Native and microbial proteases in milk and their role in the gelation of UHT milk

Karen Reichard Kohlmann, Purdue University

Abstract

This work has expanded the understanding of the gelation problem in UHT milk. It has been suggested that proteolytic enzymes have an important role in the breakdown of the milk casein proteins. Proteolysis may lead to protein rearrangement and subsequent gelation of UHT milk. Enzymes in milk may be native, as in the plasmin system, or introduced into the milk from psychrotrophic microorganisms which grow and produce enzymes prior to pasteurization. Serine proteolytic enzymes (trypsin and plasmin), along with an activator (urokinase) and an inhibitor (soybean trypsin inhibitor), were aseptically added to commercially processed UHT milk. The amount of proteolysis that resulted did not correlate with gelation, but a low level of added plasminogen (0.3 mg/l) resulted in gelation of the milk. Results hypothesized that native plasminogen activators might be present in the milk. A low level of plasmin (0.15 mg/l) was aseptically added to UHT milk and also resulted in gelation of the milk. The second part of this study examined the relationship between a microbial protease and the plasmin system. Two (of six initial) strains, Pseudomonas fragi K1/22 and Pseudomonas fluorescens M3/6, were selected for continued work based on ability to grow in non-fat dry milk at 7$\sp\circ$C and to produce extracellular proteases. Isolation of the extracellular protease from P. fluorescens M3/6 used (NH$\sb4)\sb2$SO$\sb4$ fractionation, a DE 52 ion exchange column and a G-100 gel filtration column. These steps resulted in an increase in specific activity of 582 and a purification factor of 107. Indications of purity included single bands upon silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Protease characterization found the molecular weight to be 45,500 daltons, and the isoelectric point near pH 8.25. The optimum pH range for activity was between pH 6.0 and 8.0, and the optimum temperature range for activity was between 23$\sp\circ$C and 41$\sp\circ$C. There was activity present at 7$\sp\circ$C. The purified protesase had approximately 40% activity remaining after pasteurization and was thought to be a metalloprotease. The protease was found to have activity on S-2251 (plasmin-like activity), was able to hydrolyze $\alpha$, $\beta$, and $\kappa$ casein, but was not able to activate bovine blood plasminogen.

Degree

Ph.D.

Advisors

Nielsen, Purdue University.

Subject Area

Food science

COinS