We determined the binding sites of curcumin (cur), resveratrol (res), and genistein (gen) with milk β-lactoglobulin (β-LG) at physiological conditions. Fourier transform infrared spectroscopy, circular dichroism, and fluorescence spectroscopic methods as well as molecular modeling were used to determine the binding of polyphenol–protein complexes. Structural analysis showed that polyphenols bind β-LG via both hydrophilic and hydrophobic contacts with overall binding constants of K_{curcumin–β-LG} = 4.4 (± .4) × 10⁴ M⁻¹, K_{resveratrol–β-LG} = 4.2 (± .2) × 10⁴ M⁻¹, and K_{genistein–β-LG} = 1.2 (± .2) × 10⁴ M⁻¹. The number of polyphenol molecules bound per protein (n) was 1 (cur), 1.1 (res), and 1 (gen). Molecular modeling showed the participation of several amino acid residues in polyphenol–protein complexation with the free binding energy of −12.67 (curcumin–β-LG), −12.60 (resveratrol–β-LG), and −10.68 kcal/mol (genistein–β-LG). The order of binding was cur > res > gen. Alteration of the protein conformation was observed in the presence of polyphenol with a major reduction of β-sheet and an increase in turn structure, causing a partial protein structural destabilization. β-LG might act as a carrier to transport polyphenol in vitro.

We report the binding of milk β-lactoglobulin (β-LG) with PEG-3000, PEG-6000 and methoxypoly(ethylene glycol) anthracene (mPEG-anthracene) in aqueous solution at pH 7.4, using multiple spectroscopic methods, thermodynamic analysis, transmission electron microscopy (TEM) and molecular modeling. Thermodynamic and spectroscopic analysis showed that polymers bind β-LG via van der Waals interactions, hydrogen bonding and hydrophobic interactions, with overall binding constants K_{PEG-3000–β-LG} = 9.2 (±0.9) × 10³ M⁻¹, K_{PEG-6000–β-LG} = 9.7 (±0.7) × 10³ M⁻¹ and K_{mPEG-anthracene–β-LG} = 5.5 (±0.5) × 10⁴ M⁻¹. The binding affinity was mPEG-anthracene > PEG-6000 > PEG-3000. Transmission electron microscopy analysis showed significant changes in protein morphology as polymer–protein complexation occurred, with a major increase in the diameter of the protein aggregate. Modeling showed several hydrogen bonding systems between PEG and the different amino acid stabilized polymer–β-LG complexes. The free binding energy indicated that the interaction process is spontaneous at room temperature. Furthermore, mPEG-anthracene is a stronger protein binder than PEG-3000 and PEG-6000, due to its major hydrophobic characteristics.