Effects of pulsed electric field parameters on anionic peptide migration during Electromembrane separation of a whey...

Protein-rich byproducts such as whey represent valuable sources of bioactive peptides, but their selective separation remains a major challenge in sustainable bioprocessing. However, electrodialysis with ultrafiltration membrane (EDUF) offers a promising route for peptide fractionation, and recent evidence suggests that pulsed electric field (PEF) can modulate migration efficiency and selectivity. However, the mechanistic links between PEF parameters, peptide physicochemical characteristics and migration outcomes remain poorly resolved. This study evaluated six pulse/pause combinations (1 s/1 s, 5 s/1 s, 5 s/5 s, 10s/1 s, 10s/5 s and 10s/10s) during EDUF of a whey protein hydrolysate and combined peptidomics with machine learning-based regression tree analysis to identify peptide physicochemical characteristics governing their migration. The regression tree models (R² = 49.0-74.9%) demonstrated hydrophobicity, leucine content, molecular weight and Kappa 1 as the most discriminant descriptors, with the lower prediction accuracy reflecting the complex interplay between peptide physicochemical properties and the dynamic interfacial conditions generated by PEF. After comprehensive analysis of the regression trees, three main mechanistic groups were defined and representative peptides were then tracked across all PEF conditions to reveal their migration behaviors. Group 1 peptides (no migration) did not migrate primarily by steric hindrance and electrostatic repulsion due to their high MW and/or high content in acidic residues preventing meaningful approach to the negatively charged PES 50 kDa UF membrane interface. Group 2 peptides (intermediate migration) migrating only when the time-averaged electric driving and the transiently favorable interfacial conditions created by PEF were jointly sufficient to overcome their moderate diffusive and hydrodynamic constraints combined with a diffusion boundary layer (DBL) state that remains partially destabilized. Group 3 peptides (high migration), small and highly hydrophobic, migrated efficiently across all conditions due to their low size and weak solvation, and in some cases through peptide-peptide hydrophobic interactions. Altogether, these results highlight that tuning PEF parameters, according to peptides physicochemical characteristics, enables fine control of peptide migration and selectivity in EDUF guiding the targeted recovery of bioactive fractions from complex hydrolysates.