Electro-acoustic coupling of sequential pulsed electric field-ultrasound treatment: A novel technique for modification of protein isolate extracted from Capsicum annum L. seeds.

The agro-industrial valorization of pepper (Capsicum annum L.) seeds as a sustainable protein resource requires innovative processing strategies to overcome the limitations of their native protein structure and enhance their techno-functional properties for food applications. The study aims to investigate the impact of electro-acoustic coupling between sequential pulsed electric field (PEF) and ultrasound (US) treatment on the in vitro digestibility, structural, functional, and antioxidant properties of underexploited pepper seed protein isolate (PSPI). The sequential treatment caused significant conformational and structural changes, as shown by increased surface hydrophobicity and free-SH content, decreased particle size (604.82 ± 7 to 275.97 ± 4 nm), and an increase in the magnitude of zeta potential (negative), indicating improved dispersion stability. Intrinsic fluorescence presented a decrease in intensity and a distinct red shift, suggesting exposure of aromatic residues and conformational unfolding. FTIR analysis verified the rearrangements of secondary structure with an increase in random coil and α-helix contents. The scanning electron microscope confirmed these findings, showing significant fragmentation and disruption in SEM images. XRD analysis indicated a reduction in peak intensity (2θ) from 20.12° to 19.71°, showing decreased crystallinity and molecular order. Thermal properties analysis by differential scanning calorimetry revealed a decrease in the enthalpy change (126.75 ± 0.65 to 100.27 ± 0.84 ΔH J/g), indicating reduced thermal stability due to structural modifications. These structural and molecular modifications significantly increased the solubility (48.4 ± 0.59 to 75.2 ± 0.65 %), oil-holding capacity (5.68 ± 0.06 to 7.30 ± 0.07 g/g), water-holding capacity (2.69 ± 0.04 to 3.82 ± 0.05 g/g), emulsification, and foaming properties, while reducing turbidity. Moreover, sequential treatment also significantly improved in vitro protein digestibility and antioxidant activity compared with individual PEF and US treatments. Conclusively, the effectiveness of sequential PEF and US as a novel green and sustainable industrial tool to effectively modify PSPI structure to upgrade it into a high-value functional ingredient for food applications.