Unraveling Pectin-Protein Interactions in Milk-Based Cocoa Beverages: Molecular Docking and Microstructure

This study investigated the synergistic effects of pectin concentration (0.038-0.462%) and homogenization pressure (5.85-34.1 MPa) on the physical and microstructural stability of milk-based cocoa beverages. A Central Composite Rotatable Design (CCRD) was employed to optimize these parameters by evaluating critical quality attributes, including water-holding capacity (WHC), sedimentation, creaming index, apparent viscosity, and colloidal stability. To elucidate the underlying stabilization mechanisms, molecular docking simulations were integrated to assess the thermodynamic binding affinities between pectin (D-galacturonic acid) and major milk proteins. Numerical optimization identified 0.40% pectin and a homogenization pressure of 10 MPa as the optimal formulation and processing conditions. Under these conditions, the beverage achieved a maximum WHC of 86.04%, complete elimination of macroscopic phase separation (n.d. sedimentation and creaming), and an apparent viscosity of 67.39 mPa·s. Mechanistically, molecular docking revealed strong, multifaceted hydrogen-bonding and electrostatic interactions between pectin and milk proteins, with β-lactoglobulin exhibiting the strongest predicted thermodynamic binding affinity (-6.3 kcal/mol). This macromolecular complexation promoted pronounced steric and electrostatic stabilization, as supported by the highly negative zeta potential (-30.8 mV) and enhanced microstructural uniformity observed through scanning electron microscopy-energy-dispersive X-ray spectroscopy (SEM-EDS) analysis. A rigorous 9-month storage study further demonstrated prolonged kinetic stability. Structural integrity was largely maintained, and macroscopic phase separation was substantially delayed despite time-dependent rheological rearrangements during storage. Furthermore, sensory profiling demonstrated high consumer acceptability, particularly for smoothness and homogeneity, with the optimized formulation outperforming the evaluated commercial counterparts. Collectively, these findings demonstrate the effectiveness of pectin in such beverage system and provide practical formulation and processing strategies for improving the physicochemical stability and shelf life of complex dairy-based dispersions.