This standard supersedes “National Food Safety Standard - Determination of Free Biotin in Foods for Infants and Young Children, Milk and Milk Products” (GB 5413.19-2010)
Compared with GB 5413.19-2010, the main changes of this standard are as follows:
——the standard name is revised as "National Food Safety Standard - Determination of Biotin in Foods";
——the expression manner for preparation of standard curved tube and tube for solution to be determined is modified;
——the expression manner for determination procedures is modified;
——the processing procedure for foods of cereals, potatoes, meat, fresh fruit and vegetables, alga samples, eggs, peas, nuts, entrails and those to strengthen biotin.
——the description of all-purpose glassware in the instruments and equipment is deleted.
NATIONAL STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
中華人民共和國(guó)國(guó)家標(biāo)準(zhǔn)
GB5009.259-2016
National Food Safety Standard -
Determination of Biotin in Foods
食品安全國(guó)家標(biāo)準(zhǔn)
食品中生物素的測(cè)定
1 Scope
This Standard specifies determination method of biotin in foods.
This Standard is applicable to determination of biotin in foods.
2 Principles
Biotin is the nutrient necessary for growth of lactobacillus plantarum. In the biotin determination culture medium, the growth of lactobacillus plantarum is linearly related to the content of biotin in the specimen to be determined, so the content of the substance to be determined in the specimen can be calculated by comparing the light transmittance and standard work curve.
3 Reagents and Materials
Unless otherwise specified, analytically-pure reagents and Grade II water (defined in GB/T 6682) are adopted for the purposes of this method.
3.1 Reagents
3.1.1 Dehydrated alcohol (C2H6O).
3.1.2 Sodium hydroxide (NaOH).
3.1.3 Hydrochloric acid (HCl).
3.1.4 Citrate.
3.1.5 α-amylase ≥ 1.5U/mg.
3.1.6 Papayotin: ≥ 5U/mg.
3.1.7 Sulfuric acid (H2SO4).
3.2 Preparation of reagents
3.2.1 Ethanol solution (50%): measure 500mL of dehydrated alcohol and mix it well with 500mL of water;
3.2.2 Sodium hydroxide solution (0.5 mol/L): weigh 20g of sodium hydroxide, dissolve it in 1,000mL of water and mix well.
3.2.3 Sodium chloride solution (0.85 %): weigh 8.5g of sodium chloride, dissolve it in water and dilute to 1,000mL, and mix well.
3.2.4 Hydrochloric acid solution (1mol/L): pipet 83 mL of hydrochloric acid, dilute with water to 1,000mL, and mix well.
3.2.5 Citrate buffer solution (pH 4.5): weigh 1.5g of citric acid and put it in a beaker (100mL) with magnetic stirrer, add about 50mL of distilled water into it for dissolution and then 12 mL of NaOH (1mol/L) solution, after that adjust the pH value to 4.5 with HCl (0.1 mol / L) solution. Transfer the solution to 100mL volumetric flask and scale the volume with distilled water. The buffer solution can be stored for 3d at 2℃ ~ 8℃.
3.2.6 Papayotin-amylase solution: weigh papayotin and α-amylase, 200 mg each, add in 20mL of water and grind them to homogenate. Centrifuge the homogenate at the speed of 3,000 r/min for 5min ~10 min. Prepare immediately before use.
3.2.7 Sulphuric acid solution (3%): measure 30mL of sulfuric acid, add it into 1,000mL of water, and mix well.
3.3 Standard product
Standard product of biotin (d-Biotin or Vitamin H) (C10H16N2O3S): purity ≥ 99%.
3.4 Preparation of standard solution
3.4.1 Biotin standard stock solution (100 μg/mL): accurately weigh 100 mg of biotin standard sample, dissolve it with ethanol solution (50%), then transfer it to 1,000mL volumetric flask, and dilute to the scale. Keep it in a brown bottle and preserve in the refrigerator (2℃~4℃) for 12 months.
3.4.2 Biotin standard intermediate solution (1.0 μg/mL): accurately pipet 1.00 mL of biotin standard stock solution, put it into 100mL brown volumetric flask and dilute it with ethanol solution (50%) to the scale. Mix it well, store in bottles and preserve in the refrigerator (2℃ ~ 4℃) for 6 months.
3.4.3 Biotin standard working solution (10ng/mL): accurately pipet 1.00 mL of biotin standard intermediate solution and put it into 100mL volumetric flask, dilute it with water to the scale, then mix well. Prepare immediately before use.
3.4.4 Standard working solution (10 ng/mL): prepare the solutions in two concentrations, with the higher one of 0.2 μg/mL and the lower one of 0.1 ng/mL. Pipet 5mL of working solution for twice, and dilute them with water to 250 mL and 500mL respectively.
3.5 Culture medium
3.5.1 Lactobacillus agar medium: prepare according to Appendix A.
3.5.2 Lactobacillus broth medium: prepare according to Appendix A.
3.5.3 Medium for biotin determination: prepare according to Appendix A.
Note: some merchandized synthetic mediums are effective, and they can be prepared according to descriptions on the label.
4 Instruments and Apparatus
4.1 Balance: with the sensibility of 0.1mg.
4.2 Constant temperature incubator: 37℃±1℃.
4.3 Pressure steam sterilizer: 121℃(0.10mPa~0.12mPa).
4.4 Vortex oscillator.
4.5 Centrifuge: rotation speed ≥2,000r/min.
4.6 Liquor separator: 0mL~10mL.
4.7 Adjustable electric furnace.
4.8 pH meter: with the precision of ± 0.01.
4.9 Spectrophotometer.
4.10 Super-clean bench.
Note: prior to use of glassware instrument, clean the hard glass measuring tubes and other necessary glassware with active agent (made by adding 1-dodecane sulfonic acid sodium salt or household detergent into washing water), and subject them to dry heat (200℃) for 2h.
5 Preparation and Preservation of the Bacteria Strain
5.1 Bacteria strain
Lactobacillus plantarum (ATCC8014).
5.2 Preparation of bacteria strain for reservation
5.2.1 Transfer the bacterial strain lactobacillus plantarum (ATCC 8014) to lactobacillus agar medium, culture in the constant temperature incubator (37℃±1℃) for 20h~24h, and then take out to preserve it in the refrigerator (2℃ ~4℃) as reserved bacterial strain, which shall be reproduced at least once a month.
5.2.2 Inoculate the reserved bacterial strain to lactobacillus agar medium, and culture in the constant temperature incubator (37℃±1℃) for 20h~24h to activate it for preparation of inoculation fluid. The reserved bacteria which has been preserved for a few weeks can't be used immediately for inoculation fluid preparation, and it shall reproduce continuously for 2~3 generations before test to ensure bacteria activity.
5.3 Preparation of inoculation fluid
Inoculate the activated bacterial strain to the sterilized lactobacillus broth with transfer loop and culture in the constant temperature incubator (37℃±1℃) for 16h~20h. Take out the bacterial suspension, centrifuge it and discard the supernatant; then vibrate uniformly with sodium chloride solution (0.85 %), centrifuge it and discard the supernatant, and repeat the operations for three times. Finally, dilute it with sodium chloride solution (0.85 %) until its light transmittance reaches 80 %.
Contents
Foreword i
1 Scope
2 Principles
3 Reagents and Materials
4 Instruments and Apparatus
5 Preparation and Preservation of the Bacteria Strain
6 Analysis Steps
7 Precision
8 Others
Appendix A Medium and Reagents
