GB 5009.5-2025 National food safety standard - Determination of protein in food
1 Scope
This standard specifies methods for the determination of protein in food.
This standard applies to the determination of protein in food.
Method I Kjeldahl method for nitrogen determination
2 Principle
Proteins in food are broken down under catalytic heating conditions and the resulting ammonia combines with sulfuric acid to form ammonium sulfate. Alkaline distillation frees ammonia, which is absorbed with boric acid and titrated with sulfuric acid or hydrochloric acid standard volumetric solution. The nitrogen content is calculated according to the consumption of sulfuric acid or hydrochloric acid, and then multiplied by the conversion factor to obtain the protein content.
3 Reagents and materials
Unless otherwise specified, analytical grade reagents and Grade 3 water defined in GB/T 6682 are adopted for the purpose of this method.
3.1 Reagents
3.1.1 Copper sulfate (CuSO4·5H2O).
3.1.2 Potassium sulphate (K2SO4).
3.1.3 Sodium hydroxide (NaOH).
3.1.4 Sulfuric acid (H2SO4): ≥98%.
3.1.5 Boric acid(H3BO3).
3.1.6 Alcohol (C2H5OH): 95%.
3.1.7 Methyl red (C15H15N3O2).
3.1.8 Bromocresol green (C21H14Br4O5S).
3.1.9 Methylene blue (C16H18ClN3S·3H2O).
3.1.10 pH test paper: with the measuring range of 0~14.
3.2 Preparation of reagents
3.2.1 Boric acid solution (20g/L): Take 20g of boric acid, dissolve with water and dilute to 1,000mL.
3.2.2 Sodium hydroxide solution (400g/L): Take 40g of sodium hydroxide and dissolve with water, cool and dilute to 100mL.
3.2.3 Sulfuric acid standard volumetric solution or hydrochloric acid standard volumetric solution [c(HCl)] 0.10 mol/L. Prepare and standardize as specified in GB/T 601, or use volumetric solution reference materials nationally certified and granted with the Reference Material Certificate.
3.2.4 Sulfuric acid standard volumetric solution or hydrochloric acid standard volumetric solution [c(HCl)] 0.05 mol/L: Pipette 50mL of 0.10 mol/L sulfuric acid standard volumetric solution or hydrochloric acid standard volumetric solution [c(HCl)] to the volumetric flask, dilute to 100mL. This solution shall be prepared immediately before use, and re-standardized if necessary. Alternatively, volumetric solution reference materials nationally certified and granted with the Reference Material Certificate may be used.
3.2.5 Methyl red indicator (1g/L): Take 0.1g methyl red, dissolve in 95% alcohol, dilute to 100mL, store at 10°C~30°C for up to 12 months.
3.2.6 Methylene blue indicator (1g/L): Take 0.1g of methylene blue, dissolve in 95% alcohol, dilute to 100mL, store at 10°C~30°C for up to 12 months.
3.2.7 Bromocresol green indicator (1g/L): Take 0.1g of bromocresol green, dissolve in 95% alcohol, dilute to 100mL, store at 10°C~30°C for up to 12 months.
3.2.8 Mixed indicator solution A: Mix 2 portions of methyl red-alcohol solution and 1 portion of methylene blue-alcohol solution well. Store at 10°C~30°C for up to 12 months.
3.2.9 Mixed indicator solution B: Mix 1 portions of methyl red-alcohol solution and 5 portion of bromocresol green-alcohol solution well. Store at 10°C~30°C for 2 months.
Foreword i
1 Scope
Method I Kjeldahl method for nitrogen determination
2 Principle
3 Reagents and materials
4 Instruments and apparatus
5 Analysis procedure
6 Expression of analysis results
7 Accuracy
8 Miscellaneous
Method II Spectrophotometry method
9 Principle
10 Reagents and materials
11 Instruments and apparatus
12 Analysis procedure
13 Expression of analysis results
14 Accuracy
15 Miscellaneous
Method III Combustion method
16 Principle
17 Reagents and materials
18 Instruments and apparatus
19 Specimen preparation
20 Moisture determination
21 Determination procedure
22 Expression of analysis results
23 Accuracy
24 Miscellaneous
Annex A Example of nitrogen determination distillation unit
Annex B Correction curves for the combustion method
Annex C Conversion factors for converting nitrogen to protein in common foods
GB 5009.5-2025 National food safety standard - Determination of protein in food
1 Scope
This standard specifies methods for the determination of protein in food.
This standard applies to the determination of protein in food.
Method I Kjeldahl method for nitrogen determination
2 Principle
Proteins in food are broken down under catalytic heating conditions and the resulting ammonia combines with sulfuric acid to form ammonium sulfate. Alkaline distillation frees ammonia, which is absorbed with boric acid and titrated with sulfuric acid or hydrochloric acid standard volumetric solution. The nitrogen content is calculated according to the consumption of sulfuric acid or hydrochloric acid, and then multiplied by the conversion factor to obtain the protein content.
3 Reagents and materials
Unless otherwise specified, analytical grade reagents and Grade 3 water defined in GB/T 6682 are adopted for the purpose of this method.
3.1 Reagents
3.1.1 Copper sulfate (CuSO4·5H2O).
3.1.2 Potassium sulphate (K2SO4).
3.1.3 Sodium hydroxide (NaOH).
3.1.4 Sulfuric acid (H2SO4): ≥98%.
3.1.5 Boric acid(H3BO3).
3.1.6 Alcohol (C2H5OH): 95%.
3.1.7 Methyl red (C15H15N3O2).
3.1.8 Bromocresol green (C21H14Br4O5S).
3.1.9 Methylene blue (C16H18ClN3S·3H2O).
3.1.10 pH test paper: with the measuring range of 0~14.
3.2 Preparation of reagents
3.2.1 Boric acid solution (20g/L): Take 20g of boric acid, dissolve with water and dilute to 1,000mL.
3.2.2 Sodium hydroxide solution (400g/L): Take 40g of sodium hydroxide and dissolve with water, cool and dilute to 100mL.
3.2.3 Sulfuric acid standard volumetric solution or hydrochloric acid standard volumetric solution [c(HCl)] 0.10 mol/L. Prepare and standardize as specified in GB/T 601, or use volumetric solution reference materials nationally certified and granted with the Reference Material Certificate.
3.2.4 Sulfuric acid standard volumetric solution or hydrochloric acid standard volumetric solution [c(HCl)] 0.05 mol/L: Pipette 50mL of 0.10 mol/L sulfuric acid standard volumetric solution or hydrochloric acid standard volumetric solution [c(HCl)] to the volumetric flask, dilute to 100mL. This solution shall be prepared immediately before use, and re-standardized if necessary. Alternatively, volumetric solution reference materials nationally certified and granted with the Reference Material Certificate may be used.
3.2.5 Methyl red indicator (1g/L): Take 0.1g methyl red, dissolve in 95% alcohol, dilute to 100mL, store at 10°C~30°C for up to 12 months.
3.2.6 Methylene blue indicator (1g/L): Take 0.1g of methylene blue, dissolve in 95% alcohol, dilute to 100mL, store at 10°C~30°C for up to 12 months.
3.2.7 Bromocresol green indicator (1g/L): Take 0.1g of bromocresol green, dissolve in 95% alcohol, dilute to 100mL, store at 10°C~30°C for up to 12 months.
3.2.8 Mixed indicator solution A: Mix 2 portions of methyl red-alcohol solution and 1 portion of methylene blue-alcohol solution well. Store at 10°C~30°C for up to 12 months.
3.2.9 Mixed indicator solution B: Mix 1 portions of methyl red-alcohol solution and 5 portion of bromocresol green-alcohol solution well. Store at 10°C~30°C for 2 months.
Contents of GB 5009.5-2025
Foreword i
1 Scope
Method I Kjeldahl method for nitrogen determination
2 Principle
3 Reagents and materials
4 Instruments and apparatus
5 Analysis procedure
6 Expression of analysis results
7 Accuracy
8 Miscellaneous
Method II Spectrophotometry method
9 Principle
10 Reagents and materials
11 Instruments and apparatus
12 Analysis procedure
13 Expression of analysis results
14 Accuracy
15 Miscellaneous
Method III Combustion method
16 Principle
17 Reagents and materials
18 Instruments and apparatus
19 Specimen preparation
20 Moisture determination
21 Determination procedure
22 Expression of analysis results
23 Accuracy
24 Miscellaneous
Annex A Example of nitrogen determination distillation unit
Annex B Correction curves for the combustion method
Annex C Conversion factors for converting nitrogen to protein in common foods
