior for protein isolates obtained via HIUS-assisted extraction has been reported for quinoa, black bean, lentil (Quintero-Quiroz et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and soy (Rahman \u0026amp; Lamsal, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eSecondary structure\u003c/h2\u003e \u003cp\u003eThe secondary structure of proteins refers to the local folding patterns that depend on both the amino acid sequence and interactions between atoms in the polypeptide backbone. Experimentally, it is determined by evaluating the relative proportions of α-helix, β-sheet, β-turn, and random coil structures (Rahman \u0026amp; Lamsal, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). CPI exhibited the characteristic amide I band of proteins, which, due to its sensitivity to conformational changes, allows the study of folding\u0026ndash;unfolding and aggregation processes (Fern\u0026aacute;ndez Sosa et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). Deconvolution of the amide I band enabled calculation of the relative content of secondary structural elements (α-helix, β-sheet, β-turns, and random coil) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The I6.4 showed high proportions of β-sheets and β-turns, with very low α-helix and random coil contents, indicating a highly ordered and compact conformation with limited flexibility. Increasing the extraction pH significantly raised α-helix content at the expense of β-structures (β-turns and β-sheets) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), suggesting that alkaline conditions promoted partial unfolding and refolding of the protein backbone. Nevertheless, all CPI obtained via the pH-shift process retained a predominance of β-sheet structures, indicating that the overall organization remained largely governed by intermolecular hydrogen-bonding typical of legume storage proteins. Similar β-sheet dominance has been reported for CPI regardless of extraction pH (8.0 and 10.0) (G\u0026oacute;mez et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and for commercial soy protein isolates (Hu et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe positions and the relative content of secondary structure under the amide I region of cowpea protein isolates.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eβ-sheet\u003c/p\u003e \u003cp\u003e(1610\u0026ndash;1640 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1680\u0026ndash;1695 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eα-Helix\u003c/p\u003e \u003cp\u003e(1650\u0026ndash;1660 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eβ-Turn\u003c/p\u003e \u003cp\u003e(1660\u0026ndash;1680 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRandom coil\u003c/p\u003e \u003cp\u003e(1640\u0026ndash;1650 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI6.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI6.4-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e49.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3.81c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.24\u0026thinsp;\u0026plusmn;\u0026thinsp;3.46de\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.38\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI7.5-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI8-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.44c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI9-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.14\u0026thinsp;\u0026plusmn;\u0026thinsp;3.40de\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.86\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.32\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eData are the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Different letters in a column indicate significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between samples.\u003c/p\u003e \u003cp\u003eHIUS-assisted extraction further modified CPI secondary structure in a pH-dependent manner (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). I6.4-US exhibited increased α-helix and random coil contents, with reduced β-turns and β-sheets, indicating partial unfolding and a more flexible conformation. In I7.5-US, HIUS decreased α-helix and β-sheet contents while increasing random coil and β-turn structures, resulting in a more disordered state. Conversely, I8-US showed a more ordered structure, with higher α-helix and lower random coil contents. I9-US displayed variable effects on α-helix and β-turn contents, maintaining an inverse relationship between these elements. Hydrogen bonds and electrostatic interactions stabilize protein secondary structures, and HIUS likely disrupts these interactions, inducing conformational changes (Rahman \u0026amp; Lamsal, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Comparable effects have been observed in commercial soy protein isolates treated with HIUS (400 W, 30 min) (Hu et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and in whey protein concentrate, where α-helix content slightly increased while β-sheet and β-turn contents slightly decreased (450 W, 30 min) as determined by circular dichroism (Chandrapala et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Adal (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) further noted that, although proteins can partially refold after HIUS, Faba bean protein isolates do not regain the native structure of untreated samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eTertiary structure\u003c/h2\u003e \u003cp\u003eTryptophan residues, the primary intrinsic fluorophores in proteins, exhibit a maximum emission wavelength (λ\u003csub\u003emax\u003c/sub\u003e) in water at 348 nm (Fern\u0026aacute;ndez Sosa et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). The I6.4 exhibited a λ\u003csub\u003emax\u003c/sub\u003e of 340.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 nm (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), which describes a polar environment (Rahman \u0026amp; Lamsal, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The pH-shift extraction did not result in significant changes in λ\u003csub\u003emax\u003c/sub\u003e compared to the I6.4 (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, a slight red shift tendency (higher λ\u003csub\u003emax\u003c/sub\u003e values; major exposure to a more polar environment) was observed at pH 7.5 and 8.0, whereas a slight blue shift tendency (lower λ\u003csub\u003emax\u003c/sub\u003e values; major exposure to a less polar environment) was noted at pH 9.0 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Peyrano et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) reported similar behavior for CPI when the extraction pH increased from 8.0 to 10.0.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFluorescence spectroscopy and surface hydrophobicity of cowpea protein isolates.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eλ\u003csub\u003em\u0026aacute;x\u003c/sub\u003e (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFi (Fi/mg/mL protein)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eH\u003csub\u003e0\u003c/sub\u003e (Fi/mg/mL protein)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI6.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e340.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.53x10\u003csup\u003e4\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;9.63x10\u003csup\u003e1\u003c/sup\u003ebc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1584\u0026thinsp;\u0026plusmn;\u0026thinsp;11ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI6.4-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e340.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.34x10\u003csup\u003e4\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;5.26x10\u003csup\u003e2\u003c/sup\u003ea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1706\u0026thinsp;\u0026plusmn;\u0026thinsp;25c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e340.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.56x10\u003csup\u003e4\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;2.46x10\u003csup\u003e2\u003c/sup\u003ebc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1487\u0026thinsp;\u0026plusmn;\u0026thinsp;11ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI7.5-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e339.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.66x10\u003csup\u003e4\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00x10\u003csup\u003e3\u003c/sup\u003ec\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1957\u0026thinsp;\u0026plusmn;\u0026thinsp;23cd\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e340.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.42x10\u003csup\u003e4\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;9.46x10\u003csup\u003e1\u003c/sup\u003eab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1333\u0026thinsp;\u0026plusmn;\u0026thinsp;186a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI8-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e339.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.42x10\u003csup\u003e4\u003c/sup\u003e \u0026plusmn;1.21x10\u003csup\u003e2\u003c/sup\u003eab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2190\u0026thinsp;\u0026plusmn;\u0026thinsp;66de\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e339.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.55x10\u003csup\u003e4\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07x10\u003csup\u003e3\u003c/sup\u003ebc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2473\u0026thinsp;\u0026plusmn;\u0026thinsp;11ef\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI9-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e339.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.51x10\u003csup\u003e4\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;3.59x10\u003csup\u003e2\u003c/sup\u003eabc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2693\u0026thinsp;\u0026plusmn;\u0026thinsp;53f\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFi: Intrinsic intensity; H\u003csub\u003e0\u003c/sub\u003e: Surface hydrophobicity. λmax: maximum emission wavelength.\u003c/p\u003e \u003cp\u003eData are the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Different letters in a column indicate significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between samples.\u003c/p\u003e \u003cp\u003eThe HIUS-protein extraction at pH 6.4 resulted in non-significant differences in λ\u003csub\u003emax\u003c/sub\u003e but a significant decrease in Fi, which could be attributed to an increased distance between Tryptophan and Tyrosine residues due to the unfolding process (Fern\u0026aacute;ndez Sosa et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e), by reducing the energy transfer and fluorescence intensity. The HIUS-assisted protein extraction at pH 7.5 and 8.0 resulted in a significant decrease in λ\u003csub\u003emax\u003c/sub\u003e of CPI obtained, suggesting a major exposure of Tryptophan residues to a more hydrophobic environment, reaching similar values to those obtained for I9 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The high alkaline concentration during protein extraction at this pH could modify the microenvironment of Tryptophan toward a more shielded one, modifying its sensitivity to treatments (Peyrano et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and resulting in non-significant differences of λ\u003csub\u003emax\u003c/sub\u003e between protein isolates extracted with and without HIUS. When HIUS-assisted protein extraction was performed at alkaline pH, an additional protein unfolding and reorganization of protein structure may have occurred. This could explain the absence of significant changes in Fi spectra compared to CPI obtained without HIUS.\u003c/p\u003e \u003cp\u003eThe measurement of protein surface hydrophobicity (H\u003csub\u003e0\u003c/sub\u003e) provides insight into the number of hydrophobic groups exposed on the molecular surface in a polar aqueous environment. It is commonly used to assess changes in protein conformation and stability, as well as their impact on techno-functional properties (Tang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hu et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The pH-shift extraction at pH 9.0 resulted in a significant increase in the H\u003csub\u003e0\u003c/sub\u003e of I9 compared to the other protein isolates (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Peyrano et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) also reported a significant increase in the H\u003csub\u003e0\u003c/sub\u003e of CPI when the extraction pH increased from 8.0 to 10.0. Besides, the HIUS-assisted protein extraction resulted in a significant increase in H\u003csub\u003e0\u003c/sub\u003e at pH 6.4 (7.67%), pH 7.5 (31.60%), and pH 8.0 (64.28%) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), meanwhile, non-significant changes were observed at pH 9.0. These results are consistent with Fi data, indicating that HIUS-assisted extraction at these pH levels induces a certain degree of protein unfolding, resulting in greater exposure of hydrophobic groups to the polar environment. In contrast, at pH 9.0, a structural refolding likely occurs, preventing further modification of this property. The H\u003csub\u003e0\u003c/sub\u003e values obtained also explain structural modifications that influence the a\u003csub\u003ew\u003c/sub\u003e values reported (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Rahman \u0026amp; Lamsal, (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported an increase in H\u003csub\u003e0\u003c/sub\u003e of soy proteins induced by HIUS, which was more pronounced with increasing treatment time and power. On the other hand, the H\u003csub\u003e0\u003c/sub\u003e values of I9-US and I9 were not significantly different, but were the highest, and similar to that reported by Peyrano et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) for CPI obtained at pH 10.0 without HIUS.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eThermal properties\u003c/h2\u003e \u003cp\u003eThe thermal stability of proteins is commonly assessed by differential scanning calorimetry (DSC), where an endothermic peak reflects alterations in the higher-order structure of the protein (Garcia de Oliveira et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Thermograms of all CPI exhibited a single major endothermic peak (data not shown), corresponding to the 7S globulins, the predominant fraction identified in the electrophoretic profiles. This peak was asymmetric and spanned a broad temperature range, which is attributed to the overlap of multiple denaturation events occurring in the same region, a typical feature of seed-derived proteins (Garcia de Oliveira et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Lef\u0026egrave;vre et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mession et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe DSC parameters are presented in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. ΔH represents the energy required to induce denaturation, while T\u003csub\u003ed\u003c/sub\u003e indicates the temperature at which the protein denatures (Mir et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The highest ΔH value was recorded for I6.4, indicating greater structural stability under this condition. Alkaline extraction resulted in a reduction in ΔH, suggesting a decreased structural integrity, possibly due to protein unfolding or conformational changes induced. The T\u003csub\u003ed\u003c/sub\u003e of I6.4 was 86.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u0026deg;C, which is consistent with values previously reported for cowpea protein concentrates (Garcia de Oliveira et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The extraction at pH 7.5 and pH 8.0 did not significantly affect the T\u003csub\u003ed\u003c/sub\u003e; however, extraction at pH 9.0 resulted in greater sensitivity to thermal treatment, as evidenced by a significant decrease in T\u003csub\u003ed\u003c/sub\u003e to 85.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u0026deg;C. This value is higher than that reported by (Peyrano et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) for CPI, which can be attributed to differences in protein concentration and heating rate during the calorimetric analysis (Peyrano et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThermal properties of cowpea protein isolates.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eΔH (J/g dry protein)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT\u003csub\u003ed\u003c/sub\u003e (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDD (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI6.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e86.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI6.4-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e86.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04bc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e86.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI7.5-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e86.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08cd\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e86.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI8-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e87.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07bc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI9-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e86.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eΔH: enthalpy change of transition; Td: denaturation temperature; DD: degree of protein denaturation.\u003c/p\u003e \u003cp\u003eData are the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Different letters in a column indicate significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between samples.\u003c/p\u003e \u003cp\u003eHIUS-assisted extraction significantly reduced ΔH across all evaluated pH values (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating enhanced protein denaturation associated with structural and conformational alterations in CPI caused by bond disruption. I9-US exhibited the lowest ΔH values and the highest DD (32.10%). As mentioned in the previous section, this isolate presented the highest H\u003csub\u003e0\u003c/sub\u003e, suggesting that the high concentration of OH⁻ during extraction may have already promoted prior protein denaturation. This increased exposure of hydrophobic regions upon HIUS-assisted extraction may have favored the formation of insoluble aggregates through hydrophobic and protein\u0026ndash;protein interactions, thereby limiting the recovery of soluble proteins. As a consequence, HIUS-assisted extraction did not affect the extraction yields of I9-US and had no significant effect on T\u003csub\u003ed\u003c/sub\u003e of I6.4-US and I7.5-US; however, HIUS increased T\u003csub\u003ed\u003c/sub\u003e values of I8-US and I9-US. This suggests that HIUS may induce a distinct reorganization of denatured proteins under highly alkaline conditions, enhancing thermal stability compared to samples extracted without HIUS, with potential implications for food formulation and processing strategies.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eTechno-functional properties\u003c/h2\u003e \u003cp\u003ePlant proteins have gained attention as promising food ingredients owing to their functional properties, such as high solubility and emulsifying capacity, among others (Thompson et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Fern\u0026aacute;ndez Sosa et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e; Peyrano et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The operating procedures used to extract proteins from plants influence their molecular structure and aggregation state, which is mainly related to their functional performance (Hadidi et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eColor\u003c/h2\u003e \u003cp\u003eFreeze-dried CPI can be used as an ingredient in a wide range of food formulations. Since color is a key sensory attribute that strongly influences consumer acceptance, the evaluation of CPI colour obtained is of considerable importance (Ma et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). All CPI samples obtained presented a light brown color, with positive values for both a* and b* parameters (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The markedly higher b* values contributed to a yellowish appearance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The I6.4 exhibited the highest L* and the lowest BI value; meanwhile, I7.5 and I9 presented the higher differences of ΔE* and BI with non-significant differences between them (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), suggesting that the extraction pH has a significant and variable effect on the color parameters.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eColor parameters of the cowpea protein isolates.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ea*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eb*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eL*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eΔE*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBI\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI6.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e86.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI6.4-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e23.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22g\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e77.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15fg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI7.5-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e78.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e19.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI8-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e81.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e76.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52f\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI9-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e77.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eΔE*: Total color difference; BI: Browning index.\u003c/p\u003e \u003cp\u003eData are the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Different letters in a column indicate significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between samples.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHIUS-protein extraction associated with pH resulted in varied color parameter changes. I6.4-US showed a decrease in L* and an increment of BI, with ΔE* value of 8.19, which could be attributed to the extraction of non-protein components mentioned in section 3.1. I7.5-US and I8-US showed an increase in L* and a significant reduction in both BI and ΔE* (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and I9-US presented non-significant changes in L* and ΔE*, but a decrease in the BI was found (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Adal (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) reported that ΔE* and BI reduction may be attributed to HIUS, since sonication induced pigment degradation and a decrease in protein particle size, which enhances multiple light scattering and consequently increases sample whiteness.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eProtein solubility (S\u003csub\u003e0\u003c/sub\u003e)\u003c/h2\u003e \u003cp\u003eS\u003csub\u003e0\u003c/sub\u003e is a critical techno-functional property, as it directly influences other properties of interest to the food industry, such as gelation, emulsification, and foaming capacity (Marinacci et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Consequently, numerous structural modification strategies have been employed to improve protein solubility. Understanding the pH-dependent behavior of plant protein solubility is therefore essential for developing efficient extraction and isolation methods from plant-based raw materials. All S\u003csub\u003e0\u003c/sub\u003e profiles of CPI exhibited a similar pattern as a function of pH, following a characteristic U-shaped curve between pH 2.0 and pH 10.0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Maximum S\u003csub\u003e0\u003c/sub\u003e (~\u0026thinsp;100%) was observed between pH 8.0 and pH 10.0, whereas the minimum S\u003csub\u003e0\u003c/sub\u003e (~\u0026thinsp;3.5%) occurred between pH 3.0 and pH 5.0. Similar results were reported by G\u0026oacute;mez et al., (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and Shevkani et al., (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). HIUS-assisted extraction significantly improved S\u003csub\u003e0\u003c/sub\u003e at pH 4.0 of I6.4-US (16%), I7.5-US (64%), and I9-US (64%), and at pH 6.0 of I6.4-US (64%), I7.5-US (23%), I8-US (28%) and I9-US (29%), which is particularly relevant given that most food systems have a pH close to neutrality.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eCharacteristics of emulsions: particle size distribution\u003c/h2\u003e \u003cp\u003eThe ability of plant proteins to form and stabilize emulsions is highly dependent on the nature of the proteins and on the extraction method used (McClements et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The size of the dispersed particles also plays a main role in emulsion stability (Thompson et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). All emulsions exhibited a monomodal particle size distribution, both in the absence and presence of 1% SDS. All emulsions were formulated at pH 7.0, ensuring that proteins were almost fully dissolved in the aqueous phase during homogenization (S\u003csub\u003e0\u003c/sub\u003e: 86.09\u0026ndash;95.23%). This high S\u003csub\u003e0\u003c/sub\u003e enhanced protein migration and adsorption at the oil\u0026ndash;water interface, thereby promoting both emulsion formation and stabilization. No noticeable changes in the visual appearance of the formulated emulsions were observed as pH increased.\u003c/p\u003e \u003cp\u003eFreshly prepared emulsions with pH-shift protein extraction showed a decrease in D\u003csub\u003e3.2\u003c/sub\u003e, D\u003csub\u003e4.3\u003c/sub\u003e, and FI\u003csub\u003e0\u003c/sub\u003e values when pH increased (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), which is in accordance with the protein modifications mentioned in sections 3.2.2 and 3.3.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParticle size, span, flocculation index, and coalescence index of o:w emulsions stabilized with cowpea protein isolates.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD\u003csub\u003e3,2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD\u003csub\u003e4,3\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSpan\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFI\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFI\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCI\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI6.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.979\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.234\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.182\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.104\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009dA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.126\u0026thinsp;\u0026plusmn;\u0026thinsp;0.036A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.000\u0026thinsp;\u0026plusmn;\u0026thinsp;0.000a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI6.4-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.822\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.046\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.178\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.303\u0026thinsp;\u0026plusmn;\u0026thinsp;0.017eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.075\u0026thinsp;\u0026plusmn;\u0026thinsp;0.039A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.246\u0026thinsp;\u0026plusmn;\u0026thinsp;0.018b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.902\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.159\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.180\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.079\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002bcA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.083\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.000\u0026thinsp;\u0026plusmn;\u0026thinsp;0.000a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI7.5-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.796\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.005\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.195\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.044\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007aA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.038\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.005\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.830\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.049\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.189\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.085\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004cdA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.072\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.012\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI8-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.757\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.957\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.225\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.057\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011aA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.051\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.000\u0026thinsp;\u0026plusmn;\u0026thinsp;0.000a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.810\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.018\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.193\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.060\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004abA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.067\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.000\u0026thinsp;\u0026plusmn;\u0026thinsp;0.000a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI9-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.823\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.027\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.175\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.058\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009aA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.055\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.000\u0026thinsp;\u0026plusmn;\u0026thinsp;0.000a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eD\u003csub\u003e3,2\u003c/sub\u003e: surface average diameter of freshly o:w emulsions; D\u003csub\u003e4,3\u003c/sub\u003e: volume average diameter of freshly o:w emulsions; FI\u003csub\u003e0\u003c/sub\u003e: flocculation index of freshly o:w emulsions of o:w emulsions; FI\u003csub\u003e1\u003c/sub\u003e: Flocculation index of o:w emulsions at one day of storage; CI\u003csub\u003e1\u003c/sub\u003e:Coalescence index of o:w emulsions at one day of storage of o:w emulsions.\u003c/p\u003e \u003cp\u003eData are the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Different lowercase letters within a column indicate significant differences and different uppercase letters between rows indicate significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eEmulsions formulated with I6.4-US, I7.5-US, and I8-US exhibited significantly lower D\u003csub\u003e3.2\u003c/sub\u003e and D\u003csub\u003e4.3\u003c/sub\u003e values, indicating greater efficiency in forming emulsions with smaller droplet sizes. Moreover, the FI\u003csub\u003e0\u003c/sub\u003e decreased in emulsions prepared with I7.5-US and I8-US. As detailed in Section 3.2.3, HIUS-assisted extraction increased H\u003csub\u003e0\u003c/sub\u003e, particularly at pH 7.5 and 8.0. The enhanced exposure of hydrophobic groups on the protein surface facilitated their adsorption at the oil\u0026ndash;water interface, promoting the formation of emulsions with smaller droplet sizes and probably greater stability.\u003c/p\u003e \u003cp\u003eThe analysis of emulsion stability after 24 h revealed that, except I6.4-US, all samples exhibited a CI equal to or close to zero. Regarding the FI, a decrease was observed in I6.4-US, whereas an increase was observed in I7.5. No significant changes in FI were observed in the remaining samples, suggesting consistent stability over 24 h.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eHIUS-assisted protein extraction notably improved protein yield of I6.4-US, I7.5-US, and I8-US, by enhancing mass transfer and disrupting plant cell structures, with extraction yields and protein solubility comparable to or higher than those obtained for I9, while avoiding the excessive use of highly alkaline solutions that may induce protein denaturation and reduce nutritional quality. However, it also promoted the co-extraction of non-protein components, particularly in I6.4-US.\u003c/p\u003e \u003cp\u003eStructural analyses revealed that HIUS altered the secondary and tertiary structures of proteins by inducing partial unfolding, increasing surface hydrophobicity, and decreasing thermal stability, effects that were also dependent on pH. These conformational changes correlated with the improvement in solubility at pH 6.0 and the enhancement of emulsifying capacity, especially for I7.5-US and I8-US, where smaller emulsion droplet sizes were obtained. Overall, the combined application of HIUS and pH-shift extraction emerges as a promising strategy to obtain plant protein isolates with techno-functional properties.\u003c/p\u003e \u003cp\u003eAdditionally, HIUS-assisted extraction at pH 6.4 represents a promising alternative to pH shift method, combining efficiency, functionality, and sustainability, and supporting the development of plant-based protein ingredients with minimal chemical intervention.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e2-ME\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2-mercaptoethanol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eANS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e1,8-aniline\u0026ndash;naphthalene-sulfonate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eANOVA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAnalysis of Variance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecoalescence index\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCPI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecowpea protein isolates\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eD\u003csub\u003e3,2\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esurface average diameter\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eD\u003csub\u003e4,3\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003evolume average diameter\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eΔH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eenthalpy change of transition\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edegree of protein denaturation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eflocculation index\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFi\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efluorescence intensity\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFTIR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFourier transform infrared\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHIUS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehigh-intensity ultrasound treatment\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eH\u003csub\u003e0\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esurface hydrophobicity\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eλ\u003csub\u003emax\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emaximum emission wavelength\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMW\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emolecular weight\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eS\u003csub\u003e0\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eprotein solubility\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSDS-PAGE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esodium dodecyl sulfate-polyacrylamide gel electrophoresis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eT\u003csub\u003ed\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edenaturation temperature\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by Universidad Nacional del Nordeste (UNNE), Argentina (Grant numbers 20F0009 and 24F010). Carlos Fernando Calgaro acknowledges CONICET (Argentina) for the award of a doctoral fellowship.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eCarlos Fernando Calgaro: Investigation, Data curation, Formal analysis, Methodology, Validation, Visualization, Writing \u0026ndash; Original Draft Preparation.Bel\u0026eacute;n Andrea Acevedo: Conceptualization, Data curation, Funding acquisition, Project administration, Resources, Supervision, Validation, Visualization, Writing \u0026ndash; Review \u0026amp; Editing.Felicitas Peyrano: Conceptualization, Investigation, Data curation, Formal analysis, Methodology, Resources, Validation, Visualization, Writing \u0026ndash; Review \u0026amp; Editing.Mercedes Carolina Rasia: Investigation, Data curation, Resources, Validation, Writing \u0026ndash; Review \u0026amp; Editing.Mar\u0026iacute;a Guadalupe Chaves: Conceptualization, Data curation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing \u0026ndash; Review \u0026amp; Editing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors wish to thank Andrea Gisella Gomez for her kind help and suggestions during the SDS-PAGE and FTIR assays.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eAdal, E. 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Comparative study on molecular and higher-order structures of legume seed protein isolates: Lentil, mungbean and yellow pea. \u003cem\u003eFood Chemistry\u003c/em\u003e, \u003cem\u003e411\u003c/em\u003e, 135464. https://doi.org/10.1016/j.foodchem.2023.135464\u003c/p\u003e\n\u003cp\u003eSuchintita Das, R., Zhu, X., Hannon, S., Mullins, E., Alves, S., Garcia-Vaquero, M., \u0026amp; Tiwari, B. K. (2023). Exploring Osborne fractionation and laboratory/pilot scale technologies (conventional extraction, ultrasound-assisted extraction, high-pressure processing and hydrodynamic cavitation) for protein extraction from faba bean (\u003cem\u003eVicia faba\u003c/em\u003e L.), \u003cem\u003eInnovative Food Science \u0026amp; Emerging Technologies\u003c/em\u003e, 89, 103487, https://doi.org/10.1016/j.ifset.2023.103487.\u003c/p\u003e\n\u003cp\u003eSultan, Z., Ashfaq, A., Jahan, K., Qadri, O. S., Younis, K., \u0026amp; Yousuf, O. (2024). pH shift extraction technique for plant proteins: A promising technique for sustainable development. \u003cem\u003eEnergy Nexus\u003c/em\u003e, \u003cem\u003e16\u003c/em\u003e, 100329. https://doi.org/10.1016/j.nexus.2024.100329\u003c/p\u003e\n\u003cp\u003eTang, S., Li, J., Huang, G., \u0026amp; Yan, L. (2021). Predicting protein surface property with its surface hydrophobicity. \u003cem\u003eProtein and Peptide Letters, 28\u003c/em\u003e(8), 938\u0026ndash;944. https://doi.org/10.2174/0929866528666210222160603\u003c/p\u003e\n\u003cp\u003eThompson, C. M. B., Acevedo, B. A., A\u0026ntilde;\u0026oacute;n, M. C., \u0026amp; Avanza, M. V. (2023). Emulsifying Capacity of Cowpea Protein Isolates. Effect of Thermal and Hydrolytic Treatment. \u003cem\u003ePlant Foods for Human Nutrition\u003c/em\u003e, \u003cem\u003e78\u003c/em\u003e(2), 366\u0026ndash;374. https://doi.org/10.1007/s11130-023-01072-8\u003c/p\u003e\n\u003cp\u003eYusoff, I. M., Mat Taher, Z., Rahmat, Z., \u0026amp; Chua, L. S. (2022). A review of ultrasound-assisted extraction for plant bioactive compounds: Phenolics, flavonoids, thymols, saponins and proteins. \u003cem\u003eFood Research International\u003c/em\u003e, \u003cem\u003e157\u003c/em\u003e, 111268. https://doi.org/10.1016/j.foodres.2022.111268\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Protein extraction yield, Surface hydrophobicity, Denaturation degree , Protein solubility, Emulsifying capacity","lastPublishedDoi":"10.21203/rs.3.rs-8744459/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8744459/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe growing demand for plant-based proteins has prompted the exploration of alternative sources such as cowpea (\u003cem\u003eVigna unguiculata\u003c/em\u003e), due to its high protein content (23-25%). This study compared two protein extraction methods: 1-the pH-shift method at pH 7.5, 8.0, and 9.0, and at the natural pH of the cowpea flour–water dispersion (pH 6.4); 2- pH-shift protein extraction assisted by high-intensity ultrasound (HIUS; 400 W, 60 min, pulse 2 s on/2 s off, T \u0026lt; 30 °C). Protein yield, structural modifications, thermal stability, and techno-functional properties were assessed. Protein content ranged from 77.85% to 83.0%. HIUS increased yield at all pH conditions, including at pH 6.4. HIUS induced partial unfolding, increased surface hydrophobicity, and decreased thermal stability as a function of pH-shift extraction. These structural changes improved solubility at pH 6.0 and enhanced emulsifying capacity, especially in I7.5-US and I8-US, forming emulsions with smaller droplet sizes. I6.4-US was particularly attractive, providing yields and solubility comparable to or higher than I9 while avoiding the use of highly alkaline solutions. Although HIUS-assisted extraction slightly reduced protein purity due to co-extraction of non-protein compounds, it represents a simpler and more sustainable approach. This strategy supports the development of plant-based protein ingredients with high functionality and reduced chemical usage.\u003c/p\u003e","manuscriptTitle":"Impact of high-intensity ultrasound on cowpea protein extractability, structural, and techno- functional properties","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-05 09:27:06","doi":"10.21203/rs.3.rs-8744459/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-23T18:13:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-21T04:15:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-09T07:46:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"45711689350755665054786441202991293572","date":"2026-02-09T02:02:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"158374364626439950286386866681047544728","date":"2026-02-08T15:12:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-08T01:22:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"11701532239313271420363561039931340782","date":"2026-02-06T09:08:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"224994346491275139805716412168151286268","date":"2026-02-05T14:39:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"133669670826981225805183460999017487994","date":"2026-02-05T03:48:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42762756059704056530725583076314314499","date":"2026-02-04T19:32:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"328066209718800275007880380652543622635","date":"2026-02-04T06:42:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"324549728028602169945987251887007053616","date":"2026-02-04T02:21:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"140511185235433310022398216910981410707","date":"2026-02-03T17:09:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"280467109495686161462163510740931002625","date":"2026-02-03T13:22:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"181187294497627147603714299640264400222","date":"2026-02-03T13:15:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"154389566554187623966372415382098676859","date":"2026-02-03T13:13:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-03T12:38:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-03T02:45:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-03T01:21:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food and Bioprocess Technology","date":"2026-01-30T19:06:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fdc0cbc4-08e4-4495-8ca1-c38aba3e803a","owner":[],"postedDate":"February 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-30T10:56:00+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-05 09:27:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8744459","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8744459","identity":"rs-8744459","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}