Determination of Proteolysis by Dye Binding

Determination of Proteolysis by Dye BindingDye-binding methods have been advocated for rapid quantitative measurement of milk protein (2, 8~ 11).This method can also be used as a simple sensitive test for proteo]ysis particularly useful in screening microorganisms for their ability to digest milk proteins. MethodsOrange G dye was dried to constant weight at 110 C.An aqueous solution eontMning 1.0638 g of 94% Orange G and 0.3020 g of oxalic acid dihydrate (0.05 ~) per liter was prepared (9).Enough of this solution should be prepared in one batch to complete the experiment, because the purity of dye varies from lot to lot.Milk samples of 1 ml were pipetted carefully into test tubes.In the experiments in which bacterial proteolysis was studied, sterile milk was inoculated with bacterial culture, and aliquots were aseptically transferred into sterile screw-cap tubes.Uninoculated control tubes also were prepared.The milk was incubated at 37 C and, at appropriate times, 15 ml of the Orange G solution was pipctted into selected tubes.Control milk should be incubated under the same conditions for the same times, since the dye binding of sterile milk seems to change slightly with time.The milk was thoroughly mixed with the Orange G solution by using a mechanical stirrer operating at 1,800 rpm.Care was taken to break up any coagulum and loosen curd particles adhering to the tube walls.The milk was left to equilibrate at least 30 min but, frequently, overnight periods were used.The reaction mixtures were then centrifuged.The absorbance of the supernatant dye solution was measured in a Beckman DU spectrophotometer in a special flow-through cuvette.The cuvette had a path length of 0.75 mm and was modeled ~fter that of Ashworth and Seals (1, 2).The euvette was designed to fit into a regular Beckman DU euvette holder from which one of the partitions had been removed.Teflon spacers, cemented to the cell with epoxy resin, positioned the cuvette snugly and reproducibly in its holder.The outside of all portions of the cuvette extending from the cell compartment of the spcetrophotometer was painted with a flatblack paint.A special lid that contained slots to accommodate the cuvette's inlet and outlet tubes was made for the speetrophotometer sample chamber.When making absorbance readings, the top of the cuvette was covered with a black Journal Paper J-5333 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa.Project 1488.