GB 5009.224-2016National food safety standard - Determination of trypsin inhibitor activity in soy products (English PDF)

3.1 Reagents

3.1.1 Hydrochloric acid (HCl).

3.1.2 Glacial acetic acid (CH3COOH).

3.1.3 Sodium hydroxide (NaOH).

3.1.4 Calcium chloride (CaCl2·2H2O).

3.1.5 Trypsin: freezing storage at -20°C.

3.1.6 Benzoyl-L-arginine-p-nitroaniline (L-BAPA).

3.1.7 Tris [NH2C(CH2OH)3, Tris].

3.1.8 Dimethyl sulfoxide (C2H6OS, DMOS).

5.1 Sample preparation

For powder sample, take at least 200 g of representative sample. Fully mix for use. For bulk or granular sample, take at least 200 g of representative sample.

Crush to below 425 µm. Fully mix for use. Avoid sample is over-heated during crushing process.

5.2 Sample extraction

Weigh 1g ~ 10g (to the nearest of 0.001 g) of well-prepared sample (5.1) in a

100 mL conical flask. Add 50 mL of 0.01 mol/L sodium hydroxide solution. Mix well. Use 1 mol/L hydrochloric acid solution and 0.1 mol/L hydrochloric acid solution to adjust pH to 9.5±0.1. Place in a 0°C~4°C refrigerator for 15h ~ 24h.

Take sample extract out and place at room temperature. Transfer to a 100 mL volumetric flask. Use water to set volume to scale. Shake well. After 15 min of precipitation, as required, dilute sample extract. Dilution depends on the expected TIA value of sample. Store extract in a 0°C~4°C refrigerator. It can be stored for 1 d.

5.3 Dilution of sample extract

Refer to dilution plan in Table A.1 of Annex A. Dilute sample extract into three different dilution concentrations. Ensure that at least determination result of one

TIA value in three inhibition percentages is within 40%~60%.

If three determination results are not within this range, it needs to change dilution for re-determination.

5.4 Determination of trypsin use solution activity

L-BAPA solution (3.2.11) 5 5 5 5

Sample dilution solution (5.3) 0 0 1 1

Water 3 3 2 2

Acetic acid solution (3.2.5) 1 0 1 0

Use vortex oscillator (4.5) to mix solutions in centrifuge tubes well. Place in

37°C constant-temperature water bath (4.6). Insulate for 10 min.

Respective add 1 mL of trypsin use solution (3.2.9) into four centrifuge tubes.

Use vortex oscillator to mix solutions in tubes well. Put test tubes back into constant-temperature water bath (4.6). Insulate in 37°C water bath for

10min±5s. Add 1 mL of acetic acid solutions (3.2.5) into standard tube and sample tube. Mix well.

Place centrifuge tube in centrifuge (4.4). Perform centrifugation at a speed of

4000 r/min for 10 min.

Use visible spectrophotometer (4.1). At a wavelength of 410 nm, use 10 mm cuvette, use water to perform zeroing. Determine supernatant absorbance.

This solution can remain stable within 2 h.

6.1 Inhibition rate of sample extract

Inhibition rate of sample extract is calculated according to formula (1):

Where, i - inhibition rate;

Ar - absorbance of standard solution;

Abr - absorbance of standard blank solution;

As - absorbance of sample solution;

Abs - absorbance of sample blank solution;

100% - conversion factor.

6.2 Trypsin inhibitor activity

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