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GB 5009.5-2025: National food safety standard - Determination of protein in food Delivery: 9 seconds. True-PDF full-copy in English & invoice will be downloaded + auto-delivered via email. See step-by-step procedure Status: Valid GB 5009.5: Historical versions
Similar standardsGB 5009.5-2025: National food safety standard - Determination of protein in food---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/GB5009.5-2025GB NATIONAL STANDARD OF THE PEOPLE’S REPUBLIC OF CHINA National Food Safety Standard – Determination of Protein in Foods Issued on. MARCH 16, 2025 Implemented on. SEPTEMBER 16, 2025 Issued by. National Health Commission of the People’s Republic of China; State Administration for Market Regulation. Table of ContentsForeword... 3 1 Scope... 4 2 Principle... 4 3 Reagents and Materials... 4 4 Instruments and Equipment... 5 5 Analysis Steps... 6 6 Expression of Analysis Results... 8 7 Precision... 9 8 Others... 9 9 Principle... 9ForewordThis Standard replaced GB 5009.5-2016 National Food Safety Standard - Determination of Protein in Foods. Compared with GB 5009.5-2016, the major changes of this Standard are as follows. --- Modify the scope of the standard; --- Modify Method I the sampling volume and standard titration solution concentration of the Kjeldahl method; --- Add Method II the storage conditions and time of the standard solution and color developer; --- Modify Method III the scope and detection limit of the combustion method; --- Add Appendix B combustion method calibration curve; --- Modify the expression of analysis results and Appendix C protein conversion coefficient table; --- Modify the precision. National Food Safety Standard – Determination of Protein in Foods1 ScopeThis Standard specifies the method for determination of protein in foods. This Standard is applicable to the determination of protein in foods. Method I Kjeldahl Method2 PrincipleThe protein in food is decomposed under catalytic heating conditions; and the produced ammonia combines with sulfuric acid to form ammonium sulfate. Alkalinization distillation frees the ammonia, which is absorbed by boric acid and titrated with sulfuric acid or hydrochloric acid standard titration solution. The nitrogen content is calculated based on the consumption of sulfuric acid or hydrochloric acid, and then multiplied by the conversion coefficient to obtain the protein content.3 Reagents and MaterialsUnless otherwise specified, all reagents used in this method are analytically pure, and water is Grade-3 water specified in GB/T 6682. 3.1 Reagents 3.2 Preparation of reagents 3.2.1 Boric acid solution (20 g/L). Weigh 20 g of boric acid; dissolve in water and dilute to 1,000 mL. 3.2.2 Sodium hydroxide solution (400 g/L). Weigh 40 g of sodium hydroxide; dissolve in water; cool and dilute to 100 mL.4 Instruments and Equipment4.1 Analytical balance. With sensitivity of 1 mg. 4.2 Nitrogen determination distillation apparatus. as shown in Appendix A. 4.3 Pipette. 10 mL, 25 mL and 50 mL. 4.4 Nitrogen determination bottle. 100 mL, 250 mL and 500 mL. 4.5 Digestion furnace. ≥420℃. 4.6 Semi-automatic Kjeldahl nitrogen analyzer or fully automatic Kjeldahl nitrogen analyzer. 4.7 Homogenizer. 4.8 Crusher.5 Analysis Steps5.1 Kjeldahl method 5.2 Fully automatic or semi-automatic Kjeldahl method Weigh 0.2 g ~ 2 g of solid specimen, 2 g ~ 5 g of semi-solid specimen, (accurate to 0.001 g), 10 g (mL) ~ 25 g (mL) (equivalent to 30 mg ~ 40 mg nitrogen) of liquid specimen; and then add 0.4 g of copper sulfate, 6 g of potassium sulfate and 20 mL of sulfuric acid to the digestion furnace for digestion. When the temperature of the digestion furnace reaches 420 ℃, continue digestion for at least 1 h.6 Expression of Analysis ResultsThe protein content in the specimen is calculated according to Formula (1). When the protein content is ≥1 g/100 g or 1 g/100 mL, the result shall be retained 3 significant figures; when the protein content is < 1 g/100 g or 1 g/100 mL, the result shall be retained 2 significant figures.7 PrecisionWhen the protein content in the sample is ≤10 g/100 g or 10 g/100 mL, the absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean.8 OthersWhen using 0.05 mol/L hydrochloric acid titration solution, the sample weight is 5.0 g or 5.0 mL; and the detection limit of nitrogen in this method is 0.008 g/100 g or 0.008 g/100 mL. Method II Spectrophotometry9 PrincipleThe protein in food is decomposed under catalytic heating conditions. The ammonia produced by the decomposition combines with sulfuric acid to form ammonium sulfate, which reacts with acetylacetone and formaldehyde in a sodium acetate-acetic acid buffer solution at pH 4.8 to form a yellow 3,5-diacetyl-2,6-dimethyl-1,4-dihydropyridine compound. 10 Reagents and Materials Unless otherwise specified, the reagents used in this method are analytically pure, and the water is Grade-3 water specified in GB/T 6682. 10.1 Reagents 10.2 Preparation of reagents 10.4 Preparation of standard solutions 10.4.1 Standard ammonium sulfate stock solution (by nitrogen) (1.0 g/L). Weigh 0.4720 g of ammonium sulfate dried at 105°C for 2 h; dissolve in water and dilute to 100 mL; and mix well. Each milliliter of this solution is equivalent to 1.0 mg of nitrogen. It can be stored at 4°C for 1 month. 10.4.2 Standard ammonium sulfate working solution (0.10 mg/mL). Accurately pipette 10.00 mL of standard ammonium sulfate stock solution (1.0 g/L) into a 100 mL volumetric flask; dilute to the mark with water; and mix well. Each milliliter of this solution is equivalent to 0.1 mg of nitrogen. Prepare it before use. 11 Instruments and Equipment 11.1 Spectrophotometer. 11.2 Electro-thermostatic water bath. 100℃ ±1℃. 11.3 pH meter. With accuracy of 0.01. 11.4 10 mL stoppered glass colorimetric tube. 11.5 Analytical balance. With sensitivity of 1 mg and 0.1 mg. 11.6 Nitrogen determination bottle. 100 mL, 250 mL and 500 mL. 11.7 Homogenizer. 11.8 Crusher. 12 Analysis Steps 12.1 Preparation of specimen The same as 5.1.1. 12.2 Determination of moisture The same as 5.1.2. 12.3 Digestion of specimen Weigh 0.1 g ~ 0.5 g of solid specimen, 0.2 g ~ 1 g (accurate to 0.01 g) of semi-solid specimen, 1 mL(g) ~ 5 mL(g) of liquid specimen; transfer into a dry 100 mL or 250 mL nitrogen determination bottle. Add 0.1 g of copper sulfate, 1 g of potassium sulfate and 5 mL of sulfuric acid; shake gently; place a small funnel at the mouth of the bottle; and place the nitrogen determination bottle at a 45° angle on an asbestos net with small holes. Heat slowly; and when the contents are completely carbonized and the foam stops completely, increase the fire and keep the liquid in the bottle slightly boiling until the liquid becomes blue-green, clear and transparent; and continue heating for 0.5 h~1 h. Remove the nitrogen determination bottle and cool it to room temperature. Carefully add 20 mL of water and transfer all the contents to a 100 mL volumetric flask. Wash the inner wall of the nitrogen determination bottle with a small amount of water; add the washing solution to the volumetric flask; add water to the scale, mix well and set aside. Perform a blank test at the same time. 12.5 Drawing of standard curve Pipette 0.00 mL, 0.05 mL, 0.10 mL, 0.20 mL, 0.40 mL, 0.60 mL, 0.80 mL and 1.00 mL of standard ammonium sulfate working solution (equivalent to 0.00 μg, 5.00 μg, 10.0 μg, 20.0 μg, 40.0 μg, 60.0 μg, 80.0 μg and 100.0 μg of nitrogen) and place them in 10 mL colorimetric tubes respectively. Add 4.0 mL of sodium acetate-acetic acid buffer solution and 4.0 mL of color developer; dilute to the mark with water; and mix well. Heat in a 100 ℃ water bath for 15 min. 12.6 Determination of specimen Pick 0.50 mL ~ 2.00 mL (equivalent to nitrogen < 100 μg) of specimen solution and the same amount of reagent blank solution; and place them in 10 mL colorimetric tubes, respectively. Add 4.0 mL of sodium acetate-acetic acid buffer solution and 4.0 mL of color developer; dilute to the scale with water; and mix well. Heat in a 100 ℃ water bath for 15 min. 13 Expression of Analysis Results The protein content in the specimen is calculated according to Formula (2). 14 Precision When the protein content in the sample is ≤10 g/100 g or 10 g/100 mL, the absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. 15 Others When the sample weight is 5.0g or 5.0mL, the detection limit of nitrogen in this method is 0.0001g/100g or 0.0001g/100mL. Method III Combustion Method 16 Principle The specimen burns at a high temperature of 900℃~1,200℃. During the combustion process, a mixed gas is generated. Interfering gases such as carbon and sulfur and salts are absorbed by the absorption tube; and nitrogen oxides are completely reduced to nitrogen. The formed nitrogen gas flow is detected by a thermal conductivity detector (TCD). 17 Reagents and Materials 17.1 Oxygen (O2). purity ≥99.995%. 17.2 Carrier gas (CO2, He, Ar, etc.). Purity ≥99.995%. 17.3 Nitrogen-containing standard substances. aspartic acid (C4H7NO4), urea (CH4N2O), purity ≥99%. Or nitrogen-containing standard substances certified by the state and awarded with standard substance certificates. 17.4 Nitrogen-free aluminum foil, tin capsule, nitrogen-free plastic capsule or stainless-steel crucible. ......Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al. |
