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Biotechnology and Applied Biochemistry (2001) 33, (75–83) (Printed in Great Britain)

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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

Renato Froidevaux*, Naïma Nedjar-Arroume*¹, Luc Choisnard*, Muriel Bigan† and Didier Guillochon*

*Labotatoire de Technologie des Substances Naturelles, IUT 'A' Lille 1 – BP 179, 59653 Villeneuve d'Ascq Cedex, France, and †Laboratoire de Chimie Organique et Environnement, Bat. C4 – 59655 Villeneuve d'Ascq Cedex, France

Abbreviations used: LVVh-7, LVV-haemorphin-7; VVh-7, VV-haemorphin-7; TCA, trichloroacetic acid.

¹ To whom correspondence should be addressed (e-mail LTSN@univ-lille1.fr).

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.

Received 25 September 2000/23 November 2000; accepted 27 November 2000

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