Investigating the plasmin system: From skim milk to milk protein concentrate

In milk protein concentrate manufacturing, activators and inhibitors of the plasmin system are concentrated together, thus working against one another and thus making predictions for levels of plasmin-driven proteolysis in MPCs difficult. Moreover, it is reasonable to hypothesize that the concentrated plasmin system enzymes could have induced alterations in their individual activities. Therefore, it is impossible to predict the net effect of milk protein concentration on the potential or risks of proteolysis of model beverages made from MPC. The aim of this work is to characterize plasmin-system activities in MPC85, a common milk-beverage ingredient. In this study, MPC85 powder was manufactured using a pilot-scale membrane ultrafiltration unit and a mini-spray dryer. Fluorescence of N-Succinyl-Ala-Phe-Lys-7-amido-4-methylcoumarin (Suc-Ala-Phe-Lys-AMC) was used to assess apparent plasmin activity in MPC85 and its processing streams. Additionally, the extent of casein hydrolysis at different aging periods (d 0, 3, 7, and 14 at 37°C) was determined using liquid chromatography. The UF+DF process and spray drying, respectively, did not impact apparent plasmin activities on a solids basis. However, at the early storage stage (d 3), casein proteolysis in MPC85 powder (∼76%) was not statistically different from the feed milk (∼67%). After 14 d, casein proteolysis was nearly complete (>90%) for all samples. These obtained results suggest that concentration of milk proteins enhances competitive inhibition in fluorescent plasmin assays, and that the true rate of casein hydrolysis by plasmin does not greatly change in MPC85 relative to milk. Moreover, both plasmin and plasminogen activator activities rose during storage, likely due to plasminogen activation and conversion of the activator to its 2-chain form by plasmin. These findings illustrate the impacts of MPC processing on the dynamics of plasmin activity and proteolysis over a controlled storage period. This knowledge may guide and motivate further research on improving MPC85 quality attributes by reducing its proteolytic potential in diverse food formulas.