Abstract: In this study, binary and ternary complexes were prepared by heat treatment or combined heat and ultrasonic treatment using casein micelles (MC) as matrix and proanthocyanidin (PC), catechin (Cat) and chlorophyllin sodium copper salt (Chl) or their binary combinations as ligands. The interactions between PC, Cat, Chl or their binary combinations and MC were analyzed, and the binding capacity of MC to the various active molecules was evaluated. In addition, the structure, microscopic morphology and thermal stability of the complexes were characterized, and the antioxidant properties and in vitro digestibility of the ternary complexes were investigated as well. The results showed that PC, Cat and Chl formed binary complexes with MC through hydrophobic interactions, among which MC showed the highest affinity for Chl. Under heat treatment conditions, MC bound one molecule through hydrophobic interaction and another one by van der Waals forces and hydrogen bonds. At 298 K, the binding of the first molecule improved the binding constant and the number of binding sites of the second one. The binding of PC, Cat, Chl or their binary complexes had no significant effect on the spatial structure or microscopic morphology of MC. The thermal stability of MC was enhanced after the binding of bioactive molecules under heat treatment conditions. In terms of antioxidant properties, heat treatment promoted the 2,2’-azino bis-(3-ethylbenzthiazoline-6-sulfonicacid) (ABTS) radical cation scavenging activity of the ternary complexes, while combined heat and ultrasonic treatment promoted their 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. In addition, the loading of active molecules improved the digestibility of MC in simulated intestinal fluid. This study could provide a reference for the application of MC in the loading of bioactive molecules.

Key words: proanthocyanidin; catechin; chlorophyllin sodium copper salt; casein micelle; interaction; antioxidant properties; digestibility