Thermal effect on the activity of plasmin system components

Plasmin is an indigenous milk proteinase that can affect the quality of dairy products, either positively (e.g. accelerating cheese ripening), or negatively (e.g. reducing the shelf-life of ultra-high temperature milk). Plasmin activity increases when its zymogen plasminogen (PG) is converted to PL by the action of plasminogen activators (tissue-type, t-PA; or urokinase-type, u-PA). Plasmin and PAs are inhibited by plasmin inhibitor (PI) and plasminogen activator inhibitor (PAI), respectively. Previous research on the thermal stability of the PL system has been conducted. However, previous studies have one or more of the following limitations: (1) Water- and oil-baths were used for thermal treatments; (2) Enzymes were treated in buffers versus milk; (3) Direct measure of PI and PAI was not conducted; and (4) Differentiation of t-PA and u-PA thermal stabilities was not completed. Therefore, the objective of this research was to determine the effect of thermal treatments on the activity of PL system components in milk heated (65-90°C, 15-30 s) with a pilot scale plate heat exchanger. Plasmin system components were isolated from heated milks and quantified using spectrophotometry and specialized electrophoresis. Further characterization of PI was carried out using immunochemical and enzyme kinetic assays. Plasmin inhibitor was more stable to pasteurization (75°C, 15 s) than PAI, retaining 64 versus 20% of their original activities in milk, respectively. The milk PI was identified as α2-antiplasmin, as suggested by immunoblotting and competitive inhibition of PL. The activities of bovine PL, PG, t-PA and u-PA were not significantly affected by pasteurization. At temperatures greater than 75°C, u-PA was significantly more stable than t-PA. The inactivation of PAI after pasteurization, coupled with the relative thermal stability of bovine PAs, may lead to increased PL via PG activation upon storage of pasteurized milk, thus affecting the quality of stored milk and dairy products.