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GB 5413.14-2010 PDF in English


GB 5413.14-2010 (GB5413.14-2010) PDF English
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GB 5413.14-2010: PDF in English

GB 5413.14-2010
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. Draw standard
curve, based on the transmittance of the different-concentration standard solution
against the concentration of various concentration-level’s standard substances;
according to the standard curve, then calculate the content of vitamin B12 in the
sample.
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.
4.3.2 Lactobacillus broth culture medium. see Appendix A.
4.3.3 Vitamin B12 determination culture medium. see Appendix A.
NOTE. Some products’ synthetic culture medium has good effect; preparation of such
products’ synthetic culture medium shall conform to the labelled instructions.
4.4 9g/L Normal saline (physiological saline)
Weigh 9g of NaCl to dissolve into 1000mL of water; respectively place it into the tubes
with plugs; each tube is for 10mL; sterilize for 15min at 121°C.
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.
4.9.3 Vitamin B12 work solution (1ng/mL). dilute 5 mL of intermediate solution (4.9.2)
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.
NOTE. All standard solutions should be stored in refrigerator. Solutions in 4.9.1, 4.9.2 and
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.
5.3 Spectrophotometer.
5.4 Vortex mixer.
5.5 Centrifuge. ≥2000r/min.
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.
5.10 Conical flask. 200mL.
5.11 Volumetric flask (Class-A). 100 mL, 250 mL, 500 mL.
5.12 Single-scale pipette (Class-A). capacity is 5 mL.
5.13 Hopper. diameter is 90 mm.
5.14 Quantitative filter paper. diameter is 90 mm.
5.15 Tube. 18 mm×180 mm.
NOTE. When preparing the glass apparatus, use active agent to clean the hard glass
measuring tube and other necessary glassware; after cleaning, it is required to dry
and heat for 2h at 200°C.
6 Analytical Procedure
6.1 Preparation of the test bacteria solution
6.1.1 After activating the Lactobacillus leichmannii (ATCC 7830) freeze-dried strain
(4.1); inoculate it onto the Lactobacillus broth culture medium (4.3.1); incubate for 24
h at 36°C±1°C. Then continue to inoculate for 2-3 generations to enhance the vitality.
product as X-axis, the transmittance as Y-axis, then draw standard curve.
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. If the number of the
tubes conforming to this requirements is less than 2/3 total tube number of the four
numbered tubes, then the data used for calculating the specimen content is
insufficient, and it needs re-determination. If the number of the tubes conforming to
this requirements is more than 2/3 total tube number of the four numbered tubes, then
recalculate the average content of vitamin B12 in each liter’s solution under test in each
numbered valid specimen tube. Use this average value to calculate the total average
value of all numbered tubes, Cx, which is used for calculating the content of vitamin
B12 in the specimen.
Note. Use the transmittance (T%), or the absorbance (A) to draw standard curve.
4 Result Expression
Calculate the content of vitamin B12 in specimen according to Formula (1).
X = Cx/m × f/1000 ×100 ... (1)
Where,
X - Content of vitamin B12 in sample, µg/100g;
Cx - The total average value calculated in 6.8.5, ng;
m – Mass of the specimen, g;
f -- Dilution multiple.
It shall be expressed, under repeatability conditions, by the arithmetic mean of two
independent determination results; the determination results shall remain two
significant figures.
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.2.1 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, water 1000
mL, pH 6.8 ± 0.1 (25°C ± 5°C).
A.2.2 Preparation
Dissolve all ingredients (A.2.1) into water; adjust pH; boil the ...
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Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.