Unmasking the internal structure of casein micelles through enzymatic hydrolysis: A SAXS study

Abstract

Casein micelles, one of the most studied natural association colloids, are supramolecular assemblies of caseins and colloidal calcium phosphate that constitute the fundamental building blocks of dairy matrices. Despite extensive investigation, the internal structure of casein micelles remains debated. While $κ$-casein is known to ensure colloidal stability of casein micelle suspension, the spatial organization of casein fractions and salts is still unresolved, and several structural models coexist. Small-angle scattering is a method of choice to probe biological colloids \emph{in situ}, yet interpretation of scattering data remains challenging due to the hierarchical nature of casein micelles. Here we address this issue by probing micelle structure during enzymatic gelation induced by chymosin, which cleaves $κ$-casein and triggers aggregation. Using time-resolved SAXS, we probe structural changes throughout the sol-gel transition over length scales from 3 nm to 3 $μ$m. First, we show that enzyme-driven aggregation, counterintuitively, reveals information about the internal organization of casein micelles: the reduction of specific surface area during gelation unmasks a high-q structural peak previously observed under contrast-matching conditions. Second, we report that $κ$-casein cleavage leads to a gradual disappearance of the structural feature at intermediate scales. Analysis of the disappearance kinetics and comparison to structural models reveal that $κ$-casein cleavage induces a progressive relaxation of the colloidal porous substructure, providing direct evidence for its contribution to micellar organization. More broadly, these results demonstrate that the gelation process provides unique access to the internal structure of biological colloids and offers new perspectives for interpreting scattering data in complex soft-matter systems.