1 Scope
This standard specifies the determination method of free biotin in foods for infants and young children, milk and milk products.
This standard is applicable to the determination of free biotin in foods for infants and young children, milk and milk products.
2 Normative References
The normative documents in this standard are essential to the application of this standard. For dated references, only the dated edition of the normative document referred to applies. For undated references, the latest edition of the normative document (including all the amendments) referred to applies.
3 Principles
The content of materials to be tested in the specimen is determined quantitatively by virtue of the specificity and sensitivity of lactobacillus plantarum to free biotin. In the culture medium containing all the nutrient contents except the material to be tested, microorganism growing and content of material to be tested are in the linear relationship. Therefore the content of material to be tested in the specimen may be calculated according to the comparison between light transmittance and standard working curve.
4 Reagents and Materials
Unless otherwise specified, reagents used in this method are all analytically pure, and water is the Grade 2 water as specified in GB/T 6682.
4.1 Bacterial strain: lactobacillus plantarum, ATCC 8014.
4.2 d-Biotin or Vitamin H standard substance (C10H16N2O3S): purity≥99%.
4.3 Culture medium
4.3.1 Lactobacillus agar medium: see Appendix A.
4.3.2 Lactobacillus broth medium: see Appendix A.
4.3.3 Culture medium for determining biotin: see Appendix A.
Note: some commercialized synthetic media are featured by favorable effects, and commercialized synthetic media shall be prepared according to the instructions of the labels.
4.4 Ethanol solution (50%).
4.5 Sulfuric acid solution (3%): measure 3mL sulfuric acid, add it into water, and make it to 100mL constant volume.
4.6 Sodium hydroxide solution (0.5mol/L): weigh 20g sodium hydroxide and dissolve it into 1000mL water.
4.7 Sodium chloride solution (9 g/L): weigh 9.0g sodium chloride, dissolve it in 1000mL water, put each 10mL into a test tube respectively, and sterilize for 15min.
