Interactions of isoliquiritigenin, genistein and baicalein with β-lactoglobulin: A study of the binding mechanism and protective effects

β-Lg can simultaneously load and protect the three flavonoid compounds, namely Isl, Gen, and Bai. • In terms of stability, the β-Lg-multilinked compound shows a better protective effect against the three types of flavonoids. • A spectroscopic approach was adopted to elucidate the binding mechanisms of three flavonoids with β-Lg. • The conformational characteristics of the complexes were investigated through molecular docking and dynamics simulations. This study elucidates the interaction mechanisms of Isoliquiritigenin (Isl), Genistein (Gen) and Baicalein (Bai) with β-lactoglobulin (β-Lg), with a focus on the protein's capacity for the co-encapsulation and stabilization of these flavonoids. The binding mechanisms were characterized using fluorescence spectroscopy, high-performance liquid chromatography, non-radiative energy transfer analysis, molecular simulations. The results demonstrated that the binding affinity ( K a ) followed the order Bai > Gen > Isl. Accordingly, the calculated binding distances for Isl, Gen and Bai were 5.36 nm, 4.49 nm, and 4.20 nm, respectively. β-Lg is capable of co-encapsulating two or three flavonoids simultaneously. The most stable ternary complex (β-Lg-Isl-Gen-Bai) formed when the flavonoids were added in the sequence of Bai, Isl, and then Gen. The β-Lg multi-ligand complex provided superior protection for Isl, Gen, and Bai compared to their respective single-ligand complexes across all tested parameters: stability, antioxidant capacity, and α-amylase inhibition. For Isl, the retention rate improvement under storage stability is the largest, reaching 73.86 ± 1.04%; for Gen, the retention rate improvement under light stability is the largest, reaching 75.22 ± 0.77%; and for Bai, the retention rate improvement under thermal stability is the largest, reaching 71.90 ± 1.02%. β-Lg simultaneously encapsulates and stabilizes three flavonoids Isl, Gen, Bai, serves as a natural and potent protein carrier for these bioactive compounds, and offers theoretical support for their protection against degradation.