Soluble protein classes
The classic Osborne soluble protein fractions (albumin, globulin,
prolamin, glutelin) were extracted sequentially from 10 g of ground,
defatted silflower seed by using water, dilute saline, ethanol, and
dilute alkali, and adapting the method used previously for milkweed seed
(Hojilla-Evangelista, Evangelista and Wu, 2009). The modified method
used higher solvent:solid ratios, longer extraction times, and only a
single extraction stage per solvent, which led to faster completion of
the extraction series. The changed ratio and shaking time for each
solvent are as follows: water - 40 mL/g, 30 min; 0.5 M NaCl - 40 mL/g,
30 min; 70% aqueous ethanol - 30 mL/g, 25 min; and 0.1 M NaOH - 30
mL/g, 25 min. Each extract was collected, centrifuged and analyzed for
soluble protein content by using the colorimetric Biuret method with
bovine serum albumin (BSA) as standard. Protein content of the solid
residue obtained after the extraction series was determined as described
for proximate composition. Water-extract was freeze-dried, while the
other extracts were first dialyzed (Spectra/Por membrane tubing, 34 mm
dia., 3.5 kDa molecular weight cut-off (MWCO), Spectrum Labs, Rancho
Dominguez, CA, against deionized water for three days and then
freeze-dried. Protein yield (%) was calculated as [(mass extracted ×
% protein in sample) / (starting mass × % protein in starting
sample)] ×100. The determination of soluble protein classes was done
in duplicate.