SAMPLING TECHNIQUES AND COMPARATIVE EXTRACTION PROCEDURES FOR QUANTITATIVE-DETERMINATION OF INTRACELLULAR AND EXTRACELLULAR METABOLITES IN FILAMENTOUS FUNGI

Two methods for rapid sampling and three procedures for extraction of metabolites from the filamentous fungus Monascus ruber were compared. It is shown that arrest of metabolism by either dropping the mycelial cultures in liquid nitrogen or by spraying them on a 60% solution of m ethanol kept at -40 degrees C followed by rapid centrifugation at -10 degrees C were equally effective. Metabolites were extracted from myce lia using different procedures including acid and alkaline treatments; permeabilization by cold chloroform and extraction by boiling buffere d ethanol, to demonstrate that the litter method gave the best results both in terms of recovery and stability of metabolites. In addition, this method is very simple to handle and allows the use of very low am ounts (i.e. 10-20 mg dry mass) of cellular material since the removal of ethanol by evaporation after extraction results in a concentration step of metabolites. (C) 1998 Federation of European Microbiological S ocieties. Published by Elsevier Science B.V. All rights reserved.