Methodology for Anti-nutritional Factor

Phytic acid

Phytic acid (1, 2, 3, 4, 5, 6-hexakis dihydrogen phosphate myoinositol) is a common storage form of phosphorus in seeds and is also considered as an antinutritional factor. The complexing of phytic acid with nutritionally essential elements and the possibility of interference with proteolytic digestion have been suggested as responsible for antinutritional activity. The phosphorus in phytic acid is not nutritionally available to the monogastric animals. Phytic acid also interferes with calcium and iron absorption. Hence, estimation of phytic acid in food grains becomes essential especially in cereals.





Principle

The phytate is extracted with trichloroacetic acid and precipitated as ferric salt. The iron content of the precipitate is determined colorimetrically and the phytate phosphorus content calculated from this value assuming a constant 4 Fe: 6 P molecular ratio in the precipitate.


Materials
» 3% Trichloroacetic Acid
» 3% Sodium Sulphate in 3% TCA
» 1.5NNaOH
» 3.2NHNO3
» FeCl3 Solution (Dissolve 583mg FeCl3 in 100mL of 3% TCA).
» 1.5 M Potassium thiocynate (KSCN)
   (Dissolve 29.15g in 200mL water)
» Standard Fe(NO3)3 solution




Procedure
1.
Weigh a finely ground (40 mesh) sample estimated to contain 5 to 30mg phytate P into a 125mL Erlenmeyer flask.
2.
Extract in 50mL 3% TCA for 30 min with mechanical shaking or with occasional swirling by hand for 45 min.
3.
Centrifuge the suspension and transfer a 10mL aliquot of the supernatant to a 40mL conical centrifuge tube.
4.
Add 4mL of FeCl3 solution to the aliquot by blowing rapidly from the pipette.
5.
Heat the contents in a boiling water bath for 45 min. If the supernatant is not clear after 30 min, add one or two drops of 3% sodium sulphate in 3% TCA and continue heating.
6.
Centrifuge (10 to 15 min) and carefully decant the clear supernatant.
7.
Wash the precipitate twice by dispersing well in 20 to 25mL 3% TCA, heat in boiling-water for 5 to 10 min and centrifuge.
8.
Repeat washing with water.
9.
Disperse the precipitate in a few mL of water and add 3mL 1.5N NaOH with mixing.
10.
Bring volume to approximately 30mL with water and heat in boiling water for 30 min.
11.
Filter hot (quantitatively) through a moderately retentive paper Whatman No. 2.
12.
Wash the precipitate with 60 - 70mL hot water and discard the filtrate.
13.
Dissolve the precipitate from the paper with 40mL hot 3.2 N HNO3 into a 100mL volumetric flask.
14.
Wash paper with several portions of water, collecting the washings in the same flask.
15.
Cool flask and contents to room temperature and dilute to volume with water.
16.
Transfer a 5mL aliquot to another 100mL volumetric flask and dilute to approximately 70mL.
17.
Add 20mL of 1.5M KSCN dilute to volume, and read color immediately (within 1 min) at 480nm.
18.
Run a reagent blank with each set of samples.

Standard
Weigh accurately 433mg Fe(NO3)3 and dissolve in 100mL distilled water in a volumetric flask. Dilute 2.5mL of this stock standard and make up to 250mL in a volumetric flask. Pipette out 2.5,5,10,15 and 20mL of this working standard into a series of 100mL volumetric flasks and proceed from step 16.


Calculation
Find out the mg iron present in the test from the standard curve, and calculate the phytate P as per the equation.
Phytate P mg/100g sample =
mg Fe x 15
Weight of sample (g)




References

1. Wheeler, E L and Ferrel, R E (1971) Cereal Chem 48 312.