manual of methods for determining micronutrients in fortified foods

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D. Determination of folic acid (pteroylglutamic acid) in fortified foods by microbiology I. References Horwitz W and GW Latimer (Eds.). AOAC Official Method 944.12.Folic Acid (Pteroylglutamic Acid) in Vitamin Preparations. 45.2.03. Official Methods of Analysis of AOAC International. 18 th ed, Revision 2, 2007. Maryland. II. Principle The microbiological assay to determine folic acid in fortified foods is based on the requirement of Enterococcushirae (ATCC 8043) for free folic acid, which must be taken from the culture environment. The concentration of folic acid in the samples is measured indirectly by the growth of the microorganism, considering that the content of folic acid in the standards or samples is the limiting factor for the microorganism growth. This growth is compared with that in a folic acid standard solution. Turbidity is measured in the solutions and based on the response obtained for the standard solutions the folic acid concentration in the samples is calculated. The implementation of the folic acid assay requires several steps prior to testing samples. The bacteria (Enterococcushirae) must be propagated and maintained for the analysis. The optimum growth time must be determined in order to get an adequate response and spectrophotometer should be calibrated for the turbidity assay. III. Critical Points • The method is not applicable when extraneous turbidity or color interferes with the turbidimetric measurements. • Protect solutions from undue exposure to light throughout all stages. • Media is highly hygroscopic, therefore they must be stored in cool dry places or refrigeration. • When preparing media, cool down rapidly after sterilization to avoid pH changes, unwanted precipitation, loss of nutrients and darkening of the medium due to Maillard reaction. • Media from Hi Media have been used in the implementation of the method. Other brands can be used provided that they meet the nutrient requirements described in the AOAC method for the bacteria propagation, maintenance, inoculum and assay, but the bacteria growth may vary and the method might need to be standardized again with the new media brand. • Glassware used in the analysis must be washed with a neutral soap that do not stimulate the bacterial growth. After soaking the glassware in soap, it must be rinsed thoroughly with ultrapure water and heated in the oven to eliminate any trace of contamination. • Water used in the analysis must be ultrapure and treated with activated charcoal to eliminate any color impurities that might affect the analysis. • Use of inoculated and non-inoculated blanks is essential as controls to check contamination. • Periodically, check the purity of the inoculum by culturing some cells in Blood Agar and verify that only Enterococcushirae has grown. A Gram staining is recommended. - 50 -
IV. Equipment − Analytical balance (± 0.0001 g) − Autoclave − Automatic pipette − Automatic pipette 1 mL − Automatic pipette 50 µL − Blender − Bunsen burner − Centrifuge (5000 rpm) − Dessicator − Freezer (-10 to -20 °C) − Glass or disposiblecuvettes for readings in visible light − Hot plate with agitator − Incubator (35-37 °C) − pH meter (pH 1-14) − Quart cuvettes, 1 mL for readings in UV light − Refrigerator (4-8 °C) − UV/Visible Spectrophotometer − Thermometer (0-100 °C) − Vortex mixer − Water bath for incubation (35-40 °C) V. Materials − Beakers (50, 250, 600 mL) − Erlenmeyers (250, 500, 1000 mL) − Glass sterile vials of 5 mL − Membranes of 0.45 µm − Parafilm − Plastic sterile tubes for centrifuge (10, 25 and 50 mL) − Quantitative filter paper Whatman No. 1. − Quantitative filter paper Whatman No. 5. − Sterile pipette tips 2-200 µL − Sterile pipette tips for 50-1000 µL − Test tube rack − Test tubes 20mm x 150 mm with caps − Volumetric flasks (100, 200, 250, 500 mL) − Volumetric pipettes (1, 2, 3, 4 and 5 mL) VI. Reagents − BacteriaEnterococcushirae ATCC 8043 − α-Amylase from Bacillus sp. − Hydrochloric acid (HCl) p.a., 37%, mol wt. 36.46, d=1.19 g/mL, − Folic acid, grade USP. (C 19 H 19 N 7 O 6 ), mol wt. 441.4 − Broth for inoculum preparation for microbiological assays of vitamins (Micro vitamin test inoculum broth, Hi Media M133) - 51 -
Page 1 and 2: MANUAL OF METHODS FOR DETERMINING M
Page 3 and 4: - 3 -
Page 5 and 6: I. Introduction Food fortification
Page 7 and 8: A. Qualitative method to determine
Page 9 and 10: VII. Interpretation • Unfortified
Page 11 and 12: − − − − − − Refrigerato
Page 13 and 14: 3. Stir with a magnetic stirrer for
Page 15 and 16: IX. Calculations 1. Plot a graph of
Page 17 and 18: − 1,10-phenanthroline-monohydrate
Page 19 and 20: . Preparation of the ash solution 1
Page 21 and 22: III. Methods to determine vitamin A
Page 23 and 24: IV. Equipment and materials − 50
Page 25 and 26: VIII. Qualitycontrol Run the flour
Page 27 and 28: − Volumetric flasks (100 and 10 m
Page 29 and 30: VII. Procedure a. Preparation of sa
Page 31 and 32: c. Clean-up and concentration proce
Page 33 and 34: VIII. Calculations Results can be c
Page 35 and 36: IV. Methods to determine water-solu
Page 37 and 38: A. Method to determine riboflavin i
Page 39 and 40: VI. Standard solutions 1. Riboflavi
Page 41 and 42: 8. Pipette exactly 4.0 mL of the fi
Page 43 and 44: B. Determination of thiamin in flou
Page 45 and 46: In a 25-mL beaker weigh 0.9602 g so
Page 47 and 48: C. Determination of niacin in flour
Page 49: 7. Transfer the extract to a 250 mL
Page 53 and 54: 6. Ammonium hydroxide-0.1M: In a 10
Page 55 and 56: VIII. Procedure A. Strain propagati
Page 57 and 58: Note: Prior to start analyzing samp
Page 59 and 60: IX. Calculations 1. Calculate the a
Page 61 and 62: B. Verification 1. In an Erlenmeyer
Page 63 and 64: V. References • Bowman BA and Rus

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