GB/T 5413.14-1997 Determination of vitamin B12 in infant formula and milk powder

This standard specifies the method for the determination of vitamin B12 by microbiological method. This standard is applicable to the determination of vitamin B12 in infant formula and milk powder. GB/T 5413.14-1997 Determination of vitamin B12 in infant formula and milk powder GB/T5413.14-1997 Standard download decompression password: www.bzxz.net

GB/T5413.14—1997
This standard is equivalent to the method of the American Association of Public Analytical Chemists (AOAC), which is accurate and highly sensitive. This series of standards will replace GB5413-85 from the date of implementation. This standard is proposed by the China Light Industry General Association.
This standard is under the jurisdiction of the National Dairy Standardization Center. The responsible drafting unit of this standard: National Dairy Quality Supervision and Inspection Center. The participating drafting units of this standard: Food Hygiene Supervision and Inspection Institute of the Ministry of Health, Zhejiang Light Industry Research Institute, Harbin Morinaga Dairy Co., Ltd., Nestle (China) Investment Service Co., Ltd. The main drafters of this standard: Zhang Yujie, Wang Yun, Dian Xiaohong, Lv Weiqun. 277
National Standard of the People's Republic of China
Infant formula foods and milk powder
Determination of vitamin Br2 content
Milk powder and formula foods for infant and young childrenDetermination of vitamin Br2 content1Scope
This standard specifies the method for the determination of vitamin B12 by microbiological method. This standard is applicable to the determination of vitamin B12 in infant formula foods and milk powder. 2 Principle of the method
GB/T 5413.14—1997
Replaces GB5413--85bzxZ.net
Lactobacillus leichmanni has extremely high sensitivity to the presence of vitamin Biz. This specificity can be used to quantitatively determine the content of vitamin B12 in a sample. 3 Reagents, strains and culture media
All reagents, if the specifications are not specified, are of analytical grade; all experimental water, if no other requirements are specified, is grade tertiary water. 3.1 Physiological saline: 9g/L.
3.2 Ethanol: 25% by volume.
3.3 Anhydrous disodium hydrogen phosphate.
3.4 ​​Anhydrous sodium metabisulfite.
3.5 Citric acid (containing one crystal water).
3.6 Strain: Lactobacillus leichmannii. 3.7 Culture media.
3.7.1 Lactobacillus agar medium: 15g photolyzed Chen, 5g yeast extract, 10g glucose, 100mL tomato juice, 2g potassium dihydrogen phosphate, 1g polysorbate monooleate, 10g agar, 1000mL distilled water, pH 6.8±0.2 (25℃). 3.7.2 Lactobacillus broth medium: 15g photolyzed Chen, 5g yeast extract, 10g glucose, 100mL tomato juice, 2g potassium dihydrogen phosphate, 1g polysorbate monooleate, 1000mL distilled water, pH 6.8±0.2 (25℃). 3.7.3 Culture medium for vitamin B12 determination: 15 g of vitamin-free acid hydrolyzed casein, 40 g of glucose, 0.2 g of asparagine, 20 g of sodium citrate, 4 g of ascorbic acid, 0.4 g of L-cystine, 0.4 g of DL-tryptophan, 20 mg of adenine sulfate, 20 mg of guanine hydrochloride, 20 mg of uracil, 20 mg of xanthine, 1 mg of riboflavin, 1 mg of thiamine hydrochloride, 10 μg of biotin, 2 mg of nicotinic acid, 2 mg of p-aminobenzoic acid, 1 mg of calcium pantothenate, 4 mg of pyridoxine hydrochloride, 4 mg of pyridoxal hydrochloride, 800 μg of pyridoxamine hydrochloride, 200 μg of folic acid, 1 g of potassium dihydrogen phosphate, 1 g of dipotassium hydrogen phosphate, 0.4 mg of magnesium sulfate, 20 mg of sodium fluoride, 20 mg of ferrous sulfate, 20 mg of manganese sulfate, 2 g of polysorbate monooleate, 1000 mL of steamed stuffing water, pH 6.0 ± 0. 2 (25℃).
3.8 Vitamin B12: standard substance.
Approved by the State Administration of Technical Supervision on May 28, 1997 278
Implemented on September 1, 1998
4 Instruments
Common laboratory instruments and:
Spectrophotometer.
5 Preparation
5.1 Preparation of strains
GB/T5413.141997
5.1.1 Inoculate the Lactobacillus leishmanii preserved in the straight column lactobacillus agar medium into a new medium. After culturing, transfer it once again, and then transfer it from the solid medium to the lactobacillus broth medium for culturing. 5.1.2 Centrifuge the culture solution in the lactobacillus broth at 2000r/min for 2-3min, pour out the supernatant, add 10mL of physiological saline (3.1), stir well, and centrifuge for 2-3min. Wash 3-4 times in this way, and pipette 0.4mL of the bacterial suspension into 10mL of saline (3.1) for testing. 5.1.3 Use a spectrophotometer, with physiological saline (3.1) as a blank, to measure the transmittance of the bacterial suspension in 5.1.2 at a wavelength of 550nm, which should be between 60% and 80%.
5.2 Preparation of standard solution
5.2.1 Vitamin B12 standard stock solution, with a concentration of 100ng/mL. Accurately weigh the vitamin B1z standard and dilute it with ethanol (3.2) to a vitamin B12 concentration of 100ng/mL. 5.2.2 Vitamin B12 intermediate stock solution, concentration is 1 ng/mL Use ethanol (3.2) to make up 10 mL of standard stock solution (5.2.1) to 1000 mL. 5.2.3 Standard working solution, divided into two concentrations: high concentration - vitamin B12 concentration is 0.02 ng/mL, low concentration - vitamin B12 concentration is 0.01 ng/mL. Pipette two 5 mL from the intermediate solution (5.2.2) and make up to 250 mL and 500 mL respectively with distilled water. 6 Operation steps
6.1 Sample treatment
6.1.1 Dissolve 1.3 g of anhydrous disodium hydrogen phosphate (3.3), 1.0 g of anhydrous sodium metabisulfite (3.4), and 1.2 g of citric acid (containing one crystal water) (3.5) in 100 mL of distilled water.
6.7.2 Weigh a certain amount of sample (containing about 50-100 ng of vitamin B12), mix it with 10 mL of the above solution (6.1.1), then add 150 mL of distilled water, hydrolyze it at 121°C for 10 min, adjust the pH to 4.5 after cooling, and dilute it with distilled water so that the mass concentration of vitamin B12 in the final solution is about 0.01-0.02 ng/mL, and the mass concentration of sodium metabisulfite is less than 0.03 mg/mL. 6.2 Preparation of standard curve
Add distilled water, standard solution and vitamin B12 determination culture medium into the culture tube in the order of Table 1, in triplicate. Table 1
Test tube No.
Distilled water, mL
Standard solution", mL
Culture medium, mL
1) Add low concentration standard solution to test tubes No.3~7; add high concentration standard solution to test tubes No.8~10. 6.3 Determination solution
In the order of Table 2, distilled water, sample solution and vitamin B12 determination medium are placed in the culture tube in triplicate. &
6.4 Inoculation
Test tube No.
Distilled water, mL
Sample, mL
Culture medium, mL
GB/T 5413. 14-1997
Sterilize all the test tubes in 6.2 and 6.3 at 121℃ for 5 minutes, and then cool quickly. Use a capillary pipette to add 1 drop of the bacterial suspension in 6.1.2 to each of the above test tubes (except No.1 in the standard curve tube), mix well, and incubate at 37℃±0.5℃ for 19~20h. 6.5 Determination
Use the inoculated blank tube as a control, measure the T of the standard sample tube with the highest concentration, and read the value again after two hours. If the T value of the secondary result is ≤2%, take out all the test tubes and measure their T and record it.
7 Expression of analysis results
Take the amount of vitamin B12 contained in the standard sample as The horizontal axis and the T value are used as the vertical axis to make a working curve. According to the T value measured by the sample, the average value of the vitamin B12 content is obtained from the standard curve, and then the actual content per 100 grams (or milliliters) of the sample is calculated according to formula (1): Vitamin Br2 in the sample (μg/100g (mL)) = m
In the formula: - the average content of vitamin B12 in the sample obtained from the curve, ng, F-dilution factor,
the mass (or volume) of the sample, g (or mL). m-
8 allowable difference
The difference between the two measured values ​​of the same sample shall not exceed 10% of the average value of the two measurements. 280
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