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Simple Method for Detecting Amylase Inhibitors in Biological Materials¹

Department of Microbiology and Immunology, Veterinary College of Norway, Oslo, Norway, and Department of Food Science and Technology, University of California, Davis, California 95616

A simple semiquantitative method for detecting amylase or amylase inhibitors in biological materials is described. The substrate consists of a 0.25% starch-1.5% agar gel slab buffered at pH 6.5 with 0.1 M phosphate-0.01 M sodium chloride. Three millimeters wide cellulose strips saturated with a solution to be examined for inhibitor are placed parallel on the gel slab for 2 hours at 37°. The strips are removed and other 3-mm wide cellulose strips saturated with amylase solutions are placed at right angles across the first strips. The system is incubated for 6 or 18 hours at 37°. After flooding the slab with Lugol (an iodine containing) solution, amylase activity is shown by clear lysis zones on a deep-purple background. Presence of inhibitors is indicated by interruption or narrowing of the lysis zone where the inhibitor-containing and amylase-containing strips crossed. A variation of this method using amylase or amylase-inhibitor mixtures placed into 7-mm wells cut into the starch-agar gel slab is also described. The starch-agar gel slab methods were compared with the Bernfeld method of determining amylase activity.

KEY WORDS: • amylase • amylase inhibitor

¹ The authors are grateful to the Agricultural Research Council of Norway for financial support and to Professor Sandvik and the Department of Microbiology and Immunology, Veterinary College of Norway for use of facilities.

Manuscript received 17 January 1974.