The alkaline solution was changed every 2 h. After removal of the supernatant, the alkali-treated samples were washed with distilled water until the neutral pH was reached. To extract PSC, the pre-treated eggshell membranes were soaked in 0.5 mol/l acetic acid with pepsin (15–90 U/mg defatted skin) for 12–72 h with gentle stirring. The amount of pepsin was calculated on the basis of activity. The mixture was filtered with two layers of cheesecloth
to remove undissolved debris. The solution was salted out by adding NaCl to a final concentration of 2 mol/l in the presence of 0.05 mol/l Tris (hydroxymethyl) amino methane (pH 7.0). The resulting precipitate was collected by centrifuging at 30,000g for 40 min at 5 C; the precipitate was then dissolved in 0.5 mol/lacetic acid at a sample/solution ratio of 1:10 (w/v). The final solution was dialyzed against cold distilled water using a dialysis membrane with molecular weight cut-off of 12 kDa for 12 h at 4 C, with a change of solution every 4 h. The solution was then lyophilized using a freeze dryer (ALPHA 2–4; Christ, Harz, Germany). From this point it was referred to as pepsin soluble collagen
(PSC). The yield of PSC was calculated on the basis of eggshell membrane weight after cleaning and expressed as a weight percentage.