GB 5009.224-2016 PDF English
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National food safety standard - Determination of trypsin inhibitor activity in soy products
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GB 5009.224-2016: National food safety standard - Determination of trypsin inhibitor activity in soy products
---This is an excerpt. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.), auto-downloaded/delivered in 9 seconds, can be purchased online: https://www.ChineseStandard.net/PDF.aspx/GB5009.224-2016
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard - Determination of
trypsin inhibitor activity in soy products
ISSUED ON: AUGUST 31, 2016
IMPLEMENTED ON: MARCH 01, 2017
Issued by: National Health and Family Planning Commission of the
People's Republic of China
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Principle ... 4
3 Reagents and materials ... 4
4 Instruments and equipment ... 6
5 Analysis steps ... 6
6 Expression of analysis results ... 8
7 Precision ... 9
8 Other ... 9
Annex A Dilution plan for sample extract ... 10
National Food Safety Standard - Determination of
trypsin inhibitor activity in soy products
1 Scope
This Standard specifies determination method for trypsin inhibitor activity (TIA)
in soy products.
This Standard is applicable to determination for trypsin inhibitor activity in soy
products.
2 Principle
Trypsin can react with benzoyl-L-arginine-p-nitroanilide (L-BAPA) to form p-
nitroaniline. This substance has characteristic absorption at 410 nm. Trypsin
inhibitor activity in soy products can inhibit this reaction to decrease absorbance
value. The degree of decline is proportional to the trypsin inhibitory activity. Use
spectrophotometer to determine absorbance values before and after this
reaction at 410 nm. Quantitatively analyze trypsin inhibitor activity.
3 Reagents and materials
Unless otherwise stated, reagents used in this method are analytically-pure and
water is grade-three water specified in GB/T 6682.
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).
4 Instruments and equipment
4.1 Visible spectrophotometer.
4.2 Analytical balance: resolutions are 0.01 g, 0.001 g, 0.0001 g respectively.
4.3 PH meter: precision is 0.05.
4.4 Centrifuge: speed ≥4000 r/min.
4.5 Vortex oscillator.
4.6 Constant-temperature water bath: precision is ±0.1°C.
5 Analysis steps
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 Expression of analysis results
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
......
Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
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