GB 5009.211-2022National food safety standard - Determination of folates in food (English PDF)

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 phosphate dodecahydrate (Na3PO4  12H2O).

3.1.5 Disodium 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 Lyophilized chicken pancreas powder. containing -glutamyl hydrolase.

3.2.3 Sodium hydroxide ethanol solution (0.01 mol/L). weigh-take 0.4 g of sodium hydroxide;

use 20% ethanol solution to dissolve and reach a constant volume of 1 L; mix it up.

3.2.4 Sodium hydroxide solution (1 mol/L). weigh-take 40 g of sodium hydroxide; add water

to dissolve and reach a constant volume of 1 L; mix it up.

3.2.5 Hydrochloric acid soaking solution. measure-take 100 mL of hydrochloric acid

(concentration. 36% ~ 38%); mix it up with 50 times of water.

3.3 Culture Medium

3.3.1 Agar medium for strain stock. in accordance with A.1, prepare it.

3.4 Reference Substance

Folates reference substance (C19H19N7O6, CAS. 59-30-3). purity  97%, or reference substances

certified by the state and awarded with a reference substance certificate.

3.5 Preparation of Standard Solutions

4.1 Balance. with a division value of 0.1 mg and 1 mg.

4.3 Autoclave.

4.4 Vortex oscillator.

4.5 Centrifuge. 3,000 r/min.

4.6 Inoculating loops and needles.

4.8 Tissue shredder and grinder.

4.9 UV-visible spectrophotometer.

4.10 Ultra-clean workbench.

4.11 Ultrasonic oscillator.

4.12 Microplate reader.

4.13 Centrifuge tube.

4.14 Microplate (sterile).

4.15 Filter membrane (0.22 m).

4.16 Volumetric flask.

5.1 Strains

Lactobacillus rhamnosus (ATCC 7469) or equivalent strains.

5.3 Preparation of Inoculum

One day before the experiment, take 2 mL of the folates standard working solution and mix it

with 4 mL of the medium for folates determination, then, divide it into two test tubes.

6.2 Specimen Extraction

6.2.1 Direct extraction method

When determining the content of folates added in the sample, the direct extraction method can

be adopted.

Accurately weigh-take 0.1 g ~ 2 g of solid specimen or 0.5 mL ~ 2 mL of liquid specimen,

accurate to 0.001 g; transfer it into a conical flask. Add 80 mL of sodium hydroxide ethanol

solution, with a stopper. Perform ultrasonic oscillation for 0.5 h ~ 4 h, until the specimen is

completely dissolved or dispersed, then, transfer it into a 100 mL volumetric flask; use water

to dilute to the scale.

6.2.2 Enzymatic extraction method

The enzymatic extraction method should be adopted for the naturally occurring folates in food

specimens, such as. cereals, potatoes, meat, eggs, dairy, fruits, vegetables, bacteria, algae, beans

and nuts, etc.

6.3 Dilution

In accordance with the folates content in the specimen, use water to appropriately dilute the

specimen extract, so that the folates content in the specimen diluent is within the range of 0.2 ng/mL ~ 0.3 ng/mL.

6.4 Specimen Determination

6.4.1 Test tube method

6.4.1.1 Specimen and enzyme blank series tubes

Take three test tubes; respectively add 1.0 mL, 2.0 mL and 3.0 mL of specimen diluent (Vx);

add water to 5.0 mL; mix it up. Take another three test tubes; adopt the same method to add

enzyme blank solution. For each gradient, perform 2 parallels.

6.4.1.4 Inoculation and culture

After the series tubes used for determination are cooled down to room temperature, under the

conditions of aseptic operation, add 40 L of inoculum to each 10 mL of the medium for folates

determination; mix it up. Add 5 mL of the inoculated medium for folates determination to each

determination tube; mix it up. Place it in a constant-temperature incubator at 36 C  1 C to

culture for 20 h ~ 40 h. When the maximum turbidity is obtained, terminate the culture. Prepare

another standard 0 tube (containing 0.00 ng of folates) that is not inoculated and regard it as the

6.4.1.5 Determination

Use a vortex oscillator to mix the cultured standard series tubes, specimens and enzyme blank

series tubes. Use a 1 cm cuvette, at 540 nm, adjust the light transmittance to 100% (or the

absorbance value is 0) with the 0 control tube that is not inoculated; successively determine the

light transmittance (or the absorbance value) of the standard series tubes, specimens and

enzyme blank series tubes. If the 0 control tube is turbid, it suggests that it may be contaminated

with bacteria, and the experiment needs to be re-performed.

6.4.2 Microplate method

6.4.2.1 Specimen series tubes

Firstly, under aseptic conditions, use a sterile aqueous filter (0.22 m) to filter and sterilize the

specimen diluent in 6.3.Take three 1.5 mL sterile centrifuge tubes; respectively add 100 L,

6.4.2.2 Standard series tubes

7.1 Standard Curve

Take the folates content of the standard series tubes as the x-coordinate; take the average value

of light transmittance (or absorbance value) of each standard point as the y-coordinate; draw a

standard curve.

7.2 Result Calculation

From the standard curve, calculate the corresponding content of folates (cx) in the specimens or

the enzyme blank series tubes. If the folates content of two of the three specimen series tubes

is within the range of 0.10 ng ~ 0.80 ng, and the deviation of folates content per milliliter of

specimen extract between the tubes is less than 10%, then, continue the result calculation in

accordance with Formula (1), Formula (2) and Formula (3), otherwise, re-sample and determine it.