manual of methods for determining micronutrients in fortified foods

Note: Prior to start analyzing samples, determine the optimum growth time and calibrate the
spectrophotometer following the steps described in Steps X and XI.
E. Sample preparation
1. Grind solid samples and homogenize them.
2. In a 250-mL Erlenmeyer flask, weigh 2 g wheat flour.
3. Add 50 mL distilled water treated with activated carbon and mix well.
4. Add 5 mL amylase-10% to the cereal based samples or 1 mL pancreatine-4% to the dairy samples.
Cover with a 50-mL beaker or with aluminum paper and incubate at 37°C overnight.
5. Sterilize the solution in an autoclave at 121-123°C for 15 minutes.
6. Mix well and cool the flasks down in a water bath with ice until they reach room temperature.
7. Transfer to a 200 mL volumetric flask and bring to volume with distilled water treated with activated
charcoal and agitate the solution.
8. Centrifuge 25 mLsolution at 3,000 rpm for 5 minutes.
9. Take 5 mL supernatant and transfer to a 100 mL volumetric flask with distilled water treated with
activated charcoal and agitate.
10. If the solution is turbid, filter through filter paper Whatman No. 5 and if turbidity persists, filter
through a 0.45 µm membrane.
F. Microbiological assay
1. Prepare the following tubes with screw cap:
• 3 empty tubes for uninoculated blanks which will be used as sterilization controls.
• 3 empty tubes for inoculated blanks. Controls to check chemical contamination of reagents, media
and water used in the analysis, as well as the inoculum purity.
• 10 tubes containing 0.5, 1, 2, 3, 4 and 5 mL folic acid working standard solution-1 ng/mL, respectively.
Prepare each volume in duplicate.
• Per sample: 10 tubes containing 0.5, 1, 2, 3 and 4 mL sample final extract, in duplicate.
2. Add distilled water treated with activated charcoal to the tubes to get a final volume of 5 mL, as
indicated in the following table:
- 57 -