Sonication integrated with micronization: structural modification to improve the in-vitro digestibility, functional, and antioxidant properties of red pepper seed protein isolate

The study aimed to extract protein from red pepper seeds and to probe the effects of micronization (MN) at 15,000 rpm for 3 and 6 min, ultrasound (US) at 720 W, 40 kHz for 10 and 15 min, and their combined impact on red pepper seed protein isolate (RPSPI). The combined treatment (MN 6 min and US 10 min) substantially reduced particle size and increased -ve zeta potential, thereby significantly increasing RPSPI stability compared to untreated and individual treatments. Structural results showed significant molecular changes: increased free-SH content and surface hydrophobicity, and decreased intrinsic fluorescence intensity, indicating improved exposure of buried residues and partial unfolding. Fourier Transform Infrared peak spectra results verified secondary structural modification, evidenced by a significant increase in random coil and α-helix content. The disruption and significant fragmentation observed in Scanning Electron Microscope micrographs confirmed these findings. Thermograms from Differential Scanning Calorimetry and X-ray diffraction patterns showed decreases in the thermal transition temperature and crystallinity, respectively, indicating weak intermolecular interactions. These structural changes in RPSPI significantly increased the water solubility, emulsifying activity, foaming capacity, and water and oil holding capacity, while decreasing the turbidity. Also, combined treatment significantly enhanced the ABTS, DPPH, and OH scavenging abilities (%) of RPSPI and in vitro protein digestibility. Conclusively, results revealed that this extremely underexploited by-product protein can develop into a value-added ingredient by improving its techno-functional and bioactive properties through these non-thermal technologies.