SAMPLING AND ANALYSIS OF OILSEED BY-PRODUCTS

AOCS Official Method Ba 9-58

Reapproved 2009

Urease Activity
DEFINITION
This method determines the presence of residual urease in soybean products under the conditions of the test
(References, 1).
SCOPE
Applicable to soybean meals, soy flour and to soybean mill feeds, except when urea has been added.

APPARATUS
1. Water bath—capable of being maintained at a temperature of 30 ± 0.5°C.
2. pH Meter—equipped with glass and calomel electrodes and capable of testing 5 mL of solutions. It should be a precision
instrument with a temperature compensator having a sensitivity of ± 0.02 pH units or better. Follow manufacturer’s instruc-
tions for the operation, standardization, and determination of pH. Calibrate the meter with standard buffers with values at
or near the range at which measurements are to be made (see Notes, 1).
3. Test tubes—20 mm × 150 mm, fitted with rubber stoppers.

REAGENTS
1. Phosphate buffer solution (0.05 M)—Dissolve 3.403 g of reagent-grade monobasic potassium phosphate (KH2PO4) in
approximately 100 mL of freshly distilled water. Dissolve 4.355 g of reagent-grade dibasic potassium phosphate (K2HPO4)
in approximately 100 mL of water. Combine the two solutions and make to exactly 1000 mL. If reagents are pure, pH
should be at 7.0. If it is not, adjust to 7.0 with a solution of an acid or base before using. The useful life of the buffer ­solution,
prepared as described, is less than 90 days.
2. Buffered urea solution—Dissolve 15 g urea (reagent grade) in 500 mL of the phosphate buffer solution. Add 5 mL of toluene
(see Notes, Caution) to serve as a preservative and to prevent mold formation. Adjust the pH of the urea solution to 7.0 as
in Reagents, 1.

PREPARATION OF TEST SAMPLE

1. Grind the laboratory sample as finely as possible without raising the temperature, and mix completely. At least 60% of the
grind should pass a US no. 40 standard sieve (particle size 0.425 mm or less). Soy flour requires no grinding, but make
­certain it is well mixed.

PROCEDURE
1. Weigh 0.200 g (± 0.001 g) of the test sample into a test tube and add 10 mL of the buffered urea solution. Stopper, mix, and
place in water bath at 30°C. Do not invert the tube during the process of mixing.
2. Prepare a blank by weighing 0.200 g (± 0.001 g) test sample into a test tube and add 10 mL of the phosphate buffer solution.
Stopper, mix, and place in water bath at 30°C. Allow a time interval of 5 min between the preparation of the test and the
blank portions. Agitate the contents of each tube at 5-min intervals.
3. Remove the test and blank portions from the water bath after 30 min. Transfer the supernatant liquids to a 5.0 mL beaker,
maintaining the 5 min interval between the test and blank. Determine the pH of the supernatant liquids at exactly 5 min
after removal from the bath (see Notes, 1).

CALCULATIONS
1. A difference between the pH of the test sample and the pH of the blank is an indication and an index of urease activity.

NOTES
Caution
Toluene is flammable and a dangerous fire risk. Explosive limits in air are 1.27–7%. It is toxic by ingestion, inhalation, and skin
absorption. The TLV is 100 ppm in air. A fume hood should be used at all times when using toluene.

NUMBERED NOTES
1. Care must be exercised to prevent contamination of all glassware or electrodes. Should the pH instrument fail to deliver
a prompt and stable reading, investigate. Frequently, the flow of electrolyte through the porous fibers in the calomel electrode
may be retarded by a coating of the soluble fraction from soybean.

REFERENCES
1. This method is a modification of the procedure of Caskey, C.D., and F.C. Knapp, Ind. Eng. Chem., Anal. Ed. 16:640 (1944).

Page 1 of 1