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 replaces "Determination of Folates in Foods" (GB/T 5009.211-2008).
The following changes have been made with respect to GB/T 5009.211-2006:
——the name of this standard is revised as "National Food Safety Standard - Determination of Folates in Foods";
——the name of strain is modified from lactobacillus casei to lactobacillus casei spp.rhamnosus;
——the supply information of medium and chicken pancreas used for folate degradation is deleted;
——the preparation method for casein hydrolysate is deleted from Annex B;
——the detection limit and quantitation limit are added.
National Food Safety Standard - Determination of Folates in Foods
1 Scope
This standard specifies determination method of folates in foods.
This standard is applicable to determination of folates in foods.
2 Principle
Folate is a necessary nutrient for growth of Lactobacillus casei spp.rhamnosus (ATCC 7469). Under certain control condition, inoculate Lactobacillus casei spp.rhamnosus in culture solution containing sample solution; after a certain period of cultivation , determine light transmittance (or absorbance); then calculate the content of folate in the sample according to the standard curve of folate content and light transmittance (or absorbance).
3 Reagents and Materials
Note: unless otherwise specified, all reactants adopted for this method are analytically pure and water is Grade II water as specified in GB/T 6682.
3.1 Reagents
3.1.1 Hydrochloric acid (HCl).
3.1.2 Sodium hydroxide (NaOH).
3.1.3 Sodium chloride (NaCl).
3.1.4 Sodium phorhoate tribaric dodecahydrate (Na3PO4·12H2O).
3.1.5 Disodium hydrogen phosphate heptahydrate (Na2HPO4·7H2O).
3.1.6 Dipotassium phosphate (K2HPO4).
3.1.7 Monopotassium phosphate trihydrate (KH2PO4·3H2O).
3.1.8 Magnesium sulfate heptahydrate (MgSO4·7H2O).
3.1.9 Ferrous sulfate heptahydrate (FeSO4·7H2O).
3.1.10 Manganese sulfate monohydrate (MnSO4·H2O).
3.1.11 Sodium acetate trihydrate (CH3CooNa·3H2O).
3.1.12 Glucose (C6H12O6).
3.1.13 Ascorbic acid (C6H8O6).
3.1.14 Methylbenzene (C7H8).
3.1.15 Anhydrous ethanol (C2H6O).
3.1.16 Chicken pancreas dry powder: containing γ-glutamoyl hydrolase.
3.1.17 Papayotin: enzyme activity ≥5U/mg.
3.1.18 Α-amylase: enzyme activity ≥1.5U/mg.
3.1.19 Peptone: nitrogen content ≥10%.
3.1.20 Yeast extract (dry powder): nitrogen content ≥10%.
3.1.21 Agar.
3.2 Reagent preparation
3.2.1 Phosphate buffer (0.05 mol/L, pH 6.8): respectively weigh 4.35g of sodium phorhoate tribaric dodecahydrate and 10.39g of disodium hydrogen phosphate heptahydrate, dissolve them and dilute the solution with water to 1L, then mix uniformly. Add 2mL of methylbenzene and preserve in ambient temperature. Add ascorbic acid as folate protecting anent in the proportion of about 5 mg/mL just before use, in order to make pH 6.8.
3.2.2 Ethanol solution(20% in volume fraction): measure 200 mL of absolute alcohol and 800 mL of water, then mix them uniformly.
3.2.3 Sodium hydroxide ethanol solution (0.01 mol/L): weigh 0.4 g of sodium hydroxide, dissolve it with ethanol solution, dilute to 1 L, then mix it uniformly.
3.2.4 Sodium hydroxide solution (1 mol/L): weigh 40 g of sodium hydroxide, dissolve with water, then dilute to 1L, then mix it uniformly.
3.2.5 Hydrochloric acid solution (1 mol/L): take 83.3 mL of hydrochloric acid, dilute it to 1 L with water, then mix uniformly.
3.2.6 Hydrochloric acid soak solution: measure 100mL of hydrochloric acid to mix with water (volume of 50 times).
3.2.7 Chicken pancreas solution: weigh out 100mg of chicken pancreas dry powder, add 20mL of phosphate buffer, and shake up. Prepare fresh solution before use.
3.2.8 Papayotin-amylase solution: respectively weigh out papayotin and α-amylase 200 mg each, add 20mL of phosphate buffer and grind it to homogenate. Centrifuge the homogenate at the speed of 3000 r/min for 5min. Prepare fresh solution before use.
3.3 Culture medium
3.3.1 Salt solution A: respectively weigh out 25 g of dipotassium hydrogen phosphate and 25 g of monopotassium phosphate trihydrate, dissolve it with water and dilute it to 500mL, and mix it uniformly. Add 1mL of methylbenzene; it may be preserved for 1 year in a 2℃ ~ 4℃ refrigerator.
3.3.2 Salt solution B: respectively weigh out 10g of magnesium sulfate heptahydrate, 0.5 g of sodium chloride, 0.5 g of ferrous sulfate heptahydrate and 0.5 g of manganese sulfate monohydrate, dissolve it with water and dilute it to 500mL. Add 5 drops of hydrochloric acid; it may be preserved for 1 year in a 2℃ ~ 4℃ refrigerator.
3.3.3 Agar culture medium: weigh or pipet reagents according to the requirements specified in Table 1, add water to make the volume to 100mL, mix it up; heat the solution in the boiling water bath to make the agar dissolved entirely. Adjust the pH to 6.8 ± 0.1 with 1 mol/L of hydrochloric acid solution and 1 mol/L of sodium hydroxide solution while it is hot. Separately put it into test tubes, about 3mL ~ 5mL for each test tube according to the inner diameter, liquid level shall not be less than 2cm. Plug with tampon and autoclave at 121 ℃ (0.10 MPa ~ 0.12 MPa) for 15 min. Take it out, put the test tube uprightly; after cooling, preserve it in refrigerator for future use.
Table 1 Preparation of Agar Medium for Bacteria Reserve
Reagent Usage
Glucose /g 1.0
Peptone /g 0.8
Yeast extract dry powder /g 0.2
Sodium acetate trihydrate /g 1.7
Salt solution A /mL 0.2
Salt solution B /mL 0.2
Agar /g 1.2
3.3.4 Culture solution for folate determination: prepare culture solution for folate determination according to Annex A, or directly purchase medium for folate determination with equivalent effect from the reagent supplier, and prepare it according to the instructions before use.
3.4 Standard sample
Folate standard sample (C19H19N7O6): purity≥99.0%.
3.5 Preparation of standard solution
3.5.1 Folate standard stock solution (20.0 μg/mL): accurately weigh out 20.0 mg of folate standard sample, dissolve it with sodium hydroxide ethanol solution, transfer it a 1 000mL volumetric flask and scale the volume to the scale.
Concentration calibration of folate standard stock solution: accurately pipet 1.0 ml of standard stock solution to a 5mL volumetric flask, and dilute with sodium hydroxide solution to the scale. In the condition of cuvette thickness 1 cm and wave length 256 nm, apply ultraviolet and visible spectrophotometer to test the absorbance values of the standard solution for three times after zeroing with sodium hydroxide ethanol solution; take the average and calculate the concentration of the folate standard stock solution according to Formula (1).
(1)
Where,
c1——the concentration of folate in the standard stock solution, μg/mL;
——the average absorbance value;
E——the molar extinction coefficient (24,500);
M——the molecular weight of folate (441.42);
5——the dilution ratio;
1000——the conversion coefficient of g/L converted to μg/mL.
Store the calibrated folate standard stock solution into a brown bottle, it may be preserved for two years in a 2℃ ~ 4℃ refrigerator.
3.5.2 Folate standard intermediate solution (0.200 μg/mL): pipet exactly 1.00 mL of folate standard stock solution, put it into a 100mL brown volumetric flask, and dilute with sodium hydroxide ethanol solution and dilute with water to the scale. Mix it well, store in bottles; it may be preserved for 1 year in a 2℃ ~ 4℃ refrigerator.
3.5.3 Folate standard working solution (0.200 ng/mL): accurately pipet 1.00 mL of folate standard intermediate solution, put into a 1,000mL volumetric flask, dilute with water to the scale, then mix uniformly. Prepare fresh solution before use.
Foreword
1 Scope
2 Principle
3 Reagents and Materials
4 Instruments and Equipment
5 Preparation and Preservation of Bacteria
6 Analytical Procedure (All Operations shall be Performed in a Dark Place)
7 Accuracy
8 Other
Annex A Preparation Method of Culture Solution for Folate Determination
Standard
GB 5009.211-2014 National Food Safety Standard-Determination of folates in food (English Version)
Standard No.
GB 5009.211-2014
Status
superseded
Language
English
File Format
PDF
Word Count
5000 words
Price(USD)
80.0
Implemented on
2016-3-21
Delivery
via email in 1 business day
Detail of GB 5009.211-2014
Standard No.
GB 5009.211-2014
English Name
National Food Safety Standard-Determination of folates in food
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 replaces "Determination of Folates in Foods" (GB/T 5009.211-2008).
The following changes have been made with respect to GB/T 5009.211-2006:
——the name of this standard is revised as "National Food Safety Standard - Determination of Folates in Foods";
——the name of strain is modified from lactobacillus casei to lactobacillus casei spp.rhamnosus;
——the supply information of medium and chicken pancreas used for folate degradation is deleted;
——the preparation method for casein hydrolysate is deleted from Annex B;
——the detection limit and quantitation limit are added.
National Food Safety Standard - Determination of Folates in Foods
1 Scope
This standard specifies determination method of folates in foods.
This standard is applicable to determination of folates in foods.
2 Principle
Folate is a necessary nutrient for growth of Lactobacillus casei spp.rhamnosus (ATCC 7469). Under certain control condition, inoculate Lactobacillus casei spp.rhamnosus in culture solution containing sample solution; after a certain period of cultivation , determine light transmittance (or absorbance); then calculate the content of folate in the sample according to the standard curve of folate content and light transmittance (or absorbance).
3 Reagents and Materials
Note: unless otherwise specified, all reactants adopted for this method are analytically pure and water is Grade II water as specified in GB/T 6682.
3.1 Reagents
3.1.1 Hydrochloric acid (HCl).
3.1.2 Sodium hydroxide (NaOH).
3.1.3 Sodium chloride (NaCl).
3.1.4 Sodium phorhoate tribaric dodecahydrate (Na3PO4·12H2O).
3.1.5 Disodium hydrogen phosphate heptahydrate (Na2HPO4·7H2O).
3.1.6 Dipotassium phosphate (K2HPO4).
3.1.7 Monopotassium phosphate trihydrate (KH2PO4·3H2O).
3.1.8 Magnesium sulfate heptahydrate (MgSO4·7H2O).
3.1.9 Ferrous sulfate heptahydrate (FeSO4·7H2O).
3.1.10 Manganese sulfate monohydrate (MnSO4·H2O).
3.1.11 Sodium acetate trihydrate (CH3CooNa·3H2O).
3.1.12 Glucose (C6H12O6).
3.1.13 Ascorbic acid (C6H8O6).
3.1.14 Methylbenzene (C7H8).
3.1.15 Anhydrous ethanol (C2H6O).
3.1.16 Chicken pancreas dry powder: containing γ-glutamoyl hydrolase.
3.1.17 Papayotin: enzyme activity ≥5U/mg.
3.1.18 Α-amylase: enzyme activity ≥1.5U/mg.
3.1.19 Peptone: nitrogen content ≥10%.
3.1.20 Yeast extract (dry powder): nitrogen content ≥10%.
3.1.21 Agar.
3.2 Reagent preparation
3.2.1 Phosphate buffer (0.05 mol/L, pH 6.8): respectively weigh 4.35g of sodium phorhoate tribaric dodecahydrate and 10.39g of disodium hydrogen phosphate heptahydrate, dissolve them and dilute the solution with water to 1L, then mix uniformly. Add 2mL of methylbenzene and preserve in ambient temperature. Add ascorbic acid as folate protecting anent in the proportion of about 5 mg/mL just before use, in order to make pH 6.8.
3.2.2 Ethanol solution(20% in volume fraction): measure 200 mL of absolute alcohol and 800 mL of water, then mix them uniformly.
3.2.3 Sodium hydroxide ethanol solution (0.01 mol/L): weigh 0.4 g of sodium hydroxide, dissolve it with ethanol solution, dilute to 1 L, then mix it uniformly.
3.2.4 Sodium hydroxide solution (1 mol/L): weigh 40 g of sodium hydroxide, dissolve with water, then dilute to 1L, then mix it uniformly.
3.2.5 Hydrochloric acid solution (1 mol/L): take 83.3 mL of hydrochloric acid, dilute it to 1 L with water, then mix uniformly.
3.2.6 Hydrochloric acid soak solution: measure 100mL of hydrochloric acid to mix with water (volume of 50 times).
3.2.7 Chicken pancreas solution: weigh out 100mg of chicken pancreas dry powder, add 20mL of phosphate buffer, and shake up. Prepare fresh solution before use.
3.2.8 Papayotin-amylase solution: respectively weigh out papayotin and α-amylase 200 mg each, add 20mL of phosphate buffer and grind it to homogenate. Centrifuge the homogenate at the speed of 3000 r/min for 5min. Prepare fresh solution before use.
3.3 Culture medium
3.3.1 Salt solution A: respectively weigh out 25 g of dipotassium hydrogen phosphate and 25 g of monopotassium phosphate trihydrate, dissolve it with water and dilute it to 500mL, and mix it uniformly. Add 1mL of methylbenzene; it may be preserved for 1 year in a 2℃ ~ 4℃ refrigerator.
3.3.2 Salt solution B: respectively weigh out 10g of magnesium sulfate heptahydrate, 0.5 g of sodium chloride, 0.5 g of ferrous sulfate heptahydrate and 0.5 g of manganese sulfate monohydrate, dissolve it with water and dilute it to 500mL. Add 5 drops of hydrochloric acid; it may be preserved for 1 year in a 2℃ ~ 4℃ refrigerator.
3.3.3 Agar culture medium: weigh or pipet reagents according to the requirements specified in Table 1, add water to make the volume to 100mL, mix it up; heat the solution in the boiling water bath to make the agar dissolved entirely. Adjust the pH to 6.8 ± 0.1 with 1 mol/L of hydrochloric acid solution and 1 mol/L of sodium hydroxide solution while it is hot. Separately put it into test tubes, about 3mL ~ 5mL for each test tube according to the inner diameter, liquid level shall not be less than 2cm. Plug with tampon and autoclave at 121 ℃ (0.10 MPa ~ 0.12 MPa) for 15 min. Take it out, put the test tube uprightly; after cooling, preserve it in refrigerator for future use.
Table 1 Preparation of Agar Medium for Bacteria Reserve
Reagent Usage
Glucose /g 1.0
Peptone /g 0.8
Yeast extract dry powder /g 0.2
Sodium acetate trihydrate /g 1.7
Salt solution A /mL 0.2
Salt solution B /mL 0.2
Agar /g 1.2
3.3.4 Culture solution for folate determination: prepare culture solution for folate determination according to Annex A, or directly purchase medium for folate determination with equivalent effect from the reagent supplier, and prepare it according to the instructions before use.
3.4 Standard sample
Folate standard sample (C19H19N7O6): purity≥99.0%.
3.5 Preparation of standard solution
3.5.1 Folate standard stock solution (20.0 μg/mL): accurately weigh out 20.0 mg of folate standard sample, dissolve it with sodium hydroxide ethanol solution, transfer it a 1 000mL volumetric flask and scale the volume to the scale.
Concentration calibration of folate standard stock solution: accurately pipet 1.0 ml of standard stock solution to a 5mL volumetric flask, and dilute with sodium hydroxide solution to the scale. In the condition of cuvette thickness 1 cm and wave length 256 nm, apply ultraviolet and visible spectrophotometer to test the absorbance values of the standard solution for three times after zeroing with sodium hydroxide ethanol solution; take the average and calculate the concentration of the folate standard stock solution according to Formula (1).
(1)
Where,
c1——the concentration of folate in the standard stock solution, μg/mL;
——the average absorbance value;
E——the molar extinction coefficient (24,500);
M——the molecular weight of folate (441.42);
5——the dilution ratio;
1000——the conversion coefficient of g/L converted to μg/mL.
Store the calibrated folate standard stock solution into a brown bottle, it may be preserved for two years in a 2℃ ~ 4℃ refrigerator.
3.5.2 Folate standard intermediate solution (0.200 μg/mL): pipet exactly 1.00 mL of folate standard stock solution, put it into a 100mL brown volumetric flask, and dilute with sodium hydroxide ethanol solution and dilute with water to the scale. Mix it well, store in bottles; it may be preserved for 1 year in a 2℃ ~ 4℃ refrigerator.
3.5.3 Folate standard working solution (0.200 ng/mL): accurately pipet 1.00 mL of folate standard intermediate solution, put into a 1,000mL volumetric flask, dilute with water to the scale, then mix uniformly. Prepare fresh solution before use.
Contents of GB 5009.211-2014
Foreword
1 Scope
2 Principle
3 Reagents and Materials
4 Instruments and Equipment
5 Preparation and Preservation of Bacteria
6 Analytical Procedure (All Operations shall be Performed in a Dark Place)
7 Accuracy
8 Other
Annex A Preparation Method of Culture Solution for Folate Determination
