Multiscale quantitative characterization of demineralized casein micelles: How the partial excision of nano-clusters leads to the aggregation during rehydration

The amount of colloidal calcium phosphate (CCP) nanoclusters modifies the structure and functional properties of the casein micelles. This study aims to analyze the structural changes in partially demineralized casein micelles (D-CMs) and compare them with native casein micelles (CMs) in order to provide new insights towards the subsequent changes in rehydration behavior. Two rehydration strategies were applied. First is the fast rehydration process (50 degrees C/1 h), close to the industrial practice, while the second one (25 degrees C/24 h), a slow rehydration process, used as control. The presence of large (>10 mu m) and stirring resistant aggregates in D-CMs were evidenced by Static Light Scattering, being significantly higher for D-CMs rehydrated at 50 degrees C/1 h. As evidence from the electrophoresis results, the non-covalent interactions play a major role in aggregate formation. The decrease in the CCP concentration (-15%) leads to a more loosely packed/porous structure of D-CMs (Transmission Electronic Microscopy/Cryo-Fracture), and (as observed using Nuclear Magnetic Resonance and Small Angle X-ray Scattering) the demineralization also contributes to increase disorder in the structure of the micelle. Fluorescence Spectroscopy data reveals the presence of casein-tryptophan residues in a more hydrophobic environment shedding light on the importance of hydrophobic interactions for the aggregation of D-CMs. In summary, the study sketches a clear picture of internal rearrangement of CMs structures, in partially demineralized conditions, followed by fast rehydration at high temperatures.