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GB 5009.211-2014 English PDF (GB5009.211-2014)

GB 5009.211-2014 English PDF (GB5009.211-2014)

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GB 5009.211-2014: National Food Safety Standard -- Determination of folates in food

This Standard specifies the method for determination of folates in foods. This Standard applies to the determination of folates in foods.
GB 5009.211-2014
National Food Safety Standard
Determination of Folates in Food
Issued by. National Health and Family Planning Commission of the
PEOPLE Republic of China
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Principle ... 4
3 Reagents and materials ... 4
4 Apparatuses and instruments ... 8
5 Strains preparation and storage ... 8
6 Analytical steps (all operations are conducted in dark) ... 9
7 Precision... 13
8 Other ... 13
Appendix A Preparation methods for folates determination medium ... 14 Foreword
This Standard replaces GB/T 5009.211-2008 Determination of folates in foods. Compared with GB/T 5009.211-2008, main changes of this Standard are as follows. ?€? MODIFY the standard name to ?€?National food safety standard - Determination of folates in foods?€?;
?€? MODIFY the strains name, from lactobacillus casei to lactobacillus
?€? DELETE the supply information of the medium and the chicken pancreas
which is used for folate degradation;
?€? DELETE the preparation method of enzyme casein solution in Appendix B; ?€? ADD detection limit and quantification limit.
National food safety standard
Determination of folates in foods
1 Scope
This Standard specifies the method for determination of folates in foods. This Standard applies to the determination of folates in foods.
2 Principle
Folate is a nutrient which is necessary for the growth of Lactobacillus casei spp. Rhamnosus (ATCC 7469). Under certain controlled conditions, INOCULATE the lactobacillus rhamnosus into a sample solution-containing culture medium; CULTURE it for a period of time; MEASURE the light transmittance (or absorbance). Based on the standard curve of folate content and light transmittance (or absorbance), CALCULATE the folates content in the sample.
3 Reagents and materials
Note. Unless otherwise noted, the reagent used in this method is analytically pure; AND the water is grade II 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 Dipotassium phosphate (K2HPO4).
3.1.7 Potassium dihydrogen phosphate trihydrate (KH2PO4??3H2O).
3.1.8 Magnesium sulphate heptahydrate (MgSO4??7H2O).
3.1.9 Ferrous sulfate heptahydrate (FeSO4??7H2O).
4 Apparatuses and instruments
4.1 Balance. sensitivity is 0.1mg and 1mg.
4.2 Incubator. 37??C ?? 1??C.
4.3 Pressure steam sterilizer. 121??C (0.10MPa ~ 0.12MPa).
4.4 Vortex.
4.5 Centrifuge. Speed ??? 3000r/min.
4.6 Inoculating loop.
4.7 pH meter. accuracy of ?? 0 1.
4.8 UV - visible spectrophotometer.
4.9 Clean benches.
4.10 Ultrasonic wave oscillator.
5 Strains preparation and storage
5.1 Strains
Lactobacillus casei spp. rhamnosus (ATCC 7469).
5.2 Preparation of stock strains
TRANSFER the lactobacillus rhamnosus strain to the agar medium; CULTURE it in 37??C ?? 1??C incubator for 20h ~ 24h, so to realize continuous propagation for 2 ~ 3 generations. TAKE it out; PLACE it into 2??C ~ 4??C refrigerator as stock strain. PROPAGATE at least once a month, AND it can propagate 30 generations.
Before the experiment, INOCULATE the stock strain into agar medium; CULTURE it in 37??C ?? 1??C incubator for 20h ~ 24h to activate the strains, which will be used for inoculation liquid preparation.
Note. As for the stock strain which is preserved for several weeks, it is not allowed to use it for inoculating liquid preparation, AND it is preferable to continuously propagate for 2 ~ 3 generations, so as to ensure the viability of bacteria.
5.3 Inoculating liquid preparation
One day before experiment, TAKE 2mL of folates standard working solution to mix uniformly with 4mL of folate determination medium, DISPENSE it into two 5mL for 15min.
TAKE the sample out; COOL it to room temperature; ADD 1mL of chicken pancreas; for the protein/starch-containing sample, ADD additionally 1mL of protease- amylase solution; MIX it. After adding 3 ~ 5 drops of toluene, PLACE it in 37??C ?? 1??C incubator for enzymatic extraction for 16h ~ 20h. TAKE it out; TRANSFER it into 100mL volumetric flask; ADD water to make it reach to the mark; FILTER it. TAKE another conical flask; USE same sample treatment steps; MAKE it reach to the 100mL; USE it as an enzyme blank solution.
NOTE. As for the cereal/milk-based formula food, if it is needed to measure the folate content of the matrix background, it is allowed to use enzymatic extraction method. 6.3 Dilution
Based on the folate content in the sample, USE water to appropriately dilute the sample extract, so as to make the folate content in the sample extract in the range of 0.2ng/mL ~ 0.6ng/mL.
6.4 Preparation of determination test tube series
Before using any test tubes, WASH it to clean; MAKE it subject to boiling water bath for 30min; DRAIN it to dry; PLACE it into hydrochloric acid soaking solution for 2h; DRY it at 170??C ?? 2??C for 3h.
6.4.1 Sample and enzyme blank tube series
TAKE 3 tubes; respectively ADD 0.5mL, 1.0mL, 2.0mL sample dilution (Vx); ADD water to 5.0mL. ADD 5.0mL of folate determination medium; MIX it uniformly. TAKE another 3 tubes; ADD enzyme blank solution by same method.
6.4.2 Standard series test tubes
TAKE test tubes; respectively ADD folate standard working solution 0.00mL, 0.25mL, 0.50mL, 1.00mL, 1.50mL, 2.00mL, 2.50mL, 3.00mL, 4.00mL and 5.00mL; ADD water to 5.00mL, equivalent to folates content (in standard series test tubes) of 0.00ng, 0.05ng, 0.10ng, 0.20ng, 0.30ng, 0.40ng, 0.50ng, 0.60ng, 0.80ng, and 1.00ng; then ADD 5.0mL of folates determination medium; MIX it uniformly. To ensure linear relation of standard curve, it shall prepare 2 ~ 3 sets of standard series test tubes; when drawing standard curve, CONDUCT calculation based on the mean value of each standard point.
6.5 Sterilization
STUFF with tampon for all determination series test tubes; at 121??C (0.10MPa ~ 0.12MPa), AUTOCLAVE for 15min.
Appendix A
Preparation methods for folates determination medium
A.1 Reagents
A.1.1 Absolute ethanol (C2H6O).
A.1.2 Sodium bicarbonate (NaHCO3).
A.1.3 Hydrochloric acid (HCl).
A.1.4 Sodium hydroxide (NaOH).
A.1.5 Toluene (C7H8).
A.1.6 Glacial acetic acid (C2H4O2).
A.1.7 Active carbon. particle size of 0.05mm ~ 0.074mm.
A.1.8 Adenine sulphate (C10H10N10??H2SO4).
A.1.9 Hydrochloric acid guanine (C5H5N5O5??HCl).
A.1.10 Uracil (C4H4N2O2).
A.1.11 Xanthine (C5H4N4O2).
A.1.12 Ammonia (NH5O).
A.1.13 Sodium acetate trihydrate (C2H3O2Na??3H2O).
A.1.14 Riboflavin (C17H20N4O6).
A.1.15 Biotin (C10H16N2O3S).
A.1.16 PABA (C7H7NO2).
A.1.17 Pyridoxine hydrochloride (C8H11NO3??HCl).
A.1.18 Thiamine hydrochloride (C12H17ClN4OS??HCl).
A.1.19 Calcium pantothenate (C18H32CaN2O10).
A.1.20 Niacin (C6H5NO2).
A.1.21 Polysorbate 80 (Tween 80).
A.1.22 Glutathione (C10H17N3O6S).
A.1.23 L - aspartic acid (C4H7NO4).
A.1.24 L - tryptophan (C11H12N2O2).
A.1.25 L - cysteine hydrochloride (C3H7NO2S??HCl).
A.1.26 Anhydrous glucose (C6H12O6).
A.1.27 Vitamin free casein (vitamin free casein).
A.2 Reagent preparation
A.2.1 Sodium hydroxide solution (10mol/L). WEIGH 40g of sodium hydroxide; USE 100mL of water to dissolve it.
A.2.2 Sodium hydroxide solution (1mol/L). WEIGH 4g of sodium hydroxide; USE 100mL of water to dissolve it.
A.2.3 Casein solution. WEIGH 50g of vitamin free casein; PLACE it into a 500mL beaker; ADD 200mL of hydrochloric acid solution; MAKE it subject to high pressure hydrolysis at 121??C (0.10MPa ~ 0.12MPa) for 6h. TRANSFER the hydrolysate into evaporating dish; in a boiling water bath, EVAPORATE to a paste form. ADD 200mL of water to dissolve it; EVAPORATE it into paste form again; REPEAT this cycle for 3 times to remove hydrochloric acid. USE 10mol/L sodium hydroxide to adjust the pH to 3.5 ?? 0.1. ADD 20g of activated carbon; SHAKE it for about 20min; FILTER it. REPEAT activated carbon treatment, until the filtrate is pale yellow or colorless. ADD water into the filtrate to dilute it to 1000mL; ADD 1mL ~ 3mL of toluene; it can be preserved in 2??C ~ 4??C refrigerator for 1 year.
NOTE. As for the evaporation, it is not allowed to evaporate to dry or char, so as to avoid the contained nutrients from being damaged. It is also allowed to directly purchase the acid hydrolysis vitamin-free casein of equivalent effect.
A.2.4 Adenine - guanine - uracil solution. Respectively WEIGH 0.1g of adenine sulphate, hydrochloric acid guanine, and uracil; PLACE them into a 250mL beaker; ADD 75mL of water and 2mL of hydrochloric acid; HEAT it to dissolve completely; COOL it down. If precipitate is produced, ADD additionally several drops of hydrochloric acid; HEAT it; REPEAT this cycle until there is no precipitate produced; ADD water to 100mL. ADD 3 ~ 5 drops of toluene; STORE it in a brown reagent bottle; it can be preserved in 2??C ~ 4??C refriger...

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