Aggregation behavior of micellar casein during in vitro simulated digestion triggered by serum Ca²⁺ in the gastric fluids

The gastric aggregation characteristics of micellar casein (MC) are closely related to the surrounding digestive environment. Together with pepsin and gastric acid, the effect of serum Ca²⁺ in a digestive fluid is inevitable for the formation of those aggregates. In this study, MC powders with different levels of colloidal calcium phosphate (CCP) were controlled by quick-freezing process. The unique aggregation behaviors accompanied by the addition of exogenous Ca²⁺ during in vitro simulated digestion were investigated. The results showed that quick-freezing could significantly reduce the CCP level up to 50 % compared to controls. The dissociated micelle structure provided plenty of exposed binding sites, which was prone to recombine with serum Ca²⁺ in gastric fluids and thus formed the oversized nanoclusters and massive aggregates in stomach. Only about 40 % of the aggregates were dissolved and hydrolyzed after 120 min of gastric digestion. The results of wide-angle coupled small-angle X-ray scattering demonstrated that serum Ca²⁺ triggered aggregation behaviors largely depend on the pepsin and gastric acids. The para-κ-casein produced by pepsin could be quickly cross-linked by Ca²⁺-induced connections; while the exposed phosphoserine residues caused by gastric acids allowed more monomers to become nanoclusters by the effect of Ca²⁺ bridges.