Comparison of methods for analysis of proteolysis by plasmin in milk

Sensitive methods that are currently used to monitor proteolysis by plasmin in milk are limited due to their high cost and lack of standardisation for quality assurance in the various dairy laboratories. In this study, four methods, trinitrobenzene sulphonic acid (TNBS), reverse phase high pressure liquid chromatography (RP-HPLC), gel electrophoresis and fluorescamine, were selected to assess their suitability for the detection of proteolysis in milk by plasmin. Commercial UHT milk was incubated with plasmin at 37°C for one week. Clarification was achieved by isoelectric precipitation (pH 4.6 soluble extracts) or 6% (final concentration) trichloroacetic acid (TCA). The pH 4.6 and 6% TCA soluble extracts of milk showed high correlations (R2 > 0.93) by the TNBS, fluorescamine and RP-HPLC methods, confirming increased proteolysis during storage. For gel electrophoresis, extensive proteolysis was confirmed by the disappearance of α- and β-casein bands on the seventh day, which was more evident in the highest plasmin concentration. This was accompanied by the appearance of α- and β-casein proteolysis products with higher intensities than on previous days, implying that more products had been formed as a result of casein breakdown. The fluorescamine method had a lower detection limit compared with the other methods, whereas gel electrophoresis was the best qualitative method for monitoring β-casein proteolysis products. Although HPLC was the most sensitive, the TNBS method is recommended for use in routine laboratory analysis on the basis of its accuracy, reliability and simplicity.