Enzymes

AACC Method 22-90 Page 1 of 2

Measurement of Urease Activity

Final approval May 1, 1969; Reapproval November 3, 1999

Objective The method is a modified Caskey-Knapp method that yields residual urease present in soybean products under conditions of the test. It is applicable to soybean meals, soy flour, and millfeeds. Apparatus 1. Water bath capable of being maintained at 30 0.5. 2. pH meter equipped with glass and calomel electrodes and with provision for testing 5 ml of solutions. It should be a precision instrument with sensitivity of 0.02 pH units or better, and with temperature compensator. 3. Test tubes, 20 150 mm, fitted with rubber stoppers. Reagents 1. Phosphate buffer solution, 0.05M. Dissolve 3.403 g KH2PO4 in approximately 100 ml freshly distilled water. Dissolve 4.355 g K2HPO4 in approximately 100 ml water. Combine the two solutions and make to 1000 ml. Adjust pH of solution to 7.0 with solution of strong acid or base before using. Useful life of buffer solution, prepared as described, is less than 90 days. 2. Buffered urea solution. Dissolve 15 g urea in 500 ml phosphate buffer solution. Add 5 ml toluene to serve as preservative. Adjust pH of urea solution to 7.0 as for reagent 1. Procedure 1. Grind sample as fine as possible without raising temperature. It is preferable that at least 60% of sample pass a no. 40 U.S. Standard sieve. Soy flour requires no further grinding. 2. Weigh 0.200 g of prepared sample into test tube. Add 10 ml buffered urea solution, stopper, mix, and place in water bath at 30. Note time. (Test sample.) 3. Prepare blank by weighing 0.200 g of prepared sample into test tube. Add 10 ml phosphate buffer solution, stopper, mix, and place in water bath at 30. Note time. (Blank.) 4. Allow 5-min interval between preparing test and blank. 5. In both test and blank runs, mix contents of tubes every 5 min during digestion period, remove tubes from water bath at end of 30 min, and determine pH of supernatant liquids. 6. Take pH reading at exactly 5 min after removal of tubes from bath. See Note 1. Calculation Record difference in terms of pH between test and blank runs as pH increase or urease activity.

Enzymes

AACC Method 22-90 Page 2 of 2

Measurement of Urease Activity (continued)

Precision Intralaboratory precision is 0.03 and 0.10 pH units on cooked and uncooked material, respectively. Interlaboratory precision should be 0.05 and 0.15 pH units, respectively. Note Care must be exercised to prevent contamination of all glassware or electrodes. Should the pH instrument fail to deliver a prompt stable reading, check into the difficulty. Occasionally trouble is caused by a coating from the soluble fraction of the soybeans on the fiber in the calomel electrode. Reference
Caskey, C. D., Jr., and Knapp, F. C. 1944. Method for detecting inadequately heated soybean oil meal. Ind. Eng. Chem., Anal. Ed. 16:640.