Enzymes AACC Method 22-90Page 1 of 2

Measurement of Urease ActivityFinal approval May 1, 1969; Reapproval November 3, 1999

ObjectiveThe method is a modified Caskey-Knapp method that yields residual urease

present in soybean products under conditions of the test. It is applicable to soy-bean meals, soy flour, and millfeeds.

Apparatus1. 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 of0.02 pH units or better, and with temperature compensator.

3. Test tubes, 20 150 mm, fitted with rubber stoppers.

Reagents1. Phosphate buffer solution, 0.05M. Dissolve 3.403 g KH2PO4 in approxi-

mately 100 ml freshly distilled water. Dissolve 4.355 g K2HPO4 in approxi-mately 100 ml water. Combine the two solutions and make to 1000 ml. AdjustpH of solution to 7.0 with solution of strong acid or base before using. Usefullife of buffer solution, prepared as described, is less than 90 days.

2. Buffered urea solution. Dissolve 15 g urea in 500 ml phosphate buffer solu-tion. Add 5 ml toluene to serve as preservative. Adjust pH of urea solution to 7.0as for reagent 1.

Procedure1. 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 requiresno further grinding.

2. Weigh 0.200 g of prepared sample into test tube. Add 10 ml buffered ureasolution, 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. Add10 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 diges-

tion period, remove tubes from water bath at end of 30 min, and determine pH ofsupernatant liquids.

6. Take pH reading at exactly 5 min after removal of tubes from bath. SeeNote 1.

CalculationRecord difference in terms of pH between test and blank runs as pH increase

or urease activity.

Enzymes AACC Method 22-90Page 2 of 2

Measurement of Urease Activity (continued)Precision

Intralaboratory precision is 0.03 and 0.10 pH units on cooked and uncookedmaterial, respectively. Interlaboratory precision should be 0.05 and 0.15 pHunits, respectively.

NoteCare must be exercised to prevent contamination of all glassware or elec-

trodes. Should the pH instrument fail to deliver a prompt stable reading, checkinto the difficulty. Occasionally trouble is caused by a coating from the solublefraction of the soybeans on the fiber in the calomel electrode.

ReferenceCaskey, C. D., Jr., and Knapp, F. C. 1944. Method for detecting inadequately heated soybean oil

meal. Ind. Eng. Chem., Anal. Ed. 16:640.