Codeofchina.com is in charge of this English translation. In case of any doubt about the English translation, the Chinese original shall be considered authoritative.
This standard supersedes GB/T 5413.20-1997 "Milk Powder and Formula Foods for Infant and Young Children — Determination of Choline". Compared with GB/T 5413.20-1997, the main changes in this standard are as follows:
— The standard name was modified;
— Developer composition used for enzyme reaction was modified;
— Method II Reinecke's Salt Spectrophotometry was added.
National Food Safety Standard —
Foods and Milk Products for Infant and Young Children — Determination of Choline
1 Scope
This standard specifies the method for determination of choline in foods and milk products for infants and young children. This standard is applicable to the determination of choline in foods and milk products for infants and young children.
Method 1: Enzyme-colorimetric Method
2 Principle
Choline in the sample will be change into free form after acid hydrolysis, then after enzymatic oxidation and react with developer to produce coloring matter, which of the colour concentration within a certain range is proportional to the content of choline.
3 Reagents and Materials
Note: Unless otherwise specified, reagents used in this method are all analytical reagent, water is Grade 3 water specified in GB/T 6682.
3.1 Reagents
3.1.1 Tris [(CH2OH)3CNH2].
3.1.2 Phenosalyl (C6H5OH).
3.1.3 Concentrated hydrochloric acid (HCl).
3.1.4 Sodium hydroxide (NaOH ).
3.1.5 Choline oxidase: stored at -20 ℃.
3.1.6 Peroxidase: stored at 2 ℃ to 8 ℃.
3.1.7 4-aminoantipyrine (C11H13N3O) .
3.1.8 Phospholipase D: stored at -20 ℃.
3.2 Reagent preparation
3.2.1 Hydrochloric acid (1 mol/L): measuring off 85 mL concentrated hydrochloric acid and dilute to 1000 mL with water.
3.2.2 Hydrochloric acid (3 mol/L): measuring off 125 mL concentrated hydrochloric acid and dilute to 500 mL with water.
3.2.3 Tris buffer solution(0.05 mol/L): pH =8.0 ± 0.2.
Dissolve 6.057 g tris into 500 mL distilled water, neutralize with 1 mol/L hydrochloric acid to pH value 8.0 ± 0.2, and then dilute to constant volume (1000 mL) with distilled water. This solution may be stored in refrigerator at 4℃ for a month.
3.2.4 Developers used for enzyme reaction: take choline oxidase of enzyme activity 100 to 200, peroxidase of enzyme activity 250 to 280, phospholipase D of enzyme activity 75 to 100, 15 mg 4-aminoantipyrine, 50 mg phenosalyl and put them into a volumetric flask, then dilute to the scale with 0.05 mol/L Tris buffer solution. Prepare it at instant use.
3.2.5 Sodium hydroxide solution (500g/L): dissolve 500g sodium hydroxide into water and dilute to 1000 mL.
3.3 Standard
Choline Bitartrate standard (C9H19NO7): purity≥99%.
3.4 Preparation of standard solution
3.4.1 Choline hydroxide standard stock solution (2.5 mg/mL): Bake choline bitartrate to constant weight at 102 ℃ ± 2℃ and dissolve 523 mg in a 100mL volumetric flask, and then dilute to the scale with distilled water. Store it in 4℃ ± 2 ℃ refrigerator and preserve it for no more than one week.
3.4.2 Choline hydroxide standard working solution (250 µg/mL): take 10.0 mL standard stock solution and transfer to a 100mL volumetric flask, and then dilute with water to the scale. Prepare at instant use.
4 Apparatus
4.1 Balance: the sensibility is 0.01g and 0.1mg.
4.2 Constant temperature water bath kettle: the temperature may be controlled at 70℃ ± 2℃ and 37℃ ± 2℃.
4.3 PH meter: with a precision of 0.01.
4.4 Spectrophotometer.
5 Analytical Procedures
5.1 Sample preparation
5.1.1 Solid sample
Weigh 5 g (to the accuracy of 0.01 g) well-mixed sample in a 100mL conical flask, and add 30 mL hydrochloric acid solution (3.2.1).
5.1.2 Liquor sample
Weigh 20 g (to the accuracy of 0.01 g) well-mixed sample in a 100mL conical flask, and add 10 mL hydrochloric acid solution (3.2.2).
5.1.3 Hydrolization
Place the vessel filled with sample into 70℃ water bath, cover it and mix uniformly. Hydrolyzing for 3 h (shake it at intervals of 30 min), cool down. Calibrate the pH value as 3.5 to 4.0 with sodium hydroxide solution, and then transfer to a 50mL volumetric flask and dilute to the scale with distilled water.
5.1.4 Filtration
Filter hydrolysate (5.1.3). The filtered solution shall be clear, otherwise re-filter with a 0.45 μm filter membrane. Filtered solution may be stored 3 d in refrigerator at 4℃.
5.2 Determination
5.2.1 Standard curve making
Take respectively 2 mL, 4 mL, 6 mL and 8 mL choline hydroxide standard working solution in 10mL volumetric flasks, then dilute to the scale with distilled water. Prepare 6 cuvettes, one foe reagent blank (A), the rest numbered respectively as 1 to 5 and used for standard solution and four dilution of standard solution. Put in reagents according to those specified in Table 1.
Codeofchina.com is in charge of this English translation. In case of any doubt about the English translation, the Chinese original shall be considered authoritative.
This standard supersedes GB/T 5413.20-1997 "Milk Powder and Formula Foods for Infant and Young Children — Determination of Choline". Compared with GB/T 5413.20-1997, the main changes in this standard are as follows:
— The standard name was modified;
— Developer composition used for enzyme reaction was modified;
— Method II Reinecke's Salt Spectrophotometry was added.
National Food Safety Standard —
Foods and Milk Products for Infant and Young Children — Determination of Choline
1 Scope
This standard specifies the method for determination of choline in foods and milk products for infants and young children. This standard is applicable to the determination of choline in foods and milk products for infants and young children.
Method 1: Enzyme-colorimetric Method
2 Principle
Choline in the sample will be change into free form after acid hydrolysis, then after enzymatic oxidation and react with developer to produce coloring matter, which of the colour concentration within a certain range is proportional to the content of choline.
3 Reagents and Materials
Note: Unless otherwise specified, reagents used in this method are all analytical reagent, water is Grade 3 water specified in GB/T 6682.
3.1 Reagents
3.1.1 Tris [(CH2OH)3CNH2].
3.1.2 Phenosalyl (C6H5OH).
3.1.3 Concentrated hydrochloric acid (HCl).
3.1.4 Sodium hydroxide (NaOH ).
3.1.5 Choline oxidase: stored at -20 ℃.
3.1.6 Peroxidase: stored at 2 ℃ to 8 ℃.
3.1.7 4-aminoantipyrine (C11H13N3O) .
3.1.8 Phospholipase D: stored at -20 ℃.
3.2 Reagent preparation
3.2.1 Hydrochloric acid (1 mol/L): measuring off 85 mL concentrated hydrochloric acid and dilute to 1000 mL with water.
3.2.2 Hydrochloric acid (3 mol/L): measuring off 125 mL concentrated hydrochloric acid and dilute to 500 mL with water.
3.2.3 Tris buffer solution(0.05 mol/L): pH =8.0 ± 0.2.
Dissolve 6.057 g tris into 500 mL distilled water, neutralize with 1 mol/L hydrochloric acid to pH value 8.0 ± 0.2, and then dilute to constant volume (1000 mL) with distilled water. This solution may be stored in refrigerator at 4℃ for a month.
3.2.4 Developers used for enzyme reaction: take choline oxidase of enzyme activity 100 to 200, peroxidase of enzyme activity 250 to 280, phospholipase D of enzyme activity 75 to 100, 15 mg 4-aminoantipyrine, 50 mg phenosalyl and put them into a volumetric flask, then dilute to the scale with 0.05 mol/L Tris buffer solution. Prepare it at instant use.
3.2.5 Sodium hydroxide solution (500g/L): dissolve 500g sodium hydroxide into water and dilute to 1000 mL.
3.3 Standard
Choline Bitartrate standard (C9H19NO7): purity≥99%.
3.4 Preparation of standard solution
3.4.1 Choline hydroxide standard stock solution (2.5 mg/mL): Bake choline bitartrate to constant weight at 102 ℃ ± 2℃ and dissolve 523 mg in a 100mL volumetric flask, and then dilute to the scale with distilled water. Store it in 4℃ ± 2 ℃ refrigerator and preserve it for no more than one week.
3.4.2 Choline hydroxide standard working solution (250 µg/mL): take 10.0 mL standard stock solution and transfer to a 100mL volumetric flask, and then dilute with water to the scale. Prepare at instant use.
4 Apparatus
4.1 Balance: the sensibility is 0.01g and 0.1mg.
4.2 Constant temperature water bath kettle: the temperature may be controlled at 70℃ ± 2℃ and 37℃ ± 2℃.
4.3 PH meter: with a precision of 0.01.
4.4 Spectrophotometer.
5 Analytical Procedures
5.1 Sample preparation
5.1.1 Solid sample
Weigh 5 g (to the accuracy of 0.01 g) well-mixed sample in a 100mL conical flask, and add 30 mL hydrochloric acid solution (3.2.1).
5.1.2 Liquor sample
Weigh 20 g (to the accuracy of 0.01 g) well-mixed sample in a 100mL conical flask, and add 10 mL hydrochloric acid solution (3.2.2).
5.1.3 Hydrolization
Place the vessel filled with sample into 70℃ water bath, cover it and mix uniformly. Hydrolyzing for 3 h (shake it at intervals of 30 min), cool down. Calibrate the pH value as 3.5 to 4.0 with sodium hydroxide solution, and then transfer to a 50mL volumetric flask and dilute to the scale with distilled water.
5.1.4 Filtration
Filter hydrolysate (5.1.3). The filtered solution shall be clear, otherwise re-filter with a 0.45 μm filter membrane. Filtered solution may be stored 3 d in refrigerator at 4℃.
5.2 Determination
5.2.1 Standard curve making
Take respectively 2 mL, 4 mL, 6 mL and 8 mL choline hydroxide standard working solution in 10mL volumetric flasks, then dilute to the scale with distilled water. Prepare 6 cuvettes, one foe reagent blank (A), the rest numbered respectively as 1 to 5 and used for standard solution and four dilution of standard solution. Put in reagents according to those specified in Table 1.
