Asymmetric flow field flow fractionation for the investigation of caseins cross-linked by microbial transglutaminase

The cross-linking of caseins with microbial transglutaminase (mTGase) was investigated using asymmetric flow field flow fractionation in combination with static and dynamic light scattering detections (AF4-MALS-DLS). This approach allows determining molar mass, molecular size, scaling properties, and apparent densities of casein aggregates prior to and after enzymatic treatment. The results show that, as a consequence of non-covalent interactions, casein molecules associate to form elongated aggregates with a molar mass (M_w) ranging from approx. 4 × 10⁵ to 1.2 × 10⁶ g mol⁻¹, and a mean radius of gyration (R_g,z) of approx. 16 nm. Upon enzymatic treatment with mTGase, casein aggregates become more compact as M_w increases but R_g decreases with ongoing cross-linking reaction. In contrast, the hydrodynamic radius (R_h) distribution determined by online DLS is rather unaffected by enzymatic cross-linking, indicating that mTGase cross-links casein molecules mainly within distinct aggregates. Furthermore, extensive enzymatic treatments with mTGase change the molecular shapes of casein aggregates towards more compact and denser spherical structures.