Using an experimental design for the optimization of LVV-haemorphin-7 and VV-haemorphin-7 extraction by an organic solvent mixture in the course of bovine haemoglobin peptic hydrolysis.

Mixture design was used to improve the extraction of two opioid peptides (LVV-haemorphin-7 and VV-haemorphin-7) by water-immiscible solvents in the course of bovine haemoglobin peptic hydrolysis. Because of the loss of the peptic activity, these two haemorphins did not appear in the aqueous phase when the peptic reaction was achieved in the presence of butan-2-ol alone. We have shown that it is possible to use octan-1-ol, as a co-solvent, to recover the peptic activity. However, when the hydrolysis was achieved with octan-1-ol alone, the two haemorphins appeared in the aqueous phase, but were not extracted by the organic phase. We therefore investigated haemorphin extraction in the course of the hydrolysis reaction by a mixture of butan-2-ol and octan-1-ol. We have determined the optimal conditions with respect to the extraction of opioid peptides and the stability of the pepsin. To design a future continuous-stirred-tank reactor, we propose a biphasic system composed of 45% water, 45% butan-2-ol and 10% octan-1-ol for the extraction of the two haemorphins in the course of bovine haemoglobin peptic hydrolysis.