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GB 5009.211-2022: PDF in English
GB 5009.211-2022
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard - Determination of Folates in
Food
ISSUED ON: JUNE 30, 2022
IMPLEMENTED ON: DECEMBER 30, 2022
Issued by: National Health Commission of the People’s Republic of China;
State Administration for Market Regulation.
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Principle ... 4 
3 Reagents and Materials ... 4 
4 Instruments and Equipment ... 6 
5 Preparation and Stock of Strains ... 7 
6 Analytical Procedures (all operations must be performed in the dark) ... 7 
7 Expression of Analytical Results ... 10 
8 Precision ... 12 
9 Others ... 12 
Appendix A Preparation of Culture Medium ... 13 
National Food Safety Standard - Determination of Folates in
Food
1 Scope
This Standard specifies the methods of determining folates in food.
This Standard is applicable to the determination of folates in food.
2 Principle
Folates is an essential nutrient for the growth of Lactobacillus rhamnosus. Under certain
controlled conditions, inoculate the Lactobacillus rhamnosus bacterial solution into the medium
containing the specimen solution; after culturing for a period of time, determine the light
transmittance (or absorbance value). Within a certain determination range, in accordance with
the standard curve of folates content and light transmittance (or absorbance value), the folates
content in the specimen can be calculated.
3 Reagents and Materials
Unless it is otherwise specified, the reagents used in this Method are analytically pure; the water
is Grade-2 water 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 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.
3.1.10 Papain: enzyme activity  5 U/mg.
3.1.11 -amylase: enzyme activity  1.5 U/mg.
3.2 Preparation of Reagents
3.2.1 Phosphate buffer (0.05 mol/L, pH 6.8): respectively weigh-take 4.35 g of sodium
phosphate dodecahydrate and 10.39 g of disodium hydrogen phosphate heptahydrate; add water
to dissolve and reach a constant volume of 1 L; mix it up. Add 2 mL of toluene; preserve it at
room temperature. Before use, at a ratio of approximately 5 mg/mL, add L-ascorbic acid as a
protectant of the folates; adjust pH to 6.8  0.1.
3.2.2 20% ethanol solution (2 + 8): measure-take 200 mL of anhydrous ethanol and 800 mL of
water; mix it up.
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.
3.2.6 Chicken pancreas solution: weigh-take 100 mg of lyophilized chicken pancreas powder;
add 20 mL of phosphate buffer; mix it up. Prepare it right before use.
3.2.7 Protease-amylase solution: respectively weigh-take 200 mg of papain and -amylase; add
20 mL of phosphate buffer; grind to homogenate. At 3,000 r/min, centrifuge for 5 min. Prepare
it right before use.
3.3 Culture Medium
3.3.1 Agar medium for strain stock: in accordance with A.1, prepare it.
3.3.2 Medium for folates determination: in accordance with A.2, prepare it.
NOTE: the above mediums may also be commercialized synthetic or finished medium, which shall
be prepared in accordance with the instructions before use.
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
3.5.1 Folates standard stock solution (20.0 g/mL): accurately weigh-take 20.0 mg of folates
reference substance; use sodium hydroxide ethanol solution to dissolve it; transfer it and reach
and pulverizing method may be adopted to mix them up. Before use, liquid specimens shall be
shaken to mix up. The specimens prepared above can be stored in the refrigerator at 2 C ~ 4
C for 1 week.
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.
Accurately weigh-take an appropriate amount of specimen (containing 0.2 g ~ 2 g of folates),
accurate to 0.001 g. General cereals, potatoes, meat, dairy, fresh fruits and vegetables, bacteria
and algae specimens: 2 g ~ 5 g; eggs, beans, nuts, offal and dried specimens: 0.2 g ~ 2 g; liquid
or semi-liquid specimens: 5 g ~ 10 g. Transfer it into a 100 mL conical flask; add 30 mL of
phosphate buffer. Shake it for 5 min, with a stopper. At 121 C (0.10 MPa ~ 0.12 MPa),
hydrolyze it under high pressure for 15 min.
After the specimen is taken out, cool it down to room temperature. Add 1 mL of chicken
pancreas solution, 1 mL of protease-amylase solution; mix it up. Add 3 ~ 5 drops of toluene,
then, place it in a constant-temperature incubator at 36 C  1 C to perform enzymatic
hydrolysis for 16 h ~ 20 h. Take it out and transfer it into a 100 mL volumetric flask; add water
to reach a constant volume, then, filter it.
Take another conical flask. DO NOT add the specimen. The other steps are the same as the
operation of the specimen. Regard it as the enzyme blank solution.
NOTE: For formula foods based on grains and milk powder, if the background folates content of
the matrix needs to be calculated, the enzymatic extraction method may be adopted.
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.
6.4.1.2 Standard series tubes
Take the test tubes and respectively add 0.00 mL, 0.25 mL, 0.50 mL, 1.00 mL, 1.50 mL, 2.00
mL, 2.50 mL, 3.00 mL, 4.00 mL and 5.00 mL of the folates standard working solution; add
water to 5.00 mL, which is equivalent to the folates content in the standard series tubes: 0.00
ng, 0.05 ng, 0.10 ng, 0.20 ng, 0.30 ng, 0.40 ng, 0.50 ng, 0.60 ng, 0.80 ng and 1.00 ng; mix it up.
Prepare 2 ~ 3 sets of the standard series tubes. When drawing the standard curve, calculate by
the average value of each standard point.
6.4.1.3 Sterilization
Perform autoclaving on all the series tubes used for determination and the medium for folates
determination at 121 C (0.10 MPa ~ 0.12 MPa) for 15 min (or sterilize in accordance with the
requirements of the medium).
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
0 control tube.
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.
NOTE: an appropriate spectral range for the determination is 540 nm ~ 610 nm.
6.4.2 Microplate method
Appendix A
Preparation of Culture Medium
A.1 Agar Medium for Strain Stock
A.1.1 Reagents
A.1.1.1 Dipotassium hydrogen phosphate (K2HPO4).
A.1.1.2 Potassium dihydrogen phosphate trihydrate (KH2PO4  3H2O).
A.1.1.3 Magnesium sulfate heptahydrate (MgSO4  7H2O).
A.1.1.4 Ferrous sulfate heptahydrate (FeSO4  7H2O).
A.1.1.5 Manganese sulfate monohydrate (MnSO4  H2O).
A.1.1.6 Sodium acetate trihydrate (CH3COONa  3H2O).
A.1.1.7 Glucose (C6H12O6).
A.1.1.8 Peptone: nitrogen content  10%.
A.1.1.9 Yeast extract (dry powder): nitrogen content  10%.
A.1.1.10 Agar.
A.1.1.11 Hydrochloric acid solution (1 mol/L): measure-take 83.3 mL of hydrochloric acid
(concentration: 36% ~38%); use water to reach a constant volume of 1,000 mL; mix it up.
A.1.2 Preparation of reagents
A.1.2.1 Formate salt solution: respectively weigh-take 25 g of dipotassium hydrogen phosphate
and 25 g of potassium dihydrogen phosphate trihydrate; add water to dissolve it and reach a
constant volume of 500 mL; mix it up. Add 1 mL of toluene; mix it up. This solution can be
stored in a refrigerator at 2 C ~ 4 C for 1 year.
A.1.2.2 Ethyl salt solution: respectively weigh-take 10 g 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; add water to dissolve it and reach a constant volume of 500 mL. Add 5 drops of
1 mol/L hydrochloric acid solution; mix it up. This solution can be stored in a refrigerator at 2
C ~ 4 C for 1 year.
A.1.2.3 Agar medium for strain stock: in accordance with Table A.1, weigh-take or absorb-take
each reagent; add water to 100 mL; mix it up. Heat it up in a boiling water bath, until the agar
is completely dissolved. While it is still hot, use 1 mol/L hydrochloric acid solution or 1 mol/L
A.2.2.4 Adenine-guanine-uracil solution: respectively weigh-take 0.1 g of adenine sulfate,
guanine hydrochloride and uracil into a 250 mL beaker; add 75 mL of water and 2 mL of
hydrochloric acid; heat up to completely dissolve it, then, cool it down. If precipitation is
generated, add a few drops of hydrochloric acid, then, heat it up. Repeat, until no precipitation
is generated after cooling, then, add water to 100 mL. Add 3 ~ 5 drops of toluene and store in a
brown reagent bottle. It can be stored in a refrigerator at 2 C ~ 4 C for 1 year.
A.2.2.5 Xanthine (C5H4N4O2) solution: weigh-take 0.4 g of xanthine, add 10 mL of ammonia;
heat it up to dissolve it; add 100 mL of water. Add 3 ~ 5 drops of toluene; store it in a brown
reagent bottle. It can be stored in a refrigerator at 2 C ~ 4 C for 1 year.
A.2.2.6 Acetic acid buffer (1.6 mol/L, pH 4.5): weigh-take 63 g of sodium acetate trihydrate;
use 200 mL of water to dissolve it. Add about 20 mL of glacial acetic acid to adjust pH to 4.5 
0.1. Mix it up, then, use water to dilute to 500 mL.
A.2.2.7 Vitamin solution: weigh-take 100 mg of riboflavin and use 400 mL of acetic acid buffer
to dissolve it. Take 25 mg of sodium bicarbonate and dissolve in 500 mL of water. Add 2 mg of
biotin, 200 mg of para-aminobenzoic acid, 400 mg of pyridoxine hydrochloride, 40 mg of
thiamine hydrochloride, 80 mg of calcium pantothenate and 80 mg of niacin to dissolve it. Mix
the above-mentioned two solutions, then, add water to 1,000 mL. Add 3 ~ 5 drops of toluene;
store it in a brown reagent bottle. It can be stored in a refrigerator at 2 C ~ 4 C for 1 year.
A.2.2.8 Polysorbate-80 solution (tween-80): dissolve 10 g of polysorbate-80 in absolute ethanol
and dilute it to 100 mL. Store it in a refrigerator at 2 C ~ 4 C.
A.2.2.9 Reduced glutathione (C10H17N3O6S) solution: weigh-take 0.1 g of reduced glutathione;
add 100 mL of water to dissolve it; store it in a brown bottle. Prepare right before use.
A.2.2.10 Phosphate buffer (0.05 mol/L, pH 6.8): in accordance with 3.2.1, prepare it.
A.2.2.11 Hydrochloric acid solution (1 mol/L): in accordance with A.1.11, prepare it.
A.2.2.12 Ethyl salt solution: in accordance with A.1.2.2, prepare it.
A.2.3 Medium for folates determination
Prepare 1,000 mL of the culture medium for folates determination. In accordance with Table
A.2, absorb-take the liquid reagent. After mixing it up, add 300 mL of water; successively add
the solid reagents; boil and stir it for 2 min. Use 1 mol/L sodium hydroxide solution and 1 mol/L
hydrochloric acid solution to adjust pH to 6.8  0.1. Add 20 mL of ethyl salt solution; use
phosphate buffer to supplement to 1,000 mL. When preparing, it can be proportionally
increased or decreased in accordance with the dosage. Prepare it right before use.
......

BASIC DATA
Standard ID GB 5009.211-2022 (GB5009.211-2022)
Description (Translated English) National Food Safety Standard - Determination of folates in food
Sector / Industry National Standard
Classification of Chinese Standard X09
Word Count Estimation 12,129
Date of Issue 2022-06-30
Date of Implementation 2022-12-30
Older Standard (superseded by this standard) GB 5009.211-2014
Administrative Organization National Health Commission
Issuing agency(ies) State Administration for Market Regulation