Thermal-induced covalent binding of mung bean protein and epigallocatechin gallate: Binding sites identification and emulsifying properties evaluation

The interaction between mung bean protein fraction (MBP) and epigallocatechin gallate (EGCG) under different heating durations remains unclear. This study investigated the time-dependent changes in covalent binding sites between MBP and EGCG at 70 °C, as well as the corresponding evolution of protein structure and function. Results showed free amino groups of MBP mainly participated in covalent binding. Prolonged thermal induction resulted in increased content of EGCG derivatives (i.e., gallocatechin gallate and gallic acid) but a decrease in that of EGCG. Covalent binding bands were at ∼50 kDa and ∼64 kDa, corresponding to β chain-like precursor, β chain-like and α′ chain of 8S vicilin. When heat induction extended to ≥240 min, ∼50 kDa bands partially polymerized into conjugate aggregates (>250 kDa). Mass spectrometry identified lysine and arginine as core modification sites. Extending heat induction from 120 to 240 min decreased lysine modifications (33 → 27) but increased arginine modifications (16 → 25). Appropriate heat induction promoted MBP-EGCG conjugate formation, inducing conformational changes (cross-β-sheet structures and increased surface hydrophobicity), thereby enhancing antioxidant activity and emulsifying properties. Among these, PE-60 (conjugate formed by heating 60 min) showed the optimal emulsifying property (12.17 m²/g) and good antioxidant capacity (777.69 μmol/g protein). Emulsions prepared by MBP-EGCG conjugates showed excellent storage stability, with the creaming index of 0 after 120 storage days, which may be closely related to the dense viscoelastic network structure of the emulsion stabilized by conjugates. This study clarified polyphenol-protein covalent binding mode in heated systems, providing theoretical support for developing functional mung bean protein-based dairy products.