Microstructural organization and functionality of transglutaminase-crosslinked casein micelle-based emulsions under pH variation: insights from X-ray microtomography

This study investigated the potential of X-ray microtomography (μCT) to characterize a simple emulsion model, as the transglutaminase (TGase) treated casein micelle emulsions under different pH conditions (3.5-9.0). Particular emphasis was placed on understanding how pH-induced molecular rearrangements are translated into 3D microstructural organization and macroscopic emulsion properties. A multiscale approach was employed, combining μCT, emulsifying and rheological measurements, and spectroscopic analyses of protein secondary and tertiary conformations. The results showed that pH significantly modulated protein conformation, leading to distinct microstructural organizations and macroscopic behaviors of TGase-treated casein micelle emulsion. At native and alkaline pH, increased electrostatic repulsion and molecular flexibility promoted efficient interfacial adsorption, resulting in finer droplet dispersion and high emulsifying activity. In contrast, near the isoelectric point (pH 4.6), extensive protein aggregation led to the formation of dense and interconnected microstructures, which enhanced emulsion stability despite reduced emulsifying capacity. Furthermore, the studied microstructural parameters showed strongly correlation with emulsifying properties (emulsifying activity, emulsifying stability, and destabilization indices) and rheological properties, confirming the key role of droplet organization in governing global emulsion behavior. Moreover, these relationships revealed that pH-dependent molecular changes directly govern the development and stability of the emulsion network. These findings demonstrate that X-ray microtomography is a powerful and suitable tool for characterizing fat droplet volume, spatial distribution, shape, and network organization in simple emulsion systems, comparable to more complex food emulsions. The study provides new insights into how pH governs the structure–function relationships of TGase-treated casein micelle-based emulsions.