Role of Ca²⁺ in pepsin-induced coagulation and hydrolysis of casein micelles during dynamic in vitro gastric digestion

The structural properties of the pepsin-induced milk curd in the gastric environment could impact the emptying of the stomach contents and release of nutrients. Calcium is an important composition in milk. The effect of Ca²⁺ on pepsin-induced hydrolysis of κ-casein and subsequent coagulation of casein micelles was studied in a micellar casein solution at pH ~ 6.0 at 37 °C. An NaCl-supplemented micellar casein was used as a positive control to assess the effect of increased ionic strength after CaCl2 addition. Quantitative determination of the released para-κ-casein during the reaction using reverse-phase high-performance liquid chromatography showed that specific hydrolysis of κ-casein by pepsin was little affected by the addition of either CaCl₂ or NaCl. However, rheological properties and the microstructure of curds induced by pepsin hydrolysis depended markedly on the addition of salts. Addition of CaCl₂ up to 17.5 mM facilitated aggregation, with decreases in coagulation time and critical hydrolysis degree, and increases in firming rate and maximum storage modulus (Gʹ_max); further addition of CaCl₂ (22.5 mM) resulted in a lower Gʹ_max. Increased ionic strength to 52.5 mM by adding NaCl retarded coagulation and resulted in a looser curd structure. In a human gastric simulator, micellar casein, without the addition of CaCl₂, did not coagulate until the pH decreased to ~ 5.0 after ~ 50 min of digestion. Addition of CaCl₂ facilitated coagulation of casein micelles and resulted in more cohesive curds with dense structures during digestion, which slowed general hydrolysis of the caseins by pepsin. At the same CaCl₂ concentration, a sample with higher ionic strength resulted in slower coagulation. This study provides further understanding on the effect of divalent (Ca²⁺) ions and ionic strength on the coagulation of casein micelles and the in vitro digestion behavior of milk.