Green Recovery of Rosmarinic Acid via Whey Soy Protein-Mediated Foam Fractionation: Molecular Mechanisms and Enhanced Antioxidant Activity

The sustainable isolation of nonamphiphilic phytochemicals remains a formidable challenge in biochemical engineering. In this study, a highly efficient and solvent free foam fractionation process was developed for recovering rosmarinic acid from botanical extracts. By systematically screening diverse biological surfactants, whey soy protein emerged as an exceptionally robust dual functional frother and nanoscale collector. Response surface methodology optimized the operational parameters to 850 mg/L protein concentration, pH 2.5, and a gas flow rate of 470 mL/min, yielding an outstanding target recovery of 93.08 percent alongside an enrichment ratio of 1.81. This macroscopic separation superiority was comprehensively elucidated at the molecular level through multiple spectroscopic techniques and computational modeling. Results confirmed a spontaneous static quenching complexation driven by synergistic noncovalent forces, predominantly hydrogen bonding, van der Waals interactions, π-stacking, and salt bridges. These interactions induced targeted conformational unfolding within the protein backbone, exposing hydrophobic domains that drastically elevated the thermodynamic affinity for the ascending gas-liquid interface. Furthermore, the concentrated product exhibited an antioxidant capacity enhancement exceeding 3.6 times compared to the crude extract, a result attributed to selective enrichment combined with the structural shielding effect provided by the protein macromolecule. Ultimately, this work provides critical mechanistic insights and establishes a scalable technological framework for the green purification of highly valuable botanical compounds.