Thermal inactivation of the plasmin system in MPC85 dispersions

Despite its historical attention in research, the plasmin (PL) protease system remains a critical issue in milk protein beverage stability. PL system enzymes are known to remain active after intense heat treatments and result in proteolysis in beverages with fortified milk proteins during shelf-life. In this work we studied the inactivation kinetics of PL and plasminogen activators (PA) in MPC85 protein dispersions. An oil bath was used to heat-treat MPC85 samples stored in industry-relevant stainless-steel tubes at temperatures from 83°C to 121°C. Remaining PL activities after heating were determined using the fluorescent substrate N-Succinyl-Ala-Phe-Lys 7-amido-4-methylcoumarin. The obtained results showed that PAs were more heat stable than PL in MPC85 dispersions. Obtained D-values followed known thermodynamic behavior for PL in the presence of whey proteins, suggesting a minor protective mechanism against inactivation not normally present in milk samples. Furthermore, at high temperatures there was an increase in the z-values for PL and PG inactivation, suggesting a change in underlying mechanisms. HPLC was used to characterize proteolysis during early storage to verify heat treatment efficacy on preventing proteolysis. Results showed reductions in proteolysis in heat-treated samples, suggesting the conducted heat treatments were effective for inactivating PL activity, consistent with the observed limited PL activity as analyzed by the fluorescent substrate assay. Given the established inactivation parameters in this study, we suggest heating temperatures of 92-97°C for optimal PL inactivation. These findings may motivate future research on new thermal processing conditions for improving PL inactivation and proteolytic stability of milk protein beverages.