The binding mechanism of puerarin (PUE) to β-lactoglobulin (β-lg) was investigated by fluorescence spectroscopy, molecular docking, and molecular dynamics simulation. The fluorescence spectroscopy results showed that PUE statically quenched the fluorescence of β-lg. At 25, 35, and 45 ℃, the binding constants were 7.24 × 104, 1.34 × 105, and 2.18 × 105 L/mol, and the numbers of binding sites were 1.02, 1.18, and 1.15, respectively, indicating there was only one binding site or class of binding sites. Synchronous fluorescence and three-dimensional fluorescence spectroscopy indicated that the binding of PUE to β-lg resulted in an increase in the polarity of the microenvironment of β-lg, thereby weakening the hydrophobic force. Molecular docking results showed that PUE bound to the hydrophobic cavity of β-lg, forming hydrophobic interactions with six amino acid residues of β-lg and short hydrogen bonds with five amino acid residues. The molecular dynamics results showed that the root mean square deviation (RMSD), radius of gyration (Rg), and solvent accessible surface area (SASA) of the complex were (0.17 ± 0.02) nm, (1.47 ± 0.01) nm, and (88.94 ± 2.05) nm2, and those of β-lg were (0.22 ± 0.03) nm, (1.48 ± 0.01) nm, and (90.09 ± 1.73) nm2, respectively. The root mean square fluctuation (RMSF) of the complex was lower than that of β-lg, suggesting that the PUE/β-lg complex has better stability at the molecular level. This study is of reference significance for increasing the bioavailability of PUE
