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Protein Technology Topic: Determination of Urease Activity in Soybean Meal
First, the purpose of the experiment
Master the basic principles and method steps of various methods for determining urease activity.
Second, the experimental principle
The pulverized soy product was mixed with a neutral urea buffer solution, and after 30 minutes at 30 ° C, urease catalyzed the hydrolysis of urea to produce ammonia. The produced ammonia was neutralized with an excess of hydrochloric acid solution, and then returned dropwise with a standard solution of hydrogen hydroxide.
Third, the experimental equipment
1. Sample sieve: pore size 200μm;
2, acidity meter: accuracy 0.02pH, with a magnetic stirrer and titration device;
3, constant temperature water bath: temperature control 30 ± 0.5 ° C;
4, test tube: diameter 18mm, length 1 50mm, there is a grinding plug;
5, chronograph;
6, pulverizer: should not produce strong heat when smashing (such as ball mill
7. Analyze the balance. Sensing amount of 0.1mg;
8, pipette; 10mL.
Fourth, the experimental content
1. Weigh about 0.2g of the pulverized sample, weigh it to 0.1mg, and transfer it to the test tube. (If the sample with high activity is only 0.05g sample), transfer it to 10ml urea buffer solution and cover it immediately. The test tube was shaken vigorously and immediately placed in a constant temperature water bath of 30±0.5°C, and the accurate time was kept for 30 minutes. Immediately transfer 10 ml of hydrochloric acid solution and rapidly cool to 20 °C.
2. Transfer the contents of the tube to the beaker, rinse the tube twice with 5 ml of water, and immediately titrate to pH 4.70 with sodium hydroxide standard solution.
3. Take another test tube for blank test, transfer 10 ml of urea buffer, 10 ml of hydrochloric acid solution. A sample equivalent to the above sample amount was weighed and weighed to 0.1 mg, which was quickly added to the test tube. Immediately cover the tube and shake vigorously. Place the tube in a constant temperature water bath at 30±0.5°C, hold it for exactly 20min, cool to 20°C, transfer the contents of the tube to the beaker, rinse the tube twice with 5mL of water, and use hydrogen peroxide. The sodium standard solution was titrated to pH 4.70.
4. The urease activity (UA) is expressed as the amount of ammonia released per gram of soy product per minute, which can be calculated as follows:
If the sample is pre-dried before pulverization, it is calculated as follows:
The difference between the results of one or two consecutive measurements by an analyst using the same method should not exceed 10% of the average and the results are reported as their arithmetic mean.