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Determination of Proteolysis by Dye Binding¹

Department of Dairy and Food Industry Iowa State University, Ames

ABSTRACT

Dye-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 proteolysis particularly useful in screening microorganisms for their ability to digest milk proteins.

Methods

Orange G dye was dried to constant weight at 110 C. An aqueous solution containing 1.0638 g of 94% Orange G and 0.3020 g of oxalic acid dihydrate (0.05 M) 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 pipetted into selected tubes.

¹ Journal Paper J-5333 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa, Project 1488.