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Hydrolysis-induced coagulation of casein micelles by pepsin occurs during the digestion of milk. In this study, the effect of pH (6.7-5.3) and pepsin concentration (0.110-2.75 U/mL) on the hydrolysis of κ-casein and the coagulation of the casein micelles in bovine skim milk was investigated at 37°C using reverse-phase HPLC, oscillatory rheology, and confocal laser scanning microscopy. The hydrolysis of κ-casein followed a combined kinetic model of first-order hydrolysis and putative pepsin denaturation. The hydrolysis rate increased with increasing pepsin concentration at a given pH, was pH dependent, and reached a maximum at pH ∼6.0. Both the increase in pepsin concentration and decrease in pH resulted in a shorter coagulation time. The extent of κ-casein hydrolysis required for coagulation was independent of the pepsin concentration at a given pH and, because of the lower electrostatic repulsion between para-casein micelles at lower pH, decreased markedly from ∼73% to ∼33% when pH decreased from 6.3 to 5.3. In addition, the rheological properties and the microstructures of the coagulum were markedly affected by the pH and the pepsin concentration. The knowledge obtained from this study provides further understanding on the mechanism of milk coagulation, occurring at the initial stage of transiting into gastric conditions with high pH and low pepsin concentration.

The effect of whey proteins and heat treatment (90 °C, 5 min) on pepsin-induced hydrolysis of κ-casein, and subsequent coagulation of casein micelles, was investigated at pH 6.3 and 6.0 using reverse-phase HPLC, oscillatory rheology, and confocal laser scanning microscopy. Whey proteins did not affect the hydrolysis of κ-casein but retarded the coagulation process. Heat treatment did not affect the hydrolysis kinetics in whey protein (WP)-free samples, but slightly impaired the hydrolysis rate in WP-containing samples. The coagulation process of WP-free samples was little affected by heat-treatment. However, compared with unheated WP-contained sample at the same pH, the coagulation process of the heated sample was retarded at pH 6.3 but enhanced at pH 6.0. The curd in heated samples with smaller pores had higher water holding capacity. This knowledge provides further understanding on the role of whey proteins and heat treatment on the coagulation mechanisms of milk under gastric conditions.