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

Abstract

The effect of Ca²⁺ on pepsin-induced hydrolysis of κ-casein and subsequent coagulation of casein micelles was studied in a micellar casein (MC) solution at pH ≈ 6.0 at 37 °C without stirring. An NaCl-supplemented MC solution 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 microstructures of curds induced by pepsin hydrolysis depended markedly on the addition of salts. Addition of CaCl₂ up to 17.5 mM facilitated coagulation, with decreases in coagulation time and critical hydrolysis degree, and increases in firming rate and maximum storage modulus (G′_max); further addition of CaCl₂ (22.5 mM) resulted in a lower G′_max. Increased ionic strength to 52.5 mM by adding NaCl retarded the coagulation and resulted in a looser curd structure. In a human gastric simulator, MC, 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 the emptying rate of caseins. At the same CaCl₂ concentration, a sample with higher ionic strength coagulated more slowly. This study provides further understanding on the effect of divalent (Ca²⁺) ions and ionic strength on the coagulation of casein micelles and the digestion behavior of milk.