GB 5009.259-2016_English: PDF (GB5009.259-2016)
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National food safety standard -- Determination of free biotin in foods for infants and young children, milk and milk products
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GB 5009.259-2016
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| GB 5009.259-2023 | English | 260 |
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National food safety standard - Determination of biotin in foods
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GB 5009.259-2023
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Newer version: GB 5009.259-2023 Standards related to: GB 5009.259-2023
| Standard ID | GB 5009.259-2016 (GB5009.259-2016) | | Description (Translated English) | National food safety standard -- Determination of free biotin in foods for infants and young children, milk and milk products | | Sector / Industry | National Standard | | Classification of Chinese Standard | X09 | | Word Count Estimation | 8,854 | | Date of Issue | 2016-08-31 | | Date of Implementation | 2017-03-01 | | Older Standard (superseded by this standard) | GB 5413.19-2010 | | Regulation (derived from) | State Health and Family Planning Commission Notice No.11 of 2016 | GB 5009.259-2016
GB
NATIONAL STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
National food safety standard
Determination of biotin in foods
ISSUED ON. AUGUST 31, 2016
IMPLEMENTED ON. MARCH 01, 2017
Issued by. National Health and Family Planning Commission of the PRC
Table of contents
Foreword ... 3
1 Scope ... 4
2 Principles ... 4
3 Reagents and materials ... 4
4 Instruments and equipment ... 6
5 Preparation and preservation of strains ... 7
6 Analytical procedures ... 7
7 Precision... 11
8 Others ... 11
Appendix A Medium and reagent ... 12
National food safety standard
Determination of Biotin in foods
1 Scope
This standard specifies the method for determination of biotin in food.
This standard applies to the determination of biotin in food.
2 Principles
Biotin is a nutrient necessary for the growth of Lactobacillus plantarum. In the
biotin determination medium, the growth of Lactobacillus plantarum is in linear
relation with the biotin content in the sample to be determined; AND the
content of the substance to be determined in the sample is calculated by
comparing the light transmittance with the standard working curve.
3 Reagents and materials
Unless otherwise stated, the reagents used in this method are of analytical
grade AND water is level II water as specified in GB/T 6682.
3.1 Reagents
3.1.1 Anhydrous ethanol (C2H6O).
3.1.2 Sodium hydroxide (NaOH).
3.1.3 Hydrochloric acid (HCl).
3.1.4 Citrate.
3.1.5 α-amylase. ≥ 1.5 U/mg.
3.1.6 Papain. ≥ 5 U/mg.
3.1.7 Sulfuric acid (H2SO4).
3.2 Reagent preparation
3.2.1 Ethanol solution (50%). MEASURE 500 mL of anhydrous ethanol; MIX it
uniformly with 500 mL of water.
3.2.2 Sodium hydroxide solution (0.5 mol/L). WEIGH 20 g of sodium hydroxide;
DISSOLVE it into 1000 mL of water; MIX it uniformly.
3.2.3 Sodium chloride solution (0.85%). WEIGH 8.5 g of sodium chloride; ADD
water to dissolve and dilute it to 1000 mL; MIX it uniformly.
3.2.4 Hydrochloric acid solution (1 mol/L). ABSORB 83 mL of hydrochloric acid;
USE water to dilute it to 1000 mL; MIX it uniformly.
3.2.5 Citrate buffer solution (pH 4.5). WEIGH 1.5 g of citric acid into a 100 mL
beaker with a magnetic stirrer; ADD about 50 mL of distilled water to dissolve it;
then ADD 12 mL of NaOH (1 mol/L); ADJUST the pH to 4.5 (with 0.1 mol/L
HCl); TRANSFER the solution into a 100 mL volumetric flask; USE distilled
water to make the volume reach to the mark. AND this butter solution can be
preserved for 3 d at 2 °C ~ 8 °C.
3.2.6 Protease-amylase solution. Respectively WEIGH 200 mg of papain and
α-amylase; ADD 20 mL of water and GIND it to homogenate; CENTRIFUGE it
at 3000 r/min for 5 min ~ 10 min; PREPARE it before use.
3.2.7 Sulfuric acid solution (3%). MEASURE 30 mL of sulfuric acid; ADD it to
1000 mL of water; MIX it uniformly.
3.3 Standard substance
Biotin (d-Biotin or Vitamin H) standard substance (C10H16N2O3S). purity ≥ 99%.
3.4 Standard solution preparation
3.4.1 Biotin standard stock solution (100 µg/mL). Accurately WEIGH 100 mg of
biotin standard substance; USE ethanol solution (50%) to dissolve and transfer
it into a 1000 mL volumetric flask; MAKE its volume reach to the mark.
PRESERVE it in a brown bottle, which can be stored in a 2 °C ~ 4 °C
refrigerator for 12 months.
3.4.2 Biotin standard intermediate solution (1.0 µg/mL). Accurately PIPETTE
1.00 mL of biotin standard stock solution into a 100 mL brown volumetric flask;
USE ethanol solution (50%) to dissolve it and MAKE its volume reach to the
mark. PRESERVE it in a brown bottle, which can be stored in a 2 °C ~ 4 °C
refrigerator for 6 months.
3.4.3 Biotin standard working solution (10 ng/mL). Accurately PIPETTE 1.00
mL of biotin standard intermediate solution into a 100 mL volumetric flask; USE
machine; such samples as fruit and vegetable and semi-solid need to be mixed
uniformly into homogenate; AND the liquid sample needs to be shaken before
use to mix it, which is then preserved in a 4 °C refrigerator AND determined
within 1 week.
6.2 Sample extraction
6.2.1 Potato, meat, dairy, fresh fruits and vegetables, algae samples, eggs,
beans, nuts, animal offal and other natural foods. Accurately WEIGH an
appropriate amount of homogeneous sample (m) (containing about 0.2 μg ~
0.5 μg biotin), accurate to 0.001 g; PLACE it into a 50 mL volumetric flask;
ADD 30 mL of citric acid buffer solution; SHAKE it and MAKE it subjected to
high pressure hydrolysis at 121 °C for 15 min. TAKE out the sample;
immediately COOL it to room temperature; ADD 1 mL of protease-amylase
solution; PLACE it into a 36 °C ± 1 °C constant temperature incubator for
incubation and enzymolysis for 16 h ~ 20 h; HEAT it in the 95 °C water batch
for 30 min; then rapidly COOL it to room temperature; TRANSFER it into a 100
mL volumetric flask; USE water to make the volume reach to the mark (V1).
6.2.2 Infant formula, cereals and other products (including raw and added
biotin). Accurately WEIGH an appropriate amount of sample (m) (containing
about 0.2 μg ~ 0.5 μg biotin), accurate to 0.001g; PLACE it into a 250 mL
volumetric flask; ADD 100 mL of sulfuric acid solution; MAKE it subjected to
hydrolysis at 121 °C for 30 min. After cooling, USE the sodium hydroxide
solution to adjust pH to 4.5 ± 0.2; TRANSFER it into a 250 mL volumetric flask;
USE water to make the volume reach to the mark; MIX it thoroughly. USE filter
paper to filter it; DISCARD the first few milliliters; ABSORB 5 mL of the filtrate;
ADD about 20 mL of water; USE sodium hydroxide solution to adjust the pH to
6.8 ± 0.2; TRANSFER it into a 100 mL volumetric flask; USE water to make the
volume reach to the mark (V1).
6.2.3 Strengthened biotin beverages or vitamin premixes and other samples.
as for the liquid beverage, ADD 5 mL ~ 10 mL of sample to a 100 mL
volumetric flask; ADD 50 mL of water; MIX it uniformly; TRANSFER it into a
100 mL volumetric flask; USE water to make the volume reach to the mark (V1);
as for the vitamin premixes. Accurately WEIGH appropriate amount of sample
(m), accurate to 0.001 g; PLACE it into a 500 mL volumetric flask; ADD about
300 mL of water; MIX it uniformly. ADJUST the pH to 8.0 ± 0.2; TRANSFER it
into a 1000 mL volumetric flask; USE water to make the volume reach to the
mark (V1).
6.3 Dilution
Appendix A
Medium and reagent
A.1 Lactobacillus agar medium
A.1.1 Component
TAKE 15.0 g of peptone milk, 5.0 g of yeast extract, 10.0 g of glucose, 100 mL
of tomato juice, 2.0 g of potassium dihydrogen phosphate, and 1.0 g of
polysorbate monooleate; ADD water to 1000 mL; ADJUST the pH to 6.8 ± 0.2
(25 °C ± 5 °C).
A.1.2 Preparation method
ADD 10.0 g of agar into A.1.1; HEAT to boil it to dissolve the agar; after mixing
it uniformly, CONTAIN it into different test tubes, 10 mL for each tube.
STERILIZE it at 121 °C under high pressure for 15 min; PREPARE for use.
A.2 Lactobacillus broth medium
A.2.1 Ingredients
TAKE 15.0 g of peptone milk, 5.0 g of yeast extract, 10.0 g of glucose, 100 mL
of tomato juice, 2.0 g of potassium dihydrogen phosphate, and 1.0 g of
polysorbate monooleate; ADD water to 1000 mL; ADJUST the pH to 6.8 ± 0.2
(25 °C ± 5 °C).
A.2.2 Preparation method
HEAT the ingredients in A.2.1 to boil it; MIX it uniformly and CONTAIN it into
different test tubes, 10 mL for each tube. STERILIZE it at 121 °C under high
pressure for 15 min
A.3 Biotin determination medium
A.3.1 Ingredients
TAKE 12.0 g of casein amino acid, 40.0 g of glucose, 20.0 g of sodium acetate,
0.2 g of L-cystine, 0.2 g of DL-tryptophane, 20.0 mg of adenine sulfate, 20.0
mg of guanine hydrochloride, 20.0 mg of uracil , 2.0 mg of thiamine
hydrochloride, 2.0 mg of riboflavin, 2.0 mg of nicotinic acid, 2.0 mg of
pantothenate, 4.0 mg of pyridoxine hydrochloride, 200.0 g of ρ-aminobenzoic
acid, 1.0 g of dipotassium hydrogen phosphate, 1.0 g of magnesium sulfate,
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