manual of methods for determining micronutrients in fortified foods

B. Verification
1. In an Erlenmeyer flask containing 300 mL medium for folic acid assay (HiMedia 126) aseptically
add 1 mL standard folic acid solution-100 µg/mL.
2. Sterilize in autoclave for 5 minutes at 121°C and cool down.
3. Add 1 mL working inoculum suspension.
4. Incubate for 16 hours at 37 °C.
5. Aseptically, centrifuge at 3000 rpm for 5 minutes and wash with 50 mL0.9% sterile saline solution.
Repeat this step three times and resuspend the cells in 25 mL isotonic sterile solution.
6. In aseptic conditions and using a sterile volumetric pipette, take 10 mL suspension prepared in step
5 and dispense it in a porcelain crucible.
7. Evaporate this aliquot in a water bath at 100°C.
8. Dry to constant weight in a vacuum oven at 110 ºC.
9. Considering that 10 mL saline solution have 0.09 g sodium chloride, calculate the weight of bacteria
(W B
) (g) using the following formula:
W B
dry(g) = (Crucible weight + dry solids) − Crucible weight − 0.09
10. Prepare 50 mL bacteria solution where every mL is equivalent to 0.5 mg dry cells. For this, take an
aliquot of solution prepared in step 4. Calculate the volume of this aliquot with the following equation:
€
10 mL
Aliquot volume (mL) = 50mL x
W B
dry (g) x 1 g 0.5 mg
×
1000 mg 1 mL
11. In different tubes, add 0 (Blank), 1, 1.5, 2, 2.5, 3, 4 and 5 mL diluted aliquot in step 10. Carry out
this step in duplicate. Prepare two tubes for blanks without inoculum.
12. Add
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0.9%-sterile saline solution to the tubes as necessary to complete the volume to 5 mL.
13. Add 5 mL medium for folic acid assays (HiMedia 126).
14. Agitate and read at 550 nm, setting 100% transmittance with the tubes with 0 mL or blanks.
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