Before dissolution, chicken feathers were washed with water, filtered, dried and
cut into small pieces or milled. Reactions were performed in a round flask containing
1.0 g chicken feathers, 40 g ionic liquid, 1.0 g NaHSO3 and 500 l water. The mixture
was agitated at 80 ◦C for 4 h. After the process was finalized, water (20 g, 50% wt,
based on the weight of ionic liquid) was added to the mixture and tri-phase systems
containing ionic liquid/parts of insolubilized feathers/keratin solution were
formed after centrifugation. Then the system of keratin solution was filtered, and
the filtrate was transferred to dialysis bag (MWCO 3500–5000 Da) and extensively
dialyzed against distilled water for 48 h. The protein concentration of the extracts
was measured by the Bradford protein assay method (Bio–Rad), using bovine serum
albumin as standard. The amount of protein dissolved was expressed as a percentage
of the total weight of dried feathers used. In addition, the other two systems
were vacuumized for 6 h (70 ◦C) and introduced into the fresh substrates for the
next batch. The recovery of ionic liquid was expressed as a percentage of the total
weight of it used.