Association of Enzymatically and Nonenzymatically Functionalized Caseins Analyzed by Size-Exclusion Chromatography and Light-Scattering Techniques

The influence of covalent protein modifications resulting from the Maillard reaction (glycation) of casein and lactose on the noncovalent association behavior of the protein was studied. Nonenzymatic cross-linking with methylglyoxal (MGO) and glutaraldehyde (GTA) as well as enzymatic cross-linking with microbial transglutaminase (mTG) was investigated in comparison. Molar mass, particle size, and conformational characteristics of nonmicellar casein associates as well as the extent of intraparticle protein cross-linking were examined utilizing size-exclusion chromatography (SEC) combined with UV detection and static and dynamic light scattering. Cross-linking resulted in the stabilization of a certain fraction of casein associates, with particle sizes of approximately 30 nm in radius of gyration (R_g), and promoted an incorporation of further casein molecules into those particles, yielding molar masses (M_w) of 1.0–1.2 × 10⁶ g/mol. When caseins were additionally conjugated with lactose during the early Maillard reaction, a further growth of the associates up to approximately 50 nm in R_g with a M_w of 2.1 × 10⁶ g/mol was observed. Furthermore, glycation reactions induced a transition from slightly elongated, random-coil structures toward more anisotropic conformations. Associates consisting of caseins cross-linked with GTA appeared to preserve the original particle conformation.