As the concentration of ethanol increased up to 75 vol.%, the yield
of EpSS reached 74.5%. By the addition of ethanol into the HS solution,
SS and ethanol both compete to hydrogen bond with water
molecules. While ethanol is fully miscible with water, SS is not. Precipitation
of SS in water could be observed when the solution was
left at room temperature for few days. Therefore ethanol wins this
competition resulting in the dehydration of SS and in the increase
in protein–protein interactions. As a result, SS precipitates by the
addition of ethanol, and the tendency increases with the amount
of ethanol added to the SS solution. Meanwhile, the yield of EpSS is
not dependent on stirring time (data not shown).