Spectroscopy and Computational Analysis of β‐Lactoglobulin Interactions With Catechin: A Comparative Study of Single, Double, and Triple Catechin Complexes

ABSTRACT Numerous studies have been conducted on the interaction of β‐lactoglobulin (β‐LG) with catechins. However, the comparative discussion of multiple catechins interacting simultaneously with β‐LG remains unknown. In this study, we comparatively investigated the interaction of three catechins with β‐LG in various combinations, such as β‐LG‐EGCG ((−)‐epigallocatechin gallate), β‐LG‐EGCG‐EGC ((−)‐epigallocatechin), and β‐LG‐EGCG‐EGC‐EC ((−)‐epicatechin), and their underlying mechanisms through a series of spectroscopic analyses, molecular docking, and molecular dynamics simulations. When EGCG is added alone, the particle size and absolute value of the ζ potential increase with increasing concentration; however, when two or three catechins coexist, this increase slows significantly, which may be related to the unique galloyl group and additional hydroxyl groups of EGCG. Addition of an appropriate amount of EGC increased the binding efficiency of EGCG to protein to 94.13% ± 1.24%. Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) spectroscopy showed that EGC could help EGCG increase the α‐helix content of β‐LG from 52.1% to 67.3% and disrupt the β‐sheet from 3.8% to 0.2%. Molecular dynamics (MD) simulations further revealed that in the triple catechin complex, interactions among catechins altered the conformational stability of β‐LG. This study provides new insights into the binding mechanism between catechins and protein based on the full consideration of the complexity of the food system. At the same time, it lays a solid theoretical foundation for the development of functional dairy products and for the construction of synergistic delivery systems for multiple catechins.