GB 5413.14-2010National food safety standard -- Determination of vitamin B12 in foods for infants and young children, milk and milk products (English PDF)
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Issued by
Ministry of Health of the People's Republic of China
Level / Type
National · Mandatory
Issue date
March 26, 2010
Implementation date
June 1, 2010
Scope
GB 5413.14-2010 (National food safety standard -- Determination of vitamin B12 in foods for infants and young children, milk and milk products) is available as an English-translated PDF.
GB 5413.14-2010 — This Standard specifies the method for determination of vitamin B12 in foods for infants and young children, milk and milk products. This Standard is applicable to determination of vitamin B12 in foods for infants and young children, milk and milk products.
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Document preview — GB 5413.14-2010
National Standard of the People's Republic of China
Issued by: Ministry of Health of the People's Republic of China
Contents
- Foreword3
- 1 Scope4
- 2 Normative References4
- 3 Principle4
- 4 Reagents and Materials4
- 5 Apparatuses and Instruments6
- 6 Analytical Procedure6
- 4 Result Expression9
- 7 Precision10
- 8 Others10
- Appendix A (Normative) Culture Medium and Reagent11
Foreword
This Standard replaces GB/T 5413.14-1997 Milk Powder and Formula Foods for Infant and Young Children - Determination of Vitamin B12 Content.
Compared with GB/T 5413.14-1997, this Standard mainly has the following changes.
--- Change the standard name into Determination of Vitamin B12 in Foods for Infants and Young Children, Milk and Milk Products.
This Standard's Appendix A is normative.
The historical editions replaced by this Standard are as follows.
--- GB 5413-1985, GB/T 5413.14-1997.
National Food Safety Standard Determination of Vitamin B12 in Foods for Infants and Young Children, Milk and Milk Products
1 Scope
This Standard specifies the method for determination of vitamin B12 in foods for infants and young children, milk and milk products.
This Standard is applicable to determination of vitamin B12 in foods for infants and young children, milk and milk products.
2 Normative References
The following documents are essential to the application of this document. For the dated documents, only the versions with the dates indicated are applicable to this document; for the undated documents, only the latest version (including all the amendments) are applicable to this Standard.
3 Principle
Utilize Lactobacillus leichmannii's specificity and sensitivity to vitamin B12;
quantitatively determine content of vitamin B12 in sample. In the medium for determination, SUPPLY all nutrients except vitamin B12, so the transmittance generated by microbial growth shall be corresponding to the content of Vitamin B12 in standard curve work solution and unknown to-be-measured solution.
4 Reagents and Materials
All reagents, if not specified otherwise, refer to analytic reagent; all experiment water, if not specified otherwise, refers to grade 2 water in GB/T 6682.
4.1 Strain. Lactobacillus leichmannii ATCC 7830.
4.2 Vitamin B12 (Vitamin B12 or Cyanocobalamin) standard products. its molecular
formula is C63H88CoN14O14P; purity >=99%.
4.3 Culture medium
4.3.1 Lactobacillus agar culture medium. see Appendix A.
4.5 Ethanol solution. volume fraction is 25%.
4.6 Anhydrous sodium hydrogen phosphate (Na2HPO4).
4.7 Anhydrous sodium metabisulfite (Na2S2O5).
4.8 Citric Acid (with a hydrate) (C6H8O7-H2O).
4.9 Preparation of standard solution
4.9.1 Vitamin B12 stock solution (10µg/mL). accurately weigh vitamin B12 standard
product (4.2); dilute with ethanol solution (4.5) to fix the volume to that vitamin B12 concentration is 10µg/mL.
4.9.2 Vitamin B12 intermediate solution (100ng/mL). dilute 5 mL of stock solution
(4.9.1) with ethanol solution (4.5) to 500mL.
4.9.4 Standard curve work solution. absorb twice the 5mL of Vitamin B12 work
solution (4.9.3) respectively; place them into 250mL and 500mL flasks; use water to fix volume to the scale. The concentration of high-concentration solution is 0.02ng/mL;
while the concentration of low-concentration solution is 0.01ng/mL.
4.9.3 can be stored for three months. Solutions in 4.9.4 shall be prepared just before use.
5 Apparatuses and Instruments
In addition to the microbiological laboratory conventional sterilization and cultivation devices, other devices and materials are as follows.
5.1 Balance. sensitivity is 0.1mg.
5.2 pH meter. accuracy <=0.01.
5.6 Thermostatic incubator. 36°C±1°C.
5.7 Refrigerator. 2°C ~ 5°C.
5.8 Sterile pipette. 10 mL (with 0.1 mL scale) or micro-pipette and sucker.
5.9 Dispenser. 0 mL~10 mL.
5.14 Quantitative filter paper. diameter is 90 mm.
5.15 Tube. 18 mm×180 mm.
6 Analytical Procedure
6.1 Preparation of the test bacteria solution
6.1.1 After activating the Lactobacillus leichmannii (ATCC 7830) freeze-dried strain
6.2 Treatment of specimen
6.2.1 Weigh 1.3g of anhydrous sodium hydrogen phosphate (4.6), 1.0g of anhydrous sodium metabisulfite (4.7), 1.2 g of citric acid (with a hydrate) (4.8); use 100 mL of water to dissolve.
6.2.2 Weigh certain amount of sample (accurate to 0.1mg), it contains about 50 ~
100ng of vitamin B12; use 10mL of above solution (6.2.1) to mix; then add 150mL of water. Hydrolyze for 10min at 121°C; then cool down and adjust pH to 4.5± 0.2.
6.3 Preparation of standard curve
According to the sequence in Table 1, ADD water, standard curve work solution (4.9.4) and vitamin B12 determination used culture medium (4.3.3) INTO culture tubes, make triplicate.
6.4 Preparation of solution under test
Tube No.
6.5 Sterilization
Cap all tubes (6.3 and 6.4); sterilize for 5min at 121°C (products' culture medium shall be sterilized according to the labelled instructions)
6.6 Inoculation
Cool down above-mentioned tubes rapidly to below 30°C. Use dropper or pipette to add a drop (about 50µL) of bacterial solution (6.1.2) into each of above tubes (in which, except the blank S1 in the standard curve tubes).
6.7 Incubating
Put the tube into the thermostatic incubator, then incubate for 19 ~ 20h at 36°C±1°C.
6.8 Determination
After the completion of incubating, visually inspect each tube; in not-inoculated tube, the incubating liquid should be clear. If there occurs turbid, then the determination is invalid.
6.8.1 Take inoculated blank tube as a reference, determine the transmittance of maximum-concentration standard curve tube. Re-determine this tube again after 2h. If the transmittance's difference between these two results is <=2%, then take out all the tubes and determine their transmittance.
6.8.3 After using a vortex mixer (5.4) to well-mix each test tube (it may also add a
drop of antifoaming agent), immediately transfer the culture solution into the cuvette to conduct determination; read the transmittance after 30s at wavelength 550nm. Each tube should have the same stability time. Take the content of vitamin B12 standard Tube No.
6.8.4 According to the transmittance of solution under test, obtain the concentration
of vitamin B12 in this solution under test from the standard curve; Then calculate the content of vitamin B12 in the sample according to the dilution factor and sample weighing amount. For the test tubes of which the transmittance is beyond the range of standard curve tubes S3 ~ S10, it shall be abandoned.
6.8.5 As for each numbered test tube, use the transmittance of each tube to calculate the concentration of vitamin B12 in each liter's corresponding numbered solution under test; and calculate the average concentration of vitamin B12 value in the same numbered tubes; the concentration measured in each tube shall not exceed ±15% average value; abandon those ones that exceed the limit.
4 Result Expression
Calculate the content of vitamin B12 in specimen according to Formula (1).
7 Precision
Under the repeatability condition, the absolute difference between the two independent determination results shall not exceed 10% arithmetic mean.
8 Others
This Standard's limit of detection is 0.1µg/100g.
Appendix A
(Normative)
Culture Medium and Reagent A.1 Lactobacillus agar culture medium A.1.1 Ingredient Tomato juice 100 mL, No.3 peptone 7.5 g, yeast extract 7.5 g, glucose 10.0 g, potassium dihydrogen phosphate 2.0 g, poly sorbitol monostearate 1.0 g, agar 14.0 g, water 1000 mL, pH 6.8± 0.1 (25°C ± 5°C).
A.1.2 Preparation
Dissolve all ingredients except agar into water; adjust pH; then add agar. Boil the medium to dissolve thoroughly. Dispense each tube 10mL; sterilize for 15min at 121°C; store for later use.
A.2 Lactobacillus broth culture medium A.2.2 Preparation Dissolve all ingredients (A.2.1) into water; adjust pH; boil the medium to dissolve thoroughly. Dispense each tube 10mL; sterilize for 15min at 121°C; store for later use.
A.3 Vitamin B12 determine medium A.3.1 Ingredient Vitamin-free acid hydrolysis casein 15.0 g, glucose 40.0 g, asparagine 0.2 g, sodium acetate, 20.0 g, ascorbic acid 4.0 g, L-cystine 0.4 g, DL-tryptophan 0.4 g, adenine sulfate 20.0 mg, guanine hydrochloride 20.0 mg, uracil 20.0 mg, xanthine 20.0 mg, A.3.2 Preparation Dissolve above ingredients into water, adjust pH, and store for later use.
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