UNCORRECTED PROOF FOOD MICROBIOLOGY www.elsevier.nl/locate/jnlabr/yfmic Food Microbiology ] (]]]]) ]]]–]]] Identification of yeasts isolated from wine-related environments and capable of producing 4-ethylphenol L. Dias a , S. Dias a , T. Sancho a , H. Stender b , A. Querol c , M. Malfeito-Ferreira a, *, V. Loureiro a a Laborat ! orio de Microbiologia, Departamento de Bot # anica e Engenharia Biol ! ogica, Instituto Superior de Agronomia, 1349-017 Lisbon, Portugal b AdvanDx Inc., Concord, MA, USA c Departamento de Biotecnologia, Instituto de Agroqu ! ımica y Tecnologia de Alimentos, P.O.Box 73, 46100 Burjassot, Valencia, Spain Received 4 June 2002; accepted 4 November 2002 Abstract The ability to produce 4-ethylphenol from the substrate p-coumaric acid in synthetic media was evaluated for several yeast species associated with wine production. Molar conversion rates as high as 90% were found by only Dekkera bruxellensis, D. anomala and by some unidentified strains isolated from wine-related environments. Other unidentified strains produced traces of 4-ethylphenol. All unidentified strains showed the same cultural characteristics as D. bruxellensis when grown on DBDM (Dekkera/Brettanomyces differential medium) agar. The determination of long-chain fatty acid compositions and the utilization of peptide nucleic acid (PNA) probes specific for D. bruxellensis showed that the unidentified strains did not belong to this species. Further identification, by restriction pattern generated from PCR-amplification of the 5.8S rRNA gene and the two internal transcribed spacers (ITS), assigned the unidentified strains to Candida cantarelli, C. wickerhamii, Debaryomyces hansenii, Kluyveromyces lactis and Pichia guilliermondii. However, only some strains of P. guilliermondii were capable of converting p-coumaric acid into 4-ethylphenol with efficiencies close to those observed in D. bruxellensis and D. anomala. r 2002 Elsevier Science Ltd. All rights reserved. 1. Introduction The volatile phenols are a group of compounds with a strong influence on wine flavour. The main molecules are 4-ethylphenol and 4-ethylguaiacol, in red wines, and 4-vinylphenol and 4-vinylguaiacol, in white wines (Chatonnet et al., 1992). In particular, high amounts of 4-ethylphenol in red wines result an unpleasant taint described as ‘‘phenolic odour’’ (Chatonnet et al., 1992; Rodrigues et al., 2001). The relation between high concentrations of 4-ethylphenol in wines and the activity of yeasts of the genus Dekkera, or its anamorph Brettanomyces, has been largely debated and was recently established (Chatonnet et al., 1995; Chatonnet et al., 1997). The fermenting yeast Saccharomyces cerevisiae, other yeast species or lactic bacteria are regarded as weak and non-ethylphenol producers under wine conditions (Chatonnet et al., 1992; Chatonnet et al., 1995; Chatonnet et al., 1997). The isolation of Dekkera/Brettanomyces sp. from wine-related environments is not easy due to their slow growth rates and low relative occurrence (Fugelsang, 1997; Kunkee and Bisson, 1993). To overcome such difficulties several selective and differential media have been developed (see references cited in Rodrigues et al., 2001) and recently one medium (named DBDM, after Dekkera/Brettanomyces differential medium) was re- ported as being able to recover Dekkera sp. from wines where these yeasts were present in proportions lower than 1% of the total microbial flora (Rodrigues et al., 2001). Assuming that these yeasts were the single 4- ethylphenol producers isolated from wines, as stated by Chatonnet et al. (1992, 1995, 1997), the isolates obtained with this medium were assigned to Dekkera/Brettano- myces sp. according to their colonial characteristics and after the assessment of 4-ethylphenol production (Ro- drigues et al., 2001). Therefore, these probable Dekkera sp. strains lacked identity confirmation. ARTICLE IN PRESS 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 3B2v7:51c GML4:3:1 YFMIC : 574 Prod:Type:COM pp:128ðcol:fig::3Þ ED:MariammaOmmen PAGN: gopal SCAN: Shobha *Corresponding author. Tel.: +351-21-3653100; fax: +351-21- 3635031. E-mail address: mmalfeito@isa.utl.pt (M. Malfeito-Ferreira). 0740-0020/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0740-0020(02)00152-1