Poultry Science: Outreach & Extension: Soybeans
Urease Assay
Described by: Association of Official Analytical Chemists, 1980. Official Methods of Analysis. 13th ed. Association of Official Analytical Chemists, Washington, D.C.
Basis of assay: Determines the activity of residual urease in soybean products
for use as an indicator of sufficient heat treatment; urease catalyzes a breakdown
of urea and results in a pH rise due to the generation of ammonia.
(NH2)2 - C = O + H2O ? CO2 + NH3
Urea
Procedure:
Place approximately 0.200 g (± 0.001 g) of sample into a test tube
(sample should be finely ground - grind large particles in coffee grinder).
Add 10 mL of the buffered urea solution - this will be called the test
sample
Stopper, mix/swirl gently (do not invert tube) and place in water bath at
30o C noting the time put into the bath.
Note: Allow a five minute interval between the preparation of the test and
blank samples.
Place approximately 0.200 g (± 0.001 g) of sample into a test tube
(sample should be finely ground - grind large particles in coffee grinder).
Add 10 mL of the phosphate buffer solution - this will be called the blank
sample.
Stopper, mix/swirl gently (do not invert tube) and place in water bath at
30o C noting the time put into the bath.
In both test and blank samples tubes, mix/swirl contents of tubes approximately
every five minutes during the required time in the water bath.
At end of 30 minutes, remove tubes (each tube will have a different removal
time from the water bath depending on time put in) and mix/swirl contents
one last time.
Allow tube to stand for a few minutes and transfer approximately 5 mL of the
supernatant liquid into a small vial or beaker (vial or beaker must provide
for electrode of pH meter to fit into it and to be covered with the liquid).
Determine the pH of the supernatant liquid approximately five minutes after
removal from the water bath.
Calculate differences between pH of test and pH of blank as an index of urease
activity.
Solution preparation:
Phosphate buffer solution (0.05 M phosphate buffer):
Dissolve 3.403 g of monobasic potassium phosphate (KH2PO4 , AR grade) in approximately
100 mL of distilled water. Dissolve 4.355 g of dibasic potassium phosphate
(K2HPO4, AR grade) in approximately 100 mL of distilled water. Combine the
two solutions and dilute to 1000 mL. Use stir bar and stir plate to mix solutions.
Adjust the pH of the buffer solution to 7.0 with a strong acid or base before
using. Use buffer within 90 days.
Buffered urea solution:
Dissolve 15 g of urea (AR grade) in 500 mL of the phosphate buffer solution.
Add 5 mL of toluene* to serve as a preservative and to prevent mold formation.
Use stir bar and stir plate to mix solutions. Adjust the pH of the urea solution
to 7.0 with a strong acid or base before using.
* Note - toluene is not needed if solution is to be used promptly; if used,
solution must be stirred for the duration of use to prevent separation into
specific layers.
Calculation Example:
pH of test sample - 9.2
pH of blank sample - 6.8
Urease Index (pH change) = (pH of test sample) - (pH of blank
sample)
= 9.2 - 6.8
= 2.4