2.6.2. Determination of phytate content

Phytate content was determined by the method described by Vaintraub and Lapteva (¹⁹⁸⁸). About 0.100 g of sample was extracted with 10 ml of 2.4% HCl in a mechanical shaker (Hy‐2(C), Shanghai, China) for 1 hr at a room temperature. The extract was centrifuged (Sigma 2‐16KC, UK) at 3,000 rpm for 30 min. The clear supernatant was used for phytate estimation. One ml of wade reagent (containing 0.03% solution of FeCl₃.6H₂O and 0.3% of sulfosalicylic acid in water) was added to 3 ml of the sample solution (supernatant), and the mixture was mixed on a vortex mixer for 5 s. The absorbance of the sample solutions was measured at 500 nm using UV‐VIS spectrophotometer (JASCO V‐630, Shimadzu Corporation, Tokyo, Japan).). A series of standard solutions from sodium salt of phytic acid were prepared to contain 0.0, 4.5, 9.0, 18.0, 27.0, and 36.0 μg/ml of phytic acid (analytical grade sodium phytate) in 0.2N HCl. One ml of the wade reagent was added to each test tube, and the solution was mixed on a Vortex mixer for 5 s. The mixture was centrifuged for 10 min, and the absorbance of the sample and standard was measured at 500 nm by using deionized water as a blank. The phytate content was determined from standard curve of sodium salt of phytic acid, and result was reported in mg /100g.