Effect of leucine on aroma volatiles production from Ceratocystis fimbriata grown in liquid culture L. Sanchez 1,2, * ,  , R.L. Seila 3 , P. Christen 4 and S. Revah 1 1 Department of Chemical Engineering, Universidad Auto ´noma Metropolitana-Iztapalapa, AP 55-534, CP 09340, Me´xico City, Me´xico 2 Department of Chemical Engineering, Facultad de Estudios Superiores-Zaragoza, Universidad Nacional Auto ´noma de Me´xico, Me´xico City, Me´xico 3 US Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA 4 IRD (Institut Francais de Recherche Scientifique pour le De´velopment en Coope´ration), Cicero ´n 609, CP 11530, Me´xico City, Me´xico *Author for correspondence: Tel.: +52-5-6926162, Fax: +52-5-7736331, E-mail: leosgf_50@yahoo.com   Present address: Fernando Lizardi No. 14, Colonial Iztapalapa, CP 09270, Iztapalapa DF, Mexico Received 13 June 2001; accepted 1 December 2001 Keywords: Aroma, Ceratocystis fimbriata, flavouring, leucine, synthetic liquid culture, volatiles Summary Aroma volatiles produced by Ceratocystis fimbriata on a defined liquid synthetic medium with and without the addition of leucine were identified by gas chromatography–mass spectrometry and quantified by gas chromato- graphy-flameionisationdetectionintheliquidmediumaswellasintheheadspace.Volatileswereextractedfromthe liquid by simultaneous steam distillation–solvent extraction. Ceratocystis fimbriata produced a complex set of volatileintermediarymetabolites,ofwhichethanolwasthedominantcompound(92–95%oftotalvolatiles).Low molecular weight esters, alcohols, aldehydes, ketones, alkanes, and carboxylic acids were identified in the liquid broth. Alcohols and esters were the most abundant aroma volatiles. Leucine addition effected further growth and higher volatiles production. In the headspace, ethanol and ethyl acetate accounted for 92% of total volatiles over the synthetic medium and 89% when leucine was added. Aroma perception (fruity and banana) correlated closely with liquid and headspace total volatiles. Introduction Flavours and fragrances are important for the food, feed, cosmetic, chemical and pharmaceutical industries. Most available flavour compounds are currently pro- duced via chemical synthesis or plant extraction. The formationofundesirableby-productsandtheconsumer aversion to chemicals added to food, cosmetics and other household products suggests investigation of biological sources of flavour compounds. Thus, flavour compound synthesis by biotechnological processes is beginningtoplayanincreasingroleinthefoodindustry. Micro-organisms are an alternative to plant sources, whicharestronglydependentonfactorsthataredifficult tocontrol,suchasweather,riskofplantdiseases,socio- political instability of major supplying areas, and trade restrictions (Janssens et al. 1992; Bernd & Berger 1994; Fabre et al. 1995, 1999; Krings & Berger 1998). Several authors have reported fungi as aroma pro- ducers: Trichoderma viride forcoconutflavour(Serrano- Carreon, et al. 1992), Penicillium decumbens for rose, pine and fruity flavours (Kaminski et al. 1974), and P. roqueforti forbluecheeseflavour(Revah&Lebeault 1989; Larroche et al. 1994). Ceratocystis fimbriata is a filamentous fungus, char- acterized by the production of a fruit-like aroma (Sprecher & Hanssen, 1983). A liquid nutritive medium was optimized for flavour production from C. fimbriata (Christen & Raimbault, 1991). Christen et al. (1997) investigated the effect of various supports and sub- strates,aeration,andadditionofaminoacidprecursors on fruity aroma production by C. fimbriata on solid state fermentation media. They found that volatiles production was dependent on the composition of the mediumandcultureconditions;andthattheadditionof the amino acids leucine and valine enhanced flavour production, because they are precursors of 2- and 3- methylbutanol which have a banana aroma and pro- moteacetateesterswhichhaveanevenstrongerbanana aroma (Meza et al. 1998). Christen et al. (1997) identified only headspace vola- tiles of C. fimbriata grown in liquid media by gas World Journal of Microbiology & Biotechnology 18: 231–237, 2002. 231 Ó 2002 Kluwer Academic Publishers. Printed in the Netherlands.