*4.4. Assay for Seed Phytate Content*

In order to measure the content of phytic acid we used Megazyme's kit K-PHYT 11/15 (Astori-Tecnica). Flour samples were obtained using a ball mill (Retsch MM200, Retsch GmbH Germany), grinding the seeds for 1 minute at 21 oscillations s<sup>−</sup><sup>1</sup> frequency. For each sample, in a beaker we added 20 mL of hydrochloric acid (0.66 M) to 1 g of flour that was vigorously stirred overnight at room temperature. 1 mL of extract was transferred into a 1.5 mL microfuge tube and centrifuged at 13,000 rpm for 10 min; then 0.5 mL of supernatant was transferred in a fresh microfuge tube and neutralized by the addition of 0.5 mL of sodium hydroxide solution (0.75 M). The neutralized extracts were submitted to enzymatic dephosphorylation reaction using the solutions supplied by the kit and

trichloroacetic acid (50% *w*/*v*). The reactions were done in duplicate, to determine free phosphorus and total phosphorus. For colorimetric determination of phosphorus, 1 mL of sample extract was transferred into a 1.5 mL microfuge tube with 0.50 mL of color reagen<sup>t</sup> (Ascorbic acid: 10%, Sulfuric acid: 1 M, Ammonium molybdate: 5%). The samples were mixed by vortex and incubated in a water bath at 40 ◦C for 1 h.

The standard phosphorus solutions were prepared as described in manufacturers' instructions, with the only modification that after preparation the standards were not treated as samples, i.e., incubated at 40 ◦C, but were left at room temperature for 1 hour. The phytic acid content was quantified by using a spectrophotometer (λ 655 nm). The data obtained with the Megazyme software were expressed as g phytic acid/100 g of flour that we converted into mg phytic <sup>P</sup>/g of flour.
