GB/T 23195-2008 in English

《GB/T 23195-2008蜂花粉中过氧化氢酶的测定方法 紫外分光光度法》由442（中华全国供销合作总社）归口，主管部门为中华全国供销合作总社。
本标准规定了蜂花粉中过氧化氢酶的测定方法。
本标准适用于蜂花粉中过氧化氢酶的测定。

Scope

The absorbance of the liquid varies with reaction time within a certain range, using deionized water as reagent. 4.1 pH Buffer Solution: Accurately weigh 0.5 g disodium EDTA and dissolve it in 500 mL deionized water, then mix well; accurately weigh 0.1 g KCl and add it, measure 5 mL NaOH solution, add it to a total of 150 mL, dilute to the mark, and mix well.

4.2 Hydrogen Peroxide Solution: Accurately take 10.0 mL from it for mixing, then accurately measure 0.07 mL hydrogen peroxide solution for analysis.

4.3 Liquid Nitrogen.

5 Equipment:
5.1 UV Spectrophotometer;
5.2 Refrigerated Centrifuge;
5.3 Water Bath Incubator;
5.4 pH Meter;
5.5 Analytical Balance (sensitivity 0.1 mg);
5.6 Micropipette (100 μL and 5000 μL).

6 Preparation of the Test Solution: Weigh 1 g sample, accurate to 1 mg, place it in a mortar that has been pre-cooled with an appropriate amount of liquid nitrogen, grind while adding liquid nitrogen. After the sample is thoroughly ground, transfer it to a beaker and thaw until it warms up, then add about 10 mL freshly prepared phosphate buffer (4.1) to dissolve it. Transfer it to a 25 mL volumetric flask, rinse the mortar with phosphate buffer several times, combine the washings and dilute to the mark. Mix well and place the volumetric flask in the refrigerator for 30 minutes. Take the supernatant, centrifuge at 4°C, 12000 rpm for 15 minutes on a refrigerated centrifuge, then take the clear liquid as the test solution.

7 Analysis Procedure:
7.1 UV Spectrophotometer Settings:
- Mode: Time Scan;
- Wavelength: 240 nm;
- Measurement time: 120 s;
- Reading interval: 5 s.

7.2 Place the sample and hydrogen peroxide solution (4.2) separately in a 40°C water bath for 10 minutes. Accurately take 0.1 mL of the sample into a quartz cuvette, place the quartz cuvette back into the slot (the instrument has already been zero-point calibrated before placing the quartz cuvette), then accurately measure and add 2.9 mL of hydrogen peroxide solution (4.2) to the sample, mix well, start reading immediately.

7.3 From 0s~90s, select the linear data for about 1 minute and calculate the difference in absorbance.

8 Calculation and Expression:
The enzyme activity corresponding to a decrease of 0.01 absorbance at 240 nm wavelength is one enzyme unit (U). Peroxidase activity (U/g) is calculated by formula (1):
\[
U = \frac{A_{min}}{V}
\]
where:
- A is the selected absorbance difference for 1 minute in the results;
- V is the total volume of the sample, units are milliliters (mL);
- 0.01 refers to one enzyme unit (U) per decrease of 0.01 absorbance at 240 nm wavelength;
- t is 1 minute;
- V_sample is the volume of the test solution used for measurement, in milliliters (mL);
- m is the mass of the sample, units are grams (g).

The calculation result is expressed to the nearest whole number and reported as the arithmetic mean of parallel determinations.

9 Allowable Deviation: The relative standard deviation of parallel or repeated measurements within the same laboratory should not exceed 10%.