Kinetics of pepsin-induced hydrolysis and the coagulation of milk proteins

Abstract

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.

Gastric digestion of cow milk, almond milk and oat milk in rats

“…pH 6). 54,55 This confirmed that the early coagulation of the milk proteins was induced by the hydrolysis of κ-CN by gastric pepsin, which destabilized the structural stability of the casein micelles. 56 The α s -CNs and β-CN bands were present during the entire digestion period and showed little degradation, even after 120 min of gastric digestion.…”

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

“…The release of para-κ-casein was quantified by reverse-phase high-performance liquid chromatography (RP-HPLC). 6 After the addition of pepsin and shaking for 20 s, the mixed samples were transferred into nine different test tubes (0.2 mL per tube) and incubated in a water bath at 37 °C. HPLC buffer solution (0.8 mL, 6 M guanidinium hydrochloride, 0.1 M bis-Tris, 19.5 mM DLdithiothreitol, and 5.37 mM sodium citrate, pH 7) was added at different time points (1,2,5,10,20,30,50,70, and 120 min) to stop the hydrolysis reaction.…”

“…Microstructural characterization of curd. The microstructure of the pepsin-induced curd was examined using confocal laser scanning microscopy (CLSM) 6 and scanning electron microscopy (SEM). Fast Green fluorescent dye [30 µL; 1% (wt/vol), in Milli-Q water] was added to the pre-warmed sample (37 °C, 1 mL) in an Eppendorf tube.…”

“…G′ then reached a plateau value, or slightly decreased due to syneresis. 6 The shear stress profiles as a function of strain are shown in Fig. 2 (B and D).…”

“…[3][4][5] Previous studies have suggested that the coagulation of milk in a gastric environment at pH > 6 is due to specific hydrolysis of κ-casein by the action of pepsin, which broadly resembles the action of chymosin on κ-casein that occurs during cheese manufacture. [6][7][8] The addition of Ca 2+ , which is known to improve the texture of chymosininduced milk curd (cheese), might also affect the pepsininduced milk coagulation and the behavior during gastric digestion.…”