METHOD 27:0
Printed with effect from 1st January 2003
UREASE ACTIVITY
1: Scope and Field of Application
This method is for the determination of urease activity of products derived from soya. It
can also be used to determine whether these products have been cooked for a sufficient
length of time.
2: Principle
The urease activity is determined by the amount of ammoniacal nitrogen liberated per
minute by 1g of the product at 30oC from a solution of urea.
3: Reagents
3:1 Hydrochloric acid solution, 0.1N.
3:2 Sodium hydroxide solution, 0.1N.
3:3 Phosphate buffer solution, 0.05M: dissolve 4.45g disodium hydrogen phosphate (Na2HPO4.
2H20) and 3.40g potassium dihydrogen phosphate (KH2PO4) in water and dilute to
1,000ml.
3:4 Urea releasing agent: dissolve 30.0g urea, in 1.000ml phosphate buffer solution (3.3).
Prepare immediately before use.
4: Apparatus
4:1 Potentiometric titration apparatus or high-sensitivity pH-meter (0.02 pH).
4:2 Water bath controlled at 30oC.
4:3 Test-tubes, 150x18mm, provided with ground-glass stoppers.
5: Procedure
5:1 Sample preparation
Weigh approximately 10g of the sample and grind so that it passes through a sieve having
apertures of 0.2mm.
5:2 Enzymolysis and titration
Weigh to the nearest 0.001g, approximately 0.2g of the prepared sample and transfer to a
test-tube provided with a ground-glass stopper (4.3). Add 10ml urea releasing agent (3.4),
stopper immediately and shake vigorously. Allow the tube to stand for exactly 30 minutes
in a water bath set at 30oC (4.2). Immediately add by pipette 10ml 0.1N hydrochloric acid
solution (3.1) and cool rapidly to 20oC. Transfer the contents of the tube quantitatively to a
titration vessel, rinsing twice with 5ml of water. Titrate immediately using the
potentiometric apparatus (4.1) to pH 4.7, using 0.1N sodium hydroxide solution (3.2).
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5:3 Blank test
Weigh to the nearest 0.001g, approximately 0.2g of the prepared sample and transfer to a
test-tube provided with a ground-glass stopper (4.3). Add by pipette 10ml 0.1N
hydrochloric acid solution (3.1) and 10ml of urea releasing agent (3.4). Immediately cool
the tube in ice water for 30 minutes. Continue as in 5.2 from ".... Transfer the contents of
the tube quantitatively....".
NOTE: For urease activities greater than 1mg of N/g/minute at 30oC, the sample size
should be reduced to 50mg. Products containing more than 10% by weight of crude fatty
substances should be defatted cold before analysis.
6: Expression of the Results
The urease activity is calculated by using for formula:
mgN/g/minute at 30oC = 1.4 (b - a)
30 x E
where:
a = ml 0.1N sodium hydroxide solution used in the titration of the sample;
b = ml 0.1N sodium hydroxide solution used in the titration of the blank test; and
E = weight of sample in grams.
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