Heat-induced formation of β-lactoglobulin–milk fat globule membrane complexes: A molecular analysis

Abstract The heat-induced formation of molecular polymers between β-lactoglobulin (β-LG) and the milk fat globule membrane (MFGM) has been widely recognized; nevertheless, the molecular mechanisms underlying this formation remain unclear. This study aimed to investigate the binding mechanisms and physicochemical properties of complexes formed between β-LG and MFGM under two distinct heat treatment conditions: 63 °C for 30 min and 90 °C for 15 min. Differential scanning calorimetry analysis indicated that heating the complexes at 90 °C for 15 min enhanced their thermal stability. Following this treatment, MFGM-protein complexes exhibited decreased intrinsic fluorescence intensity and a blue shift in the maximum emission wavelength. To elucidate the molecular mechanisms underlying complex formation, blocking agents including dithiobis-nitrobenzoic acid, N-ethylmaleimide, and dithioerythritol were applied at both 63 °C/30 min and 90 °C/15 min, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results demonstrate that β-LG–MFGM complex formation involves disulfide bonds (notably Cys121), hydrogen bonding, hydrophobic interactions, and electrostatic interactions, in addition to general intermolecular forces. Heat treatment was found to promote both the formation and thermal stability of these complexes. These findings provide a mechanistic basis for further investigation of how heat processing influences the digestive behavior and functional properties of β-LG–MFGM complexes.