Unraveling how phytochemical saponins suppress α-Lactalbumin thermal aggregation: A Multi-Dimensional Spectrometric Approach

This study investigated how tea saponin (Ts), gypenoside (Gyp), mogroside V(Mog), glycyrrhizic acid (GA) and stevioside (STE) affected the heat-induced aggregates of α-lactalbumin (Ala), focusing on inhibition mechanisms of disulfide cross-linking and their impact on structural and physicochemical properties. SDS-PAGE and size exclusion chromatography (SEC) confirmed that saponins probably bound Ala monomers via hydrophobic forces and hydrogen bonds, inhibiting the formation of disulfide bonds in heat-induced Ala (H-Ala) and effectively decreasing polymer generation, with the inhibitory effect in the order of Gyp, GA, Ts, STE, Mog. LC-MS/MS analysis revealed that Ala (6), Ala (91), Ala (111) and Ala (120) were the most active sites for the disulfide crosslinking reaction in H-Ala. Specifically, the number of intramolecular disulfide cross-linked peptides of heat-induced Ala-Mog, Ala-STE, Ala-GA, Ala-Ts and Ala-Gyp complexes significantly decreased by 5, 6, 7, 8 and 10, respectively, compared with H-Ala. Notably, the heat-induced Ala-Gyp complex exhibited the highest absolute zeta potential, smallest particle size, lowest hydrophobicity, which correlated with its superior inhibition of aggregation. The study highlighted the potential of saponins as natural additives for the stabilization of heat-sensitive proteins during the thermal processing, offering new insights for food industry applications.