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This standard replaces GB 5009.211-2014 National food safety standard - Determination of folic acid in foods.
The following main changes have been made with respect to GB 5009.211-2014:
——The microplate-based screening method is added;
——The requirements for precision are modified;
——Annex A is modified.
National food safety standard -
Determination of folic acid in foods
1 Scope
This standard specifies the method for determination of folic acid in foods.
This standard is applicable to the determination of folic acid in foods.
2 Principle
Folic acid is an essential nutrient for the growth of lactobacillus rhamnosus. Under certain control conditions, lactobacillus rhamnosus solution is inoculated into the culture medium containing specimen solution, and the light transmittance (or absorbance value) is measured after a period of culture. In a certain measurement range, the folic acid content in the specimen can be calculated according to the standard curve between folic acid content and light transmittance (or absorbance value).
3 Reagents and materials
Unless otherwise specified, analytically-pure reagents and Class-II water specified in GB/T 6682 are used for the purpose of this method.
3.1 Reagents
3.1.1 Hydrochloric acid (HCl).
3.1.2 Sodium hydroxide (NaOH).
3.1.3 Sodium chloride (NaCl).
3.1.4 Trisodium phosphate dodecahydrate (Na3PO4·12H2O).
3.1.5 Di-Sodium hydrogen phosphate heptahydrate (Na2HPO4·7H2O).
3.1.6 L-Ascorbic acid (C6H8O6).
3.1.7 Toluene (C7H8).
3.1.8 Anhydrous ethanol (C2H6O).
3.1.9 Freeze-dried pancreas powder: containing γ-glutamyl hydrolase.
3.1.10 Papain: enzyme activity ≥ 5 U/mg.
3.1.11 α-amylase: enzyme activity ≥ 1.5 U/mg.
3.2 Preparation of reagents
3.2.1 Phosphate buffered saline (0.05mol/L, pH6.8): weigh 4.35g of trisodium phosphate dodecahydrate and 10.39g of di-sodium hydrogen phosphate heptahydrate, dissolve them in water, dilute the solution to 1L, and shake it well. Add 2mL of toluene and store the reagent at room temperature. Before use, add l-ascorbic acid as folic acid protectant at a ratio of about 5mg/mL, and adjust the pH value to 6.8±0.1.
3.2.2 20% ethanol solution (2+8): measure 200mL of ethanol and mix it with 800mL water uniformly.
3.2.3 Sodium hydroxide ethanol solution (0.01mol/L): weigh 0.4g of sodium hydroxide, dissolve it with 20% ethanol solution, dilute the resulted solution to 1L, and shake it well.
3.2.4 Sodium hydroxide solution (1mol/L) : weigh 40g of sodium hydroxide, add water to dissolve it, dilute the resulted solution to 1L and shake it well.
3.2.5 Hydrochloric acid soak solution: weigh 100mL of hydrochloric acid (with a concentration of 36%~38%) and mix it with water 50 times the amount of the solution.
3.2.6 Chicken pancreas solution: weigh 100mg of freeze-dried pancreas powder, dissolve it into 20mL of phosphate buffered saline and shake well. It shall be freshly prepared before use.
3.2.7 Protease-amylase solution: weigh 200mg of papain and α-amylase respectively into 20mL of phosphate buffered saline, grind the mixture to homogenate, and then centrifuge it at 3,000r/min for 5min. It shall be freshly prepared before use.
3.3 Culture media
3.3.1 Agar medium reserved for bacteria species: prepared according to A.1.
3.3.2 Medium for folic acid determination: prepared according to A.2.
Note: the above media may also be commercial synthetic or prepared media, to be prepared according to the instructions before use.
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3.4 Standard sample
Standard folic acid sample (C19H19N7O6, CAS: 59-30-3): purity ≥97%, or its standard sample certified by the state and issued with a reference material certificate.
3.5 Preparation of standard solutions
3.5.1 Standard folic acid reserve solution (20.0μg/mL): accurately weigh 20.0mg of standard folic acid sample, dissolve it in sodium hydroxide ethanol solution, transfer the mixture and dilute it in a 1,000mL brown volumetric flask. Transfer the solution to a brown glass container and store it in dark place at 2℃~4℃ in a refrigerator, with a shelf life of 2 years.
3.5.2 Standard folic acid intermediate solution (0.200μg/mL): accurately pipette 1.00mL of standard folic acid reserve solution into a 100mL brown volumetric flask, dilute it with sodium hydroxide ethanol solution to the scale, and mix it well. Transfer the solution to a brown glass container and store it in dark place at 2℃~4℃ in a refrigerator, with a shelf life of 1 year.
3.5.3 Standard folic acid working solution (0.200ng/mL) : accurately pipette 1.00mL of standard folic acid intermediate solution into a 1,000mL volumetric flask, dilute it with water to the scale, and mix it well. It shall be freshly prepared before use.
4 Instruments and equipment
4.1 Balances: with sensitivities of 0.1mg and 1mg, respectively.
4.2 Thermostatic incubator: 36℃±1℃.
4.3 Autoclave.
4.4 Vortex mixer.
4.5 Centrifuge: 3,000r/min
4.6 Inoculating loops and needles.
4.7 pH meter: with an accuracy of ±0.1.
4.8 Tissue crusher and grinder.
4.9 Ultraviolet-visible spectrophotometer.
4.10 Superclean bench.
4.11 Ultrasonic mixer.
4.12 Microplate reader.
4.13 Centrifuge tube.
4.14 Microplate (sterile).
4.15 Filter membrane (0.22μm).
4.16 Volumetric flask.
Note: the glassware used shall be cleaned with hydrochloric acid soak solution or sodium lauryl sulfonate detergent, and then dried and heated at 250℃ for 1h~2h before use.
Foreword II
1 Scope
2 Principle
3 Reagents and materials
4 Instruments and equipment
5 Preparation and storage of bacteria species
6 Analysis steps (all operations shall be carried out in dark place)
7 Expression of analysis results
8 Precision
9 Others
Annex A Medium preparation
