Chemical analysis
Moisture, ash and total protein content of whole seed and meal were
analysed according to AOAC Official Method 934.01, AOAC Method 942.05
and AOAC Method 990.03 with combustion-based nitrogen content using 6.25
as a conversion factor (Latimer & Horwitz, 2005), respectively. The
level of phytate was determined using the method described by Oomah et
al. (2008) with modifications. 2% (v/v) HCl and centrifugation at 1000
× g for 20 min were used to obtain the extract containing
phytate. The anion exchange column (AG-1-X8, Bio-k Laboratories Inc.)
was equilibrated with 0.08% (v/v) HCl before addition of extract and
the eluate (150 μL) was mixed with Wade reagent (50 μL;
FeCl3∙6H2O + sulfosalicylic acid) in a
microplate well. The extraction of phenolic compounds was carried out
using 70% aqueous acetone (1:30, w:v, meal-to-solvent ratio) at room
temperature for 1 h. The extract was then centrifuged at 10,000 ×g for 20 min and the absorbance of the recovered supernatant was
measured at 326 nm (Bio-Rad xMarkTM microplate
spectrophotometer, Bio-Rad Laboratories Inc.). Total phenolic content
was expressed as sinapic acid equivalent/g of meal as per Oomah et al.
(2010). Meal amino acid content (18 amino acids, asparagine and
glutamine combined with aspartic and glutamic acid, respectively) was
evaluated using AOAC Method 994.12 (Latimer & Horwitz, 2005).