GB 5413.14-2010 PDF English
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| Standard ID | Contents [version] | USD | STEP2 | [PDF] delivery | Name of Chinese Standard | Status |
| GB 5413.14-2010 | English | 70 |
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National food safety standard -- Determination of vitamin B12 in foods for infants and young children, milk and milk products
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| GB/T 5413.14-1997 | English | 199 |
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Milk powder and formula foods for infant and young children--Determination of vitamin B12 content
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GB 5413.14-2010: National food safety standard -- Determination of vitamin B12 in foods for infants and young children, milk and milk products
---This is an excerpt. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.), auto-downloaded/delivered in 9 seconds, can be purchased online: https://www.ChineseStandard.net/PDF.aspx/GB5413.14-2010
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard
Determination of Vitamin B12 in Foods for Infants
and Young Children, Milk and Milk Products
Issued on. MARCH 26, 2010
Implemented on. JUNE 1, 2010
Issued by. Ministry of Health of the People’s Republic of China
Table of Contents
Foreword... 3
1 Scope... 4
2 Normative References... 4
3 Principle... 4
4 Reagents and Materials... 4
5 Apparatuses and Instruments... 6
6 Analytical Procedure... 6
4 Result Expression... 9
7 Precision... 10
8 Others... 10
Appendix A (Normative) Culture Medium and Reagent... 11
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.
......
Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
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