pH-Induced Structural Transitions during Complexation and Coacervation of â-Lactoglobulin and Acacia Gum

pH-induced structural changes during complex coacervation between â-lactoglobulin (BLG)and Acacia gum (AG)in aqueous solutions were determined by coupling slow in situ acidification of BLG/AG mixed dispersions and different experimental methods. The combined signal evolution of dynamic light scattering at 90° scattering angle (I90), electrophoretic mobility, turbidimetry (ô), circular dichroism, and phase contrast microscopy allowed the distinction of critical structural transitions and the definition of their corresponding pH. The formation of soluble BLG/AG complexes was initiated at pHsc (4.90), since I90 and ô significantly increased from the baseline. In parallel or just following complexation, a conformational change of BLG was detected at pHpct (4.8). An increase in positive charge density of BLG induced complex aggregation atpHca (4.7). More efficient charge neutralization of aggregated complexes, especially through the lowering of thenumberofAGnegative charges, promoted initiation of phase separation atpHpsi (4.4).Mixeddispersions became unstable and phase separation occurred at pHps (4.2). The phase separation of mixed dispersions was suggested by the maximum value of scattered light, by an important acceleration of the dispersion turbidity, by a strong increase of hydrod dynamic radii, and by the first appearance of light fluctuations as observed by phase contrast microscopy. At the microscopic level, the first coacervates were observed at pHcoa (4.0), near the pH of the maximum of turbidity. It was also noticed that, from the onset of interactions between biopolymers, the pH decrease led to (i)a gradual homogenization of particle size in the mixed dispersion as suggested by the decrease of dispersion polydispersity and (ii)conformational transitions of the protein (a loss of R-helix structure at pHpct and a gain in protein secondary structure near pHcoa, probably involving â-sheet components).