2.4. Antinutritional components: Phytic acid and total phenolic content
The phytic acid content (PAC) was determined using the phytic acid assay kit (Megazyme Inc., Sydney, Australia). All measurements were done in triplicate (n=3) and reported as the percentage of phytic acid on a dry weight basis (d.b.).
The total phenolic content (TPC) was determined using the Folin-Ciocalteu method according to Olukomaiya et al. (2020) with some modifications. In brief, 0.5 g of sample was extracted using 5 mL 80% (v/v) methanol for 15 min (stirring at 500 rpm and room temperature). The supernatant was collected after centrifugation at 5000 × g for 10 min and filtered with the Whatman No. 1 filter paper (Whatman International Ltd., Maidstone, UK). The remaining pellet was extracted again using 80% (v/v) methanol, followed by centrifugation and filtration under the same conditions to obtain an aqueous phenolic extract for analysis. A 0.1 mL aliquot of the extract was mixed with 2.5 mL 10% (v/v) Folin-Ciocalteu reagent. After a 2-min reaction time, 2.5 mL 7.5% (w/w) sodium carbonate was immediately added to the sample mixture and allowed to incubate in the dark for 30 min at room temperature. The absorbance was measured at 750 nm using a spectrophotometer (Genesys 10S UV-VIS, Thermo Scientific, Madison, WI, USA). Gallic acid (reagent grade) was used as the standard. All measurements were done with triplicate independent assays (n=3) and reported as milligrams of gallic acid equivalents (GAE) per gram of dry meal (mean ± standard deviation).